STEM CELLS最新文献

Background: The function and mechanism of pri-miRNA N6-methyladenosine (m6A) modification in promoting miRNA maturation and regulating osteoblastic differentiation are not fully understood. The aim of this study was to investigate the role and regulatory mechanism of miRNA shear maturation regulated by methyltransferase like 3 (METTL3) in human adipose-derived stem cell (hASC) osteogenesis.

Methods and results: First, we found METTL3 promoted osteogenesis both in vivo and in vitro. Subsequently, 3 pri-miRNAs with the most significant methylated peaks were identified through methylated RNA immunoprecipitation sequencing. Through quantitative real-time polymerase chain reaction, MeRIP-qPCR, and co-immunoprecipitation, it was determined that METTL3 promoted the processing of hsa-miR-4526 by mediating pri-miR4526/5190 m6A modification. Subsequent in vivo and in vitro experiments demonstrated that hsa-miR-4526 promoted osteogenesis. Dual luciferase reporter assay was performed to verify that hsa-miR-4526 regulated osteogenic differentiation through TUBB3. It was found that TUBB3 can inhibit hASC osteogenesis. Further rescue experiments confirmed that METTL3 inhibited TUBB3 expression through hsa-miR-4526, thereby regulating osteogenic differentiation. RNA-seq revealed that TUBB3 may be involved in cell metabolism, calcium enrichment, osteoclast differentiation, and other pathways.

Conclusion: Our study is the first to investigate the mechanism of pri-miRNA m6A modification in regulating hASC osteogenesis, presenting a novel idea and method for repairing bone defects.

Myocardial infarction can lead to the loss of billions of cardiomyocytes, and while cell-based therapies are an option, immature nature of in vitro-generated human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) is a roadblock to their development. Existing iPSC differentiation protocols don't go beyond producing fetal iCMs. Recently, adult extracellular matrix (ECM) was shown to retain tissue memory and have some success driving tissue-specific differentiation in unspecified cells in various organ systems. Therefore, we focused on investigating the effect of adult human heart-derived extracellular matrix (ECM) on iPSC cardiac differentiation and subsequent maturation. By preconditioning iPSCs with ECM, we tested whether creating cardiac environments around iPSCs would drive iPSCs toward cardiac fate and which ECM components might be involved. We report novel high- and low-abundance proteomes of young, adult, and aged human hearts, with relative abundances to total proteins and each other. We found that adult ECM had extracellular galactin-1, fibronectin, fibrillins, and perlecan (HSPG2) which are implicated in normal heart development. We also showed preconditioning iPSCs with adult cardiac ECM resulted in enhanced cardiac differentiation, yielding iCMs with higher functional maturity, more developed mitochondrial network and coverage, enhanced metabolic maturity, and shift towards more energetic profile. These findings demonstrate the potential use of cardiac ECM in iCM maturation and as a promising strategy for developing iCM-based therapies, disease modeling, and drug screening studies. Upon manipulating ECM, we concluded that the beneficial effects observed were not solely due to the ECM proteins, which might be related to the decorative units attached.

Background: When pigs underwent apical resection (AR) on postnatal day (P) 1 (ARP1) followed by myocardial infarction (MI) on P28, the hearts had little evidence of scarring; meanwhile, hearts underwent MI on P28 without ARP1 showed large infarcts on P56; and the improvement of ARP1 hearts was driven primarily by cardiomyocyte proliferation. AR and MI were performed ~5 mm (AR) and ~20 mm (MI) above the heart apex; thus, we hypothesize that ARP1 preserved the cardiomyocytes cell-cycle throughout the left ventricle, rather than only near the resection site.

Methods: Sections of cardiac tissue were collected from the left ventricle of uninjured pigs and from both the border zone (BZ) of AR and uninjured regions (remote zone, [RZ]) in ARP1 hearts. Cardiomyocyte proliferation was evaluated via immunofluorescence analysis of phosphorylated histone 3 [PH3] and symmetric Aurora B (sAuB). Single nucleus RNA sequencing (snRNAseq) data collected from the hearts of fetal pigs, uninjured pigs, and the BZ and RZ of ARP1 pigs was evaluated via our cell-cycle-specific autoencoder to identify proliferating cardiomyocytes.

Results: Cardiomyocyte PH3 and sAuB expression, and percentage of proliferating cardiomyocytes in snRNA data was significantly more common in both BZ and RZ of ARP1 than uninjured hearts but did not differ significantly between the ARP1-BZ and ARP1-RZ at any time point. Heat shock proteins HSPA5 and HSP90B1 were overexpressed at both ARP1-BZ and ARP1-RZ. In AC16 cell, overexpression (and knockdown) of HSPA5-HSP90B1 increased (and decrease) cell-cycle activity.

Conclusion: ARP1 preserved proliferative capacity of cardiomyocytes located throughout the left ventricle.

Non-lymphoid immunoregulatory cells, including mesenchymal stem cells (MSCs), myeloid-derived suppressor cells (MDSCs), regulatory macrophages (Mregs), and tolerogenic dendritic cells (Tol-DCs), play critical roles in maintaining immune homeostasis. However, their therapeutic application in autoimmune diseases and graft-versus-host disease (GVHD) has received comparatively less attention. Induced pluripotent stem cells (iPSCs) offer a promising platform for cell engineering, enabling superior quality control, scalable production, and large-scale in vitro expansion of iPSC-derived non-lymphoid immunoregulatory cells. These advances pave the way for their broader application in autoimmune disease and GVHD therapy. Recent innovations in iPSC differentiation protocols have facilitated the generation of these cell types with functional characteristics akin to their primary counterparts. This review explores the unique features and generation processes of iPSC-derived non-lymphoid immunoregulatory cells, their therapeutic potential in GVHD and autoimmune disease, and their progress toward clinical translation. It emphasizes the phenotypic and functional diversity within each cell type and their distinct effects on disease modulation. Despite these advancements, challenges persist in optimizing differentiation efficiency, ensuring functional stability, and bridging the gap to clinical application. By synthesizing current methodologies, preclinical findings, and translational efforts, this review underscores the transformative potential of iPSC-derived non-lymphoid immunoregulatory cells in advancing cell-based therapies for alloimmune and autoimmune diseases.

Over the last 2 decades, research has increasingly focused on cancer stem cells (CSCs), considered responsible for tumor formation, resistance to therapies, and relapse. The traditional "static" CSC model used to describe tumor heterogeneity has been challenged by the evidence of CSC dynamic nature and plasticity. A comprehensive understanding of the mechanisms underlying this plasticity, and the capacity to unambiguously identify cancer markers to precisely target CSCs are crucial aspects for advancing cancer research and introducing more effective treatment strategies. In this context, Raman spectroscopy (RS) and specific Raman schemes, including CARS, SRS, SERS, have emerged as innovative tools for molecular analyses both in vitro and in vivo. In fact, these techniques have demonstrated considerable potential in the field of cancer detection, as well as in intraoperative settings, thanks to their label-free nature and minimal invasiveness. However, the RS integration in pre-clinical and clinical applications, particularly in the CSC field, remains limited. This review provides a concise overview of the historical development of RS and its advantages. Then, after introducing the CSC features and the challenges in targeting them with traditional methods, we review and discuss the current literature about the application of RS for revealing and characterizing CSCs and their inherent plasticity, including a brief paragraph about the integration of artificial intelligence with RS. By providing the possibility to better characterize the cellular diversity in their microenvironment, RS could revolutionize current diagnostic and therapeutic approaches, enabling early identification of CSCs and facilitating the development of personalized treatment strategies.

Pluripotent stem cells provide opportunities for treating injuries and previously incurable diseases. A major concern is the immunogenicity of stem cells and their progeny. Here, we have dissected the molecular mechanisms that allow natural killer (NK) cells to respond to human pluripotent stem cells, investigating a wide selection of activating and inhibitory NK-cell receptors and their ligands. Reporter cells expressing the activating receptor NKG2D responded strongly to embryonic stem (ES) cell lines and induced pluripotent stem (iPS) cell lines, whereas reporter cells expressing the activating receptors NKp30, NKp46, KIR2DS1, KIR2DS2, and KIR2DS4 did not respond. Human ES and iPS cells invariably expressed several ligands for NKG2D. Expression of HLA-C and HLA-E was lacking or low, insufficient to trigger reporter cells expressing the inhibitory receptors KIR2DL1, -2DL2, or -2DL3. Similar results were obtained for the pluripotent embryonic carcinoma cell lines NTERA-2 and 2102Ep, and also iPS-cell-derived neural progenitor cells. Importantly, neural progenitor cells and iPS-cell-derived motoneurons also expressed B7H6, the ligand for the activating receptor NKp30. In line with these observations, IL-2-stimulated NK cells showed robust cytotoxic responses to ES and iPS cells as well as to iPS-cell-derived motoneurons. No significant differences in cytotoxicity levels were observed between KIR/HLA matched and mismatched combinations of NK cells and pluripotent targets. Together, these data indicate that pluripotent stem cells and their neural progeny are targets for NK-cell killing both by failing to sufficiently express ligands for inhibitory receptors and by expression of ligands for activating receptors.

Aims: Bone marrow mononuclear cells (BM-MNCs) are a rich source of hematopoietic stem cells that have been widely used in experimental therapies for patients with various diseases, including fractures. Activation of angiogenesis is believed to be one of the major modes of action of BM-MNCs; however, the essential mechanism by which BM-MNCs activate angiogenesis remains elusive. This study aimed to demonstrate that BM-MNCs promote bone healing by enhancing angiogenesis through direct cell-to-cell interactions via gap junctions, in addition to a previously reported method.

Methods: Using a murine fracture model, we aimed to elucidate the relationship between gap junction-mediated cell-to-cell interactions and enhanced fracture healing after BM-MNC transplantation. We evaluated the transfer of substances from BM-MNCs to vascular endothelial cells and osteoblasts in the tissues surrounding the fracture site and assessed the effects of BM-MNC transplantation on bone healing, angiogenesis, and osteogenesis.

Results: Bone marrow mononuclear cells transferred substances to vascular endothelial cells and osteoblasts in the tissues surrounding the fracture site. Moreover, BM-MNC transplantation promoted bone healing via gap junction-mediated cell-to-cell interactions, accelerating both angiogenesis and osteogenesis.

Conclusions: Our findings provide a novel understanding of fracture healing mechanisms and suggest that BM-MNC transplantation enhances bone healing through gap junction-mediated cell-to-cell interactions, contributing to the development of regenerative medicine strategies targeting bone repair.

Heart disease, particularly resulting from myocardial infarction (MI), continues to be a leading cause of mortality, largely due to the limited regenerative capacity of the human heart. Current therapeutic approaches seek to generate new cardiomyocytes from alternative sources. Direct cardiac reprogramming, which converts fibroblasts into induced cardiomyocytes (iCMs), offers a promising alternative by enabling in situ cardiac regeneration and minimizing tumorigenesis concerns. Here we review recent advancements in the understanding of transcriptional and epigenetic mechanisms underlying cardiac reprogramming, with a focus on key early-stage molecular events, including epigenetic barriers and regulatory mechanisms that facilitate reprogramming. Despite substantial progress, human cardiac fibroblast reprogramming and iCM maturation remain areas for further exploration. We also discuss the combinatorial roles of reprogramming factors in governing transcriptional and epigenetic changes. This review consolidates current knowledge and proposes future directions for promoting the translational potential of cardiac reprogramming techniques.

Cell replacement therapies using human pluripotent stem cell-derived ventral midbrain (VM) dopaminergic (DA) progenitors are currently in clinical trials for treatment of Parkinson's disease (PD). Recapitulating developmental patterning cues, such as fibroblast growth factor 8 (FGF8), secreted at the midbrain-hindbrain boundary (MHB), is critical for the in vitro production of authentic VM DA progenitors. Here, we explored the application of alternative MHB-secreted FGF-family members, FGF17 and FGF18, for VM DA progenitor patterning. We show that while FGF17 and FGF18 both recapitulated VM DA progenitor patterning events, FGF17 induced expression of key VM DA progenitor markers at higher levels than FGF8 and transplanted FGF17-patterned progenitors fully reversed motor deficits in a rat PD model. Early activation of the cAMP pathway mimicked FGF17-induced patterning, although strong cAMP activation came at the expense of EN1 expression. In summary, we identified FGF17 as a promising alternative FGF candidate for robust VM DA progenitor patterning.

Pluripotent stem cell-derived retinal organoids (ROs) have been investigated for applications in regenerative medicine, retinal disease models, and compound safety evaluation. Although the development of 3D organoids has provided novel opportunities for innovation, some unresolved limitations continue to exist in organoid research; the passive diffusion of oxygen and nutrients limits the growth and functional gain of organoids. Vascularization may circumvent these problems because it allows oxygen and nutrients to enter the organoid core. In the present study, we generate the vascularized retinal organoids (vROs) from healthy human induced pluripotent stem cells. vROs are created from ROs by co-culturing them with vascular organoid (VO)-derived vascular endothelial cells/pericytes. The expression of mature neuronal markers is markedly higher in the vROs than in the ROs. When vROs are cultured under diabetic conditions, their size and the number of retinal ganglion cells are significantly decreased. In conclusion, the co-culture of ROs with VO-derived cells enables the production of ROs with vascular-like structures, and the vROs respond to severe diabetic retinopathy conditions. In summary, our findings underscore the potential of vROs as invaluable tools for elucidating disease mechanisms and screening therapeutic interventions for retinal vascular disorders, thereby paving the way for personalized medicine approaches in ophthalmology.