Identification of differentially regulated secretome components during skeletal myogenesis

Myogenesis is a well-characterized program of cellular differentiation that is exquisitely sensitive to the extracellular milieu. Systematic characterization of the myogenic secretome (i.e. the ensemble of secreted proteins) is, therefore, warranted for the identification of novel secretome components that regulate both the pluripotency of these progenitor mesenchymal cells, and also their commitment and passage through the differentiation program. Previously, we have successfully identified 26 secreted proteins in the mouse skeletal muscle cell line C2C12 (1). In an effort to attain a more comprehensive picture of the regulation of myogenesis by its extracellular milieu, quantitative profiling employing stable isotope labeling by amino acids in cell culture was implemented in conjunction with two parallel high throughput online reverse phase liquid chromatography-tandem mass spectrometry systems. In summary, 34 secreted proteins were quantified, 30 of which were shown to be differentially expressed during muscle development. Intriguingly, our analysis has revealed several novel up- and down-regulated secretome components that may have critical biological relevance for both the maintenance of pluripotency and the passage of cells through the differentiation program. In particular, the altered regulation of secretome components, including follistatin-like protein-1, osteoglycin, spondin-2, and cytokine-induced apoptosis inhibitor-1, along with constitutively expressed factors, such as fibulin-2, illustrate dynamic changes in the secretome that take place when differentiation to a specific lineage occurs.     

Mitophagy in hematopoietic stem cells

Hematopoietic stem cells (HSCs) are inherently quiescent and self-renewing, yet can differentiate and commit to multiple blood cell types. Intracellular mitochondrial content is dynamic, and there is an increase in mitochondrial content during differentiation and lineage commitment in HSCs. HSCs reside in a hypoxic niche within the bone marrow and rely heavily on glycolysis, while differentiated and committed progenitors rely on oxidative phosphorylation. Increased oxidative phosphorylation during differentiation and commitment is not only due to increased mitochondrial content but also due to changes in mitochondrial cytosolic distribution and efficiency. These changes in the intracellular mitochondrial landscape contribute signals toward regulating differentiation and commitment. Thus, a functional relationship exists between the mitochondria in HSCs and the state of the HSCs (i.e., stemness vs. differentiated). This review focuses on how autophagy-mediated mitochondrial clearance (i.e., mitophagy) may affect HSC mitochondrial content, thereby influencing the fate of HSCs and maintenance of hematopoietic homeostasis.     

MicroRNA‐31a‐5p from aging BMSCs links bone formation and resorption in the aged bone marrow microenvironment

The alteration of age‐related molecules in the bone marrow microenvironment is one of the driving forces in osteoporosis. These molecules inhibit bone formation and promote bone resorption by regulating osteoblastic and osteoclastic activity, contributing to age‐related bone loss. Here, we observed that the level of microRNA‐31a‐5p (miR‐31a‐5p) was significantly increased in bone marrow stromal cells (BMSCs) from aged rats, and these BMSCs demonstrated increased adipogenesis and aging phenotypes as well as decreased osteogenesis and stemness. We used the gain‐of‐function and knockdown approach to delineate the roles of miR‐31a‐5p in osteogenic differentiation by assessing the decrease of special AT‐rich sequence‐binding protein 2 (SATB2) levels and the aging of BMSCs by regulating the decline of E2F2 and recruiting senescence‐associated heterochromatin foci (SAHF). Notably, expression of miR‐31a‐5p, which promotes osteoclastogenesis and bone resorption, was markedly higher in BMSCs‐derived exosomes from aged rats compared to those from young rats, and suppression of exosomal miR‐31a‐5p inhibited the differentiation and function of osteoclasts, as shown by elevated RhoA activity. Moreover, using antagomiR‐31a‐5p, we observed that, in the bone marrow microenvironment, inhibition of miR‐31a‐5p prevented bone loss and decreased the osteoclastic activity of aged rats. Collectively, our results reveal that miR‐31a‐5p acts as a key modulator in the age‐related bone marrow microenvironment by influencing osteoblastic and osteoclastic differentiation and that it may be a potential therapeutic target for age‐related osteoporosis.     

人类胚胎干细胞系H9培养和分化中细胞异质性的发现

目的:在人类胚胎干细胞系H9培养和分化过程中探讨该细胞系的异质性。方法:对人类胚胎干细胞系H9进行体外未分化培养和诱导分化,鉴定其多潜能性和分化状态;在诱导其向拟胚体细胞的分化过程中,检测多潜能相关基因及分化特异基因的表达情况。结果:发现多潜能相关基因(Oct4、SOX2和Nanog)和种系特异性基因(Cdx2、Bachurary、SOX1、Fgf5和AFP)并不限于分别在未分化细胞和分化细胞中表达。结论:提示H9细胞系在培养过程中的非基因异质性现象,为进一步认识胚胎干细胞的自我更新和多潜能性提供了有意义的参考。     

The therapeutic effect of bone marrow–derived mesenchymal stem cells on osteoarthritis is improved by the activation of the KDM6A/SOX9 signaling pathway caused by exposure to hypoxia

Abnormal expression of KDM6A and SOX9 is a key factor in the pathogenesis of osteoarthritis (OA). Cellular treatments of OA with articular cartilage chondrocytes (ACCs) and bone marrow mesenchymal stem cells (BMSCs) are promising, but their underlying mechanisms remain to be explored. The pellet size, weight and sulfated glycosaminoglycan/DNA content of ACCs were measured to evaluate the effect of BMSCs on the chondrogenic differentiation of SCCs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to analyze the proliferation of ACCs cultured along or cocultured with BMSCs. Quantitative polymerase chain reaction (qPCR) was performed to evaluate the messenger RNA expression of KDM6A, SOX9, type2 collagen, and Aggrecan in ACCs and OA rats. Western blot and immunohistochemistry were performed to analyze the expression of KDM6A and SOX9 proteins. Bisulfite sequencing PCR was performed to assess the DNA methylation level of the SOX9 promoter. Flow cytometry was used to evaluate the apoptotic status of ACCs. The chondrogenic differentiation of ACCs was significantly enhanced by coculturing with BMSCs, especially under a hypoxic condition. The expression of KDM6A, SOX9, type2 collagen, and Aggrecan was remarkably elevated in ACCs cocultured with BMSCs. Also, the DNA methylation of SOX9 promoter was decreased in ACCs cocultured with BMSCs, along with notably reduced apoptosis. Moreover, ACCs cocultured with BMSCs could repair cartilage lesions and prevent the abnormal expression of KDM6A, SOX9, type2 collagen, and Aggrecan in OA rats. In this study, we cocultured ACCs with BMSCs and used them to treat OA rats. Our findings presented a mechanistic basis for explaining the therapeutic effect of BMSCs on OA treatment.     

Synthetic osteogenic growth peptide promotes differentiation of human bone marrow mesenchymal stem cells to osteoblasts via RhoA/ROCK pathway

The osteogenic growth peptide (OGP) is a naturally occurring tetradecapeptide that has attracted considerable clinical interest as a bone anabolic agent and hematopoietic stimulator. In vitro studies have demonstrated that OGP directly regulates the bone marrow mesenchymal stem cells' (BMSCs) differentiation into osteoblasts. However, the exact mechanism of this process remains unknown. In the present study, we investigated the role of RhoA/ROCK signaling in differentiation along this lineage using human BMSCs. OGP treatment increased the mRNA level of bone morphogenetic protein-2 and alkaline phosphatase activity after osteogenic induction. Analysis of BMSCs induced in the presence of OGP revealed an increase in RhoA activity, and phosphorylation of FAK and cofilin. The ROCK-specific inhibitors, Y27632, blocked the OGP-induced regulation of BMSC differentiation. Taken together, these data suggest that OGP not only acts on BMSCs to stimulate osteogenic differentiation, but also in a dose-dependent manner, and this effect is mediated via the activation of RhoA/ROCK pathway.     

Critical role of filamin-binding LIM protein 1 (FBLP-1)/migfilin in regulation of bone remodeling

Bone remodeling is a complex process that must be precisely controlled to maintain a healthy life. We show here that filamin-binding LIM protein 1 (FBLP-1, also known as migfilin), a kindlin- and filamin-binding focal adhesion protein, is essential for proper control of bone remodeling. Genetic inactivation of FBLIM1 (the gene encoding FBLP-1) in mice resulted in a severe osteopenic phenotype. Primary FBLP-1 null bone marrow stromal cells (BMSCs) exhibited significantly reduced extracellular matrix adhesion and migration compared with wild type BMSCs. Loss of FBLP-1 significantly impaired the growth and survival of BMSCs in vitro and decreased the number of osteoblast (OB) progenitors in bone marrow and OB differentiation in vivo. Furthermore, the loss of FBLP-1 caused a dramatic increase of osteoclast (OCL) differentiation in vivo. The level of receptor activator of nuclear factor κB ligand (RANKL), a key regulator of OCL differentiation, was markedly increased in FBLP-1 null BMSCs. The capacity of FBLP-1 null bone marrow monocytes (BMMs) to differentiate into multinucleated OCLs in response to exogenously supplied RANKL, however, was not different from that of WT BMMs. Finally, we show that a loss of FBLP-1 promotes activating phosphorylation of ERK1/2. Inhibition of ERK1/2 activation substantially suppressed the increase of RANKL induced by the loss of FBLP-1. Our results identify FBLP-1 as a key regulator of bone homeostasis and suggest that FBLP-1 functions in this process through modulating both the intrinsic properties of OB/BMSCs (i.e., BMSC-extracellular matrix adhesion and migration, cell growth, survival, and differentiation) and the communication between OB/BMSCs and BMMs (i.e., RANKL expression) that controls osteoclastogenesis.     

Neuronal differentiation of bone marrow-derived stromal stem cells involves suppression of discordant phenotypes through gene silencing

Tissue engineering involves the construction of transplantable tissues in which bone marrow aspirates may serve as an accessible source of autogenous multipotential mesenchymal stem cells. Increasing reports indicate that the lineage restriction of adult mesenchymal stem cells may be less established than previously believed, and stem cell-based therapeutics await the establishment of an efficient protocol capable of achieving a prescribed phenotype differentiation. We have investigated how adult mouse bone marrow-derived stromal cells (BMSCs) are guided to neurogenic and osteogenic phenotypes. Na?ve BMSCs were found surprisingly active in expression of a wide range of mRNAs and proteins, including those normally reported in terminally differentiated neuronal cells and osteoblasts. The na?ve BMSCs were found to exhibit voltage-dependent membrane currents similar to the neuronally guided BMSCs, although with smaller amplitudes. Once BMSCs were exposed to the osteogenic culture condition, the neuronal characteristics quickly disappeared. Our data suggest that the loss of discordant phenotypes during BMSC differentiation cannot be explained by the selection and elimination of unfit cells from the whole BMSC population. The percent ratio of live to dead BMSCs examined did not change during the first 8-10 days in either neurogenic or osteogenic differentiation media, and cell detachment was estimated at <1%. However, during this period, bone-associated extracellular matrix genes were selectively down-regulated in neuronally guided BMSCs. These data indicate that the suppression of discordant phenotypes of differentiating adult stem cells is achieved, at least in part, by silencing of superfluous gene clusters.