A novel system to study early hematopoietic development is described. This report documents the in vitro capacity of murine embryonic stem (ES) cells to differentiate into hematopoietic precursors of most, if not all, of the colony-forming cells found in normal bone marrow. This system is used to correlate the genetic expression of cytokines, their receptors, the 13-globins, and the hematopoietic cell surface markers throughout the time course of ES cell differentiation with the hematopoietic development that occurs in these cultures. Our results indicate that there is a strong transcriptional activation, in a well-defined temporal order, of most of these genes including erythropoietin (Epo), CSF-1, IL-4, IL-6, I~-globins, as well as the receptors for Epo, CSF-I, and IL-4. IL-3 and GM-CSF were not expressed during the first 24 days of ES cell differentiation. In contrast, the Steel (S/) factor (SLF) was expressed early and underwent substantial up-regulation during this differentiation, and its receptor, c-kit, was expressed relatively constantly throughout the culture period. Our results are consistent with the conclusion that SLF, Epo, IL-4, and IL-6 are important during the early stages of ES cell differentiation and hematopoietic development. Furthermore, these results argue strongly that IL-3 and GM-CSF are not critical to early hematopoiesis. This system offers a unique in vitro model for studying hematopoietic development at the earliest possible stages.

To study hematopoietic differentiation a variety of in vitro systems have been established using hematopoietic precursors derived from various explanted adult and fetal tissues. In this prospective we describe and discuss the potential of a novel system for studying the earliest stages of hematopoietic development. In addition, some of the applications of this system as a unique in vitro model for studying other developmental systems are discussed. Murine embryonic stem cells (ESC), which are totipotent and can be maintained undifferentiated indefinitely in vitro, have the capacity to differentiate in vitro into hematopoietic precursors of most, if not all, of the colony forming cells found in normal bone marrow. This potential can be exploited to study the control of the early stages of hematopoietic induction and differentiation. Recent results have indicated that there is a strong transcriptional activation, in a well defined temporal order, of many of the hematopoietically relevant genes. Examples of the genes expressed early during the induction of hematopoiesis include erythropoietin (Epo) and its receptor as well as the Steel (SI) factor (SLF) and its receptor (c-kit). Several other genes, including CSF-1, IL-1, and G-CSF were expressed during the later stages of hematopoietic differentiation. Contrasting with these observations, IL-3 and GM-CSF were not expressed during the first 24 days of ES cell differentiation suggesting that neither factor is necessary for the induction of hematopoietic precursors. Although these studies are just beginning, this system is easily manipulated and gives us an approach to understanding the control of the induction and differentiation of the hematopoietic system in ways not previously possible.

Preodontoblasts divide asynchronously and their terminal differentiation occurs gradually. Experimental data suggested that the expression of competence by preodontoblasts to respond to specific epigenetic signals, triggering their overt differentiation, requires a minimal number of cell cycles. The intrinsic timing mechanism could imply division counting and preodontoblasts of juxtaposed cell generations might sequentially withdraw from the last physiological cycle. To test such an hypothesis, embryonic mouse lower incisors were cultured in vitro and treated sequentially with nocodazole in order to induce a transitory synchronization of the dividing preodontoblasts and preameloblasts. This synchronization led to a disorganization of the physiological gradual terminal differentiation of the odontoblasts, giving rise to three distinct domains comprising respectively: 1) odontoblasts with altered polarization and predentin secretion; 2) odontoblasts demonstrating equivalent polarization and predentin deposition; and 3) preodontoblasts-odontoblasts involved in gradual terminal differentiation. These results strongly suggest that the gradient of odontoblast functional differentiation results from sequential withdrawal from asynchronous cell cycles of competent cells able to overtly differentiate.