Embryonal carcinoma stem cell differentiation: Influence of retinoic acid and cell/extracellular matrix interactions

Differentiation of teratocarcinoma stem cells provides an opportunity to study the mechanisms that govern restriction of stem cell potential and emergence of mature cell types. The working hypothesis of this thesis was that manipulation of environmental factors, identical or analogous to factors present during early embryogenesis, can restrict the differentiation potential of multipotent embryonal carcinoma. These experiments describe the differentiation of a multipotent embryonal carcinoma, PCC4azal, in culture, into neuron-like cells after treatment with retinoic acid and dibutyryl cyclic adenosinemonophosphate (dbcAMP). After 5 days of drug treatment, approximately 60% to 80% of the cells in the culture displayed multiple long processes. The phenotype of these cells was characterized using monoclonal antibodies to the type II beta tubulin isotype, MAP2, tau, and neurofilaments. A small number of cells ({dollar}<{dollar}5%) which expressed glial fibrillary acidic protein were identified after 15 days in culture. The restriction of stem cell potential was dependent upon the initial subculture density. PCC4azal stem cells were malignant in vivo, but treatment with retinoic acid and dbcAMP for 5 days prior to placement in vivo abolished tumorigenicity. Laminin potentiated neural differentiation when it was used as a substrate. Laminin promoted greater cell attachment, spreading, and culture confluence than fibronectin, type I collagen substrates, and glass substrates. Only laminin induced more rapid neural differentiation, cell body clustering, neurite growth, and neurite fasciculation than glass substrates. The interaction between differentiating cells and laminin was shown to be due, at least in part, to a specific interaction with the Try-Ile-Gly-Ser-Arg (YIGSR) in the B1 chain of laminin. PCC4azal cells synthesized laminin A, B1 and B2 subunits after treatment with retinoic acid, concurrent with neural differentiation. These results are consistent with the hypotheses that: (1) retinoic acid induces differentiation of PCC4azal embryonal carcinoma without influencing the types of cells which differentiate; (2) the restriction of stem cell differentiation to neuron-like cells is greatly enhanced by concurrent treatment with dbcAMP; (3) the restriction is dependent upon initial subculture density; (4) laminin is at least permissive for restriction of stem cell potential; and (5) the specific interactions between cells and laminin during neural differentiation occur, at least in part through interaction with the YIGSR sequence in laminin.