Sorting human skeletal muscle-derived myoendothelial cells and perivascular stem cells by multiparameter fluorescence-activated cell sorting

doi:10.3969/j.issn.2095-4344.0900

Abstract

Abstract:

BACKGROUND: At present, the preplate method is widely used for the separation of skeletal muscle-derived stem/progenitor cells. However, little is reported on the sorting of human skeletal muscle-derived stem/progenitor cells using multiparameter fluorescence-activated cell sorting (MP-FACS).
OBJECTIVE: To explore the method of sorting high-purity myoendothelial cells and perivascular stem cells from human skeletal muscle tissues by MP-FACS.
METHODS: Human skeletal muscle tissue was collected and digested with collagenase (type I and type IV) and dispase, to collect mononuclear cells. Myoendothelial cells with phenotype (CD56⁺CD34⁺CD144⁺CD45^-) and perivascular stem cells with phenotype (CD146⁺CD45^-CD34^-CD144^-CD56^-) were sorted by MP-FACS.
RESULTS AND CONCLUSION: Fourteen samples of MECs and PSCs were sorted by MP-FACS. Myoendothelial cells and perivascular stem cells accounted for (0.04±0.01)% and (0.30±0.07)% in human skeletal muscle mononuclear cells, respectively. After the freshly sorted cells were re-analyzed by flow cytometry, the purity of myoendothelial cells and perivascular stem cells was up to (96.80±1.16)% and (92.50±0.50)%, respectively, and the survival rate of myoendothelial cells and perivascular stem cells reached (95.58±1.10)% and (95.15±0.67)%, respectively. After primary culture, myoendothelial cells and perivascular stem cells grew well, and furthermore, they could be successfully induced to differentiate into osteoblasts and adipocytes in vitro, respectively, indicating that myoendothelial cells and perivascular stem cells have good multi-differentiation ability. To conclude, the myoendothelial cells and perivascular stem cells obtained by MP-FACS have high purity and good activity.

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: Muscle, Skeletal, Endothelial Cells, Flow Cytometry, Tissue Engineering

CLC Number:

R394.2

Gu Jing-jing, Yang Ji-hui, Yang Ting-ting, Yang Xiao-ping, Zheng Bo. Sorting human skeletal muscle-derived myoendothelial cells and perivascular stem cells by multiparameter fluorescence-activated cell sorting[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(21): 3357-3364.

Figures/Tables 3

2.1 人骨骼肌单个核细胞分离共分离人骨骼肌肉组织14例，男8例，女6例，年龄(46.36±18.61)岁(20-77岁)；分离肌肉质量为(4.67±1.06) g；每克肌肉可分离出单个核细胞(2.63±0.34)×106。分离步骤可参照图1。 2.2 多参数流式分选图将抗体标记好的样本上机，根据空细胞管、同型对照管和单阳管校正FACS参数。首先根据7AAD选出活细胞(图2A)，再根据前向和侧向散射角圈定细胞群(图2B)，通过多色标记，从圈定的细胞群中先选出CD45-细胞(图2C)，然后从CD45-细胞选出CD56-和CD56+细胞(图2D)，从CD56+细胞群中选出CD34+CD144+细胞群即CD56+CD34+CD144+CD45-的MECs(图2G)，从CD56-细胞群中选出CD34-CD144-细胞群(图2E)，再从CD34-CD144-细胞群中选出CD146+细胞群即CD146+ CD45-CD34-CD144-CD56-的PSCs(图2F)。对14例多参数流式细胞分选结果进行统计：MECs阳性率为(0.04± 0.01)%，分选得到细胞数为(1.22±0.34)×104；PSCs阳性率为(0.30±0.07)%，分选得到细胞数为(3.49±0.79)×104(表1)。 2.3 新分选后MECs和PSCs回测纯度吸取新分选的MECs和PSCs悬液各100 µL，用DPBS稀释成300 µL，上机对分选后的细胞进行纯度回测。MECs回测纯度为98.1%(图3A)，PSCs回测纯度为92.6%(图3B)。对FACS的14例MECs和PSCs均进行纯度回测，统计得MECs纯度为(96.80±1.16)%，PSCs纯度为(92.50±0.50)%(表1)。 2.4 新分选MECs和PSCs存活率用细胞计数板计数并根据被锥虫蓝染色的细胞为死细胞，未被染色的细胞为活细胞来计算存活率。对FACS的14例MECs和PSCs均进行存活率测定，统计得MECs存活率为(95.58±1.10)%，PSCs存活率为(95.15±0.67)%(表1)。 2.5 分选出的MECs和PSCs的细胞形态倒置相差显微镜下观察细胞形态，刚分选的细胞呈圆球型、透亮、均匀分布在培养板中(图4A，B)；3 d左右，培养板中贴壁生长的长梭形细胞，细胞两端较长突起，胞体中央膨大，为细胞核；培养至7 d左右，细胞形态逐渐变为均一的长梭形，胞质丰富(图4C，D)；14 d左右细胞融合达70%-80%，可进行传代继续培养。目前已经传代至第8代，细胞状态良好，形态规则。 2.6 MECs和PSCs向成骨细胞和成脂细胞诱导分化鉴定结果 MECs和PSCs分别在成骨细胞和成脂细胞诱导剂下培养，能成功诱导为成骨细胞和成脂细胞。成骨诱导后实验组茜素红染色呈阳性反应，胞质内可见到大量暗红色颗粒状样物质(图5A，E)，证实为钙盐沉积，而对照组(未进行成骨诱导)在镜下并未见到暗红色颗粒状物质(图5B，F)；成脂诱导后实验组油红O染色可见胞浆内有大量红色的脂滴形成(图5C，G)，而对照组(未进行成脂诱导)镜下观察未见有红色脂滴形成(图5D，H)。以上结果证明MEC和PSCs具有良好的多向分化能力。"

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