Human induced pluripotent stem cells-derived liver organoids grown on a Biomimesys® hyaluronic acid-based hydroscaffold as a new model for studying human lipoprotein metabolism.

The liver plays a key role in the metabolism of lipoproteins, controlling both production and catabolism. To accelerate the development of new lipid-lowering therapies in humans, it is essential to have a relevant in vitro study model available. The current hepatocyte-like cells (HLCs) models derived from hiPSC can be used to model many genetically driven diseases but require further improvement to better recapitulate the complexity of liver functions. Here, we aimed to improve the maturation of HLCs using a three-dimensional (3D) approach using Biomimesys®, a hyaluronic acid-based hydroscaffold in which hiPSCs may directly form aggregates and differentiate toward a functional liver organoid model. After a 28-day differentiation 3D protocol, we showed that many hepatic genes were upregulated in the 3D model (liver organoids) in comparison with the 2D model (HLCs). Liver organoids, grown on Biomimesys®, exhibited an autonomous cell organization, were composed of different cell types and displayed enhanced cytochromes P450 activities compared to HLCs. Regarding the functional capacities of these organoids, we showed that they were able to accumulate lipids (hepatic steatosis), internalize low-density lipoprotein and secrete apolipoprotein B. Interestingly, we showed for the first time that this model was also able to produce apolipoprotein (a), the apolipoprotein (a) specific of Lp(a). This innovative hiPSC-derived liver organoid model may serve as a relevant model for studying human lipopoprotein metabolism, including Lp(a).

Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis.

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

FACS-assisted CRISPR-Cas9 genome editing of human induced pluripotent stem cells.

This manuscript proposes an efficient and reproducible protocol for the generation of genetically modified human induced pluripotent stem cells (hiPSCs) by genome editing using CRISPR-Cas9 technology. Here, we describe the experimental strategy for generating knockout (KO) and knockin (KI) clonal populations of hiPSCs using single-cell sorting by flow cytometry. We efficiently achieved up to 15 kb deletions, molecular tag insertions, and single-nucleotide editing in hiPSCs. We emphasize the efficacy of this approach in terms of cell culture time. For complete details on the use and execution of this protocol, please refer to Canac et al. (2022) and Bray et al. (2022).

Identification of a Gain-of-Function LIPC Variant as a Novel Cause of Familial Combined Hypocholesterolemia.

BACKGROUND: Atherosclerotic cardiovascular disease is the main cause of mortality worldwide and is strongly influenced by circulating low-density lipoprotein (LDL) cholesterol levels. Only a few genes causally related to plasma LDL cholesterol levels have been identified so far, and only 1 gene, ANGPTL3, has been causally related to combined hypocholesterolemia. Here, our aim was to elucidate the genetic origin of an unexplained combined hypocholesterolemia inherited in 4 generations of a French family. METHODS: Using next-generation sequencing, we identified a novel dominant rare variant in the LIPC gene, encoding for hepatic lipase, which cosegregates with the phenotype. We characterized the impact of this LIPC-E97G variant on circulating lipid and lipoprotein levels in family members using nuclear magnetic resonance-based lipoprotein profiling and lipidomics. To uncover the mechanisms underlying the combined hypocholesterolemia, we used protein homology modeling, measured triglyceride lipase and phospholipase activities in cell culture, and studied the phenotype of APOE*3.Leiden.CETP mice after LIPC-E97G overexpression. RESULTS: Family members carrying the LIPC-E97G variant had very low circulating levels of LDL cholesterol and high-density lipoprotein cholesterol, LDL particle numbers, and phospholipids. The lysophospholipids/phospholipids ratio was increased in plasma of LIPC-E97G carriers, suggestive of an increased lipolytic activity on phospholipids. In vitro and in vivo studies confirmed that the LIPC-E97G variant specifically increases the phospholipase activity of hepatic lipase through modification of an evolutionarily conserved motif that determines substrate access to the hepatic lipase catalytic site. Mice overexpressing human LIPC-E97G recapitulated the combined hypocholesterolemic phenotype of the family and demonstrated that the increased phospholipase activity promotes catabolism of triglyceride-rich lipoproteins by different extrahepatic tissues but not the liver. CONCLUSIONS: We identified and characterized a novel rare variant in the LIPC gene in a family who presents with dominant familial combined hypocholesterolemia. This gain-of-function variant makes LIPC the second identified gene, after ANGPTL3, causally involved in familial combined hypocholesterolemia. Our mechanistic data highlight the critical role of hepatic lipase phospholipase activity in LDL cholesterol homeostasis and suggest a new LDL clearance mechanism.

PCSK9 regulates the NODAL signaling pathway and cellular proliferation in hiPSCs.

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a key regulator of low-density lipoprotein (LDL) cholesterol metabolism and the target of lipid-lowering drugs. PCSK9 is mainly expressed in hepatocytes. Here, we show that PCSK9 is highly expressed in undifferentiated human induced pluripotent stem cells (hiPSCs). PCSK9 inhibition in hiPSCs with the use of short hairpin RNA (shRNA), CRISPR/cas9-mediated knockout, or endogenous PCSK9 loss-of-function mutation R104C/V114A unveiled its new role as a potential cell cycle regulator through the NODAL signaling pathway. In fact, PCSK9 inhibition leads to a decrease of SMAD2 phosphorylation and hiPSCs proliferation. Conversely, PCSK9 overexpression stimulates hiPSCs proliferation. PCSK9 can interfere with the NODAL pathway by regulating the expression of its endogenous inhibitor DACT2, which is involved in transforming growth factor (TGF) ß-R1 lysosomal degradation. Using different PCSK9 constructs, we show that PCSK9 interacts with DACT2 through its Cys-His-rich domain (CHRD) domain. Altogether these data highlight a new role of PCSK9 in cellular proliferation and development.

[Hepatic organoids: What are the challenges?] / Les organoïdes hépatiques - Quels sont les enjeux ?

The study and understanding of liver organogenesis have allowed the development of protocols for pluripotent stem cells differentiation to overcome the lack of primary cells, providing an almost unlimited source of liver cells. However, as their differentiation in conventional 2D culture systems has shown serious limits, hepatic organoids derived from human pluripotent stem cells represent a promising alternative. These complex and organized structures, containing one or more cell types, make it possible to recapitulate in vitro some of the organ functions, thus enabling numerous applications such as the study of the liver development, the mass production of functional liver cells for transplantation or the development of bioartificial livers, as well as the in vitro modeling of hepatic pathologies allowing high throughput applications in drug screening or toxicity studies. Economic and ethical issues must also be taken into account before using these organoids in therapeutic applications.

TITLE: Les organoïdes hépatiques - Quels sont les enjeux ? ABSTRACT: L'étude et la compréhension de l'organogenèse du foie ont permis le développement de protocoles de différenciation des cellules souches pluripotentes afin de pallier le manque de cellules primaires, offrant ainsi une source quasi illimitée de cellules hépatiques. La différenciation de ces cellules dans des systèmes de culture conventionnels en deux dimensions (2D) ayant cependant montré ses limites, des organoïdes hépatiques ont été dérivés de cellules souches pluripotentes humaines et représentent désormais une alternative prometteuse. Ces structures 3D, complexes et organisées, intégrant un ou plusieurs types cellulaires, permettent de reproduire in vitro une ou plusieurs fonctions de l'organe, et ouvrent ainsi la voie à de nombreuses applications, comme l'étude du développement du foie, la production en masse de cellules hépatiques fonctionnelles pour la transplantation ou le développement de foies bioartificiels, sans oublier la modélisation de pathologies hépatiques permettant le criblage à haut débit de médicaments ou des études de toxicité. Des enjeux économiques et éthiques doivent également être pris en considération avant une utilisation de ces organoïdes pour des applications thérapeutiques.

A dominant vimentin variant causes a rare syndrome with premature aging.

Progeroid syndromes are a group of rare genetic disorders, which mimic natural aging. Unraveling the molecular defects in such conditions could impact our understanding of age-related syndromes such as Alzheimer's or cardiovascular diseases. Here we report a de novo heterozygous missense variant in the intermediate filament vimentin (c.1160 T > C; p.(Leu387Pro)) causing a multisystem disorder associated with frontonasal dysostosis and premature aging in a 39-year-old individual. Human vimentin p.(Leu387Pro) expression in zebrafish perturbed body fat distribution, and craniofacial and peripheral nervous system development. In addition, studies in patient-derived and transfected cells revealed that the variant affects vimentin turnover and its ability to form filaments in the absence of wild-type vimentin. Vimentin p.(Leu387Pro) expression diminished the amount of peripilin and reduced lipid accumulation in differentiating adipocytes, recapitulating key patient's features in vivo and in vitro. Our data highlight the function of vimentin during development and suggest its contribution to natural aging.

Human Induced Pluripotent Stem (hiPS) Cells from Urine Samples: A Non-Integrative and Feeder-Free Reprogramming Strategy.

Human induced pluripotent stem (hiPS) cell technology has already revolutionized some aspects of fundamental and applied research such as study of disease mechanisms and pharmacology screening. The first clinical trial using hiPS cell-derived cells began in Japan, only 10 years after the publication of the proof-of concept article. In this exciting context, strategies to generate hiPS cells have evolved quickly, tending towards non-invasive protocols to sample somatic cells combined with "safer" reprogramming strategies. In this unit, we describe a protocol combining both of these advantages to generate hiPS cells with episomal plasmid transfection from urine samples of individuals carrying the desired genotype. Based on previous published works, this simplified protocol requires minimal equipment and reagents, and is suitable both for scientists familiar with the hiPS cells technology and neophytes. HiPS cells displaying classical features of pluripotency and suitable for all desired downstream applications are generated rapidly (<10 weeks) and with high efficiency. © 2017 by John Wiley & Sons, Inc.

Urine-sample-derived human induced pluripotent stem cells as a model to study PCSK9-mediated autosomal dominant hypercholesterolemia.

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a critical modulator of cholesterol homeostasis. Whereas PCSK9 gain-of-function (GOF) mutations are associated with autosomal dominant hypercholesterolemia (ADH) and premature atherosclerosis, PCSK9 loss-of-function (LOF) mutations have a cardio-protective effect and in some cases can lead to familial hypobetalipoproteinemia (FHBL). However, limitations of the currently available cellular models preclude deciphering the consequences of PCSK9 mutation further. We aimed to validate urine-sample-derived human induced pluripotent stem cells (UhiPSCs) as an appropriate tool to model PCSK9-mediated ADH and FHBL. To achieve our goal, urine-sample-derived somatic cells were reprogrammed into hiPSCs by using episomal vectors. UhiPSC were efficiently differentiated into hepatocyte-like cells (HLCs). Compared to control cells, cells originally derived from an individual with ADH (HLC-S127R) secreted less PCSK9 in the media (-38.5%; P=0.038) and had a 71% decrease (P<0.001) of low-density lipoprotein (LDL) uptake, whereas cells originally derived from an individual with FHBL (HLC-R104C/V114A) displayed a strong decrease in PCSK9 secretion (-89.7%; P<0.001) and had a 106% increase (P=0.0104) of LDL uptake. Pravastatin treatment significantly enhanced LDL receptor (LDLR) and PCSK9 mRNA gene expression, as well as PCSK9 secretion and LDL uptake in both control and S127R HLCs. Pravastatin treatment of multiple clones led to an average increase of LDL uptake of 2.19 ± 0.77-fold in HLC-S127R compared to 1.38 ± 0.49 fold in control HLCs (P<0.01), in line with the good response to statin treatment of individuals carrying the S127R mutation (mean LDL cholesterol reduction=60.4%, n=5). In conclusion, urine samples provide an attractive and convenient source of somatic cells for reprogramming and hepatocyte differentiation, but also a powerful tool to further decipher PCSK9 mutations and function.

Toward Personalized Medicine: Using Cardiomyocytes Differentiated From Urine-Derived Pluripotent Stem Cells to Recapitulate Electrophysiological Characteristics of Type 2 Long QT Syndrome.

BACKGROUND: Human genetically inherited cardiac diseases have been studied mainly in heterologous systems or animal models, independent of patients' genetic backgrounds. Because sources of human cardiomyocytes (CMs) are extremely limited, the use of urine samples to generate induced pluripotent stem cell-derived CMs would be a noninvasive method to identify cardiac dysfunctions that lead to pathologies within patients' specific genetic backgrounds. The objective was to validate the use of CMs differentiated from urine-derived human induced pluripotent stem (UhiPS) cells as a new cellular model for studying patients' specific arrhythmia mechanisms. METHODS AND RESULTS: Cells obtained from urine samples of a patient with long QT syndrome who harbored the HERG A561P gene mutation and his asymptomatic noncarrier mother were reprogrammed using the episomal-based method. UhiPS cells were then differentiated into CMs using the matrix sandwich method.UhiPS-CMs showed proper expression of atrial and ventricular myofilament proteins and ion channels. They were electrically functional, with nodal-, atrial- and ventricular-like action potentials recorded using high-throughput optical and patch-clamp techniques. Comparison of HERG expression from the patient's UhiPS-CMs to the mother's UhiPS-CMs showed that the mutation led to a trafficking defect that resulted in reduced delayed rectifier K(+) current (IKr). This phenotype gave rise to action potential prolongation and arrhythmias. CONCLUSIONS: UhiPS cells from patients carrying ion channel mutations can be used as novel tools to differentiate functional CMs that recapitulate cardiac arrhythmia phenotypes.

Role of PCSK9 beyond liver involvement.

PURPOSE OF REVIEW: Proprotein convertase subtilisin kexin type 9 (PCSK9) acts as an endogenous natural inhibitor of the LDL receptor pathway, by targeting the receptor to lysosomes for degradation. Beside the liver, PCSK9 is also expressed at significant levels in other tissues, where its function remains unclear. The current review focuses on the extrahepatic actions of PCSK9. RECENT FINDINGS: The generation of liver-specific PCSK9 knockout mice has clearly indicated that PCSK9 affects cholesterol homeostasis via its action on extrahepatic organs. PCSK9 is highly expressed in the intestine, where it controls the production of triglyceride-rich lipoproteins and the transintestinal cholesterol excretion. The role of PCSK9 in the endocrine pancreas and glucose homeostasis remains unclear because conflicting data exist concerning the metabolic phenotype of PCSK9-deficient mice. Sparse data suggest that PCSK9 might also play a role in kidneys, vascular smooth muscle cells, and neurons. SUMMARY: Based on the virtuous combination of genetic and pharmacological approaches, the major function of PCSK9 as a key regulator of hepatic LDL receptor metabolism had quickly emerged. Accumulating evidence indicates that intestinal PCSK9 is also involved in the modulation of lipid homeostasis. Additional studies are warranted to decipher the physiological function of PCSK9 in other extrahepatic tissues and thus to better assess the safety of PCSK9 inhibitors.

Human induced pluripotent stem cells in hepatology: beyond the proof of concept.

The discovery of the wide plasticity of most cell types means that it is now possible to produce virtually any cell type in vitro. This concept, developed because of the possibility of reprogramming somatic cells toward induced pluripotent stem cells, provides the opportunity to produce specialized cells that harbor multiple phenotypical traits, thus integrating genetic interindividual variability. The field of hepatology has exploited this concept, and hepatocyte-like cells can now be differentiated from induced pluripotent stem cells. This review discusses the choice of somatic cells to be reprogrammed by emergent new and nonintegrative strategies, as well as the application of differentiated human induced pluripotent stem cells in hepatology, including liver development, disease modeling, host-pathogen interactions, and drug metabolism and toxicity. The actual consensus is that hepatocyte-like cells generated in vitro present an immature phenotype. Currently, developed strategies used to resolve this problem, such as overexpression of transcription factors, mimicking liver neonatal and postnatal modifications, and re-creating the three-dimensional hepatocyte environment in vitro and in vivo, are also discussed.

Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

BACKGROUND: Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. RESULTS: We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. CONCLUSIONS: We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

Differential expression of E-cadherin, ß-catenin, and Lewis x between invasive hydatidiform moles and post-molar choriocarcinomas.

Trophoblast cell adhesion and migration are carefully coordinated during normal placental development. We have compared the expression of three adhesion molecules, E-cadherin, ß-catenin, and Lewis x, by immunohistochemistry during normal trophoblast differentiation, and in hydatidiform moles and choriocarcinomas. Both E-cadherin and ß-catenin were expressed in normal placenta cytotrophoblast, and this expression decreased with trophoblast maturation. E-cadherin was mainly localized along the contact between cytotrophoblast and syncytiotrophoblast, which indicates its role in the differentiation of the syncytial layer. Lewis x disappeared progressively during differentiation of normal villous vessels, and was expressed in molar pregnancies. Interestingly, whereas choriocarcinomas were not, or poorly, stained, invasive hydatidiform moles (invHMs) strongly expressed Lewis x in vascular structures. This observation correlated well with E-cadherin and ß-catenin expression and suggests that these three markers are associated with the invasive transformation. The presence of robust endothelial structures in invHMs could also explain their ability to maintain organized villous architecture (contrary to metastatic choriocarcinomas) during their invasion of extrauterine tissues such as the lung or the brain after dissemination through the blood flow. In our hands, Lewis x appeared to be a new, reliable marker that can be used to clearly distinguish invHMs from choriocarcinomas.

Comparison of cardiomyogenic potential among human ESC and iPSC lines.

We recently reported that, following induction of clumps of pluripotent H1 human embryonic stem cells (hESCs) with activin-A and Bmp4 in defined medium for 5 days, widespread differentiation of rhythmically contracting cardiomyocytes occurs within 3-4 weeks. In this study, the same approach was used to assess whether human induced pluripotent stem cells (hiPSCs), which may theoretically provide an unlimited source of patient-matched cells for transplantation therapy, can similarly undergo cardiomyocyte differentiation. Differentiation of four pluripotent cell lines (H1 and H9 hESCs and C2a and C6a hiPSCs) was compared in parallel by monitoring rhythmic contraction, morphologic differentiation, and expression of cardiomyogenic genes. Based on expression of the cardiomyogenic lineage markers MESP1, ISL1, and NKX2-5, all four cell lines were induced into the cardiomyogenic lineage. However, in contrast to the widespread appearance of striations and rhythmic contractility seen in H9 and especially in H1 hESCs, both hiPSC lines exhibited poor terminal differentiation. These findings suggest that refined modes of generating hiPSCs, as well as of inducing cardiomyogenesis in them, may be required to fulfill their potential as agents of cardiac regeneration.

JD induced pluripotent stem cell-derived hepatocytes faithfully recapitulate the pathophysiology of familial hypercholesterolemia.

UNLABELLED: Elevated levels of low-density lipoprotein cholesterol (LDL-C) in plasma are a major contributor to cardiovascular disease, which is the leading cause of death worldwide. Genome-wide association studies (GWAS) have identified 95 loci that associate with control of lipid/cholesterol metabolism. Although GWAS results are highly provocative, direct analyses of the contribution of specific allelic variations in regulating LDL-C has been challenging due to the difficulty in accessing appropriate cells from affected patients. The primary cell type responsible for controlling cholesterol and lipid flux is the hepatocyte. Recently, we have shown that cells with hepatocyte characteristics can be generated from human induced pluripotent stem cells (iPSCs). This finding raises the possibility of using patient-specific iPSC-derived hepatocytes to study the functional contribution of GWAS loci in regulating lipid metabolism. To test the validity of this approach, we produced iPSCs from JD a patient with mutations in the low-density lipoprotein receptor (LDLR) gene that result in familial hypercholesterolemia (FH). We demonstrate that (1) hepatocytes can be efficiently generated from FH iPSCs; (2) in contrast to control cells, FH iPSC-derived hepatocytes are deficient in LDL-C uptake; (3) control but not FH iPSC-derived hepatocytes increase LDL uptake in response to lovastatin; and (4) FH iPSC-derived hepatocytes display a marked elevation in secretion of lipidated apolipoprotein B-100. CONCLUSION: Cumulatively, these findings demonstrate that FH iPSC-derived hepatocytes recapitulate the complex pathophysiology of FH in culture. These results also establish that patient-specific iPSC-derived hepatocytes could be used to definitively determine the functional contribution of allelic variation in regulating lipid and cholesterol metabolism and could potentially provide a platform for the identification of novel treatments of cardiovascular disease. (HEPATOLOGY 2012).

Induction of cardiomyogenesis in human embryonic stem cells by human embryonic stem cell-derived definitive endoderm.

We previously reported that chick anterolateral endoderm (AL endoderm) induces cardiomyogenesis in mouse embryoid bodies. However, the requirement to micro-dissect AL endoderm from gastrulation-stage embryos precludes its use to identify novel cardiomyogenic factors, or to scale up cardiomyocyte numbers for therapeutic experiments. To circumvent this problem we have addressed whether human definitive endoderm (hDE) cells, which can be efficiently generated in large numbers from human embryonic stem cells (hESCs), can mimic the ability of AL endoderm to induce cardiac myogenesis. Results demonstrate that both hDE cells and medium conditioned by them induce cardiac myogenesis in pluripotent hESCs, as indicated by rhythmic beating and immunohistochemical/quantitative polymerase chain reaction monitoring of marker gene expression. The cardiomyogenic effect of hDE is enhanced when pluripotent hESCs are preinduced to the mes-endoderm state. Because this approach is tractable and scalable, it may facilitate identification of novel hDE-secreted factors for inclusion in defined cardiomyogenic cocktails.

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

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.