Bone Marrow-derived Mesenchymal Stem Cells Research Articles

Precision Delivery of Human Bone Marrow-Derived Mesenchymal Stem Cells Into the Pancreas Via Intra-arterial Injection Prevents the Onset of Diabetes.

Mesenchymal stem cells (MSCs) are a promising therapy to potentially treat diabetes given their potent anti-inflammatory and immune-modulatory properties. While these regenerative cells have shown considerable promise in cell culture, their clinical translation has been challenging. In part, this can be attributed to these cells not reaching the pancreas to exert their regenerative effects following conventional intravenous (IV) injection, with the majority of cells being trapped in the lungs in the pulmonary first-pass effect. In the present study, we will therefore examine whether direct delivery of MSCs to the pancreas via an intra-arterial (IA) injection can improve their therapeutic efficacy. Using a mouse model, in which repetitive low doses of STZ induced a gentle, but progressive, hyperglycemia, we tested bone marrow-derived MSCs (BM-MSCs) which we have shown are enriched with pro-angiogenic and immunomodulatory factors. In cell culture studies, BM-MSCs were shown to preserve islet viability and function following exposure to proinflammatory cytokines (IFN-γ, IL-1β, and TNF-α) through an increase in pAkt. When tested in our animal model, mice receiving IV BM-MSCs were not able to mitigate the effects of STZ, however those which received the same dose and batch of cells via IA injection were able to maintain basal and dynamic glycemic control, to similar levels as seen in healthy control animals, over 10 days. This study shows the importance of considering precision delivery approaches to ensure cell-based therapies reach their intended targets to enable them to exert their therapeutic effects.

Stem cell grafts enhance endogenous extracellular vesicle expression in the stroke brain

Endogenous brain repair occurs following an ischemic stroke but is transient, thus unable to fully mount a neuroprotective response against the evolving secondary cell death. Finding a treatment strategy that may render robust and long-lasting therapeutic effects stands as a clinically relevant therapy for stroke. Extracellular vesicles appear to be upregulated after stroke, which may represent a candidate target for neuroprotection. In this study, we probed whether transplanted stem cells could enhance the expression of extracellular vesicles to afford stable tissue remodeling in the ischemic stroke brain. Aged rats were initially exposed to the established ischemic stroke model of middle cerebral artery occlusion then received intravenous delivery of either bone marrow-derived mesenchymal stem cell transplantation or vehicle. A year later, the animals were assayed for brain damage, inflammation, and extracellular vesicle expression. Our findings revealed that while core infarction was not reduced, the stroke animals transplanted with stem cells displayed a significant reduction in peri-infarct cell loss that coincided with downregulated Iba1-labeled inflammatory cells and upregulated CD63-positive extracellular vesicles that appeared to be co-localized with GFAP-positive astrocytes. Interestingly, grafted stem cells were not detected at one year post-transplantation period, suggesting that the extracellular vesicles likely originated within the host brain. That long-lasting functional benefits persisted in the absence of surviving transplanted stem cells, but with upregulation of endogenous extracellular vesicles, advances the concept that transplantation of stem cells acutely after stroke propels host extracellular vesicles to the ischemic brain, altogether promoting chronic brain remodeling.

Mesenchymal stem cells biological and biotechnological advances: Implications for clinical applications

BackgroundMesenchymal stem cells (MSCs) are multipotent stem cells that are able to differentiate into multiple lineages including bone, cartilage, muscle and fat. They hold immunomodulatory properties and therapeutic ability to treat multiple diseases, including autoimmune and chronic degenerative diseases. In this article, we reviewed the different biological properties, applications and clinical trials of MSCs. Also, we discussed the basics of manufacturing conditions, quality control, and challenges facing MSCs in the clinical setting. MethodsExtensive review of the literature was conducted through the databases PubMed, Google Scholar, and Cochrane. Papers published since 2015 and covering the clinical applications and research of MSC therapy were considered. Furthermore, older papers were considered when referring to pioneering studies in the field. ResultsThe most widely studied stem cells in cell therapy and tissue repair are bone marrow-derived mesenchymal stem cells. Adipose tissue-derived stem cells became more common and to a lesser extent other stem cell sources e.g., foreskin derived MSCs. MSCs therapy were also studied in the setting of COVID-19 infections, ischemic strokes, autoimmune diseases, tumor development and graft rejection. Multiple obstacles, still face the standardization and optimization of MSC therapy such as the survival and the immunophenotype and the efficiency of transplanted cells. MSCs used in clinical settings displayed heterogeneity in their function despite their extraction from healthy donors and expression of similar surface markers. ConclusionMesenchymal stem cells offer a rising therapeutic promise in various diseases. However, their potential use in clinical applications requires further investigation.

Aqueous Supramolecular Transformations of Motor Bola-Amphiphiles at Multiple Length-Scale.

Molecular motor amphiphiles have already been widely attempted for dynamic nanosystems across multiple length-scale for developments of small functional materials, including controlling macroscopic foam properties, amplifying motion as artificial molecular muscles, and serving as extracellular matrix mimicking cell scaffolds. However, limiting examples of bola-type molecular motor amphiphiles are considered for constructing macroscopic biomaterials. Herein, this work presents the designed two second generation molecular motor amphiphiles, motor bola-amphiphiles (MBAs). Aside from the photoinduced motor rotation of MBAs achieved in both organic and aqueous media, the rate of recovering thermal helix inversion step can be controlled by the rotor part with different steric hindrances. Dynamic assembled structures of MBAs are observed under (cryo)-transmission electron microscopy (TEM). This dynamicity assists MBAs in further assembling as macroscopic soft scaffolds by applying a shear-flow method. Upon photoirradiation, the phototropic bending function of MBA scaffolds is observed, demonstrating the amplification of molecular motion into macroscopic phototropic bending functions at the macroscopic length-scale. Since MBAs are confirmed with low cytotoxicity, human bone marrow-derived mesenchymal stem cells (hBM-MSCs) can grow on the surface of MBA scaffolds. These results clearly demonstrate the concept of designing MBAs for developing photoresponsive dynamic functional materials to create new-generation soft robotic systems and cell-material interfaces.

Bibliometric Analysis of MSC-based Therapies for Non-COVID-19 ARDS

Background: Acute respiratory distress syndrome (ARDS) poses a significant challenge as it lacks specific treatments and can occur due to various etiologies. Mesenchymal stem cells (MSCs) have emerged as a promising cell-based therapy for ARDS due to their immunomodulatory, anti-inflammatory, and anti-fibrotic properties. Despite encouraging findings from preclinical studies, clinical evidence supporting the efficacy of MSCs in non-COVID-19 ARDS remains insufficient. Methods: We conducted a systematic search of three major databases (Web of Science Core Collection, Scopus, and PubMed) to identify original articles focusing on MSCs in non-COVID-19 ARDS. Subsequently, we employed the bibliometric package in R Studio to analyze and visualize bibliometric indicators derived from the retrieved articles. Results: Our analysis of 244 original studies revealed a notable trend in research on MSCs and non-COVID-19 ARDS. While the number of publications in this area saw an increase beginning in 2007, it exhibited a decline after 2019, with only 20 articles published in 2022. Notably, a significant proportion (131/244) of these studies originated from Chinese scholars. MSC derivatives emerged as a recent research focus due to their unique advantages as an alternative to MSCs. Specifically, umbilical cord/placental-derived MSCs have gained traction, surpassing the use of bone marrow-derived MSCs by 2022. The route of delivery is still mainly intravenous. Despite the potential advantages of the intratracheal route for lung-related diseases, the intravenous route remains the preferred mode of drug delivery. Conclusion: Research on non-COVID-19 ARDS deserves further attention and investments. Existing studies have primarily focused on MSC derivatives that have shown clinical efficacy. Furthermore, umbilical cord/placental-derived MSCs are expected to replace traditional bone marrow- derived MSCs in research. Intratracheal delivery, which offers advantages for treating pulmonary diseases, still requires extensive experiments to validate.

Vascular endothelial growth factor secretion and immunosuppression are distinct potency mechanisms of human bone marrow mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are investigated as cellular therapeutics for inflammatory bowel diseases and associated perianal fistula, although consistent efficacy remains a concern. Determining host factors that modulate MSCs' potency including their secretion of angiogenic and wound-healing factors, immunosuppression, and anti-inflammatory properties are important determinants of their functionality. We investigated the mechanisms that regulate the secretion of angiogenic and wound-healing factors and immune suppression of human bone marrow MSCs. Secretory analysis of MSCs focusing on 18 angiogenic and wound-healing secretory molecules identified the most abundancy of vascular endothelial growth factor A (VEGF-A). MSC viability and secretion of other angiogenic factors are not dependent on VEGF-A secretion which exclude the autocrine role of VEGF-A on MSC's fitness. However, the combination of inflammatory cytokines IFNγ and TNFα reduces MSC's VEGF-A secretion. To identify the effect of intestinal microvasculature on MSCs' potency, coculture analysis was performed between human large intestine microvascular endothelial cells (HLMVECs) and human bone marrow-derived MSCs. HLMVECs do not attenuate MSCs' viability despite blocking their VEGF-A secretion. In addition, HLMVECs neither attenuate MSC's IFNγ mediated upregulation of immunosuppressive enzyme indoleamine 2,3-dioxygenase nor abrogate suppression of T-cell proliferation despite the attenuation of VEGF-A secretion. We found that HLMVECs express copious amounts of endothelial nitric oxide synthase and mechanistic analysis showed that pharmacological blocking reverses HLMVEC-mediated attenuation of MSC's VEGF-A secretion. Together these results suggest that secretion of VEGF-A and immunosuppression are separable functions of MSCs which are regulated by distinct mechanisms in the host.

BMSC loaded photo-crosslinked hyaluronic acid/collagen hydrogel incorporating FG4592 for enhanced cell proliferation and nucleus pulposus differentiation

Intervertebral disc degeneration arises from damage or degeneration of the nucleus pulposus (NP). In this study, we developed a photo-crosslinkable hydrogel incorporating FG4592 to support the growth and differentiation of bone-marrow-derived mesenchymal stem cells (BMSC). Initially, hyaluronic acid was modified with tyramine and combined with collagen to introduce riboflavin as a photo-crosslinker. This hydrogel transitioned from liquid to gel upon exposure to blue light in 3 min. The results showed that the hydrogel was biodegradable and had mechanical properties comparable to those of human NP tissues. Scanning electron microscopy after BMSC seeding in the hydrogel revealed an even distribution, and cells adhered to the collagen fibers in the hydrogel with minimal cell mortality. The effect of FG4592 on BMSC proliferation and differentiation was examined, revealing the capability of FG4592 to promote BMSC proliferation and direct differentiation resembling human NP cells. After cultivating BMSCs in the photo-crosslinked hydrogel, there was an upregulation in the expression of glycosaminoglycans, aggrecan, type II collagen, and keratin 19 proteins. Cross-species analyses of rat and human BMSCs revealed consistent results. For potential clinical applications, BMSC loaded with photo-crosslinked hydrogels can be injected into damaged intervertebral disc to facilitate NP regeneration.

Advantages of pooling of human bone marrow-derived mesenchymal stromal cells from different donors versus single-donor MSCs

Mesenchymal stromal cells (MSC) from adult bone marrow are the most commonly used cells in clinical trials. MSCs from single donors are the preferred starting material but suffer from a major setback of being heterogeneous that results in unpredictable and inconsistent clinical outcomes. To overcome this, we developed a method of pooling MSCs from different donors and created cell banks to cater clinical needs. Initially, the master cell banks (MCBs) were created at passage 1 (P1) from the bone marrow MSCs isolated from of nine different donors. At this stage, MCBs from three different donors were mixed in equal proportion and expanded till P3 to create working cell banks. Further, the pooled cells and individual donor MSCs were expanded till P5 and cryopreserved and extensively characterised. There was a large heterogeneity among the individual donor MSCs in terms of growth kinetics (90% Coefficient of variation (CV) for cell yield and 44% CV for population doubling time at P5), immunosuppressive ability (30% CV at 1:1 and 300% CV at 1:10 ratio), and the angiogenic factor secretion potential (20% CV for VEGF and71% CV for SDF-1). Comparatively, the pooled cells have more stable profiles (60% CV for cell yield and 7% CV for population doubling time at P5) and exhibit better immunosuppressive ability (15% CV at 1:1 and 32% CV at 1:10 ratio ) and consistent secretion of angiogenic factors (16% CV for VEGF and 51% CV for SDF-1). Further pooling does not compromise the trilineage differentiation capacity or phenotypic marker expression of the MSCs. The senescence and in vitro tumourigenicity characteristics of the pooled cells are also similar to those of individual donor MSCs. We conclude that pooling of MSCs from three different donors reduces heterogeneity among individual donors and produces MSCs with a consistent secretion and higher immunosuppressive profile.

Cytokines secreted from bone marrow-derived mesenchymal stem cells promote apoptosis of CD34+ leukemic stem cells as anti-cancer therapy

ObjectiveThe effect of mesenchymal stem cells (MSCs) on the immortal characteristics of malignant cells, particularly hematologic cancer cells, remains a topic of debate, with the underlying mechanisms still requiring further elucidation. We explored the in vitro effect of the bone marrow-derived MSCs (BM-MSCs) on CD34+ leukemic stem cells (LSCs) enriched from the KG1-a cell line by assessing apoptosis, measuring cytokine levels, and examining TERT protein expression. Additionally, the potential signaling pathways implicated in this process, such as P53, PTEN, NF-κB, ERK1/2, Raf-1, and H-RAS, were also investigated. MethodsCD34+ LSCs were enriched from the KG1-a cell line with the magnetic activated cell sorting (MACS) method. Two cell populations (BM-MSCs and CD34+ LSCs) were co-cultured on trans well plates for seven days. Next, CD34+ LSCs were collected and subjected to Annexin V/PI assay, cytokine measurement, and western blotting. ResultsBM-MSCs caused a significant increase in early and late apoptosis in the CD34+LSCs. The significant presence of interleukin (IL)-2 and IL-4 was evident in the co-cultured media. In addition, BM-MSCs significantly increased the protein expression of P53, PTEN, NF-κB, and significantly decreased p-ERK1/2, Raf-1, H-RAS, and TERT. ConclusionThe mentioned effects of IL-2 and IL-4 cytokines released from BM-MSCs on CD34+ LSCs as therapeutic agents were applied by the components of P53, PTEN, NF-κB, p-ERK1/2, Raf-1, and H-RAS signaling pathways.

Exosomes isolated from TNF-α-treated bone marrow mesenchymal stem cells ameliorate pelvic floor dysfunction in rats.

Exosomes derived from bone marrow-derived mesenchymal stem cells (BMSCs) can alleviate the symptoms of pelvic floor dysfunction (PFD) in rats. However, the potential therapeutical effects of exosomes derived from BMSCs treated with tumour necrosis factor (TNF)-α on the symptoms of PFD in rats are unknown. Exosomes extracted from BMSCs treated with or without TNF-α were applied to treat PFD rats. Our findings revealed a significant elevation in interleukin (IL)-6 and TNF-α, and matrix metalloproteinase-2 (MMP2) levels in the vaginal wall tissues of patients with pelvic organ prolapse (POP) compared with the control group. Daily administration of exosomes derived from BMSCs, treated either with or without TNF-α (referred to as Exo and TNF-Exo), resulted in increased void volume and bladder void pressure, along with reduced peak bladder pressure and leak point pressure in PFD rats. Notably, TNF-Exo treatment demonstrated superior efficacy in restoring void volume, bladder void pressure and the mentioned parameters compared with Exo treatment. Importantly, TNF-Exo exhibited greater potency than Exo in restoring the levels of multiple proteins (Elastin, Collagen I, Collagen III, IL-6, TNF-α and MMP2) in the anterior vaginal walls of PFD rats. The application of exosomes derived from TNF-α-treated BMSCs holds promise as a novel therapeutic approach for treating PFD.

Biocompatible 3D-Printed Devices With Adipose Stem Cells in the Regenerative Process of Sciatic Nerve Lesions in Rodent Models: An Experimental Study.

Peripheral nerve injuries are a significant clinical challenge. The rat sciatic nerve serves as an ideal model for studying nerve regeneration. Extensive research has been conducted to unravel the intricate mechanisms involved in peripheral nerve regeneration, aiming to develop effective therapeutic strategies for nerve injury patients. Research including different types of materials that can be used as nerve guides like synthetic polymershave been investigated for their biocompatibility and molding properties. Among multiple stem cell types, adipose-derived stem cells (ASCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), and induced pluripotent stem cells (iPSCs) have shown neuroprotective and regenerative important properties. The purposes of our study were to develop a protocol for rat sciatic nerve injury treated with 3D-printed guide and adipose stem cells to investigate nerve regeneration through histologic examination and biomechanical characteristics of muscular tissue. We use 20 (100%) male Wistar rats, measuring between 350 g ± 35 g, whounderwent complete transection of the right sciatic nerve, resulting in a 1 cm defect. The group was separated into three subgroups: the first subgroup (n = 8) was treated with a 3D-printed guide with adiposestem cells, the second subgroup (n = 8) was treated with a 3D-printed guide without adiposestem cells, and the third subgroup (n = 4) was the control group. At four, eight, and 12 weeks, we measured with ultrasonography the grade of muscular atrophy. At 12 weeks, we harvested the sciatic nerve and performed a histological examination and mechanical investigation of the tibialis anterior muscle. On the examined specimen of the first subgroup, cross-sectioned nerve structures were present, surrounded by a mature fibro-adipose connective tissue, with blood vessels. In the second subgroup, no nerve structure was observed on the examined sections, but in the polymorphic inflammatory infiltrate and control group, no signs of regeneration were found. The present study shows a promising potential when utilizing adipose stem cell-based therapies for promoting peripheral nerve regeneration following large (>1 cm) nerve defects knowing that at this size, regeneration is impossible with known treatments.

Comparison of infant bone marrow- and umbilical cord-derived mesenchymal stem cells in multilineage differentiation

We compared infant bone marrow-derived mesenchymal stem cells (infant BMSCs) with umbilical cord-derived mesenchymal stem cells (UCSCs) by assessing multilineage differentiation. Proliferation was gauged through changes in cell numbers and doubling time. Senescence-related genes (p16, p21, and p53), senescence-associated β-galactosidase (SA-β-gal), and γH2AX immunofluorescence determined senescence presence. Superoxide dismutases (SODs) and genes related to various differentiations were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Differentiation was confirmed through histochemical, immunohistochemical, and immunofluorescence staining. Infant BMSCs surpassed UCSCs in proliferation. Infant BMSCs exhibited lower senescence-related gene expression at late passages, upregulated antioxidant enzymes during early passages, and reduced SA-β-gal staining. Chondrogenic gene expression (SOX9, COL2, and COL10) was enhanced in infant BMSCs, along with improved immunohistochemical staining. Infant BMSCs showed higher expression of osteogenic (ALP and OCN) and adipogenic (PPARγ and LPL) genes, confirmed by histochemical staining. However, UCSCs had higher expression of tenogenic genes (MMP3, SCX, DCN, and TNC). Hepatogenic differentiation potential was similar, with no significant difference in hepatogenic gene expression (ALB and TAT). Compared to UCSCs, infant BMSCs demonstrated superior proliferation, reduced senescence, increased antioxidant capacity, and enhanced differentiation potential toward chondrogenic, osteogenic, and adipogenic lineages.

Fabrication and in vitro biological performance of a double-layered nanoparticles-microarc oxidation composite coating on titanium for dental implant application

Sufficient residual alveolar bone volume plays an important role in the success rate and service life of dental implants. However, alveolar bone deficiency is a common clinical phenomenon, and the alveolar bone would be further absorbed by peri-implant infection. Therefore, it is highly desirable to promote peri-implant alveolar bone regeneration and inhibit alveolar bone resorption when the alveolar bone mass is insufficient. For this purpose, a pH-sensitive double-layered nanoparticles-microarc oxidation (MAO) composite coating was fabricated on titanium for dental implant application in this study. The pH-sensitive double-layered nanoparticles were prepared by a poly(L-lactic acid) inner layer and a chitosan outer shell, containing stromal-cell derived factor-1, recombinant human bone morphogenetic protein 2 and osteoprotegerin. The composite coating was fabricated on MAO coating by cross-linking the pH-sensitive double-layered nanoparticles with gelatin. The surface morphology of the composite coating showed that the pH-sensitive double-layered nanoparticles were well distributed and tightly cross-linked in the pores of MAO coating. The composite coating could sustain release the three drugs for more than 30 days. With decreasing pH, the release of osteoprotegerin from the composite coating increased (p < 0.05). In vitro biological studies suggested that the composite coating exhibited no cytotoxicity, and can recruit bone marrow-derived mesenchymal stem cells (BMSCs), promote BMSC differentiation into osteoblasts, and inhibit osteoclast generation. Moreover, with decreasing pH, the inhibitory effect on osteoclast generation was enhanced (p < 0.05). It can be concluded that the fabricated composite coating, which can promote bone regeneration and inhibit bone resorption, has the potential to be applied on the surface of dental implant, especially when the residual alveolar bone is in poor condition.

Atoh1 overexpression promotes Guinea pig bone marrow mesenchymal stem cells to differentiate into neural stem cell

Sensorineural hearing loss (SNHL) is a prevalent condition in otolaryngology. A key obstacle is finding effective strategies for regenerating damaged cochlear hair cells in adult animals. A practical and reliable approach has been developed to create a superior cell source for stem cell transplantation in the inner ear to treat SNHL. Atoh1 is involved in the differentiation of neurons, intestinal secretory cells, and mechanoreceptors including auditory hair cells, and thus plays an important role in neurogenesis. Lentivirus-mediated transfection of bone marrow mesenchymal stem cells (BMSCs) was utilized to achieve stable expression of the essential transcription factor Atoh1, which is crucial for developing auditory hair cells without compromising cell survival. By manipulating the induction conditions through altering the cell growth environment using anti-adherent culture, the synergistic impact of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) was effectively applied to significantly improve the differentiation efficiency of bone marrow-derived mesenchymal stem cells (BMSC) into neural stem cells (NSCs) following Atoh1 transfection, thereby reducing the induction time. The study indicated that the newly proposed transdifferentiation method effectively transformed BMSCs into NSCs in a controlled environment, presenting a potential approach for stem cell transplantation to promote hair cell regeneration.

Sustainably cultured coral scaffold supports human bone marrow mesenchymal stromal cell osteogenesis

The current gold standard grafting material is autologous bone due to its osteoinductive and osteoconductive properties. Autograft harvesting results in donors site morbidity. Coral scaffolds offer a natural autograft alternative, sharing the density and porosity of human bone. This study investigated the biocompatibility and osteogenic potential of a novel, sustainably grown Pocillopora scaffold with human bone marrow-derived mesenchymal stromal cells (MSCs). The coral-derived scaffold displays a highly textured topography, with concavities of uniform size and a high calcium carbonate content. Large scaffold samples exhibit compressive and diametral tensile strengths in the range of trabecular bone, with strengths likely increasing for smaller particulate samples. Following the in vitro seeding of MSCs adjacent to the scaffold, the MSCs remained viable, continued proliferating and metabolising, demonstrating biocompatibility. The seeded MSCs densely covered the coral scaffold with organized, aligned cultures with a fibroblastic morphology. In vivo coral scaffolds with MSCs supported earlier bone and blood vessel formation as compared to control constructs containing TCP-HA and MSCs. This work characterized a novel, sustainably grown coral scaffold that was biocompatible with MSCs and supports their in vivo osteogenic differentiation, advancing the current repertoire of biomaterials for bone grafting.

IL-1β + TGF-β2 dual-licensed mesenchymal stem cells have reduced major histocompatibility class I expression and positively modulate tenocyte migration, metabolism, and gene expression.

The study objectives were to 1) determine the mesenchymal stem cell (MSC) surface expression of major histocompatibility complex (MHC) class I and transcriptome-wide gene expression changes following IL-1β + TGF-β2 dual licensing and 2) evaluate if IL-1β + TGF-β2 dual-licensed MSCs had a greater ability to positively modulate tenocyte function compared to naive MSCs. Equine bone marrow-derived MSCs from 6 donors and equine superficial digital flexor tenocytes from 3 donors. Experiments were performed in vitro. Flow cytometry and bulk RNA sequencing were utilized to determine naive and dual-licensed MSC phenotype and transcriptome-wide changes in gene expression. Conditioned media were generated from MSCs and utilized in tenocyte cell culture assays as a method to determine the effect of MSC paracrine factors on tenocyte function. Dual-licensed MSCs have a reduced expression of MHC class I and exhibit enrichment in functional pathways associated with the extracellular matrix, cell signaling, and tissue development. Additionally, dual-licensed MSC-conditioned media significantly improved in vitro tenocyte migration and metabolism to a greater degree than naive MSC-conditioned media. In tenocytes exposed to IL-1β, dual-licensed conditioned media also positively modulated tenocyte gene expression. Our data indicate that conditioned media containing paracrine factors secreted from dual-licensed MSCs significantly modulates in vitro tenocyte function, which may confer benefits in vivo to healing tendons following injury. Additionally, due to reduced MHC class I expression in dual-licensed MSCs, this technique may also provide an avenue to provide an effective "off-the-shelf" allogenic source of MSCs.

Equine mesenchymal stem cell-derived extracellular vesicle productivity but not overall yield is improved via 3-D culture with chemically defined media.

Mesenchymal stem cell (MSC) extracellular vesicles (EVs) have emerged as a biotherapeutic for osteoarthritis; however, manufacturing large quantities is not practical using traditional monolayer (2-D) culture. We aimed to examine the effects of 3-D and 2-D culture 2 types of media: Dulbecco modified Eagle medium and a commercially available medium (CM) on EV yield. Banked bone marrow-derived MSCs (BM-MSCs) from 6 healthy, young horses were used. 4 microcarriers (collagen-coated polystyrene, uncoated polystyrene, collagen-coated dextran, and uncoated dextran) were tested in static and bioreactor cultures, and the optimal microcarrier was chosen. The BM-MSCs were inoculated into a bioreactor with collagen-coated dextran microcarriers at 5,000 cells/cm2 or onto culture dishes at 4,000 cells/cm2 in either Dulbecco modified Eagle medium or CM media. Supernatants were obtained for metabolite and pH analysis. The BM-MSCs were expanded until confluent (2-D) or for 7 days (3-D) when the 48-hour EV collection period commenced using EV-depleted media. Extracellular vesicles were isolated and characterized via nanoparticle tracking analysis, Western blot, transmission electron microscopy, and protein quantification. The BM-MSCs were harvested, quantified, and immunophenotyped. The number of EVs isolated was not improved by 3-D culture or CM media, however, the CM 3-D condition improved the number of EVs produced per BM-MSC over the CM 2-D condition (mean ± SD: 306 ± 99 vs 37 ± 22, respectively). Glucose decreased and lactate and ammonium accumulated in 3-D culture. Surface markers of stemness exhibited reduced expression in 3-D culture. Optimization of our 3-D culture methods could improve BM-MSC expansion and thus EV yield.

Bifunctional mesoporous glasses for bone tissue engineering: Biological effects of doping with cerium and polyphenols in 2D and 3D in vitro models

This study evaluates the cytocompatibility of cerium-doped mesoporous bioactive glasses (Ce-MBGs) loaded with polyphenols (Ce-MBGs-Poly) for possible application in bone tissue engineering after tumour resection. We tested MBGs powders and pellets on 2D and 3D in vitro models using human bone marrow-derived mesenchymal stem cells (hMSCs), osteosarcoma cells (U2OS), and endothelial cells (EA.hy926). Promisingly, at a low concentration in culture medium, Poly-loaded MBGs powders containing 1.2 mol% of cerium inhibited U2OS metabolic activity, preserved hMSCs viability, and had no adverse effects on EA.hy926 migration. Moreover, the study discussed the possible interaction between cerium and Poly, influencing anti-cancer effects. In summary, this research provides insights into the complex interactions between Ce-MBGs, Poly, and various cell types in distinct 2D and 3D in vitro models, highlighting the potential of loaded Ce-MBGs for post-resection bone tissue engineering with a balance between pro-regenerative and anti-tumorigenic activities.

Knockdown the moyamoya disease susceptibility gene, RNF213, upregulates the expression of basic fibroblast growth factor and matrix metalloproteinase-9 in bone marrow derived mesenchymal stem cells.

Moyamoya disease (MMD) is a chronic, progressive cerebrovascular occlusive disease. Ring finger protein 213 (RNF213) is a susceptibility gene of MMD. Previous studies have shown that the expression levels of angiogenic factors increase in MMD patients, but the relationship between the susceptibility gene RNF213 and these angiogenic mediators is still unclear. The aim of the present study was to investigate the pathogenesis of MMD by examining the effect of RNF213 gene knockdown on the expression of matrix metalloproteinase-9 (MMP-9) and basic fibroblast growth factor (bFGF) in rat bone marrow-derived mesenchymal stem cells (rBMSCs). Firstly, 40 patients with MMD and 40 age-matched normal individuals (as the control group) were enrolled in the present study to detect the levels of MMP-9 and bFGF in serum by ELISA. Secondly, Sprague-Dawley male rat BMSCs were isolated and cultured using the whole bone marrow adhesion method, and subsequent phenotypic analysis was performed by flow cytometry. Alizarin red and oil red O staining methods were used to identify osteogenic and adipogenic differentiation, respectively. Finally, third generation rBMSCs were transfected with lentivirus recombinant plasmid to knockout expression of the RNF213 gene. After successful transfection was confirmed by reverse transcription-quantitative PCR and fluorescence imaging, the expression levels of bFGF and MMP-9 mRNA in rBMSCs and the levels of bFGF and MMP-9 protein in the supernatant of the culture medium were detected on the 7th and 14th days after transfection. There was no significant difference in the relative expression level of bFGF among the three groups on the 7th day. For the relative expression level of MMP-9, there were significant differences on the 7th day and 14th day. In addition, there was no statistically significant difference in the expression of bFGF in the supernatant of the RNF213 shRNA group culture medium, while there was a significant difference in the expression level of MMP-9. The knockdown of the RNF213 gene affects the expression of bFGF and MMP-9. However, further studies are needed to determine how they participate in the pathogenesis of MMD. The findings of the present study provide a theoretical basis for clarifying the pathogenesis and clinical treatment of MMD.

Cell-Specific Impacts of Surface Coating Composition on Extracellular Vesicle Secretion.

Biomaterial properties have recently been shown to modulate extracellular vesicle (EV) secretion and cargo; however, the effects of substrate composition on EV production remain underexplored. This study investigates the impacts of surface coatings composed of collagen I (COLI), fibronectin (FN), and poly l-lysine (PLL) on EV secretion for applications in therapeutic EV production and to further understanding of how changes in the extracellular matrix microenvironment affect EVs. EV secretion from primary bone marrow-derived mesenchymal stromal cells (BMSCs), primary adipose-derived stem cells (ASCs), HEK293 cells, NIH3T3 cells, and RAW264.7 cells was characterized on the different coatings. Expression of EV biogenesis genes and cellular adhesion genes was also analyzed. COLI coatings significantly decreased EV secretion in RAW264.7 cells, with associated decreases in cell viability and changes in EV biogenesis-related and cell adhesion genes at day 4. FN coatings increased EV secretion in NIH3T3 cells, while PLL coatings increased EV secretion in ASCs. Surface coatings had significant effects on the capacity of EVs derived from RAW264.7 and NIH3T3 cells to impact in vitro macrophage proliferation. Overall, surface coatings had different cell-specific effects on EV secretion and in vitro functional capacity, thus highlighting the potential of substrate coatings to further the development of clinical EV production systems.