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Hyperactivation of the transcription factor Stat5 leads to various leukemias. Stat5 activity is regulated by the protein phosphatase SHP-1 in a phospholipase C (PLC)-β3-dependent manner. Thus,PLC-β3-deficient mice develop myeloproliferative neoplasm,like Lyn (Src family kinase)- deficient mice. Here we show that Lyn/PLC-β3 doubly deficient lyn(-/-);PLC-β3(-/-) mice develop a Stat5-dependent,fatal myelodysplastic/myeloproliferative neoplasm,similar to human chronic myelomonocytic leukemia (CMML). In hematopoietic stem cells of lyn(-/-);PLC-β3(-/-) mice that cause the CMML-like disease,phosphorylation of SHP-1 at Tyr(536) and Tyr(564) is abrogated,resulting in reduced phosphatase activity and constitutive activation of Stat5. Furthermore,SHP-1 phosphorylation at Tyr(564) by Lyn is indispensable for maximal phosphatase activity and for suppression of the CMML-like disease in these mice. On the other hand,Tyr(536) in SHP-1 can be phosphorylated by Lyn and another kinase(s) and is necessary for efficient interaction with Stat5. Therefore,we identify a novel Lyn/PLC-β3-mediated regulatory mechanism of SHP-1 and Stat5 activities. View Publication

Because primary myelofibrosis (PMF) originates at the level of the pluripotent hematopoietic stem cell (HSC),we examined the effects of various therapeutic agents on the in vitro and in vivo behavior of PMF CD34(+) cells. Treatment of PMF CD34(+) cells with chromatin-modifying agents (CMAs) but not hydroxyurea,Janus kinase 2 (JAK2) inhibitors,or low doses of interferon-α led to the generation of greater numbers of CD34(+) chemokine (C-X-C motif) receptor (CXCR)4(+) cells,which were capable of migrating in response to chemokine (C-X-C motif) ligand (CXCL)12 and resulted in a reduction in the proportion of hematopoietic progenitor cells (HPCs) that were JAK2V617F(+). Furthermore,sequential treatment of PMF CD34(+) cells but not normal CD34(+) cells with decitabine (5-aza-2'-deoxycytidine [5azaD]),followed by suberoylanilide hydroxamic acid (SAHA; 5azaD/SAHA),or trichostatin A (5azaD/TSA) resulted in a higher degree of apoptosis. Two to 6 months after the transplantation of CMAs treated JAK2V617F(+) PMF CD34(+) cells into nonobese diabetic/severe combined immunodeficient (SCID)/IL-2Rγ(null) mice,the percentage of JAK2V617F/JAK2(total) in human CD45(+) marrow cells was dramatically reduced. These findings suggest that both PMF HPCs,short-term and long-term SCID repopulating cells (SRCs),are JAK2V617F(+) and that JAK2V617F(+) HPCs and SRCs can be eliminated by sequential treatment with CMAs. Sequential treatment with CMAs,therefore,represents a possible effective means of treating PMF at the level of the malignant SRC. View Publication

Human-induced pluripotent stem cells (iPSCs) generated from human adult somatic cells through reprogramming hold great promises for future regenerative medicine. However,exposure of human iPSCs to animal feeder and serum in the process of their generation and maintenance imposes risk of transmitting animal pathogens to human subjects,thus hindering the potential therapeutic applications. Here,we report the successful generation of human iPSCs in a feeder-independent culture system with defined factors. Two stable human iPSC lines were established from primary human dermal fibroblasts of two healthy volunteers. These human iPSCs expressed a panel of pluripotency markers including stage-specific embryonic antigen (SSEA)-4,tumor-rejection antigen (TRA)-1-60,TRA-1-81,and alkaline phosphatase,while maintaining normal karyotypes and the exogenous reprogramming factors being silenced. In addition,these human iPSCs can differentiate along lineages representative of the three embryonic germ layers upon formation of embryoid bodies,indicating their pluripotency. Furthermore,subcutaneous transplantation of these cells into immunodeficient mice resulted in teratoma formation in 6 to 8 weeks. Our findings are an important step toward generating patient-specific iPSCs in a more clinically compliant manner by eliminating the need of animal feeder cells and animal serum. View Publication

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS),and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage,resolution,cost,concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls,the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This,along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states,identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression. View Publication

The Sox6 transcription factor plays critical roles in various cell types,including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells,we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar,evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo,and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied,in erythroid cells,by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation,mediated by the double Sox6 binding site within its own promoter,may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation. View Publication

Despite many years of intensive effort,there is surprisingly little consensus on the most suitable markers with which to locate and isolate stem cells from adult tissues. By comparison,the study of cancer stem cells is still in its infancy; so,unsurprisingly,there is great uncertainty as to the identity of these cells. Stem cell markers can be broadly categorized into molecular determinants of self-renewal,clonogenicity,multipotentiality,adherence to the niche,and longevity. This review assesses the utility of recognizing cancer stem cells by virtue of high expression of aldehyde dehydrogenases (ALDHs),probably significant determinants of cell survival through their ability to detoxify many potentially cytotoxic molecules,and contributing to drug resistance. Antibodies are available against the ALDH enzyme family,but the vast majority of studies have used cell sorting techniques to enrich for cells expressing these enzymes. Live cells expressing high ALDH activity are usually identified by the ALDEFLUOR kit and sorted by fluorescence activated cell sorting (FACS). For many human tumours,but notably breast cancer,cell selection based upon ALDH activity appears to be a useful marker for enriching for cells with tumour-initiating activity (presumed cancer stem cells) in immunodeficient mice,and indeed the frequency of so-called ALDH(bri) cells in many tumours can be an independent prognostic indicator. View Publication

The Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor that is activated by phosphorylation at S102 whereby it induces the expression of growth promoting genes such as EGFR and HER-2. We recently illustrated by an in vitro kinase assay that a novel peptide to YB-1 was highly phosphorylated by the serine/threonine p90 S6 kinases RSK-1 and RSK-2,and to a lesser degree PKCα and AKT. Herein,we sought to develop this decoy cell permeable peptide (CPP) as a cancer therapeutic. This 9-mer was designed as an interference peptide that would prevent endogenous YB-1(S102) phosphorylation based on molecular docking. In cancer cells,the CPP blocked P-YB-1(S102) and down-regulated both HER-2 and EGFR transcript level and protein expression. Further,the CPP prevented YB-1 from binding to the EGFR promoter in a gel shift assay. Notably,the growth of breast (SUM149,MDA-MB-453,AU565) and prostate (PC3,LNCap) cancer cells was inhibited by ∼90% with the CPP. Further,treatment with this peptide enhanced sensitivity and overcame resistance to trastuzumab in cells expressing amplified HER-2. By contrast,the CPP had no inhibitory effect on the growth of normal immortalized breast epithelial (184htert) cells,primary breast epithelial cells,nor did it inhibit differentiation of hematopoietic progenitors. These data collectively suggest that the CPP is a novel approach to suppressing the growth of cancer cells while sparing normal cells and thereby establishes a proof-of-concept that blocking YB-1 activation is a new course of cancer therapeutics. View Publication

Human pluripotent stem (hPS) cells such as human embryonic stem (hES) and induced pluripotent stem (hiPS) cells are vulnerable under single cell conditions,which hampers practical applications; yet,the mechanisms underlying this cell death remain elusive. In this paper,we demonstrate that treatment with a specific inhibitor of non-muscle myosin II (NMII),blebbistatin,enhances the survival of hPS cells under clonal density and suspension conditions,and,in combination with a synthetic matrix,supports a fully defined environment for self-renewal. Consistent with this,genetically engineered mouse embryonic stem cells lacking an isoform of NMII heavy chain (NMHCII),or hES cells expressing a short hairpin RNA to knock down NMHCII,show greater viability than controls. Moreover,NMII inhibition increases the expression of self-renewal regulators Oct3/4 and Nanog,suggesting a mechanistic connection between NMII and self-renewal. These results underscore the importance of the molecular motor,NMII,as a novel target for chemically engineering the survival and self-renewal of hPS cells. View Publication

INTRODUCTION: The physiological signals that direct the division and differentiation of the zygote to form a blastocyst,and subsequent embryonic stem cell division and differentiation during early embryogenesis,are unknown. Although a number of growth factors,including the pregnancy-associated hormone human chorionic gonadotropin (hCG) are secreted by trophoblasts that lie adjacent to the embryoblast in the blastocyst,it is not known whether these growth factors directly signal human embryonic stem cells (hESCs).backslashnbackslashnMETHODS: Here we used hESCs as a model of inner cell mass differentiation to examine the hormonal requirements for the formation of embryoid bodies (EB's; akin to blastulation) and neuroectodermal rosettes (akin to neurulation).backslashnbackslashnRESULTS: We found that hCG promotes the division of hESCs and their differentiation into EB's and neuroectodermal rosettes. Inhibition of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) signaling suppresses hESC proliferation,an effect that is reversed by treatment with hCG. hCG treatment rapidly upregulates steroidogenic acute regulatory protein (StAR)-mediated cholesterol transport and the synthesis of progesterone (P4). hESCs express P4 receptor A,and treatment of hESC colonies with P4 induces neurulation,as demonstrated by the expression of nestin and the formation of columnar neuroectodermal cells that organize into neural tubelike rosettes. Suppression of P4 signaling by withdrawing P4 or treating with the P4-receptor antagonist RU-486 inhibits the differentiation of hESC colonies into EB's and rosettes.backslashnbackslashnCONCLUSIONS: Our findings indicate that hCG signaling via LHCGR on hESC promotes proliferation and differentiation during blastulation and neurulation. These findings suggest that trophoblastic hCG secretion and signaling to the adjacent embryoblast could be the commencement of trophic support by placental tissues in the growth and development of the human embryo. View Publication

The C-X-C-type chemokine Cxcl12,also known as stromal cell-derived factor-1,plays a critical role in hematopoiesis during fetal development. However,the functional requirement of Cxcl12 in the adult hematopoietic stem/progenitor cell (HSPC) regulation was still unclear. In this report,we developed a murine Cxcl12 conditional deletion model in which the target gene can be deleted at the adult stage. We found that loss of stroma-secreted Cxcl12 in the adult led to expansion of the HSPC population as well as a reduction in long-term quiescent stem cells. In Cxcl12-deficient bone marrow,HSPCs were absent along the endosteal surface,and blood cell regeneration occurred predominantly in the perisinusoidal space after 5-fluorouracil myelosuppression challenge. Our results indicate that Cxcl12 is required for HSPC homeostasis regulation and is an important factor for osteoblastic niche organization in adult stage bone marrow. View Publication