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Bone marrow-derived mesenchymal stem cells (MSC) have been defined as primitive,undifferentiated cells,capable of self-renewal and with the ability to give rise to different cell lineages,including adipocytes,osteocytes,fibroblasts,chondrocytes,and myoblasts. MSC are key components of the hematopoietic microenvironment. Several studies,including some from our own group,suggest that important quantitative and functional alterations are present in the stroma of patients with myelodysplasia (MDS). However,in most of such studies the stroma has been analyzed as a complex network of different cell types and molecules,thus it has been difficult to identify and characterize the cell(s) type(s) that is (are) altered in MDS. In the present study,we have focused on the biological characterization of MSC from MDS. As a first approach,we have quantified their numbers in bone marrow,and have worked on their phenotypic (morphology and immunophenotype) and cytogenetic properties. MSC were obtained by a negative selection procedure and cultured in a MSC liquid culture medium. In terms of morphology,as well as the expression of certain cell markers,no differences were observed between MSC from MDS patients and those derived from normal marrow. In both cases,MSC expressed CD29,CD90,CD105 and Prolyl-4-hydroxylase; in contrast,they did not express CD14,CD34,CD68,or alkaline phosphatase. Interestingly,in five out of nine MDS patients,MSC developed in culture showed cytogenetic abnormalities,usually involving the loss of chromosomal material. All those five cases also showed cytogenetic abnormalities in their hematopoietic cells. Interestingly,in some cases there was a complete lack of overlap between the karyotypes of hematopoietic cells and MSC. To the best of our knowledge,the present study is the first in which a pure population of MSC from MDS patients is analyzed in terms of their whole karyotype and demonstrates that in a significant proportion of patients,MSC are cytogenetically abnormal. Although the reason of this is still unclear,such alterations may have an impact on the physiology of these cells. Further studies are needed to assess the functional integrity of MDS-derived MSC. View Publication

The beta-globin locus control region (LCR) is a large DNA element that is required for high-level expression of beta-like globin genes from the endogenous mouse locus or in transgenic mice carrying the human beta-globin locus. The LCR encompasses 6 DNaseI hypersensitive sites (HSs) that bind transcription factors. These HSs each contain a core of a few hundred base pairs (bp) that has most of the functional activity and exhibits high interspecies sequence homology. Adjoining the cores are 500- to 1000-bp flanks" with weaker functional activity and lower interspecies homology. Studies of human beta-globin transgenes and of the endogenous murine locus show that deletion of an entire HS (core plus flanks) moderately suppresses expression. However� View Publication

OBJECTIVE: Various preparative protocols have been proposed for the acquisition and cultivation of mesenchymal stem cells (MSC). Whereas surface antigen markers have failed to precisely define this population,microarray analysis might provide a better tool for characterization of MSC. METHODS: In this study,we have analyzed global gene expression profiles of human MSC isolated from adipose tissue (AT),from umbilical cord blood (CB),and from bone marrow (BM) under two growth conditions and have compared them to terminally differentiated human fibroblasts (HS68). Profiles were compared using our Human Genome Microarray representing 51.144 different cDNA clones. RESULTS: Cultured with the appropriate conditions,osteogenic and adipogenic differentiation could be confirmed in all MSC preparations but not in fibroblasts. No phenotypic differences were observed by flow cytometry using a panel of 22 surface antigen markers. Whereas MSC derived from different donors using the same culture procedure yielded a consistent and reproducible gene expression profile,many genes were differentially expressed in MSC from different ontogenetic sources or from different culture conditions. Twenty-five genes were overlapping and upregulated in all MSC preparations from AT,CB,and BM as compared to HS68 fibroblasts. These genes included fibronectin,ECM2,glypican-4,ID1,NF1B,HOXA5,and HOXB6. Many genes upregulated in MSC are involved in extracellular matrix,morphogenesis,and development,whereas several inhibitors of the Wnt pathway (DKK1,DKK3,SFRP1) were highly expressed in fibroblasts. CONCLUSION: Our results have provided a foundation for a more reproducible and reliable quality control using genotypic analysis for defining MSC. View Publication

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step,we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells,and then,expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123(low)HLA-DR- phenotype,expressed myeloperoxidase,and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules,CD1a,CD11c,CD80,CD86,DC-SIGN,and CD40; and were capable of Ag processing,triggering naive T cells in MLR,and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-alpha coexpressed a high level of CD14,and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition,because ES cells can be expanded without limit,they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance. View Publication

Feeder-independent culture of human embryonic stem cells. View Publication

Small molecules will need to be identified and/or developed that target protein classes limiting reprogramming efficiency. A specific class of proteins includes epigenetic regulators that silence,or minimize expression,of pluripotency genes in differentiated cells. To better understand the role of specific epigenetic modulators in reprogramming,we have used shRNA delivered by lentivirus to assess the significance of individual epi-proteins in reprogramming pluripotent gene expression. View Publication

BACKGROUND AND PURPOSE: Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here,we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals. This study was undertaken to explore the adverse effects of cytosine arabinoside (Ara-C) on randomly differentiated hESCs.backslashnbackslashnEXPERIMENTAL APPROACH: Human embryonic stem cells were used to investigate the effects of a developmental toxicant Ara-C. Sublethal concentrations of Ara-C were given for two time points,day 7 and day 14 during the differentiation. Gene expression was assessed with microarrays to determine the dysregulated transcripts in presence of Ara-C.backslashnbackslashnKEY RESULTS: Randomly differentiated hESCs were able to generate the multilineage markers. The low concentration of Ara-C (1 nM) induced the ectoderm and inhibited the mesoderm at day 14. The induction of ectodermal markers such as MAP2,TUBB III,PAX6,TH and NESTIN was observed with an inhibition of mesodermal markers such as HAND2,PITX2,GATA5,MYL4,TNNT2,COL1A1 and COL1A2. In addition,no induction of apoptosis was observed. Gene ontology revealed unique dysregulated biological process related to neuronal differentiation and mesoderm development. Pathway analysis showed the axon guidance pathway to be dysregulated.backslashnbackslashnCONCLUSIONS AND IMPLICATIONS: Our results suggest that hESCs in combination with toxicogenomics offer a sensitive in vitro developmental toxicity model as an alternative to traditional animal experiments. View Publication

With regard to human induced pluripotent stem cells (hiPSCs),in which adult cells are reprogrammed into embryonic-like cells using defined factors,their functional and transcriptional expression pattern during endothelial differentiation has yet to be characterized. In this study,hiPSCs and human embryonic stem cells (hESCs) were differentiated using the embryoid body method,and CD31(+) cells were sorted. Fluorescence activated cell sorting analysis of hiPSC-derived endothelial cells (hiPSC-ECs) and hESC-derived endothelial cells (hESC-ECs) demonstrated similar endothelial gene expression patterns. We showed functional vascular formation by hiPSC-ECs in a mouse Matrigel plug model. We compared the gene profiles of hiPSCs,hESCs,hiPSC-ECs,hESC-ECs,and human umbilical vein endothelial cells (HUVECs) using whole genome microarray. Our analysis demonstrates that gene expression variation of hiPSC-ECs and hESC-ECs contributes significantly to biological differences between hiPSC-ECs and hESC-ECs as well as to the distances" among hiPSCs� View Publication

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

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

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