The identification of endothelial progenitors in the adult circulation by Asahara and colleagues 4
has challenged the dogma that vasculogenesis is restricted to embryogenesis. Adult endothelial progenitors are found in association with the hematopoietic compartment 1, 2
. However whether endothelial and hematopoietic progenitors have a common origin or represent independent lineages within the bone marrow remains a topic of debate. Interestingly, a study involving the analysis of blood and bone marrow samples obtained from chronic myeloid leukemia (CML) revealed the presence of BCR/ABL
, the oncoprotein encoded by the Philadelphia chromosome, in both hematopoietic and endothelial cells 34
. This is particularly relevant since detection of the Philadelphia chromosome in all blood lineages of individuals with CML has been considered as direct evidence for the existence of the HSC in humans 35
. Stronger evidence for an association between these two lineages in postnatal life was provided by Grant and colleagues 36
. By transplanting individual Sca-1+
BM HSCs from GFP transgenic mice into irradiated recipients, these authors demonstrated that upon hematopoietic engraftment, donor-derived GFP+
cells contributed to the endothelial lineage when mice were subjected to retinal ischemia 36
. Similar results were observed following the transplantation of cord blood CD34+
cells into NOD/scid
mice. These results were corroborated by another study demonstrating that infusion of individual Sca-1+
BM HSCs resulted in broad incorporation of donor-derived cells into the endothelial compartment even in the absence of additional vascular injury, which was not a result of cell fusion 37
. Further studies by this group suggest that endothelial engraftment resulted from the stem cell pool or from a common myeloid progenitor but not from a lymphoid progenitor 38
. Although all these findings support the hypothesis that endothelial progenitors arise within the hematopoietic compartment, it is still unclear which cell type in the hematopoietic hierarchy gives rise to the endothelial lineage.
By using a combination of surface markers, single cell sorting assays, and subsequent replating studies, here we demonstrate for the first time, the existence of an adult common precursor for endothelial, myeloid, and lymphoid progenitors in human cord blood. Unexpectedly, this bipotent precursor resides within the CD34+CD45+CD133+CD38+ fraction, downstream of CD34+CD38− cells, suggesting that a multipotent progenitor, not the hematopoietic stem cell, is the point of divergence of the endothelial and hematopoietic lineages.
Despite the evident ability of CD34+CD45+CD133+CD38+ cells to differentiate into hematopoietic and endothelial cells in vitro, hematopoietic differentiation predominates in ischemic transplantation. This could be attributed to two major issues: i) hemangioblasts may not be endowed with in vivo vascularization potential since so far, this ability has not been investigated in ES- or embryo-derived hemangioblasts; ii) an ischemic xenogeneic mouse environment is not appropriate or sufficient to induce the differentiation of this human precursor into endothelial cells.
Since our goal here was to investigate whether hematopoietic cells, in particular CD133+
, have the ability to differentiate into endothelial cells, we did not address the existence of other angiogenic cells outside of the hematopoietic compartment, as suggested by other investigators 26, 27, 39
. It might be the case that these endothelial progenitors are endowed with robust in vivo
revascularization potential, although this has yet to be determined in animal models of ischemia. Mouse-to-mouse transplantation experiments involving the analyses of mice that had been subjected to unilateral femoral artery occlusion following the engraftment of BM cells isolated from transgenic mice expressing enhanced green fluorescent protein (GFP) revealed that donor GFP+
cells fail to incorporate into the adult growing vasculature, but were detectable around growing collateral arteries 40
. A similar outcome was obtained following the transplantation of hematopoietic stem cells isolated from GFP transgenic mice directly into ischemic myocardium of wild-type mice 41
. Interestingly here we observed a similar engraftment pattern following the transplantation of freshly isolated CD34+
cells into hind-limb ischemic mice. In these engrafted mice, human CD31+
cells were found in large quantities surrounding recipient’s vasculature, suggesting that the improved perfusion observed in transplanted mice may be due mostly due to a paracrine pro-angiogenic function of these cells. This would be in agreement with recent studies involving the transplantation of human CD133+
cells 42, 43
or EPCs 44, 45
, which indicate that these cell types secrete angiogenic factors 40, 44
On the other hand, when CD34+CD45+CD133+CD38+ cells are allowed to differentiate into endothelial cells in vitro, and then injected into mice using the Matrigel plug assay or the ischemia model, these cells give rise to functional human-derived and chimeric mouse-human blood vessels. Taken together our data reveals that the CD34+CD45+CD133+CD38+ cell fraction, which per se may have an angiogenic effect in vivo, is endowed with the ability to differentiate in vitro into myeloid, lymphoid, and endothelial lineages. The endothelial cells generated from this common precursor are able to participate in new blood vessel formation, providing a rationale for the use of this cell population, as well as their progenitor, for therapeutic applications in ischemia conditions.
NOVELTY AND SIGNIFICANCE
What Is Known?
- Endothelial progenitor cells (EPCs) are not restricted to embryogenesis.
- In postnatal life, EPCs are present in hematopoietic tissues, and are endowed with in vivo regenerative potential.
- Origin of endothelial progenitors within hematopoietic tissues is still unknown.
What New Information Does This Article Contribute?
- Identification of a multipotent hematopoietic progenitor in cord blood that gives rise to endothelial precursors.
- This multipotent progenitor can be isolated based on the expression of CD34, CD45, CD133, and CD38 antigens.
- Transplantation of freshly isolated CD34+CD45+CD133+CD38+ cells into ischemic hind-limbs results in improved perfusion, possibly through a paracrine effect.
- Transplantation of endothelial cells differentiated in vitro from the CD34+CD45+CD133+CD38+ cell fraction gives rise to functional human-derived and chimeric mouse-human blood vessels.
Recent investigations have highlighted the potential of EPCs for therapeutic applications in vascular medicine. Hematopoietic tissues from adults have been the primary cell source in these laboratory studies and early clinical trials. Most previous investigations have involved the infusion of heterogeneous populations of cells. To date, it is not clear which cell type within these hematopoietic tissues is responsible for vascular regeneration. We demonstrate here through clonal analysis that at least one source of endothelial progenitor cells in human cord blood can be traced back to a common progenitor of lymphoid and myeloid hematopoietic cells, a cell marked by the antigens, CD34, CD45, CD133, and CD38. While injection of this freshly-isolated progenitor cell into ischemic hind-limbs of immunodeficient mice results in improved recovery from ischemia, engrafted cells do not adopt an endothelial fate directly in this in vivo environment, but rather adopt a pro-angiogenic hematopoietic fate. However when this progenitor is first differentiated in vitro into endothelial cells, these cells generate functional human-derived and chimeric mouse-human blood vessels. These findings provide insights into the identity of EPCs and their positioning with regard to the hematopoietic hierarchy, giving scientific rationale for future clinical applications in patients with ischemic vascular diseases.