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After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34+ cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgene-free iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future.

Somatic cells can be converted to an embryonic-like state by introducing a defined set of factors. Induced pluripotent stem (iPS) cells raise new hopes for regenerative medicine. What are the hurdles that need to be overcome to take advantage of this technique? Induced pluripotent stem (iPS) cells are human somatic cells that have been reprogrammed to a pluripotent state. There are several hurdles to be overcome before iPS cells can be considered as a potential patient-specific cell therapy, and it will be crucial to characterize the developmental potential of human iPS cell lines. As a research tool, iPS-cell technology provides opportunities to study normal development and to understand reprogramming. iPS cells can have an immediate impact as models for human diseases, including cancer.

An early stage in mouse blood development is reconstructed from gene expression data on thousands of single cells. Reconstruction of the molecular pathways controlling organ development has been hampered by a lack of methods to resolve embryonic progenitor cells. Here we describe a strategy to address this problem that combines gene expression profiling of large numbers of single cells with data analysis based on diffusion maps for dimensionality reduction and network synthesis from state transition graphs. Applying the approach to hematopoietic development in the mouse embryo, we map the progression of mesoderm toward blood using single-cell gene expression analysis of 3,934 cells with blood-forming potential captured at four time points between E7.0 and E8.5. Transitions between individual cellular states are then used as input to develop a single-cell network synthesis toolkit to generate a computationally executable transcriptional regulatory network model of blood development. Several model predictions concerning the roles of Sox and Hox factors are validated experimentally. Our results demonstrate that single-cell analysis of a developing organ coupled with computational approaches can reveal the transcriptional programs that underpin organogenesis.

Poor survival of human embryonic stem (hES) cells after cell dissociation is an obstacle to research, hindering manipulations such as subcloning. Here we show that application of a selective Rho-associated kinase (ROCK) inhibitor 1 , 2 , Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency (from ∼1% to ∼27%) and facilitates subcloning after gene transfer. Furthermore, dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture 3 and form floating aggregates. We demonstrate that the protective ability of Y-27632 enables SFEB-cultured hES cells to survive and differentiate into Bf1 + cortical and basal telencephalic progenitors, as do SFEB-cultured mouse ES cells.

The first haematopoietic stem cells (HSCs) appear in the aorta-gonad-mesonephros (AGM) region, major vitelline and umbilical vessels, and placenta; however, whether they arise locally or from immigrant yolk sac precursor cells remains unclear. This issue is best addressed by measuring cell-lineage relationships rather than cell potentials. To undertake long-term in vivo tracing of yolk sac cells, we designed a non-invasive pulse-labelling system based on Cre/ loxP recombination. Here we show that in Runx1 +/- (runt-related transcription factor 1) heterozygous mice, yolk sac cells expressing Runx1 at embryonic day 7.5 develop into fetal lymphoid progenitors and adult HSCs. During mid-gestation the labelled (embryonic day 7.5) yolk sac cells colonize the umbilical cord, the AGM region and subsequently the embryonic liver. This raises the possibility that some HSCs associated with major embryonic vasculature are derived from yolk sac precursors. We observed virtually no contribution of the labelled cells towards the yolk sac vasculature, indicating early segregation of endothelial and haematopoietic lineages. Something in the blood There are two recognized phases of blood formation (haematopoiesis) during embryonic development: an initial 'primitive' stage that takes place in the extraembryonic yolk sac and provides nutrients to the early embryo, then a 'definitive' stage that begins in a region of the embryo known as the aorta-gonad-mesonephros. There has been prolonged debate over whether the source of the definitive haematopoietic population is local or external, deriving from inwardly migrating precursors from the yolk sac. A new non-invasive cell tracing study contradicts the current orthodoxy and suggests that the yolk sac is indeed the source of adult haematopoietic stem cells. Knowledge of the origins of the blood supply is relevant to work on the generation of blood stem cells in culture, with the potential for therapeutic applications. Genetic marking is used to perform a non-invasive cell tracing technique, labelling cells of the definitive haematopoetic system and observed the migration of haematopoetic progenitors from the yolks sac to the fetal liver and thymus.

During development, the retinal vasculature grows toward hypoxic areas in an organized fashion. By contrast, in ischemic retinopathies, new blood vessels grow out of the retinal surfaces without ameliorating retinal hypoxia. Restoration of proper angiogenic directionality would be of great benefit to reoxygenize the ischemic retina and resolve disease pathogenesis. Here, we show that binding of the semaphorin 3E (Sema3E) ligand to the transmembrane PlexinD1 receptor initiates a signaling pathway that normalizes angiogenic directionality in both developing retinas and ischemic retinopathy. In developing mouse retinas, inhibition of VEGF signaling resulted in downregulation of endothelial PlexinD1 expression, suggesting that astrocyte-derived VEGF normally promotes PlexinD1 expression in growing blood vessels. Neuron-derived Sema3E signaled to PlexinD1 and activated the small GTPase RhoJ in ECs, thereby counteracting VEGF-induced filopodia projections and defining the retinal vascular pathfinding. In a mouse model of ischemic retinopathy, enhanced expression of PlexinD1 and RhoJ in extraretinal vessels prevented VEGF-induced disoriented projections of the endothelial filopodia. Remarkably, intravitreal administration of Sema3E protein selectively suppressed extraretinal vascular outgrowth without affecting the desired regeneration of the retinal vasculature. Our study suggests a new paradigm for vascular regeneration therapy that guides angiogenesis precisely toward the ischemic retina.

Human induced pluripotent stem cells (hiPSCs) possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs). These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay): soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×10⁴ RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research.

Preparation of specific lineages at high purities from embryonic stem (ES) cells requires both selective culture conditions and markers to guide and monitor the differentiation. In this study, we distinguished definitive and visceral endoderm by using a mouse ES cell line that bears the gfp and human IL2Rα (also known as CD25 ) marker genes in the goosecoid ( Gsc ) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc + Sox17 + definitive endoderm and Gsc − Sox17 + visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, we identified seven surface molecules that are expressed differentially in the two populations. One of the seven markers, Cxcr4, to which a monoclonal antibody is available allowed us to monitor and purify the Gsc + population from genetically unmanipulated ES cells under the condition that selects definitive endoderm.

In mice, coat pigmentation requires a stem cell (SC) system in which the survival, proliferation, and differentiation of melanocytes (MCs) are regulated by microenvironments in hair follicles (HFs). In vitro systems are required to analyze the behavior of single melanocyte stem cells (MCSCs) and their potential to form SC systems in vivo. We describe here an experimental system for the isolation, self-renewal, and differentiation of MCSCs, as well as an in vivo reconstitution assay for assessing their potential. Using Dcttm1(Cre)Bee/CAG-CAT-GFP mice, we show that, in the presence of stem cell factor and basic fibroblast growth factor and the XB2 feeder cell line, purified MCSCs can undergo clonogenic proliferation, resulting in c-Kitlow side scatterlow cells. In culture, these cells maintain their capacity to differentiate and reconstitute an MCSC system in HFs. As these cells are present in the upper part of the HF near the bulge region, express only low levels of housekeeping genes, and are resistant to neonatal treatment with ACK2, it is likely that only MCSCs that are quiescent in vivo have clonogenic activity in vitro. We also found that MCSCs can be purified from wild-type mice by fluorescent cell sorting and can be characterized in vitro.

Vitrification and slow-freezing methods have been used for the cryopreservation of human pluripotent stem cells (hPSCs). Vitrification requires considerable skill and post-thaw recovery is low. Furthermore, it is not suitable for cryopreservation of large numbers of hPSCs. While slow-freezing methods for hPSCs are easy to perform, they are usually preceded by a complicated cell dissociation process that yields poor post-thaw survival. To develop a robust and easy slow-freezing method for hPSCs, several different cryopreservation cocktails were prepared by modifying a commercially available freezing medium (CP-1™) containing hydroxyethyl starch (HES), and dimethyl sulfoxide (DMSO) in saline. The new freezing media were examined for their cryopreservation efficacy in combination with several different cell detachment methods. hPSCs in cryopreservation medium were slowly cooled in a conventional -80°C freezer and thawed rapidly. hPSC colonies were dissociated with several proteases. Ten percent of the colonies were passaged without cryopreservation and another 10% were cryopreserved, and then the recovery ratio was determined by comparing the number of Alkaline Phosphatase-positive colonies after thawing at day 5 with those passaged without cryopreservation at day 5. We found that cell detachment with Pronase/EDTA followed by cryopreservation using 6% HES, 5% DMSO, and 5% ethylene glycol (EG) in saline (termed CP-5E) achieved post-thaw recoveries over 80%. In summary, we have developed a new cryopreservation medium free of animal products for slow-freezing. This easy and robust cryopreservation method could be used widely for basic research and for clinical application.