技术资料

Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising can- didate cell sources for regenerative medicine. However,despite the common ability of hiPSCs and hESCs to dif- ferentiate into all 3 germ layers,their functional equivalence at the single cell level remains to be demonstrated. Moreover,single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here,we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs,suggesting that hiPSCs occupy an alternate,less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exer- cised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs,particularly when producing differentiated cells for regenerative medicine aims. View Publication

Human embryonic stem (hES) cells have a potential use for the repair and regeneration of injured tissues. However,teratoma formation can be a major obstacle for hES-mediated cell therapy. Therefore,tracking the fate and function of transplanted hES cells with noninvasive imaging could be valuable for a better understanding of the biology and physiology of teratoma formation. In this study,hES cells were stably transduced with a double fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein. Following bioluminescence imaging and histology,we demonstrated that engraftment of hES cells was followed by dramatically increasing signaling and led to teratoma formation confirmed by histology. Studies of the angiogenic processes within teratomas revealed that their vasculatures were derived from both differentiated hES cells and host. Moreover,FACS analysis showed that teratoma cells derived from hES cells expressed high levels of CD56 and SSEA-4,and the subcultured SSEA-4(+) cells showed a similar cell surface marker expression pattern when compared to undifferentiated hES cells. We report here for the first time that SSEA-4(+) cells derived from teratoma exhibited multipotency,retained their differentiation ability in vivo as confirmed by their differentiation into representative three germ layers. View Publication

Patients with Fanconi anemia (FA) have a high risk of developing acute myeloid leukemia (AML). In this study,we attempted to identify cell-surface markers for leukemia-initiating cells in FA-AML patients. We found that the IL-3 receptor-α (IL-3Rα) is a promising candidate as an leukemia-initiating cell-specific antigen for FA-AML. Whereas IL-3Rα expression is undetectable on normal CD34(+)CD38(-) HSCs,it is overexpressed on CD34(+)CD38(-) cells from FA patients with AML. We examined the leukemia-initiating cell activity of IL-3Rα-positive FA-AML cells in a humanized" FA xenotransplant model in which we separated AML cells into IL-3Rα-positive and IL-3Rα-negative CD34 fractions and transplanted them into irradiated recipient mice. In all 3 FA-AML samples� View Publication

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease,characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene,resulting in the generation of progerin,a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin,and more importantly,lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs,progerin and its ageing-associated phenotypic consequences are restored. Specifically,directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally,our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs,also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing,our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing. View Publication

Purpose: The bone marrow microenvironment is considered a critical component in the dissemination and fate of cancer cells in the metastatic process. We explored the possible correlation between bone marrow mesenchymal stem cells (BM-MSC) and disseminated breast cancer-initiating cells (BCIC) in primary breast cancer patients. Experimental design: Bone marrow mononuclear cells (BM-MNC) were collected at the time of primary surgery in 12 breast cancer patients. BM-MNC was immunophenotyped and BCIC was defined as epithelial cells (CD326+CD45-) with a stem-like" phenotype (CD44+CD24low/-� View Publication

Here,we show that as human embryonic stem cells (ESCs) exit the pluripotent state,NANOG can play a key role in determining lineage outcome. It has previously been reported that BMPs induce differentiation of human ESCs into extraembryonic lineages. Here,we find that FGF2,acting through the MEK-ERK pathway,switches BMP4-induced human ESC differentiation outcome to mesendoderm,characterized by the uniform expression of T (brachyury) and other primitive streak markers. We also find that MEK-ERK signaling prolongs NANOG expression during BMP-induced differentiation,that forced NANOG expression results in FGF-independent BMP4 induction of mesendoderm,and that knockdown of NANOG greatly reduces T induction. Together,our results demonstrate that FGF2 signaling switches the outcome of BMP4-induced differentiation of human ESCs by maintaining NANOG levels through the MEK-ERK pathway. View Publication

Hematopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors' effort,which,in concert with microRNAs,drives cell fate and responds to promiscuous patterns of gene expression by turning on lineage-specific genes and repressing alternate lineage transcripts. We obtained microRNA profiles from human CD34+ hematopoietic progenitor cells and in vitro differentiated erythroblasts,megakaryoblasts,monoblasts and myeloblast precursors that we analyzed together with their gene expression profiles. The integrated analysis of microRNA-mRNA expression levels highlighted an inverse correlation between microRNAs specifically upregulated in one single-cell progeny and their putative target genes,which resulted in downregulation. Among the upregulated lineage-enriched microRNAs,hsa-miR-299-5p emerged as having a role in controlling CD34+ progenitor fate,grown in multilineage culture conditions. Gain- and loss-of-function experiments revealed that hsa-miR-299-5p participates in the regulation of hematopoietic progenitor fate,modulating megakaryocytic-granulocytic versus erythroid-monocytic differentiation. View Publication

MicroRNAs (miRNAs) are small,noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-,maturation-,and disease-specific miRNA expression has been described,miRNA-dependent phenotypes and miRNA-regulated signaling in hematopoietic cells are largely unknown. Combining functional genomics,biochemical analysis,and unbiased and hypothesis-driven miRNA target prediction,we show that lentivirally over-expressed miR-125b blocks G-CSF-induced granulocytic differentiation and enables G-CSF-dependent proliferation of murine 32D cells. In primary lineage-negative cells,miR-125b over-expression enhances colony-formation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression,respectively. However,gene-specific RNAi reveals that this reduction,alone and in combination,is not sufficient to block G-CSF-dependent differentiation. STAT3 protein expression,DNA-binding,and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression,indicating miR-125b-mediated reduction of one or more STAT3 cofactors. Indeed,we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/miR-125b cells. Interestingly,gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells. View Publication

Adipose tissue is an abundantly available source of proliferative and multipotent mesenchymal stem cells with promising potential for regenerative therapeutics. We previously demonstrated that both human and mouse adipose-derived stem cells (ASCs) can be reprogrammed into induced pluripotent stem cells (iPSCs) with efficiencies higher than those that have been reported for other cell types. The ASC-derived iPSCs can be generated in a feeder-independent manner,representing a unique model to study reprogramming and an important step toward establishing a safe,clinical grade of cells for therapeutic use. In this study,we provide a detailed protocol for isolation,preparation and transformation of ASCs from fat tissue into mouse iPSCs in feeder-free conditions and human iPSCs using feeder-dependent or feeder/xenobiotic-free processes. This protocol also describes how ASCs can be used as feeder cells for maintenance of other pluripotent stem cells. ASC derivation is rapid and can be completed in textless1 week,with mouse and human iPS reprogramming times averaging 1.5 and 2.5 weeks,respectively. View Publication

One of the key consequences of exposure of human cells to genotoxic agents is the activation of DNA damage responses (DDR). While the mechanisms underpinning DDR in fully differentiated somatic human cells have been studied extensively,molecular signaling events and pathways involved in DDR in pluripotent human embryonic stem cells (hESC) remain largely unexplored. We studied changes in the human genome-wide transcriptome of H9 hESC line following exposures to 1Gy of gamma-radiation at 2h and 16h post-irradiation. Quantitative real-time PCR was performed to verify the expression data for a subset of genes. In parallel,the cell growth,DDR kinetics,and expression of pluripotency markers in irradiated hESC were monitored. The changes in gene expression in hESC after exposure to ionizing radiation (IR) are substantially different from those observed in somatic human cell lines. Gene expression patterns at 2h post-IR showed almost an exclusively p53-dependent,predominantly pro-apoptotic,signature with a total of only 30 up-regulated genes. In contrast,the gene expression patterns at 16h post-IR showed 354 differentially expressed genes,mostly involved in pro-survival pathways,such as increased expression of metallothioneins,ubiquitin cycle,and general metabolism signaling. Cell growth data paralleled trends in gene expression changes. DDR in hESC followed the kinetics reported for human somatic differentiated cells. The expression of pluripotency markers characteristic of undifferentiated hESC was not affected by exposure to IR during the time course of our analysis. Our data on dynamics of transcriptome response of irradiated hESCs may provide a valuable tool to screen for markers of IR exposure of human cells in their most naive state; thus unmasking the key elements of DDR; at the same time,avoiding the complexity of interpreting distinct cell type-dependent genotoxic stress responses of terminally differentiated cells. View Publication

Although it has been observed that aggregate size affects cardiac development,an incomplete understanding of the cellular mechanisms underlying human pluripotent stem cell-derived cardiomyogenesis has limited the development of robust defined-condition cardiac cell generation protocols. Our objective was thus to elucidate cellular and molecular mechanisms underlying the endogenous control of human embryonic stem cell (hESC) cardiac tissue development,and to test the hypothesis that hESC aggregate size influences extraembryonic endoderm (ExE) commitment and cardiac inductive properties. hESC aggregates were generated with 100,1000,or 4000 cells per aggregate using microwells. The frequency of endoderm marker (FoxA2 and GATA6)-expressing cells decreased with increasing aggregate size during early differentiation. Cardiogenesis was maximized in aggregates initiated from 1000 cells,with frequencies of 0.49±0.06 cells exhibiting a cardiac progenitor phenotype (KDR(low)/C-KIT(neg)) on day 5 and 0.24±0.06 expressing cardiac Troponin T on day 16. A direct relationship between ExE and cardiac differentiation efficiency was established by forming aggregates with varying ratios of SOX7 (a transcription factor required for ExE development) overexpressing or knockdown hESCs to unmanipulated hESCs. We demonstrate,in a defined,serum-free cardiac induction system,that robust and efficient cardiac differentiation is a function of endogenous ExE cell concentration,a parameter that can be directly modulated by controlling hESC aggregate size. View Publication

Overexpression of high mobility group AT-hook 2 (HMGA2) is found in a number of benign and malignant tumors,including the clonal PIGA(-) cells in 2 cases of paroxysmal nocturnal hemoglobinuria (PNH) and some myeloproliferative neoplasms (MPNs),and recently in hematopoietic cell clones resulting from gene therapy procedures. In nearly all these cases overexpression is because of deletions or translocations that remove the 3' untranslated region (UTR) which contains binding sites for the regulatory micro RNA let-7. We were therefore interested in the effect of HMGA2 overexpression in hematopoietic tissues in transgenic mice (ΔHmga2 mice) carrying a 3'UTR-truncated Hmga2 cDNA. ΔHmga2 mice expressed increased levels of HMGA2 protein in various tissues including hematopoietic cells and showed proliferative hematopoiesis with increased numbers in all lineages of peripheral blood cells,hypercellular bone marrow (BM),splenomegaly with extramedullary erythropoiesis and erythropoietin-independent erythroid colony formation. ΔHmga2-derived BM cells had a growth advantage over wild-type cells in competitive repopulation and serial transplantation experiments. Thus overexpression of HMGA2 leads to proliferative hematopoiesis with clonal expansion at the stem cell and progenitor levels and may account for the clonal expansion in PNH and MPNs and in gene therapy patients after vector insertion disrupts the HMGA2 locus. View Publication