Endothelial progenitor cells proliferated via MEK-dependent p42 MAPK signaling pathway

Abstract

Endothelial progenitor cells (EPCs) clinical applications have been well reported. However, due to low number of EPCs that could be isolated, EPCs expansion study became one of the main focuses. Some optimized mediums to culture EPCs were currently available. However, the proliferation signaling pathway is not clearly disclosed yet. Peripheral blood was collected from eight healthy subjects, followed by mononuclear cells (MNCs) isolation. MNCs were then prepared and cultured for 2 days. After that, non-adherent cells were harvested and further cultured for 3 days. Resulted colony-forming unit (CFU)-Hill colonies were documented and enumerated under an inverted light microscope. To detect membrane markers, immunofluorescence was performed to detect CD34, VEGFR-2, and CD133. Cell documentation was conducted under a fluorescence microscope. To check cell proliferation, XTT Cell Proliferation Assay Kit was used according to kit insert. To detect possible activation of p44/42 MAPK, western blot was performed to detect p44/42 MAPK and phosphorylated p44/42 MAPK. All visualized bands were captured and quantified. Our results showed that EPCs markers (CD34, CD133 and VEGFR-2) were detected in 3 days culture. From XTT cell proliferation assay and CFU enumeration results, we found that EPCs proliferated significantly (p = 0.012) with addition of supplement. Phosphorylated-p42 MAPK expression of EPCs treated with supplement was significantly higher than the one of EPCs without treatment. Significant inhibition of p42 MAPK phosphorylation by U0126 was observed (p = 0.012). By pretreatment of U0126, number of viable cells and CFUs treated with supplement was significantly decreased (p = 0.012). Our results showed that MEK-dependent p42 MAPK pathway might play an important role in EPCs proliferation.

Caspase Inhibitor Diminishes Caffeic Acid-induced Apoptosis in Osteosarcoma Cells

“…DAPI staining was performed as described by Sandra, et al (14,15) Cells were seeded onto coverslips, stimulated, rinsed in phosphate buffered saline (PBS) and fixed with icecold tricholoroacetic acid and gradient ethanol. Then cells were permeabilized with 0.1% BSA and 0.1% Triton X-100 for 3 minutes, and stained with 1 μg/ml 4',6-Diamidino-2-phenylindole (Calbiochem) for 3 minutes.…”

Preliminary Study: Purple Sweet Potato Extract Seems to Be Superior to Increase the Migration of Impaired Endothelial Progenitor Cells Compared to l-Ascorbic Acid

“…Peripheral blood mononuclear cells (PBMCs) were isolated from the blood sample by Ficoll Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA). To isolate EPCs from PBMCs, a standard protocol was conducted as described previously [22]. Briefly, 5 × 10 5 cells/mL PBMCs were cultured in the fibronectin-coated 6-well plate with basal stemline II hematopoietic stem cell expansion medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 15% fetal bovine serum and 40 ng/mL vascular endothelial growth factor.…”

Role of Herbal Extract in Stem Cell Development

“…13 The application of stem cell therapy in the medical field are rapidly developing. Efforts to achieve the applications also vary, such as the development of stem cell and its microenvironment isolation methods 14,15 , development of methods to increase the number of stem cells [16][17][18][19][20][21][22] , the development of stem cell differentiation methods 23 (including cell reprogramming) 24,25 , stem cell storage methods 26,27 , the development of stem cell transplantation methods 16,28 , etc. Since then, some particular stem cell types with their potential differentiation capacities can be isolated and cultured to meet the requirement of each clinical study to be explored.…”

“…Meanwhile, in some circumstances, cell number is not enough, so therefore cell number should be enriched in an in vitro stem cell culture setting. 16,17,[19][20][21][22] In an in vitro stem cell culture, besides suitable and sterile equipment, reagents such as culture medium, serum and antibiotics are all important. Although all those criteria are fulfilled, somehow stem cell enrichment cannot be achieved, cell number is still below the target.…”