THE DEVELOPMENT IN VITRO OF CHIMERIC AGGREGATES OF DISSOCIATED EMBRYONIC CHICK AND MOUSE CELLS

Various embryonic tissues and organ rudiments can be dissociated into suspensions of discrete, viable cells following treatment with Caand Mg-free saline and trypsin.1-3 When cultivated in vitro under appropriate conditions, such cells reaggregate into compact clusters (Figs. 1-6), which subsequently re-establish tissue-like relationships and differentiate histotypically.2 3 These findings, originally established for chondrogenic, nephrogenic, and myogenic cells, have recently been extended to other embryonic tissues.3-8 If two different types of embryonic chick cells are intermingled in the same suspension, the resulting aggregates incorporate both types of cells; however, in the course of the further development of such heterotypic aggregates, t the diverse types of cells form distinct, histogenetically uniform groupings.2 The problem of grouping of animal cells in its relation to morphogenesis was discussed in detail by Weiss9 in reference to the concepts of "affinities"10 and "coaptation" ;ll its experimental implications were explored in the chick embryo12 and in amphibian embryos3 "4 and larvae"5-t7 and also under conditions of tissue culture.3' 18-20 Several of these studies strongly suggested that cells of diverse lineages manifested characteristic preferences in establishing intercellular contacts and tissue contiguity. This view was further supported by the results of recent experiments on heterotypic aggregates of chick cells2 which convincingly demonstrated a type-specific grouping of cells in the formation and development of such aggregates. These observations fell short of proof, however, due to the difficulty of identifying early embryonic chick cells when dissociated into discrete units in suspension; under these conditions, nearly all types of such cells look alike, and their identities in heterotypic mixtures are therefore not readily determined. The obvious solution to this impasse was to have cells marked in a way which would make them individually distinguishable in a mixed population. In searching for suitable "marker cells," an attempt was made to exploit the morphological differences between chick and mouse cells; mouse cell neclei are larger than chick cell nuclei and stain differ-, ently with basic stains and hematoxylin. Previous studies have shown that mouse and chick tissues can be successfully cultured in heterologous media21 22 and maintained simultaneously in culture without apparent incompatibility;23-25 it has further been noticed that under such conditions the differences of size and staining properties of the cells and nuclei of the two species are retained. Accordingly, the feasibility of obtaining composite aggregates, consisting of both chick and mouse cells, was explored. Preliminary experiments26 demonstrated that aggregates formed in suspensions of intermingled chick and mouse cells incorporated, under appropriate conditions, cells of both species. Upon further cultivation, such heterologous aggregates developed histogenetically in accordance with the origin of their cellular components. Due to the differences in size and the