Showing papers by "Peter W. Andrews published in 2014"

Aneuploidy induces profound changes in gene expression, proliferation and tumorigenicity of human pluripotent stem cells

Abstract: Human pluripotent stem cells (hPSCs) tend to acquire genomic aberrations in culture, the most common of which is trisomy of chromosome 12. Here we dissect the cellular and molecular implications of this trisomy in hPSCs. Global gene expression analyses reveal that trisomy 12 profoundly affects the gene expression profile of hPSCs, inducing a transcriptional programme similar to that of germ cell tumours. Comparison of proliferation, differentiation and apoptosis between diploid and aneuploid hPSCs shows that trisomy 12 significantly increases the proliferation rate of hPSCs, mainly as a consequence of increased replication. Furthermore, trisomy 12 increases the tumorigenicity of hPSCs in vivo, inducing transcriptionally distinct teratomas from which pluripotent cells can be recovered. Last, a chemical screen of 89 anticancer drugs discovers that trisomy 12 raises the sensitivity of hPSCs to several replication inhibitors. Together, these findings demonstrate the extensive effect of trisomy 12 and highlight its perils for successful hPSC applications.

Time-Lapse Analysis of Human Embryonic Stem Cells Reveals Multiple Bottlenecks Restricting Colony Formation and Their Relief upon Culture Adaptation

Abstract: Using time-lapse imaging, we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells (hESCs) and that are relieved by karyotypically abnormal variants that are selected by prolonged culture. Only a minority of karyotypically normal cells divided after plating, and these were mainly cells in the later stages of cell cycle at the time of plating. Furthermore, the daughter cells showed a continued pattern of cell death after division, so that few formed long-term proliferating colonies. These colony-forming cells showed distinct patterns of cell movement. Increasing cell density enhanced cell movement facilitating cell:cell contact, which resulted in increased proportion of dividing cells and improved survival postplating of normal hESCs. In contrast, most of the karyotypically abnormal cells reentered the cell cycle on plating and gave rise to healthy progeny, without the need for cell:cell contacts and independent of their motility patterns.

Harmonizing standards for producing clinical-grade therapies from pluripotent stem cells

Abstract: volume 32 NumBeR 8 AuGuST 2014 nature biotechnology conceived for somatic-cell therapies will have to be modified5,6. For example, use of iPSCs may require special guidance with respect to tumorigenicity, genetic integrity, release assays and sterility/aseptic processes. Confusion will arise if existing guidelines are inappropriately adapted or protocols are inadequately generalized to all cell types7–9. It is important to recognize that producing a clinical-grade, PSC-based therapy involves more than complying with cGMP and CMC manufacturing standards. Additional issues that must be considered include regulations on sourcing of donor tissue, ethical guidelines, intellectual-property law and data sharing. Figure 1 outlines the hurdles that arise at different stages of product development. Many of the issues summarized in Figure 1 remain unresolved. For example, in manufacturing, new reference or control material is needed to generate convincing data on in-process testing, lot-to-lot variability and release assays. Guidelines for tissue collection, ownership and payment for PSC generation are in flux10. Equally important, questions regarding consent for the hundreds of thousands of existing samples that could be used as a source of iPSCs must be addressed. More generally, there are uncertainties in how to extend the regulations and standards of institutional review boards, HIPPA (Health Insurance Portability and Privacy Act) and OHRP (Office for Human Research Protection) to PSC-based therapies10,11. This new class of therapy may also require new interpretations of ethical guidelines12, patent law13,14 and the unique propertyrights issues that arise for cells that can make gametes15. To the Editor: Generating clinical-grade cells from pluripotent stem cells (PSCs) for use in patients is not simply a matter of complying with current good manufacturing practices (cGMPs) and chemistry and manufacturing controls (CMCs). A range of other issues demand careful attention, including accessing tissue in an ethical manner and adhering to the varied rules and regulations of specific local and national jurisdictions. The current patchwork of practices represents a major hindrance to progress in regenerative medicine. We propose the establishment of an international body tasked with developing, evaluating and harmonizing the technical, ethical, legal and regulatory frameworks that govern the production of therapies based on PSCs. All PSC-based therapies involve the in vitro conversion of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into differentiated cells that migrate, integrate, survive and function therapeutically in patients1,2. These therapies will be administered by different routes, alone or in combination with biologic or synthetic materials, for a variety of indications, and will require different final-product formulations. Although no PSC-based therapy has yet been approved, at least six or seven groups have commenced or are planning early-stage clinical trials. Unlike somatic cells, PSCs are immortal and have the potential to make any differentiated cell type. These differences have important consequences at all stages of clinical translation, manufacture and commercialization, including requirements for shipping, tracking and identity specifications. Therapies based on somatic cells (including multipotent stem cells or other nonpluripotent cell types) involve collecting cells from a particular donor followed by limited growing, testing, storing and banking of the cells. Manufacture of somatic cell–based therapies involves myriad challenges, including compliance with cGMP and CMC regulations, scale-up and scale-out, and appropriate in-process testing and sterility and potency assays. But therapies based on PSCs bring additional layers of complexity. The cells must undergo extensive expansion and long differentiation procedures to generate appropriate phenotypes while eliminating unwanted phenotypes, including residual pluripotent cells3,4. The use of iPSCs as an autologous product that may be genetically modified raises further issues related to small lot sizes and lack of a master cell bank as in allogeneic therapies. The unique challenges associated with PSC-based therapies are summarized in Table 1. In our view, the manufacturing challenges specific to these therapies mean that existing cGMP and CMC regulations

Aneuploidy in pluripotent stem cells and implications for cancerous transformation

Abstract: Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation.

Aza-deoxycytidine induces apoptosis or differentiation via DNMT3B and targets embryonal carcinoma cells but not their differentiated derivatives.

Abstract: Aza-deoxycytidine induces apoptosis or differentiation via DNMT3B and targets embryonal carcinoma cells but not their differentiated derivatives

Corrigendum: Harmonizing standards for producing clinical-grade therapies from pluripotent stem cells

Abstract: Nat. Biotechnol. 32, 724–726 (2014); published online 7 August 2014; corrected after print 10 October 2014; 10.1038/nbt.2973 In the version of this article initially published, two author names were misspelled: the correct names are Cavagnaro, not Cavanagro; Feigal not Feigel. In addition, the name for the Health Insurance Portability and Accountability Act (HIPAA) was given as Health Insurance Portability and Privacy Act (HIPPA).