Human cardiac extracellular matrix supports myocardial lineage commitment of pluripotent stem cells.
OBJECTIVE
Cross-talk between organ-specific extracellular matrix (ECM) and stem cells is often assumed but has not been directly demonstrated. We developed a protocol for the preparation of human cardiac ECM (cECM) and studied whether cECM has effects on pluripotent stem cell differentiation that may be useful for future cardiac regeneration strategies in patients with end-stage heart failure.


METHODS
Of note, 0.3 mm-thick cECM slices were prepared from samples of myocardium from patients with end-stage non-ischaemic dilated cardiomyopathy, using a three-step protocol involving hypotonic lysis buffer, sodium dodecyl sulphate (SDS) and foetal bovine serum (FBS). Murine embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and mesenchymal stromal cells (MSCs) were seeded and grown in standard culture, on cECM or on non-specific ECM preparations (Matrigel® or Geltrex®). Cell attachment, apoptosis induction (Caspase 3/7 activity) and metabolic activity (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium conversion) were followed. Transcriptional activation of genes involved in pluripotency; early and late myocardial development; and endothelial, ectodermal or endodermal commitment were monitored by quantitative real-time polymerase chain reaction (rtPCR). Protein expression of selected markers was confirmed by immunohistology.


RESULTS
cECM supported the proliferation of ESCs and iPSCs, and Caspase 3/7 activity was significantly lower compared with standard culture. Cardiac lineage commitment was favoured when ESCs or iPSCs were grown on cECM, as evidenced by the significantly increased mRNA expression of cardiac alpha myosin heavy polypeptide 6 (Myh6), cardiac troponin T2 (Tnnt2) and NK2 homeobox 5 (Nkx2.5) as well as positive immunohistology for cardiac troponin T and heavy-chain cardiac myosin protein. In contrast, Matrigel or Geltrex did not induce cardiac-specific markers. MSCs showed no evidence of cardiomyocyte differentiation.


CONCLUSIONS
Human cardiac ECM seems to direct differentiation of pluripotent stem cells towards a cardiomyocyte phenotype. This phenomenon supports the use of cardiac ECM preparations for guided stem cell differentiation and myocardial repair, and may ultimately increase the therapeutic efficacy of cell therapy in heart failure patients.
Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité Campus Virchow-Klinikum, Berlin, Germany.
Barbara
B
Oberwallner
Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité Campus Virchow-Klinikum, Berlin, Germany.
Andreja
A
Brodarac
Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité Campus Virchow-Klinikum, Berlin, Germany.
Petra
P
Anić
Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Center, University of Cologne, Cologne, Germany.
Tomo
T
Šarić
Deutsches Herzzentrum Berlin (DHZB), Berlin, Germany.
Katharina
K
Wassilew
Department of Cardiac and Thoracic Surgery, Heart Center, University of Cologne, Cologne, Germany.
Klaus
K
Neef
Department of Cardiac and Thoracic Surgery, Heart Center, University of Cologne, Cologne, Germany.
Yeong-Hoon
YH
Choi
Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité Campus Virchow-Klinikum, Berlin, Germany Deutsches Herzzentrum Berlin (DHZB), Berlin, Germany stamm@dhzb.de.
Christof
C
Stamm
D000818:Animals; D017209:Apoptosis; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D024722:Cardiac Myosins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D005109:Extracellular Matrix / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D051379:Mice; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism* / Q000502:physiology; D039904:Pluripotent Stem Cells / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism* / Q000502:physiology; D020107:Troponin T / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
D015415:Biomarkers; D020107:Troponin T; D024722:Cardiac Myosins
Differentiation; Extracellular matrix; Pluripotent cells; Recellularization; Stem cells