Bone Marrow-derived Mesenchymal Stem Cells Research Articles

Abstract Tu002: Mesenchymal Stem Cell-Derived Extracellular Vesicles Mitigate Mitochondrial DNA-Induced Activation of Porcine Peripheral Blood Mononuclear Cells

Objective: Systemic inflammation is a well-established component of post-cardiac arrest syndrome (PCAS), a condition responsible for significant morbidity and mortality in patients that are initially resuscitated from sudden cardiac arrest. Immune cell activation is pivotal in PCAS pathophysiology, with emerging evidence implicating mitochondrial DNA (mtDNA) as a potent pro-inflammatory stimulus in this context. Given the paucity of available interventions to inhibit mtDNA-induced immune cell activation, we investigated the therapeutic potential of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) to modulate the inflammatory response of porcine peripheral blood mononuclear cells (PBMCs) activated by mtDNA in vitro . Methods: Porcine bone marrow-derived mesenchymal stem cells (MSCs; P2) were cultured for 5 days and serum-starved for 3 days to collect MSC-EVs. These EVs were isolated from conditioned media (CM) using size exclusion chromatography and ultracentrifugation, then characterized by nanoparticle tracking analysis (ZetaView) and ExoCheck. Naïve PBMCs from healthy swine were cultured for 1 week before stimulation with lipofectamine 2000 (TR-control) or 2 µg/mL mtDNA+TR. MSC-EVs were added to activated PBMCs (2.5e8–2e9 EVs/well) and inflammatory surface marker expression (flow cytometry), inflammatory cytokine transcripts (qPCR) and release (ELISA), and reactive oxygen/nitrogen species (ROS/RNS) production were assessed 24 hours later. Results: mtDNA-stimulated PBMC populations exhibited altered surface marker expression consistent with enrichment of inflammatory granulocytes (CD172+CD163-), macrophages (CD14+CD163+), and adhesion molecules (CD172+) compared to TR-control (all p<0.05). Addition of MSC-EVs significantly reduced the number of all inflammatory cell populations, with the most prominent effects achieved at the highest EV dose (2e9 EVs/well). Likewise, MSC-EVs significantly attenuated mtDNA-activated PBMC expression and secretion of TNFα, IL-1β, and IL-6 ( Figure) and decreased ROS/RNS production in a dose-dependent manner. Conclusion: MSC-EVs attenuate mtDNA-induced activation of PBMCs in a dose-dependent manner, as determined by reductions in pro-inflammatory surface marker expression, inflammatory cytokine gene expression and secretion, and ROS/RNS production. These findings indicate that MSC-EVs may be a viable therapeutic for the inhibition of mtDNA-mediated immune activation in PCAS.

Exploring the relationship between pyroptosis and inflammatory bone loss: Evidence from a cigarette smoke-induced osteoporosis mouse model

Smoking is by far one of the greatest public health threats and is recognized as an important predisposing factor for osteoporosis. Exposure to cigarette smoke (CS) has been reported to be associated with inflammation-associated diseases through the induction of pyroptosis. Nevertheless, the correlation between pyroptosis and bone loss induced by CS remains uninvestigated. Here, a mouse model of mainstream smoke exposure-induced osteoporosis was established. μCT, biomechanical testing, and immunohistochemical staining of bone tissue were used to assess the deleterious effects of CS on bone metabolism. In vitro, the effects of cigarette smoke extracts (CSE) on mouse primary bone marrow-derived mesenchymal stem cells (BMSCs) were tested by cell viability assays, gene and protein expression assays, and alizarin red staining. The utilization of the pyroptosis inhibitor MCC950 served to confirm the critical role of BMSCs pyroptosis in CS-induced osteoporosis. Our results indicated that exposure to mainstream smoke led to a notable decrease in the quantity of osteoblasts and hindered the process of osteogenic differentiation in mice. Additionally, there was a significant increase in the expression of pyroptosis-related proteins in the bone marrow. The inhibitory effects of CSE on cell viability and osteogenic differentiation of BMSCs were found to be dose-dependent in vitro. However, the presence of the pyroptosis inhibitor MCC950 significantly improved the impaired osteogenic differentiation and bone mineralization caused by CSE. These results highlight the crucial involvement of BMSCs pyroptosis in the development of bone loss induced by CS. In summary, the findings of this study provide novel evidence that CS exerts a detrimental effect on the process of osteogenesis in BMSCs through the induction of pyroptosis, ultimately leading to bone loss. Inhibition of pyroptosis effectively attenuated the toxicological effects of CS on BMSCs, providing a new target for preventing inflammatory osteoporosis.

Inhibition Effect of Resveratrol on Spontaneous Senescence of Human Bone Marrow Derived Mesenchymal Stem Cells

To explore whether Resveratrol (RSV) can inhibit the spontaneous senescence of human bone marrow-derived mesenchymal stem cells (MSC). MSC were serially cultured to passage 13 and passage 15 to establish model groups exhibiting spontaneous senescence, respectively. MSC at passage 13 and passage 15 were treated with 5 nmol/L RSV for 48 h to establish the RSV-treated groups. SA-β-Gal staining was used to detect cell senescence. MTT assay was used to detect cell proliferation. RT-PCR method was used to detect senescenceassociated telomerase activity. Western blot was used to detect the senescence-associated protein level of the phosphorylated-mTOR. SA-β-Gal staining showed that the senescent cells of MSC in RSV-treated group was significantly less than those in the model group (RSV group compared with model group at passage 13, P < 0.05; RSV group compared with model group at passage 15, P < 0.01). The cell proliferation ability of MSC in RSV-treated group was significantly higher than those in model group, at 72 h in passage 13, there was significant difference between RSV-treated group and model group (P < 0.05). RT-PCR results showed that the hTERT mRNA expression of MSC in RSV-treated group was higher than that in model group, which was significantly different between RSV-treated group and model group at passage 13 (P < 0.05). Western blot results showed that the phosphorylated (Ser2448)-mTOR level of MSC in RSV-treated group was lower than that in model group, which was significantly different between RSV-treated group and model group at passage 13 (P < 0.05). RSV can inhibit the spontaneous senescence of human MSC by mediating mTOR activity.

Expression of the HIF-1α/VEGF pathway is upregulated to protect alveolar bone density reduction in nasal-obstructed rats.

Hypoxia and mouth breathing are closely related to maxillofacial bone metabolism and are characteristic of obstructive sleep apnea-hypopnea syndrome (OSAHS). Being key factors in the hypoxia response, hypoxia-inducible factor 1α (HIF-1α) and HIF-responsive gene vascular endothelial growth factor (VEGF) are essential for bone remodeling. This study focuses on the role of the HIF-1α/VEGF pathway in alveolar bone metabolism during OSAHS. 36 three-week-old male Wistar rats were divided into three groups: twelve control rats, twelve bilateral nasal obstructed (BNO) rats, twelve BNO rats treated with intraperitoneal injection of Dimethyloxalylglycine (DMOG). After two weeks, the microstructure and bone mineral density (BMD) of alveolar bone were evaluated using micro-computed tomography (micro-CT). The expressions of HIF-1α and VEGF in the alveolar bone were then assessed via immunohistochemistry staining, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Alkaline phosphatase (ALP) staining and Alizarin red S staining were performed to evaluate osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). Significant reductions in alveolar bone density were noted in BNO rats. Bilateral nasal obstruction increased the expressions of HIF-1α and VEGF in alveolar bone. With upregulation of HIF-1α/VEGF via DMOG, alveolar bone density of BNO rats increased. Furthermore, DMOG promoted the osteogenic differentiation of BMSCs by stabilizing the HIF-1α protein and increasing the expression of VEGF. Bilateral nasal obstruction changes alveolar bone structure and leads to a reduction in alveolar bone density. Moreover, the expression of the HIF-1α/VEGF signaling pathway increases to protect alveolar bone density reduction in BNO rats.

Mesenchymal stromal/stem cell tissue source and in vitro expansion impact extracellular vesicle protein and miRNA compositions as well as angiogenic and immunomodulatory capacities.

Recently, therapies utilizing extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) have begun to show promise in clinical trials. However, EV therapeutic potential varies with MSC tissue source and in vitro expansion through passaging. To find the optimal MSC source for clinically translatable EV-derived therapies, this study aims to compare the angiogenic and immunomodulatory potentials and the protein and miRNA cargo compositions of EVs isolated from the two most common clinical sources of adult MSCs, bone marrow and adipose tissue, across different passage numbers. Primary bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (ASCs) were isolated from adult female Lewis rats and expanded in vitro to the indicated passage numbers (P2, P4, and P8). EVs were isolated from the culture medium of P2, P4, and P8 BMSCs and ASCs and characterized for EV size, number, surface markers, protein content, and morphology. EVs isolated from different tissue sources showed different EV yields per cell, EV sizes, and protein yield per EV. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of proteomics data and miRNA seq data identified key proteins and pathways associated with differences between BMSC-EVs and ASC-EVs, as well as differences due to passage number. In vitro tube formation assays employing human umbilical vein endothelial cells suggested that both tissue source and passage number had significant effects on the angiogenic capacity of EVs. With or without lipopolysaccharide (LPS) stimulation, EVs more significantly impacted expression of M2-macrophage genes (IL-10, Arg1, TGFβ) than M1-macrophage genes (IL-6, NOS2, TNFα). By correlating the proteomics analyses with the miRNA seq analysis and differences observed in our in vitro immunomodulatory, angiogenic, and proliferation assays, this study highlights the trade-offs that may be necessary in selecting the optimal MSC source for development of clinical EV therapies.

Therapeutic potential of Chinese medicinal herbs stimulating osteogenic differentiation of bone marrow-derived mesenchymal stem cells in osteoporosis.

Osteoporosis (OP) is a common and complex chronic metabolic disease with an increasing incidence rate, which has markedly increased the human health burden worldwide. The predominant cause of OP is an imbalance between osteoblasts (OB) and osteoclasts (OC). Studies on the correlation between bone marrow-derived mesenchymal stem cells (BMSCs) and OP have indicated that BMSCs-induced OB differentiation is an important pathway for bone tissue renewal. Chinese medicinal herbs have been used for centuries to treat various types of OPs because they are safer and more effective. The in vivo and in vitro experiments have confirmed that these herbs or their primary phytochemicals may exert therapeutic effects by stimulating BMSCs differentiation, which restores OB and OP balance, inhibits adipocyte differentiation, exerts anti-inflammatory and antioxidant effects, regulates the immune system, etc. This review summarizes the research on how Chinese medicinal herbs or their primary phytochemicals treat OP by stimulating BMSC differentiation and provides a scientifically reliable basis and perspective for their future clinical application.

Characterization of MSCs expressing islet neogenesis associated protein (INGAP): INGAP secretion and cell survival in vitro and in vivo

Mesenchymal stem/stromal cells (MSCs) are emerging as a new therapy for diabetes. Here we investigate the properties of MSCs engineered to express Islet Neogenesis Associated Protein (INGAP) previously shown to reverse diabetes in animal models and evaluate their potential for anti-diabetic applications in mice. Mouse bone marrow-derived MSCs retrovirally transduced to co-express INGAP, Firefly Luciferase and EGFP (INGAP-MSCs), were characterized in vitro and implanted intraperitoneally (IP) into non-diabetic and diabetic C57BL/6 mice (Streptozotocin model) and tracked by live bioluminescence imaging (BLI). Distribution and survival of IP injected INGAP-MSCs differed between diabetic and non-diabetic mice, with a rapid clearance of cells in the latter, and a stronger retention (up to 4 weeks) in diabetic mice concurring with homing towards the pancreas. Interestingly, INGAP-MSCs inhibited the progression of hyperglycemia starting at day 3 and lasting for the entire 6 weeks of the study. Pursuing greater retention, we investigated the survival of INGAP-MSCs in hydrogel matrices. When mixed with Matrigel™ and injected subcutaneously into non-diabetic mice, INGAP-MSCs remained in the implant up to 16 weeks. In vitro tests in three matrices (Matrigel™, Type I Collagen and VitroGel®-MSC) demonstrated that INGAP-MSCs survive and secrete INGAP, with best results at the density of 1–2 x 106 cells/mL. However, all matrices induced spontaneous adipogenic differentiation of INGAP-MSCs in vitro and in vivo, which requires further investigation of its potential impact on MSC therapeutic properties. In summary, based on their ability to stop the rise in hyperglycemia in STZ-treated mice, INGAP-MSCs are a promising therapeutic tool against diabetes but require further research to improve cell delivery and survival.

Mesenchymal stem cell-derived small extracellular vesicles enhance the therapeutic effect of retinal progenitor cells in retinal degenerative disease rats.

Our previous study demonstrated that combined transplantation of bone marrow-derived mesenchymal stem cells and retinal progenitor cells in rats has therapeutic effects on retinal degeneration that are superior to transplantation of retinal progenitor cells alone. Bone marrow- derived mesenchymal stem cells regulate and interact with various cells in the retinal microenvironment by secreting neurotrophic factors and extracellular vesicles. Small extracellular vesicles derived from bone marrow-derived mesenchymal stem cells, which offer low immunogenicity, minimal tumorigenic risk, and ease of transportation, have been utilized in the treatment of various neurological diseases. These vesicles exhibit various activities, including anti-inflammatory actions, promotion of tissue repair, and immune regulation. Therefore, novel strategies using human retinal progenitor cells combined with BMSC-derived small extracellular vesicles may represent an innovation in stem cell therapy for retinal degeneration. In this study, we developed such an approach utilizing retinal progenitor cells combined with bone marrow-derived mesenchymal stem cell-derived small extracellular vesicles to treat retinal degeneration in Royal College of Surgeons rats, a genetic model of retinal degeneration. Our findings revealed that the combination of bone marrow-derived mesenchymal stem cell-derived small extracellular vesicles and retinal progenitor cells significantly improved visual function in these rats. The addition of bone marrow-derived mesenchymal stem cell-derived small extracellular vesicles as adjuvants to stem cell transplantation with retinal progenitor cells enhanced the survival, migration, and differentiation of the exogenous retinal progenitor cells. Concurrently, these small extracellular vesicles inhibited the activation of regional microglia, promoted the migration of transplanted retinal progenitor cells to the inner nuclear layer of the retina, and facilitated their differentiation into photoreceptors and bipolar cells. These findings suggest that bone marrow-derived mesenchymal stem cell-derived small extracellular vesicles potentiate the therapeutic efficacy of retinal progenitor cells in retinal degeneration by promoting their survival and differentiation.

LINC00963 Represses Osteogenic Differentiation of hBMSCs via the miR-10b-5p/RAP2A/AKT Axis.

Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is important for human bone formation. Long non-coding RNAs (lncRNAs) are critical regulators in osteogenic differentiation. This study aimed to explore the function and mechanisms of long intergenic non-protein coding RNA 963 (LINC00963) in affecting osteogenesis. Cell differentiation was assessed by alkaline phosphatase (ALP) activity detection and ALP staining assay. Meanwhile, levels of osteogenic marker genes, including RUNX family transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN), were detected by RT-qPCR and western blot. Cell proliferation and apoptosis were measured using CCK-8 assay and flow cytometry analysis. RNA immunoprecipitation (RIP), RNA pull-down and luciferase reporter assays were used to investigate the interaction between genes. LINC00963 expression was down-regulated in hBMSCs treated with osteogenic induction. LINC00963 overexpression inhibited hBMSCs differentiation, proliferation, and elevated apoptosis. LINC00963 acted as a competing endogenous RNA (ceRNA) to interact with miR-10b-5p and thereby regulated the expression level of Ras-related protein Rap-2a (RAP2A). LINC00963 regulated RAP2A to inhibit the level of phosphorylated AKT (p-AKT). LINC00963 inhibited hBMSCs differentiation, proliferation, and elevated apoptosis via the miR-10b-5p/RAP2A/AKT signaling, which might help improve the treatment of osteoporosis.

Advancing cellular insights: Super-resolution STORM imaging of cytoskeletal structures in human stem and cancer cells

Fluorescence microscopy is an important tool for cell biology and cancer research. Present-day approach of implementing advanced optical microscopy methods combined with immunofluorescence labelling of specific proteins in cells is now able to deliver optical super-resolution up to ∼25 nm. Here we perform super-resolved imaging using standard immunostaining protocol combined with easy stochastic optical reconstruction microscopy (easySTORM) to observe structural differences of two cytoskeleton elements, actin and tubulin in three different cell types namely human bone marrow-derived mesenchymal stem cells (MSCs), human glioblastoma (U87MG) and breast cancer (MDAMB-231) cells. The average width of the actin bundle obtained from STORM images of stem cells is observed to be larger than the same for U87MG and MDAMB-231 cells. No significant difference is however noticed in the width of the tubulin within the same cells. We also study the functional effect on the 2D migration potential of MDAMB-231 cells silenced for NICD1 and β-catenin. Although similar migration speed is observed for cells with the above two conditions compared to their control cells, easySTORM images show that widths of the actin in MDAMB-231 cells in β-catenin silenced is significantly lower than the same in control cells. Such minute differences however are not observable in widefield images. The outcome of our easySTORM investigation should benefit the researchers carrying out detailed investigations of the cellular structure and potential therapeutic applications.

Perinatal nicotine vaping exposure induces pro-myofibroblastic phenotype in rat bone marrow-derived mesenchymal stem cells

Perinatal nicotine exposure via tobacco smoking results in increased proclivity to chronic lung disease (CLD); however, the underlying molecular mechanisms remain incompletely understood. We previously demonstrated that in addition to nicotine’s direct effects on the developing lung, there are also adverse molecular alterations in bone marrow-derived mesenchymal stem cells (BMSCs), which are vital to lung injury repair. Whether perinatal nicotine exposure via electronic-cigarette (e-cig) vaping also adversely affects BMSCs is unknown. This is highly relevant due to marked increase in e-cig vaping including by pregnant women. Hypothesizing that perinatal nicotine exposure via e-cig vaping predisposes BMSCs to a pro-myofibroblastic phenotype, pregnant rat dams were exposed to fresh air (control), vehicle (e-cig without nicotine), or e-cig (e-cig with nicotine) daily during pregnancy and lactation. At postnatal day 21, offspring BMSCs were isolated and studied for cell proliferation, migration, wound healing response, and expression of key Wnt and PPARγ signaling intermediates (β-catenin, LEF-1, PPARγ, ADRP and C/EBPα) and myogenic markers (fibronectin, αSMA, calponin) proteins using immunoblotting. Compared to controls, perinatal e-cig exposure resulted in significant decrease in BMSC proliferation, migration, and wound healing response. The expression of key Wnt signaling intermediates (β-catenin, LEF-1) and myogenic markers (fibronectin, αSMA, calponin) increased significantly, while PPARγ signaling intermediates (PPARγ, ADRP, and C/EBPα) decreased significantly. Based on these data, we conclude that perinatally e-cig exposed BMSCs demonstrate pro-myofibroblastic phenotype and impaired injury-repair potential, indicating a potentially similar susceptibility to CLD following perinatal nicotine exposure via vaping as seen following parenteral perinatal nicotine exposure.

Multifunctional Sr,Mg-Doped Mesoporous Bioactive Glass Nanoparticles for Simultaneous Bone Regeneration and Drug Delivery.

Mesoporous bioactive glass nanoparticles (MBGNs) doped with therapeutical ions present multifunctional systems that enable a synergistic outcome through the dual delivery of drugs and ions. The aim of this study was to evaluate influence of co-doping with strontium and magnesium ions (SrMg-MBGNs) on the properties of MBGNs. A modified microemulsion-assisted sol-gel synthesis was used to obtain particles, and their physicochemical properties, bioactivity, and drug-loading/release ability were evaluated. Indirect biological assays using 2D and 3D cell culture models on human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and endothelial EA.hy926 cells, respectively, were used to determine biocompatibility of MBGNs, their influence on alkaline phosphatase (ALP) production, calcium deposition, and cytoskeletal organization. Results showed that Sr,Mg-doping increased pore volume and solubility, and changed the mesoporous structure from worm-like to radial-dendritic, which led to a slightly accelerated drug release compared to pristine MBGNs. Biological assays confirmed that particles are biocompatible, and have ability to slightly induce ALP production and calcium deposition of hBM-MSCs, as well as to significantly improve the proliferation of EA.hy926 compared to biochemical stimulation via vascular endothelial growth factor (VEGF) administration or regular media. Fluorescence staining revealed that SrMg-MBGNs had a similar effect on EA.hy926 cytoskeletal organization to the VEGF group. In conclusion, Sr,Mg-MBGNs might be considered promising biomaterial for biomedical applications.

Transcriptional profiling of fracture-associated cytokines and growth factors identifies transcriptional regulation of osteogenic genes by recombinant-human IL1β

Signaling molecules secreted during bone fracture healing stimulate molecular and cellular changes to promote healing and tissue regeneration. Molecular characterization of cytokines and growth factors can provide insight into their function and potential application as biologics or drug targets for bone tissue regeneration. Previous studies have identified the expression of IL1B, IL6, IL34, IGF1, ANGPT1, and TGFB3 during osteogenic differentiation and facture healing. In this study, we evaluated the transcriptome of adipose-derived mesenchymal stromal cells (AMSCs) following stimulation with these biological factors and examined their role in osteogenic differentiation. Stimulation of AMSCs with recombinant human (rh)-IL1B led to the largest gene expression changes compared to the other signaling factors including 1411 induced and 541 repressed genes (>2-fold). Rh-IL1B regulated genes included inflammatory (IL1B, IL6, CCL2) and osteogenic (BMP2, RUNX2, TWIST2) genes. Rh-IL1B also induced the expression of BMP2 in bone marrow-derived mesenchymal stromal cells (BMSCs) and bone digest cells. Although no changes in osteogenic differentiation of AMSCs was observed, low concentrations of rh-IL1B (0.1 ng/mL) promoted alkaline phosphatase activity of differentiated bone digest cells. Further, rh-IL1B significantly induced the secretion of BMP2 from these cells. Together this data provides insight into the molecular function of fracture-associated biological factors.

A tetrahedral DNA nanostructure-mediated miRNA inhibitor delivery system: Type H vessel-related bone healing during distraction osteogenesis

The clinical treatment of critical bone defects presents a great challenge, and distraction osteogenesis (DO) emerges as a highly effective strategy. However, prolonged consolidation periods resultant complications limit its clinical applicability. MicroRNA (miR) therapeutic strategies, with a notable focus on miR-205, demonstrate significant potential during DO. Nevertheless, the limited cellular uptake and facile biodegradation of miRNA constrain its applications. To address these challenges, we introduce tetrahedral DNA nanostructure (TDN), an innovative nanomaterial, engineered as gene carriers. TDN delivers the miR-205 inhibitor (TDN@miR205) to synthesize the novel nanoparticles that support bone healing for the first time. TDN@miR205 enhances the osteogenic potential of bone marrow-derived mesenchymal stem cells (BMSCs), whereas TDN alone is ineffective. Further exploration reveals TDN-modulated angiogenesis-osteogenesis communication, establishing a crucial intercellular regulatory mechanism. Notably, TDN@miR205 expedites bone formation by promoting type H vessels-related angiogenesis, secreting SLIT3 to inhibit osteoclasts, inducing SDF1 homing of BMSCs, then upregulating osteogenic genes and proteins via the TGF-β/BMP pathway, facilitating early bony ossification both in vitro and in vivo. In conclusion, TDN@miR205, the multifunctional nanoparticle-mediated vascular bone regeneration significantly contributes to ossification in DO and establishes this platform as a promising and versatile strategy for addressing DO and other complex bone diseases.

Biological characteristics of tissue engineered-nerve grafts enhancing peripheral nerve regeneration

BackgroundA favorable regenerative microenvironment is essential for peripheral nerve regeneration. Neural tissue-specific extracellular matrix (ECM) is a natural material that helps direct cell behavior and promote axon regeneration. Both bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived mesenchymal stem cells (ADSCs) transplantation are effective in repairing peripheral nerve injury (PNI). However, there is no study that characterizes the in vivo microenvironmental characteristics of these two MSCs for the early repair of PNI when combined with neural tissue-derived ECM materials, i.e., acellular nerve allograft (ANA).MethodsIn order to investigate biological characteristics, molecular mechanisms of early stage, and effectiveness of ADSCs- or BMSCs-injected into ANA for repairing PNI in vivo, a rat 10 mm long sciatic nerve defect model was used. We isolated primary BMSCs and ADSCs from bone marrow and adipose tissue, respectively. First, to investigate the in vivo response characteristics and underlying molecular mechanisms of ANA combined with BMSCs or ADSCs, eighty-four rats were randomly divided into three groups: ANA group, ANA+BMSC group, and ANA+ADSC group. We performed flow cytometry, RT-PCR, and immunofluorescence staining up to 4 weeks postoperatively. To further elucidate the underlying molecular mechanisms, changes in long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) were systematically investigated using whole transcriptome sequencing. We then constructed protein–protein interaction networks to find 10 top ranked hub genes among differentially expressed mRNAs. Second, in order to explore the effectiveness of BMSCs and ADSCs on neural tissue-derived ECM materials for repairing PNI, sixty-eight rats were randomized into four groups: ANA group, ANA+BMSC group, ANA+ADSC group, and AUTO group. In the ANA+BMSC and ANA+ADSC groups, ADSCs/BMSCs were equally injected along the long axis of the 10-mm ANA. Then, we performed histological and functional assessments up to 12 weeks postoperatively.ResultsThe results of flow cytometry and RT-PCR showed that ANA combined with BMSCs exhibited more significant immunomodulatory effects, as evidenced by the up-regulation of interleukin (IL)-10, down-regulation of IL-1β and tumor necrosis factor-alpha (TNF-α) expression, promotion of M1-type macrophage polarization to M2-type, and a significant increase in the number of regulatory T cells (Tregs). ANA combined with ADSCs exhibited more pronounced features of pro-myelination and angiogenesis, as evidenced by the up-regulation of myelin-associated protein gene (MBP and MPZ) and angiogenesis-related factors (TGF-β, VEGF). Moreover, differentially expressed genes from whole transcriptome sequencing results further indicated that ANA loaded with BMSCs exhibited notable immunomodulatory effects and ANA loaded with ADSCs was more associated with angiogenesis, axonal growth, and myelin formation. Notably, ANA infused with BMSCs or ADSCs enhanced peripheral nerve regeneration and motor function recovery with no statistically significant differences.ConclusionsThis study revealed that both ANA combined with BMSCs and ADSCs enhance peripheral nerve regeneration and motor function recovery, but their biological characteristics (mainly including immunomodulatory effects, pro-vascular regenerative effects, and pro-myelin regenerative effects) and underlying molecular mechanisms in the process of repairing PNI in vivo are different, providing new insights into MSC therapy for peripheral nerve injury and its clinical translation.

Retinitis pigmentosa and stem cell therapy.

Retinitis pigmentosa (RP) is a group of genetic disorders characterized by progressive degeneration of photoreceptors and retinal pigment epithelium (RPE) cells. Its main clinical manifestations include night blindness and progressive loss of peripheral vision, making it a prevalent debilitating eye disease that significantly impacts patients' quality of life. RP exhibits significant phenotypic and genetic heterogeneity. For instance, numerous abnormal genes are implicated in RP, resulting in varying clinical presentations, disease progression rates, and pathological characteristics among different patients. Consequently, gene therapy for RP poses challenges due to these complexities. However, stem cells have garnered considerable attention in the field of RPE therapy since both RPE cells and photoreceptors can be derived from stem cells. In recent years, a large number of animal experiments and clinical trials based on stem cell transplantation attempts, especially cord blood mesenchymal stem cell (MSC) transplantation and bone marrow-derived MSC transplantation, have confirmed that stem cell therapy can effectively and safely improve the outer retinal function of the RP-affected eye. However, stem cell therapy also has certain limitations, such as the fact that RP patients may involve multiple types of retinal cytopathia, which brings great challenges to stem cell transplantation therapy, and further research is needed to solve various problems faced by this approach in the clinic. Through comprehensive analysis of the etiology and histopathological changes associated with RP, this study substantiates the efficacy and safety of stem cell therapy based on rigorous animal experimentation and clinical trials, while also highlighting the existing limitations that warrant further investigation.

Photo-Clickable Triazine-Trione Thermosets as Promising 3D Scaffolds for Tissue Engineering Applications.

There is an overwhelming demand for new scaffolding materials for tissue engineering (TE) purposes. Polymeric scaffolds have been explored as TE materials; however, their high glass transition state (Tg) limits their applicability. In this study, a novel materials platform for fabricating TE scaffolds is proposed based on solvent-free two-component heterocyclic triazine-trione (TATO) formulations, which cure at room temperature via thiol-ene/yne photochemistry. Three ester-containing thermosets, TATO-1, TATO-2, and TATO-3, are used for the fabrication of TE scaffolds including rigid discs, elastic films, microporous sponges, and 3D printed objects. After 14 days' incubation the materials covered a wide range of properties, from the soft TATO-2 having a compression modulus of 19.3MPa and a Tg of 30.4°C to the hard TATO-3 having a compression modulus of 411MPa and a Tg of 62.5°C. All materials exhibit micro- and nano-surface morphologies suited for bone tissue engineering, and in vitro studies found them all to be cytocompatible, supporting fast cell proliferation while minimizing cell apoptosis and necrosis. Moreover, bone marrow-derived mesenchymal stem cells on the surface of the materials are successfully differentiated into osteoblasts, adipocytes, and neuronal cells, underlining the broad potential for the biofabrication of TATO materials for TE clinical applications.

Enhanced Tissue Regeneration Through Immunomodulatory Extracellular Vesicles Encapsulated in Diversified Silk-Based Injectable Hydrogels.

Mesenchymal stem cells (MSCs) immunologically trained using lipopolysaccharide (LPS) display enhanced immunomodulatory capabilities. Extracellular vesicles (EVs) derived from MSCs are widely used in regenerative medicine owing to their bioactive properties without the drawbacks of cell therapy. However, it remains unclear whether EVs derived from LPS-stimulated (trained) MSCs (L-EVs) inherit the enhanced reparative potential from their parent cells. Thus, this study first aims to explore the effect of immunological training on the bioactivity of L-EVs. LPS-trained bone marrow-derived MSCs (BMSCs) secrete more EVs, and these EVs significantly promote M2 macrophage polarization. Subsequently, hydrogel systems based on thixotropic injectable silk fibroin are prepared for in vivo EV delivery. These hydrogels have controllable gelation time and exhibit outstanding reparative effects on rat skin wounds and alveolar bone defects. Finally, it is revealed that L-EVs promote M2 macrophage polarization by inhibiting the nuclear translocation of PKM2. Overall, this study shows that the immunological training of BMSCs effectively improves the therapeutic effects of their EVs and provides a convenient and diversified EV delivery strategy using an injectable silk fibroin hydrogel. This strategy has broad clinical application prospects for tissue regeneration.

Synthesis and Systematic Investigation of Lepidiline A and Its Gold(I), Silver(I), and Copper(I) Complexes Using In Vitro Cancer Models and Multipotent Stem Cells.

The imidazole alkaloid lepidiline A from the root of Lepidium meyenii has a moderate to low in vitro anticancer effect. Our aim was to extend cytotoxicity investigations against a panel of cancer cells, including multidrug-resistant cancer cells, and multipotent stem cells. Lepidiline A is a N-heterocyclic carbene precursor, therefore a suitable ligand source for metal complexes. Thus, we synthesized lepidiline A and its copper(I), gold(I), and silver(I) complexes and tested them against ovarian, gastrointestinal, breast, and uterine cancer cells and bone marrow-derived and adipose-derived mesenchymal stem cells. Lepidiline A and its copper complex demonstrated moderate cytotoxicity, while silver and gold complexes exhibited significantly enhanced and consistent cytotoxicity against both cancer and stem cell lines. ABCB1 in the multidrug-resistant uterine sarcoma line conferred significant resistance against lepidiline A and the copper-lepidiline A complex, but not against the silver and gold complexes. Our results indicate that only the copper complex induced a significant and universal increase in the production of reactive oxygen species within cells. In summary, binding of metal ions to lepidiline A results in enhanced cytotoxicity with the nature of the metal ion playing a critical role in determining its properties.

Improving Fertility in Non-obstructive Azoospermia: Results from an Autologous Bone Mar-row-Derived Mesenchymal Stromal/Stem Cell Phase I Clinical Trial.

In this phase I clinical trial, our primary objective was to develop an innovative therapeutic approach utilizing autologous bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs) for the treatment of nonobstructive azoospermia (NOA). Additionally, we aimed to assess the feasibility and safety of this approach. We recruited 80 participants in this non-randomized, open-label clinical trial, including patients undergoing NOA treatment using autologous BM-MSCs (n=40) and those receiving hormone therapy as a control group (n=40). Detailed participant characteristics, such as age, baseline hormonal profiles, etiology of NOA, and medical history, were thoroughly documented. Autotransplantation of BM-MSCs into the testicular network was achieved using microsurgical testicular sperm extraction (microTESE). Semen analysis and hormonal assessments were performed both before and six months after treatment. Additionally, we conducted an in-silico analysis to explore potential protein-protein interactions between exosomes secreted from BM-MSCs and receptors present in human seminiferous tubule cells. Our results revealed significant improvements following treatment, including increased testosterone and inhibin B levels, elevated sperm concentration, and reduced levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin. Notably, in nine patients (22.5%) previously diagnosed with secondary infertility and exhibiting azoospermia before treatment, the proposed approach yielded successful outcomes, as indicated by hormonal profile changes over six months. Importantly, these improvements were achieved without complications. Additionally, our in-silico analysis identified potential binding interactions between the protein content of BM-MSC-derived exosomes and receptors integral to spermatogenesis. Autotransplantation of BM-MSCs into the testicular network using microTESE in NOA patients led to the regeneration of seminiferous tubules and the regulation of hormonal profiles governing spermatogenesis. Our findings support the safety and effectiveness of autologous BM-MSCs as a promising treatment modality for NOA, with a particular focus on the achieved outcomes in patients with secondary infertility (registration number: IRCT20190519043634N1).