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Biologie

人类多能干细胞的高效分化为心肌细胞，并鉴定流式细胞仪

Published: September 23, 2014
doi:
10.3791/52010
, , , , , , ,
1Department of Biochemistry,Medical College of Wisconsin, 2Stanford Cardiovascular Institute,Stanford University School of Medicine, 3Department of Anesthesiology,Medical College of Wisconsin, 4Stem Cell and Regenerative Medicine Consortium, LKS Faculty of Medicine,Hong Kong University, 5Division of Cardiology,Johns Hopkins University School of Medicine, 6Cardiovascular Research Center, Biotechnology and Bioengineering Center,Medical College of Wisconsin

Summary

该文章描述的详细方法通过选择性调节Wnt通路，随后通过流式细胞仪的参考标记分析来评估群体的均匀性和同一性的有效分化人多能干细胞分化为心肌细胞。

Abstract

目前迫切需要开发用于修复受损的心脏，发现没有对心脏毒性作用的新的治疗药物，改善策略，以准确地模拟心脏疾病的方法。利用人类诱导多能干细胞（hiPSC）技术，“一盘”为这些应用程序生成心肌的潜力继续产生很高的热情。近年来，为了有效地生成人类多能干细胞（hPSCs）心肌细胞的能力有很大的提高，为我们提供了新的机会，以模拟人类心脏发育的早期阶段，否则无法访问。相反，许多以前的方法中，心肌细胞的分化这里描述的协议不需要的细胞聚集或添加激活素A或BMP4和鲁棒生成细胞的，这对于心肌肌钙蛋白高度阳性培养物I和T（TNNI3，TNNT2），iroquois-一流的同源蛋白IRX-4（IRX4），肌球蛋白调节轻链2，室/心肌亚型（MLC2v）和肌球蛋白调节轻链2，心房异构体（MLC2a）由10天对所有的人胚胎干细胞（胚胎干细胞）和hiPSC行测试，以日期。细胞可以传代并保持在培养90天以上。该战略在技术上实现简单，具有成本效益。从多能干细胞的心肌细胞的特性通常包括参考标记的分析，无论是在mRNA和蛋白质水平。对于蛋白质分析，流式细胞仪是评估细胞的培养质量，确定亚群同质化的强大的分析工具。然而，在样品制备技术变化可能显著影响流式细胞仪检测数据的质量。因此，染色方法的标准化应该促进各种差异化策略之间的比较。因此，对于IRX4，MLC2v，MLC2a，TNNI3和TNN的分析而优化的染色方法T2通过流式细胞术进行了描述。

Introduction

心肌细胞从hPSCs，包括胚胎干细胞和hiPSC，生成可作为非常早期的人类心脏发育过程中的体外模型，提供了洞察阶段不进行机理研究，否则访问。该模型系统提供了独特的机会来研究控制心脏谱系的承诺和细胞命运规范的分子途径。近年来，为了有效地生成心肌细胞从hPSCs的能力大大改善了1-15。然而，其中的协议存在的细胞系的变化相对于在产生心肌细胞和定时，在该细胞表达腔特异性标记物（ 例如 ，心室和心房）的效率。理想情况下，该模型系统的未来应用，功能上定义的细胞更均匀种群是期望的。与以前的方法中，心肌细胞的分化这里描述的协议不要求CELL聚集或添加激活素A和骨形态发生蛋白4（BMP4）和强劲的生成TNNI3，TNNT2，IRX4，MLC2v和MLC2a文化非常积极的一天10细胞穿过测试日的所有人类胚胎干细胞和hiPSC线。该策略在技术上容易实现，尤其是相对于三维培养，大量培养，或胚状体基础的策略4-9，并且在一项研究描述了一种小分子具有选择性毒性，对hPSCs（Boheler 等人最近被限定。 ）65。该协议的功能包括使用人类胚胎干细胞资格基质（基质胶）的单层，利用小分子完全定义的媒体来调节Wnt信号（类似，但有别于1,2,7,13）单层培养hPSCs的分化，流式细胞仪染色方法的分化效率和小区标识评价的优化流程。综上所述，本协议的优点相比之前的报道，包括其高性价比eness，重现性，以及其高效率，用于产生在多个HPSC线，包括胚胎干细胞和hiPSC线心肌细胞。

流式细胞仪是评估细胞的培养质量，确定亚群同质化的强大的分析工具，以及适当的实验设计，可以提供定量测量。与所有的抗体为基础的策略，实验结果准确解释要求的试验设计元素，包括抗体浓度和固定和通透条件（针对细胞内的抗原时），经过认真的检查每一个抗体作为次优条件显著影响抗体的效结合，因此，结果的解释。重要的是，如果需要进行定量的，单克隆抗体是至关重要的，因为多克隆抗体可以识别多个表位，并且容易发生批次到批次的变化。目前，各种抗体（POlyclonal和单克隆）和染色方案已经描述了用于在体外分化的评估，从而难以比较中协议1,2,9,11 cardiomyogenesis的效率。出于这个原因，单克隆抗体用于仅当对于所有流式细胞仪分析。展望未来，预计这些染色方法的标准化，特别是关于定量，最好允许差异化战略之间的比较。

标记物的选择，以及它们相应的抗体，用于评估在体外 cardiomyogenesis纯度之间的报告而有所不同。 TNNT2一直被认为是细胞致力于心肌命运的指标和常规用于评价心肌分化方法的效率。然而，TNNT2也早期小鸡和大鼠发展16,17表达在骨骼肌中，它是存在于人平滑肌18 </sup>。因此，TNNT2不一定是人类cardiomyogenesis 在体外的特异性标志物。 MLC2v和MLC2a通常用于为心室和心房亚型的替代指标，分别为。然而，与依托MLC2v和MLC2a确定在体外分化的上下文心肌亚型挑战源自以下事实：这些基因产物可以不通过成人限于整个心脏发育的特定腔室，从心脏管。在啮齿动物心脏循环，MLC2a基因是主要的心房/流道面积和MLC2v基因主要是在室/流出道地区。在环状心脏，共表达MLC2a和MLC2v的mRNA中的流入道，房室通道，并流出道19,20进行观察。由出生后3天，MLC2v mRNA表达被限制在心室和由出生后10天，MLC2a被限制到心房中的新生大鼠心脏19。因此，解释关于cardiomyogenesis效率和亚型的身份不但要考虑的参考标志物水平的存在和数量，但必须考虑到发展阶段（县）的分化所分析的时间点对应的数据。这是特别重要的考虑是由hPSCs的体外分化产生心肌细胞的成熟阶段最相似的那些胚胎/胎儿发育21-25。因此，依靠标记的空间表现在出生后心脏可能不适合HPSC源性细胞的评估，至少在某些情况下。

在努力促进更具体标准的发展，用于定义在体外心肌细胞的身份，TNNI3被认为是一种有价值的标志物，用于评估cardiomyogenesis 体外 ，它被限制为心肌整个胚胎发育中的小鸡和斑马鱼15,20并且不存在于人胎儿骨骼肌26。而TNNI1是存在于人类胎儿心脏，TNNI3是唯一TNNI存在于正常成人心脏27,28亚型。对于心肌细胞亚型的身份，IRX4 29-31是细胞与心室命运的信息标记。在蛋白质水平上，IRX4最近已显示被限制在从线性心脏管通过新生儿阶段的心室中的鼠标32。因此，优化的染色方法为TNNI3的通过流式细胞术分析和IRX4进行说明。就我们所知，这是高效率的基于抗体染色并在人心肌IRX4水平的流式细胞仪分析的方法的第一次描述。

Protocol

1，解决方案和媒体准备人类胚胎干细胞合格基质涂层原液慢慢解冻胚胎干细胞在冰上合格基质（5毫升）中的4ºC过夜。分配等份放入预冷的1.5毫升的无菌微量离心管中并立即存储于-20℃。 注：在等分试样的体积将根据许多变化，并且通常为270〜350微升。制造商提供的步骤2.1中所述关于实现稀释后的1X浓度为25毫升需要等分的音量的详细信息。 HPSC媒体原液使?…

Representative Results

在第0天，将细胞100％汇合以紧凑的形态和最小的细胞碎片。于第1-2天，通常观察显著细胞死亡（40-50％），但附着的细胞将保留紧凑的形态（ 图1A）。在这段时间内，媒体是橙色和浑浊的。粉红媒体表示过度的细胞死亡，并且在这种情况下，确认用锥虫蓝和停止，如果细胞死亡高于70％。细胞会在3-4天恢复和密度就会增加。在5-6天，最小的细胞死亡，并出现密集的修补程序可能开始出…

Discussion

关键的分化方案的成功是利用hPSCs的高品质文化已在微分之前的开始传代在单细胞水平为至少五个通道。类似的分化效率都在各种HPSC行常规观察到，如果他们是100％汇合，在分化开始时，独立细胞系。如果细胞在分化开始时的汇合是≤95％或> 100％的次最佳效率是观察。因此，接种密度和传代的定时应仔细优化为每个线路达到100％汇合的单层细胞在分化的开始。在这方面，镀槽的明确数目/孔，?…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

这项研究是由美国国立卫生研究院4R00HL094708-03支持，MCW的研究事务委员会新教师奖和克恩基金会（启动资金）在威斯康星医学院（RLG）;香港的主题为基础的研究计划T13-706 / 11（KRB）的研究资助局; U01 HL099776，CIRM TR3-05556，CIRM DR2A-05394，和美国心脏协会研究者贡献奖（JCW）;美国心脏协会博士后奖学金12POST12050254（印刷电路板）。我们感谢希望坎贝尔在威斯康星血液研究所的流式细胞仪的核心与数据收集和仔细审查稿件的援助。

Materials

Name of Reagent / Equipment Company Catalog Number Comments/Description
Cell Culture
BD Matrigel, hESC-qualified matrix BD Biosciences 354277
Accutase cell detachment solution Stem Cell Technologies 7920
Dubelcco's phosphate buffered saline (DPBS), no calcium, no magnesium Life Technologies 14190-136
Dimethyl sulfoxide (DMSO, Hybri-Max, sterile-filtered) Sigma Aldrich D2650
Sodium bicarbonate Sigma Aldrich 53817
Citric acid Sigma Aldrich C2404-100G
Y-27632 dihydrochloride selective p160ROCK inhibitor R&D Systems 1254
L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate Sigma Aldrich A8960-5G
Sodium selenite Sigma Aldrich S5261-10G
Transferrin (Optiferrin – Defined, Animal-free, Recombinant Human) Invitria 777TRF029
Fibroblast growth factor 2 (FGF2) R&D Systems 4144-TC-01M
Transforming growth factor beta 1 (TGFβ1) Peprotech 100-21
DMEM/F12 with L-Glutamine and 15 mM HEPES Life Technologies 11330-032
Insulin Sigma Aldrich I9278-5ML
CHIR-99021 HCl Sellekchem S2924
IWR-1 Sigma Aldrich I0161
RPMI 1640 with L-Glutamine Life Technologies 11875-093
B-27 Supplement Minus Insulin Life Technologies A1895601
B-27 Supplement (with Insulin) Life Technologies 17504-044
Fetal bovine serum Life Technologies 10437
Flow cytometry reagents
Phosphate buffered saline (10X) Quality Biological Inc. 119-069-151
Hank's balanced salt solution without Ca2+ and Mg2+ Life Technologies 14175-095
BD Cytofix BD Biosciences 554655
BD Phosflow perm buffer III BD Biosciences 558050
16% Paraformaldehyde Thermo Scientific 28906
Methanol Sigma Aldrich 179957-4L
Acetone Mallenckrodt Chemicals  2440-16
Triton X-100 Amresco M236-10ML
Goat serum Life Technologies 16210-064
Trypan blue solution, 0.4% Life Technologies 15250-061
Materials
5 ml round bottom polystyrene tube (without cap) Corning 352008 for preparation of cells for flow cytometry
5 ml round bottom polystyrene tube (with 35 µM cell strainer snap cap) Corning 352235 for preparation of cells for flow cytometry
2 ml rubber bulbs Fischer Scientific 03-448-24
9" borosilicate unplugged glass Pasteur pipets BioExpress P-2904-2
0.65 mL microcentrifuge tubes, graduated, green GeneMate C-3259-G
1.5 mL microcentrifuge tubes, graduated, red GeneMate C-3260-R
TC20 automated cell counter BioRad 145-0102
Counting slides for TC20 BioRad 145-0011
6 well flat bottom tissue-culture treated plate Corning 353046
20 mL syringes BD Plastipak 300613 For filter sterilization of aliquots
Sterile syringe filters, 0.2 µm polyethersulfone VWR 28145-501 For filter sterilization of aliquots
250 mL filter system, 0.22 µm polyethersulfone, sterilizing, low binding  Corning 431096 For filter sterilization of bulk media 
500 mL filter system, 0.22 µm polyethersulfone, sterilizing, low binding  Corning 431097 For filter sterilization of bulk media 
Antibodies and Isotype Controls
Anti-TNNI3 (clone: 284 (19C7)) Abcam ab19615 Primary antibody
Anti-TNNT2 (clone: 1C11) Thermo Scientific MA1-16687 Primary antibody
Anti-MYL2 (MLC2v, clone: 330G5) Synaptic Systems 310111 Primary antibody
Anti-MYL7 (MLC2a, clone: 4E7) Abcam AB131661 Primary antibody
Anti-IRX4   Bioss BS-9464R Primary antibody
Alexa Fluor 488 Goat anti-mouse IgG1  Life Technologies A21121 Secondary antibody
Alexa Fluor 647 Goat anti-mouse IgG2a Life Technologies A21241 Secondary antibody
Alexa Fluor 488 Goat anti-mouse IgG2b Life Technologies A21141 Secondary antibody
Phycoerythrin(PE) Goat anti-rabbit IgG R&D Systems F0110 Secondary antibody
Mouse IgG1 BD Biosciences 557273 Isotype Control
Mouse IgG2a eBiosciences 16-4724-81 Isotype Control
Rabbit IgG-PE R&D Systems IC105P Isotype Control
TaqMan Probesets for qRT-PCR
ACTB Life Technologies Hs01060665
Brachyury T Life Technologies Hs00610080
CASQ2 Life Technologies Hs00154286
IRX4 Life Technologies Hs01100809
ISL1 Life Technologies Hs00158126
MESP1 Life Technologies Hs01001283
MYH11 (SMMHC) Life Technologies Hs00224610
MYH6 Life Technologies Hs01101425
MYL2 (MLC2v) Life Technologies Hs00166405
MYL7 (MLC2a) Life Technologies Hs01085598
MYOG Life Technologies Hs01072232
NKX2.5 Life Technologies Hs00231763
NPPA Life Technologies Hs01081097
POU5F1 Life Technologies Hs04260367
SOX17 Life Technologies HS00751752
TNNI1 Life Technologies Hs00913333
TNNI2 Life Technologies Hs00268536
TNNI3 Life Technologies HS00165957
TNNT2 Life Technologies Hs00165960
DOWNLOAD MATERIALS LIST

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Tags

Human Pluripotent Stem CellsCardiomyocyte DifferentiationCardiac DevelopmentCardiac MarkersFlow CytometryIRX4MLC2vMLC2aTNNI3TNNT2

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Bhattacharya, S., Burridge, P. W., Kropp, E. M., Chuppa, S. L., Kwok, W., Wu, J. C., Boheler, K. R., Gundry, R. L. High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry. J. Vis. Exp. (91), e52010, doi:10.3791/52010 (2014).
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