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  • Özet
  • Özet
  • Giriş
  • Protokol
  • Sonuçlar
  • Tartışmalar
  • Açıklamalar
  • Teşekkürler
  • Malzemeler
  • Referanslar
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Özet

Burada, fonksiyonel, sözleşme, cardiomyocyte benzeri hücreler üretmek için kardiyak farklılaşma potansiyeli insan Mezenkimal kök hücrelerin genç kaynaklarının verimli koşum için bir yöntem mevcut tüp bebek.

Özet

Miyokard İnfarktüsü ve sonraki iskemik art arda sıralı cardiomyocytes, konjestif kalp yetmezliği, ölüm dünya çapında önde gelen nedenidir önde gelen geniş kaybına neden. Mezenkimal Kök hücre (MSCs) geçerli, invaziv teknikler değiştirmek hücre tabanlı terapiler için umut verici bir seçenek vardır. MSCs kardiyak hücre tipleri, dahil olmak üzere Mezenkimal soy ayırabilirsiniz ama fonksiyonel hücrelere tam farklılaşma değil henüz elde. Önceki yöntemler farklılaşma farmakolojik ajanlar veya büyüme faktörleri dayalı. Ancak, daha fizyolojik ilgili stratejileri cardiomyogenic dönüşüme MSCs etkinleştirmesini de isteyebilirsiniz. Burada, cardiomyocyte gibi sözleşme hücreleri üretmek için cardiomyocyte besleyici katmanlar üzerinde MSC toplamları kullanarak bir farklılaşma yöntem mevcut.

İnsan göbek kordonu Perivasküler hücreleri (HUCPVCs) daha ayrıştırması daha potansiyel için gösterilmiştir kemik iliği MSCs (BMSCs) gibi MSC türleri yaygın olarak araştırıldı. Ontogenetically genç bir kaynağı olarak, biz büyük kaynaklarına göre birinci üç aylık dönem (FTM) HUCPVCs cardiomyogenic potansiyelini araştırdık. FTM HUCPVCs çoğu zaman kültürlü rahim içinde immunoprivileged özellikleri korumak MSCs roman, zengin bir kaynağıdır vitro. Bu farklılaşma protokolü, FTM ve terim kullanarak HUCPVCs BMSCs için karşılaştırıldığında önemli ölçüde artış cardiomyogenic farklılaşma cardiomyocyte işaretleri (Yani, myocyte artırıcı bir faktör 2 C, kardiyak troponin T, ağır zincir kardiyak myosin, sinyal düzenleyici protein α ve connexin 43) artan ifade tarafından belirtildiği şekilde elde. Onlar da kendi alt HLA-A ifade ve daha yüksek HLA-G ifade tarafından gösterdiği olarak anlamlı derecede düşük immünojenisite muhafaza. Farklılaşma toplamak tabanlı uygulama, FTM HUCPVCs potansiyel ve oluşturulan ortak kültür kardiyak besleyici katmanlar üzerinde 1 hafta içinde hücre kümeleri müteahhitlik, bunu yapmak için ilk MSC türü haline artan toplama oluşumu gösterdi.

Bu farklılaşma stratejisi etkili FTM HUCPVCs gibi genç MSCs cardiomyogenic potansiyelini koşum ve o vitro öncesi farklılaşma onların rejeneratif etkinliği içinde vivoartırmak için potansiyel bir strateji olabilir öneriyor bizim sonuçlar gösterilmektedir.

Giriş

Konjestif kalp yetmezliği (CHF) morbidite ve mortalite dünya çapında önde gelen bir nedeni olarak devam ederse. En düşük CHF kez cardiomyocytes büyük kaybı ve akut miyokard infarktüsü (mı)1patolojik sonucu olarak skar dokusu boş hücre gelişimi takip oluşur. Kalp kısmen kendi kendini yenileyerek bir organ olmakla birlikte, bereket ve fonksiyon kez yaralanma sonra en iyi kurtarma için yetersiz hale yaşlı hastalarda ikamet kök ve progenitör hücre havuz önemli ölçüde doku rejenerasyonu yürütmek için sorumlu azalır. Böylece, sağlıklı donör hücre nakli hasarlı Miyokardiyum içine dahil deneysel tedaviler geliştirmede büyük bir ilgi vardır. Donör hücreleri doku yapısı geri kalmayıp aynı zamanda etkilenen Miyokardiyum fonksiyonel iyileşme elde etmek olduğunu zorunludur.

Yerel kalp kalp doku yerleşik istihdam ve endojen kemik iliği kökenli kök hücre sonrası yaralanma için2,3,4onarmak. Rejeneratif hücreleri-host ve donör-kaynaklı hem-must uygun fenotip ve yetenek verimli ve güvenli bir şekilde kayıp hücreleri değiştirmek için birlikte remodeling Miyokardiyum microenvironment işlevinde elde etmek için kapasite var. Vitro farklılaştırma yöntemleri yaygın yüksek verimli, kök hücre tabanlı cardiomyocyte üretim5,6elde etmek için kullanılmaktadır. Kardiyak lineage işaretleri ifade profili kardiyak lineage7karşı kök hücre farklılaşma süreci tanımlamak için kullanılır. Erken ayırt etme belirteçleri, NKX2.5, myocyte enhancer factor 2 C gibi (Mef2c) ve GATA48,9, cardiomyogenic sürecinin başlangıç belirtisi olabilir. Olgun cardiomyocyte işaretleri farklılaşma etkinliğini değerlendirmek için yaygın olarak kullanılan sinyal düzenleyici protein α (SIRPA)10, kardiyak troponin T (cTnT)11, ağır zincir kardiyak myosin (MYH6)8,12,13ve connexin 43 (Cx43)14,15,16' dır. Embriyonik kök hücreler (ESCs) ve pluripotent kök hücreler (PSC) kullanarak yöntemler iyice en iyi duruma getirilmiş ve endüktif faktörler, oksijen ve besin gradyan ayrıntılarını ve eylem5,6,7,17,18tam zamanlaması ile ilgili ele. Yine de, ESC ve PSC temel teknolojileri hala birden çok etik ve güvenlik endişeleri, suboptimal Elektrofizyolojik ve immünolojik özellikleri19,20birlikte mevcut. Bu hücreler ile sık sık nakledilen ev sahipliği yapan immunorejection deneyim ve kalıcı immünosupresyon gerektirir. Bu esas olarak MHC kompleksi (MHC) molekülleri ana bilgisayar ve donör ve elde edilen T hücreli yanıt21eşleşmeyebilir nedeniyle. Bireysel MHC sınıf ı eşleştirme olası bir çözüm, daha erişilebilir bir klinik pratikte immunoprivileged ret endişe üstesinden gelmek için evrensel bir hücre kaynağı gerektirir.

Klinik uygulamalar, MSCs ve özellikle, BMSCs, kullanmak için bir alternatif hücre kaynağı olarak kullanılmak üzere doku rejenerasyonu 199522onların ilk açıklama beri araştırdı. MSCs hemen hemen her bozukluklarına doku23' bulunabilir yerleşik rejeneratif hücreleri olduğuna inanılmaktadır. Yalıtım istenilen kaynaktan üzerine MSCs kültüründe kolayca genişletilebilir, geniş parakrin kapasitesine sahip ve genellikle immunoprivileged ya da immunomodulatory özellikleri24,25sahip. Onların güvenliği ve etkinliği zaten birkaç önceden klinik çalışmalarda, özellikle kalp yenilenme3,26için gösterilmiştir.

Birçok MSC farklılaşma stratejileri farmakolojik ajanlar, 5-azacytidine22 ve DMSO27ve büyüme veya BMP5,7,28,29 veya Anjiyotensin-II gibi30, morphogenic faktörler gibi değişken verimlilik ile kullanmaktadır. Bu stratejiler, saf rejeneratif hücre posta veya yaralanma siteye teslim sonra karşılaşmak muhtemeldir engelleri Ancak, temel alan değil içinde vivo. Daha fizyolojik ilgili stratejileri, süre daha zor için tanımlamak ve işlemek, MSC farklılaşma doku microenvironment gelen sinyalleri aracılığıyla bağlı olmak öncül dayalı. Önceki çalışmalarda bu maruz kalma kardiyak hücre lysates31 veya Ventriküler Miyokardiyum32,33göstermiştir veya doğrudan birincil cardiomyocytes vitro15,34, kişiyle MSCs kardiyak işaretlerinin ifade artırabilir. Kısmen, BMSCs ve cardiomyocytes39,40 füzyon doğmakta olan Miyokardiyum oluşturulan, ancak diğerleri ile MSCs35,36,37,38, kardiyak yaralanmaları tedavi sonra spontan cardiomyogenesis göstermiştir. Bilgimizi, fonksiyonel, kendiliğinden sözleşme cardiomyocytes insan MSCs (hMSCs) herhangi bir doku kaynağı üzerinden henüz bildirilmiştir değil.

Geçerli fikir birliği tüm MSCs Perivasküler hücreleri23ortaya olduğunu. Genç MSCs pericyte özelliklere sahip insan göbek kordonu doku41,42,43Perivasküler bölgesinden ayrılmış olabilir. BMSCs ile karşılaştırıldığında, HUCPVCs sahip artan farklılaşma potansiyeli ve birçok Rejeneratif avantajı, her iki vitro41,44 ve in vivo45,46,47. Özellikle, maternal-fetal arabirim olan kaynak, önemli ölçüde daha düşük immünojenisite MSCs yetişkin kaynaklarına göre HUCPVCs var. Bizim araştırma karakterizasyonu ve FTM HUCPVCs, araştırdık, MSCs en genç kaynak önceden klinik uygulamaları hangi biz daha önce proliferatif ve daha yüksek multilineage arttı göstermiştir odaklanır ayırt etme kapasiteleri, cardiomyogenic soy41yılında da dahil olmak üzere.

Burada, endüktif Kuvvetleri MSCs. toplamları tam cardiomyogenic farklılaşma ulaşmak için daha iyi koşullar içinde vivo 2D yapisan kültürlere göre modeller 3D bir çevre sağlamak olarak toplama oluşumu ve birincil kalp hücresi besleyici katmanlar birleştirir bir iletişim kuralı mevcut. Kardiyak besleyici katmanlar kullanarak son nakli site için MSCs temsilcisidir bir ortam sağlar. Biz öncesi ya da doğum sonrası göbek bağları izole MSCs genç kaynakları formu toplamları ve yetişkin BMSCs için hala onların bağışıklık ayrıcalık koruyarak karşılaştırıldığında kalp fenotip ulaşmak için daha yüksek bir kapasiteye sahip göstermek. Kardiyak lineage marker genler ve hücre içi indüklenen ifade dik yükselmesine dışında (Yani, cTnT ve MYH6) ve hücre yüzey proteinleri (Yani, SIRPA ve Cx43) belirli cardiomyocytes için biz göstermek FTM HUCPVCs farklılaşma potansiyeli bu yöntemle harnessed olabilir ve onlar kendiliğinden cardiomyocyte benzeri hücreler sözleşme için artış verebilir.

Protokol

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.

Sonuçlar

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...

Tartışmalar

Kök hücre kardiyak farklılaşma geliştirme için 2 yılda altında MSC kaynaklardan cardiomyocyte benzeri hücreleri oluşturmak için kullanılan birkaç farklı stratejileri ile olmuştur. Bu stratejiler, birçoğu ancak, verimsiz, ve kullanılan koşulları genellikle çevre nakledilen hücreler karşılaşma içinde vivotemsilcisi değildir.

Mevcut yöntemler aksine burada sunulan Protokolü birincil kardiyak besleyici katmanlar ve MSC toplama oluşumu bir arada kullanır. Bir...

Açıklamalar

Dr. Clifford L. Librach olduğunu ortak patent sahibinin: Kanada ve Avustralya'verilen yöntemleri yalıtım ve ilk üç aylık göbek kordonu dokusundan elde edilen hücre kullanımı,.

Teşekkürler

Yazarlar aşağıdaki personel üye teşekkür ve araştırma katkılarından dolayı personel: Matthew Librach, Leila Maghen, Tanya A. Baretto, Shlomit Kenigsberg ve Andrée Gauthier-Fisher. Bu eser Ontario araştırma fonu tarafından - araştırmada mükemmellik (ORF-RE, yuvarlak #7) ve Program A.ş. oluşturmak destek verdi

Malzemeler

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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