In vitro cultivation and differentiation of fetal liver stem cells from mice

During embryonic development, pluripotent endoderm tissue in the developing foregut may adopt pancreatic fate or hepatic fate depending on the activation of key developmental regulators. Transdifferentiation occurs between hepatocytes and pancreatic cells under specific conditions. Hepatocytes and pancreatic cells have the common endodermal progenitor cells. In this study we isolated hepatic stem/progenitor cells from embryonic day (ED) 12-14 Kun-Ming mice with fluorescence-activated cell sorting (FACS). The cells were cultured under specific conditions. The cultured cells deploy dithizone staining and immunocytochemical staining at the 15th, 30th and 40th day after isolation. The results indicated the presence of insulin-producing cells. When the insulin-producing cells were transplanted into alloxaninduced diabetic mice, the nonfasting blood glucose level was reduced. These results suggested that fetal liver stem/progenitor cells could be converted into insulin-producing cells under specific culture conditions. Fetal liver stem/progenitor cells could become the potential source of insulin-producing cells for successful cell transplantation therapy strategies of diabetes.     

The role of HGF on invasive properties and repopulation potential of human fetal hepatic progenitor cells

The success of hepatocyte transplantation has been limited by the low efficiency of transplanted cell integration into liver parenchyma. Human fetal hepatic progenitor cells (hepatoblasts) engraft more effectively than adult hepatocytes in mouse livers. However, the signals required for their integration are not yet fully understood. We investigated the role of HGF on the migration and invasive ability of human hepatic progenitors in vitro and in vivo.Hepatoblasts were isolated from the livers of human fetuses between 10 and 12 weeks of gestation. Their invasive ability was assessed in the presence or absence of HGF. These cells were also transplanted into immunodeficient mice and analyzed by immunohistochemistry.In contrast to TNF-alpha, HGF increased the motogenesis and invasiveness of hepatoblasts, but not of human adult hepatocytes, via phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. The invasive ability of human hepatoblasts correlated with the expression and secretion of matrix metalloproteinases (MMPs). Hepatoblasts stimulated with HGF prior transplantation into newborn mice migrated from the portal area into the hepatic parenchyma.Conclusions: In contrast to adult hepatocytes, hepatoblasts display invasive ability that can be modulated by HGF in vitro and in vivo.     

Mouse hepatoblasts at distinct developmental stages are characterized by expression of EpCAM and DLK1: Drastic change of EpCAM expression during liver development

Hepatoblasts are hepatic progenitor cells that expand and give rise to either hepatocyte or cholangiocytes during liver development. We previously reported that delta-like 1 homolog (DLK1) is expressed in the mouse liver primordium at embryonic day (E) 10.5 and that DLK1⁺ cells in E14.5 liver contain high proliferative and bipotential hepatoblasts. While the expression of epithelial cell adhesion molecule (EpCAM) in hepatic stem/progenitor cells has been reported, its expression profile at an early stage of liver development remains unknown. In this study, we show that EpCAM is expressed in mouse liver bud at E9.5 and that EpCAM⁺DLK1⁺ hepatoblasts form hepatic cords at the early stage of hepatogenesis. DLK1⁺ cells of E11.5 liver were fractionated into EpCAM⁺ and EpCAM⁻ cells; one forth of EpCAM⁺DLK1⁺ cells formed a colony in vitro whereas EpCAM⁻DLK1⁺ cells rarely did it. Moreover, EpCAM⁺DLK1⁺ cells contained cells capable of forming a large colony, indicating that EpCAM⁺DLK1⁺ cells in E11.5 liver contain early hepatoblasts with high proliferation potential. Interestingly, EpCAM expression in hepatoblasts was dramatically reduced along with liver development and the colony-forming capacities of both EpCAM⁺DLK1⁺ and EpCAM⁻DLK1⁺ cells were comparable in E14.5 liver. It strongly suggested that most of mouse hepatoblasts are losing EpCAM expression at this stage. Moreover, we provide evidence that EpCAM⁺DLK1⁺ cells in E11.5 liver contain extrahepatic bile duct cells as well as hepatoblasts, while EpCAM⁻DLK1⁺ cells contain mesothelial cell precursors. Thus, the expression of EpCAM and DLK1 suggests the developmental pathways of mouse liver progenitors.     

Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells

Hedgehog (Hh) signaling plays crucial roles in development and homeostasis of various organs. In the adult liver, it regulates proliferation and/or viability of several types of cells, particularly under injured conditions, and is also implicated in stem/progenitor cell maintenance. However, the role of this signaling pathway during the normal developmental process of the liver remains elusive. Although Sonic hedgehog (Shh) is expressed in the ventral foregut endoderm from which the liver derives, the expression disappears at the onset of the liver bud formation, and its possible recurrence at the later stages has not been investigated. Here we analyzed the activation and functional relevance of Hh signaling during the mouse fetal liver development. At E11.5, Shh and an activation marker gene for Hh signaling, Gli1, were expressed in Dlk⁺ hepatoblasts, the fetal liver progenitor cells, and the expression was rapidly decreased thereafter as the development proceeded. In the culture of Dlk⁺ hepatoblasts isolated from the E11.5 liver, activation of Hh signaling stimulated their proliferation and this effect was cancelled by a chemical Hh signaling inhibitor, cyclopamine. In contrast, hepatocyte differentiation of Dlk⁺ hepatoblasts in vitro as manifested by the marker gene expression and acquisition of ammonia clearance activity was significantly inhibited by forced activation of Hh signaling. Taken together, these results demonstrate the temporally restricted manner of Hh signal activation and its role in promoting the hepatoblast proliferation, and further suggest that the pathway needs to be shut off for the subsequent hepatic differentiation of hepatoblasts to proceed normally.     

Wnt antagonist SFRP3 inhibits the differentiation of mouse hepatic progenitor cells

Wnt/β‐catenin pathway plays an important role in regulating embryonic development. Hepatocytes differentiate from endoderm during development. Hepatic progenitor cells (HPCs) have been isolated from fetal liver and extrahepatic tissues. Most current studies in liver development and hepatic differentiation have been focused on Wnts, β‐catenin, and their receptors. Here, we sought to determine the role of Wnt antagonists in regulating hepatic differentiation of fetal liver‐derived HPCs. Using mouse liver tissues derived from embryonic day E12.5 to postnatal day (PD) 28, we found that 13 of the 19 Wnt genes and almost all of Wnt receptors/co‐receptors were expressed in most stages. However, Wnt antagonists SFRP2, SFRP3, and Dkk2 were only detected in the early stages. We established and characterized the reversible stable HPCs derived from E14.5 mouse fetal liver (HP14.5). HP14.5 cells were shown to express high levels of early liver progenitor cell markers, but low levels or none of late liver markers. HP14.5 cells were shown to differentiate into mature hepatocytes upon dexamethasone (Dex) stimulation. Dex‐induced late marker expression and albumin promoter activity in HP14.5 cells were inhibited by exogenous expression of SFRP3. Furthermore, Dex‐induced glycogen synthesis of PAS‐positive HP14.5 cells was significantly inhibited by SFRP3. Therefore, our results have demonstrated that the expression of Wnt antagonists decreases as hepatic differentiation progresses, suggesting that a balanced Wnt signaling may be critical during mouse liver development and hepatic differentiation. J. Cell. Biochem. 108: 295–303, 2009. © 2009 Wiley‐Liss, Inc.     

Embryonic Hematopoietic Progenitor Cells Reside in Muscle before Bone Marrow Hematopoiesis

In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of Gata2, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, exo utero transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.     

New primary culture systems to study the differentiation and proliferation of mouse fetal hepatoblasts

Hepatoblasts have the potential to differentiate into both hepatocytes and biliary epithelial cells through a differentiation program that has not been fully elucidated. With the aim to better define the mechanism of differentiation of hepatoblasts, we isolated hepatoblasts and established new culture systems. We isolated hepatoblasts from E12.5 fetal mouse liver by using E-cadherin. The E-cadherin+ cells expressed alpha-fetoprotein (AFP) and albumin (Alb) but not cytokeratin 19 (CK19). Transplantation of the E-cadherin+ cells into mice that had been subjected to liver injury or biliary epithelial injury led to differentiation of the cells into hepatocytes or biliary epithelial cells, respectively. In a low-cell-density culture system in the absence of additional growth factors, E-cadherin+ cells formed colonies of various sizes, largely comprising Alb-positive cells. Supplementation of the culture medium with hepatocyte growth factor and epidermal growth factor promoted proliferation of the cells. Thus the low-cell-density culture system should be useful to identify inductive factors that regulate the proliferation and differentiation of hepatoblasts. In a high-cell-density system in the presence of oncostatin M+dexamethasone, E14.5, but not E12.5, E-cadherin+ cells differentiated into mature hepatocytes, suggesting that unidentified factors are involved in hepatic maturation. Culture of E-cadherin+ cells derived from E12.5 or E14.5 liver under high-cell-density conditions should allow elucidation of the mechanism of hepatic differentiation in greater detail. These new culture systems should be of use to identify growth factors that induce hepatoblasts to proliferate or differentiate into hepatocytes and biliary epithelial cells.     

TGF-beta-1 up-regulates extra-cellular matrix production in mouse hepatoblasts

Fetal liver is the major embryonic hematopoietic organ and is extrinsically colonized by circulating hematopoietic stem cells (HSCs). Integrin beta-1 expression on HSCs is crucial for colonization, suggesting that interaction of Integrin beta-1 with extra-cellular matrix (ECM) factors promotes HSC adherence to fetal liver. However, little is known about how ECM production is regulated in fetal liver. Here we used flow cytometry to sort fetal liver compartments and detected ECM gene and protein expression predominantly in sorted hepatoblasts. mRNA and protein analysis suggested that TGF-beta-1 expressed by hepatoblasts, sinusoid endothelial cells and hematopoietic cells, binds to the TGF-beta receptor type-2 expressed on hepatoblasts to stimulate ECM production. Intra-cardiac injection of TGF-inhibitors into mouse embryos dramatically decreased fetal liver ECM gene expression. Taken together, our observations suggest that hepatoblasts predominantly produce ECM factors under control of TGF-beta-1 in fetal liver.     

人鼠肝组织嵌合体模型的制备

目的研究制备人鼠肝组织嵌合小鼠模型。方法将人骨髓干细胞直接注射到一定日龄胎鼠肝组织,每只注射移植约1×109人骨髓干细胞。用免疫组化对出生一定日龄移植小鼠肝脏进行甲胎蛋白免疫组织化学检测,检定分析人肝细胞在小鼠体内嵌合生长情况。结果移植人骨髓干细胞胎鼠出生2月龄、12月龄可检测到甲胎蛋白。结论将人骨髓干细胞移植小鼠肝脏内能够存活并分化成人肝细胞并能够长期存活。     

A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver

We previously reported a new in vivo model named as "GFP/CCl(4) model" for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific "niche" made by CCl(4) induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.     

Notch is the key factor in the process of fetal liver stem/progenitor cells differentiation into hepatocytes

Cell transplantation is efficient method to therapy end-stage liver disease (ESLD). How to punctually induce stem cell differentiation into hepatocyte is still a challenge. Notch plays important roles in embryonic development and cell differentiation. However, during the differentiation process from fetal liver stem/progenitor cells (FLSPCs) to mature hepatocytes, the contribution of Notch, especially which Notch receptor is primarily responsible, is unknown. First, specific Notch receptor responsible for FLSPCs differentiation was identified. On both tissue level and cell level, we found that Notch3 was the only receptor greater expressed in liver tissue at embryonic day (ED) 14 and FLSPCs, compared with the adult liver and BRL cells, respectively. Second, morphological phenotypic and functional aspects were analyzed to evaluate whether Notch inhibition by GSIs (γ-secretase inhibitors, inhibitor of Notch) promotes the differentiation of FLSPCs into hepatocytes. Results showed that N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) as GSIs was able to induce FLSPCs differentiation into hepatocytes. The differentiated FLSPCs showed similar morphology to mature hepatocytes, expressed hepatic markers indicative of a mature developmental stage, and displayed similar functionality to mature hepatocytes. The differentiation efficiency by GSIs was similar to that by hepatocyte growth factor (HGF) induction. More specifically, as the differentiation of FLSPCs progressed towards hepatocytes, the expression of Notch3 was gradually down-regulated, consistent with the down-regulation of other stem cell markers. These findings imply that Notch3 may not only be a regulator of FLSPCs differentiation into hepatocytes, but also be a potential marker of FLSPCs.     

Differentiation and Selection of Hepatocyte Precursors in Suspension Spheroid Culture of Transgenic Murine Embryonic Stem Cells

Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for “live” monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in in vitro screening assays.     

The road to regenerative liver therapies: The triumphs,trials and tribulations

The liver is one of the few organs that possess a high capacity to regenerate after liver failure or liver damage. The parenchymal cells of the liver, hepatocytes, contribute to the majority of the regeneration process. Thus, hepatocyte transplantation presents an alternative method to treating liver damage. However, shortage of hepatocytes and difficulties in maintaining primary hepatocytes still remain key obstacles that researchers must overcome before hepatocyte transplantation can be used in clinical practice. The unique properties of pluripotent stem cells (PSCs) and induced pluripotent stem cells (iPSCs) have provided an alternative approach to generating enough functional hepatocytes for cellular therapy. In this review, we will present a brief overview on the current state of hepatocyte differentiation from PSCs and iPSCs. Studies of liver regenerative processes using different cell sources (adult liver stem cells, hepatoblasts, hepatic progenitor cells, etc.) will be described in detail as well as how this knowledge can be applied towards optimizing culture conditions for the maintenance and differentiation of these cells towards hepatocytes. As the outlook of stem cell-derived therapy begins to look more plausible, researchers will need to address the challenges we must overcome in order to translate stem cell research to clinical applications.     

Extracellular matrix is required for the survival and differentiation of transplanted hepatic progenitor cells

Engelbreth-Holm-Swarm (EHS) gel has been reported to maintain the mature hepatocyte phenotypes in primary cultured hepatocytes. We investigated the effect of EHS gel on the differentiation of fetal liver cells, which contain stem/progenitor cells. The isolated fetal liver cells cultured on EHS gel formed a spherical shape and increased liver-specific gene expressions compared with cells cultured on collagen. The hepatic progenitor cells that were transplanted subcutaneously to BALB/c nude mice could survive and express hepatocyte marker alpha-fetoprotein when the cells were suspended with EHS gel. These findings demonstrate that EHS gel supports cytodifferentiation from immature progenitor cells to hepatocytes and maintain its differentiated phenotypes in vitro and in vivo.     

Immunohistochemical analyses of cell–cell interactions during hepatic organoid formation from fetal mouse liver cells cultured in vitro

Cell–cell interactions among cell types constituting the fetal liver such as hepatoblasts, stellate cells and endothelial cells lead to functional lobule development. The present study was undertaken to investigate hepatic histogenesis in the primary culture of E12.5 mouse livers, including cell–cell and cell–matrix interactions. Fetal livers were dispersed with protease treatment and cultured for 5 days. Cellular adhesion of each hepatic cell type, gene expression and extracellular matrix deposition were analyzed by immunohistochemistry and immunoblotting. Immunohistochemical analysis demonstrated that the primary culture of fetal liver cells contained at least hepatoblasts, mesenchymal cells, endothelial cells, hemopoietic cells and Kupffer cells. Although hepatoblasts, mesenchymal cells, and endothelial cells aggregated separately in the initial step, they then formed a spheroid together, adhering to the glass slide, which led to the formation of flattened hepatic organoids. Hepatoblasts more preferentially adhered to mesenchymal cells than endothelial cells. Several extracellular matrix depositions were seen in aggregates consisting of at least hepatoblasts and mesenchymal cells within 12 h, but were poor in those lacking hepatoblasts. These data show that the primary culture of fetal liver cells contains most cell types constituting fetal livers, and may be useful for studying cell–cell interactions during liver development.     

Osterix-Cre Labeled Progenitor Cells Contribute to the Formation and Maintenance of the Bone Marrow Stroma

We have carried out fate mapping studies using Osterix-EGFPCre and Osterix-CreERt animal models and found Cre reporter expression in many different cell types that make up the bone marrow stroma. Constitutive fate mapping resulted in the labeling of different cellular components located throughout the bone marrow, whereas temporal fate mapping at E14.5 resulted in the labeling of cells within a region of the bone marrow. The identity of cell types marked by constitutive and temporal fate mapping included osteoblasts, adipocytes, vascular smooth muscle, perineural, and stromal cells. Prolonged tracing of embryonic precursors labeled at E14.5dpc revealed the continued existence of their progeny up to 10 months of age, suggesting that fate mapped, labeled embryonic precursors gave rise to long lived bone marrow progenitor cells. To provide further evidence for the marking of bone marrow progenitors, bone marrow cultures derived from Osterix-EGFPCre/Ai9 mice showed that stromal cells retained Cre reporter expression and yielded a FACS sorted population that was able to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro and into osteoblasts, adipocytes, and perivascular stromal cells after transplantation. Collectively, our studies reveal the developmental process by which Osterix-Cre labeled embryonic progenitors give rise to adult bone marrow progenitors which establish and maintain the bone marrow stroma.     

Derivation and Characterization of Hepatic Progenitor Cells from Human Embryonic Stem Cells

The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell–derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.     

Robust differentiation of fetal hepatocytes from human embryonic stem cells and iPS

Hepatocyte transplantation is considered as an alternative to organ transplantation in particular for the treatment of liver metabolic diseases. However, due to the difficulties to obtain a large number of hepatocytes, new sources of cells are needed. These cells could be either of hepatic origin (hepatic stem cells) or extrahepatic such as mesenchymal stem cells or pluripotent stem cells (human embryonic stem cells [hESC] or iPS). We developed a new method to differentiate hESCs into fetal hepatocytes. These conditions recapitulate the main liver developmental stages, using fully defined medium devoid of animal products or unknown factors. The differentiated cells express many fetal hepatocytes markers (cytochrome P450 3A7, albumin, alpha-1-antitrypsin, etc.). The cells display specific hepatic functions (ammonia metabolism, excretion of indocyanin green) and are capable to engraft and express hepatic proteins two months after transplantation into newborn uPAxrag2gc-/- mouse liver. We have also showed that this approach is transposable to human iPS, and further studies on animal models will allow us to compare the in vivo potential of these two sources of pluripotent cells. Finally, only studies on large animals such as nonhuman primates will validate an eventual clinical application.