Expanding the Boundaries of Embryonic Stem Cells

The boundaries of embryonic stem cell (ESC) research have extended considerably in recent years in several important ways. Alongside a deeper understanding of the pluripotent state, ESCs have been successfully integrated into various fields, such as genomics, epigenetics, and disease modeling. Significant progress in cell fate control has pushed directed differentiation and tissue engineering further than ever before and promoted clinical trials. The geographical distribution of research activity has also expanded, especially for human ESCs. This review outlines these developments and future challenges that remain

TeratoScore: Assessing the Differentiation Potential of Human Pluripotent Stem Cells by Quantitative Expression Analysis of Teratomas

SummaryTeratoma formation is the gold standard assay for testing the capacity of human pluripotent stem cells to differentiate into all embryonic germ layers. Although widely used, little effort has been made to transform this qualitative assay into a quantitative one. Using gene expression data from a wide variety of cells, we created a scorecard representing tissues from all germ layers and extraembryonic tissues. TeratoScore, an online, open-source platform based on this scorecard, distinguishes pluripotent stem cell-derived teratomas from malignant tumors, translating cell potency into a quantitative measure (http://benvenisty.huji.ac.il/teratoscore.php). The teratomas used for the algorithm also allowed us to examine gene expression differences between tumors with a diploid karyotype and those initiated by aneuploid cells. Chromosomally aberrant teratomas show a significantly different gene expression signature from that of teratomas originating from diploid cells, particularly in central nervous system-specific genes, congruent with human chromosomal syndromes

Identification of Novel Imprinted Differentially Methylated Regions by Global Analysis of Human-Parthenogenetic-Induced Pluripotent Stem Cells

Parental imprinting is an epigenetic phenomenon by which genes are expressed in a monoallelic fashion, according to their parent of origin. DNA methylation is considered the hallmark mechanism regulating parental imprinting. To identify imprinted differentially methylated regions (DMRs), we compared the DNA methylation status between multiple normal and parthenogenetic human pluripotent stem cells (PSCs) by performing reduced representation bisulfite sequencing. Our analysis identified over 20 previously unknown imprinted DMRs in addition to the known DMRs. These include DMRs in loci associated with human disorders, and a class of intergenic DMRs that do not seem to be related to gene expression. Furthermore, the study showed some DMRs to be unstable, liable to differentiation or reprogramming. A comprehensive comparison between mouse and human DMRs identified almost half of the imprinted DMRs to be species specific. Taken together, our data map novel DMRs in the human genome, their evolutionary conservation, and relation to gene expression

Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells

AbstractHuman embryonic stem (ES) cells are pluripotent cell lines that have been derived from the inner cell mass (ICM) of blastocyst stage embryos [1–3]. They are characterized by their ability to be propagated indefinitely in culture as undifferentiated cells with a normal karyotype and can be induced to differentiate in vitro into various cell types [1, 2, 4–6]. Thus, human ES cells promise to serve as an unlimited cell source for transplantation. However, these unique cell lines tend to spontaneously differentiate in culture and therefore are difficult to maintain. Furthermore, colonies may contain several cell types and may be composed of cells other than pluripotent cells [1, 2, 6]. In order to overcome these difficulties and establish lines of cells with an undifferentiated phenotype, we have introduced a reporter gene that is regulated by a promoter of an ES cell-enriched gene into the cells. For the introduction of DNA into human ES cells, we have established a specific transfection protocol that is different from the one used for murine ES cells. Human ES cells were transfected with enhanced green fluorescence protein (EGFP), under the control of murine Rex1 promoter. The transfected cells show high levels of GFP expression when in an undifferentiated state. As the cells differentiate, this expression is dramatically reduced in monolayer cultures as well as in the primitive endoderm of early stage (simple) embryoid bodies (EBs) and in mature EBs. The undifferentiated cells expressing GFP can be analyzed and sorted by using a Fluorescence Activated Cell Sorter (FACS). Thus, we have established lines of human ES cells in which only undifferentiated cells are fluorescent, and these cells can be followed and selected for in culture. We also propose that the pluripotent nature of the culture is made evident by the ability of the homogeneous cell population to form EBs. The ability to efficiently transfect human ES cells will provide the means to study and manipulate these cells for the purpose of basic and applied research

Reversion of FMR1 Methylation and Silencing by Editing the Triplet Repeats in Fragile X iPSC-Derived Neurons

SummaryFragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from a CGG repeat expansion in the fragile X mental retardation 1 (FMR1) gene. Here, we report a strategy for CGG repeat correction using CRISPR/Cas9 for targeted deletion in both embryonic stem cells and induced pluripotent stem cells derived from FXS patients. Following gene correction in FXS induced pluripotent stem cells, FMR1 expression was restored and sustained in neural precursor cells and mature neurons. Strikingly, after removal of the CGG repeats, the upstream CpG island of the FMR1 promoter showed extensive demethylation, an open chromatin state, and transcription initiation. These results suggest a silencing maintenance mechanism for the FMR1 promoter that is dependent on the existence of the CGG repeat expansion. Our strategy for deletion of trinucleotide repeats provides further insights into the molecular mechanisms of FXS and future therapies of trinucleotide repeat disorders

Publishing SNP Genotypes of Human Embryonic Stem Cell Lines: Policy Statement of the International Stem Cell Forum Ethics Working Party

Novel methods and associated tools permitting individual identification in publicly accessible SNP databases have become a debatable issue. There is growing concern that current technical and ethical safeguards to protect the identities of donors could be insufficient. In the context of human embryonic stem cell research, there are no studies focusing on the probability that an hESC line donor could be identified by analyzing published SNP profiles and associated genotypic and phenotypic information. We present the International Stem Cell Forum (ISCF) Ethics Working Party’s Policy Statement on “Publishing SNP Genotypes of Human Embryonic Stem Cell Lines (hESC)”. The Statement prospectively addresses issues surrounding the publication of genotypic data and associated annotations of hESC lines in open access databases. It proposes a balanced approach between the goals of open science and data sharing with the respect for fundamental bioethical principles (autonomy, privacy, beneficence, justice and research merit and integrity)

Assessing the Safety of Human Pluripotent Stem Cells and Their Derivatives for Clinical Applications

Pluripotent stem cells may acquire genetic and epigenetic variants during culture following their derivation. At a conference organized by the International Stem Cell Initiative, and held at The Jackson Laboratory, Bar Harbor, Maine, October 2016, participants discussed how the appearance of such variants can be monitored and minimized and, crucially, how their significance for the safety of therapeutic applications of these cells can be assessed. A strong recommendation from the meeting was that an international advisory group should be set up to review the genetic and epigenetic changes observed in human pluripotent stem cell lines and establish a framework for evaluating the risks that they may pose for clinical use.Funding for The International Stem Cell Initiative and for this meeting were provided by the International Stem Cell Forum (http://www.stem-cell-forum.net). Additional support for the meeting was provided by the Pluripotent Stem Cell Platform, a Hub funded by the UK Regenerative Medicine Platform, grant ref. MR/L012537/1, and also by Ajinomoto Inc., Stem Cell Technologies Inc. and Thermo Fisher Scientific. B.R. is founder, chief scientific officer, shareholder, and has interests in Cell Cure Neurosciences Ltd

Assessing the Safety of Stem Cell Therapeutics

Unprecedented developments in stem cell research herald a new era of hope and expectation for novel therapies. However, they also present a major challenge for regulators since safety assessment criteria, designed for conventional agents, are largely inappropriate for cell-based therapies. This article aims to set out the safety issues pertaining to novel stem cell-derived treatments, to identify knowledge gaps that require further research, and to suggest a roadmap for developing safety assessment criteria. It is essential that regulators, pharmaceutical providers, and safety scientists work together to frame new safety guidelines, based on “acceptable risk,” so that patients are adequately protected but the safety “bar” is not set so high that exciting new treatments are lost

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