Stem Cell Research & Therapy最新文献

Background: Dental pulp stem cells (DPSCs) have acquired noteworthy attention for their application in treating ischemic diseases and facilitating tissue regeneration. However, the host's immune response following allogenic DPSC transplantation often handicaps the long-term survival of transplanted cells, thereby limiting the application of DPSCs in cell therapy. This study aims to investigate whether genetic modification can alleviate the immunogenicity of DPSCs.

Methods: Beta 2-microglobulin (B2M) and the class II histocompatibility complex transactivator (CIITA) were individually knocked down in DPSCs by lentiviral particles encoding short hairpin (sh) RNAs. The self-renewal capacity and pluripotency of DPSCs-shB2M (B2M silenced DPSCs) and DPSCs-shCIITA (CIITA silenced DPSCs) were evaluated by CCK8 and differentiation assays including osteogenesis, adipogenesis, and neurogenesis. The expression of HLA-I and HLA-II in DPSCs-shB2M and DPSCs-shCIITA after IFN-γ treatment were analyzed by western blotting, immunofluorescence, and flow cytometry. The function of genetically modified cells was assessed by leukocyte-mediated cytotoxicity and T-cell proliferation assays.

Results: Western blotting, immunofluorescence, and flow cytometry revealed that DPSCs-shB2M and DPSCs-shCIITA exhibited impaired IFN-γ inducible HLA-I and HLA-II expression. There were no significant differences in the self-renewal capacity and pluripotency among DPSCs-shB2M, DPSCs-shCIITA, and control groups (p > 0.05). Lower leukocyte-mediated cytotoxicity and higher cell survival rates were found in DPSCs-shB2M and DPSCs-shCIITA groups compared to the control (p < 0.05). T cell proliferation was significantly inhibited in both DPSCs-shB2M and DPSCs-shCIITA groups (p < 0.05).

Conclusion: Genetic knockdown of B2M or CIITA in DPSCs substantially reduced their immunogenicity without compromising their stemness, thereby broadening the clinical application of DPSCs in cell therapy and tissue regeneration.

Background: The interaction between mesenchymal stem cells (MSC) and Glioblastoma (GBM), although potentially of the highest importance, is ill-understood. This is due, in part, to the lack of relevant experimental models. The similarity between the in vitro situations and the in vivo situation can be improved by 3D co-culture as it reproduces key cell-cell interactions between the tumor microenvironment (TME) and cancer cells.

Methods: MSC Can acquired characteristics of cancer associated fibroblasts (CAF) by being cultured with conditioned medium from GBM cultures and thus are called MSC^CAF. We co Cultured MSC^CAF with patient derived GBM in a scaffold 3D bioprinted model. We studied the response to current GBM therapy (e.g. Temozolomide + /Radiation) on the co cultures by bulk transcriptomic (RNA Seq) and epigenetic (ATAC Seq) analyses RESULTS: The transcriptomic modifications induced by standard GBM treatment in bioprinted scaffolds of mono- or co-cultures of GBM ± MSC can be analyzed. We found that mitochondrial encoded OXPHOS genes are overexpressed under these conditions and are modified by both co-culture and treatment (chemotherapy ± radiation). We have identified two new markers of MSC/GBM interactions, one epigenetically regulated (i.e. TREM-1) associated with an increased overall survival in GBM patients and another implicated in post-transcriptional regulation (i.e. the long non-coding RNA, miR3681HG), which is associated with a reduced overall survival in GBM patients.

Background: The secretome of primary bovine mammosphere-derived epithelial cells (MDECs) has been shown to exert antimicrobial, regenerative, and immunomodulatory properties in vitro, which warrants its study as a potential biologic treatment with the potential to be translated to human medicine. Currently, the use of the MDEC secretome as a therapy is constrained by the limited life span of primary cell cultures and the decrease of secretome potency over cell passages.

Methods: To address these limitations, early-passage bovine MDECs were immortalized using hTERT, a human telomerase reverse transcriptase. The primary and immortal MDECs were compared morphologically, transcriptomically, and phenotypically. The functional properties and proteomic profiles of the secretome of both cell lines were evaluated and compared. All experiments were performed with both low and high passage cell cultures.

Results: We confirmed through in vitro experiments that the secretome of immortalized MDECs, unlike that of primary cells, maintained antimicrobial and pro-migratory properties over passages, while pro-angiogenic effects of the secretome from both primary and immortalized MDECs were lost when the cells reached high passage. The secretome from primary and immortalized MDECs, at low and high passages exerted immunomodulatory effects on neutrophils in vitro.

Conclusions: High passage immortalized MDECs retain a bioactive secretome with antimicrobial, regenerative, and immunomodulatory properties, suggesting they may serve as a consistent cell source for therapeutic use.

Background: Injection of autologous adipose-derived regenerative cells (ADRCs) combined with lipotransfer has been suggested to alleviate symptoms in diseases including breast cancer-related lymphedema (BCRL). We recently performed a randomized controlled trial injecting lipoaspirate with ADRCs into the axilla of BCRL patients, and here we aimed in the intervention group to define in an unbiased fashion whether ADRC injection was linked to the clinical outcome.

Methods: 39 BCRL patients received lipotransfer assisted with autologous ADRCs (4.20 × 10⁷ ± 1.75 × 10⁷ cells) whereas 41 BCRL patients were included for placebo treatment. At 12 month follow-up, we assessed quality of life, lymphangiography, and bioimpedance enclosing 59 outcome parameters. Multifactorial analysis of clinical outcomes was used to define responders and non-responders to the intervention, and collected ADRCs from these patient groups were analyzed by single cell RNA sequencing (scRNAseq).

Results: Unbiased multifactorial analysis ranked and defined the clinical outcomes (Sf36 physical change, L-Dex Lymph Change, ICG mdanderson change) with the highest effect on BCRL patients. The 10 patients with the highest- and lowest effect (five responders and five non-responders) were included in the study. No difference between non-responders and responders were observed for injected ADRC number/size/viability (p > 0.05). In scRNAseq, we did not find any major difference (p > 0.05) between groups in ADRC composition regarding adipose derived stem cells, endothelial-, smooth muscle-, T-, B-, mast cells as well as macrophages, which was verified by flow cytometry. Differential subcluster gene expression between groups were for 92.5% of genes, including those encoding secretory proteins, below the threshold of 1.5, and thus neglible. Together this suggested that the ADRC phenotype was indistinguishable between BCRL responders and non-responders to the intervention.

Conclusion: Our data suggest that the ADRC injection and ADRC phenotype or heterogeneity have no effect on the clinical outcomes on BCRL, and ADRC assisted lipotranfer for BCRL should therefore not be considered currently.

Background: Chronic cerebral ischemia (CCI) is a significant health issue characterized by hypoperfusion due to damage or occlusion of the cerebral or carotid arteries. CCI may lead to progressive cognitive impairment that is considered as a prelude to neurodegenerative diseases, including dementia and Alzheimer's disease (AD). Endothelial progenitor cells (EPCs) have been implicated in vascular repair in ischemic cerebrovascular diseases, primarily by differentiating into endothelial cells (ECs) or through paracrine effects. However, the clinical transplantation of stem cell therapies remains limited. In this study, we investigated the effects of EPC-derived conditioned medium (EPC-CM) on the impaired vasculature and neurological function in a rodent model of CCI and the mechanism involved.

Methods: EPC-CM was analyzed by cytokine array to identify key factors involved in angiogenesis and cellular senescence. The effects and mechanism of the candidate factors in the EPC-CM were validated in vitro using oxygen-glucose deprivation (OGD)-injured ECs and EPCs. The therapeutic effects of EPC-CM and the identified key factor were further examined in a rat model of CCI, which was induced by bilateral internal carotid artery ligation (BICAL). EPC-CM was administered via intracisternal injection one week post BICAL. The cerebral microvasculature and neurobehavior of the rats were examined three weeks after BICAL.

Results: Macrophage migration inhibitory factor (MIF) was identified as a key factor in the EPC-CM. Recombinant MIF protein promoted angiogenesis and prevented senescence in the injured EPCs and ECs. The effect was similar to that of the EPC-CM. These therapeutic effects were diminished when the EPC-CM was co-treated with MIF-specific antibody (Ab). Additionally, the vascular, motor, and cognitive improvements observed in the BICAL rats treated with EPC-CM were abolished by co-treated with MIF Ab. Furthermore, we found MIF promoted angiogenesis and anti-senescence via activating the AKT pathway. Inhibition of the AKT pathway diminished the protective effects of MIF in the in vitro study.

Conclusions: We demonstrated that EPC-CM protected the brain from chronic ischemic injury and promoted functional recovery through MIF-mediated AKT pathway. These findings suggest EPC-CM holds potential as a novel cell-free therapeutic approach for treating CCI through the actions of MIF.

Background: Although mesenchymal stem cells (MSCs) have been proven to be appropriate candidates for the treatment of AKI-CKD, their efficacy is limited and variable. Astilbin (AST) had a protective effect on MSCs from oxidative stress via ROS-scavenging, however, whether it can improve MSCs' renoprotection and the underlying mechanism need to be elucidated.

Methods: AST-pretreated MSCs were administered intravenously into the ischemia-reperfusion injury mice models and the renal function, pathological changes and inflammation. Were evaluated. In addition, DARTS, molecular docking, surface plasma resonance(SPR), dual-luciferase reporter gene assay and the ChIP-PCR were utilized to explore the potential signaling pathways through which AST exert renal protective effects on MSCs.

Results: AST-pretreated MSCs markedly improved kidney function, reduced kidney pathological injury and inflammation in AKI and AKI-CKD mice. RNA-seq results showed that PTGS2 related pathway was significantly up-regulated in MSCs after AST pretreatment. DARTS assay, molecular docking and SPR assay revealed that AST could bind with the transcriptional factor of Kruppel-Like Factor 4(KLF4) protein. The promoter of PTGS2 had the binding and transcriptional activation by KLF4. Furthermore, AST pretreatment promoted the secretion of PGE2 in MSCs. And then the westren blot results showed that the protein levels of CD163 and CD206 were upregulated after coculture in AST-pretreated MSCs, indicating that the polarization of RAW264.7 cells towards M2-like macrophages was induced. Knockdown of PTGS2 reversed the ability of AST-pretreated MSCs in converting macrophages to M2 phenotype and reducing their therapeutic effects on AKI-CKD mice.

Conclusion: AST pretreatment enhances the efficacy of MSCs on AKI and AKI-CKD mice by inducing of M2-like phenotype polarization in macrophages through the PTGS2-mediated pathway. This approach not only provides a novel strategy to strengthen the capability of MSCs but also helps elucidate the beneficial effects of the Chinese herbal medicine AST.

Background: Hypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal disability and mortality. Although intensive studies and therapeutic approaches, there are limited restorative treatments till now. Human embryonic stem cell (hESCs)-derived cortical neural progenitors have shown great potentials in ischemic stroke in adult brain. However, it is unclear whether they are feasible for cortical reconstruction in immature brain with hypoxic-ischemic encephalopathy.

Methods: By using embryonic body (EB) neural differentiation method combined with DAPT pre-treatment and quantitative cell transplantation, human cortical neuroblasts were obtained and transplanted into the cortex of hypoxic-ischemic injured brain with different dosages 2 weeks after surgery. Then, immunostaining, whole-cell patch clamp recordings and behavioral testing were applied to explore the graft survival and proliferation, fate commitment of cortical neuroblasts in vitro, neural circuit reconstruction and the therapeutic effects of cortical neuroblasts in HIE brain.

Results: Transplantation of human cortical neural progenitor cells (hCNPs) in HIE-injured cortex exhibited long-term graft overgrowth. DAPT pre-treatment successfully synchronized hCNPs from different developmental stages (day 17, day 21, day 28) to deep layer cortical neuroblasts which survived well in HIE injured brain and greatly prevented graft overgrowth after transplantation. Importantly, the cortical neuroblasts primarily differentiated into deep-layer cortical neurons and extended long axons to their projection targets, such as the cortex, striatum, thalamus, and internal capsule in both ipsilateral and contralateral HIE-injured brain. The transplanted cortical neurons established synapses with host cortical neurons and exhibited spontaneous excitatory or inhibitory post-synaptic currents (sEPSCs or sIPSCs) five months post-transplantation. Rotarod and open field tests showed greatly improved animal behavior by intra-cortex transplantation of deep layer cortical neuroblasts in HIE injured brain.

Conclusions: Transplanted hESCs derived cortical neuroblasts survive, project to endogenous targets, and integrate into host cortical neural circuits to rescue animal behavior in the HIE-injured brain without graft overgrowth, providing a novel and safe cell replacement strategy for the future treatment of HIE.

Background: Hair follicles (HFs) are dynamic structures which are readily accessible within the skin that contain various pools of stem cells with broad regenerative potential, such as dermal papilla cells (DPCs), dermal sheath cells, and epithelial HF stem cells. DPCs act as signalling centres for HF regeneration. The current method for isolating human DPCs are inefficient. These methods struggle to obtain freshly isolated original DPCs and do not maintain the characteristics of DPCs effectively.

Methods: In this study, two simple but more efficient methods were explored. Force-triggered density gradient sedimentation (FDGS) and cocktail enzyme digestion treatment (CEDT) were used to isolate purified DP spheres from human HFs, obtaining purified freshly isolated original DPCs from DP spheres. The expression profiles of isolated DPCs were tested, and gene expression of DPC-specific markers were analyzed using immunofluorescence staining, RT-qPCR and western blot.

Results: The 10% Ficoll PM400 was determined as the optimal concentration for FDGS method. Primary DPCs, DSCs and HFSCs were isolated simultaneously using the FDGS and CEDT method. The expression profiles of fresh DPCs isolated using the FDGS and CEDT methods were similar to those of traditionally isolated DPCs. DP-specific markers were expressed at significantly higher levels in freshly isolated DPCs than in traditionally isolated DPCs.

Conclusions: Compared to traditional methods, the presented laboratory protocols were able to isolate fresh DPCs with high efficiency, thereby improving their research potential.

Background: The efficiency of mesenchymal stem cells (MSCs) in treating myocardial infarction (MI) remains inconsistent, which limits their therapeutic applications. Therefore, exploring the mechanism for the inconsistent efficacy of MSCs and identification the criteria for screening MSCs are important for improving the efficiency of MSCs.

Methods: Mouse model after MI was utilized to test the role of MSCs from different donors and the functional subpopulation in improving cardiac function. Heterogeneity of MSCs was identified using single-cell RNA sequencing (scRNA-seq) of MSC-GY. GSEA and Scissor analyses were used to find the functional subpopulations of MSCs that promote angiogenesis. The role of functional subpopulations in promoting angiogenesis was verified by detecting the secretory proteins, the ratio of N-CADHERIN⁺/CD168^- subpopulations in MSCs, and the tube formation, migration, and proliferation of HUVECs after treatment with conditional medium (CM) derived from different MSCs.

Results: We found that umbilical cord-derived MSCs (UC-MSCs) from different donors have varied therapeutic efficacy in MI mice and UC-MSCs with higher therapeutic effectiveness exhibited the most potent pro-angiogenic effects by secreting elevated levels of angiogenesis-related proteins, such as MYDGF, VEGFA, and FGF2. ScRNA-seq of 10,463 UC-MSCs revealed that the N-CADHERIN⁺/CD168^- subpopulation was closely associated with pro-angiogenic effects, and the ratio of this cell subpopulation was positively correlated with the angiogenic potential of MSCs. We also found that the N-CADHERIN⁺/CD168^- subpopulation was the functional subpopulation of MSCs in improving cardiac function of MI mice.

Conclusions: Our study identified that the N-CADHERIN⁺/CD168^- subpopulation was the functional subpopulation of MSCs in treating MI, which was essential for the development and utilization of MSCs in MI treatment.

Background: Although stem cell transplantation is a promising approach for the treatment of myocardial infarction (MI), there are still some problems faced such as the low survival rate of stem cells. Here, we investigated the role of Notoginsenoside R1 (NGR1) pretreatment in improving the effects of neonatal rat bone marrow mesenchymal stem cell (MSC) transplantation for treatment of MI.

Methods: Cardiac functions were detected by echocardiography and the myocardial infarct size was determined by Masson's trichrome staining in a rat model of MI. The cardioprotective effects of NGR1/LY294002 co-pretreated MSCs was evaluated to explore the underlying mechanism. The angiogenesis was determined by vWF and α-SMA immunofluorescence staining and cell apoptosis was detected by TUNEL. In vitro, the effects of NGR1 on stem cell proliferation was examined by CCK-8 and levels of P-Akt, P-CREB, P-FoxO1 were detected by western blot. Apoptosis, ROS content, and cytokine levels were examined by DAPI and TUNEL staining, a ROS assay kit, and ELISA, respectively.

Results: NGR1 elevated the therapeutic effect of MSC transplantation on infarction by preserving cardiac function, increasing angiogenesis and expressions of IGF-1, VEGF, and SDF-1, and reducing cell apoptosis, whereas the addition of LY294002 prior to NGR1 treatment significantly counteracted the foregoing effects of NGR1. NGR1 pretreatment and SC79 pretreatment were similar in that both significantly increased P-Akt and P-FoxO1 levels in MSC and did not affect P-CREB levels. Besides, both NGR1 and SC79 promoted VEGF, SCF and bFGF levels in MSC cultures, and significantly reduced ROS accumulation and the attenuated cell apoptosis in MSC triggered by H₂O₂. Similarly, addition of LY294002 before NGR1 treatment significantly counteracted the aforementioned effects of NGR1 in vitro.

Conclusions: NGR1 pretreatment enhances the effect of MSC transplantation for treatment of MI through paracrine signaling, and the mechanism underlying this effect may be associated with PI3K/Akt/FoxO1 signaling pathways.