Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an efficient three-step protocol

Liver transplantation is the only definitive treatment for end-stage cirrhosis and fulminant liver failure, but the lack of available donor livers is a major obstacle to liver transplantation. Recently, induced pluripotent stem cells (iPSCs) derived from the reprogramming of somatic fibroblasts, have been shown to resemble embryonic stem (ES) cells in that they have pluripotent properties and the potential to differentiate into all cell lineages in vitro, including hepatocytes. Thus, iPSCs could serve as a favorable cell source for a wide range of applications, including drug toxicity testing, cell transplantation, and patient-specific disease modeling. Here, we describe an efficient and rapid three-step protocol that is able to rapidly generate hepatocyte-like cells from human iPSCs. This occurs because the endodermal induction step allows for more efficient and definitive endoderm cell formation. We show that hepatocyte growth factor (HGF), which synergizes with activin A and Wnt3a, elevates the expression of the endodermal marker Foxa2 (forkhead box a2) by 39.3% compared to when HGF is absent (14.2%) during the endodermal induction step. In addition, iPSC-derived hepatocytes had a similar gene expression profile to mature hepatocytes. Importantly, the hepatocyte-like cells exhibited cytochrome P450 3A4 (CYP3A4) enzyme activity, secreted urea, uptake of low-density lipoprotein (LDL), and possessed the ability to store glycogen. Moreover, the hepatocyte-like cells rescued lethal fulminant hepatic failure in a nonobese diabetic severe combined immunodeficient mouse model. Conclusion: We have established a rapid and efficient differentiation protocol that is able to generate functional hepatocyte-like cells from human iPSCs. This may offer an alternative option for treatment of liver diseases.     

Hepatocyte differentiation from embryonic stem cells and umbilical cord blood cells

With the development of regeneration medicine, many researchers have attempted hepatic differentiation from nonhepatic-origin cell sources. The differentiation of embryonic stem (ES) cells into hepatocyte-like cells has been reported in several papers. Mouse ES cells have shown a potential to develop into hepatocyte-like cells in vitro on the basis of hepatic gene expression after adding several growth factors. We transplanted cultured embryoid body (EB) cells (male) into female mice. A liver specimen of the recipient was examined by immunohistochemical staining for albumin and fluorescence in situ hybridization for the Y chromosome after transplantation. Both Y chromosome- and albumin-positive cells were recognized in the recipient female liver, and were considered to be hepatocyte-like cells derived from ES cells containing the Y chromosome. Many groups, including ourselves, have studied hepatocyte-like cell differentiation from umbilical cord blood cells (UBCs). We cultured nucleated cells isolated from UBCs. Using immunostaining, ALB-positive and CK-19-positive cells were recognized in the culture. Dual staining of ALB and CK-19 demonstrated that ALB was coexpresed with CK-19, suggesting the existence of hepatic progenitors. In this review, we consider recent studies of the differentiation of hepatocytes from nonhepatic origins, especially ES cells and umbilical cord blood.     

内脏内胚层样END-2细胞体外诱导鼠胚胎干细胞分化为心肌细胞的实验研究

目的探讨内脏内胚层样END-2细胞体外诱导胚胎干细胞（embryon ic stem cells,ESCs）分化为心肌细胞的特征。方法用鼠胚胎成纤维细胞（mouse embryon ic fibrob lasts,MEF）作为饲养层促进ESCs增殖并抑制其分化,先将ESCs悬浮培养形成23 d的拟胚体（embryoid bod ies,EBs）,再和END-2细胞共培养诱导向心肌细胞分化。实验分4组。第1,2组EBs分别和END-2细胞或END-2细胞条件培养液共培养;第3组EBs和表面铺有一层琼脂糖的END-2细胞共培养;第4组自然分化组为对照组。相差显微镜下观察分化细胞的形态学变化,免疫细胞荧光技术检测心肌细胞特异性肌钙蛋白T（TnT）的表达;透射电镜观察分化心肌细胞的超微结构。结果各实验组均可见自发节律性收缩的拟胚体。随着培养的延长,自发节律性收缩的拟胚体数目也增加,均表达心肌细胞特异性蛋白TnT,以及观察到心肌样超微结构。在和END-2细胞直接接触的诱导条件下,分化的细胞形态较单一。结论END-2细胞通过分泌可溶性细胞因子可诱导ESCs向心肌细胞分化,直接接触在END-2细胞诱导作用中并不是必要的,但可诱导出较单一的细胞。     

Thinking outside the liver: Induced pluripotent stem cells for hepatic applications

The discovery of induced pluripotent stem cells (iPSCs) unraveled a mystery in stem cell research, after identification of four re-programming factors for generating pluripotent stem cells without the need of embryos. This breakthrough in generating iPSCs from somatic cells has overcome the ethical issues and immune rejection involved in the use of human embryonic stem cells. Hence, iPSCs form a great potential source for developing disease models, drug toxicity screening and cell-based therapies. These cells have the potential to differentiate into desired cell types, including hepatocytes, under in vitro as well as under in vivo conditions given the proper microenvironment. iPSC-derived hepatocytes could be useful as an unlimited source, which can be utilized in disease modeling, drug toxicity testing and producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to cell transplantation. In this review, we discuss the induction methods, role of reprogramming factors, and characterization of iPSCs, along with hepatocyte differentiation from iPSCs and potential applications. Further, we discuss the location and detection of liver stem cells and their role in liver regeneration. Although tumor formation and genetic mutations are a cause of concern, iPSCs still form a promising source for clinical applications.     

In vitro differentiation of mouse bone marrow mononuclear cells into hepatocyte-like cells.

AIM:: It is imperative to explore some ways to gain the functional hepatocytes for hepatocyte transplantation. Bone marrow stem cells can differentiate into hepatocytes in vivo and in vitro. We select fibroblast growth factor-4 (FGF-4), oncostatin M (OSM), hepatocyte growth factor (HGF) and epithermal growth factor (EGF) as differentiation factors, and design the appropriate directed differentiation medium in order to gain hepatocytes through directed differentiation of bone marrow stem cells. METHODS:: Bone marrow mononuclear cells (BMMCs) were cultured in the directed differentiation media including FGF-4, OSM, HGF and EGF. In the course of cell differentiation, cell morphology was observed, and the expression patterns of some genes of the hepatocyte were validated and confirmed by RT-PCR. The ALB-, and CK18-expressed cells were gone further step to be confirmed by Western blot analysis, immunofluorescence and flow cytometric analysis. Hepatocyte functional activity, including glycogen synthesis and urea production, were confirmed by periodic acid-Shiff (PAS) staining and urea assay. RESULTS:: Some epithelial-like cells or polygonal cells appeared in the directed differentiation medium within 12 days, and the number and sizes of colonies of epithelial-like cells or polygonal cells increased in the course of the cell directed differentiation. AFP, HNF-3ss, ALB and CK18 mRNA expressions first appeared within day 7, and lasted throughout the later directed differentiation. TTR, G-6-P and TAT mRNA expressions could be detected within day 14, and their expressions lasted in the course of the later directed differentiation. ALB and CK18 were confirmed to exist in the differentiated BMMCs by Western blot analysis. ALB was found in the cytoplasm and cell membrane, while CK18 scattered in the cytoplasm by immunofluorescent staining. On day 21,the ratio of ALB-positive cells was 69.45%, and the ratio of CK18-positive cells was 67.36%. The accumulation of glucogen was detected in the cytoplasm of the differentiated cells. The directed differentiated BMMCs produced urea 3 days later, and they produced urea in a time-dependent manner. CONCLUSIONS:: BMMCs could differentiate into hepatocytes or hepatocyte-like cells in the differentiation media including HGF, FGF-4, EGF, and OSM. These hepatocyte-like cells were identified at the gene level and protein level. Furthermore, these hepatocyte-like cells had some hepatocellular synthesis and metabolism functions.     

Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver

We established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate injured liver. Using mouse ES cells transfected with the green fluorescent protein (GFP) reporter gene regulated by albumin (ALB) enhancer/promoter, we found that a serum-free chemically defined medium supports formation of embryoid bodies (EBs) and differentiation of hepatic lineage cells in the absence of exogenous growth factors or feeder cell layers. The first GFP+ cells expressing ALB were detected in close proximity to "beating" myocytes after 7 days of EB cultures. GFP+ cells increased in number, acquired hepatocyte-like morphology and hepatocyte-specific markers (i.e., ALB, AAT, TO, and G6P), and by 28 days represented more than 30% of cells isolated from EB outgrowths. The FACS-purified GFP+ cells developed into functional hepatocytes without evidence of cell fusion and participated in the repairing of diseased liver when transplanted into MUP-uPA/SCID mice. The ES cell-derived hepatocytes were responsive to normal growth regulation and proliferated at the same rate as the host hepatocytes after an additional growth stimulus from CCl(4)-induced liver injury. The transplanted GFP+ cells also differentiated into biliary epithelial cells. In conclusion, a highly enriched population of committed hepatocyte precursors can be generated from ES cells in vitro for effective cell replacement therapy.     

大鼠诱导多能干细胞向肝系细胞的诱导分化

目的本研究探讨利用诱导因子将大鼠诱导多能干细胞(induced pluripotent stem cells,iPSCs)快速高效诱导分化为肝细胞样细胞(hepatocyte-like cells,HLCs)。方法利用诱导因子将iPSCs向肝系细胞方向分化,采用免疫细胞化学染色和RT-PCR方法检测培养20 d时细胞各种肝系细胞标志表达。结果培养分化20 d的iPSCs形成肝细胞样集落,表达肝系细胞特征性标志,并且具有尿素合成和糖原贮备能力。结论利用诱导因子可有效地将大鼠iPSCs诱导分化为HLCs,有望为细胞移植治疗各种肝脏疾病提供种子细胞。     

HNF-4α determines hepatic differentiation of human mesenchymal stem cells from bone marrow

AIM: To investigate the differentiation status and key factors to facilitate hepatic differentiation of human bone-marrow-derived mesenchymal stem cells (MSCs). METHODS: Human MSCs derived from bone marrow were induced into hepatocyte-like cells following a previously published protocol. The differentiation status of the hepatocyte-like cells was compared with various human hepatoma cell lines. Overexpression of hepatocyte nuclear factor (HNF)-4α was mediated by adenovirus infection of these hepatocyte-like...     

Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes

Embryonic stem (ES) cells have a potential to differentiate into various progenitor cells. Here we investigated the differentiation capacity of mouse ES cells into hepatocytes both in vitro and in vivo. During the culture of embryoid bodies (EBs) derived from ES cells, albumin (ALB) messenger RNA (mRNA) was expressed within 12 days after removal of leukemia inhibitory factor, and alpha-fetoprotein (AFP) mRNA was observed within 9 days without additional exogenous growth factors. In ES cells and early EBs, by contrast, neither ALB mRNA nor AFP mRNA was observed. ALB protein was first detected at day 15 and the level increased with the culture period. The differentiation of EBs facilitated the synthesis of urea with the culture period, whereas early EBs and ES cells produced no urea. These results suggest that cultured EBs contain hepatocytes capable of producing ALB and urea. ES cells and the isolated cells from EBs were transplanted through portal vein to the liver after 30% partial hepatectomy of female mice pretreated with 2-acetylaminofluorene. Four weeks after transplantation with isolated cells from day-9 EBs, ES-derived cells containing Y-chromosome in the liver were positive for ALB (0.2% of total liver cells), whereas teratoma was found in mice transplanted with ES cells or EBs up to day 6. The incidence of teratoma was decreased with the culture duration and no teratoma was observed in the liver transplanted with isolated cells from day-9 EBs. In conclusion, our in vitro and in vivo experiments revealed that cultured EBs contain functional hepatocytes or hepatocyte-like cells.     

Directed differentiation of human embryonic stem cells into functional hepatic cells

The differentiation capacity of human embryonic stem cells (hESCs) holds great promise for therapeutic applications. We report a novel three-stage method to efficiently direct the differentiation of human embryonic stem cells into hepatic cells in serum-free medium. Human ESCs were first differentiated into definitive endoderm cells by 3 days of Activin A treatment. Next, the presence of fibroblast growth factor-4 and bone morphogenetic protein-2 in the culture medium for 5 days induced efficient hepatic differentiation from definitive endoderm cells. Approximately 70% of the cells expressed the hepatic marker albumin. After 10 days of further in vitro maturation, these cells expressed the adult liver cell markers tyrosine aminotransferase, tryptophan oxygenase 2, phosphoenolpyruvate carboxykinase (PEPCK), Cyp7A1, Cyp3A4 and Cyp2B6. Furthermore, these cells exhibited functions associated with mature hepatocytes including albumin secretion, glycogen storage, indocyanine green, and low-density lipoprotein uptake, and inducible cytochrome P450 activity. When transplanted into CCl4 injured severe combined immunodeficiency mice, these cells integrated into the mouse liver and expressed human alpha-1 antitrypsin for at least 2 months. In addition, we found that the hESC-derived hepatic cells were readily infected by human immunodeficiency virus-hepatitis C virus pseudotype viruses. CONCLUSION: We have developed an efficient way to direct the differentiation of human embryonic stem cells into cells that exhibit characteristics of mature hepatocytes. Our studies should facilitate searching the molecular mechanisms underlying human liver development, and form the basis for hepatocyte transplantation and drug tests.     

Biochemical and molecular characterization of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells on a novel three-dimensional biocompatible nanofibrous scaffold

Background:  There is significant interest in using nanofibers in tissue engineering from stem cells. The transdifferentiation of mesenchymal stem cells into the hepatic lineage in a nanofibrous structure has not been reported. In this study, a three dimensional nanofibrous scaffold is introduced for differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs) into hepatocytes.
Methods:  A scaffold composed of Poly (ε-caprolactone), collagen and polyethersulfone was fabricated by the electrospinning technique. After characterization of isolated hBMSCs, the performance of the cells on the scaffold was evaluated by Scanning Electron Microscopy (SEM) and MTT assay. Cytological, molecular and biochemical markers were measured to confirm differentiation potential of hBMSCs into hepatocytes.
Results:  The isolated cells possessed the basic properties of mesenchymal stem cells (MSCs). Based on scanning electron microscope (SEM) analysis and MTT assay, it was shown that the cells adhere, penetrate and proliferate on the nanofibers. Cultured cells on the nanofibers differentiated into hepatocyte-like cells and expressed hepatocyte specific markers such as albumin, α-fetoprotein, cytokeratin-18, cytokeratin-19 and cytochrome P450 3A4 at mRNA levels. Appearance of a considerable number of albumin-positive cells cultivated on the scaffold (47 ± 4%) as compared to the two-dimensional culture system (28 ± 6%) indicates the supporting role of the scaffold. The efficiency of the cells to produce albumin, urea, transferrin, serum glutamic pyruvic transaminase and serum oxaloacetate aminotransferase in hepatocytes on the scaffold further attest to the functionality of the cells.
Conclusion:  The data presented in this study show that the engineered nanofibrous scaffold is a conductive matrix which supports and enhances MSC development into functional hepatocyte-like cells.     

Cell therapy from bench to bedside: Hepatocytes from fibroblasts - the truth and myth of transdifferentiation

Hepatocyte transplantation is an alternative to liver transplantation in certain disorders such as inherited liver diseases and liver failure. It is a relatively less complicated surgical procedure, and has the advantage that it can be repeated several times if unsuccessful. Another advantage is that hepatocytes can be isolated from partly damaged livers which are not suitable for liver transplantation. Despite these advantages hepatocyte transplantation is less popular. Important issues are poor engraftment of the transplanted cells and the scarcity of donor hepatocytes. Generation of “hepatocyte like cells”/iHeps from embryonic stem cells (ES) and induced pluripotent stem cells (iPSCs) by directed differentiation is an emerging solution to the latter issue. Direct conversation or trans-differentiation of fibroblasts to “hepatocyte like cells” is another way which is, being explored. However this method has several inherent and technical disadvantages compared to the directed differentiation from ES or iPSC. There are several methods claiming to be “highly efficient” for generating “highly functional” “hepatocyte like cells”. Currently different groups are working independently and coming up with differentiation protocols and each group claiming an advantage for their protocol. Directed differentiation protocols need to be designed, compared, analyzed and tweaked systematically and logically than empirically. There is a need for a well-coordinated global initiative comparable to the Human Genome Project to achieve this goal in the near future.     

Origin of hepatocellular carcinoma: role of stem cells

The question of whether hepatocellular carcinoma (HCC) arises from the differentiation block of stem cells or dedifferentiation of mature cells remains controversial. Recently, researchers suggested that HCC may originate from the transdifferentiation of bone marrow cells. Interestingly, there are four levels of cells in the hepatic stem cell lineage: bone marrow cells, hepato-pancreas stem cells, oval cells and hepatocytes. Hematopoietic stem cells and the liver are known to have a close relationship in early development. Bone marrow stem cells could differentiate into oval cells, which could differentiate into hepatocytes and duct cells. The development of pancreatic and liver buds in embryogenesis suggests the existence of a common progenitor cell to both the pancreas and liver. Cellular events during hepatocarcinogenesis illustrate that HCC may arise from cells at various stages of differentiation in the hepatic stem cell lineage.     

Human embryonic stem cells and liver diseases: from basic research to future clinical application

Human embryonic stem cells (hESC) provide access to the earliest stages of human development and because of their high proliferation capability, pluripotency and low immunogenicity may serve as a potential source of specialized cells for regenerative medicine. hESC-derived hepatocyte-like cells exhibit characteristic hepatocyte morphology, express hepatocyte markers and are capable of executing a range of hepatocyte functions. However, there are many challenges and obstacles to be overcome before the use of hESC and hESC-derived hepatocyte-like cells in clinical practice can be realized. Here, we highlight some of the recent efforts in this area, in hope of providing insights toward this complex yet important area of therapeutical modality for treating patients with liver disease.     

In vitro hepatic differentiation of human mesenchymal stem cells

This study examined whether mesenchymal stem cells (MSCs), which are stem cells originated from embryonic mesoderm, are able to differentiate into functional hepatocyte-like cells in vitro. MSCs were isolated from human bone marrow and umbilical cord blood, and the surface phenotype and the mesodermal multilineage differentiation potentials of these cells were characterized and tested. To effectively induce hepatic differentiation, we designed a novel 2-step protocol with the use of hepatocyte growth factor and oncostatin M. After 4 weeks of induction, cuboidal morphology, which is characteristic of hepatocytes, was observed, and cells also expressed marker genes specific of liver cells in a time-dependent manner. Differentiated cells further demonstrated in vitro functions characteristic of liver cells, including albumin production, glycogen storage, urea secretion, uptake of low-density lipoprotein, and phenobarbital-inducible cytochrome P450 activity. In conclusion, human MSCs from different sources are able to differentiate into functional hepatocyte-like cells and, hence, may serve as a cell source for tissue engineering and cell therapy of hepatic tissues. Furthermore, the broad differentiation potential of MSCs indicates that a revision of the definition may be required.     

Progress in stem cell biology in regenerative medicine for liver disease

Regenerative medicine using stem cells has attracted much attention, since stem cells are responsible for highly proliferative activity and multipotential ability of differentiation. Induced pluripotent stem cells and embryonic stem cells or the adult stem cells such as bone marrow-derived stem cells and adipose tissue-derived stem cells have been expected as a cell source of regenerative medicine. Since differentiating methods of human stem cells into the defined lineage of cells remains to be developed, we focus on the differentiating strategies of pluripotent stem cells and mesenchymal stem cells into liver lineage, especially on cytokine function and gene expression during hepatic differentiation. The survey of previously published papers discloses that the protocols that mimic the liver developmental process seem to be effective in obtaining functional hepatocytes. However, in order to develop hepatic regenerative medicine that is useful in a clinical setting, more effective and potent strategies that obtain mature hepatocytes are required.