Laminin-221-derived recombinant fragment facilitates isolation of cultured skeletal myoblasts.

Introduction: Laminin is a major component of the basement membrane, containing multiple domains that bind integrin, collagen, nidogen, dystroglycan, and heparan sulfate. Laminin-221, expressed in skeletal and cardiac muscles, has strong affinity for the cell-surface receptor, integrin α7X2ß1. The E8 domain of laminin-221, which is essential for cell integrin binding, is commercially available as a purified recombinant protein fragment. In this study, recombinant E8 fragment was used to purify primary rodent myoblasts. We established a facile and inexpensive method for primary myoblast culture exploiting the high affinity binding of integrin α7X2ß1 to laminin-221. Methods: Total cell populations from dissociated muscle tissue were enzymatically digested and seeded onto laminin-221 E8 fragment-coated dishes. The culture medium containing non-adherent floating cells was removed after 2-hour culture at 37 °C. The adherent cells were subjected to immunofluorescence staining of desmin, differentiation experiments, and gene expression analysis. Results: The cells obtained were 70.3 ± 5.49% (n = 5) desmin positive in mouse and 67.7 ± 1.65% (n = 3) in rat. Immunofluorescent staining and gene expression analyses of cultured cells showed phenotypic traits of myoblasts. Conclusion: This study reports a novel facile method for primary culture of myoblasts obtained from mouse and rat skeletal muscle by exploiting the high affinity of integrin α7X2ß1 to laminin-221.

Microencapsulated Sertoli cells sustain myoblast proliferation without affecting the myogenic potential. In vitro data.

Sertoli cells (SeC) isolated from porcine testes have shown direct effects on muscle precursor cells sustaining C2C12 myoblasts proliferation and inhibiting oxidative stress and apoptosis in the early phase of the differentiation process, and stimulating myoblast fusion into myotubes and the expression of markers of myogenic differentiation in the late phase. This suggested that the cocktail of factors secreted by SeC stimulates proliferation in myoblasts without weakening their myogenic potential resulting in the formation of the critical myoblast amount necessary to rebuild the required muscle mass upon a damage. Here, we show that co-culturing C2C12 myoblasts with high doses of SeC microencapsulated in clinical grade alginate-based microcapsules (MC-SeC) for three days in differentiation medium (DM) translates into increased cell numbers and almost absence of myotube formation. However, after removal of MC-SeC, an intense fusion activity into myotubes was observed culminating in a fusion index similar to that of control after additional three days of culture in DM. These data definitely demonstrate that SeC-derived factors preserve the myogenic potential while sustaining cell proliferation in C2C12 myoblasts.

Beta-agonist drugs modulate the proliferation and differentiation of skeletal muscle cells in vitro.

Essentially employed for the treatment of airway obstructions in humans, ß-agonists are also known to have an anabolic effect in animals' skeletal muscle. In vivo and in vitro studies have attested the increase in animal body mass and the hypertrophy of muscle cells following the administration of specific ß-agonists. However, the contribution of ß-agonists to C2C12 myoblasts growth remains obscure. We therefore aimed to investigate the impact of ß1-and ß2-agonist drugs on the proliferation and differentiation of skeletal muscle cells. Direct observations and cytotoxicity assay showed that clenbuterol, salbutamol, cimaterol and ractopamine enhanced muscle cell growth and viability during the proliferation stage. Structural examinations coupled to Western blot analysis indicated that salbutamol and cimaterol triggered a decrease in myotube formation. A better comprehension of the effect of ß-agonists on myogenic regulatory genes in the muscle cells is crucial to establish a specific role of ß-agonists in muscle development, growth, and regeneration.

Osteogenic differentiation enhances the MC3T3-E1 secretion of glycosaminoglycans with an affinity for basic fibroblast growth factor and bone morphogenetic protein-2.

INTRODUCTION: It is generally recognized that a wide variety of morphogens and growth factors bind to the glycosaminoglycans (GAG) of proteoglycans (PG) to affect their bioavailability to ligands. Many growth factors involving in osteogenic differentiation require the GAG side chains to facilitate their interaction to the cell surface receptors and the biosynthesis of osteogenic proteins. The objective of this study is to investigate the secretion of GAG from MC3T3-E1 pre-osteoblasts of a murine bone calvaria during the osteogenic differentiation. METHODS: When MC3T3-E1 cells were cultured in the differentiation medium (DM) containing bone morphogenetic protein (BMP)-2, the alkaline phosphatase activity, calcium content and the amount of basic fibroblast growth factor (bFGF)- or BMP-2-bound sulfated GAG were determined. Moreover, the disaccharide analysis of the GAG was performed. RESULTS: When MC3T3-E1 cells were cultured in the differentiation medium (DM) containing bone morphogenetic protein (BMP)-2, the alkaline phosphatase activity and calcium content were significantly enhanced compared with those of the BMP-2-free DM and normal medium with or without BMP-2. Significantly higher amount of GAG secreted was detected for cells cultured in the DM containing BMP-2, in contrast to other culture conditions. The GAG secreted had an affinity for BMP-2 and basic fibroblast growth factor (bFGF). The disaccharide analysis of GAG demonstrated that the percentage of ΔHexA α1,4GlcNSO3 and ΔHexA (2-OSO3) α1,4GlcNSO3 increased, but that of ΔHexA α1,4GlcNSO3(6-OSO3) decreased (ΔHexA: unsaturated uronic acid residue, GlcNSO3: N-sulfated glucosamine, ΔHexA (2-OSO3): unsaturated uronic acid 2-sulfate residue, GlcNSO3(6-OSO3): N-sulfated glucosamine 6-sulfated). CONCLUSION: It was found that the osteogenic differentiation allowed cells to enhance the secretion of GAG with an affinity for BMP-2 and bFGF.

Formation of Human Colonic Crypt Array by Application of Chemical Gradients Across a Shaped Epithelial Monolayer.

BACKGROUND & AIMS: The successful culture of intestinal organoids has greatly enhanced our understanding of intestinal stem cell physiology and enabled the generation of novel intestinal disease models. Although of tremendous value, intestinal organoid culture systems have not yet fully recapitulated the anatomy or physiology of the in vivo intestinal epithelium. The aim of this work was to re-create an intestinal epithelium with a high density of polarized crypts that respond in a physiologic manner to addition of growth factors, metabolites, or cytokines to the basal or luminal tissue surface as occurs in vivo. METHODS: A self-renewing monolayer of human intestinal epithelium was cultured on a collagen scaffold microfabricated with an array of crypt-like invaginations. Placement of chemical factors in either the fluid reservoir below or above the cell-covered scaffolding created a gradient of that chemical across the growing epithelial tissue possessing the in vitro crypt structures. Crypt polarization (size of the stem/proliferative and differentiated cell zones) was assessed in response to gradients of growth factors, cytokines, and bacterial metabolites. RESULTS: Chemical gradients applied to the shaped human epithelium re-created the stem/proliferative and differentiated cell zones of the in vivo intestine. Short-chain fatty acids applied as a gradient from the luminal side confirmed long-standing hypotheses that butyrate diminished stem/progenitor cell proliferation and promoted differentiation into absorptive colonocytes. A gradient of interferon-γ and tumor necrosis factor-α significantly suppressed the stem/progenitor cell proliferation, altering crypt formation. CONCLUSIONS: The in vitro human colon crypt array accurately mimicked the architecture, luminal accessibility, tissue polarity, cell migration, and cellular responses of in vivo intestinal crypts.

Optimization of in vitro cultivation strategies for human adipocyte derived stem cells.

With adipose-derived stem cells being in the focus of research in regenerative medicine, the need arises for fast reliable cultivation protocols. We have tested the cultivation of human adipose-derived stem cells in endothelial cell growth medium prior to induction and differentiation, against the long-established use of DMEM/F12 medium-based cultivation protocols. We found that cultivation in endothelial cell growth medium not only accelerates growth before induction and differentiation, but also allows shorter induction and differentiation times than those following precultivation with DMEM/F12 medium with regard to the formation of mature adipocytes and to the viability undifferentiated cells. These results were first observed morphologically but could be confirmed by performing adiponectin ELISA and cell proliferation assays.