Generation of functional murine cardiac myocytes from induced pluripotent stem cells.
BACKGROUND
The recent breakthrough in the generation of induced pluripotent stem (iPS) cells, which are almost indistinguishable from embryonic stem (ES) cells, facilitates the generation of murine disease- and human patient-specific stem cell lines. The aim of this study was to characterize the cardiac differentiation potential of a murine iPS cell clone in comparison to a well-established murine ES cell line.


RESULTS
With the use of a standard embryoid body-based differentiation protocol for ES cells, iPS cells as well as ES cells were differentiated for 24 days. Although the analyzed iPS cell clone showed a delayed and less efficient formation of beating embryoid bodies compared with the ES cell line, the differentiation resulted in an average of 55% of spontaneously contracting iPS cell embryoid bodies. Analyses on molecular, structural, and functional levels demonstrated that iPS cell-derived cardiomyocytes show typical features of ES cell-derived cardiomyocytes. Reverse transcription polymerase chain reaction analyses demonstrated expression of marker genes typical for mesoderm, cardiac mesoderm, and cardiomyocytes including Brachyury, mesoderm posterior factor 1 (Mesp1), friend of GATA2 (FOG-2), GATA-binding protein 4 (GATA4), NK2 transcription factor related, locus 5 (Nkx2.5), T-box 5 (Tbx5), T-box 20 (Tbx20), atrial natriuretic factor (ANF), myosin light chain 2 atrial transcripts (MLC2a), myosin light chain 2 ventricular transcripts (MLC2v), alpha-myosin heavy chain (alpha-MHC), and cardiac troponin T in differentiation cultures of iPS cells. Immunocytology confirmed expression of cardiomyocyte-typical proteins including sarcomeric alpha-actinin, titin, cardiac troponin T, MLC2v, and connexin 43. iPS cell cardiomyocytes displayed spontaneous rhythmic intracellular Ca(2+) fluctuations with amplitudes of Ca(2+) transients comparable to ES cell cardiomyocytes. Simultaneous Ca(2+) release within clusters of iPS cell-derived cardiomyocytes indicated functional coupling of the cells. Electrophysiological studies with multielectrode arrays demonstrated functionality and presence of the beta-adrenergic and muscarinic signaling cascade in these cells.


CONCLUSIONS
iPS cells differentiate into functional cardiomyocytes. In contrast to ES cells, iPS cells allow derivation of autologous functional cardiomyocytes for cellular cardiomyoplasty and myocardial tissue engineering.
Mauritz
Christina
C
Leibniz Research Laboratories for Biotechnology and Artificial Organs, Carl-Neuberg Strasse 1, 30625 Hannover, Germany.
Schwanke
Kristin
K
Reppel
Michael
M
Neef
Stefan
S
Katsirntaki
Katherina
K
Maier
Lars S
LS
Nguemo
Filomain
F
Menke
Sandra
S
Haustein
Moritz
M
Hescheler
Juergen
J
Hasenfuss
Gerd
G
Martin
Ulrich
U
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D015536:Down-Regulation; D005455:Fluorescent Antibody Technique; D023281:Genomics; D018398:Homeodomain Proteins / Q000235:genetics; D008564:Membrane Potentials / Q000502:physiology; D008648:Mesoderm / Q000166:cytology / Q000502:physiology; D051379:Mice; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000502:physiology; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000502:physiology; D018342:Receptors, Adrenergic, beta-1 / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050794:STAT1 Transcription Factor / Q000502:physiology; D023822:Tissue Engineering / Q000379:methods*
D015415:Biomarkers; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; D018342:Receptors, Adrenergic, beta-1; D011976:Receptors, Muscarinic; D050794:STAT1 Transcription Factor