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

Human embryonic stem cells (hESCs) are emerging as an attractive alternative source for cell replacement therapy since the cells can be expanded in culture indefinitely and differentiated into any cell types in the body. In order to optimize cell-to-cell interaction,cell proliferation and differentiation into specific lineages as well as tissue organization,it is important to provide a microenvironment for the hESCs which mimics the stem cell niche. One approach is to provide a three-dimensional (3D) environment such as encapsulation. We present an approach to culture and differentiate hESCs into midbrain dopamine (mdDA) neurons in a 3D microenvironment using alginate microcapsules for the first time. A detailed gene and protein expression analysis during neuronal differentiation showed an increased gene and protein expression of various specific DA neuronal markers,particularly tyrosine hydroxylase (TH) by textgreater100 folds after 2weeks and at least 50% higher expression after 4weeks respectively,compared to cells differentiated under conventional two-dimensional (2D) platform. The encapsulated TH+ cells co-expressed mdDA neuronal markers,forkhead box protein A-2 (FOXA2) and pituitary homeobox-3 (PITX3) after 4weeks and secreted approximately 60pg/ml/106 cells higher DA level when induced. We propose that the 3D platform facilitated an early onset of DA neuronal generation compared to that with conventional 2D system which also secretes more DA under potassium-induction. It is a very useful model to study the proliferation and directed differentiation of hESCs to various lineages,particularly to mdDA neurons. This 3D system also allows the separation of feeder cells from hESCs during the process of differentiation and also has potential for immune-isolation during transplantation studies. ?? 2013 Elsevier B.V. View Publication

Optimization of pluripotent stem cell expansion and differentiation is facilitated by biological tools that permit non-invasive and dynamic monitoring of pluripotency,and the ability to select for an undifferentiated input cell population. Here we report on the generation and characterisation of clonal human embryonic stem (HES3,H9) and human induced pluripotent stem cell lines (UQEW01i-epifibC11) that have been stably modified with an artificial EOS(C3+) promoter driving expression of EGFP and puromycin resistance-conferring proteins. We show that EGFP expression faithfully reports on the pluripotency status of the cells in these lines and that antibiotic selection allows for an efficient elimination of differentiated cells from the cultures. We demonstrate that the extinction of the expression of the pluripotency reporter during differentiation closely correlates with the decrease in expression of conventional pluripotency markers,such as OCT4 (POU5F1),TRA-1-60 and SSEA4 when screening across conditions with various levels of pluripotency-maintaining or differentiation-inducing signals. We further illustrate the utility of these lines for real-time monitoring of pluripotency in embryoid bodies and microfluidic bioreactors. View Publication

Previous studies have shown the relevance of bone marrow-derived MSCs (BM-MSCs) in controlling graft-versus-host disease (GVHD) after allogeneic transplantation. Since adipose tissue-derived MSCs (Ad-MSCs) may constitute a good alternative to BM-MSCs,we have expanded MSCs derived from human adipose tissue (hAd-MSCs) and mouse adipose tissue (mAd-MSCs),investigated the immunoregulatory properties of these cells,and evaluated their capacity to control GVHD in mice. The phenotype and immunoregulatory properties of expanded hAd-MSCs were similar to those of human BM-MSCs. Moreover,hAd-MSCs inhibited the proliferation and cytokine secretion of human primary T cells in response to mitogens and allogeneic T cells. Similarly,ex vivo expanded mAd-MSCs had an equivalent immunophenotype and exerted immunoregulatory properties similar to those of hAd-MSCs. Moreover,the infusion of mAd-MSCs in mice transplanted with haploidentical hematopoietic grafts controlled the lethal GVHD that occurred in control recipient mice. These findings constitute the first experimental proof that Ad-MSCs can efficiently control the GVHD associated with allogeneic hematopoietic transplantation,opening new perspectives for the clinical use of Ad-MSCs. View Publication

Megakaryoblastic leukemia 1 (MKL1),identified as part of the t(1;22) translocation specific to acute megakaryoblastic leukemia,is highly expressed in differentiated muscle cells and promotes muscle differentiation by activating serum response factor (SRF). Here we show that Mkl1 expression is up-regulated during murine megakaryocytic differentiation and that enforced overexpression of MKL1 enhances megakaryocytic differentiation. When the human erythroleukemia (HEL) cell line is induced to differentiate with 12-O-tetradecanoylphorbol 13-acetate,overexpression of MKL1 results in an increased number of megakaryocytes with a concurrent increase in ploidy. MKL1 overexpression also promotes megakaryocytic differentiation of primary human CD34(+) cells cultured in the presence of thrombopoietin. The effect of MKL1 is abrogated when SRF is knocked down,suggesting that MKL1 acts through SRF. Consistent with these findings in human cells,knockout of Mkl1 in mice leads to reduced platelet counts in peripheral blood,and reduced ploidy in bone marrow megakaryocytes. In conclusion,MKL1 promotes physiologic maturation of human and murine megakaryocytes. View Publication

Oncogenic Kit mutations are found in somatic gastrointestinal (GI) stromal tumors (GISTs) and mastocytosis. A mouse model for the study of constitutive activation of Kit in oncogenesis has been produced by a knock-in strategy introducing a Kit exon 11-activating mutation into the mouse genome based on a mutation found in a case of human familial GIST syndrome. Heterozygous mutant KitV558Delta/+ mice develop symptoms of disease and eventually die from pathology in the GI tract. Patchy hyperplasia of Kit-positive cells is evident within the myenteric plexus of the entire GI tract. Neoplastic lesions indistinguishable from human GISTs were observed in the cecum of the mutant mice with high penetrance. In addition,mast cell numbers in the dorsal skin were increased. Therefore KitV558Delta/+ mice reproduce human familial GISTs,and they may be used as a model for the study of the role and mechanisms of Kit in neoplasia. Importantly,these results demonstrate that constitutive Kit signaling is critical and sufficient for induction of GIST and hyperplasia of interstitial cells of Cajal. View Publication

Spinal muscular atrophy (SMA),a motor neuron disease (MND) and one of the most common genetic causes of infant mortality,currently has no cure. Patients with SMA exhibit muscle weakness and hypotonia. Stem cell transplantation is a potential therapeutic strategy for SMA and other MNDs. In this study,we isolated spinal cord neural stem cells (NSCs) from mice expressing green fluorescent protein only in motor neurons and assessed their therapeutic effects on the phenotype of SMA mice. Intrathecally grafted NSCs migrated into the parenchyma and generated a small proportion of motor neurons. Treated SMA mice exhibited improved neuromuscular function,increased life span,and improved motor unit pathology. Global gene expression analysis of laser-capture-microdissected motor neurons from treated mice showed that the major effect of NSC transplantation was modification of the SMA phenotype toward the wild-type pattern,including changes in RNA metabolism proteins,cell cycle proteins,and actin-binding proteins. NSC transplantation positively affected the SMA disease phenotype,indicating that transplantation of NSCs may be a possible treatment for SMA. View Publication

Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP),we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region and binding site levels,IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3' UTR-enriched targets. RNA Bind-N-seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes,including a reduction in cell adhesion and increase in cell death. For cell adhesion,we find IMP1 maintains levels of integrin mRNA specifically regulating RNA stability of ITGB5 in hPSCs. Additionally,we show that IMP1 can be linked to hPSC survival via direct target BCL2. Thus,transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. View Publication

Generation of induced pluripotent stem cells (iPSCs) from human urine-derived cells (hUCs) provides a convenient and non-invasive way to obtain patient-specific iPSCs. However,many isolated hUCs exhibit very poor proliferation and are difficult to reprogram. In this study,we optimized reprogramming approaches for hUCs with very poor proliferation. We report here that a compound cocktail containing cyclic pifithrin-a (a P53 inhibitor),A-83-01,CHIR99021,thiazovivin,NaB,and PD0325901 significantly improves the reprogramming efficiency (170-fold more) for hUCs. In addition,we showed that replacement of Matrigel with autologous hUC feeders can overcome the reprogramming failure due to the massive cell death that occurs during delivery of reprogramming factors. In summary,we describe improved approaches to enable iPSC generation from hUCs that were otherwise difficult to reprogram,a valuable asset for banking patient-specific iPSCs. View Publication

Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells,but how best to select iPSC clones suitable for this purpose from among the many clones that can be simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors,we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation,reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a(+)CD42b(+) platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b(+) platelets were present in thrombi after laser-induced vessel wall injury. In contrast,sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression,decreased GATA1 expression,and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression,particularly its later decline,is key to producing functional platelets from selected iPSC clones. View Publication

AIM: Dendritic cell (DC)-based vaccines are designed to exploit the intrinsic capacity of these highly effective antigen presenting cells to prime and boost antigen-specific T-cell immune responses. Successful development of DC-based vaccines will be dependent on the ability to utilize and harness the full potential of these potent immune stimulatory cells. Recent advances to generate DCs derived from human embryonic stem cells (hESCs) that are suitable for clinical use represent an alternative strategy from conventional approaches of using patient-specific DCs. Although the differentiation of hESC-derived DCs in serum-free defined conditions has been established,the stimulatory potential of these hESC-derived DCs have not been fully evaluated. METHODS: hESC-derived DCs were differentiated in serum-free defined culture conditions. The delivery of antigen into hESC-derived DCs was investigated using mRNA transfection and replication-deficient adenoviral vector transduction. hESC-derived DCs modified with antigen were evaluated for their capacity to stimulate antigen-specific T-cell responses with known HLA matching. Since IL-12 is a key cytokine that drives T-cell function,further enhancement of DC potency was evaluated by transfecting mRNA encoding the IL-12p70 protein into hESC-derived DCs. RESULTS: The transfection of mRNA into hESC-derived DCs was effective for heterologous protein expression. The efficiency of adenoviral vector transduction into hESC-derived DCs was poor. These mRNA-transfected DCs were capable of stimulating human telomerase reverse transcriptase antigen-specific T cells composed of varying degrees of HLA matching. In addition,we observed the transfection of mRNA encoding IL-12p70 enhanced the T-cell stimulation potency of hESC-derived DCs. CONCLUSION: These data provide support for the development and modification of hESC-derived DCs with mRNA as a potential strategy for the induction of T-cell-mediated immunity. View Publication

Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adeno- associated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However,natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs),which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities,and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (˜50%) to hPSCs,which are importantly accompanied by a considerable increase in gene-targeting frequencies,up to 0.12%. While this level is likely sufficient for numerous applications,we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (textgreater1%) in the presence of targeted double- stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus,this study demonstrates that under appropriate selective pressures,AAV vectors can be created to mediate efficient gene targeting in hPSCs,alone or in the presence of ZFN- mediated double-stranded DNA breaks. View Publication

The advance in stem cell research relies largely on the efficiency and biocompatibility of technologies used to manipulate stem cells. In our previous study,we had designed an amphipathic peptide RV24 that can deliver proteins into cancer cell lines efficiently without significant side effects. Encouraged by this observation,we moved forward to test whether RV24 could be used to deliver proteins into human embryonic stem cells and human induced pluripotent stem cells. RV24 successfully mediated protein delivery into these pluripotent stem cells,as well as their derivatives including neural stem cells and dendritic cells. Based on NMR studies and particle surface charge measurements,we proposed that hydrophobic domain of RV24 interacts with ??-sheet structures of the proteins,followed by formation of peptide cage" to facilitate delivery across cellular membrane. These findings suggest the feasibility of using amphipathic peptide to deliver functional proteins intracellularly for stem cell research. ?? 2012 Elsevier Inc." View Publication