Bone Marrow-derived Mesenchymal Stromal Stem Research Articles

The antiapoptotic effects of conditioned medium from bone marrow-derived mesenchymal stromal stem cells on cyclophosphamide-induced testicular damage in rat: An experimental study.

Cyclophosphamide (CP) has some negative effects on the reproductive system. Stem cells and their metabolites are being utilized to enhance fertility after chemotherapy. This study aimed to investigate the impact of conditioned medium (CM) derived from bone marrow mesenchymal stromal stem cells (BM-MSCs) on the toxic effects of CP on testicles. BM-MSCs were isolated, a CM was collected and 25-fold concentrated. 24 male Wistar rats (8 wk, 200-250 gr) were randomly divided into following groups: control, CP, CP+ Dulbecco's Modified Eagle Medium (DMEM), CP+CM. CP was given at a single dose of 100 mg/kg. 2 wk after the CP administration, CM was injected into the testicular efferent duct. Sperm parameters, testicular histopathology, and the level of testosterone were analyzed 2 months after treatment. The expression of B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (Bax) genes were evaluated by real-time polymerase chain reaction. CP had a negative effect on testis histology (p 0.001) and sperm quality (p 0.001). It changed the expression of genes associated with apoptosis (p 0.001). Treatment with CM reduced the expression of Bax (p 0.001), while significantly increasing the expression of Bcl2 (p = 0.01). It improved sperm count (p = 0.03), viability (p 0.001), motility (p 0.001), spermatogonial count (p 0.001), and epithelial thickness of testicular tubules (p = 0.02). These findings suggest that CM produced from BM-MSCs may be valuable for therapeutic approaches in reproductive medicine and may lessen the side effects of CP.

Recent Approaches to Isolating and Culturing Mouse Bone Marrowderived Mesenchymal Stromal Stem Cells.

The isolation and culture of murine Bone Marrow-derived Mesenchymal stromal Stem Cells (mBMSCs) have attracted great interest in terms of the pre-clinical applications of stem cells in tissue engineering and regenerative medicine. In addition, culturing mBMSCs is important for studying the molecular mechanisms of bone remodeling using relevant transgenic mice. Several factors have created challenges in the isolation and high-yield expansion of homogenous mBMSCs; these factors include low frequencies of Bone Marrow-derived mesenchymal stromal Stem Cells (BMSCs) in bone marrow, variation among inbred mouse strains, contamination with Haematopoietic Progenitor Cells (HPCs), the replicative senescence phenotype and cellular heterogeneity. In this review, we provide an overview of nearly all protocols used for isolating and culturing mBMSCs with the aim of clarifying the most important guidelines for culturing highly purified mBMSC populations retaining in vitro and in vivo differentiation potential.

Total body irradiation tremendously impair the proliferation, differentiation and chromosomal integrity of bone marrow-derived mesenchymal stromal stem cells.

Total body irradiation (TBI) is frequently used in hematopoietic stem cell transplantation (HSCT) and is associated with many complications due to radiation injury to the normal cells, including normal stem cells. Nevertheless, the effects of TBI on the mesenchymal stromal stem cell (MSC) are not fully understood. Bone marrow-derived MSCs (BM-MSCs) isolated from normal adults were irradiated with 200cGy twice daily for consecutive 3days, a regimen identical to that used in TBI-conditioning HSCT. The characteristics, differentiation potential, cytogenetics, hematopoiesis-supporting function, and carcinogenicity of the irradiated BM-MSCs were then compared to the non-irradiated control. The irradiated and non-irradiated MSCs shared similar morphology, phenotype, and hematopoiesis-supporting function. However, irradiated MSCs showed much lower proliferative and differentiative potential. Irradiation also induced clonal cytogenetic abnormalities of MSCs. Nevertheless, the carcinogenicity of irradiated MSCs is low in vitro and in vivo. In parallel with the ex vivo irradiation experiments, decreased proliferative and differentiative abilities and clonal cytogenetic abnormalities can also be found in MSCs isolated from transplant recipients who had received TBI-based conditioning previously. Thus, TBI used in HSCT drastically injury MSCs and may contribute to the development of some long-term complications associated with clonal cytogenetic abnormality and poor adipogenesis and osteogenesis after TBI.

Articular Cartilage Repair with Mesenchymal Stem Cells After Chondrogenic Priming: A Pilot Study.

Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. The objective of this study was to assess a protocol that involved autologous transplantation of BMSCs into full-thickness cartilage defects in sheep following isolation, expansion, and a short period (4 days) of chondrogenic priming. The impact of oxygen tension during preimplantation culture was investigated. It was hypothesized that chondrogenically primed BMSCs would produce superior cartilaginous repair tissue relative to control defects, and that culture under hypoxia would yield improved repair tissue in comparison to normoxia. Ovine BMSCs were isolated, expanded to passage 2, seeded within a hyaluronic acid (HYAFF) scaffold, and primed ex vivo in chondrogenic medium for 4 days under normoxia (21% oxygen) or hypoxia (3% oxygen). Full-thickness, 7-mm-diameter articular cartilage defects were created in the femoral condyles of five sheep. Twenty defects were treated with normoxia-cultured, autologous BMSC-seeded scaffolds (eight); hypoxia-cultured, autologous BMSC-seeded scaffolds (eight); cell-free scaffolds (two); or no implants (two). Preimplantation priming was evaluated through gene expression analysis using reverse transcription quantitative polymerase chain reaction. After 6 months, histological assessment was performed on repair tissues with a modified O'Driscoll scoring system and tissue dimension analysis. Priming of preimplantation BMSC-seeded scaffolds in chondrogenic medium for 4 days resulted in significantly increased gene expression of hyaline cartilage-related collagen II and aggrecan relative to unprimed BMSCs (p < 0.05). Defects implanted with chondrogenically primed BMSC-seeded scaffolds developed cartilaginous repair tissues that contained safranin O-positive proteoglycans, and had significantly larger repair tissue areas, higher percentages of defect fill, and improved histological scores than cell-free controls (p < 0.05). Although hypoxic culture improved the preimplantation gene expression profile, a consistent difference in histological scores was not found between normoxia- and hypoxia-seeded BMSC-seeded scaffolds after 6 months (p = 0.90). This study demonstrates in a sheep model that (1) chondrogenic priming ex vivo improves the gene expression profile of BMSCs; (2) chondrogenically primed BMSCs are associated with the development of superior cartilaginous tissue to cell-free controls within cartilage defects; and (3) oxygen tension during preimplantation ex vivo culture does not consistently modulate cartilaginous repair tissue formation following BMSC transplantation into cartilage defects.

Effects of Carthamus Tinctorius Extract on Adipogenic Differentiation of Mouse Bone Marrow-Derived Mesenchymal Stromal Stem Cells

Effects of Carthamus Tinctorius Extract on Adipogenic Differentiation of Mouse Bone Marrow-Derived Mesenchymal Stromal Stem Cells

146 - Intracoronary Bone Marrow-Derived Mesenchymal Stromal Stem Cells Injection for Ischemic Cardiomyopathy Patient: A Case Report from Makati Medical Center, Philippines

146 - Intracoronary Bone Marrow-Derived Mesenchymal Stromal Stem Cells Injection for Ischemic Cardiomyopathy Patient: A Case Report from Makati Medical Center, Philippines

Optimal Seeding Densities for In Vitro Chondrogenesis of Two- and Three-Dimensional-Isolated and -Expanded Bone Marrow-Derived Mesenchymal Stromal Stem Cells Within a Porous Collagen Scaffold.

Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. The objective of this study was to assess the impact of cell seeding density within a collagen I scaffold on in vitro BMSC chondrogenesis following isolation and expansion in two-dimensional (2D) and three-dimensional (3D) environments. It was hypothesized that both expansion protocols would produce BMSCs capable of hyaline-like chondrogenesis with an optimal seeding density of 10 × 10(6) cells/cm(3). Ovine BMSCs were isolated in a 2D environment by plastic adherence, expanded to passage two in flasks containing an expansion medium, and seeded within collagen I scaffolds at densities of 50, 10, 5, 1, and 0.5 × 10(6) BMSCs/cm(3). For 3D isolation and expansion, aspirates containing known quantities of mononucleated cells (bone marrow-derived mononucleated cells [BMNCs]) were seeded on scaffolds at 50, 10, 5, 1, and 0.5 × 10(6) BMNCs/cm(3) and cultured in the expansion medium for an equivalent duration to 2D expansion. Constructs were differentiated in vitro in the chondrogenic medium for 21 days and assessed with reverse-transcription quantitative polymerase chain reaction, safranin O staining, histological scoring using the Bern Score, collagen immunofluorescence, and glycosaminoglycan (GAG) quantification. Two-dimensional-expanded BMSCs seeded at all densities were capable of proteoglycan production and displayed increased expressions of aggrecan and collagen II messenger RNA (mRNA) relative to predifferentiation controls. Collagen II deposition was apparent in scaffolds seeded at 0.5-10 × 10(6) BMSCs/cm(3). Chondrogenesis of 2D-expanded BMSCs was most pronounced in scaffolds seeded at 5-10 × 10(6) BMSCs/cm(3) based on aggrecan and collagen II mRNA, safranin O staining, Bern Score, total GAG, and GAG/deoxyribonucleic acid (DNA). For 3D-expanded BMSC-seeded scaffolds, increased aggrecan and collagen II mRNA expressions relative to controls were noted with all densities. Proteoglycan deposition was present in scaffolds seeded at 0.5-50 × 10(6) BMNCs/cm(3), while collagen II deposition occurred in scaffolds seeded at 10-50 × 10(6) BMNCs/cm(3). The highest levels of aggrecan and collagen II mRNA, Bern Score, total GAG, and GAG/DNA occurred with seeding at 50 × 10(6) BMNCs/cm(3). Within a collagen I scaffold, 2D- and 3D-expanded BMSCs are capable of hyaline-like chondrogenesis with optimal cell seeding densities of 5-10 × 10(6) BMSCs/cm(3) and 50 × 10(6) BMNCs/cm(3), respectively.

Hypoxic culture of bone marrow-derived mesenchymal stromal stem cells differentially enhances in vitro chondrogenesis within cell-seeded collagen and hyaluronic acid porous scaffolds.

IntroductionThe quality of cartilaginous tissue derived from bone marrow mesenchymal stromal stem cell (BMSC) transplantation has been correlated with clinical outcome. Therefore, culture conditions capable of modulating tissue phenotype, such as oxygen tension and scaffold composition, are under investigation. The objective of this study was to assess the effect of hypoxia on in vitro BMSC chondrogenesis within clinically approved porous scaffolds composed of collagen and hyaluronic acid (HA). It was hypothesized that hypoxic isolation/expansion and differentiation would improve BMSC chondrogenesis in each construct.MethodsOvine BMSCs were isolated and expanded to passage 2 under hypoxia (3% oxygen) or normoxia (21% oxygen). Cell proliferation and colony-forming characteristics were assessed. BMSCs were seeded at 10 million cells per cubic centimeter on cylindrical scaffolds composed of either collagen I sponge or esterified HA non-woven mesh. Chondrogenic differentiation was performed in a defined medium under hypoxia or normoxia for 14 days. Cultured constructs were assessed for gene expression, proteoglycan staining, glycosaminoglycan (GAG) quantity, and diameter change.ResultsIsolation/expansion under hypoxia resulted in faster BMSC population doublings per day (P <0.05), whereas cell and colony counts were not significantly different (P = 0.60 and 0.30, respectively). Collagen and HA scaffolds seeded with BMSCs that were isolated, expanded, and differentiated under hypoxia exhibited superior aggrecan and collagen II mRNA expressions (P <0.05), GAG quantity (P <0.05), and proteoglycan staining in comparison with normoxia. GAG/DNA was augmented with hypoxic isolation/expansion in all constructs (P <0.01). Comparison by scaffold composition indicated increased mRNA expressions of hyaline cartilage-associated collagen II, aggrecan, and SOX9 in collagen scaffolds, although expression of collagen X, which is related to hypertrophic cartilage, was also elevated (P <0.05). Proteoglycan deposition was not significantly improved in collagen scaffolds unless culture involved normoxic isolation/expansion followed by hypoxic differentiation. During chondrogenesis, collagen-based constructs progressively contracted to 60.1% ± 8.9% of the initial diameter after 14 days, whereas HA-based construct size was maintained (109.7% ± 4.2%).ConclusionsHypoxic isolation/expansion and differentiation enhance in vitro BMSC chondrogenesis within porous scaffolds. Although both collagen I and HA scaffolds support the creation of hyaline-like cartilaginous tissue, variations in gene expression, extracellular matrix formation, and construct size occur during chondrogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0075-4) contains supplementary material, which is available to authorized users.

Stemness enhancement of human neural stem cells following bone marrow MSC coculture.

Rapid loss of stemness capacity in purified prototype neural stem cells (NSCs) remains a serious challenge to basic and clinical studies aiming to repair the central nervous system. Based on the essential role of mesodermal guidance in the process of neurulation, we hypothesized that coculture of human NSCs (hNSCs) with human bone marrow-derived mesenchymal stromal stem cells (hMSCs) could enhance the stemness of hNSCs through Notch-1 signaling. We have now tested the hypothesis by assessing behaviors of hNSCs and hMSCs under systematically designed coculture conditions relative to monocultures, with or without Notch-1 manipulation in vitro. Our data demonstrate that expression levels of Notch-1 and Hes-1 as determined by immunocytochemistry are significantly higher in hNSCs cocultured with hMSCs than those of controls. Furthermore, coculturing significantly increases immunoreactivity of CD15, a neural stemness marker, but decreases CD24, a marker of neural/neuronal commitment in hNSCs. The effect is independent from the physical status of cell growth since coculture and notch signaling actually promotes hNSC adhesion. Importantly, coculture with hMSCs markedly augments hNSC proliferation rate (e.g., higher yield in G2/M phase subpopulation in a notch-dependent manner detected by flow cytometry) without diminishing their lineage differentiation capabilities. The results suggest that coculture of hNSCs with hMSCs enhances stemness biology of hNSCs partially via activation of Notch-1 signal transduction. Our finding sheds new light on mesoderm-ectoderm cell fate determination via contact-based hMSC-hNSC interactions and provides mechanistic leads for devising effective regimens to sustain and augment stemness of in vitro established hNSC and hMSC lines for basic science, translational and clinical applications.

Porous Scaffold Seeding and Chondrogenic Differentiation of BMSC-seeded Scaffolds.

Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects (Bornes et al., 2014). BMSCs can be seeded within porous biomaterial scaffolds that support three-dimensional cell organization, chondrogenic differentiation and extracellular matrix deposition for the creation of engineered cartilage. This protocol describes our defined methods for isolation and expansion of human and ovine BMSCs, seeding of BMSCs within porous scaffolds and in vitro chondrogenic differentiation (Adesida et al., 2012; Bornes et al., 2015).

Age-related CXC chemokine receptor-4-deficiency impairs osteogenic differentiation potency of mouse bone marrow mesenchymal stromal stem cells

Cysteine (C)-X-C chemokine receptor-4 (CXCR4) is the primary transmembrane receptor for stromal cell-derived factor-1 (SDF-1). We previously reported in mouse or human bone marrow-derived mesenchymal stromal stem cells (BMSCs) that deleting or antagonizing CXCR4 inhibits bone morphogenetic protein-2 (BMP2)-induced osteogenic differentiation. The goal of this study was to determine whether CXCR4-deficiency in BMSCs is an age-related effect in association with impaired osteogenic differentiation potency of aged BMSCs. Using BMSCs derived from C57BL/6J wild type mice at ages ranging from 3 to 23 months old, we detected decreased CXCR4 mRNA and protein expression as well as SDF-1 secretion with advancing aging. Moreover, CXCR4-deficient BMSCs from elderly vs. young mice exhibited impaired osteogenic differentiation in response to BMP2 stimulation or when cultured in dexamethasone (Dex)-containing osteogenic medium, evidenced by decreased alkaline phosphatase activity, osteocalcin synthesis, and calcium deposition (markers for immature and mature osteoblasts). Mechanistically, impaired BMP2- or Dex-osteoinduction in BMSCs of elderly mice was mediated by inhibited phosphorylation of intracellular R-Smads and Erk1/2 or Erk1/2 and p38 proteins, and decreased Runx2 and Osx expression (osteogenesis “master” regulators) were also detected. Furthermore, adenovirus-mediated repair of CXCR4 expression in BMSCs of elderly mice restored their osteogenic differentiation potentials to both BMP2 treatment and osteogenic medium. Collectively, our results demonstrate for the first time that CXCR4 expression in mouse BMSCs declines with aging, and this CXCR4-deficiency impairs osteogenic differentiation potency of aged BMSCs. These findings provide novel insights into mechanisms underlying age-related changes in BMSC-osteogenesis, and will potentiate CXCR4 as a therapeutic target to improve BMSC-based bone repair and regeneration in broad orthopedic situations.