Program: Oral and Poster Abstracts
Session: 501. Hematopoietic Stem and Progenitor Biology: Poster II
First of all, we investigated the candidate gene RNA expression in purified murine hematopoietic cell populations. Interestingly, we observed that Evl and miR-342 are not highly expressed in murine Lineage-Sca1+ckit+ (LSK) cells but their expression increases profoundly with blood cell differentiation. While Evl expression was highest in lymphocytes (20-30 fold higher as compared to LSK cells), miR-342 was expressed at the highest level in macrophages (300fold higher compared to LSK cells). To study the role of the candidates in hematopoiesis, we overexpressed Evl and miR-342 by using lentiviral vectors in murine primary LSK cells. Gene expression profiling of LSK cells overexpressing Evl revealed that 32% (62 out of 190) of the deregulated transcripts were involved in hematopoietic system development and function. Moreover, the top deregulated canonical pathways detected are essential for the development of B-cells (p=2.59*10-11). However, pathways important for myeloid cells such as immune cell trafficking and, more specifically, granulocytic adhesion and diapedesis (p=2.59*10-3) were significantly upregulated within miR-342- positive LSK cells. Functional analysis showed that Evl overexpression leads to a three- to fourfold increase of preB-cell colonies compared to control vector-transduced LSK cells. By contrast, miR-342 overexpressing cells formed a twofold higher number of myeloid colonies in semisolid medium. Next, the influence of Evl and its intronic miRNA on self-renewal and multilineage differentiation in vivo was investigated in serial bone marrow (BM) transplantation experiments. Within the PB of primary recipient mice, we detected a decrease of Evl-positive cells over time (week 4: 20.6 ± 9.6%; week 20: 4.7 ± 2.4%). Within the spleens a significantly higher donor-derived B-cell frequency was detectable (Evl: 63.6 ± 17.1%; Mock: 39.9 ± 18.3%). In line with our in vitro results, we detected a 4.3 fold higher frequency of Evl positive B-cells four weeks after secondary transplantation (Evl: 68.1 ± 9.8 %; Mock: 15.9 ± 7.5%).
In summary, our data show that different hematopoietic differentiation programs are driven by one common gene locus depending on the expressed gene product. While the protein-coding gene Evl drives B-cell lymphopoiesis, its intronic miR-342 promotes myeloid differentiation.
Disclosures: No relevant conflicts of interest to declare.
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