Phenotype and hematopoietic potential of side population cells throughout embryonic development

Abstract

Adult murine bone marrow hematopoietic stem cells (HSCs) can be purified by sorting Hoechst 33342-extruding side population (SP) cells. Herein we investigated whether SP cells reside within embryonic tissues and exhibit hematopoietic progenitor activity. We isolated yolk sac (YS) and embryonic tissues 7.5 to 11.5 days after coitus (dpc), resolved an SP in each, and demonstrated that these SP cells exhibit distinct phenotypic and functional characteristics throughout development. YS and embryonic SP isolated 8.0 dpc expressed vascular endothelial-cadherin (VE-cadherin) and vascular endothelial receptor 2 (Flk-1), markers not expressed by bone marrow SP but expressed by endothelial cells and progenitors. SP at this stage did not express CD45 or produce hematopoietic colonies in vitro. In contrast, SP isolated 9.5 to 11.5 dpc contained a significantly higher proportion of cells expressing cKit and CD45, markers highly expressed by bone marrow SP. Furthermore, YS SP isolated 9.5 to 11.5 dpc demonstrated 40-to 90-fold enrichment for hematopoietic progenitor activity over unfractionated tissue. Our data indicate that YS and embryonic SP cells detected prior to the onset of circulation express the highest levels of endothelial markers and do not generate blood cells in vitro; however, as development progresses, they acquire hematopoietic potential and phenotypic characteristics similar to those of bone marrow SP.

Isolation of Murine Embryonic Hemogenic Endothelial Cells

“…In addition, we have also shown that hemogenic endothelial cells express markers of both endothelial and stem cells (Flk1 and cKit, respectively), but do not express the hematopoietic lineage marker, CD45 5,11,12 . Thus, hemogenic endothelial cells can be identified and isolated by FACS as Flk1+/cKit+/CD45-SP cells, and we have shown that these cells give rise to HSPC contained within the Flk1-/cKit+/CD45+ SP fraction of yolk sac and AGM cells 5,11,12 . Hemogenic endothelial cells and HSPC can be identified and isolated from yolk sac or AGM tissues harvested from either freshly euthanized embryos, or from embryos cultured for up to 48 hours in ex vivo embryo culture (as depicted in Figure 1).…”

“…Towards this goal, we have defined the phenotype of hemogenic endothelial cells, on a clonal level, within the murine yolk sac 11 and AGM 12 , two major sites of definitive HSPC production during embryogenesis. Like HSPC within adult bone marrow 17 , embryonic hemogenic endothelial cells and HSPC exhibit Hoechst dye efflux properties and therefore appear within the "side population" (SP) of cells on a FACS plot 5,11,12 (as shown in Figure 3). In addition, we have also shown that hemogenic endothelial cells express markers of both endothelial and stem cells (Flk1 and cKit, respectively), but do not express the hematopoietic lineage marker, CD45 5,11,12 .…”

“…Like HSPC within adult bone marrow 17 , embryonic hemogenic endothelial cells and HSPC exhibit Hoechst dye efflux properties and therefore appear within the "side population" (SP) of cells on a FACS plot 5,11,12 (as shown in Figure 3). In addition, we have also shown that hemogenic endothelial cells express markers of both endothelial and stem cells (Flk1 and cKit, respectively), but do not express the hematopoietic lineage marker, CD45 5,11,12 . Thus, hemogenic endothelial cells can be identified and isolated by FACS as Flk1+/cKit+/CD45-SP cells, and we have shown that these cells give rise to HSPC contained within the Flk1-/cKit+/CD45+ SP fraction of yolk sac and AGM cells 5,11,12 .…”

“…At the earliest stages of blood development (primitive hematopoiesis), the origin of erythroblasts remains vigorously debated 1 . In contrast, at later stages of blood cell development (definitive hematopoiesis), it has become increasingly clear that multi-lineage HSPC arise from specialized vascular endothelial cells that acquire blood forming potential (hemogenic endothelial cells) within the yolk sac, placenta, and AGM [2][3][4][5] , as well as in the vitelline and umbilical vessels, the embryonic endocardium 6 , and head vasculature 7 . The specification of hemogenic endothelial cells within these distinct tissues occurs at specific stages of development; for example, within the yolk sac at ~E8.25 and within the AGM at ~E10 [8][9][10][11][12] .…”

Qualitative and quantitative analysis of rabbit's fat mesenchymal stem cells

“…The HSC are derived from mesoderm 5 . The hematopoietic progenitors accumulate around the blood islands initially, migrating to the fetal liver reaching the bone marrow 6,7 .…”