Proliferation Of Mesenchymal Stromal Cells Research Articles

Effects of aging on the biomechanical properties of the lung extracellular matrix: dependence on tissular stretch.

Introduction: Aging induces functional and structural changes in the lung, characterized by a decline in elasticity and diminished pulmonary remodeling and regenerative capacity. Emerging evidence suggests that most biomechanical alterations in the lung result from changes in the composition of the lung extracellular matrix (ECM), potentially modulating the behavior of pulmonary cells and increasing the susceptibility to chronic lung diseases. Therefore, it is crucial to investigate the mechanical properties of the aged lung. This study aims to assess the mechanical alterations in the lung ECM due to aging at both residual (RV) and functional (FV) lung volumes and to evaluate their effects on the survival and proliferation of mesenchymal stromal cells (MSCs). Methods: The lungs from young (4-6-month-old) and aged (20-24-month-old) mice were inflated with optimal cutting temperature compound to reach FV or non-inflated (RV). ECM proteins laminin, collagen I and fibronectin were quantified by immunofluorescence and the mechanical properties of the decellularized lung sections were assessed using atomic force microscopy. To investigate whether changes in ECM composition by aging and/or mechanical properties at RV and FV volumes affects MSCs, their viability and proliferation were evaluated after 72h. Results: Laminin presence was significantly reduced in aged mice compared to young mice, while fibronectin and collagen I were significantly increased in aged mice. In RV conditions, the acellular lungs from aged mice were significantly softer than from young mice. By contrast, in FV conditions, the aged lung ECM becomes stiffer than that of in young mice, revealing that strain hardening significantly depends on aging. Results after MSCs recellularization showed similar viability and proliferation rate in all conditions. Discussion: This data strongly suggests that biomechanical measurements, especially in aging models, should be carried out in physiomimetic conditions rather than following the conventional non-inflated lung (RV) approach. The use of decellularized lung scaffolds from aged and/or other lung disease murine/human models at physiomimetic conditions will help to better understand the potential role of mechanotransduction on the susceptibility and progression of chronic lung diseases, lung regeneration and cancer.

Process development for the production of mesenchymal stromal cell-derived extracellular vesicles in conventional 2D systems

In recent years, the importance of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) has increased significantly. For their widespread use, a standardized EV manufacturing is needed which often includes conventional, static 2D systems. For these system critical process parameters need to be determined. We studied the impact of process parameters on MSC proliferation, MSC-derived particle production including EVs, EV- and MSC-specific marker expression, and particle functionality in a HaCaT cell migration assay. We found that cell culture growth surface and media affected MSCs and their secretory behavior. Interestingly, the materials that promoted MSC proliferation did not necessarily result in the most functional MSC-derived particles. In addition, we found that MSCs seeded at 4 × 103 cells cm−2 produced particles with improved functional properties compared to higher seeding densities. MSCs in a highly proliferative state did not produce the most particles, although these particles were significantly more effective in promoting HaCaT cell migration. The same correlation was found when investigating the cultivation temperature. A physiological temperature of 37°C was not optimal for particle yield, although it resulted in the most functional particles. We observed a proliferation-associated particle production and found potential correlations between particle production and glucose consumption, enabling the estimation of final particle yields. Our findings suggest that parameters, which must be defined prior to each individual cultivation and do not require complex and expensive equipment, can significantly increase MSC-derived particle production including EVs. Integrating these parameters into a standardized EV process development paves the way for robust and efficient EV manufacturing for early clinical phases.

Formation of osteoconductive biograft with bioorganic scaffold, human mesenchymal stromal cells, and platelet-rich plasma with its evaluation in vitro

BackgroundComplex graft bioengineering is an actual topic in bone defects’ repair. For those, different scaffolds may be seeded with mesenchymal stromal cells and growth / differentiation factors. The natural role of platelet factors in reparative processes justifies the possibility of its usage for mesenchymal stromal cell proliferation and differentiation into osteoblasts in vitro in terms of the scaffold-based bioengineering. To develop and evaluate in vitro biocompatibility and osteoconductivity of a complex biograft based on a bioorganic scaffold seeded with human bone marrow mesenchymal stromal cells and saturated with growth and differentiation factors of allogeneic platelet-rich plasma. ResultsThe properties of viability and adhesion of human bone marrow mesenchymal stromal cells in four types of bioorganic scaffolds were evaluated with biochemical and immunomorphological methods. Scaffold with the least cytotoxicity was used as a basis for complex biograft formation, so as a carrier for cells and platelet-derived factors. Then, cell proliferation activity and osteogenic differentiation were estimated with biochemical, morphological, histochemical and molecular-biological methods. The study showed high viability of cells in all bioorganic scaffolds but the least cytotoxicity was the one based on xenogeneic collagen sponge. We also found that allogeneic platelet-rich plasma positively affects the proliferation and osteogenic differentiation of bone marrow mesenchymal stromal cells in a complex biograft in vitro. ConclusionsThe properties of the developed complex biograft characterize its biocompatibility and osteoconductivity and make it potentially suitable for regenerative medicine, particularly for reconstructive surgery of bone defects.How to cite: Danilkovich NN, Kosmacheva SM, Ionova AG, et al. Formation of osteoconductive biograft with bioorganic scaffold, human mesenchymal stromal cells, and platelet rich plasma with its evaluationin vitro. Electron J Biotechnol 2024;69. https://doi.org/10.1016/j.ejbt.2024.01.004.

Cigarette smoke and nicotine effect on human mesenchymal stromal cell wound healing and osteogenic differentiation capacity.

Mesenchymal stromal cells (MSCs) play a crucial role in promoting tissue regeneration and healing, particularly in bone tissue. Both smoking and nicotine use are known to delay and inhibit the healing process in patients. This study aims at delineating these cellular effects by comparing the impact of nicotine alone to cigarette smoke with equivalent nicotine content, and shedding light on potential differences in the healing process. We examined how cigarette smoke and nicotine affect the migration, proliferation, and osteogenic differentiation of human patient-derived MSCs in vitro, as well as the secretion of cytokines IL-6 and IL-8. We measured nicotine concentration of the cigarette smoke extract (CSE) to clarify the role of the nicotine in the effect of the cigarette smoke. MSCs exposed to nicotine-concentration-standardized CSE exhibited impaired wound healing capability, and at high concentrations, increased cell death. At lower concentrations, CSE dose-dependently impaired migration, proliferation, and osteogenic differentiation, and increased IL-8 secretion. Nicotine impaired proliferation and decreased PINP secretion. While there was a trend for elevated IL-6 levels by nicotine in undifferentiated MSCs, these changes were not statistically significant. Exposure of MSCs to equivalent concentrations of nicotine consistently elicited stronger responses by CSE and had a more pronounced effect on all studied parameters. Our results suggest that the direct effect of cigarette smoke on MSCs contributes to impaired MSC function, that adds to the nicotine effects. Cigarette smoke extract reduced the migration, proliferation, and osteogenic differentiation in MSCs in vitro, while nicotine alone reduced proliferation. Cigarette smoke impairs the osteogenic and regenerative ability of MSCs in a direct cytotoxic manner. Cytotoxic effect of nicotine alone impairs regenerative ability of MSCs, but it only partly explains cytotoxic effects of cigarette smoke. Direct effect of cigarette smoke, and partly nicotine, on MSCs could contribute to the smoking-related negative impact on long-term bone health, especially in bone healing.

Platelet gel and factors determining its biological activity

A platelet gel (PG) derived from platelet concentrate (PC) is considered as a perspective therapeutic agent with hemostatic and regenerative properties. PG was obtained from PC separated from human peripheral blood by automatic apheresis by adding human thrombin (30 U/ml). We compared the proliferation of human mesenchymal stromal cells (MSCs) in vitro in the presence of PG and the dependence of gel density on excess of fibrinogen, the presence of calcium chloride, calcium gluconate, and aprotinin. PG was formed from CT in the presence of thrombin during 5–10 minutes. PG as gel-like fibrin membrane contained platelets and an admixture of leukocytes, and was capable to enhance the proliferation of MSCs in vitro. The presence of calcium gluconate (10 mg/ml) increased in the presence of PG the rate of MSCs proliferation in vitro. The presence of aprotinin in PG at a concentration of 10–1000 KIU/ml caused a dose-dependent decrease in the rate of gel biodegradation and did not affect the ability of PG to stimulate the proliferation of human MSCs in vitro.

THE EFFECT OF CHRONOLOGICAL AGE AND GENDER AND DIFFERENT SOURCE OF TISSUE ON THE IN VITRO PROPERTIES OF MESENCHYMAL STROMAL CELLS

AbstractObjectivesOsteoarthritis is a common articular cartilage disorder and causes a significant global disease burden. Articular cartilage has a limited capacity of repair and there is increasing interest in the use of cell-based therapies to facilitate repair including the use of Mesenchymal Stromal Cells (MSCs). There is some evidence in the literature that suggests that advancing age and gender is associated with declining MSC function, including reduced proliferation and differentiation potential, and greater cellular apoptosis. In our study, we first performed a systematic review of the literature to determine the effects of chronological age and gender on the in vitro properties of MSCs, and then performed a laboratory study to investigate these properties.Methods and ResultsWe initially conducted a PRISMA systematic review of the literature to review the evidence base for the effects of chronological age and gender on the in vitro properties of MSCs including cell numbers, expansion, cell surface characterization and differentiation potential. This was followed by laboratory-based experiments to assess these properties. Compare the extent of the effect of age on MSC cell marker expression, proliferation and pathways. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the synovium, fat pad and bone fragments using a method developed in our laboratory. The growth kinetics was determined by calculating the population doublings per day. Following expansion in culture, MSCs at P2 were characterised for a panel of cell surface markers using flow cytometry. The cells were positive for CD73, CD90 and CD105, and negative for antibody cocktail (eg included CD34, CD45). The differentiation potential of the MSCs was assessed through tri-lineage differentiation assays. At P2 after extracting RNA, we investigate the gene analysis using Bulk seq. Clear differences between the younger and older patients and gender were indicated.ConclusionsChronological age and gender-related changes in MSC function have important implications on the use of these cells in clinical applications for an ageing population. The results from this study will be used to plan further work looking at the effects of chronological age and gender on cellular senescence and identify pathways that could be targeted to potentially reverse any age and gender-related changes.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

EV products obtained from iPSC-derived MSCs show batch-to-batch variations in their ability to modulate allogeneic immune responses in vitro.

Mesenchymal stromal cells (MSCs) have demonstrated therapeutic potential in diverse clinical settings, largely due to their ability to produce extracellular vesicles (EVs). These EVs play a pivotal role in modulating immune responses, transforming pro-inflammatory cues into regulatory signals that foster a pro-regenerative milieu. Our previous studies identified the variability in the immunomodulatory effects of EVs sourced from primary human bone marrow MSCs as a consistent challenge. Given the limited proliferation of primary MSCs, protocols were advanced to derive MSCs from GMP-compliant induced pluripotent stem cells (iPSCs), producing iPSC-derived MSCs (iMSCs) that satisfied rigorous MSC criteria and exhibited enhanced expansion potential. Intriguingly, even though obtained iMSCs contained the potential to release immunomodulatory active EVs, the iMSC-EV products displayed batch-to-batch functional inconsistencies, mirroring those from bone marrow counterparts. We also discerned variances in EV-specific protein profiles among independent iMSC-EV preparations. Our results underscore that while iMSCs present an expansive growth advantage, they do not overcome the persistent challenge of functional variability of resulting MSC-EV products. Once more, our findings accentuate the crucial need for batch-to-batch functional testing, ensuring discrimination of effective and ineffective MSC-EV products for considered downstream applications.

Silver-releasing bioactive glass nanoparticles for infected tissue regeneration

Bacterial infections represent a formidable challenge, often leaving behind significant bone defects post-debridement and necessitating prolonged antibiotic treatments. The rise of antibiotic-resistant bacterial strains further complicates infection management. Bioactive glass nanoparticles have been presented as a promising substitute for bone defects and as carriers for therapeutic agents against microorganisms. Achieving consistent incorporation of ions into BGNs has proven challenging and restricted to a maximum ion concentration, especially when reducing the particle size. This study presents a notable achievement in the synthesis of 10 nm-sized Ag-doped bioactive glass nanoparticles (Ag-BGNs) using a modified yet straightforward Stöber method. The successful incorporation of essential elements, including P, Ca, Al, and Ag, into the glass structure at the intended concentrations (i.e., CaO wt% above 20 %) was confirmed by EDS, signifying a significant advancement in nanoscale biomaterial engineering. While exhibiting a spherical morphology and moderate dispersity, these nanoparticles tend to form submicron-sized aggregates outside of a solution state. The antibacterial effectiveness against MRSA was established across various experimental conditions, with Ag-BGNs effectively sterilizing planktonic bacteria without the need for antibiotics. Remarkably, when combined with oxacillin or fosfomycin, Ag-BGNs demonstrated a potent synergistic effect, restoring antibacterial capabilities against MRSA strains resistant to these antibiotics when used alone. Ag-BGNs exhibited potential in promoting human mesenchymal stromal cell proliferation, inducing the upregulation of osteoblast gene markers, and significantly contributing to bone regeneration in mice. This innovative synthesis protocol holds substantial promise for the development of biomaterials dedicated to the regeneration of infected tissue.

Reduced Bone Regeneration in Rats With Type 2 Diabetes Mellitus as a Result of Impaired Stromal Cell and Osteoblast Function-A Computer Modeling Study.

Bone has the fascinating ability to self-regenerate. However, under certain conditions, such as type 2 diabetes mellitus (T2DM), this ability is impaired. T2DM is a chronic metabolic disease known by the presence of elevated blood glucose levels that is associated with reduced bone regeneration capability, high fracture risk, and eventual non-union risk after a fracture. Several mechanical and biological factors relevant to bone regeneration have been shown to be affected in a diabetic environment. However, whether impaired bone regeneration in T2DM can be explained due to mechanical or biological alterations remains unknown. To elucidate the relevance of either one, the aim of this study was to investigate the relative contribution of T2DM-related alterations on either cellular activity or mechanical stimuli driving bone regeneration. A previously validated in silico computer modeling approach that was capable of explaining bone regeneration in uneventful conditions of healing was further developed to investigate bone regeneration in T2DM. Aspects analyzed included the presence of mesenchymal stromal cells (MSCs), cellular migration, proliferation, differentiation, apoptosis, and cellular mechanosensitivity. To further verify the computer model findings against in vivo data, an experimental setup was replicated, in which regeneration was compared in healthy and diabetic after a rat femur bone osteotomy stabilized with plate fixation. We found that mechanical alterations had little effect on the reduced bone regeneration in T2DM and that alterations in MSC proliferation, MSC migration, and osteoblast differentiation had the highest effect. In silico predictions of regenerated bone in T2DM matched qualitatively and quantitatively those from ex vivo μCT at 12 weeks post-surgery when reduced cellular activities reported in previous in vitro and in vivo studies were included in the model. The presented findings here could have clinical implications in the treatment of bone fractures in patients with T2DM. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Substrate mechanical properties bias MSC paracrine activity and therapeutic potential.

Mesenchymal stromal cells (MSCs) have significant therapeutic potential due to their ability to differentiate into musculoskeletal lineages suitable for tissue-engineering, as well as the immunomodulatory and pro-regenerative effects of the paracrine factors that these cells secrete. Cues from the extracellular environment, including physical stimuli such as substrate stiffness, are strong drivers of MSC differentiation, but their effects upon MSC paracrine activity are not well understood. This study, therefore sought to determine the impact of substrate stiffness on the paracrine activity of MSCs, analysing both effects on MSC fate and their effect on T-cell and macrophage activity and angiogenesis. The data show that conditioned medium (CM) from MSCs cultured on 0.2 kPa (soft) and 100 kPa (stiff) polyacrylamide hydrogels have differing effects on MSC proliferation and differentiation, with stiff CM promoting proliferation whilst soft CM promoted differentiation. There were also differences in the effects upon macrophage phagocytosis and angiogenesis, with the most beneficial effects from soft CM. Analysis of the media composition identified differences in the levels of proteins including IL-6, OPG, and TIMP-2. Using recombinant proteins and blocking antibodies, we confirmed a role for OPG in modulating MSC proliferation with a complex combination of factors involved in the regulation of MSC differentiation. Together the data confirm that the physical microenvironment has an important influence on the MSC secretome and that this can alter the differentiation and regenerative potential of the cells. These findings can be used to tailor the culture environment for manufacturing potent MSCs for specific clinical applications or to inform the design of biomaterials that enable the retention of MSC activity after delivery into the body. STATEMENT OF SIGNIFICANCE: • MSCs cultured on 100 kPa matrices produce a secretome that boosts MSC proliferation • MSCs cultured on 0.2 kPa matrices produce a secretome that promotes MSC osteogenesis and adipogenesis, as well as angiogenesis and macrophage phagocytosis • IL-6 secretion is elevated in MSCs on 0.2 kPa substrates • OPG, TIMP-2, MCP-1, and sTNFR1 secretion are elevated in MSCs on 100 kPa substrates.

Investigation of the effect of natural astaxanthin on cytotoxicity of mesenchymal stromal cells

The search for new promising dental products that have anti-inflammatory, wound-healing, antibacterial and immunomodulatory effects and at the same time exhibit minimal side effects is quite an urgent task of modern scientific and practical medicine. As part of this work, we have produced four samples of an antioxidant dental gel containing natural astaxanthin. Astaxanthin, which is part of the dental gel, by its chemical structure belongs to the class of carotenoids and is a natural antioxidant present in various amounts in living organisms.
 The first gel sample we made consisted of natural astaxanthin. An additional substance of the second sample of the preventive gel was interferon [12]. Interferon α has the ability to stimulate the phagocytic activity of macrophages, as well as the cytotoxic activity of T cells and NK cells. It has an indirect antiviral effect, increasing the body’s resistance to viral infections and modulating the immune system’s response aimed at neutralizing viruses or destroying infected cells. The third sample of dental gel contained polyprenol. Polyprenols are precursors of the most important transport lipid of all living organisms, dolichol. Since 2012, polyprenols have been included in the list of the most important essential components of nutrition for normal life support. Polyprenols are included in the list of vital components for the human body, as they are the building material for damaged cells. They help to stabilize cell membranes and restore cells of the brain, heart, liver and other organs, stimulate energy exchange and regenerative potential of body tissues. Polyprenols interact perfectly with antioxidants, increasing the efficiency of their work. The fourth sample included scallop extract as an enhancer of natural astaxanthin.
 Objective: to evaluate the effect of antioxidant gel on the survival and proliferation of mesenchymal stromal cells (MSCs) of the human umbilical cord.
 Materials and methods. Antioxidant gel with astaxanthin, astaxanthin with interferon, astaxanthin with polyprenol and astaxanthin with scallop extract were examined for the viability of MSCs in the MTT test and in the PI test.
 Results. The antioxidant gel with interferon and polyprenol has a proliferative effect on MSCs compared to control samples, which confirms its potential of side effects, antitoxic and wound healing effect. When studying the induction of apoptosis, we found that the percentage of apoptotic cells according to the PI test did not differ from the cells of the control group, which may indicate the absence of apoptosis-inducing effect of drugs on mesenchymal stromal cells.
 Conclusion. Studying the effects of the developed preventive antioxidant gel with astaxanthin, astaxanthin and interferon, astaxanthin and polyprenol, astaxanthin and scallop extract on the stromal cells of the human umbilical cord, no toxic and side effects were recorded.

Influence of Rho/ROCK inhibitor Y-27632 on proliferation of equine mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) isolated form bone marrow and adipose tissue are the most common cells used for cell therapy of orthopedic diseases. MSC derived from different tissues show differences in terms of their proliferation, differentiation potential and viability in prolonged cell culture. This suggests that there may be subtle differences in intracellular signaling pathways that modulate these cellular characteristics. The Rho/ROCK signaling pathway is essential for many cellular functions. Targeting of this pathway by the ROCK inhibitor Y-27632 has been shown to be beneficial for cell viability and proliferation of different cell types. The aim of this study was to investigate the effects of Rho/ROCK inhibition on equine MSC proliferation using bone marrow-derived MSC (BMSC) and adipose-derived MSC (ASC). Primary ASC and BMSC were stimulated with or without 10 ng/mL TGF-β3 or 10 μM Y-27632, as well as both in combination. Etoposide at 10 μM was used as a positive control for inhibition of cell proliferation. After 48 h of stimulation, cell morphology, proliferation activity and gene expression of cell senescence markers p53 and p21 were assessed. ASC showed a trend for higher basal proliferation than BMSC, which was sustained following stimulation with TGF-β3. This included a higher proliferation with TGF-β3 stimulation compared to Y-27632 stimulation (p < 0.01), but not significantly different to the no treatment control when used in combination. Expression of p21 and p53 was not altered by stimulation with TGF-β3 and/or Y-27632 in either cell type. In summary, the Rho/ROCK inhibitor Y-27632 had no effect on proliferation activity and did not induce cell senescence in equine ASC and BMSC.

Effect of La3+ and Mg2+ combined system on bioactivity and osteogenesis of bioinspired La-doped magnesium phosphate composites prepared utilizing the precursor method

Lanthanum (La) compounds have attracted much attention because of low toxicity and biocompatibility. In this study, a series of La-doped magnesium phosphate composites bone repair materials (LMPCs) were prepared by the precursor method, in which the molar ratio of lanthanum was 1%, 3% and 5%, respectively. The results of X-ray powder diffraction, scanning electron microscope and compressive strength test show that La-doped magnesium phosphates (LMPs) synthesized by the precursor method have uniform particle distribution, high purity and excellent degradability. In vitro research results show that LMPCs exhibited reliable biosafety and excellent bioactivity. The introduction of lanthanum enhances the antibacterial activity of Escherichia coli and Staphylococcus aureus of the composite. Furthermore, the combined system of released La3+ and Mg2+ is more conducive to promoting the proliferation of mesenchymal stromal cells (MSC) and angiogenesis of human umbilical vein endothelial cells (HUVECs) when the content of lanthanum is 3% (referred to as LMPC-2). By up-regulating the expression of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2), LMPC-2 is prominent in promoting osteogenic differentiation, and mineralized calcium deposition in vitro. Moreover, the results of micro-CT, H&E and Masson staining confirmed the beneficial effect of the combined system of La3+ and Mg2+ on the repair of rat skull defects in vivo. These achievements will provide new strategies for improving the biological activity of LMPCs and provide reference value for bone repairing in the clinic.

Inhibition of ERK1/2 signaling prevents bone marrow fibrosis by reducing osteopontin plasma levels in a myelofibrosis mouse model

Clonal myeloproliferation and development of bone marrow (BM) fibrosis are the major pathogenetic events in myelofibrosis (MF). The identification of novel antifibrotic strategies is of utmost importance since the effectiveness of current therapies in reverting BM fibrosis is debated. We previously demonstrated that osteopontin (OPN) has a profibrotic role in MF by promoting mesenchymal stromal cells proliferation and collagen production. Moreover, increased plasma OPN correlated with higher BM fibrosis grade and inferior overall survival in MF patients. To understand whether OPN is a druggable target in MF, we assessed putative inhibitors of OPN expression in vitro and identified ERK1/2 as a major regulator of OPN production. Increased OPN plasma levels were associated with BM fibrosis development in the Romiplostim-induced MF mouse model. Moreover, ERK1/2 inhibition led to a remarkable reduction of OPN production and BM fibrosis in Romiplostim-treated mice. Strikingly, the antifibrotic effect of ERK1/2 inhibition can be mainly ascribed to the reduced OPN production since it could be recapitulated through the administration of anti-OPN neutralizing antibody. Our results demonstrate that OPN is a novel druggable target in MF and pave the way to antifibrotic therapies based on the inhibition of ERK1/2-driven OPN production or the neutralization of OPN activity.

Circulating Endothelial Progenitor and Mesenchymal Stromal Cells as Biomarkers for Monitoring Disease Status and Responses to Exercise.

Noncommunicable chronic diseases, such as obesity, cardiovascular disease (CVD), and type 2 diabetes (T2D), pose significant health challenges globally. Important advances have been made in the understanding of the pathophysiologal mechanisms and treatment of noncommunicable diseases in recent years. Lack of physical activity is a primary contributor to many noncommunicable diseases including metabolic syndrome, T2D, CVD, and obesity. Certain diabetes medications and non-pharmaceutical interventions, such as physical activity and exercise, are shown to be effective in decreasing the CVD risks associated with heart disease, stroke, obesity, prediabetes, and T2D. The ability to measure and analyze circulating adult stem cells (ASCs) has gained particular interest due to their potential to identify at-risk individuals and implications in various therapeutics. Therefore, the purpose of this narrative review is to (1) provide an overview of ASCs; specifically endothelial progenitor cells (EPCs) and mesenchymal stromal cells (MSCs), (2) describe the responses of these cells to acute and chronic exercise, and (3) highlight the potential effect of exercise on EPCs and MSCs in aging and disease. EPCs are circulating cells, abundantly available in peripheral blood, bone marrow, and umbilical cord, and are defined by cell surface markers such as CD34+. EPCs are expected to play an important role in angiogenesis and neovascularization and have been implicated in the treatment of CVD. MSCs are essential for maintaining tissue and organ homeostasis. MSCs are defined as multipotent heterogeneous cells that can proliferate in vitro as plastic-adherent cells, have fibroblast-like morphology, form colonies in vitro, and can differentiate into ostyeoblasts, adipocytes, chondroblasts, and myoblasts. In the presence of aging and disease, EPCs and MSCs decrease in quantity and functional capacity. Importantly, exercise facilitates EPC differentiation and production from bone marrow and also helps to promote migration and homing to the hypoxic and damaged tissue which in turn improve angiogenesis and vasculogenesis. Similarly, exercise stimulates increases in proliferation and migratory activity of MSCs. Despite the reported benefits of exercise on EPC and MSC number and function, little is known regarding the optimal exercise prescription for aging and clinical populations. Moreover, the interactions between medications and exercise on EPCs and MSCs is currently unclear. Use of ASCs as a biomarker have the potential to revolutionize the management of patients with a variety of metabolic and obesity related disorders and also pro-inflammatory diseases. Further investigation of clinical entities are urgently needed to understand the implications of interventions such as exercise, diet, and various medications on EPC and MSC quantity and function in aging and clinical populations.

Injectable nanoporous microgels generate vascularized constructs and support bone regeneration in critical-sized defects

Large and aberrant bone fractures require ossification and concomitant vascularization for proper healing. Evidence indicates that osteogenesis and vessel growth are coupled in bone fractures. Although the synergistic role of endothelial cells has been recognized, vascularizing large bone grafts remains a challenge and has apprehended the clinical translation of engineered bone constructs. Here, we describe a facile method to fabricate vascularized constructs using chitosan and gelatin-based microgels that promote osteogenesis of human mesenchymal stromal cells (MSC) while supporting endothelial sprouting and network formation. The microgels are enzymatically degradable and had a high hydration rate with a volume swelling ratio of ~ 493% and a polymer density of ~ 431 mg/cm3, which is comparable to that of native skeletal tissues. AFM indentation of the surface showed an average Young’s modulus of 189 kPa, falling in a range that is conducive to both osteogenesis and vasculogenesis. The osteogenic microgel containing chitosan, gelatin, and hydroxyapatite, mimicking the bone matrix, supported robust attachment, proliferation, and differentiation of MSC. On the other hand, the vasculogenic microgels containing only gelatin, enriched endothelial phenotype and enabled vascular networks formation when embedded in 3D matrices. Combining the two types of microgels created a hybrid construct that sustained the functions of both osteogenic and vasculogenic microgels and enhanced one another. Using a murine model, we also show that the osteogenic microgels regenerate bone in a critical-sized defect with > 95% defect closure by week 12. These multifunctional microgels can be administered minimally invasively and can conformally fill large bone defects. This work lays the foundation to establish principles of designing multiphasic scaffolds with tissue-specific biophysical and biochemical properties for regenerating vascularized and interfacial tissues.

Abstract P3081: Glypican 3 Regulates Diabetes Induced Mesenchymal Stromal Cells Dysfunctions

Mesenchymal stromal cells (MSC) based therapies are considered as an ideal stem cell-based treatment for cardiovascular diseases due to their immunosuppressive characteristics, anti-inflammatory properties, and differentiation potential. Diabetic microenvironment encompassing high glucose, inflammation, hypoxic conditions, and reactive oxygen species content has deleterious effects on the functionality of MSCs. The purpose of this study was to understand the molecular basis of diabetes-induced MSC dysfunction and potentially reversing these dysfunctions to enhance cell-based therapeutics for myocardial repair in diabetic patients. we isolated MSCs from bone marrow of mice from db/+ non-diabetic (WT-MSC) and diabetic mice (db/db-MSC) and examined the effect of diabetes on MSC differentiation, proliferation, angiogenesis, and immunomodulation in vitro, and their reparative functions on myocardial injury repair post-MI in mice. Compared to WT-MSC, MSCs isolated from bone marrow of diabetic mice showed impaired differentiation, decreased proliferation, reduced immunomodulatory activities and impairment to promote endothelial cell tubulogenesis. Our data also shows diminished functional activity of diabetic MSCs to improve post-MI cardiac functions. Furthermore, we found that glypican-3 (GPC3), a heparan sulfate proteoglycan, is highly upregulated in diabetic MSCs when compared to WT-MSC. GPC3 overexpression in WT-MSC showed decreased proliferation, lowers the immunosuppression activity, and reduced vascular tube formation. Interestingly, GPC3 knockdown in WT-MSC showed increased proliferation, improved the immunosuppression activity, and enhanced vascular tube formation. Our data also showed that, knockdown of GPC3 in diabetic MSCs restore their functions. In conclusion, these data suggest that diabetes increases GPC3 expression in MSC thereby impairs MSC functions under diabetic condition and knockdown of GPC3 in diabetic MSC rescued diabetes-induced dysfunctions. Ongoing studies are testing whether ex vivo GPC3 knockdown in db/db MSCs will rescue their defective cardiac reparative activities, post-MI.

Engineered Cell‐Secreted Extracellular Matrix Modulates Cell Spheroid Mechanosensing and Amplifies Their Response to Inductive Cues for the Formation of Mineralized Tissues (Adv. Healthcare Mater. 10/2022)

Mesenchymal Stromal Cells In article number 2102337 by J. Kent Leach and co-workers, an engineered cell-secreted extracellular matrix (ECM) is incorporated into mesenchymal stromal cell (MSC) spheroids to promote cell–cell and cell–matrix interactions and enhance MSC survival and differentiation. Activation of YAP through integrin binding to the incorporated ECM promotes MSC proliferation and osteogenic differentiation, highlighting the therapeutic potential of ECM-loaded spheroids as building blocks for bone repair.

Hypoxia preconditioning elicit differential response in tissue-specific MSCs via immunomodulation and exosomal secretion.

Mesenchymal stromal cells (MSCs) are emerging as an ideal candidate for regenerative medicine. It is known that the culture conditions impact the cellular properties of MSCs and their therapeutic behavior. Moreover, maintenance of MSCs in low oxygen tension for a short duration has shown to be beneficial for MSCs as it is similar to that of their physiological niche. However, the precise mechanism through which hypoxia pre-conditioning affects MSCs is not clear yet. Thus, in this study, we have investigated the effect of hypoxia exposure (1% O2) on tissue-specific MSCs over a period of time under serum-free culture conditions and evaluated the changes in expression of immuno-modulatory molecules and exosome biogenesis and secretion markers. It was observed that all MSCs responded differentially towards hypoxia exposure as indicated by the expression of HIF-1α. Moreover, this short-term exposure did not induce any changes in MSCs cellular morphology, proliferation rate, and surface marker profiling. In addition, we observed an enhancement in the expression of immunomodulatory factors (HLA-G, PGE-2, and IDO) after hypoxia exposure of 12 to 24h in all tissue-specific MSCs. Interestingly, we have also observed the upregulation in exosome secretion that was further corelated to the upregulation of expression of exosome biogenesis and secretion markers (ALIX, TSG101, RAB27a, RAB27b). Though there was a differential response of MSCs where WJ-MSCs and BM-MSCs showed upregulation of these markers at 6-12h of hypoxia pre-conditioning, while AD-MSCs showed similar changes beyond 24h of hypoxia exposure.

Improvement of Mesenchymal Stromal Cell Proliferation and Differentiation via Decellularized Extracellular Matrix on Substrates With a Range of Surface Chemistries.

Decellularized extracellular matrix (dECM) deposited by mesenchymal stromal cells (MSCs) has emerged as a promising substrate for improved expansion of MSCs. To date, essentially all studies that have produced dECM for MSC expansion have done so on tissue culture plastic or glass. However, substrate surface chemistry has a profound impact on the adsorption of proteins that mediate cell-material interactions, and different surface chemistries can cause changes in cell behavior, ECM deposition, and the in vivo response to a material. This study tested the hypothesis that substrate surface chemistry impacts the deposition of ECM and its subsequent bioactivity. This hypothesis was tested by producing glass surfaces with various surface chemistries (amine, carboxylic acid, propyl, and octyl groups) using silane chemistry. ECM was deposited by an immortalized MSC line, decellularized, and characterized through SDS-PAGE and immunofluorescence microscopy. No significant difference was observed in dECM composition or microarchitecture on the different surfaces. The decellularized surfaces were seeded with primary MSCs and their proliferation and differentiation were assessed. The presence of dECM improved the proliferation of primary MSCs by ~100% in comparison to surface chemistry controls. Additionally, the adipogenesis increased by 50–90% on all dECM surfaces in comparison to surface chemistry controls, and the osteogenesis increased by ~50% on the octyl-modified surfaces when dECM was present. However, no statistically significant differences were observed within the set of dECM surfaces or control surfaces. These results support the null hypothesis, meaning surface chemistry (over the range tested in this work) is not a key regulator of the composition or bioactivity of MSC-derived dECM. These results are significant because they provide an important insight into regenerative engineering technologies. Specifically, the utilization of dECM in stem cell manufacturing and tissue engineering applications would require the dECM to be produced on a wide variety of substrates. This work indicates that it can be produced on materials with a range of surface chemistries without undesired changes in the bioactivity of the dECM.