the ability of hESC and hiPSCs to differentiated into various cell types in vitro, including hepatocytes (Hannan et al., 2013), neural progenitors (Dhara et al., 2008), cardiomyocytes (Mummery et al., 2012) and smooth muscle (Cheung et al., 2011), and these have been extensively reviewed. However, in terms of vascular regeneration and stimulation of angiogenesis, hPSC-derived endothelial cells (hPSC-ECs) are thought to have the greatest potential, although methods of derivation still remain suboptimal.
1.6.1 In vitro differentiation of pluripotent stem cells to
endothelium
Thus far, there have been a large number of publications describing protocols for the derivation of ECs from hPSCs, and these have been reviewed extensively (Descamps et al., 2012). Within this, there are two main approaches which have
been taken when generating hESC-ECs; 3D embyroid body (EB)-based culture systems and 2D monolayer culture systems.
Endothelial-associated genes, including Pecam-1 (CD31), VE-Cadherin (CD144) and CD34, are increased during spontaneous EB-based differentiation of hESCs (Levenberg et al., 2002). However, the efficiency of these differentiations is low, with only ~2% of cells expressing CD31 on their cell surface when analysed by FACS. Other studies showed that addition of VEGF into the system can increase the numbers of cells expressing CD31 and CD144 (Nourse et al., 2010), and although these cells can be isolated and cultured to obtain higher percentages of CD31+ cells (Levenberg et al., 2002), more direct differentiation systems have been investigated to increase initial hPSC-EC generation efficiency.
3D EB-based direct differentiation protocols have been efficient in generating hESC-EC or hiPSC-ECs. These protocols are efficient, generating anywhere between 5-50% hPSC-ECs or hPSC-EC precursors in the first instance, before sorting and further culture to obtain cultures of 100% functional hPSC-ECs (Rufaihah et al., 2011, Costa et al., 2013, White et al., 2013). Rufaihah et al. used hiPSCs to generate cells which were positive for CD31, CD144, endothelial nitric oxide synthase (eNOS) and von Willibrand Factor (vWF), and were able to perform functionally as ECs in terms of acetylated low-density lipoprotein (acLCL) incorporation and tubule formation assays. When hiPSC-ECs were transplanted into a murine model of hind limb ischemia, there was a significant increase in both blood perfusion and capillary density, possibly as a result of detected higher levels of angiogenic cytokines, such as Angiopoetin-1 and VEGF- A and -C (Rufaihah et al., 2011). Another published EB-based EC differentiation assay involved the isolation of KDR+ (CD309; VEGFR2) precursor cells on day 6 of differentiation. On day 6, approximately 44% of cells were positive for KDR, and only 12 or 13% of these expressed CD31 or CD144 respectively. When isolated cells were plated onto fibronectin (FN), cultured in endothelial medium, they exhibited a high proliferative capacity and after 14 days contained 97% or 93% CD144+CD31+ cells when hESCs or hiPSCs were used respectively. Like the previous study, cells derived using this protocol displayed a cobblestone-like morphology, were functional in in vitro assays, and were able to form functional capillaries which linked with the endogenous circulatory system in a murine matrigel plug assay (White et al., 2013). Similarly, Costa et al. isolated
CD34+KDR+ endothelial precursors on day 6 of an EB-based differentiation system, and continued their culture in a monolayer where, by day 12 of differentiation, cells uniformly expressed CD31, CD144, KDR, CD34 and other EC markers. Again, these cells were able to form tubules in vitro and incorporate acLDL, and the study also demonstrated a high degree of transcriptional similarity between hESC-ECs and post-natal ECs (Costa et al., 2013).
Recently, studies have focused on both increasing hESC-EC differentiation efficiency, i.e. the percentage of ECs generated, and scalability, i.e. the number of ECs generated. Although highly efficient, 3D EB-based culture systems are difficult to scale up to produce clinically relevant numbers of cells due to methods of EB formation, and because of this, monolayer-based protocols have also been investigated. An early 2D monolayer protocol used a co-culture system, whereby H9 hESCs were plated onto MEF and in hESC differentiation medium (Wang et al., 2007). Using this protocol, cells were shown to upregulate endothelial-associated CD31 and downregulate pluripotency-associated genes such as Oct4. However, the percentage of CD34+ cells before selection was low (~5-10%), and the use of MEFs results in the inability of this protocol to be clinically transferrable. Kane et al. developed a fully defined, feeder- and serum-free 21 day monolayer-based protocol, yielding approximately 80% CD144+CD31+ cells in the SA461 and SA121 hESC lines. These cells expressed high levels of endothelial-associated genes, and performed functionally in wound healing assays in vitro and in a murine model of hind limb ischemia in vivo (Kane et al., 2010). Recently, a method for simultaneous derivation of ECs and pericytes from hiPSCs was published (Orlova et al., 2014a, Orlova et al., 2014c). The protocol is a fully defined, monolayer-based protocol, allowing for easy large scale adaptation. Differentiation efficiency was consistent amongst different hPSC lines, including both hESC and hiPSC, with 10-30% of cells expressing CD31 (Pecam-1) and CD34, which were also show to express comparable levels of other endothelial markers, including CD144 and CD309 (KDR; VEGFR2). The cells could be isolated and expanded, and analysis showed cells were able to perform functionally in an in vivo zebrafish xenograft model, and form interactions with pericytes – derived simultaneously – in vitro, promoting further differentiation of pericytes to smooth muscle cells. Furthermore, Patsch and colleagues also described a highly efficient monolayer-
based system for the derivation of ECs from hPSCs, which they developed alongside a protocol for VSMC differentiation (Patsch et al., 2015). Using two hESC lines (SA001 and Hues9) and two hiPSC lines, it was demonstrated that between 61.8% and 88.8% of generated cells were CD144+, when assessed by flow cytometry. Additionally, the CD144+ cells also expressed other endothelial- associated cell surface markers, including CD31, KDR, CD34, CD105 and von Willibrand factor (vWF). The group also showed that the CD144+ hPSC-ECs could be purified on day 6, using magnetic activated cell sorting (MACS), to obtain virtually pure (~96%) hPSC-EC cultures. Moreover, analysis of the transcriptional signature and metabolomics profiles revealed high levels of similarity between the generated CD144+ hPSC-ECs and primary vascular cells.
Encouragingly, the studies described, using in vivo assays, showed largely positive results, with transplantation of hESC-ECs resulting in an increase in revascularisation and angiogenesis. These data are promising for the potential translation of these technologies into the clinic, as possible treatments for PAD and CLI.
1.6.2 In vivo development of the vasculature
During embryonic development in vivo the vascular and hematopoietic systems are thought to be closely linked, with the first ECs originating from the lateral and posterior mesoderm, before migrating towards the yolk sac, where they differentiate into ECs and hematopoietic cells (HCs) (Medvinsky et al., 2011). The close relationship between the developing endothelial and hematopoietic systems led to the suggestions that they share a common mesodermal ancestor – the hemangioblast – and this common progenitor was observed in vitro in studies involving murine ESCs (Nishikawa et al., 1998). Another theory, however, suggests that the first hematopoietic stem cells (HSCs) are derived from ECs with hematopoietic potential, known as hemogenic endothelium (HE). In vivo studies have shown, using time lapse imagining in both mouse and zebrafish, that HCs originate from VE-Cadherin (CD144) expressing ECs in the dorsal aorta (Zovein et al., 2008, Bertrand et al., 2010, Boisset et al., 2010, Kissa et al., 2010). It has, however, been demonstrated that these two separate hypotheses can be merged to form a single linear developmental process, stating that hemangioblasts
generate HCs via the formation of intermediate hemogenic endothelial populations (Lancrin et al., 2009).
1.6.2.1 In vitro formation of hemogenic endothelium
As well as direct hPSC-EC differentiation systems, there have been a number of publications showing simultaneous derivation of ECs and hematopoietic cells through a common HE progenitor, mimicking conditions in vivo.
Originally, the Vodyanik group had demonstrated the ability of both hESCs and hiPSCs to differentiate to EC and hematopoietic cells simultaneously in vitro (Vodyanik et al., 2006, Choi et al., 2009). Cells were co-cultured with mouse bone marrow stromal cell line OP9 for 8 days, after which time both CD43+
hematopoietic cells and CD31+CD43- ECs were detected. Recently, Choi et al. attempted to identify and classify HE populations present within the OP9 hPSC differentiation system (Choi et al., 2012). By day 5 of differentiation 3 distinct CD144+ cell populations were identified; CD144+CD235a+CD43+CD41+, CD144+CD73+ and CD144+CD73-CD235a-CD43-. All 3 of these subsets exhibited a similar endothelial phenotype, and were functional, capable of acLDL uptake. CD117 (c-Kit), a angiohematopoietic progenitor marker, was expressed at different levels in the different CD144+ subpopulations, with the highest expression in the CD144+CD73+ cells, the lowest in the
CD144+CD235a+CD43+CD41+ cells, and an intermediate level in the CD144+CD73- CD235a-CD43- cells. Further functional and phenotypic analysis allowed for their
definition of the following EC populations; HE progenitors are CD144+CD73−
CD235a/CD43−CD117intermediate with primary endothelial characteristics but can
generate blood and endothelial cells, and CD144+CD73+CD235a/CD43- non-HE progenitors, which have all the functional and phenotypic characteristics of endothelial cells. Extensive functional characterisations of EC populations, including in vivo assays, were, however, not performed within this study.
The assay developed by Choi et al. involves co-culture with the OP9 mouse bone marrow stromal cell line, introducing an undefined factor into the system. Recently, a system, using fully defined conditions, generating both distinct hemogenic and arterial vascular endothelium has been published (Ditadi et al., 2015), although cells are cultured on feeders before being transferred to
Matrigel – an undefined cell matrix – 24 hrs prior to differentiation. In this protocol, a previously defined CD34+CD43- population, with the additional cell surface profile of CD31+CD144+KDR+CD117low and the ability to generate T-
lymphoid, erythroid and myeloid cells (Kennedy et al., 2012), were isolated and cultured on Matrigel in hematopoietic supportive conditions. It was demonstrated that these cells underwent endothelial to hematopoietic transition (EHT) and formed CD45+ hematopoietic cells, which were then able to undergo further definitive hematopoiesis. Definitive venous and arterial ECs were identified from the CD34+CD43- fraction as CD73hiCD184- and CD73medCD184+ fractions respectively. Both of these cells types were shown to form functional vessels when transplanted into immunocompromised mice, subcutaneously in a Matrigel plug.
Combinations of direct and indirect differentiation systems are valuable tools for studying the mechanisms driving development of ECs in vitro and in vivo.