In vitro development of blastema cells in axolotl limb regeneration: effect of insulin and nerve extracts on cellular proliferation

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Abstract
For the purpose of investigating the nature of the nervous factor which controls cell proliferation in limb blastema of Newts, we have cultured primary mesenchymous cells from limb blastemas of Axolotl. The cultures were carried out in Petri dishes (Primaria, Falcon) with a basal medium with contained diluted MEM supplemented with hormones (insulin, somatotropin, hydrocortisone and thyroxine). In this medium, the cells disperse from the explant from the 4th day of culture and begin to divide from the 7th day; 3 weeks later the culture begins to decline. During the course of culture, beginning at the 8th day, differentiation of myotubes and chondrogenesis occur. The mitotic index, measured on the 16th day after 48 hr of colchicine treatment, is about 1.6%. Addition of foetal calf serum to the basal medium favors cell migration and survival and stimulates proliferation (mitotic: index 6%); beef embryo extract has no effect on cell migration and a small effect on proliferation (mitotic index: 2.3%). Addition to the basal medium of insulin or nerve extracts (brain and spinal cord of adult newts, brain of 12 days chick embryos) 6 days before we measure the mitotic index stimulates proliferation in proportion to the dose, up to 6 times the mitotic index in basal medium. These results are discussed with respect to the problem of cell proliferation control during limb regeneration.

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