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Identification of prevalent specific markers is crucial to stem/progenitor cell purification. Determinants such as the surface antigens CD34 and CD38 are traditionally used to analyze and purify hematopoietic stem/progenitor cells (HSCs/HPCs). However,the variable expression of these membrane antigens poses some limitations to their use in HSC/HPC purification. Techniques based on drug/stain efflux through the ATP-binding cassette (ABC)G2 pump (side population [SP] phenotype) or on detection of aldehyde dehydrogenase (ALDH) activity have been independently developed and distinguish the SP and ALDH(Bright) (ALDH(Br)) cell subsets for their phenotype and proliferative capability. In this study,we developed a multiparametric flow cytometric method associating both SP and ALDH activities on human lineage negative (Lin(-)) bone marrow cells and sorted different cell fractions according to their SP/ALDH activity level. We find that Lin(-)CD34(+)CD38(Low/-) cells are found throughout the spectrum of ALDH expression and are enriched especially in ALDH(Br) cells when associated with SP functionality (SP/ALDH(Br) fraction). Furthermore,the SP marker identified G(0) cells in all ALDH fractions,allowing us to sort quiescent cells regardless of ALDH activity. Moreover,we show that,within the Lin(-)CD34(+)CD38(-)ALDH(Br) population,the SP marker identifies cells with higher primitive characteristics,in terms of stemness-related gene expression and in vitro and in vivo proliferative potential,than the Lin(-)CD34(+) CD38(-)ALDH(Br) main population cells. In conclusion,our study shows that the coexpression of SP and ALDH markers refines the Lin(-)CD34(+)CD38(-) hematopoietic compartment and identifies an SP/ALDH(Br) cell subset enriched in quiescent primitive HSCs/HPCs. View Publication

Surface marker expression forms the basis for characterization and isolation of human embryonic stem cells (hESCs). Currently,there are few well-defined protein epitopes that definitively mark hESCs. Here we combine immunotranscriptional profiling of hESC lines with membrane-polysome translation state array analysis (TSAA) to determine the full set of genes encoding potential hESC surface marker proteins. Three independently isolated hESC lines (HES2,H9,and MEL1) grown under feeder and feeder-free conditions were sorted into subpopulations by fluorescence-activated cell sorting based on coimmunoreactivity to the hESC surface markers GCTM-2 and CD9. Colony-forming assays confirmed that cells displaying high coimmunoreactivity to GCTM-2 and CD9 constitute an enriched subpopulation displaying multiple stem cell properties. Following microarray profiling,820 genes were identified that were common to the GCTM-2(high)/CD9(high) stem cell-like subpopulation. Membrane-polysome TSAA analysis of hESCs identified 1,492 mRNAs encoding actively translated plasma membrane and secreted proteins. Combining these data sets,88 genes encode proteins that mark the pluripotent subpopulation,of which only four had been previously reported. Cell surface immunoreactivity was confirmed for two of these markers: TACSTD1/EPCAM and CDH3/P-Cadherin,with antibodies for EPCAM able to enrich for pluripotent hESCs. This comprehensive listing of both hESCs and spontaneous differentiation-associated transcripts and survey of translated membrane-bound and secreted proteins provides a valuable resource for future study into the role of the extracellular environment in both the maintenance of pluripotency and directed differentiation. View Publication

Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations,including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin axis,causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro,osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore,the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy,bone marrow transplantation,or gene therapy resulted in full recovery of the altered bone parameters. Remarkably,untreated ADA-severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. View Publication

Human immunodeficiency virus type 1 (HIV-1) vectors transduce rhesus blood cells poorly due to a species-specific block by TRIM5alpha and APOBEC3G,which target HIV-1 capsid and viral infectivity factor (Vif),respectively. We sought to develop a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV-1 and simian immunodeficiency virus (SIV),including SIV capsid (sCA) and SIV Vif. A chimeric HIV-1 vector including sCA (chiHIV) was superior to the conventional SIV in transducing a human blood cell line and superior to the conventional HIV-1 vector in transducing a rhesus blood cell line. Among human CD34(+) hematopoietic stem cells (HSCs),the chiHIV and HIV-1 vectors showed similar transduction efficiencies; in rhesus CD34(+) HSCs,the chiHIV vector yielded superior transduction rates. In in vivo competitive repopulation experiments with two rhesus macaques,the chiHIV vector demonstrated superior marking levels over the conventional HIV-1 vector in all blood lineages (first rhesus,15 to 30% versus 1 to 5%; second rhesus,7 to 15% versus 0.5 to 2%,respectively) 3 to 7 months postinfusion. In summary,we have developed an HIV-1-based lentiviral vector system that should allow comprehensive preclinical testing of HIV-1-based therapeutic vectors in the rhesus macaque model with eventual clinical application. View Publication

Development of non-invasive and accurate methods to track cell fate after delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. In this protocol,we describe the in vivo monitoring of stem cell survival,proliferation and migration using reporter genes. We established stable ES cell lines constitutively expressing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein,firefly luciferase and herpes simplex virus thymidine kinase (HSVtk)) reporter genes using lentiviral transduction. We used fluorescence-activated cell sorting to purify these populations in vitro,bioluminescence imaging and positron emission tomography (PET) imaging to track them in vivo and fluorescence immunostaining to confirm the results ex vivo. Unlike other methods of cell tracking,such as iron particle and radionuclide labeling,reporter genes are inherited genetically and can be used to monitor cell proliferation and survival for the lifetime of transplanted cells and their progeny. View Publication

Platelet factor 4 (PF4) is a negative regulator of megakaryopoiesis,but its mechanism of action had not been addressed. Low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) has been shown to mediate endothelial cell responses to PF4 and so we tested this receptor's importance in PF4's role in megakaryopoiesis. We found that LRP1 is absent from megakaryocyte-erythrocyte progenitor cells,is maximally present on large,polyploidy megakaryocytes,and near absent on platelets. Blocking LRP1 with either receptor-associated protein (RAP),an antagonist of LDL family member receptors,or specific anti-LRP1 antibodies reversed the inhibition of megakaryocyte colony growth by PF4. In addition,using shRNA to reduce LRP1 expression was able to restore megakaryocyte colony formation in bone marrow isolated from human PF4-overexpressing mice (hPF4(High)). Further,shRNA knockdown of LRP1 expression was able to limit the effects of PF4 on megakaryopoiesis. Finally,infusion of RAP into hPF4(High) mice was able to increase baseline platelet counts without affecting other lineages,suggesting that this mechanism is important in vivo. These studies extend our understanding of PF4's negative paracrine effect in megakaryopoiesis and its potential clinical implications as well as provide insights into the biology of LRP1,which is transiently expressed during megakaryopoiesis. View Publication

Enumeration of human embryonic stem cell (hESC) numbers through single cell digestion can be time consuming especially in high-throughput or multi-factorial analysis containing 50+ samples. We have developed a reproducible,cost-effective method of counting hESCs in clumps circumventing the need to manually dissociate each sample to single cells. The method is based on the DNA binding capacity of propidium iodide (PI) and subsequent fluorescent signal detection. Standard curves generated for cell numbers versus PI fluorescence as single cells or clumps showed an almost identical relationship in the lines of best fit. The reproducibility of the assay was first demonstrated by seeding hESC clumps at specific cell densities ranging 0.05[x02013]2x105 cells/well and then secondly by using the assay to count cell numbers after different growth conditions. Validation tests showed that consistent seeding densities are important in maintaining undifferentiated hESC culture and that the assay can be used to estimate relative cell numbers and growth curves with high accuracy. View Publication

MLL rearrangements are hallmark genetic abnormalities in infant leukemia known to arise in utero. They can be induced during human prenatal development upon exposure to etoposide. We also hypothesize that chronic exposure to etoposide might render cells more susceptible to other genomic insults. Here,for the first time,human embryonic stem cells (hESCs) were used as a model to test the effects of etoposide on human early embryonic development. We addressed whether: (i) low doses of etoposide promote MLL rearrangements in hESCs and hESCs-derived hematopoietic cells; (ii) MLL rearrangements are sufficient to confer hESCs with a selective growth advantage and (iii) continuous exposure to low doses of etoposide induces hESCs to acquire other chromosomal abnormalities. In contrast to cord blood-derived CD34(+) and hESC-derived hematopoietic cells,exposure of undifferentiated hESCs to a single low dose of etoposide induced a pronounced cell death. Etoposide induced MLL rearrangements in hESCs and their hematopoietic derivatives. After long-term culture,the proportion of hESCs harboring MLL rearrangements diminished and neither cell cycle variations nor genomic abnormalities were observed in the etoposide-treated hESCs,suggesting that MLL rearrangements are insufficient to confer hESCs with a selective proliferation/survival advantage. However,continuous exposure to etoposide induced MLL breaks and primed hESCs to acquire other major karyotypic abnormalities. These data show that chronic exposure of developmentally early stem cells to etoposide induces MLL rearrangements and make hESCs more prone to acquire other chromosomal abnormalities than postnatal CD34(+) cells,linking embryonic genotoxic exposure to genomic instability. View Publication

Acute myelogenous leukemia is driven by leukemic stem cells (LSCs) generated by mutations that confer (or maintain) self-renewal potential coupled to an aberrant differentiation program. Using retroviral mutagenesis,we identified genes that generate LSCs in collaboration with genetic disruption of the gene encoding interferon response factor 8 (Irf8),which induces a myeloproliferation in vivo. Among the targeted genes,we identified Mef2c,encoding a MCM1-agamous-deficiens-serum response factor transcription factor,and confirmed that overexpression induced a myelomonocytic leukemia in cooperation with Irf8 deficiency. Strikingly,several of the genes identified in our screen have been reported to be up-regulated in the mixed-lineage leukemia (MLL) subtype. High MEF2C expression levels were confirmed in acute myelogenous leukemia patient samples with MLL gene disruptions,prompting an investigation of the causal interplay. Using a conditional mouse strain,we demonstrated that Mef2c deficiency does not impair the establishment or maintenance of LSCs generated in vitro by MLL/ENL fusion proteins; however,its loss led to compromised homing and invasiveness of the tumor cells. Mef2c-dependent targets included several genes encoding matrix metalloproteinases and chemokine ligands and receptors,providing a mechanistic link to increased homing and motility. Thus,MEF2C up-regulation may be responsible for the aggressive nature of this leukemia subtype. View Publication

The role of miRNAs in regulating megakaryocyte differentiation was examined using bipotent K562 human leukemia cells. miR-34a is strongly up-regulated during phorbol ester-induced megakaryocyte differentiation,but not during hemin-induced erythrocyte differentiation. Enforced expression of miR-34a in K562 cells inhibits cell proliferation,induces cell-cycle arrest in G(1) phase,and promotes megakaryocyte differentiation as measured by CD41 induction. miR-34a expression is also up-regulated during thrombopoietin-induced differentiation of CD34(+) hematopoietic precursors,and its enforced expression in these cells significantly increases the number of megakaryocyte colonies. miR-34a directly regulates expression of MYB,facilitating megakaryocyte differentiation,and of CDK4 and CDK6,to inhibit the G(1)/S transition. However,these miR-34a target genes are down-regulated rapidly after inducing megakaryocyte differentiation before miR-34a is induced. This suggests that miR-34a is not responsible for the initial down-regulation but may contribute to maintaining their suppression later on. Previous studies have implicated miR-34a as a tumor suppressor gene whose transcription is activated by p53. However,in p53-null K562 cells,phorbol esters induce miR-34a expression independently of p53 by activating an alternative phorbol ester-responsive promoter to produce a longer pri-miR-34a transcript. View Publication

BACKGROUND: The presence of chromosomal abnormalities could have a negative impact for human embryonic stem cell (hESC) applications both in regenerative medicine and in research. A biomarker that allows the identification of chromosomal abnormalities induced in hESC in culture before they take over the culture would represent an important tool for defining optimal culture conditions for hESC. Here we investigate the expression of CD30,reported to be a biomarker of hESCs with abnormal karyotype,in undifferentiated and spontaneously differentiated hESC.backslashnbackslashnMETHODS AND RESULTS: hESC were derived and cultured on mouse fibroblasts in KO-SR containing medium (serum free media) and passaged mechanically. Our results based on analysis at mRNA (RT-PCR) and protein (fluorescence-activated cell sorting and immunocytochemistry) level show that CD30 is expressed in undifferentiated hESC,even at very early passages,without any correlation with the presence of chromosomal anomalies. We also show that the expression of CD30 is rapidly lost during early spontaneous differentiation of hESC.backslashnbackslashnCONCLUSION: We conclude that CD30 expression in hESC cultures is probably a consequence of culture conditions,and that KO-SR may play a role. In addition,the expression of so-called 'stemness' markers does not change in undifferentiated hESC during long-term culture or when cells acquire chromosomal abnormalities. View Publication