Vascular Precursor Cells

Abstract

Understanding the mechanisms that regulate the proliferation and differentiation of human stem and progenitor cells is critically important for the development and optimization of regenerative medicine strategies. For vascular regeneration studies, specifically, a true “vascular stem cell” population has not yet been identified. However, a number of cell types that exist endogenously, or can be generated or propagated ex vivo, function as vascular precursor cells and can participate in and/or promote vascular regeneration. Herein, we provide an overview of what is known about the regulation of their differentiation specifically toward a vascular endothelial cell phenotype.

Introduction

Regenerative medicine encompasses strategies designed to enable the repair or replacement of tissues and organs that are damaged through insult or defective at birth. These include the localized delivery of reparative stem or progenitor cells,^1,2 the promotion of endogenous repair mechanisms via biologically active molecules^3,4 or scaffold placement,^5,6 and transplantation of in vitro engineered organs and tissues.^7,8 Such emerging technologies have the potential to solve critical problems associated with organ shortage and transplant rejection. Greater understanding of the molecular regulation and biology of human stem and progenitor cell populations is therefore of growing importance to both the clinical and the translational research communities.

In this review, we focus on cell sources for vascular regeneration. A true “vascular stem cell” population, defined as an immediate precursor to all vascular endothelial and/or mural cells, has not yet been identified in vivo. However, significant progress has been made in deriving functional vascular precursors and fully differentiated vascular cell types in vitro from a variety of multipotent cell sources, including adult progenitor cells, embryonic stem cells, and induced pluripotent stem cells, which will be defined and discussed herein.

Adult-Derived Vascular Progenitors

Numerous studies suggest the existence of adult vascular progenitor cells, which are thought to contribute to neovascularization in both physiological and pathophysiological conditions. Adult vascular progenitors were first isolated from human and mouse peripheral blood by Asahara et al. ⁹ in 1997 on the basis of CD34 and Flk-1 expression. These cells were shown to be capable of differentiating into endothelial cells in vitro and incorporating into sites of active neovascularization in vivo. Adult vascular progenitors capable of giving rise to, or promoting the generation of, differentiated vascular cell types in vivo have since been isolated from bone marrow, peripheral blood, umbilical cord blood, adipose tissue, and various vascular beds.^9-16 It is hypothesized that both tissue-resident and circulating vascular progenitors are specifically recruited to sites of neovascularization in response to tissue injury. Thus, these vascular progenitors could be isolated for use in autologous stem cell transplant applications to promote therapeutic neovascularization. However, to date, adult vascular progenitors remain an incompletely characterized phenotype and are variously isolated on the basis of expression of a variety of cell surface markers, including CD34, Flk-1, CD133, and CD14 ¹⁷ ; the specific function of these populations is not entirely clear. More thorough reviews of this topic have been previously published.^18,19

Embryonic Stem Cell–Derived Vascular Cells

Embryonic stem (ES) cells are totipotent or pluripotent cells with the ability to undergo self-renewal for an indefinite period of time, giving rise to both pluripotent and differentiated daughter cells via asymmetric division. These cells are currently derived from the inner cell mass of mammalian embryos.^20-22 Cells with similar stem-like properties have also been derived from the reprogramming of somatic cells to generate induced pluripotent stem (iPS) cells,^23,24 which are discussed in a later section. Such pluripotent stem cells can be induced to differentiate into all cell types of the adult body, including vascular endothelial and mural cells.

Vascular differentiation of ES cells, specifically, is most commonly induced via in vitro culture as embryoid bodies (EB), ²⁵ which undergo spontaneous differentiation into tissue-specific progenitors representing all 3 embryonic germ layers. ²⁶ CD34⁺CD31⁺ vascular progenitors are then isolated from spontaneously differentiated EB and cultured under conditions that promote vascular differentiation and proliferation. ²⁷ Another routine approach is to co-culture ES cells on murine OP9 bone marrow stromal cells, a technique originally developed to differentiate ES cells into hematopoietic cells^28,29 and more recently found to induce vascular endothelial cell differentiation. ³⁰ Both protocols have shortcomings for understanding the molecular regulation of endothelial cell differentiation, including the presence of additional cell types and undefined serum-derived factors. As a result, the field is now moving toward the development of feeder- and xeno-free culture and differentiation systems, in which ES cells are maintained on cell-free extracellular matrices with chemically defined media and growth factor supplementation.^31,32

Molecular Regulation of ES-Derived Endothelial Cell Differentiation

Endothelial cell differentiation is a prerequisite for the formation of blood vessels during embryonic development and likely contributes to postnatal neovascularization. In the developing mouse, primordial endothelium first arises as a subset of Flk-1⁺CD31⁺CD45^– mesodermal derivatives. ³³ Endodermally derived soluble factors, such as Indian hedgehog (Ihh),^34,35 vascular endothelial growth factor (VEGF),^36,37 and basic fibroblast growth factor (bFGF),^38,39 then act upon specific receptors localized within the mesoderm to promote the differentiation of vascular progenitors (further reviewed by Iacobas et al. ⁴⁰ ). Global gene expression analysis of the developing vasculature during mouse embryogenesis also suggests a role for Wnt signaling in promoting vascular differentiation from murine ES cells, ⁴¹ although the interactions among these pathways are not clearly defined.

Multiple development models including avian, zebrafish, and mouse embryos have been studied in an attempt to understand the molecular regulation of endothelial cell differentiation in vitro. Endothelial differentiation from human embryonic stem (hES) cells has been assessed by a number of groups at different time points during the differentiation process.^25,29,30,42 Expression of surface markers such as Flk-1, CD31, VE-cad, Tie-2, CD34, and CD45; soluble factors including Ihh and VEGF; and downstream target genes such as Wnt/Dkk2, BMP2, BMP4, eNOS, HDAC, HIF-1α, and JAK/STAT3/STAT5 signaling have been assessed for their potential roles in this process.

In the hES cell model, Ihh promotes endothelial marker expression (e.g., CD31, VE-cadherin) and endothelial cell differentiation through BMP4 signaling,^30,43 whereas VEGF promotes the proliferation of CD31-expressing endothelial cells (25). BMP2 also plays a role in endothelial cell development in other model systems.^44,45 In a rat model, recombinant human-BMP2 enhanced the expression of hypoxia inducible factor (HIF-1α) and inhibitor of DNA binding gene (Id1) through the stimulation of JAK2/STAT3/STAT5 signaling pathways. ⁴⁵ HIF-1α plays an important role in promoting VEGF transcription, ⁴⁶ which is evident in HIF-1α-null murine embryos that have lethal vascular malformations. ⁴⁷ Although Min and coworkers ⁴⁸ have shown a role for DKK2, a Wnt pathway regulator, in promoting murine endothelial morphogenesis and angiogenesis, it is not clear whether or how these pathways interact.

Rossig and co-workers showed an important role for acetylation in aiding differentiation toward vascular phenotypes, demonstrating a role for histone deacetylase (HDAC) activity in promoting expression of the homeobox transcription factor HoxA9. ⁴⁹ HoxA9 was shown to be involved in the transcriptional regulation of key endothelial genes such as eNOS, Flk-1, and VE-cadherin in cultured endothelial cells. At a global level, the group observed severe impairment in recovery after hind-limb ischemia in HoxA9- deficient mice. The investigators proposed that shear stress-induced maturation through HoxA9-mediated genes may influence endothelial monolayer recovery after injury. Whether similar regulation occurs in the hES cell system to promote endothelial cell differentiation is yet to be determined.

Induced Pluripotent Stem Cells

ES cells have the potential to treat a host of human diseases, but progress has been constrained by ethical implications regarding the use of human embryos, as well as the possibility of tissue rejection following transplantation. Induced pluripotent stem (iPS) cells have the potential to mitigate these complications, since they may be derived from patient-specific somatic cells by enforced reexpression of a specific subset of “master” pluripotency genes. In 2006, mouse iPS cells were first generated by the Yamanaka group, using retroviruses to transduce mouse fibroblasts with Oct3/4, Sox2, c-Myc, and Klf4. ⁵⁰ A year later, human iPS cells were independently derived by both the Thomson and Yamanaka laboratories by transducing human dermal fibroblasts with virally encoded transcriptional regulators of pluripotency.^23,24 The reprogrammed human iPS cells were shown to be functionally and phenotypically similar to undifferentiated human ES cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. Furthermore, these cells could be differentiated into cell types representing all 3 embryonic germ layers in vitro and in vivo. ²³

From a clinical perspective, it is important to note the disadvantages of using viruses to reprogram somatic cells. Extensive work is being conducted to develop clinically feasible methods for deriving iPS cells suitable for human therapeutic application. New ways of inducing pluripotency in somatic cells have been established, including adenoviral transduction and plasmid transfection, both of which rely on transient expression of reprogramming factors without integration of ectopic DNA into the host genome.^51-55

Mouse iPS Cell–Derived Vascular Progenitors

In 2008, 2 groups independently observed differentiation of mouse iPS cells into endothelial cells via EB formation and culture on collagen matrices.^56,57 Mouse iPS cells have been used by a number of groups to study the biology of neovascularization and to develop clinically relevant vascular regenerative therapies. A role for iPS cell–derived Flk-1 progenitors in the treatment of acute myocardial infarction was recently described. ⁵⁸ Investigators observed favorable myocardial remodeling, improved left ventricular function, and increased tissue vascularization, suggesting a relevant role for these cells in engraftment strategies. Xie et al. ⁵⁹ also demonstrated a role for mouse iPS cells in vascular tissue engineering through the differentiation of these cells into smooth muscle cells. Retinoic acid treatment of iPS cells led to SMC differentiation in 3D scaffolds in vitro and in vivo, pointing the way to a potential patient-specific vessel regeneration protocol.

Human iPS Cell–Derived Vascular Progenitors

In terms of self-renewal and pluripotency, hiPS cells share many functional and phenotypic similarities with hES cells.^23,24 However, despite their many similarities, iPS cells demonstrate some key molecular differences compared with ES cells. Numerous groups have reported similarities between the 2 cell types at the transcriptional level while pointing out some key differences.^23,60-63 It is important to note that heterogeneity in gene expression levels also exists between different hiPS cell types themselves. ⁶¹ It is difficult to predict whether such differences will be of any consequence under physiological and pathophysiological transplant conditions or will influence the molecular regulation of specific iPS cell lines. Using EB formation assays, hiPS cells have been found to be differentiated into vascular and hematopoietic cell types similar to those derived from hES cells ⁶³ ; however, little is known about their molecular regulation and whether it is similar to those derived from hES cells.

Summary

Adult and embryonic stem/progenitor cell studies are gaining attention and provide hope for the development of regenerative medicine strategies. This is especially true since the establishment of human iPS cells, which can be tailor-made for patients to meet individual needs, thus eliminating the problems attached with transplant rejection. The murine system has been useful in understanding the mechanisms that govern differentiation and development, and give us insight into the various issues involved with stem cell research and therapy. However, not all studies in such animal models are applicable to humans; thus, investigating human cell development and differentiation using human pluripotent cell sources will be key for continued development and optimization of clinical strategies.