Ablation of Wnt signaling in bone marrow stromal cells overcomes microenvironment-mediated drug resistance in acute myeloid leukemia.

The survival of leukemic cells is significantly influenced by the bone marrow microenvironment, where stromal cells play a crucial role. While there has been substantial progress in understanding the mechanisms and pathways involved in this crosstalk, limited data exist regarding the impact of leukemic cells on bone marrow stromal cells and their potential role in drug resistance. In this study, we identify that leukemic cells prime bone marrow stromal cells towards osteoblast lineage and promote drug resistance. This biased differentiation of stroma is accompanied by dysregulation of the canonical Wnt signaling pathway. Inhibition of Wnt signaling in stroma reversed the drug resistance in leukemic cells, which was further validated in leukemic mice models. This study evaluates the critical role of leukemic cells in establishing a drug-resistant niche by influencing the bone marrow stromal cells. Additionally, it highlights the potential of targeting Wnt signaling in the stroma by repurposing an anthelmintic drug to overcome the microenvironment-mediated drug resistance.

[Latest Findings on the Role of RUNX1 in Bone Development and Disorders]. / RUNX1åœ¨éª¨å‘è‚²åŠéª¨ç›¸å ³ç–¾ç— ä¸­çš„ä½œç”¨ç ”ç©¶è¿›å±•.

Runt-related transcription factor (RUNX1) is a transcription factor closely involved in hematopoiesis. RUNX1 gene mutation plays an essential pathogenic role in the initiation and development of hematological tumors, especially in acute myeloid leukemia. Recent studies have shown that RUNX1 is also involved in the regulation of bone development and the pathological progression of bone-related diseases. RUNX1 promotes the differentiation of mesenchymal stem cells into chondrocytes and osteoblasts and modulates the maturation and extracellular matrix formation of chondrocytes. The expression of RUNX1 in mesenchymal stem cells, chondrocytes, and osteoblasts is of great significance for maintaining normal bone development and the mass and quality of bones. RUNX1 also inhibits the differentiation and bone resorptive activities of osteoclasts, which may be influenced by sexual dimorphism. In addition, RUNX1 deficiency contributes to the pathogenesis of osteoarthritis, delayed fracture healing, and osteoporosis, which was revealed by the RUNX1 conditional knockout modeling in mice. However, the roles of RUNX1 in regulating the hypertrophic differentiation of chondrocytes, the sexual dimorphism of activities of osteoclasts, as well as bone loss in diabetes mellitus, senescence, infection, chronic inflammation, etc, are still not fully understood. This review provides a systematic summary of the research progress concerning RUNX1 in the field of bone biology, offering new ideas for using RUNX1 as a potential target for bone related diseases, especially osteoarthritis, delayed fracture healing, and osteoporosis.

[Integrin and N-cadherin Co-Regulate the Polarity of Mesenchymal Stem Cells via Mechanobiological Mechanisms]. / æ•´åˆç´ å’ŒN-é’™é»ç´ ååŒè°ƒæŽ§é—´å è´¨å¹²ç»†èƒžæžæ€§çš„åŠ›å­¦ç”Ÿç‰©å­¦æœºåˆ¶ç ”ç©¶.

Objective: To investigate the synergistic regulation of the polarization of mesenchymal stem cells by integrin and N-cadherin-mediated mechanical adhesion and the underlying mechanobiological mechanisms. Methods: Bilayer polyethylene glyeol (PEG) hydrogels were formulated and modified with RGD and HAVDI peptides, respectively, to achieve mechanical adhesion to integrin and N-cadherin and to replicate the integrin-mediated mechanical interaction between cells and the extracellular matrix and the N-cadherin-mediated cell-cell mechanical interaction. The polar proteins, phosphatidylinositol 3-kinase (PI3K) and phosphorylated myosin light chain (pMLC), were characterized through immunofluorescence staining in individual cells with or without contact with HAVDI peptides under integrin-mediated adhesion, N-cadherin-mediated adhesion, and different intracellular forces. Their expression levels and polar distribution were analyzed using Image J. Results: Integrin-mediated adhesion induced significantly higher polar strengths of PI3K and pMLC in the contact group than in those in the no contact group, resulting in the concentration of the polar angle of PI3K to ß-catenin in the range of 135° to 180° and the concentration of the polar angle of pMLC to ß-catenin in the range of 0° to 45° in the contact group. Inhibition of integrin function led to inhibition of the polarity distribution of PI3K in the contact group, but did not change the polarity distribution of pMLC protein. The effect of N-cadherin on the polarity distributions of PI3K and pMLC was similar to that of integrin. However, inhibition of the mechanical adhesion of N-cadherin led to inhibition of the polarity intensity and polarity angle distribution of PI3K and pMLC proteins in the contact group. Furthermore, inhibition of the mechanical adhesion of N-cadherin caused weakened polarity intensity of integrin ß1, reducing the proportion of cells with polarity angles between integrin ß1 and ß-catenin concentrating in the range of 135° to 180°. Additionally, intracellular forces influenced the polar distribution of PI3K and pMLC proteins. Reducing intracellular forces weakened the polarity intensity of PI3K and pMLC proteins and their polarity distribution, while increasing intracellular forces enhanced the polarity intensity of PI3K and pMLC proteins and their polarity distribution. Conclusion: Integrin and N-cadherin co-regulate the polarity distribution of cell proteins and N-cadherin can play an important role in the polarity regulation of stem cells through local inhibition of integrin.

Effect of metabolically divergent pig breeds and tissues on mesenchymal stem cell expression patterns during adipogenesis.

BACKGROUND: Unraveling the intricate and tightly regulated process of adipogenesis, involving coordinated activation of transcription factors and signaling pathways, is essential for addressing obesity and related metabolic disorders. The molecular pathways recruited by mesenchymal stem cells (MSCs) during adipogenesis are also dependent on the different sources of the cells and genetic backgrounds of donors, which contribute to the functional heterogeneity of the stem cells and consequently affect the developmental features and fate of the cells. METHODS: In this study, the alteration of transcripts during differentiation of synovial mesenchymal stem cells (SMSCs) derived from fibrous synovium (FS) and adipose synovial tissue (FP) of two pig breeds differing in growth performance (German Landrace (DL)) and fat deposition (Angeln Saddleback (AS)) was investigated. SMSCs from both tissues and breeds were stimulated to differentiate into adipocytes in vitro and sampled at four time points (day 1, day 4, day 7 and day 14) to obtain transcriptomic data. RESULTS: We observed numerous signaling pathways related to the cell cycle, cell division, cell migration, or cell proliferation during early stages of adipogenesis. As the differentiation process progresses, cells begin to accumulate intracellular lipid droplets and changes in gene expression patterns in particular of adipocyte-specific markers occur. PI3K-Akt signaling and metabolic pathways changed most during adipogenesis, while p53 signaling and ferroptosis were affected late in adipogenesis. When comparing MSCs from FS and FP, only a limited number of differentially expressed genes (DEGs) and enriched signaling pathways were identified. Metabolic pathways, including fat, energy or amino acid metabolism, were highly enriched in the AS breed SMSCs compared to those of the DL breed, especially at day 7 of adipogenesis, suggesting retention of the characteristic metabolic features of their original source, demonstrating donor memory in culture. In contrast, the DL SMSCs were more enriched in immune signaling pathways. CONCLUSIONS: Our study has provided important insights into the dynamics of adipogenesis and revealed metabolic shifts in SMSCs associated with different cell sources and genetic backgrounds of donors. This emphasises the critical role of metabolic and genetic factors as important indications and criteria for donor stem cell selection.

Synergistic effect of human uterine cervical mesenchymal stem cell secretome and paclitaxel on triple negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer and, despite its adverse effects, chemotherapy is the standard systemic treatment option for TNBC. Since, it is of utmost importance to consider the combination of different agents to achieve greater efficacy and curability potential, MSC secretome is a possible innovative alternative. METHODS: In the present study, we proposed to investigate the anti-tumor effect of the combination of a chemical agent (paclitaxel) with a complex biological product, secretome derived from human Uterine Cervical Stem cells (CM-hUCESC) in TNBC. RESULTS: The combination of paclitaxel and CM-hUCESC decreased cell proliferation and invasiveness of tumor cells and induced apoptosis in vitro (MDA-MB-231 and/or primary tumor cells). The anti-tumor effect was confirmed in a mouse tumor xenograft model showing that the combination of both products has a significant effect in reducing tumor growth. Also, pre-conditioning hUCESC with a sub-lethal dose of paclitaxel enhances the effect of its secretome and in combination with paclitaxel reduced significantly tumor growth and even allows to diminish the dose of paclitaxel in vivo. This effect is in part due to the action of extracellular vesicles (EVs) derived from CM-hUCESC and soluble factors, such as TIMP-1 and - 2. CONCLUSIONS: In conclusion, our data demonstrate the synergistic effect of the combination of CM-hUCESC with paclitaxel on TNBC and opens an opportunity to reduce the dose of the chemotherapeutic agents, which may decrease chemotherapy-related toxicity.

MSC-derived mitochondria promote axonal regeneration via Atf3 gene up-regulation by ROS induced DNA double strand breaks at transcription initiation region.

BACKGROUND: The repair of peripheral nerve injury poses a clinical challenge, necessitating further investigation into novel therapeutic approaches. In recent years, bone marrow mesenchymal stromal cell (MSC)-derived mitochondrial transfer has emerged as a promising therapy for cellular injury, with reported applications in central nerve injury. However, its potential therapeutic effect on peripheral nerve injury remains unclear. METHODS: We established a mouse sciatic nerve crush injury model. Mitochondria extracted from MSCs were intraneurally injected into the injured sciatic nerves. Axonal regeneration was observed through whole-mount nerve imaging. The dorsal root ganglions (DRGs) corresponding to the injured nerve were harvested to test the gene expression, reactive oxygen species (ROS) levels, as well as the degree and location of DNA double strand breaks (DSBs). RESULTS: The in vivo experiments showed that the mitochondrial injection therapy effectively promoted axon regeneration in injured sciatic nerves. Four days after injection of fluorescently labeled mitochondria into the injured nerves, fluorescently labeled mitochondria were detected in the corresponding DRGs. RNA-seq and qPCR results showed that the mitochondrial injection therapy enhanced the expression of Atf3 and other regeneration-associated genes in DRG neurons. Knocking down of Atf3 in DRGs by siRNA could diminish the therapeutic effect of mitochondrial injection. Subsequent experiments showed that mitochondrial injection therapy could increase the levels of ROS and DSBs in injury-associated DRG neurons, with this increase being correlated with Atf3 expression. ChIP and Co-IP experiments revealed an elevation of DSB levels within the transcription initiation region of the Atf3 gene following mitochondrial injection therapy, while also demonstrating a spatial proximity between mitochondria-induced DSBs and CTCF binding sites. CONCLUSION: These findings suggest that MSC-derived mitochondria injected into the injured nerves can be retrogradely transferred to DRG neuron somas via axoplasmic transport, and increase the DSBs at the transcription initiation regions of the Atf3 gene through ROS accumulation, which rapidly release the CTCF-mediated topological constraints on chromatin interactions. This process may enhance spatial interactions between the Atf3 promoter and enhancer, ultimately promoting Atf3 expression. The up-regulation of Atf3 induced by mitochondria further promotes the expression of downstream regeneration-associated genes and facilitates axon regeneration.

Young osteocyte-derived extracellular vesicles facilitate osteogenesis by transferring tropomyosin-1.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can undergo inadequate osteogenesis or excessive adipogenesis as they age due to changes in the bone microenvironment, ultimately resulting in decreased bone density and elevated risk of fractures in senile osteoporosis. This study aims to investigate the effects of osteocyte senescence on the bone microenvironment and its influence on BMSCs during aging. RESULTS: Primary osteocytes were isolated from 2-month-old and 16-month-old mice to obtain young osteocyte-derived extracellular vesicles (YO-EVs) and senescent osteocyte-derived EVs (SO-EVs), respectively. YO-EVs were found to significantly increase alkaline phosphatase activity, mineralization deposition, and the expression of osteogenesis-related genes in BMSCs, while SO-EVs promoted BMSC adipogenesis. Neither YO-EVs nor SO-EVs exerted an effect on the osteoclastogenesis of primary macrophages/monocytes. Our constructed transgenic mice, designed to trace osteocyte-derived EV distribution, revealed abundant osteocyte-derived EVs embedded in the bone matrix. Moreover, mature osteoclasts were found to release osteocyte-derived EVs from bone slices, playing a pivotal role in regulating the functions of the surrounding culture medium. Following intravenous injection into young and elderly mouse models, YO-EVs demonstrated a significant enhancement of bone mass and biomechanical strength compared to SO-EVs. Immunostaining of bone sections revealed that YO-EV treatment augmented the number of osteoblasts on the bone surface, while SO-EV treatment promoted adipocyte formation in the bone marrow. Proteomics analysis of YO-EVs and SO-EVs showed that tropomyosin-1 (TPM1) was enriched in YO-EVs, which increased the matrix stiffness of BMSCs, consequently promoting osteogenesis. Specifically, the siRNA-mediated depletion of Tpm1 eliminated pro-osteogenic activity of YO-EVs both in vitro and in vivo. CONCLUSIONS: Our findings suggested that YO-EVs played a crucial role in maintaining the balance between bone resorption and formation, and their pro-osteogenic activity declining with aging. Therefore, YO-EVs and the delivered TPM1 hold potential as therapeutic targets for senile osteoporosis.

BMP7-Loaded Human Umbilical Cord Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Ameliorate Liver Fibrosis by Targeting Activated Hepatic Stellate Cells.

Purpose: Human umbilical cord mesenchymal stem cell (hucMSC)-derived small extracellular vesicles (sEVs) are natural nanocarriers with promising potential in treating liver fibrosis and have widespread applications in the fields of nanomedicine and regenerative medicine. However, the therapeutic efficacy of natural hucMSC-sEVs is currently limited owing to their non-specific distribution in vivo and partial removal by mononuclear macrophages following systemic delivery. Thus, the therapeutic efficacy can be improved through the development of engineered hucMSC-sEVs capable to overcome these limitations. Patients and Methods: To improve the anti-liver fibrosis efficacy of hucMSC-sEVs, we genetically engineered hucMSC-sEVs to overexpress the anti-fibrotic gene bone morphogenic protein 7 (BMP7) in parental cells. This was achieved using lentiviral transfection, following which BMP7-loaded hucMSC-sEVs were isolated through ultracentrifugation. First, the liver fibrosis was induced in C57BL/6J mice by intraperitoneal injection of 50% carbon tetrachloride (CCL4) twice a week for 8 weeks. These mice were subsequently treated with BMP7+sEVs via tail vein injection, and the anti-liver fibrosis effect of BMP7+sEVs was validated using small animal in vivo imaging, immunohistochemistry (IHC), tissue immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Finally, cell function studies were performed to confirm the in vivo results. Results: Liver imaging and liver histopathology confirmed that the engineered hucMSC-sEVs could reach the liver of mice and aggregate around activated hepatic stellate cells (aHSCs) with a significantly stronger anti-liver fibrosis effect of BMP7-loaded hucMSC-sEVs compared to those of blank or negative control-transfected hucMSC-sEVs. In vitro, BMP7-loaded hucMSC-sEVs promoted the phenotypic reversal of aHSCs and inhibited their proliferation to enhance the anti-fibrotic effects. Conclusion: These engineered BMP7-loaded hucMSC-sEVs offer a novel and promising strategy for the clinical treatment of liver fibrosis.

Functional diversity of apoptotic vesicle subpopulations from bone marrow mesenchymal stem cells in tissue regeneration.

Apoptosis releases numerous apoptotic vesicles that regulate processes such as cell proliferation, immunity, and tissue regeneration and repair. Now, it has also emerged as an attractive candidate for biotherapeutics. However, apoptotic vesicles encompass a diverse range of subtypes, and it remains unclear which specific subtypes play a pivotal role. In this study, we successfully isolated different apoptotic vesicle subtypes based on their sizes and characterized them using NTA and TEM techniques, respectively. We compared the functional variances among the distinct subtypes of apoptotic vesicles in terms of stem cell proliferation, migration, and differentiation, as well as for endothelial cell and macrophage function, effectively identifying subtypes that exhibit discernible functional differences. ApoSEV (with diameter <1000 nm) promoted stem cell proliferation, migration, and multi-potent differentiation, and accelerated skin wound healing of diabetes mouse model, while apoBD (with diameter >1000 nm) played the opposite effect on cell function and tissue regeneration. Lastly, employing protein analysis and gene sequencing techniques, we elucidated the intrinsic mechanisms underlying these differences between different subtypes of apoEVs. Collectively, this study identified that apoptotic vesicle subtypes possessed distinct bio-functions in regulating stem cell function and behaviour and modulating tissue regeneration, which primarily attribute to the distinct profiling of protein and mRNA in different subtypes. This comprehensive analysis of specific subtypes of apoEVs would provide novel insights for potential therapeutic applications in cell biology and tissue regeneration.

The potency of mesenchymal stem/stromal cells: does donor sex matter?

Mesenchymal stem/stromal cells (MSCs) are a promising therapeutic tool in cell therapy and tissue engineering because of their multi-lineage differentiation capacity, immunomodulatory effects, and tissue protective potential. To achieve optimal results as a therapeutic tool, factors affecting MSC potency, including but not limited to cell source, donor age, and cell batch, have been investigated. Although the sex of the donor has been attributed as a potential factor that can influence MSC potency and efficacy, the impact of donor sex on MSC characteristics has not been carefully investigated. In this review, we summarize published studies demonstrating donor-sex-related MSC heterogeneity and emphasize the importance of disclosing donor sex as a key factor affecting MSC potency in cell therapy.

[Research Progress in the Regulatory Role of circRNA-miRNA Network in Bone Remodeling]. / circRNA-miRNAç½‘ç»œè°ƒæŽ§éª¨é‡å¡‘çš„ç ”ç©¶è¿›å±•.

The dynamic balance between bone formation and bone resorption is a critical process of bone remodeling. The imbalance of bone formation and bone resorption is closely associated with the occurrence and development of various bone-related diseases. Under both physiological and pathological conditions, non-coding RNAs (ncRNAs) play a crucial regulatory role in protein expression through either inhibiting mRNAs translation or promoting mRNAs degradation. Circular RNAs (circRNAs) are a type of non-linear ncRNAs that can resist the degradation of RNA exonucleases. There is accumulating evidence suggesting that circRNAs and microRNAs (miRNAs) serve as critical regulators of bone remodeling through their direct or indirect regulation of the expression of osteogenesis-related genes. Additionally, recent studies have revealed the involvement of the circRNAs-miRNAs regulatory network in the process by which mesenchymal stem cells (MSCs) differentiate towards the osteoblasts (OB) lineage and the process by which bone marrow-derived macrophages (BMDM) differentiate towards osteoclasts (OC). The circRNA-miRNA network plays an important regulatory role in the osteoblastic-osteoclastic balance of bone remodeling. Therefore, a thorough understanding of the circRNA-miRNA regulatory mechanisms will contribute to a better understanding of the regulatory mechanisms of the balance between osteoblastic and osteoclastic activities in the process of bone remodeling and the diagnosis and treatment of related diseases. Herein, we reviewed the functions of circRNA and microRNA. We also reviewed their roles in and the mechanisms of the circRNA-miRNA regulatory network in the process of bone remodeling. This review provides references and ideas for further research on the regulation of bone remodeling and the prevention and treatment of bone-related diseases.

Stepwise combined cell transplantation using mesenchymal stem cells and induced pluripotent stem cell-derived motor neuron progenitor cells in spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is an intractable neurological disease in which functions cannot be permanently restored due to nerve damage. Stem cell therapy is a promising strategy for neuroregeneration after SCI. However, experimental evidence of its therapeutic effect in SCI is lacking. This study aimed to investigate the efficacy of transplanted cells using stepwise combined cell therapy with human mesenchymal stem cells (hMSC) and induced pluripotent stem cell (iPSC)-derived motor neuron progenitor cells (iMNP) in a rat model of SCI. METHODS: A contusive SCI model was developed in Sprague-Dawley rats using multicenter animal spinal cord injury study (MASCIS) impactor. Three protocols were designed and conducted as follows: (Subtopic 1) chronic SCI + iMNP, (Subtopic 2) acute SCI + multiple hMSC injections, and (Main topic) chronic SCI + stepwise combined cell therapy using multiple preemptive hMSC and iMNP. Neurite outgrowth was induced by coculturing hMSC and iPSC-derived motor neuron (iMN) on both two-dimensional (2D) and three-dimensional (3D) spheroid platforms during mature iMN differentiation in vitro. RESULTS: Stepwise combined cell therapy promoted mature motor neuron differentiation and axonal regeneration at the lesional site. In addition, stepwise combined cell therapy improved behavioral recovery and was more effective than single cell therapy alone. In vitro results showed that hMSC and iMN act synergistically and play a critical role in the induction of neurite outgrowth during iMN differentiation and maturation. CONCLUSIONS: Our findings show that stepwise combined cell therapy can induce alterations in the microenvironment for effective cell therapy in SCI. The in vitro results suggest that co-culturing hMSC and iMN can synergistically promote induction of MN neurite outgrowth.

A de novo dominant-negative variant is associated with OTULIN-related autoinflammatory syndrome.

OTULIN-related autoinflammatory syndrome (ORAS), a severe autoinflammatory disease, is caused by biallelic pathogenic variants of OTULIN, a linear ubiquitin-specific deubiquitinating enzyme. Loss of OTULIN attenuates linear ubiquitination by inhibiting the linear ubiquitin chain assembly complex (LUBAC). Here, we report a patient who harbors two rare heterozygous variants of OTULIN (p.P152L and p.R306Q). We demonstrated accumulation of linear ubiquitin chains upon TNF stimulation and augmented TNF-induced cell death in mesenchymal stem cells differentiated from patient-derived iPS cells, which confirms that the patient has ORAS. However, although the de novo p.R306Q variant exhibits attenuated deubiquitination activity without reducing the amount of OTULIN, the deubiquitination activity of the p.P152L variant inherited from the mother was equivalent to that of the wild-type. Patient-derived MSCs in which the p.P152L variant was replaced with wild-type also exhibited augmented TNF-induced cell death and accumulation of linear chains. The finding that ORAS can be caused by a dominant-negative p.R306Q variant of OTULIN furthers our understanding of disease pathogenesis.

Vegfc-expressing cells form heterotopic bone after musculoskeletal injury.

Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels.

Regulation of muscle hypertrophy through granulin: Relayed communication among mesenchymal progenitors, macrophages, and satellite cells.

Skeletal muscles exert remarkable regenerative or adaptive capacities in response to injuries or mechanical loads. However, the cellular networks underlying muscle adaptation are poorly understood compared to those underlying muscle regeneration. We employed single-cell RNA sequencing to investigate the gene expression patterns and cellular networks activated in overloaded muscles and compared these results with those observed in regenerating muscles. The cellular composition of the 4-day overloaded muscle, when macrophage infiltration peaked, closely resembled that of the 10-day regenerating muscle. In addition to the mesenchymal progenitor-muscle satellite cell (MuSC) axis, interactome analyses or targeted depletion experiments revealed communications between mesenchymal progenitors-macrophages and macrophages-MuSCs. Furthermore, granulin, a macrophage-derived factor, inhibited MuSC differentiation, and Granulin-knockout mice exhibited blunted muscle hypertrophy due to the premature differentiation of overloaded MuSCs. These findings reveal the critical role of granulin through the relayed communications of mesenchymal progenitors, macrophages, and MuSCs in facilitating efficient muscle hypertrophy.

Toll-like receptor activation regulates the paracrine effect of adipose-derived mesenchymal stem cells on reversing osteoarthritic phenotype of chondrocytes.

BACKGROUND: The therapeutic efficacy of intra-articular mesenchymal stem cells (MSCs) injection for patients with osteoarthritis (OA) currently exhibits inconsistency, and the underlying mechanism remains elusive. It has been postulated that the immunomodulatory properties and paracrine activity of MSCs might be influenced by the inflammatory micro-environment within osteoarthritic joints, potentially contributing to this observed inconsistency. METHODS: Adipose-derived MSCs (ADSCs) were isolated from SD rats and pre-treated with Toll-like receptor 3 (TLR3) agonist Poly I:C or Toll-like receptor 4 (TLR4) agonist LPS. The pre-treated ADSCs were then co-cultured with IL-1ß-induced osteoarthritic chondrocytes using a Transwell system to analyze the paracrine effect of ADSCs on reversing the osteoarthritic phenotype of chondrocytes. RESULTS: RT-PCR and Western blot analysis revealed that Poly I:C and LPS pre-treatments up-regulated the expression of IL-10 and IL-6 in ADSCs, respectively. Furthermore, only Poly I:C-preconditioned ADSCs significantly promoted proliferation while inhibiting apoptosis in IL-1ß-treated chondrocytes. Additionally, Poly I:C-preconditioned ADSCs downregulated MMP13 expression while upregulating aggrecan and collagen II expression levels in IL-1ß-treated chondrocytes. CONCLUSIONS: TLR3 activation polarizes ADSCs into an immunomodulatory phenotype distinct from TLR4 activation, exerting differential effects on reversing the osteoarthritic phenotype of chondrocytes; thus indicating that MSCs' paracrine effect regulated by TLRs signaling impacts the efficacy of intra-articular MSCs injection.

Human umbilical cord mesenchymal stem cell-derived exosomes alleviate the severity of experimental autoimmune encephalomyelitis and enhance lag-3 expression on foxp3 + CD4 + T cells.

BACKGROUND: Multiple sclerosis (MS) is a complex autoimmune disease that affects the central nervous system, causing inflammation, demyelination, and neurodegeneration. Understanding the dysregulation of Tregs, dynamic cells involved in autoimmunity, is crucial in comprehending diseases like MS. However, the role of lymphocyte-activation gene 3 (Lag-3) in MS remains unclear. METHODS: In this study, we explore the potential of exosomes derived from human umbilical cord mesenchymal stem cells (hUMSCs-Exs) as an immune modulator in experimental autoimmune encephalomyelitis (EAE), a model for MS. RESULTS: Using flow cytometry, our research findings indicate that groups receiving treatment with hUMSC-Exs revealed a significant increase in Lag-3 expression on Foxp3 + CD4 + T cells. Furthermore, cell proliferation conducted on spleen tissue samples from EAE mice using the CFSE method exposed to hUMSC-Exs yielded relevant results. CONCLUSIONS: These results suggest that hUMSCs-Exs could be a promising anti-inflammatory agent to regulate T-cell responses in EAE and other autoimmune diseases. However, further research is necessary to fully understand the underlying mechanisms and Lag-3's precise role in these conditions.

Effects of bone marrow mesenchymal stem cell-conditioned medium on the proliferation and migration of liposarcoma cells.

INTRODUCTION: Liposarcoma constitutes a prevalent subtype of soft tissue sarcoma, represents approximately 20% of all sarcomas. However, conventional chemotherapeutic agents have shown restricted effectiveness in treating liposarcoma patients. Accumulating evidence indicates that mesenchymal stem cells (MSCs) have the characteristic of migration to tumor site, promote or suppress tumors. How human bone marrow mesenchymal stem cells (BMSCs) contribute to liposarcoma phenotype remains poorly understood. This study aims to investigate the effects of human bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on the proliferation and migration of liposarcoma cell lines 93T449 and SW872, as well as explore potential underlying mechanisms of BMSC-CM action on these cells. MATERIALS AND METHODS: We transfected BMSCs with lentiviral constructs to knock down the transcriptional co-activator Yes-associated protein 1 (YAP1), conditioned medium (CM) obtained from BMSCs and shYAP1-BMSC, respectively. Liposarcoma cell lines 93T449 and SW872 were co-cultured with BMSC-CM or shYAP1-BMSC-CM. Cell proliferation ability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was evaluated using flow cytometric assay. A wound healing assay was used to analyze cell migration. The expression levels of YAP1, Bcl-2, and matrix metalloproteinase-2 (MMP-2) were determined by western blot assay. RESULTS: Co-culturing liposarcoma cell lines 93T449 and SW872 with BMSC-CM promoted tumor cell proliferation, while shYAP1-BMSC-CM significantly inhibited cell viability and migration, induced apoptosis, and downregulated Bcl-2 and MMP-2 expression. CONCLUSIONS: These findings provide new insights into the impact of BMSC-CM on liposarcoma and suggest its possible involvement in liposarcoma cell growth.

Age-related Morphofunctional Changes in Sickle Cell Mice Bone Marrow Mesenchymal Stromal Cells.

BACKGROUND AND OBJECTIVES: Bone marrow mesenchymal stromal cells (BM-MSCs) are key elements of the hematopoietic niche and participate in the regulatory mechanisms of hematopoietic stem cells (HSCs). Hematological diseases can affect MSCs and their functions. However, the dysregulations caused by sickle cell disease (SCD) are not fully elucidated. This work explored changes in BM-MSCs and their relationship with age using sickle cell mice (Townes-SS). MATERIALS AND METHODS: BM-MSCs were isolated from Townes-SS, and control groups 30- and 60-day-old Townes-AA and C57BL/6 J. RESULTS: The BM-MSCs showed no morphological differences in culture and demonstrated a murine MSC-like immunophenotypic profile (Sca-1+, CD29+, CD44+, CD90.2+, CD31-, CD45-, and CD117-). Subsequently, all BM-MSCs were able to differentiate into adipocytes and osteocytes in vitro. Finally, 30-day-old BM-MSCs of Townes-SS showed higher expression of genes related to the maintenance of HSCs (Cxcl12, Vegfa, and Angpt1) and lower expression of pro-inflammatory genes (Tnfa and Il-6). However, 60-day-old BM-MSCs of Townes-SS started to show expression of genes related to reduced HSC maintenance and increased expression of pro-inflammatory genes. CONCLUSION: These results indicates age as a modifying factor of gene expression of BM-MSCs in the context of SCD.

Specific lipid magnetic sphere sorted CD146-positive bone marrow mesenchymal stem cells can better promote articular cartilage damage repair.

BACKGROUND: The characteristics and therapeutic potential of subtypes of bone marrow mesenchymal stem cells (BMSCs) are largely unknown. Also, the application of subpopulations of BMSCs in cartilage regeneration remains poorly characterized. The aim of this study was to explore the regenerative capacity of CD146-positive subpopulations of BMSCs for repairing cartilage defects. METHODS: CD146-positive BMSCs (CD146 + BMSCs) were sorted by self-developed CD146-specific lipid magnetic spheres (CD146-LMS). Cell surface markers, viability, and proliferation were evaluated in vitro. CD146 + BMSCs were subjected to in vitro chondrogenic induction and evaluated for chondrogenic properties by detecting mRNA and protein expression. The role of the CD146 subpopulation of BMSCs in cartilage damage repair was assessed by injecting CD146 + BMSCs complexed with sodium alginate gel in the joints of a mouse cartilage defect model. RESULTS: The prepared CD146-LMS had an average particle size of 193.7 ± 5.24 nm, an average potential of 41.9 ± 6.21 mv, and a saturation magnetization intensity of 27.2 Am2/kg, which showed good stability and low cytotoxicity. The sorted CD146 + BMSCs highly expressed stem cell and pericyte markers with good cellular activity and cellular value-added capacity. Cartilage markers Sox9, Collagen II, and Aggrecan were expressed at both protein and mRNA levels in CD146 + BMSCs cells after chondrogenic induction in vitro. In a mouse cartilage injury model, CD146 + BMSCs showed better function in promoting the repair of articular cartilage injury. CONCLUSION: The prepared CD146-LMS was able to sort out CD146 + BMSCs efficiently, and the sorted subpopulation of CD146 + BMSCs had good chondrogenic differentiation potential, which could efficiently promote the repair of articular cartilage injury, suggesting that the sorted CD146 + BMSCs subpopulation is a promising seed cell for cartilage tissue engineering.