A comparative study on in vitro proliferation, differentiation, transportation and immunocompatibility of mesenchymal stem cells derived from human amniotic fluid, umbilical cord, and placenta

doi:10.3969/j.issn.2095-4344.0960

Abstract

Abstract:

BACKGROUND: With the advancement of regenerative medicine, the use frequency of stem cells has gradually increased in the treatment of diseases. At present, several factors such as the number of cells, the ability of differentiation and the possibility of immunological rejection during allogeneic inoculation are taken into consideration in the clinical use of stem cells. Accordingly, it is very meaningful to screen out the more suitable type of stem cells for clinical use.
OBJECTIVE: To discuss the differences of mesenchymal stem cells (MSCs) derived from human amniotic fluid, umbilical cord and placenta in terms of the ability to proliferate in vitro, the ability to differentiate, the possibility of transport and the possibility of allogeneic inoculation, thereby providing evidence for the clinical application of MSCs.
METHODS: Three kinds of MSCs were isolated and cultured at first, and they were then identified by immunofluorescence and induced into osteoblasts, adipocytes and neuroblasts in vitro. qPCR was used to detect the expression of Nestin, NT-3, and MANF after neural differentiation. HLA-ABC and HLA-DR expression in these three kinds of MSCs was quantitatively analyzed by flow cytometry. Finally, the number of living cells was calculated at 24, 48 and 72 hours after they were placed in the cell transport solution at room temperature.
RESULTS AND CONCLUSION: (1) The adipogenic ability of placenta-derived MSCs and the osteogenic capability of umbilical cord-derived MSCs were the best among three kinds of MSCs. All three kinds of cells expressed Nestin and GFAP, which are markers of neural stem cells. (2) NT-3 was highly expressed in umbilical cord-derived MSCs after neural induction, while MANF was highly expressed in placenta-derived MSCs after neural induction. They could be used to repair different types of nerve injuries. (3) The human amniotic fluid-derived MSCs were more suitable for allogeneic inoculation because they expressed HLA-ABC in a low level and did not express HLA-DR which showed the lowest possibility to cause immune rejection. (4) The survival rate of amniotic fluid-derived MSCs was the highest after a 72 hours induction in the cell transport fluid. In conclusion, as there is a slight difference in biological characteristics of human amniotic fluid, umbilical cord and placenta-derived MSCs, these three kinds of MSCs can be used for clinical treatment of different diseases.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Amniotic Fluid, Placenta, Umbilical Cord, Cell Proliferation, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Zhao Shu-can, Zheng Gui-chun, Lin Lian-peng, Mei Zhan-tu, Wang Bing-yun, Chen Sheng-feng, Chen Zhi-sheng. A comparative study on in vitro proliferation, differentiation, transportation and immunocompatibility of mesenchymal stem cells derived from human amniotic fluid, umbilical cord, and placenta[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 4028-4034.

Figures/Tables 2

2.1 细胞生长曲线、细胞可获得数量以及群体倍增时间第3代hUC-MSCs的生长速度较快，但是传至第6代时，hAFSCs增殖速度明显快于hUC-MSCs，hP-MSCs生长速度最慢(图1A，B)，经多次传代纯化后，第6代细胞群体倍增时间更低(图1C)。假设第1代能获得1×106个细胞(不同羊水样本的密度、脐带样本的长短和粗细、胎盘样本的大小和质量不能保证完全一致，所以不选择这些因素做比较)，统计第1-12代可获得的细胞总数(图1D)，可知hUC-MSCs的生长速度较快，hAFSCs次之，hP-MSCs最慢。 2.2 细胞的特异性表面标记鉴定和成脂、成骨分化能力 3种细胞均表达CD44(图2A-C)、CD90(图2D-F)，不表达CD34(图2G-I)。 hP-MSCs在成脂诱导17 d发现有少许脂滴，hAFSCs、hUC-MSCs均在成脂诱导19 d时出现少许脂滴。在第21天进行油红O染色，可见胞浆内有被染红的脂滴，分别对染色后有明显红色脂滴的视野进行拍照(图3A-C)，经Image ProPlus软件分析成脂部分的面积(即成脂能力)：hUC-MSCs>hAFSCs>hP-MSCs(图4)。 3种细胞在成骨诱导约1周时逐渐由长梭形变为方形、鳞片形。hP-MSCs、hUC-MSCs、hAFSCs分别在14，16，19 d出现钙结节，在第21天进行茜素红染色，可见红色矿化沉着物(图3C-E)，每组随机选取6个不同视野的照片，经Image ProPlus软件分析成骨部分的面积(即成骨能力)：hP-MSCs>hUC-MSCs>hAFSCs(图4)。 2.3 细胞的神经分化能力 3种细胞经诱导后均表达神经干细胞标记物Nestin以及GFAP(图5)。经诱导后3种细胞Nestin、NT-3、MANF基因表达均上升且差异有显著性意义(P < 0.05)，hP-MSCs的NT-3基因表达显著升高，hUC-MSCs的MANF基因表达较其他2种细胞升高明显(图6)。 2.4 细胞免疫相容性抗体HLA-ABC、HLA-DR的表达低表达HLA-ABC、HLA-DR可显示出间充质干细胞具有较低的免疫排斥反应。选取第4代3种细胞进行流式细胞仪分析HLA-ABC、HLA-DR以及对应的同型对照IgG1、IgG2的表达(图7，8)，结果显示HLA-ABC的表达量hUC-MSCs> hAFSCs>hP-MSCs，HLA-DR的表达量hP-MSCs> hUC-MSCs>hAFSCs。 2.5 3种细胞在运输液内常温放置的活细胞数量在室温放置24，48，72 h后，计算活细胞数量(图9)，可见hAFSCs更适合于储存液的运输，3 d后细胞数量可达到73%。"

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