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Current induction schemes directing hematopoietic differentiation of human embryonic stem cells (hESCs) are not well defined to mimic the sequential stages of hematopoietic development in vivo. Here,we report a 3-stage method to direct differentiation of hESCs toward hematopoietic progenitors in chemically defined mediums. In the first 2 stages,we efficiently generated T-positive primitive streak/mesendoderm cells and kinase domain receptor-positive (KDR(+)) platelet-derived growth factor receptor α-negative (PDGFRα(-)) hemato-vascular precursors sequentially. In the third stage,we found that cells in a spontaneous differentiation condition mainly formed erythroid colonies. Addition of all-trans retinoic acid (RA) greatly enhanced generation of hematopoietic progenitors in this stage while suppressing erythroid development. The RA-treated cells highly expressed definitive hematopoietic genes,formed large numbers of multilineage and myeloid colonies,and gave rise to greater than 45% CD45(+) hematopoietic cells. When hematopoietic progenitors were selected with CD34 and C-Kit,greater than 95% CD45(+) hematopoietic cells could be generated. In addition,we found that endogenous RA signaling at the second stage was required for vascular endothelial growth factor/basic fibroblast growth factor-induced hemato-vascular specification,whereas exogenously applied RA efficiently induced KDR(-)PDGFRα(+) paraxial mesoderm cells. Our study suggests that RA signaling plays diverse roles in human mesoderm and hematopoietic development. View Publication

Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However,our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore,we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types,n = 792) by immunohistochemical staining. Using the ALDEFUOR assay,ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition,an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct,and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn't significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers,we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439,p = 0.0036). Finally,ALDH(br) tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker,ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast,lung,ovarian or colon cancer. View Publication

Trib1,Trib2,and Trib3 are mammalian homologs of Tribbles,an evolutionarily conserved Drosophila protein family that mediates protein degradation. Tribbles proteins function as adapters to recruit E3 ubiquitin ligases and enhance ubiquitylation of the target protein to promote its degradation. Increased Trib1 and Trib2 mRNA expression occurs in human myeloid leukemia and induces acute myeloid leukemia in mice,whereas Trib3 has not been associated with leukemia. Given the high degree of structural conservation among Tribbles family members,we directly compared the 3 mammalian Tribbles in hematopoietic cells by reconstituting mice with hematopoietic stem cells retrovirally expressing these proteins. All mice receiving Trib1 or Trib2 transduced hematopoietic stem cells developed acute myeloid leukemia,whereas Trib3 mice did not. Our previous data indicated that Trib2-mediated degradation of the transcription factor,CCAAT/enhancer-binding protein-alpha (C/EBPalpha),is important for leukemogenesis. Similar to Trib2,Trib1 induced C/EBPalpha degradation and inhibited its function. In contrast,Trib3 failed to inactivate or promote efficient degradation of C/EBPalpha. These data reveal that the 3 Tribbles homologs differ in their ability to promote degradation of C/EBPalpha,which account for their differential ability to induce leukemia. View Publication

Accidental insertional activation of proto-oncogenes and potential vector mobilization pose serious challenges for human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviral vectors have elucidated distinct target site preferences,highlighting vectors based on the alpharetrovirus Rous sarcoma virus (RSV) as those with the most neutral integration spectrum. To date,alpharetroviral vector systems are based mainly on single constructs containing viral coding sequences and intact long terminal repeats (LTR). Even though they are considered to be replication incompetent in mammalian cells,the transfer of intact viral genomes is unacceptable for clinical applications,due to the risk of vector mobilization and the potentially immunogenic expression of viral proteins,which we minimized by setting up a split-packaging system expressing the necessary viral proteins in trans. Moreover,intact LTRs containing transcriptional elements are capable of activating cellular genes. By removing most of these transcriptional elements,we were able to generate a self-inactivating (SIN) alpharetroviral vector,whose LTR transcriptional activity is strongly reduced and whose transgene expression can be driven by an internal promoter of choice. Codon optimization of the alpharetroviral Gag/Pol expression construct and further optimization steps allowed the production of high-titer self-inactivating vector particles in human cells. We demonstrate proof of principle for the versatility of alpharetroviral SIN vectors for the genetic modification of murine and human hematopoietic cells at a low multiplicity of infection. View Publication

The developmental switch from human fetal (gamma) to adult (beta) hemoglobin represents a clinically important example of developmental gene regulation. The transcription factor BCL11A is a central mediator of gamma-globin silencing and hemoglobin switching. Here we determine chromatin occupancy of BCL11A at the human beta-globin locus and other genomic regions in vivo by high-resolution chromatin immunoprecipitation (ChIP)-chip analysis. BCL11A binds the upstream locus control region (LCR),epsilon-globin,and the intergenic regions between gamma-globin and delta-globin genes. A chromosome conformation capture (3C) assay shows that BCL11A reconfigures the beta-globin cluster by modulating chromosomal loop formation. We also show that BCL11A and the HMG-box-containing transcription factor SOX6 interact physically and functionally during erythroid maturation. BCL11A and SOX6 co-occupy the human beta-globin cluster along with GATA1,and cooperate in silencing gamma-globin transcription in adult human erythroid progenitors. These findings collectively demonstrate that transcriptional silencing of gamma-globin genes by BCL11A involves long-range interactions and cooperation with SOX6. Our findings provide insight into the mechanism of BCL11A action and new clues for the developmental gene regulatory programs that function at the beta-globin locus. View Publication

Microfluidic image cytometry (MIC) has been developed to study phenotypes of various hPSC lines by screening several chemically defined serum/feeder-free conditions. A chemically defined hPSC culture was established using 20 ng mL(-1) of bFGF on 20 microg mL(-1) of Matrigel to grow hPSCs over a week in an undifferentiated state. Following hPSC culture,we conducted quantitative MIC to perform a single cell profiling of simultaneously detected protein expression (OCT4 and SSEA1). Using clustering analysis,we were able to systematically compare the characteristics of various hPSC lines in different conditions. View Publication

PURPOSE: The existence of cancer stem cells (CSCs) in breast cancer has profound implications for cancer prevention. In this study,we evaluated sulforaphane,a natural compound derived from broccoli/broccoli sprouts,for its efficacy to inhibit breast CSCs and its potential mechanism. EXPERIMENTAL DESIGN: Aldefluor assay and mammosphere formation assay were used to evaluate the effect of sulforaphane on breast CSCs in vitro. A nonobese diabetic/severe combined immunodeficient xenograft model was used to determine whether sulforaphane could target breast CSCs in vivo,as assessed by Aldefluor assay,and tumor growth upon cell reimplantation in secondary mice. The potential mechanism was investigated using Western blotting analysis and beta-catenin reporter assay. RESULTS: Sulforaphane (1-5 micromol/L) decreased aldehyde dehydrogenase-positive cell population by 65% to 80% in human breast cancer cells (P textless 0.01) and reduced the size and number of primary mammospheres by 8- to 125-fold and 45% to 75% (P textless 0.01),respectively. Daily injection with 50 mg/kg sulforaphane for 2 weeks reduced aldehyde dehydrogenase-positive cells by textgreater50% in nonobese diabetic/severe combined immunodeficient xenograft tumors (P = 0.003). Sulforaphane eliminated breast CSCs in vivo,thereby abrogating tumor growth after the reimplantation of primary tumor cells into the secondary mice (P textless 0.01). Western blotting analysis and beta-catenin reporter assay showed that sulforaphane downregulated the Wnt/beta-catenin self-renewal pathway. CONCLUSIONS: Sulforaphane inhibits breast CSCs and downregulates the Wnt/beta-catenin self-renewal pathway. These findings support the use of sulforaphane for the chemoprevention of breast cancer stem cells and warrant further clinical evaluation. View Publication

BACKGROUND: We recently reported that gastrointestinal (GI) cancer cells can be reprogrammed to a pluripotent state by the ectopic expression of defined embryonic stem (ES)-like transcriptional factors. The induced pluripotent cancer (iPC) cells from GI cancer were sensitized to chemotherapeutic agents and differentiation-inducing treatment during a short-term culture,although a phenotype induced by long-term culture needs to be studied. METHODS: A long-term cultured (Lc)-iPC cells were produced in GI cancer cell lines by virus-mediated introduction of four ES-like genes-c-MYC,SOX2,OCT3/4,and KLF4-followed by a culture more than three months after iPC cells induction. An acquired state was studied by expression of immature-related surface antigens,Tra-1-60,Tra-1-81,Tra-2-49,and Ssea-4; and epigenetic trimethyl modification at lysine 4 of histone H3. Sensitivity to chemotherapeutic agents and tumorigenicity were studied in Lc-iPC cells. RESULTS: Whereas the introduction of defined factors of iPC cells once induced an immature state and sensitized cells to therapeutic reagents,the endogenous expression of the ES-like genes except for activated endogenous c-MYC was down-regulated in a long-term culture,suggesting a high magnitude of the reprogramming induction by defined factors and the requirement of therapeutic maintenance in Lc-iPC cells from cholangiocellular carcinoma HuCC-T1 cells,which harbor TP53(R175H) and KRAS(G12D). The Lc-iPC cells showed resistance to 5-fluorouracil in culture,and high tumorigenic ability with activated endogenous c-MYC in immunodeficient mice. CONCLUSION: The Lc-iPC cells from HuCC-T1 might be prone to an undesirable therapeutic response because of an association with the activated endogenous c-MYC. To consider the possible therapeutic approach in GI cancer,it would be necessary to develop a predictive method for evaluating the improper reprogramming-associated aggressive phenotype of iPC cells. View Publication

Metabolic studies of human embryonic stem cells (hESCs) can provide important information for stem cell bioprocessing. To this end,we have examined growth and metabolism of hESCs in both traditional 2-dimensional (2D) colony cultures and 3-dimensional microcarrier cultures using a conditioned medium and 3 serum-free media. The 2D colony cultures plateaued at cell densities of 1.1-1.5 × 10�?� cells/mL at day 6 due to surface limitation. Microcarrier cultures achieved 1.5-2 × 10�?� cells/mL on days 8-10 before reaching a plateau; this growth arrest was not due to surface limitation,but probably due to metabolic limitations. Metabolic analysis of the cultures showed that amino acids (including glutamine) and glucose are in excess and are not limiting cell growth; on the other hand,the high levels of waste products (25 mM lactate and 0.8 mM ammonium) and low pH (6.6) obtained at the last stages of cell propagation could be the causes for growth arrest. hESCs cultured in media supplemented with lactate (up to 28 mM) showed reduced cell growth,whereas ammonium (up to 5 mM) had no effect. Lactate and,to a lesser extent,ammonia affected pluripotency as reflected by the decreasing population of cells expressing pluripotent marker TRA-1-60. Feeding hESC cultures with low concentrations of glucose resulted in lower lactate levels (∼10%) and a higher pH level of 6.7,which leads to a 40% increase in cell density. We conclude that the high lactate levels and the low pH during the last stages of high-density hESC culture may limit cell growth and affect pluripotency. To overcome this limitation,a controlled feed of low levels of glucose and online control of pH can be used. View Publication

Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state,instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date,only mouse iPSC lines are known to be truly pluripotent. However,initial mouse iPSC lines failed to form chimeric offspring,but did generate teratomas and differentiated embryoid bodies,and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore,there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1,SOX2,NANOG,KLF4,LIN28,and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high,85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies,genetic engineering,and other aspects of stem cell and developmental biology. View Publication

The study of human erythropoiesis in health and disease requires a robust culture system that consistently and reliably generates large numbers of immature erythroblasts that can be induced to differentiate synchronously. We describe a culture method modified from Leberbauer et al. (2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34(+) cells. This pure population of immature erythroblasts can be expanded to obtain 4x10(8) erythroblasts from 1x10(8) PBMC after 13-14 days in culture. Upon synchronized differentiation,high levels of enucleation (80-90%) and low levels of cell death (textless10%) are achieved. We compared the yield of erythroblasts obtained from PBMC,CD34(+) cells or PBMC depleted of CD34(+) cells and show that CD34(-) cells represent the most significant early erythroid progenitor population. This culture system may be particularly useful for investigating the pathophysiology of anemic patients where only small blood volumes are available. View Publication

Unlocking the clinical potential of stem cell based therapies requires firstly elucidation of the biological mechanisms which direct stem cell fate decisions and thereafter,technical advances which allow these processes to be driven in a fully defined culture environment. Strategies for the generation of defined surfaces for human embryonic stem cell (hESC) and mesenchymal stem cell (MSC) culture remain in their infancy. In this paper we outline a simple,effective and efficient method for presenting proteins or peptides on an otherwise non-fouling Layer-by-Layer (LbL) self-assembled surface of hyaluronic acid (HA) and chitosan (CHI). We are able to generate a surface that has both good temporal stability and the ability to direct biological outcomes based on its defined surface composition. Surface functionalization is achieved through suspending the selected extracellular matrix (ECM) protein domain or extracted full-length protein in buffer containing a cross-linking agent (N-hydroxysulfosuccinimide/N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) over the LbL HA-CHI surface and then allowing the solvent to evaporate overnight. This simple,but important step results in remarkable protein deposition efficiencies often exceeding 50%,whereas traditional cross-linking methods result in such poor deposition of non-collagenous proteins that a.) quantification of bound amounts of protein is outside the resolution of commonly utilized protein assays,and b.) these surfaces are both unable to support cell attachment and growth. The utility of the protein-modified HA-CHI surfaces is demonstrated through the identification of specific hESC attachment efficiencies and through directing MSC osteogenic outcomes on these fully defined surfaces. This simple and scalable method is shown to enable the development of defined stem cell culture conditions,as well as the elucidation of the fundamental biological processes necessary for the realization of stem cell based therapies. View Publication