BLOOD ISLAND FORMATION IN ATTACHED CULTURES OF MURINE EMBRYONIC STEM-CELLS

Differentiation of murine embryonic stem cells in suspension culture r esults in the formation of cystic embryoid bodies that develop blood i slands, In this study pre-cystic embryoid bodies were attached to a su bstratum, and the program of differentiation was monitored. The attach ed ES cell cultures formed blood islands on a cell layer that migrated out from the center of attachment and beneath a mesothelial-like cell layer. Morphological and in situ marker analysis showed benzidine-pos itive hematopoietic cells surrounded by vascular endothelial cells tha t expressed PECAM and took up DiI-Ac-LDL. Waves of morphological diffe rentiation were evident, suggesting a graded response to differentiati on signals. Electron microscopy of the blood islands showed that they were similar to blood islands of cystic embryoid bodies and mouse yolk sacs, and cell-cell junctions were evident among the blood island cel ls. RNA expression analysis was consistent with the presence of hemato poietic precursor cells of several lineages and a primitive vascular e ndothelium in the cultures. Thus a program of vascular and hematopoiet ic development can be elaborated in attached ES cell cultures, and the se blood islands are accessible to experimental manipulation. (C) 1995 Wiley-Liss, Inc.