搜索结果: 'gmp t cell activator'

Unlocking the clinical potential of stem cell based therapies requires firstly elucidation of the biological mechanisms which direct stem cell fate decisions and thereafter,technical advances which allow these processes to be driven in a fully defined culture environment. Strategies for the generation of defined surfaces for human embryonic stem cell (hESC) and mesenchymal stem cell (MSC) culture remain in their infancy. In this paper we outline a simple,effective and efficient method for presenting proteins or peptides on an otherwise non-fouling Layer-by-Layer (LbL) self-assembled surface of hyaluronic acid (HA) and chitosan (CHI). We are able to generate a surface that has both good temporal stability and the ability to direct biological outcomes based on its defined surface composition. Surface functionalization is achieved through suspending the selected extracellular matrix (ECM) protein domain or extracted full-length protein in buffer containing a cross-linking agent (N-hydroxysulfosuccinimide/N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) over the LbL HA-CHI surface and then allowing the solvent to evaporate overnight. This simple,but important step results in remarkable protein deposition efficiencies often exceeding 50%,whereas traditional cross-linking methods result in such poor deposition of non-collagenous proteins that a.) quantification of bound amounts of protein is outside the resolution of commonly utilized protein assays,and b.) these surfaces are both unable to support cell attachment and growth. The utility of the protein-modified HA-CHI surfaces is demonstrated through the identification of specific hESC attachment efficiencies and through directing MSC osteogenic outcomes on these fully defined surfaces. This simple and scalable method is shown to enable the development of defined stem cell culture conditions,as well as the elucidation of the fundamental biological processes necessary for the realization of stem cell based therapies. View Publication

Adhesion properties of hematopoietic stem cells (HSCs) in the bone marrow (BM) niches control their migration and affect their cell-cycle dynamics. The serum response factor (Srf) regulates growth factor-inducible genes and genes controlling cytoskeleton structures involved in cell spreading,adhesion,and migration. We identified a role for Srf in HSC adhesion and steady-state hematopoiesis. Conditional deletion of Srf in BM cells resulted in a 3-fold expansion of the long- and short-term HSCs and multipotent progenitors (MPPs),which occurs without long-term modification of cell-cycle dynamics. Early differentiation steps to myeloid and lymphoid lineages were normal,but Srf loss results in alterations in mature-cell production and severe thrombocytopenia. Srf-null BM cells also displayed compromised engraftment properties in transplantation assays. Gene expression analysis identified Srf target genes expressed in HSCs,including a network of genes associated with cell migration and adhesion. Srf-null stem cells and MPPs displayed impair expression of the integrin network and decreased adherence in vitro. In addition,Srf-null mice showed increase numbers of circulating stem and progenitor cells,which likely reflect their reduced retention in the BM. Altogether,our results demonstrate that Srf is an essential regulator of stem cells and MPP adhesion,and suggest that Srf acts mainly through cell-matrix interactions and integrin signaling. View Publication

This unit describes the generation of tetracycline-inducible short hairpin RNA interference (shRNAi) human embryonic stem cell (hESC) lines. Using this vector-based approach enables stable and long-term expression of target hairpins under the control of doxycycline/tetracycline. Target degradation can be controlled in both a dose- and time-dependent manner that can even be switched off,depending upon the particular requirements of the study. View Publication

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration,but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs),endothelial cells (hiPSC-ECs),and smooth muscle cells (SMCs) are often limited by low yield,purity,and/or poor phenotypic stability. Here,we present novel protocols for generating hiPSC-CMs,-ECs,and -SMCs that are substantially more efficient than conventional methods,as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells,by sealing the needle track to prevent the cells from being squeezed out of the myocardium,and cell survival,by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury,the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch,and treatment with both the cells and the patch,but not with the cells alone,was associated with significant improvements in cardiac function and infarct size. View Publication

Human pluripotent stem cells derived cardiomyocytes (PSC-CMs) have been widely used for disease modeling,drug safety screening,and preclinical cell therapy to regenerate myocardium. Most studies have utilized PSC-CM grown in vitro for a relatively short period after differentiation. These PSC-CMs demonstrated structural,electrophysiological,and mechanical features of primitive cardiomyocytes. A few studies have extended in vitro PSC-CM culture time and reported improved maturation of structural and electromechanical properties. The degree of mitochondrial maturation,however,remains unclear. This study characterized the development of mitochondria during prolonged in vitro culture. PSC-CM demonstrated an improved mitochondrial maturation with prolonged culture,in terms of increased mitochondrial relative abundance,enhanced membrane potential,and increased activity of several mitochondrial respiratory complexes. These are in parallel with the maturation of other cellular components. However,the maturation of mitochondria in PSC-CMs grown for extended in vitro culture exhibits suboptimal maturation when compared with the maturation of mitochondria observed in the human fetal heart during similar time interval. View Publication