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In questo articolo

  • Riepilogo
  • Abstract
  • Introduzione
  • Protocollo
  • Risultati
  • Discussione
  • Divulgazioni
  • Riconoscimenti
  • Materiali
  • Riferimenti
  • Ristampe e Autorizzazioni

Riepilogo

Qui, presentiamo un metodo per sfruttare in modo efficiente il potenziale di differenziazione cardiache di giovane fonti di cellule staminali mesenchimali umane al fine di generare cellule funzionali, contraenti, del cardiomyocyte-come in vitro.

Abstract

Infarto miocardico e la successiva cascata ischemica causare la vasta perdita di cardiomiociti, che porta a insufficienza cardiaca congestizia, la principale causa di mortalità in tutto il mondo. Cellule staminali mesenchimali (MSCs) sono un'opzione di promessa per le terapie basate sulle cellule sostituire tecniche attuali, invasive. MSCs possono differenziare in lignaggi mesenchymal, inclusi i tipi di cellule cardiache, ma completa differenziazione in cellule funzionali non è ancora stato raggiunto. Metodi precedenti di differenziazione sono stati basati su agenti farmacologici o fattori di crescita. Tuttavia, le strategie più fisiologicamente pertinenti anche possono abilitare MSCs per subire la trasformazione cardiomiogenici. Qui, presentiamo un metodo di differenziazione utilizzando MSC aggregati sugli strati dell'alimentatore del cardiomyocyte per produrre del cardiomyocyte-come le cellule contraenti.

Cellule perivascolari del cordone ombelicale umano (HUCPVCs) sono state indicate per avere una maggiore differenziazione potenziale più comunemente studiato tipi di MSC, quali midollo osseo (BMSC) MSCs. Come fonte di ontogeneticamente più giovane, abbiamo studiato il potenziale di cardiomiogenici di HUCPVCs di primo-acetonide (FTM) rispetto alle più vecchie fonti. HUCPVCs FTM sono una romanzo, ricca fonte di MSCs che mantengono la loro nell'utero nuova proprietà una volta coltivate in vitro. Utilizzando questo protocollo di differenziazione, FTM e termine HUCPVCs raggiunto cardiomiogenici significativamente maggiore differenziazione rispetto alle BMSCs, come indicato da aumentata espressione di marcatori dei cardiomiociti (cioè, il fattore di rinforzatore del miocita 2C, troponina cardiaca T, miosina cardiaca catena pesante, segnale proteina regolatrice α e connexin 43). Mantennero anche significativamente più bassa immunogenicità, come dimostrato dalla loro espressione di HLA-A più bassa e più alta espressione di HLA-G. L'applicazione basata su aggregazione differenziazione, FTM HUCPVCs ha mostrato formazione aumentata aggregata potenziale e generato contraenti cluster di cellule entro 1 settimana di co-coltura su livelli cardiaci alimentatore, diventando il primo tipo MSC a farlo.

I nostri risultati dimostrano che questa strategia di differenziazione può sfruttare efficacemente il potenziale cardiomiogenici di giovani cellule staminali mesenchimali, quali HUCPVCs FTM e suggerisce che in vitro pre-differenziazione potrebbe essere una strategia potenziale per aumentare la loro efficacia rigenerativa in vivo.

Introduzione

Insufficienza cardiaca congestizia (CHF) persiste come delle cause principali di morbilità e mortalità in tutto il mondo. Il CHF accade spesso seguendo la perdita massiccia di cardiomiociti e lo sviluppo del tessuto di cicatrice senza cellula come risultato patologico di un infarto miocardico (MI)1. Mentre il cuore è un organo parzialmente autorinnovabile, piscina di cella residente, staminali e progenitrici, responsabile per l'esecuzione di rigenerazione tissutale significativamente diminuisce in abbondanza e la funzione in pazienti invecchiati, diventando spesso insufficiente per un recupero ottimale dopo la ferita. Così, c'è grande interesse nello sviluppo di trattamenti sperimentali che coinvolgono il trapianto delle cellule del donatore sano nel miocardio danneggiato. È imperativo che le cellule del donatore non solo ripristino la struttura del tessuto, ma anche realizzare il recupero funzionale del miocardio interessato.

Cuore natale impiega cuore tessuto residenti e cellule staminali endogene del midollo osseo-originario di alberino-ferita riparazione2,3,4. Donatore-derivati rigenerativa e di cellule-host allo stesso modo, deve avere la capacità di ottenere il fenotipo appropriato e funzione nel microambiente del miocardio che ritocco, insieme alla capacità di efficienza e sicurezza sostituire le cellule perse. Metodi di differenziazione in vitro sono stati ampiamente utilizzati per realizzare dei cardiomiociti ad alta efficienza, basati su cellule staminali produzione5,6. Il profilo di espressione di marcatori di linea cardiaca viene utilizzato per definire il processo di differenziazione delle cellule staminali verso la linea cardiaca7. Marcatori di precoce differenziazione, quali NKX 2.5, fattore di rinforzatore del miocita 2C (Mef2c) e GATA48,9, può essere un'indicazione dell'inizio del processo di cardiomiogenici. Marcatori del cardiomyocyte maturo comunemente usati per valutare l'efficacia di differenziazione sono segnale proteina regolatrice α (SIRPA)10, troponina cardiaca T (cTnT)11, catena pesante della miosina cardiaca (MYH6)8,12,13e connessina 43 (Cx43)14,15,16. I metodi di utilizzo di cellule staminali embrionali (ESCs) e cellule staminali pluripotenti (PSC) sono stati accuratamente ottimizzati e discussi per quanto riguarda i dettagli di fattori induttivi, ossigeno e nutrienti gradienti e l'esatta tempistica di azione5,6,7,17,18. Ciò nonostante, tecnologie basate su ESC e PSC presentano ancora più preoccupazioni etiche e sicurezza, insieme non ottimali caratteristiche elettrofisiologiche ed immunologiche19,20. Padroni di casa trapiantati con queste cellule spesso esperienza immunorejection e richiedono l'immunosoppressione permanente. Ciò è dovuto principalmente molecole di istocompatibilità (MHC) complesse nell'ospite ed il donatore e il risultante della T-cellula risposta21di disadattamento. Mentre singoli MHC di classe I di corrispondenza è una possibile soluzione, una pratica clinica più accessibile richiederebbe una fonte di cellule che universalmente è nuova per superare la preoccupazione del rifiuto.

Come fonte alternativa cellulare per l'uso in applicazioni cliniche, MSCs e in particolare, BMSC, sono stati studiati per l'uso nella rigenerazione del tessuto dalla loro descrizione iniziale nel 199522. MSCs sono creduti per essere cellule rigenerative residenti che possono essere trovate in quasi qualsiasi tessuto vascolarizzato23. Isolamento dalla sorgente desiderata, MSCs possono essere facilmente espanse in coltura, hanno capacità estesa paracrine e possiedono spesso nuova o immunomodulatori proprietà24,25. Loro sicurezza ed efficacia hanno già dimostrati in parecchi studi pre-clinici, in particolare per la rigenerazione cardiaca3,26.

Molte strategie di differenziazione di MSC utilizzano agenti farmacologici, quali 5-azacytidine22 e DMSO27e crescita o fattori morfogenetici, come BMPs5,7,28,29 o dell'angiotensina-II30, con efficienza variabile. Queste strategie, tuttavia, non sono basate sugli ostacoli che una cella rigenerativa ingenuo è probabile incontrare dopo homing o recapitati al sito della lesione in vivo. Strategie più fisiologicamente rilevanti, mentre più difficile da definire e manipolare, si basano sulla premessa che MSC differenziazione può essere indotta attraverso segnali dal microambiente del tessuto stesso. Gli studi precedenti hanno dimostrato che l'esposizione alla cellula cardiaca lisati31 o miocardio ventricolare32,33, o diretto contatto con primaria cardiomiociti in vitro15,34, può aumentare l'espressione di marcatori cardiaci in MSCs. Gli altri hanno dimostrato cardiomiogenesi spontanea dopo trattamento di lesioni cardiache con MSCs35,36,37,38, anche se in parte, la fusione di BMSC e cardiomiociti39,40 generato il miocardio nascente. A nostra conoscenza, non sono ancora stati segnalati cardiomiociti funzionali, spontaneamente contraenti da MSCs umane (hMSCs) di qualsiasi origine di tessuto.

Il consenso corrente è che tutti i MSCs derivano da perivascolari cellule23. MSCs giovane con proprietà Pericita può essere isolato dalla regione perivascolare del cordone ombelicale umano tessuto41,42,43. Rispetto alle BMSCs, HUCPVCs possiedono una maggiore differenziazione potenziale e diversi altri vantaggi rigenerative, entrambi in vitro41,44 ed in vivo45,46,47. In particolare, la fonte che è l'interfaccia materno-fetale, HUCPVCs le immunogenicità significativamente più basso rispetto alle fonti di adulti di MSCs. La nostra ricerca si concentra sulla caratterizzazione e applicazioni pre-cliniche di FTM HUCPVCs, la fonte più giovane di MSCs studiato, che precedentemente abbiamo indicato sono aumentati multilineage proliferative e superiore capacità di differenziazione, tra cui nel lignaggio cardiomiogenici41.

Qui, presentiamo un protocollo che unisce formazione aggregata e strati dell'alimentatore primario delle cellule cardiache come forze induttive per raggiungere la differenziazione di cardiomiogenici completa di MSCs. aggregati forniscono un ambiente 3D, che modelle migliori condizioni in vivo rispetto al 2D culture aderente. Utilizzando livelli cardiaci alimentatore fornisce un ambiente che è rappresentante del sito ultimate trapianto per MSCs. Dimostriamo che più giovane fonti di cellule staminali mesenchimali isolate da cordone ombelicale pre- o post-natali hanno una maggiore capacità di formare aggregati e raggiungere il fenotipo cardiaco rispetto al BMSCs adulto, mantenendo comunque il loro privilegio immunitario. Oltre la ripida elevazione dei geni marcatori di linea cardiaca e l'espressione indotta di intracellulare (cioè, cTnT e MYH6) e proteine di superficie cellulare (vale a dire, SIRPA e Cx43) specifico per cardiomiociti, indichiamo che il potenziale di differenziazione di FTM HUCPVCs possa essere imbrigliato con questo metodo e che possono dare origine a spontaneamente contraenti del cardiomyocyte-come le cellule.

Protocollo

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.

Risultati

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...

Discussione

La differenziazione delle cellule staminali cardiaca è stato in sviluppo per oltre 2 decenni, con diverse strategie diverse, viene utilizzate per generare del cardiomyocyte-come le cellule da fonti di MSC. Molte di queste strategie, tuttavia, sono inefficienti, e le condizioni utilizzate spesso non sono rappresentative dell'ambiente trapiantate cellule incontro in vivo.

In contrasto con i metodi esistenti, il protocollo presentato qui utilizza una combinazione di strati di alimentato...

Divulgazioni

Dr. Clifford L. Librach è contitolare del brevetto: metodi di isolamento e di uso di cellule derivate da tessuti di cordone ombelicale primo acetonide, concessa in Canada e in Australia.

Riconoscimenti

Gli autori ringraziare i seguenti membri del personale e personale per i loro contributi di ricerca: Matthew Librach, Leila Maghen, Tanya A. Baretto, Shlomit Kenigsberg e Andrée Gauthier-Fisher. Questo lavoro è stato supportato dal fondo di ricerca The Ontario - l'eccellenza della ricerca (ORF-RE, Round #7) e creare programma Inc.

Materiali

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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