Differentiation in Culture of Mixed Aggregates of Dissociated Tissue Cells.

Our concepts of the process of determination in embryonic development have been based largely on results obtained by extirpation, transplantation, and explantation of tissue masses consisting of many cells. These methods have revealed an apparent restriction of potencies during development. The varying results obtained with different methods, however, have led to serious questioning of the conclusions. For, as Harrison1 pointed out, "there always may be new conditions, not yet tested, under which other potencies might be revealed." An important factor influencing the degree and variety of differentiation in tissue culture is the size of the tissue mass involved.25 Thus small pieces from the node region of the chick blastoderm (definitive primitive streak stage) fail to differentiate in culture, while larger pieces invariably form brain tissue.' Explants of presumptive head mesoderm from a single early gastrula of Triton taeneatus form only muscle in vitro, but larger masses consisting of identical cells, derived from the fusion of several of these same primordia, ofrmmuscle, chorda brain sensory primordia, and ectoderm.5 It is evident from these observations that a description of the potencies of a given tissue mass composed of many cells does not necessarily reveal the capacities of the individual constituent cells. These studies raise the possibility that, when an intact tissue mass is judged to be determined by all the usual criteria, individual cells within the mass may retain wider potencies. Perhaps in transferring a mass of cells to a new site the immediate environment of the inner cells is but slightly altered. Such a change would be sufficient to cause a shift in the direction of cell differentiation in the earliest phases of development(e.g., early amphibian gastrula); but in more advanced stages, generally considered to be determined, a more acute change may be necessary. Accordingly, in the latter instance, fixity of the mass under the imposed conditions could not be taken as evidence of cell fixity. With the discovery that it is possible to dissociate vertebrate tissue cells by treatment with high pH,' trypsin,7 8 or versene9 and, after aggregation, to obtain characteristic differentiation in vitro, we have a promising method for attacking this problem. By mixing dissociated cells derived from different primordia (or even from differentiated tissues) and combining them into complex tissue masses by aggregation, we may drastically alter the immediate environment of embryonic tissue cells. The differentiation of such complex aggregates may then provide important information on the potencies of these cells. It was with this hope that we began the present investigation. Our method has been to combine cells derived from two different organs at various advanced stages of organogenesis and study the differentiation in vitro of the complex aggregates.