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

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

Most primitive hematopoietic cells appear to be normally quiescent in vivo,whereas their leukemic counterparts in patients with chronic myeloid leukemia (CML) are maintained in a state of rapid turnover. This difference is also seen in the long-term culture system,where control of primitive hematopoietic progenitor proliferation is mediated by interactions of these cells with marrow-derived mesenchymal cells of the fibroblast lineage. We now show that exogenous addition of macrophage inflammatory protein 1 alpha (MIP-1 alpha) to normal long-term cultures can reversibly and specifically block the activation of primitive" (high proliferative potential)� View Publication

Various combinations of antibodies directed to cell surface markers have been used to isolate human and rhesus macaque hematopoietic stem cells (HSCs). These protocols result in poor enrichment or require multiple complex steps. Recently,a simple phenotype for HSCs based on cell surface markers from the signaling lymphocyte activation molecule (SLAM) family of receptors has been reported in the mouse. We examined the possibility of using the SLAM markers to facilitate the isolation of highly enriched populations of HSCs in humans and rhesus macaques. We isolated SLAM (CD150(+)CD48(-)) and non-SLAM (not CD150(+)CD48(-)) cells from human umbilical cord blood CD34(+) cells as well as from human and rhesus macaque mobilized peripheral blood CD34(+) cells and compared their ability to form colonies in vitro and reconstitute immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 γc receptor(null),NSG) mice. We found that the CD34(+) SLAM population contributed equally or less to colony formation in vitro and to long-term reconstitution in NSG mice compared with the CD34(+) non-SLAM population. Thus,SLAM family markers do not permit the same degree of HSC enrichment in humans and rhesus macaques as in mice. View Publication

BACKGROUND Previous studies have shown that foamy viral (FV) vectors are a promising alternative to gammaretroviral and lentiviral vectors and also that insulators can improve FV vector safety. However,in a previous analysis of insulator effects on FV vector safety,strong viral promoters were used to elicit genotoxic events. In the present study,we developed and analyzed the efficacy and safety of a high-titer,clinically relevant FV vector driven by the housekeeping promoter elongation factor-1alpha$ and insulated with an enhancer blocking A1 insulator (FV-EGW-A1). METHODS Human CD34+ cord blood cells were exposed to an enhanced green fluorescent protein expressing vector,FV-EGW-A1,at a multiplicity of infection of 10 and then maintained in vitro or transplanted into immunodeficient mice. Flow cytometry was used to measure engraftment and marking in vivo. FV vector integration sites were analyzed to assess safety. RESULTS FV-EGW-A1 resulted in high-marking,multilineage engraftment of human repopulating cells with no evidence of silencing. Engraftment was highly polyclonal with no clonal dominance and a promising safety profile based on integration site analysis. CONCLUSIONS An FV vector with an elongation factor-1alpha$ promoter and an A1 insulator is a promising vector design for use in the clinic. View Publication

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse,but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed textless10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However,the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses textless20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells,mES cells lacking H2AX,a histone protein involved in the DNA damage response,were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining,H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs),an increase in dsb rejoining rate,and a decrease in Ku70/80. Unlike mouse ES,human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells,they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM,and they add to the growing list of differences in the way rodent and human cells deal with DNA damage. View Publication

We analyzed 112 patients with high-risk acute myeloid leukemia (61 in complete remission [CR]; 51 in relapse),who received human leukocyte-antigen (HLA)-haploidentical transplants from natural killer (NK) alloreactive (n = 51) or non-NK alloreactive donors (n = 61). NK alloreactive donors possessed HLA class I,killer-cell immunoglobulin-like receptor (KIR) ligand(s) which were missing in the recipients,KIR gene(s) for missing self recognition on recipient targets,and alloreactive NK clones against recipient targets. Transplantation from NK-alloreactive donors was associated with a significantly lower relapse rate in patients transplanted in CR (3% versus 47%) (P textgreater .003),better event-free survival in patients transplanted in relapse (34% versus 6%,P = .04) and in remission (67% versus 18%,P = .02),and reduced risk of relapse or death (relative risk versus non-NK-alloreactive donor,0.48; 95% CI,0.29-0.78; P textgreater .001). In all patients we tested the missing ligand" model which pools KIR ligand mismatched transplants and KIR ligand-matched transplants from donors possessing KIR(s) for which neither donor nor recipient have HLA ligand(s). Only transplantation from NK-alloreactive donors is associated with a survival advantage." View Publication

Pluripotent or multipotent cell-based therapeutics are vital for skeletal reconstruction in non-healing critical-sized defects since the local endogenous progenitor cells are not often adequate to restore tissue continuity or function. However,currently available cell-based regenerative strategies are hindered by numerous obstacles including inadequate cell availability,painful and invasive cell-harvesting procedures,and tumorigenesis. Previously,we established a novel platform technology for inducing a quiescent stem cell-like stage using only a single extracellular proteoglycan,fibromodulin (FMOD),circumventing gene transduction. In this study,we further purified and significantly increased the reprogramming rate of the yield multipotent FMOD reprogrammed (FReP) cells. We also exposed the 'molecular blueprint' of FReP cell osteogenic differentiation by gene profiling. Radiographic analysis showed that implantation of FReP cells into a critical-sized SCID mouse calvarial defect,contributed to the robust osteogenic capability of FReP cells in a challenging clinically relevant traumatic scenario in vivo. The persistence,engraftment,and osteogenesis of transplanted FReP cells without tumorigenesis in vivo were confirmed by histological and immunohistochemical staining. Taken together,we have provided an extended potency,safety,and molecular profile of FReP cell-based bone regeneration. Therefore,FReP cells present a high potential for cellular and gene therapy products for bone regeneration. View Publication

Engineered neural stem cells (NSCs) are a promising approach to treating glioblastoma (GBM). The ideal NSC drug carrier for clinical use should be easily isolated and autologous to avoid immune rejection. We transdifferentiated (TD) human fibroblasts into tumor-homing early-stage induced NSCs (h-iNSC(TE)),engineered them to express optical reporters and different therapeutic gene products,and assessed the tumor-homing migration and therapeutic efficacy of cytotoxic h-iNSC(TE) in patient-derived GBM models of surgical and nonsurgical disease. Molecular and functional analysis revealed that our single-factor SOX2 TD strategy converted human skin fibroblasts into h-iNSC(TE) that were nestin(+) and expressed pathways associated with tumor-homing migration in 4 days. Time-lapse motion analysis showed that h-iNSC(TE) rapidly migrated to human GBM cells and penetrated human GBM spheroids,a process inhibited by blockade of CXCR4. Serial imaging showed that h-iNSC(TE) delivery of the proapoptotic agent tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) reduced the size of solid human GBM xenografts 250-fold in 3 weeks and prolonged median survival from 22 to 49 days. Additionally,h-iNSC(TE) thymidine kinase/ganciclovir enzyme/prodrug therapy (h-iNSC(TE)-TK) reduced the size of patient-derived GBM xenografts 20-fold and extended survival from 32 to 62 days. Mimicking clinical NSC therapy,h-iNSC(TE)-TK therapy delivered into the postoperative surgical resection cavity delayed the regrowth of residual GBMs threefold and prolonged survival from 46 to 60 days. These results suggest that TD of human skin into h-iNSC(TE) is a platform for creating tumor-homing cytotoxic cell therapies for cancer,where the potential to avoid carrier rejection could maximize treatment durability in human trials. View Publication

Stem cells hold great promise for therapies aimed at regenerating damaged tissue,drug screening and studying in vitro models of human disease. However,many challenges remain before these applications can become a reality. One such challenge is developing chemically defined and scalable culture conditions for derivation and expansion of clinically viable human pluripotent stem cells,as well as controlling their differentiation with high specificity. Interaction of stem cells with their extracellular microenvironment plays an important role in determining their differentiation commitment and functions. Regenerative medicine approaches integrating cell-matrix and cell-cell interactions,and soluble factors could lead to development of robust microenvironments to control various cellular responses. Indeed,several of these recent developments have provided significant insight into the design of microenvironments that can elicit the targeted cellular response. In this article,we will focus on some of these developments with an emphasis on matrix-mediated expansion of human pluripotent stem cells while maintaining their pluripotency. We will also discuss the role of matrix-based cues and cell-cell interactions in the form of soluble signals in directing stem cell differentiation into musculoskeletal lineages. View Publication

Air-liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling,infection or inflammation confounding PCD diagnostic results. ALI culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI culture method adopted from April 2018 across three collaborating PCD diagnostic sites,including current University Hospital Southampton COVID-19 risk mitigation measures,and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI culture and 199 (82.9{\%}) were ciliated. Fifty-four of 83 (63.9{\%}) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen; 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated,scanning electron microscopy demonstrated excellent ciliation,and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary,our ALI culture protocol provides high ciliation rates across three centres,minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful,facilitating PCD research. View Publication

Epstein-Barr virus (EBV) persistently infects more than 90% of the human population,causing infectious mononucleosis,susceptibility to autoimmune diseases and multiple malignancies of epithelial or B cell-origin. EBV infects epithelial cells and B cells through interaction between viral glycoproteins and different host receptors,but it has remained unknown whether a common receptor mediates infection of its two major host cell targets. Here,we establish R9AP as a crucial EBV receptor for entry into epithelial and B cells. R9AP silencing or knockout,R9AP-derived peptide and R9AP monoclonal antibody each significantly inhibit,whereas R9AP overexpression promotes,EBV uptake into both cell types. R9AP binds directly to the EBV glycoprotein gH/gL complex to initiate gH/gL-gB-mediated membrane fusion. Notably,the interaction of R9AP with gH/gL is inhibited by the highly competitive gH/gL-neutralizing antibody AMMO1,which blocks EBV epithelial and B cell entry. Moreover,R9AP mediates viral and cellular membrane fusion in cooperation with EBV gp42-human leukocyte antigen class II or gH/gL-EPHA2 complexes in B cells or epithelial cells,respectively. We propose R9AP as the crucial common receptor of B cells and epithelial cells and a potential prophylactic and vaccine target for EBV. View Publication

The neurosphere assay can detect and expand neural stem cells (NSCs) and progenitor cells,but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall,we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 mum coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay,the neural colony forming cell assay (N-CFCA),and label-retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis,with the number of neurosphere-forming cells detected in individual 400 mum sections varying from a minimum of eight to a maximum of 891 depending upon the rostral-caudal coordinate assayed. Moreover,the greatest variability occurred in the rostral portion of the lateral ventricles,thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 +/- 276) or colonies (4275 +/- 124) we detected along the neuraxis did not differ significantly,LRC numbers were significantly reduced (1186 +/- 188,7 month chase) in comparison to both total colonies and neurospheres. Moreover,approximately two orders of magnitude fewer NSC-derived colonies (50 +/- 10) were detected using the N-CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU+/Ki-67+) or competent to divide (BrdU+/Mcm-2+),and proliferate upon transfer to culture,it is unclear whether this technique selectively detects endogenous NSCs. Overall,caution should be taken with the interpretation and employment of all these techniques. View Publication