Mesenchymal Stromal Progenitor Cells Research Articles

Senescence during early differentiation reduced the chondrogenic differentiation capacity of mesenchymal progenitor cells.

Introduction: Mesenchymal stromal/progenitor cells (MSCs) are promising for cartilage cell-based therapies due to their chondrogenic differentiation capacity. However, MSCs can become senescent during in vitro expansion, a state characterized by stable cell cycle arrest, metabolic alterations, and substantial changes in the gene expression and secretory profile of the cell. In this study, we aimed to investigate how senescence and the senescence-associated secretory phenotype (SASP) affect chondrogenic differentiation of MSCs. Methods: To study the effect of senescence, we exposed MSCs to gamma irradiation during expansion or during chondrogenic differentiation (the pellet culture). Western blot analysis was used to evaluate MSCs response to the chondrogenic inductor TGF-β. Results: When senescence was induced during expansion or at day 7 of chondrogenic differentiation, we observed a significant reduction in the cartilage matrix. Interestingly, when senescence was induced at day 14 of differentiation, chondrogenesis was not significantly altered. Moreover, exposing chondrogenic pellets to the medium conditioned by senescent pellets had no significant effect on the expression of anabolic or catabolic cartilage markers, suggesting a neglectable paracrine effect of senescence on cartilage generation in our model. Finally, we show that senescent MSCs showed lower phosphorylated SMAD2 levels after TGFβ1 stimulation than control MSCs. Conclusion: Overall, these results suggest that the occurrence of senescence in MSCs during expansion or early differentiation could be detrimental for cartilage tissue engineering.

Activation PDGFR-α/AKT Mediated Signaling Pathways in Oral Squamous Cell Carcinoma by Mesenchymal Stem/Stromal Cells Promotes Anti-apoptosis and Decreased Sensitivity to Cisplatin.

Desmoplasia, a hallmark of a head and neck cancer, has both biologic and physiologic effects on cancer progression and chemotherapeutic response. Mesenchymal stem/stromal cells (MSCs), also known as mesenchymal stromal progenitor cells, have been shown to play a role in cancer progression, alter apoptotic responses, and confer resistance to chemotherapy in various carcinomas. The pathophysiology of MSCs with respect to tumorigenesis is widely reported in other cancers and is sparsely reported in oral squamous cell carcinomas (OSCCs). We previously reported paracrine mediated PDGF-AA/PDGFR-α signaling to underlie MSCs chemotaxis in OSCC. Given the poor clinical response to primary chemotherapy, we hypothesized that MSCs may alter cancer cell sensitivity to cisplatin through activation of PDGFR-α mediated signaling pathways. Co-culture of MSCs with human derived OSCC cell lines, JHU-012 and −019, resulted in a significant increase in the production of PDGF-AA and MCP-1 compared to cancer cells grown alone (p < 0.005) and was accompanied by an increase in the phosphorylation state of PDGFR-α (p < 0.02) and downstream target AKT at S473 (p < 0.025) and T308 (p < 0.02). JHU-012 and −019 cancer cells grown in co-culture were significantly less apoptotic (p < 0.001), expressed significantly higher levels of Bcl-2 (p < 0.04) with a concomitant significant decrease in bid expression (p < 0.001) compared to cancer cells grown alone. There was a significant increase in the cisplatin dose response curve in cancer cell clones derived from JHU-012 and 019 cancer cells grown in co-culture with MSCs compared to clones derived from cancer cells grown alone (p < 0.001). Moreover clones derived from JHU-012 cells grown in co-culture with MSCs were significantly more susceptible to cisplatin following pretreatment with, crenolanib, a PDGFR inhibitor, compared to cancer cells grown alone or in co-culture with MSCs (p < 0.0001). These findings suggest that crosstalk between cancer cells and MSCs is mediated, at least in part, by activation of autocrine PDGF-AA/PDGFR-α loop driving AKT-mediated signaling pathways, resulting in reduced cancer cell sensitivity to cisplatin through alterations in apoptosis.

Inhibition of overactive TGF-\u03b2 attenuates progression of heterotopic ossification in mice

Acquired heterotopic ossification (HO) is a painful and debilitating disease characterized by extraskeletal bone formation after injury. The exact pathogenesis of HO remains unknown. Here we show that TGF-β initiates and promotes HO in mice. We find that calcified cartilage and newly formed bone resorb osteoclasts after onset of HO, which leads to high levels of active TGF-β that recruit mesenchymal stromal/progenitor cells (MSPCs) in the HO microenvironment. Transgenic expression of active TGF-β in tendon induces spontaneous HO, whereas systemic injection of a TGF-β neutralizing antibody attenuates ectopic bone formation in traumatic and BMP-induced mouse HO models, and in a fibrodysplasia ossificans progressive mouse model. Moreover, inducible knockout of the TGF-β type II receptor in MSPCs inhibits HO progression in HO mouse models. Our study points toward elevated levels of active TGF-β as inducers and promoters of ectopic bone formation, and suggest that TGF-β might be a therapeutic target in HO.

Cellular players of hematopoietic stem cell mobilization in the bone marrow niche.

Hematopoietic stem cells (HSC) reside in perivascular regions of the bone marrow (BM) embedded within a complex regulatory unit called the niche. Cellular components of HSC niches include vascular endothelial cells, mesenchymal stromal progenitor cells and a variety of mature hematopoietic cells such as macrophages, neutrophils, and megakaryocytes-further regulated by sympathetic nerves and complement components as described in this review. Three decades ago the discovery that cytokines induce a large number of HSC to mobilize from the BM into the blood where they are easily harvested, revolutionised the field of HSC transplantation-curative for immune-deficiencies and some malignancies. However, despite now routine use of granulocyte-colony stimulating factor (G-CSF) to mobilise HSC for transplant, only in last 15years has research on the mechanisms behind why and how HSC can be induced to move into the blood began. These studies have revealed the complexity of the niche that retains HSC in the BM. This review describes how BM niches and HSC themselves change during administration of G-CSF-or in the recovery phase of chemotherapy-to facilitate movement of HSC into the blood, and research now leading to development of novel therapeutics to further boost HSC mobilization and transplant success.

EphB4 enhances the process of endochondral ossification and inhibits remodeling during bone fracture repair

Previous reports have identified a role for the tyrosine kinase receptor EphB4 and its ligand, ephrinB2, as potential mediators of both bone formation by osteoblasts and bone resorption by osteoclasts. In the present study, we examined the role of EphB4 during bone repair after traumatic injury. We performed femoral fractures with internal fixation in transgenic mice that overexpress EphB4 under the collagen type 1 promoter (Col1-EphB4) and investigated the bone repair process up to 12 weeks postfracture. The data indicated that Col1-EphB4 mice exhibited stiffer and stronger bones after fracture compared with wild-type mice. The fractured bones of Col1-EphB4 transgenic mice displayed significantly greater tissue and bone volume 2 weeks postfracture compared with that of wild-type mice. These findings correlated with increased chondrogenesis and mineral formation within the callus site at 2 weeks postfracture, as demonstrated by increased safranin O and von Kossa staining, respectively. Interestingly, Col1-EphB4 mice were found to possess significantly greater numbers of clonogenic mesenchymal stromal progenitor cells (CFU-F), with an increased capacity to form mineralized nodules in vitro under osteogenic conditions, when compared with those of the wild-type control mice. Furthermore, Col1-EphB4 mice had significantly lower numbers of TRAP-positive multinucleated osteoclasts within the callus site. Taken together, these observations suggest that EphB4 promotes endochondral ossification while inhibiting osteoclast development during callus formation and may represent a novel drug target for the repair of fractured bones.

Circulating Mesenchymal Stromal Cells As a New Prospective Cancer Marker,

Abstract 3404Mobilization of progenitor cells is implicated in pathology and can be indicative of disease progression. Recently, we reported the influence of body mass index (BMI) on level of circulating progenitor cells (Bellows et al., Obesity 2011). Comparative analysis of peripheral blood mononuclear cells (PBMC) from 12 non-obese (BMI < 30) and 14 obese (BMI > 30) disease-free donors by flow cytometry revealed that obesity is associated with a 10-fold increased frequency of circulating mesenchymal stromal progenitor cells (MSC), which circulate at a very low level in healthy lean individuals. We showed that obesity is also associated with a 5-fold increased frequency of circulating progenitor cells (CPC), a population consisting of hematopoietic and endothelial precursors, while the frequencies of mature endothelial cells (EC) and CD34-bright leukocytes (CD34b LC) are unaffected by BMI. Here, we followed up on the assessment of circulating MSC as a potential surrogate pathology marker by analyzing the frequency of circulating CD34-positive progenitor and endothelial cells in a cohort of colorectal cancer patients. PBMC were collected from 45 obese and lean cancer patients and compared to control cancer-free donors. Flow cytometric enumeration of cells was performed based on established immunophenotypes: CD34brightCD31dimCD45dim (CPC), CD34dimCD31brightCD45- (EC), CD34brightCD31-CD45- (MSC) and CD34brightCD45bright CD34b (LC). Groups were compared using multivariate regression analysis. After adjusting for co-founders such as age and BMI, the mean frequencies of MSC and CD34bLC, but not of CPC and EC, were found to be significantly higher in the circulation of CRC patients compared to cancer-free donors. Interestingly, the frequency of circulating MSC, but not of the other cell populations, was also found to be significantly higher in the circulation of obese CRC patients compared to lean CRC patients and obese cancer-free controls. We conclude that markedly increased frequency of MSC in the peripheral blood may represent a new diagnostic CRC marker. BMI-dependent changes in circulating MSC, potentially mobilized from adipose tissue may reveal their trafficking to tumors, which could be one of the mechanistic links between obesity and cancer progression. Validation of MSC as a new surrogate marker of cancer could provide a tool for determining prognosis, predicting response to therapy, and detecting relapse following treatment. Disclosures:No relevant conflicts of interest to declare.

Influence of BMI on Level of Circulating Progenitor Cells

Obesity complicates a number of diseases through mechanisms that are poorly defined. Mobilization and recruitment of progenitor cells to pathological sites is an important factor in disease progression. Here, we analyzed the influence of obesity on the systemic circulation of CD34+ cell populations and correlated frequencies of cells displaying previously established cell marker signatures with the BMI. Comparative analysis of peripheral blood mononuclear cells (PBMC) from 12 nonobese (BMI <30 kg/m2) and 14 obese (BMI >30 kg/m2) disease-free donors by flow cytometry revealed that obesity is associated with a fivefold increased frequency of circulating progenitor cells (CPC), a population consisting of hematopoietic and endothelial precursors. Our data also indicate that obesity is associated with increased frequency of circulating mesenchymal stromal progenitor cells (MSC). In contrast, the frequencies of mature endothelial cells (EC) and CD34-bright leukocytes are unaffected by obesity. Combined, our results indicate that obesity promotes mobilization of progenitor cells, which may have clinical relevance.

Development of Myocardial Constructs Using Modulus-Matched Acrylated Polypropylene Glycol Triol Substrate and Different Nonmyocyte Cell Populations

Tissue engineering approaches are currently being investigated for the restoration of myocardial function in heart failure patients, most commonly by combining cells with a substrate to form myocardial-like constructs (MCs). The final properties of these constructs are dependant on the characteristics of both the substrate and the cells used for fabrication. To create a construct with the appropriate mechanical properties required for any future therapeutic, we tailored an acrylated polypropylene glycol triol (aPPGT) substrate to the elastic modulus of heart tissue and then investigated the fabrication of MCs. We first assessed the aPPGT substrate alone in vivo, both under normal conditions and in an infarct model in mice, and found that there was a mild foreign body response with good integration of the substrate into the epicardial surface in mice hearts. We next studied the fabrication and properties of MCs by culturing mouse embryonic cardiomyocytes on the aPPGT substrate. To achieve myocardial-like concentrically contractile constructs, cocultures with supportive stromal cells were found to be essential and both mouse heart-derived stromal cells or bone-derived mouse mesenchymal stromal progenitor cells (mMSCs) could be used. These different stromal cell types produced MCs with different properties. The average beating rate of the constructs formed from mouse heart-derived stromal cells was significantly higher those constructs formed using mMSCs. Conversely, the constructs formed using mMSCs had reduced fibrotic extracellular matrix secretion and increased hepatocyte growth factor expression. Both of these mMSC construct properties may enhance integration and therapeutic efficacy of the construct postimplantation on the surface of the infarcted heart. This study thus demonstrates the formation of MCs using mechanically tailored aPPGT substrate and also demonstrates the effects of different stromal cell populations have on the properties of the resultant MCs, both of which are critical for future applications of tissue engineering in heart failure patients.

BMP12 and BMP13 gene transfer induce ligamentogenic differentiation in mesenchymal progenitor and anterior cruciate ligament cells

To date there are only very few data available on the ligamentogenic differentiation capacity of mesenchymal stromal/progenitor cells (MSC) and anterior cruciate ligament (ACL) fibroblasts. We describe the in vitro potential of MSC and ACL cells to undergo ligamentogenic differentiation upon transduction with adenoviral vectors encoding the human cDNA for bone morphogenetic protein (BMP) 12 and BMP13, also known as growth and differentiation factors (GDF) 6 and 7, respectively. Transgene expression for at least 14 days was confirmed by Western blot analyzes. After 21 days of cell culture within collagen type I hydrogels, histochemical (hematoxylin/eosin (H&E), Azan and van Gieson), immunohistochemical and polymerase chain reaction (PCR) analyzes of the genetically modified constructs of both cell types revealed elongated, viable fibroblast-like cells embedded in a ligament-like matrix rich in collagens, vimentin, fibronectin, decorin, elastin, scleraxis, tenascin, and tenomodulin. It appears that both MSC and ACL fibroblasts are capable of ligamentogenic differentiation with these factors. This information may aid in the development of biologic approaches to repair and restore ACL after injury.