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Dans cet article

  • Résumé
  • Résumé
  • Introduction
  • Protocole
  • Résultats
  • Discussion
  • Déclarations de divulgation
  • Remerciements
  • matériels
  • Références
  • Réimpressions et Autorisations

Résumé

Ici, nous présentons une méthode pour exploiter efficacement le potentiel de différenciation cardiaques des jeunes sources de cellules souches mésenchymateuses humaines afin de générer des cellules fonctionnelles, contractants, cardiomyocyte-comme in vitro.

Résumé

Infarctus du myocarde et la cascade ischémique ultérieure entraîner la perte étendue de cardiomyocytes, conduisant à l’insuffisance cardiaque congestive, la principale cause de mortalité dans le monde. Cellules souches mésenchymateuses (CSM) sont une option prometteuse pour les thérapies à base de cellules remplacer les techniques actuelles, envahissantes. MSCs peuvent se différencier en lignées mésenchymateuses, y compris les types de cellules cardiaques, mais complète différenciation en cellules fonctionnelles n’a pas encore été atteint. Les méthodes précédentes de différenciation reposaient sur des agents pharmacologiques ou de facteurs de croissance. Cependant, les stratégies pertinentes plus physiologiquement peuvent également activer MSCs subir des transformations cardiomyogénique. Nous présentons ici une méthode de différenciation utilisant des agrégats MSC sur couches de cardiomyocyte mangeoire pour produire des cellules maître cardiomyocyte.

Cordon ombilical humain périvasculaires cellules (HUCPVCs) ont démontré avoir une différenciation plus grande potentielle que couramment étudié les types de MSC, comme la moelle osseuse MSCs (BMSC). Comme une source ontologiquement plus jeune, nous avons étudié le potentiel de cardiomyogénique de HUCPVCs de (FTM) au premier trimestre par rapport à des sources plus anciennes. FTM HUCPVCs constituent une source originale, riche de MSCs qui conservent leurs in utero immunoprivileged propriétés lorsque cultivées in vitro. Utilisant ce protocole de différenciation, la FTM et le terme HUCPVCs atteint cardiomyogénique une augmentation significative de différenciation par rapport à BMSC, comme en témoigne l’augmentation de l’expression des marqueurs de cardiomyocytes (c.-à-d., myocytes enhancer facteur 2C, troponine T, la myosine cardiaque de chaîne lourde, signal protéine régulatrice α et connexin 43). Ils ont soutenu également significativement plus faible immunogénicité, tel que démontré par leur plus faible expression de HLA-A et l’expression de HLA-G plus élevée. Application de différentiation axée sur l’agrégat, FTM HUCPVCs a montré formation globale accrue potentiels et généré contractantes des amas de cellules dans la semaine suivant la co-culture sur couches alimentation cardiaque, devenant le premier type MSC de le faire.

Nos résultats démontrent que cette stratégie de différenciation permet d’exploiter efficacement le potentiel de cardiomyogénique du jeunes MSCs, tels que les FTM HUCPVCs et suggère que cette différenciation in vitro préliminaire pourrait être une stratégie possible pour augmenter leur efficacité régénératrice in vivo.

Introduction

Insuffisance cardiaque congestive (ICC) persiste comme des principales causes de morbidité et de mortalité dans le monde. CHF survient souvent suite à la perte massive des cardiomyocytes et le développement du tissu de cicatrice acellulaire pathologique suite à un infarctus du myocarde (im)1. Alors que le cœur est un organe partiellement autorenouvellement, le résident souches et progénitrices cell pool chargé d’exécuter la régénération tissulaire significativement diminue dans l’abondance et la fonction chez les patients âgés, devenant souvent insuffisante pour une récupération optimale après une blessure. Il y a donc beaucoup d’intérêt dans le développement des traitements expérimentaux qui impliquent la transplantation de cellules de donneur sain dans le myocarde endommagé. Il est impératif que les cellules du donneur non seulement restaurer la structure du tissu, mais aussi réaliser la récupération fonctionnelle du myocarde touché.

Le cœur natif emploie coeur tissu-résident et endogènes provenant de la moelle osseuse des cellules souches pour blessure après réparation2,3,4. Régénératrice des cellules hôtes et donateurs dérivés-alike-doit avoir la capacité d’obtenir le phénotype approprié et la fonction dans le micro-environnement du myocarde retouche, ainsi que la capacité de façon efficace et sécuritaire remplacer les cellules perdues. Méthodes de différenciation in vitro ont servi largement à atteindre de haut rendement, sur les cellules souches cardiomyocyte production5,6. Le profil d’expression des marqueurs de la lignée cardiaque sert à définir le processus de différenciation des cellules souches vers la lignée cardiaque7. Marqueurs de différenciation précoce, tels que NKX2.5, facteur de renforceur de myocyte 2C (Mef2c) et GATA48,9, peuvent être une indication de l’ouverture du processus cardiomyogénique. Marqueurs de cardiomyocyte mature couramment utilisées pour évaluer l’efficacité de différenciation sont signal protéine régulatrice α (SIRPA)10, troponine T (cTnT)11, la chaîne lourde de la myosine cardiaque (MYH6)8,12,13et connexine 43 (Cx43)14,15,16. Les méthodes utilisant des cellules souches embryonnaires (CSE) et les cellules souches pluripotentes (CSP) ont été soigneusement optimisés et discutés concernant les détails des facteurs inductifs, de l’oxygène et de nutriments dégradés et le moment exact de l’action5,6,7,17,18. Néanmoins, ESC et CFP-axée sur les technologies présentent toujours plusieurs préoccupations éthiques et de sécurité, ainsi que des caractéristiques électrophysiologiques et immunologiques sous-optimale19,20. Hôtes transplantés ces cellules souvent expérience immunorejection et nécessitent une immunosuppression permanente. C’est principalement en raison d’une non-concordance de majeur d’histocompatibilité (MHC) des molécules complexes dans l’hôte et les bailleurs de fonds et à la résultante lymphocytes réponse21. Tout en individuel MHC classe I correspondant est une solution possible, une pratique clinique plus accessible nécessiterait une source de cellules qui est universellement immunoprivileged à surmonter la crainte de rejet.

Comme une source de cellule de rechange pour l’usage dans des applications cliniques, MSCs et en particulier, BMSC, ont été étudiées pour une utilisation dans la régénération des tissus depuis leur description initiale en 1995,22. MSCs sont censés être résidents cellules régénératrices qui peuvent être trouvés dans presque n’importe quel tissu vascularisé23. Sur l’isolation de la source souhaitée, MSCs peuvent facilement être étendus dans la culture, ont paracrine vaste capacité et possèdent souvent immunoprivileged ou immunomodulateurs propriétés24,25. Leur innocuité et l’efficacité ont déjà été démontrés dans plusieurs études précliniques, en particulier pour la régénération cardiaque3,26.

Plusieurs stratégies de différenciation des MSC utilisent des agents pharmacologiques, tels que la 5-azacytidine22 et27de DMSO et croissance ou morphogéniques facteurs, tels que BMP5,7,28,29 ou l’angiotensine-II30, avec une efficacité variable. Ces stratégies, cependant, ne sont pas fondées sur les obstacles qu’une cellule régénératrice de naïve est susceptible de rencontrer après domiciliation ou remis à l’endroit de la blessure in vivo. Des stratégies plus physiologiquement pertinents, bien que plus difficiles à définir et manipuler, reposent sur la prémisse que la différenciation MSC peut être induite par le biais de signaux provenant du microenvironnement tissu lui-même. Des études antérieures ont montré que l’exposition à la cellule cardiaque lysats31 ou le myocarde ventriculaire32,33, ou directement auprès des cardiomyocytes primaire in vitro15,34, peut augmenter l’expression des marqueurs cardiaques dans MSCs. D’autres ont démontré cardiomyogénèse spontanée après le traitement de lésions cardiaques avec MSCs35,36,37,38, bien qu’en partie, la fusion du BMSC et cardiomyocytes39,40 généré le myocarde naissant. À notre connaissance, des cardiomyocytes fonctionnels, contractants spontanément de MSCs humaines (CSM) de n’importe quelle source de tissus n’ont pas encore été signalées.

Le consensus actuel est que MSCs tous les proviennent de cellules périvasculaires23. MSCs Young avec des propriétés de pericyte peuvent être isolés de la région périvasculaire du cordon ombilical humain tissu41,42,43. En comparaison avec BMSC, HUCPVCs possèdent la différenciation accrue potentielle et plusieurs autres avantages régénératrices, les deux in vitro41,44 et in vivo45,46,47. En particulier, la source étant l’interface materno-foetale, HUCPVCs ont significativement plus faible immunogénicité comparée aux sources adultes de MSCs. Nos recherches portent sur la caractérisation et applications précliniques de FTM HUCPVCs, la plus jeune source de MSCs, objet d’une enquête, dont nous avons déjà montré a augmenté de multilineage proliférative et plu les capacités de différenciation, y compris dans la lignée de cardiomyogénique41.

Nous présentons ici un protocole qui combine formation globale et couches de cellule cardiaque primaire chargeur inductifs forces pour atteindre la différenciation cardiomyogénique complète d’agrégats MSCs. fournir un environnement 3D, qui modélise mieux des conditions in vivo par rapport aux cultures adhérentes 2D. Utilisant des couches cardiaque chargeur fournit un environnement qui soit représentative du site transplantation ultime pour les MSCs. Nous démontrons que les plus jeunes sources de MSCs isolées de cordons ombilicaux de pré ou post natals ont une plus grande capacité à former des agrégats et rejoindre le phénotype cardiaque comparativement à BMSC adultes, tout en conservant leur privilège immunitaire. Outre l’élévation abrupte de gènes marqueurs cardiaques lignage et l’expression induite d’intracellulaire (c.-à-d. cTnT et MYH6) et les protéines de surface cellulaire (c.-à-d., SIRPA et Cx43) spécifique pour les cardiomyocytes, nous montrons que le potentiel de différenciation des FTM HUCPVCs peut être exploité grâce à cette méthode et qu’ils peuvent donner lieu au contractant spontanément cellules cardiomyocytes.

Protocole

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.

Résultats

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...

Discussion

La différenciation cardiaque de cellules souches a été en développement depuis plus de 2 décennies, avec plusieurs différentes stratégies utilisées pour générer des cellules cardiomyocytes provenant de sources MSC. Beaucoup de ces stratégies, cependant, sont inefficaces, et les conditions d’utilisation ne sont souvent pas représentatifs du milieu transplanté des cellules rencontre in vivo.

Contrairement aux méthodes existantes, le protocole présenté ici utilise une c...

Déclarations de divulgation

Dr. Clifford L. Librach est cotitulaire du brevet : méthodes d’isolement et d’utilisation de cellules provenant de tissus de cordon ombilical premier trimestre, accordé au Canada et en Australie.

Remerciements

Les auteurs remercie les membres du personnel suivants et recherche du personnel pour leur contribution : Matthew Librach, Leila Maghen, Tanya A. Baretto, Shlomit Kenigsberg et Andrée Gauthier-Fisher. Ce travail a été soutenu par le Fonds de recherche The Ontario - Excellence de la recherche (ER-FRO, tour #7) et créer Program Inc.

matériels

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

Références

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