搜索结果: 'NeuroCult Proliferation'

Basic fibroblast growth factor (bFGF) is a crucial factor sustaining human pluripotent stem cells (hPSCs). We designed this study to search the substitutive factors other than bFGF for the maintenance of hPSCs by using human placenta-derived conditioned medium without exogenous bFGF (hPCCM-),containing chemokine (C-X-C motif) receptor 2 (CXCR2) ligands,including interleukin (IL)-8 and growth-related oncogene $\$(GRO$\$),which were developed on the basis of our previous studies. First,we confirmed that IL-8 and/or GRO$\$ independent roles to preserve the phenotype of hPSCs. Then,we tried CXCR2 blockage of hPSCs in hPCCM- and verified the significant decrease of pluripotency-associated genes expression and the proliferation of hPSCs. Interestingly,CXCR2 suppression of hPSCs in mTeSR™1 containing exogenous bFGF decreased the proliferation of hPSCs while maintaining pluripotency characteristics. Lastly,we found that hPSCs proliferated robustly for more than 35 passages in hPCCM- on a gelatin substratum. Higher CXCR2 expression of hPSCs cultured in hPCCM- than those in mTeSR™1 was observable. Our findings suggest that CXCR2 and its related ligands might be novel factors comparable to bFGF supporting the characteristics of hPSCs and hPCCM- might be useful for the maintenance of hPSCs as well as for the accurate evaluation of CXCR2 role in hPSCs without the confounding influence of exogenous bFGF. View Publication

Umbilical cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells (HSCs) for transplantation to treat various hematological disorders. The major limitation to the use of UCB-derived HSCs (UCB-HSCs) in transplantation,however,is the low numbers of HSCs in a unit of cord blood. To overcome this limitation,various cytokines or small molecules have been used to expand UCB-HSCs ex vivo. In this study,we investigated a synergistic effect of the combination of HIL-6,SR1,and UM171 on UCB-HSC culture and found that this combination resulted in the highest number of CD34+ cells. These results suggest that the combination of SR1,UM171 and HIL-6 exerts a synergistic effect in the proliferation of HSCs from UCB and thus,SR1,UM171 and HIL-6 is the most suitable combination for obtaining HSCs from UCB for clinical transplantation. View Publication

Emerging evidence indicates that in myelodysplastic syndromes (MDS),the bone marrow (BM) microenvironment may also contribute to the ineffective,malignant haematopoiesis in addition to the intrinsic abnormalities of haematopoietic stem precursor cells (HSPCs). The BM microenvironment influences malignant haematopoiesis through indirect mechanisms,but the processes by which the BM microenvironment directly contributes to MDS initiation and progression have not yet been elucidated. Our previous data showed that BM-derived stromal cells (BMSCs) from MDS patients have an abnormal expression of focal adhesion kinase (FAK). In this study,we characterise the morpho-phenotypic features and the functional alterations of BMSCs from MDS patients and in FAK knock-downed HS-5 cells. The decreased expression of FAK or its phosphorylated form in BMSCs from low-risk (LR) MDS directly correlates with BMSCs' functional deficiency and is associated with a reduced level of haemoglobin. The downregulation of FAK in HS-5 cells alters their morphology,proliferation,and differentiation capabilities and impairs the expression of several adhesion molecules. In addition,we examine the CD34+ healthy donor (HD)-derived HSPCs' properties when co-cultured with FAK-deficient BMSCs. Both abnormal proliferation and the impaired erythroid differentiation capacity of HD-HSPCs were observed. Together,these results demonstrate that stromal adhesion mechanisms mediated by FAK are crucial for regulating HSPCs' homeostasis. View Publication

Human pluripotent stem cells (hPSCs) display a very short G1 phase and rapid proliferation kinetics. Regulation of the cell cycle,which is linked to pluripotency and differentiation,is dependent on the stem cell environment,particularly on culture density. This link has been so far empirical and central to disparities in the growth rates and fractions of self-renewing hPSCs residing in different cycle phases. In this study,hPSC cycle progression in conjunction with proliferation and differentiation were comprehensively investigated for different culture densities. Cell proliferation decelerated significantly at densities beyond 50×10(4) cells/cm(2). Correspondingly,the G1 fraction increased from 25% up to 60% at densities greater than 40×10(4) cells/cm(2) while still hPSC pluripotency marker expression was maintained. In parallel,expression of the cycle inhibitor CDKN1A (p21) was increased,while that of p27 and p53 did not change significantly. After 4 days of culture in an unconditioned medium,greater heterogeneity was noted in the differentiation outcomes and was limited by reducing the density variation. A quantitative model was constructed for self-renewing and differentiating hPSC ensembles to gain a better understanding of the link between culture density,cycle progression,and stem cell state. Results for multiple hPSC lines and medium types corroborated experimental findings. Media commonly used for maintenance of self-renewing hPSCs exhibited the slowest kinetics of induction of differentiation (kdiff),while BMP4 supplementation led to 14-fold higher kdiff values. Spontaneous differentiation in a growth factor-free medium exhibited the largest variation in outcomes at different densities. In conjunction with the quantitative framework,our findings will facilitate rationalizing the selection of cultivation conditions for the generation of stem cell therapeutics. View Publication

Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here,we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks,enhancing the reconstitution capacity of edited HSPCs. However,this results in lower HDR efficiency,rendering ex vivo culture necessary yet detrimental. Mechanistically,ex vivo activation triggers a multi-step process initiated by p38 mitogen-activated protein kinase (MAPK) phosphorylation,which generates mitogenic reactive oxygen species (ROS),promoting fast cell-cycle progression and subsequent proliferation-induced DNA damage. Thus,p38 inhibition before gene editing delays G1/S transition and expands transcriptionally defined HSCs,ultimately endowing edited cells with superior multi-lineage differentiation,persistence throughout serial transplantation,enhanced polyclonal repertoire,and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications. View Publication

Multiple myeloma (MM) is the second most common hematological malignancy,characterized by a clonal expansion of malignant plasma cells in bone marrow. Monoclonal gammopathy of undetermined significance (MGUS) is the premalignant condition of MM. The tumor microenvironment is thought to influence the progression from premalignant conditions. Myeloid‐derived suppressor cells (MDSCs) are a heterogenous group of different cellular subsets with myeloid origin,characterized by their ability to inhibit T‐cell responses. MDSC are thought to play an important immunoregulatory role in different diseases,and in many cancers their levels seem to correlate with a poor prognosis. There are three different subsets,the neutrophil‐like polymorphonuclear (PMN)‐MDSC,the monocyte‐like (M)‐MDSC,and the immature early (e)MDSC. In this study,we investigate the levels and functions of all MDSC subsets in the bone marrow of both MGUS and MM patients and compare it to blood MDSC. We found that MDSC levels are not increased in neither the blood nor bone marrow of MGUS or MM patients,and they lack strong T‐cell suppressive abilities. Blood PMN‐MDSC seems to have a small inhibitory effect,but mature neutrophils were more suppressive. Interestingly,eMDSC levels were decreased in the blood of MM patients. Our data indicate that MDSC are not key players in the pathogenesis of MM,but that mature neutrophils may be more important as they have a stronger immunoregulatory effect. View Publication

Cell therapy offers an innovative approach for treating enteric neuropathies. Postnatal gut-derived enteric neural stem/progenitor cells (ENSCs) represent a potential autologous source,but have a limited capacity for proliferation and neuronal differentiation. Since serotonin (5-HT) promotes enteric neuronal growth during embryonic development,we hypothesized that serotonin receptor agonism would augment growth of neurons from transplanted ENSCs. Postnatal ENSCs were isolated from 2 to 4 week-old mouse colon and cultured with 5-HT4 receptor agonist (RS67506)-loaded liposomal nanoparticles. ENSCs were co-cultured with mouse colon explants in the presence of RS67506-loaded (n = 3) or empty nanoparticles (n = 3). ENSCs were also transplanted into mouse rectum in vivo with RS67506-loaded (n = 8) or blank nanoparticles (n = 4) confined in a thermosensitive hydrogel,Pluronic F-127. Neuronal density and proliferation were analyzed immunohistochemically. Cultured ENSCs gave rise to significantly more neurons in the presence of RS67506-loaded nanoparticles. Similarly,colon explants had significantly increased neuronal density when RS67506-loaded nanoparticles were present. Finally,following in vivo cell delivery,co-transplantation of ENSCs with 5-HT4 receptor agonist-loaded nanoparticles led to significantly increased neuronal density and proliferation. We conclude that optimization of postnatal ENSCs can support their use in cell-based therapies for neurointestinal diseases. View Publication

Magnesium (Mg) is a promising biodegradable metallic material for applications in cellular/tissue engineering and biomedical implants/devices. To advance clinical translation of Mg-based biomaterials,we investigated the effects and mechanisms of Mg degradation on the proliferation and pluripotency of human embryonic stem cells (hESCs). We used hESCs as the in vitro model system to study cellular responses to Mg degradation because they are sensitive to toxicants and capable of differentiating into any cell types of interest for regenerative medicine. In a previous study when hESCs were cultured in vitro with either polished metallic Mg (99.9% purity) or pre-degraded Mg,cell death was observed within the first 30 hours of culture. Excess Mg ions and hydroxide ions induced by Mg degradation may have been the causes for the observed cell death; hence,their respective effects on hESCs were investigated for the first time to reveal the potential mechanisms. For this purpose,the mTeSR®1 hESC culture media was either modified to an alkaline pH of 8.1 or supplemented with 0.4-40 mM of Mg ions. We showed that the initial increase of media pH to 8.1 had no adverse effect on hESC proliferation. At all tested Mg ion dosages,the hESCs grew to confluency and retained pluripotency as indicated by the expression of OCT4,SSEA3,and SOX2. When the supplemental Mg ion dosages increased to greater than 10 mM,however,hESC colony morphology changed and cell counts decreased. These results suggest that Mg-based implants or scaffolds are promising in combination with hESCs for regenerative medicine applications,providing their degradation rate is moderate. Additionally,the hESC culture system could serve as a standard model for cytocompatibility studies of Mg in vitro,and an identified 10 mM critical dosage of Mg ions could serve as a design guideline for safe degradation of Mg-based implants/scaffolds. View Publication

AbstractThe generation of chimeric antigen receptor?modified natural killer (CAR?NK) cells using induced pluripotent stem cells (iPSCs) has emerged as one of the paradigms for manufacturing off?the?shelf universal immunotherapy. However,there are still some challenges in enhancing the potency,safety,and multiple actions of CAR?NK cells. Here,iPSCs were site?specifically integrated at the ribosomal DNA (rDNA) locus with interleukin 24 (IL24) and CD19?specific chimeric antigen receptor (CAR19),and successfully differentiated into iPSC?derived NK (iNK) cells,followed by expansion using magnetic beads in vitro. Compared with the CAR19?iNK cells,IL24 armored CAR19?iNK (CAR19?IL24?iNK) cells showed higher cytotoxic capacity and amplification ability in vitro and inhibited tumor progression more effectively with better survival in a B?cell acute lymphoblastic leukaemia (B?ALL) (Nalm?6 (Luc1))?bearing mouse model. Interestingly,RNA?sequencing analysis showed that IL24 may enhance iNK cell function through nuclear factor kappa B (NF?B) pathway?related genes while exerting a direct effect on tumor cells. This study proved the feasibility and potential of combining IL24 with CAR?iNK cell therapy,suggesting a novel and promising off?the?shelf immunotherapy strategy. Zhang et al. successfully regenerated iNK cells from human iPSCs with rDNA locus gene editing. IL24 enhances the antitumor activity and proliferation of armored CAR?iNK cells,which may be involved in cellular?positive upregulation and adhesion pathways. View Publication

Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation,inhibits proliferation,and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects,C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5'-flanking region and enhances the promoter activity of Gfi-1. Moreover,in K562 cells,RNA interference-mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1,but not of a transcriptional repressor mutant (Gfi-1P2A),inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast,Gfi-1 short hairpin RNA-tranduced CD34(+) chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together,these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells. View Publication

Human embryonic stem cells (hESC) have a unique capacity to self-renew and differentiate into all the cell types found in human body. Although the transcriptional regulators of pluripotency are well studied,the role of cytoplasmic regulators is still poorly characterized. Here,we report a new stem cell-specific RNA-binding protein L1TD1 (ECAT11,FLJ10884) required for hESC self-renewal and cancer cell proliferation. Depletion of L1TD1 results in immediate downregulation of OCT4 and NANOG. Furthermore,we demonstrate that OCT4,SOX2,and NANOG all bind to the promoter of L1TD1. Moreover,L1TD1 is highly expressed in seminomas,and depletion of L1TD1 in these cancer cells influences self-renewal and proliferation. We show that L1TD1 colocalizes and interacts with LIN28 via RNA and directly with RNA helicase A (RHA). LIN28 has been reported to regulate translation of OCT4 in complex with RHA. Thus,we hypothesize that L1TD1 is part of the L1TD1-RHA-LIN28 complex that could influence levels of OCT4. Our results strongly suggest that L1TD1 has an important role in the regulation of stemness. View Publication

Cytokines play a crucial role in the maintenance of polyclonal naive and memory T cell populations. It has previously been shown that ex vivo,the IL-7 cytokine induces the proliferation of naive recent thymic emigrants (RTE) isolated from umbilical cord blood but not mature adult-derived naive and memory human CD4(+) T cells. We find that the combination of IL-2 and IL-7 strongly promotes the proliferation of RTE,whereas adult CD4(+) T cells remain relatively unresponsive. Immunological activity is controlled by a balance between proliferation and apoptotic cell death. However,the relative contributions of IL-2 and IL-7 in regulating these processes in the absence of MHC/peptide signals are not known. Following exposure to either IL-2 or IL-7 alone,RTE,as well as mature naive and memory CD4(+) T cells,are rendered only minimally sensitive to Fas-mediated cell death. However,in the presence of the two cytokines,Fas engagement results in a high level of caspase-dependent apoptosis in both RTE as well as naive adult CD4(+) T cells. In contrast,equivalently treated memory CD4(+) T cells are significantly less sensitive to Fas-induced cell death. The increased susceptibility of RTE and naive CD4(+) T cells to Fas-induced apoptosis correlates with a significantly higher IL-2/IL-7-induced Fas expression on these T cell subsets than on memory CD4(+) T cells. Thus,IL-2 and IL-7 regulate homeostasis by modulating the equilibrium between proliferation and apoptotic cell death in RTE and mature naive and memory T cell subsets. View Publication