Summary

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Published: March 01, 2016
doi:

Summary

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Abstract

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues.

To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRPα and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.

Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

Introduction

心脏组织工程已在过去十年中极大地推进,以多组发布最近来自鼠心肌细胞1-6和,充分发挥功能,打浆组织,人干细胞衍生的心肌细胞7-12的结果。心脏组织工程领域是由两个主要和基本上独立目标驱动:1),以开发可移植到衰竭心脏改善功能4-6外源性移植物;和2) 的体外模型开发用于研究生理学和疾病,或作为筛选工具用于治疗发展2,7。

三维(3-D)的细胞培养物被认为是开发下一代筛选工具,作为3维矩阵反映更自然心脏微环境比传统的二维单层细胞培养至关重要;的确细胞生物学的一些方面是在2-D对的3-D培养物13,14根本不同</suP>。细胞外基质,及细胞群:此外,工程化的心脏组织被从完全定义的组件构成。对于传统的设计的人心脏组织中,而细胞外基质组合物(通常为纤维蛋白9或胶原7,8,10)被严格控制,输入单元组合物不太明确,与细胞的整个混合物从一个定向心脏分化任胚胎干细胞(ESC 7,9)或诱导的多能干细胞(IPSC的10,12)被添加到组织中。取决于具体的细胞系和所使用的分化方案的效率,所得到的心肌细胞的百分比可以从低于25%的范围内,以90%以上,具体的心肌的表型( ,ventricular-,atrial-或起搏器状)也可以有所不同,甚至非心肌分数可以是高度异质15,16和改变的分化心肌米到期yocytes 17。

最近心脏组织工程工作一直试图控制单元的输入口,与任一心脏记者人胚胎干细胞系8或细胞表面标记18被用来分化的心肌细胞成分分离。虽然最初只心肌细胞组成的组织,似乎是理想的,这是事实上并非如此;完全的心肌细胞组成hECTs无法压缩成功能的组织,具有一定的群体找到一个3:心肌细胞比为1:成纤维细胞产生了最高的抽搐力8。通过使用各种小区选择方法,其中包括表面标记活细胞分选,可以创建具有限定的细胞群体hECTs。而非心脏间质细胞的标记物已经有一段时间,如推定的成纤维细胞标记物CD90 19,20,心肌细胞的表面标记已经比较困难鉴别。 SIRPα是确定用于人类心肌细胞18中的第一心脏表面标记之间并已被证明是心脏谱系高度选择性。最近,我们已发现,双分拣SIRPα+和CD90 细胞产生几乎纯的心肌细胞,与CD90 +群体表现出成纤维细胞样表型(Josowitz,未发表的观察结果)。基于这些发现收集,在此,我们介绍了如何创建使用3 hECTs:SIRPα+ / CD90的结合1 心肌细胞和CD90 +成纤维细胞。

工程师一个完全确定的人类心脏组织的能力,不仅是用于创建健壮的筛选工具,而且还为开发模型系统研究新兴细胞和基于基因的心脏治疗至关重要。在心脏衰竭特别是,众多的细胞疗法,利用细胞类型,包括间质干细胞(MSC)21 </SUP>,心脏干细胞22和骨髓单核细胞23-25,已经在临床试验中进行测试。虽然许多初步结果已被看好21,23,25,最初的好处往往削弱随着时间的推移26-29。类似的趋势已经报道鼠工程心脏组织,这显示显著功能的好处,由于MSC的补充,但效益长期培养1期间不会持续。基础的亚最佳性能是我们有限理事细胞疗法的机制的知识。如何治疗细胞发挥其有益的影响,以及肌细胞nonmyocyte相互作用的潜在的负面影响更深的了解,将有助于提高治疗的临床收益和显著效益的持续发展,以最小的副作用,患者的心脏衰竭。

在这里,我们描述了使用定义hECTs来interrog吃的基于细胞的疗法的机制。受控组织构成是必要的识别影响心肌性能的具体因素。直接补充hECTs与感兴趣的治疗性细胞类型( 例如 ,干细胞),可以揭示在心肌细胞性能的影响,如我们在大鼠的ECT 1已经证明。

以下多步协议开始指示心脏干细胞的分化,其次多的组织的生物反应器的制造,并与组织结构和功能分析的说明结束。我们的实验中使用的是美国国立卫生研究院批准H7人类胚胎干细胞(hESC细胞)线进行。但是,以下的协议也已使用额外的人类胚胎干细胞系,并与类似的结果3诱导多能干细胞(hiPSC)行测试。我们已发现,在心肌细胞的分化和成功HECT制造效率可以细胞系相关的,特别是FO从个别病人衍生řhiPSC线。按照此协议,两个6孔皿镀有共168万人类胚胎干细胞(每孔14万个细胞),它区分了20天,分选,足以花费六个定义组织后产生大约250万细胞。

Protocol

注:执行使用HEPA过滤II级生物安全柜在无菌条件下,所有的细胞操纵和由通过0.2μm过滤器过滤他们消毒所有的解决方案。无论是在相同的无菌条件或层流罩进行组织结构和功能测试。 1.在心脏分化准备H7人类胚胎干细胞的播种 (1天)准备基底膜基质解冻的hESC合格基底膜基质的150微升等分试样在冰上过夜,在4℃。 在涂层板胚胎干细胞(0-4天),电…

Representative Results

以获得心肌细胞,的Boheler和廉分化方法略加修改使用30,31。当务之急是在分化过程中的细胞生长的对数阶段开始,也即初始种群足够汇合排序(约75%是最优的)后,得到的细胞的可用数目。典型地,对于H7的hESCs,在保持在37℃的每一个的6孔培养皿的孔140000人类胚胎干细胞中必不可少8媒体和5%CO 2培养箱的密度电镀后4天产生充分融合培养开始分化,如所示在图1A中- <s…

Discussion

定义人工程心脏组织(HECT)的建设可以提供人体心肌细胞的功能更一致和可靠的模型。关键的是,系统中的所有细胞和细胞外成分是已知的并且根据需要,从而消除从分化过程产生的其他未知的细胞类型的混杂影响可以被操纵。以平衡快速的细胞生长和高收率,优选的分化开始在人类胚胎干细胞的75%汇合时,理想的电镀后四天。此外,这两个排序后的细胞解离和再凝集期间使用ROCK抑制剂Y-27632?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作是由美国国立卫生研究院(1F30HL118923-01A1)到TJC,NIH / NHLBI PEN合同HHSN268201000045C到KDC,香港TRS T13-706 / 11(KDC),美国国立卫生研究院(R01 HL113499),以BDG,美国的研究资助局的支持心脏协会(12PRE12060254)到RJ和香港研究资助局(TBRS,T13-706 / 11)RL额外的资金由美国国立卫生研究院DRB 5T32GM008553-18并在系统和发展NIDCR,跨学科培训实习提供给TJC生物学与出生缺陷T32HD075735。作者还希望衷心感谢亚瑟Autz在纽约城市学院的赞恩中心协助加工技术援助生物反应器马姆杜Eldaly。我们也感谢肯尼斯Boheler对心脏分化咨询医生,和Joshua野兔博士慷慨地提供人类骨髓间充质干细胞。

Materials

Cell Culture Company Catalog Number Comments
Amphotericin B Sigma-Aldrich A2411 Prepare a 2.5 mg/ml stock in DMSO and filter-sterilize
B27 with Insulin Life Technologies 17505055
B27 without Insulin Life Technologies A1895601
CHIR99021 Stemgent 04-0004 Create 6 μM stock, then aliquot and store at -20 °C.
Essential 8 Media Life Technologies A1517001
H7 Human Embryonic Stem Cells WiCell WA07
hESC Qualified Matrix, Corning Matrigel Corning 354277 Thaw on ice at 4 °C overnight then aliquot 150 μl into separate tubes and store at -20 °C.
IWR-1 Sigma-Aldrich I0161 Create 10 mM stock and aliquot. Store at -20 °C
Neonatal Calf Serum Life Technologies 16010159
Non-enzymatic Dissociation Reagent: Gentle Cell Dissociation Reagent Stem Cell Technologies 7174
Penicillin-Streptomycin Corning 30-002-CI
RPMI 1640 Life Technologies 11875-093 Keep refrigerated
Y-27632 (ROCK Inhibitor) Stemgent 04-0012 Resuspend to a 10 mM stock concentration, aliquot and store at -20 °C.  Avoid freeze thaw cycles.
Cell Sorting Company Catalog Number Comments
4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) Life Technologies D1306
CD90-FITC BioLegend 328107
Enzymatic Dissociation Reagent: Cell Detach Kit I (0.04 % Trypsin/ 0.03% EDTA, Trypsin neutralization solution and Hanks Buffered Salt Solution)  PromoCell C-41200
Fetal Bovine Serum Atlanta Biologics S11250
SIRPα-PE/Cy7 BioLegend 323807
Tissue Construction Company Catalog Number Comments
0.25% Trypsin/0.1% EDTA Fisher Scientific 25-053-CI Optional: For collection of supplemental cells of interest
10x MEM Sigma-Aldrich M0275-100ML
10X PBS Packets Sigma-Aldrich P3813
Collagen, Bovine Type I Life Technologies A10644-01 Keep on ice
DMEM/F12 Life Technologies 11330057
Dulbecco’s Modified Eagles Medium (DMEM), High Glucose Sigma-Aldrich D5648
Polydimethylsiloxane (PDMS) Dow Corning Sylgard 184
Sodium HEPES Sigma-Aldrich H3784
Sodium Hydroxide Sigma-Aldrich 221465
Materials Company Catalog Number Comments
1.5 ml microcentrifuge tubes Fisher Scientific NC0536757
15 ml polyproylene centrifuge tube Corning 352096
5 ml Polystyrene Round-Bottom Tube Corning 352235 With integrated 35 μm cell strainer
50 ml polyproylene centrifuge tube Corning 352070
6-well flat bottom tissue-culture treated plate Corning 353046
Cell Scraper, Disposable Biologix 70-2180
Polysulfone McMaster-Carr
Polytetrafluoroethylene (Teflon) McMaster-Carr
Equipment Company Catalog Number Comments
Dissecting Microscope Olympus SZ-61 Or similar, must have a mount for the high speed camera to attach
Electrical Pacing System Astro-Med, Inc Grass S88X Stimulator
High Speed Camera Pixelink PL-B741U Or similar, but must be capable of 100 frames per second for accurate data acquisition
Plate Temperature Control Used to maintain media temperature during data acqusition.
Custom Materials Company Catalog Number Comments
LabView Post-tracking Program available upon request from the authors

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Cite This Article
Cashman, T. J., Josowitz, R., Gelb, B. D., Li, R. A., Dubois, N. C., Costa, K. D. Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy. J. Vis. Exp. (109), e53447, doi:10.3791/53447 (2016).

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