Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cells: the matrix sandwich method.
BACKGROUND
Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research, including disease modeling, and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix is also fundamentally involved in cardiac development from the earliest morphogenetic events, such as gastrulation.


OBJECTIVE
We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using extracellular matrix in combination with growth factors known to promote cardiogenesis.


RESULTS
PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms.


CONCLUSIONS
Dynamic extracellular matrix application promoted epithelial-mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.
Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA.
Jianhua
J
Zhang
Matthew
M
Klos
Gisela F
GF
Wilson
Amanda M
AM
Herman
Xiaojun
X
Lian
Kunil K
KK
Raval
Matthew R
MR
Barron
Luqia
L
Hou
Andrew G
AG
Soerens
Junying
J
Yu
Sean P
SP
Palecek
Gary E
GE
Lyons
James A
JA
Thomson
Todd J
TJ
Herron
José
J
Jalife
Timothy J
TJ
Kamp
D028341:Activins / Q000494:pharmacology; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D003094:Collagen; D004338:Drug Combinations; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects / Q000502:physiology; D005109:Extracellular Matrix / Q000502:physiology*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D007797:Laminin; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects; D011509:Proteoglycans; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
C525809:BMP4 protein, human; D055415:Bone Morphogenetic Protein 4; D004338:Drug Combinations; D036341:Intercellular Signaling Peptides and Proteins; D007797:Laminin; D011509:Proteoglycans; C422405:activin A; D016222:Fibroblast Growth Factor 2; D028341:Activins; C059485:matrigel; D003094:Collagen