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

The prostate is a gland that contributes to men's fertility. It is highly responsive to androgens and is often the site of carcinogenesis,as prostate cancer is the most frequent cancer in men in over a hundred countries. To study the normal prostate,few in vitro models exist,and most of them do not express the androgen receptor (AR). To overcome this issue,prostate epithelial cells can be grown in primary culture ex vivo in 2- and 3-dimensional culture (organoids). However,methods to purify these cells often require flow cytometry,thus necessitating specialized instruments and expertise. Herein,we present a detailed protocol for the harvest,purification,and primary culture of mouse prostate epithelial cells to grow prostate organoids ex vivo. This protocol does not require flow cytometry approaches,facilitating its implementation in most research laboratories,and organoids grown with this protocol are highly responsive to androgens. In summary,we present a new simple method that can be used to grow prostate organoids that recapitulate the androgen response of this gland in vivo. View Publication

In recent years,the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated,but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs,and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kit(pos) cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor,Gleevec (imatinib mesylate; Novartis International,Basel,Switzerland,http://www.novartis.com),to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (Kit(W)/Kit(W-v)) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5,sarcomeric proteins beta-MHC and MLC-2v,and ANF and connexin-43. Immunostaining revealed alpha-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy,the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating,myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kit(pos) bone marrow hematopoietic cells as the source of the myogenic clusters. View Publication

Definitive hematopoietic stem and progenitor cells (HSCs/Ps) originating from the yolk sac and/or para-aorta-splanchno-pleura/aorta-gonad-mesonephros are hypothesized to colonize the fetal liver,but mechanisms involved are poorly defined. The Rac subfamily of Rho GTPases has been shown to play essential roles in HSC/P localization to the bone marrow following transplantation. Here,we study the role of Rac1 in HSC/P migration during ontogeny and seeding of fetal liver. Using a triple-transgenic approach,we have deleted Rac1 in HSCs/Ps during very early embryonic development. Without Rac1,there was a decrease in circulating HSCs/Ps in the blood of embryonic day (E) 10.5 embryos,while yolk sac definitive hematopoiesis was quantitatively normal. Intraembryonic hematopoiesis was significantly impaired in Rac1-deficient embryos,culminating with absence of intra-aortic clusters and fetal liver hematopoiesis. At E10.5,Rac1-deficient HSCs/Ps displayed decreased transwell migration and impaired inter-action with the microenvironment in migration-dependent assays. These data suggest that Rac1 plays an important role in HSC/P migration during embryonic development and is essential for the emergence of intraembryonic hematopoiesis. View Publication

Survivin,which is the smallest member of the inhibitor of apoptosis protein (IAP) family,is a chromosomal passenger protein that mediates the spindle assembly checkpoint and cytokinesis,and also functions as an inhibitor of apoptosis. Frequently overexpressed in human cancers and not expressed in most adult tissues,survivin has been proposed as an attractive target for anticancer therapies and,in some cases,has even been touted as a cancer-specific gene. Survivin is,however,expressed in proliferating adult cells,including human hematopoietic stem cells,T-lymphocytes,and erythroid cells throughout their maturation. Therefore,it is unclear how survivin-targeted anticancer therapies would impact steady-state blood development. To address this question,we used a conditional gene-targeting strategy and abolished survivin expression from the hematopoietic compartment of mice. We show that inducible deletion of survivin leads to ablation of the bone marrow,with widespread loss of hematopoietic progenitors and rapid mortality. Surprisingly,heterozygous deletion of survivin causes defects in erythropoiesis in a subset of the animals,with a dramatic reduction in enucleated erythrocytes and the presence of immature megaloblastic erythroblasts. Our studies demonstrate that survivin is essential for steady-state hematopoiesis and survival of the adult,and further,that a high level of survivin expression is critical for proper erythroid differentiation. View Publication

Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58{\%} Venus(+) HSPCs with 6-16{\%} human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD),caused by mutations in the gp91phox subunit of the NADPH oxidase,TI of a gp91phox transgene into AAVS1 resulted in ∼15{\%} gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs,4-11{\%} of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases. View Publication

Cardiac malformations and disease are the leading causes of death in the United States in live-born infants and adults,respectively. In both of these cases,a decrease in the number of functional cardiomyocytes often results in improper growth of heart tissue,wound healing complications,and poor tissue repair. The field of cardiac tissue engineering seeks to address these concerns by developing cardiac patches created from a variety of biomaterial scaffolds to be used in surgical repair of the heart. These scaffolds should be fully degradable biomaterial systems with tunable properties such that the materials can be altered to meet the needs of both in vitro culture (e.g. disease modeling) and in vivo application (e.g. cardiac patch). Current platforms do not utilize both structural anisotropy and proper cell-matrix contacts to promote functional cardiac phenotypes and thus there is still a need for critically sized scaffolds that mimic both the structural and adhesive properties of native tissue. To address this need,we have developed a silk-based scaffold platform containing cardiac tissue-derived extracellular matrix (cECM). These silk-cECM composite scaffolds have tunable architectures,degradation rates,and mechanical properties. Subcutaneous implantation in rats demonstrated that addition of the cECM to aligned silk scaffold led to 99% endogenous cell infiltration and promoted vascularization of a critically sized scaffold (10 × 5 × 2.5 mm) after 4 weeks in vivo. In vitro,silk-cECM scaffolds maintained the HL-1 atrial cardiomyocytes and human embryonic stem cell-derived cardiomyocytes and promoted a more functional phenotype in both cell types. This class of hybrid silk-cECM anisotropic scaffolds offers new opportunities for developing more physiologically relevant tissues for cardiac repair and disease modeling. View Publication

CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as $\beta$-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex,consisting of purified Cas9 protein and guide RNA,into HSPCs. Because $\beta$-hemoglobinopathies are most prevalent in developing countries,it is desirable to have a reliable,efficient,easy-to-use and cost effective delivery method. With this goal in mind,we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF),a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale. View Publication