Ultrastructural analysis of differentiation of rat endoderm in vitro. Adipose vascular-stromal cells induce endoderm differentiation, which in turn induces differentiation of the vascular-stromal cells into chondrocytes.

Isolated definitive endoderm from 9-day-old rat embryos was cultivated up to 24 days in plastic and glass petri dishes and on developing vascular-stromal cells (mesenchymal cells) from epididymal white and interscapular brown adipose tissue of 4-week-old male rats. Explants were analyzed histologically and ultrastructurally. Endoderm attached to the bottom of the glass or petri dishes degenerated under one week of cultivation. Endoderm free floating in the culture medium developed into unilaminar vesicles whose flat epithelium did not differentiate. However, endoderm inoculated on developing mesenchymal cells differentiated into glandular explants or into ciliated pseudostratified columnar respiratory epithelium. The glandular explants were made up of at least four different kinds of cells whose cytoplasm showed predominantly: a) polyribosomes, b) lysosomes, c) mitochondria or d) cytoskeletal filaments. Endodermal cells differentiated only if, during cultivation, they were in contact with or in close proximity to developing mesenchymal cells. Endoderm differentiating into the respiratory epithelium in turn directed differentiation of the underlying vascular-stromal cells into lamina propria cells and chondrocytes. Cultivated vascular-stromal cells in the upper layers became thicker, ellipsoid in shape and with enlarged intercellular space. They appeared to be lamina propria cells and, together with the respiratory epithelium, built folds of respiratory mucosa. The vascular-stromal cells in the layers close to the bottom developed into chondrocytes; i.e., the cells became oval and agglomerated in nest like structures with a defined extracellular matrix. Their cytoplasm contained abundant cisternae of rough endoplasmic reticulum and numerous vacuoles with PAS positive substance. These observations showed that even developing vascular-stromal cells from adipose tissue from postlactating rats can trigger the process of definitive endoderm differentiation. Once triggered, differentiating endoderm influenced differentiation of the vascular-stromal cells into the cells and tissues of a wall of the respiratory tract.