搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Gain of 20q11.21 is a chromosomal abnormality that is recurrently found in human pluripotent stem cells and cancers,strongly suggesting that this mutation confers a proliferative or survival advantage to these cells. In this work we studied three human embryonic stem cell (hESC) lines that acquired a gain of 20q11.21 during in vitro culture. The study of the mRNA gene expression levels of the loci located in the common region of duplication showed that HM13,ID1,BCL2L1,KIF3B and the immature form of the micro-RNA miR-1825 were up-regulated in mutant cells. ID1 and BCL2L1 were further studied as potential drivers of the phenotype of hESC with a 20q11.21 gain. We found no increase in the protein levels of ID1,nor the downstream effects expected from over-expression of this gene. On the other hand,hESC with a gain of 20q11.21 had on average a 3-fold increase of Bcl-xL (the anti-apoptotic isoform of BCL2L1) protein levels. The mutant hESC underwent 2- to 3-fold less apoptosis upon loss of cell-to-cell contact and were ∼2-fold more efficient in forming colonies from a single cell. The key role of BCL2L1 in this mutation was further confirmed by transgenic over-expression of BCL2L1 in the wild-type cells,leading to apoptosis-resistant cells,and BCL2L1-knock-down in the mutant hESC,resulting in a restoration of the wild-type phenotype. This resistance to apoptosis supposes a significant advantage for the mutant cells,explaining the high frequency of gains of 20q11.21 in human pluripotent stem cells. View Publication

Astrocytes are instrumental to major brain functions,including metabolic support,extracellular ion regulation,the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental,psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes),we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP,S100$\$,NFIA and ALDH1L1. In addition,mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion,the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy. View Publication

Pluripotent stem cell markers can be useful for diagnostic evaluation of human tumors. The novel pluripotent marker stage-specific embryonic antigen-5 (SSEA-5) is expressed in undifferentiated human induced pluripotent cells (iPSCs),but little is known about SSEA-5 expression in other primitive tissues (e.g.,human tumors). We evaluated SSEA-5 immunoreactivity patterns in human tumors,cell lines,teratomas,and iPS cells together with another pluripotent cell surface marker L1 cell adhesion molecule (L1CAM). We tested two hypotheses: (1) SSEA-5 and L1CAM would be immunoreactive and colocalized in human tumors; (2) SSEA-5 and L1CAM immunoreactivity would persist in iPSCs following retinal differentiating treatment. SSEA-5 immunofluorescence was most pronounced in primitive tumors,such as embryonal carcinoma. In tumor cell lines,SSEA-5 was highly immunoreactive in Capan-1 cells,while L1CAM was highly immunoreactive in U87MG cells. SSEA-5 and L1CAM showed colocalization in undifferentiated iPSCs,with immunopositive iPSCs remaining after 20 days of retinal differentiating treatment. This is the first demonstration of SSEA-5 immunoreactivity in human tumors and the first indication of SSEA-5 and L1CAM colocalization. SSEA-5 and L1CAM warrant further investigation as potentially useful tumor markers for histological evaluation or as markers to monitor the presence of undifferentiated cells in iPSC populations prior to therapeutic use. View Publication

Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a mitochondrial disorder characterized by myoclonus epilepsy,generalized seizures,ataxia and myopathy. MERRF syndrome is primarily due to an A to G mutation at mtDNA 8344 that disrupts the mitochondrial gene for tRNA(Lys). However,the detailed mechanism by which this tRNA(Lys) mutation causes mitochondrial dysfunction in cardiomyocytes or neurons remains unclear. In this study,we generated human induced pluripotent stem cells (hiPSCs) that carry the A8344G genetic mutation from patients with MERRF syndrome. Compared with mutation-free isogenic hiPSCs,MERRF-specific hiPSCs (MERRF-hiPSCs) exhibited reduced oxygen consumption,elevated reactive oxygen species (ROS) production,reduced growth,and fragmented mitochondrial morphology. We sought to investigate the induction ability and mitochondrial function of cardiomyocyte-like cells differentiated from MERRF-hiPSCs. Our data demonstrate that that cardiomyocyte-like cells (MERRF-CMs) or neural progenitor cells (MERRF-NPCs) differentiated from MERRF-iPSCs also exhibited increased ROS levels and altered antioxidant gene expression. Furthermore,MERRF-CMs or -NPCs contained fragmented mitochondria,as evidenced by MitoTracker Red staining and transmission electron microscopy. Taken together,these findings showed that MERRF-hiPSCs and MERRF-CM or -NPC harboring the A8344G genetic mutation displayed contained mitochondria with an abnormal ultrastructure,produced increased ROS levels,and expressed upregulated antioxidant genes. View Publication

The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDH(hi)) activity,a progenitor cell function conserved between several lineages. BM ALDH(hi) cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDH(hi) cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDH(hi) cells,mice transplanted with purified ALDH(hi) cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDH(hi) cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue,suggesting that transient ALDH(hi) cell engraftment stimulated endogenous revascularization. Thus,human BM ALDH(hi) cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans. View Publication

Embryonic and induced pluripotent stem cells (IPSCs) derived from mammalian species are valuable tools for modeling human disease,including retinal degenerative eye diseases that result in visual loss. Restoration of vision has focused on transplantation of neural progenitor cells (NPCs) and retinal pigmented epithelium (RPE) to the retina. Here we used transgenic common marmoset (Callithrix jacchus) and human pluripotent stem cells carrying the enhanced green fluorescent protein (eGFP) reporter as a model system for retinal differentiation. Using suspension and subsequent adherent differentiation cultures,we observed spontaneous in vitro differentiation that included NPCs and cells with pigment granules characteristic of differentiated RPE. Retinal cells derived from human and common marmoset pluripotent stem cells provide potentially unlimited cell sources for testing safety and immune compatibility following autologous or allogeneic transplantation using nonhuman primates in early translational applications. View Publication

Activin/Nodal signaling via SMAD2/3 maintains human embryonic stem cell (hESC) pluripotency by direct transcriptional regulation of NANOG or,alternatively,induces mesoderm and definitive endoderm (DE) formation. In search of an explanation for these contrasting effects,we focused on SNON (SKIL),a potent SMAD2/3 corepressor that is expressed in hESCs but rapidly down-regulated upon differentiation. We show that SNON predominantly associates with SMAD2 at the promoters of primitive streak (PS) and early DE marker genes. Knockdown of SNON results in premature activation of PS and DE genes and loss of hESC morphology. In contrast,enforced SNON expression inhibits DE formation and diverts hESCs toward an extraembryonic fate. Thus,our findings provide novel mechanistic insight into how a single signaling pathway both regulates pluripotency and directs lineage commitment. View Publication

INTRODUCTION: The reprogramming of a patient's somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming,however,represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA),an excisable lentiviral reprogramming vector,provides,in our hands,the most consistent reprogramming approach that addresses this safety concern. Nevertheless,most viral integrations occur in genes,and exactly how the integration,epigenetic reprogramming,and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known. METHODS: In this study,we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry,karyotyping and teratoma formation,and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions. RESULTS: We found that a STEMCCA-derived iPSC line that contains a single integration,found to be located in an intronic location in an actively transcribed gene,PRPF39,displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs,differentiated them into multiple clinically relevant cell types (including oligodendrocytes,hepatocytes,and cardiomyocytes),and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status. CONCLUSION: For the first time,these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and,in light of the limited availability of current good manufacturing practice cellular manufacturing facilities,highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics. View Publication

Targeted delivery of siRNA controlled by near-infrared light using hollow gold nanoshells has been demonstrated in cancer and stem cells models. Here,a universal surface module and several functionalization rules for the maximized delivery of short nucleic acids (here,siRNA) applicable for diverse gold nanocarriers are described. Streptavidin is devised as a handle to assemble biotinylated cell penetrating peptides (e.g.,transactivating transcriptional activator (TAT)),as well as an insulator between the positive charge of TAT and the dense negative charge of RNA. However,direct linking of streptavidin to functional siRNA inhibits its silencing activity. The approach then involves the orthogonal assembly of two types of RNA strands: one with biotin modification for cell targeting and penetration (scaffold RNA); the other without biotin as functional RNA (i.e.,siRNA). Initially,flexible single-stranded RNA is used for dense surface-packing,followed by hybridization with the complementary RNA strand to maximize the assembly of the targeting peptide for cellular uptake and siRNA delivery. This orthogonal approach for the delivery of short oligonucleotides,together with novel surface functionalization rules discovered here,should enable the use of these materials for nanomedicinal research and applications. View Publication

The recent identification of hemogenic endothelium (HE) in human pluripotent stem cell (hPSC) cultures presents opportunities to investigate signaling pathways that are essential for blood development from endothelium and provides an exploratory platform for de novo generation of hematopoietic stem cells (HSCs). However,the use of poorly defined human or animal components limits the utility of the current differentiation systems for studying specific growth factors required for HE induction and manufacturing clinical-grade therapeutic blood cells. Here,we identified chemically defined conditions required to produce HE from hPSCs growing in Essential 8 (E8) medium and showed that Tenascin C (TenC),an extracellular matrix protein associated with HSC niches,strongly promotes HE and definitive hematopoiesis in this system. hPSCs differentiated in chemically defined conditions undergo stages of development similar to those previously described in hPSCs cocultured on OP9 feeders,including the formation of VE-Cadherin(+)CD73(-)CD235a/CD43(-) HE and hematopoietic progenitors with myeloid and T lymphoid potential. View Publication

The t(16:16) and inv(16) are associated with FAB M4Eo myeloid leukemias and result in fusion of the CBFB gene to the MYH11 gene (encoding smooth muscle myosin heavy chain [SMMHC]). Knockout of CBFbeta causes embryonic lethality due to lack of definitive hematopoiesis. Although knock-in of CBFB-MYH11 is not sufficient to cause disease,expression increases the incidence of leukemia when combined with cooperating events. Although mouse models are valuable tools in the study of leukemogenesis,little is known about the contribution of CBFbeta-SMMHC to human hematopoietic stem and progenitor cell self-renewal. We introduced the CBFbeta-MYH11 cDNA into human CD34+ cells via retroviral transduction. Transduced cells displayed an initial repression of progenitor activity but eventually dominated the culture,resulting in the proliferation of clonal populations for up to 7 months. Long-term cultures displayed a myelomonocytic morphology while retaining multilineage progenitor activity and engraftment in NOD/SCID-B2M-/- mice. Progenitor cells from long-term cultures showed altered expression of genes defining inv(16) identified in microarray studies of human patient samples. This system will be useful in examining the effects of CBFbeta-SMMHC on gene expression in the human preleukemic cell,in characterizing the effect of this oncogene on human stem cell biology,and in defining its contribution to the development of leukemia. View Publication