Jixuan Li

Highly efficient generation of human hepatocyte–like cells from induced pluripotent stem cells

There exists a worldwide shortage of donor livers available for orthotropic liver transplantation and hepatocyte transplantation therapies. In addition to their therapeutic potential, primary human hepatocytes facilitate the study of molecular and genetic aspects of human hepatic disease and development and provide a platform for drug toxicity screens and identification of novel pharmaceuticals with potential to treat a wide array of metabolic diseases. The demand for human hepatocytes, therefore, heavily outweighs their availability. As an alternative to using donor livers as a source of primary hepatocytes, we explored the possibility of generating patient‐specific human hepatocytes from induced pluripotent stem (iPS) cells. Conclusion: We demonstrate that mouse iPS cells retain full potential for fetal liver development and describe a procedure that facilitates the efficient generation of highly differentiated human hepatocyte‐like cells from iPS cells that display key liver functions and can integrate into the hepatic parenchyma in vivo. (HEPATOLOGY 2010.)

GATA6 Is Essential for Embryonic Development of the Liver but Dispensable for Early Heart Formation

ABSTRACT Several lines of evidence suggest that GATA6 has an integral role in controlling development of the mammalian liver. Unfortunately, this proposal has been impossible to address directly because mouse embryos lacking GATA6 die during gastrulation. Here we show that the early embryonic deficiency associated with GATA6-knockout mice can be overcome by providing GATA6-null embryos with a wild-type extraembryonic endoderm with the use of tetraploid embryo complementation. Analysis of rescued Gata6 − / − embryos revealed that, although hepatic specification occurs normally, the specified cells fail to differentiate and the liver bud does not expand. Although GATA6 is expressed in multiple tissues that impact development of the liver, including the heart, septum transversum mesenchyme, and vasculature, all are relatively unaffected by loss of GATA6, which is consistent with a cell-autonomous requirement for GATA6 during hepatogenesis. We also demonstrate that a closely related GATA factor, GATA4, is expressed transiently in the prehepatic endoderm during hepatic specification and then lost during expansion of the hepatic primordium. Our data support the proposal that GATA4 and GATA6 are functionally redundant during hepatic specification but that GATA6 alone is available for liver bud growth and commitment of the endoderm to a hepatic cell fate.

Loss of both GATA4 and GATA6 blocks cardiac myocyte differentiation and results in acardia in mice.

Despite significant advances in identifying signaling molecules that induce cardiogenesis in mammals, the transcription factors that control the onset of cardiac myocyte gene expression have remained elusive. Candidates include the zinc finger transcription factors GATA binding proteins 4 and 6 (GATA4, GATA6). The individual loss of either protein in mice results in lethality prior to the onset of heart development due to defects in the extra-embryonic endoderm; however, when this extra-embryonic deficiency is circumvented using tetraploid embryo complementation, cardiac myocyte differentiation initiates normally. Here we show that these factors have redundant roles in controlling the onset of cardiac myocyte differentiation. As a consequence, Gata4(-/-)Gata6(-/-) embryos completely lack hearts, although second heart field progenitor cells are still generated. Our data support a model whereby GATA4 or GATA6 are essential for expression of the network of transcription factors that regulate the onset of cardiac myocyte gene expression during mammalian development.

Development of the mammalian liver and ventral pancreas is dependent on GATA4

BackgroundIn the mouse, the parenchyma of both the liver and ventral pancreas is specified from adjacent domains of the ventral foregut endoderm. GATA4, a zinc finger transcription factor, is strongly expressed in these endodermal domains and molecular analyses have implicated GATA4 in potentiating liver gene expression during the onset of hepatogenesis. We therefore hypothesized that GATA4 has an integral role in controlling the early stages of pancreatic and liver development.ResultsTo determine whether GATA4 contributes to development of either the pancreas or liver we characterized the formation of pancreatic and hepatic tissues in embryos derived from Gata4-/- ES cells by tetraploid embryo complementation. In the absence of GATA4, development of the liver and ventral pancreas was disrupted. At embryonic day (E) 9.5, the liver bud failed to expand although, contrary to expectations, the hepatic endoderm was able to form a pseudo-stratified epithelial liver bud that expressed hepatic genes. Moreover, as we had shown previously, the embryos lacked septum transversum mesenchyme suggesting that liver defects may be cell non-autonomous. Analyses of pancreatic development revealed a complete absence of the ventral but not the dorsal pancreas in Gata4-/- embryos. Moreover, Gata6-/- embryos displayed a similar, although less dramatic phenotype, suggesting a critical role for multiple GATA factors at the earliest stages of ventral pancreas development.ConclusionThis study defines integral roles for GATA factors in controlling early development of the mammalian liver and pancreas.

Hepatocyte nuclear factor 4α is implicated in endoplasmic reticulum stress-induced acute phase response by regulating expression of cyclic adenosine monophosphate responsive element binding protein H

Loss of the nuclear hormone receptor hepatocyte nuclear factor 4α (HNF4α) in hepatocytes results in a complex pleiotropic phenotype that includes a block in hepatocyte differentiation and a severe disruption to liver function. Recent analyses have shown that hepatic gene expression is severely affected by the absence of HNF4α, with expression of 567 genes reduced by ≥2.5‐fold (P ≤ 0.05) in Hnf4α−/− fetal livers. Although many of these genes are direct targets, HNF4α has also been shown to regulate expression of other liver transcription factors, and this raises the possibility that the dependence on HNF4α for normal expression of some genes may be indirect. We postulated that the identification of transcription factors whose expression is regulated by HNF4α might reveal roles for HNF4α in controlling hepatic functions that were not previously appreciated. Here we identify cyclic adenosine monophosphate responsive element binding protein H (CrebH) as a transcription factor whose messenger RNA can be identified in both the embryonic mouse liver and adult mouse liver and whose expression is dependent on HNF4α. Analyses of genomic DNA revealed an HNF4α binding site upstream of the CrebH coding sequence that was occupied by HNF4α in fetal livers and facilitated transcriptional activation of a reporter gene in transient transfection analyses. Although CrebH is highly expressed during hepatogenesis, CrebH−/− mice were viable and healthy and displayed no overt defects in liver formation. However, upon treatment with tunicamycin, which induces an endoplasmic reticulum (ER)–stress response, CrebH−/− mice displayed reduced expression of acute phase response proteins. Conclusion: These data implicate HNF4α in having a role in controlling the acute phase response of the liver induced by ER stress by regulating expression of CrebH. (HEPATOLOGY 2008.)

Hepatocyte nuclear factor 4Α is implicated in endoplasmic reticulum stress–induced acute phase response by regulating expression of cyclic adenosine monophosphate responsive element binding protein H Potential conflict of interest: Nothing to report.

Loss of the nuclear hormone receptor hepatocyte nuclear factor 4α (HNF4α) in hepatocytes results in a complex pleiotropic phenotype that includes a block in hepatocyte differentiation and a severe disruption to liver function. Recent analyses have shown that hepatic gene expression is severely affected by the absence of HNF4α, with expression of 567 genes reduced by ≥2.5‐fold (P ≤ 0.05) in Hnf4α−/− fetal livers. Although many of these genes are direct targets, HNF4α has also been shown to regulate expression of other liver transcription factors, and this raises the possibility that the dependence on HNF4α for normal expression of some genes may be indirect. We postulated that the identification of transcription factors whose expression is regulated by HNF4α might reveal roles for HNF4α in controlling hepatic functions that were not previously appreciated. Here we identify cyclic adenosine monophosphate responsive element binding protein H (CrebH) as a transcription factor whose messenger RNA can be identified in both the embryonic mouse liver and adult mouse liver and whose expression is dependent on HNF4α. Analyses of genomic DNA revealed an HNF4α binding site upstream of the CrebH coding sequence that was occupied by HNF4α in fetal livers and facilitated transcriptional activation of a reporter gene in transient transfection analyses. Although CrebH is highly expressed during hepatogenesis, CrebH−/− mice were viable and healthy and displayed no overt defects in liver formation. However, upon treatment with tunicamycin, which induces an endoplasmic reticulum (ER)–stress response, CrebH−/− mice displayed reduced expression of acute phase response proteins. Conclusion: These data implicate HNF4α in having a role in controlling the acute phase response of the liver induced by ER stress by regulating expression of CrebH. (HEPATOLOGY 2008.)

Design of the Artificial Acellular Feeder Layer for the Efficient Propagation of Mouse Embryonic Stem Cells

Embryonic stem (ES) cells are pluripotent-undifferentiated cells that have a great interest for the investigation of developmental biology. Murine ES cells maintain their pluripotency by the supplementation of the leukemia inhibitory factor (LIF). LIF is reported to act as a matrix-anchored form, and immobilized cytokines are useful to sustain their signaling on target cells. In this study, we used the immobilizable fusion protein composed of LIF and IgG-Fc region, which was used as a model of the matrix-anchored form of LIF to establish a novel system for ES cell culture and to investigate the effect of immobilized LIF on maintenance of ES cell pluripotency. Mouse ES cells maintained their undifferentiated state on the surface coated with LIF-Fc. Furthermore, when cultured on the co-immobilized surface with LIF-Fc and E-cadherin-Fc, mouse ES cells showed characteristic scattering morphologies without colony formation, and they could maintain their undifferentiated state and pluripotency without additional LIF supplementation. The activation of LIF signaling was sustained on the co-immobilized surface. These results indicate that immobilized LIF and E-cadherin can maintain mouse ES cells efficiently and that the immobilizable LIF-Fc fusion protein is useful for the investigation of signaling pathways of an immobilized form of LIF in the maintenance of ES cell pluripotency.

Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation

BackgroundTransgenic mice have been used extensively to analyze gene function. Unfortunately, traditional transgenic procedures have only limited use in analyzing alleles that cause lethality because lines of founder mice cannot be established. This is frustrating given that such alleles often reveal crucial aspects of gene function. For this reason techniques that facilitate the generation of embryos expressing such alleles would be of enormous benefit. Although the transient generation of transgenic embryos has allowed limited analysis of lethal alleles, it is expensive, time consuming and technically challenging. Moreover a fundamental limitation with this approach is that each embryo generated is unique and transgene expression is highly variable due to the integration of different transgene copy numbers at random genomic sites.ResultsHere we describe an alternative method that allows the generation of clonal mouse embryos harboring a single-copy transgene at a defined genomic location. This was facilitated through the production of Hprt negative embryonic stem cells that allow the derivation of embryos by tetraploid embryo complementation. We show that targeting transgenes to the hprt locus in these ES cells by homologous recombination can be efficiently selected by growth in HAT medium. Moreover, embryos derived solely from targeted ES cells containing a single copy LacZ transgene under the control of the α-myosin heavy chain promoter exhibited the expected cardiac specific expression pattern.ConclusionOur results demonstrate that tetraploid embryo complementation by F3 hprt negative ES cells facilitates the generation of transgenic mouse embryos containing a single copy gene at a defined genomic locus. This approach is simple, extremely efficient and bypasses any requirement to generate chimeric mice. Moreover embryos generated by this procedure are clonal in that they are all derived from a single ES cell lines. This facilitates the comparative analysis of lethal alleles and thereby advances our ability to analyze gene function in mammals.