技术资料

Cell-based therapies are considered as one of the most promising approaches for the treatment of degenerating pathologies including muscle disorders and dystrophies. Advances in the approach of reprogramming somatic cells into induced pluripotent stem (iPS) cells allow for the possibility of using the patient's own pluripotent cells to generate specific tissues for autologous transplantation. In addition,patient-specific tissue derivatives have been shown to represent valuable material for disease modeling and drug discovery. Nevertheless,directed differentiation of pluripotent stem cells into a specific lineage is not a trivial task especially in the case of skeletal myogenesis,which is generally poorly recapitulated during the in vitro differentiation of pluripotent stem cells.Here,we describe a practical and efficient method for the derivation of skeletal myogenic precursors from differentiating human pluripotent stem cells using controlled expression of PAX7. Flow cytometry (FACS) purified myogenic precursors can be expanded exponentially and differentiated in vitro into myotubes,enabling researchers to use these cells for disease modeling as well as therapeutic purposes. View Publication

Recently,induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer,both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore,the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front,although donor cell types for generating iPSCs are wide-ranging,T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells,which can be applied to novel immunotherapies. In the present study,we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining,had a normal karyotype,and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined,animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application. View Publication

We report generation of induced pluripotent stem cell (iPSC) lines from ten Parkinson's disease (PD) patients carrying SNCA,PARK2,LRRK2,and GBA mutations,and one age-matched control. After validation of pluripotency,long-term genome stability,and integration-free reprogramming,eight of these lines (one of each SNCA,LRRK2 and GBA,four PARK2 lines,and the control) were differentiated into neural stem cells (NSC) and subsequently to dopaminergic cultures. We did not observe significant differences in the timeline of neural induction and NSC derivation between the patient and control line,nor amongst the patient lines,although we report considerable variability in the efficiency of dopaminergic differentiation among patient lines. We performed whole genome expression analyses of the lines at each stage of differentiation (fibroblast,iPSC,NSC,and dopaminergic culture) in an attempt to identify alterations by large-scale evaluation. While gene expression profiling clearly distinguished cells at different stages of differentiation,no mutation-specific clustering or difference was observed,though consistent changes in patient lines were detected in genes associated mitochondrial biology. We further examined gene expression in a stress model (MPTP-induced dopaminergic neuronal death) using two clones from the SNCA triplication line,and detected changes in genes associated with mitophagy. Our data suggested that even a well-characterized line of a monogenic disease may not be sufficient to determine the cause or mechanism of the disease,and highlights the need to use more focused strategies for large-scale data analysis. View Publication

The differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) towards functional neurons particularly hold great potential for the cell-based replacement therapy in neurodegenerative diseases. Here,we describe a stepwise differentiation protocol that mimics the early stage of neural development in human to promote the generation of neuroprogenitors at a high yield. Both the hESCs and hiPSCs are initially cultured in an optimized feeder-free condition,which offer an efficient formation of aggregates. To specify the neuroectodermal specification,these aggregates are differentiated in a defined neural induction medium to develop into neural rosettes-like structures. The rosettes are expanded into free-floating sphere and can be further propagated or developed into variety of neuronal subtypes. View Publication

Despite major progress in the management of human liver disease,the only cure for a critically failing organ is liver transplantation. While a highly successful approach,the use of cadaveric organs as a routine treatment option is severely limited by organ availability. Therefore,the use of cell-based therapies has been explored to provide support for the failing liver. In addition to developing new treatments,there is also an imperative to develop better human models 'in a dish'. Such approaches will undoubtedly lead to a better understanding of the disease process,offering new treatment or preventative strategies. With both approaches in mind,we have developed robust hepatocyte differentiation methodologies for use with pluripotent stem cells. Importantly,our procedure is highly efficient (∼ 90%) and delivers active,drug-inducible,and predictive human hepatocyte populations. View Publication

The recent development of HIV-1 lentiviral vectors is especially useful for gene transfer because they achieve efficient integration into nondividing cell genomes and successful long-term expression of the transgene. These attributes make the vector useful for gene delivery,mutagenesis,and other applications in mammalian systems. Here we describe two HIV-1-based lentiviral vector derivatives,pZR-1 and pZR-2,that can be used in gene-trap experiments in mammalian cells in vitro and in vivo. Each lentiviral gene-trap vector contains a reporter gene,either beta-lactamase or enhanced green fluorescent protein (EGFP),that is inserted into the U3 region of the 3' long terminal repeat. Both of the trap vectors readily integrate into the host genome by using a convenient infection technique. Appropriate insertion of the vector into genes causes EGFP or beta-lactamase expression. This technique should facilitate the rapid enrichment and cloning of the trapped cells and provides an opportunity to select subpopulations of trapped cells based on the subcellular localization of reporter genes. Our findings suggest that the reporter gene is driven by an upstream,cell-specific promoter during cell culture and cell differentiation,which further supports the usefulness of lentivirus-based gene-trap vectors. Lentiviral gene-trap vectors appear to offer a wealth of possibilities for the study of cell differentiation and lineage commitment,as well as for the discovery of new genes. View Publication

There is potential interest for combining allogeneic hematopoietic cell transplantation (HCT),and particularly allogeneic HCT with a nonmyeloablative regimen,to the tyrosine kinase inhibitor imatinib (Glivec; Novartis,Basel,Switzerland,http://www.novartis.com) in order to maximize anti-leukemic activity against Philadelphia chromosome-positive leukemias. However,because imatinib inhibits c-kit,the stem cell factor receptor,it could interfere with bone marrow engraftment. In this study,we examined the impact of imatinib on normal progenitor cell function. Imatinib decreased the colony-forming capacity of mobilized peripheral blood human CD133(+) cells but not that of long-term culture-initiating cells. Imatinib also decreased the proliferation of cytokine-stimulated CD133(+) cells but did not induce apoptosis of these cells. Expression of very late antigen (VLA)-4,VLA-5,and CXCR4 of CD133(+) cells was not modified by imatinib,but imatinib decreased the ability of CD133(+) cells to migrate. Finally,imatinib did not decrease engraftment of CD133(+) cells into irradiated nonobese diabetic/severe combined immunodeficient/beta2m(null) mice conditioned with 3 or 1 Gy total body irradiation. In summary,our results suggest that,despite inhibition of hematopoietic progenitor cell growth in vitro,imatinib does not interfere with hematopoietic stem cell engraftment. View Publication

Genetic changes in human pluripotent stem cells (hPSCs) gained during culture can confound experimental results and potentially jeopardize the outcome of clinical therapies. Particularly common changes in hPSCs are trisomies of chromosomes 1,12,17,and 20. Thus,hPSCs should be regularly screened for such aberrations. Although a number of methods are used to assess hPSC genotypes,there has been no systematic evaluation of the sensitivity of the commonly used techniques in detecting low-level mosaicism in hPSC cultures. We have performed mixing experiments to mimic the naturally occurring mosaicism and have assessed the sensitivity of chromosome banding,qPCR,fluorescence in situ hybridization,and digital droplet PCR in detecting variants. Our analysis highlights the limits of mosaicism detection by the commonly employed methods,a pivotal requirement for interpreting the genetic status of hPSCs and for setting standards for safe applications of hPSCs in regenerative medicine. View Publication

BACKGROUND: Circulation of mature fetal blood cells in the maternal blood for a certain postpartum period has been verified,but detailed study of the fetal HPCs has not been reported. The objective of this study was to evaluate the frequency and clearance of these cells in the peripheral blood of puerperal women. STUDY DESIGN AND METHODS: PBMNCs from 15 puerperal women who gave birth to male infants were cultured in semi-solid medium containing hematopoietic stimulating factors. Colonies formed in the medium were individually characterized,collected,and subjected to PCR amplification of the SRY gene on Y chromosome to confirm fetal origin. RESULTS: The mean numbers of fetal progenitor cell colonies isolated per mL of maternal blood were 1.63,2.48,0.56,0.12,and 0 on the day of delivery,at 4 days,1 month,6 months,and 1 year after delivery,respectively. There was no difference in the ratio of fetal versus maternal colonies between erythroid and granulocyte/macrophage lineages. CONCLUSION: The present study demonstrated that a significant number of fetal HPCs circulate in the maternal blood for a duration of at least 6 months after delivery. View Publication

This study investigated the factors responsible for migration and homing of magnetically labeled AC133(+) cells at the sites of active angiogenesis in tumor. AC133(+) cells labeled with ferumoxide-protamine sulfate were mixed with either rat glioma or human melanoma cells and implanted in flank of nude mice. An MRI of the tumors including surrounding tissues was performed. Tumor sections were stained for Prussian blue (PB),platelet-derived growth factor (PDGF),hypoxia-inducible factor-1alpha (HIF-1alpha),stromal cell derived factor-1 (SDF-1),matrix metalloproteinase-2 (MMP-2),vascular endothelial growth factor (VEGF),and endothelial markers. Fresh snap-frozen strips from the central and peripheral parts of the tumor were collected for Western blotting. MRIs demonstrated hypointense regions at the periphery of the tumors where the PB(+)/AC133(+) cells were positive for endothelial cells markers. At the sites of PB(+)/AC133(+) cells,both HIF-1alpha and SDF-1 were strongly positive and PDGF and MMP-2 showed generalized expression in the tumor and surrounding tissues. There was no significant association of PB(+)/AC133(+) cell localization and VEGF expression in tumor cells. Western blot demonstrated strong expression of the SDF-1,MMP-2,and PDGF at the peripheral parts of the tumors. HIF-1alpha was expressed at both the periphery and central parts of the tumor. This work demonstrates that magnetically labeled cells can be used as probes for MRI and histological identification of administered cells. View Publication

Although many acute myeloid leukemia (AML) colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) directly isolated from patients are actively cycling,quiescent progenitors are present in most samples. In the current study,(3)H-thymidine ((3)H-Tdr) suicide assays demonstrated that most NOD/SCID mouse leukemia-initiating cells (NOD/SL-ICs) are quiescent in 6 of 7 AML samples. AML cells in G(0),G(1),and S/G(2)+M were isolated from 4 of these samples using Hoechst 33342/pyroninY staining and cell sorting. The progenitor content of each subpopulation was consistent with the (3)H-Tdr suicide results,with NOD/SL-ICs found almost exclusively among G(0) cells while the cycling status of AML CFCs and LTC-ICs was more heterogeneous. Interestingly,after 72 hours in serum-free culture with or without Steel factor (SF),Flt-3 ligand (FL),and interleukin-3 (IL-3),most G(0) AML cells entered active cell cycle (percentage of AML cells remaining in G(0) at 72 hours,1.2% to 37%,and 0% to 7.6% in cultures without and with growth factors [GFs],respectively) while G(0) cells from normal lineage-depleted bone marrow remained quiescent in the absence of GF. All 4 AML samples showed evidence of autocrine production of 2 or more of SF,FL,IL-3,and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition,3 of 4 samples contained an internal tandem duplication of the FLT3 gene. In summary,quiescent leukemic cells,including NOD/SL-ICs,are present in most AML patients. Their spontaneous entry into active cell cycle in short-term culture might be explained by the deregulated GF signaling present in many AMLs. View Publication

Since mouse embryonic stem (ES) cells was first derived in 1981,the ability of this unprecedented cell type to self-renew and differentiate without limit has revolutionized the discovery tools that are used to study gene functions and development. Furthermore,they have inspired others to hunt for similar cells from other species. The derivation of human ES cells in 1998 has accelerated these discoveries and has also widely provoked public interest,due to both the scientific significance of these cells for human tissue regeneration and the ethical disputes over the use of donated early human embryos. However,this is no longer a barrier,with the recent discovery of methods that can convert differentiated somatic cells into ES-like cells or induced pluripotent stem (iPS) cells,by using defined reprogramming factors. This review attempts to summarize the progresses in the derivation of ES cells (as well as other embryo-derived pluripotent cells) and iPS cells from various species. We will focus on the molecular and biological features of the cells,as well as the different determinants identified thus far to sustain their pluripotency. View Publication