Embryonal subregion-derived stromal cell lines from novel temperature-sensitive SV40 T antigen transgenic mice support hematopoiesis

Abstract

Throughout life, the hematopoietic system requires a supportive microenvironment that allows for the maintenance and differentiation of hematopoietic stem cells (HSC). To understand the cellular interactions and molecules that provide these functions, investigators have previously established stromal cell lines from the late gestational stage and adult murine hematopoietic microenvironments. However, the stromal cell microenvironment that supports the emergence, expansion and maintenance of HSCs during mid-gestational stages has been largely unexplored. Since several tissues within the mouse embryo are known to harbor HSCs (i.e. aortagonads-mesonephros, yolk sac, liver), we generated numerous stromal cell clones from these mid-gestational sites. Owing to the limited cell numbers,isolations were performed with tissues from transgenic embryos containing the ts SV40 Tag gene (tsA58) under the transcriptional control of constitutive and ubiquitously expressing promoters. We report here that the growth and cloning efficiency of embryonic cells (with the exception of the aorta) is increased in the presence of the tsA58 transgene. Furthermore, our results show that the large panel of stromal clones isolated from the different embryonal subregions exhibit heterogeneity in their ability to promote murine and human hematopoietic differentiation. Despite our findings of heterogeneity in hematopoietic growth factor gene expression profiles, high-level expression of some factors may influence hematopoietic differentiation. Interestingly, a few of these stromal clones express a recently described chordin-like protein, which is an inhibitor of bone morphogenic proteins and is preferentially expressed in cells of the mesenchymal lineage.

PDGFRβ+ cells play a dual role as hematopoietic precursors and niche cells during mouse ontogeny

“…Signals from the microenvironment are required for HSPC generation. In vitro studies have shown that stromal cell lines derived from E11 AGMs support hematopoiesis in co-cultures with BM HSPCs (Oostendorp et al, 2002a(Oostendorp et al, , 2002b. This was further confirmed in vivo in 2013, when Richard et al (2013) demonstrated that the interaction between the subaortic mesenchyme and the endothelium in the chick embryo was necessary to induce Runx1 expression, a transcription factor required for HSPC generation (Chen et al, 2009).…”

Comparative analysis of extracellular vesicle isolation methods from human AML bone marrow cells and AML cell lines

“…MOLM-13 cells were seeded at a density of 1×10 6 cells/ml in RPMI medium (Gibco, Life Technologies) with 10% FBS (Gibco, Life Technologies) and 20 U/ml P/S. The murine embryonic stromal cell line EL08-1D2 ( 39 , 40 ) was cultured on gelatin-coated flasks in alphaMEM medium [Gibco, MEM Alpha Medium (1×) + Glutamax I] containing 15% FBS, 5% horse serum, 20 U/ml P/S, and 10 µM 2-mercaptoethanol. Seeding density was 1,000 cells/cm 2 .…”

Mesenchymal stem cells: paradoxes of passaging

“…This layer consists morphologically of a densely packed, round cell layer in the mesenchyme that does not express hematopoietic or endothelial markers, but it does express extracellular matrix proteins suggestive of stroma. In addition, there have been a number of studies in which stromal cell lines have been produced by culturing AGM cells using isolation and culture techniques (i.e., plastic adherence and growth in 10-20% serum containing media) similar to those used for adult MSC [26][27][28]. These stromal cell lines have been primarily characterized on the basis of their ability to support embryonic or adult hematopoiesis and have undergone little phenotypic characterization or analysis of potentiality.…”