
==== Front
Stem Cell Res Ther
Stem Cell Res Ther
Stem Cell Research & Therapy
1757-6512
BioMed Central London

39300579
3934
10.1186/s13287-024-03934-7
Research
Therapeutic efficacy and in vivo distribution of human umbilical cord-derived mesenchymal stem cell spheroids transplanted via B-Ultrasound-guided percutaneous portal vein puncture in rhesus monkey models of liver fibrosis
Li Shanshan 1
Fu Xufeng 12
Wang Junfeng 3
Yang Hongju 4
Wang Dan 1
Dong Xudong 5
Duan Yanchao 1
Wang Hong 1
Yan Yaping yanyp@lpbr.cn

1
http://orcid.org/0000-0003-0861-0682
Si Wei siw@lpbr.cn

1
1 https://ror.org/00xyeez13 grid.218292.2 0000 0000 8571 108X State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, 650504 China
2 https://ror.org/02h8a1848 grid.412194.b 0000 0004 1761 9803 Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, 750004 China
3 https://ror.org/00c099g34 grid.414918.1 Department of Hepatic and Bile Duct Surgery, The First People’s Hospital of Yunnan Province, Kunming, 650105 China
4 https://ror.org/02g01ht84 grid.414902.a 0000 0004 1771 3912 Geriatric Medical Center, Division of geriatric Gastroenterology, The First Affiliated Hospital of Kunming Medical University, Kunming, 650032 Yunnan China
5 https://ror.org/00c099g34 grid.414918.1 Department of Obstetrics, The First People’s Hospital of Yunnan Province, Kunming, 650105 China
19 9 2024
19 9 2024
2024
15 31524 6 2024
10 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

Liver fibrosis can progress to end-stage cirrhosis and liver cancer. Mesenchymal stem cells (MSCs) were considered the most promising therapeutic strategy, but most of the MSCs injected intravenously traditionally are trapped in the lungs, rapidly reducing their survival ability. MSC spheroids cultured in 3D have shown higher tolerance to fluid shear stress and better survival than dissociated MSCs. Simulating the route of orthotopic liver transplantation, transplanting MSC spheroids into the liver via hepatic portal vein may impact superior therapeutic effects.

Methods

In the present study, human umbilical cord-derived MSC spheroids (hUC-MSCsp) were transplanted into rhesus monkey models of liver fibrosis via B-ultrasound-guided percutaneous portal vein puncture with minimized body invasion. The therapeutic effect is evaluated through hematology, ultrasound, and pathology. To study the effect of hUC-MSCsp on gene expression in rhesus monkeys with liver injury, transcriptome sequencing analysis was performed on the livers of rhesus monkeys. The distribution of transplanted hUC-MSCsp was traced with RNA scope technology.

Results

We found that hUC-MSCsp significantly restored liver function, including ALT, AST, ALB, GLOB and bilirubin. hUC-MSCsp also significantly reduced liver collagen deposition and inflammatory infiltration, and promote dismission of liver ascites. Subsequently, the therapeutic effects were further validated in TGF-β1/Smad pathway by global transcription profile. The distribution of transplanted hUC-MSCsp were also tracked, and we found that hUC-MSCsp distributed in the liver in a sphere status at 1 h after transplantation. After 16 days, the hUC-MSCsp were dispersed into dissociated cells that were predominantly distributed in the spleen, and a significant number of dissociated cells were still present in the liver.

Conclusions

This study reveals the distributions of transplanted hUC-MSCsp after liver portal vein transplantation, and provides a novel approach and new insights into the molecular events of potential molecular events underlying the treatment of liver fibrosis with hUC-MSCsp.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-024-03934-7.

Keywords

Liver fibrosis
Rhesus monkey
hUC-MSC spheroids
TGF-β1/Smad signaling pathway
Cell tracing in vivo
National Key Research and Development Program of China2018YFA0801400 Si Wei http://dx.doi.org/10.13039/501100005273 Natural Science Foundation of Yunnan Province 202202AG050018 Si Wei Yunnan Provincial Key Research and Development Plan202403AC100020 Yang Hongju Famous Medical Specialist of High-level Talent Training Support Program of Yunnan ProvinceRLMY20190004 Yang Hongju Yunnan Health Training Project of High Level TalentsL-2019016 Wang Junfeng Yunnan High-level Talent Cultivation Support Plan of Famous Doctor ProjectKH-SWR-MY-2020-001 Wang Junfeng issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcBackground

Liver fibrosis is a fibrogenic and inflammatory process resulting from hepatocyte injury, characterized subsequent hepatic architectural distortion and resultant loss of liver function [1]. The primary pathophysiology underlying liver fibrosis involves increased deposition of type I and III collagen in the extracellular matrix (ECM) [2]. Currently, liver fibrosis affects approximately 500 million individuals worldwide, and if left untreated, it can progress to irreversible end-stages such as liver cirrhosis or hepatocellular carcinoma [3]. Liver cirrhosis may give rise of various complications including ascites and hepatic encephalopathy. Therefore, early intervention in liver fibrosis is crucial for preventing the development of liver cirrhosis [4, 5]. Although orthotopic liver transplantation is currently considered the most efficacious therapeutic approach, its clinical application remains limited due to organ donor scarcity, exorbitant costs, and requirement for lifelong immunosuppression [6]. Consequently, there is an urgent need for alternative therapeutic approaches and regenerative strategies in the treatment of liver fibrosis.

The transplantation of mesenchymal stem cells (MSCs) has emerged as the most promising therapeutic approach for liver fibrosis considering recent advancements in regenerative medicine [7–9]. Many studies have explored the mechanisms of MSCs in treating liver fibrosis from different perspectives. MSCs may differentiate into hepatocytes or fuse with existing hepatocytes after being introduced into damaged liver tissue [10]. Moreover, MSCs can facilitate the regeneration of damaged liver tissue by producing various cytokines, growth factors, and extracellular vesicles [11], but the precise mechanism underlying the therapeutic effect of MSCs on liver fibrosis remains elusive. Despite the favorable therapeutic effects demonstrated by MSCs in liver fibrosis [12], further investigation is required to understand their distribution and homing mechanisms in vivo before their widespread clinical application. In our previous study, we observed that one hour after intravenous infusion of MSCs into healthy monkeys, MSCs primarily localized in lungs and heart, with limited cell presence in liver [13]. A major challenge for clinical application of MSCs lies in their low homing efficiency. Considering that liver transplantation typically involves injecting through human portal vein [14], we speculate that transplanting MSCs via hepatic portal vein may enhance their retention within the liver and improve therapeutic efficacy. Moreover, traditional 2D monolayer-cultured MSC therapy has limitations, including restricted pluripotency and paracrine function of MSCs, accelerated cellular aging, and compromised therapeutic outcomes [15, 16]. Previous studies have demonstrated that 3D cultured MSC spheroids (MSCsp) promote intercellular signal transmission, enhance cell migration and proliferation, augment paracrine function, and achieve effective therapeutic outcomes even at low cell dose [17, 18].

In our previous study, we have previously demonstrated the safety of intravenous injection of human mesenchymal stem cell spheroids (∼ 450 μm) into healthy monkeys [13]. However, the therapeutic effect of MSCsp remain unknown, including their retention in the liver and effectiveness in treating liver fibrosis. Unfortunately, ethical constraints, safety concerns and sampling difficulty hinder these investigations from being conducted in humans. Therefore, it is crucial to establish appropriate models that accurately simulate human liver disease for evaluating the therapeutic potential and elucidating the mechanisms underlying MSC therapy. For cell therapy in mouse models, due to concerns about the small size of blood vessels, the usual approach is to intravenously inject dissociated cells instead of spheres. Macaques have larger blood vessel diameters, and when simulating human disease conditions, monkeys resemble humans more in terms of body size, physiological parameters, and immune and therapeutic responses than mice. In addition, non-human primate models are more suitable for evaluating the safety of cell therapy, including dosage and route of administration, which can be easily inferred to humans [13, 19, 20]. Therefore, in the present study, we established a rhesus monkey model of liver fibrosis induced by N-Nitrosodimethylamine (NDMA) and further evaluated therapeutic efficacy of transplantation of hUC-MSCsp via B-ultrasound-guided percutaneous portal vein puncture. Our results demonstrate that hUC-MSCsp ameliorated liver fibrosis and promoted hepatic ascites absorption of by downregulating the TGF-β1/smad signaling pathway. Furthermore, we tracked the hUC-MSCsp injected via portal vein infusion in monkeys and quantified their presence in major organs by using qPCR analysis of the human-specific gene ARHGAP11B [13]. Our findings provide valuable theoretical, technical, and practical insights for clinical trials involving hUC-MSCsp as a potential therapy for liver fibrosis. Moreover, our study sheds light on the mechanism by which hUC-MSCsp home to liver via portal vein infusion and ameliorates liver fibrosis.

Methods

hUC-MSCs isolation, identification and generation of hUC-MSCsp

Cell isolation and culture

The collection and application of umbilical cords samples from healthy volunteers in this study was approved by Ethics Committee of Yunnan first people’s Hospital (KHLL2019-KY052). The hUC-MSCs were obtained by direct explanted method with donated umbilical cords. All donors gave their informed consents prior to tissue donation. The tissue of umbilical cord was cut into small fragments and explanted in 150-cm2 plates and cultured in DMEM medium supplemented with 12% fetal bovine serum and 1% penicillin/ streptomycin incubate in a CO2 incubator at 37 °C. The morphology of the hUC-MSCs crawled out from the umbilical cord explant were examined and photographed using a light microscope (Leica, Germany).

Characterization of surface markers of MSCs

The identification of hUC-MSCs were conducted by the surface marker characterization with BD MSCs identification Kit (BD Bioscience) by flow cytometry. Briefly, the MSCs were dissociated with 0.25% trypsin and rinsed with 1 × DPBS two times. Afterward, the cells were adjusted to 1 × 106 cells/mL per tube and incubated with each antibody on ice for 30 min, the antibodies including CD44, CD90, CD73, CD105 and negative cocktail including CD45, CD34, CD11b, CD19, and HLA-DR. The isotype control antibody cocktail was used as a negative control. The cells were rinsed with 1 × DPBS twice and processed the measurement with FACS Calibur system (BD Bioscience, USA). The data were further analyzed with flowjo software (Ver.10).

Cell differentiation

The hUC-MSCs were tested for the capability of tri-lineage differentiation to adipocytes, osteocytes, and chondrocytes with the commercial differentiation kits (R&D Systems, USA) according to the manufacturer’s manual. Briefly, for the adipogenic differentiation, the MSCs were plate in the 6-well plate with 1 × 106 cells/well, the adipogenic differentiation medium were changed every other day for 21 days. The cells were fixed with 4% paraformaldehyde (PFA) for 30 min at 4 °C, then the cells were assessed by staining the cells with 0.5% Oil Red-O solution. For osteogenic differentiation, MSCs were administered with osteogenic differentiation medium for 21 days, and the mineralization of differentiated cells were assessed by staining with Alizarin red. For chondrogenic differentiation, 2 × 106 MSCs were centrifuged at 1000 rpm to form the cell pellets. These pellets were then cultured in chondrogenic differentiation medium for 21 days, fixed in 4% PFA and embedded in OCT (Sakura, Japan). The samples were sliced into 7 μm sections with a cryostat (Leica, Germany), and the sections were subsequently stained with Toluidine Blue [21]. All dyes were purchased from Sigma Aldrich (St. Louis, MO, USA).

Formation of hUC-MSCsp

hUC-MSCs were allowed to form spheroids as described previously [22]. The MSCs were dissociated with 0.25% trypsin and rinsed twice with 1 × DPBS. The cell count was then adjusted 106 cells in 25 ml of complete culture medium. The cell suspension was gently mixed, and 25 µL of the mixture was pipetted onto the surface of a 150 mm culture dish. The dish was inverted and incubated in an incubator for 48 h. Spheroids were formed from the dissociated cells in these hanging drops containing about 1 × 104 cells/drop. Then, the cell spheres were rinsed with DPBS and transferred to a 50 ml centrifuge tube. The spheroids were allowed to settle by gravity before being resuspended in physiological saline.

Evaluation of hUC-MSCsp in the treatment of liver fibrosis in rhesus monkeys

Generation of liver fibrosis model in rhesus monkeys

A total of 7 healthy rhesus monkeys were used for this study (Table S1). All the monkeys were individual caged. The animal room was set on a 12-hours light and 12-hours darkness cycle. The temperature and the humidity of the animal room were kept at 18 °C to 26 °C and 40–70%, respectively. Briefly, 5 mg/kg dimethylaniline (NDMA, Wako, Japan) once every 3 days was injected intraperitoneally to the monkeys for a total of 60 injections. Fibrosis stage was determined according to baseline Ishak, and monkeys with an Ishak score greater than 2 were included in the following study [23]. The evaluation of liver fibrosis progression after grouping is scored by three professional veterinarians who are not involved in this project and are not familiar with animal grouping. The macaques were fed with monkey chow twice a day and supplemented with fresh fruits and vegetables once per day. All experimental procedures were reviewed and approved under protocol # PZWH-K2019-0007 by the Institutional Animal Care and Use Committee of Kunming University of Science and Technology and were performed in accordance with the Guide for the Care and Use of Laboratory Animals Eighth Edition. The veterinarian followed ARRIVE guidelines to ensure appropriate handling procedures for immobilization, sedation, and anesthesia. Abnormal postures, anorexia, vocalization, lethargy, and self-directed behaviors are indicators to evaluate pain or distress. New toys, cage changes, or comforting foods (such as peanuts, raisins or candies) are employed to moderate stress without compromising the scientific aspects of the experiments. The work has been reported in line with the ARRIVE guidelines 2.0.

hUC-MSCsp transplantation via portal vein

Six macaques were successfully induced into liver fibrosis models and grouped using a computer-based random order generator. Three of them were randomly selected to receive MSC injection, and the remaining three received saline injection. Therefore, the rhesus monkeys with liver fibrosis were divided into four groups, which was referred as the monkeys before NDMA administration (Normal, n = 6), NDMA administrated (Model, n = 6), MSCsp transplanted (MSCsp, n = 3), and saline infused (Saline, n = 3). For the MSCsp group, 3 × 103 hUC-MSCsp (about 3 × 107 cells) suspended in 30 mL saline were injected into the hepatic portal vein with PTC needle (Hakko Co.,Ltd, Japan) under the guidance of B-ultrasound. For the sham group, an equivalent amount of saline solution was similarly injected into the hepatic portal vein. The MSCsp and saline were injected in triplets with a one-month interval. We can only choose the smallest sample size because rhesus monkeys are very precious, and at least 3 monkeys in each group have completed statistical analysis to obtain data for in vivo evaluation of treatment effects.

Biochemical analysis of serum

Peripheral venous blood (2–3 mL) was collected after fasting for 8 h for biochemical analysis one month after the third cell transplantation. Briefly, serum was separated by centrifugation at 3,000 rpm for 5 min and stored at -80℃ until analysis. Biochemical analysis was performed using the automatic biochemical analyzer (Mindray, BS-2000 M) along with its accompanying reagents. Daily quality control was conducted in accordance with the 1–3/2-2s quality control rules, using Bole quality control products. Liver enzymes (e.g., ALT, AST, AST / ALT, r-GT, ALP, LDH), plasma proteins (e.g., TP, ALB, GLOB), bilirubin (e.g., TBIL, DBIL, IBIL), liver fiber index (e.g., PIIINP, IV-C, LN, CG, HA) and liver cancer indexes (e.g., AFP, TSGF) was examined in each monkey.

Ultrasound examination and histological evaluation of monkey liver

Ultrasound testing and liver tissue collection are both conducted on an empty stomach in animals, with each fixed time starting at 9am. Rhesus monkeys were anesthetized by intramuscular injection of ketamine (0.5 mL/monkey). Ultrasound examination (Siemens, Japan) was performed on the liver of each monkey. Liver samples were biopsied from the right lobe by using a biopsy gun (MG16; BARD, USA) under the guide of B-ultrasound one month after the third cell transplantation. Liver samples were harvested at the indicated time points before or after MSCs injection and fixed in 4% paraformaldehyde. Liver Sect. (7 μm in thickness) were subjected to hematoxylin and eosin (H&E) staining and Masson staining. The sections were observed under an inverted microscope (Olympus, Japan).

Library preparation and transcriptome sequencing

To study the effect of MSCsp on gene expression in rhesus monkeys with liver fibrosis, transcriptome sequencing analysis was performed on the livers from the four groups of rhesus monkeys. RNA of each sample was extracted from liver tissues at different time periods. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. We use HISAT2 v2.0.5 compare the paired end clean reads with the reference genome, feature Counts v1.5.0-p3 was used to count the reads numbers mapped to each gene. Then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.

Differential expression gene (DEG) analysis

DEG analysis was performed using the DESeq2 R package (1.16.1). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting p-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted p-value < 0.05 found by DESeq2 were assigned as differentially expressed.

Gene expression analysis by qRT-PCR

For the qRT-PCR, total RNA was extracted from each liver tissues using Trizol® reagent (Thermo Fisher Scientific, USA). The collected RNA was dissolved in sterile diethyl pyrocarbonate-treated water (Sangon Biotech, China), and cDNA was synthesized using a Prime-Script RT reagent kit (Takara, Japan) at 37 °C for 5 min and 85 °C for 5 s. Quantification of specific genes was performed using SYBR® Premix Ex TaqTM II kit (Takara, Japan) and CXF real-time PCR system (Bio-Rad Laboratories, USA) at particular condition (Stage 1: 95 °C for 3 s; Stage 2: 95 °C for 3 s; 60 °C for 30 s, and repeat for 37 cycles). All experiments were performed in triplicate, and the data were analyzed using the 2−∆∆Ct method. All the primers were commercially synthesized (Sangon Biotech, China), and the primers’ sequences were shown in Table S2.

Protein extraction and western blot analysis

Liver tissue was lysed with RIPA buffer (Beyotime, China) with PMSF (Solarbio, China). The total soluble protein was quantified with a BCA Protein Assay Kit (Beyotime, China). Protein samples were loaded onto 10% SDS-PAGE gels, electrophoresis, and then transformed to PVDF membrane after fixation. The following antibodies were applied: anti- TGFβ (1:1000, NOVUS, USA, NBP2-45137), anti- Smad2 (1:1000 CST, USA, 5339 S), anti- Smad3 (1:1000, CST, USA, 9523 S), anti- Snail (1:1000, CST, USA, 3879 S), anti- GAPDH (1:1000, Abcam, UK, ab8245), goat anti-rabbit IgG-HRP and Cy3-conjugated goat anti-mouse (1:5000, CST, Beverly, MA, USA). The membranes were treated following the ECL WB Protocol (Bio-Rad, Milan, Italy). The original images were recorded and analysed with Odyssey Infrared Imaging System.

Tracing of hUC-MSCsp in major organs of rhesus monkeys

One rhesus monkey with chronic liver fibrosis (Ishak Score = 2) was used for the tracing of transplanted hUC-MSCsp. Two check points were set to track MSCsp in vivo, including 1 h (short time) and 16 days (long time) post cell transplantation in this monkey. Liver tissue was biopsied 1 h after MSCsp transplantation. After 16 days, the rhesus monkey was euthanized by intravenous injection of barbiturates (100 mg/kg) and cell tracing was conducted on the liver, heart, spleen, lung and kidney.

H&E and Masson staining

Liver tissues were fixed in 4% paraformaldehyde, and then were embedded in paraffin for slicing. Liver Sect. (7 μm of thickness) were subjected to hematoxylin and eosin (H&E) and Masson staining. The sections were observed under an inverted microscope (Olympus, Japan).

Immunofluorescence staining

The tissues collected from the liver, heart, spleen, lung and kidney were fixed with 4% paraformaldehyde and embedded with OCT (sukula, Japan), and were sliced (10 μm of thickness) for immunofluorescence staining. Immunofluorescence staining was performed as previously reported in the literature. The primary antibodies used were anti-ARHGAP11B (1:100, Novus, USA, 05888). The secondary antibodies used were an Alexa-Fluor®488-conjugated goat anti-rabbit antibody (1:500, Invitrogen, USA). Sections were observed under a confocal fluorescence microscope (Leica, USA).

RNA-scope

The obtained tissues were immediately frozen with liquid nitrogen. RNA-scope (Advanced Cell Diagnostics, USA) was performed using probes targeting the human specific gene ARHGAP11B (NM_001039841.3) on slides, according to the manufacturer’s protocol. The RNA-scope procedure included the following steps: RNA-scope (Advanced Cell Diagnostics, USA) was performed using probes targeting the human specific gene ARHGAP11B (NM_001039841.3) on slides, according to the manufacturer’s protocol. Specifically, (1) Deparaffinize FFPE sections: the frozen sections were fixed (4% PFA, 4 °C, 15 min), dehydrated (50%, 70%, 100% ethanol, 5 min). (2) Apply RNAscope Hydrogen Peroxide: Lay the deparaffinized slides on the bench, and add ∼ 5–8 drops of RNAscope Hydrogen Peroxide to cover each section. Immediately insert the slide into a Tissue-Tek Slide Rack submerged in a Tissue-Tek Staining Dish filled with distilled water. (3) Apply RNAscope Protease Plus: Add ∼ 5 drops of RNAscope Protease Plus to entirely cover each Sect. 4) Proceed to the RNAscope Assay: signals were amplified and generated with an RNAscope Multiplex Fluorescent Reagent Kit v2 (ACD, Catalog No. 323100).

Statistical analysis

Student’s t test or one-way ANOVA was employed for statistical comparisons (Spss16.0). The results of the bioinformatics analysis were visualized using GraphPad Prism 5 software. The statistical significances were calculated as p values, and p < 0.05 was considered as statistical significance. Student’s t-test for comparison between two groups and one-way ANOVA for comparison among three and more groups. All data were expressed the means ± SEM. Model assumptions were checked using the Levene’s Test for homogeneity of variance.

Results

Preparation of hUC-MSCsp and injections of hUC-MSCsp into monkeys via B-ultrasound-guided portal vein puncture

The morphology, immunophenotype profiles and tri-lineage differentiation potential of hUC-MSCs were examined at passage 4. The results of immunophenotype profiles analyzed by flow cytometry showed that hUC-MSCs expressed high levels of the positive markers CD44, CD73 and CD90 (Fig. 1A). The hUC-MSCs could differentiate into osteocytes, adipocytes, and chondrocytes in vitro (Fig. 1B). Then, hUC-MSCs was formed into spheroids by hanging drop culture, and the hUC-MSCsp were characterized by tight texture and uniform size (1 × 104 cell/ spheroid, about 350 μm in diameter) (Fig. 1C).

The development of liver fibrosis in rhesus monkeys was scored by Ishak [23], and liver with an Ishak score at 2 or greater were included in subsequent studies. The six rhesus monkeys with liver fibrosis were randomly divided into two groups, including the MSCsp group (average ishak score of 3.3) and the saline sham group (average ishak score of 3). The experimental flow chart was shown in Fig. 1D. The hUC-MSCsp were injected into the monkeys through the portal vein under the guide of ultrasound guidance, and obvious granular strong echogenic points indicating the distribution of MSCsp were observed in the liver under ultrasound (Fig. 1E-F).

Fig. 1 Formation of hUC-MSCsp and injection into rhesus monkeys via portal vein. A-B. Immunophenotypes and tri-lineage differentiation potential of hUC-MSCs. C. Morphology of hUC-MSCsp. D. Experimental flow chart. The blue arrows showed the fibrotic nodules. E. Liver morphology under ultrasound prior to MSCsp transplantation. F. Liver morphology under ultrasound after hUC-MSCsp transplantation. When hUC-MSCsp entered the liver via the portal vein and displayed granular hyperechoic points under ultrasound (yellow arrow), PTC needle (red arrow)

Serological indicators related to liver function and liver fibrosis were restored in rhesus monkeys after hUC-MSCsp transplantation

In term of serum parameters, the results showed that the liver enzymes, plasma proteins, bilirubin, liver fibrosis and liver cancer indexes were worsened by DMNA administration but ameliorated after hUC-MSCsp transplantation. The liver enzymes including glutamic pyruvic transaminase (ALT), glutamic oxalacetic transaminase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (γ-GT) and lactic dehydrogenase (LDH) increased in the model group, but significantly decreased after hUC-MSCsp transplantation compared to the saline group (Fig. 2A). The plasma protein including total protein (TP) and albumin (ALB) decreased and globulin (GLOB) was increased in the model group, while these indexes significantly improved in the hUC-MSCsp group compared to the saline group (Fig. 2B). The bilirubin indexes including total bilirubin (TBIL), direct Bilirubin (DBIL) and indirect bilirubin (IBIL) increased in the model groups, while significantly improved by hUC-MSCsp transplantation (Fig. 2C). The liver fibrosis indexes (type II collagen propeptide (PIINP), type IV collagen (IV-C), laminin (LN), hyaluronic acid (HA) and glycocholic acid (CG)) and liver cancer indexes (alpha fetoprotein (AFP) and tumor specific growth factor (TSGF)) increased in the model group, and these indices continually increased in the saline groups. However, both liver fibrosis and liver cancer indexes significantly reduced after hUC-MSCsp transplantation (Fig. 2D and E). These results on serological indicators suggest that the transplantation of hUC-MSCsp can significantly improve liver function and ameliorated liver fibrosis in the rhesus monkey models.

Fig. 2 hUC-MSCsp restored serological indicators in rhesus monkey models of liver fibrosis. (A) Liver enzymes. (B) Plasma protein indexes. (C) Bilirubin indexes. (D) Liver fiber indexes. (E) liver cancer indexes. *: a significant difference (p < 0.05). **: a significant difference (p < 0.01). ***: a significant difference (p < 0.001)

hUC-MSCsp injections significantly ameliorated liver fibrosis and promoted the absorption of hepatic ascites in rhesus monkeys

Compared to control group, the liver pathology of rhesus monkeys from the model group showed chronic inflammatory infiltration in the portal area, destruction of the hepatic lobule structure, dense deposition of collagen fibers (Fig. 3A), and liver parenchymal echo thickening with diffuse lesions and severe hepatic ascites (Fig. 3B). After hUC-MSCsp spheroids transplantation via portal vein, the inflammatory infiltration, deposition of collagen fibers and diffuse lesions were ameliorated in the MSCsp group compared to the saline group (Fig. S1, Fig. 3A) (Table S3). More significantly, the hepatic ascites completely disappeared after cell transplantation (Fig. 3C). These results indicate that the liver fibrosis of rhesus monkeys is ameliorated by hUC-MSCsp.

Fig. 3 hUC-MSCsp ameliorated liver fibrosis in rhesus monkeys. A. H&E and Masson and staining of liver sections from biopsy tissue. B: Ultrasonic examination of the livers, the red arrows pointing to the location of hepatic ascites

hUC-MSCsp ameliorated liver fibrosis in rhesus monkey models through TGF-β1/Smad pathway

To validate the alterations of gene expression profile of the livers after NDMA modeling or MSCsp administration. Bulk transcriptome sequencing was performed on the liver tissues from the four groups. The PCA result showed significant differences among the four groups and were used for subsequent analysis (Fig. S2A). Meanwhile, the heatmaps of cluster analysis on DEGs also showed significant differences among the four groups (Fig. S2B-S2J). Further enrichment analysis was performed on the DEGs, and the results showed that the key genes including TGF-β1, Smad2, Smad3, Snail, Mmp2 and Mmp9 from TGF-β1/Smad signaling pathway highly expressed in the model group and the saline group. In contrast, the expressions of these genes were significantly downregulated and approached the levels of the normal group after MSCsp transplantation (Fig. 4A and B). Further confirmation of these genes expressions was performed by using RT-PCR and western blotting. Similarly, the expression of TGF-β1, Smad2, Smad3 and Snail increased in the model group, and reached to the highest level in the saline group. After MSCsp transplantation, the expression levels of these genes all decreased to normal levels (Fig. 4C). As far as the protein level, the expression of phosphorylated smad2 and smad3 significantly decreased in the MSCsp group compared to the saline group (Fig. 4D). These results suggested that hUC-MSCsp might alleviate liver fibrosis of rhesus monkeys though inhibiting TGF-β1/Smad signaling pathway and improve epithelial mesenchymal transition (EMT) of liver cells and hepatic stellate cells (Fig. 4E).

Fig. 4 hUC-MSCsp ameliorated liver fibrosis of rhesus monkeys by inhibiting TGF-β1/Smad pathway. (A) Heatmap of TGF-β1, Smad2, Smad3, Snail expression. (B) Heatmap of TGF-β1, Smad2, Smad3, Snail expression in each individual in the four groups. Red denoting upregulated genes, and blue denoting downregulated genes. (C) Expression of TGF-β1, Smad2, Smad3 andSnail verified by qRT-PCR. *: significant difference (p < 0.05). **: significant difference (p < 0.01). ***: a significant difference (p < 0.001). (D) Expression of TGF-β1, Smad2, Smad3 and Snail verified by Western blot. Full-length blots are presented in Additional Fig. S3. E: Diagram of hUC-MSCs inhibiting TGFβ-1/Smad signaling pathway

Short-term and long-term tracing of hUC-MSCsp distribution in rhesus monkeys

In many previous studies, MSCs have been reported to alleviate liver diseases in rodent models and humans, but the ultimate fate of the injected cells has not yet been elucidated. In our previous study, MSCs were found to be nearly undetectable at 21 days after following intravenous injection of MSCsp. Therefore, we set two time points including 1 h (short time) and 16 days after cell injection (long time) to track the distribution of injected cells in a liver fibrosis monkey model administrated with hUC-MSCsp (Fig. 5A). The histomorphology of the biopsied liver tissue showed that spheroids could be observed around the portal vein around the hepatic portal area at one hour after hUC-MSCsp transplantation (Fig. 5B). In order to verify that the observed spheres were the injected hUC-MSCsp, we performed RNA-scope analysis on the liver slices using human specific gene ARHGAP11B, which is express in hUC-MSCsp but not in any tissues of rhesus monkeys (Fig. 6A). At the same time, we also observed that spheroids around the portal vein of liver by immunofluorescence at 1 h after hUC-MSCsp transplantation (Fig. 5B). However, the spheroids disappeared around the portal vein, but replaced by single cells instead at 16 days after hUC-MSCsp transplantation (Fig. 5C).

The largest number of single cells appeared in the spleen at 16 days after hUC-MSCsp transplantation detected by the RNA-scope results of ARHGAP11B (Fig. 6B). The quantized expression of ARHGAP11B in main organs by using qRT-PCR at 16 days after MSCsp transplantation showed that the expression of ARHGAP11B was the highest in spleen but was the lowest in heart (Fig. 6C). These results indicated that a significant amount of hUC-MSCsp enters the liver at the beginning of infusion through the hepatic portal vein, however, most of the MSCsp is gradually dispersed over time and eventually distributed into the spleen mostly.

Fig. 5 hUC-MSCsp and dissociated MSCs were found in liver fibrosis of rhesus monkeys. (A) Cell tracing flow chart. (B) hUC-MSCsp were found around the hepatic portal vein 1 h after cell injection. (C) Dissociated MSCs were found around the hepatic portal vein by RNA scope 16 days after transplantation

Fig. 6 Distribution of hUC-MSCsp in 5 organs at 16 days after cell transplantation. (A) Gel electrophoresis images of ARHGAP11B expression detected by RT-PCR in human MSCs and 5 rhesus monkey tissues. Full-length gels are presented in Additional Fig. S4. (B) Immunofluorescence maps of ARHGAP11B in 5 organs by RNA-scope. (C) Expression of ARHGAP11B in the 5 organs detected by qRT-PCR

Discussion

Although numerous studies have demonstrated the therapeutic effects of MSCs on liver fibrosis, efficacy and safety evaluation based on idealized animal disease model is one of the prerequisites for clinical practice of MSC therapy. The large species difference between rodents and human extremely hindering the understanding of the pathophysiological processes [24]. In this study, we induced liver fibrosis in rhesus monkey with NDMA and then transplanted hUC-MSCsp instead of dissociated MSCs into the liver of rhesus monkeys through the portal vein to evaluate the therapeutic outcomes. We also track the distribution of the transplanted cell spheres in in both the short and long term. The results found that transplantation of hUC-MSCsp via B-ultrasound-guided portal vein puncture is safe and effective in treating liver fibrosis in rhesus monkey, and did not cause abnormalities or death in the animals after cell transplantation. More importantly, hUC-MSCsp transplantation significantly ameliorated liver fibrosis and diminished ascites in rhesus monkey. One hour after hUC-MSCsp transplantation, Previously, we found that hUC-MSCsp were still distributed in the liver of monkeys in the form of spheres. Even 16 days after cell transplantation, a significant number of hUC-MSCsp were found to remain in the spleen and liver. Therefore, our results can provide a new approach for clinical MSCs treatment of liver fibrosis, and provide references for the fate of transplanted cells.

Limited cell viability following transplantation compromises the clinical benefits of MSCs. MSCs and other therapeutic cell types are rarely able to survive in the host after systemic infusion, primarily due to clearance by lung occlusion and immediate inflammatory responses. Improving the ability of MSCs to home in the injury site is crucial for enabling MSCs to exert therapeutic effects [25, 26]. Previous studies have demonstrated that macroautophagy/autophagy is highly induced and ROS production is suppressed in 3D-cutured MSCs (MSCsp) compared to 2D-cultured MSCs (MSCdiss). Additionally, MSCsp enhanced immunosuppressive effect, suppressed TNF-α expression, and increased TGF-β expression [27]. MSCsp also enhance the resistance to oxidative stress-induced apoptosis, and exhibited significant ability to prevent apoptosis compared to MSCdiss by inhibiting CASP3 and inducing of SOD2 expression [28]. Therefore, MSCsp offer superior immune suppression and higher survival rates compared to MSCdiss. Our previous study demonstrated that 3D cultured MSCsp showed higher tolerated fluidic shear stress, better survival, less lung entrapment and higher efficacy than dissociated MSCs. MSCsp has tight shell and avoid mass aggregation, and the big diameters of large blood vessels in monkeys and allow sufficient time for MSCs spheroids to dissociate into single cells and passing through small vessels without causing embolism, which has been demonstrated in our previous study [13]. In addition, MSCs spheroids showed a good therapeutic effect on the treatment of hepatic fibrosis in mice by enhancing the expression of antifibrotic factors [29]. However, MSCs transplanted into cynomolgus monkeys via intravenous injection, either single cells or cell spheres, were entrapped in the lung and heart, and rarely found in the bodies 21 days later [13]. Transplanted intravenously MSCs trapped in the lungs and cleared, and encounter a highly oxidative, inflammatory microenvironment that substantially impairs cell engraftment and survival [27]. Therefore, intravenous injection of MSCs may result in poor therapeutic effects in the treatment of diseases due to the insufficient cell homing ability. The continuous progression of liver fibrosis will lead to cirrhosis [3–5], only liver transplantation can be effective for the treatment of late stage of cirrhosis, while liver cell transplantation is mainly achieved through portal vein injection [14]. In order to minimize the invasion, we developed a procedure that MSCsp could be injected into the liver via B-ultrasound-guided percutaneous portal vein puncture. Our results demonstrated that the degree of fibrosis in rhesus monkeys was significantly ameliorated, and the ascites were completely absorbed. Therefore, our results provide a novel approach for the clinical treatment of liver fibrosis with MSCs, which may mimic orthotopic liver transplantation by injecting MSCsp into the human liver through the portal vein to achieve better therapeutic effects but minimize physiological invasion.

MSCs have shown good results in treating liver fibrosis in both mice and humans, but little is known about the mechanism of MSCs in treating liver fibrosis [7–9]. To further investigate the therapeutic mechanism, transcriptomic sequencing of biopsied liver tissue revealed a large number of relevant genes affected by DMNA modeling and recovered by hUC-MSCsp administration. By further gene enrichment analysis, we found that TGF-β1/Smad signaling pathway was inhibited in the process of hUC-MSCsp ameliorating liver fibrosis of monkey models, which was further confirmed by the transcription and translation levels. TGF-β1 is closely related to liver fibrosis, and is considered as one of the most important fibrotic cytokines known to date. TGF-β binds to aheteromeric complex of type I and type II transmembrane receptors that phosphorylate and activate receptor-regulated R-Smads (Smad2/3). Phosphorylated Smad2/3 complex activates hepatic stellate cells proliferate and express extracellular matrix (ECM) proteins. Hepatic stellate cells also produce large amounts of chemokines and cytokines, which links hepatic inflammation to fibrogenesis [30, 31]. Similarly, our study also showed that hUC-MSCsp effectively inhibited the TGFβ-1/Smad pathway and might subsequently reduce EMT of hepatocytes and hepatic stellate cells, thereby alleviated liver fibrosis.

To locate the distribution of hUC-MSCsp via the hepatic portal vein into the body of monkeys, the granuliform were appeared around the portal vein in the hepatic portal area detected by B-ultrasound echo signal and histomorphologic analysis. Meanwhile, a large number of human MSCs was also found in liver tissue by immunofluorescence detection of human specific gene ARHGAP11B after 1 h of transplantation. After 16 days of transplantation, scattered MSCs were found in the heart, liver, spleen, lung and kidney, mostly in the spleen. The results indicate that a large amounts of hUC-MSCsp did enter the liver at the beginning of infusion through the hepatic portal vein and played therapeutic roles. However, most MSCsp gradually dispersed into individual cells over time, and may eventually end up in the spleen. In our previous study, human MSC spheroids were injected intravenously into monkeys and mainly distribute in lung and heart after 1 h, but almost no MSCs were appeared in other tissues after 21 days [13]. A study of MSCs treating mouse model of liver fibrosis showed that only a small amount of MSCs retained in the liver fibrotic area after 7 days of infusion through the tail vein, and most of the MSCs were trapped in the lung [32]. The main reason for the differences compared to the present study may be the different infusion routes of MSCs, and it may also be related to the different homing and chemotactic mechanisms of MSC spheroids in healthy and liver injured monkeys.

Although combining our previous research with this study, we have found that portal vein transplantation of hUC-MSCsp can induce more cells to homing to the liver and significantly alleviate liver ascites and fibrosis in macaques. However, due to a severe shortage of experimental monkeys and budget constraints, our study was conducted with a relatively small sample size, which may affect the statistical results. Our study did not include a comparative analysis with dissociated hUC -MSCs transplantation or alternative stem cell sources via portal vein puncture, which is beyond the scope of the current study. In future research, larger sample sizes should be utilized to validate these findings and conduct long-term effectiveness and safety tracking.

Conclusions

In summary, we successfully generated a liver fibrosis model of rhesus monkeys and transplanted 3D cultured hUC-MSCsp via B-ultrasound-guided percutaneous portal vein puncture, which significantly improved the liver function and ameliorated liver fibrosis in rhesus monkey. The therapeutic effects were further validated on TGF-β1/Smad pathway by global transcription profile and the expression of relevant genes was also confirmed by qRT-PCR and Western blotting. Finally, the distributions of transplanted hUC-MSCsp were revealed. This study not only demonstrated the safety and efficacy of the infusion hUC-MSCsp through the hepatic portal vein in monkeys for the treatment of liver fibrosis, but also provided new insights into the underlying molecular events of MSCs for the treatment of liver fibrosis. Moreover, this study demonstrates the promising nature of our therapeutic strategy, which requires further investigations and validation before it can be applied in clinical practice.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Acknowledgements

The authors would like to thank the veterinarians for taking care of experimental animals. The authors declare that they have not used Artificial Intelligence in this study.

Author contributions

LS, FX, WJ and YY performed the all experiments. LS, FX, YY and SW wrote the paper. LS, YH and WD performed cell experiments and prepared figures and/or table. DX did the statistical analyses. DY and WH performed animal experiments. YY and SW conceived and designed the experiments and reviewed drafts of the paper. SW and YH contributed to reagents and materials.

Funding

This study was supported by grants from the Natural Science Foundation of Yunnan Province of 202202AG050018 (W.S.), the National Key Research and Development Program of China of 2018YFA0801400 (W.S.), the Yunnan Provincial Key Research and Development Plan of 202403AC100020 (HJ. Y.) and the Famous Medical Specialist of High-level Talent Training Support Program of Yunnan Province of RLMY20190004 (HJ. Y.), the Medical leading talents Project in Yunnan Province L-2019016 (JF. W.), the Yunnan Province High-level personnel training support Program famous Medical Project YNWR-MY-2020-035(JF. W.).

Data availability

The obtained transcriptome data have been uploaded into the SRA database and are accessible via the accession number PRJNA1114939 (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA1114939).

Declarations

Ethics approval and consent to participate

All animal work was compliant with the protocol of Institutional Animal Care and Use Committees (IACUC) at Kunming University of Technology (PZWH-K2019-0007). Date of approval is Jan 2, 2020. Title of the approved project: Therapeutic efficacy and distribution of human umbilical cord-derived mesenchymal stem cell spheroids transplanted via the portal vein into rhesus monkey models of liver fibrosis. Usage of human sample (n = 3, from healthy donors) and the protocol used in this study are approved by Ethics Committee of Yunnan first people’s Hospital (KHLL2019-KY052). Date of approval is Dec 25, 2019. All the donors (n = 3) provided informed consents prior to tissue donation. Title of the approved project: Establishment of key clinical applicability technologies for MSCs derived from human umbilical cord and application in the treatment of liver fibrosis in macaques.

Consent for publication

Not applicable.

Competing interests

The authors have declared that no competing interest exists.

Abbreviations

MSCs Mesenchymal stem cells

hUC-MSCsp hUC-MSC spheroids

ECM Extracellular matrix

NDMA N-Nitrosodimethylamine

hUC-MSC Human umbilical cord-derived MSC

ALT Glutamic pyruvic transaminase

AST Glutamic oxalacetic transaminase

ALP Alkaline phosphatase

γ-GT γ-glutamyltransferase

LDH Lactic dehydrogenase

TP Total protein

ALB Albumin

GLOB Globulin

TBIL Total bilirubin

DBIL Direct Bilirubin

IBIL Indirect bilirubin

PIINP Type II collagen propeptide

IV-C Type IV collagen

LN Laminin

HA Hyaluronic acid

CG Glycocholic acid

AFP Alpha fetoprotein

TSGF Tumor specific growth factor

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Shanshan Li, Xufeng Fu and Junfeng Wang contributed equally to this work.
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