Jacques Samarut

VIP and PACAP induce selective neuronal differentiation of mouse embryonic stem cells.

The capacity of embryonic stem cells (ES cells) to differentiate into neuronal cells represents a potential source for neuronal replacement and a model for studying factors controlling early stages of neuronal differentiation. Various molecules have been used to induce such differentiation but so far neuropeptides acting via functional G‐protein‐coupled receptors (GPCRs) have not been investigated. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase‐activating polypeptide (PACAP) are neuropeptides expressed in early development which affect neuronal precursor proliferation and neuronal differentiation. VIP and PACAP share two common receptors (VPAC1 and VPAC2 receptors) while only PACAP binds with high affinity to PAC1 receptors. The aim of the study was to determine whether VIP and PACAP could produce functional neuronal differentiation of ES cells. Mouse ES cells were allowed to aggregate in embryoid bodies (EBs) in the presence or not of VIP and PACAP for 1u2003week. VIP and PACAP potently increased the proportion of EB‐derived cells expressing specifically a neuronal phenotype shown by immunocytochemistry and neurite outgrowth without altering glial cell number. Binding and RT‐PCR analyses demonstrated the presence of VPAC2 and PAC1 receptors on ES cells. Accordingly, both peptides increased cyclic AMP and intracellular calcium. In contrast, EB‐derived cells only expressed a functional PAC1 receptor, suggesting a switch in GPCR phenotype during ES cell differentiation. These original data demonstrate that functional GPCRs for VIP and PACAP are present on ES cells and that these neuropeptides may induce their differentiation into a neuronal phenotype. It opens an exciting new field for neuropeptide regulation of tissue ontogenesis.

Effects of T3Rα1 and T3Rα2 Gene Deletion on T and B Lymphocyte Development

Thyroid hormones bind to several nuclear receptors encoded by T3Rα and T3Rβ genes. There is now accumulating evidence that thyroid hormones act on the immune system. Indeed, mice deficient for thyroid hormones show a reduction in lymphocyte production. However, the mechanisms involved and, in particular, the role of the different thyroid hormone receptors in lymphocyte development have not been investigated. To address that question, we have studied lymphocyte development in mice deficient for the T3Rα1 and T3Rα2 gene products. A strong decrease in spleen cell numbers was found compared with wild-type littermates, B lymphocytes being more severely affected than T lymphocytes. A significant decrease in splenic macrophage and granulocyte numbers was also found. In bone marrow, a reduction in CD45+/IgM− pro/pre-B cell numbers was found in these mice compared with wild-type littermates. This decrease seems to result from a proliferation defect, as CD45+/IgM− cells incorporate less 5-bromo-2′-deoxyuridine in vivo. To define the origin of the bone marrow development defect, chimeric animals between T3Rα−/− and Rag1−/− mice were generated. Results indicate that for B cells the control of the population size by T3Rα1 and T3Rα2 is intrinsic. Altogether, these results show that T3Rα1 or T3Rα2 gene products are implicated in the control of the B cell pool size.

A novel growth-factor–dependent myeloid cell line derived from mouse bone marrow cells contains progenitors endowed with high proliferative potential

Constitutive expression of human colony-stimulating factor-1 receptor (CSF-1R) confers long-lasting CSF-1-dependent proliferation to mouse myeloid cell lines. We developed mice transgenic for human CSF-1R because mouse CSF-1 cannot activate human CSF-1R. Then bone marrow cells from transgenic mice were plated onto MS-5 stromal cells expressing the membrane form of human CSF-1 (2M-1 cells) in order to combine the hematopoietic supporting properties of stromal cells and the proliferative effects of CSF-1. Thus, we were able to derive a hematopoietic cell line, called 47.10, that grew indefinitely under these conditions, whereas no cell line could be developed from nontransgenic mice. Proliferation of 47.10 cells is severely affected by neutralizing anti-CSF-1R monoclonal antibodies. Morphologic and cytofluorometry analysis established that most 47.10 cells are immature myelomonocytic cells. Consistent with this phenotype, the myeloid transcription factor PU.1, but not the erythroid transcription factor GATA-1, is expressed in 47.10 cells. A few 47.10 cells (3-5%) do not express lineage specific markers; they differentiate spontaneously to lineage-positive cells after replating on 2M-1 cells. In agar cultures, 47.10 cells form 7- and 14-day colonies in response to a cocktail of granulocyte/macrophage colony-stimulating factor (2.5 ng/mL), interleukin-3 (1 ng/mL), and mouse CSF-1 (10 ng/mL). Under these conditions, about 0.5% of 47.10 cells formed large 14-day colonies (>1 mm) composed of mature monocytes and granulocytes, reflecting the presence of progenitors endowed with high proliferative potential (HPP-47.10 cells). In conclusion, we have characterized a novel continuous myeloid cell line presenting a hierarchical structure similar to that of the bone marrow progenitor cell compartment.