Ex vivo reconstitution of arterial endothelium by embryonic stem cell-derived endothelial progenitor cells in baboons

Abstract

There is an increasing need for an animal model that can be used to translate basic research into clinical therapy. We documented the differentiation and functional competence of embryonic stem cell (ESC)-derived endothelial cells in baboons. Baboon angioblasts were sequentially differentiated from embryoid body cultures for 9 days in an angioblast differentiation medium with varying concentrations of BMP-4, FLT-3 ligand, stem cell factor, thrombopoietin, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and knockout serum replacement. Real-time polymerase chain reaction results showed that ESC-derived angioblasts downregulated NANOG and OCT3/4, upregulated T-brachyury and GATA2, and moderately expressed CD34; they did not express CD144, TEK, or VWF, and varied in levels of CD31 expression. Several populations of putative angioblasts appeared 3 days and 9 days after differentiation, as identified by flow cytometry. Angioblasts at this stage exhibited dual paths of differentiation toward hematopoietic and vascular fates. To examine whether derived angioblasts could reconstitute the endothelium, we built an ex vivo culture system and seeded fluorescently labeled angioblast cultures onto a denuded segment of the femoral artery. We found that the seeded cells were able to grow into the endothelium on the interior surface of denuded artery segments within 5 days after seeding. After 14 days of ex vivo culture, the transplanted cells expressed CD31, an endothelial marker. The control arteries, seeded with vehicle only, did not harbor cells with endothelial markers. We conclude that ESC-derived angioblasts are promising therapeutic agents for repairing damaged vasculature, and that the baboon model will be vital for optimizing therapies for human clinical studies.

FIG. 1.

Experimental design for generating angioblasts from ESCs. Undifferentiated ESCs were cultured with feeder cells under routine conditions. The embryoid body (EB) method was used to generate angioblasts from ESCs. After EBs were formed, they were suspended in ESC/ADM media with varying ratios for indicated times. At the end of angioblast induction, they were transferred to collagen-coated plates to reach confluence. The reconstitutive ability of derived cells was evaluated by an ex vivo culture system. (Endothelial cells, inner layer; smooth muscle cells, intermediate layer cells; fibroblasts and pericyte, outer layer cells). ESCs, embryonic stem cells; ADM, angioblast differentiation medium.

FIG. 2.

Characteristics of pluripotent baboon stem cells. Pluripotency of baboon ESCs was confirmed by growth behavior in colonies [(a) 100×] and positive immunostaining for NANOG [(b) 200×], OCT-4 [(c) 200×], SSEA-4 [(d) 400×], and histochemical staining for alkaline phosphatase [(e) 100×]. (f ) was isotype image (200×).

FIG. 3.

Angioblast formation from EBs under ADM culturing. (A) Morphological development of angioblasts under ADM culturing. Undifferentiated ESCs were induced to form EBs using AggreWellÔ plates; 5,000–10,000 cells were contained in each EB (a). After 9 days of culture in ESC/ADM media at various ratios as indicated Fig. 1, EBs were transferred onto collagen-coated plates and their morphological changes during differentiation were observed. Images (b) through (g) are representative of their growth features. One day after initiation of the monolayer culture (b), some cells grew out from EBs and continued to cover the plate after 3 days (c) and 5 days (d); the cultures became confluent after 9 days (e). Angioblast cultures appeared to have typical cobblestone morphology under phase-contrast microscopy from day 5 (f ) and retained that morphology for 3 weeks (g). (a–e), 100×. (f–g) 400×. (B) Gene expression analysis by real time-polymerase chain reaction (RT-PCR) of EB cultures grown in ADM for 5 and 9 days. Total RNA was isolated from ESC, EC, and EB cultures at day 5 (D5) and day 9 (D9). Expression of 10 angioblast marker genes, together with a housekeeping gene (GAPDH) was analyzed by quantitative real-time PCR. All mRNA expression levels are expressed as cycle threshold values. (C) Immunohistochemical staining of mesodermal nuclear transcription factors T-brachyury and GATA2, and membrane KDR in ESCs (a–c) and EBs at day 5 (d–f ). (g), isotype control. Nuclei are stained blue with DAPI. 400×. (D) The kinetics of generation of angioblasts during culture of EBs in ADM. Flow cytometry results indicate the characteristics of phenotypic expression of several progenitor markers in pluripotent ESCs (a, d, g), in ESC-derived cells after 3 days differentiation (b, e, h), and in ESC-derived cells after 9 days differentiation (c, f, i). (E) Dual differentiation of angioblasts toward hematopoietic and vascular lineages. When angioblasts were cultured in monolayer, a mixed culture of suspended cells and attached cells (a) appeared 1–2 days after cell seeding. Some cells spontaneously formed vascular structures consisting of lumen and capillary (b). After 5–7 days, we observed cell clusters with a red grape-shape (c); immunohistochemical staining confirmed the presence of CD235a [(f ), red], an early erythrocyte marker. Spindle-shaped colony cells also existed (d); some of them were positively stained for smooth muscle α-actin [(g), green], either in assembled fiber form or unassembled premature molecules. Distinct cobblestone-shaped cultures were also observed (e); they were strongly positive for CD34 [(h), red]. All cell nuclei were stained with DAPI (blue). Isotype-matched staining is shown in (i). (a–e), 100×. (f–i), 400×. EC, endothelial cell; DAPI, 4,6-diamidino-2-phenylindole.

FIG. 3.

Angioblast formation from EBs under ADM culturing. (A) Morphological development of angioblasts under ADM culturing. Undifferentiated ESCs were induced to form EBs using AggreWellÔ plates; 5,000–10,000 cells were contained in each EB (a). After 9 days of culture in ESC/ADM media at various ratios as indicated Fig. 1, EBs were transferred onto collagen-coated plates and their morphological changes during differentiation were observed. Images (b) through (g) are representative of their growth features. One day after initiation of the monolayer culture (b), some cells grew out from EBs and continued to cover the plate after 3 days (c) and 5 days (d); the cultures became confluent after 9 days (e). Angioblast cultures appeared to have typical cobblestone morphology under phase-contrast microscopy from day 5 (f ) and retained that morphology for 3 weeks (g). (a–e), 100×. (f–g) 400×. (B) Gene expression analysis by real time-polymerase chain reaction (RT-PCR) of EB cultures grown in ADM for 5 and 9 days. Total RNA was isolated from ESC, EC, and EB cultures at day 5 (D5) and day 9 (D9). Expression of 10 angioblast marker genes, together with a housekeeping gene (GAPDH) was analyzed by quantitative real-time PCR. All mRNA expression levels are expressed as cycle threshold values. (C) Immunohistochemical staining of mesodermal nuclear transcription factors T-brachyury and GATA2, and membrane KDR in ESCs (a–c) and EBs at day 5 (d–f ). (g), isotype control. Nuclei are stained blue with DAPI. 400×. (D) The kinetics of generation of angioblasts during culture of EBs in ADM. Flow cytometry results indicate the characteristics of phenotypic expression of several progenitor markers in pluripotent ESCs (a, d, g), in ESC-derived cells after 3 days differentiation (b, e, h), and in ESC-derived cells after 9 days differentiation (c, f, i). (E) Dual differentiation of angioblasts toward hematopoietic and vascular lineages. When angioblasts were cultured in monolayer, a mixed culture of suspended cells and attached cells (a) appeared 1–2 days after cell seeding. Some cells spontaneously formed vascular structures consisting of lumen and capillary (b). After 5–7 days, we observed cell clusters with a red grape-shape (c); immunohistochemical staining confirmed the presence of CD235a [(f ), red], an early erythrocyte marker. Spindle-shaped colony cells also existed (d); some of them were positively stained for smooth muscle α-actin [(g), green], either in assembled fiber form or unassembled premature molecules. Distinct cobblestone-shaped cultures were also observed (e); they were strongly positive for CD34 [(h), red]. All cell nuclei were stained with DAPI (blue). Isotype-matched staining is shown in (i). (a–e), 100×. (f–i), 400×. EC, endothelial cell; DAPI, 4,6-diamidino-2-phenylindole.

FIG. 3.

Angioblast formation from EBs under ADM culturing. (A) Morphological development of angioblasts under ADM culturing. Undifferentiated ESCs were induced to form EBs using AggreWellÔ plates; 5,000–10,000 cells were contained in each EB (a). After 9 days of culture in ESC/ADM media at various ratios as indicated Fig. 1, EBs were transferred onto collagen-coated plates and their morphological changes during differentiation were observed. Images (b) through (g) are representative of their growth features. One day after initiation of the monolayer culture (b), some cells grew out from EBs and continued to cover the plate after 3 days (c) and 5 days (d); the cultures became confluent after 9 days (e). Angioblast cultures appeared to have typical cobblestone morphology under phase-contrast microscopy from day 5 (f ) and retained that morphology for 3 weeks (g). (a–e), 100×. (f–g) 400×. (B) Gene expression analysis by real time-polymerase chain reaction (RT-PCR) of EB cultures grown in ADM for 5 and 9 days. Total RNA was isolated from ESC, EC, and EB cultures at day 5 (D5) and day 9 (D9). Expression of 10 angioblast marker genes, together with a housekeeping gene (GAPDH) was analyzed by quantitative real-time PCR. All mRNA expression levels are expressed as cycle threshold values. (C) Immunohistochemical staining of mesodermal nuclear transcription factors T-brachyury and GATA2, and membrane KDR in ESCs (a–c) and EBs at day 5 (d–f ). (g), isotype control. Nuclei are stained blue with DAPI. 400×. (D) The kinetics of generation of angioblasts during culture of EBs in ADM. Flow cytometry results indicate the characteristics of phenotypic expression of several progenitor markers in pluripotent ESCs (a, d, g), in ESC-derived cells after 3 days differentiation (b, e, h), and in ESC-derived cells after 9 days differentiation (c, f, i). (E) Dual differentiation of angioblasts toward hematopoietic and vascular lineages. When angioblasts were cultured in monolayer, a mixed culture of suspended cells and attached cells (a) appeared 1–2 days after cell seeding. Some cells spontaneously formed vascular structures consisting of lumen and capillary (b). After 5–7 days, we observed cell clusters with a red grape-shape (c); immunohistochemical staining confirmed the presence of CD235a [(f ), red], an early erythrocyte marker. Spindle-shaped colony cells also existed (d); some of them were positively stained for smooth muscle α-actin [(g), green], either in assembled fiber form or unassembled premature molecules. Distinct cobblestone-shaped cultures were also observed (e); they were strongly positive for CD34 [(h), red]. All cell nuclei were stained with DAPI (blue). Isotype-matched staining is shown in (i). (a–e), 100×. (f–i), 400×. EC, endothelial cell; DAPI, 4,6-diamidino-2-phenylindole.

FIG. 4.

Ex vivo bioreactor. (A) Diagram of bioreactor for evaluating the reconstitutive ability of derived angioblasts. The bioreactor has 2 parts: a rotary cylinder (a) with 4 stainless steel tubes on both sides (b), and a laminar perfusion system (c) driven by a digital peristaltic pump. The cells to be tested were seeded onto the lumen of the denuded artery, and then the 2 ends of the segment were ligated with plastic tubing to construct a circuit. Endothelial differentiation medium in a reservoir (d) was perfused through the lumen of the vessel using a digital peristaltic pump. The vessel was submerged in medium and placed in an incubator. (B) Photograph of the bioreactor.

FIG. 5.

Reconstitution of angioblast cultures derived from ESCs in ex vivo culture system. (A) Scanning electron microscopy images showing re-endothelialization of denuded endothelium layer by inoculated ESC-derived endothelial progenitor cells. Normal endothelium (a); denuded artery (b); growth status of ESC-derived EPCs 3 days after seeding (c) and 5 days after seeding (d) on denuded blood vessels; vehicle control without cell seeding after 5 days (e). 100×. (B) Attachment and growth of angioblasts on denuded artery surface. When ESC-derived angioblasts are transplanted into an ex vivo culture system, the cells attach, grow, and mature on the interior surface of denuded blood vessels. The transplanted cells marked with CellTracker probe (in red) integrated into the lumen of the denuded vessel surface, thus demonstrating their ability to reconstitute damaged blood vessels (c, d); the vehicle control group did not show any presence of fluorescence probe (a, b). Prolonged culture of angioblasts for 2 weeks expressed CD31 antigen [in purple (d)], while short-term culture for 5 days (c) did not show CD31 positivity. Nuclei are stained blue with DAPI. 400×. EPC, endothelial progenitor cell.

FIG. 5.

Reconstitution of angioblast cultures derived from ESCs in ex vivo culture system. (A) Scanning electron microscopy images showing re-endothelialization of denuded endothelium layer by inoculated ESC-derived endothelial progenitor cells. Normal endothelium (a); denuded artery (b); growth status of ESC-derived EPCs 3 days after seeding (c) and 5 days after seeding (d) on denuded blood vessels; vehicle control without cell seeding after 5 days (e). 100×. (B) Attachment and growth of angioblasts on denuded artery surface. When ESC-derived angioblasts are transplanted into an ex vivo culture system, the cells attach, grow, and mature on the interior surface of denuded blood vessels. The transplanted cells marked with CellTracker probe (in red) integrated into the lumen of the denuded vessel surface, thus demonstrating their ability to reconstitute damaged blood vessels (c, d); the vehicle control group did not show any presence of fluorescence probe (a, b). Prolonged culture of angioblasts for 2 weeks expressed CD31 antigen [in purple (d)], while short-term culture for 5 days (c) did not show CD31 positivity. Nuclei are stained blue with DAPI. 400×. EPC, endothelial progenitor cell.