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

Improvement of methods to produce endoderm-derived cells from pluripotent stem cells is important to realize high-efficient induction of endodermal tissues such as pancreas and hepatocyte. Difficulties hampering such efforts include the low efficiency of definitive endoderm cell induction and establishing appropriate defined culture conditions to ensure a safe cell source for human transplantation. Based on previous studies,we revised the experimental condition of definitive endoderm induction in feeder- and serum-free culture. Our results suggested that CHIR99021 is more effective than Wnt3A ligand in feeder- and serum-free conditions. In addition,keeping cell density low during endoderm induction is important for the efficiency. On the other hand,we showed that overtreatment with CHIR99021 converted the cells into BRACHYURY-expressing posterior mesoderm cells rather than endoderm,indicating strict CHIR99021 treatment requirements for endoderm differentiation. Nevertheless,these results should enable better control in the production of definitive endoderm-derived cells. View Publication

BACKGROUND: Serum-free cultures supplemented with stem cell factor (SCF) and IL-6 is reported to support the extensive growth of less functional human cord blood-derived mast cells. OBJECTIVE: To obtain more functional mast cells from cord blood,we developed a culture system combining a serum-free condition for 0-8 weeks of culture,and followed by a serum-supplemented culture condition and examined the function of the cells compared to the cells cultured continuously in serum-free condition. METHODS: Human cord blood progenitors were purified with anti-CD133 antibody. They were cultured in a serum-free medium StemSpan supplemented with SCF at 100 ng/ml and IL-6 at 50 ng/ml for 8 weeks. Then,an aliquot of the cultured cells were cultured in the above condition but further supplemented with 10% fetal calf serum (FCS). RESULTS: The addition of FCS after 8 weeks of culture significantly increased the amount of histamine per mast cell (3.8 pg/cell) when compared to the serum-free condition (0.7 pg/cell). The cells cultured with FCS after 8 weeks expressed more FcvarepsilonRI alpha and released textgreater30% of the histamine content upon anti-IgE stimulation than those cultured without serum. CONCLUSION: It is uncertain why FCS enhanced the functional maturation of mast cells when added after week 8 of culture but suppressed mast cell development when added at day 0 of culture. Yet,the present method combining a serum-free culture system with a serum-supplemented culture system seems to be beneficial for most of the laboratories to obtain functional human mast cells. View Publication

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs),and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo,but is essential for normal HSC/HPC differentiation. In addition,Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased β1 integrin and Cxcr4 expression. Moreover,deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and,subsequently,improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult. View Publication

It is likely that arrhythmias should be avoided for therapies based on human pluripotent stem cell (hPSC)-derived cardiomyocytes (CM) to be effective. Towards achieving this goal,we introduced light-activated channelrhodopsin-2 (ChR2),a cation channel activated with 480 nm light,into human embryonic stem cells (hESC). By using in vitro approaches,hESC-CM are able to be activated with light. ChR2 is stably transduced into undifferentiated hESC via a lentiviral vector. Via directed differentiation,hESCChR2-CM are produced and subjected to optical stimulation. hESCChR2-CM respond to traditional electrical stimulation and produce similar contractility features as their wild-type counterparts but only hESCChR2-CM can be activated by optical stimulation. Here it is shown that a light sensitive protein can enable in vitro optical control of hESC-CM and that this activation occurs optimally above specific light stimulation intensity and pulse width thresholds. For future therapy,in vivo optical stimulation along with optical inhibition could allow for acute synchronization of implanted hPSC-CM with patient cardiac rhythms. View Publication

How BMP signaling integrates into and destabilizes the pluripotency circuitry of human pluripotent stem cells (hPSCs) to initiate differentiation into individual germ layers is a long-standing puzzle. Here we report muscle segment homeobox 2 (MSX2),a homeobox transcription factor of msh family,as a direct target gene of BMP signaling and a master mediator of hPSCs' differentiation to mesendoderm. Enforced expression of MSX2 suffices to abolish pluripotency and induce directed mesendoderm differentiation of hPSCs,while MSX2 depletion impairs mesendoderm induction. MSX2 is a direct target gene of the BMP pathway in hPSCs,and can be synergistically activated by Wnt signals via LEF1 during mesendoderm induction. Furthermore,MSX2 destabilizes the pluripotency circuitry through direct binding to the SOX2 promoter and repression of SOX2 transcription,while MSX2 controls mesendoderm lineage commitment by simultaneous suppression of SOX2 and induction of NODAL expression through direct binding and activation of the Nodal promoter. Interestingly,SOX2 can promote the degradation of MSX2 protein,suggesting a mutual antagonism between the two lineage-specifying factors in the control of stem cell fate. Together,our findings reveal crucial new mechanisms of destabilizing pluripotency and directing lineage commitment in hPSCs. View Publication

We employed Fourier transform infrared (FTIR) microspectroscopy to investigate the effects of different tissue culture environments on the FTIR spectra of undifferentiated human embryonic stem cells (hESCs) and their differentiated progeny. First we tested whether there were any possible spectral artifacts resulting from the use of transflectance measurements by comparing them with transmission measurements and found no evidence of these concluding that the lack of any differences resulted from the homogeneity of the dried cytospun cellular monolayers. We found that hESCs that were enzymatically passaged onto mouse embryonic fibroblasts (MEFs) in KOSR based hESC medium,hESCs enzymatically passaged onto Matrigel in mTESR medium and hESCs mechanically passaged onto MEFs in KOSR-based hESC medium,possessed unique FTIR spectroscopic signatures that reflect differences in their macromolecular chemistry. Further,these spectroscopic differences persisted even upon differentiation towards mesendodermal lineages. Our results suggest that FTIR microspectroscopy is a powerful,objective,measurement modality that complements existing methods for studying the phenotype of hESCs and their progeny,particularly changes induced by the cellular environment. View Publication

Hematopoietic stem cells (HSCs) are enriched for aldehyde dehydrogenase (ALDH) activity and ALDH is a selectable marker for human HSCs. However,the function of ALDH in HSC biology is not well understood. We sought to determine the function of ALDH in regulating HSC fate. Pharmacologic inhibition of ALDH with diethylaminobenzaldehyde (DEAB) impeded the differentiation of murine CD34(-)c-kit(+)Sca-1(+)lineage(-) (34(-)KSL) HSCs in culture and facilitated a ninefold expansion of cells capable of radioprotecting lethally irradiated mice compared to input 34(-)KSL cells. Treatment of bone marrow (BM) 34(-)KSL cells with DEAB caused a fourfold increase in 4-week competitive repopulating units,verifying the amplification of short-term HSCs (ST-HSCs) in response to ALDH inhibition. Targeted siRNA of ALDH1a1 in BM HSCs caused a comparable expansion of radioprotective progenitor cells in culture compared to DEAB treatment,confirming that ALDH1a1 was the target of DEAB inhibition. The addition of all trans retinoic acid blocked DEAB-mediated expansion of ST-HSCs in culture,suggesting that ALDH1a1 regulates HSC differentiation via augmentation of retinoid signaling. Pharmacologic inhibition of ALDH has therapeutic potential as a means to amplify ST-HSCs for transplantation purposes. View Publication

Recently it has been demonstrated that CD30 expression was rather specific for transformed than for normal human ES cells and therefore CD30 maybe suggested as a potential marker for human ES cells bearing chromosomal abnormalities. Using immunohistochemistry and RT-PCR analysis we examined �?¡D30 expression in 10 hESCs lines with normal and abberant karyotypes. All hESC lines expressed CD30 antigen and RNA in undifferentiated state whether cell line beared chromosomal abnormalities or not. In contrast to previous notions our data demonstrate that CD30 could be considered as marker of undifferentiated hESCs without respect to karyotype changes. View Publication

Hematopoiesis is tightly controlled by transcription regulatory networks,but how and when specific transcription factors control lineage commitment are still largely unknown. Within the hematopoietic stem cell (Lin(-)Sca-1(+)c-Kit(+)) compartment these lineage-specific transcription factors are expressed at low levels but are up-regulated with the process of lineage specification. CCAAT/enhancer binding protein α (C/EBPα) represents one of these factors and is involved in myeloid development and indispensable for formation of granulocytes. To track the cellular fate of stem and progenitor cells,which express C/EBPα,we developed a mouse model expressing Cre recombinase from the Cebpa promoter and a conditional EYFP allele. We show that Cebpa/EYFP(+) cells represent a significant subset of multipotent hematopoietic progenitors,which predominantly give rise to myeloid cells in steady-state hematopoiesis. C/EBPα induced a strong myeloid gene expression signature and down-regulated E2A-induced regulators of early lymphoid development. In addition,Cebpa/EYFP(+) cells compose a fraction of early thymic progenitors with robust myeloid potential. However,Cebpa/EYFP(+) multipotent hematopoietic progenitors and early thymic progenitors retained the ability to develop into erythroid and T-lymphoid lineages,respectively. These findings support an instructive but argue against a lineage-restrictive role of C/EBPα in multipotent hematopoietic and thymic progenitors. View Publication

The mechanisms that regulate hematopoietic stem cell (HSC) fate decisions between proliferation and multilineage differentiation are unclear. Members of the Wnt family of ligands that activate the canonical Wnt signaling pathway,which utilizes beta-catenin to relay the signal,have been demonstrated to regulate HSC function. In this study,we examined the role of noncanonical Wnt signaling in regulating HSC fate. We observed that noncanonical Wnt5a inhibited Wnt3a-mediated canonical Wnt signaling in HSCs and suppressed Wnt3a-mediated alterations in gene expression associated with HSC differentiation,such as increased expression of myc. Wnt5a increased short- and long-term HSC repopulation by maintaining HSCs in a quiescent G(0) state. From these data,we propose that Wnt5a regulates hematopoiesis by the antagonism of the canonical Wnt pathway,resulting in a pool of quiescent HSCs. View Publication

Using time-lapse imaging,we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells (hESCs) and that are relieved by karyotypically abnormal variants that are selected by prolonged culture. Only a minority of karyotypically normal cells divided after plating,and these were mainly cells in the later stages of cell cycle at the time of plating. Furthermore,the daughter cells showed a continued pattern of cell death after division,so that few formed long-term proliferating colonies. These colony-forming cells showed distinct patterns of cell movement. Increasing cell density enhanced cell movement facilitating cell:cell contact,which resulted in increased proportion of dividing cells and improved survival postplating of normal hESCs. In contrast,most of the karyotypically abnormal cells reentered the cell cycle on plating and gave rise to healthy progeny,without the need for cell:cell contacts and independent of their motility patterns. ?? 2014 The Authors. View Publication