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IntroductionHuman amniotic fluid stem (hAFS) cells have been shown to differentiate into multiple lineages, including myoblasts. However, molecular mechanisms underlying the myogenic differentiation of hAFS cells and their regenerative potential for muscle injury remain to be elucidated.MethodsIn order to induce myogenic differentiation of hAFS cells, lentiviruses for MYOD were constructed and transduced into hAFS cells. Formation of myotube-like cells was analyzed by immunocytochemistry, and expression of molecular markers for myoblasts was analyzed by reverse transcription polymerase chain reaction and Western blotting. For in vivo muscle regeneration, MYOD transduced hAFS cells were injected into left tibialis anterior (TA) muscles injured with cardiotoxin, and muscle regeneration was analyzed using hematoxylin and eosin, immunocytochemistry and formation of neuro-muscular junction.ResultsMYOD expression in hAFS cells successfully induced differentiation into multinucleated myotube-like cells. Consistently, significant expression of myogenic marker genes, such as MYOG, DES, DMD and MYH, was induced by MYOD. Analysis of pre-myogenic factors showed that expression of PAX3, MEOX1 and EYA2 was significantly increased by MYOD. MYOD was phosphorylated and localized in the nucleus. These results suggest that in hAFS cells, MYOD is phosphorylated and localized in the nucleus, thus inducing expression of myogenic factors, resulting in myogenic differentiation of hAFS cells. To test regenerative potential of MYOD-transduced hAFS cells, we transplanted them into injured muscles of immunodeficient BALB/cSlc-nu mice. The results showed a substantial increase in the volume of TA muscle injected with MYOD-hAFS cells. In addition, TA muscle tissue injected with MYOD-hAFS cells has more numbers of neuro-muscular junctions compared to controls, indicating functional restoration of muscle injury by MYOD-hAFS cells.ConclusionsCollectively, our data suggest that transduction of hAFS cells with MYOD lentiviruses induces skeletal myogenic differentiation in vitro and morphological and functional regeneration of injured muscle in vivo.

The tectorial membrane (TM) is an apical extracellular matrix (ECM) that hovers over the cochlear sensory epithelium and plays an essential role in auditory transduction. The TM forms facing the luminal endolymph-filled space and exhibits complex ultrastructure. Contrary to the current extracellular assembly model, which posits that secreted collagen fibrils and ECM components self-arrange in the extracellular space, we show that surface tethering of α-tectorin (TECTA) via a glycosylphosphatidylinositol anchor is essential to prevent diffusion of secreted TM components. In the absence of surface-tethered TECTA, collagen fibrils aggregate randomly and fail to recruit TM glycoproteins. Conversely, conversion of TECTA into a transmembrane form results in a layer of collagens on the epithelial surface that fails to form a multilayered structure. We propose a three-dimensional printing model for TM morphogenesis: A new layer of ECM is printed on the cell surface concomitant with the release of a preestablished layer to generate the multilayered TM.

There is growing interest in bioactive substances from marine organisms for their potential use against diverse human diseases. Osteoporosis is a skeletal disorder associated with bone loss primarily occurring through enhanced osteoclast differentiation and resorption. Recently, we reported the anti-osteoclastogenic activity of fermented Pacific oyster (Crassostrea gigas) extract (FO) in vitro. The present study focused on investigating the anti-osteoporotic efficacy of FO in bone loss prevention in an experimental animal model of osteoporosis and elucidating the mechanism underlying its effects. Oral administration of FO significantly decreased ovariectomy-induced osteoclast formation and prevented bone loss, with reduced serum levels of bone turnover biomarkers including osteocalcin and C-terminal telopeptide fragment of type I collagen C-terminus (CTX). FO significantly suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts and attenuated the induction of osteoclast-specific genes required for osteoclastogenesis and bone resorption. Furthermore, FO inhibited RANKL-mediated IκBα and p65 phosphorylation in BMMs. Taken together, these results demonstrate that FO effectively suppresses osteoclastogenesis in vivo and in vitro, and that FO can be considered as a potential therapeutic option for the treatment of osteoporosis and osteoclast-mediated skeletal diseases.