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Stem Cell Therapy for Autoimmune Disease 1st Edition
Richard K. Burt (Author) Digital Instant Download
Author(s): Richard K. Burt (Author)
ISBN(s): 9781587060311, 1000724670
Edition: 1
File Details: PDF, 110.93 MB
Year: 2004
Language: english
Stem Cell Therapy
for Autoimmune Disease
Stem Cell Therapy
for Autoimmune Disease

Richard K. Burt
Chief, Division o f Im m unotherapy
D epartm ent of M edicine
Feinberg School o f M edicine
N orthw estern University
Chicago, Illinois, U.S.A.

Alberto M. Marmont
Professor Em eritus
Division of H em atology and Stem Cell T ransplantation
San M artin o ’s H ospital
Genoa, Italy

Boca Raton London New York

CRC Press is an imprint of the

Taylor & Francis Group, an informa business
 STEM CELL THERAPY FOR AUTOIMMUNE DISEASE

First published 2004 by Landes Bioscience

Published 2019 by CRC Press

Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742

© 2004 by Taylor & Francis Group, LLC

CRC Press is an imprint of Taylor & Francis Group, an Informa business

No claim to original U.S. Government works

ISBN 13: 978-1-58706-031-l (hbk)

This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish
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Library of Congress Cataloging-in-Publication Data

Stem cell therapy for autoimmune disease/ [edited by] Richard K. Burt,
Alberto M. Marmont.
p.; cm.
Includes index.
Includes bibliographical references and index.
ISBN 1-58706-031-0
I. Autoimmune diseases--T reatment. 2. Stem cells--T ransplantation.
[DNLM: 1. Autoimmune Diseases--cherapy. 2. Stem Cell
Transplantation. WD 305 S824 2004] I. Burt, Richard K., 1956- II.
Marmont, A. M. (Alberto M.)
RC600.S746 2004
616.97'806--dc22
2003026938
 T his b o o k is in appreciation o f the support, encouragem ent,
and patience o f m y w ife, Shalina, children, M ichael, Rajan, Reena, Shantha,
and m y patients w ho have allow ed m e the priviledge to be their doctor.
A ll o f w hom have been the inspiration to continue.
Richard K Burt

T his b ook is dedicated to Paul Ehrlich, w h o created the

fail-safe dictum o f "horror autotoxicus", but also appreciated that
violation o f this concept was conducive to disease.
Alberto M. M armont
 CONTENTS
1. When is a Stem Cell Really a Stem Cell? 1 13. The Extracellular Matrix as a Substrate
Gerald J. Spangrude for Stem Cell Growth and Development
and Tissue Repair 87
2. Embryonic Stem Cells: Unique Potential Stephen F. Badylak and M ervin C. Yoder
to Treat Autoimmune Diseases 5
D an S. K aufm an a nd James A. Thomson 14. Gene Transfer into Human Hematopoietic
Stem Cells: Problems and Perspectives 92
3. Neural Stem Cells and Oligodendrocyte Serguei Kisselev, Tatiana Seregina ,
Progenitors in the Central Nervous System 11 Richard K. B urt and Charles /. Link
Jennifer A. Jackson and D iana L. Clarke
15. The Etiopathogenesis of Autoimmunity 106
4. Turning Blood into Liver 18 Howard A m ital and Yehuda Shoenfeld
Bryon E. Petersen and N eil D. Theise
16. Overview of Immune Tolerance Strategies 113
5. Adipose Tissue-Derived Adult Stem Cells: Charles J. H ackett and Helen Quill
Potential for Cell Therapy 24
Laura Aust, Lyndon Cooper, Blythe Devlin , 17. Death Receptor-Mediated Apoptosis
Tracey du Laney, Sandra Foster, and Lymphocyte Homeostasis 119
Jeffrey M . Gimble, Farshid Guilaky Lixin Zhengy Richard M. Siegely
Yuan D i C. Halvorseny Kevin Hicoky Jagan R. M uppidi, Felicita H ornung
A m y Kloster, H enry E. Rice, A nindita Sen, a nd Michael J. Lenardo
Robert W. Storms and William O. Wilkison
18. Shifting Paradigms in Peripheral Tolerance 132
6. Hematopoietic Stem Cell Biology: Jonathan D. Powell and Ronald H. Schwartz
Relevance to Autoimmunity 31
Richard J. Jones 19. Dendritic Cells Control the Balance
between Tolerance and Autoimmunity 139
7. Properties and Therapeutic Potentials Sim on W. F. M illing and G. Gordon MacPherson
o f Adult Stem Cells from Bone Marrow
Stroma (MSCs) 35 20. CD4+ T Regulatory Cells and Modulation
D arw in J. Prockop of Undesired Immune Responses 148
Rosa Bacchetta, Megan K Levings
8. Regeneration of Cardiomyocytes from Bone and Maria-Grazia Roncarolo
Marrow Stem Cells and Application
to Cell Transplantation Therapy 39 21. Major Histocompatibility Complex
Keiichi Fukuda and Autoimmune Disease 155
Ursula Holzer and Gerald T. N epom
9. Clinical Trials of Hematopoietic Stem Cells
for Cardiac and Peripheral Vascular Diseases 49 22. Analyzing Complex Polygenic Traits:
Hiroaki M atsubara The Role of Non-HLA Genes in the
Susceptibility to Autoimmune Disorders 164
10. The Stem Cell Continuum: A Plastic Plasticity 55 Bernard R. Lauwerys and Edward K. Wakeland
Peter J. Quesenberry ; Jean-Francois Lamberty
Gerald A. Colvin , M ark Doonerf 23. Drug-Induced Autoimmunity 173
Christine I. McAuliJfet Mehrdad Abedi , Robert L. Rubin and Anke K retz-Romm el
Deborah Greer, Delia Demers , Jan Cerny,
Brian E. Moore , Evangelos Badiavas 24. Evidence for a Role of Infections
and Vincent Falanga in the Activation of Autoreactive T Cells
and the Pathogenesis of Autoimmunity 182
11. Adult Stem Cell Plasticity 59 /. Ludovic Croxford and Stephen D. Miller
Sean Lee and Diane S. Krause
25. Molecular Analysis of Immunity 194
12. Collection and Expansion of Stem Cells 73 D aniel Douek
Linda Kelley and Ian McNiece
26. Immune Reconstitution after Hematopoietic 37. Hematopoietic Stem Cell Transplantation
Stem Cell Transplantation 206 for Multiple Sclerosis: Finding Equipoise 309
Andreas Thiel, Tobias Alexander, Athanasios Fassas and Richard K. B urt
Christian A. Schm idt, Falk Hiepe,
Renate Arnold, Andreas Radbruch, 38. Molecular and Cellular Pathogenesis
Larissa Verda and Richard K. B urt o f Systemic Lupus Erythematosus 320
George C. Tsokos, Yuang-Taungluang,
27. Historical Perspective and Rationale Christos G. Tsokos
o f HSCT for Autoimmune Diseases 223 a nd M adhusoodana P. N am biar
Alberto M . M a rm o n t
39. Definition, Classification, Activity
28. High-Dose Immune Suppression and Damage Indices in Systemic Lupus
without Hematopoietic Stem Cells Erythematosus 328
for Autoimmune Diseases 232 Jennifer M. Grossman and Kenneth C. Kalunian
Robert A. Brodsky
40. Lupus Nephritis 339
29. Autologous Stem Cell Transplantation A nnie Y. Suh and Robert M . Rosa
in Animal Models o f Autoimmune
Diseases 237 41. Hematopoietic Stem Cell Transplantation
D.W. van B ekkum for Systemic Lupus Erythematosus 347
A n n E. Traynor, Richard K. B urt
30. Allogeneic Hemopoietic Stem Cell and Alberto M arm ont
Transplantation in Animal Models
o f Autoimmune Disease 245 42. Treatment of Rheumatoid Arthritis 358
Susum u Ikehara Stuart Weisman a nd A rth u r Kavanaugh

31. Mobilization and Conditioning Regimens 43. Haemopoietic Stem Cell Transplantation
in Stem Cell Transplant for Autoimmune for Rheumatoid Arthritis—World
Diseases 253 Experience and Future Trials 367
Ewa Carrier and Richard K. B urt John A. Snowden, John J. Moore,
Sarah J. Bingham, Steve Z . Pavletic
32. Infection in the Hematopoeitic Stem Cell a nd Richard K. B urt
Transplant Recipient with Autoimmune
Disease 44. Autologous Stem Cell Transplantation
262
Valentina Stosor and Teresa R. Zem bower for Refractory Juvenile Idiopathic
Arthritis (JIA) 378
33. Immunological Aspects of Multiple Sclerosis Nico W ulffraat
with Emphasis on the Potential Use
o f Autologous Hemopoietic Stem Cell 45. Immunology of Scleroderma 388
Carol M. A rtlett
Transplantation 277
Paolo A. Muraro, H enry F. McFarland
and Roland M artin
46. Hematopoietic Stem Cell Transplantation
for Systemic Sclerosis 398
34. Axonal Injury and Disease Progression A ndrew M. Yeager, D iane BuchBarker,
Thom as A. Medsger, Jr.
in Multiple Sclerosis 284 a nd A lbert D. Donnenberg
Carl B jartm ar a n d Bruce D. Trapp

47. High-Dose Immunosuppressive

35. Monitoring Disease Activity
Chemotherapy with Autologous Stem
in Multiple Sclerosis 290
Cell Support for Chronic Autoimmune
Lorri Lobeck
Thrombocytopenia 404
Richard D. H uhn, Patrick F. Fogarty,
36. Intense Immunosuppression Followed
Ryotaro N akam ura a nd Cynthia E. D unbar
by Autologous Stem Cell Transplantation
in Severe Multiple Sclerosis Cases: 48. High-Dose Chemotherapy
MRI and Clinical Data 303 with Haematopoietic Stem Cell
G.L. Mancardi, R. Saccardi, A. M urialdo,
Transplantation in Primary Systemic
F. Pagliai, F. Gualandi, A. M arm ont,
M. Inglese, P. Bruzzi, M.P. Sormani,
Vasculitis, Behçet’s Disease
M.G. Marrosu, G. Meucci, L. Massacesi, and Sjogren’s Syndrome 411
A. Bertolotto, A. Lugaresi, E. Merelli, Christoph Fiehn and M anfred Hensel
M . Filippi a nd the Italian Gitm o-Neuro
Intergroup on A S C T fo r M ultiple Sclerosis
49. Hematopoietic Stem Cell Transplantation 54. Autologous Hematopoietic Stem Cell
in the Treatment of Chronic Inflammatory Transplantation for Crohn’s Disease 448
Demyelinating Polyradiculoneuropathy 419 Robert M . Craig and Richard K. B u rt
George H utton, Yu Oyama, Richard K. B urt
and Uday Popat 55. Bronchial Asthma and Idiopathic
Pulmonary Fibrosis as Potential Targets
50. Hematopoietic Stem Cell Therapy for Hematopoietic Stem Cell
for Patients with Refractory Myasthenia Transplantation 457
Gravis 429 Julio C. Voltarelli, Eduardo A. D onadi,
Richard K. B urt José A. B. M artinez, Elcio O. Vianna
and Willy Sarti
51. Hematopoietic Stem Cell Transplantation
in Patients with Autoimmune Bullous 56. Autologous Stem Cell Transplantation
Skin Disorders 434 in Relapsing Polychondritis 468
Joan G uitart and Richard K. B urt FalkH iepe, Andreas Thiel, Oliver Rosen,
Gero Massenkeil, Gerd-Rüdiger Burmester,
Andreas Radbruch a n d Renate Arnold
52. Idiopathic Inflammatory Myositis 437
Yu Oyama, Walter G. Barr and Richard K B urt
57. Allogeneic Hematopoietic Stem Cell
53. Hematopoietic Stem Cell Transplantation Transplantation for Autoimmune Diseases 474
as Treatment for Type 1 Diabetes 442 Shim on Slavin, Alberto M arm ont
and Richard K B urt
Julio C. Voltarelli, Richard K. Burt,
N orm a Kenyon, D ixon B. Kaufman
and Elizabeth C. Squiers
Index 481
 EDITORS =
Richard K. Burt
Chief, Division of Immunotherapy
Department of Medicine
Feinberg School of Medicine
Northwestern University
Chicago, Illinois, U.S.A
Chapter 14, 26, 3 1 37, 41, 43, 49, 50, 51, 52, 53, 54, 5 7

Alberto M. Marmont
Professor Emeritus
Division of Hematology and Stem Cell Transplantation
San Martino’s Hospital
Genoa, Italy
Chapter 27, 36, 41, 5 7

CONTRIBUTORS
Mehrdad Abedi Evangelos Badiavas
Research Department Research Department
Roger Williams Medical Center Roger Williams Medical Center
Providence, Rhode Island, U.S .A. Providence, Rhode Island, U.S.A.
Chapter 10 Chapter 10

Tobias Alexander Stephen F. Badylak

Clinical Immunology Department of Biomedical Engineering
German Rheumatism Research Centre Berlin Purdue University
Berlin, Germany West Lafayette, Indiana, U.S.A.
Chapter 2 6 Chapter 13

Howard Amital Walter G. Barr

Center for Autoimmune Diseases Division of Rheumatology
Department of Medicine ‘B’ Feinberg School of Medicine
Sheba Medical Center Northwestern University
Tel-Hashomer Chicago, Illinois, U.S.A.
Tel-Aviv University Chapter 52
Tel Aviv, Israel
Chapter 15 Antonio Bertolotto
Department of Neurology
Renate Arnold San Luigi Gonzaga Hospital
Department of Haematology and Oncology Orbassano, Italy
University Hospital Charité Chapter 3 6
Berlin, Germany
Chapter 26, 5 6 Sarah J. Bingham
Rheumatology Research Unit
Carol M. Artlett University of Leeds
Division of Rheumatology Leeds, U.K.
Jefferson Medical College Chapter 43
Thomas Jefferson University
Philadelphia, Pennsylvania, U.S.A. Carl Bjartmar
Chapter 45 Department of Neurosciences
Lerner Research Institute
Laura Aust Cleveland Clinic Foundation
Artecel Sciences, Inc. Cleveland, Ohio, U.S.A.
Durham, North Carolina, U.S.A. Chapter 3 4
Chapter 5
Robert A. Brodsky
Rosa Bacchetta Division of Hematologic Malignancies
San Raffaele Telethon Institute for Gene Therapy Sidney Kimmel Comprehensive Cancer Center
Université Vita-Salute San Raffaele Johns Hopkins School of Medicine
Milan, Italy Baltimore, Maryland, U.S.A.
Chapter 20 Chapter 28
Paolo Bruzzi Delia Demers
Unit of Clinical Epidemiology and Trials Research Department
National Cancer Institute Roger Williams Medical Center
Genova, Italy Providence, Rhode Island, U.S.A.
Chapter 3 6 Chapter 10
Diane BuchBarker
Stem Cell Transplantation Program Blythe Devlin
Division of Hematology/Oncology Artecel Sciences, Inc.
Department of Medicine Durham, North Carolina, U.S.A.
University of Pittsburgh School of Medicine Chapter 5
Pittsburgh, Pennsylvania, U.S.A.
Chapter 46 Eduardo A. Donadi
Division of Clinical Immunology
Gerd-Rüdiger Burmester University Hospital
Department of Internal Medicine School of Medicine of Ribeiräo Preto
Rheumatology and Clinical Immunology University of Sao Paulo
University Hospital Charité Ribeiräo Preto, Brazil
Berlin, Germany Chapter 55
Chapter 5 6
Albert D. Donnenberg
Ewa Carrier
Stem Cell Transplantation Program
Blood and Marrow Transplant Program
Division of Hematology/Oncology
University of California San Diego
Department of Medicine
San Diego, California, U.S.A.
University of Pittsburgh School of Medicine
Chapter 31
Pittsburgh, Pennsylvania, U.S.A.
Chapter 4 6
Jan Cerny
Research Department
Mark Dooner
Roger Williams Medical Center
Research Department
Providence, Rhode Island, U.S.A.
Roger Williams Medical Center
Chapter 10
Providence, Rhode Island, U.S.A.
Chapter 10
Diana L. Clarke
ES Cell International
Daniel Douek
Cambridge, Massachusetts, U.S.A. National Institute of Allergy and Infectious Disease
Chapter 3
National Institutes of Health
Bethesda, Maryland, U.S.A.
Gerald A. Colvin
Chapter 25
Research Department
Roger Williams Medical Center
Tracey du Laney
Providence, Rhode Island, U.S.A.
Artecel Sciences, Inc.
Chapter 10
Durham, North Carolina, U.S.A.
Chapter 5
Lyndon Cooper
Artecel Sciences, Inc.
Cynthia E. Dunbar
Durham, North Carolina, U.S.A.
National Heart, Lung and Blood Institute
Chapter 5
National Institutes of Health
Robert Craig Bethesda, Maryland, U.S.A.
Division of Immunotherapy and Gastroenterology Chapter 4 7
Feinberg School of Medicine
Northwestern University Vincent Falanga
Chicago, Illinois, U.S.A. Research Department
Chapter 54 Roger Williams Medical Center
Providence, Rhode Island, U.S.A.
J. Ludovic Croxford Chapter 10
Department of Microbiology-Immunology
Interdepartmental Immunobiology Center Athanasios Fassas
Feinberg School of Medicine Department of Hematology
Northwestern University The George Papanicolaou General Hospital
Chicago, Illinois, U.S.A. Thessaloniki, Greece
Chapter 24 Chapter 3 7
Christoph Fiehn Joan Guitart
University of Heidelberg Department of Dermatology
Department of Hematology, Oncology Feinberg School of Medicine
and Rheumatology Northwestern University and Northwestern
Heidelberg, Germany Memorial Hospital
Chapter 48 Chicago, Illinois, U.S.A.
Chapter 51
Massimo Filippi
Neuroimaging Research Unit Charles J. Hackett
Department of Neuroscience Division of Allergy, Immunology and Transplantation
Scientific Institute National Institute of Allergy and Infectious Diseases
Ospedale San Raffaele National Institutes of Health
Milan, Italy Bethesda, Maryland, U.S.A.
Chapter 3 6 Chapter 16

Patrick F. Fogarty Yuan Di C. Halvorsen

National Heart, Lung and Blood Institute Artecel Sciences, Inc.
National Institutes of Health Durham, North Carolina, U.S.A.
Bethesda, Maryland, U.S.A. Chapter 5
Chapter 4 7
Manfred Hensel
Sandra Foster University of Heidelberg
Artecel Sciences, Inc. Department of Hematology, Oncology
Durham, North Carolina, U.S.A. and Rheumatology
Chapter 5 Heidelberg, Germany
Chapter 48
Keiichi Fukuda
Institute for Advanced Cardiac Therapeutics Kevin Hicok
Keio University School of Medicine Artecel Sciences, Inc.
Tokyo, Japan Durham, North Carolina, U.S.A.
Chapter 8 Chapter 5

Jeffrey Gimble Falk Hiepe

Artecel Sciences, Inc. Department of Rheumatology
Durham, North Carolina, U.S.A. University Hospital Charité
Chapter 5 Berlin, Germany
Chapter 26, 5 6
Deborah Greer
Research Department Ursula Holzer
Roger Williams Medical Center Benaroya Research Institute
Providence, Rhode Island, U.S.A. Virginia Mason Research Center
Chapter 10 Seattle, Washington, U.S.A.
Chapter 21
Jennifer M. Grossman
Department of Medicine/Rheumatology Felicita Hornung
University of California at Los Angeles Cell Biology Section
Los Angeles, California, U.S.A. Laboratory of Viral Diseases
Chapter 39 National Institute of Allergy and Infectious Diseases
National Institutes of Health
Francesca Gualandi Bethesda, Maryland, U.S.A.
Division of Hematology and Stem Cell Chapter 17
Transplantation
San M artino’s Hospital Richard D. Huhn
Genova, Italy Coriell Institute
Chapter 3 6 Camden, New Jersey, U.S.A.
Chapter 47
Farshid Guilak
Artecel Sciences, Inc.
Durham, North Carolina, U.S.A.
Chapter 5
George J. Hutton Dixon B. Kaufman
Department of Neurology Division of Transplant Surgery
Baylor International MS Center Department of Surgery
Baylor College of Medicine Feinberg School of Medicine
Houston, Texas, U.S.A. Northwestern University
Chapter 49 Chicago, Illinois, U.S.A.
Chapter 53
Susumu Ikehara
First Department of Pathology Arthur Kavanaugh
Transplantation Center Center for Innovative Therapy
Regeneration Research Center for Intractable Diseases Division of Rheumatology, Allergy and Immunology
Kansai Medical University University of California, San Diego
Moriguchi City, Osaka, Japan La Jolla, California, U.S.A.
Chapter 30 Chapter 42

Matilde Inglese Linda Kelley

Neuroimaging Research Unit University of Utah
Department of Neuroscience Salt Lake City, Utah, U.S.A.
Scientific Institute Chapter 12
Ospedale San Raffaele
Milan, Italy Norma Kenyon
Chapter 3 6 Diabetes Research Institute
Department of Surgery
Jennifer A. Jackson University of Miami School of Medicine
Curis Inc. Miami, Florida, U.S.A.
Cambridge, Massachusetts, U.S.A. Chapter 53
Chapter 3
Serguei Kisselev
Richard J. Jones Stoddard Cancer Research Institute
Sidney Kimmel Comprehensive Cancer Center Iowa Methodist Medical Center
Johns Hopkins School of Medicine Des Moines, Iowa, U.S.A.
Baltimore, Maryland, U.S.A. Chapter 14
Chapter 6
Amy Kloster
Yuang-Taung Juang Artecel Sciences, Inc.
Department of Cellular Injury Durham, North Carolina, U.S.A.
Walter Reed Army Institute of Research Chapter 5
Silver Spring, Maryland, U.S.A.
and Diane S. Krause
Department of Medicine Department of Laboratory Medicine
Uniformed Services University of the Health Sciences Yale University School of Medicine
Bethesda, Maryland, U.S.A. New Haven, Connecticut, U.S.A.
Chapter 38 Chapter 11

Kenneth C. Kalunian Anke Kretz-Rommel

Division of Rheumatology, Allergy and Immunology Alexon Antibody Technologies
UCSD Division of Rheumatology San Diego, California, U.S.A.
University of California, San Diego Chapter 23
La Jolla, California, U.S.A.
Chapter 39 Jean-Francois Lambert
Research Department
Dan S. Kaufman Roger Williams Medical Center
University of Wisconsin Providence, Rhode Island, U.S.A.
Madison, Wisconsin, U.S.A. Chapter 10
Chapter 2
Bernard R. Lauwerys
Service de Rhumatologie
Cliniques Universitaires Saint-Luc
Brussels, Belgium
Chapter 22
Sean Lee José A. B. Martinez
Department of Laboratory Medicine Pulmonary Division
Yale University School of Medicine University Hospital
New Haven, Connecticut, U.S.A. School of Medicine of Ribeirâo Preto
Chapter 11 University of Sao Paulo
Ribeirâo Preto, Brazil
Michael J. Lenardo Chapter 55
Laboratory of Immunology
National Institute of Allergy and Infectious Diseases Luca Massacesi
National Institutes of Health Department of Neurological and Psychiatric Sciences
Bethesda, Maryland, U.S.A. University of Firenze
Chapter 17 Firenze, Italy
Chapter 3 6
Megan K. Levings
San Raffaele Telethon Institute for Gene Therapy Gero Massenkeil
Université Vita-Salute San Raffaele Departments of Internal Medicine
Milan, Italy Rheumatology and Clinical Immunology
Chapter 20 Hematology and Oncology
Berlin, Germany
Charles Link Chapter 5 6
Stoddard Cancer Research Institute
Iowa Methodist Medical Center Hiroaki Matsubara
Des Moines, Iowa, U.S.A. Department of Medicine and Cardiovascular Division
Chapter 14 Kyoto Prefecture University School of Medicine
Kamigyo-ku, Kyoto, Japan
Lorri Lobeck Chapter 9
Department of Neurology
Medical College of Wisconsin Christine I. McAuliffe
Milwaukee, Wisconsin, U.S.A. Research Department
Chapter 35 Roger Williams Medical Center
Providence, Rhode Island, U.S.A.
Alessandra Lugaresi Chapter 10
Department of Oncology and Neurosciences
University of Chieti Henry F. McFarland
Chieti, Italy National Institute of Neurologic Disease and Stroke
Chapter 3 6 National Institutes of Health
Bethesda, Maryland, U.S.A.
G. Gordon MacPherson Chapter 33
Sir William Dunn School of Pathology
University of Oxford Ian McNiece
Oxford, U.K. Johns Hopkins Oncology Center
Chapter 19 Baltimore, Maryland, U.S.A.
Chapter 12
Giovanni Luigi Mancardi
Department of Neurological Sciences, Opthalmology Thomas A. Medsger, Jr.
and Genetics Stem Cell Transplantation Program
University of Genova Division of Rheumatology
Genova, Italy Department of Medicine
Chapter 3 6 University of Pittsburgh School of Medicine
Pittsburgh, Pennsylvania, U.S.A
Maria Giovanna Marrosu Chapter 4 6
Department of Neurosciences
University of Cagliari Elisa Merelli
Cagliari, Italy Department of Neurology
Chapter 3 6 University of Modena
Modena, Italy
Roland Martin Chapter 3 6
National Institute of Neurologic Disease and Stroke
National Institutes of Health
Bethesda, Maryland, U.S.A.
Chapter 33
Giuseppe Meucci Madhusoodana P. Nambiar
Department of Neurology Department of Cellular Injury
Hospital of Livorno Walter Reed Army Institute of Research
Livorno, Italy Silver Spring, Maryland, U.S.A.
Chapter 3 6 and
Department of Medicine
Stephen D. Miller Uniformed Services University of the Health Sciences
Department of Microbiology-Immunology Bethesda, Maryland, U.S.A.
Interdepartmental Immunobiology Center Chapter 38
Feinberg School of Medicine
Northwestern University Gerald T. Nepom
Chicago, Illinois, U.S.A. Benaroya Research Institute
Chapter 24 Virginia Mason Research Center
Seattle, Washington, U.S.A.
Simon W. F. Milling Chapter 21
Sir William Dunn School of Pathology
University of Oxford Yu Oyama
Oxford, U.K. Division of Immunotherapy
Chapter 19 Feinberg School of Medicine
Northwestern University
Brian E. Moore Chicago, Illinois, U.S.A.
Research Department Chapter 49, 52
Roger Williams Medical Center
Providence, Rhode Island, U.S.A. Francesca Pagliai
Chapter 10 Careggi Hospital
Bone Marrow Transplantation Unit
John J. Moore Firenze, Italy
Department of Haematology Chapter 3 6
St. Vincent’s Hospital
Sydney, Australia Steve Z. Pavletic
Chapter 43 Department of Internal Medicine
University of Nebraska Medical Center
Jagan R. Muppidi Omaha, Nebraska, U.S.A.
Laboratory of Immunology Chapter 43
Autoimmunity Branch
National Institute of Arthritis and Musculoskeletal Bryon E. Petersen
and Skin Diseases Department of Pathology, Immunology
National Institutes of Health and Laboratory Medicine
Bethesda, Maryland, U.S.A. University of Florida
Chapter 17 Gainesville, Florida, U.S.A.
Chapter 4
Paolo A. Muraro
National Institute of Neurologic Disease and Stroke Uday Popat
National Institutes of Health Department of Medicine
Bethesda, Maryland, U.S.A. Center for Cell and Gene Therapy
Chapter 33 Baylor College of Medicine
Houston, Texas, U.S.A.
Alessandra Murialdo Chapter 49
Department of Neurological Sciences, Opthalmology
and Genetics Jonathan D. Powell
University of Genova Departments of Oncology and Pharmacology
Genova, Italy Johns Hopkins School of Medicine
Chapter 3 6 Baltimore, Maryland, U.S.A.
Chapter 18
Ryotaro Nakamura
City of Hope Darwin J. Prockop
Duarte, California, U.S.A. Center for Gene Therapy
Chapter 47 Tulane University Health Sciences Center
New Orleans, Louisiana, U.S.A.
Chapter 7
Peter J. Quesenberiy Willy Sarti
Research Department Division of Clinical Immunology
Roger Williams Medical Center University Hospital
Providence, Rhode Island, U.S.A. School of Medicine of Ribeirao Preto
Chapter 10 University of Sao Paulo
Ribeirao Preto, Brazil
Helen Quill Chapter 55
Division of Allergy, Immunology and Transplantation
National Institute of Allergy and Infectious Diseases Christian A. Schmidt
National Institutes of Health Department of Haematology and Oncology
Bethesda, Maryland, U.S.A. University Hospital Greifswald
Chapter 16 Greifswald, Germany
Chapter 2 6
Andreas Radbruch
Cell Biology Group Ronald H. Schwartz
German Rheumatism Research Centre National Institute of Allergy and Infectious Disease
Berlin, Germany National Institutes of Health
Chapter 26 , 5 6 Bethesda, Maryland, U.S.A.
Chapter 18
Henry E. Rice
Artecel Sciences, Inc. Anindita Sen
Durham, North Carolina, U.S.A. Artecel Sciences, Inc.
Chapter 5
Durham, North Carolina, U.S.A.
Chapter 5
Maria-Grazia Roncarolo
San Raffaele Telethon Institute for Gene Therapy Tatiana Seregina
Université Vita-Salute San Raffaele Stoddard Cancer Research Institute
Milan, Italy Iowa Methodist Medical Center
Chapter 20
Des Moines, Iowa, U.S.A.
Chapter 14
Robert Rosa
Division of Nephrology and Hypertension Yehuda Shoenfeld
Feinberg School of Medicine Center for Autoimmune Diseases
Northwestern University Department of Medicine ‘B’
Chicago, Illinois, U.S.A Sheba Medical Center
Chapter 40
Tel-Hashomer
Tel-Aviv University
Oliver Rosen Tel Aviv, Israel
Department of Internal Medicine Chapter 15
Rheumatology and Clinical Immunology
Hematology and Oncology Richard M. Siegel
University Hospital Charité Autoimmunity Branch
Berlin, Germany National Institute of Arthritis and Musculoskeletal
Chapter 5 6
and Skin Diseases
National Institutes of Health
Robert L. Rubin Bethesda, Maryland, U.S.A.
Department of Molecular Genetics and Microbiology Chapter 17
University of New Mexico School of Medicine
Albuquerque, New Mexico, U.S.A. Shimon Slavin
Chapter 25 Department of Bone Marrow Transplantation
and Cancer Immunotherapy
Riccardo Saccardi
Hadassah University Hospital
Careggi Hospital
Jerusalem, Israel
Bone Marrow Transplantation Unit Chapter 5 7
Firenze, Italy
Chapter 3 6
John A. Snowden
Department of Haematology and Division
of Genomic Medicine
Royal Hallamshire Hospital
Sheffield, U.K.
Chapter 43
Maria Pia Sormani Ann E. Traynor
Unit of Clinical Epidemiology and Trials Division of Immunotherapy
National Cancer Institute Feinberg School of Medicine
Genova, Italy Northwestern University
Chapter 3 6 Chicago, Illinois, U.S.A.
Chapter 41
Gerald J. Spangrude
Departments of Oncological Sciences, Pathology Christos G. Tsokos
and Medicine Department of Cellular Injury
Division of Hematology Walter Reed Army Institute of Research
University of Utah Silver Spring, Maryland, U.S.A.
Salt Lake City, Utah, U.S.A. and
Chapter 1 Department of Medicine
Uniformed Services University of the Health Sciences
Elizabeth C. Squiers Bethesda, Maryland, U.S.A.
Genzyme/SangStat Chapter 38
Fremont, California, U.S.A.
Chapter 53 George C. Tsokos
Department of Cellular Injury
Robert W. Storms Walter Reed Army Institute of Research
Artecel Sciences, Inc. Silver Spring, Maryland, U.S.A.
Durham, North Carolina, U.S.A. and
Chapter 5 Department of Medicine
Uniformed Services University of the Health Sciences
Valentina Stosor Bethesda, Maryland, U.S.A.
Division of Infectious Diseases Chapter 38
Feinberg School of Medicine
Northwestern University D.W. van Bekkum
Chicago, Illinois, U.S.A. Leiden, The Netherlands
Chapter 31 Chapter 29

Annie Y. Suh Larissa Verda

Division of Nephrology and Hypertension Division of Immunotherapy for Autoimmune Diseases
Feinberg School of Medicine Feinberg School of Medicine
Northwestern University Northwestern University
Chicago, Illinois, U.S.A. Chicago, Illinois, U.S.A.
Chapter 40 Chapter 2 6

Neil D. Theise Elcio O. Vianna

Department of Pathology Pulmonary Division
New York University School of Medicine University Hospital
New York, New York, U.S.A. School of Medicine of Ribeiräo Preto
Chapter 4 University of Sao Paulo
Ribeiräo Preto, Brazil
Andreas Thiel Chapter 55
Clinical Immunology
German Rheumatism Research Centre Berlin Julio C. Voltarelli
Berlin, Germany Division of Clinical Immunology
Chapter 26, 5 6 Bone Marrow Transplantation Unit
University Hospital
James A. Thomson School of Medicine of Ribeiräo Preto
University of Wisconsin University of Säo Paulo
Madison, Wisconsin, U.S.A. Ribeiräo Preto, Brazil
Chapter 2 Chapter 53, 55

Bruce D. Trapp Edward K. Wakeland

Department of Neurosciences Center for Immunology
Lerner Research Institute Southwestern Medical Center
Cleveland Clinic Foundation University of Texas
Cleveland, Ohio, U.S.A. Dallas, Texas, U.S.A.
Chapter 3 4 Chapter 22
Stuart Weisman Mervin C. Yoder
Center for Innovative Therapy Cancer Research Institute
Division of Rheumatology, Allergy and Immunology Indiana University School of Medicine
University of California, San Diego Indianapolis, Indiana, U.S.A.
La Jolla, California, U.S.A. Chapter 13
Chapter 42
Teresa R. Zembower
William O. Wilkison Division of Infectious Diseases
Artecel Sciences, Inc. Feinberg School of Medicine
Durham, North Carolina, U.S.A. Northwestern University
Chapter 5 Chicago, Illinois, U.S.A.
Chapter 32
Nico Wulffraat
Pediatric BMT Unit Lixin Zheng
University Medical Center Utrecht Laboratory of Immunology
Utrecht, The Netherlands National Institute of Allergy and Infectious Diseases
Chapter 44
National Institutes of Health
Bethesda, Maryland, U.S.A.
Andrew M. Yeager Chapter 17
Stem Cell Transplantation Program
Division of Hematology/Oncology
Department of Medicine
University of Pittsburgh School of Medicine
Pittsburgh, Pennsylvania, U.S.A.
Chapter 4 6
Representation of Shiva. ©2001 Indra Sharma/Mandala Publishing. Used with permission.
 PREFACE
The Immune System and Its Treatment with Stem Cell Therapy:
Creator, Preserver, and Destroyer

T
he creation of something new means loss of the old, while tense immune suppressive transplant regimens. While as yet not
destruction means, for better or worse, a new beginning. delineated, some possible mechanisms and pathways leading to
This duality of existence is an element of Eastern religions tolerance after hematopoietic stem cell transplantation are sug-
and philosophies. For example, Shiva, the Hindu Deity of creation gested in these chapters. Tissue regeneration from blood stem cells
and preservation, is also the God of destruction. The paradox and is also suggested by animal experiments on stem cell plasticity or
complexity of the immune system is that it embodies these same metamoirosis (i.e., change in fate) as described within this text-
attributes. It is the protector and preserver without which human book. Ongoing early clinical trials on tissue regeneration from
life is impossible, as well as in the case of autoimmune disease, the blood stem cells are described in the chapter on stem cell therapy
destroyer of that which it protects. for cardiac and peripheral vascular disease. Whether autologous
Shiva represents contradictions that coexist in conscious re- hematopoietic stem cells, through the process of mobilization and
ality. The many appendages are images for contradictory and var- reinfusion, may be manipulated to contribute to tissue repair in au-
ied functions within one consciousness. The hands hold objects toimmune diseases is a future area for translational research.
representing these attributes, a flame for destruction, the trident Allogeneic stem cell sources include siblings or other related
for creation, and a snake as imagery for mastery over nature. Shiva’s donors, unrelated donors, cord blood, and finally although not yet
duality is sometimes also manifest in gender, pictured not as male in clinical trial, embryonic stem cells. Allogeneic stem cells offer
or female but as a “Half Woman Lord”. So it is with the immune the advantage of changing genetic susceptibility to autoimmune
system, autoimmune diseases more common in women, and tasks disease. In addition they possess the ability of eradicating the au-
that are interrelated with numerous arms and opposing functions. toimmune clones which survive the conditioning regimens, which
Attempts at treating one component invariably affects, not uncom- in these patients are preferably of the nonmyeloablative (NST)
monly in an adverse way, other circuits or components of immune modality. Thus, a Graft-versus-Autoimmunity effect is elicited, as
surveillance. The immune system interacts with nature to hold discussed in the chapters by Marmont and Slavin et al.
mastery over environmental pathogens, destroying or coaxing them Embryonic stem cells are being applied in vitro and in ani-
to live in peaceful coexistence, but other times influenced by patho- mal models to both tolerance and regenerative medicine. In ani-
gens, xenobiotics, gender hormones, or other environmental trig- mal models, embryonic stem cells have been used as an alternate
gers to destroy the host it also protects. marrow donor source resulting in engraftment occurring across
Central tolerance, that is negative selection within the thy- major histocompatibility barriers without graft versus host dis-
mus, eliminates T cells with opposite avidities. T cells expressing ease (Burt et al, unpublished). When working with embryonic stem
either too strong or having no affinity for self-epitopes undergo cells, simultaneous creation, preservation, and destruction are not
apoptotic destruction, while those with mild/moderate affinity for just metaphysical discussions but real issues yet to be resolved by
self-peptides are positively selected. These T cells may in turn me- society.
diate autoimmunity and/or down regulate autoimmune responses Embryonic stem cell lines have been developed from blas-
through regulatory T cells. The process of thymic education that tocysts that were otherwise destined to be destroyed following in
selects for repertoires with mild to moderate self-recognition re- vitro fertilization. In vitro fertilization involves the collection of
sults in an autoimmune duality. Autoimmune cells are physiologic numerous oocytes that are fertilized not within the fallopian tubes
while autoimmune disease is pathologic. Autoimmunity is nor- but rather in a Petri dish containing even more numerous donor
mal, but autoimmune disease is abnormal. Unlike a malignancy, spermatocytes. While numerous blastocysts (pre-implantation
in which a tum or cell is always viewed as pathologic, for autoim- embryos) form, only one will be implanted in the uterus, the oth-
mune disease, treatment must also preserve that which the therapy ers are cryopreserved. When the couple no longer desires children,
is designed to destroy. It is for these reasons that this textbook on what becomes of the remaining blastocysts? Paradoxically, in the
stem cell therapy for autoimmune disease has numerous chapters philosophical spirit of attempting to preserve the life of the un-
devoted to basic immunology. The clinical art of balance in treating born, blastocysts are currently destroyed rather than being allowed
an intertwined and at times contradictory immune system begins to live on as embryonic stem cell lines that contribute to the tissue
with the science of immunology. and existence of another individual. Does the blastocyst have a
Stem cell tra n sp la n ta tio n may be com plicated by soul and if so, is it better to allow its existence to continue in an-
treatment-related mortality and like the immune system that it other individual or to destroy it entirely?
regenerates has equal potential to either create and preserve or Hematopoietic stem cells as a therapeutic tool to induce tol-
destroy. The dual nature that defines stem cells is differentiation erance were first applied to human autoimmune disorders in 1996.
that ultimately leads to death and self-renewal which leads to im- Since then, the door has opened on trials involving numerous au-
mortality. What types of stem cells are there? How are they col- toimmune diseases and allogeneic as well as autologous hemato-
lected? What are their attributes and characteristics? This textbook poietic stem cells. Stem cells are becoming a new therapeutic tool
devotes many chapters to familiarize the reader with the basic sci- to supplement or replace traditional approaches of surgery, phar-
ence, clinical aspects, and new questions being raised in the field macy, and radiotherapy. As the embryonic stem cell-related ethi-
of stem cell biology. Blood stem cells for tolerance and tissue re- cal issues involving creation and destruction are resolved, their
generation are a rapidly developing research and clinical field that clinical application may follow the clinical paths, trails, and trials
is being applied to autoimmune diseases. already being explored with hematopoietic stem cells.
In clinical trials, autologous hematopoietic (blood) stem cells
are being used to reduce the cytopenic interval following in - Richard K B u r t , M .D .
Alberto M . M arm ont , M .D .
 CHAPTER 1

When is a Stem Cell Really a Stem Cell?

Gerald J. Spangrude

Introduction

I
n recent years, data from numerous experimental studies has changed our expectations for the applications of this type of
suggested that the potential uses of stem cells in medicine therapy,9 even though many questions have been raised by these
may reach far beyond bone marrow transplantation. How interesting findings.10 Second, the derivation of totipotent hu-
applicable is recent research to modern medicine, and how soon man stem cells from both embryonic and fetal sources has intro-
might we expect to see stem cells applied to tissue engineering duced a potential new source of tissue for engineering applica-
problems? These and other questions are explored in this intro- tions. Equally important, this new technology marks the genesis
ductory chapter. It is altogether fitting that a discussion of the of a new level of conflict between science and religion that sur-
therapeutic potentials of stem cell therapy be grounded in our passes that raised by older questions of creationism versus evolu-
field, being the first to apply stem cell therapy to the clinical tion. The potential use of stem cells derived from adult tissues
management of acquired and inherited diseases. But what is a introduces yet another side to this complex story. How are we to
stem cell? In the context of bone marrow transplantation, we define when a stem cell is a stem cell? It is in this vein that I
understand the answer to this question in a concrete and func- examine a few of the historical aspects of stem cell biology in
tional sense due to decades of research and clinical applications order to better understand where we have come from at this stage
that grew out of the need to understand the effects of ionizing in the development of the stem cell field.
radiation on biological systems. In the years following the Sec-
ond World War, a considerable am ount of scientific effort was Em bryonic Stem Cells: A T im elin e
focused on the prevention and treatment o f radiation sickness. Lewis11 has correctly identified the origins of the stem cell
From these studies came the observation that transplants of spleen biology field in the work of Leroy Stevens, a developmental bi-
or bone marrow cells contribute to cellular recovery following ologist who identified frequent testicular tumors arising sponta-
lethal radiation.1 Almost 50 years after this dramatic insight, we neously in strain 129 mice at the Jackson Laboratories. This work
now understand that the ability of such transplants to reconsti- was published to little fanfare beginning in 1958.12 However, the
tute hematopoiesis following radiation depends upon the pres- curiosity of Mintz and Illmensee led to a startling observation.
ence of extremely rare stem cells found predominantly in the W hen malignant teratocarcinoma cells were mixed into develop-
bone marrow but capable of mobilization into peripheral tissues ing mouse embryos, the environment of the embryo harnessed
via the blood vascular system.2 the unregulated growth of the tumor and directed these cells to
After many years shrouded in mystery and controversy, the proper channels of proliferation and differentiation.13 The result
characteristics of blood stem cells were gradually revealed through was chimeric mice in which a significant portion of the body
novel assays3'5 and methods for isolation of these rare cells.6,7 We mass was derived from the teratocarcinoma. This startling dis-
now understand that the definition o f a stem cell must include covery was viewed at the time as evidence for environmental regu-
the two essential characteristics of self-renewal (cellular division lation of malignant growth, but the potential of these cells was
maintains stem cell potential) and multipotency (differentiation certainly not overlooked by developmental biologists. Embryonic
into functionally distinct lineages). To complicate matters, it is stem cell lines were derived from the inner cell mass of mouse
clear that progenitor cells, which are m ultipotent but lack blastocysts in 1981,14,15 as shown in Figure 1. These cells were
self-renewal potential, are often difficult to distinguish from true adapted for growth in culture without differentiation, but could
stem cells.8 Finally, at least some confusion persists in the tissue differentiate into mesoderm, endoderm, and ectoderm in vitro
stem cell field, where unipotent precursor cells which maintain a and in vivo. The derivation of embryonic stem cell lines was rap-
tissue through a self-renewing process are often considered stem idly exploited to give birth to the field of targeted mutagenesis,16, 7
cells. an entirely new approach to the investigation of complex mam-
The general field of stem cell biology has been the subject of malian biological systems. Today, it is difficult for scientists to
intense public interest in recent years for several reasons. First, imagine a world in which the genome could not be mutated in a
the demonstration that recipients of bone marrow transplants specific manner. The true power of stem cell biology was revealed
harbor donor-derived cells in a variety of tissues has radically to the world at large with the announcement that the transfer of

Adapted with permission from Bone Marrow Transplantation, VoL 32, Supplement 1, Aug 2003.

Stem Cell Therapyfor Autoimmune Diseasef edited by Richard K. Burt and Alberto M. Marmont. ©2004 Landes Bioscience/Eurekah.com.
2 Stern Ceil Therapy for Autoimmune Disease

Figure 1. From the initial descriptions of the ability of testicular carcinoma cells to produce pluripotent embryonic stem cells, these cells
have since been derived from blastocysts as well as the primordial germ cells in the developing genital ridge in both mouse and man. Figure
courtesy of Terese Winslow, used with permission of the artist.

nuclei derived from adult somatic cells into enucleated oocytes blastocyst-derived embryonic stem cells but lacking apparent tu-
produced, at a low frequency, viable offspring clonally derived morigenic potential. This combination o f multipotential differ-
from the donor o f the nuclei.18 entiation in the absence of tumor formation has lead to the pro-
posed use of these cells in clinical trials to treat spinal cord injury,
M ou se to M an Parkinsons disease, and other cell-based therapies. W ith the specter
The successful application of targeted mutagenesis in the mouse of the cloning of human beings looming before us, the National
was not the only useful application of embryonic stem cell lines. Academy of Sciences initiated a comprehensive analysis of this
A variety of investigators utilized these cell lines to model the brave new world.21 The current state of federal funding will sup-
development of the early embryo in culture systems, and success- port the utilization of fetal-derived embryonic germ cells in clini-
fully recapitulated several aspects of embryogenesis. W hen the cal applications, most likely because the derivation and use of
application of in vitro fertilization to the clinical problem of in- these cells avoids some of the concerns raised by the concept of
fertility resulted in the birth of the first test-tube baby in 1978, frozen embryos as sources of embryonic stem cells. Embryonic
the stage was set for the eventual derivation of human embryonic germ cells are unable to be implanted into a surrogate mother to
stem cells from embryos fertilized in vitro but not implanted into produce a genetically normal human, unlike the embryos formed
a w om b.19 Since these early embryos are frozen in quantities that during in vitro fertalization. As such, the only embryos that might
exceed clinical need, large banks o f fertilized embryos destined be formed by embryonic germ cells would be genetic mosaics of
for destruction now exist around the world as a consequence of the germ cell and a blastocyst in which such cells might be intro-
the widespread application of in vitro fertilization. Some of these duced, or would be the product of somatic cell nuclear transfer.
embryos have been cultured to derive embryonic stem cell lines, Since the latter process can be performed using a wide variety of
however the derivation of cell lines in addition to those already in cell types, the embryonic germ cell provides no special advantage
existence has been deemed unnecessary and will not be supported in this sense.
by federal funding agencies in the United States.
A second approach to the application of stem cell technology A dult Stem C ells
in humans utilizes tissue derived from the genital ridge of aborted Undifferentiated cells that are found in a differentiated adult
first trimester fetuses (Fig. I).20 These cells, which normally de- tissue are considered adult stem cells, particularly when these cells
velop into mature gametes, can be cultured under specific condi- contribute to ongoing tissue maintenance or repair. These cells
tions to produce cell lines with all known characteristics of may be capable of self-renewal, but do not replicate indefinitely
When is a Stem Ceil Really a Stem Cell? 3

in culture. Adult stem cells may differentiate to produce progenitor, Recent advances in vector design have made gene correction a
precursor, and mature cells, but these activities are usually lim- feasible approach to the treatment of a number of genetic diseases,
ited to the cells contained in the tissue o f origin. Adult stem cells including X-linked severe combined immunodeficiency disease,
usually comprise a small minority of the total tissue mass, and as hemophilia, and a number of autoimmune disorders. Clinical
such are usually quite difficult to identify and isolate. Adult stem application o f stem cell therapy depends on robust self-renewal
cells have been described in regenerating tissues such as the liver, and differentiation of the transplanted cells, and in this sense
epithelium and muscle, as well as in tissues like the brain, which trans-differentiation must be sufficiently frequent and robust to
previously was thought not to possess extensive regenerative prop- achieve enough tissue replacement to be clinically useful. How-
erties. By far, the most well-characterized example of adult stem ever, if harnessed and properly regulated, it is not difficult to imag-
cells is that of the hematopoietic system. ine the application of stem cell therapy in diverse settings such as
diabetes (generation of islet cells from stem cells), repair of dam-
H em atop oietic Stem Cells: Paradigms for Stem aged heart muscle, and rebuilding the nervous system after injury
or age-related decline.26
C ell B iology
The limited life-span of most blood cells demands that a con-
tinual source of these cells be assured throughout life. It is likely R egulatory and Funding Issues
for this reason that the hematopoietic system has so readily lent The United States government now provides some funding
itself to applications involving clinical transplantation. Indeed, for human stem cell research. While N IH funds cannot be used
the challenges faced by cells utilized in bone marrow transplanta- for derivation of human ES lines, this type o f research can be
tion are not so different than the normal physiologic role played performed using private sources of funding if proper informed
by these cells during maintenance of hematopoiesis over the life- consent is obtained under a protocol approved by an institutional
time o f the normal mammal. Compared to the hematopoietic review board according to N IH guidelines. N IH funds can be
system, other tissues of the adult mammal display relatively lim- used for research that utilizes existing embryonic stem cell lines,
ited potential for replacement from endogenous stem cells in re- as well as for derivation and use of embryonic germ cells from
sponse to tissue injury. Furthermore, no other tissue is character- fetal tissues. This apparent discrepancy in policy arises due to the
ized by such a wide variety of different cell lineages which all arise consideration that established stem cell lines and aborted fetal
from a common stem cell in a developmental process that con- tissues are not embryos and can not, by themselves, develop into
tinues throughout life. The ability to model many of these differ- human beings. The N IH guidelines and the FDA regulate ex-
entiation pathways under controlled conditions in vitro, and the perimental and clinical use of human pluripotent stem cells and
availability of recombinant proteins that select or direct differen- fetal tissues. As of September 25, 2002, 5 N IH grants have been
tiation along specific lineages makes the hematopoietic system approved and funded for a total of $4.2 million, and administra-
the premier paradigm for the field of stem cell biology. tive supplements for embryonic stem cell research have been
awarded to 30 additional investigators.27
P lasticity
The concept of stem cell plasticity refers to the phenomenon
T h e Future o f Stem C ell T herapy
of trans-differentiation, which is the ability of an adult stem cell A number of challenges remain before the promise of stem
from one tissue to differentiate as a specialized cell type of an- cell therapy can be translated into application. First and fore-
other tissue. A recent study showed that neural stem cells were most, the political and ethical conflicts that surround the use of
capable of regenerating blood lineages in transplant recipients,22 human embryonic and fetal tissue for medical applications must
and the field rapidly advanced as examples of muscle, epithe- be resolved. The concept that stem cells derived from adult tis-
sues will substitute for those obtained from fetal or embryonic
lium, liver, and other tissues derived from heterologous stem cells
sources is simply too premature to be used as a basis for legisla-
(usually bone marrow-derived) were reported.23 W ith few excep-
tion and regulation. While the combination of gene therapy with
tions, these studies involved transplantation of large numbers of
stem cell therapy has proven to be effective for certain diseases,
cells, leaving open the possibility that distinct classes of stem cells
methods of gene delivery must be improved to prevent unpre-
were responsible for regeneration of the different tissues. Fur-
dictable adverse events. Animal models must be refined to allow
thermore, the magnitude of tissue replacement has often been
comprehensive analysis of potential risks and benefits prior to
minor, suggesting that this approach to tissue engineering will
clinical application. These and other barriers stand before us,
require extensive optimization in order to be clinically useful. Fi-
marking the path toward new applications in clinical medicine.
nally, technical artifacts24 and difficulty in reproducing some of
the reported findings25 suggest that caution is indicated in inter-
References
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14. Evans MJ, Kaufman M H. Establishment in culture o f pluripotential Nature 2002; 416:485-487.
cells from mouse embryos. Nature 1981; 292:154-156. 25. Morshead CM, Benveniste P, Iscove N N et al. Hematopoietic
15. Martin GR. Isolation o f a pluripotent cell line from early mouse competence is a rare property o f neural stem cells that may depend
embryos cultured in medium conditioned by teratocarcinoma stem on genetic and epigenetic alterations. Nat Med 2002; 8:268-273.
cells. Proc Natl Acad Sci USA 1981; 78:7634-7638. 26. Department o f Health and Human Services. Stem cells: Scientific
16. Doetschman T, Gregg RG, Maeda N et al. Targeted correction of a progress and future research directions. National Institutes o f Health
mutant HPRT gene in mouse embryonic stem cells. Nature 1987; 2001. http://www.nih.gov/news/stemcell/scireport.htm.
330:576-578. 27. Zerhouni E. Stem cell research. Senate Appropriations Subcommittee
17. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene on Labor H H S Education. W ashington, DC: 2002. http ://
targeting in mouse embryo-derived stem cells. Cell 1987; 51:503-512. www.nih.gov/about/director/092502sctestimony.htm.
 CHAPTER 2 =

Embryonic Stem Cells: Unique Potential to Treat

Autoimmune Diseases
D an S. Kaufman and James A. T hom son

Introduction

E
mbryonic stem (ES) cells are pluripotent cells that can be prolonged periods as cells that maintain multipotent potential
maintained indefinitely in culture as undifferentiated cell without evidence of differentiation down a particular develop-
lines, yet retain their ability to form any cell type. The mental lineage. However, in the proper environment or with the
derivation of human embryonic stem cells provides a new model proper stimuli, a stem cell retains the ability to form more spe-
system to learn about human developmental biology. These cells cialized cells such as blood, muscle, liver, or skin. Broadly speak-
may also be a suitable starting point to produce novel cell-based ing, there are two main categories of stem cells: “adult” stem cells
therapies to treat a variety of diseases. This chapter describes the and embryonic stem cells. Adult stem cells are derived from
characteristics of human embryonic cells and the potential of post-natal tissue and are typically thought to have a limited de-
these cells to be used for hematopoietic cell transplantation, tol- velopmental potential. Hematopoietic stem cells (HSCs) found
erance induction, and treatment of autoimmune diseases. in the bone marrow produce blood cells, neural stem cells (NSCs)
This book, “Stem Cell Therapy for Autoimmune Disease,” found in the central nervous system give rise to neurons and glial
reflects the recently growing interest in stem cells and cell-based cells, hepatic stem cells found in the liver, produce hepatocytes
therapies. Among the various medical specialties, hematologists and biliary cells are all examples of adult stem cells. In contrast,
are probably the most familiar with the concept of stem cells. ES cells are derived from preimplantation blastocysts and have
Many of the diseases seen in the clinical fields of hematology and the potential to form any cell type in the body.
hematopoietic stem cell transplantation (HSCT) are clonal in
nature and demonstrate how a defect in a single early precursor Research Prior to D erivation o f H um an ES Cells
cell can lead to a systemic disease. For example, in chronic myel- While the derivation of human ES cells has recently sparked
ogenous leukemia (CML), the transformation of a single hemato- considerable enthusiasm (and some debate) over the potential of
poietic progenitor leads to an overwhelming production of ma- these cells to be used to treat human disease, it is important to
ture and immature hematopoietic cells. If untreated, this “stem recognize that decades of basic scientific research preceded the
cell malignancy” will lead to death of the afflicted individual within isolation and characterization of human ES cells. Studies on mouse
a few years. However, H SCT can cure CML and other hemato- and human embryonal carcinoma (EC) cell lines were a crucial
logical malignancies by replacement of the abnormal clone with precedent to work on ES cells. Differences and similarities be-
normal hematopoietic stem cells (HSCs). Additionally, it is also tween human EC cells, murine ES cells, and human ES cells are
possible to treat many nonmalignant hematologic conditions such listed in Table 1. EC cells are multipotent malignant precursor
as congenital immunodeficiencies and sickle cell anemia with cells derived from teratocarcinomas.4 Mouse and human EC cells
HSCT.1,2 The ability to use H SCT to treat severe autoimmune can be isolated from these tumors and be maintained in vitro as
disease (as described in this book) is one of the newest applica- undifferentiated cells. W hen treated with agents to induce differ-
tions o f stem cell biology.3 Indeed, the autoimmune process can entiation (such as retinoic acid) or reimplanted into immunode-
be considered another form of stem cell abnormality. In many of ficient animals, multiple differentiated cell lineages can be de-
these cases, a clone of self-reacting lymphocytes become abnor- rived. EC cells have provided important insights regarding the
mally activated, leading to the destruction of normal cells and
mechanisms of normal mammalian development and abnormal
tissues. Autologous or allogeneic H SCT aims to alleviate this pro-
tumorigenesis. However, the use and interpretation of work on
cess by the elimination or suppression of these rogue lympho-
these cells is limited because EC cells typically have genetic ab-
cytes. As described in this chapter, the derivation of human ES
cells may eventually lead to novel methods of stem cell therapies normalities, and their developmental potential is often restricted
for autoimmune diseases. to a few cell lineages. For these reasons, it became important to
Stem cells are defined as specific cell types that have two im- derive and characterize embryonic stem cells, the normal non-
portant properties: self-renewal and differentiation. Self-renewal malignant counterpart to EC cells. This breakthrough was first
refers to the ability of these cells to undergo cell division for reported by two groups in 1981.5,6

Stem Cell Therapyfor Autoimmune Disease, edited by Richard K. Burt and Alberto M. Marmont. ©2004 Landes Bioscience/Eurekah.com.
6 Stem Ceil Therapy for Autoimmune Disease

T able 1. C om parison o f p rim o rd ia l c e ll lin e s

Murine ES Cells Human EC Cells Human ES Cells

Source Peri-Im plantation Embryo Teratocarcinom as Pre-Im plantation B lastocysts

Karyotype N orm al A n eu p loid Normal

Surface a n tig en s SSEA1+ SSEA1' SSEA1'
SSEA3" SSEA3+ SSEA3+
SSEA4" SSEA4+ SSEA4+
TRA-1-60" T R A -l-6 0 + T R A -l-6 0 +
TR A -1-81' T R A -1-81+ TR A -1-81+
CD133+
Culture con d ition s R equires LIF or o th e r LIF in d e p e n d e n t, Require MEF fe e d e r c e lls or MEF
to m aintain self-ren ew al g p l 3 0 / s t a t 3 a g o n is t m ost fe e d e r -in d e p e n d e n t c o n d itio n e d m edia
D oes n o t require LIF
In vivo p o ten tia l T o tip o te n t L im ited lin e a g e s Unknown

Abbreviations: ES= embryonic stem; EC= embryonal carcinoma; LIF= leukemia inhibitory factor; MEF= mouse embryonic fibroblast;
SSEA= stage specific embryonal antigens; TRA= transcription factor.

Mouse ES cells are typically derived from the inner cell mass tion to delete specific genes within ES cells. For example, dele-
(ICM) o f early preimplantation stage blastocysts. This preim- tion of the genes for the vascular endothelial growth factor (VEGF)
plantation stage of development occurs after fertilization of the receptors flk-1 and fit-1 leads to death at approximately day 9 of
oocyte and before attachment to the uterus. During this time, mouse embryogenesis. Analysis of these mice demonstrates lack
the zygote undergoes several rounds of cell division. The cells of normal hematopoietic and endothelial cell development and
produced at this early stage are not committed to become any leads to the presumption that these receptors (and VEGF) are
particular part o f the body. Indeed, it is possible to split the de- required for normal hematopoiesis.13,14 These results correspond
veloping embryo in half at this stage, and each half has the ability to other studies that find flk-1 is expressed on HSCs.15
to develop normally. The first cell differentiation is evident after While research on mouse ES cells has been fruitful to eluci-
5-6 days of development with the appearance of the outer cell date mechanisms of mammalian development, mouse and hu-
mass (trophectoderm) and the inner cell mass (ICM) (Fig. 1). man embryogenesis are distinctly different. For example, the rela-
The trophectoderm develops into the outer layers of the placenta; tive size and structure of the placenta and other fetal structures
whereas the ICM , a small cluster o f about a dozen cells, will even- are quite dissimilar.16 These discrepancies lead to the very likely
tually derive all the cells o f the fetal and adult body, and some possibility that mouse ES cells may not always closely model nor-
extraembryonic structures. ES cells are derived by careful isola- mal human developmental biology. Indeed, the yolk sac, an im-
tion of the ICM and culturing these cells under conditions that portant organ of early hematopoiesis, is quite different between
permit them to divide without undergoing differentiation down mouse and m an.17 Therefore, it is valuable to produce a model
specific developmental lineages. Under proper conditions, ES cells system that more closely resembles human development. Toward
can be maintained for months or years as undifferentiated cells this goal, our group (Thomson) derived ES cells from nonhu-
without evidence o f senescence. ES cells naturally express high man primates: rhesus monkeys and common marmosets.18,19 Im-
levels of telomerase and maintain a normal karyotype (unlike EC portantly, the earliest stages of embryogenesis is very analogous
cells).7 However, under specific conditions, the ES cells can be between humans and nonhuman primates.16
induced to differentiate into specific cell types of interest. This
potential is best demonstrated by inducing mouse ES cells to form Characteristics o f Human ES Cells
chimeras with an intact embryo. Careful analysis and breeding of Using lessons learned from derivation of nonhuman primate
the resulting chimeras show that all cells within an adult organ- ES cells, it became feasible to generate ES cells from human pre-
ism can be formed from a single ES cell.8 implantation blastocysts. W ith informed consent and protocols
Work with mouse ES cells has unraveled some of the basics of approved by the local institutional review board, fertilized oo-
mammalian developmental biology and genetics. Not surprisingly, cytes no longer desired by couples undergoing in vitro fertiliza-
research using mouse ES cells has been particularly amenable to tion (and destined to be discarded) were donated to this research
defining mechanisms o f hematopoietic development. Work by endeavor. The technique used to derive human ES cells was simi-
many groups has demonstrated that mouse ES cells can be in- lar to that used for nonhuman primate ES cells. The oocytes were
duced to differentiate into hematopoietic lineages in vitro.9 Intri- cultured to blastocyst stage and immunosurgery used to isolate
cate time-course experiments show the regulation of specific genes the ICM, which is cultured on mitotically-inactivated mouse em-
during specific developmental stages.10,11 O ther studies carefully bryonic fibroblast feeder cells. The ICM-derived cells divided and
alter external stimuli (cytokines, adhesion molecules) to deter- were serial passed without evidence of differentiation— thereby
mine how these factors affect hematopoiesis.12 Perhaps the most established into lines of human ES cells. The multipotent nature
precise experiments have used genetic homologous recombina- of these cells was initially demonstrated by intramuscular injec-
Embryonic Stem Cells: Unique Potential to Treat Autoimmune Diseases 7

pause, a state of suspended embryo growth prior to uterine im-

plantation, requires LIF or other gpl30 agonists. Humans do
not have such a capacity and presumably do not require LIF or
similar molecules to regulate embryonic development.
Mouse and human ES cells also differ in the expression of
particular glycolipids on the cell surface. Undifferentiated hu-
man ES and EC cells express the stage-specific embryonal anti-
gens (SSEA-3 and SSEA-4), as well as TRA -1-60 and TRA-1-81,
but do not express SSEA-1.20 In contrast, mouse ES cells express
SSEA-1, but not SSEA-3, SSEA-4, TRA-1-60, orTR A -1-81.16
These differences again reflect a fundamental embryological dif-
ference between mouse and human development. The enzymes
that produce the glycosylation events which lead to production
of these glycolipids are regulated differently between species,
though the reason for this difference remains unclear.
Recently, the culture conditions required for growth of undif-
ferentiated human ES cells have been refined. Whereas the origi-
nal derivation of these cells used irradiated mouse embryonic fi-
broblast (MEF) feeder cells and media containing fetal bovine
serum, it is now possible to grow these cells in serum-free media
and on plates coated with either Matrigel® or laminin.25 Growth
without feeder cells still requires “conditioned media” taken from
cultures of MEFs. The requirement for “conditioned media” sug-
gests that soluble factor(s) produced by the MEFs are essential
for maintenance o f undifferentiated ES cell growth. These refine-
ments in the culture methods are the start of a process to identify
the conditions that are essential for maintenance of human ES
cell self-renewal. Eventually, use o f a chemically defined media
will aide in large-scale growth of human ES cells as a prelude to
better cell-based therapies.
The excitement surrounding the derivation and characteriza-
tion of human ES cells stems from the potential of these cells to
be induced to produce any cell type in the body. These cells may
become an important source to replace diseased or damaged cells
Figure 1. Derivation of human ES cell lines. Human blastocysts were
and tissues. For example, insulin producing pancreatic beta- cells
grown from cleavage-stage embryos produced by in vitro fertiliza-
may be derived to treat diabetics, dopaminergic neurons may be
tion. Inter cell mass (ICM) cells were separated from trophectoderm
produced to treat patients with Parkinsons disease, and hemato-
by immunosurgery and plated onto irradiated mouse fibroblast feeder
poietic cells may be developed for H SCT and transfusion medi-
cells. Colonies were then expanded and cloned with maintenance of
cine. However, before any of these treatments become a reality,
undifferentiated state (reproduced with permission from Stem Cells).
several barriers must be over come (more extensively reviewed
elsewhere, ref. 26). Foremost among these barriers is to derive
tion into immunodeficient mice. Derivatives of all three embry-
efficient means to induce differentiation of human ES cells to the
onic lineages (endoderm, ectoderm, and mesoderm) could be
cell types of interest. The fundamental differences between mouse
easily identified within these teratomas.20
and human ES cells strongly suggests that mechanisms to induce
Since the initial description of human ES cells, several other
differentiation o f mouse ES cells may not work equally well with
groups have established similar lines.21,22 By convention, all hu-
human ES cells. Indeed, one main strategy to induce differentia-
man ES cell lines should meet the following criteria: (1) deriva-
tion of mouse ES cells is withdrawal of LIF from the culture me-
tion from preimplantation embryo; (2) prolonged undifferenti-
ated p ro liferatio n ; (3) ability to differentiate into cells dia. Since human and rhesus ES cells do not require LIF for un-
representative of all three embryonic germ layers, and (4) main- differentiated growth, elimination of LIF is not a suitable means
tenance of normal karyotype. Importantly, clonally derived lines to induce differentiation.
of human ES cells that meet the above criteria have also been Despite the still relatively recent isolation of human ES cells
established. This provides unambiguous evidence that a single and the potential difficulties in inducing differentiation of these
human ES cell can produce all cell lineages.23 cells down defined pathways, it is remarkable that several types of
The characterization of human ES cells immediately demon- differentiated cell lineages already have been described. To
strated fundamental differences between these cells and mouse varying degrees, hematopoietic27 (Fig. 2), neural,28,29 insulin-
ES cells. Leukemia inhibitory factor (LIF) or other agonists of producing pancreatic beta cells,26,30 cardiac muscle,31 and hépa-
the gpl30-STAT3 signaling cascade are required for maintenance tocytes32 have all been characterized. Next, it will be crucial to
of mouse ES cells as undifferentiated cells. Removal of LIF rap- demonstrate normal physiologic function of these cells in vitro
idly induces differentiation of these cells. In contrast, LIF is not and in vivo. This in vivo testing may be done initially using trans-
sufficient to sustain human (and nonhuman primate) ES cells.18,20 plantation into immunodeficient mice, or neonatal mice. How-
Recent studies suggest this species difference may reflect the abil- ever, the availability of rhesus ES cells presents the opportunity
ity of the mouse reproductive cycle to undergo diapause.24 Dia- to derive nonhuman primate models for preclinical testing. In-
8 Stem Celt Therapy for Autoimmune Disease

Table 2. Strategies to prevent immune-mediated

rejection of human ES cell-derived cells
and tissues

• Immunosuppressive medications
• HLA-defined ES cell "banks"
• Deletion/insertion of HLA genes
• Insertion of genes for immune-modifying proteins
• Deletion of genes for co-stimulatory immune response
proteins
• Nuclear reprogramming to produce HLA-matched ES cells
• Hematopoietic chimerism

The development of transplantable HSCs from human ES cells

may be quite difficult. However, the enormous benefits that may
be gained makes the potential for this development extremely
exciting. There are thousands of patients a year who would ben-
efit if such a breakthrough is accomplished. Even if it does not
Figure 2. Undifferentiated and differentiated human ES cells. A) become readily feasible to produce these transplantable blood cells
Undifferentiated Human embryonic stem (ES) cells maintained on from human ES cells, the basic research to understand the mecha-
irradiated mouse fibroblast feeder cells. Colony of cells with uniform nisms of the earliest stages of human hematopoietic cell develop-
morphology and large nuclei characteristic of human ES cells, origi- ment may be applied toward better growth of other alternative
nal magnification 200X. B) Cobblestone area-forming cells (CAFC) sources of HSCs. For example, cord blood expansion may be-
derived from human ES cells. Human ES cells were allowed to dif- come more feasible if we more clearly understand the particular
ferentiate on S17 mouse bone marrow stromal feeder cells with ap- genes and proteins that maintain the long-term engraftment po-
pearance of CAFC typical of hematopoietic precursor cells, original
tential of HSCs.
magnification 200X. C) Colony-forming unit-granulocyte, macroph-
If (perhaps when) human ES cell-derived HSCs can be used
age (CFU-GM) and D) Burst-forming unit-erythroid (BFU-E) de-
for HSCT, these cells may have important clinical implications
rived colonies from human ES cells. Undifferentiated human ES
beyond the current indications of HSCT. There is good reason to
cells allowed to differentiate on S17 cells were harvested and plated
conjecture that these ES cell-derived HSCs can be used to create
in methylcelloulose based media supplemented with hematopoietic
tolerance for transplantation of other ES cell-derived cells and
cytokines resulting the appearance of typical CFU-GM and BFU-E
tissues. Avoidance o f im m une-m ediated rejection o f ES
(red, hemoglobinized cells) derived colonies. Original magnification
cell-derived cells will be one of the important barriers to over-
of C is 50X and D is 100X. More complete description of derivation
come prior to routine clinical use of these cells. Various strategies
of hematopoietic colonies from human ES cells in ref. 27.
to prevent rejection have been outlined elsewhere26,45 (and Table
2). O ne intriguing strategy takes advantage of the multipotent
deed, rhesus monkey models for hematopoietic cell transplanta-
nature of ES cells to derive HSCs and a second cell line of interest
tion, diabetes mellitus and Parkinson’s disease already exist.26,33
(beta cell, for example) from the same parental ES cell line. Trans-
plantation of the ES cell-derived HSCs would be used to induce
H u m an ES C ells, H em atop oietic C ell a state of hematopoietic chimerism in a patient. Then, this pa-
Transplantation and Tolerance tie n t should be im m unologically tolerant to the second
The development o f hematopoietic cells from human ES cells HLA-matched cell lineage derived from the same parental ES cell
is a natural extension of attempts over the past thirty years to line.
produce new and/or improved sources of cells for hematopoietic This hypothesis has precedent in many clinical cases where
cell transplantation. H SCT is typically used to treat hematologic patients who have undergone allogeneic HSCT subsequently re-
malignancies such as leukemia, lymphoma, or multiple myeloma. ceive a second organ (typically a kidney) from the same donor as
While transplantation of HSCs from a donor (allogeneic HSCT) the hematopoietic cells. Due to immune reconstitution from
often affords the best chance to cure these otherwise fatal dis- HSCs that are a perfect tissue match for kidney, often no addi-
eases, too frequently a suitable HLA-matched sibling or unre-
tional immune suppression is needed, and in many cases, immu-
lated donor cannot be found. Indeed, it has been estimated that
nosuppressive medications can be discontinued without loss of
only approximately one-third of patients who would benefit from
the bone marrow or solid organ graft.46 Animal models more
an allogeneic H SC T actually receive a transplant.34"36 The re-
clearly demonstrate that transplantation of highly purified HSCs
maining patients either undergo autologous H SCT (using their
will induce tolerance to HLA-matched organs, but these animals
own HSCs) or further chemotherapy. Often, these alternatives
do not produce as high a cure rate as allogeneic HSCT,37,38 lead- will still reject third-party, non HLA matched grafts.47,48 Studies
ing to efforts to find alternative sources of HSCs. These alterna- using large animals (nonhuman primates, dogs, miniature swine)
tives include umbilical cord blood cells,39,40 activated autologous show nonmyeloablative conditions followed by allogeneic HSCT
lymphocytes,41 or other ex vivo expanded HSCs.42,43 Moreover, can create a state of hematopoietic mixed chimerism. These chi-
the growing use of nonmyeloablative allogeneic H SCT is expand- meric animals are tolerant to solid organ grafts from the hemato-
ing the potential pool o f patients who may benefit from these poietic cell donor.49 Prospective studies are now being done in
treatments.
44 human trials that combine nonmyeloablative allogeneic HSCT
Embryonic Stem Cells: Unique Potential to Treat Autoimmune Diseases 9

with transplantation of a kidney from the same donor. Initial 5. Evans MJ, Kaufman MH. Establishment in culture of pluripotential
reports of these studies are promising.50 cells from mouse embryos. Nature 1981; 292:154-156.
6. Martin GR. Isolation of a pluripotent cell line from early mouse
This potential to use human ES cell-derived HSCs to induce embryos cultured in medium conditioned by teratocarcinoma stem
hematopoietic chimerism and tolerance has particular relevance cells. Proc Nat Acad Sci USA 1981; 78:7634-7638.
to treatment of autoimmune disease. One of the major goals of 7. Smith A. Embryonic stem cells. In: Marshak DR, Gardner R,
human ES cell-based research is to develop replacement cells that Gottlieb D, eds. Stem cell biology Cold Spring Harbor, NY: Cold
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8. Nagy A, Rossant J, Nagy R et al. Derivation o f completely cell culture
fore, devising methods to induce human ES cells to produce pan-
derived mice from early-passage embryonic stem cells. Proc Natl Acad
creatic islet cells to treat diabetics or oligodendrocytes to benefit Sci USA 1993; 90:8424-8428.
patients with multiple sclerosis seems quite promising. However, 9. Keller GM. In vitro differentiation of embryonic stem cells. Curr
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11. Robertson SM, Kennedy M, Shannon JM et al. A transitional stage
jected if immunosuppressive drugs are not used. O f course drugs in the commitment o f mesoderm to hematopoiesis requiring the
such as cyclosporin, tacrolimus, and corticosteroids have signifi- transcription factor scl/tal-1. Development 2000; 127:2447-2459.
cant complications such as infections, renal failure, diabetes, and 12. Nishikawa SI, Nishikawa S, Hirashima M et al. Progressive lineage
lymphoproliférative disorders. However, the unique characteris- analysis by cell sorting and culture identifies flkl+ve-cadherin+ cells
tics of ES cells may allow methods to permit transplantation of at a diverging point of endothelial and hemopoietic lineages.
these cells without need for immunosuppressive medications.26,45 Development 1998; 125:1747-1757.
13. Shalaby F, Rossant J, Yamaguchi TP et al. Failure o f blood-island
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ming may one day also be able to accomplish this feat.51 How- Nature 1995; 376:66-70.
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the underlying autoimmune process may continue to pose a prob- defining hematopoietic stem cells. Science 1999; 285:1553-1558.
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progenitor or stem cell clones contribute to hematopoiesis in Replacing high-dose cytotoxic therapy with graft-versus-tumor effects.
nonhuman primates. Blood 2000; 96:1-8. Blood 2001; 97:3390-3400.
34. Oudshoorn M, Cornelissen JJ, Fibbe WE et al. Problems and possible 45. Kaufman DS, Odorico JS, Thomson JA. Transplantation therapies
solutions in finding an unrelated bone marrow donor. Results o f from human embryonic stem cells- circumventing immune rejection,
consecutive searches for 240 Dutch patients. Bone Marrow Transplant e-biomed: J Regenerative Medw 2000; 1:11-15.
1997; 20:1011-1017. 46. Dey B, Sykes M, Spitzer TR. Outcomes of recipients of both bone
35. Howe CWS, Radde-Stepanick T. Hematopoietic cell donor registries. marrow and solid organ transplants. A Review Medicine (Baltimore)
In: Thomas ED, Blume KG, Forman SJ, eds. Hematopoietic cell 1998; 77:355-369.
transplantation. Malden, MA: Blackwell Sciences, 1999:2:503-512. 47. Gandy KL, Weissman IL. Tolerance of allogeneic heart grafts in mice
36. Patients proceeding to stem cell transplant: National Marrow Donor simultaneously reconstituted with purified allogeneic hematopoietic
Program 2 0 0 1 . http://w w w .m arrow .org/N M D P /SL ID E SE T / stem cells. Transplantation 1998; 65:295-304.
sld025.htm. 48. Shizuru JA, Weissman IL, Kernoff R et al. Purified hematopoietic
37. Silver RT, W oolf SH, Hehlmann R et al. An evidence-based analysis stem cell grafts induce tolerance to alloantigens and can mediate
of the effect of busulfan, hydroxyurea, interferon, and allogeneic bone positive and negative t cell selection. Proc Natl Acad Sci USA 2000;
marrow transplantation in treating the chronic phase o f chronic 97:9555-9560.
m yeloid leukemia: Developed for the American Society o f 49. Wekerle T, Sykes M. Mixed chimerism as an approach for the
Hematology. Blood 1999; 94:1517-1536. induction o f transplantation tolerance. Transplantation 1999;
38. Stockerl-Goldstein KE, Blume KG. Allogeneic hematopoietic cell
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transplanation for adult patients with acute myeloid leukemia. In:
50. Spitzer TR, Delm onico F, Tolkoff-Rubin N et al. Combined
Thomas ED, Blume KG, Forman SJ, eds. Hematopoietic Cell
histocompatibility leukocyte antigen-matched donor bone marrow
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39. Laughlin MJ, Barker J, Bambach B et al. Hematopoietic engraftment and renal transplantation for multiple myeloma with end stage renal
and survival in adult recipients o f umbilical-cord blood from disease: The induction o f allograft tolerance through mixed
unrelated donors. N Engl J Med 2001; 344:1815-1822. lymphohematopoietic chimerism. Transplantation 1999; 68:480-484.
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Eurocord Transplant Group and the European Blood and Marrow 17:1171-1174.
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41. Lum LG, LeFever AV, Treisman JS et al. Immune modulation in pancreas transplants. Diabetes 1989; 38:85-87.
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2001; 24:408-419.
 CHAPTER 3

Neural Stem Cells and Oligodendrocyte Progenitors

in the Central Nervous System
Jennifer A. Jackson and D iana L. Clarke

Introduction

T
he adult vertebrate central nervous system (CNS) consists which contacts and repeatedly envelopes a stretch of axon with
of four major differentiated cell types: neurons, astrocytes, subsequent condensation of its spiraling plasma membrane. This
oligodendrocytes and ependymal cells. Historically, there myelination of axons imparts insulating properties that allow the
has been disagreement on how these differentiated cell rapid propagation of action potentials throughout the CNS with-
types
are generated in the CNS. Progress remains hindered by the out continuous regeneration of the action potential along each
complexity of cell structure in this system, the lack of specific segment of an axon.
cell surface markers to identify distinct cell types and the presence
o f numerous transit amplifying cell populations that rapidly O rigin o f Neural and G lial Progenitors
generate early progenitors. At present it is clear that some cells, in the C N S
termed neural stem cells, can generate neurons as well as astrocytes
All cells comprising the CNS are originally derived from the
and oligodendrocytes of the glial lineage both in vitro and in
early neuroepithelium that forms as the neural plate along the
vivo. However, controversy still exists over whether the majority
midline of the developing embryo. As development proceeds, this
of glia in the CNS are generated by multipotential stem cells or
single layer of pseudostratified epithelium folds to form the neu-
progenitor cells that were born as committed glioblasts. Neverthe-
ral tube (Fig. 1). The differentiation of the neuroepithelial stem
less, the existence of stem cells in the CNS has important impli-
cells into neurons and glia then proceeds in a temporal specific
cations for understanding both the mechanisms that generate
manner that is specific for each region of the developing neural
neural diversity during embryonic development and the recruitment
tu b e.4,5 G enerally neurogenesis occurs first, followed by
and differentiation o f neural stem cells present in the adult. This
gliogenesis. This patterning of the neural tube is thought to be-
review summarizes our knowledge of stem cells that comprise
gin at the neural plate stage of development through inductive
the CNS and examines the broad plasticity reported for adult
interactions that create organizing centers at the dorsal and ven-
CNS stem cell populations.
tral poles.6'8 These specialized neuroepithelial cells generate sig-
nals that induce, often in a concentration dependent manner, the
Interdependence o f N eurons and G lia expression of patterning genes in adjacent neuroepithelial cells.
in the C N S These patterning genes generally encode homeodomain transcrip-
Glial cells constitute the majority of the cells in the CNS. These tion factors, and their expression patterns divide the cells in the
cells provide the structural scaffolding that is important for mi- neuroepithelium into different domains along the rostral-caudal
gration of early neuroblasts, are a major source of adhesion mol- and dorso-ventral axes of the neural tube.9,16 These patterning
ecules that participate in the formation of neural networks, form genes are thought to specify neuronal subtype identity and con-
limiting membranes that separate the CNS from other tissues trol the duration of specific types of neurogenesis occurring in
and aid in the rapid conduction of electrical impulses down axons the brain and spinal cord during each developmental stage.
of mature neurons. There is now growing recognition that glia, The exact mechanisms underlying the developmental changes
possibly through their immense glial networks, may possess com- in the stem cell and precursor population in any given region of
munication skills that complement those of neurons themselves.1,2 the neural tube are not understood. However, by midgestation in
Astroglia are stellate, branched supporting cells that contact the the developing cortex of the brain, for example, young neurons
soma, dendrites and axons of neurons, the soma and processes of have migrated beyond the germinal ventricular zone (VZ) of the
oligodendrocytes and associate intricately with other astrocytes. neuroepithelium with the aid of newly formed glial cells in this
Their close association with the surface of various neurons is region (Fig. 2). These radial glia contact the inner ventricular
thought to mediate the exchange of substances between these two surface and the outer pial surface of the neural tube, guiding neu-
cell types.3Their multiple processes also contact, induce and main- ronal migration away from the VZ and forming the second ger-
tain the tight junctions in endothelial cells that effectively form minal zone, the subventricular zone (SVZ). W hen early neuro-
the blood-brain barrier. Oligodendroglia, also a supporting glial blast formation has ceased, the neuroepithelial stem cells begin to
cell type present in the CNS, extend many processes, each of differentiate into glioblasts. Clonal studies suggest that most glia

Stem Cell Therapy for Autoimmune Disease, edited by Richard K. Burt and Alberto M. Marmont. ©2004 Landes Bioscience/Eurekah.com.
12 Stem Cell Therapy for Autoimmune Disease

Figure 1. Early development of the central nervous system (CNS). Cross-section through the head anlage of a developing chick embryo:
Initially, cells forming the neural plate lie on both sides of the primitive streak. As the primitive streak migrates caudally, the mesoderm
invaginates, and the neural plate forms the neural groove in the midline of the embryo. Changes in cell shape cause the neural groove to
expand upward and fold inward to form the neural tube. Cells lining the neural tube are the early neuroepithelial stem cells.

originate from stem cells in the neuroepithelium.11 These cells cells isolated from the spinal cord generate spinal cord progeny.19
migrate out into the adjacent SVZ where they proliferate and Stem cells isolated from the basal forebrain generate more GABA
become astrocytes and oligodendrocytes. Lineage tracing studies containing neurons than stem cells derived from dorsal regions.20
using stereotactically injected retrovirus support the view that the
majority of progenitors within this germinal matrix are glial pre- A d ult Neural Stem C ells
cursors that generate either astrocytes or oligodendrocytes. 2,13 Proliferative stem cell compartments are not exclusive to the
Some SVZ cells give rise to both oligodendrocytes and astrocytes, developing CNS. However, after embryonic development, the
and a rare cell will develop into both neurons and glia,14 athough exact characteristics of the neural stem cell in the CNS have re-
this remains a controversial issue.15 mained elusive and controversial. Numerous pioneering experi-
W hen glioblast formation ceases shortly after birth, the ger- ments have demonstrated that specific regions of the mammalian
minal VZ disappears throughout the neuroaxis and many of the CNS undergo a moderate, yet continuous level of neurogenesis
remaining neuroepithelial cells become ependymal cells. The postnatally and throughout adult life21'24 (Fig. 3). To date,
ependymal cells persist throughout adulthood lining the luminal neurogenesis in the adult mammalian CNS is known to utilize at
surface of the ventricular system of the brain and the central ca- least one dividing progenitor cell population25 and two different
nal of the spinal cord. These cells possess multiple cilia on their multipotent stem cell populations.2 28 The putative progenitor
apical surface that effectively move the cerebral spinal fluid cell population resides in the subgranular zone of the dentate gy-
throughout these regions. Similarly, the SVZ, decreases in size rus located in the hippocampus, the region of the brain involved
and persists immediately adjacent to the ependymal cell layer in learning and memory. The two remaining stem cell popula-
throughout most of the ventricular regions o f the brain. How- tions have been reported to exist in and near the anterior lateral
ever, a SVZ region is not present in the developing or mature ventricular wall of the cerebral cortex, both of which exist in the
regions of the spinal cord. adult as highly differentiated glial cell types; SVZ astrocytes and
As compartmentalization of the CNS becomes apparent, neu- ventricular ependymal cells. While there is only a limited amount
ral stem cells in the early mammalian CNS are considered to be of neurogenesis that occurs in the adult hippocampus, stem cells
concentrated in seven major areas: the olfactory bulb, VZ and located in the SVZ are thought to continuously replace interneu-
SVZ of the forebrain; the hippocampus, cerebellum, cerebral cor- rons in the olfactory bulb. It remains controversial whether the
tex and the spinal cord. Their number and pattern of develop- rapidly dividing multipotent stem cells in the SVZ are a distinct
ment vary in different species.4,16,17 However, once the pattern- stem cell population that contributes to the generation o f olfac-
ing of the different CNS compartments is in place, it is believed tory interneurons. It has been suggested that the adjacent ependy-
that stem cells located in these different regions of the developing mal cell layer, which has been shown to divide at a comparatively
CNS are develop mentally distinct and are not a single popula- slower rate in vivo, may give rise to the SVZ cells.26,29 Although
tion of stem cells that are dispersed over multiple sites.18 Stem glial cells in the SVZ are derived from the VZ during early
Neural Stem Cells and Oligodendrocyte Progenitors in the Central Nervous System 13

Figure 2. Early differentiation of the neuroepithelium in the neocortex of the brain. Early in development, neuroepithelial stem cells reside
in the luminal cellular layer of the neural tube in an area generally termed the ventricular zone (VZ). These cells begin to divide rapidly,
initially generating radial glia and early restricted neuroblasts. The neuroblasts exit from the cell cycle, delaminate from the neuroepithelium,
accumulate within the ventricular zone and activate a number of genes indicative of generic neuronal differentiation. Activation of these
genes is thought to put into motion the expression of specific cascades of factors that specify both neuronal determination and differentiation
in different regions of the cortex. As the neuroblasts mature, radial glia provide a primary substrate for the migration of post-mitotic neurons
from the germinal ventricular zone to the emerging layers of the neocortex. After neuroblast formation subsides, neuroepithelial stem cells
in the ventricular zone generate glioblasts. These glioblasts migrate out into the adjacent subventricular zone (SVZ) where they proliferate
and become primarily astrocytes and oligodendrocytes. These cells then migrate away from this region to populate other regions of the
developing cortex.

embryonic development, a lineage relationship between these two promote the production or activity of proneural transcription
multipotent adult cell types has not been firmly established. How- factors. Similarly, they can differentiate into glia in response to a
ever, the location of these two adult neural stem cell populations variety of extracellular signals such as ciliary neurotrophic factor
has some surprising parallels to early neurogenic development of (CN TF), bone morphogenic proteins (BMPs), transforming
the ventricular regions in the embryonic brain. The ependymal growth factor-a (TG Fa), and neuregulin-1 (Nrg-1)/ glial growth
cells, within the lateral ventricular wall, occupy a position analo- factor-2 (GGF2). However it is not known whether these same
gous to the embryonic VZ cells and are thought to be derived factors directly induce glial differentiation in vivo. Because envi-
directly from a subset of embryonic VZ cells. W hile the ependy- ronmental conditions can be provided in vitro by adding specific
mal cells are highly differentiated glial cells that line the luminal trophic factors to the culture medium, neural stem cells differen-
surface of the adult ventricular system, these seemingly differen- tiating in cultures have been shown to exhibit considerable plas-
tiated cells express several proteins expressed by neural stem cells ticity not normally observed in vivo. Thus, understanding the
during normal development including nestin, musashi, and Notch environmental conditions necessary to promote specific types of
1 receptors. differentiation from a stem cell may show that the limitations
While the identification and existence of adult neural stem that these cells perceive in vivo are controlled by environmental
cells in the ventricular and SVZ was a surprising finding, even cues and not necessarily by intrinsic commitment of the stem cell
more surprising is the suggestion that they are highly differenti- itself.
ated glial cell types and not remnants of nascent undifferentiated
stem cells that are specified early during embryonic development. O ligodendroglial Progenitors
Numerous trophic factors have been shown to influence the ac- The multipotent neural stem cells or early neural progenitor
tual developmental fate of a progenitor or multipotent stem cell cells present in the central nervous system of embryonic, neona-
from these regions, which may differ from its developmental po- tal and adult animals can also differentiate into intriguing
tential.30,31 In culture, cells from these regions can differentiate lineage-restricted progenitors termed oligodendroglial progenitors.
into neurons in response to instructive extracellular signals that Most oligodendrocyte precursor cells in the developing central
14 Stem Ceil Therapy for Autoimmune Disease

Figure 3. Neurogenic regions of the mammalian CNS. Neural stem cells in the adult CNS have been identified in four different regions:
the ependymal cell layer lining the lateral ventricular regions of the brain, the adjacent subventricular zone, the hippocampus and the spinal
cord. The ventricular system of the brain is continuous with the central canal of the spinal cord (A). Coronal sections through different
regions of the brain show the location of the hippocampus near the posterior region of the cerebral hemispheres (B). The ependymal cell
layer lining the luminal surface of the lateral ventricles and the adjacent subventricular zone is shown in C.

nervous system terminally differentiate into oligodendrocytes that progenitors to differentiate into oligodendrocytes is intrinsic to
myelinate axons. Terminally differentiated oligodendrocytes do the lineage.36 However, co-culture with neurons increases myelin
not divide, dedifferentiate, or reenter the cell cycle. However, pre- gene expression, such as PLP, MBP and MAG.
cursors to oligodendrocytes do exist and their division persists,
albeit at a slow rate, throughout life, with a certain potentiality P lasticity o f the O ligodendrocyte Lineage
bias for myelin repair. Recently, mouse oligodendroglial progenitors derived from
Oligodendrocyte progenitors have been characterized in ro- to tip o ten t em bryonic stem cells were transplanted into a
dent species by their bipolar morphology and by the presence of myelin-deficient rat model.37 These cells were able to interact
specific markers. In vitro studies as well as in vivo experiments with the host neurons and efficiently myelinate axons in the brain
have shown that these cells are actively proliferating and posses and spinal cord. Similarly, oligodendrocyte progenitors have been
migratory properties. Oligodendrocyte progenitors arise from isolated from the adult brain. These cells have been propagated
m ultip o ten tial cells in spatially restricted germ inal zones extensively in vitro as neurospheres— clonal spheroid cell aggre-
thoughout the brain and spinal cord. These progenitors migrate gates that detach from the tissue culture dish and grow in suspen-
long distances away from these zones in the CNS before they sion 21— to generate a large number of multipotent stem cell prog-
settle along fiber tracts o f the future white matter and then trans- eny that maintain their myelinating potential.38 Phenotypic
form into preoligodendrocytes. The pre-oligodendrocyte is a characterization of these cells has indicated that these oligoden-
multiprocessed, post-migratory cell that retains the ability to di- drocyte progenitors resemble neonatal rather than adult progeni-
vide but is not responsive to the mitogen, platelet-derived growth tors. The ability to generate such cells from both embryonic stem
factor (PDGF).32-34 These cells can be identified by their acquisi- cells and the adult brain opens many possibilities to explore the
tion of the marker, 0 4 .35 The preoligodendrocyte can further potential of these cells for repairing myelin disorders. Moreover,
differentiate into an immature oligodendrocyte, characterized in the ability of the adult-derived oligodendrocyte progenitors to
the rat by the appearance o f the marker GalC, and the loss of apparently dedifferentiate to a more primative state, suggests that
expression of GD3 and A2B5 antigens on the cell surface. CNP these cells may also represent a unique progenitor cell population
(2’,3’-cyclic nucleotide 3’-phosphodiesterase), the earliest known that may be permissive to manipulation both in vitro and in vivo.
myelin-specific protein to be synthesized by developing oligo-
dendrocytes also appears at this time. Are Neural Stem C ells D erived from the A dult
The mature oligodendrocyte is characterized by the expression C N S Irreversibly D eterm ined?
of myelin basic protein (MBP), myelin associated-glycoprotein Adult spinal-cord-derived cells, which normally generate only
(MAG) and myelin proteolipid protein (PLP). In vitro analyses glia4,26 can make interneurons if injected into the adult hippoc-
suggest that maturation of oligodendrocytes from the precursor stage ampus.39 Similarly, adult hippocampal-derived stem cells can make
to the mature cell is identical in culture, even in the absence of olfactory interneurons after transplantation into the SVZ.40
neurons, as in intact tissue. Thus the capacity of oligodendrocyte However, the ability of adult-derived cells to produce complex
Neural Stem Celts and Oligodendrocyte Progenitors in the Central Nervous System 15

projection neurons that span long distances in the mature CNS, differentiation of these cells to myocytes, a process which required
has not been demonstrated. direct contact between the neural stem cell and the muscle cells.47
Only recently have people investigating the differentiation A conceptually different approach has also demonstrated the
potential of adult neural stem cells discovered that their differen- myogenic differentiation potential of neural stem cells.48 This ap-
tiation repertoire extends well beyond the boundaries of the cell proach took advantage of the large number of inductive signals
types identified in the CNS. This revelation has caused quite a present in embryonic stem cells undergoing differentiation in vitro,
stir among traditional developmental biologists and many valid a process that ultimately results in the differentiation of the ES
arguments have been raised and remain to be answered.20,41 If cells to various cell lineages. In order to expose neural stem cells
this newly discovered potentiality is true, it may cause us to to such a milieu, the cells were cultured in close proximity to
re-evaluate or define the methods that are used to determine differentiating embryoid bodies. In these experiments, differen-
whether a neural stem cell is truly irreversibly specified or not. It tiated neural stem cells gave rise primarily to myocytes, identified
is possible that embryonic or adult neural stem cells, when cul- by the expression o f the markers desmin and myosin heavy chain,
tured in vitro, exhibit a renewed potentiality that has not been and exhibited morphological features such as syncytium formation.
tested or observed in direct transplantation assays. This renewed Two separate methods have been used to simultaneously dem-
potential may involve complex inter- and intracellular mecha- onstrate the broad differentiation potential of neural stem cells.48
nisms that may have been unwittingly imparted to these cells in These experiments have relied heavily on the abundance of fac-
culture. Alternatively, the need to restrict the potential of a stem tors present in the early embryonic environment to demonstrate
cell may decrease as an organism matures.42 Thus, during embry- such potential. In a separate set of experiments, adult neural stem
onic development, cells may be exposed to overlapping sets of cells were injected into early developing chick or mouse embryos.
extracellular signals. This may initially necessitate their use of In the developing chick, neurospheres or clusters o f
cell-autonomous mechanisms to restrict their differentiation. clonally-derived neural stem cells were placed directly on or near
Therefore, transplanting restricted cells from one location to an- the primitive streak of a gastrulating embryo. This allowed the
other does not affect their differentiation potential. However, once neural stem cells to integrate into the epiblast, move with the
an organism matures, stem cells in different tissues may be spa- primitive ectoderm cells through the primitive streak during gas-
tially segregated into specific niches where they no longer actively trulation and be distributed throughout the three definitive germ
encounter signals that restrict their differentiation potential. These layers. In the mouse, dissociated neural stem cells, or small
cells may be released from their cell autonomous programs making neurospheres, were aggregated with morulae or injected into blas-
them more responsive to environmental cues that can influence tocysts. In both of these early embryo models, neural stem cell
their differentiation. progeny were found in numerous tissues of the host, in environ-
ments that inevitably had exposed them to a large number of
D ifferentiation to H em atopoietic C ells different inductive signals. The neural stem cells integrated in a
Intriguingly, in the past few years several reports have indi- mosaic pattern into many tissues and were morphologically in-
cated that both hematopoietic and neural stem cells both dem- distinguishable from neighboring host cells. Contributions to tis-
onstrate surprising plasticity.43 Bone marrow derived cells have sues were large and occasionally comprised as much as 30% of
been shown to generate cells expressing neuronal markers in the the entire organ. Cell type specific antibodies were used to verify
brain.44,45 Similarly, embryonic or adult-derived neural stem cells whether the neural stem cell derived cells had remained neural or
from the brain, when injected intravenously into sublethally irra- if they had taken on a phenotype appropriate for the tissue into
diated mice, have been shown to generate hematopoietic deriva- which they had integrated. In both the chick and mouse assays,
tives.46 In this study, in vitro clonogenic assays, immunocytochem- neural stem cell progeny had indeed differentiated to many em-
istry and flow ctyometric analysis were used to test whether these bryonic cell types including hépatocytes, cardiomyocytes and
cells had adopted a hematopoietic identity. In the clonogenic as- epidermal cells, thus representing cells that originate from all three
says, cells from the bone marrow of transplanted animals were germ layers. Importantly, in experiments where multipotent sec-
plated in the presence of defined cytokines. Colonies founded by ondary clonal cultures were established, it was shown that a single
single hematopoietic precursors of neural stem cell origin were neural stem cell, had the potential to generate progeny for all
13% pure granulocyte, 30% granulocyte-macrophage, 22% pure three germ layers.
macrophage and 19% mixed colonies. A few colonies did not While many of the transplantation and embryonic differen-
originate from the donor stem cell population confirming that tiation studies have demonstrated the ability of adult neural stem
some endogenous hematopoietic progenitors had survived the cell populations to populate and differentiate into cell types spe-
irradiation. Importantly, none of the neural stem cell cultures or cific for a particular tissue in vivo, their longevity and functional
progeny o f clones used in the transplantation proliferated or contribution to a living organism have not been demonstrated.
formed colonies in the clonogenic assays. The neural stem cells Many of the obstacles hampering the definitive detection of do-
used in this study also generated neurons, astrocytes and oligo- nor cells in these early experiments can now be addressed as new
dendrocytes in vitro, arguing against the possibility that the blood genetically marked strains of mice and methods increasing the
forming cells from the brain were stray hematopoietic stem cells efficiency of cellular contribution have been identified. It will be
that contributed to the repopulation of the hematopoietic lineages. of particular interest to determine whether the contribution of a
donor stem cell to a particular adult stem cell niche will allow the
D ifferentiation to O ther Som atic C ell Lineages original donor stem cell to retain its ability to self-renew, con-
The myogenic potential o f adult neural stem cells has also tinuously contributing to the neogenesis of cell types particular
been recently demonstrated. Co-culture of primary mouse or to that tissue. It will also be of interest to determine whether stem
human neural stem cells with myoblasts or injection of the neu- cells introduced into the early embryonic environment exhibit
ral stem cells into skeletal muscle of adult mice resulted in the germline contribution.
16 Stem Ceil Therapy for Autoimmune Disease

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Exploring the Variety of Random
Documents with Different Content
In presence of the magistrates of the Canongate,
Edinburgh, ‘William Heriot, younger, baxter, 1568-9. Jan. 10.
became, out of his awn free motive will, cautioner
for George Heriot, that the said George sall remove furth of this
burgh and freedom thereof, within the space of fifteen days next,
and nae be fund thereintill, in case the said George associate not
himself to the religion of Christ’s kirk, and satisfie the kirk in making
of repentance, as effeirs.’—C. C. R.
This was a part of the process of completing the Reformation.

‘... the Regent made progress first to Stirling,

where four priests of Dumblane were condemnit to 1569. May.
the death, for saying of mess against the act of
parliament; but he remittit their lives, and causit 1569. Aug. 16.
them to be bund to the mercat cross with their
vestments and chalices in derision, where the people cast eggs and
other villanie at their faces, by the space of ane hour; and thereafter
their vestments and chalices were burnt to ashes. From that he
passed to Sanctandrois, where a notable sorcerer called Nic Neville
was condemnit to the death and brunt; and a Frenchman callit Paris,
wha was ane of the devisers of the king’s death, was hangit in
Sanctandrois, and with him William Stewart, Lyon King of Arms, for
divers points of witchcraft and necromancy.’—H. K. J.
The Diurnal of Occurrents relates the Regent’s proceedings against
the powers of the other world in this journey in a style equally cool
and laconic. ‘In my Lord Regent’s passing to the north, he causit
burn certain witches in Sanctandrois, and in returning he causit burn
ane other company of witches in Dundee.’
The Regent once more set out on an expedition
against the Border thieves, attended by a hundred Sep.
men of war. In the words of a poetical panegyrist:

‘... having established all thing in this sort,

To Liddesdale again he did resort.
Through Ewesdale, Esdale, and all the dales rade he,
And also lay three nichts in Cannobie,
Where nae prince lay thir hunder years before;
Nae thief durst stir, they did him fear so sore;
And that they should nae mair their theft allege,
Threescore and twelve he brought of them in pledge,
Syne warded them, whilk made the rest keep order,
Than might the rash buss keep kye on the Border.’68

It is said that no former ruler had ever so thoroughly awed the

Border men. On his return to Edinburgh in November, he distributed
the hostages among certain barons of the realm.
This, however, was the last of Moray’s expeditions against the
thieves. He was approaching the end of his career, doomed by party
hatred in conjunction with private malice.
‘The Earl of Moray, the Good Regent, was slain in
Linlithgow by James Hamilton of Bothwell-haugh, 1569-70. Jan.
who shot the said Regent with a gun out at ane 23.
window, and presently thereafter fled out at the
back, and leapt on a very good horse, which the Hamiltons had
ready waiting for him; and, being followed speedily, after that spur
and wand had failed him, he drew forth his dagger, and struck his
horse behind; whilk causit the horse to leap a very broad stank; by
whilk means he escaped.’—Bir.
 REGENCIES OF LENNOX AND MAR:
1570-1572.
The death of the Regent Moray proved a great blow to the infant
king’s party, for there was no man of equal mark and energy to take
his place. The friends of the exiled queen raised their heads again,
and in a force which might well give the ruling party some anxiety.
Seeing the imminence of the danger, Elizabeth yielded to the wishes
of Mary’s enemies, and sent an army under the Earl of Sussex into
Scotland in April, who ‘burnt, herrit, and destroyit sae mickle of the
Merse and Teviotdale as they might be masters of, asseizit the castle
of Farniehirst, and demolishit the same, and thereafter past to
Hawick and to Branksholm, and burnt and herrit the same,’ thus
punishing the Scotts, Kerrs, and others who had lately made a
hostile incursion in Mary’s behalf into England. Towards the end of
the month, they besieged and took Hume Castle. A similar army
under Sir William Drury entered Scotland in the ensuing month, and
committed the like havock in Lanarkshire, so as to disable the
queen’s friends of the house of Hamilton. The sufferings thus
occasioned in certain districts were dreadful, and the principal
sufferers were the poor. In Hume Castle, when taken by Sussex,
‘was the hale guids and gear perteining to the hale tenants of my
Lord Hume, wherethrough the saids puir tenants were allutterly
herrit.’ The devastation at Hamilton was ‘in sic sort and maner as the
like in this realm has not been heard before.’ And when the English
troops came thence to Linlithgow on their return, ‘they herrit all the
Monkland, the Lord Fleming’s bounds, my Lord Livingstone’s bounds,
together with all their puir tenants and friends, in sic maner that nae
heart can think thereon but the same must be dolorous.’—D. O. Yet
this was but a foretaste of the woes which a disputed succession
was now for three years to lay upon the land.
At the dictation of Elizabeth—for the Protestant lords in Scotland
were wholly subservient to her—Matthew, Earl of Lennox, paternal
grandfather of the young king, was elected Regent (July 17, 1570).
The real ruling spirit was the Earl of Morton, who lost no time in
proceeding against some friends of Queen Mary in the north. Taking
the town of Brechin, which had been held for her, he caused thirty-
one of the garrison to be mercilessly put to death. ‘The deaths of
thir persons were greatomly bewailit by mony.’ At the same time, the
Earl of Sussex made an inroad into Dumfriesshire, cast down many
houses of Mary’s friends, ‘burnt certain houses in the town of
Dumfries, and reft and spulyit all that they micht get.’ Three
considerable districts in Scotland were this summer reduced to a
desert.
The Gordon power in the north, that of the Hamiltons and Argyle in
the west, and the Border chiefs, formed the great centres of Mary’s
party, which altogether was so strong, that it must have triumphed
but for the backing which the other party received from England. As
matters stood, the king’s friends were able to maintain themselves in
possession of the country at large, holding Stirling as the seat of
government, while Kirkaldy of Grange, governor of the Castle of
Edinburgh, unexpectedly went over to the queen’s side, as did
Maitland of Lethington, and some others lately arrayed against her.
Edinburgh and its castle consequently became a centre of operations
for that party. Then commenced an intestine war, at first consisting
of mutual devastations on each other’s lands, but soon assuming a
sanguinary character. It is not consistent with our design to relate it
in detail; but a few characteristic proceedings are given in the
chronicle, usually in the simple and pathetic language of the time.
Lennox being killed in a surprise at Stirling (September 3, 1571), the
Earl of Mar was chosen to the vacant regency. Under him the war
advanced with even increased ferocity, until it came to be a rule that
no quarter should be given on either side. In little more than a
twelvemonth, this gentle-natured noble sunk under the burden of
government; ‘the maist cause of his deid was that he lovit peace,
and could not have the same.’—D. O. The Earl of Morton, the ablest
man of the whole party since Moray, but merciless and greedy in the
extreme, succeeded, with the full approbation of the Mistress of the
Protestant party of Scotland.

Lord Fleming being a conspicuous leader on the

queen’s side, and captain of Dumbarton Castle, his 1570. May.
lands in the counties of Lanark and Dumbarton
were amongst those which fell under the vengeance of the ruling
party. As one of the enormities perpetrated by the Earl of Lennox
and his men on Lord Fleming’s estates—‘they have slain and
destroyit the deer of his forest of Cumbernauld, and the white kye
and bulls of the said forest, to the great destruction of policy and
hinder of the commonweal. For that kind of kye and bulls has been
keepit thir mony years in the said forest, and the like was not
maintenit in ony other parts of the Isle of Albion, as is weel
knawn.’69
The ‘white kye and bulls’ here spoken of are
believed to have been a remnant of the original 1570.
wild cattle of the Caledonian forest. Boece
describes them as white, with lion-like manes, fierce, untamable,
and shunning human society—so misanthropical, indeed, that they
would eat nothing which the hand of man had touched. He, like the
writer quoted above, says that none of them were left but only in
Cumbernauld. Leslie, however, tells us that they also existed in the
parks of Stirling and Kincardine. Latterly, there have been herds of
the same oxen (but perhaps imported) in the Duke of Hamilton’s
park of Cadzow, in Lanarkshire; in the Duke of Buccleuch and
Queensberry’s at Drumlanrig; and in Lord Tankerville’s park at
Chillingham, in Northumberland.
Perhaps the severities of the English army on this occasion were only
what her Scottish allies would themselves have practised against
their opponents. What follows, however, seems to have gone a little
beyond the bounds of partisan vengeance, while it not less illustrates
the sacrifice of national dignity at which the enemies of Mary were
content to purchase the aid of the English queen.
Sir William Drury, returning with the English army
from the devastation of the Duke of Chatelherault’s May 29.
country in Lanarkshire, resolved to destroy the
town of Linlithgow, in retribution for its having proved a harbour for
the enemies of Elizabeth and of her ally the young Scottish king. It
seemed but right that the scene of the murder of Moray should thus
suffer. He therefore called the provost of the burgh before him, and
announced his intention, saying, however, that he would allow time
for the carrying away of any women in childbed or impotent people,
and also conceding that a place should be appointed, to which the
goods belonging to the citizens should be brought for preservation.
‘The time being come for this execution, the Earl of Morton, that still
accompanied the English general, offered himself as an intercessor
to entreat and sue for a pardon, bringing afore the general a
multitude of wailing people, whose mournful and most piteous cries
was lamentable and very importunate.’
Drury insisted that justice demanded an example
being made of Linlithgow; but ‘the people of all 1570. July 4.
sorts so pressed about him, and made such pitiful
cries and sorrowful noise, with children sucking of their mothers’
breasts, that he, taking ruth of their miserable estates, at this their
lamentable suit, especially at the great instance of the Earl of
Morton, who came bareheaded to speak for them, the general was
content to save the town and people therein.’ He took assurance
from them, however, that the chief inhabitants should follow his
camp to Berwick, and there wait the clemency of the queen of
England.—Holinshed.
‘... at 10 hours at night, there was ane earthquake in the city of
Glasgow, and lastit but ane short space; but it causit the inhabitants
of the said city to be in great terror and fear.’—D. O.

‘In this time there was ane monstrous fish seen in Loch Fyne, having
great een in the head thereof, and at some times wald stand aboon
the water as high as the mast of a ship; and the said [creature] had
upon the head thereof [twa crowns, ane] aboon little, and the
downmaist crown meikle; whilk was reportit by wise men, that the
same was ane sign and taiken of ane sudden alteration within this
realm.’—D. O.
The low intelligence of the age is seen in nothing more
conspicuously than in the numerous tales of animals alleged to have
been seen, with peculiarities impossible in nature, and believed to be
ominous of public calamity. The appearance of a similar animal in
another of the Argyleshire lochs in 1510 is noted by Hector Boece,
on the information of Duncan Campbell, a noble knight. This ‘terrible
beast’ was ‘of the bigness of a greyhound, and footed like a gander.
Issuing out of the water early in the morning about midsummer,’ he
‘did very easily and without any force or straining of himself
overthrow huge oaks with his tail, and therewith killed outright three
men that hunted him with three strokes of his said tail, the rest of
them saving themselves in trees thereabouts, whilst the aforesaid
monster returned to the water. Those that are given to the
observation of rare and uncouth sights, believe that this beast is
never seen but against some great trouble and mischief to come
upon the realm of Scotland.’70
In Holinshed’s Chronicle (1577), the Firth of Forth is said
occasionally to contain ‘sundry fishes of a monstrous shape, with
cowls hanging over their heads like unto monks, and in the rest
resembling the body of man. They shew themselves above the water
to the navel, howbeit they never appear but against some great
pestilence of men or murrain of cattle; wherefore their only sight
doth breed great terror to the Scottish nation, who are very great
observers of uncouth signs and tokens.’
On the whole, it is most likely that some species of the cetacea or
phocidæ was concerned in giving rise to these tales of sea-monsters.
Sir William Sinclair of Roslin, who was living at this
time, thus notes the appearance of an 1570.
extraordinary animal in the year 1500: ‘Hutcheon
Frizell in Glenconie, the best and maist in estimation of the Lord
Lovat’s kin, he and ane servand with him, being at the hunting on
ane hie land amang very rank heather, twa arrow-draught frae him
he heard like the call of ane ratch approaching near and near, while
[till] at the last he saw it, and shot at it ane dead straik with ane
arrow; where it lap and welterit up and down ane spear length of
breadth and length. The heather and bent being mair nor ane foot
of height, it being in the deid-thraw, brint all to the eird [earth], as it
had been muirburn. It was mair nor twa eln of length, as great as
the coist of ane man, without feet, having ane mickle fin on ilk side,
with ane tail and ane terrible head. His great deer-dogs wald not
come near it. It had great speed. They callit it ane dragon.’71
He commemorates a sea-animal not less wonderful, which was
thrown upon the coast of Northumberland in 1544. ‘At the sea-side
at Bamburgh, there was nae kind of fish ta’en by the space of twa
year; but the sea made ane great routing and horrible noise, which
was by [beside] custom and use. So it chancit, at the hie spring
[tide], that ane terrible beast was casten in dead, of the quantity
[bulk] of ane man. Nae man could devise ane thing mair terrible,
with horns on the head of it, red een, with misshapen face, with
lucken [webbed] hands and feet, and ane great rumple hinging to
the eird. It consumit and stinkit sae, that in short time nae man nor
beast might come near it; but all the country about saw it before,
and sundry took great fear and dreadour for the sicht of it a lang
space after. It was callit a Sea-devil. Witness the Laird of Mow.’72
‘The summer right guid, and all victuals guid cheap; the August right
fair and guid weather.’—C. F.

An extraordinary act of Gilbert, Earl of Cassillis,

sometimes called King of Carrick, on account of the Sep. 1. 1570.
great power which he possessed in that district.
The revenues of the abbey of Crossraguel, in Carrick, had been
bestowed upon Master Allan Stewart. The earl had got a feu of the
abbey from a predecessor of Stewart, but it never was confirmed.
After some fruitless endeavours to obtain a confirmation from
Stewart, the earl inveigled him to the castle of Dunure, a strong
fortalice situated on a rocky part of the coast overlooking the
Atlantic.
Here the Commendator was honourably entertained—‘gif a prisoner
can think ony entertainment pleasing. But after that certain days
were spent, and that the earl could not obtain the feus of
Crossraguel according to his awn appetite, he determined to prove
gif a collation could work that which neither dinner nor supper could
do of a long time. And so the said Master was carried to a secret
chalmer [according to Stewart’s own account, to a house called the
Black Voute (Vault) of Dunure; there is something horribly suitable in
the name]. With him passit the honourable earl, his worshipful
brother, and sic as was appointed to be servants at that banquet. In
the chalmer there was a great iron chimney, under it a fire; other
great provision was not seen. The first course was: “My lord abbot
(said the earl), it will please you confess here, that with your awn
consent ye remain in my company, because ye dare not commit you
to the hands of others.” The abbot answered: “Wald ye, my lord,
that I should make a manifest leasing for your pleasure? The truth
is, my lord, it is against my will that I am here; neither yet have I
ony pleasure in your company.” “But ye sall remain with me at this
time,” said the earl. “I am not able to resist your will and pleasure,”
said the abbot, “in this place.” “Ye maun then obey me,” said the
earl. And with that were presented unto him certain letters to
subscrive, amongst which there was a five-year tack [lease] and a
nineteen-year tack, and a charter of feu of all the lands of
Crossraguel, with all the clauses necessar for the earl to haste him to
hell! For gif adultery, sacrilege, oppression, barbarous cruelty, and
theft heaped upon theft, deserve hell, the great King of Carrick can
no more escape hell, for ever, nor the imprudent abbot escaped the
fire for a season, as follows.
‘After that the earl espied repugnance, and that he
could not come to his purpose by fair means, he 1570.
commandit his cooks to prepare the banquet. And
so first they flayit the sheep, that is, they took off the abbot’s
claithes, even to his skin; and next they band him to the chimney,
his legs to the one end and his arms to the other; and so they began
to beet the fire, sometimes to his buttocks, sometimes to his legs,
sometimes to his shoulders and arms. And that the roast should not
burn, but that it might roast in sop, they spared not flamming with
oil. (Lord, look thou to sic cruelty!) And, that the crying of the
miserable man sould not be heard, they closed his mouth.... In that
torment they held the poor man, while that ofttimes he cried “for
God’s sake to dispatch him; he had as meikle gold in his awn purse
as wald buy powder eneugh to shorten his pain.”
‘The famous King of Carrick and his cooks, perceiving the roast to be
eneugh, commandit it to be tane from the fire, and the earl himself
began the grace in this manner: “Benedicite, Jesus, Maria! you are
the most obstinate man that ever I saw! Gif I had known that ye had
been so stubborn, I wold not for a thousand crowns handled you so.
I never did so to man, before you.”‘—Ban.
The abbot’s own account, in the complaint which he afterwards
rendered to the privy-council, is different, in as far as it describes
him as now yielding to the earl’s desire, in order to save his life and
free himself from the pain he was suffering. He also says that he at
this time subscribed the papers presented by the earl, though, it
would appear, in an incomplete manner. He goes on—‘which being
done, the earl causit the tormentors of me sweir upon ane bible
never to reveal ane word of this my unmerciful handling to ony
person or persons.
‘Yet he, not being satisfied with their proceedings, came again upon
the 7 day of the month, bringing with him the same charter and
tack, which he compellit me to subscrive, and required me to ratify
and approve the same before notar and witnesses; which alluterly I
refused. And therefore he, as of before, band me, and put me to the
same manner of tormenting, and I said, notwithstanding, “he should
first get my life ere ever I agreed to his desire;” and being in so
great pain as I trust never man was in, with his life, I cried: “Fye
upon you! will ye ding whingers in me, and put me out of this world!
Or else put a barrel of powder under me, rather nor be demeaned in
this unmerciful manner!” The earl hearing me cry, bade his servant,
Alexander Richard, put ane serviette [towel] in my throat, which he
obeyed; the same being performed at 11 hours at night; wha then
seeing that I was in danger of my life, my flesh consumed and burnt
to the bones, and that I wald not condescend to their purpose, I was
releivit of that pain; wherethrough I will never be able nor well in my
life time.’
The abbot was relieved from Dunure by the Laird
of Bargeny, an enemy of Cassillis. The government 1570.
was too weak and in too much trouble to avenge
his cause against the earl, who thenceforth continued to draw the
revenues of Crossraguel. But ‘my lord gave the abbot some money
to live upon, whilk contentit him all his days.’—Hist. Ken.

Robert Hepburn, second son of the Laird of

Waughton, was a partisan of the queen. Travelling Sep. 7.
to visit his friends in Lothian, he was betrayed by a companion to
the knowledge of a party of the king’s friends, consisting of the
Lairds of Applegarth and Carmichael, who consequently made an
attempt to lay hold of him as he was passing Bathgate. ‘He, being
alone with ane boy, fled, and they chasit him continually fra the said
place while he come to the castle of Edinburgh, wherein he was
resavit with great difficulty; for when the said Robert was passand in
at the castle-yett, his adversaries were at Patrick Edgar his house-
end. Ane thing to be wonderit at that he could escape the hands of
the said persons, considering their multitude and [their being] as
weel horsit as he was; and he being riding upon ane brown naig,
could never have space to change off the same upon his led horse,
but continually rade while he come to the castle foresaid; but his
pursuers not only changit horse, but also did cast from them saddles
and other gear, to mak licht for pursuing of him.’73—D. O.

John Kello, minister of Spott, in Haddingtonshire,

was executed in Edinburgh for the murder of his Oct. 4. 1570.
wife. The confession of this wretched man shews
that he was tempted to the horrible act by a desire to marry more
advantageously, his circumstances being somewhat straitened. He
deliberated on the design for forty days; tried poison, which failed;
then accomplished it by strangulation. His confession admits the
amiable character of the victim; nay, he tells that, ‘in the verie death,
she could not believe I bure her onie evil will, but was glad, as she
then said, to depart, gif her death could do me either vantage or
pleasure.’74 According to a contemporary recital, ‘he stranglit her in
her awn chamber, and thereafter closit the ordinar door that was
within the house for his awn passage, and sae finely seemit to
colour that purpose after he had done it, that immediately he passed
to the kirk, and in the presence of the people made sermon as if he
had done nae sic thing. And when he was returnit hame, he brought
some neighbours into his house to vissie his wife, and callit at the
ordinar door, but nae answer was made. Then he passed to another
back passage with the neighbours, and that was fund open, and she
hinging stranglit at the roof of the house. Then, with admiration, he
cryit, as though he had knawn naething of the purpose, and they for
pity in like manner cryit out. But, in [the] end, finding himself prickit
with the judgments of God, of the grievous punishment wherewith
transgressors have been plagued in time bygane, he thought gude
to communicate his fact to ane of his brether in office, wha then was
schoolmaster at Dunbar.’—H. K. J.
To resume his own confession: ‘Mr Andrew Simson, minister of
Dunbar, did so lively rype furth the inward cogitations of my heart,
and discover my mind so plainly, that I persuaded myself God spak
in him ... he remembered me of ane dream which in my great
sickness did apparently present the self. “Brother,” said he, “I do
remember when I visited you in time of your sickness, ye did expose
to me this vision, that ye were carried by ane great man before the
face of ane terrible judge, and to escape his fury, ye did precipitate
yourself in ane deep river, when his angels and messengers did
follow you with two-edged swords, and sae when they struck at you,
ye did decline and jouk in the water, while in the end, by ane way
unknown to you, ye did escape. This vision I do interpret, that ye
are the author yourself of this cruel murder then conceived in your
heart, and ye were carried before the terrible judgments of God in
your awn conscience, which now stands in God’s presence to accuse
you; the messengers of God is the justice of the country before the
which ye sall be presented; the water wherein ye stood is that vain
hypocrisy of your awn, and feigned blasphemy of God’s name,
whereby ye purpose to colour your impiety; your deliverance sall be
spiritual.”... At this time did God move my heart to acknowledge the
horror of my awn offence, and how far Sathan had obteinit victory
ower me.’—Ban. J. ‘Briefly, by his awn confession, being clearly
convict, he was condemnit to be hangit, and his body to be casten in
the fire and brynt to ashes, and so to die without any burial. And
thus he departit this life, with an extreme penitent and contrite
heart, baith for this and all other his offences in general, to the great
gude example and comfort of all beholders.’—H. K. J.

In those days, while as yet there were not only no

newspapers, but no ready means of conveying Oct. 1570.
letters, true intelligence made its way slowly, and
the most ridiculous rumours obtained circulation. For example, on
John Knox being at this time struck with apoplexy, ‘a bruit [report]
went through Scotland and England, that he was become the most
deformed creature that ever was seen; that his face was turned
awry to his neck; and that he would never preach or speak again.’ In
the ensuing year, while the venerable reformer lived at St Andrews,
it was rumoured, and very generally believed as a serious truth, that
he had been banished from the town, ‘because in his yard he had
raised some sancts, among whom came up the devil with horns;
which, when his servant, Richard Bannatyne, saw, he ran wood, and
so died.’ It is stated that Lady Hume and some others thronged
round the postman of St Andrews, with anxious inquiries whether it
was true that Knox was banished from St Andrews, and that
Bannatyne had run mad in consequence of seeing the devil raised.75

At this time, the witches of Athole are spoken of as noted

personages. In the late and present civil dissensions they sided with
the unfortunate queen, having probably too much Highland feeling
to dissent from the great man of the district, the Earl of Athole, who
was one of her majesty’s warmest friends. About the time indicated,
a present was sent to Mary, supposed to be from this uncanny
portion of her late subjects. It was ‘a pretty hart horn, not exceeding
in quantity the palm of a man’s hand, covered with gold, and
artificially wrought. In the head of it were curiously engraven the
arms of Scotland; in the nether part of it a throne, and a
gentlewoman sitting in the same, in a robe-royal, with a crown upon
her head. Under her feet was a rose environed with a thistle. Under
that were two lions, the one bigger, the other lesser. The bigger lion
held his paw upon the face of the other, as his lord and commander.
Beneath all were written these words:

“Fall what may fall,

The lion shall be lord of all.”

This was evidently designed to convey a hope and

wish that Mary should erelong, in spite of all 1570.
contrarious circumstances, be in possession of
England as well as of her native dominions.76 In the same spirit was
a rhymed prophecy which, at the same time, came into circulation,
but which was quickly falsified:

‘The howlet shall lead the bear to his bane,

The queen of England shall die the twelfth year of her reign;
The court of England that is so wanton,
Shall shortly be brought to confusion.’—Cal.

A sad picture of civil war is presented by the so-

called Harrying of Bothwell Moor. ‘Captain Andrew Nov. 12.
Cunningham and Captain Thomas Crawford,
accompanied with certain men of weir, departit of Glasgow, and
passed in the night to Bothwell Moor, where they reft and spulyit all
the inhabitants and tenants thereof; and because the Hamiltons was
gathering to rescue the said guids, they fearit to return again to the
said town of Glasgow, but came to Edinburgh with the same. They
brought to the said burgh of Edinburgh 400 kye and oxen, 600
sheep, and 60 mares and staigs [colts]; this done, they passed to
my Lord Regent, he being in Dalkeith, and knew his mind, whither
they should take ane composition from the poor tenants, awners of
the same, or not; but the matter was sae unmercifully handled, that
the said guids were proclaimit by sound of drum and trumpet, to be
sauld [to] whatsomever persons wald buy the same.... To hear the
lamentable crying of the said poor tenants, for the unmerciful
robbery and oppression committit upon the said persons by the men
of weir, it wald made ane stane-heartit man to greit and bewail. But
cry what they wald cry, and lament as they pleasit, there was nane
that obtainit comfort at their unmerciful hands; for when the said
poor creatures made their complaint to the Regent, he wald not hear
them, while [till] the oppression was cryit out upon by John Craig,
minister. And then the Regent and lords of secret council ordainit
that ane half of the guids be renderit again to the said poor tenants;
but ere this time, the men of weir had sparfilit the best of them, and
then the poor tenants were constrainit either to take again the ane
half of the warst of the said guids that were left behind, or else they
wald not have gotten naething.’—D. O.

‘... there was ane day of law betwixt the

Hoppringles and Elliots in Edinburgh, wherein the Dec. 7. 1570.
ane party set upon the other, and, had not the Dec. 9.
town of Edinburgh redd [separated] them, there
had been great slauchter done the said day.’—D. O.
‘Patrick Moscrop, son to John Moscrop, advocate, and Eupham
M’Calyean, only apparent heir to Mr Thomas M‘Calyean, ane of the
senators of the College of Justice, were married in the said Thomas
M‘Calyean’s house within Edinburgh, but nocht by permission of the
kirk, and that for fear of tumult to be made by Archibald Ruthven,
brother to William Lord Ruthven, wha allegit he had the first promise
of her.... This order of marriage endured in ane manner ane slander
to the kirk of God.’77—D. O.
From this day till the 22d March, ‘great frost, that
nae plews gaed while aucht days; and men might 1570-1. Jan. 15.
pass and repass on the ice of Lyon the 3d day of
March.’ February 22, after noon, ‘there came ane great storm, and
snaw and hail and wind, that nae man nor beast might take up their
heads, nor gang, nor ride, and mony beasts, and mony men and
women, were perished in sundry parts, and all kind of victuals right
dear, and that because nae mills might grind for the frost.’—C. F.

A General Assembly sitting in Edinburgh issued an

order that adulterers, murderers, and others guilty 1571. Mar.
of heinous offences, who might desire to be
received back into Christian fellowship, should first appear penitently
before their respective ministers, and then present themselves in
linen clothes, bareheaded and barefooted, before the synod of their
district. It was presently found, however, that divers of these
penitents were too far distant from the meeting-places of the
synods, and others were in such poverty, or under such terror of
enemies, that they could not, or durst not travel through the
country.
This fact verifies to us a passage in a contemporary historian: ‘The
haill realm of Scotland was sae divided in factions, that it was hard
for any peaceable man as he rade out the hie way, to profess himself
openly, either to be a favourer of the king or queen. All the people
were casten sae lowss, and were become of sic dissolute minds and
actions, that nane was in account but he that could either kill or
rieve his neighbour.’—H. K. J.
Incidents characteristic of such a time abound in
the contemporary diarists. ‘March 27, David Lawtie, 1571.
writer to the signet [in Edinburgh] was invaded by
Thomas Douglas, and the maist part of his fore-finger strucken fra
him.’—D. O. October 30, ‘There was ane combat betwixt Campbell
on the king’s part, and ane Smith, a lieutenant or servant within
Edinburgh. Campbell strack him twice through the body without
blood drawn upon himself, except a scrape upon the thumb.’—Ban.

The castle of Dumbarton being taken by surprise,

great joy was experienced by the king’s party on Apr. 7.
finding John Hamilton, archbishop of St Andrews,
among the prisoners. The primate, a zealous adherent of the ancient
faith, and partisan of the queen, was suspected of various crimes
against the Protestant cause; so no mercy was to be expected for
him. Then was seen the remarkable spectacle of the head of the
church in Scotland—he whom Jerome Cardan travelled from London
to Scotland only to cure of some trifling ailments—dragged with but
little ceremony to a scaffold and put to a dog’s death—a victim of
the frightful passions excited by civil war. In answer to a dittay which
George Buchanan assisted in bringing against him at Stirling, he
denied everything but a foreknowledge of and participation in the
death of Moray, ‘of whilk he repentit, and askit God mercy. Being
further accusit gif ony of his surname or friends were upon the
counsel thereof, he answerit that he wold accuse nae man at that
time but himself. As touching his religion,’ says this chronicler, ‘I
reasonit with him, and could find naething but that he was ane
papist, and exhortit sic as were near hand upon the scaffold to abide
at the Catholic faith—sae he termed the papistry. In the castle, he
desirit some papist priest to whom he micht confess him, and of
whom he micht resave consolation [absolution] of his sins, according
to the order of the kirk (as he spak); and sae he continuit to the
death in the papistry, as he livit. As the bell struck at six hours at
even, he was hangit at the mercat-cross of Stirling, upon ane gibbet,
on whilk was written thir twa verses following:

“Cresce diu, felix arbor, semperque vireto

Frondibus, ut nobis talia poma feras.”—D. O.
At this time, Mr William Collace was first regent in
St Leonard’s College, St Andrews; he ‘had the 1571.
estimation of being the maist solid and learnit in
Aristotle’s philosophy.’ James Melville gives an interesting picture of
this learned person, to whose class he came at fifteen years of age,
so ill prepared for understanding the language (Latin) in which the
prelections took place, that ‘I did naething,’ says Melville, ‘but
bursted and grat at his lessons, and was of mind to have gone home
again, were [it] not the loving care of that man comforted me; [he]
took me in his awn chalmer, causit me to lie with himself, and every
night teached me in private till I was acquainted with the matter.
Then he gave us ane compend of his awn of philosophy and the
parts thereof ... whilk I thought I understood better. About the whilk
time my father, coming to the town, begoud to examine me, and,
finding some beginning, was exceeding rejoiced, and uttered
sweeter affection to me than ever before. He enterteinit my regent
very heartily in his lodging, and gave him great thanks; he sent me
to him, after he had taken leave, with twa pieces of gold in a napkin;
but the gentleman was sae honest and loving, that he wald have
none of his gold, but with austere countenance sent me back with it;
nay, never wald receive gold or silver all the time of my course.’
Melville mentions having frequent opportunities at this time of
seeing and hearing John Knox, who had taken refuge in St Andrews,
while Edinburgh was possessed by the queen’s party. ‘Mr Knox wald
some time come in and repose him in our college yard, and call us
scholars unto him and bless us, and exhort us to knaw God and his
wark in our country, and stand by the gude cause, and follow the
guid example of our masters.’
‘I saw him every day of his doctrine go hooly and fair [softly and
fairly] with a furring of matricks about his neck, a staff in ane hand,
and guid godly Richard Ballanden, his servant, halding up the other
oxter [armpit] from the abbey to the parish kirk, and by the said
Richard and another servant, lifted up to the pulpit, whaur he
behovit to lean at his first entry, but ere he had done with his
sermon, he was sae active and vigorous, that he was like to ding
that pulpit in blads [knock the pulpit in splinters], and flie out of it.’
He adds: ‘This year, in the month of July, Mr John Davidson, ane of
our regents, made a play at the marriage of Mr John Colvin, whilk I
saw playit in Mr Knox’s presence, wherein, according to Mr Knox’s
doctrine, the castle of Edinburgh was besieged, taken, and the
captain, with ane or twa with him, hangit in effigy.’
This dramatic performance represented an
unfulfilled prophecy of the reformer. When Kirkaldy 1571.
of Grange, after many years of zealous service in
the reforming cause, declared for the Queen, and held out
Edinburgh Castle against the Regent, Knox, who had loved him
much, was deeply grieved. He felt, however, no doubt as to the
ultimate triumph of his own cause against all such opposition, and it
was perhaps no great venture for so acute a person to utter the
prediction that, notwithstanding the trust which Kirkaldy put in that
powerful fortress, it should yet run like a sand-glass; it should spew
out the captain with shame; he should not come out at the gate, but
over the walls. Mr Robert Hamilton, minister of St Andrews, asking
his warrant for this vaticination, he said: ‘God is my warrant, and ye
shall see it.’ ‘As the other was scarcely satisfied,’ says James Melville,
‘the next sermon from the pulpit, he repeats the threatenings, and
adds thereto: “Thou that will not believe my warrant, shall see it
with thy e’es that day, and shall say: ‘What have I to do here?’” This
sermon the said Mr Robert’s servant wrote....’—Ja. Mel.

This year ‘great weirs in the north land betwixt the Gordons and
Forbeses, and the Forbeses put till the warst, and mony slain of
them, and towns wasted and burnt.’—C. F.
Adam Gordon, brother of the Earl of Huntly, was a leader in these
broils, and of some avail in supporting the queen’s cause. He stained
his name by a frightful act of cruelty. The house of Towie, belonging
to Alexander Forbes, was maintained by his lady against Gordon. On
his sending to demand its surrender, the brave dame answered that
she could not give it up without direction from her husband. Gordon
then set fire to it, and burnt the heroic woman, her children and
servants—twenty-seven persons in all!

July. 1571.
The queen’s party, after holding a parliament in Edinburgh, where
they affected formally to re-establish her government, sent a
pursuivant to Jedburgh, ‘to proclaim the new erected authority,’
probably thinking that the man would be safe in the performance of
his duty at that town through the favour of Kerr of Ferniehirst, their
fellow-partisan. They little reckoned on the spirit of the Border
burghers. ‘He was suffered to read his letters till he came to this
point, that the lords assembled in Edinburgh had found all the
proceedings against the queen null, and that all men should obey
her only. Then the provost caused the pursuivant to come down
from the cross, and eat his letters. Thereafter, [he] caused loose
down his points, and gave him his wages —— with a bridle; and
threatened that if ever he came again, he should lose his life.
Ferniehirst threatened the town: but they gave him the defiance.’—
Cal.
A few months after, Ferniehirst and Buccleuch mustered a great
multitude of the Border thieves, and came to take vengeance on the
burghers of Jedburgh. The town, assisted by Kerr of Cessford, stood
to its defence; and when Lord Ruthven came with a party of horse
to aid them, they were able to beat back the assailants, many of
whom fell into their hands.

‘There was ane sow farried in William Davidson’s

house, flesher in Edinburgh, of thirteen gryces Aug. 1.
[pigs], of the whilk there was ane a monster. It
had the gruntle [snout] in the heich of the heid, and under that it
had twa een, ane nose and mouth, ane brow, ane cheek, ane
tongue, and lugs like to the similitude of man in all sorts; the
remanent thereof was like ane other gryce without hair. This
portendit some mischief to this burgh.’—D. O.

The Earl of Argyle, Robert Lord Boyd, and some

other nobles, lately friends of the queen, were now Aug.
brought over to the king’s side, after sundry
meetings and discussions with the Regent. ‘The greedy and
insatiable appetite of benefices was the maist cause thereof, for
there was nane that was brought under the king’s obedience but for
reward either given or promised. Als he [the Earl of Argyle] was
greatumly persuadit hereto by Lord Boyd, wha persuadit the kirk to
part the said earl and his wife, and [the earl] to marry his [Lord
Boyd’s] daughter, wha was married upon the young laird of
Cunninghamheid of before.’—D. O.
After these particulars, it is instructive to read the epitaph inscribed
on Lord Boyd’s tomb in the Laigh Kirk (Burns’s Laigh Kirk) of
Kilmarnock:

‘Heir lyis yt godlie, noble, wyis Lord Boyd,

Quha kirk and king and commonweil decoird,
Quhilk war (quhill they yis jowell all injoyd)
Defendit, counsaild, governd, by that lord.’ &c.

The Regent Lennox held a parliament at Stirling,

where he made an oration to the nobility. The Aug. 28.
king, five years old, was present, and, while his
grandfather was speaking, he looked up and espied a hole in the
roof, occasioned by ‘the lack of some sclates.’ At the conclusion of
the harangue, the child remarked: ‘I think there is ane hole in this
parliament.’
‘In effect, his majesty’s words came true; for the
same month, about the end of the parliament 1571.
(September 3), there came to Striviling in the
night, ere the nobility or town knew, the Earl of Huntly, the queen’s
lieutenant, Claud Hamilton, with the Lairds of Buccleuch and
Ferniehirst, who, ere day brake, had possessed themselves of the
town, crying “God and the Queen!” so that those that were for the
King and his Regent could not, for the multitude of enemies, come
to a head. Wherever they could see any that belonged to the
Regent, him they killed without mercy. The Regent being taken
prisoner by the Laird of Buccleuch, and horsed behind him, ane
wicked fellow lift up his jack, and shot him through the body with a
pistol.... [On a counter-surprise, the queen’s party] departed the
town immediately. The Earl of Mar was declared Regent, and
concluded the parliament. This was the hole which the young king
did see in the parliament, although he meant nothing less.’—Bal.

Robert Lord Boyd entered this day into a bond of

manred with William Fairly, brother of David Fairly Nov. 10.
of that Ilk. Manred, properly, is a service of
allegiance; but in Scotland it had come, in the course of time, to be
an agreement, sometimes between a great man and a less,
sometimes between two or more equally great men, to stand by
each other in all contingencies of war and law, excepting only (and
perhaps it was but a hypocritical exception) where the king’s majesty
and his commands were concerned. It was an arrangement dictated
by the exigencies of a rude time, when law was but partial and
uncertain in its actings, and natural feeling often called for
something being done, whether the law would or no. As something
not very consonant with good government, or even such attempts at
the same as might be made in those days, manred had been
denounced by a statute so long ago as 1457, when it was enacted
‘that nae man dwelling within burgh be fund in manrent, nor ride in
rout in feir of weir with nae man, but with the king or his officers, or
with the lord of the burgh.’ But acts of parliament were voices crying
in the wilderness in Scotland, and manred still continued to have its
place in the economy of life in this age.
On this occasion, William Fairly binds and obliges
himself to be ‘man and subject servant’ to Lord 1571.
Boyd and his heirs, ‘aefaldly and truly to serve
them upon their retinue and expenses in household and out of
household, as best sall please them in all their affairs, and as weel in
defence as pursuit, with whom or against whom it sall happen them
to have action and ado,’ the king excepted. He is likewise to help
them with his good counsel, ‘and sall never hear nor know their
hurt, damage, nor skaith, in ony sort, but sall diligently sift out the
same, and mak true declaration thereof.’
The consideration for all this service is the possession of ‘the thretty-
shilling land of auld extent of Byrehill.’
This was but the first of a long series of similar engagements which
Lord Boyd formed down to his death in 1589.78 For a forty-shilling
land, the Laird of Fergushill becomes bound, October 26, 1572, in
the same way as William Fairly, and to take part with the said lord
and his heirs, in all their actions, quarrels, questions, and debates.
The Laird of Lochrig, the Laird of Rowallan, Andrew Macfarlane of
Arroquhar, and the Laird of Camstroddan, all in succession put
themselves in this relation to his lordship. In March 1575, the Laird
of Blair engaged with his friends, tenants, and servants, to ‘ride,
gang, and assist with the said lord, in all kind of leeful conventions.’
It was with such satellites that a great man of that age, if to be tried
on any criminal charge, appeared at the place of law, professedly
that he might be sure of fair-play, but in reality with the effect of
overbearing justice. It was with such assistants that two or three
lords were sometimes enabled to take possession of the
government, and for a time rule all at their pleasure. Amongst the
most curious things in the early history of the reformed religion, are
the occasions when it was manifestly indebted for its progress to
associations of this irregular kind.

About this time, there was apprehended ‘one that

keepit ane hostelry at Brechin, who before, at Dec. 24.
divers times, had murdered sundry that came to
lodge with him, the wife being also as busy as the man with a mell
[mallet], to fell their guests sleeping in their beds.’—Ban.

Among numberless skirmishes, surprises, and

barbarous ravagings which marked the struggle 1571-2. Jan.
between the friends of the queen and those of the Feb. 8.
infant king, was an affair of several parts or acts in
this and the ensuing month. Lord Maxwell being contracted in
marriage to a sister of the Earl of Angus, the lady’s relation, the Earl
of Morton, proposed to give a banquet on the occasion at Dalkeith
Castle. The wine required at the feast was to be brought in carts
from Leith, together with some venison and a quantity of silver
plate. Kirkaldy and his friends in the castle hearing of this, sent out a
party of horse, which surprised Morton’s servants, and took as spoil
the materials of the proposed banquet. Morton who, it was said,
smarted more from the loss of the plate than the killing of a few of
his servants in the struggle, immediately sent a party to requite
Kirkaldy’s attack by laying waste his estate in Fife. Kirkaldy, again,
repaid these attentions by sending a party a few nights after to set
fire to the town of Dalkeith. On this occasion, he killed ten of
Morton’s people, and took nine prisoners. ‘In their return [they]
perceived fifty-sax horses from Dalkeith to Leith, passing laded with
ale; they brake the barrels, and made prey of the horses, and
brought into Edinburgh many kye and oxen forth of that lordship for
supply of their scant and hunger.’—H. K. J. ‘These three scuffles
went all under one name, and were ever after called Lord Maxwell’s
Handfasting.’79

The condition of the ordinary places of worship in

this time of civil war is sketched in the 1572. Mar.
Lamentation of Lady Scotland, printed by
Lekprevik in 1572.

‘The rooms appointit people to consider,

To hear God’s word, where they suld pray together,
Are now convertit in sheep-cots and faulds,
Or else are fallen, because nane them uphalds.
The parish kirks, I ween, they sae misguide,
That nane for wind and rain therein may bide:
Therefore nae pleasure tak they of the temple,
Nor yet to come where nocht is to contemple,
But craws and dows, cryand and makand beir,
That nane throuchly the minister may hear.
But feathers, filth, and dung does lie abroad,
Where folk should sit to hear the word of God;
Whilk is occasion to the adversaries,
To mock and scorn sic things before your eyes.
Thus to disdain the house of orison,
Does mak folk cauld to their devotion;
And als they do disdain to hear God’s word,
Thinking the same to be ane jesting bourd;
They go to labour, drinking, or to play,
And not to you,80 upon the Sabbath day.’
The civil war told nowhere with more severity than
on Edinburgh, which was the scene of the principal 1572.
transactions. The bringing of victuals or coal to the
city was forbidden by the beleaguering troops under pain of death,
and the penalty was exacted in many instances. The consequence
was ‘great penury and scant of vivres, sae that all was at ane
exceeding dearth.’—D. O. In May, oatmeal was nine shillings of the
native money per peck; eleven ounces of wheaten bread cost 8d.,
‘and baps of nine [ounces] for 12d.’ It was found necessary to
demolish some houses for the sake of the wood, to be used as fuel.
At the commencement of a truce on the 22d of July, the meal had
risen to twelve shillings, the boll of wheat to ten pounds, and a
carcass of beef to sixteen pounds. On that day, ‘after noon, the
victuals whilk was keepit to ane dearth was brought to Leith and
sauld, the meal for five shillings the peck, ... and [sae] very mickle
bread baken, that it that was sauld for sixteen pennies was sauld for
six pennies. Thanks to God.’ During the scarcity, ale not being to be
had, a drink of vinegar and water was substituted.—D. O.
‘Nochttheless,’ if we are to believe the same chronicle, ‘the remainers
therein [that is, in Edinburgh] abade patiently and were of good
comfort, and usit all pleasures whilk were wont to be usit in the
month of May in auld times, viz., Robin Hood and Little John.’
From the day here noted to the 8th of June, the
war between the queen’s party in Edinburgh and Apr. 16.
the king’s beyond the city was conducted on the
principle of No quarter. All who were taken on either side were
presently put to death. The common belief was, that this frightful
system originated with Morton, who conceived that by such severity
the war would sooner cease. In the end, both parties, ‘wearied of
execution daily made, were content to cease from such rigour, and
use fair wars, as in former times.’—Spot.
‘... there was ane minister [named Robert Waugh]
hangit in Leith (and borne to the gibbet, because Apr. 21.
he was birsit81 with the boots82). The principal
cause was that he said to the Earl of Morton, that he defended ane
unjust cause, and that he wald repent when nae time was to repent.
And when he was required by whom he was commanded to say the
same, he answered and said: “By the haly spreit.”‘—D. O.
In the same year, Mr Andrew Douglas, minister of Dunglass, was
first tortured, and then hanged, for publicly rebuking Morton on
account of his living with the widow of Captain Cullen.
Another characteristic incident of the time, but of a
somewhat mysterious character, occurred in a July 19. 1572.
southern burgh. James Tweedie, burgess of
Peebles, John Wightman, Martin Hay, and John Bower there, and
Thomas Johnston, son to Thomas Johnston of Craigieburn, were
tried for being concerned in ‘the cruel slaughter of the umwhile John
Dickison of Winkston; committit within the town of Peebles on the
1st of Julii instant.’ They were acquitted. The fact is only worth
mentioning here, to afford an opportunity of illustrating the long
perseverance of tradition in certain favouring circumstances. In his
youth, which was passed in the town referred to, the author
distinctly remembers hearing an aged person speak of how Provost
Dickison was long ago ‘stickit’ at the back of the Dean’s Well in the
High Street. The event was then 240 years past.

‘The Earl of Mar, Regent, ended his life, about

three hours in the morning. It was constantly Oct. 29.
affirmed, that about the time of his death, the
trough of the water of Montrose, where it runneth through his lands,
was dry, the water running nevertheless above [higher up]. At the
same time, a violent wind drave a great number of sheep from the
links of Montrose into the sea.’—Cal.
Some events of the kind did certainly occur about the time of the
Regent’s death; but, contrary to all rule in such matters, they came
after that event, if we are to believe another historian, who places
them under November, and describes them as follows:
‘In this mean time was ane great ferly in Montrose. By the space of
six hours, the water thereof was dry in the sea, and during the whilk
space the people past within the said sea, and got sundry fishes....
After the whilk space, the people on the sands perceiving the water
as ane popill pitt, frae the whilk they fled to land, and syne it was
sea again suddenly, and never nane perishit hereinto. Also there was
ane hill callit ... , whilk burnt by the said space; men riding by the
way, the manes and coils of their horses burnt, the wands of their
hands burnt; poor men passing on the way, the staves in their hands
burnt, and when they wald dight [wipe] off the fire thereof, it wald
entres again.’—D. O.
 REGENCY OF MORTON: 1572-1578.
The Earl of Morton had no sooner assumed the reins of government,
than his vigorous talents began to be felt. The chief strength of
Mary’s friends was in Edinburgh Castle, held for her by Kirkaldy of
Grange. All the means at the Regent’s command proving insufficient
to reduce this fortress, he obtained from England an army of 1500
men, commanded by Sir William Drury, and provided with artillery.
The castle stood a siege of three weeks, and was then obliged to
yield (May 29, 1573). With mean vindictiveness, Morton sent the
gallant Kirkaldy to the gallows. Maitland of Lethington might have
shared the same fate, if it had not been anticipated either by a
natural death or suicide. The other chiefs of the queen’s party were
spared. After this event, the friends of Mary could no longer make
an appearance anywhere in her favour. The new government
remained triumphant, and peace was restored to a bleeding and
exhausted country.
Morton was, on the whole, a serviceable, though not a just or
clement ruler. It was his policy, arising from his love of money, to
punish his adversaries rather by fines than bloodshed. All the
persons of note who had befriended the queen, he caused to give
security for their future behaviour. The smallest offence forfeited the
pledge, and the cautioners were then mulcted without mercy. Under
this ruling passion, he tampered with the coin, sold justice, and
cheated the church of its revenues. It was supposed that he had
concealed large treasures in his castle of Dalkeith; but we have no
certain account of their ever being found, and probably the popular
notions on the subject were exaggerated.
Under Morton, a slight move was made towards the establishment of
a kind of episcopacy in the church, though the persons he appointed
to the sees were mere creatures who consented to be receivers of
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