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本文内容

  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

在这里，我们提出一种方法，有效地利用的心肌细胞分化潜力，年轻的人骨髓间充质干细胞的来源以生成功能、 订约、 心肌样细胞体外。

摘要

心肌梗死和随后的缺血级联导致大量伤亡，心肌细胞，导致充血性心衰，全世界死亡的主要原因。骨髓间充质干细胞 (MSCs) 是一种有前景的细胞为基础的疗法，以取代当前的侵入性技术。骨髓间充质干细胞可以分化成间充质血统，其中包括心脏的细胞类型，但尚未实现完整功能的细胞分化。以前的分化的方法基于药物制剂或生长因子。然而，更多生理有关的战略也可以启用骨髓间充质干细胞向心肌细胞转化。在这里，我们提出一种利用心肌细胞饲养层上 MSC 聚合产生订约心肌样细胞的分化方法。

人脐带血管周围细胞 (HUCPVCs) 已被证明有更大的分化潜能比一般调查 MSC 类型，如骨髓间充质干细胞 (BMSCs)。作为一个 ontogenetically 年轻的来源，我们调查了早孕 (FTM) HUCPVCs 相比更旧的来源的心肌样潜力。FTM HUCPVCs 是新颖、 丰富的来源，保留其在子宫内理想属性时培养的间充质干细胞在体外。HUCPVCs 使用此分化协议、 FTM 和期限取得显著增加向心肌细胞分化与骨髓间充质干细胞，相比，增加表达的心肌细胞标记 （即，肌细胞增强因子 2 C、 心肌肌钙蛋白 T、 重链心肌肌球蛋白、 信号调节蛋白 α 和缝隙连接蛋白 43） 所示。他们还保持显著低免疫原性，证明了他们低 HLA A 表达和更高的 HLA — G 表达这一点。应用基于聚合的分化，FTM HUCPVCs 表明增加聚合形成潜在和生成的共培养对心脏饲养层 1 星期内收缩细胞集群，成为第一的 MSC 类型，这样做。

我们的结果表明，这种分化策略可以有效利用向心肌细胞的潜力，年轻的骨髓间充质干细胞，如 FTM HUCPVCs，表明那种体外预分化可能是一个潜在的战略，以增加其再生效果的体内。

引言

充血性心力衰竭 (CHF) 仍是全球发病率和死亡率的主要原因。CHF 经常发生后心肌细胞的巨大损失和心肌梗死 (MI)1的病理结果无细胞瘢痕组织的发展。虽然心脏是部分自我更新的器官，驻地干、 祖细胞池负责执行组织再生显著降低中丰度和老年患者，往往变得不足以最佳恢复的损伤后的功能。因此，是极大的兴趣发展涉及的健康供者细胞移植到受损心肌的实验性治疗。势在必行的是供者细胞不仅恢复结构的组织，但也达到心肌功能恢复的影响。

本机的心员工心组织居民和损伤后内源性的骨髓源性干细胞修复2，，34。蓄热式导出细胞宿主和捐助者都必须有获得适当的表型和功能的重塑心肌，并有能力以高效、 安全地更换丢失的细胞微环境的能力。在体外分化方法曾经广泛实现高效率、 基于干细胞的心肌细胞生产5，6。表达谱的心脏沿袭标记用于定义向心脏沿袭7干细胞分化的过程。早期分化标记，如 NKX2.5，肌细胞增强因子 2 C (Mef2c) 和 GATA48，9，可以指示开始向心肌细胞进程。成熟的心肌细胞标记通常用于评估分化效果是信号调节蛋白 α （关于）10、 心肌肌钙蛋白 T （肌钙）11、 重链心肌肌球蛋白 (MYH6)8，，1213和缝隙连接蛋白 43 (Cx43)14，，1516。利用胚胎干细胞 (Esc) 和多能性干细胞 (Psc) 的方法彻底优化并讨论了有关的诱导因素，氧气和养分的渐变，细节和行动5，，67，，1718的确切时间。尽管如此，基于 ESC 和 PSC 的技术仍然存在多个的伦理和安全问题，和次优的电生理及免疫学特征19，20。主机经常用这些细胞移植免疫排斥的经验和需要永久免疫抑制。这主要是由于失主要组织相容性复合体 (MHC) 分子在东道国和捐助和由此产生的 T 细胞反应21。同时个别 MHC I 类匹配是一个可能的解决方案，更易于访问的临床实践需要普遍是理想来克服排斥反应的关切的细胞来源。

作为替代细胞来源，用于在临床应用中，骨髓间充质干细胞和骨髓间充质干细胞，特别被进行了自其最初被描述在 1995年22组织再生使用。骨髓间充质干细胞据说是驻地的再生细胞在几乎任何带血管蒂的组织23中可以发现。骨髓间充质干细胞后从所需的源的隔离，很容易可以扩大在文化，有广泛的旁分泌能力，经常拥有理想或免疫调节属性24，25。其安全性和有效性已经进行了几个临床前研究，特别是心脏再生3，，26。

许多 MSC 分化策略利用药理剂 5-氮杂胞苷22和二甲基亚砜27，和增长或人骨的因素，像 BMPs5，7，，2829或血管紧张素 II30，与变量效率。这些策略，不过，不基于天真再生细胞有望后归巢或传递到损伤的部位，遇到的障碍体内。更多生理有关的战略，同时更难定义和操纵，都基于的前提下，可以通过从组织微环境本身的信号诱导 MSC 分化。先前的研究显示，暴露于心肌细胞裂解物31或左室心肌32，33，或直接与原发性心肌细胞体外15，34，接触可以增加心肌标志物检测在骨髓间充质干细胞中表达。别人有后，表现出自发再生治疗心肌损伤与骨髓间充质干细胞35，36，，3738，尽管部分，骨髓间充质干细胞和心肌细胞39，40融合生成新生心肌。我们所知，从人骨髓间充质干细胞 (hMSCs) 的任何组织源的功能、 自发订约心肌细胞尚未报告。

目前的共识是所有骨髓间充质干细胞产生的血管周围细胞23。年轻骨髓间充质干细胞与周细胞属性可以从人脐带组织41，，4243的血管周围地区隔离。与骨髓间充质干细胞，HUCPVCs 拥有更多的分化潜能和几个其他再生的优势，两个体外41，44和体内45，，4647。值得注意的是，被母胎界面的来源，HUCPVCs 有显著较低免疫原性相比成人骨髓间充质干细胞的来源。我们的研究重点的表征和 FTM HUCPVCs，年轻源间充质干细胞研究，临床前应用，我们以前已经增加了增殖能力和较高的 multilineage 分化的能力，包括在向心肌细胞谱系41。

在这里，我们提出一种协议，结合聚合形成和原发性心脏细胞饲养层，归纳部队达到完成向心肌细胞分化的骨料骨髓间充质干细胞。 提供 3D 的环境，更好地模型条件体内相比 2D 遗民文化。利用心脏饲养层提供一种环境，是对骨髓间充质干细胞的最终移植网站的代表。我们证明年轻来源的间充质干细胞分离产前或产后的脐带有较高的能力，与窗体集料，达到相比成人骨髓间充质干细胞，同时仍然保持其免疫特权心肌表型。除了陡海拔及心脏沿袭标记基因诱导的表达的细胞内 （即， cTnT 与 MYH6） 和细胞表面蛋白 (即，关于和 Cx43) 具体为心肌细胞，我们表明可以用这种方法利用 FTM HUCPVCs 分化潜力，它们可以导致自发性收缩心肌样细胞。

研究方案

All studies involving animals were conducted and reported according to ARRIVE guidelines48. All studies were performed with institutional research ethics board approval (REB number 454-2011, Sunnybrook Research Institute; REB 29889, University of Toronto, Toronto, Canada). All animal procedures were approved by the Animal Care Committee of the University Health Network (Toronto, Canada), and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, 8th edition (National Institutes of Health 2011).

1. Tissue Culture

  1. Culture FTM HUCPVCs, term HUCPVCs (previously established, n ≥ 3 independent lines for each)42 and commercially available BMSCs in alpha-minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and a 1% penicillin/streptomycin (P/S) cocktail. Culture rat primary cardiomyocytes and MSC-cardiomyocyte co-cultures in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM-F12) containing 10% FBS and 1% P/S.
    NOTE: Sterilize the medium using a 0.2-µm filter. Store prepared medium solutions at 4 °C for up to 3 weeks.
  2. Maintain cell cultures in humidified incubators (95% relative humidity, 37 °C, and 5% CO2) and passage at 70-80% confluency, determined by phase-contrast microscopy. Use appropriate volumes of medium for the size of tissue culture dish used (e.g., 10 mL in a 10-cm dish and 2 mL per well in 6-well tissue culture plate). Use these culture conditions for the duration of the protocol.
  3. Dissociate MSC monolayers for passaging or MSC-cardiomyocyte co-culture establishment using a dissociation enzyme solution (2 mL/well in a 6-well plate) and incubate at 37 °C for 4 min.
  4. Transfer the dissociated cells to a 15-mL tube and centrifuge at 400 x g for 5 min.
  5. Aspirate the supernatant without disrupting the cell pellet and resuspend the cells in 1 mL of a culture medium appropriate for counting using an automated cell counter. Seed the cells as described in the following protocol sections.

2. Preparation of Primary Rat Cardiomyocyte-MSC Co-cultures

  1. Obtain heart tissue for primary cardiomyocyte isolation.
    1. Euthanize rat pups (5-6 days postnatal) using CO2 asphyxiation. Set CO2 chambers to 20% gas replacement (flow rate = 0.2 x chamber volume per min). Confirm exitus by the absence of the pinch reflex.
    2. Remove the atria with the connecting major blood vessels using sterilized instruments (i.e., forceps and curved scissors)41. Transfer the hearts to 50-mL tubes containing sterile PBS with1% P/S (PBS-P/S) on ice.
    3. Cut the ventriculi in half and let the blood wash out in a 10-cm dish with 10 mL of PBS-P/S on ice. Cut the ventricular walls into small pieces (diameter = 2-3 mm) using curved scissors.
    4. Transfer the heart pieces from 10-12 animals to a 50-mL tube using a serological pipette and let them settle.
    5. Remove as much PBS-P/S as possible without removing any heart pieces. Add 10 mL of new PBS-P/S.
  2. Digest the heart tissue to isolate the cardiomyocytes.
    1. Allow the heart pieces to settle. Replace the PBS-P/S with 10 mL of 0.15% trypsin in PBS and shake at 37 °C for 10 min.
    2. Discard the supernatant. Repeat the digestion described in step 2.2.1 three more times, but decant the supernatants into 50-mL collection tubes containing 10 mL of 100% FBS.
  3. Centrifuge the cells (400 x g, 5 min) and aspirate the supernatant. Resuspend the cells in DMEM-F12 containing 10% FBS and 1% P/S and seed onto a 6-well plate (1 x 105 cells/cm2, 2 mL of medium per well).
  4. After 1 h, transfer the medium containing non-attached cells to a 50-mL tube and discard the attached cells. Count the cells in suspension and re-plate them into new 6-well plates (1 x 105 cells/cm2, 2 mL of DMEM-F12 containing 10% FBS and 1% P/S per well).
  5. Inhibit cell proliferation with bromodeoxyuridine (BrdU).
    Caution: BrdU is a strong teratogen and suspected mutagen. Please ensure proper training is provided and refer to the safety data sheet before use.
    1. Once cells have attached, replace the medium in the 6-well plate with DMEM-F12 containing 10% FBS, 1% P/S (2 mL of medium per well), and 5 µM BrdU. Incubate for 16 h (37 °C, 5% CO2).
    2. Remove the BrdU-containing medium and replace with DMEM-F12 containing 10% FBS and 1% P/S (2 mL of medium per well).
  6. Prepare pre-stained MSCs.
    1. Once MSC cultures are at 70-80% confluency in 10-cm dishes, remove the culture medium and add 3 mL of cell dissociation solution. Incubate the dish at 37 °C and 5% CO2 for 5 min.
    2. Transfer the dissociated cells to a 15-mL tube and centrifuge at 400 x g for 5 min.
    3. Aspirate the supernatant without disrupting the cell pellet and resuspend the cells in 1 mL of DMEM-F12 containing 10% FBS and 1% P/S for counting using an automated cell counter.
    4. Dilute the cells to a concentration of 1 x 106 MSC/mL of DMEM-F12 containing 10% FBS and 1% P/S.
    5. Incubate the MSCs with viable, non-transferable fluorescent dye (5 µM, 30 min, 37 °C, 5% CO2) in 1.5-mL centrifuge tubes for 1 h.
    6. Centrifuge the tubes at 400 x g for 5 min. Aspirate the supernatant and resuspend the pellet in DMEM-F12 containing 10% FBS and 1% P/S for a cell concentration of 1 x 106 MSC/mL. Repeat this a total of 3 times.
  7. Transfer the MSCs onto cardiomyocytes (step 2.5.2) at a concentration of 10 x 104 cells per well of the 6-well plate.

3. Preparation of Aggregate Co-cultures

  1. Prepare a single-cell suspension of MSCs (2 x 104 cells/mL of medium, passage # ≤ 6) in alpha-MEM supplemented with 10% FBS and 1% P/S (see step 2.6).
    NOTE: Refer to section 1 of the protocol for the passaging of cells. Alternatively, pre-stain MSCs as per step 2.6.
  2. Initiate aggregate formation by placing 25-µL drops of cell suspension (500 cells) on the inner surface of the lids of 10-cm tissue culture dishes (up to 50 drops per lid). Place the lids on their bottom counterparts containing PBS-P/S. Incubate at 37 °C and 5% CO2.
    NOTE: Place 5-7 mL of PBS-P/S into the culture dish below the hanging drops to avoid drop evaporation.
  3. Observe aggregate formation in the drops after 3 days using a stereomicroscope. If over 40 out of 50 drops contain formed aggregates, collect the drops from the lids using a 1-mL micropipette and transfer the aggregates directly onto primary rat cardiomyocyte monolayers (prepared in steps 2.1-2.7; 10 drops/well). Avoid vigorous pipetting to preserve aggregate integrity.
  4. Keep aggregate co-cultures in the incubators for up to 2 weeks, changing the full volume of medium (2 mL of DMEM-F12 containing 10% FBS and 1% P/S per well) every 72 h.
    1. Daily observe aggregates attaching on feeder cell layers using bright-field microscopy. Record contracting aggregates when observed.
  5. Prepare aggregates for analysis.
    1. Remove the medium and add 2 mL of PBS per well of a 6-well tissue culture dish. Remove the PBS and add 2 mL of dissociation solution per well. Incubate for 3 min at 37 °C and 5% CO2.
    2. Centrifuge at 400 x g for 5 min to obtain a cell pellet. Resuspend in medium, as specified for the applications described in the subsequent steps (see steps 4.1, 5.1, and 6.1) and pass through a 70-µm cell strainer.

4. Flow Cytometry (FC) and Fluorescence-activated Cell Sorting (FACS)

  1. Incubate cell suspensions (1 x 105 cells in 200 µL of PBS containing 3% FBS) with fluorophore-conjugated (FITC or APC) primary antibodies (i.e., CD49f, Cx43, TRA-1-85, HLA-A, HLA-G, and SIRPA for FC or TRA-1-85 for FACS; 1:40) at 4 °C for 30 min, protected from light.
  2. Centrifuge (400 x g, 5 min) and resuspend the cells in 1 mL of PBS with 3% FBS for FC or PBS with 0.5% FBS for FACS.
    NOTE: The FC of MSCs was optimized by Hong et al.41.
  3. Maintain the cells at 4 °C in the dark until they are ready to be analyzed by FC (at least 1 x 104 events) or FACS. Sort the cells as described41. Re-plate TRA-1-85 high-positive sorted cells in 6-well plates (1 x 104 cells/well, 2 mL of DMEM-F12 containing 10% FBS and 1% P/S) within 1 h.
    NOTE: For the gating strategy of the TRA-1-85 human cell surface antigen, see the Supplementary Figure.

5. Immunocytochemistry (ICC) and Microscopy

  1. Re-plate the cell suspensions obtained from the co-cultures (step 3.5.2) or FACS (section 4) onto chamber slides (1 x 104 cells/well, 2 mL of DMEM-F12 containing 10% FBS and 1% P/S per well). Let the cells attach overnight in a tissue culture incubator (see section 1 for the conditions).
  2. Fix the cells using 3 mL of 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature. Wash 3 times with 3 mL of PBS containing 1% bovine serum albumin (BSA; PBS-BSA) for 5 min per wash.
    Caution: Wear appropriate personal protective equipment when handling PFA.
  3. Permeabilize the cells in 3 mL of PBS-BSA with 0.1% Triton X-100. Incubate at room temperature for 10 min for intracellular antigens (i.e., alpha sarcomeric actinin (aSarc) and Cx43), or 25 min for intra-nuclear antigens (i.e., Mef2c and human nuclear antigen (HuNu)). Wash 3 times with 3 mL of PBS-BSA for 5 min per wash.
  4. Block the samples against non-specific antibody reactions with 3 mL of PBS containing 5% normal goat serum (NGS) and 1% BSA for 15 min at room temperature. Wash 3 times with 3 mL of PBS-BSA for 5 min per wash.
  5. Incubate the cells in the primary antibodies (i.e., Mef2c, aSarc, Cx43, and HuNu) diluted 1:200 in 3 mL of PBS-BSA at 4 °C overnight.
  6. Wash 3 times with 3 mL of PBS-BSA for 5 min per wash and incubate with secondary antibodies for 30 min at room temperature. Wash 3 times with 3 mL of PBS-BSA for 5 min per wash.
  7. Store the stained specimens in 3 mL of of mounting medium.
  8. Acquire images using a fluorescence microscope. Use a 10X objective (NA = 0.3), and a 20X objective (NA = 0.45) for lower-magnification imaging. Use fluorescence filter cubes and wavelengths for GFP (ex = 470/22 nm, em = 525/50 nm) and RFP (ex = 531/40 nm, em = 593/40 nm) for the secondary antibodies used (see the Materials and Equipment Table).
  9. Quantify images using imaging software (see the Materials and Equipment Table for the recommended software). Normalize the fluorescence intensity readings to the secondary control acquisitions.

6. RNA Isolation and Quantitative RT-PCR

  1. Prepare RNA samples from undifferentiated MSC cultures or MSCs sorted from co-cultures using column-based RNA isolation, according to the manufacturer's instructions. Prepare 1 x 104 to 1 x 106 cells in 0.7 mL of cell lysis buffer (provided with the RNA isolation kit) per sample.
  2. Prepare cDNA from up to 2 µg of RNA per 100-µL RT reaction.
  3. Perform qPCR using 10 ng of cDNA per reaction (40 cycles, 60 °C annealing/extending temperature).
    1. Use primers for human MY6H and cTnT in a 500-nM concentration and 1-100 ng of cDNA per reaction (see the Materials and Equipment Table). Use GAPDH, ACTB, and HPRT as internal housekeeping normalizers. Use commercially available human-induced pluripotent stem cell-derived cardiomyocytes as a positive control.
      NOTE: Express the fold-change of expression compared to undifferentiated MSC-derived cDNA samples.

结果

HUCPVCs Display Higher Aggregate-formation Potential and CD49f Expression Levels Compared to BMSCs:

To induce the differentiation of hMSCs (i.e., FTM HUCPVCs, term HUCPVCs, and BMSCs), single-cell suspensions of undifferentiated MSCs or MSC-containing hanging drops (Table 1) were transferred onto rat primary cardiomyocyte monolayers to establish direct co-cultures or aggregate co-cultur...

讨论

2 年来，发展与几个不同的策略被用来从 MSC 来源生成心肌样细胞受到了心脏干细胞分化。许多这些策略，然而，是低效的而且使用的条件往往不代表环境移植细胞遇到的体内。

与现有的方法，相比议定书 》 在这里提出了一种利用原发性心脏饲养层和 MSC 聚合形成的组合。原发性心脏饲养层提供类似于间充质干细胞移植后暴露的分化环境。聚合生成环境与氧气和养分的...

披露声明

克利福德 · L.Librach 博士是联合专利持有人:隔离和使用来自第一孕期胎儿脐带组织细胞的方法，授予在加拿大和澳大利亚。

致谢

作者感谢以下人员和研究人员所作的贡献: 马修 Librach、 莱拉 Maghen、 坦尼娅 A.Baretto，Shlomit Kenigsberg 和安德鲁 Gauthier 费舍尔。这项工作被支持由安大略省研究基金-研究卓越 （ORF RE，圆 #7） 和创建程序公司。

材料

NameCompanyCatalog NumberComments
0.25% Trypsin/EDTAGibco25200056For cell dissociation
Alpha-MEMGibco12571071For HUCPVC and BMSC culture media.
PE-conjugated anti-human/mouse CD49f antibodyBiolegend313612Integrin marker for FC
APC-conjugated human Cx43/GJA1 antibodyR&D SystemsFAB7737AConnexin 43 marker for FC
FITC-conjugated HLA-A2 antibodyGenway Biotech Inc.GWB-66FBD2Immunogenicity marker for FC
FITC-conjugated anti-HLA-G [MEM-G/9] antibodyAbcamab7904Immunogenicity marker for FC
FITC-conjugated mouse anti-human SIRPA/CD172a antibodyAbD Serotec/Bio-RadMCA2518FCardiac marker for FC
APC-conjugated human TRA-1-85/CD147 antibodyR&D SystemsFAB3195AHuman cell marker for FC and FACS
FITC-conjugated human TRA-1-85/CD147 antibodyR&D SystemsFAB3195FHuman cell marker for FC and FACS
Anti-connexin 43/GJA1 antibodyAbcamab11370Cx43. For ICC
Goat anti-rabbit IgG (H+L) cross-absorbed secondary antibody, Alexa Fluor 555Life TechnologiesA-21428For ICC
Anti-sarcomeric alpha actinin [EA-53] antibodyAbcamab9465aSARC. For ICC
Goat anti-mouse IgM heavy chain cross-absorbed secondary antibody, Alexa Fluor 555Life TechnologiesA-21426For ICC
Mef2C (D80C1) XP rabbit antibodyNew England BioLabs Ltd.5030SFor ICC
Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488Life TechnologiesA-21206For ICC
Anti-nuclei (HuNu) (clone 235-1) antibodyEMD MilliporeMAB1281For ICC
MZ9.5 StereomicroscopeLeicaFor imaging aggregates.
1.5 ml centrifuge microtubesAxygenMCT-150-CFor staining MSCs with fluorescent dye.
ImageJOpen source image processing software.
Aria II BDUHN SickKids FC Facility. For cell sorting.
Bone marrow mesechymal stromal cellsLonzaPT-2501BMSCs
Bovine serum albuminSigma-AldrichA7030-100GBSA. To prepare solutions for ICC
BrdUEMD MilliporeMAB3424Caution: BrdU is a strong teratogen and suspected mutagen. Please ensure proper training and refer to the SDS before use.
Canto IIBDUHN SickKids FC Facility. For flow cytometry.
cDNA EcoDry PremixClontech/Takara639570For preparation of cDNA for qPCR
CellTracker Green CMFDA DyeLife TechnologiesC7025Fluorescent imaging of cell cytoplasm
Countess automated cell counterInvitrogen Inc.C10227For cell counting
DMEM-F12Sigma-AldrichD6421For rat primary cardiomyocyte culture medium.
Dulbecco's Phosphate Buffered SalineGibco10010023D-PBS, without Ca2+, Mg2+
EVOSLife TechnologiesIn-house fluorescent microscope
FACSCaliburBDIn-house. For flow cytometry.
Fetal bovine serum (Hyclone)GE HealthcareSH3039603FBS. Component of cell culture medium.
IDT Prime Time qPCR probesIntegrated Data TechnologiesFAM fluorophorehttp://www.idtdna.com/pages/products/gene-expression/primetime-qpcr-assays-and-primers
Lab Vision PermaFluor Aqueous Mounting MediumThermoScientificTA-030-FMFor storage of cells to undergo ICC
LSR II BDUHN SickKids FC Facility. For flow cytometry.
MoFlo AstriosBeckman CoulterUHN SickKids FC Facility. For cell sorting.
Normal goat serumCell Signaling Technology5425SNGS. Used in blocking solution for ICC
Nunc Lab-Tek II Chamber Coverglass, 8-wellsThermo Scientific Nunc155409To prepare samples for ICC
OmniPur Triton X-100 SurfactantEMD Millipore9410-OPAs a component of permeabilizing solution when preparing cells for ICC
Paraformaldehyde, 16% Solution, EM GradeElectron Microscopy Sciences15710For fixing cells for ICC.
Penicillin/streptomycinGibco15140122Component of cell culture medium.
PrimersSigmaCustom Standard DNA Oligos, Desalted, 0.2 μmolCTnT_F: GGC AGC GGA AGA GGA TGC TGA A; CTnT_R: GAG GCA CCA AGT TGG GCA TGA ACG A; MYH6 F: GCA AAG TAC TGG ATG ACA CGC T; MYH6 R: GTC ATT GCT GAA ACC GAG AAT G
Quorum Spinning Disk ConfocalZeissSickKids Imaging Facility
ReproCardio hiPS cell derived cardiomyocytesReproCellRCD001NPositive control for qPCR
RNeasy mini kitQiagen74106To isolate RNA for qPCR
Rotor-Gene SYBR Green PCR KitQiagen204074For qPCR with master mix
RPMI 1640GibcoA1049101For MSC, monocyte coculture medium.
TaqMan qPCR primer assaysThermo Fisher Scientific4444556For qPCR
Trypan BlueLife TechnologiesT10282Staining of cells for viability and counting
TrypsinGibco272500108For cell dissociation
VolocityPerkin-ElmerVolocity 6.3Imaging software
0.2 μm pore filterThermo Fisher Scientific566-0020For sterilizing tissue culture media
HERAcell 150i CO2 IncubatorThermo Fisher Scientific51026410For incubating cells
Dulbecco's phosphate buffered salineSigma-AldrichD8537PBS. 1X, Without calcium chloride and magnesium chloride
ForcepsAlmedic7727-A10-704For handing rat heart. Can use any similar forceps.
ScissorsFine Science Tools14059-11For mincing rat heart. Curved scissors recommended.
50 mL tubeBD Falcon352070For collection during cardiomyocyte collection and general tissue culture procedures
15 mL tubeBD Falcon352096For general tissue culture procedures
6-well platesThermo Scientific NuncCA73520-906For tissue culture
10 cm tissue culture dishesCorning25382-428For aggregate formation
Axiovert 40C MicroscopeZeissFor bright-field imaging through out tissue culture and the rest of the protocol
70 μm cell strainerFisherbrand22363548To ensure a single cell suspension before flow cytometry or sorting
Triton X-100EMD Millipore9410-1LUsed in permeabilization solution for ICC
Hoechst 33342Thermo Fisher ScientificH1399Stain used during visualization of Cx43 localization

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