3.1 Introduction
3.1.1 Differentiation potential of stem cells
One of definitions of stem cells is multidifferentiation potential. Also the degree of their stemness is determined by their differentiation potential. According to results of section 2, sphere forming cells expressed pluripotent cell markers. Therefore in this section, we aimed to confirm their differentiation potential in vivo and in vitro.
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3.2 Experimental<→Experiment>
3.2.1 In vitro Differentiation Assays.
In vitro differentiation assays were examined following the published differentiation culture conditions for murin ES cells.
Mesoderm lineage differentiation assay. Dissociated muscle cells were stained with anti-αmooth muscle actin antibody, anti-Myosin antibody and anti-Desmin antibody. Chondrocyte were stained with Safranin-0 and Fast Green. Osteocytes were stained with ALIZARIN RED S. After 21 days, adipocytes were stained with Oil Re 0.
Ectoderm lineage (Neural lineage) differentiation assay. Cells were plated on ortinin-coated chamber slides and incubated with anti-βIII Tubuin mouse monoclonal, anti-O4 mouse monoclonal antibody and anti-GFAP mouse monoclonal antibody.
Endoderm lineage (Hepatic) differentiation assay. Differentiated cells were detected by immunohistochemistory using anti-αfetoprotein mouse monoclonal antibody, anti-Albumin goat polyclonal antibody and anti-Cytokeratin 18 mouse monoclonal antibody. Results from immunohistochemistry were confirmed by RT-PCR.
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3.2.2 In Vivo Differentiation.
Spheres were seeded onto biodegradable scaffolds and implanted into subcutaneous of NOD/SCID mice (Charles River laboratories). After 6 weeks, the implants were harvested and fixed with 10% formaldehyde, then examined by immunocytochemistry.
