Hepatocyte-like cells derived from human embryonic stem cells specifically via definitive endoderm and a progenitor stage.

Human embryonic stem cells offer a potential unlimited supply for functional hepatocytes, since they can differentiate into hepatocyte-like cells displaying a characteristic hepatic morphology and expressing various hepatic markers. These cells could be used in various applications such as studies of drug metabolism and hepatotoxicity, which however, would require a significant expression of drug metabolizing enzymes. To derive these cells we use a stepwise differentiation protocol where growth- and maturation factors are added. The first phase involves the formation of definitive endoderm. Next, these cells are treated with factors known to promote the induction and proliferation towards hepatic progenitor cell types. In the last phase the cells are terminally differentiated and maturated into functional hepatocyte-like cells. The cultures were characterized by analysis of endodermal or hepatic markers and compared to cultures derived without induction via definitive endoderm. Hepatic functions such as urea secretion, glycogen storage, indocyanine green uptake and secretion, and cytochrome P450-expression and activity were evaluated. The DE-Hep showed a hepatocyte morphology with sub-organized cells and exhibited many liver-functions including transporter activity and capacity to metabolize drugs specific for important cytochrome P450 sub-families. This represents an important step in differentiation of hESC into functional hepatocytes.

Transcriptional profiling of human embryonic stem cells differentiating to definitive and primitive endoderm and further toward the hepatic lineage.

Human embryonic stem cells (hESC) can differentiate into a variety of specialized cell types, and they constitute a useful model system to study embryonic development in vitro. In order to fully utilize the potential of these cells, the mechanisms that regulate the developmental processes of specific lineage differentiation need to be better defined. The aim of this study was to explore the molecular program involved in the differentiation of hESC toward definitive endoderm (DE) and further into the hepatic lineage, and to compare that with primitive endoderm (PrE) differentiation. To that end, we applied two protocols: a specific DE differentiation protocol and an intrinsic differentiation protocol that mainly mediates PrE formation. We collected hESC, hESC-derived DE, DE-derived hepatocyte-progenitors (DE-Prog), DE-derived hepatocyte-like cells (DE-Hep), and the corresponding PrE derivatives. The samples were analyzed using microarrays, and we identified sets of genes that were exclusively up-regulated in DE derivatives (compared to PrE derivatives) at discrete developmental stages. We also investigated known protein interactions among the set of up-regulated genes in DE-Hep. The results demonstrate important differences between DE and PrE differentiation on the transcriptional level. In particular, our results identify a unique molecular program, exclusively activated during development of DE and the subsequent differentiation of DE toward the hepatic lineage. We identified key genes and pathways of potential importance for future efforts to improve hepatic differentiation from hESC. These results reveal new opportunities for rational design of specific interventions with the purpose of generating enriched populations of DE derivatives, including functional hepatocytes.

Glutathione transferases in hepatocyte-like cells derived from human embryonic stem cells.

Human embryonic stem cells (hESCs) offer a potential unlimited source for functional human hepatocytes, since hESCs can differentiate into hepatocyte-like cells displaying a characteristic hepatic morphology and expressing several hepatic markers. These hepatocyte-like cells could be used in various human in vitro hepatocyte assays, e.g. as a test system for studying drug metabolism and drug-induced hepatotoxicity. Since the toxic effect of a compound is commonly dependent on biotransformation into metabolites, the presence of drug metabolising enzymes in potential test systems must be evaluated. We have investigated the presence of glutathione transferases (GSTs) in hepatocyte-like cells by immunocytochemistry and Western blotting. Results show that these cells have high levels of GSTA1-1, whereas GSTP1-1 is not present in most cases. GSTM1-1 is detected by immunocytochemistry but not by Western blotting. In addition, GST activity is detected in hepatocyte-like cells at levels comparable to human hepatocytes. These results indicate that the hepatocyte-like cells have characteristics that closely resemble those of human adult hepatocytes.

Clonal derivation and characterization of human embryonic stem cell lines.

Human embryonic stem cells (hESC) are isolated as clusters of cells from the inner cell mass of blastocysts and thus should formally be considered as heterogeneous cell populations. Homogenous hESC cultures can be obtained through subcloning. Here, we report the clonal derivation and characterization of two new hESC lines from the parental cell line SA002 and the previously clonally derived cell line AS034.1, respectively. The hESC line SA002 was recently reported to have an abnormal karyotype (trisomy 13), but within this population of cells we observed rare individual cells with an apparent normal karyotype. At a cloning efficiency of 5%, we established 33 subclones from SA002, out of which one had a diploid karyotype and this subline was designated SA002.5. From AS034.1 we established one reclone designated AS034.1.1 at a cloning efficiency of 0.1%. These two novel sublines express cell surface markers indicative of undifferentiated hESC (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), Oct-4, alkaline phosphatase, and they display high telomerase activity. In addition, the cells are pluripotent and form derivatives of all three embryonic germ layers in vitro as well as in vivo. These results, together with the clonal character of SA002.5 and AS034.1.1 make these homogenous cell populations very useful for hESC based applications in drug development and toxicity testing. In addition, the combination of the parental trisomic hESC line SA002 and the diploid subclone SA002.5 provides a unique experimental system to study the molecular mechanisms underlying the pathologies associated with trisomy 13.

Signals from the embryonic mouse pancreas induce differentiation of human embryonic stem cells into insulin-producing beta-cell-like cells.

The recent success in restoring normoglycemia in type 1 diabetes by islet cell transplantation indicates that cell replacement therapy of this severe disease is achievable. However, the severe lack of donor islets has increased the demand for alternative sources of beta-cells, such as adult and embryonic stem cells. Here, we investigate the potential of human embryonic stem cells (hESCs) to differentiate into beta-cells. Spontaneous differentiation of hESCs under two-dimensional growth conditions resulted in differentiation of Pdx1(+)/Foxa2(+) pancreatic progenitors and Pdx1(+)/Isl1(+) endocrine progenitors but no insulin-producing cells. However, cotransplantation of differentiated hESCs with the dorsal pancreas, but not with the liver or telencephalon, from mouse embryos resulted in differentiation of beta-cell-like cell clusters. Comparative analysis of the basic characteristics of hESC-derived insulin(+) cell clusters with human adult islets demonstrated that the insulin(+) cells share important features with normal beta-cells, such as synthesis (proinsulin) and processing (C-peptide) of insulin and nuclear localization of key beta-cell transcription factors, including Foxa2, Pdx1, and Isl1.

Derivation, characterization, and differentiation of human embryonic stem cells.

The derivation of human embryonic stem (hES) cells establishes a new avenue to approach many issues in human biology and medicine for the first time. To meet the increased demand for characterized hES cell lines, we present the derivation and characterization of six hES cell lines. In addition to the previously described immunosurgery procedure, we were able to propagate the inner cell mass and establish hES cell lines from pronase-treated and hatched blastocysts. The cell lines were extensively characterized by expression analysis of markers characteristic for undifferentiated and differentiated hES cells, karyotyping, telomerase activity measurement, and pluripotency assays in vitro and in vivo. Whereas three of the cell lines expressed all the characteristics of undifferentiated pluripotent hES cells, one cell line carried a chromosome 13 trisomy while maintaining an undifferentiated pluripotent state, and two cell lines, one of which carried a triploid karyotype, exhibited limited pluripotency in vivo. Furthermore, we clonally derived one cell line, which could be propagated in an undifferentiated pluripotent state.

Glial cells generate neurons: the role of the transcription factor Pax6.

Radial glial cells, ubiquitous throughout the developing CNS, guide radially migrating neurons and are the precursors of astrocytes. Recent evidence indicates that radial glial cells also generate neurons in the developing cerebral cortex. Here we investigated the role of the transcription factor Pax6 expressed in cortical radial glia. We showed that radial glial cells isolated from the cortex of Pax6 mutant mice have a reduced neurogenic potential, whereas the neurogenic potential of non-radial glial precursors is not affected. Consistent with defects in only one neurogenic lineage, the number of neurons in the Pax6 mutant cortex in vivo is reduced by half. Conversely, retrovirally mediated Pax6 expression instructs neurogenesis even in astrocytes from postnatal cortex in vitro. These results demonstrated an important role of Pax6 as intrinsic fate determinant of the neurogenic potential of glial cells.