Horseshoe Crab Peri-Vitelline Fluid Triggers the Human Bone Marrow Stem Cell Differentiation into Cardiomyocyte In Vitro

Abstract

In the developing eggs of the horseshoe crab (Tachypleus gigas, Muller), the peri-vitelline fluid (PVF) occurs at the 21st stage of embryogenesis. Besides maintaining the homeostasis balance inside the developing eggs it also helps in organogenesis. Considering PVF assisting cell differentiation and proliferation during embryonic development, we made an attempt to examine its activity in triggering human bone marrow stem cells transforming into cardiomyocyte. Out of ten-peak PVF profile obtained from FPLC analysis, only the eighth fraction (PVF-8) showed highest activity on the differentiation of human bone marrow stem cell into myocyte differentiation. By FACS analysis, the optimum dose of PVF-8 was 0.1 mg/ml where maximum rate of bone marrow stem cells differentiation into myocyte observed. Since PVF-8 was showing the highest myocyte differentiation activity it was further analysed for its purity on SDSPAGE where a single band of 29 kDa molecular weight was obtained. The protein sequencing of the PVF-8 showed the presence of 122 amino acids. In order to identify the myocytes present in the colonies formed after the longtermed culture of bone marrow stem cells, cells expressing myosin were identified both by immunohistochemical and FACS analysis. In immunehistochemical analysis, PVF-8 was found to induce a great expression of myosin in some adherent’s cells incubated with it as compared to control cells cultured in presence of VEGF or b-FGF. Moreover, in presence of PVF-8, peroxidase activity in the exposed cells surpassed those in the control group. These results confirmed that PVF-8 effectively improved the rate of bone marrow stem cell differentiation.