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

Insight into the normal function of PrP(C),and how it can be subverted to produce neurotoxic effects,is provided by PrP molecules carrying deletions encompassing the conserved central region. The most neurotoxic of these mutants,Δ105-125 (called ΔCR),produces a spontaneous neurodegenerative illness when expressed in transgenic mice,and this phenotype can be dose-dependently suppressed by co-expression of wild-type PrP. Whether the toxic activity of ΔCR PrP and the protective activity or wild-type PrP are cell-autonomous,or can be exerted on neighboring cells,is unknown. To investigate this question,we have utilized co-cultures of differentiated neural stem cells derived from mice expressing ΔCR or wild-type PrP. Cells from the two kinds of mice,which are marked by the presence or absence of GFP,are differentiated together to yield neurons,astrocytes,and oligodendrocytes. As a surrogate read-out of ΔCR PrP toxicity,we assayed sensitivity of the cells to the cationic antibiotic,Zeocin. In a previous study,we reported that cells expressing ΔCR PrP are hypersensitive to the toxic effects of several cationic antibiotics,an effect that is suppressed by co-expression of wild type PrP,similar to the rescue of the neurodegenerative phenotype observed in transgenic mice. Using this system,we find that while ΔCR-dependent toxicity is cell-autonomous,the rescuing activity of wild-type PrP can be exerted in trans from nearby cells. These results provide important insights into how ΔCR PrP subverts a normal physiological function of PrP(C),and the cellular mechanisms underlying the rescuing process. View Publication

INTRODUCTION: Ischemic stroke is a leading cause of death and disability,but treatment options are severely limited. Cell therapy offers an attractive strategy for regenerating lost tissues and enhancing the endogenous healing process. In this study,we investigated the use of human embryonic stem cell-derived neural precursors as a cell therapy in a murine stroke model.backslashnbackslashnMETHODS: Neural precursors were derived from human embryonic stem cells by using a fully adherent SMAD inhibition protocol employing small molecules. The efficiency of neural induction and the ability of these cells to further differentiate into neurons were assessed by using immunocytochemistry. Whole-cell patch-clamp recording was used to demonstrate the electrophysiological activity of human embryonic stem cell-derived neurons. Neural precursors were transplanted into the core and penumbra regions of a focal ischemic stroke in the barrel cortex of mice. Animals received injections of bromodeoxyuridine to track regeneration. Neural differentiation of the transplanted cells and regenerative markers were measured by using immunohistochemistry. The adhesive removal test was used to determine functional improvement after stroke and intervention.backslashnbackslashnRESULTS: After 11 days of neural induction by using the small-molecule protocol,over 95% of human embryonic stem-derived cells expressed at least one neural marker. Further in vitro differentiation yielded cells that stained for mature neuronal markers and exhibited high-amplitude,repetitive action potentials in response to depolarization. Neuronal differentiation also occurred after transplantation into the ischemic cortex. A greater level of bromodeoxyuridine co-localization with neurons was observed in the penumbra region of animals receiving cell transplantation. Transplantation also improved sensory recovery in transplant animals over that in control animals.backslashnbackslashnCONCLUSIONS: Human embryonic stem cell-derived neural precursors derived by using a highly efficient small-molecule SMAD inhibition protocol can differentiate into electrophysiologically functional neurons in vitro. These cells also differentiate into neurons in vivo,enhance regenerative activities,and improve sensory recovery after ischemic stroke. View Publication

Sickle cell disease (SCD) is the most common human genetic disease which is caused by a single mutation of human β-globin (HBB) gene. The lack of long-term treatment makes the development of reliable cell and gene therapies highly desirable. Disease-specific patient-derived human induced pluripotent stem cells (hiPSCs) have great potential for developing novel cell and gene therapies. With the disease-causing mutations corrected in situ,patient-derived hiPSCs can restore normal cell functions and serve as a renewable autologous cell source for the treatment of genetic disorders. Here we successfully utilized transcription activator-like effector nucleases (TALENs),a recently emerged novel genome editing tool,to correct the SCD mutation in patient-derived hiPSCs. The TALENs we have engineered are highly specific and generate minimal off-target effects. In combination with piggyBac transposon,TALEN-mediated gene targeting leaves no residual ectopic sequences at the site of correction and the corrected hiPSCs retain full pluripotency and a normal karyotype. Our study demonstrates an important first step of using TALENs for the treatment of genetic diseases such as SCD,which represents a significant advance toward hiPSC-based cell and gene therapies. View Publication

Gaucher disease (GD) is caused by insufficient activity of acid $\$-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD,induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs,NPCs,and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition,GD2 neurons showed increased $\$-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons,but intriguingly,those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes,sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings,providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge,this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease. View Publication

OBJECTIVE As an inhibitor of the AhR signalling pathway,StemRegenin 1 (SR1) not only promotes the expansion of CD34+ cells but also increases CD34- cell numbers. These CD34- cells influenced the ex vivo expansion of CD34+ cells. In this work,the effects of periodically removing CD34- cells combined with SR1 addition on the ex vivo expansion and biological functions of HSCs were investigated. MATERIALS AND METHODS CD34- cells were removed periodically with SR1 addition to investigate cell subpopulations,cell expansion,biological functions,expanded cell division mode and supernatant TGF-$\beta$1 contents. RESULTS After 10-day culture,the expansion of CD34+ cells in the CD34- cell removal plus SR1 group was significantly higher than that in the control group and the SR1 group. Moreover,periodically removing CD34- cells with SR1 addition improved the biological function of expanded CD34+ cells and significantly increased the percentage of self-renewal symmetric division of CD34+ cells. In addition,the concentration of total TGF-$\beta$1 and activated TGF-$\beta$1 in the supernatant was significantly lower than those in the control group and the SR1 group. RT-qPCR results showed that the periodic removal of CD34- cells with cooperation from SR1 further reduced the expression of AhR-related genes. CONCLUSIONS Periodic removal of CD34- cells plus cooperation with SR1 improved the expansion of CD34+ cells,maintained better biological function of expanded CD34+ cells and reduced the TGF-$\beta$1 contents by downregulating AhR signalling. View Publication

Spinal cord injury (SCI) is one of the most debilitating injuries,and transplantation of stem cells in a scaffold is a promising strategy for treatment. However,stem cell treatment of SCI has been severely impaired by the increased generation of reactive oxygen species in the lesion microenvironment,which can lead to a high level of stem cell death and dysfunction. Herein,a MnO2 nanoparticle (NP)-dotted hydrogel is prepared through dispersion of MnO2 NPs in a PPFLMLLKGSTR peptide modified hyaluronic acid hydrogel. The peptide-modified hydrogel enables the adhesive growth of mesenchymal stem cells (MSCs) and nerve tissue bridging. The MnO2 NPs alleviate the oxidative environment,thereby effectively improving the viability of MSCs. Transplantation of MSCs in the multifunctional gel generates a significant motor function restoration on a long-span rat spinal cord transection model and induces an in vivo integration as well as neural differentiation of the implanted MSCs,leading to a highly efficient regeneration of central nervous spinal cord tissue. Therefore,the MnO2 NP-dotted hydrogel represents a promising strategy for stem-cell-based therapies of central nervous system diseases through the comprehensive regulation of pathological microenvironment complications. 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

Autoantibodies against envelope (Env) protein encoded by human endogenous retrovirus group K (HERV-K) are prevalent in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE),but it remains unclear which proviruses are responsible for this autoantigen. It also remains poorly understood how the transcription of HERV-K loci is regulated in cells that can produce Env.ResultsWe aligned our neutrophil RNA sequencing data to the new telomere-to-telomere reference genome and found uniquely mapping transcripts from HERV-K101,K102,K104,K108,K109,K117 and ERVK5,of which only K102,K108,and K109 encode an intact Env. Expression of K102 and K108 were higher in SLE than in healthy donors or RA (padj < 0.05). Transcripts from these proviruses increased in response to interferon-α in monocytes and neutrophils from RA patients and healthy donors,but not in SLE,presumably because they have chronically elevated type I interferons in vivo. Indeed,HERV-K expression was significantly higher in SLE patients with high type I interferon gene signature. Tumor necrosis factor-α and other cytokines and TLR ligands also induced HERV-K102 and K108 transcripts. Interferon-α also increased detectable Env protein in monocytes,macrophages,and neutrophils from RA patients. Among the genes for epigenetic silencers of HERV-K,only TRIM28 was significantly decreased in SLE patients with high interferons (padj = 0.00024).ConclusionsOur data establish a role for interferons in maintaining increased HERV-K expression in SLE and suggest that interferons or other cytokines can upregulate HERV-K to similar levels in RA. A transient increase may also accompany normal immune responses,suggesting that endogenous retroviruses may have been co-opted for efficient immune responses.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13100-025-00371-y. View Publication

Myeloproliferative Neoplasms (MPN) are malignancies of hematopoietic stem and progenitor cells (HSPCs) that lead to the overproduction of mature blood cells. These disorders include Essential Thrombocythemia (ET),Polycythemia Vera (PV),and Primary Myelofibrosis (PMF),primarily driven by somatic mutations such as JAK2 V617F . Research indicates that mesenchymal stromal cells (MSCs) support fibrosis in PMF,though their role in ET and PV remains less clear. Furthermore,in vivo studies of ET/PV HSPCs remain a challenge due to low engraftment levels in xenograft models. We employed a 3D scaffold model to create an MPN humanized xenograft mouse model,enabling in vivo functional studies of primary MPN progenitor cells and the supportive role of human MSCs. Using this model,we first demonstrated robust hematopoietic support of healthy (HD) HSPCs by PV and ET MSCs. We then investigated the role of MSCs in sustaining JAK2 V617F mutant cells by using a CRISPR‐Cas9 editing model,along with primary PV and ET HSPCs. Our results showed consistent engraftment of CRISPR‐edited JAK2 V617F mutant HSPCs and PV and ET patient‐derived HSPCs in scaffolds seeded with HD,PV,and ET stroma,providing the first in vivo evidence that PV and ET MSCs can sustain both healthy and MPN‐associated hematopoiesis. Furthermore,HD MSCs were also capable of sustaining PV and ET HSPCs in vivo. Overall,we present the first humanized MPN xenograft model that offers valuable insights into how human BM MSCs interact with JAK2 V617F mutant clones. View Publication

PURPOSE: The purpose of this study was to determine whether histone deacetylase (HDAC) inhibitors (HDACI) such as vorinostat or entinostat (SNDX-275) could increase the lethality of the dual Bcr/Abl-Aurora kinase inhibitor KW-2449 in various Bcr/Abl(+) human leukemia cells,including those resistant to imatinib mesylate (IM). EXPERIMENTAL DESIGN: Bcr/Abl(+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) cells,including those resistant to IM (T315I,E255K),were exposed to KW-2449 in the presence or absence of vorinostat or SNDX-275,after which apoptosis and effects on signaling pathways were examined. In vivo studies combining HDACIs and KW2449 were carried out by using a systemic IM-resistant ALL xenograft model. RESULTS: Coadministration of HDACIs synergistically increased KW-2449 lethality in vitro in multiple CML and Ph(+) ALL cell types including human IM resistant cells (e.g.,BV-173/E255K and Adult/T315I). Combined treatment resulted in inactivation of Bcr/Abl and downstream targets (e.g.,STAT5 and CRKL),as well as increased reactive oxygen species (ROS) generation and DNA damage (γH2A.X). The latter events and cell death were significantly attenuated by free radical scavengers (TBAP). Increased lethality was also observed in primary CD34(+) cells from patients with CML,but not in normal CD34(+) cells. Finally,minimally active vorinostat or SNDX275 doses markedly increased KW2449 antitumor effects and significantly prolonged the survival of murine xenografts bearing IM-resistant ALL cells (BV173/E255K). CONCLUSIONS: HDACIs increase KW-2449 lethality in Bcr/Abl(+) cells in association with inhibition of Bcr/Abl,generation of ROS,and induction of DNA damage. This strategy preferentially targets primary Bcr/Abl(+) hematopoietic cells and exhibits enhanced in vivo activity. Combining KW-2449 with HDACIs warrants attention in IM-resistant Bcr/Abl(+) leukemias. View Publication

OBJECTIVES: Recent data show that Imatinib mesylate (IM) also affects haematopoietic stem cells (HSC),T lymphocytes and dendritic cells that do not harbour constitutively active tyrosine kinases. MATERIALS AND METHODS: We evaluated possible effects of IM on human bone marrow-derived mesenchymal stem cells (MSC) in vitro. RESULTS: Screening the activity of 42 receptor tyrosine kinases revealed an exclusive inhibition of platelet-derived growth factor receptorbeta (PDGFRbeta). Analysis of downstream targets of PDGFRbeta demonstrated IM-mediated reduction of Akt and Erk1/2 phosphorylation. Culture of MSC with IM led to the reversible development of perinuclear multi-vesicular bodies. The proliferation and clonogenicity of MSC were significantly reduced compared to control cultures. IM favoured adipogenic differentiation of MSC whereas osteogenesis was suppressed. The functional deficits described led to a 50% reduction in the support of clonogenic haematopoietic stem cells,cultured for 1 month on a monolayer of MSC with IM. CONCLUSION: In summary,inhibition of PDGFRbeta and downstream Akt and Erk signalling by IM has a significant impact on proliferation and differentiation of human MSC in vitro. View Publication

Human embryonic stem cells (hESC) are capable to give rise to all cell types in the human body during the normal course of development. Therefore,these cells hold a great promise in regenerative cell replacement based therapeutical approaches. However,some controversy exists in literature concerning the ultimate fate of hESC after exposure to genotoxic agents,in particular,regarding the effect of DNA damaging insults on pluripotency of hESC. To comprehensively address this issue,we performed an analysis of the expression of marker genes,associated with pluripotent state of hESC,such as Oct-4,Nanog,Sox-2,SSEA-4,TERT,TRA-1-60 and TRA-1-81 up to 65h after exposure to ionizing radiation (IR) using flow cytometry,immunocytochemistry and quantitative real-time polymerase chain reaction techniques. We show that irradiation with relatively low doses of gamma-radiation (0.2Gy and 1Gy) does not lead to loss of expression of the pluripotency-associated markers in the surviving hESC. While changes in the levels of expression of some of the pluripotency markers were observed at different time points after IR exposure,these alterations were not persistent,and,in most cases,the expression of the pluripotency-associated markers remained significantly higher than that observed in fully differentiated human fibroblasts,and in hESCs differentiated into definitive endodermal lineage. Our data suggest that exposure of hESC to relatively low doses of IR as a model genotoxic agent does not significantly affect pluripotency of the surviving fraction of hESC. View Publication