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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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Related in: MedlinePlus

Specification of angioblasts induced by endothelial growth factors. (A) Proliferative potential of EPCs derived from ESCs. Morphologic features of angioblast cultures under EGM-2 differentiation are shown at day two (a), day nine (b) and day 15 (c). Panel d indicates the average cell number per colony during the culture. Significant differences existed between ADM and EGM-2 or ECGS; results are expressed as mean ± SD, t-test, n = 20. (B) Antigenic expression of angioblasts specified by different culture media. Flow cytometry results of specified EPCs together with HPP-ECFCs are presented. Single-cell suspensions were stained with the indicated antibody combination; all cells were gated as CD45−. Isotype-matched control antibody staining was performed and used to process the data. (C) Comparison of EGM-2 and ECGS in angioblast specification. Antigenic expression pattern by flow cytometry reveals the differences of angioblast specification under EGM-2 and ECGS, using HPP-ECFCs as a reference. (D) LDL uptake by angioblasts specified by three media. ESC-derived cells were grown in ADM (b), EGM-2 (c) and ECGS (d); and incubated with Dil-LDL to measure their uptake ability. Dil-LDL is labelled in red. Nuclei are stained blue with DAPI 400×. (E) Cellular reactivity to 10 ng/ml TNF-α. Specified EPCs under EGM-2 (dark blue), ECGS (orange), ADM (maroon), or control EC (light blue), were cultured in monolayer and treated with TNF-α for 4 hrs; corresponding controls were treated with an equal amount of PBS. Flow cytometry to detect CD54, CD62E and CD106 was conducted. Percentage positivity is shown. Results were calculated from three experiments and expressed as mean ± SD. ★ indicates significant difference between TNF-α-treated cells and corresponding controls (P < 0.05). (F) Vascular-like and microvessel formation on Matrigel. (a) endothelial cells isolated from blood vessels; (b and e) specified EPCs from EGM-2; (c and f) specified EPCs from ECGS; (d) specified EPCs from ADM. a–d, 100×; e–f, 400×.
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fig02: Specification of angioblasts induced by endothelial growth factors. (A) Proliferative potential of EPCs derived from ESCs. Morphologic features of angioblast cultures under EGM-2 differentiation are shown at day two (a), day nine (b) and day 15 (c). Panel d indicates the average cell number per colony during the culture. Significant differences existed between ADM and EGM-2 or ECGS; results are expressed as mean ± SD, t-test, n = 20. (B) Antigenic expression of angioblasts specified by different culture media. Flow cytometry results of specified EPCs together with HPP-ECFCs are presented. Single-cell suspensions were stained with the indicated antibody combination; all cells were gated as CD45−. Isotype-matched control antibody staining was performed and used to process the data. (C) Comparison of EGM-2 and ECGS in angioblast specification. Antigenic expression pattern by flow cytometry reveals the differences of angioblast specification under EGM-2 and ECGS, using HPP-ECFCs as a reference. (D) LDL uptake by angioblasts specified by three media. ESC-derived cells were grown in ADM (b), EGM-2 (c) and ECGS (d); and incubated with Dil-LDL to measure their uptake ability. Dil-LDL is labelled in red. Nuclei are stained blue with DAPI 400×. (E) Cellular reactivity to 10 ng/ml TNF-α. Specified EPCs under EGM-2 (dark blue), ECGS (orange), ADM (maroon), or control EC (light blue), were cultured in monolayer and treated with TNF-α for 4 hrs; corresponding controls were treated with an equal amount of PBS. Flow cytometry to detect CD54, CD62E and CD106 was conducted. Percentage positivity is shown. Results were calculated from three experiments and expressed as mean ± SD. ★ indicates significant difference between TNF-α-treated cells and corresponding controls (P < 0.05). (F) Vascular-like and microvessel formation on Matrigel. (a) endothelial cells isolated from blood vessels; (b and e) specified EPCs from EGM-2; (c and f) specified EPCs from ECGS; (d) specified EPCs from ADM. a–d, 100×; e–f, 400×.

Mentions: As demonstrated previously, our angioblast differentiation protocol can generate angioblasts via mesodermal intermediates and the cells demonstrate dual potential to differentiate into haematopoietic and vascular lineages [21]. To direct angioblast differentiation towards functional EPCs, we tested whether endothelial growth media for mature endothelial cultures could specify and drive the angioblasts into EPCs more efficiently. In this study, we chose two media, EGM-2 and ECGS, and continued to culture angioblasts in ADM as a reference control. EGM-2 contains defined growth factors, whereas ECGS contains bovine pituitary extracts. After angioblast differentiation, we transferred EBs onto collagen IV-coated plates and cultured them for 12 days. Figure 2 illustrates the dynamic process of angioblast specifications.


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)

Specification of angioblasts induced by endothelial growth factors. (A) Proliferative potential of EPCs derived from ESCs. Morphologic features of angioblast cultures under EGM-2 differentiation are shown at day two (a), day nine (b) and day 15 (c). Panel d indicates the average cell number per colony during the culture. Significant differences existed between ADM and EGM-2 or ECGS; results are expressed as mean ± SD, t-test, n = 20. (B) Antigenic expression of angioblasts specified by different culture media. Flow cytometry results of specified EPCs together with HPP-ECFCs are presented. Single-cell suspensions were stained with the indicated antibody combination; all cells were gated as CD45−. Isotype-matched control antibody staining was performed and used to process the data. (C) Comparison of EGM-2 and ECGS in angioblast specification. Antigenic expression pattern by flow cytometry reveals the differences of angioblast specification under EGM-2 and ECGS, using HPP-ECFCs as a reference. (D) LDL uptake by angioblasts specified by three media. ESC-derived cells were grown in ADM (b), EGM-2 (c) and ECGS (d); and incubated with Dil-LDL to measure their uptake ability. Dil-LDL is labelled in red. Nuclei are stained blue with DAPI 400×. (E) Cellular reactivity to 10 ng/ml TNF-α. Specified EPCs under EGM-2 (dark blue), ECGS (orange), ADM (maroon), or control EC (light blue), were cultured in monolayer and treated with TNF-α for 4 hrs; corresponding controls were treated with an equal amount of PBS. Flow cytometry to detect CD54, CD62E and CD106 was conducted. Percentage positivity is shown. Results were calculated from three experiments and expressed as mean ± SD. ★ indicates significant difference between TNF-α-treated cells and corresponding controls (P < 0.05). (F) Vascular-like and microvessel formation on Matrigel. (a) endothelial cells isolated from blood vessels; (b and e) specified EPCs from EGM-2; (c and f) specified EPCs from ECGS; (d) specified EPCs from ADM. a–d, 100×; e–f, 400×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814022&req=5

fig02: Specification of angioblasts induced by endothelial growth factors. (A) Proliferative potential of EPCs derived from ESCs. Morphologic features of angioblast cultures under EGM-2 differentiation are shown at day two (a), day nine (b) and day 15 (c). Panel d indicates the average cell number per colony during the culture. Significant differences existed between ADM and EGM-2 or ECGS; results are expressed as mean ± SD, t-test, n = 20. (B) Antigenic expression of angioblasts specified by different culture media. Flow cytometry results of specified EPCs together with HPP-ECFCs are presented. Single-cell suspensions were stained with the indicated antibody combination; all cells were gated as CD45−. Isotype-matched control antibody staining was performed and used to process the data. (C) Comparison of EGM-2 and ECGS in angioblast specification. Antigenic expression pattern by flow cytometry reveals the differences of angioblast specification under EGM-2 and ECGS, using HPP-ECFCs as a reference. (D) LDL uptake by angioblasts specified by three media. ESC-derived cells were grown in ADM (b), EGM-2 (c) and ECGS (d); and incubated with Dil-LDL to measure their uptake ability. Dil-LDL is labelled in red. Nuclei are stained blue with DAPI 400×. (E) Cellular reactivity to 10 ng/ml TNF-α. Specified EPCs under EGM-2 (dark blue), ECGS (orange), ADM (maroon), or control EC (light blue), were cultured in monolayer and treated with TNF-α for 4 hrs; corresponding controls were treated with an equal amount of PBS. Flow cytometry to detect CD54, CD62E and CD106 was conducted. Percentage positivity is shown. Results were calculated from three experiments and expressed as mean ± SD. ★ indicates significant difference between TNF-α-treated cells and corresponding controls (P < 0.05). (F) Vascular-like and microvessel formation on Matrigel. (a) endothelial cells isolated from blood vessels; (b and e) specified EPCs from EGM-2; (c and f) specified EPCs from ECGS; (d) specified EPCs from ADM. a–d, 100×; e–f, 400×.
Mentions: As demonstrated previously, our angioblast differentiation protocol can generate angioblasts via mesodermal intermediates and the cells demonstrate dual potential to differentiate into haematopoietic and vascular lineages [21]. To direct angioblast differentiation towards functional EPCs, we tested whether endothelial growth media for mature endothelial cultures could specify and drive the angioblasts into EPCs more efficiently. In this study, we chose two media, EGM-2 and ECGS, and continued to culture angioblasts in ADM as a reference control. EGM-2 contains defined growth factors, whereas ECGS contains bovine pituitary extracts. After angioblast differentiation, we transferred EBs onto collagen IV-coated plates and cultured them for 12 days. Figure 2 illustrates the dynamic process of angioblast specifications.

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