Cognitive aging is a leading public health concern with the increasing aging population, but there is still lack of specific interventions directed against it. Recent studies have shown that ...cognitive function is intimately affected by systemic milieu in aging brain, and improvement of systemic environment in aging brain may be a promising approach for rejuvenating cognitive aging. Here, we sought to study the intervention effects of clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on cognitive aging in a murine model of aging. The conventional aging model in mice induced by d-galactose (d-gal) was employed here. Mice received once every two weeks intraperitoneal administration of hUC-MSCs. After 3 months of systematical regulation of hUC-MSCs, the hippocampal-dependent learning and memory ability was effectively improved in aged mice, and the synaptic plasticity was remarkably enhanced in CA1 area of the aged hippocampus; moreover, the neurobiological substrates that could impact on the function of hippocampal circuits were recovered in the aged hippocampus reflecting in: dendritic spine density enhanced, neural sheath and cytoskeleton restored, and postsynaptic density area increased. In addition, the activation of the endogenic neurogenesis which is beneficial to stabilize the neural network in hippocampus was observed after hUC-MSCs transplantation. Furthermore, we demonstrated that beneficial effects of systematical regulation of hUC-MSCs could be mediated by activation of mitogen-activated protein kinase (MAPK)-ERK-CREB signaling pathway in the aged hippocampus. Our study provides the first evidence that hUC-MSCs, which have the capacity of systematically regulating the aging brain, may be a potential intervention for cognitive aging.
Human embryonic stem cells (hESCs) have become an ideal cell source for the ex vivo generation of megakaryocyte (MK) and platelet products for clinical applications. However, an ongoing challenge is ...to establish scalable culture systems to maximize the yield of stem cell‐derived MKs that release platelets. We defined a specific dynamic 3D manufacturing system in a baffled‐flow manner that could remarkably facilitate megakaryopoiesis and increase the yield of platelet‐producing MKs from hESCs within a 12‐day induction period. Additionally, an increased number of >16N ploidy MKs, proplatelets, and platelets were generated from induced cells harvested on Day 12 using the specific dynamic culture method. The specific dynamic culture method significantly enhanced endothelium‐to‐haematopoietic transition and early haematopoiesis. More importantly, MK fate was significantly facilitated in a specific dynamic manner during early haematopoiesis. Mechanistically, this dynamic culture significantly enhanced mitochondrial function via the oxidative phosphorylation pathway and caused differentiation skewing of hESCs toward megakaryopoiesis. This study can aid in the automatic and scalable production of MKs from stem cells using baffled‐flow bioreactors and assist in the manufacturing of hESC‐derived MK and platelet products.
Human embryonic stem cells (hESCs) have become an ideal cell source for ex vivo generation of megakaryocyte (MK) and platelet products for clinical applications. We defined a specific dynamic culture system in a baffled‐flow manner that enabled mass production of MKs from hESCs within 12 days. The specific dynamic culture method significantly enhanced the endothelium‐to‐haematopoietic transition, early haematopoiesis and MK fate specification. Mechanistically, this dynamic culture significantly enhanced mitochondrial function via the oxidative phosphorylation pathway and caused differentiation skewing of hESCs toward megakaryopoiesis. This study could aid in the automatic and scalable production of MKs from stem cells using baffled‐flow bioreactors, and could also assist in the manufacturing of hESC‐derived MK and platelet products.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Ionizing radiation (IR) activate the early growth response-1 (Egr-1) promoter by production of radical oxygen intermediates (ROIs). Egr-EF, an expression vector pCIneo containing Egr-1 promoter ...cloned upstream of the cDNA for Flt3 ligand, was used to treat hematopoietic damage. 5-fluorouracil, a commonly used chemotherapeutic agent, cause tumor cell death by producing DNA damage and generating ROIs. We therefore hypothesized that clinically employed chemotherapeutic agents that increase ROIs could also be employed to activate Egr-EF in a chemoinducible gene therapy strategy. The goal of this study was to explore the effect of Flt3 Ligand gene transcription regulated by fluorouracil-induced Egr-1 promoter on hematopoietic recovery.
Human Flt3 Ligand (FL) cDNA and enhanced green fluorescent protein (EGFP) cDNA were linked together with IRES and inserted into the expression vector pCI-neo under control of the Egr-1 promoter (Egr-EF). The vector was transfected into the HFCL human bone marrow stromal cell line, and these cells were exposed to 5-FU, a chemotherapeutic drug. Expression of FL by HFCL/EF cells after 5-FU treatment was determined with ELISA, western blot and RT-PCR assays. In addition, the effect of FL from HFCL/EF cell culture supernatants on growth of CD34+ cells from cord blood was also studied. HFCL/EF cells were injected into CB-17 combined immunodeficient (SCID) mice with B16 melanoma. 5-FU was given three days after injection of the HFCL/EF cells. In the recipient mice, white blood cell levels in peripheral blood and expression of EGFP and FL in human stromal cells were measured. Tumor volumes in tumor-bearing mice were also measured.
5-FU treatment increased EGFP levels and secreted FL levels in HFCL/EF cells. Supernatants from HFCL/EF cell cultures treated with 5-FU increased CD34+ cell growth significantly. HFCL/EF exhibited an increase in the number of white blood cells after chemotherapy.
The data presented here support the use of transcriptional control mediated by chemoinducible gene therapy to reduce hematopoietic injury associated with 5-FU.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Trillium tschonoskii is a medicinal plant known to biosynthesize steroidal saponins. A phytochemical investigation of the rhizomes of T. tschonoskii led to the isolation of nine new furostanol ...saponins (1–9) and 11 known analogues (10–20). Five of these new compounds were shown to have hydroxy groups at the C-5 and C-6 positions, while two possess a rare aglycone containing carbonyl groups at the C-16 and C-22 positions as well as a Δ17(20) double bond, and the others have conjugated double bonds in the E-ring or have different sugar chains at the C-3 position. All the isolates were tested for their effect on the expansion of human cord blood (CB) CD34+ hematopoietic stem and progenitor cells. It was found that CB CD34+ cells treated with compounds 6, 7, 9, 10, 14, 15, and 19 showed increased numbers of rigorously phenotype-defined hematopoietic stem cells. Notably, compounds 9, 10, 13, and 14 demonstrated an enhanced ability to increase the percentages and numbers of CB CD34+CD38– cells and multipotential progenitors. The present study is the first to report that furostanol saponins from T. tschonoskii rhizomes can promote hematopoietic stem/progenitor cell (HSPC) expansion.
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IJS, KILJ, NUK, PNG, UL, UM
Mesenchymal stem cells (MSCs) are heterogeneous populations with broad application prospects in cell therapy, and using specific subpopulations of MSCs can enhance their particular capability under ...certain conditions and achieve better therapeutic effects. However, no studies have reported how to obtain high‐quality specific MSC subpopulations in vitro culture. Here, for the first time, we established a general operation process for obtaining high‐quality clinical‐grade cell subpopulations from human umbilical cord MSCs (hUC‐MSCs) based on particular markers. We used the MSC‐CD106+ subpopulations, whose biological function has been well documented, as an example to explore and optimize the crucial links of primary preparation, pre‐treatment, antibody incubation, flow sorting, quality and function test. After comprehensively evaluating the quality and function of the acquired MSC‐CD106+ subpopulations, including in vitro cell viability, apoptosis, proliferation, marker stability, adhesion ability, migration ability, tubule formation ability, immunomodulatory function and in vivo wound healing ability and proangiogenic activity, we defined an important pre‐treatment scheme which might effectively improve the therapeutic efficiency of MSC‐CD106+ subpopulations in two critical clinical application scenarios—direct injection after cell sorting and post‐culture injection into bodies. Based on the above, we tried to establish a general five‐step operation procedure for acquiring high‐quality clinical‐grade MSC subpopulations based on specific markers, which cannot only improve their enrichment efficiency and the reliability of preclinical studies, but also provide valuable methodological guidance for the rapid clinical transformation of specific MSC subpopulations.
We established an optimized operation process for obtaining human umbilical cord mesenchymal stem cells (hUC‐MSC) subpopulations with specific markers, which is also suitable for MSCs derived from other tissues.
Step One. Preparation of clinical‐grade hUC‐MSCs. The clinical‐grade hUC‐MSCs obtained fully comply with the cGMP guidelines and meet the quality standards of the NIFDC. Step Two. Removal of non‐specific sites and poor‐quality cells without cellular damage. Treatment with MACS was proved as a safe and effective pre‐treatment by verifying multiple core indicators related to subpopulation quality. Step Three. Incubation with the antibodies against specific functional markers. Select the optimum condition of specific antibodies to ensure the purity of subsets obtained by subsequent sorting. Step Four. Flow Sorting and obtaining hUC‐MSC subpopulations with specific markers. This step has higher technical requirements related to flow cytometry. Step Five. Quality test and function research of hUC‐MSC subpopulations with specific markers. Do more research and verification to ensure the efficiency of clinical transformation of the obtained MSC subpopulations. Application Scenes. The clinical‐grade MSC subpopulations obtained through the above‐optimized operation process do not only guarantee the quality of the cells in basic research and preclinical research but also provide safety assurance for clinical application.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Ex vivo red blood cell (RBC) production generates unsatisfactory erythroid cells. A deep exploration into terminally differentiated cells is required to understand the impairments for RBC generation ...and the underlying mechanisms. Here, we mapped an atlas of terminally differentiated cells from umbilical cord blood mononuclear cells (UCBMN) and pluripotent stem cells (PSC) and observed their dynamic regulation of erythropoiesis at single‐cell resolution. Interestingly, we detected a few progenitor cells and non‐erythroid cells from both origins. In PSC‐derived erythropoiesis (PSCE), the expression of haemoglobin switch regulators (BCL11A and ZBTB7A) were significantly absent, which could be the restraint for its adult globin expression. We also found that PSCE were less active in stress erythropoiesis than in UCBMN‐derived erythropoiesis (UCBE), and explored an agonist of stress erythropoiesis gene, TRIB3, could enhance the expression of adult globin in PSCE. Compared with UCBE, there was a lower expression of epigenetic‐related proteins (e.g., CASPASE 3 and UBE2O) and transcription factors (e.g., FOXO3 and TAL1) in PSCE, which might restrict PSCE's enucleation. Moreover, we characterized a subpopulation with high proliferation capacity marked by CD99high in colony‐forming unit‐erythroid cells. Inhibition of CD99 reduced the proliferation of PSC‐derived cells and facilitated erythroid maturation. Furthermore, CD99–CD99 mediated the interaction between macrophages and erythroid cells, illustrating a mechanism by which macrophages participate in erythropoiesis. This study provided a reference for improving ex vivo RBC generation.
scRNA‐seq and cell typing of late‐stage umbilical cord blood mononuclear cells‐ and pluripotent stem cell (PSC)‐derived cells were conducted. Stress erythropoiesis was more active in UCBMN‐derived erythropoiesis (UCB‐E) than in PSC‐derived erythropoiesis (PSC‐E). Regulators that impair adult globin expression and enucleation between UCB‐E and PSC‐E were compared at single cell level. CD99high progenitor cells were a proliferating colony forming unit erythroid subpopulation.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
MicroRNAs (miRNAs) are small, noncoding RNAs which can often act as an oncogene or a tumor suppressor. Several miRNAs are associated with the development of hepatocellular carcinoma (HCC). We ...demonstrated that miR-125b significantly suppresses HCC cell proliferation and promotes apoptosis by inhibiting the gene expression of the anti-apoptotic protein, Bcl-2. Bioinformatic analysis indicated that the 3′UTR of Bcl-2 has binding sites for miR-125b. Luciferase reporter assay confirmed the ability of miR-125b to dramatically suppress Bcl-2 transcription, suggesting that Bcl-2 is a target gene for miR-125b. We concluded that miR-125b acts as a tumor suppressor in hepatic tumor development by targeting Bcl-2 and inducing cancer cell apoptosis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Gap junction-mediated cell-cell interactions are highly conserved and play essential roles in cell survival, proliferation, differentiation and patterning. We report that Connexin 32 (Cx32)-mediated ...gap junctional intercellular communication (GJIC) is necessary for human embryonic stem cell-derived hepatocytes (hESC-Heps) during step-wise hepatic lineage restriction and maturation. Vitamin K2, previously shown to promote Cx32 expression in mature hepatocytes, up-regulated Cx32 expression and GJIC activation during hepatic differentiation and maturation, resulting in significant increases of hepatic markers expression and hepatocyte functions. In contrast, negative Cx32 regulator 2-aminoethoxydiphenyl borate blocked hESC-to-hepatocyte maturation and muted hepatocyte functions through disruption of GJIC activities. Dynamic gap junction organization and internalization are phosphorylation-dependent and the p38 mitogen-activated protein kinases pathway (MAPK) can negatively regulate Cxs through phosphorylation-dependent degradation of Cxs. We found that p38 MAPK inhibitor SB203580 improved maturation of hESC-Heps correlating with up-regulation of Cx32; by contrast, the p38 MAPK activator, anisomycin, blocked hESC-Heps maturation correlating with down-regulation of Cx32. These results suggested that Cx32 is essential for cell-cell interactions that facilitate driving hESCs through hepatic-lineage maturation. Regulators of both Cx32 and other members of its pathways maybe used as a promising approach on regulating hepatic lineage restriction of pluripotent stem cells and optimizing their functional maturation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
To build a protocol of separation and induction of megakaryocytes derived from cord blood mononuclear cells.
Red blood cells were precipitated by hydroxyethyl starch (HES). Mononuclear cells were ...obtained by density gradient centrifugation with Ficoll. The inducing efficiencies of megakaryocytes by using of different cytokine cocktails and culture media were analyzed.
The best choice for erythrocyte sedimentation and high efficiency of nucleated cells retrieving were obtained by using of 1.5% HES. The isolated cord blood mononuclear cells were cultured with domestic serum-free medium supplemented with 116t (IL-11, IL-6, TPO), st36(SCF, TPO, IL-3, IL-6), pt36 (PDGF,TPO,IL-3,IL-6) or pst36 for 7 days. St36 group (50 ng/ml SCF, 50 ng/ml TPO, 20 ng/ml IL-3 and 50 ng/ml IL-6) yielded the most CD41/CD61 positive (6.79±1.97)×10⁴. The cell viability (82.85 ± 0.64)% of st36 group by using of imported serum-free medium was better than (60.90±6.93)% that in domestic medium on day 7 after induction, and CD41/CD61 p
Q42; The availability of a well-characterized animal brain tumor model will play an important role in identifying treatments for human brain tumors. Wistar rats bearing 9L glioma cells can develop ...solid, well-circumcised tumors, and may be a useful animal model for the evaluation of various therapeutic approaches for gliosarcomas. In this study, the 9L/Wistar rat glioma model was produced by intracerebral implantation of 9LLUC glioma cells syngenic to Fischer 344 (F344) rats. Bioluminescence imaging showed that tumors progressively grew from day 7 to day 21 in 9LLUC/F344 rats, and tumor regression was found in some 9LLUC/Wistar rats. Hematoxylin-eosin staining verified that intracranial tumors were gliomas. Immunohistochemistry results demonstrated that no CD4- and CD8-positive cells were found in the syngeneic 9LLUC/F344 model. However, many infiltrating CD4- and CD8-positive cells were observed within the tumors of the 9LLUC/Wistar model. Our data suggests that compared with 9L/F344 rats, 9L glioma Wistar rats may not be suitable for evaluating brain glioma immunotherapies, even though the model induced an immune response and exhibited tumor regression.
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