Early B-Cell Factor 2 Represents A Novel Marker of Highly Purified Messenchymal Stem-Like Cells in Mouse Bone Marrow.

Abstract 252

The future therapeutic use of mesenchymal stem cells (MSCs) for human depends on the establishment of preclinical studies with other mammals such as mouse. However, purification and characterization of mouse MSCs from bone marrow (BM) remain poorly documented. The lack of MSC-specific markers for isolation and characterization has been a main obstacle to both research and clinical application with MSCs. The isolation method based on the adherence properties of MSCs in culture has proven ineffective because of large contamination of various hematopoietic lineages and potential phenotypic changes in cultured cells. Consequently, there is very little knowledge about the precise properties of a MSC in its native environment. In the present study, we have utilized a bacterial artificial chromosomes (BAC) transgenic reporter mouse line expressing enhanced green fluorescent protein (EGFP) under the control of the regulatory elements of the Ebf2 gene. Early B cell factor 2 (EBF2) , is a member of the EBF family of transcription factors and has been shown to be expressed in the endosteal niche (Kieslinger et al 2005), a region where MSCs may be defined. The Ebf2-EGFP expressing stromal cells (CD45⁻TER119⁻GFP⁺) are composed 0.002% of total BM mononuclear cells and could be sorted by fluorescence-activated cell sorter (FACS) from adult Tg (Ebf2-EGFP)FB58Gsat/Mmcd mice. The fidelity of GFP expression to that of the endogenous Ebf2 gene was confirmed by quantitative real time PCR providing evidences for that the reporter gene expression marked a defined population of CD45⁻ cells. GFP⁺ cells could not be found in the hematopoietic cell compartments (CD45⁺TER119⁺), indicating a unique expression of EBF2 in stromal cells. In addition, a 10-fold reduction in frequency of CD45⁻TER119⁻GFP⁺ cells in marrow cells compared to that in bone suggested a preferential distribution of the GFP⁺ stromal cells in endosteal area of the bone. Colony forming unit-fibroblast (CFU-F) assay of the sorted cells revealed that the frequency of CFU-Fs in CD45⁻TER119⁻GFP⁺ cells reached as high as 1 out of 15 whereas only around1/4000 could be detected in CD45⁻TER119⁻GFP⁻ cells, indicating the GFP⁺ EBF2 expressing cells are enriched for primitive stromal progenitor cells in BM. Morphologically, CFU-Fs derived from the GFP⁺ cells are mostly spindle-shaped and mononuclear. In contrast, the CFU-Fs derived from the GFP- cells are enriched with big cells containing multiple nuclei and differentiated stromal cells. In line with the function and morphological data, Microarray data obtained from two independent experiments revealed a dramatic downregulation of cell cycle genes including Cdc6, Cdca2-4, −7,−8 and Ki67, Cdk4-6) and up-regulation of Cdkis such as p57 and p21 in the GFP⁺ cells, compared to the GFP⁻ cells, indicating quiescence state of GFP⁺ cells. Even though the GFP⁺ cells functionally appeared more primitive than the GFP⁻ cells, multiple lineage associated genes specific for osteoblasts, adipocyte, chondrocyte and myocyte were relatively higher expressed in the GFP⁺ cell population compared to the GFP⁻ cells, possibly indicating lineage priming events. To test the expression profiles of cell surface antigens that has been studied in the MSCs selected in culture, we performed multiple-color FACS analysis of expressions of CD34, SCA1, CD44 and CD29 within CD45⁻TER119⁻GFP⁺ cells. In order to exclude contamination of hematopoietic cells, we add additional markers B220/CD19 in separated channels during the FACS analysis. Consistent with the Microarray data, we found that GFP⁺ cells are 100% CD29⁺, but 100% CD44⁻. In addition, they express higher levels of CD34, SCA1, compared to the GFP⁻ cells. This is in contrast to the previous studies showing that the MSCs from culture express higher level of CD44 and most of them are CD34⁻. Thus, using the transgenic EBF2 reporter mouse model we have been able to prospectively isolate an MSC like cell directly from the adult mouse BM largely increasing the possibilities to investigate phenotypic and molecular characteristics of the BM primitive mesenchymal progenitor cells ex vivo.