Identification of CD141+ vasculogenic precursor cells from human bone marrow and their endothelial engagement in the arteriogenesis by co-transplantation with mesenchymal stem cells

Abstract

Background Critical limb ischemia (CLI) is a condition characterized by insufficient blood flow to the lower limbs, resulting in severe ischemia and potentially leading to amputation. This study aims to identify novel vasculogenic precursor cells (VPCs) in human bone marrow and evaluate their efficacy in combination with bone marrow-derived mesenchymal stem cells (BM-MSCs) for the treatment of CLI. Methods Ex vivo cultured VPCs and BM-MSCs from bone marrow were characterized and their effects on neovascularization and long-term tissue regeneration were tested in a mouse CLI model. Results VPCs, expressing high levels of hepatocyte growth factor and c-MET, were identified from human bone marrow aspirates. These cells exhibited strong vasculogenic capacity in vitro but possessed a cellular phenotype distinct from those of previously reported endothelial precursor cells in circulation or cord blood. They also expressed most surface markers of BM-MSCs and demonstrated multipotent differentiation ability. Screening of 376 surface markers revealed that VPCs uniquely display CD141 (thrombomodulin). CD141+VPCs are present in BM aspirates as a rare population and can be expanded ex vivo with a population doubling time of approximately 20 h, generating an elaborate vascular network even under angiogenic factor-deficient conditions and recruiting BM-MSCs to the network as pericyte-like cells. Intramuscular transplantation of a combination of human CD141+VPCs and BM-MSCs at a ratio of 2:1 resulted in limb salvage, blood flow recovery, and regeneration of large vessels in the femoral artery-removed CLI model, with an efficacy superior to that of singular transplantation. Importantly, large arteries and arterioles in dual cell transplantation expressed human CD31 in the intima and human α-smooth muscle actin in media layer at 4 weeks post-transplantation, indicating direct engagement of transplanted cells in arteriogenesis. Conclusions CD141+VPCs represent a novel bone marrow-derived vasculogenic precursor cell population with potent therapeutic potential for CLI when combined with BM-MSCs, offering a promising cell-based strategy for vascular regeneration.

1. Introduction

Endothelial precursor cells (EPCs) were initially characterized by Asahara et al. [1]. EPCs isolated from the umbilical cord and peripheral blood (PB) mononuclear cells (MNCs) exhibit vasoreparative ability in vitro and in vivo but have dissimilar cellular and molecular properties [2, 3]. Early EPCs, myeloid angiogenic cells (MACs), and circulating angiogenic cells (CACs) have myeloid and hematopoietic origins; they exhibit limited proliferative potential and cannot generate tube-like structures in vitro but can stimulate the formation of new blood vessels by secreting angiogenic growth factors, chemokines, and cytokines rather than directly integrating into vascular networks [4]. Outgrowth endothelial cells (OECs) or endothelial colony forming cells (ECFCs), derived from adherent cells between 7 and 21 days, exhibit endothelial-lineage commitment [4, 5] but are not originated from the bone marrow [6].

Protein C receptor (CD201, also known as EPCR)-expressing endothelial cells (ECs) and CD157 (BST-1)-expressing side population cells, which were recently identified as tissue-resident vascular endothelial stem cells (VESCs) in the development of mouse mammary fat pads [7] and in the intima of large vessels of the mouse liver and other tissues, are expected to be involved in homeostatic and regenerative events or pathological disease and cancer development [2, 8]. Accordingly, mouse VESCs in the vessel intima and OECs isolated from the blood, expressing most endothelial surface markers, such as CD31, vascular endothelial (VE)-cadherin, and vascular endothelial growth factor receptor 2 (VEGFR2), may be classified as endothelial-lineage-committed tissue stem cells or activated ECs. However, human VESCs are yet to be identified, and consistent endothelial-lineage commitment efficacy of OECs is lacking. Moreover, the definitive phenotype of EPC-like or vasculogenic cells derived from a variety of tissues remains elusive, which has led to a considerable debate regarding the selection of specific surface markers.

Peripheral artery disease (PAD) refers to atherosclerosis affecting arteries of the lower extremities, which results in reduced blood flow and can progress to critical limb ischemia (CLI), a severe condition with high risk of amputation. Current treatments are limited, and cell-based therapies using endothelial progenitor cells or mesenchymal stem cells have shown promise but with inconsistent outcomes. Therefore, identifying novel vasculogenic cell populations with robust regenerative potential is crucial for developing effective therapies for CLI.