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

The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-,atrial-,and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives,which in turn promoted cardiomyocyte differentiation. Moreover,a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation,improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation,measuring action potentials,and intracellular Ca(2+) dynamics. These findings are relevant for improving our understanding on human heart development,and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes,similar to those observed in adult cardiac myocytes. View Publication

The t(12;21)(p13;q22) translocation is the most common chromosomal abnormality yet identified in any pediatric leukemia and gives rise to the TEL-AML1 fusion product. To investigate the effects of TEL-AML1 on hematopoiesis,fetal liver hematopoietic progenitor cells (HPCs) were transduced with retroviral vectors expressing this fusion protein. We show that TEL-AML1 dramatically alters differentiation of HPCs in vitro,preferentially promoting B-lymphocyte development,enhancing self-renewal of B-cell precursors,and leading to the establishment of long-term growth factor-dependent pre-B-cell lines. However,it had no effect on myeloid development in vitro. Further experiments were performed to determine whether TEL-AML1 also demonstrates lineage-specific activity in vivo. TEL-AML1-expressing HPCs displayed a competitive advantage in reconstituting both B-cell and myeloid lineages in vivo but had no effect on reconstitution of the T-cell lineage. Despite promoting these alterations in hematopoiesis,TEL-AML1 did not induce leukemia in transplanted mice. Our study provides a unique insight into the role of TEL-AML1 in leukemia predisposition and a potential model to study the mechanism of leukemogenesis associated with this fusion. View Publication

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into peripheral blood by the cytokine granulocyte colony-stimulating factor (G-CSF) has become the preferred source of HSPCs for stem cell transplants. However,G-CSF fails to mobilize sufficient numbers of stem cells in up to 10% of donors,precluding autologous transplantation in those donors or substantially delaying transplant recovery time. Consequently,new regimens are needed to increase the number of stem cells in peripheral blood upon mobilization. Using a forward genetic approach in mice,we mapped the gene encoding the epidermal growth factor receptor (Egfr) to a genetic region modifying G-CSF-mediated HSPC mobilization. Amounts of EGFR in HSPCs inversely correlated with the cells' ability to be mobilized by G-CSF,implying a negative role for EGFR signaling in mobilization. In combination with G-CSF treatment,genetic reduction of EGFR activity in HSPCs (in waved-2 mutant mice) or treatment with the EGFR inhibitor erlotinib increased mobilization. Increased mobilization due to suppression of EGFR activity correlated with reduced activity of cell division control protein-42 (Cdc42),and genetic Cdc42 deficiency in vivo also enhanced G-CSF-induced mobilization. Our findings reveal a previously unknown signaling pathway regulating stem cell mobilization and provide a new pharmacological approach for improving HSPC mobilization and thereby transplantation outcomes. View Publication

Stable pluripotent feeder-free propagation of human embryonic stem cells (hESCs) prior to their therapeutic applications remains a major challenge. Matrigel™ (BD Singapore) is a murine sarcoma-derived extracellular matrix (ECM) widely used as a cell-free support combined with conditioned or chemically defined media; however,inherent xenogenic and immunological threats invalidate it for clinical applications. Using human fibrogenic cells to generate ECM is promising but currently suffers from inefficient and time-consuming deposition in vitro. We recently showed that macromolecular crowding (MMC) accelerated ECM deposition substantially in vitro. In the current study,we used dextran sulfate 500 kDa as a macromolecular crowder to induce WI-38 fetal human lung fibroblasts at 0.5% serum condition to deposit human ECM in three days. After decellularization,the generated ECMs allowed stable propagation of H9 hESCs over 20 passages in chemically-defined medium (mTEsR1) with an overall improved outcome compared to Matrigel in terms of population doubling while retaining teratoma formation and differentiation capacity. Of significance,only ECMs generated by MMC allowed the successful propagation of hESCs. ECMs were highly complex and in contrast to Matrigel,contained no vitronectin but did contain collagen XII,ig-h3 and novel for hESC-supporting human matrices,substantial amounts of transglutaminase 2. Genome-wide analysis of promoter DNA methylation states revealed high overall similarity between human ECM- and Matrigel-cultured hESCs; however,distinct differences were observed with 49 genes associated with a variety of cellular functions. Thus,human ECMs deposited by MMC by selected fibroblast lines are a suitable human microenvironment for stable hESC propagation and clinically translational settings. View Publication

The pluripotent property of hESCs makes them attractive for treatment of degenerative diseases such as diabetes. We have developed a stage-wise directed differentiation protocol to produce alginate-encapsulated islet-like cells derived from hESCs,which can be directly implanted for diabetes therapy. The advantage of alginate encapsulation lies in its capability to immunoisolate,along with the added possibility of scalable culture. We have evaluated the possibility of encapsulating hESCs at different stages of differentiation. Encapsulation of predifferentiated cells resulted in insufficient cellular yield and differentiation. On the other hand,encapsulation of undifferentiated hESCs followed by differentiation induction upon encapsulation,resulted in the highest viability and differentiation. More striking was that alginate encapsulation resulted in a much stronger differentiation compared to parallel 2D cultures,resulting in 20-fold increase in c-peptide protein synthesis. To elucidate the mechanism contributing to encapsulation-mediated enhancement in hESC maturation,investigation of the signaling pathways revealed interesting insight. While the phospho-protein levels of all the tested signaling molecules were lower under encapsulation,the ratio of pSMAD/pAKT was significantly higher,indicating a more efficient signal transduction under encapsulation. These results clearly demonstrate that alginate encapsulation of hESCs and differentiation to islet-cells types provides a potentially translatable treatment option for type1 diabetes. View Publication

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step,we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells,and then,expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123(low)HLA-DR- phenotype,expressed myeloperoxidase,and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules,CD1a,CD11c,CD80,CD86,DC-SIGN,and CD40; and were capable of Ag processing,triggering naive T cells in MLR,and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-alpha coexpressed a high level of CD14,and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition,because ES cells can be expanded without limit,they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance. View Publication

Embryoid bodies (EBs) can be generated by culturing human pluripotent stem cells in ultra-low attachment culture vessels,under conditions that are adverse to pluripotency and proliferation. EBs generated in suspension cultures are capable of differentiating into cells of the ectoderm,mesoderm,and endoderm. In this chapter,we describe techniques for generation of EBs from human pluripotent stem cells. Once formed,the EBs can then be dissociated using specific enzymes to acquire a single cell population that has the potential to differentiate into cells of all three germ layers. This population can then be cultured in specialized conditions to obtain progenitor cells of specific lineages. Pure populations of progenitor cells generated on a large scale basis can be used for research,drug discovery/development,and cellular transplantation therapy. View Publication

BACKGROUND: Human embryonic stem cells (hESCs) are a potentially inexhaustible source of cells for replacement therapy. However,successful preclinical and clinical progress requires efficient and controlled differentiation towards the specific differentiated cell fate. METHODS: We previously developed a strategy to generate blast cells (BCs) from hESCs that were capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Although the BCs were shown to repair damaged vasculature in multiple animal models,the large-scale generation of cells under these conditions was challenging. Here we report a simpler and more efficient method for robust generation of hemangioblastic progenitors. RESULTS: In addition to eliminating several expensive factors that are unnecessary,we demonstrate that bone morphogenetic protein (BMP)-4 and VEGF are necessary and sufficient to induce hemangioblastic commitment and development from hESCs during early stages of differentiation. BMP-4 and VEGF significantly upregulate T-brachyury,KDR,CD31 and Lmo2 gene expression,while dramatically downregulating Oct-4 expression. The addition of basic FGF during growth and expansion was found to further enhance BC development,consistently generating approximately 1 x 10(8) BCs from one six well plate of hESCs. CONCLUSION: This new method represents a significantly improved system for generating hemangioblasts from hESCs,and although simplified,results in an eightfold increase in cell yield. View Publication

We have demonstrated previously that cord blood CD133(+) cells isolated in the G(0) phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity,in contrast to CD133(+)G(1) cells. Here,we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133(+)G(0) cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133(+)G(1) counterparts (13.5 and 4.1%,respectively). This suggests that stem cell activity resides in the CD133(+)G(0) population. In long-term BM cultures,the CD133(+)G(0) cells generate significantly more progenitors than the CD34(+)G(0) population (Ptextless0.001) throughout the culture period. Furthermore,a comparison of CD133(+)G(0) versus CD133(+)G(1) cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G(0) cells. We conclude that CD133(+) cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G(1) counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols. View Publication

Inefficient neural differentiation of human induced pluripotent stem cells (hiPSCs) motivates recent investigation of the influence of biophysical characteristics of cellular microenvironment,in particular nanotopography,on hiPSC fate decision. However,the roles of geometry and dimensions of nanotopography in neural lineage commitment of hiPSCs have not been well understood. The objective of this study is to delineate the effects of geometry,feature size and height of nanotopography on neuronal differentiation of hiPSCs. HiPSCs were seeded on equally spaced nanogratings (500 and 1000nm in linewidth) and hexagonally arranged nanopillars (500nm in diameter),each having a height of 150 or 560nm,and induced for neuronal differentiation in concert with dual Smad inhibitors. The gratings of 560nm height reduced cell proliferation,enhanced cytoplasmic localization of Yes-associated protein,and promoted neuronal differentiation (up to 60% βIII-tubulin(+) cells) compared with the flat control. Nanograting-induced cell polarity and cytoplasmic YAP localization were shown to be critical to the induced neural differentiation of hiPSCs. The derived neuronal cells express MAP2,Tau,glutamate,GABA and Islet-1,indicating the existence of multiple neuronal subtypes. This study contributes to the delineation of cell-nanotopography interactions and provides the insights into the design of nanotopography configuration for pluripotent stem cell neural lineage commitment. View Publication

Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties,we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR,a fluorescent substrate for ALDH,and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56(+) fraction in those cells,but,we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly,ALDH activity and Aldh1a1 expression levels were very low in mouse,rat,rabbit and non-human primate myoblasts. Using different approaches,from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde,an inhibitor of class I ALDH,to cell fractionation by flow cytometry using the ALDEFLUOR assay,we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H(2) O(2) )-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity,as a purification strategy,could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage. View Publication