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.
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3.3Results
3.3.1 Differentiation potential of cells in vitro
When representative bone marrow derived spheres were dissociated into single cells and exposed to three different differentiation media, the cells differentiated to express specific genes of the three lineages, Map2 (ectoderm), MyoD (mesoderm) and alpha-fetoprotein (AFP, endoderm) (Fig. 10).The addition of a neural differentiation medium to the in vitro environment of cells from bone marrow spheres, resulted in expression of pIII tubulin (a marker for neuron) (Fig. 11).
Alternatively, the addition of 20% fetal calf serum to the media resulted in the expression of markers representative of mesoderm; that is, a-smooth muscle actin (Fig. 11) as well as the mesenchymal cells, chondrocytes, osteocytes and adipocytes (Fig. 12). Thus, cells from spheres differentiated into all cell types of neural (neurons, oligodendrocytes and ghas) and mesenchymal stem cell lineage (chondrocytes, osteocytes and adipocytes). When exposed to a hepatocyte differentiation media the expression of a-fetoprotein (Fig. 11), was seen, suggestive of differentiation into endodermal tissue.
