定义人工程心脏组织建设探讨心肌细胞疗法的机理
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（ 即 ，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
Representative Results
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 |
References
- Serrao, G. W., et al. Myocyte-depleted engineered cardiac tissues support therapeutic potential of mesenchymal stem cells. Tissue Eng. Part A. 18 (13-14), 1322-1333 (2012).
- Hansen, A., et al. Development of a drug screening platform based on engineered heart tissue. Circ. Res. 107 (1), 35-44 (2010).
- Fink, C., Ergün, S., Kralisch, D., Remmers, U., Weil, J., Eschenhagen, T. Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement. FASEB J. 14 (5), 669-679 (2000).
- Yildirim, Y., et al. Development of a biological ventricular assist device: preliminary data from a small animal model. Circulation. 116, 16-23 (2007).
- Sekine, H., et al. Cardiac Cell Sheet Transplantation Improves Damaged Heart Function via Superior Cell Survival in Comparison with Dissociated Cell Injection. Tissue Eng. Part A. 17 (23-24), 2973-2980 (2011).
- Lesman, A., et al. Transplantation of a tissue-engineered human vascularized cardiac muscle. Tissue Eng. Part A. 16 (1), 115-125 (2010).
- Turnbull, I. C., et al. Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium. FASEB J. 28 (2), 644-654 (2014).
- Thavandiran, N., Dubois, N., et al. Design and formulation of functional pluripotent stem cell-derived cardiac microtissues. Proc. Natl. Acad. Sci. U. S. A. 110 (49), 4698-4707 (2013).
- Schaaf, S., et al. Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology. PloS One. 6 (10), e26397 (2011).
- Tulloch, N. L., et al. Growth of engineered human myocardium with mechanical loading and vascular coculture. Circ. Res. 109 (1), 47-59 (2011).
- Nunes, S. S., et al. Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes. Nature Methods. 10 (8), 781-787 (2013).
- Ma, Z., et al. Three-dimensional filamentous human diseased cardiac tissue model. Biomaterials. 35 (5), 1367-1377 (2014).
- Baker, B. M., Chen, C. S. Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues. J. Cell Sci. 125 (13), 3015-3024 (2012).
- Pontes Soares, C., Midlej, V., de Oliveira, M. E. W., Benchimol, M., Costa, M. L., Mermelstein, C. 2D and 3D-organized cardiac cells shows differences in cellular morphology, adhesion junctions, presence of myofibrils and protein expression. PloS One. 7 (5), e38147 (2012).
- Burridge, P. W., Keller, G., Gold, J. D., Wu, J. C. Production of De Novo Cardiomyocytes: Human Pluripotent Stem Cell Differentiation and Direct Reprogramming. Cell Stem Cell. 10 (1), 16-28 (2012).
- Mummery, C. L., Zhang, J., Ng, E. S., Elliott, D. A., Elefanty, A. G., Kamp, T. J. Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. Circ. Res. 111 (3), 344-358 (2012).
- Kim, C., et al. Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation. Stem Cells Dev. 19 (6), 783-795 (2010).
- Dubois, N. C., et al. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nature Biotechnol. 29 (11), 1011-1018 (2011).
- Hudon-David, F., Bouzeghrane, F., Couture, P., Thibault, G. Thy-1 expression by cardiac fibroblasts: lack of association with myofibroblast contractile markers. J. Mol. Cell. Cardiol. 42 (5), 991-1000 (2007).
- Gago-Lopez, N., et al. THY-1 receptor expression differentiates cardiosphere-derived cells with divergent cardiogenic differentiation potential. Stem Cell Reports. 2 (5), 576-591 (2014).
- Hare, J. M., Traverse, J. H., et al. A randomized, double-blind, placebo-controlled, dose-escalation study of intravenous adult human mesenchymal stem cells (prochymal) after acute myocardial infarction. J. Am. Coll. Cardiol. 54 (24), 2277-2286 (2009).
- Bolli, R., et al. Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial. Lancet. 378 (9806), 1847-1857 (2011).
- Wollert, K. C., et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet. 364 (9429), 141-148 (2004).
- Hirsch, A., et al. Intracoronary infusion of autologous mononuclear bone marrow cells or peripheral mononuclear blood cells after primary percutaneous coronary intervention: rationale and design of the HEBE trial–a prospective, multicenter, randomized trial. Am. Heart. J. 152 (3), 434-441 (2006).
- Jeevanantham, V., Butler, M., Saad, A., Abdel-Latif, A., Zuba-Surma, E. K., Dawn, B. Adult bone marrow cell therapy improves survival and induces long-term improvement in cardiac parameters: a systematic review and meta-analysis. Circulation. 126 (5), 551-568 (2012).
- Meyer, G. P., et al. Intracoronary bone marrow cell transfer after myocardial infarction: 5-year follow-up from the randomized-controlled BOOST trial. Eur. Heart J. 30 (24), 2978-2984 (2009).
- Meyer, G. P., et al. Intracoronary bone marrow cell transfer after myocardial infarction: eighteen months’ follow-up data from the randomized, controlled BOOST (BOne marrOw transfer to enhance ST-elevation infarct regeneration) trial. Circulation. 113 (10), 1287-1294 (2006).
- Hirsch, A., et al. Intracoronary infusion of mononuclear cells from bone marrow or peripheral blood compared with standard therapy in patients after acute myocardial infarction treated by primary percutaneous coronary intervention: results of the randomized controlled HEBE trial. Eur. Heart J. 32 (14), 1736-1747 (2011).
- Simari, R. D., et al. Bone marrow mononuclear cell therapy for acute myocardial infarction: a perspective from the cardiovascular cell therapy research network. Circ. Res. 114 (10), 1564-1568 (2014).
- Bhattacharya, S., et al. High efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry. J. Vis. Exp. (91), e52010 (2014).
- Lian, X., et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc. Natl. Acad. Sci. U. S. A. 109 (27), 1848-1857 (2012).
- Burridge, P. W., et al. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PloS One. 6 (4), e18293 (2011).
- Kean, T. J., Lin, P., Caplan, A. I., Dennis, J. E. MSCs: Delivery Routes and Engraftment, Cell-Targeting Strategies, and Immune Modulation. Stem Cells Int. , 732742 (2013).
- Trachtenberg, B., et al. Rationale and design of the Transendocardial Injection of Autologous Human Cells (bone marrow or mesenchymal) in Chronic Ischemic Left Ventricular Dysfunction and Heart Failure Secondary to Myocardial Infarction (TAC-HFT) trial: A randomized, double-blind, placebo-controlled study of safety and efficacy. Am. Heart J. 161 (3), 487-493 (2011).
- Williams, A. R., et al. Intramyocardial stem cell injection in patients with ischemic cardiomyopathy: functional recovery and reverse remodeling. Circ. Res. 108 (7), 792-796 (2011).
- Razeghi, P., Young, M. E., Alcorn, J. L., Moravec, C. S., Frazier, O. H., Taegtmeyer, H. Metabolic gene expression in fetal and failing human heart. Circulation. 104 (24), 2923-2931 (2001).
- Rajabi, M., Kassiotis, C., Razeghi, P., Taegtmeyer, H. Return to the fetal gene program protects the stressed heart: a strong hypothesis. Heart Fail. Rev. 12 (3-4), 331-343 (2007).
- Taegtmeyer, H., Sen, S., Vela, D. Return to the fetal gene program: a suggested metabolic link to gene expression in the heart. Ann. N. Y. Acad. Sci. 1188, 191-198 (2010).
