Neta Lavon

Induced Pluripotent Stem Cells and Embryonic Stem Cells Are Distinguished by Gene Expression Signatures

Induced pluripotent stem cells (iPSCs) outwardly appear to be indistinguishable from embryonic stem cells (ESCs). A study of gene expression profiles of mouse and human ESCs and iPSCs suggests that, while iPSCs are quite similar to their embryonic counterparts, a recurrent gene expression signature appears in iPSCs regardless of their origin or the method by which they were generated. Upon extended culture, hiPSCs adopt a gene expression profile more similar to hESCs; however, they still retain a gene expression signature unique from hESCs that extends to miRNA expression. Genome-wide data suggested that the iPSC signature gene expression differences are due to differential promoter binding by the reprogramming factors. High-resolution array profiling demonstrated that there is no common specific subkaryotypic alteration that is required for reprogramming and that reprogramming does not lead to genomic instability. Together, these data suggest that iPSCs should be considered a unique subtype of pluripotent cell.

Identification and Classification of Chromosomal Aberrations in Human Induced Pluripotent Stem Cells

Because of their somatic cell origin, human induced pluripotent stem cells (HiPSCs) are assumed to carry a normal diploid genome, and adaptive chromosomal aberrations have not been fully evaluated. Here, we analyzed the chromosomal integrity of 66 HiPSC and 38 human embryonic stem cell (HESC) samples from 18 different studies by global gene expression meta-analysis. We report identification of a substantial number of cell lines carrying full and partial chromosomal aberrations, half of which were validated at the DNA level. Several aberrations resulted from culture adaptation, and others are suspected to originate from the parent somatic cell. Our classification revealed a third type of aneuploidy already evident in early passage HiPSCs, suggesting considerable selective pressure during the reprogramming process. The analysis indicated high incidence of chromosome 12 duplications, resulting in significant enrichment for cell cycle-related genes. Such aneuploidy may limit the differentiation capacity and increase the tumorigenicity of HiPSCs.

Study of hepatocyte differentiation using embryonic stem cells.

The liver has many crucial functions including metabolizing dietary molecules, detoxifying compounds, and storing glycogen. The hepatocytes, comprising most of the liver organ, progressively modify their gene expression profile during the fetal development according to their roles in the different phases of development. Embryonic stem (ES) cells serve as a major tool in understanding liver development. These cells may also serve as a source of hepatic cells for cellular therapy. In this review, we aim to summarize the research that has been performed in the field of hepatocyte differentiation from mouse and human ES cells. We discuss the various methodologies for the differentiation of ES cells towards hepatic cells using either spontaneous or directed differentiation protocols. Although many protocols for differentiating ES cells to hepatic cells have been developed, the analysis of their status is not trivial and can lead to various conclusions. Hence, we discuss the issues of analyzing hepatocytes by means of the specificity of the markers for hepatocytes and the status of the cells as fetal or adult hepatocytes.

The Effect of Overexpression of Pdx1 and Foxa2 on the Differentiation of Human Embryonic Stem Cells into Pancreatic Cells

Human embryonic stem cells (HESCs) are pluripotent cells that may serve as a source of cells for transplantation medicine and as a tool to study human embryogenesis. Using genetic manipulation methodologies, we have investigated the potential of HESCs to differentiate into the various pancreatic cell types. We initially created various HESCs carrying the enhanced green fluorescent protein (eGFP) reporter gene under the control of either the insulin promoter or the pancreatic and duodenal homeobox factor‐1 (Pdx1) promoter. Our analysis revealed that during the differentiation of HESCs into embryoid bodies (EBs), we could detect green fluorescent cells when eGFP is regulated by Pdx1 promoter but not by insulin promoter. To examine whether we can induce differentiation into pancreatic cells, we have established human embryonic stem cell lines that constitutively express either Pdx1 or the endodermal transcription factor Foxa2. Following differentiation into EBs, the constitutive expression of Pdx1 enhanced the differentiation of HESCs toward pancreatic endocrine and exocrine cell types. Thus, we have demonstrated expression of several transcription factors that are downstream of Pdx1 and various molecular markers for the different pancreatic cell types. However, the expression of the insulin gene could be demonstrated only when the cells differentiated in vivo into teratomas. We conclude that although overexpression of Pdx1 enhanced expression of pancreatic enriched genes, induction of insulin expression may require additional signals that are only present in vivo.

Human Embryonic Stem Cells as Models for Aneuploid Chromosomal Syndromes

Syndromes caused by chromosomal aneuploidies are widely recognized genetic disorders in humans and often lead to spontaneous miscarriage. Preimplantation genetic screening is used to detect chromosomal aneuploidies in early embryos. Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may serve as models for human syndromes caused by aneuploidies. We have established 25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro development. The hESC lines exhibited morphology and expressed markers typical of hESCs. They demonstrated long‐term proliferation capacity and pluripotent differentiation. Karyotype analysis revealed that two‐third of the cell lines carry a normal euploid karyotype, while one‐third remained aneuploid throughout the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X (Turner syndrome). On the basis of the level of single nucleotide polymorphism heterozygosity in the aneuploid chromosomes, we determined whether the aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene expression profiles of the trisomic cell lines suggested that all three chromosomes are actively transcribed. Our analysis allowed us to determine which tissues are most affected by the presence of a third copy of either chromosome 13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic development. The results presented here suggest that aneuploid embryos can serve as an alternative source for either normal euploid or aneuploid hESC lines, which represent an invaluable tool to study developmental aspects of chromosomal abnormalities in humans.STEM CELLS 2010; 28:1530–1540.

Derivation of Euploid Human Embryonic Stem Cells from Aneuploid Embryos

Human embryonic stem cells (HESCs) are pluripotent cells derived from the inner cell mass of preimplantation embryos. In this study, to isolate new lines of HESCs, we used blastocyst‐stage embryos diagnosed as aneuploid in preimplantation genetic screening (PGS). During in vitro fertilization treatments, PGS is widely applied to identify chromosomal aneuploidies, especially in cases of advanced maternal age. Embryos that are detected as carrying aneuploidies are destined to be discarded unless donated for research. From 74 fresh PGS‐defined aneuploid embryos, we derived seven HESC lines. Most of the embryos were left to hatch spontaneously through the hole created for blastomere biopsy and further treated by immunosurgery. The seven HESC lines exhibited morphology and markers typical of HESCs and the capacity for long‐term proliferation. The derived HESC lines manifested pluripotent differentiation potential both in vivo and in vitro. Surprisingly, karyotype analysis of the HESC lines that were derived from these aneuploid embryos showed that the cell lines carry a normal euploid karyotype. We show that the euploidy was not achieved through chromosome duplication. Alternatively, we suggest that the euploid HESC lines originated from mosaic embryos consisting of aneuploid and euploid cells, and in vitro selection occurred to favor euploid cells. We assume that aneuploid HESC lines could be isolated mostly from embryos that are uniform for the aneuploidy. These results led us to conclude that the aneuploid mosaic embryos that are destined to be discarded can serve as an alternative source for normal euploid HESC lines.

Variations of X Chromosome Inactivation Occur in Early Passages of Female Human Embryonic Stem Cells

X chromosome inactivation (XCI) is a dosage compensation mechanism essential for embryonic development and cell physiology. Human embryonic stem cells (hESCs) derived from inner cell mass (ICM) of blastocyst stage embryos have been used as a model system to understand XCI initiation and maintenance. Previous studies of undifferentiated female hESCs at intermediate passages have shown three possible states of XCI; 1) cells in a pre-XCI state, 2) cells that already exhibit XCI, or 3) cells that never undergo XCI even upon differentiation. In this study, XCI status was assayed in ten female hESC lines between passage 5 and 15 to determine whether XCI variations occur in early passages of hESCs. Our results show that three different states of XCI already exist in the early passages of hESC. In addition, we observe one cell line with skewed XCI and preferential expression of X-linked genes from the paternal allele, while another cell line exhibits random XCI. Skewed XCI in undifferentiated hESCs may be due to clonal selection in culture instead of non-random XCI in ICM cells. We also found that XIST promoter methylation is correlated with silencing of XIST transcripts in early passages of hESCs, even in the pre-XCI state. In conclusion, XCI variations already take place in early passages of hESCs, which may be a consequence of in vitro culture selection during the derivation process. Nevertheless, we cannot rule out the possibility that XCI variations in hESCs may reflect heterogeneous XCI states in ICM cells that stochastically give rise to hESCs.

Human embryonic stem cells from aneuploid blastocysts identified by pre-implantation genetic screening

Human embryonic stem cells are derived from the inner cell mass of pre-implantation embryos. The cells have unlimited proliferation potential and capacity to differentiate into the cells of the three germ layers. Human embryonic stem cells are used to study human embryogenesis and disease modeling and may in the future serve as cells for cell therapy and drug screening. Human embryonic stem cells are usually isolated from surplus normal frozen embryos and were suggested to be isolated from diseased embryos detected by pre-implantation genetic diagnosis. Here we report the isolation of 12 human embryonic stem cell lines and their thorough characterization. The lines were derived from embryos detected to have aneuploidy by pre-implantation genetic screening. Karyotype analysis of these cell lines showed that they are euploid, having 46 chromosomes. Our interpretation is that the euploid cells originated from mosaic embryos, and in vitro selection favored the euploid cells. The undifferentiated cells exhibited long-term proliferation and expressed markers typical for embryonic stem cells such as OCT4, NANOG, and TRA-1-60. The cells manifested pluripotent differentiation both in vivo and in vitro. To further characterize the different lines, we have analyzed their ethnic origin and the family relatedness among them. The above results led us to conclude that the aneuploid mosaic embryos that are destined to be discarded can serve as source for normal euploid human embryonic stem cell lines. These lines represent various ethnic groups; more lines are needed to represent all populations.

Generation of Hepatocytes from Human Embryonic Stem Cells

Human embryonic stem cells (HESCs) are pluripotent cells having a self-renewal capacity. These unique characteristics of HESCs allow them to be an unlimited source of cells that was shown to differentiate into many cell types, among them hepatocytes. The creation of hepatocytes in culture will allow us to further understand the mechanisms involved in the embryogenesis of hepatocytes in humans and to study pathologies related to aberrant differentiation of these cells. The resultant hepatocytes may serve as a source of cells for transplantation and as cells for toxicological studies and drug screening. In the past 10 years, since the derivation of HESCs, various protocols for the differentiation of HESCs to hepatic-like cells were published. In this chapter we detail our protocol for differentiating HESCs into hepatic-like cells through embryoid bodies. We further describe the method for the genetic labeling of the hepatic-like cells derived from the HESCs and their isolation by fluorescence-activated cell sorter. We also summarize the published protocols for differentiation of HESCs into hepatic-like cells.

Derivation, expansion, and characterization of human embryonic stem cell lines from aneuploid embryos.

Human embryonic stem cells (hESCs) are an invaluable cell source to study human embryogenesis and development and for exploring the nature of human diseases. Moreover, hESCs can serve as an unlimited source of cells for cell therapy. The first hESC lines were derived from frozen blastocyst-stage embryos. In the past 12 years, the field evolved and the hESC lines are derived from pre-embryos in various developmental stages using several techniques. In parallel, the wide use of hESCs triggered the development of materials and methods for expansion of the cell lines derived. Here, we describe our method for derivation, expansion, and characterization of hESC lines from pre-embryos that were diagnosed to carry aneuploid cells and were destined to be discarded.