HNF4A is essential for specification of hepatic progenitors from human pluripotent stem cells. Development

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Development (Impact Factor: 6.46). 08/2011; 138(19):4143-53. DOI: 10.1242/dev.062547
Source: PubMed


The availability of pluripotent stem cells offers the possibility of using such cells to model hepatic disease and development. With this in mind, we previously established a protocol that facilitates the differentiation of both human embryonic stem cells and induced pluripotent stem cells into cells that share many characteristics with hepatocytes. The use of highly defined culture conditions and the avoidance of feeder cells or embryoid bodies allowed synchronous and reproducible differentiation to occur. The differentiation towards a hepatocyte-like fate appeared to recapitulate many of the developmental stages normally associated with the formation of hepatocytes in vivo. In the current study, we addressed the feasibility of using human pluripotent stem cells to probe the molecular mechanisms underlying human hepatocyte differentiation. We demonstrate (1) that human embryonic stem cells express a number of mRNAs that characterize each stage in the differentiation process, (2) that gene expression can be efficiently depleted throughout the differentiation time course using shRNAs expressed from lentiviruses and (3) that the nuclear hormone receptor HNF4A is essential for specification of human hepatic progenitor cells by establishing the expression of the network of transcription factors that controls the onset of hepatocyte cell fate.

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    • "In addition, HNF4a was found to be essential for the specification of hepatic progenitors from human pluripotent stem cells (iPSCs) [15]. The homeobox transcription factor Prox1 (prospero-related homeobox 1), an early specific marker during the development of liver and pancreas from the foregut endoderm, is a co-regulator of HNF4a and human liver receptor homolog-1 (hLRH-1) [16] [17]. "
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    ABSTRACT: Background & aims: Hepatocyte-like cells, differentiated from different stem cell sources, are considered to have a range of possible therapeutic applications, including drug discovery, metabolic disease modelling, and cell transplantation. However, little is known about how stem cells differentiate into mature and functional hepatocytes. Methods: Using transcriptomic screening, a transcription factor, liver X receptor α (NR1H3), was identified as increased during HepaRG cell hepatogenesis; this protein was also upregulated during embryonic stem cell and induced pluripotent stem cell differentiation. Results: Overexpressing NR1H3 in human HepaRG cells promoted hepatic maturation; the hepatocyte-like cells exhibited various functions associated with mature hepatocytes, including cytochrome P450 (CYP) enzyme activity, secretion of urea and albumin, upregulation of hepatic-specific transcripts and an increase in glycogen storage. Importantly, the NR1H3-derived hepatocyte-like cells were able to rescue lethal fulminant hepatic failure using a non-obese diabetic/severe combined immunodeficient mouse model. Conclusions: In this study, we found that NR1H3 accelerates hepatic differentiation through an HNF4α-dependent reciprocal network. This contributes to hepatogenesis and is therapeutically beneficial to liver disease.
    Journal of Hepatology 07/2014; 61(6). DOI:10.1016/j.jhep.2014.07.025 · 11.34 Impact Factor
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    • "To generate hepatic progenitor cells, Activin A was removed from the medium and replaced with BMP4 and FGF2. After 5 days in culture nearly all of the cells expressed HNF4a, which is a transcription factor that is expressed in hepatic progenitor cells and is essential for their differentiation to hepatocytes (Figure  2, day 10) [15]. At this stage, some cells also began to express low levels of alpha-fetoprotein (AFP), which is another marker of the early hepatic lineage. "
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    ABSTRACT: Background The characterization of induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) routinely includes analyses of chromosomal integrity. The belief is that pluripotent stem cells best suited to the generation of differentiated derivatives should display a euploid karyotype; although, this does not appear to have been formally tested. While aneuploidy is commonly associated with cell transformation, several types of somatic cells, including hepatocytes, are frequently aneuploid and variation in chromosomal content does not contribute to a transformed phenotype. This insight has led to the proposal that dynamic changes in the chromosomal environment may be important to establish genetic diversity within the hepatocyte population and such diversity may facilitate an adaptive response by the liver to various insults. Such a positive contribution of aneuploidy to liver function raises the possibility that, in contrast to existing dogma, aneuploid iPSCs may be capable of generating hepatocyte-like cells that display hepatic activities. Results We examined whether a human iPSC line that had multiple chromosomal aberrations was competent to differentiate into hepatocytes and found that loss of normal chromosomal content had little impact on the production of hepatocyte-like cells from iPSCs. Conclusions iPSCs that harbor an abnormal chromosomal content retain the capacity to generate hepatocyte–like cells with high efficiency.
    BMC Research Notes 07/2014; 7(1):437. DOI:10.1186/1756-0500-7-437
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    • "To elucidate the molecular mechanisms of liver development, both conditional knockout mouse models and cell culture systems are useful. For example, DeLaForest et al. demonstrated the role of HNF4α in hepatocyte differentiation using hESC culture systems (DeLaForest et al., 2011). The technology for inducing hepatocyte differentiation from hESCs has recently been dramatically advanced (Takayama et al., 2012a). "
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    ABSTRACT: Human embryonic stem cells (hESCs) and their derivatives are expected to be used in drug discovery, regenerative medicine and the study of human embryogenesis. Because hepatocyte differentiation from hESCs has the potential to recapitulate human liver development in vivo, we employed this differentiation method to investigate the molecular mechanisms underlying human hepatocyte differentiation. A previous study has shown that a gradient of transforming growth factor beta (TGFβ) signaling is required to segregate hepatocyte and cholangiocyte lineages from hepatoblasts. Although CCAAT/enhancer binding proteins (c/EBPs) are known to be important transcription factors in liver development, the relationship between TGFβ signaling and c/EBP-mediated transcriptional regulation in the hepatoblast fate decision is not well known. To clarify this relationship, we examined whether c/EBPs could determine the hepatoblast fate decision via regulation of TGFβ receptor 2 (TGFBR2) expression in the hepatoblast-like cells differentiated from hESCs. We found that TGFBR2 promoter activity was negatively regulated by c/EBPα and positively regulated by c/EBPβ. Moreover, c/EBPα overexpression could promote hepatocyte differentiation by suppressing TGFBR2 expression, whereas c/EBPβ overexpression could promote cholangiocyte differentiation by enhancing TGFBR2 expression. Our findings demonstrated that c/EBPα and c/EBPβ determine the lineage commitment of hepatoblasts by negatively and positively regulating the expression of a common target gene, TGFBR2, respectively.
    Development 11/2013; 141(1). DOI:10.1242/dev.103168 · 6.46 Impact Factor
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