MiR-210-3p inhibits osteogenic differentiation and promotes adipogenic differentiation correlated with Wnt signaling in ERα-deficient rBMSCs

MicroRNAs (miRNAs) regulate activities in living organisms through various signaling pathways and play important roles in the development and progression of osteoporosis. The balance between osteogenic and adipogenic differentiation of rBMSCs is closely related to the occurrence of osteoporosis. ERα regulates bone metabolism in various tissues. However, the correlation among ERα, miRNAs, and the differentiation of rBMSCs is still unclear. In this study, we used lentivirus transfection into rBMSCs to construct an ERα-deficient model, analyzed the differences in expressed miRNAs between control and ERα-deficient rBMSCs. The results revealed that the expression of 25 miRNAs were upregulated, 164 miRNAs were downregulated, and some of the regulated miRNAs such as miR-210-3p and miR-214-3p were related to osteogenic or adipogenic differentiation, as well as to particular signaling pathways. Next, we overexpressed miR-210-3p to evaluate its effects on the osteogenic and adipogenic differentiation of rBMSCs, and identified the relationship among miR-210-3p, Wnt signaling pathway, and the differentiation of rBMSCs. The results indicated that ERα-deficient inhibited osteogenic differentiation, promoted adipogenic differentiation, and regulated the expression of some miRNAs. Meanwhile, overexpression of miR-210-3p promoted osteogenic differentiation and inhibited adipogenic differentiation of rBMSCs, processes likely to be related to the Wnt signaling pathway. In conclusion, we identified a group of upregulated and downregulated miRNAs in ERα-deficient rBMSCs that might play a vital role in regulating osteogenic or adipogenic differentiation. One of these, miR-210-3p, inhibited osteogenic differentiation and promoted adipogenic differentiation correlated with the Wnt signaling pathway in ERα-deficient rBMSCs, providing new insight into the regulation of bone metabolism.     

miR-21 regulates osteogenic and adipogenic differentiation of BMSCs by targeting PTEN

Objective:To explore the effects and mechanism of miR-21 on the osteogenic/adipogenic differentiation of mouse BMSCs.Methods:The bilateral ovaries of C57BL/6J mice (n=24) were removed to construct an osteoporosis model. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-21, osteogenic/adipogenic genes, and PTEN. ALP and ARS and ORO staining were used to detect the formation of calcium nodules and lipid droplets in BMSCs. Western blot was used to detect the expression of PTEN.Results:miR-21 was significantly down-regulated in osteoporotic mice. The expression of miR-21 was significantly up-regulated after the osteogenic induction of BMSCs, and the expression of miR-21 was significantly down-regulated after the adipogenic induction. Overexpression of miR-21 significantly promoted the osteogenic differentiation of BMSCs and inhibits the adipogenic differentiation of BMSCs.Conclusion:MiR-21 can promote osteogenic differentiation of BMSCs and inhibit their adipogenic differentiation by negatively regulating PTEN.     

Modulating Human Mesenchymal Stem Cell Plasticity Using Micropatterning Technique

In our previous work, we have reported that enforced elongation of human mesenchymal stem cells (hMSCs) through micropatterning promoted their myocardial lineage commitment. However, whether this approach is robust enough to retain the commitment when subsequently subjected to different conditions remains unsolved. This de-differentiation, if any, would have significant implication on the application of these myocardial-like hMSCs either as tissue engineered product or in stem cell therapy. Herein, we investigated the robustness of micropatterning induced differentiation by evaluating the retention of myocardial differentiation in patterned hMSCs when challenged with non-myocardial differentiation cues. Altogether, we designed four groups of experiments; 1) Patterned hMSCs cultured in normal growth medium serving as a positive control; 2) Patterned hMSCs cultured in normal growth medium for 14 days followed by osteogenic and adipogenic media for next 7 days (to study the robustness of the effect of micropatterning); 3) Patterned hMSCs (initially grown in normal growth medium for 14 days) trypsinized and recultured in different induction media for next 7 days (to study the robustness of the effect of micropatterning without any shape constrain) and 4) Patterned hMSCs cultured in osteogenic and adipogenic media for 14 days (to study the effects of biochemical cues versus biophysical cues). It was found that hMSCs that were primed to commit to myocardial lineage (Groups 2 and 3) were able to maintain myocardial lineage commitment despite subsequent culturing in osteogenic and adipogenic media. However, for hMSCs that were not primed (Group 4), the biochemical cues seem to dominate over the biophysical cue in modulating hMSCs differentiation. It demonstrates that cell shape modulation is not only capable of inducing stem cell differentiation but also ensuring the permanent lineage commitment.     

Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients.     

LncRNA SNHG1 modulates adipogenic differentiation of BMSCs by promoting DNMT1 mediated Opg hypermethylation via interacting with PTBP1

Recent evidence indicates that the abnormal differentiation of bone marrow‐derived mesenchymal stem cells (BMSCs) plays a pivotal role in the pathogenesis of osteoporosis. LncRNA SNHG1 has been found to be associated with the differentiation ability of BMSCs. In this study, we aimed to elucidate the role of lncRNA SNHG1 and its associated pathway on the differentiation of BMSCs in osteoporosis. Mice that underwent bilateral ovariectomy (OVX) were used as models of osteoporosis. Induced osteogenic or adipogenic differentiation was performed in mouse BMSCs. Compared to sham animals, lncRNA SNHG1 expression was upregulated in OVX mice. Also, the in vitro expression of SNHG1 was increased in adipogenic BMSCs but decreased in osteogenic BMSCs. Moreover, overexpression of SNHG1 enhanced the adipogenic capacity of BMSCs but inhibited their osteogenic capacity as determined by oil red O, alizarin red, and alkaline phosphatase staining, while silencing of SNHG1 led to the opposite results. LncRNA SNHG1 interacting with the RNA‐binding polypyrimidine tract‐binding protein 1 (PTBP1) promoted osteoprotegerin (Opg) methylation and suppressed Opg expression via mediating DNA methyltransferase (DNMT) 1. Furthermore, Opg was showed to regulate BMSC differentiation. Knockdown of SNHG1 decreased the expressions of adipogenic related genes but increased that of osteogenic related genes. However, the knockdown of Opg partially reversed those effects. In summary, lncRNA SNHG1 upregulated the expression of DNMT1 via interacting with PTBP1, resulting in Opg hypermethylation and decreased Opg expression, which in turn enhanced BMSC adipogenic differentiation and contributed to osteoporosis.     

Morphologic and transcriptomic comparison of adipose- and bone-marrow-derived porcine stem cells cultured in alginate hydrogels

Advances in bioengineering, material chemistry, and developmental biology have led to the design of three-dimensional (3D) culture systems that better resemble the surrounding structure and chemistry of the in situ niches of cells in tissues. This study was designed to characterize and compare porcine adipose-derived stem cells (ADSC) and bone-marrow-derived stem cells (BMSC) induced to differentiate toward osteogenic and adipogenic lineages in vitro by using a 3D alginate hydrogel. The morphology and gene expression of the two cell populations during differentiation were analyzed. Both ADSC and BMSC showed morphological evidence of osteogenic and adipogenic differentiation. Expression patterns of genes characteristic of the onset of osteogenic differentiation (ALP, COL1A1, SPARC, SPP1) were low at the beginning of culture and generally increased during the period of differentiation up to 28 days in culture. Expression of genes associated with adipogenic differentiation (ACSL1, ADFP, ADIPOQ, CD36, DBI, DGAT2, PPARG, SCD) was consistently increased in ADSC cultured in alginate hydrogel relative to the start of differentiation. However, adipogenic gene expression of BMSC cultured in alginate hydrogel was more limited when compared with that of ADSC. Evaluation of cell numbers (via the MTT staining assay) suggested a greater viability of BMSC under osteogenic conditions in alginate hydrogels than under adipogenic conditions, whereas ADSC had greater viability under adipogenic conditions than under osteogenic conditions. This study thus provides an important initial evaluation of ADSC and BMSC seeded and differentiated toward the osteogenic and adipogenic cell lineages in a 3D alginate hydrogel in vitro.     

Histone demethylase KDM7A reciprocally regulates adipogenic and osteogenic differentiation via regulation of C/EBPα and canonical Wnt signalling

Recent emerging evidences revealed that epigenetic methylation of histone and DNA regulates the lineage commitment of mesenchymal progenitor cells. This study was undertaken to delineate the actions of histone lysine demethylase 7A (KDM7A) on osteogenic and adipogenic differentiation. Kdm7a expression was up‐regulated in primary marrow stromal cells and established stromal ST2 line after adipogenic and osteogenic treatment. Silencing of endogenous Kdm7a in the cells blocked adipogenic differentiation whereas promoted osteogenic differentiation. Conversely, overexpression of wild‐type Kdm7a in the progenitor cells enhanced adipogenic differentiation whereas inhibited osteogenic differentiation. However, the effect of KDM7A on cell differentiation was largely attenuated when the point mutation was made that abolishes enzymatic activity of KDM7A. Mechanism investigations revealed that silencing of Kdm7a down‐regulated the expression of the CCAAT/enhancer binding protein α (C/EBPα) and secreted frizzled‐related protein 1 (Sfrp1). Chromatin immunoprecipitation (ChIP) assay revealed that KDM7A directly binds to the promoters of C/EBPα and Sfrp1 and removes the histone methylation marks H3K9me2 and H3K27me2. Furthermore, silencing of Kdm7a activated canonical Wnt signalling. Thereafter, activation of canonical Wnt signalling through silencing of Sfrp1 in ST2 attenuated the stimulation of adipogenic differentiation and inhibition of osteogenic differentiation by KDM7A. Our study suggests that KDM7A balances adipogenic and osteogenic differentiation from progenitor cells through epigenetic control of C/EBPα and canonical Wnt signalling and implicates that control of KDM7A action has an epigenetic perspective of curtailing metabolic disorders like osteoporosis.     

Low frequency mechanical stimulation inhibits adipogenic differentiation of C3H10T1/2 mesenchymal stem cells

Oscillatory mechanical stimulation at relatively high frequencies (0.1 Hz) has been shown to inhibit adipogenic and promote osteogenic differentiation of mesenchymal stem cells. However, for physiological interpretations and ease of implementation it is of interest to know whether different rates of mechanical stimulation can produce similar results. We hypothesized that relatively low frequency mechanical stimulation (0.01 Hz) can inhibit adipogenic differentiation of C3H10T1/2 mouse mesenchymal stem cells, even in a potent adipogenic differentiation medium. C3H10T1/2 cells were cultured in adipogenic medium under control (non-mechanically stimulated) conditions and under oscillatory surface stretch with 10% amplitude and 0.01 Hz frequency for 6h per day for up to 5 days. Cell population was assessed by counting and adipogenic differentiation was assessed by real-time quantitative PCR (qPCR) analysis of peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid binding protein 4 (FABP4) after 3 and 5 days. Involvement of the ERK signaling pathway was assessed by Western blot. Low frequency mechanical stimulation significantly decreased expression of PPARγ after 3 days and FABP4 after 3 and 5 days versus non-stimulated culture. ERK signaling was decreased in mechanically-stimulated culture, indicating a role in the inhibition of adipogenic differentiation. Application of this study: Low frequency mechanical stimulation may provide a technically simple means for control of mesenchymal stem cell differentiation in cell-based therapies, particularly for inhibition of differentiation toward undesired adipogenic lineages.     

Analysis of matrix metalloproteinase activity during differentiation of mesenchymal stem cells isolated from different tissues of one donor

Mesenchymal stem cells established from bone marrow (FetMSC) and limb bud (M-FetMSC) of early human embryo, as well as spheroids derived these cells, were induced to undergo osteogenic and adipogenic differentiation. Differentiated cells exhibited the activity of metalloproteinase (MMP)-9, -2, and -1. Its activity was different in osteogenic and adipogenic cells, as well as in monolayer cultures (2D) and cell spheroids (3D). The direct correlation between the level of adipogenic differentiation and gelatinases MMP-9 and MMP-2 activities in both cell lines in 2D and 3D culture was shown. M-FetMSC cells in 2D culture 12 days in culture during showed low potential for adipogenesis and reduced activity of MMP-2 and MMP-9. The low level of adipogenic differentiation in 2D M-FetMSC culture was accompanied with increased MMP-1 activity and enhanced differentiation (3D culture) resulted in a significant increase of both MMP activities. MMP-1 activity varied oppositely. MMP-1 activity declined in 3D cultures with a higher level of adipogenic differentiation. The level of osteogenic differentiation was similar in both cell lines during 2D and 3D cultivation. MMP-1 and -9 activities in both cell lines were not associated with osteogenic differentiation. MMP-2 and MMP-2 activity in these cells remained unchanged. The results suggest MMP implication in FetMSC and М-FetMSC differentiation. The difference in MMP activities during the cell differentiation may be caused by variations in the microenvironment or ECM properties in 2D and 3D cultures.     

Effects of iron oxide incorporation for long term cell tracking on MSC differentiation in vitro and in vivo

Successful cell therapy will depend on the ability to monitor transplanted cells. With cell labeling, it is important to demonstrate efficient long term labeling without deleterious effects on cell phenotype and differentiation capacity. We demonstrate long term (7 weeks) retention of superparamagnetic iron oxide particles (SPIO) by mesenchymal stem cells (MSCs) in vivo, detectable by MRI. In vitro, multilineage differentiation (osteogenic, chondrogenic and adipogenic) was demonstrated by histological evaluation and molecular analysis in SPIO labeled and unlabeled cells. Gene expression levels were comaparable to unlabeled controls in adipogenic and chondrogenic conditions however not in the osteogenic condition. MSCs seeded into a scaffold for 21 days and implanted subcutaneously into nude mice for 4 weeks, showed profoundly altered phenotypes in SPIO labeled samples compared to implanted unlabeled control scaffolds, indicating chondrogenic differentiation. This study demonstrates long term MSC traceability using SPIO and MRI, uninhibited multilineage MSC differentiation following SPIO labeling, though with subtle but significant phenotypical alterations.     

Osteogenic differentiation of stem cells derived from human periodontal ligaments and pulp of human exfoliated deciduous teeth

Multipotent stem cells derived from periodontal ligaments (PDLSC) and pulp of human exfoliated deciduous teeth (SHED) represent promising cell sources for bone regeneration. Recent studies have demonstrated that retinoic acid (RA) and dexamethasone (Dex) induce osteogenesis of postnatal stem cells. The objective of this study was to examine the effects of RA and Dex on the proliferation and osteogenic differentiation of SHED and PDLSC and to compare the osteogenic characteristics of SHED and PDLSC under RA treatment. SHED and PDLSC were treated with serum-free medium either alone or supplemented with RA or Dex for 21 days. The proliferation of SHED and PDLSC was significantly inhibited by both RA and Dex. RA significantly upregulated gene expression and the activity of alkaline phosphatase in SHED and PDLSC. Positive Alizarin red and von Kossa staining of calcium deposition was seen on the RA-treated SHED and PDLSC after 21 days of culture. The influences of RA on the osteogenic differentiation of SHED and PDLSC were significantly stronger than with Dex. Supplemention with insulin enhanced RA-induced osteogenic differentiation of SHED. Thus, RA is an effective inducer of osteogenic differentiation of SHED and PDLSC, whereas RA treatment in combination with insulin supplementation might be a better option for inducing osteogenic differentiation. Significantly higher cell proliferation of PDLSC results in greater calcium deposition after 3-week culture, suggesting that PDLSC is a better osteogenic stem cell source. This study provides valuable information for efficiently producing osteogenically differentiated SHED or PDLSC for in vivo bone regeneration.     

Cryopreservation does not alter karyotype, multipotency, or NANOG/SOX2 gene expression of amniotic fluid mesenchymal stem cells

Cryopreservation of mesenchymal stem cells from amniotic fluid is of clinical importance, as these cells can be harvested during the prenatal period and stored for use in treatments. We examined the behavior of mesenchymal stem cells from human amniotic fluid in culture that had been subjected to cryopreservation. We assessed chromosomal stability through karyotype analysis, determined whether multipotent capacity (differentiation into adipogenic, chondrogenic, and osteogenic cells) is maintained, and analyzed SOX2 and NANOG expression after thawing. Five amniotic fluid samples were cryopreserved for 150 days. No chromosomal aberrations were observed. The expression levels of NANOG and SOX2 also were quite similar before and after cryopreservation. Capacity for differentiation into adipogenic, chondrogenic, and osteogenic tissues also remained the same. We conclude that cryopreservation of amniotic fluid does not alter karyotype, NANOG/SOX2 gene expression, or multipotent capacity of stem cells that have been collected from amniotic fluid during pregnancy.     

Systems-level analysis of gene expression data revealed NR0B2/SHP as potential tumor suppressor in human liver cancer

Nuclear receptors (NRs) play pivotal roles in cell growth, proliferation, differentiation and homeostasis. Recent progress demonstrates that NR is tightly linked to human disease such as cancer, diabetes and obesity. Here we explore NR expression profiles in human tissue using systematic approaches. NR gene profiles reveal that individual NR has its own gene expression signature depending on tissue type. Of many organs, NRs expression is enriched in liver. Expression of many NRs was significantly changed in liver cancer. Notably, NR0B2/SHP expression level was significantly decreased in human liver cancer but not in normal liver. In addition, expression of SHP is well associated with good prognosis. SHP gene network analysis based on microarray data in liver cancer shows that SHP regulates cell proliferation and metabolism related gene sets. Our systematic approaches suggest that loss of SHP expression in liver might be key genetic events during hepatocarcinogenesis.