Differentiation of bone marrow-derived mesenchymal stem cells into hepatocyte-like cells in an alginate scaffold

Objectives: Alginate scaffolds are the most frequently investigated biomaterials in tissue engineering. Tissue engineering techniques that generate liver tissue have become important for treatment of a number of liver diseases and recent studies indicate that bone marrow‐derived stem cells (BMSCs) can differentiate into hepatocyte‐like cells. The goal of the study described here, was to examine in vitro hepatic differentiation potential of BMSCs cultured in an alginate scaffold. Materials and methods: To investigate the potential of BMSCs to differentiate into hepatocyte‐like cells, we cultured BMSCs in alginate scaffolds in the presence of specific growth factors including hepatocyte growth factor, epidermal growth factor and fibroblast growth factor‐4. Results: We can demonstrate that alginate scaffolds are compatible for growth of BMSCs and when cultured in alginate scaffolds for several days they display several liver‐specific markers and functions. Specifically, they expressed genes encoding alpha‐foetoprotein, albumin (ALB), connexin 32 and CYP7A1. In addition, these BMSCs produced both ALB and urea, expressed cytokeratin‐18 (CK‐18) and were capable of glycogen storage. Percentage of CK‐18 positive cells, a marker of hepatocytes, was 56.7%. Conclusions: Our three‐dimensional alginate scaffolds were highly biocompatible with BMSCs. Furthermore, culturing induced their differentiation into hepatocyte‐like cells. Therefore, BMSCs cultured in alginate scaffolds may be applicable for hepatic tissue engineering.     

Clonal mesenchymal stem cells derived from human bone marrow can differentiate into hepatocyte‐like cells in injured livers of SCID mice

There is increasing evidence that human mesenchymal stem cells (hMSCs) can be a valuable, transplantable source of hepatocytes. Most of the hMSCs preparations used in these studies were likely heterogeneous cell populations, isolated by adherence to plastic surfaces or by density gradient centrifugation. Therefore, the participation of other unknown trace cell populations cannot be rigorously discounted. Here we report the isolation and establishment of a cloned human MSC line (chMSC) from human bone marrow primary culture, through which we confirmed the hepatic differentiation capability of authentic hMSCs. chMSCs expressed markers of mesenchymal cells, but not markers of hematopoietic stem cells. In vitro, chMSCs can differentiate into either mesenchymal cells or cells exhibiting hepatocyte‐like phenotypes. When transplanted intrasplentically into carbon tetrachloride‐injured livers of SCID mice, EGFP‐tagged chMSCs engrafted into the host liver parenchyma, exhibited typical hepatocyte morphology, form a three‐dimensional architecture, and differentiate into hepatocyte‐like cells expressing human albumin and α‐1‐anti‐trypsin. By confocal microscopy, ultrafine intercellular nanotubular structures were visible between adjacent transplanted and host hepatocytes. We postulate that these structures may assist in the phenotype conversion of chMSCs, possibly by exchange of cytoplasmic components between native hepatocytes and transplanted cells. Thus, a clonal pure population of hMSCs, which can be expanded in culture, may have potential as a cellular source for substitution damaged cells in hepatic injury. J. Cell. Biochem. 108: 693–704, 2009. © 2009 Wiley‐Liss, Inc.     

An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13^highCD133⁺ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.     

Human amniotic fluid-derived stem cells can differentiate into hepatocyte-like cells in vitro and in vivo

Although human amniotic fluid is an attractive source of multipotent stem cells, the potential of amniotic fluid stem cells (AFSCs) to differentiate into hepatic cells has not been extensively evaluated. In this study, we examined whether human AFSCs can differentiate into a hepatic cell lineage in vitro and in vivo. After being treated with cytokines (fibroblast growth factor 4, basic fibroblast growth factor, hepatocyte growth factor, and oncostatin), AFSCs developed a morphology similar to that of hepatocytes. RT-PCR and immunofluorescence analysis showed that the treated AFSCs expressed the hepatocyte-specific markers albumin, cytokeratin 18, and alpha-fetoprotein. The differentiated cells also developed hepatocyte-specific functions, i.e., they secreted albumin, absorbed indocyanine green, and stored glycogen. When transplanted into CCl(4)-injured immunodeficient mice, undifferentiated AFSCs were integrated into the liver tissue, and they expressed markers characteristic of mature human hepatocytes. Although integration of AFSCs into the liver was limited (0.1-0.3% of hepatocytes), histological analysis showed that the recipient mice recovered more rapidly from CCl(4) injury than CCl(4)-injured mice that did not receive AFSCs. AFSCs can differentiate into hepatocyte-like cells in vitro and in vivo and can represent an easily accessible source of progenitor cells for hepatocyte regeneration and liver cell transplantation.     

Human liver model systems in a dish

The human adult liver has a multi‐cellular structure consisting of large lobes subdivided into lobules containing portal triads and hepatic cords lined by specialized blood vessels. Vital hepatic functions include filtering blood, metabolizing drugs, and production of bile and blood plasma proteins like albumin, among many other functions, which are generally dependent on the location or zone in which the hepatocyte resides in the liver. Due to the liver's intricate structure, there are many challenges to design differentiation protocols to generate more mature functional hepatocytes from human stem cells and maintain the long‐term viability and functionality of primary hepatocytes. To this end, recent advancements in three‐dimensional (3D) stem cell culture have accelerated the generation of a human miniature liver system, also known as liver organoids, with polarized epithelial cells, supportive cell types and extra‐cellular matrix deposition by translating knowledge gained in studies of animal organogenesis and regeneration. To facilitate the efforts to study human development and disease using in vitro hepatic models, a thorough understanding of state‐of‐art protocols and underlying rationales is essential. Here, we review rapidly evolving 3D liver models, mainly focusing on organoid models differentiated from human cells.     

Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo

Embryonic stem cells (ES cells), bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and hepatic stem cells in liver have been known as a useful source that can induce to differentiate into hepatocytes. In this study, we examined whether human adipose tissue-derived stromal cells (hADSC) can differentiate into hepatic lineage in vitro. hADSC, that were induced to differentiate into hepatocyte-like cells by the treatment of HGF and OSM, had morphology similar to hepatocytes. Addition of DMSO enhanced differentiation into hepatocytes. RT-PCR and immunocytochemical analysis showed that hADSC express albumin and alpha-fetoprotein during differentiation. Differentiated hADSC showed LDL uptake and production of urea. Additionally, transplanted hADSC to CCl4-injured SCID mouse model were able to be differentiated into hepatocytes and they expressed albumin in vivo. Mesenchymal stem cells isolated from human adipose tissue are immunocompatible and are easily isolated. Therefore, hADSC may become an alternative source to hepatocyte regeneration or liver cell transplantation.     

Differentiation of human embryonic stem cells to hepatocyte-like cells on a new developed xeno-free extracellular matrix

Human embryonic stem cells (hESCs) provide a new source for hepatocyte production in translational medicine and cell replacement therapy. The reported hESC-derived hepatocyte-like cells (HLCs) were commonly generated on Matrigel, a mouse cell line-derived extracellular matrix (ECM). Here, we performed the hepatic lineage differentiation of hESCs following a stepwise application of growth factors on a newly developed serum- and xeno-free, simple and cost-benefit ECM, designated “RoGel,” which generated from a modified conditioned medium of human fibroblasts. In comparison with Matrigel, the differentiated HLCs on both ECMs expressed similar levels of hepatocyte-specific genes, secreted α-fetoprotein, and metabolized ammonia, showed glycogen storage activity as well as low-density lipoprotein and indocyanine green uptake. The transplantation of hESC–HLCs into the carbon tetrachloride-injured liver demonstrated incorporation of the cells into the host mouse liver and the expression of albumin. The results suggest that the xeno-free and cost-benefit matrix may be applicable in bioartificial livers and also may facilitating a clinical application of human pluripotent stem cell-derived hepatocytes in the future.     

Establishment and characterization of hepatic stem-like cell lines from normal adult rat liver

The liver is a unique organ with the potential to regenerate from injury. Hepatic stem cells contribute to liver regeneration when surviving hepatocytes in injured liver are unable to proliferate. To investigate the mechanism of liver regeneration in vitro, we established hepatic stem cell lines named HY1, HY2 and HY3, derived from a healthy liver of adult rat. HY cells showed an expression pattern similar to oval cells, and efficiently induced hepatic differentiation following sequential treatment with type I collagen, transforming growth factor-beta1 (TGF-beta1), and hepatocyte growth factor (HGF) or oncostatin M (OSM). These results suggested that HY cells are liver stem cells representing an excellent tool for in vitro studies on liver regeneration.     

Hepatogenic differentiation of mesenchymal stem cells induced by insulin like growth factor-I

AIM:To improve hepatic differentiation of human mesenchymal stem cell(MSC)using insulin growth factor 1(IGF-Ⅰ),which has important role in liver development,hepatocyte differentiation and function.METHODS:Bone marrow of healthy donors was aspirated from the iliac crest.The adherent cells expanded rapidly and were maintained with periodic passages until a relatively homogeneous population was established.The identification of these cells was carried out by immunophenotype analysis and differentiation potential into osteocytes and adipocytes.To effectively induce hepatic differentiation,we designed a protocol based on a combination of IGF-Ⅰ and liver specificfactors(hepatocyte growth factor,oncostatin M and dexamethasone).Morphological features,hepatic functions and cytological staining were assessed to evaluate transdifferentiation of human marrow-derived MSCs.RESULTS:Flow cytometric analysis and the differentiation potential into osteoblasts and adipocytes showed that more than 90% of human MSCs which were isolated and expanded were positive by specif ic markers and functional tests.Morphological assessment and evaluation of glycogen storage,albumin and α-feto protein expression,as well as albumin and urea secretion revealed a statistically signif icant difference between the experimental groups and control.CONCLUSION:In vitro differentiated MSCs using IGF-Ⅰwere able to display advanced liver metabolic functions,supporting the possibility of developing them as potential alternatives to primary hepatocytes.     

Generation,characterization and potential therapeutic applications of mature and functional hepatocytes from stem cells

Liver cancer is the sixth most common tumor in the world and the majority of patients with this disease usually die within 1 year. The effective treatment for end‐stage liver disease (also known as liver failure), including liver cancer or cirrhosis, is liver transplantation. However, there is a severe shortage of liver donors worldwide, which is the major handicap for the treatment of patients with liver failure. Scarcity of liver donors underscores the urgent need of using stem cell therapy to the end‐stage liver disease. Notably, hepatocytes have recently been generated from hepatic and extra‐hepatic stem cells. We have obtained mature and functional hepatocytes from rat hepatic stem cells. Here, we review the advancements on hepatic differentiation from various stem cells, including hepatic stem cells, embryonic stem cells, the induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, and probably spermatogonial stem cells. The advantages, disadvantages, and concerns on differentiation of these stem cells into hepatic cells are highlighted. We further address the methodologies, phenotypes, and functional characterization on the differentiation of numerous stem cells into hepatic cells. Differentiation of stem cells into mature and functional hepatocytes, especially from an extra‐hepatic stem cell source, would circumvent the scarcity of liver donors and human hepatocytes, and most importantly it would offer an ideal and promising source of hepatocytes for cell therapy and tissue engineering in treating liver disease. J. Cell. Physiol. 228: 298–305, 2013. © 2012 Wiley Periodicals, Inc.     

Simulated microgravity promoted differentiation of bipotential murine oval liver stem cells by modulating BMP4/Notch1 signaling

Faster growth and differentiation of liver stem cells to hepatocyte is one of the key factors during liver regeneration. In recent years, simulated microgravity, a physical force has shown to differentially regulate the differentiation and proliferation of stem cells. In the present work, we studied the effect of simulated microgravity on differentiation and proliferation of liver stem cells. The cells were subjected to microgravity, which was simulated using indigenously fabricated 3D clinostat. Proliferation, apoptosis, immunofluorescence assays and Western blot analysis were carried out to study the effects of simulated microgravity on liver stem cells. Microgravity treatment for 2 h enhanced proliferation of stem cells by twofold without inducing apoptosis and compromising cell viability. Analysis of hepatocyte nuclear factor 4‐α (HNF4‐α) expression after 2 h of microgravity treatment revealed that microgravity alone can induce the differentiation of stem cells within 2–3 days. Probing bone morphogenic protein 4 (BMP4) and Notch1 in microgravity treated stem cells elaborated downregulation of Notch1 and upregulation of BMP4 after 2 days of incubation. Further, blocking BMP4 using dorsomorphin and chordin conditioned media from chordin plasmid transfected cells attenuated microgravity mediated differentiation of liver stem cells. In conclusion, microgravity interplays with BMP4/Notch1 signaling in stem cells thus inducing differentiation of stem cells to hepatocytes. Present findings can be implicated in clinical studies where microgravity activated stem cells can regenerate the liver efficiently after liver injury. J. Cell. Biochem. 112: 1898–1908, 2011. © 2011 Wiley‐Liss, Inc.     

Characterization and hepatogenic differentiation of mesenchymal stem cells from human amniotic fluid and human bone marrow: a comparative study

Since stem cells can differentiate into hepatocyte, stem cell-based therapy becomes a potential alternative treatment for terminal liver diseases. However, an appropriate source of human mesenchymal stem cells (hMSCs) for hepatocytes has not yet been clearly elucidated. The aim of the present study was to investigate the in vitro biological characterization and hepatic differentiation potential of human amniotic fluid-derived mesenchymal stem cells (AF-hMSCs) and human bone marrow-derived mesenchymal stem cells (BM-hMSCs). Our results show that AF-hMSCs possess higher proliferation and self-renewal capacity than BM-hMSCs. Cytogenetic studies indicate that AF-hMSCs are as genetically stabile as BM-hMSCs. Following incubation with specific hepatogenic agents, AF-hMSCs showed a higher hepatic differentiation potential than BM-hMSCs. Expression of several liver-specific markers was significantly greater in AF-hMSCs than in BM-hMSCs, as shown by real time RT-PCR and immunofluorescence (IF). In conclusion, AF-hMSCs possess superior potential for hepatic differentiation, making them more suitable for diverse terminal liver diseases.     

Effects of extracellular matrixes and growth factors on the hepatic differentiation of human embryonic stem cells

Hepatocytes derived from human embryonic stem cells (hESCs) are a potential cell source for regenerative medicine. However, the definitive factors that are responsible for hepatic differentiation of hESCs remain unclear. We aimed to evaluate the effects of various extracellular matrixes and growth factors on endodermal differentiation and to optimize the culture conditions to induce hepatic differentiation of hESCs. The transgene vector that contained enhanced green fluorescent protein (EGFP) under the control of human alpha-fetoprotein (AFP) enhancer/promoter was transfected into hESC lines. The transgenic hESCs were cultured on extracellular matrixes (collagen type I, laminin, and Matrigel) in the presence or absence of growth factors including hepatocyte growth factor (HGF), bone morphogenetic protein 4, fibroblast growth factor 4, all-trans-retinoic acid, and activin A. The expression of AFP-EGFP was measured by flow cytometry. The culture on Matrigel-coated dishes with 100 ng/ml activin A showed 19.5% of EGFP-positive proportions. Moreover, the sequential addition of 100 ng/ml activin A and 20 ng/ml HGF resulted in 21.7% and produced a higher yield of EGFP-positive cells than the group stimulated by activin A alone. RT-PCR and immunocytochemical staining revealed these EGFP-positive cells to differentiate into mesendoderm-like cells by use of activin A and then into hepatic endoderm cells by use of HGF. Two other hESC lines also differentiated into endoderm on the hepatic lineage by our method. In conclusion, we therefore found this protocol to effectively differentiate multiple hESC lines to early hepatocytes using activin A and HGF on Matrigel.     

Signalling pathways involved in the process of mesenchymal stem cells differentiating into hepatocytes

End‐stage liver disease can be the termination of acute or chronic liver diseases, with manifestations of liver failure; transplantation is currently an effective treatment for these. However, transplantation is severely limited due to the serious lack of donors, expense, graft rejection and requirement of long‐term immunosuppression. Mesenchymal stem cells (MSCs) have attracted considerable attention as therapeutic tools as they can be obtained with relative ease and expanded in culture, along with features of self‐renewal and multidirectional differentiation. Many scientific groups have sought to use MSCs differentiating into functional hepatocytes to be used in cell transplantation with liver tissue engineering to repair diseased organs. In most of the literature, hepatocyte differentiation refers to use of various additional growth factors and cytokines, such as hepatocyte growth factor (HGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), oncostatin M (OSM) and more, and most are involved in signalling pathway regulation and cell–cell/cell–matrix interactions. Signalling pathways have been shown to play critical roles in embryonic development, tumourigenesis, tumour progression, apoptosis and cell‐fate determination. However, mechanisms of MSCs differentiating into hepatocytes, particularly signalling pathways involved, have not as yet been completely illustrated. In this review, we have focused on progress of signalling pathways associated with mesenchymal stem cells differentiating into hepatocytes along with the stepwise differentiation procedure.     

胚胎干细胞向肝实质细胞体外定向诱导分化过程中的肝卵圆细胞

如果肝脏严重受损致使肝细胞大部分坏死,或由于某些原因 ( 肝毒性物质、致癌物质的作用 ) 抑制残存肝细胞增殖时,肝内前体／干细胞———肝卵圆细胞便被激活并分化生成肝细胞和胆管细胞等以参与肝修复 . 基于此理论,人们建立了啮齿类动物肝卵圆细胞诱导实验模型 . 但显然上述模型不适用于人类,所以有必要开发一种适用于人类的、高效的肝卵圆细胞的新诱导模型 . 选用小鼠胚胎干细胞,转成拟胚体分化 3 天后分组,诱导组添加肝细胞生长因子 (HGF) 、表皮生长因子 (EGF) 作定向诱导分化 . 其间用免疫细胞化学 (ICC) 检测肝卵圆细胞标志物 A6 等的表达,用流式细胞仪筛选肝卵圆细胞并行 RT-PCR 、透射电镜检测 . 所筛选的肝卵圆细胞进一步体外培养并进行 ICC 和 RT-PCR ,检测其分化生成成熟的肝细胞和胆管细胞的能力 . 研究证实胚胎干细胞体外定向诱导生成肝实质细胞的过程中,存在着有双向分化能力的肝卵圆细胞这个中间分化阶段 . 诱导组肝卵圆细胞分化率均显著地高于对照组,最高时可达 6.11% 左右 . HGF 和 EGF 能显著性诱导胚胎干细胞源性卵圆细胞的生成 . 流式细胞仪筛选 Sca-1+/CD34+ 细胞占总细胞数的 4.59% ,其中 A6 阳性肝卵圆细胞占 90.81% 左右 . 使用流式细胞仪可获得高富集的 A6+/Sca-1+/CD34+ 肝卵圆细胞 . 提供了一种可适用于人类的肝卵圆细胞的新诱导模型 .