心肌致密化不全患者特异性诱导多潜能干细胞及心肌样细胞的构建

刘奥怡; 张蕾; 龚梦嘉; 田杰; 吕铁伟

doi:10.16016/j.1000-5404.201907075

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心肌致密化不全患者特异性诱导多潜能干细胞及心肌样细胞的构建

基础医学 | 更新时间：2026-01-09

- 心肌致密化不全患者特异性诱导多潜能干细胞及心肌样细胞的构建
- Generation of induced pluripotent stem cells and cardiac myocyte-like cells from a patient with noncompaction of ventricular myocardium
- 第三军医大学学报 2019年41卷第24期页码：2401-2408
- 作者机构：
  
  400014 重庆, 重庆医科大学附属儿童医院心血管内科，儿童发育疾病研究教育部重点试验室，国家儿童健康与疾病临床医学研究中心，儿童发育重大疾病国家国际科技合作基地，儿科学重庆市重点试验室
- 作者简介：
  
  [ 吕铁伟，E-mail:ltw200145@163.com LYU Tiewei, E-mail: ltw200145@163.com
- 基金信息：
  
  国家自然科学基金面上项目(81570218)
- DOI：10.16016/j.1000-5404.201907075
  中图分类号： R329-33;R329.24;R542.2
- 收稿：2019-07-10，
  
  修回：2019-09-24，
  
  纸质出版：2019-12-30
- 稿件说明：
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刘奥怡, 张蕾, 龚梦嘉, 等. 心肌致密化不全患者特异性诱导多潜能干细胞及心肌样细胞的构建[J]. 第三军医大学学报, 2019,41(24):2401-2408.

Aoyi LIU, Lei ZHANG, Mengjia GONG, et al. Generation of induced pluripotent stem cells and cardiac myocyte-like cells from a patient with noncompaction of ventricular myocardium[J]. Journal of Third Military Medical University, 2019, 41(24): 2401-2408.
刘奥怡, 张蕾, 龚梦嘉, 等. 心肌致密化不全患者特异性诱导多潜能干细胞及心肌样细胞的构建[J]. 第三军医大学学报, 2019,41(24):2401-2408. DOI： 10.16016/j.1000-5404.201907075.

Aoyi LIU, Lei ZHANG, Mengjia GONG, et al. Generation of induced pluripotent stem cells and cardiac myocyte-like cells from a patient with noncompaction of ventricular myocardium[J]. Journal of Third Military Medical University, 2019, 41(24): 2401-2408. DOI： 10.16016/j.1000-5404.201907075.

摘要

目的

构建心肌致密化不全(noncompaction of ventricular myocardium，NVM)患者来源的诱导多潜能干细胞(induced pluripotent stem cells

iPS)及心肌样细胞(cardiomyocytes

CMs)。

方法

用仙台病毒感染1名NVM患者(女性，16岁，2016年我科住院患者)和1名同期24岁健康男性志愿者的尿液细胞获得iPS细胞，进而将iPS细胞诱导分化为iPS-CMs。对iPS和iPS-CMs进行免疫荧光染色检测干细胞和心肌细胞标志物、碱性磷酸酶(alkaline phosphatase

AP)活性检测、核型分析、拟胚体(embryoidbodies

EBs)形成和膜片钳试验检测，同时采用全外显子测序(whole exon sequencing

WES)对NVM患者血液、iPS及iPS-CMs进行遗传学背景的比较

并筛选出可能和NVM发生相关的突变基因。

结果

成功获得NVM患者及正常人的尿液细胞和iPS

并分化为iPS-CMs。免疫荧光显示iPS表达干细胞标志Oct4、Sox2、Ssea4和TRA-1-81，iPS细胞具有高AP活性、具有正常核型、克隆能够在体外悬浮培养分化成3个胚层的细胞；iPS-CMs表达心肌细胞特异性标志物α-actinin和TNNT2，膜片钳结果显示iPS-CMs形成心肌细胞特异性钠电流及动作电位。WES结果显示患者血液、iPS及iPS-CMs三者具有相似的遗传谱，并发现可能与NVM发生相关的突变。

结论

成功构建出NVM患者的iPS-CMs，为采用iPS技术开展NVM的研究提供有力的细胞模型。

Abstract

Objective

To obtain induced pluripotent stem cells (iPS) and cardiac myocyte-like cells (CMs) from a patient with noncompaction of ventricular myocardium (NVM).

Methods

Urine cells from a 16-year-old female patient with NVM and a 24-year-old healthy male volunteer were infected with Sendai virus to obtain iPS cells

which were further induced to differentiate into iPS-CMs. Both the iPS cells and iPS-CMs were identified by immunofluorescence staining

alkaline phosphatase activity

karyotype analysis

embryoid body (EBs) formation and cell patch-clamp experiment. Whole-exome sequencing of the blood sample

iPS cells and iPS-CMs from the NVM patient was performed for genetic comparison to identify gene mutations potentially related with NVM.

Results

We successfully obtained urine cells

iPS cells and iPS-CMs from the NVM patient and the healthy volunteer. Immunofluorescence assay showed that the iPS cells expressed pluripotent stem cell markers Oct4

Sox2

Ssea4 and TRA-1-81. iPS cells exhibited a high alkaline phosphatase activity with a normal karyotype

and could be induced to differentiate into 3 germ layers

in vitro

. Immunocytochemical staining revealed that iPS-CMs expressed cardiac myocyte-specific markers TNNT2 and α-actinin. Patch-clamp experiment demonstrated the presence of functional cardiac-specific voltage-gated Na

currents and action potentials in the iPS-CMs. Whole-exome sequencing showed similar genetic profiles between the blood samples

iPS cells and iPS-CMs of the NVM patient

and several gene mutations were found to have potential associations with NVM.

Conclusion

We successfully obtain iPS cells and iPS-CMs from the NVM patient

and these cells provide useful cell models for the study of NVM using iPS technology.

关键词

Keywords

references

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