Paul J. Gokhale

Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells

We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental.

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

Specific Knockdown of Oct4 and β2‐microglobulin Expression by RNA Interference in Human Embryonic Stem Cells and Embryonic Carcinoma Cells

We have used RNA interference (RNAi) to downregulate β2‐microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not β2‐microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA‐1‐60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.

OCT4 spliced variants are differentially expressed in human pluripotent and nonpluripotent cells.

OCT4 is a master regulator of self‐renewal in embryonic stem cells and can potentially encode two spliced variants, designated OCT4A and OCT4B. We have examined the expression pattern of these OCT4 isoforms in various human pluripotent and nonpluripotent cells. Our data revealed that whereas OCT4A expression is restricted to embryonic stem (ES) and embryonal carcinoma (EC) cells, OCT4B can be detected in various nonpluripotent cell types. Furthermore, we detected a novel OCT4 spliced variant, designated OCT4B1, that is expressed primarily in human ES and EC cells and is downregulated following their differentiation. We also found a significantly higher level of OCT4B1 expression in stage‐specific embryonic antigen‐3 (SSEA3)(+) compared with SSEA3(−) subpopulations of cultured ES cells. Taken together, our data demonstrated a distinctive expression pattern for OCT4 spliced variants in different cell types and highlight the necessity of defining the type of OCT4 when addressing the expression of this gene in different human cells.

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.

Signalling of DNA damage and cytokines across cell barriers exposed to nanoparticles depends on barrier thickness

The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.

Cell‐Cell Signaling Through NOTCH Regulates Human Embryonic Stem Cell Proliferation

Unlike pluripotent mouse embryonic stem (ES) cells, human ES cells and their malignant equivalents, embryonal carcinoma (EC) cells, require close cell‐cell contact for efficient growth. Signaling through the NOTCH receptor, initiated by interaction with ligands of the DELTA/JAGGED family expressed on neighboring cells, plays a role in regulating the self‐renewal of several stem cell systems. Members of the NOTCH and DELTA/JAGGED families are expressed by human EC and ES cells, and we have therefore investigated the possible role of NOTCH in the maintenance of these cells. Cleavage of both NOTCH1 and NOTCH2 to yield the intracellular domain responsible for the canonical signaling pathway of NOTCH was detected in several human EC and ES cell lines, suggesting that NOTCH signaling is active. Furthermore, the proliferation of human EC cells, as well as the expression of several downstream NOTCH target genes, was markedly reduced after small interfering RNA knockdown of NOTCH1, NOTCH2, and the canonical effector CBF‐1 or after blocking NOTCH signaling with the γ‐secretase inhibitor L‐685,458. The inhibitor also caused a reduction in the growth of human ES cells, although without evidence of differentiation. The results indicate that cell‐cell signaling through the NOTCH system provides a critical cue for the proliferation of human EC and ES cell in vitro.

Novel regulators of stem cell fates identified by a multivariate phenotype screen of small compounds on human embryonic stem cell colonies

Understanding the complex mechanisms that govern the fate decisions of human embryonic stem cells (hESCs) is fundamental to their use in cell replacement therapies. The progress of dissecting these mechanisms will be facilitated by the availability of robust high-throughput screening assays on hESCs. In this study, we report an image-based high-content assay for detecting compounds that affect hESC survival or pluripotency. Our assay was designed to detect changes in the phenotype of hESC colonies by quantifying multiple parameters, including the number of cells in a colony, colony area and shape, intensity of nuclear staining, and the percentage of cells in the colony that express a marker of pluripotency (TRA-1-60), as well as the number of colonies per well. We used this assay to screen 1040 compounds from two commercial compound libraries, and identified 17 that promoted differentiation, as well as 5 that promoted survival of hESCs. Among the novel small compounds we identified with activity on hESC are several steroids that promote hESC differentiation and the antihypertensive drug, pinacidil, which affects hESC survival. The analysis of overlapping targets of pinacidil and the other survival compounds revealed that activity of PRK2, ROCK, MNK1, RSK1, and MSK1 kinases may contribute to the survival of hESCs.

High-content screening of small compounds on human embryonic stem cells

Human ES (embryonic stem) cells and iPS (induced pluripotent stem) cells have been heralded as a source of differentiated cells that could be used in the treatment of degenerative diseases, such as Parkinsons disease or diabetes. Despite the great potential for their use in regenerative therapy, the challenge remains to understand the basic biology of these remarkable cells, in order to differentiate them into any functional cell type. Given the scale of the task, high-throughput screening of agents and culture conditions offers one way to accelerate these studies. The screening of small-compound libraries is particularly amenable to such high-throughput methods. Coupled with high-content screening technology that enables simultaneous assessment of multiple cellular features in an automated and quantitative way, this approach is proving powerful in identifying both small molecules as tools for manipulating stem cell fates and novel mechanisms of differentiation not previously associated with stem cell biology. Such screens performed on human ES cells also demonstrate the usefulness of human ES/iPS cells as cellular models for pharmacological testing of drug efficacy and toxicity, possibly a more imminent use of these cells than in regenerative medicine.

Points to consider in the development of seed stocks of pluripotent stem cells for clinical applications: International Stem Cell Banking Initiative (ISCBI)

In 2009 the International Stem Cell Banking Initiative (ISCBI) contributors and the Ethics Working Party of the International Stem Cell Forum published a consensus on principles of best practice for the procurement, cell banking, testing and distribution of human embryonic stem cell (hESC) lines for research purposes [1], which was broadly also applicable to human induced pluripotent stem cell (hiPSC) lines. Here, we revisit this guidance to consider what the requirements would be for delivery of the early seed stocks of stem cell lines intended for clinical applications. The term ‘seed stock’ is used here to describe those cryopreserved stocks of cells established early in the passage history of a pluripotent stem cell line in the lab that derived the line or a stem cell bank, hereafter called the ‘repository’.