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

This study aimed to assess the efficacy of Quality and Quantity media-cultured mononuclear cells (QQ-MNCs) for promoting nerve regeneration in a mouse sciatic nerve transection model. Human peripheral blood mononuclear cells (PB-MNCs) and QQ-MNCs derived from healthy volunteers were used/compared. The left sciatic nerve was surgically transected in 27 mice. After complete nerve transection was confirmed,end-to-end direct epineurial nerve repair was performed using 9–0 nylon. Fibrin glue was applied to the tissue around the injury site to limit diffusion of the study treatment followed by application of 0.5 ml phosphate buffered saline (PBS) or PB-MNCs (2x10 6 cells) or QQ-MNCs (2x10 6 cells) to the injury site. The skin was then closed using 6–0 nylon. Histomorphology,immunohistochemistry,electrophysiologic examination,and functional assessment were evaluated at 12-weeks followed by euthanasia and subsequent harvesting of the left sciatic nerves and the left and right gastrocnemius muscles for examination. QQ-MNCs mice exhibited significant improvement in all histomorphologic parameters (axon fiber diameter,myelin thickness,percentage of nerve density) and immunohistochemistry assays (S100,SOX10,GFAP,neurofilament,IL-1β,VEGF,anti-HNA,TNF-α,vWF) compared to PBS mice (all p < 0.05). QQ-MNCs mice also had a significantly higher Basso Mouse Scale score compared to PBS mice ( p = 0.018). The percentage of nerve density adjacent to the injury site was significantly higher in QQ-MNCs mice than in PB-MNCs mice ( p = 0.049). IL-1β expression was significantly lower in QQ-MNCs mice than in PB-MNCs mice ( p = 0.01). QQ-MNCs mice demonstrated significantly better functional and histomorphologic outcomes of nerve regeneration compared to PB-MNCs mice and PBS mice. View Publication

Induced pluripotent stem cells (IPS) are an artificially derived type of pluripotent stem cell,showing many of the same characteristics as natural pluripotent stem cells. IPS are a hopeful therapeutic model; however there is a critical need to determine their response to environmental toxins. Effects of arsenic on cells have been studied extensively; however,its effect on IPS is yet to be elucidated. Arsenic trioxide (ATO) has been shown to inhibit cell proliferation,induce apoptosis and genotoxicity in many cells. Based on ATOs action in other cells,we hypothesize that it will induce alterations in morphology,inhibit cell viability and induce a genotoxic effect on IPS. Cells were treated for 24 hours with ATO (0-9 µg/mL). Cell morphology,viability and DNA damage were documented. Results indicated sufficient changes in morphology of cell colonies mainly in cell ability to maintain grouping and ability to remain adherent. Cell viability decreased in a dose dependent manner. There were significant increases in tail length and moment as well as destruction of intact DNA as concentration increased. Exposure to ATO resulted in a reproducible dose dependent sequence of events marked by changes in morphology,decrease of cell viability,and induction of genotoxicity in IPS. View Publication

In systemic lupus erythematosus,defective clearance of apoptotic debris and activation of innate cells result in a chronically activated type 1 IFN response,which can be measured in PBMCs of most patients. Metformin,a widely used prescription drug for Type 2 diabetes,has a therapeutic effect in several mouse models of lupus through mechanisms involving inhibition of oxidative phosphorylation and a decrease in CD4+ T cell activation. In this study,we report that in CD4+ T cells from human healthy controls and human systemic lupus erythematosus patients,metformin inhibits the transcription of IFN-stimulated genes (ISGs) after IFN-alpha treatment. Accordingly,metformin inhibited the phosphorylation of pSTAT1 (Y701) and its binding to IFN-stimulated response elements that control ISG expression. These effects were independent of AMPK activation or mTORC1 inhibition but were replicated using inhibitors of the electron transport chain respiratory complexes I,III,and IV. This indicates that mitochondrial respiration is required for ISG expression in CD4+ T cells and provides a novel mechanism by which metformin may exert a therapeutic effect in autoimmune diseases. View Publication

Hematopoietic stem cells (HSCs) can be isolated from murine bone marrow by their ability to efflux the Hoechst 33342 dye. This method defines an extremely small and hematopoietically potent subset of cells known as the side population (SP). Recent studies suggest that transplanted single SP cells are capable of lymphohematopoietic repopulation at near absolute efficiencies. Here,we carefully reevaluate the hematopoietic potential of individual SP cells and find substantially lower rates of reconstitution. Our strategy involved the cotransplantation of single SP cells along with different populations of competitor cells that varied in their self-renewal capacity. Even with minimized HSC competition,SP cells were only able to reconstitute up to 35% of recipient mice. Furthermore,through immunophenotyping and clonal in vitro assays we find that SP cells are virtually homogeneous. Isolation of HSCs on the basis of Hoechst exclusion and a single cell-surface marker allows enrichment levels similar to that obtained with complex multicolor strategies. Altogether,our results indicate that even an extremely homogeneous HSC population,based on phenotype and dye efflux,cannot reconstitute mice at absolute efficiencies. View Publication

Telomere length must be tightly regulated in highly proliferative tissues,such as the lymphohematopoietic system. Under steady-state conditions,the levels and functionality of hematopoietic-committed or multipotent progenitors were not affected in late-generation telomerase-deficient mice (mTerc(-/-)) with critically short telomeres. Evaluation of self-renewal potential of mTerc(-/-) day-12 spleen colony-forming units demonstrated no alteration as compared with wildtype progenitors. However,the replating ability of mTerc(-/-) granulocyte-macrophage CFUs (CFU-GMs) was greatly reduced as compared with wildtype CFU-GMs,indicating a diminished capacity of late-generation mTerc(-/-) committed progenitors when forced to proliferate. Long-term bone marrow cultures of mTerc(-/-) bone marrow (BM) cells show a reduction in proliferative capacity; this defect can be mainly attributed to the hematopoietic,not to the stromal,mTerc(-/-) cells. In serial and competitive transplantations,mTerc(-/-) BM stem cells show reduced long-term repopulating capacity,concomitant with an increase in genetic instability compared with wildtype cells. Nevertheless,in competitive transplantations late-generation mTerc(-/-) precursors can occasionally overcome this proliferative impairment and reconstitute irradiated recipients. In summary,our results demonstrate that late-generation mTerc(-/-) BM cells with short telomeres,although exhibiting reduced proliferation ability and reduced long-term repopulating capacity,can still reconstitute myeloablated animals maintaining stem cell function. View Publication

BACKGROUND: Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs).backslashnbackslashnMETHODS AND RESULTS: To induce endothelial cell differentiation,undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days,CD31(+) cells (13.7+/-2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation,these hESC-ECs expressed endothelial specific markers such as vWF (96.3+/-1.4%),CD31 (97.2+/-2.5%),and VE-cadherin (93.7+/-2.8%),form vascular-like channels,and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward,5x10(6) hESC-ECs treated for 24 hours with nicotine (10(-8) M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 microg/ml) in the drinking water. Surprisingly,bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally,in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs).backslashnbackslashnCONCLUSIONS: This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs,and enhance their angiogenic effects in vivo. Furthermore,activation of nAChRs has anti-apoptotic,angiogenic,and proliferative effects through MAPK and Akt signaling pathways. View Publication

Teratoma formation is a critical obstacle to safe clinical translation of human embryonic stem (ES) cell-based therapies in the future. As current methods of isolation are unable to yield 100% pure population of differentiated cells from a pluripotent donor source,potential development of these tumors is a significant concern. Here we used non-invasive reporter gene imaging to investigate the relationship between human ES cell number and teratoma formation in a xenogenic model of ES cell transplantation. Human ES cells (H9 line) were stably transduced with a double fusion (DF) reporter construct containing firefly luciferase and enhanced green fluorescent protein (Fluc- eGFP) driven by a human ubiquitin promoter. Immunodeficient mice received intramyocardial (n = 35) or skeletal muscle (n = 35) injection of 1 × 102,1 × 103,1 × 104,1 × 105 or 1 × 106 DF positive ES cells suspended in saline for myocardium and Matrigel for skeletal muscle. Cell survival and proliferation were monitored via bioluminescence imaging (BLI) for an 8 week period following transplantation. Mice negative for Fluc signal after 8 weeks were followed out to day 365 to confirm tumor absence. Significantly,in this study,a minimum of 1 × 105 ES cells in the myocardium and 1 × 104 cells in the skeletal muscle was observed to be requisite for teratoma development,suggesting that human ES cell number may be a critical factor in teratoma formation. Engraftment and tumor occurrence were also observed to be highly dependent on ES cell number. We anticipate these results should yield useful insights to the safe and reliable application of human ES cell derivatives in the clinic. Keywords View Publication

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We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts,an apical domain,marked by EZRIN and atypical PKC$\$,is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly,actin polymerization,regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA),promotes lumen formation,whereas actin contraction,mediated by MYOSIN-II,inhibits this process. Finally,we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development,this system can provide a powerful model for investigation of this process in a controlled environment. Overall,our data establish that lumenogenesis is a fundamental cell biological property of human PSCs. View Publication

Background?-catenin,acting as the core effector of canonical Wnt signaling pathway,plays a pivotal role in controlling lineage commitment and the formation of definitive endoderm (DE) during early embryonic development. Despite extensive studies using various animal and cell models,the ?-catenin-centered regulatory mechanisms underlying DE formation remain incompletely understood,partly due to the rapid and complex cell fate transitions during early differentiation.ResultsIn this study,we generated new CTNNB1-/- human ES cells (hESCs) using CRISPR-based insertional gene disruption approach and systematically rescued the DE defect in these cells by introducing various truncated or mutant forms of ?-catenin. Our analysis showed that a truncated ?-catenin lacking both N- and C-terminal domains (?N148C) could robustly rescue the DE formation,whereas hyperactive ?-catenin mutants with S33Y mutation or N-terminal deletion (?N90) had limited ability to induce DE lineage. Notably,the ?N148C mutant exhibited significant nuclear translocation that was positively correlated with successful DE rescue. Transcriptomic analysis further uncovered that two weak ?-catenin mutants lacking the C-terminal transactivation domain (CTD) activated primitive streak (PS) genes,whereas the hyperactive ?-catenin mutants activated mesoderm genes.ConclusionOur study uncovered an unconventional regulatory function of ?-catenin through weak transactivation,indicating that the levels of ?-catenin activity determine the lineage bifurcation from mesendoderm into endoderm and mesoderm.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-024-01279-5. View Publication

Hematopoietic stem and progenitor cells (HSPCs) are critical for the treatment of blood diseases in clinic. However,the limited source of HSPCs severely hinders their clinical application. In the embryo,hematopoietic stem cells (HSCs) arise from hemogenic endothelial (HE) cells lining the major arteries in vivo. In this work,by engineering vascular niche endothelial cells (VN-ECs),we generated functional HSPCs in vitro from ECs at various sites,including the aorta-gonad-mesonephros (AGM) region and the placenta. Firstly,we converted mouse embryonic HE cells from the AGM region (aHE) into induced HSPCs (iHSPCs),which have the abilities for multilineage differentiation and self-renewal. Mechanistically,we found that VN-ECs can promote the generation of iHSPCs via secretion of CX3CL1 and IL1A. Next,through VN-EC co-culture,we showed that placental HE (pHE) cells,a type of extra-embryonic HE cells,were successfully converted into iHSPCs (pHE-iHSPCs),which have multilineage differentiation capacity,but exhibit limited self-renewal ability. Furthermore,comparative transcriptome analysis of aHE-iHSPCs and pHE-iHSPCs showed that aHE-iHSPCs highly expressed HSC-specific and self-renewal-related genes. Moreover,experimental validation showed that retinoic acid (RA) treatment promoted the transformation of pHE cells into iHSPCs that have self-renewal ability. Collectively,our results suggested that pHE cells possess the potential to transform into self-renewing iHSPCs through RA treatment,which will facilitate the clinical application of placental endothelial cells in hematopoietic cell generation. Subject terms: Haematopoietic stem cells,Haematopoietic stem cells View Publication