Progenitors of oligodendrocytes: limiting dilution analysis in fetal rat brain culture

In this paper, we describe the use of a combination of cell culture techniques and limiting dilution analysis to determine the number of oligodendrocyte progenitor cells and the oligodendrocyte clone size in primary dispersed cultures of 20- to 21-day-old fetal rat brain. Single-cell suspensions (1, 2, 3 x 10(6) cells/ml) were plated in either microwell or 100 mm dishes. After 22 days in culture the number of differentiated oligodendrocytes was ascertained by determining the amount of myelin basic protein by radioimmunoassay. The total amount of myelin basic protein was the same in the two types of dish, indicating that proliferation and differentiation were unaffected when oligodendrocytes were grown in microwells. The fraction (F0) of microwells containing no oligodendrocytes was determined at each cell dilution. F0 decreased exponentially with increasing total cell concentration. The linearity of the plot of ln F0 versus cell number indicates that the number of oligodendrocyte progenitor cells is limiting. From the equation describing the Poisson distribution of progenitor cells in microwells we calculate that, at the time of plating, primary cultures of fetal rat brain contain one oligodendrocyte progenitor cell per 1.3 x 10(5) brain cells, or a total population of 300-500 progenitor cells per brain. The mean oligodendrocyte clone size was determined to be approximately 825 at 22 days and close to 2000 by 35 days in culture. Therefore, each progenitor cell must undergo approximately 11 divisions, on the average, during postnatal development.