Stem cells and development最新文献

Exosomes derived from mesenchymal stem cells (MSCs) show therapeutic potential despite limited yield. In contrast, the yield of exosome-mimetic vesicles (EMVs), which share a similar structure and size with exosomes is high. Previous studies have compared their proteomic profiles, and the microRNA (miRNA) expression signatures remain unexplored. EMVs from human bone marrow MSCs were isolated through continuous extrusion and exosomes were isolated from the supernatant via differential ultracentrifugation. miRNA sequencing was performed using high-throughput sequencing, and the miRNA expression profiles of MSC-EMVs, MSC-exosomes, and MSCs were compared. Following the comparison of differentially expressed miRNAs in MSC-EMVs and MSC-exosomes, target gene prediction and functional enrichment analyses were performed. Furthermore, a trend analysis was conducted on the miRNA expression levels in the three groups to further explore the relationship between miRNA expression levels. Our study confirmed that EMVs could be stably produced and that their yield was approximately 100-fold higher than that of exosomes. A total of 763 known miRNAs were identified through comparison using the miRBase library. The miRNAs in EMVs and exosomes overlapped with those in MSCs; however, EMVs shared more miRNAs with the parent cells. Comparative analysis identified 21 upregulated and 17 downregulated miRNAs in EMVs versus exosomes, while trend analysis revealed 108 miRNAs preferentially expressed in MSCs and EMVs. Functional enrichment of differentially expressed miRNAs provides new insights for EMV-based therapies. Importantly, we demonstrated that both MSC-EMVs and MSC-exosomes significantly attenuated LPS-induced inflammation in THP-1 macrophages by modulating cytokine secretion (ELISA), suppressing iNOS expression (immunofluorescence), and inhibiting NF-κB activation (western blot). In an lipopolysaccharide (LPS)-induced acute kidney injury model, both vesicle types effectively reduced renal inflammation and tissue damage (histopathology and protein analysis). Our findings not only present the first comprehensive miRNA profiling comparison between MSC-derived EMVs and exosomes but also validate their comparable anti-inflammatory efficacy, supporting EMVs as a viable high-yield alternative for cell-free therapies.

Therapies aimed at manipulating microvasculature require the ability to generate both blood and lymphatic vessels. Adipose-derived stromal vascular fraction (SVF), consisting of endothelial cells, progenitor cells, pericytes, smooth muscle cells, fibroblasts, and immune cells, has emerged as a heterogeneous cell composition able to promote blood vessel formation and growth, but whether SVF forms lymphatic vessels remains unknown. The objective of this study was to evaluate whether SVF can form lymphatic vessels. SVF was isolated from C57BL/6 mouse inguinal adipose tissue, characterized for prevalence of blood (PECAM+) and lymphatic (Prox1+, Podoplanin+, LYVE-1+) endothelial cells and cultured with avascular mouse mesentery tissues for up to 9 days. The presence of lymphatic endothelial cells in SVF is supported by the percentages of PECAM+ cells that are also positive for lymphatic markers. By day 1 after SVF seeding, cells established PECAM+ segments, and by day 3 cell clusters with segment extensions were observed. At later time points, segments established network of blood vessels. In parallel, a subset of structures positive for lymphatic marker labeling and characterized by a rounded shape (termed "blebs") connected with nearby SVF-derived blood vessel and were changing shape over time. Our findings provoke a new research area focused on the ability for SVF to form lymphatic vessels.

The differentiation of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) into specific subtypes, including ventricular, atrial, conduction, and nodal, remains a significant challenge for in vitro disease modeling and regenerative medicine. While chemical approaches have been explored for subtype specification, these protocols often result in heterogeneous CM populations. In this study, we tested the hypothesis that differential electrical stimulation (ES) can guide/modulate the differentiation of subtype-specific CMs from hiPSCs. By varying stimulation parameters, such as frequency and onset of ES at different developmental time points, we demonstrate that ES alone promotes the differentiation of hiPSC into either ventricular or atrial CMs, without changing any chemical cues. Our results show that lower frequency stimulation earlier in development promotes atrial gene expression, while higher frequency ES later in development promotes ventricular differentiation. These findings were validated by gene expression analysis, immunostaining, and measurement of calcium signaling. This study highlights the potential of ES as a tunable tool for directing CM subtype specification, offering a promising strategy for the generation of pure populations of CM subtypes for use in precision medicine, disease modeling, and regenerative therapies.

Mesenchymal stem/stromal cells (MSCs) possess immunoregulatory capacity, which is enhanced in an inflammatory environment. Participation of extracellular vesicles (EVs) in this function is proposed, as they can transport various immunoregulatory molecules. However, the impact of the inflammatory microenvironment on the load of the different types of EVs released by these cells is not fully known. Therefore, this work analyzes in detail the temporal effect of IFN-γ, alone or in combination with TNF-α (TNF-α + IFN-γ), on the cargo of immunoregulatory molecules (programmed cell death ligand 1 [PD-L1], CD73, and intercellular adhesion molecule 1 [ICAM-1]) in large extracellular vesicles (L-EVs) released by human bone marrow mesenchymal stem cells (BM-MSCs). The presence of these molecules on the surface of L-EVs was determined by flow cytometry. Our results demonstrate that exposing BM-MSCs to TNF-α + IFN-γ for 24 h increases the percentage of PD-L1+ and CD73+ L-EVs. However, if this stimulus persists, the release of L-EVs with an immunoregulatory phenotype (PD-L1+, CD73+, and PD-L1+CD73+) decreases. The impact of pro-inflammatory cytokines on the transport of ICAM-1 by L-EVs is late, since up to 72 h of treatment with IFN-γ or TNF-α + IFN-γ, the percentage of ICAM-1+ L-EVs increases. In contrast, stimulation with IFN-γ for 72 h favors the release of CD73^high and ICAM-1^high L-EVs, but this effect also decreases in the presence of TNF-α. Our study generates novel knowledge about the impact of the inflammatory microenvironment on the cargo composition of L-EVs released by BM-MSCs and demonstrates, for the first time, that the prolonged presence of TNF-α reduces the cargo of immunoregulatory molecules in these structures.

Diabetic nephropathy (DN), recognized as the leading cause of end-stage renal disease globally, necessitates novel therapeutic development. While mesenchymal stromal cells (MSCs) demonstrate therapeutic potential in DN management, their precise mechanisms require systematic elucidation. This study investigated the link between DN and inflammation activation, as well as the pathophysiological significance of MSC-mediated macrophage polarization and podocyte injury repair during this progression. We administered MSCs to streptozotocin-induced diabetic rats via tail vein injection and co-cultured podocytes and MSCs under high-glucose (HG) conditions. Subsequently, we assessed M2-like macrophage polarization and inflammation levels both in vitro and in vivo. In addition, we observed the distribution and homing of MSCs in vivo through ⁸⁹Zr labeling. Our results revealed that HG increased podocyte apoptosis and inflammation in both podocyte and diabetic rats. Treatment with MSCs attenuated inflammation, promoted M2-like macrophage polarization in podocyte under HG conditions as well as in diabetic rats, ultimately ameliorating kidney injury. Importantly, it was observed that MSCs homed to the kidney of DN rats, thereby exerting their therapeutic effects. Collectively, our findings demonstrate that MSCs exhibit renal homing capacity in diabetic kidney and protect podocytes from inflammation by promoting M2 macrophage polarization, thereby establishing MSCs as a promising therapeutic cell-free strategy for DN.

This study aimed to investigate the effects and underlying mechanisms of compressive force on the osteogenic differentiation of human dental follicle stem cells (DFSCs) and to explore its potential role in orthodontically induced inflammatory root resorption (OIIRR). Human DFSCs (hDFSCs) were subjected to a compressive force of 2 g/cm². Western blot and quantitative real-time polymerase chain reaction were used to quantify the expression levels of biglycan (BGN), Runt related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and components of the bone morphogenetic protein (BMP)2/Smad1 signaling pathway in hDFSCs. To elucidate the regulatory role of the BGN/BMP2/Smad1 signaling pathway, a BGN overexpression plasmid and a BMP signaling activator were utilized. In addition, a mouse OIIRR model was established to determine the involvement of the BGN/BMP2/Smad1 signaling axis in vivo. Under compressive force, the mRNA and protein expression levels of ALP, RUNX2, and components of the BGN/BMP2/Smad1 signaling pathway were downregulated. Overexpression of BGN significantly upregulated BMP2 and phosphorylated Smad1 expression (P < 0.05) and enhanced the osteogenic differentiation of hDFSCs. Furthermore, activation of the BMP2/Smad1 signaling pathway using sb4 also reversed the compressive force-induced decline in osteogenic differentiation of hDFSCs. In vivo, the expression levels of the BGN/BMP2/Smad1 signaling axis and the osteogenic markers were significantly reduced on the compressive side of periodontal tissue compared with the control group (P < 0.01). BGN plays a crucial role in the osteogenic differentiation of hDFSCs under compressive force via the BMP2/Smad1 signaling axis and may contribute to the occurrence of OIIRR in mice.

Recently, cell therapies, including chimeric antigen receptor (CAR) modified T cell therapy and mesenchymal stem cell (MSC) therapy, have demonstrated considerable potential for systemic lupus erythematosus (SLE). In this study, a CAR-MSC model was constructed, combining two cell therapies. The structural domains of the CAR were designed by using the anti-CD19 scFv, targeting the CD19 antigen on the surface of B cells and the intracellular region of the interferon-gamma receptor, activating the JAK-STAT1 signaling pathway. Then we screened and identified the most effective structural domain of CAR as CAR1, as it facilitates MSCs to maintain significantly higher levels of JAK2 phosphorylation and IDO expression, as shown by western blot analysis. We also demonstrated CAR1 could be consistently and stably expressed at high levels in MSCs, and CAR1 transduction did not significantly affect the surface antigenic phenotypic criteria of MSCs via flow analysis. Furthermore, immunofluorescence results showed CAR1-MSCs could stably bind CD19 antigen, and they were activated by human CD19 antigen resulting in significantly high JAK2 phosphorylation and IDO expression via western blot analysis following co-culture. Besides, when activated peripheral blood mononuclear cells (PBMCs) were co-cultured with untransduced MSCs (UTD-MSCs) and CAR1-MSCs in vitro, respectively, the results showed that the percentage of activated CD3⁺ T cells and CD19⁺ B cells was both significantly lower after co-culturing. The percentage of activated CD19⁺ B cells was lower in the CAR1-MSCs co-culture group than in the UTD-MSCs co-culture group, whereas the percentage of activated CD3⁺ T cells was similar in the two co-culture groups. This suggests that CAR1 increased the inhibitory ability of MSCs on activated CD19⁺ B cells and had no significant effect on the ability of MSCs to inhibit activated CD3⁺ T cells. In conclusion, CAR1-MSCs were successfully constructed and demonstrated the ability to enhance the inhibitory effect of MSCs on activated human CD19⁺ B cells, facilitating SLE therapy.

Stem cell therapy holds promise for Parkinson's disease (PD). To identify optimal stem cell regimens in PD mouse models and inform translational research, we conducted a network meta-analysis (NMA). Specifically, we systematically searched for studies on stem cell therapy in PD mouse models up to September 2024 in PubMed, Embase, Scopus, Web of Science, China National Knowledge Infrastructure, WANFANG, and VIP. Based on the data collected, we conducted an NMA using GeMTC-0.14.3 software. The results of traditional meta-analysis of 148 studies demonstrated superior efficacy of most interventions versus controls at biweekly intervals (2-8 weeks post-treatment), with neural stem cells engineered with neurotrophic factors (NSC-NFs) showing the lowest weighted mean difference, indicating optimal therapeutic effect. NMA demonstrated that NF-engineered NSC therapy ranked the highest at biweekly time points (2-8 weeks post-treatment). Doses of 10⁵ cells showed optimal efficacy at 2, 4, and 6 weeks, peaking within this range, whereas doses of 10³ cells showed the best efficacy at 8 weeks. Medial forebrain bundle (MFB) administration showed superior efficacy at weeks 2 and 8, while striatum (STR) infusion showed greater therapeutic effects at weeks 4 and 6, with both approaches significantly outperforming nasal and intravenous delivery at all evaluated time points (2, 4, 6, and 8 weeks). Taken together, these results suggest that NSC-NF (dosage of 10⁵) delivered via MFB (at 2 and 8 weeks) or STR (at 4 and 6 weeks) may represent the optimal strategy. It provides important guidance for optimizing preclinical and clinical trial designs and offers valuable insights for clinical translation.

Premature ovarian failure (POF) is a significant reproductive disorder characterized by the loss of ovarian function, leading to infertility and endocrine disruption. Hormone replacement therapy (HRT) remains the most commonly used clinical treatment for POF. However, in patients with a history of ovarian or breast cancer, HRT poses significant risks, necessitating the development of alternative approaches. Stem cell-based therapy has emerged as a promising option for treating female infertility disorders such as POF. This study aimed to evaluate the therapeutic effects of ovarian granulosa-like cells (OGLCs) derived from Wharton's jelly-mesenchymal stem cells (WJ-MSCs) in a POF mouse model. WJ-MSCs were successfully differentiated into OGLCs using combination with a growth factor cocktails, as confirmed by the significant upregulation of granulosa cell-specific markers (P < 0.01). To assess their therapeutic potential, POF was induced in female mice using cyclophosphamide and busulfan, and OGLCs were injected into the ovaries. After 3 weeks, vaginal smear analysis revealed restoration of estrus cycle in OGLC-treated mice. Enzyme-linked immunosorbent assay analysis demonstrated the recovery of serum 17β-estradiol and follicle-stimulating hormone levels (P < 0.05), while histological staining confirmed increased follicular development and restoration of ovarian structure. Furthermore, real-time quantitative polymerase chain reaction analysis showed a significant upregulation of genes related to follicular development and primordial follicle activation, including downstream molecules of the mTOR/PI3K pathway, following OGLCs treatment. These findings suggest that OGLCs possess a strong potential for restoring ovarian function in POF. This study provides evidence supporting the use of OGLCs as a novel cell-based therapeutic approach for female reproductive diseases.