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Endothelial reconstitution by CD34+ progenitors derived from baboon embryonic stem cells.

Shi Q, Schatten G, Hodara V, Simerly C, VandeBerg JL - J. Cell. Mol. Med. (2013)

Bottom Line: The efficiency of generating CD34+ EPCs did not differ significantly from ECGS to EGM-2 culture media, however, angioblasts specified in ECGS medium expressed a higher percentage of CD34+/CXCR4+ cells (3.49 ± 1.32%, n = 3) than those specified in EGM-2 medium (0.49 ± 0.52%, n = 3).After 14 days of ex vivo culture, the grafted cells had attached and integrated to the denuded surface; in addition, they had matured further and expressed terminally differentiated endothelial markers including CD31 and CD146.In conclusion, we have proved that specified CD34+ EPCs are promising therapeutic agents for repairing damaged vasculature.

View Article: PubMed Central - PubMed

Affiliation: Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX 78245-0549, USA. qshi@txbiomedgenetics.org

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Experimental design for generating and identifying therapeutic EPCs from ESCs. EPCs are generated from ESCs via angioblast formation from EBs suspended in ESC/ADM media with varying ratios for indicated times, following by specification in monolayer culture on collagen-coated plates under three different media to form functional EPCs. Progenitors are matured on a microenvironment provided by injured vascular surface. Therapeutic effects are evaluated by transplanting EPCs onto a denuded vascular surface, which provides a suitable microenvironment for EPCs to mature towards functional competence.
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fig01: Experimental design for generating and identifying therapeutic EPCs from ESCs. EPCs are generated from ESCs via angioblast formation from EBs suspended in ESC/ADM media with varying ratios for indicated times, following by specification in monolayer culture on collagen-coated plates under three different media to form functional EPCs. Progenitors are matured on a microenvironment provided by injured vascular surface. Therapeutic effects are evaluated by transplanting EPCs onto a denuded vascular surface, which provides a suitable microenvironment for EPCs to mature towards functional competence.

Mentions: The BAB15 baboon ESC lines at passage 39 were obtained from the Pittsburgh Development Center, University of Pittsburgh School of Medicine; they were cultured according to the published method and used within no more than 10 passages. We confirmed the cellular pluripotency by growth behaviour; positive immunostaining for OCT-4, NANOG and SSEA-4; and histochemical staining for ALP [21]. Pooled colonies of high-proliferative-potential endothelial colony-forming cells (HPP-ECFCs), which were considered tissue-resident EPCs, were isolated and cultured according to the method described previously [22]. We modified our protocol for differentiating endothelial progenitors from ESCs based on reported methods [23–26] and developed a step-wise differentiation protocol (Fig. 1). In the first stage, we aimed at differentiating angioblasts from ESCs in three-dimensional embryoid body (EB) culture. We derived angioblasts from ESCs in three-dimensional EB culture in angioblast differentiation medium (ADM). We used AggreWell plates to form uniform EBs containing about 5000–10,000 cells and cultured them in ESC medium for 3 days. The ADM consisted of ESC medium supplemented with a cocktail of 0.5 ng/ml BMP-4, 5 ng/ml basic FGF, 10 ng/ml VEGF, 5 ng/ml stem cell factor, 5 ng/ml thrombopoietin and 10 ng/ml FLT-3 ligand. Subsequently, we added ADM to EB cultures at gradually increased ratios at the time-points indicated in Figure 1. At the end of day nine, the EBs were transferred onto collagen-coated plates (BD Biosciences, San Jose, CA, USA) for monolayer culture [21]. At this stage, we specified angioblast preparations and generated endothelial progenitors. Two specifying media were used: (1) EGM-2 medium containing EGF, hydrocortisone, VEGF, FGF-B, R3-IGF-1, ascorbic acid, heparin, gentamicin, amphotericin-B, and FCS (Lonza, detailed concentrations are provided at http://www.lonza.com), and (2) ECGS medium with endothelial growth factors from bovine pituitary extracts (Sigma-Aldrich, St. Louis, MO, USA) [27]. At the same time, some cells were allowed to continue growing in ADM as a reference control. After 12 days, endothelial lineage progenitor cells were harvested by enzymatic digestion for various tests. To determine the average cell number per colony, we fixed the cells grown on 24-well plate and stained the cells with thiazine dye (HEMA-Diff kit, StatLab, TX, USA). We counted the stained nuclei of each cell with the Objective Micrometer (Meiji Techno America, San Jose, CA, USA) under the microscope to achieve the accurate cell count in colonies.


Endothelial reconstitution by CD34+ progenitors derived from baboon embryonic stem cells.

Shi Q, Schatten G, Hodara V, Simerly C, VandeBerg JL - J. Cell. Mol. Med. (2013)

Experimental design for generating and identifying therapeutic EPCs from ESCs. EPCs are generated from ESCs via angioblast formation from EBs suspended in ESC/ADM media with varying ratios for indicated times, following by specification in monolayer culture on collagen-coated plates under three different media to form functional EPCs. Progenitors are matured on a microenvironment provided by injured vascular surface. Therapeutic effects are evaluated by transplanting EPCs onto a denuded vascular surface, which provides a suitable microenvironment for EPCs to mature towards functional competence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814022&req=5

fig01: Experimental design for generating and identifying therapeutic EPCs from ESCs. EPCs are generated from ESCs via angioblast formation from EBs suspended in ESC/ADM media with varying ratios for indicated times, following by specification in monolayer culture on collagen-coated plates under three different media to form functional EPCs. Progenitors are matured on a microenvironment provided by injured vascular surface. Therapeutic effects are evaluated by transplanting EPCs onto a denuded vascular surface, which provides a suitable microenvironment for EPCs to mature towards functional competence.
Mentions: The BAB15 baboon ESC lines at passage 39 were obtained from the Pittsburgh Development Center, University of Pittsburgh School of Medicine; they were cultured according to the published method and used within no more than 10 passages. We confirmed the cellular pluripotency by growth behaviour; positive immunostaining for OCT-4, NANOG and SSEA-4; and histochemical staining for ALP [21]. Pooled colonies of high-proliferative-potential endothelial colony-forming cells (HPP-ECFCs), which were considered tissue-resident EPCs, were isolated and cultured according to the method described previously [22]. We modified our protocol for differentiating endothelial progenitors from ESCs based on reported methods [23–26] and developed a step-wise differentiation protocol (Fig. 1). In the first stage, we aimed at differentiating angioblasts from ESCs in three-dimensional embryoid body (EB) culture. We derived angioblasts from ESCs in three-dimensional EB culture in angioblast differentiation medium (ADM). We used AggreWell plates to form uniform EBs containing about 5000–10,000 cells and cultured them in ESC medium for 3 days. The ADM consisted of ESC medium supplemented with a cocktail of 0.5 ng/ml BMP-4, 5 ng/ml basic FGF, 10 ng/ml VEGF, 5 ng/ml stem cell factor, 5 ng/ml thrombopoietin and 10 ng/ml FLT-3 ligand. Subsequently, we added ADM to EB cultures at gradually increased ratios at the time-points indicated in Figure 1. At the end of day nine, the EBs were transferred onto collagen-coated plates (BD Biosciences, San Jose, CA, USA) for monolayer culture [21]. At this stage, we specified angioblast preparations and generated endothelial progenitors. Two specifying media were used: (1) EGM-2 medium containing EGF, hydrocortisone, VEGF, FGF-B, R3-IGF-1, ascorbic acid, heparin, gentamicin, amphotericin-B, and FCS (Lonza, detailed concentrations are provided at http://www.lonza.com), and (2) ECGS medium with endothelial growth factors from bovine pituitary extracts (Sigma-Aldrich, St. Louis, MO, USA) [27]. At the same time, some cells were allowed to continue growing in ADM as a reference control. After 12 days, endothelial lineage progenitor cells were harvested by enzymatic digestion for various tests. To determine the average cell number per colony, we fixed the cells grown on 24-well plate and stained the cells with thiazine dye (HEMA-Diff kit, StatLab, TX, USA). We counted the stained nuclei of each cell with the Objective Micrometer (Meiji Techno America, San Jose, CA, USA) under the microscope to achieve the accurate cell count in colonies.

Bottom Line: The efficiency of generating CD34+ EPCs did not differ significantly from ECGS to EGM-2 culture media, however, angioblasts specified in ECGS medium expressed a higher percentage of CD34+/CXCR4+ cells (3.49 ± 1.32%, n = 3) than those specified in EGM-2 medium (0.49 ± 0.52%, n = 3).After 14 days of ex vivo culture, the grafted cells had attached and integrated to the denuded surface; in addition, they had matured further and expressed terminally differentiated endothelial markers including CD31 and CD146.In conclusion, we have proved that specified CD34+ EPCs are promising therapeutic agents for repairing damaged vasculature.

View Article: PubMed Central - PubMed

Affiliation: Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX 78245-0549, USA. qshi@txbiomedgenetics.org

Show MeSH
Related in: MedlinePlus