On the Streets of San Francisco: Highlights from the ISSCR Annual Meeting 2010

The 2010 Annual Meeting of the International Society for Stem Cell Research (ISSCR) was held in San Francisco in June with an exciting program covering a wealth of stem cell research from basic science to clinical research

Dynamic single cell imaging of direct reprogramming reveals an early specifying event

available in PMC 2010 November 1.The study of induced pluripotency often relies on experimental approaches that average measurements across a large population of cells, the majority of which do not become pluripotent. Here we used high-resolution, time-lapse imaging to trace the reprogramming process over 2 weeks from single mouse embryonic fibroblasts (MEFs) to pluripotency factor–positive colonies. This enabled us to calculate a normalized cell-of-origin reprogramming efficiency that takes into account only the initial MEFs that respond to form reprogrammed colonies rather than the larger number of final colonies. Furthermore, this retrospective analysis revealed that successfully reprogramming cells undergo a rapid shift in their proliferative rate that coincides with a reduction in cellular area. This event occurs as early as the first cell division and with similar kinetics in all cells that form induced pluripotent stem (iPS) cell colonies. These data contribute to the theoretical modeling of reprogramming and suggest that certain parts of the reprogramming process follow defined rather than stochastic steps.Burroughs Wellcome Fund (Career Award at the Scientific Interface)Pew Charitable TrustsMassachusetts Life Sciences Center (New Investigator grant)Broad Institute (Investigator of the Merkin Foundation for Stem Cell Research)Howard Hughes Medical Institute (Early Career Scientist)Alfred P. Sloan FoundationNational Institutes of Health (U.S.) (Pioneer Award

Oligodendrocyte Death in Pelizaeus-Merzbacher Disease Is Rescued by Iron Chelation.

Pelizaeus-Merzbacher disease (PMD) is an X-linked leukodystrophy caused by mutations in Proteolipid Protein 1 (PLP1), encoding a major myelin protein, resulting in profound developmental delay and early lethality. Previous work showed involvement of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways, but poor PLP1 genotype-phenotype associations suggest additional pathogenetic mechanisms. Using induced pluripotent stem cell (iPSC) and gene-correction, we show that patient-derived oligodendrocytes can develop to the pre-myelinating stage, but subsequently undergo cell death. Mutant oligodendrocytes demonstrated key hallmarks of ferroptosis including lipid peroxidation, abnormal iron metabolism, and hypersensitivity to free iron. Iron chelation rescued mutant oligodendrocyte apoptosis, survival, and differentiationin vitro, and post-transplantation in vivo. Finally, systemic treatment of Plp1 mutant Jimpy mice with deferiprone, a small molecule iron chelator, reduced oligodendrocyte apoptosis and enabled myelin formation. Thus, oligodendrocyte iron-induced cell death and myelination is rescued by iron chelation in PMD pre-clinical models.H.N. acknowledges postdoctoral fellowship support from the European Leukodystrophy Association, and career transition fellowship support from National Multiple Sclerosis Society. M.C. acknowledges funding support from Career Development Grant awarded by Cerebral Palsy Alliance Research Foundation Inc. This work was supported by funding from the National Multiple Sclerosis Foundation (to M.W., D.H. R.), the European Leukodystrophy Association and the New York Stem Cell Foundation (to M.W.), and Action Medical Research, the Adelson Medical Research Foundation, the National Institute for Health Research Cambridge Biomedical Research Centre and the European Research Council (to D.H. R)

Primary skin fibroblasts as a model of Parkinson's disease

Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts and recombinant cell lines can be used as model systems for Parkinson's disease. Skin fibroblasts present a system with defined mutations and the cumulative cellular damage of the patients. PINK1 and Parkin genes show relevant expression levels in human fibroblasts and since both genes participate in stress response pathways, we believe fibroblasts advantageous in order to assess, e.g. the effect of stressors. Furthermore, since a bioenergetic deficit underlies early stage Parkinson's disease, while atrophy underlies later stages, the use of primary cells seems preferable over the use of tumor cell lines. The new option to use fibroblast-derived induced pluripotent stem cells redifferentiated into dopaminergic neurons is an additional benefit. However, the use of fibroblast has also some drawbacks. We have investigated PARK6 fibroblasts and they mirror closely the respiratory alterations, the expression profiles, the mitochondrial dynamics pathology and the vulnerability to proteasomal stress that has been documented in other model systems. Fibroblasts from patients with PARK2, PARK6, idiopathic Parkinson's disease, Alzheimer's disease, and spinocerebellar ataxia type 2 demonstrated a distinct and unique mRNA expression pattern of key genes in neurodegeneration. Thus, primary skin fibroblasts are a useful Parkinson's disease model, able to serve as a complement to animal mutants, transformed cell lines and patient tissues

Reprogramming Primordial Germ Cells into Pluripotent Stem Cells

Background: Specification of primordial germ cells (PGCs) results in the conversion of pluripotent epiblast cells into monopotent germ cell lineage. Blimp1/Prmt5 complex plays a critical role in the specification and maintenance of the early germ cell lineage. However, PGCs can be induced to dedifferentiate back to a pluripotent state as embryonic germ (EG) cells when exposed to exogenous signaling molecules, FGF-2, LIF and SCF. Methodology and Principal Findings: Here we show that Trichostatin A (TSA), an inhibitor of histone deacetylases, is a highly potent agent that can replace FGF-2 to induce dedifferentiation of PGCs into EG cells. A key early event during dedifferentiation of PGCs in response to FGF-2 or TSA is the down-regulation of Blimp1, which reverses and apparently relieves the cell fate restriction imposed by it. Notably, the targets of Blimp1, which include c-Myc and Klf-4, which represent two of the key factors known to promote reprogramming of somatic cells to pluripotent state, are up-regulated. We also found early activation of the LIF/Stat-3 signaling pathway with the translocation of Stat-3 into the nucleus. By contrast, while Prmt5 is retained in EG cells, it translocates from the nucleus to the cytoplasm where it probably has an independent role in regulating pluripotency. Conclusions/Significance: We propose that dedifferentiation of PGCs into EG cells may provide significant mechanistic insights on early events associated with reprogramming of committed cells to a pluripotent state

Splenectomy Normalizes Hematocrit in Murine Polycythemia Vera

Splenic enlargement (splenomegaly) develops in numerous disease states, although a specific pathogenic role for the spleen has rarely been described. In polycythemia vera (PV), an activating mutation in Janus kinase 2 (JAK2V617) induces splenomegaly and an increase in hematocrit. Splenectomy is sparingly performed in patients with PV, however, due to surgical complications. Thus, the role of the spleen in the pathogenesis of human PV remains unknown. We specifically tested the role of the spleen in the pathogenesis of PV by performing either sham (SH) or splenectomy (SPL) surgeries in a murine model of JAK2V617F-driven PV. Compared to SH-operated mice, which rapidly develop high hematocrits after JAK2V617F transplantation, SPL mice completely fail to develop this phenotype. Disease burden (JAK2V617) is equivalent in the bone marrow of SH and SPL mice, however, and both groups develop fibrosis and osteosclerosis. If SPL is performed after PV is established, hematocrit rapidly declines to normal even though myelofibrosis and osteosclerosis again develop independently in the bone marrow. In contrast, SPL only blunts hematocrit elevation in secondary, erythropoietin-induced polycythemia. We conclude that the spleen is required for an elevated hematocrit in murine, JAK2V617F-driven PV, and propose that this phenotype of PV may require a specific interaction between mutant cells and the spleen

Efficacious Intermittent Dosing of a Novel JAK2 Inhibitor in Mouse Models of Polycythemia Vera

A high percentage of patients with the myeloproliferative disorder polycythemia vera (PV) harbor a Val617→Phe activating mutation in the Janus kinase 2 (JAK2) gene, and both cell culture and mouse models have established a functional role for this mutation in the development of this disease. We describe the properties of MRLB-11055, a highly potent inhibitor of both the WT and V617F forms of JAK2, that has therapeutic efficacy in erythropoietin (EPO)-driven and JAK2V617F-driven mouse models of PV. In cultured cells, MRLB-11055 blocked proliferation and induced apoptosis in a manner consistent with JAK2 pathway inhibition. MRLB-11055 effectively prevented EPO-induced STAT5 activation in the peripheral blood of acutely dosed mice, and could prevent EPO-induced splenomegaly and erythrocytosis in chronically dosed mice. In a bone marrow reconstituted JAK2V617F-luciferase murine PV model, MRLB-11055 rapidly reduced the burden of JAK2V617F-expressing cells from both the spleen and the bone marrow. Using real-time in vivo imaging, we examined the kinetics of disease regression and resurgence, enabling the development of an intermittent dosing schedule that achieved significant reductions in both erythroid and myeloid populations with minimal impact on lymphoid cells. Our studies provide a rationale for the use of non-continuous treatment to provide optimal therapy for PV patients

Freeze-Dried Somatic Cells Direct Embryonic Development after Nuclear Transfer

The natural capacity of simple organisms to survive in a dehydrated state has long been exploited by man, with lyophylization the method of choice for the long term storage of bacterial and yeast cells. More recently, attempts have been made to apply this procedure to the long term storage of blood cells. However, despite significant progress, practical application in a clinical setting is still some way off. Conversely, to date there are no reports of attempts to lyophilize nucleated somatic cells for possible downstream applications. Here we demonstrate that lyophilised somatic cells stored for 3 years at room temperature are able to direct embryonic development following injection into enucleated oocytes. These remarkable results demonstrate that alternative systems for the long-term storage of cell lines are now possible, and open unprecedented opportunities in the fields of biomedicine and for conservation strategies

Efficient Non-Viral Reprogramming of Myoblasts to Stemness with a Single Small Molecule to Generate Cardiac Progenitor Cells

The current protocols for generation of induced pluripotent stem (iPS) cells involve genome integrating viral vectors which may induce tumorgenesis. The aim of this study was to develop and optimize a non-viral method without genetic manipulation for reprogramming of skeletal myoblasts (SMs) using small molecules

Establishment of Mouse Embryonic Stem Cell-Derived Erythroid Progenitor Cell Lines Able to Produce Functional Red Blood Cells

BACKGROUND: The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If erythroid cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. To evaluate the feasibility of establishing useful erythroid cell lines, we attempted to establish such cell lines from mouse embryonic stem (ES) cells. METHODOLOGY/PRINCIPAL FINDINGS: We developed a robust method to obtain differentiated cell lines following the induction of hematopoietic differentiation of mouse ES cells and established five independent hematopoietic cell lines using the method. Three of these lines exhibited characteristics of erythroid cells. Although their precise characteristics varied, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. In addition, we did not observe formation of any tumors following transplantation of these cells. CONCLUSION/SIGNIFICANCE: To the best of our knowledge, this is the first report to show the feasibility of establishing erythroid cell lines able to produce mature RBCs. Considering the number of human ES cell lines that have been established so far, the intensive testing of a number of these lines for erythroid potential may allow the establishment of human erythroid cell lines similar to the mouse erythroid cell lines described here. In addition, our results strongly suggest the possibility of establishing useful cell lines committed to specific lineages other than hematopoietic progenitors from human ES cells