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

An urgent need exists to devise strategies to augment antiviral immune responses in patients with HIV who are virologically well controlled and immunologically stable on highly active antiretroviral therapy (HAART). The objective of this study was to compare the immunomodulatory effects of the cytokines interleukin (IL)-21 with IL-15 on CD8 T cells in patients with HIV RNA of less than 50 copies/mL and CD4 counts greater than 200 cells/mm.(3) Patient CD8 T cells displayed skewed maturation and decreased perforin expression compared with healthy controls. Culture of freshly isolated patient peripheral-blood mononuclear cells (PBMCs) for 5 hours to 5 days with IL-21 resulted in up-regulation of perforin in CD8 T cells,including memory and effector subsets and virus-specific T cells. IL-21 did not induce T-cell activation or proliferation,nor did it augment T-cell receptor (TCR)-induced degranulation. Treatment of patient PBMCs with IL-15 resulted in induction of perforin in association with lymphocyte proliferation and augmentation of TCR-induced degranulation. Patient CD8 T cells were more responsive to cytokine effects than the cells of healthy volunteers. We conclude that CD8 T cells of patients with HIV can be modulated by IL-21 to increase perforin expression without undergoing overt cellular activation. IL-21 could potentially be useful for its perforin-enhancing properties in anti-HIV immunotherapy. View Publication

Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium,functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts,we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system,these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs,we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust,scalable,and consistent methodology. In the present study,we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set,we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality,with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic screens are discussed,demonstrating the value of this biologically relevant and reproducible technology. In addition,this assay system was able to identify novel and potent inducers of differentiation and proliferation of induced pluripotent stem cell-derived cardiac progenitor cells. View Publication

The origins of the epithelial cells participating in the development,tissue homeostasis,and cancer of the human breast are poorly understood. However,emerging evidence suggests a role for adult tissue-specific stem cells in these processes. In a hierarchical manner,these generate the two main mammary cell lineages,producing an increasing number of cells with distinct properties. Understanding the biological characteristics of human breast stem cells and their progeny is crucial in attempts to compare the features of normal stem cells and cancer precursor cells and distinguish these from nonprecursor cells and cells from the bulk of a tumor. A historical overview of research on human breast stem cells in primary tissue and in culture reveals the progress that has been made in this area,whereas a focus on the cell-of-origin and reprogramming that occurs during neoplastic conversion provides insight into the enigmatic way in which human breast cancers are skewed toward the luminal epithelial lineage. View Publication

We report transgenic mouse models in which three or four reprogramming factors are expressed from a single genomic locus using a drug-inducible transgene. Multiple somatic cell types can be directly reprogrammed to generate induced pluripotent stem cells (iPSCs) by culture in doxycycline. Because reprogramming factors are carried on a single polycistronic construct,the mice can be easily maintained,and the transgene can be easily transferred into other genetic backgrounds. View Publication

Coronary heart disease (CHD) is a leading cause of death globally,while its current management is limited to reducing the myocardial infarction area without actually replacing dead cardiomyocytes. Direct cell reprogramming is a method of cellular cardiomyoplasty which aims for myocardial tissue regeneration,and CD34+ cells are one of the potential sources due to their shared embryonic origin with cardiomyocytes. However,the isolation and culture of non-adherent CD34+ cells is crucial to obtain adequate cells for high-efficiency genetic modification. This study aimed to investigate the optimal method for isolation and culture of CD34+ peripheral blood cells using certain culture media. A peripheral blood sample was obtained from a healthy subject and underwent pre-enrichment,isolation,and expansion. The culture was subsequently observed for their viability,adherence,and confluence. Day 0 observation of the culture showed a healthy CD34+ cell with a round cell shape,without any adherent cells present yet. Day 4 of observation showed that CD34+ cells within the blood plasma medium became adherent,indicated by their transformations into spindle or oval morphologies. Meanwhile,CD34+ cells in vitronectin and fibronectin media showed no adherent cells and many of them died. Day 7 observation revealed more adherent CD34+ cells in blood plasma medium,and which had 75% of confluence. In conclusion,the CD34+ cells that were isolated using a combination of density and magnetic methods may be viable and adequately adhere in culture using blood plasma medium,but not in cultures using fibronectin and vitronectin. View Publication

The C-X-C-type chemokine Cxcl12,also known as stromal cell-derived factor-1,plays a critical role in hematopoiesis during fetal development. However,the functional requirement of Cxcl12 in the adult hematopoietic stem/progenitor cell (HSPC) regulation was still unclear. In this report,we developed a murine Cxcl12 conditional deletion model in which the target gene can be deleted at the adult stage. We found that loss of stroma-secreted Cxcl12 in the adult led to expansion of the HSPC population as well as a reduction in long-term quiescent stem cells. In Cxcl12-deficient bone marrow,HSPCs were absent along the endosteal surface,and blood cell regeneration occurred predominantly in the perisinusoidal space after 5-fluorouracil myelosuppression challenge. Our results indicate that Cxcl12 is required for HSPC homeostasis regulation and is an important factor for osteoblastic niche organization in adult stage bone marrow. View Publication

The stem-cell pool is considered to be maintained by a balance between symmetric and asymmetric division of stem cells. The cell polarity model proposes that the facultative use of symmetric and asymmetric cell division is orchestrated by a polarity complex consisting of partitioning-defective proteins Par3 and Par6,and atypical protein kinase C (aPKCζ and aPKCλ),which regulates planar symmetry of dividing stem cells with respect to the signaling microenvironment. However,the role of the polarity complex is unexplored in mammalian adult stem-cell functions. Here we report that,in contrast to accepted paradigms,polarization and activity of adult hematopoietic stem cell (HSC) do not depend on either aPKCζ or aPKCλ or both in vivo. Mice,having constitutive and hematopoietic-specific (Vav1-Cre) deletion of aPKCζ and aPKCλ,respectively,have normal hematopoiesis,including normal HSC self-renewal,engraftment,differentiation,and interaction with the bone marrow microenvironment. Furthermore,inducible complete deletion of aPKCλ (Mx1-Cre) in aPKCζ(-/-) HSC does not affect HSC polarization,self-renewal,engraftment,or lineage repopulation. In addition,aPKCζ- and aPKCλ-deficient HSCs elicited a normal pattern of hematopoietic recovery secondary to myeloablative stress. Taken together,the expression of aPKCζ,aPKCλ,or both are dispensable for primitive and adult HSC fate determination in steady-state and stress hematopoiesis,contrary to the hypothesis of a unique,evolutionary conserved aPKCζ/λ-directed cell polarity signaling mechanism in mammalian HSC fate determination. View Publication

Exposure of murine bone marrow (BM) cells to ionizing radiation (IR; 4 Gy) resulted in textgreater95% inhibition of the frequency of various day types of cobblestone area-forming cells in association with the induction of apoptosis in hematopoietic stem cell alike cells (Lin(-) ScaI(+) c-kit(+) cells; IR: 64.8 +/- 0.4% versus control: 20.4 +/- 0.5%; P textless 0.001) and progenitors (Lin(-) ScaI(-) c-kit(+) cells; IR: 46.2 +/- 1.4% versus control: 7.8 +/- 0.5%; P textless 0.001). Incubation of murine BM cells with busulfan (BU; 30 micro M) for 6 h also inhibited the cobblestone area-forming cell frequency but failed to cause a significant increase in apoptosis in these two types of hematopoietic cells. After 5 weeks of long-term BM cell culture,33% and 72% of hematopoietic cells survived IR- and BU-induced damage,respectively,as compared with control cells,but they could not form colony forming units-granulocyte macrophages. Moreover,these surviving cells expressed an increased level of senescence-associated beta-galactosidase,p16(Ink4a),and p19(Arf). These findings suggest that IR inhibits the function of hematopoietic stem cell alike cells and progenitors primarily by inducing apoptosis,whereas BU does so mainly by inducing premature senescence. In addition,induction of premature senescence in BM hematopoietic cells also contributes to IR-induced inhibition of their hematopoietic function. Interestingly,the induction of hematopoietic cell senescence by IR,but not by BU,was associated with an elevation in p53 and p21(Cip1/Waf1) expression. This suggests that IR induces hematopoietic cell senescence in a p53-p21(Cip1/Waf1)-dependent manner,whereas the induction of senescence by BU bypasses the p53-p21(Cip1/Waf1) pathway. View Publication

Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation,overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities,which indicates the presence of other mechanisms for Evi-1 activation. In this study,we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL-mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population,in which Evi-1 particularly contributes to the propagation of MLL-ENL-immortalized cells. Furthermore,gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell-like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell-derived MLL leukemic cells. View Publication