a-quantitative-assessment-phagocytosis-allogeneic-xenogeneic

A Quantitative Assessment of the Phagocytosis of Allogeneic and Xenogeneic Erythrocytes by Rat Macrophages <em>In Vitro</em>

Xenogeneic cell transplantation often faces significant immune rejection, even in immunodeficient animal models. Among residual immune components, ...

University of Chinese Academy of Sciences

Hepatocyte-like cells (HLCs) can be generated through directed differentiation or transdifferentiation. Employing two strategies, we generated induced pluripotent stem cell (iPSC)-HLCs and hiHeps from the same donor cell line. Both types of HLCs clustered distinctly from each other during gene expression profiling. In particular, differences existed in gene expression for phase II drug metabolism and lipid accumulation, underpinned by H3K27 acetylation status in iPSC-HLCs and hiHeps. While distinct phenotypes were achieved in vitro, both types of HLCs demonstrated similar phenotypes following transplantation into Fah-deficient mice. In conclusion, functional HLCs can be obtained from the same donor using two strategies. Global gene expression defined the differences between those populations in vitro. Importantly, both HLCs displayed partial but markedly improved hepatic function following transplantation in vivo, demonstrating plasticity and the potential for cell-based modeling in the dish and cell-based therapy in the future.

Distinct Gene Expression and Epigenetic Signatures in Hepatocyte-like Cells Produced by Different Strategies from the Same Donor.

Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the  mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a  mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the  First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease.

Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats.

Transplantation of human hepatocytes (HHs) holds significant potential for treating liver diseases. However, the supply of transplantable HHs is severely constrained by limited donor availability and compromised capacity for in vitro expansion. In response to chronic injury, some HHs are reprogrammed into proliferative cells that express both hepatocyte and progenitor markers, suggesting exploitable strategies for expanding HHs in vitro. Here, we report defined medium conditions that allow 10,000-fold expansion of HHs. These proliferating HHs are bi-phenotypic, partially retaining hepatic features while gaining expression of progenitor-associated genes. Importantly, these cells engraft into injured mouse liver at a level comparable to primary HHs, and they undergo maturation following transplantation in vivo or differentiation in vitro. Thus, this study provides a protocol that enables large-scale expansion of transplantable HHs, which could be further developed for modeling and treating human liver disease.

In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity.

Primary hepatocytes are widely used in regenerative medicine, drug metabolism analysis, and in vitro drug screens. To overcome the shortage of liver donors, several strategies, such as differentiation of pluripotent stem cells and transdifferentiation from somatic cells, were developed to generate hepatocytes from alternative sources. Here, we describe in detail lenti-virus-based procedure for direct conversion of human fibroblasts to hepatocytes (hiHep cells) in vitro. A detailed protocol for preparation of human fibroblasts from scar tissues is also provided. Based on this protocol, FOXA3, HNF1A, and HNF4A are introduced into SV40-large-T-antigen-expressing human scar fibroblasts by lenti-virus. It usually takes about 5-7 days to get epithelial hiHep colonies. SV40-large-T-antigen-expressing hiHep (hiHep) cells are proliferative and can be expanded to a large number for potential uses.

Conversion of Fibroblasts to Hepatocytes In Vitro.

The ever-increasing therapeutic and pharmaceutical demand for liver cells calls for systems that enable mass production of hepatic cells. Here we describe a large-scale suspension system that uses human endoderm stem cells (hEnSCs) as precursors to generate functional and transplantable hepatocytes (E-heps) or cholangiocytes (E-chos). hEnSC-derived hepatic populations are characterized by single-cell transcriptomic analyses and compared with hESC-derived counterparts, in-vitro-maintained or -expanded primary hepatocytes and adult cells, which reveals that hepatic differentiation of hEnSCs recapitulates in vivo development and that the heterogeneities of the resultant populations can be manipulated by regulating the EGF and MAPK signaling pathways. Functional assessments demonstrate that E-heps and E-chos possess properties comparable with adult counterparts and that, when transplanted intraperitoneally, encapsulated E-heps were able to rescue rats with acute liver failure. Our study lays the foundation for cell-based therapeutic agents and in vitro applications for liver diseases.

Large-scale Generation of Functional and Transplantable Hepatocytes and Cholangiocytes from Human Endoderm Stem Cells.

Although liver-humanized animals are desirable tools for drug development and expansion of human hepatocytes in large quantities, their development is restricted to mice. In animals larger than mice, a precondition for efficient liver humanization remains preliminary because of different xeno-repopulation kinetics in livers of larger sizes. Since rats are ten times larger than mice and widely used in pharmacological studies, liver-humanized rats are more preferable. Here, Fah Rag2 IL2rg (FRG) rats are generated by CRISPR/Cas9, showing accelerated liver failure and lagged liver xeno-repopulation compared to FRG mice. A survival-assured liver injury preconditioning (SALIC) protocol, which consists of retrorsine pretreatment and cycling 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) administration by defined concentrations and time intervals, is developed to reduce the mortality of FRG rats and induce a regenerative microenvironment for xeno-repopulation. Human hepatocyte repopulation is boosted to 31 ± 4% in rat livers at 7 months after transplantation, equivalent to approximately a 1200-fold expansion. Human liver features of transcriptome and zonation are reproduced in humanized rats. Remarkably, they provide sufficient samples for the pharmacokinetic profiling of human-specific metabolites. This model is thus preferred for pharmacological studies and human hepatocyte production. SALIC may also be informative to hepatocyte transplantation in other large-sized species.

Survival-Assured Liver Injury Preconditioning (SALIC) Enables Robust Expansion of Human Hepatocytes in Fah Rag2 IL2rg Rats.

Cellular senescence is a persistently hypoproliferative state with diverse stressors in a specific aging microenvironment. Senescent cells have a double-edged sword effect: they can be physiologically beneficial for tissue repair, organ growth, and body homeostasis, and they can be pathologically harmful in age-related diseases. Among the hallmarks of senescence, the SASP, especially SASP-related extracellular vesicle (EV) signalling, plays the leading role in aging transmission via paracrine and endocrine mechanisms. EVs are successful in intercellular and interorgan communication in the aging microenvironment and age-related diseases. They have detrimental effects on downstream targets at the levels of immunity, inflammation, gene expression, and metabolism. Furthermore, EVs obtained from different donors are also promising materials and tools for antiaging treatments and are used for regeneration and rejuvenation in cell-free systems. Here, we describe the characteristics of cellular senescence and the aging microenvironment, concentrating on the production and function of EVs in age-related diseases, and provide new ideas for antiaging therapy with EVs.

Roles of extracellular vesicles in the aging microenvironment and age-related diseases.

Cell therapy provides hope for treatment of advanced liver failure. Proliferating human hepatocytes (ProliHHs) were derived from primary human hepatocytes (PHH) and as potential alternative for cell therapy in liver diseases. Due to the continuous decline of mature hepatic genes and increase of progenitor like genes during ProliHHs expanding, it is challenge to monitor the critical changes of the whole process. Raman microspectroscopy is a noninvasive, label free analytical technique with high sensitivity capacity. In this study, we evaluated the potential and feasibility to identify ProliHHs from PHH with Raman spectroscopy.

Single cell Raman spectroscopy to identify different stages of proliferating human hepatocytes for cell therapy.

The progression of Biliary Atresia (BA) is associated with the number of reactive ductular cells (RDCs) whose heterogeneity in origin and evolution in humans remains unknown. SOX9-positive liver progenitor-like cells (LPLCs) have been shown to participate in RDCs and new hepatocyte formation during cholestatic liver regeneration in an animal model, which implies the possibility that hepatocyte-reprogrammed LPLCs could be a source of RDCs in BA. The present study aimed to elucidate the characteristics of SOX9-positive LPLCs in BA for exploring new possible therapeutic targets by manipulating the bi-differentiation process of LPLCs to prevent disease progression.

Characteristics of SOX9-positive progenitor-like cells during cholestatic liver regeneration in biliary atresia.

Liver resection is the first-line treatment for primary liver cancers, providing the potential for a cure. However, concerns about post-hepatectomy liver failure (PHLF), a leading cause of death following extended liver resection, have restricted the population of eligible patients. Here, we engineered a clinical-grade bioartificial liver (BAL) device employing human-induced hepatocytes (hiHeps) manufactured under GMP conditions. In a porcine PHLF model, the hiHep-BAL treatment showed a remarkable survival benefit. On top of the supportive function, hiHep-BAL treatment restored functions, specifically ammonia detoxification, of the remnant liver and facilitated liver regeneration. Notably, an investigator-initiated study in seven patients with extended liver resection demonstrated that hiHep-BAL treatment was well tolerated and associated with improved liver function and liver regeneration, meeting the primary outcome of safety and feasibility. These encouraging results warrant further testing of hiHep-BAL for PHLF, the success of which would broaden the population of patients eligible for liver resection.

Reversal of liver failure using a bioartificial liver device implanted with clinical-grade human-induced hepatocytes.

Hepatocyte transplantation has the potential to treat acute liver failure and correct liver-based metabolic disorders. Proliferating human hepatocytes (ProliHHs) provide a large-scale source as an alternative to primary human hepatocytes. However, host rejection led to inefficient graft survival and function, which hindered the clinical application of cell therapy. Herein, we employed the lentiviral system to overexpress immunomodulatory factors programmed death-ligand 1 (cluster of differentiation 274) (CD274) and cluster of differentiation 47 (CD47) in ProliHHs. CD47+274 overexpression inhibited macrophage and T cell responses in vitro. After transplantation into mice via the spleen without immunosuppression, CD47+274 ProliHHs accumulation in the liver significantly increased for 48 hours compared with ProliHHs. Consistent with the in vitro results, CD47+274 ProliHHs were less aggregated and infiltrated by macrophages and also recruited fewer T cells in the liver. Seven days after transplantation, the human albumin level of engineered ProliHHs doubled compared with control group. CD47+274 ProliHHs further ameliorated the liver injury induced using concanavalin A. Overall, our results suggested CD47+274 overexpression reduced innate and adaptive immune responses during hepatocyte transplantation, and the survival rate and graft function of transplanted hepatocyte-like cells were all significantly improved.

CD47 and PD-L1 overexpression in proliferating human hepatocytes attenuated immune responses and ameliorated acute liver injury in mice.

Numerous studies have shown that hepatocyte transplantation is a promising approach for liver diseases, such as liver-based metabolic diseases and acute liver failure. However, it lacks strong evidence to support the long-term therapeutic effects of hepatocyte transplantation in clinical practice. Currently, major hurdles include availability of quality-assured hepatocytes, efficient engraftment and repopulation, and effective immunosuppressive regimens. Notably, cell sources have been advanced recently by expanding primary human hepatocytes by means of dedifferentiation in vitro. Moreover, the transplantation efficiency was remarkably improved by the established preparative hepatic irradiation in combination with hepatic mitogenic stimuli regimens. Finally, immunosuppression drugs, including glucocorticoid and inhibitors for co-stimulating signals of T cell activation, were proposed to prevent innate and adaptive immune rejection of allografted hepatocytes. Despite remarkable progress, further studies are required to improve in vitro cell expansion technology, develop clinically feasible preconditioning regimens, and further optimize immunosuppression regimens or establish ex vivo gene correction-based autologous hepatocyte transplantation.

Hepatocyte transplantation: The progress and the challenges.

Alginate-encapsulated hepatocyte transplantation is a promising strategy to treat liver failure. However, its clinical application was impeded by the lack of primary human hepatocytes and difficulty in controlling their quality. We previously reported proliferating human hepatocytes (ProliHHs). Here, quality-controlled ProliHHs were produced in mass and engineered as liver organoids to improve their maturity. Encapsulated ProliHHs liver organoids (eLO) were intraperitoneally transplanted to treat liver failure animals. Notably, eLO treatment increased the survival of mice with post-hepatectomy liver failure (PHLF) and ameliorated hyperammonemia and hypoglycemia by providing liver functions. Additionally, eLO treatment protected the gut from PHLF-augmented permeability and normalized the increased serum endotoxin and inflammatory response, which facilitated liver regeneration. The therapeutic effect of eLO was additionally proved in acetaminophen-induced liver failure. Furthermore, we performed assessments of toxicity and biodistribution, demonstrating that eLO had no adverse effects on animals and remained non-tumorigenic.

Preclinical efficacy and safety of encapsulated proliferating human hepatocyte organoids in treating liver failure.

China has a high prevalence of liver diseases, with about 300 million patients suffering from various types of liver diseases, where the incidence of severe liver diseases is 1%-3%. More than half a million people die from end-stage liver disease every year in China. In situ liver transplantation is the most effective treatment for liver failure, but only a limited number of patients undergo liver transplantation due to the shortage of donors. Hepatocyte transplantation requires only a certain number of hepatocytes rather than the whole liver, regardless of complex issues such as in vitro reconstruction of the three-dimensional structure of the liver, blood vessels and biliary ducts, enabling it to be safer and easier to promote in clinical practice than liver transplantation. This study aims to evaluate the safety and tolerability of microencapsulated hepatocyte intraperitoneal transplantation therapy in adult patients with liver failure.

Phase I safety and tolerability dose escalation study of microencapsulated hepatocyte intraperitoneal transplantation therapy in adult patients with liver failure: a study protocol.

Article	Year
Distinct Gene Expression and Epigenetic Signatures in Hepatocyte-like Cells Produced by Different Strategies from the Same Donor. Stem cell reports\| PubMed ID: 29173899	2017
Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats. The Journal of biological chemistry\| PubMed ID: 29507093	2018
In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity. Cell stem cell\| PubMed ID: 30416071	2018
Conversion of Fibroblasts to Hepatocytes In Vitro. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30536093	2019
Large-scale Generation of Functional and Transplantable Hepatocytes and Cholangiocytes from Human Endoderm Stem Cells. Cell reports\| PubMed ID: 33296648	2020
Survival-Assured Liver Injury Preconditioning (SALIC) Enables Robust Expansion of Human Hepatocytes in Fah Rag2 IL2rg Rats. Advanced science (Weinheim, Baden-Wurttemberg, Germany)\| PubMed ID: 34382351	2021
Roles of extracellular vesicles in the aging microenvironment and age-related diseases. Journal of extracellular vesicles\| PubMed ID: 34609061	2021
Single cell Raman spectroscopy to identify different stages of proliferating human hepatocytes for cell therapy. Stem cell research & therapy\| PubMed ID: 34717753	2021
Characteristics of SOX9-positive progenitor-like cells during cholestatic liver regeneration in biliary atresia. Stem cell research & therapy\| PubMed ID: 35313986	2022
Reversal of liver failure using a bioartificial liver device implanted with clinical-grade human-induced hepatocytes. Cell stem cell\| PubMed ID: 37059100	2023
CD47 and PD-L1 overexpression in proliferating human hepatocytes attenuated immune responses and ameliorated acute liver injury in mice. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons\| PubMed ID: 37532180	2023
Hepatocyte transplantation: The progress and the challenges. Hepatology communications\| PubMed ID: 37695736	2023
Preclinical efficacy and safety of encapsulated proliferating human hepatocyte organoids in treating liver failure. Cell stem cell\| PubMed ID: 38458193	2024
Phase I safety and tolerability dose escalation study of microencapsulated hepatocyte intraperitoneal transplantation therapy in adult patients with liver failure: a study protocol. BMJ open\| PubMed ID: 40233953	2025

Ludi Zhang

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