Co-Emergence of Specialized Endothelial Cells from Embryonic Stem Cells.

A well-formed and robust vasculature is critical to the health of most organ systems in the body. However, the endothelial cells (ECs) forming the vasculature can exhibit a number of distinct functional subphenotypes like arterial or venous ECs, as well as angiogenic tip and stalk ECs. In this study, we investigate the in vitro differentiation of EC subphenotypes from embryonic stem cells (ESCs). Using our staged induction methods and chemically defined mediums, highly angiogenic EC subpopulations, as well as less proliferative and less migratory EC subpopulations, are derived. Furthermore, the EC subphenotypes exhibit distinct surface markers, gene expression profiles, and positional affinities during sprouting. While both subpopulations contained greater than 80% VE-cad+/CD31+ cells, the tip/stalk-like EC contained predominantly Flt4+/Dll4+/CXCR4+/Flt-1- cells, while the phalanx-like EC was composed of higher numbers of Flt-1+ cells. These studies suggest that the tip-specific EC can be derived in vitro from stem cells as a distinct and relatively stable EC subphenotype without the benefit of its morphological positioning in the sprouting vessel

Directed Derivation of Specialized Endothelial Cells from Pluripotent Stem Cells

A well-formed and robust vasculature is critical to the health of most organ systems in the body. However, endothelial cells (EC) can exhibit a number of distinct functional subphenotypes like arterial or venous EC, as well as angiogenic tip and stalk EC. This study focused on directing the differentiation of endothelial cells subphenotypes from mouse and human embryonic stem cells (ESC), as well as, human induced pluripotent stem (iPS) cells in vitro using a staged and chemically-defined methodology. Using these methods, we discovered and characterized highly angiogenic tip/stalk-containing EC emerging as distinct from less proliferative and less migratory phalanx EC, and examined our ability to direct specification of these subphenotypes. We found that both tip/stalk-containing and phalanx-containing sub-populations were more than 80% VE-cad+ without FACS purification. These EC exhibited distinct mRNA gene expression profiles, surface marker expression, and sprouting capacity in a fibrin gel assay. The tip/stalk EC are Flt4+/Dll4+/Flt-1-/Notch-1- reflecting a migratory more VEGF responsive phenotype indicative of tip cell surface expression pattern. Phalanx ECs are more homogeneous and less responsive to VEGF signaling – comprising of higher levels of Flt-1 and Notch-1. Human stem cell derived EC required additional purification step and treatment with TGFβR1 inhibitor, small molecule SB431542 in order to maintain stability of expanded EC populations. In our attempts at directing the differentiation of EC subpopulations, many of the cell populations did not survive. Of the surviving cells, signaling from PIGF and BMP treatments exhibited the greatest potential for directing stem cells toward tip-like EC and stalk-like EC, respectively. Additionally, immobilized rhDll4 induced quiescence and high VE-cad+/CD31+ expression in the EC consistent with a phalanx-like EC subphenotype. The ability to generate these functionally distinct vascular ESC-EC in vitro is an important development in regenerative medicine

Co-Emergence of Specialized Endothelial Cells from Embryonic Stem Cells.

A well-formed and robust vasculature is critical to the health of most organ systems in the body. However, the endothelial cells (ECs) forming the vasculature can exhibit a number of distinct functional subphenotypes like arterial or venous ECs, as well as angiogenic tip and stalk ECs. In this study, we investigate the in vitro differentiation of EC subphenotypes from embryonic stem cells (ESCs). Using our staged induction methods and chemically defined mediums, highly angiogenic EC subpopulations, as well as less proliferative and less migratory EC subpopulations, are derived. Furthermore, the EC subphenotypes exhibit distinct surface markers, gene expression profiles, and positional affinities during sprouting. While both subpopulations contained greater than 80% VE-cad+/CD31+ cells, the tip/stalk-like EC contained predominantly Flt4+/Dll4+/CXCR4+/Flt-1- cells, while the phalanx-like EC was composed of higher numbers of Flt-1+ cells. These studies suggest that the tip-specific EC can be derived in vitro from stem cells as a distinct and relatively stable EC subphenotype without the benefit of its morphological positioning in the sprouting vessel

Multifactorial Optimizations for Directing Endothelial Fate from Stem Cells.

Embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells are attractive in vitro models of vascular development, therapeutic angiogenesis, and tissue engineering. However, distinct ESC and iPS cell lines respond differentially to the same microenvironmental factors. Developing improved/optimized differentiation methodologies tailored/applicable in a number of distinct iPS and ESC lines remains a challenge in the field. Currently published methods for deriving endothelial cells (EC) robustly generate high numbers of endothlelial progenitor cells (EPC) within a week, but their maturation to definitive EC is much more difficult, taking up to 2 months and requiring additional purification. Therefore, we set out to examine combinations/levels of putative EC induction factors-utilizing our stage-specific chemically-defined derivation methodology in 4 ESC lines including: kinetics, cell seeding density, matrix signaling, as well as medium treatment with vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The results indicate that temporal development in both early and late stages is the most significant factor generating the desired cells. The generation of early Flk-1+/KDR+ vascular progenitor cells (VPC) from pluripotent ESC is directed predominantly by high cell seeding density and matrix signaling from fibronectin, while VEGF supplementation was NOT statistically significant in more than one cell line, especially with fibronectin matrix which sequesters autocrine VEGF production by the differentiating stem cells. Although some groups have shown that the GSK3-kinase inhibitor (CHIR) can facilitate EPC fate, it hindered the generation of KDR+ cells in our preoptimized medium formulations. The methods summarized here significantly increased the production of mature vascular endothelial (VE)-cadherin+ EC, with up to 93% and 57% purity from mouse and human ESC, respectively, before VE-cadherin+ EC purification

Multifactorial Optimizations for Directing Endothelial Fate from Stem Cells

<div><p>Embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells are attractive in vitro models of vascular development, therapeutic angiogenesis, and tissue engineering. However, distinct ESC and iPS cell lines respond differentially to the same microenvironmental factors. Developing improved/optimized differentiation methodologies tailored/applicable in a number of distinct iPS and ESC lines remains a challenge in the field. Currently published methods for deriving endothelial cells (EC) robustly generate high numbers of endothlelial progenitor cells (EPC) within a week, but their maturation to definitive EC is much more difficult, taking up to 2 months and requiring additional purification. Therefore, we set out to examine combinations/levels of putative EC induction factors—utilizing our stage-specific chemically-defined derivation methodology in 4 ESC lines including: kinetics, cell seeding density, matrix signaling, as well as medium treatment with vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The results indicate that temporal development in both early and late stages is the most significant factor generating the desired cells. The generation of early Flk-1⁺/KDR⁺ vascular progenitor cells (VPC) from pluripotent ESC is directed predominantly by high cell seeding density and matrix signaling from fibronectin, while VEGF supplementation was NOT statistically significant in more than one cell line, especially with fibronectin matrix which sequesters autocrine VEGF production by the differentiating stem cells. Although some groups have shown that the GSK3-kinase inhibitor (CHIR) can facilitate EPC fate, it hindered the generation of KDR+ cells in our preoptimized medium formulations. The methods summarized here significantly increased the production of mature vascular endothelial (VE)-cadherin+ EC, with up to 93% and 57% purity from mouse and human ESC, respectively, before VE-cadherin+ EC purification.</p></div

Putative variables directing ESC Differentiation into EC.

<p><b>A)</b> ESC first differentiate into mesodermal Flk-1/KDR+ VPC (stage 1), and subsequently commit and mature into EC (stage 2). The Flk-1+ and KDR⁺ markers identify VPC for mouse and human cells, respectively. The subsequent EC are identified by VE-cadherin⁺ expression. <b>B-G)</b> The mouse and human ESC lines used exhibit markers consistent with pluripotency. Mouse ESC-A3 exhibited <b>B)</b> colony morphology and stained positive for <b>C)</b> Oct¾ (red) and <b>D)</b> SSEA-1 (green); counterstained for DAPI (blue);scale = 100μm. Human ESC-H7 exhibited <b>E)</b> colony morphology and stained positive for <b>F)</b> Oct¾ (red) and <b>G)</b> SSEA-4 (green); scale = 100μm. <b>H)</b> Summary of the ranges of conditions that were analyzed for induction of ESC into Flk-1/KDR+ VPC and VPC into VE-cadherin+ EC. Stage 1 conditions included matrix signaling, kinetics, cell seeding density, and amounts of VEGF supplemented in the chemically-defined cell culture medium. Stage 2 additionally examined the bFGF concentration required in the cell culture medium. Four ESC lines were used in this study: two mouse ESC lines (A3 and R1) and two human ESC lines (H7 and H9). <b>I)</b> Representative histograms of the Flk-1+ expression of mESC-A3 and KDR+ expression hESC-H9 as they change over time. These mESC-A3 were induced by seeding cells 10,000 cells/cm² on fibronectin with 20 ng/mL VEGF. The percentage of Flk-1+ from mESC-A3 increased from day 1 to a maximum at day 3. The hESC-H9 were induced by seeding cells at 10,000 cells/cm² induced on fibronectin with 15 ng/mL VEGF with the number of KDR+ cells peaking at day 12. <b>J)</b> Representative histograms of the Flk-1+ expression of mESC-A3 and KDR+ expression hESC-H9 at increasing levels of VEGF treatment. For mESC-A3, cells were induced on fibronectin at 10,000 cells/cm and data collected on day 3 of differentiation. The hESC-H9 were also induced on fibronectin at 10,000 cells/cm with the data collected on day 12 of differentiation. <b>K)</b> Representative histograms of the Flk-1+ expression of mESC-A3 and KDR+ expression human hESC-H9 as a function of cell seeding density. For mESC-A3, cells were induced on fibronectin with 20ng/ml VEGF and data collected on day 3 of differentiation. The hESC-H9 were induced on fibronectin with 15 ng/ml VEGF and the data was collected on day 12 of differentiation. Flk-1 percentages increase with higher seeding densities, based on a density of 1,000 cells/cm² to a maximum of 10,000 cells/cm². Both mESC and hESC generate the greatest percentage of VPC at cell seeding density of 10,000 cells/cm². <b>L)</b> When mESC cells were induced (stage 1) on larger 100mm² plates, Flk-1 expression increased by approximately 10% for both mESC-A3 and mESC-R1 cells.</p

Results from stage 2 inductions of mouse ESC.

<p><b>A)</b> VPC were induced into VE-cad+ EC. <b>B)</b> Results of differentiating mESC-A3 VPC on fibronectin (N = 1). The A3-ESC Flk-1+ VPC failed to adhere to the collagen type-IV substrate, so no data is available for that condition. Results of differentiating mESC-R1 VPC on <b>C)</b> fibronectin (N = 1), and <b>D)</b> collagen IV (N = 1). <b>E)</b> Based on regression analyses, P-values are provided for stage 2 mESC inductions, with statistical significance (p < 0.05, highlighted in red). The seeding density and bFGF treatment level were both found to be statistically significant variables, but not VEGF treatment levels.</p

Results from stage 1 human ESC inductions.

<p>Induction of <b>A)</b> hESC-H7 and hESC-H9 were examined for peak expression of KDR+ VPC over time and a range of cell seeding densities (N = 1). The best differentiation conditions were achieved on days 14 and 12 for hESC-H7 and hESC-H9, respectively. <b>B)</b> The human ESC were then induced for range of VEGF treatments (N = 3). <b>C)</b> Based on regression analyses, P-values are provided for stage 1 hESC inductions, with statistical significance (p < 0.05, highlighted in red). Here VEGF was significant in the hESC-H9 line only.</p

EC Maturation Over Time.

<p>After stage 2 induction of Flk-1/KDR+ VPC into VE-cad+ EC, the cells were cultured for longer periods in order to examine further maturation and/or self-purification. Averaged percentages of VE-cadherin+ for both mouse <b>A)</b> and human cells <b>B)</b> are given. <b>A)</b> The mESC-A3 were expanded on fibronectin at 10,000 cells/cm² with 10 ng/mL VEGF and 10 ng/mL bFGF. The mESC-A3 grew to a purity of 93% VE-cadherin by day 14. <b>B)</b> The hESC-H7 were cultured on fibronectin, seeded at a density of 10,000 cells/cm² with 25 ng/mL VEGF and 50 ng/mL bFGF. The hESC-H7 also increased in expression of VE-cadherin+ cells, peaking at a 57% by day 48 of total induction (14 days in stage 1 followed by 34 days in stage 2). <b>C, G, E, I)</b> Unpurified mESC-R1 derived EC <b>D, F, H, J)</b> hESC-H7 derived EC express markers and morphology consistent with EC phenotype: <b>C-D)</b> VE-cadherin (red) counterstained with phalloidin (green) and <b>E-F)</b> PECAM-1 (green) counterstained with phalloidin (red) Nuclei stained with DAPI (blue). Large mesenchymal-like cells are seen surrounding the murine cells in <b>C</b>). The mESC-A3 and hESC-H7 also <b>(G-H)</b> took up acetylated LDL and <b>(I-J)</b> formed vascular-like structures in Matrigel^™ within 24 hours.</p