3056 – NES-EGFP TRANSGENIC MICE AS A MODEL FOR IN VIVO AND IN VITRO STUDY OF BONE-MARROW-DERIVED MESENCHYMAL STEM CELLS (MSCS)

Abstract

Transgenic NES-EGFP mice developed by G. Enikolopov (Tg(Nes-EGFP)33Enik), which have a green fluorescent protein (EGFP) gene under the promoter of the nestin gene, had been previously used to show that early mesenchymal progenitors are in the population of CD45–GFP+ bone marrow (BM) cells. Here we show that NES-EGFP mice can serve as a convenient model for studying MSCs capable of transferring hematopoietic microenvironment under the renal capsule of syngeneic mice. BM from one femur of NES-EGFP mouse was used to obtain Dexter-type long-time bone marrow culture (LTBMCs, n = 5). GFP+ cells had been counted weekly over the entire area of the T25 flasks for 5 weeks after culture initiation using a fluorescence microscope. Four weeks after the initiation of LTBMCs, two day long time-lapse vital photography was performed. Ectopic foci of hematopoiesis under the renal capsule (n = 6) were obtained from the BM of NES-EGFP donors in syngeneic recipients. The number of C-kit+/–GFP+ cells in the formed foci was determined using FACS analysis. GFP+ cells with stromal phenotype in the adherent cell layer of LTBMCs had been detected during 5 weeks of cultivation. The stromal layer of LTBMC contained fibroblast-like GFP-low and GFP-hi cell subpopulations. GFP-low cells were scattered across the surface and predominated. GFP-hi cells were rare and formed colonies. Ectopic foci of hematopoiesis contained 2036 ± 734 C-kit–GFP+ stromal cells. Most of these cells had GFP-low phenotype. The cellularity of the focus strongly and positively correlated with the number of C-kit–GFP+ in it. The results show that C-kit–GFP+ BM cells of NES-EGFP mice are able to survive implantation and include MSCs and early stromal mesenchymal progenitors, the number of which determines the size of the focus. NES-EGFP mice can be used for in vitro and in vivo experiments to study functional BM-derived MSCs.