Polymorphism in fowl serum albumin. VI. Changes in in vitro protein synthesizing activity in developing embryonic fowl liver

Summary Cell-free protein synthesizing systems were prepared from the livers of chick embryos at selected ages and the characteristics of individual fractions were compared. While polysomes showed decreasing size with older embryos, isolated polysomes did not differ significantly in amino acid incorporating activity when assayed with standard cell sap. When assayed with standard polysomes, cell sap activity decreased with increasing developmental age whether incorporation was measured using [³H]lysine, [³H]leucine, or [³H]aminoacyl-tRNA. Free amino acid concentrations in the cell sap showed reproducible independent variation during development which was taken into consideration in calculating net amino acid incorporation. A large increase in ribonuclease activity was observed during development; however, nuclease inhibitor activity was absent before day 15 but increased thereafter. Aminoacyl-tRNA synthetase activity did not vary significantly. It is proposed that the observed changes in the rate of cell-free protein synthesis result not only from increasing ribonuclease activity with increasing developmental age but also from changes in the activity of other soluble factors.This is paper VI in a series; paper V is reference 6. The series title is based on earlier work with systems derived from fowl which synthesized two genetic variants of serum albumin²¹.This research was supported in part by a grant from the Damon Runyon Memorial Fund (DRG-1125). Dr. H. M. Jernigan was an N.I.H. Postdoctoral Fellow (5 F02 GM 50944-02).To whom all inquiries are to be addressed.     

Synthesis of serum proteins by cultures of chick embryo yolk sac endodermal cells

Endodermal cells were isolated from yolk sacs of 3-day chick embryos and cultured for 6 days in Eagle's minimal essential media plus 10% fetal calf serum. During this period cells rapidly lost their ability to synthesize DNA as judged by [3H]thymidine incorporation into DNA. In spite of this loss of DNA synthesis serum protein synthesis and secretion remained at a constant 45% of total protein synthesis and secretion. This was determined by immunoprecipitation of culture media using antibodies directed against embryonic chick serum proteins. Media were also analyzed for the synthesis and secretion of specific serum proteins using polyacrylamide gel electrophoresis. The relative synthesis and secretion of the individual serum proteins followed that previously observed in ovo with the exception of alpha-globulin-a which became undetectable. When culture media were supplemented with ovalbumin or insulin the relative synthesis and secretion of certin specific serum proteins were altered. However, analysis of these same media samples showed that the total amounts of serum protein synthesis and secretion were unaffected.     

In vitro production of hemopoietic stem cells: washings from organ cultures of embryonic liver]

Hemopoietic cells including CFUs could be washed off from the organ culture of fetal liver periodically for 4 weeks. Under the cultivation conditions employed this treatment did not reduce the CFUs content of the culture essentially; thus, the washings off could be used to elevate the CFUs yield per culture.     

Automated maintenance of embryonic stem cell cultures

Embryonic stem cell (ESC) technology provides attractive perspectives for generating unlimited numbers of somatic cells for disease modeling and compound screening. A key prerequisite for these industrial applications are standardized and automated systems suitable for stem cell processing. Here we demonstrate that mouse and human ESC propagated by automated culture maintain their mean specific growth rates, their capacity for multi-germlayer differentiation, and the expression of the pluripotency-associated markers SSEA-1/Oct-4 and Tra-1-60/Tra-1-81/Oct-4, respectively. The feasibility of ESC culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.     

Primary cultures of embryonic rat heart cells

Summary In contrast to neonatal rat heart cells in culture, embryonic myocardial cells form colonies. They also differ from neonatal cells in their appearance and behavior; they have fewer striations, fewer binucleated cells, loose packing and sorting from nonmuscle cells, and most of all a greater capacity to divide. This work was supported in part by Department of Energy Contract EY-76-C-03-0012 and Environmental Protection Agency Contract IAG-D5-E-681-AO. This paper is part of a dissertation submitted by M. J. O. M. in partial fulfillment of the requirement for the Ph.D. degree at the University of California.     

Adhesive forces in embryonic stem cell cultures

Most cell culture systems grow and spread as contact-inhibited monolayers on flat culture dishes, but the embryonic stem cell (ESC) is one of the cell phenotypes that prefer to self-organize as tightly packed three-dimensional (3D) colonies. ESC also readily form 3D cell aggregates, called embryoid bodies (EB) that partially mimic the spatial and temporal processes of the developing embryo. Here, the rationale for ESC aggregation, rather than “spreading” on gelatin-coated or mouse embryonic fibroblast (MEF)-coated dishes, is examined through the quantification of the expression levels of adhesion molecules on ESC and the calculation of the adhesive forces on ESC. Modeling each ESC as a dodecahedron, the adhesive force for each ESC-ESC binding was found to be 9.1 × 10⁵ pN, whereas, the adhesive force for ESC-MEF binding was found to be an order of magnitude smaller at 7.9 × 10⁴ pN. We also show that E-cadherin is the dominating molecule in the ESC-ESC adhesion and blocking E-cadherin leads to a significant reduction in colony formation. Here, we mathematically describe the preference for ESC to self-assemble into ESC-ESC aggregates and 3D colonies, rather than to bind and spread on gelatin or MEF-coated dishes, and have shown that these interactions are predominantly due to E-cadherin expression on ESC.Key words: embryonic stem cells, stem cell morphology, E-cadherin, beta-1 integrin, cell adhesive forces, quantitative flow cytometry