During the past several years, there has been intense research in the field of bone marrow-derived stem cell (BMSC) therapy to facilitate its translation into clinical setting. Although a lot has been accomplished, plenty of challenges lie ahead. Furthermore, there is a growing body of evidence showing that administration of BMSC-derived conditioned media (BMSC-CM) can recapitulate the beneficial effects observed after stem cell therapy. BMSCs produce a wide range of cytokines and chemokines that have, until now, shown extensive therapeutic potential. These paracrine mechanisms could be as diverse as stimulating receptor-mediated survival pathways, inducing stem cell homing and differentiation or regulating the anti-inflammatory effects in wounded areas. The current review reflects the rapid shift of interest from BMSC to BMSC-CM to alleviate many logistical and technical issues regarding cell therapy and evaluates its future potential as an effective regenerative therapy.
Fibroblast growth factor18 (FGF18) belongs to the FGF family and is a pleiotropic protein that stimulates proliferation in several tissues. Bone marrow mesenchymal stem cells (BMSCs) participate in the normal replacement of damaged cells and in disease healing processes within bone and the haematopoietic system. In this study, we constructed FGF18 and investigated its effects on rat BMSCs (rBMSCs). The proliferative effects of FGF18 on rBMSCs were examined using an MTS assay. To validate the osteogenic differentiation effects of FGF18, ALP and mineralization activity were examined as well as osteogenic differentiation-related gene levels. FGF18 significantly enhanced rBMSCs proliferation (p<0.001) and induced the osteogenic differentiation by elevating ALP and mineralization activity of rBMSCs (p<0.001). Furthermore, these osteogenic differentiation effects of FGF18 were confirmed via increasing the mRNA levels of collagen type I (Col I), bone morphogenetic protein 4 (BMP4), and Runt-related transcription factor 2 (Runx2) at 3 and 7 days. These results suggest that FGF18 could be used to improve bone repair and regeneration.
Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to be able to migrate towards glioma, but the molecular mechanisms responsible for this migratory behavior still require further elucidation. This study aimed to test the role of vascular endothelial growth factor (VEGF) in the C6 glioma-induced migration of BMSCs, evaluate the effect of VEGF on the migratory capacity and vascular cell adhesion molecule-1 (VCAM-1) expression of BMSCs and explore the role of VCAM-1 in the VEGF-induced migration of BMSCs. The results showed that C6 glioma cells significantly increased the migration of BMSCs in vitro, which was partially blocked by a VEGF neutralizing antibody, and 20 ng/ml recombinant rat VEGF164 incubation enhanced the migration of BMSCs. Moreover, 12 h of 20 ng/ml VEGF164 incubation upregulated the VCAM-1 expression of BMSCs and the blocking of VCAM-1 reduced the VEGF164-induced migration of BMSCs. The data also revealed that LY294002, an inhibitor of phosphoinositide-3-kinase (PI3K), decreased the VEGF-induced migration and VCAM-1 expression of BMSCs. These findings indicate that VEGF participates in mediating the C6 glioma-induced migration of BMSCs by upregulating their VCAM-1 expression, and that PI3K is involved in the signal transduction of VEGF164-induced migration and VCAM-1 expression of BMSCs.
mesenchymal stem cells; vascular endothelial growth factor; migration; glioma; vascular cell adhesion molecule-1
Sepsis causes over 200,000 deaths yearly in the US; better treatments are urgently needed. Administering bone marrow stromal cells (BMSCs—also known as mesenchymal stem cells) to mice before or shortly after inducing sepsis by cecal ligation and puncture reduced mortality and improved organ function. The beneficial effect of BMSCs was eliminated by macrophage depletion or pretreatment with antibodies specific for interleukin-10 (IL-10) or IL-10 receptor. Monocytes and/or macrophages from septic lungs made more IL-10 when prepared from mice treated with BMSCs versus untreated mice. Lipopolysaccharide (LPS)-stimulated macrophages produced more IL-10 when cultured with BMSCs, but this effect was eliminated if the BMSCs lacked the genes encoding Toll-like receptor 4, myeloid differentiation primary response gene-88, tumor necrosis factor (TNF) receptor-1a or cyclooxygenase-2. Our results suggest that BMSCs (activated by LPS or TNF-α) reprogram macrophages by releasing prostaglandin E2 that acts on the macrophages through the prostaglandin EP2 and EP4 receptors. Because BMSCs have been successfully given to humans and can easily be cultured and might be used without human leukocyte antigen matching, we suggest that cultured, banked human BMSCs may be effective in treating sepsis in high-risk patient groups.
To investigate the effect of oligodeoxynucleotides (ODNs) on the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) to osteoblasts, in order to find a candidate ODN with potential for the treatment of periodontitis, a series of ODNs were designed and selected to test their effect on the promotion of the differentiation of BMSCs to osteoblasts in vitro and on the repair of periodontal tissue in rats with periodontitis. It was found that MT01, one of the ODNs with the sequences of human mitochondrial DNA, stimulated the proliferation of BMSCs, the differentiation of BMSCs to osteoblasts and mRNA expression of bone-associated factors including Runx2, Osterix, OPG, RANKL and collagen I in vitro. In vivo study showed that MT01 prevented the loss of alveolar bone in the rats with periodontitis and induced the production of proteins of OPG and Osterix in the bone tissue. These results indicated that MT01 could induce differentiation of BMSCs to osteoblasts and inhibit the alveolar bone absorption in rats with periodontitis.
oligodeoxynucleotide; bone marrow mesenchymal stem cells; osteoblasts; differentiation; periodontitis
Bone marrow stromal cells (BMSCs) are being used for immune modulatory, anti-inflammatory and tissue engineering applications, but the properties responsible for these effects are not completely understood. Human BMSCs were characterized to identify factors that might be responsible for their clinical effects and biomarkers for assessing their quality.
Early passage BMSCs prepared from marrow aspirates of 4 healthy subjects were compared to 3 human embryonic stem cell (hESC) samples, CD34+ cells from 3 healthy subjects and 3 fibroblast cell lines. The cells were analyzed with oligonucleotide expression microarrays with more than 35,000 probes.
BMSC gene expression signatures of BMSCs differed from those of hematopoietic stem cells (HSCs), hESCs and fibroblasts. Genes up-regulated in BMSCs were involved with cell movement, cell-to-cell signaling and interaction and proliferation. The up-regulated genes most likely belonged to pathways for integrin signaling, integrin linked kinase (ILK) signaling, NFR2-mediated oxidative stress response, regulation of actin-based motility by Rho, actin cytoskeletal signaling, caveolar-mediated endocytosis, clathrin-mediated endocytosis and Wnt/β catenin signaling. Among the most highly up-regulated genes were structural extracellular (ECM) proteins:α5 and β 5 integrin chains, fibronectin, collagen type IIIα1 and Vα1; and functional EMC proteins: connective tissue growth factor (CTGF), transforming growth factor beta induced protein (TGFBI) and ADAM12.
Global analysis of human BMSCs suggests that they are mobile, metabolically active, proliferative and interactive cells that make use of integrins and integrin signaling. They produce abundant ECM proteins that may contribute to their clinical immune modulatory and anti-inflammatory effects.
Ankylosing spondylitis (AS) is a chronic autoimmune disease, and the precise pathogenesis is largely unknown at present. Bone marrow-derived mesenchymal stem cells (BMSCs) with immunosuppressive and anti-inflammatory potential and Th17/Treg cells with a reciprocal relationship regulated by BMSCs have been reported to be involved in some autoimmune disorders. Here we studied the biological and immunological characteristics of BMSCs, the frequency and phenotype of CCR4+CCR6+ Th/Treg cells and their interaction in vitro in AS.
The biological and immunomodulation characteristics of BMSCs were examined by induced multiple-differentiation and two-way mixed peripheral blood mononuclear cell (PBMC) reactions or after stimulation with phytohemagglutinin, respectively. The interactions of BMSCs and PBMCs were detected with a direct-contact co-culturing system. CCR4+CCR6+ Th/Treg cells and surface markers of BMSCs were assayed using flow cytometry.
The AS-BMSCs at active stage showed normal proliferation, cell viability, surface markers and multiple differentiation characteristics, but significantly reduced immunomodulation potential (decreased 68 ± 14%); the frequencies of Treg and Fox-P3+ cells in AS-PBMCs decreased, while CCR4+CCR6+ Th cells increased, compared with healthy donors. Moreover, the AS-BMSCs induced imbalance in the ratio of CCR4+CCR6+ Th/Treg cells by reducing Treg/PBMCs and increasing CCR4+CCR6+ Th/PBMCs, and also reduced Fox-P3+ cells when co-cultured with PBMCs. Correlation analysis showed that the immunomodulation potential of BMSCs has significant negative correlations with the ratio of CCR4+CCR6+ Th to Treg cells in peripheral blood.
The immunomodulation potential of BMSCs is reduced and the ratio of CCR4+CCR6+ Th/Treg cells is imbalanced in AS. The BMSCs with reduced immunomodulation potential may play a novel role in AS pathogenesis by inducing CCR4+CCR6+ Th/Treg cell imbalance.
Stimulatory G protein-mediated cAMP signaling is intimately involved in skeletal homeostasis. However, limited information is available on the role of the cAMP signaling in regulating the differentiation of mesenchymal stem cells into mature osteoblasts and adipocytes. To investigate this, we treated primary mouse bone marrow stromal cells (BMSCs) with forskolin to stimulate cAMP signaling and determined the effect on osteoblast and adipocyte differentiation. Exposure of differentiating osteoblasts to forskolin markedly inhibited progression to the late stages of osteoblast differentiation, and this effect was replicated by continuous exposure to PTH. Strikingly, forskolin activation of cAMP signaling in BMSCs conditioned mesenchymal stem cells (MSCs) to undergo increased osteogenic differentiation and decreased adipogenic differentiation. PTH treatment of BMSCs also enhanced subsequent osteogenesis, but promoted an increased adipogenesis as well. Thus, activation of cAMP signaling alters the lineage commitment of MSCs, favoring osteogenesis at the expense of adipogenesis.
Cyclic AMP (cAMP); Osteogenesis; Adipogenesis; Parathyroid hormone (PTH); Mesenchymal stem cells; Alkaline phosphatase
The aim of the present study was to investigate the effect of bone marrow mesenchymal stem cell (BMSC) transp1antation on lung and heart damage in a rat model of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). The animals were randomly divided into 3 groups: control, PAH and BMSC implantation groups. Structural changes in the pulmonary vascular wall, such as the pulmonary artery lumen area (VA) and vascular area (TAA) were measured by hematoxylin and eosin (H&E) staining, and the hemodynamics were detected by echocardiography. Two weeks post-operation, our results demonstrated that sublingual vein injection of BMSCs significantly attenuated the pulmonary vascular structural and hemodynamic changes caused by pulmonary arterial hypertension. The mechanism may be executed via paracrine effects.
bone marrow mesenchymal stem cells; pulmonary arterial hypertension; pulmonary vascular wall
To evaluate the efficacy of human bone marrow-derived mesenchymal stem cells (hBMSCs) as gene delivery vehicles to simultaneously express human hepatocyte growth factor (HGF) and interleukin 1 receptor antagonist (IL-1Ra) to improve the outcome of islet transplantation.
Morphology and islet-binding affinity of hBMSCs were checked by microscope. The expression of target genes and endogenous genes was determined by ELISA. Protection of islets by hBMSCs was evaluated in vitro by Calcein-AM/Propidium Iodide staining and in vivo by allogeneic islet transplantation study. Function and revascularization of islets was evaluated by immune fluorescence study.
Non-donor-specific hBMSCs showed strong binding affinity to human islets and protected viability and function. Transduction of hBMSCs with adenovirus encoding human HGF and human IL-1Ra (Adv-hHGF-hIL-1Ra) prior to co-culturing with islets further protected from apoptotic cell death, helped maintain 3D structures and morphology, and enhanced insulin secretion. Transplantation of human islets reconstituted with Adv-hHGF-hIL-1Ra transduced hBMSCs under the kidney capsule of streptozotocin-induced diabetic non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice reversed diabetes by reducing blood glucose levels to ≤200 mg/dL for up to 15 weeks and reduced the number of islets required to achieving normoglycemia. Blood glucose levels of mice transplanted with islets alone reversed to ≥500 mg/dL 4 weeks post-transplantation.
Results indentified hBMSCs as effective gene delivery vehicles to improve the outcome of islet transplantation.
adenovirus; gene therapy; islet transplantation; mesenchymal stem cells
Mesenchymal stem cells (MSCs) are multipotent and give rise to distinctly differentiated cells from all three germ layers. Neuronal differentiation of MSC has great potential for cellular therapy. We examined whether the cluster of mechanically made, not neurosphere, could be differentiated into neuron-like cells by growth factors, such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF).
Materials and Methods
BMSCs grown confluent were mechanically separated with cell scrapers and masses of separated cells were cultured to form cluster BMSCs. As described here cluster of BMSCs were differentiated into neuron-like cells by EGF, HGF, and VEGF. Differentiated cells were analyzed by means of phase-contrast inverted microscopy, reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, and immunocytochemistry to identify the expression of neural specific markers.
For the group with growth factors, the shapes of neuron-like cells was observable a week later, and two weeks later, most cells were similar in shape to neuron-like cells. Particularly, in the group with chemical addition, various shapes of filament structures were seen among the cells. These culture conditions induced MSCs to exhibit a neural cell phenotype, expressing several neuro-glial specific markers.
bone marrow-derived mesenchymal stem cells (BMSCs) could be easily induced to form clusters using mechanical scraping, not neurospheres, which in turn could differentiate further into neuron-like cells and might open an attractive possibility for clinical cell therapy for neurodegenerative diseases. In the future, we consider that neuron-like cells differentiated from clusters of BMSCs are needed to be compared and analyzed on a physiological and molecular biological level with preexisting neuronal cells, and studies on the possibility of their transplantation and differentiation capability in animal models are further required.
Neuron-like cells; mesenchymal stem cell; epidermal growth factor; vascular endothelial growth factor; hepatocyte growth factor
Background and Purpose
We compared the effect of treatment of stroke with bone marrow stromal cells from stroke rats (Isch-BMSC) and normal rats (Nor-BMSC) on functional outcome.
Isch-BMSCs and Nor-BMSCs were intravenously injected into rats 24 hours after middle cerebral artery occlusion. To test the mechanism of Isch-BMSC-enhanced neurorestoration, Isch-BMSC and Nor-BMSC cultures were used.
Isch-BMSC significantly promoted functional outcome and enhanced angiogenesis, arterial density, and axonal regeneration compared with Nor-BMSC treatment animals. Isch-BMSCs exhibited increased Angiopoietin-1, Tie2, basic fibroblast growth factor, glial cell-derived neurotrophic factor, vascular endothelial growth factor, and Flk1 gene expression compared with Nor-BMSC. Using transwell coculture of BMSCs with brain-derived endothelial cells, Isch-BMSCs increased phosphorylated-Tie2 activity in brain-derived endothelial cells and enhanced brain-derived endothelial cells capillary tube formation compared with Nor-BMSCs. Inhibition of Tie2 gene expression in brain-derived endothelial cells using siRNA significantly attenuated BMSC-induced capillary tube formation.
These data suggest that Isch-BMSCs are superior to Nor-BMSCs for the neurorestorative treatment of stroke, which may be mediated by the enhanced trophic factor and angiogenic characteristics of Isch-BMSCs.
Ang1; angiogenesis; axonal regeneration; bone marrow stromal cell; cerebral infarct; Tie2
It is well established that bone maintenance and healing is compromised in alcoholics. Adult mesenchymal stromal cells (MSCs) in bone marrow (BMSCs) and adipose tissue (ASCs) likely contribute to bone homeostasis and formation. Direct and indirect alcohol exposure inhibits osteoprogenitor cell function through a variety of proposed mechanisms. The goal of this study was to characterize the effects of chronic alcohol ingestion on the native number and in vitro growth characteristics and multipotentiality of adult BMSCs and ASCs in a rat model. Adult male Sprague-Dawley rats received a liquid diet containing 36% ethanol (EtOH) or an isocaloric substitution of dextramaltose (control). After 4, 8, or 12 weeks of the diet, ASCs were harvested from epididymal adipose tissue and BMSCs from femoral and tibial bone marrow. Cell doublings per day (CDs) and doubling times (DT) were determined for primary cells (P0) and cell passages 1 through 6 (P1-6). Fibroblastic, (CFU-F), adipogenic (CFU-Ad) and osteogenic (CFU-Ob) colony forming unit frequencies were assessed for P0, P3, and P6. The CDs and DTs were lower and higher, respectively, for ASCs and BMSCs harvested from EtOH versus control rats at all time points. The CFU-F, CFU-Ad and CFU-Ob were significantly higher in ASCs harvested from control versus EtOH rats for P0, P3, and P6 at all times. Both CFU-Ad and CFU-Ob were significantly higher in P0 BMSCs harvested from control versus EtOH rats after 12 weeks of the diet. The CFU-Ob for P3 BMSCs from control rats was significantly higher than those from ETOH rats after 8 and 12 weeks on the diet. All three CFU frequencies in ASCs from EtOH rats tended to decrease with increasing diet duration. The ASC cell and colony morphology was different between control and EtOH cohorts in culture. These results emphasize the significant detrimental effects of chronic alcohol ingestion on the in vitro expansion and multipotentiality of adult MSCs. Maintenance of the effects through multiple cell passages in vitro suggests cells may be permanently compromised.
Adipose; bone marrow; adult stromal cells; alcohol; bone
To detect the differentiation effects of retinal cells or extracts on bone marrow-derived mesenchymal stem cells (BMSC).
Human fetal BMSC were previously labelled by carboxyfluorescein succinimidyl ester (CFSE), and co-cultured with retinal pigment epithelial (RPE) cells which were pre-treated with ultraviolet irradiation at a ratio of 1:1 to induce the differentiation of BMSC for up to 14 days. In some assays, a retinal extract of bovine retinal extract (BRE) was added to detect the potential effects of retinal component on the differentiation of BMSC. In addition, Neuron-specific enolase (NSE), Nestin and Glial fibrillary acidic protein (GFAP) immunostaining were performed to determine the characteristics of BMSC.
The results indicated that by co-cultured with RPE cells, fetal BMSC were differentiated into neural-like cells expressing special neuronal markers Nestin, GFAP and NSE. And the expression of these markers was obviously increased by BRE.
Retina derived cells and extracts can induce the differentiation of BMSC into neural-like cells.
bone marrow-derived mesenchymal stem cells; retinal pigment epithelial cells; neural-like cells; bovine retinal extract
Due to its injectability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for orthopedic applications. However, a literature search revealed no report on human bone marrow mesenchymal stem cell (hBMSC) encapsulation in CPC for bone tissue engineering. The aim of this study was to encapsulate hBMSCs in alginate hydrogel beads and then incorporate them into CPC, CPC–chitosan and CPC–chitosan–fiber scaffolds. Chitosan and degradable fibers were used to mechanically reinforce the scaffolds. After 21 days, that the percentage of live cells and the cell density of hBMSCs inside CPC-based constructs matched those in alginate without CPC, indicating that the CPC setting reaction did not harm the hBMSCs. Alkaline phosphate activity increased by 8-fold after 14 days. Mineral staining, scanning electron microscopy and X-ray diffraction confirmed that apatitic mineral was deposited by the cells. The amount of hBMSC-synthesized mineral in CPC–chitosan–fiber matched that in CPC without chitosan and fibers. Hence, adding chitosan and fibers, which reinforced the CPC, did not compromise hBMSC osteodifferentiation and mineral synthesis. In conclusion, hBMSCs were encapsulated in CPC and CPC–chitosan–fiber scaffolds for the first time. The encapsulated cells remained viable, osteodifferentiated and synthesized bone minerals. These self-setting, hBMSC-encapsulating CPC-based constructs may be promising for bone tissue engineering applications.
Stem cell encapsulation; Osteogenic differentiation; Alginate hydrogel; Calcium phosphate cement scaffold; Bone marrow mesenchymal stem cells
Bisphosphonates (BPs) can be locally used to improve the osteogenesis around hydroxyapatite (HA) implants. However, there are almost no reports discussing the effects of BPs in the bonding state with HA on bone mesenchymal stromal cells (BMSCs). Clodronate is a BP widely used in clinical practice. This study was designed to evaluate the effects of clodronate combined with HA on BMSCs’ multi-directional differentiation.
Material and methods
The HA and clodronate-HA complex were prepared. BMSCs were isolated from Sprague-Dawley rat bone marrow and then the cells were cultured with both HA and clodronate-HA. The method of transcriptional and translational assay (MTT) and multi-directional induction (including osteogenic, adipogenic, and myogenic differentiation) were used to evaluate the effect of clodronate-HA on BMSC differentiation.
Scanning electron microscopy indicated active proliferation of the cells on clodronate-HA and HA. MTT of BMSCs cultured on clodronate-HA and HA demonstrated no significant differences between the two groups. BMSCs differentiated into osteocytes, adipocytes, and myocytes after being cultured with both clodronate-HA and HA. This indicated that BMSCs still retained multi-directional capability. The alkaline phosphatase activity of osteogenic induced BMSCs of both groups had no significant difference. However, there was a significant difference in total protein found between them.
The results suggest that clodronate in the bonding state with HA has no obvious inhibition of the proliferation and activity of BMSCs on the complex, and there was no evidence of a negative effect on multi-directional capability of the BMSCs.
BMSC; bisphosphonate; hydroxyapatite; multi-directional differentiation
This study evaluated the effects of bone marrow-derived mesenchymal stem cells (BMSCs) or their conditioned medium (CM) on the repair and prevention of Acute Kidney Injury (AKI) induced by gentamicin (G). Animals received daily injections of G up to 20 days. On the 10th day, injections of BMSCs, CM, CM+trypsin, CM+RNase or exosome-like microvesicles extracted from the CM were administered. In the prevention groups, the animals received the BMSCs 24 h before or on the 5th day of G treatment. Creatinine (Cr), urea (U), FENa and cytokines were quantified. The kidneys were evaluated using hematoxylin/eosin staining and immunohystochemistry. The levels of Cr, U and FENa increased during all the periods of G treatment. The BMSC transplantation, its CM or exosome injections inhibited the increase in Cr, U, FENa, necrosis, apoptosis and also increased cell proliferation. The pro-inflammatory cytokines decreased while the anti-inflammatory cytokines increased compared to G. When the CM or its exosomes were incubated with RNase (but not trypsin), these effects were blunted. The Y chromosome was not observed in the 24-h prevention group, but it persisted in the kidney for all of the periods analyzed, suggesting that the injury is necessary for the docking and maintenance of BMSCs in the kidney. In conclusion, the BMSCs and CM minimized the G-induced renal damage through paracrine effects, most likely through the RNA carried by the exosome-like microvesicles. The use of the CM from BMSCs can be a potential therapeutic tool for this type of nephrotoxicity, allowing for the avoidance of cell transplantations.
The purpose of this study was to investigate the effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) with or without osteogenic differentiation medium (ODM) on osteogenic differentiation of primary human bone-marrow-derived mesenchymal stem cells (hBMSCs) in vitro.
The hBMSCs were isolated from medullary reaming tissue. At 80% confluence, hBMSCs were treated with different concentrations of rhBMP-7 with and without ODM. Alkaline phosphatase (ALP) activity, calcium deposition and messenger RNA (mRNA) expression of osteocalcin (OC) and osteopontin (OPN) were examined.
ALP activity and calcium deposits in hBMSC culture were significantly increased by rhBMP-7 at 0.1 μg/ml (0.23 ± 0.07 IU and 28.9 ± 4.2 mg/dl) and 1.0 μg/ml (0.32 ± 0.03 IU and 38.7 ± 3.0 mg/dl), respectively, in the presence of ODM, showing a clearly dose-dependent osteoblastic differentiation. However, the same dose of 0.1 μg/ml rhBMP-7 without ODM and ODM alone induced low level of ALP and calcium deposits, indicating a synergistic effect of rhBMP-7 and ODM on committed osteogenic differentiation. Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed up-regulated OC and OPN mRNA levels, corroborating the synergistic effect of rhBMP-7 and ODM.
Our study showed that rhBMP-7 with ODM created a synergistic effect on up-regulation of osteogenic genes as well as osteogenic differentiation of primary hBMSCs in vitro. In the presence of ODM, the lowest concentration of rhBMP-7 needed to induce significant osteogenic differentiation of hBMSCs was 0.1 μg/ml.
Bone marrow stromal cells (BMSCs) are pluripotent and thereby a potential candidate for cell replacement therapy for central nervous system degenerative disorders and traumatic injury. However, the mechanism of their differentiation and effect on neural tissues has not been fully elucidated. This study evaluates the effect of BMSCs on neural cell growth and survival in a retinal ganglion cell (RGCs) model by assessing the effect of changes in the expression of a BMSC-secreted protein, thrombospondin-1 (TSP-1), as a putative mechanistic agent acting on RGCs.
Methods and Findings
The effect of co-culturing BMSCs and RGCs in vitro was evaluated by measuring the following parameters: neurite outgrowth, RGC survival, BMSC neural-like differentiation, and the effect of TSP-1 on both cell lines under basal secretion conditions and when TSP-1 expression was inhibited. Our data show that BMSCs improved RGC survival and neurite outgrowth. Synaptophysin, MAP-2, and TGF-β expression are up-regulated in RGCs co-cultured with BMSCs. Interestingly, the BMSCs progressively displayed neural-like morphology over the seven-day study period. Restriction display polymerase chain reaction (RD-PCR) was performed to screen for differentially expressed genes in BMSCs cultured alone or co-cultured with RGCs. TSP-1, a multifactorial extracellular matrix protein, is critically important in the formation of neural connections during development, so its function in our co-culture model was investigated by small interfering RNA (siRNA) transfection. When TSP-1 expression was decreased with siRNA silencing, BMSCs had no impact on RGC survival, but reduced neurite outgrowth and decreased expression of synaptophysin, MAP-2 and TGF-β in RGCs. Furthermore, the number of BMSCs with neural-like characteristics was significantly decreased by more than two-fold using siRNA silencing.
Our data suggest that the TSP-1 signaling pathway might have an important role in neural-like differentiation in BMSCs and neurite outgrowth in RGCs. This study provides new insights into the potential reparative mechanisms of neural cell repair.
Migration, proliferation, and differentiation of hematopoietic stem cells (HSCs) are dependent upon a complex three-dimensional (3D) bone marrow microenvironment. Although osteoblasts control the HSC pool, the subendosteal niche is complex and its cellular composition and the role of each cell population in HSC fate have not been established. In vivo models are complex and involve subtle species-specific differences, while bidimensional cultures do not reflect the 3D tissue organization. The aim of this study was to investigate in vitro the role of human bone marrow–derived mesenchymal stromal cells (BMSC) and active osteoblasts in control of migration, lodgment, and proliferation of HSCs.
A complex mixed multicellular spheroid in vitro model was developed with human BMSC, undifferentiated or induced for one week into osteoblasts. A clear limit between the two stromal cells was established, and deposition of extracellular matrix proteins fibronectin, collagens I and IV, laminin, and osteopontin was similar to the observed in vivo. Noninduced BMSC cultured as spheroid expressed higher levels of mRNA for the chemokine CXCL12, and the growth factors Wnt5a and Kit ligand. Cord blood and bone marrow CD34+ cells moved in and out the spheroids, and some lodged at the interface of the two stromal cells. Myeloid colony-forming cells were maintained after seven days of coculture with mixed spheroids, and the frequency of cycling CD34+ cells was decreased.
Undifferentiated and one-week osteo-induced BMSC self-assembled in a 3D spheroid and formed a microenvironment that is informative for hematopoietic progenitor cells, allowing their lodgment and controlling their proliferation.
We sought to characterize the function of bone marrow stromal cell (BMSC) populations in tumor progression. Because this function may depend on the cell-lineage and mouse strain heterogeneity, we first characterized ex vivo the BMSCs harvested from C57BL/6 versus FVB mice and established their in vivo function in tumor growth and metastasis experiments. All plastic-adherent BMSCs expressed platelet-derived growth factor receptor beta (PDGFRβ) and stem cell antigen 1 (Sca1), consistent with a mesenchymal precursor phenotype, as well as CD80. Moreover, these BMSCs were capable of differentiation along mesenchymal lineage into adipocytes, osteoblasts, chondrocytes or myofibroblasts. However, further phenotypic analysis detected a distinct populations of myeloid (CD11b+) precursor cells amongst the ex vivo expanded BMSCs –with specific surface marker phenotypes and gene expression pattern. When co-implanted with metastatic cancer cells, all the BMSCs persisted and integrated into tumor stroma, but only myeloid BMSCs significantly promoted tumor growth and metastasis. These data demonstrate the differential effect of BMSCs sub-populations on tumor progression. These results may have important implications for anti-tumor therapy and for the use of mesenchymal BMSCs as cell-based therapies.
Myeloid; mesenchymal; bone marrow-derived cells; tumor; metastasis
We sought to characterize the function of bone marrow stromal cell (BMSC) populations in tumor progression. Because this function may depend on the cell-lineage and mouse strain heterogeneity, we first characterized ex vivo the BMSCs harvested from C57BL/6 versus FVB mice and established their in vivo function in tumor growth and metastasis experiments. All plastic-adherent BMSCs expressed platelet-derived growth factor receptor beta (PDGFRβ) and stem cell antigen 1 (Sca1), consistent with a mesenchymal precursor phenotype, as well as CD80. Moreover, these BMSCs were capable of differentiation along mesenchymal lineage into adipocytes, osteoblasts, chondrocytes or myofibroblasts. However, further phenotypic analysis detected a distinct populations of myeloid (CD11b+) precursor cells amongst the ex vivo expanded BMSCs -with specific surface marker phenotypes and gene expression pattern. When co-implanted with metastatic cancer cells, all the BMSCs persisted and integrated into tumor stroma, but only myeloid BMSCs significantly promoted tumor growth and metastasis. These data demonstrate the differential effect of BMSCs sub-populations on tumor progression. These results may have important implications for anti-tumor therapy and for the use of mesenchymal BMSCs as cell-based therapies.
Myeloid; mesenchymal; bone marrow-derived cells; tumor; metastasis
Bone marrow mesenchymal stem cells (BMSCs) have been shown to ameliorate diabetes in animal models. The mechanism, however, remains largely unknown. An unanswered question is whether BMSCs are able to differentiate into β-cells in vivo, or whether BMSCs are able to mediate recovery and/or regeneration of endogenous β-cells. Here we examined these questions by testing the ability of hBMSCs genetically modified to transiently express vascular endothelial growth factor (VEGF) or pancreatic-duodenal homeobox 1 (PDX1) to reverse diabetes and whether these cells were differentiated into β-cells or mediated recovery through alternative mechanisms. Human BMSCs expressing VEGF and PDX1 reversed hyperglycemia in more than half of the diabetic mice and induced overall improved survival and weight maintenance in all mice. Recovery was sustained only in the mice treated with hBMSCs-VEGF. However, de novo β-cell differentiation from human cells was observed in mice in both cases, treated with either hBMSCs-VEGF or hBMSCs- PDX1, confirmed by detectable level of serum human insulin. Sustained reversion of diabetes mediated by hBMSCs-VEGF was secondary to endogenous β-cell regeneration and correlated with activation of the insulin/IGF receptor signaling pathway involved in maintaining β-cell mass and function. Our study demonstrated the possible benefit of hBMSCs for the treatment of insulin-dependent diabetes and gives new insight into the mechanism of β-cell recovery after injury mediated by hBMSC therapy.
Osteoinductive biomaterials are promising for bone repair. There is no direct proof that bone marrow mesenchymal stem cells (BMSCs) home to non-osseous sites and participate in ectopic bone formation induced by osteoinductive bioceramics. The objective of this study was to use a sex-mismatched beagle dog model to investigate BMSC homing via blood circulation to participate in ectopic bone formation via osteoinductive biomaterial. BMSCs of male dogs were injected into female femoral marrow cavity. The survival and stable chimerism of donor BMSCs in recipients were confirmed with polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH). Biphasic calcium phosphate (BCP) granules were implanted in dorsal muscles of female dogs. Y chromosomes were detected in samples harvested from female dogs which had received male BMSCs. At 4 weeks, cells with Y-chromosomes were distributed in the new bone matrix throughout the BCP granule implant. At 6 weeks, cells with Y chromosomes were present in newly mineralized woven bone. TRAP positive osteoclast-like cells were observed in 4-week implants, and the number of such cells decreased from 4 to 6 weeks. These results show that osteoprogenitors were recruited from bone marrow and homed to ectopic site to serve as a cell source for calcium phosphate-induced bone formation. In conclusion, BMSCs were demonstrated to migrate from bone marrow through blood circulation to non-osseous bioceramic implant site to contribute to ectopic bone formation in a canine model. BCP induced new bone in muscles without growth factor delivery, showing excellent osteoinductivity that could be useful for bone tissue engineering.
Bone repair; Osteoinductive; Calcium phosphate ceramic; Bone marrow mesenchymal stem cells; Y chromosomes; Canine model
Rationale: Bronchopulmonary dysplasia (BPD) and emphysema are characterized by arrested alveolar development or loss of alveoli; both are significant global health problems and currently lack effective therapy. Bone marrow–derived mesenchymal stem cells (BMSCs) prevent adult lung injury, but their therapeutic potential in neonatal lung disease is unknown.
Objectives: We hypothesized that intratracheal delivery of BMSCs would prevent alveolar destruction in experimental BPD.
Methods: In vitro, BMSC differentiation and migration were assessed using co-culture assays and a modified Boyden chamber. In vivo, the therapeutic potential of BMSCs was assessed in a chronic hyperoxia-induced model of BPD in newborn rats.
Measurements and Main Results: In vitro, BMSCs developed immunophenotypic and ultrastructural characteristics of type II alveolar epithelial cells (AEC2) (surfactant protein C expression and lamellar bodies) when co-cultured with lung tissue, but not with culture medium alone or liver. Migration assays revealed preferential attraction of BMSCs toward oxygen-damaged lung versus normal lung. In vivo, chronic hyperoxia in newborn rats led to air space enlargement and loss of lung capillaries, and this was associated with a decrease in circulating and resident lung BMSCs. Intratracheal delivery of BMSCs on Postnatal Day 4 improved survival and exercise tolerance while attenuating alveolar and lung vascular injury and pulmonary hypertension. Engrafted BMSCs coexpressed the AEC2-specific marker surfactant protein C. However, engraftment was disproportionately low for cell replacement to account for the therapeutic benefit, suggesting a paracrine-mediated mechanism. In vitro, BMSC-derived conditioned medium prevented O2-induced AEC2 apoptosis, accelerated AEC2 wound healing, and enhanced endothelial cord formation.
Conclusions: BMSCs prevent arrested alveolar and vascular growth in part through paracrine activity. Stem cell–based therapies may offer new therapeutic avenues for lung diseases that currently lack efficient treatments.
stem cells; aging; lung; oxygen