Stem Cells: Nuclear Reprogramming and Therapeutic Applications (Novartis Foundation Symposia)

Nuclear Cloning, Epigenetic Reprogramming, and Cellular Differentiation
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Whether iPS cell-derived neurons will always faithfully recapitulate the same degenerative processes observed in patients and serve as platforms for drug discovery relevant to common late-onset diseases remains to be determined. Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines.

2018 Demystifying Medicine: Use of induced pluripotent stem cells (iPSC) for regenerative medicine

However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem ES or induced pluripotent stem iPS cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines.

This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines. Somatic coding mutations in human induced pluripotent stem cells.

Nobel Prize in Physiology or Medicine 2012

Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem hiPS cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled an estimated six protein-coding point mutations per exome.

The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use. Impracticality of egg donor recruitment in the absence of compensation.

A functionally characterized test set of human induced pluripotent stem cells.

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Nature biotechnology. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate.

Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells ESCs. Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.

Kiskinis E, Eggan K. Progress toward the clinical application of patient-specific pluripotent stem cells. The Journal of clinical investigation. Pubmed: Kiskinis E, Eggan K. Pubmed: Induced pluripotent stem iPS cells are generated by epigenetic reprogramming of somatic cells through the exogenous expression of transcription factors.

Eggan Lab Publications

These cells, just like embryonic stem cells, are likely to have a major impact on regenerative medicine, because they self-renew and retain the potential to be differentiated into all cell types of the human body. In this Review, we describe the current state of iPS cell technology, including approaches by which they are generated and what is known about their biology, and discuss the potential applications of these cells for disease modeling, drug discovery, and, eventually, cell replacement therapy.

Egli D, Eggan K. Recipient cell nuclear factors are required for reprogramming by nuclear transfer. The cell cycle state of the donor and recipient cells, as well as their extent of differentiation, have each been cited as important determinants of reprogramming success. Here, we have used donor and recipient cells at various cell cycle and developmental stages to investigate the importance of these parameters. We found that many stages of the cell cycle were compatible with reprogramming as long as a sufficient supply of essential nuclear factors, such as Brg1, were retained in the recipient cell following enucleation.

Consistent with this conclusion, the increased efficiency of reprogramming when using donor nuclei from embryonic cells could be explained, at least in part, by reintroduction of embryonic nuclear factors along with the donor nucleus. By contrast, cell cycle synchrony between the donor nucleus and the recipient cell was not required at the time of transfer, as long as synchrony was reached by the first mitosis.

Our findings demonstrate the remarkable flexibility of the reprogramming process and support the importance of nuclear transcriptional regulators in mediating reprogramming. Shushing down the epigenetic landscape towards stem cell differentiation. Here, we review the presentations at that meeting and the questions that emerged concerning how a stem cell ;decides' to self-renew or differentiate, what their distinct properties are and how this information can be used to develop novel therapies.

Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2.

In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation. Reprogramming after chromosome transfer into mouse blastomeres.

We have recently shown that fertilized zygotes retain reprogramming activities, suggesting that such activities might also persist in cleavage-stage embryos. Here, we used chromosome transplantation techniques to investigate whether the blastomeres of two-cell-stage mouse embryos can reprogram more differentiated cells.

Embryos produced by this method could be used to derive stem cell lines and also developed to term, generating mosaic "cloned" animals. These results demonstrate that blastomeres retain reprogramming activities and support the notion that discarded human preimplantation embryos may be useful recipients for the production of genetically tailored human embryonic stem cell lines. Optimal timing of inner cell mass isolation increases the efficiency of human embryonic stem cell derivation and allows generation of sibling cell lines.

Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. We report a method to specifically capture an arbitrary subset of genomic targets for single-molecule bisulfite sequencing for digital quantification of DNA methylation at single-nucleotide resolution. To investigate epigenetic differences associated with dedifferentiation, we compared methylation in three human fibroblast lines and eight human pluripotent stem cell lines. Chromosome-wide methylation patterns were similar among all lines studied, but cytosine methylation was slightly more prevalent in the pluripotent cells than in the fibroblasts.

Induced pluripotent stem iPS cells appeared to display more methylation than embryonic stem cells. We found regions methylated differently in fibroblasts and pluripotent cells. This targeted approach should be particularly useful for analyzing DNA methylation in large genomes. A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog. However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells.

These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming.

We show that one of these molecules functions in reprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog.

Digital RNA allelotyping reveals tissue-specific and allele-specific gene expression in human. This method involves ultra-deep sequencing of padlock-captured single-nucleotide polymorphisms SNPs from the transcriptome. We characterized four cell lines established from two human subjects in the Personal Genome Project. When we applied allelotyping to two pairs of sibling human embryonic stem cell lines, the sibling lines were more similar in allele-specific expression than were the genetically unrelated lines.

We found that the variation of allelic ratios in gene expression among different cell lines was primarily explained by genetic variations, much more so than by specific tissue types or growth conditions. Comparison of expressed SNPs on the sense and antisense transcripts suggested that allelic ratios are primarily determined by cis-regulatory mechanisms on the sense transcripts.

Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation. Although methods for differentiating embryonic stem cells into specific cell types have become increasingly sophisticated, the utility of the resulting cells for modeling disease has not been determined. We have asked whether specific neuronal subtypes produced from human embryonic stem cells can be used to investigate the mechanisms leading to neural degeneration in amyotrophic lateral sclerosis ALS. We show that human spinal motor neurons, but not interneurons, are selectively sensitive to the toxic effect of glial cells carrying an ALS-causing mutation in the SOD1 gene.

Our findings demonstrate the relevance of these non-cell-autonomous effects to human motor neurons and more broadly demonstrate the utility of human embryonic stem cells for studying disease and identifying potential therapeutics. Using stem cells and reprogramming to understand disease. Regenerative medicine.

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Dr Eggan has devoted the last 7 years to performing stem cell research. He is currently leading a research group that is investigating the mechanisms regulating epigenetic reprogramming after somatic cell nuclear transfer and using nuclear transfer techniques to derive disease-specific human embryonic stem cell lines from diabetic and Parkinson's disease patients.

His accomplishments include cloning mice from olfactory sensory neurons, deriving embryonic germ cells and male gametes from embryonic stem cells and characterizing the abnormalities that sometimes arise as a result of nuclear transfer.

Human cloning - Wikipedia

Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science New York, N. These cells could in turn be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. Although recent studies have demonstrated the reprogramming of human fibroblasts to a pluripotent state, it remains unclear whether these induced pluripotent stem iPS cells can be produced directly from elderly patients with chronic disease.

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We have generated iPS cells from an year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis ALS. These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiate into motor neurons, the cell type destroyed in ALS. Mediators of reprogramming: transcription factors and transitions through mitosis. Nature reviews. Molecular cell biology. Consistent with this view, animal cloning studies demonstrated that the intact genome of a differentiated cell can be reprogrammed to support the development of an entire organism and allow the production of pluripotent stem cells.

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Recent progress in reprogramming research now points to an important role for transcription factors in the establishment and the maintenance of cellular phenotypes, and to cell division as a mediator of transitions between different states of gene expression. Short-circuiting epiblast development. Two recent papers describe the derivation of stem cell lines from mouse epiblast that may provide an explanation for the unique character of human embryonic stem cells.

Developmental reprogramming after chromosome transfer into mitotic mouse zygotes. Pubmed: Until now, animal cloning and the production of embryonic stem cell lines by somatic cell nuclear transfer have relied on introducing nuclei into meiotic oocytes. In contrast, attempts at somatic cell nuclear transfer into fertilized interphase zygotes have failed. As a result, it has generally been assumed that unfertilized human oocytes will be required for the generation of tailored human embryonic stem cell lines from patients by somatic cell nuclear transfer.

Here we report, however, that, unlike interphase zygotes, mouse zygotes temporarily arrested in mitosis can support somatic cell reprogramming, the production of embryonic stem cell lines and the full-term development of cloned animals.