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A novel lineage restricted, pericyte-like cell line isolated from human embryonic stem cells

Pericytes (PCs) are endothelium-associated cells that play an important role in normal vascular function and maintenance. We developed a method comparable to GMP quality protocols for deriving self-renewing perivascular progenitors from the human embryonic stem cell (hESC), line ESI-017. We identified a highly scalable, perivascular progenitor cell line that we termed PC-A, which expressed surface markers common to mesenchymal stromal cells. PC-A cells were not osteogenic or adipogenic under standard differentiation conditions and showed minimal angiogenic support function in vitro. PC-A cells were capable of further differentiation to perivascular progenitors with limited differentiation capacity, having osteogenic potential (PC-O) or angiogenic support function (PC-M), while lacking adipogenic potential. Importantly, PC-M cells expressed surface markers associated with pericytes. Moreover, PC-M cells had pericyte-like functionality being capable of co-localizing with human umbilical vein endothelial cells (HUVECs) and enhancing tube stability up to 6 days in vitro. We have thus identified a self-renewing perivascular progenitor cell line that lacks osteogenic, adipogenic and angiogenic potential but is capable of differentiation toward progenitor cell lines with either osteogenic potential or pericyte-like angiogenic function. The hESC-derived perivascular progenitors described here have potential applications in vascular research, drug development and cell therapy.

Pericytes (PCs) are integral to the development, maturation and stabilization of vasculature. PCs wrap around the endothelial cells (ECs) to provide scaffolding support and regulate EC behavior, such as the formation of endothelial cell-cell junctions. PCs also regulate EC migration, differentiation and stabilization through pericyte-EC direct cell contacts and paracrine signaling pathways 1 . Furthermore, PCs may function as multipotent mesenchymal stromal cells (MSCs) or perivascular stromal cells (PSCs) serving as a source of repair cells that are activated following injury. A lack of functional PCs is associated with a variety of pathologic conditions, including neurodegenerative disorders, ischemic disorders and diabetic retinopathy 2 . Preclinical studies indicate therapeutic potential of PCs for regenerative treatments for a multitude of disorders, including bone defects, limb ischemia, ischemic heart disease, muscular dystrophy and retinal vasculopathy [3][4][5][6][7] . Translation of pericyte research to the clinic will require a scalable, well defined cell source. The use of primary cells for regenerative medicine is limited because of batch to batch variation, cell heterogeneity, low replicative capacity and loss of function in culture. Moreover, the use of autologous stem cells for therapy could be limited by the age or health status of the patient. For example, MSCs lose both osteogenic and vascular support function with aging 8 . Derivation of PCs from human embryonic stem cell (hESC) lines offers the possibility of a renewable and scalable source of uniform cells for research and development of regenerative therapies.
Previous studies have identified primary pericytes and human pluripotent stem cell (hPSC) derived pericyte-like cells with both angiogenic support function and MSC-like multi-lineage potential 4,9 . However, recent mouse studies suggest that specialized subtypes of pericytes may exist with more restricted lineage potential 10 . Here we demonstrate the derivation of 3 distinct progenitor cell types from the GMP compatible hESC line, ESI-017 11 . Using a modified endothelial cell derivation protocol, we first derived a self-renewing perivascular progenitor cell type we termed PC-A. PC-A cells expressed multipotent stem cell markers like CD133 and CD34, but lacked osteogenic or adipogenic potential and angiogenic support function. Further directed differentiation of PC-A cells resulted in the generation of 2 distinct perivascular progenitor cell types; one with osteogenic potential (PC-O) and a second with pericyte-like angiogenic support function (PC-M). Both of the PC-A derived cell types failed to differentiate to adipocytes under conditions that successfully differentiated bone marrow derived mesenchymal stromal cells (BM-MSC) to adipocytes. We have thus derived a novel scalable progenitor cell from hESCs that can be used as a source of at least 2 distinct lineage restricted progenitor cell types.
We established the identity of all 3 progenitor cell types by surface marker expression. Notably, the pericyte-like cell type, PC-M cells, expressed CD146 and CD105, suggesting that these cells may have angiogenic support function similar to PCs and MSC sub-populations identified in vivo 12 . Using a modified in vitro Matrigel TM tube formation assay, we found that PC-M cells have angiogenic support function similar to or greater than primary placental pericytes (Pl-PCs) and BM-MSCs. Specifically, PC-M cells co-localized with human umbilical vein endothelial cells (HUVECs) and provided superior tube stabilization. We have thus derived a scalable, pericyte-like cell, PC-M, with angiogenic support function characteristic of pericytes. PC-M cells are a novel, well defined and highly expandable cell type with the potential to be further developed for improved in vitro angiogenesis assays, drug screening, and cell therapy applications.

Results
Derivation of self-renewing hESC-derived perivascular progenitors with stable morphology and high scalability. Multiple progenitor cell lines were derived from the human embryonic stem cell (hESC) line ESI-017 using a modified protocol previously established for the generation of endothelial progenitor cells 13 . We seeded ESI-017 cells at multiple densities to generate embryoid-bodies (EBs) in AggreWell TM plates and then transferred the EBs as single cell suspensions to adherent culture conditions, screening for differences in cell morphology (Fig. 1). The resulting cell cultures showed significantly different cell morphologies as a function of initial cell seeding density during EB generation. Adherent cell cultures derived from EBs formed at low cell hESC-derived perivascular progenitors express mesenchymal and perivascular markers. We assessed the 3 hESC-derived perivascular progenitors for cell surface markers associated with mesenchymal stromal cells, pericytes and endothelial cells (Fig. 3). All cells were assayed by flow cytometry following extended passage in their respective expansion or derivation medium ( Fig. 1, Days 8+ ). We found that all 3 perivascular progenitors were positive for CD146 and CD73 (83-100%). Furthermore, all 3 perivascular progenitors showed low or no expression of CD31 (< 10%). PC-A cells, but not PC-M or PC-O cells showed intermediate expression of CD34 (38%). Interestingly, PC-O cells were negative for CD133, while PC-A cells were positive for CD133 (> 98%) and PC-M cells showed intermediate expression (p6, 34%). Further expansion of PC-M cells resulted in loss of CD133 (p22, 4%). PC-M cells were also negative for pluripotency markers Tra-1-60 and Oct-4 (data not shown).
Flow cytometry analysis of PC-M cells at multiple passages was used to further establish cell identity. Expression of CD105, PDGFRβ and NG-2 markers in PC-M cells changed over multiple passaging events and PC-M cell expansion. We found that PC-M cells were initially positive for pericyte markers, PDGFRβ (40-50%) and NG-2 (10-20%) at passage 3 (p3, data not shown), but rapidly lost expression of both markers (p6, Supplementary Fig. S3). In contrast, PC-M cells showed an increase in expression of CD105 from intermediate to late passages (p6, 58%; p22, 99%, Fig. 3). Notably, late passage PC-M cells displayed a similar surface marker profile to pericytes derived from induced pluripotent stem cells 14,15 .

hESC-derived perivascular progenitors have restricted differentiation potential. To investi-
gate the functional multipotency of all 3 hESC-derived perivascular progenitors, we examined the capability of these cells to differentiate into osteoblasts and adipocytes in vitro. Previous studies have demonstrated that MSCs and PCs are capable of differentiation to both osteoblasts and adipocytes. Using osteogenic or adipogenic differentiation media, all 3 hESC-derived perivascular progenitors were assayed for differentiation potential (Fig. 4). For reference, primary Pl-PCs and BM-MSCs were similarly assayed (Fig. 4). The extent of calcium-rich mineralization of the cell matrix was assessed using Alizarin Red S staining after 21 days of culture in osteogenic medium (Fig. 4a). PC-A and PC-M cells were not osteogenic having little or no Alizarin Red staining under these conditions. However, PC-A cells, but not PC-M cells, showed significantly altered cell morphology in osteogenic differentiation media compared with growth media (Fig. 4a). PC-O cells, Pl-PCs and BM-MSCs displayed extensive calcification and demonstrated osteogenic differentiation potential, with Pl-PCs exhibiting the most extensive calcification. After 14 days of culture in adipogenic media the extent of lipid droplet formation was assessed by Oil Red O staining (Fig. 4b). Only BM-MSCs stained positive for accumulation of lipid droplets. Therefore, only BM-MSCs demonstrated both osteogenic and adipogenic potential.

PC-M cells stabilize endothelial cells and resulting vasculogenic tube networks in vitro .
We examined the ability of all 3 hESC-derived perivascular progenitors to support angiogenesis by seeding the cells on growth factor reduced-Matrigel in monoculture and co-culture with human umbilical vein endothelial cells (HUVECs) in vitro. In monocultures, we assessed the ability of these cells to form tube networks. At 1 day of monoculture, only PC-M cells formed tube networks with extensive branching between flat cell sheets (Fig. 5a). PC-O cells and BM-MSCs formed tube-like structures, but showed large, dense cell clusters and less branching. PC-A cells formed small cell clusters with minimal branching. Pl-PCs formed large cell clusters with no observable tube-like structures or branching. We next assessed the ability of hESC-derived perivascular progenitors or primary cells to stabilize HUVEC tube networks formed by HUVECs in co-cultures. At 1 day of co-culture, tube networks were observed in all co-cultures (Fig. 5b). The representative images of the resulting tube networks show that co-cultures containing variable test cells, have variable tube thickness, branching and cell clustering at branch points (Fig. 5b). The total tube network length, including edges of cell sheets, was not significantly different across co-culture conditions and compared with HUVECs in monoculture (Supplementary Fig. S4). However, the average total branching length was highest for co-cultures containing PC-M cells. Notably, PC-M cells were localized We hypothesized that PC-M cells may further stabilize endothelial tube networks in vitro, a hallmark of pericyte function 12,16 . We found that increasing the ratio of HUVECs to PC-M cells significantly improves the initial tube network formation, as well as long-term stability (Supplementary Fig. S5). When seeded at a ratio of 20:1, HUVECs to PC-M cells, PC-M cells improve tube network formation and stability over the course of 6 days (Fig. 6). In monoculture, HUVECs formed a complete tube network within 4-8 hours. This network remained intact at 1 day and began to degrade by 2 days (Fig. 6a), after which the HUVECs further dispersed and were unable to establish a tube network over the course of 6 days. In monoculture, PC-M cells form small clusters or remain as isolated cells over the course of 6 days when seeded at a low number correlating with the number of PC-M cells in co-culture (Fig. 6b). In co-culture, HUVECs and PC-M cells formed an extensive tube network, showing similar branching to HUVEC monocultures, but longer and thicker or denser tube-like structures (Fig. 6c). Minimal degradation of tube structures is observed in co-culture and the presence of an interconnected tube network persisted for at least 6 days without media exchange or the addition of exogenous growth factors (Fig. 6c) Fig. S6). These results demonstrate that PC-M cells are an angiogenic support cell type, consistent with pericyte cell functionality in vitro.  primary cell types, we have developed a method for the derivation of perivascular progenitors from hESCs. We hypothesized that modification of a method for deriving endothelial progenitor cells (EPCs) from hESCs could yield highly expandable, stable vascular support cell types, such as pericytes (13). We found that increasing the initial seeding density during embryoid body (EB) formation supported the derivation of a novel, perivascular progenitor cell type, termed PC-A. Further expansion of PC-A cells in mesenchymal cell culture medium or endothelial cell culture medium, was used to derive 2 additional perivascular progenitor cells, termed PC-O and PC-M, respectively. We attempted continuous culture and expansion of all 3 perivascular progenitors in their respective cell culture medium. We showed that PC-A cells and PC-M cells were scalable, therefore these cell lines may be suitable for industrial scale production for research and clinical development. In contrast, PC-O cells were not scalable, having a low population doubling rate. In the present study, we explored only 2 cell culture variables-cell seeding density during EB formation and cell culture medium. Therefore, it is probable that alternative cell density and expansion conditions exist which may yield additional cell types. Furthermore, the derivation and culture methods described here may be further modified to support production of additional cell types, including clonal cell populations of PC-A and/or PC-M cells.

Discussion
We showed that all 3 hESC-derived perivascular progenitors have unique expression of multiple cell surface markers, with similar expression observed only for surface markers CD146 and CD73 (Figs. 3 and 7). Moreover, all 3 cell types demonstrated restricted mesenchymal lineage potential (Fig. 4). These results suggest that all 3 cell lines are different perivascular cell subtypes, each having a distinct differentiation potential 10 . For example, only PC-A cells were positive for CD133, suggesting that these cells are an early, progenitor cell with high proliferation and differentiation potential 18 . However, we found that PC-A and PC-M cells were unable to differentiate toward osteoblasts or adipocytes, whereas with further differentiation PC-A derived PC-O cells were able to differentiate toward osteoblasts, but not adipocytes. The lack of adipocyte differentiation potential of PC-A and PC-M cells suggests that these cells are immature stem cell types similar to fetal mesenchymal stromal cells, which also lack adipogenic potential 19,20 . Both PC-A and PC-M cells highly expressed CD146, a cell-adhesion molecule actively involved in angiogenesis 21 . However, only PC-M cells showed similar trends of marker expression as primary pericytes such as high expression of CD105 and low expression of CD133 and CD34, suggesting that PC-M cells are pericyte-like cells with angiogenic support function 12,22 . We propose that PC-A and PC-M cells are 2 unique subsets of perivascular cells with PC-A being a more primitive self-replicating progenitor. Recently, 2 subsets of pericytes with and without angiogenic function in vitro and in vivo were identified in mice. Notably, the pro-angiogenic pericyte subset identified in mice did not undergo adipogenic differentiation in vitro 10 .
Expression of pericyte markers is highly dynamic in vitro and in vivo. In vivo, pericyte-specific markers are known to vary across cell developmental stages and in various tissue types, such that subsets of pericyte cells have different expression of markers, including PDGFRβ and NG2 1 . In vitro, these same makers may also vary as a result of in vitro culturing. Further immunophenotyping of PC-M cells showed that these cells rapidly lost expression of 2 pericyte markers, PDGFRβ and NG2, during in vitro expansion. Although PDGFRβ and NG2 are associated with adult stem and progenitor cells, particularly from brain vasculature, the loss of these specific markers in PC-M cells during expansion culture does not preclude these cells from being identified as pericyte-like cells. Expression of CD105 increased following expansion of PC-M cells. The upregulation of CD105, which is upregulated during hypoxia and highly expressed in other angiogenic cell types, suggests that PC-M cells might be angiogenic or function as angiogenic support cells 23 . After expansion, PC-M cells were positive for CD73 and lost expression of CD133 (< 5%) suggesting PC-M cells lack hematopoietic stem cells or residual undifferentiated hESCs. Importantly, the immunophenotype of PC-M cells at early and late passages showed that these cells were positive for CD146 and negative for CD34 (Fig. 3). Previously, isolation of CD146(+ )/CD34(− ) cells correlated with angiogenic support activity in pericyte cells from multiple tissues 12 . The stable expression of these markers led us to hypothesize that these were pericyte-like cells capable of supporting and stabilizing angiogenesis with high scalability needed for clinical applications.
The formation of functional and stable blood vessels in vivo depends on both endothelial cells and perivascular cells, including pericytes 1 . In the absence of definitive markers, pericytes can be identified functionally in vitro by their ability to co-localize with endothelial cells and stabilize tube network formation 12 . Vasculogenic tube assembly by endothelial cells in monoculture are unstable, with tube networks degrading after 1 day unless supported by a secondary cell type. In the present study, the second cell type provided is the hESC-derived perivascular progenitor cell, PC-M. PC-M cells in monoculture demonstrated the ability to form an independent tube network. Similar to endothelial cell tube networks in monoculture, PC-M tube networks in monoculture were unstable, with tube networks degrading after 2 days. In co-culture, PC-M cells demonstrated good angiogenic support function, stabilizing vasculogenic tube assembly by HUVECs. PC-O cells or BM-MSCs also supported stable tube formation but these were less stable than PC-M cell co-cultures. In contrast, co-cultures of HUVECs with PC-A cells or Pl-PCs were not stable (Fig. S6). The greater in vitro tube stabilization by PC-M  (Fig. 6). These results indicate that the in vitro model recapitulates the direct cell to cell contact that is important role for stabilizing tube networks in vivo. Overall, these results are consistent with a model of vascular morphogenesis wherein pericyte migration, tube assembly and/or recruitment to existing tube networks is essential for the stable formation of vasculogenic tube assembly 24 .
In summary, we have developed a process for deriving novel perivascular progenitor cell line, PC-A, from hESCs (Fig. 7). PC-A cells can further be differentiated toward PC-O cells, which are osteogenic, or PC-M cells, which are not osteogenic but show significant in vitro angiogenic support function being capable of stabilizing HUVEC tube formation ( Table 1). The methods used here may be readily scaled for basic research and clinical translation, since the source cell line, ESI-017, and culture conditions are comparable to GMP quality protocols. Notably, PC-A cells were expanded in serum-free medium prior to further differentiation. Both cell derivatives presented here have potential applications in cell therapy. PC-O cells could potentially be useful for developmental research, disease modeling, and clinical applications for bone repair and osteoporosis because of their osteogenic lineage restriction. PC-M cells may be useful for angiogenesis research, pro-angiogenic and anti-angiogenic drug screening, disease modeling and clinical applications requiring vascular support function but lacking other mesenchymal differentiation capacities. Further studies aimed at understanding the angiogenic potential of these cells in vivo using animal models of ischemic repair will be essential for establishing their potential in pro-angiogenic therapies.  Table 1. Differentiation potential and angiogenic support function hESC-derived perivascular progenitors and primary perivascular cells. Each cell type was evaluated for osteogenic and adipogenic potential; cells capable of differentiating down these linages are marked (+). Each cell type was evaluated for angiogenic support function as monitored by tube network formation for 6 days of cell culture (Figs 5, 6 and S6). Cells cocultured with human umbilical vein endothelial cells showed (−), minimal (+) or extensive (++) tube network formation over multiple days.