https://doi.org/10.

1038/s41586-025-08799-1

Supplementary information

Gene-modified pig-to-human liver

xenotransplantation
In the format provided by the
authors and unedited

Nature | www.nature.com/nature
SUPPLEMENTARY FIGURE 1
Fig. 1c

GAPDH was used as the control. For hCD46 and GAPDH, samples obtained from

liver tissues of the donor and wild-type pig were run on the same gel, and then

transferred to the same PVDF membrane, which were cropped and incubated in

different primary antibodies. For hCD55, the same amounts of samples were uploaded

on another gel.

Extended Data Fig. S1a

1
Extended Data Fig. S1b

2
The cDNA were extracted from the same samples, and then went through PCR using

the indicated primers. Then the products were running on different gels.

3
SUPPLEMENTARY FIGURE 2

GGTA1
WT pig PBMCs Blank control

WT pig PBMCs BSI-B4

Donor pig PBMCs Blank control

Donor pig PBMCs BSI-B4

The above four groups of histograms are superimposed to obtain the following figure：
4
β4GalNT4
WT pig PBMCs Blank control

WT pig PBMCs DBA

Donor pig PBMCs Blank control

Donor pig PBMCs DBA

5
The above four groups of histograms are superimposed to obtain the following figure：

Neu5Gc
WT pig PBMCs Isotype control

WT pig PBMCs Neu5GC antibody

Donor pig PBMCs Isotype control

6
Donor pig PBMCs Neu5GC antibody

The above four groups of histograms are superimposed to obtain the following figure：

CD46
WT pig PBMCs Blank control

WT pig PBMCs CD46 antibody

7
Donor pig PBMCs Blank control

Donor pig PBMCs CD46 antibody

The above four groups of histograms are superimposed to obtain the following figure：

CD55
WT pig PBMCs Blank control

8
WT pig PBMCs CD55 antibody

Donor pig PBMCs Blank control

Donor pig PBMCs CD55 antibody

The above four groups of histograms are superimposed to obtain the following figure：

9
Supplementary figure 2. The gating strategy of Fig. 1c.

These images are the source data of Fig. 1c. The four groups of histograms of the

above genes were superimposed respectively to obtain Fig. 1c.

10
SUPPLEMENTARY FIGURE 3

IgG
WT pig PBMCs IgG antibody control (Negative Control)

WT pig PBMCs and Recipient sera IgG antibody

Donor pig PBMCs IgG antibody control (Negative Control)

Donor pig PBMCs and Recipient sera IgG antibody

The above four groups of histograms are superimposed to obtain the following figure
11
IgM
WT pig PBMCs IgM antibody control (Negative Control)

WT pig PBMCs and Recipient sera IgM antibody

Donor pig PBMCs IgM antibody control (Negative Control)

Donor pig PBMCs and Recipient sera IgM antibody

12
The above four groups of histograms are superimposed to obtain the following figure

Supplementary figure 3. The gating strategy of Fig. 1e.

These images are the source data of Fig. 1e. The four groups of histograms of the

above genes were superimposed respectively to obtain Fig. 1e.

13
SUPPLEMENTARY TABLES

Table S1. Patient medication management

Category Drug Dosage Day

Proton pump 40mg bid POD: -1，1 - 10
Esomeprazole
inhibitor 40mg tid POD: 0
200mg qd POD:0, 6 - 10
Magnesium Isoglycyrrhizinate
200mg bid POD:1 - 5
Hepatoprotect
1g bid POD: 0 - 6,9-10
ive drug Ademetionine butanedisulfonate
0.5g bid POD: 7 - 8
Polyene Phosphatidylcholine 465mg bid POD: 6 - 10
Glucose and Sodium Chloride
300ml qd POD: 3 - 6
Injection
Enteral nutriti
Probiotics 500mg tid POD: 3 - 10
on
Enteral Nutritional Powder 27.9g tid POD: 5 - 6
Enteral Nutritional Powder 9.3g qd POD: 7
10% Glucose 1000ml qd POD: -1, 7 - 9
50% Glucose 200ml qd POD: -1, 7 - 9
Compound Amino Acid 500ml qd POD: -1, 7 - 9
Fat-soluble Vitamin(Ⅱ)/Water-
10ml qd POD: -1, 7 - 9
Parenteral nut soluble Vitamin
rition Alanyl-Glutamine 20g qd POD: -1, 7 - 9
250ml bid POD: 1 - 6
Fructose
500ml qd POD: 7 - 10
Adjust insulin dosage according to th
Insulin
e blood glucose level
40g qd POD: -1 - 2
60g qd POD: 3 - 8
Albutein
Protein 80g qd POD: 9
30g qd POD: 10
Human Immunoglobulin 10g qd POD: 0 - 6
3g qd POD: 0 - 2
Vitamin C
6g qd POD: 3
micronutrient 200mg qd POD: 0 - 2
s Vitamin B6
400mg qd POD: 3
1g pre-transfusion; Adjust dosage acc
Calcium Gluconate
ording to the blood calcium level
Antiviral dru
Ganciclovir 0.25g bid POD: 0 - 10
g
Antifungal dr
Caspofungin Acetate 50mg qd POD: 3 - 10
ug
14
Antimicrobial Meropenem 0.5g tid POD: -1 - 10
drug Linezolid 0.6g bid POD: 8 - 10
Anti-inflamm
Ulinastatin 0.5 MIU bid POD: -1 - 10
atory drug
Anticoagulati
Heparin Sodium Injection 12500 IU qd POD: 0 - 10
on drug
Vasoactive dr Adjust dosage according to the blood
Noradrenaline
ug pressure

Table S2. Biochemical indexes of the recipient

Sample Item category Representative indexes

Whole blood Lymphocytes T cell, B cell, CD3+CD4+T cell,

CD3+CD8+T cell

Whole blood Plasma concentration FK506

Whole blood Cytokines IL-6, TNF-α, IFN-α, IFN-γ

Serum Liver function ALT, AST, TB, DBil, IDBil, ALP,

γ-GGT

Serum Immunoglobin IgG, IgM

Serum Inflammatory mediator CRP, PCT

Serum Myocardial enzyme CK, CK-MB

Plasma Coagulation function PLT, PT, APTT, D-dimer, PTA, PTA,

INR

Plasma Coagulation factors FⅡ, FⅤ, FⅦ, FⅧ, FⅨ, FⅩ, FⅪ

Plasma Plasma concentration MMF

15
Table S3. Reagents and antibodies
Product
Reagent Dilution Ratio Clone Name Brand
Number
Bandeirea
simplicifolia
Isolectin BSI-B4 FC(1:500) L2895 SIGMA
agglutinin,
BS-I
Dolichos Biflorus
FC(1:500) FL-1031 Fluorescein Vector
Agglutinin
Neu5Gc Polyclonal
FC(1:200) 146901 Poly21469 Biolegend
antibody)
Goat Anti-Chicken
IgY H&L FC(1:200) ab96947 Abcam
(DyLight® 488)
CD55 monoclonal
5ul/106 cells SC-59092PE BRIC 216 Santa-cruz
antibody
CD46 monoclonal
10ul/106 cells MAB4439 MEM-258 Abnova
antibody
CD55 Polyclonal WB(1:5000),
26580-1-AP Proteintech
antibody IHC-P(1:200)
CD46 Polyclonal WB(1:2000),
12494-1-AP Proteintech
antibody IHC-P(1:500)
TBM monoclonal
IHC-P(1:500) 67831-1-Ig 5A4C5 Proteintech
antibody
GAPDH
Monoclonal WB(1:20000) 60004-1-Ig 1E6D9 Proteintech
antibody
Goat anti-Human
IgG (H+L)
Cross-Adsorbed
FC(1:200) A11013 invitrogen
Secondary
Antibody,
Alexa Fluor 488
Goat anti-Human
IgM (Heavy chain)
Cross-Adsorbed
FC(1:200) A21249 invitrogen
Secondary
Antibody, Alexa
Fluor 647
Pig Albumin (Alb)
CSB-E16207p CUSABIO
ELISA Kit
LEGENDplex™
740809 Biolegend
Human
16
Inflammation Panel
1 (13-plex) with
Filter Plate
C3d monoclonal
IHC-P(1:150) NBP3-07741 C3D/2891 NOVUS
antibody
C4d monoclonal
IHC-P(1:200) NBP2-34234 C4D204 NOVUS
antibody
C5b-9 Polyclonal
IHC-P(1:1000) ab55811 Abcam
antibody
IgM monoclonal
IHC-P(1:100) ab200541 IM260 Abcam
antibody
IgG monoclonal
IHC-P(1:1000) ab109489 EPR4421 Abcam
antibody
Ki-67 Polyclonal
IHC-P(1:500) 27309-1-AP Proteintech
antibody
smooth muscle actin
specific
IHC-P(1:400) 80008-1-RR 5H7 Proteintech
Recombinant
antibody
CD31 monoclonal
IF(1:50) MCA1746GA LCI-4 BIO-RAD
antibody

Table S4. Primer sequence

Virus/primer name Oligonucleotide sequence Accession numbers

porcine HQ113116.1
cytomegalovirus
PCMV-F CGCTGTGAGGACCCTATGTTG
G
PCMV-R TGGTGATGGACGCCGCTAGA
porcine endogenous PERV-A:
retrovirus ABC HQ540592.1
PERV-B:
HQ540595.1
PERV-C:
HM159246.1
PERV-ABC-F GCAGCCCCTCCAGTATTGGCC
PERV-ABC-R GGGGTGAACCGCCTGAAGGC
Porcine endogenous AM229312.2
retrovirus C
PERV-C-F GGCGCCACCTCCCGATTCGG
G

17
PERV-C-R GAGTAAGGAACCACGCGATG
G
RPL4 ribosomal 100038029
protein L4 [ Sus
scrofa (pig) ]
pRPL4-F ATGGCGTGTGCTCGTCC
pRPL4-R TTATGCAACAGCCTTCTTTTC
TTCTGT
Pig RPP30 XM_001924771
(housekeeping)
Pig RPP30- F GATTTGGACCTGCGAGCGG
Pig RPP30-R GTAAGCGGCGTTCTCCACGA
Human RPP30 NM_001104546
(housekeeping)
Human RPP30-F AGATTTGGACCTGCGAGCG
Human RPP30-R GAGCGGCTGTCTCCACAAGT

18
SUPPLEMENTARY METHODS

Postoperative management of recipient

After surgery, the recipient, whose vital signs were closely monitored, was

administered with vasoactive drugs, anticoagulation drugs, anti-inflammatory drugs,

antibiotics, hepatoprotective drugs, proton pump inhibitors, parenteral nutrition,

enteral nutrition and other supportive therapies (Table S1 of the Supplementary

Information). Meanwhile, the abdominal cavity and bile drainage were observed and

recorded. Biopsy of the xenograft (0, 2, and 10 d) and the recipient’s native liver (10 d)

were performed for hematoxylin and eosin (H&E) staining. Immunohistochemical

staining (C3d, C4d, C5b-9, IgM and IgG) of a pig liver biopsy was also performed at

specific time points (0 d, 2 h and 10 d) to test for signs of hyperacute rejection.

Concurrently, immune-related biochemical indicators in the recipient’s whole blood or

serum were measured daily beginning on 1 day before surgery (-1 d) until the trial

endpoint (10 d) (Table S2 of the Supplementary Information).

Immunosuppression regimen

The core principle of immunosuppression, to prevent recipient organ rejection and

inflammation, is derived from our previous clinical protocol. This primarily included

treatment with intravenous antihuman thymoglobulin (ATG) (75 mg) to deplete T

cells 1 day before surgery (-1 d) and on the day of surgery (0 d). Rituximab (100–200

mg) was administered intravenously 3–5 d after surgery to inhibit B cells. Eculizumab

19
(900 mg) was administered intravenously to inhibit complement on the day of surgery

(0 d). Etanercept (25 mg/0.47 ml) was injected subcutaneously on the day of surgery

(0 d) and 3 and 7 d after surgery to inhibit TNF and control inflammation. Pulsed

intravenous methylprednisolone sodium succinate (Solu-Medrol) (650 mg three times

on the day of surgery) was administered to maintain immunosuppression, in addition

to Mycophenolate mofetil (MMF, 1 g after surgery and twice daily from 1 d onwards),

Tacrolimus (FK506, 5 mg twice daily to 0.6 mg daily), and a daily tapering of

Solu-Medrol (750–190 mg daily). Recipient plasma and whole blood were assessed

daily to monitor the plasma concentrations of MMF and FK506, respectively.

Meanwhile, the recipient received a heparin sodium infusion and was adjusted to the

range of systemic anticoagulation therapy, in addition to other anti-pathogen drugs

and hepatoprotective and nutritional support therapy. The specific dosing records are

found in Extended Data Fig. 6.

Infectious disease prevention and surveillance

Porcine endogenous retrovirus (PERV) is the major pathogen transferred from

xenografts involving pigs. The donor pig in this study was bred and raised in facilities

free of designated pathogen (DPF) and regularly monitored for porcine

cytomegalovirus (PCMV), porcine circovirus (PCV) and other viruses to minimize

the risk of transmission. In this study, virus expression was monitored by qRT-PCR

and gel electrophoresis. Briefly, RNA was isolated from the PBMCs of the recipient

and the donor pig. Reverse transcription and qRT-PCR were simultaneously

20
performed to collect FAM fluorescence signals. After 40 cycles of reverse

transcription (50℃/10 min), predenaturation (95℃/10 min) and the PCR reaction

(95℃/15 sec + 60℃/1 min), virus expression was analyzed according to the Ct value.

Additionally, the products were gone through gel electrophoresis. All the sequences of

the primers are provided in Table S4 of the Supplementary Information.

Liver function assessment and histological analyses

To evaluate the pig liver function and hemodynamics, the levels of porcine albumin,

liver injury-related indicators (ALT, AST, etc.), coagulation-related indicators (PLT,

PT, APTT, etc.), and other biochemical indexes were measured in the recipient’s

peripheral blood beginning at 1 day before surgery (-1 d) (Table S2 in the

Supplementary Information). The blood flow velocity of the hepatic vein, the PV, and

the hepatic artery were also monitored by ultrasound after surgery. Liver biopsies

from the pig and recipient were H&E stained, and hemorrhage, necrosis, and cell

infiltration in the sections were semi-quantitatively analyzed at the time of surgery (0

d), 2 h post-surgery (2 h) and at the trial endpoint (10 d). The differentiation and

function of hepatocytes, hepatic stellate cells (HSCs), and liver sinusoidal endothelial

cells (LSECs) were assessed in porcine liver-tissue samples obtained during surgery

(0 h) and at the trial endpoint (10 d) using IHC testing (Ki67 and αSMA), IF staining

(CD31), and scanning electron microscopy (SEM) (Supplementary Methods).

Histology

21
Liver samples were preserved in 4% paraformaldehyde (PFA), embedded in paraffin,

sectioned, and routinely stained with H&E. Samples preserved in PFA were

embedded with an optimum cutting temperature compound and cut into 8-μm sections.

For IHC, sections were incubated at 4℃ overnight with primary antibodies, washed,

and incubated for 2 hours at room temperature with horseradish

peroxidase-conjugated secondary antibodies. The sections were then developed using

a 3,3’-diaminobenzidine substrate kit (Zhongshan Jinqiao Biotech, Beijing, China).

Immunofluorescence (IF) was conducted using fluorescent antibodies and

counter-stained with DAPI (Thermofisher). Scanning electron microscopy (SEM) and

transmission electron microscopy (TEM) were conducted as previously described. 22

All the antibodies were listed in Table S3 of the Supplementary Information.

Flow cytometry IgG and IgM binding assay

Porcine peripheral blood mononuclear cells (PBMCs) were isolated using porcine

lymphocyte separation medium (Dakewe Biotech, Shenzhen, China, Cat.

No.7411011), and PBMCs were diluted with heat-inactivated recipient serum (final

concentration of 25%. In brief, 1×105 cells were resuspended in 100 μl FACS buffer,

mixed with 100 μl recipient serum diluted 1:1) and incubated at 4℃ for 1 h. The cells

were washed twice with FACS buffer (calcium-free magnesium PBS containing 1%

BSA). After incubating PBMCs at 4℃ for 20 min using 10% heat-inactivated goat

serum as the blocking solution, PBMCs and Goat anti-Human IgG (H+L)

Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488, Invitrogen, Cat. No.

22
A11013) or Goat anti-Human IgM (Heavy chain) Cross-Adsorbed Secondary

Antibody, Alexa Fluor™ 647, Invitrogen, Cat. No. A21249) antibody was incubated

at 4℃ in the dark for 30 min. After washing twice, the cells were resuspended in

FACS buffer and assessed using a MA900 flow cytometer (Sony, MA900). The

results were analyzed using FlowJo 10.8.1 software. Results are expressed as the

specific median fluorescence intensity (MFI).

Inflammation-related multiple cytokine assays

Assays were performed according to the instructions of LEGENDplex™ Human

Inflammation Panel 1 (13-plex) with Filter Plate (BioLegend, Cat.No.740809) and the

recipient plasma (Assay Buffer) was diluted twice before testing.

23
SUPPLEMENTAL REFERENCES
22. Wang L, Wang CM, Hou LH, Dou GR, Wang YC, Hu XB, et al. Disruption of

the transcription factor recombination signal-binding protein-Jkappa (RBP-J)

leads to veno-occlusive disease and interfered liver regeneration in mice.

Hepatology 2009, 49(1): 268-277.

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SUPPLEMENTARY VIDEO

Supplementary video. Gene-modified pig to brain-dead recipient heterotopic

auxiliary liver transplantation

The video illustrates the main process of the donor organ source, the position of the

graft, the surgical operation, and the postoperative management in this porcine-brain

death receptor heterotopic auxiliary liver transplantation through cartoon.

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