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Pluripotent Stem Cells

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hES say G1-yeS!

By Stuart P. Atkinson

The potential applications of human embryonic stem cells (hESC) in regenerative medicine are far reaching, but the need for large numbers of these cells requires prolonged in vitro culture which can lead to the accumulation of unwanted genetic changes and potentially, tumorigenicity.

US give Geron Go-ahead for ESC Trial for Spinal Cord Injury

Widely reported

The US Food and Drug Administration (FDA) has authorised Geron to carry out the world's first clinical trial using human embryonic stem cells (hESC) in patients with acute spinal cord injury. GRNOPC1 contains hESC-derived oligodendrocyte progenitor cells that have been shown to have the ability to restore function in animal models of acute spinal cord injury (Keirstead et al.). The Phase I clinical trial won initial approval in January last year but was delayed due to safety concerns raised in animal studies in which a higher frequency of small cysts was observed within the injury site in the spinal cord of animals injected than in other studies. However, further studies in rats addressed these safety concerns and Geron now plans to test the therapy on around 10 volunteer patients paralysed by spinal cord injuries. The initial phase will be testing for safety and Geron has selected up to seven U.S. medical centres as candidates to participate in this study and in planned protocol extensions.

Sources

Daily Telegraph (UK)

Financial Times (UK)

Medical News Today

Associated Press

Keirstead, H.S., G. Nistor, G. Bernal, M. Totoiu, F. Cloutier, K. Sharp, and O. Steward, Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci, 2005. 25(19): p. 4694-705.

iPSC’s “Memory” Suggests Limited Use Compared to ESC

Two advance articles in Nature and Nature Biotechnology from the labs of Konrad Hochedlinger, George Daley and Andrew Feinberg have suggested that iPSC retain a “memory” of their initial state, and therefore may be of limited use in regenerative medicine. When studying murine iPSC, it was found that the epigenetic profile of the mouse donor cell wasn’t entirely “erased” and the differentiation potential of the iPSC was linked to the cell of origin. Further analysis of this type in human iPSC may decide whether iPSC technology as we know it will be a suitable replacement for hESC derivation.

Also see the following news items from BusinessWeek, Nature and The-Scientist.

Human iPSC Generated from Peripheral Blood Cells

Human iPSC Generated from Peripheral Blood Cells

From Cell Stem Cell and Stem Cells and Development

Articles in the new edition of Cell Stem Cell and an online advance report from Stem Cells and Development report the production of human induced pluripotent stem cells (iPSCs) from peripheral blood samples, suggesting another potentially easier target cell for reprogramming, rather than cells from skin biopsies which require invasive collection techniques and long culture periods before use.   Navigate to the papers from Seki et al, Loh et al, Staerk et al and Kunisato et al by clicking on the hyperlinks.   Also see here for a preview of this work by Shinya Yamanaka.

 

Stem cells made without new genes

From Nature News

Nature News reports that a group from University of Glasgow, UK, has created human pluripotent cells using a virus alone.   These findings, from Andrew Baker and his colleague Nicole Kane, were reported at the International Society for Stem Cell Research annual meeting in San Francisco, California.   Read more from Nature here.

 

 

 

 

 

 

Leap forward for LEOPARD syndrome patients

By Maria H. Ledran

Researchers, overseen by Bruce Gelb and Ihor Lemischka at the Mount Sinai School of Medicine (New York), have described the derivation of two separate induced pluripotent stem cell (iPSC) lines from LEOPARD syndrome patients.  LEOPARD syndrome is a multifaceted autosomal-dominant developmental disease (closely related to Noonan syndrome), which can result in facial dysmorphia, growth retardation, cardiac anomalies and deafness, among a plethora of other potential clinical features.  The break-though, reported in the June 9th issue of Nature, describes how, for the first time, stem cells have been differentiated in vitro into cells with a cardiomyopathay – that is, with a cardiac disease phenotype.  The vast majority of LEOPARD syndrome cases, and about half of Noonan syndrome cases result from missense mutations in the ubiquitously expressed PTPN11 gene, which encodes the protein tyrosine phosphatise SHP2 – a crucial regulator of normal development.  These mutations have been shown to result in a gain of function phenotype, by destabilising the catalytically inactive protein conformation, and thus inhibiting growth factor induced ERK1/2 signalling.  Additionally a distinct class of somatic PTPN11 mutations contribute to juvenile leukaemogenesis.  Despite the description of animal models of LEOPARD syndrome in both Drosophila and zebrafish, a detailed molecular basis of the disease phenotypes has remained obscure, but the group hopes that these disease specific cells might be able to contribute fresh insights.

Approximately 80% of LEOPARD syndrome patients exhibit hypertrophic cardiomyopathy: thickening of heart cardiomyocytes resulting from increased cell size with an enlarged sarcomeric (contractile) protein component.  Infact this aspect of the disease is the most life threatening, because of the association with cardiac arrhythmia and sudden death.  The researchers revealed that the cardiomyocytes differentiated from LEOPARD iPSCs showed a perturbed phenotype – with a significantly increased average cell size and increased sarcomere assembly – essentially recapitulating the disease state in vitro.  Upon further investigation it also became apparent that the transcription factor NFAT4 was nearly three times as likely to be localised in the nucleus of LEOPARD syndrome iPSCs compared to wild type iPSCs – highlighting the importance of the calcineurin-NFAT pathway in the regulation of cardiac hypertrophy.  Moreover, phosphorylation of EGFR and MEK1 proteins was significantly increased in LEOPARD syndrome iPSCs, making a promising future line of investigation, given that MEK1 is the upstream kinase of ERK1/2 (the protein known to be perturbed in this disease), and since RAS-mitogen-activated kinase (MAPK) is the major deregulated signalling pathway in SHP2 mutants.  Basal levels of phosphorylated ERK were increased in LEOPARD iPSCs, and giving further confidence to these disease specific stem cells as an in vitro disease model, receptor tyrosine kinase stimulation failed to increase activation of ERK, mimicking results previously obtained in an alternative SHP2 mutated model.  Taken together these findings offer insight into a previously inpenetrateable problem underlying the molecular basis of the disease phenotype and potentially implicate the perturbation of RAS-MAPK signalling from the earliest stem cell origins.

 

Of principal importance to this study and the majority of stem cell based research, is ease of access to theoretically unlimited numbers of disease affected stem cells and their derivatives: in this case of particular value when studying cardiac related phenotypes since access to diseased human heart is very limited, and cardiac cell types do not readily proliferate in vitro.  Indeed amongst the most eagerly anticipated applications of iPSC technologies include the prospects of using these types of diseased and normal cells in the large scale screening for novel drugs, for therapeutic treatment strategies and for illuminating underlying disease pathogenesis.  For instance in the case of LEOPARD syndrome this could include the identification of molecules that could block the overgrowth of cardiomyocytes, an application with a wider potential benefits in the treatment of all cardiomyopic disease.

The description of LEOPARD patient specific iPSCs also contributes to a growing body of work initiated by the first description of cellular reprogramming by Shinya Yamanaka in 2007.  Indeed, in the week that Yamanaka has  been honoured with yet another prize in recognition of his achievements (the 26th annual Kyoto Prize in Advanced Technology), it is undoubted that the insights that iPSCs already gained and likely ahead are phenomenal.  However, as the researchers behind the generation of LEOPARD iPSCs themselves note, there are still many important hurdles to overcome with this and other pluripotent cell technologies.  For instance in the case of LEOPARD iPSCs, differentiation efficiency was variable and the group was unable to generate a sufficiently pure population of cardiac cells to fully asses essential disease characteristics such as the reactivation of fetal gene programs and the assessment of cardiac protein synthesis rates.  Nonetheless, without a doubt the continued generation of patient specific iPSCs will be of chief concern for many labs around the world and is likely to give fresh insight into many disease types that would otherwise be much more difficult to study.

Dual AID for Reprogramming and DNA Methylation

By Stuart P. Atkinson

Although induced pluripotent stem cell (iPSC) generation continues to forge ahead, the early molecular mechanisms behind the reprogramming process remain poorly understood. In an attempt to understand these mechanisms, Bhutani and colleagues utilised heterokaryon formation as their model system rather than iPSC generation or somatic cell nuclear transfer (SCNT), which represent more complex, time consuming systems. The authors generated heterokaryons by fusing mouse embryonic stem cells (mESC) with a GFP reporter gene and human fibroblasts (hFFs) with a DsRed reporter gene, generating a dual-colour heterokaryon that can be sorted to high purity for analysis. In this system, the pluripotent genetic program dominates, and therefore should have an affect on the somatic human genetic program. In this way, generation of an interspecies heterokaryon may allow for reprogramming in the presence of all pluripotency factors, as supplied by the mESC genome, and synchronous initiation of reprogramming. Further, species-specific mRNA and promoter analysis allows the distinction between specific activities occurring in the mESCs and the hFFs nucleus.

Human iPSC Generation – Nothing Common in “Site” for Lentiviral Integration

by Stuart P. Atkinson

Traditional techniques for the generation of induced pluripotent stem cells (iPSCs) require the use of integrating viral particles to deliver the required factors for efficient reprogramming. Reprogramming efficiency, however, lies in the region of only 0.02 to 1% of target cells, and this falls to much lower levels when non-integrational means of reprogramming factor delivery or expression are used. The low efficiency observed with integrational delivery suggests that other factors are involved in the reprogramming process, whilst the reduction of efficiency with non-integrational methods suggests that integration of the viral vectors into the host genome may be important. This may be due to prolonged expression of integrated vectors or the lack of dilution of the vectors upon cell proliferation. Another theory is that insertion of the viral vectors into the host genome somehow enhances the efficiency of reprogramming by perturbation of nearby gene expression. This would suggest that within each target cell type, iPSCs may have common viral integration sites.

It’s DNA methylation Jim, but Not As We Know It!

By Stuart P. Atkinson

DNA methylation is an epigenetic mechanism which allows the regulation of gene expression, genome structure and genome stability. Current dogma dictates that DNA methylation mainly occurs on cytosines at CG di-nucleotides, which are found clustered together forming dense patches within the genome. Such regions are known as CpG islands and usually coincide with regulatory regions in promoter sequences, with methylation of cytosines within these regions positively correlated to gene repression. Previous studies of DNA methylation status of various cell types have provided snapshots of this epigenetic modification having mainly concerned themselves with analysing the DNA methylation status of these CpG islands.

Alarm bells for iPSC?

by Lyle Armstrong

Induced pluripotent Stem Cells (iPSC) are widely believed to share many of the characteristics of Embryonic Stem Cells (ESC) and as such have been credited with the potential to revolutionise regenerative medicine. The potential benefit of iPSC exists because of their genetic similarity to the individual from whom they were derived, implying that if differentiated and clinically useful cell types produced from iPSC were transplanted back into the individual, the likelihood of immune rejection should be greatly reduced.

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