Recapitulation of the embryonic cardiovascular progenitor cell niche
Introduction
The mammalian heart is the first organ to develop and function in the fetus. It contains three major cell lineages, cardiac myocytes (CMs), smooth muscle cells (SMCs) and endothelial cells (ECs), which are believed to derive from a common cardiovascular progenitor cell (CPC) [1], [2], [3], [4]. This progenitor represents one of the earliest stages in mesodermal specification to the cardiovascular lineage. It can be identified in mouse embryos or embryonic stem (ES) cell derivatives by expression of the kinase insert domain protein receptor-expressing (KDR, also known as Flk1). Subsequent studies have reported additional markers, defining multipotent mouse CPCs including Islet1 (Isl1+) and NK2 transcription factor-related, locus 5 (Nkx2.5+) [1], [2], [3]. We isolated Flk1+ progenitor cells from differentiating mouse ES or induced-pluripotent stem (iPS) cells that possess the ability to differentiate into all three cell types of the cardiovascular lineage as well as hematopoietic cells [5]. A comparable CPC is also present during human cardiogenesis [6]; however, this Isl1+/Flk1+ progenitor is rapidly lost postnatally and is not detectable in the adult heart.
Endogenous Isl1+ CPCs are found in discrete clusters within the developing right ventricle, the atria and outflow tracts of both human and mouse hearts [3], [6], which are reminiscent of a stem cell niche. Niches are anatomically defined, three-dimensional (3D) microenvironments that regulate stem or progenitor cell fate through cell–cell interactions, cell–matrix contacts and localized soluble factors [7], [8], [9]. Significant progress has been made in characterizing such specific microenvironments in selected stem cell compartments, including the hematopoietic, epidermal, intestinsal and neural stem cell niches; however, the characteristics of the cardiovascular niche are poorly defined [10]. A major challenge in stem cell biology is defining the components of such niches that are crucial for regulating stem/progenitor cell development and maintenance, as well as exploiting this knowledge for therapeutic potential. The interplay between stem or progenitor cells and their niche creates a dynamic system necessary for maintaining a balance between self-renewal and differentiation of the cells. Wnt/β-catenin signaling is important for the maintenance and self-renewal of stem/progenitor cells of various lineages and has also been implicated in cardiac development [11]. In the cardiovascular system, Wnt signaling through β-catenin appears to regulate both the specification and the proliferation of Isl1+ CPCs [12]. β-catenin directly binds and regulates the Isl1 promoter in cardiovascular cells [13]. Ablation of β-catenin in Isl1+ cells lead to the disruption of multiple aspects of cardiogenesis including a defective outflow tract and right ventricular development, resulting in embryonic lethality [13], [14]. These data strongly suggest that β-catenin signaling plays an important role in the specification and maintenance of Isl1+ CPCs.
Extracellular matrix (ECM) modulates cell anchorage and activity of signaling molecules by direct interaction with stem cells and their progeny [7], [8], [15]. The ECM of the heart is generally composed of collagens, particularly collagen types I (ColI; equals ∼85% of the cardiac collagens), III, IV (ColIV) and VI; fibronectin, elastin, glycoproteins and proteoglycans [8], [16]. These ECM components are expressed in precise temporal and spatial patterns, and are constantly remodeled during fetal cardiac development and in diseased states [17]. While fibrillar collagens like ColI play a major role in disease-related remodeling processes [18], ColIV and several glycoproteins, including laminins, are involved in cardiovascular development and have been implicated to contribute to the stem cell niche in the hematopoietic and nervous systems [19]. ColIV and laminin have also been identified to regulate cardiac stem cells in the adult heart [10], [17]; however, their role in regulating the cardiovascular niche in the developing heart remains unclear.
To determine if CPCs also reside within a niche in the developing heart, we examined fetal human and murine hearts for the presence of an anatomically distinct niche and attempted to characterize the ECM molecules and signaling pathways that mediate its interactions with CPCs. We demonstrate that the cardiovascular niche is enriched for the ECM protein ColIV, which plays a key role in controlling CPC fate. In addition, we show that inhibiting p300-dependent β-catenin signaling is critical for CPC in vitro expansion. To recapitulate the 3D cardiovascular niche microenvironment in vitro, we created unique electrospun scaffolds and demonstrate that three-dimensionality itself supports CPC expansion.
Section snippets
Human and murine tissue procurement and processing
First trimester (6–12 weeks of developmental age) human fetal hearts were discarded material obtained from elective terminations of pregnancies performed by Family Planning Associates in Los Angeles. Mouse embryos were isolated from CF1 mice at E15.5 gestational age. All heart tissues were immediately washed after harvest in sterile phosphate buffered saline (PBS, HyClone, Logan, UT), fixed in 10% buffered formalin for 12 h, then rinsed in tap water and transferred to 70% ethanol. Fixed
Characterization of the endogenous CPC niche
Multipotent endogenous CPCs have been identified in the fetal mouse heart based on the expression of the cardiogenic transcription factor Isl1 [2], [3]. We have previously described a system to potentiate differentiation of murine ES cells to CPCs based on culturing them on ColIV. These Flk1+ progenitor cells express Isl1 as well as other known CPC markers including Nkx2.5 (Suppl. Fig. 1A) and possess the ability to differentiate into functional cardiovascular cells, including CMs, SMCs and ECs
Discussion
It has been speculated that, similar to satellite cells in adult skeletal muscle, cardiac stem cells in the adult heart are quiescent cells, surrounded by BM proteins including ColIV and laminin, and give rise to cardiovascular cells through asymmetric division when needed for cardiac repair [10], [17]. However, these adult cardiac stem cells differ widely from the cardiac progenitor cell populations described in the fetal heart and whether a stem or progenitor cell niche existed in the
Conclusion
In this study we have characterized the CPC niche in developing human and mouse hearts. We identified crucial niche ECM proteins and demonstrated their biological relevance for controlling CPC fate. We further demonstrated that three-dimensionality is not only important for CPC expansion in vitro, but that in combination with specific inhibition of certain β-catenin signals can enhance 3D engineered ECM protein-based in vitro culture systems. While pluripotent stem cells such as ES and iPS
Acknowledgments
This work was supported by the NIH (5T32HL007895-10 to K.S-L.; P01-HL080111 and R01-HL094941 to W.R.M.), CIRM (RB1-01354), Laubisch and Cardiovascular Development Funds (to W.R.M.), Fraunhofer-Gesellschaft (Attract 692263 to K.S.-L.), Ruth Kirschstein National Research Service Award (GM007185 to B.V.H; T32HL69766 to J.M.G.) and the Alberta Heritage Foundation for Medical Research Fellowship (AHFMR to A.N.).
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