Elastic modulus of hydrogel regulates osteogenic differentiation via liquid-liquid phase separation of YAP.

Craniofacial bone defects induced by congenital malformations, trauma, or diseases frequently challenge the orthodontic or restorative treatment. Stem cell-based bone regenerative approaches emerged as a promising method to resolve bone defects. Microenvironment physical cues, such as the matrix elastic modulus or matrix topography, regulate stem cell differentiation via multiple genes. We constructed gelatin methacryloyl (GelMA), a well-known scaffold, to investigate the impact of elastic modulus on osteogenic differentiation in a three-dimensional environment. Confocal microscope was used to observe and assess the condensates fission and fusion. New bone formation was evaluated by micro-computed tomography at 6 weeks in calvarial defect rat. We found that the light curing increased elastic modulus of GelMA, and the pore size of GelMA decreased. The expression of osteogenic markers was inhibited in hBMSCs cultured in the low-elastic-modulus GelMA. In contrast, the expression of YAP, TAZ and TEAD was increased in the hBMSCs in the low-elastic-modulus GelMA. Furthermore, YAP assembled via liquid-liquid phase separation (LLPS) into condensates that were sensitive to 1'6-hexanediol. YAP recruit TAZ and TEAD4, but not RUNX2 into the condensates. In vivo, we also found that hBMSCs in high-elastic-modulus GelMA was more apt to form new bone. This study provides new insight into the mechanism of osteogenic differentiation. Reagents that can regulate the elastic modulus of substrate or LLPS may be applied to promote bone regeneration.

MetaLnc9 facilitates osteogenesis of human bone marrow mesenchymal stem cells by activating the AKT pathway.

AIM OF THE STUDY: To investigate the role of MetaLnc9 in the osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs). MATERIALS AND METHODS: We used lentiviruses to knockdown or overexpress MetaLnc9 in hBMSCs. qRT-PCR was employed to determine the mRNA levels of osteogenic-related genes in transfected cells. ALP staining and activity assay, ARS staining and quantification were used to identify the degree of osteogenic differentiation. Ectopic bone formation was conducted to examine the osteogenesis of transfected cells in vivo. AKT pathway activator SC-79 and inhibitor LY294002 were used to validate the relationship between MetaLnc9 and AKT signaling pathway. RESULTS: The expression of MetaLnc9 was significantly upregulated in the osteogenic differentiation of hBMSCs. MetaLnc9 knockdown inhibited the osteogenesis of hBMSCs, whereas overexpression of it promoted the osteogenic differentiation both in vitro and in vivo. Taking a deeper insight, we found that MetaLnc9 enhanced the osteogenic differentiation by activating AKT signaling. The inhibitor of AKT signaling LY294002 could reverse the positive effect on osteogenesis brought by MetaLnc9 overexpression, whereas the activator of AKT signaling SC-79 could reverse the negative effect caused by MetaLnc9 knockdown. CONCLUSION: Our works uncovered a vital role of MetaLnc9 in osteogenesis via regulating the AKT signaling pathway. [Figure: see text].

Knockdown of circHIPK3 promotes the osteogenic differentiation of human bone marrow mesenchymal stem cells through activating the autophagy flux.

Many circular RNAs (circRNAs) involved in the osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs) have recently been discovered. The role of circHIPK3 in osteogenesis has yet to be determined. Cell transfection was conducted using small-interfering RNAs (siRNAs). Expression of osteogenic markers were detected by quantitative reverse transcription-polymerase chain reaction, western blotting analysis, and immunofluorescence staining. Ectopic bone formation models in nude mice were used to examined the bone formation ability in vivo. The autophagy flux was examined via western blotting analysis, immunofluorescence staining and transmission electron microscopy analysis. RNA immunoprecipitation (RIP) analysis was carried out to analyze the binding between human antigen R (HUR) and circHIPK3 or autophagy-related 16-like 1 (ATG16L1). Actinomycin D was used to determine the mRNA stability. Our results demonstrated that silencing circHIPK3 promoted the osteogenesis of hBMSCs while silencing the linear mHIPK3 did not affect osteogenic differentiation, both in vivo and in vitro. Moreover, we found that knockdown of circHIPK3 activated autophagy flux. Activation of autophagy enhanced the osteogenesis of hBMSCs and inhibition of autophagy reduced the osteogenesis through using autophagy regulators chloroquine and rapamycin. We also discovered that circHIPK3 and ATG16L1 both bound to HUR. Knockdown of circHIPK3 released the binding sites of HUR to ATG16L1, which stabilized the mRNA expression of ATG16L1, resulting in the upregulation of ATG16L1 and autophagy activation. CircHIPK3 functions as an osteogenesis and autophagy regulator and has the potential for clinical application in the future.

The emerging roles of circular RNAs in regulating the fate of stem cells.

Circular RNAs(circRNAs) are a large family of RNAs shaping covalently closed ring-like molecules and have become a hotspot with thousands of newly published studies. Stem cells are undifferentiated cells and have great potential in medical treatment due to their self-renewal ability and differentiation capacity. Abundant researches have unveiled that circRNAs have unique expression profile during the differentiation of stem cells and could serve as promising biomarkers of these cells. There are key circRNAs relevant to the differentiation, proliferation, and apoptosis of stem cells with certain mechanisms such as sponging miRNAs, interacting with proteins, and interfering mRNA translation. Moreover, several circRNAs have joined in the interplay between stem cells and lymphocytes. Our review will shed lights on the emerging roles of circRNAs in regulating the fate of diverse stem cells.