The Role of Non-Myogenic Mesenchymal Stem Cells in Skeletal Muscle of Dystrophin/Utrophin Double Knockout Mice

Adult skeletal muscle possesses a remarkable regenerative ability, which largely depends on satellite cells; however, in severe muscular dystrophies, such as Duchenne muscular dystrophy (DMD), skeletal muscle integrity is compromised and the muscle is often replaced by a mixture of fibrous tissue and white adipocytes in a process termed fibro-adipogenic degeneration. The precise cellular origin and the environmental cues responsible for accumulation of fat/fibrotic tissues during the course of the disease remain unknown. Using a previously published preplate technique, two distinct populations of muscle derived cells from skeletal muscle were isolated: 1) a rapidly adhering cell population (RACs), which is non-myogenic, Pax7- and expresses the mesenchymal stem cell (MSC) marker PDGFRα, hence termed non-myogenic MSCs (nmMSCs); and 2) a slowly adhering cell population (SACs) which is Pax7+ and highly myogenic, termed myogenic progenitor cells (MPCs). In this dissertation, the role of nmMSCs in the histopathogenesis of dystrophin/utrophin double knock out (dKO) mice, an animal model of DMD, was investigated. The nmMSCs become activated during the disease progression in dKO mice, displaying increased proliferation and adipogenic, osteogenic, and fibrogenic potentials compared to age-matched WT counterparts. The activated dKO-nmMSCs also significantly reduced the myogenic potential of the dKO-MPCs, an effect, at least partially, mediated by the secreted frizzled-related protein 1 (sFRP1) released by the dKO-nmMSCs. These results suggest that nmMSCs are likely the cell source involved in the deposition of non-muscle tissues in dKO muscles and activation of nmMSCs likely exacerbates muscle wasting and degeneration during the disease progression by limiting the myogenic potential of MPCs. Interestingly, the nmMSCs also appear to play an important role in muscle regeneration after acute injury in normal WT muscle. The nmMSCs isolated from cardiotoxin injured WT muscle significantly enhanced the myogenic potential of MPCs after co-cultivation, suggesting that the nmMSCs may facilitate muscle repair in normal WT muscle. These results suggest that the muscle micro-environment plays a major role in the fate of nMMSCs. Overall, data from this dissertation proposes that a therapeutic strategy targeting nmMSCs could represent a novel treatment for DMD

Influence of cholesterol/caveolin-1/caveolae homeostasis on membrane properties and substrate adhesion characteristics of adult human mesenchymal stem cells

Abstract Background Adult mesenchymal stem cells (MSCs) are an important resource for tissue growth, repair, and regeneration. To utilize MSCs more effectively, a clear understanding of how they react to environmental cues is essential. Currently, relatively little is known about how the composition of the plasma membranes affects stem cell phenotype and properties. The presence of lipid molecules, including cholesterol in particular, in the plasma membrane plays a crucial role in regulating a variety of physiological processes in cells. In this study, we examined the effects of perturbations in cholesterol/caveolin-1 (CAV-1)/caveolae homeostasis on the membrane properties and adhesive characteristics of MSCs. Findings from this study will contribute to the understanding of how cholesterol/CAV-1/caveolae regulates aspects of the cell membrane important to cell adhesion, substrate sensing, and microenvironment interaction. Methods We generated five experimental MSC groups: 1) untreated MSCs; 2) cholesterol-depleted MSCs; 3) cholesterol-supplemented MSCs; 4) MSCs transfected with control, nonspecific small interfering (si)RNA; and 5) MSCs transfected with CAV-1 siRNA. Each cell group was analyzed for perturbation of cholesterol status and CAV-1 expression by performing Amplex Red cholesterol assay, filipin fluorescence staining, and real-time polymerase chain reaction (PCR). The membrane fluidity in the five experimental cell groups were measured using pyrene fluorescence probe staining followed by FACS analysis. Cell adhesion to collagen and fibronectin as well as cell surface integrin expression were examined. Results Cholesterol supplementation to MSCs increased membrane cholesterol, and resulted in decreased membrane fluidity and localization of elevated numbers of caveolae and CAV-1 to the cell membrane. These cells showed increased expression of α1, α4, and β1 integrins, and exhibited higher adhesion rates to fibronectin and collagen. Conversely, knockdown of CAV-1 expression or cholesterol depletion on MSCs caused a parallel decrease in caveolae content and an increase in membrane fluidity due to decreased delivery of cholesterol to the cell membrane. Cells with depleted CAV-1 expression showed decreased cell surface integrin expression and slower adhesion to different substrates. Conclusions Our results demonstrate that perturbations in cholesterol/CAV-1 levels significantly affect the membrane properties of MSCs. These findings suggest that modification of membrane cholesterol and/or CAV-1 and caveolae may be used to manipulate the biological activities of MSCs

Additional file 1: of Influence of cholesterol/caveolin-1/caveolae homeostasis on membrane properties and substrate adhesion characteristics of adult human mesenchymal stem cells

Table S1. Cell groups used in this study. Each cell group was generated by pooling MSCs from three to four donors. Donor MSCs are specified by age (number) and gender (m, male; f, female). (PDF 14 kb

Caveolin-1 mediates soft scaffold-enhanced adipogenesis of human mesenchymal stem cells

Abstract Background Human bone marrow-derived mesenchymal stem cells (hBMSCs) can differentiate into adipocytes upon stimulation and are considered an appropriate cell source for adipose tissue engineering. In addition to biochemical cues, the stiffness of a substrate that cells attach to has also been shown to affect hBMSC differentiation potential. Of note, most current studies are conducted on monolayer cultures which do not directly inform adipose tissue engineering, where 3-dimensional (3D) scaffolds are often used to create proper tissue architecture. In this study, we aim to examine the adipogenic differentiation of hBMSCs within soft or stiff scaffolds and investigate the molecular mechanism mediating the response of hBMSCs to substrate stiffness in 3D culture, specifically the involvement of the integral membrane protein, caveolin-1 (CAV1), known to regulate signaling in MSCs via compartmentalizing and concentrating signaling molecules. Methods By adjusting the photo-illumination time, photocrosslinkable gelatin scaffolds with the same polymer concentration but different stiffnesses were created. hBMSCs were seeded within soft and stiff scaffolds, and their response to adipogenic induction under different substrate mechanical conditions was characterized. The functional involvement of CAV1 was assessed by suppressing its expression level using CAV1-specific siRNA. Results The soft and stiff scaffolds used in this study had a compressive modulus of ~0.5 kPa and ~23.5 kPa, respectively. hBMSCs showed high viability in both scaffold types, but only spread out in the soft scaffolds. hBMSCs cultured in soft scaffolds displayed significantly higher adipogenesis, as revealed by histology, qRT-PCR, and immunostaining. Interestingly, a lower CAV1 level was observed in hBMSCs in the soft scaffolds, concomitantly accompanied by increased levels of Yes-associated protein (YAP) and decreased YAP phosphorylation, when compared to cells seeded in the stiff scaffolds. Interestingly, reducing CAV1 expression with siRNA was shown to further enhance hBMSC adipogenesis, which may function through activation of the YAP signaling pathway. Conclusions Soft biomaterials support superior adipogenesis of encapsulated hBMSCs in 3D culture, which is partially mediated by the CAV1-YAP axis. Suppressing CAV1 expression levels represents a robust method in the promotion of hBMSC adipogenesis

A new murine model of Barth Syndrome neutropenia links TAFAZZIN deficiency to increased ER stress induced apoptosis.

Barth syndrome is an inherited X-linked disorder that leads to cardiomyopathy, skeletal myopathy and neutropenia. These symptoms result from the loss of function of the enzyme TAFAZZIN, a transacylase located in the inner mitochondrial membrane that is responsible for the final steps of cardiolipin production. The link between defective cardiolipin maturation and neutropenia remains unclear. To address potential mechanisms of neutropenia, we examined myeloid progenitor development within the fetal liver of TAFAZZIN knock-out animals as well as within the adult bone marrow of wild-type recipients transplanted with TAFAZZIN KO hematopoietic stem cells. We also used the ER Hoxb8 system of conditional immortalization to establish a new murine model system for the ex vivo study of TAFAZZIN-deficient neutrophils. The TAFAZZIN KO cells demonstrated the expected dramatic differences in cardiolipin maturation that result from a lack of TAFAZZIN enzyme activity. Contrary to our hypothesis, we did not identify any significant differences in neutrophil development or neutrophil function across a variety of assays including phagocytosis, and the production of cytokines or reactive oxygen species. However, transcriptomic analysis of the TAFAZZIN-deficient neutrophil progenitors demonstrated an upregulation of markers of endoplasmic reticulum stress and confirmatory testing demonstrated that the TAFAZZIN-deficient cells had increased sensitivity to certain ER stress mediated and non ER stress mediated triggers of apoptosis. While the link between increased sensitivity to apoptosis and the variably penetrant neutropenia phenotype seen in some Barth syndrome patients remains to be clarified, our studies and new model system set a foundation for further investigation