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The ability of acute myeloid leukemic (AML) blasts to differentiate into leukemic dendritic cells (DC) thus acquiring the potential to present known and unknown leukemic antigens efficiently,holds promise as a possible new treatment for AML patients with minimal residual disease. Recent advances in culture methods have made the clinical use of leukemic DC feasible. However,additional measures appear to be essential in order to potentiate vaccines and to overcome the intrinsic tolerant state of the patients immune system. This review describes ways to improve AML-DC vaccines and discusses critical aspects concerning the development of clinical vaccination protocols. View Publication

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7),the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions,and currently,there are no effective forms of therapy. Nevertheless,some advances in patient therapy are being made,and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials. To establish a foundation for personalized,gene-corrected,patient-specific cell transfer,we generated induced pluripotent stem (iPS) cells from three subjects with RDEB (RDEB iPS cells). We found that Col7 was not required for stem cell renewal and that RDEB iPS cells could be differentiated into both hematopoietic and nonhematopoietic lineages. The specific epigenetic profile associated with de-differentiation of RDEB fibroblasts and keratinocytes into RDEB iPS cells was similar to that observed in wild-type (WT) iPS cells. Importantly,human WT and RDEB iPS cells differentiated in vivo into structures resembling the skin. Gene-corrected RDEB iPS cells expressed Col7. These data identify the potential of RDEB iPS cells to generate autologous hematopoietic grafts and skin cells with the inherent capacity to treat skin and mucosal erosions that typify this genodermatosis. View Publication

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH),a DA neuron marker,and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth. View Publication

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration,conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However,it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines,along with methylomes of ES cells,somatic cells,and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability,including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation,and differences in CG methylation and histone modifications. Lastly,differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency,providing an iPSC reprogramming signature that is maintained after differentiation. View Publication

Human embryonic stem (hES) cells have the dual ability to self-renew and differentiate into specialized cell types. However,in order to realize the full potential of these cells it is important to understand how the genes responsible for their unique characteristics are regulated. In this study we examine the regulation of the tropomyosin-related kinase (TRK) genes which encode for receptors important in hES cell survival and self-renewal. Although the TRK genes have been studied in many neuronal cell types,the regulation of these genes in hES cells is unclear. Our study demonstrates a novel regulatory relationship between the TRKC gene and the transcription factor SOX2. Our results found that hES cells highly express full-length and truncated forms of the TRKC gene. However,examination of the related TRKB gene showed a lower overall expression of both full-length and truncated forms. Through RNA interference,we knocked down expression levels of SOX2 in hES cells and examined the expression of TRKC,as well as TRKB. Upon loss of SOX2 we found that TRKC mRNA levels were significantly downregulated but TRKB levels remained unchanged,demonstrating an important regulatory dependence on SOX2 by TRKC. We also found that TRKC protein levels were also decreased after SOX2 knock down. Further analysis found the regulatory region of TRKC to be highly conserved among many mammals with potential SOX binding motifs. We confirmed a specific binding motif as a site that SOX2 utilizes to directly interact with the TRKC regulatory region. In addition,we found that SOX2 drives expression of the TRKC gene by activating a luciferase reporter construct containing the TRKC regulatory region and the SOX binding motif. View Publication

Human U1 small nuclear (sn)RNA,required for splicing of pre-mRNA,is encoded by genes on chromosome 1 (1p36). Imperfect copies of these U1 snRNA genes,also located on chromosome 1 (1q12-21),were thought to be pseudogenes. However,many of these variant" (v)U1 snRNA genes produce fully processed transcripts. Using antisense oligonucleotides to block the activity of a specific vU1 snRNA in HeLa cells View Publication

Progestins play a deleterious role in the onset of breast cancer,yet their influence on existing breast cancer and tumor progression is not well understood. In luminal estrogen receptor (ER)- and progesterone receptor (PR)-positive breast cancer,progestins induce a fraction of cells to express cytokeratin 5 (CK5),a marker of basal epithelial and progenitor cells in the normal breast. CK5(+) cells lose expression of ER and PR and are relatively quiescent,increasing their resistance to endocrine and chemotherapy compared to intratumoral CK5(-)ER(+)PR(+) cells. Characterization of live CK5(+) cells has been hampered by a lack of means for their direct isolation. Here,we describe optical (GFP) and bioluminescent (luciferase) reporter models to quantitate and isolate CK5(+) cells in luminal breast cancer cell lines utilizing the human KRT5 gene promoter and a viral vector approach. Using this system,we confirmed that the induction of GFP(+)/CK5(+) cells is specific to progestins,is dependent on PR,can be blocked by antiprogestins,and does not occur with other steroid hormones. Progestin-induced,fluorescence-activated cell sorting-isolated CK5(+) cells had lower ER and PR mRNA,were slower cycling,and were relatively more invasive and sphere forming than their CK5(-) counterparts in vitro. Repeated progestin treatment and selection of GFP(+) cells enriched for a persistent population of CK5(+) cells,suggesting that this transition can be semi-permanent. These data support that in PR(+) breast cancers,progestins induce a subpopulation of CK5(+)ER(-)PR(-) cells with enhanced progenitor properties and have implications for treatment resistance and recurrence in luminal breast cancer. View Publication

A commonly activated signaling cascade in many human malignancies,including glioblastoma multiforme,is the Akt pathway. This pathway can be activated via numerous upstream alterations including genomic amplification of epidermal growth factor receptor,PTEN deletion,or PIK3CA mutations. In this study,we screened phosphatidylinositol 3-kinase/Akt small-molecule inhibitors in an isogenic cell culture system with an activated Akt pathway secondary to a PIK3CA mutation. One small molecule,A-443654,showed the greatest selective inhibition of cells with the mutant phenotype. Based on these findings,this inhibitor was screened in vitro against a panel of glioblastoma multiforme cell lines. All cell lines tested were sensitive to A-443654 with a mean IC(50) of approximately 150 nmol/L. An analogue of A-443654,methylated at a region that blocks Akt binding,was on average 36-fold less active. Caspase assays and dual flow cytometric analysis showed an apoptotic mechanism of cell death. A-443654 was further tested in a rat intracranial model of glioblastoma multiforme. Animals treated intracranially with polymers containing A-443654 had significantly extended survival compared with control animals; animals survived 79% and 43% longer than controls when A-443654-containing polymers were implanted simultaneously or in a delayed fashion,respectively. This small molecule also inhibited glioblastoma multiforme stem-like cells with similar efficacy compared with traditionally cultured glioblastoma multiforme cell lines. These results suggest that local delivery of an Akt small-molecule inhibitor is effective against experimental intracranial glioma,with no observed resistance to glioblastoma multiforme cells grown in stem cell conditions. 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

Since the derivation of human embryonic stem (hES) cells,their translation to clinical therapies has been met with several challenges,including the need for large-scale expansion and controlled differentiation processes. Suspension bioreactors are an effective alternative to static culture flasks as they enable the generation of clinically relevant cell numbers with greater efficacy in a controlled culture system. We,along with other groups,have developed bioreactor protocols for the expansion of pluripotent murine ES cells. Here we present a novel bioreactor protocol that yields a 25-fold expansion of hES cells over 6 days. Using immunofluorescence,flow cytometry,and teratoma formation assays,we demonstrated that these bioreactor cultures retained high levels of pluripotency and a normal karyotype. Importantly,the use of bioreactors enables the expansion of hES cells in the absence of feeder layers or matrices,which will facilitate the adaptation of good manufacturing process (GMP) standards to the development of hES cell therapies. View Publication

Mature CD4(+)Vbeta5(+) T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or TCR revision. In Vbeta5 transgenic mice,this latter tolerance pathway results in the appearance of CD4(+)Vbeta5(-)TCRbeta(+) T cells,coinciding with Rag1,Rag2,and TdT expression and the accumulation of V(beta)-DJ(beta) recombination intermediates in peripheral CD4(+) T cells. Because postthymic RAG-dependent TCR rearrangement has remained controversial,we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We show in this study that Rag deletion in post-positive selection T cells in Vbeta5 transgenic mice blocks TCR revision in vivo and that mature peripheral T cells sorted to remove cells bearing endogenous TCRbeta-chains can express newly generated TCRbeta molecules in adoptive hosts. These findings unambiguously demonstrate postthymic,RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4(+) T cells. View Publication

The longevity-promoting NAD+-dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4,TBX3,and PAX6,which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells. View Publication