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In diesem Artikel

  • Zusammenfassung
  • Zusammenfassung
  • Einleitung
  • Protokoll
  • Ergebnisse
  • Diskussion
  • Offenlegungen
  • Danksagungen
  • Materialien
  • Referenzen
  • Nachdrucke und Genehmigungen

Zusammenfassung

Hier präsentieren wir eine Methode, um effizient die kardialen Differenzierung junge Quellen humaner mesenchymaler Stammzellen um funktionelle, Auftraggeber, Cardiomyocyte-wie Zellen erzeugen Potenzial in-vitro-.

Zusammenfassung

Myokardinfarkt und der anschließenden ischämischen Kaskade führen zu den umfangreichen Verlust von Kardiomyozyten, führt zu Herzinsuffizienz, die führende Todesursache weltweit. Mesenchymaler Stammzellen (MSCs) sind eine vielversprechende Option für zellbasierte Therapien, aktuelle, invasive Techniken zu ersetzen. MSCs mesenchymalen Abstammungen, kardiale Zelltypen, einschließlich differenzieren können, aber vollständige Differenzierung in funktionellen Zellen noch nicht erreicht. Bisherige Methoden der Differenzierung beruhten auf pharmakologische Wirkstoffe oder Wachstumsfaktoren. Mehr physiologisch relevante Strategien ermöglichen jedoch auch MSCs Cardiomyogenic Transformation unterziehen. Hier präsentieren wir Ihnen eine Differenzierung-Methode mit MSC Aggregate auf Cardiomyocyte Feeder Schichten, um Cardiomyocyte-wie auftraggebende Zellen zu produzieren.

Menschlichen Nabelschnur perivaskuläre Zellen (HUCPVCs) haben gezeigt, dass eine größere Differenzierung als potenzielle haben häufig untersucht MSC-Typen, z. B. Knochenmark MSCs (BMSCs). Als ontogenetisch jüngere Quelle untersuchten wir das Cardiomyogenic Potential des Ersttrimester-(FTM) HUCPVCs im Vergleich zu älteren Quellen. FTM HUCPVCs sind eine neuartige, reiche Quelle von MSCs, die ihre in Utero Immunabwehr Eigenschaften beim kultivierten behalten in Vitro. Mit dieser Differenzierung Protokoll, FTM und Begriff erreicht HUCPVCs deutlich erhöhte Cardiomyogenic Differenzierung im Vergleich zu BMSCs, wie durch die erhöhte Expression von Cardiomyocyte Marker (d. h. Myozyten Enhancer Faktor 2 C, kardiale Troponin T schwere Kette kardialen Myosin, Signal regulatorischen Protein α und Connexin 43) angegeben. Sie behielten auch deutlich geringer Immunogenität, wie durch ihren niedrigeren HLA-A Ausdruck und höheren Ausdruck von HLA-G. Aggregat-basierte Differenzierung anwenden, zeigte FTM HUCPVCs erhöhte Aggregatbildung Potenzial und generierten contracting Zellen Cluster innerhalb 1 Woche nach Kokultur auf kardiale Feeder Schichten, immer die erste MSC-Art, dies zu tun.

Unsere Ergebnisse zeigen, dass diese Differenzierungsstrategie effektiv das Cardiomyogenic Potential des jungen MSCs wie FTM HUCPVCs nutzen kann und deutet darauf hin, dass in-vitro- Pre-Differenzierung könnte eine mögliche Strategie, um ihre regenerativen Wirksamkeit in Vivozu erhöhen.

Einleitung

Herzinsuffizienz (CHF) bleibt als eine der Hauptursachen für Morbidität und Mortalität weltweit. CHF tritt häufig nach den massiven Verlust von Kardiomyozyten und die Entwicklung der zellfreien Narbengewebe als pathologische Ergebnis ein Myokardinfarkt (MI)1. Während das Herz ein teilweise selbst erneuernde Organ ist, verringert sich residente Stamm- und Vorläuferzellen Zelle Pool verantwortlich für die Regeneration des Gewebes deutlich Ausführung in Hülle und Fülle und Funktion bei gealterten Patienten oft nicht ausreichend für optimale Erholung nach einer Verletzung zu. So gibt es großes Interesse an der Entwicklung von experimentellen Behandlungen, bei denen die Transplantation von gesunden Spenderzellen in den Geschädigten Myokard. Es ist zwingend notwendig, dass die Spenderzellen nicht nur die Struktur des Gewebes wiederherzustellen, sondern auch die funktionelle Wiederherstellung der betroffenen Myokard.

Die native Herz beschäftigt Herz Gewebe-Resident und endogenen Knochenmark stammen Stammzellen zur posttraumatischen reparieren2,3,4. Regenerative Zellen-Host und Spender abgeleitet gleichermaßen muss haben die Fähigkeit, die entsprechenden Phänotyp und Funktion in der Mikroumgebung umgestaltet Myokard, sowie die Fähigkeit, effizient und sicher die verlorenen Zellen ersetzen zu erhalten. In Vitro Differenzierung Methoden sind weitgehend benutzt, um eine hocheffiziente, stammzellbasierte Cardiomyocyte Produktion5,6zu erreichen. Expressionsprofil kardiale Linie Marker wird verwendet, um den Prozess der Stammzell-Differenzierung in der kardialen Linie7Richtung zu definieren. Frühe Differenzierung Marker wie NKX2.5, Myozyten Enhancer Faktor 2 C (Mef2c) und GATA48,9, kann einen Hinweis über die Einleitung des Cardiomyogenic Prozesses. Reife Cardiomyocyte Marker häufig verwendet, um Differenzierung Wirksamkeit zu beurteilen sind Signal regulatorischen Protein α (SIRPA)10, kardiale Troponin T (cTnT)11, schwere Kette kardialen Myosin (MYH6)8,12,13und Connexin 43 (Cx43)14,15,16. Die Methoden, die Verwendung von embryonalen Stammzellen (WSR) und pluripotenten Stammzellen (EAP) wurden sorgfältig optimiert und diskutiert über die Details der induktive Faktoren, Sauerstoff und Nährstoff Steigungen und der genaue Zeitpunkt der Aktion5,6,7,17,18. Dennoch präsentieren ESC und PSC-basierten Technologien noch mehrere Sicherheit und ethische Bedenken zusammen mit suboptimalen elektrophysiologische und immunologischen Funktionen19,20. Gastgeber, die oft mit diesen Zellen transplantiert erleben Immunorejection und erfordern ständige Immunsuppression. Dies ist vor allem auf großen Histocompatibility Komplexes (MHC) Moleküle in dem Host und dem Spender und der daraus resultierenden T-Zell Antwort21Fehlanpassungen. Während einzelne MHC Klasse I matching ist eine mögliche Lösung, eine zugänglichere klinische Praxis würde benötigen eine Zelle, die allgemein die Immunabwehr, die Sorge der Ablehnung zu überwinden ist.

Als alternative Zelle Quelle für den Einsatz in der klinischen Anwendung, MSCs und insbesondere BMSCs, wurden für den Einsatz in Geweberegeneration seit ihrer Erstbeschreibung im Jahr 199522untersucht. MSCs werden geglaubt, um Wohnsitz regenerativen Zellen, die in nahezu jedem vaskularisierte Gewebe23gesucht werden kann. Bei der Isolierung von der gewünschten Quelle MSCs können leicht erweitert werden, in der Kultur, haben umfangreiche parakrine Kapazität und oft besitzen Immunabwehr oder immunmodulatorische Eigenschaften24,25. Ihre Sicherheit und Wirksamkeit wurden bereits in mehreren präklinischen Studien, insbesondere zur kardialen Regeneration3,26gezeigt.

Viele MSC Differenzierungsstrategien nutzen pharmakologische Wirkstoffe, wie z. B. 5-Azacytidine22 und DMSO27, und Wachstum oder morphogenetische Faktoren wie BMPs5,7,28,29 oder Angiotensin-II30, mit Variablen Wirkungsgrad. Diese Strategien basieren jedoch nicht auf die Hindernisse, die eine naiven regenerative Zelle nach Homing oder an der Stelle der Verletzung auftreten dürfte in-vivo. Mehr physiologisch relevante Strategien zwar schwieriger zu definieren und zu manipulieren, basieren auf der Prämisse, dass MSC Differenzierung durch Signale aus dem Gewebe Mikroumgebung selbst induziert werden kann. Frühere Studien haben gezeigt, dass die Exposition gegenüber der Herzmuskelzellen Lysates31 oder Linksventrikuläres Myokard32,33, oder direkt Kontakt mit primären Kardiomyozyten in-vitro-15,34, den Ausdruck der kardialen Marker im MSCs erhöhen kann. Andere zeigten spontane Cardiomyogenesis nach Behandlung von kardialen Verletzungen mit MSCs35,36,37,38, zwar einerseits die Verschmelzung von BMSCs und Herzzellen39,40 der im Entstehen begriffenen Myokard generiert. Nach unserem Kenntnisstand haben funktionelle, spontan Auftraggeber Herzzellen von menschlichen MSCs (hMSCs) von einer beliebigen Quelle Gewebe noch nicht gemeldet.

Der aktuelle Konsens ist, dass alle MSCs aus perivaskuläre Zellen23entstehen. Young-MSCs mit Pericyte Eigenschaften können aus der perivaskuläre Region der menschlichen Nabelschnur Gewebe41,42,43isoliert werden. Im Vergleich zu BMSCs besitzen HUCPVCs erhöhte Differenzierungspotenzial und einige andere regenerative Vorteile, sowohl in-vitro-41,44 und in Vivo45,46,47. Insbesondere habe die Quelle als mütterlich fötalen Schnittstelle, HUCPVCs deutlich geringer im Vergleich zu Erwachsenen Quellen von MSCs Immunogenität. Unsere Forschung konzentriert sich auf die Charakterisierung und prä-klinischen Anwendungen von FTM HUCPVCs, die jüngste Quelle der MSCs untersucht, die wir zuvor gezeigt haben, proliferative und höhere Multilineage gestiegen Differenzierung Kapazitäten, namentlich auf der Cardiomyogenic Linie41.

Hier präsentieren wir eine Protokoll, die Aggregatbildung und primäre kardiale Zellschichten Feeder verbindet wie induktive Kräfte, die komplette Cardiomyogenic Differenzierung von MSCs. Aggregate zu erreichen eine 3D Umgebung bieten die besseren Bedingungen in Vivo im Vergleich zu 2D anhaftende Kulturen Modelle. Verwendung von kardialen Feeder Schichten bietet eine Umgebung, die repräsentativ für die ultimative Transplantation-Website für die MSCs ist. Wir zeigen, dass jüngere Quellen von MSCs isoliert vor oder nach der Geburt die Nabelschnur eine höhere Kapazität, Form Aggregate und den kardialen Phänotyp im Vergleich zu Erwachsenen BMSCs haben, unter Beibehaltung ihrer immun-Privileg zu erreichen. Neben der steilen Höhe des kardialen Linie Marker-Gene und die induzierte Expression von intrazellulären (d. h. cTnT und MYH6) und Zelle-Oberfläche Proteine (z.B. SIRPA und Cx43) speziell für Kardiomyozyten, wir zeigen, dass die Differenzierung Potenzial von FTM HUCPVCs kann, mit dieser Methode genutzt werden und sie spontan Infizierung Cardiomyocyte-ähnlichen Zellen hervorrufen können.

Protokoll

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.

Ergebnisse

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...

Diskussion

Die kardialen Differenzierung von Stammzellen wird mit mehreren unterschiedlichen Strategien genutzt, um Cardiomyocyte-wie Zellen aus MSC Quellen erzeugen seit über 2 Jahrzehnten entwickelt. Viele dieser Strategien sind jedoch ineffizient und die Bedingungen sind oft nicht repräsentativ für die Umwelt transplantierten Zellen Begegnung in Vivo.

Im Gegensatz zu bestehenden Verfahren nutzt das Protokoll hier vorgestellten eine Kombination von primären kardiale Feeder Schichten und MS...

Offenlegungen

Dr. Clifford L. Librach ist Mitinhaber des Patents: Methoden der Isolation und der Verwendung von Zellen aus ersten Trimester Nabelschnur Gewebe, in Kanada und Australien gewährt.

Danksagungen

Die Autoren danken den folgenden Mitarbeiter und Forschung Personal für ihre Beiträge: Matthew Librach, Leila Maghen, Tanya A. Baretto, Shlomit Kenigsberg und Andrée Gauthier-Fisher. Diese Arbeit wurde von der Ontario Research Fund - Research Excellence (ORF-RE, Runde #7) und erstellen Programm Inc. unterstützt.

Materialien

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

Referenzen

  1. Badano, L. P., et al. Prevalence, clinical characteristics, quality of life, and prognosis of patients with congestive heart failure and isolated left ventricular diastolic dysfunction. J Am Soc Echocardiogr. 17 (3), 253-261 (2004).
  2. Leri, A., Kajstura, J., Anversa, P. Cardiac stem cells and mechanisms of myocardial regeneration. Physiol Rev. 85 (4), 1373-1416 (2005).
  3. Orlic, D., et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A. 98 (18), 10344-10349 (2001).
  4. Schuster, M. D., et al. Myocardial neovascularization by bone marrow angioblasts results in cardiomyocyte regeneration. Am J Physiol Heart Circ Physiol. 287 (2), 525-532 (2004).
  5. Zandstra, P. W., et al. Scalable production of embryonic stem cell-derived cardiomyocytes. Tissue Eng. 9 (4), 767-778 (2003).
  6. Boheler, K. R., et al. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ Res. 91 (3), 189-201 (2002).
  7. Kattman, S. J., et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell. 8 (2), 228-240 (2011).
  8. Dixon, J. E., Dick, E., Rajamohan, D., Shakesheff, K. M., Denning, C. Directed differentiation of human embryonic stem cells to interrogate the cardiac gene regulatory network. Mol Ther. 19 (9), 1695-1703 (2011).
  9. Stennard, F. A., et al. Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4, and GATA5 in regulation of gene expression in the developing heart. Dev Biol. 262 (2), 206-224 (2003).
  10. Dubois, N. C., et al. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat Biotechnol. 29 (11), 1011-1018 (2011).
  11. Panteghini, M. Present issues in the determination of troponins and other markers of cardiac damage. Clin Biochem. 33 (3), 161-166 (2000).
  12. Burridge, P. W., et al. Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability. Stem Cells. 25 (4), 929-938 (2007).
  13. Ovchinnikov, D. A., et al. Isolation of contractile cardiomyocytes from human pluripotent stem-cell-derived cardiomyogenic cultures using a human NCX1-EGFP reporter. Stem Cells Dev. 24 (1), 11-20 (2015).
  14. Moscoso, I., et al. Differentiation "in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation. Transplant Proc. 37 (1), 481-482 (2005).
  15. Ramkisoensing, A. A., et al. Gap junctional coupling with cardiomyocytes is necessary but not sufficient for cardiomyogenic differentiation of cocultured human mesenchymal stem cells. Stem Cells. 30 (6), 1236-1245 (2012).
  16. van Kempen, M., et al. Expression of the electrophysiological system during murine embryonic stem cell cardiac differentiation. Cell Physiol Biochem. 13 (5), 263-270 (2003).
  17. Mummery, C. L., et al. Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. Circ Res. 111 (3), 344-358 (2012).
  18. Puceat, M. Protocols for cardiac differentiation of embryonic stem cells. Methods. 45 (2), 168-171 (2008).
  19. Naito, H., et al. Optimizing engineered heart tissue for therapeutic applications as surrogate heart muscle. Circulation. 114, 72-78 (2006).
  20. Zimmermann, W. H., et al. Heart muscle engineering: an update on cardiac muscle replacement therapy. Cardiovasc Res. 71 (3), 419-429 (2006).
  21. Hulot, J. S., et al. Considerations for pre-clinical models and clinical trials of pluripotent stem cell-derived cardiomyocytes. Stem Cell Res Ther. 5 (1), 1 (2014).
  22. Wakitani, S., Saito, T., Caplan, A. I. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve. 18 (12), 1417-1426 (1995).
  23. Caplan, A. I. Adult Mesenchymal Stem Cells: When, Where, and How. Stem Cells Int. 2015, 628767 (2015).
  24. Burchfield, J. S., Dimmeler, S. Role of paracrine factors in stem and progenitor cell mediated cardiac repair and tissue fibrosis. Fibrogenesis Tissue Repair. 1 (1), 4 (2008).
  25. Hsiao, S. T., et al. Comparative analysis of paracrine factor expression in human adult mesenchymal stem cells derived from bone marrow, adipose, and dermal tissue. Stem Cells Dev. 21 (12), 2189-2203 (2012).
  26. Tomita, S., et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation. 100, 247-256 (1999).
  27. Skerjanc, I. S. Cardiac and skeletal muscle development in P19 embryonal carcinoma cells. Trends Cardiovasc Med. 9 (5), 139-143 (1999).
  28. Hou, J., et al. Combination of BMP-2 and 5-AZA is advantageous in rat bone marrow-derived mesenchymal stem cells differentiation into cardiomyocytes. Cell Biol Int. 37 (12), 1291-1299 (2013).
  29. Yoon, J., et al. Differentiation, engraftment and functional effects of pre-treated mesenchymal stem cells in a rat myocardial infarct model. Acta Cardiol. 60 (3), 277-284 (2005).
  30. Xing, Y., Lv, A., Wang, L., Yan, X. The combination of angiotensin II and 5-azacytidine promotes cardiomyocyte differentiation of rat bone marrow mesenchymal stem cells. Mol Cell Biochem. 360 (1-2), 279-287 (2012).
  31. Yuan, Y., et al. Differentiation of mesenchymal stem cells into cardio myogenic cells under the induction of myocardial cell lysate. Zhonghua Xin Xue Guan Bing Za Zhi. 33 (2), 170-173 (2005).
  32. Toma, C., Pittenger, M. F., Cahill, K. S., Byrne, B. J., Kessler, P. D. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation. 105 (1), 93-98 (2002).
  33. Yannarelli, G., et al. Donor mesenchymal stromal cells (MSCs) undergo variable cardiac reprogramming in vivo and predominantly co-express cardiac and stromal determinants after experimental acute myocardial infarction. Stem Cell Rev. 10 (2), 304-315 (2014).
  34. Rangappa, S., Entwistle, J. W., Wechsler, A. S., Kresh, J. Y. Cardiomyocyte-mediated contact programs human mesenchymal stem cells to express cardiogenic phenotype. J Thorac Cardiovasc Surg. 126 (1), 124-132 (2003).
  35. Bakogiannis, C., et al. Circulating endothelial progenitor cells as biomarkers for prediction of cardiovascular outcomes. Curr Med Chem. 19 (16), 2597-2604 (2012).
  36. Deb, A., et al. Bone marrow-derived cardiomyocytes are present in adult human heart: A study of gender-mismatched bone marrow transplantation patients. Circulation. 107 (9), 1247-1249 (2003).
  37. Laflamme, M. A., Myerson, D., Saffitz, J. E., Murry, C. E. Evidence for cardiomyocyte repopulation by extracardiac progenitors in transplanted human hearts. Circ Res. 90 (6), 634-640 (2002).
  38. Quaini, F., et al. Chimerism of the transplanted heart. N Engl J Med. 346 (1), 5-15 (2002).
  39. Alvarez-Dolado, M., et al. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature. 425 (6961), 968-973 (2003).
  40. Nygren, J. M., et al. Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation. Nat Med. 10 (5), 494-501 (2004).
  41. Hong, S. H., et al. Ontogeny of human umbilical cord perivascular cells: molecular and fate potential changes during gestation. Stem Cells Dev. 22 (17), 2425-2439 (2013).
  42. Sarugaser, R., Ennis, J., Stanford, W. L., Davies, J. E. Isolation, propagation, and characterization of human umbilical cord perivascular cells (HUCPVCs). Methods Mol Biol. 482, 269-279 (2009).
  43. Sarugaser, R., Lickorish, D., Baksh, D., Hosseini, M. M., Davies, J. E. Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors. Stem Cells. 23 (2), 220-229 (2005).
  44. Kadivar, M., et al. In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells. Biochem Biophys Res Commun. 340 (2), 639-647 (2006).
  45. Baksh, D., Yao, R., Tuan, R. S. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells. 25 (6), 1384-1392 (2007).
  46. Wu, K. H., et al. Cardiac potential of stem cells from whole human umbilical cord tissue. J Cell Biochem. 107 (5), 926-932 (2009).
  47. Yannarelli, G., et al. Human umbilical cord perivascular cells exhibit enhanced cardiomyocyte reprogramming and cardiac function after experimental acute myocardial infarction. Cell Transplant. 22 (9), 1651-1666 (2013).
  48. Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. J Pharmacol Pharmacother. 1 (2), 94-99 (2010).
  49. Yu, K. R., et al. CD49f enhances multipotency and maintains stemness through the direct regulation of OCT4 and SOX2. Stem Cells. 30 (5), 876-887 (2012).
  50. Lee, R. H., et al. The CD34-like protein PODXL and alpha6-integrin (CD49f) identify early progenitor MSCs with increased clonogenicity and migration to infarcted heart in mice. Blood. 113 (4), 816-826 (2009).
  51. Szaraz, P., et al. In Vitro Differentiation of First Trimester Human Umbilical Cord Perivascular Cells into Contracting Cardiomyocyte-Like Cells. Stem Cells Int. 2016, 7513252 (2016).
  52. Bauwens, C., Yin, T., Dang, S., Peerani, R., Zandstra, P. W. Development of a perfusion fed bioreactor for embryonic stem cell-derived cardiomyocyte generation: oxygen-mediated enhancement of cardiomyocyte output. Biotechnol Bioeng. 90 (4), 452-461 (2005).
  53. Jing, D., Parikh, A., Tzanakakis, E. S. Cardiac cell generation from encapsulated embryonic stem cells in static and scalable culture systems. Cell Transplant. 19 (11), 1397-1412 (2010).
  54. Hare, J. M., et al. Comparison of allogeneic vs autologous bone marrow-derived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial. JAMA. 308 (22), 2369-2379 (2012).
  55. Huang, X. P., et al. Differentiation of allogeneic mesenchymal stem cells induces immunogenicity and limits their long-term benefits for myocardial repair. Circulation. 122 (23), 2419-2429 (2010).
  56. Hare, J. M., et al. A randomized, double-blind, placebo-controlled, dose-escalation study of intravenous adult human mesenchymal stem cells (prochymal) after acute myocardial infarction. J Am Coll Cardiol. 54 (24), 2277-2286 (2009).

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