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Adipose-derived mesenchymal stem cells and endothelial cells in vitro transdifferentiation Melatonin Regulation

Author: ChenJia
Tutor: ZuoHongGang
School: Beijing Union Medical College
Course: Biomedical Engineering
Keywords: Human adipose-derived mesenchymal stem cells endothelial cells differentiation melatonin poly-ε-caprolactone
CLC: R329
Type: Master's thesis
Year: 2011
Downloads: 42
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Abstract

Objective:Human adipose-derived mesenchymal stem cells, a kind of stem cells owning tremendous growth ability and differential ability, showed a large number of advantages to attract academicians’attention, such as steady biological character, abundant source, easily culture in vitro, etc. Hence, the passage focused on:①set up a primary culture system of hADSCs and differentiation into endothelial cells in the serum free medium;②ompatibility, increasing rate and differentiation into the endothelial cells between hADSCs and material;③nfluence on the endothelial differentiation, exchange of calcium ion concentration and expression of ERK and p-ERK by MT.Methods:hADSCs were obtained from liposuction and their immuno-types were detected by flow cytometer (FCM). Adipogenic and osteogenic differentiation were carried out to prove their multi-potency. Medium containing 40ng/ml VEG F and 10ng/ml bFGF was used to differentiate hADSCs into endothelium. vWF and VE-Cadherin was examined by FCM, WP body was observed by TEM. Tube formation and Dil-Ac-LDL phagocytosis test indicated the differentiation of endothelial cells. After hADSCs implantation in the 60-80μm and 180-200μm apertures of poly-ε-caprolactone materials, comparison of seeding rate and growth were carried out by immunofluorescence with DAPI and scanning electron microscope (SEM). CCK-8 method was used to draw the growth curve. Endothelial differentiation with 40ng/ml VEGF and 10ng/ml bFGF after 50day, vWF and VE-cadherin were detected by flow cytometers and Flk-1 was observed by immunofluorescence way. The hADSC processed by different dose (10μmol/L, 1μmol/L,0.1μmol/L) were cultured until 8 days. Then vWF and Cadherin were detected by FCM, compared with the control group without melatonin and positive control containing 50μM calcium ionophore A23187. [Ca2+]i labeled with Fluo-3 was detected by laser confocal microscopy. hADSC processed by different doses of MT after 24h planting on Matrigel, tube formation was observed under the inverted phase contrast microscope. Expression of ERK and p-ERK was evaluated by western blot analysis. Results:FCM shows that CD45(-)CD14(-)CD44(+)CD29(+)CD105(+) cells are hADSCs, which are capable of differentiation into adipogenic and osteogenic cells. Over time the expression of vWF and VE-Cadherin increase. WP body can be seen by TEM, and the tube formation can be observed after 30 days of differentiation. Differentiated hADSCs could uptake Ac-LDL, similar to endothelial cells, after implantation of hADSCs, cells looked long fusiform attaching on the material by SEM. Comparatively, seeding rate of 60-80μm aperture material was (98.33±0.33)%, which was obviously more than seeding rate as(90.33±1.45)%of 180-200μm aperture material, and growth curve observed so. Hence, ehdothelial differentiation was carried out with 60-80μm aperture material. Differentiating after 50 days, positive ration of vWF was (80.9±0.90)% and VE-cadherin was (84.3±1.10)%. Flk-1 expression was observed in most of cells via immuofluorescence. Expression of vWF (13.15±4.55)%and VE-Cadherin (17.96±8.74)% of hADSCs processed by 10μmol/L MT were not obviously higher than the control group (P>0.05), while expression of vWF (19.99±1.74)%and VE-Cadherin(21.13±9.74)%of hADSCs by 1μmol/L MT, and xpression of vWF(43.66+0.89)%and VE-Cadherin(49.42+4.92)%of hADSCs by 0.1μmol/L were both obviously higher than the control group (P< 0.05). However, positive expression of v WFand VE-cadherin of hADSCs pretreated by 40μmol/L ERK pathway blockers U0126 were less than MT treatment group. [Ca2+]i of cells processing by 10μmol/L,1μmol/L,0.1μmol/L MT respectively went up by-46.02%,12.27%and 99.09%. Cells treated by 1 and 0.1μmol/L MT appeared tube formation, and the control and 10μmol/L group not, while cells pretreated by 40μmol/L U0126 could not formate tubes. Comparing with the control group, p-ERK of medium and low dosages group increased obviously (P< 0.05) and high dosage decrease obviously (P< 0.05), but there is no difference in the expression of ERK at all(P>0.05).Conclusions:①we successfully isolated hADSCs which are capable of differentiation into endothelial cells.②poly-ε-caprolactone with the aperture of 60-80μm is suitable for the growth of hADSCs which are also successfully differentiated into endothelial cells on it.③1 and 0.1μmol／L doses MT can accelerate the differention from hADSCs to endothelium obviously through increasing of[Ca2+]i by activating the ERK/MAPK pathway.

Adipose-derived mesenchymal stem cells and endothelial cells in vitro transdifferentiation Melatonin Regulation

Abstract

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