Abstract
Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.
| Original language | English (US) |
|---|---|
| Article number | e55456 |
| Journal | Journal of Visualized Experiments |
| Volume | 2017 |
| Issue number | 121 |
| DOIs | |
| State | Published - Mar 22 2017 |
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Keywords
- Cardiomyocyte
- Developmental biology
- Direct reprogramming
- Gata4
- Hand2
- Hcn4
- iAM
- iPM
- Issue 121
- iVM
- Mef2c
- Myl2
- Nppa
- Tbx5
ASJC Scopus subject areas
- Neuroscience(all)
- Chemical Engineering(all)
- Immunology and Microbiology(all)
- Biochemistry, Genetics and Molecular Biology(all)
Cite this
Assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts. / Fernandez-Perez, Antonio; Munshi, Nikhil V.
In: Journal of Visualized Experiments, Vol. 2017, No. 121, e55456, 22.03.2017.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts
AU - Fernandez-Perez, Antonio
AU - Munshi, Nikhil V.
PY - 2017/3/22
Y1 - 2017/3/22
N2 - Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.
AB - Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.
KW - Cardiomyocyte
KW - Developmental biology
KW - Direct reprogramming
KW - Gata4
KW - Hand2
KW - Hcn4
KW - iAM
KW - iPM
KW - Issue 121
KW - iVM
KW - Mef2c
KW - Myl2
KW - Nppa
KW - Tbx5
UR - http://www.scopus.com/inward/record.url?scp=85017099252&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85017099252&partnerID=8YFLogxK
U2 - 10.3791/55456
DO - 10.3791/55456
M3 - Article
C2 - 28362413
AN - SCOPUS:85017099252
VL - 2017
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
SN - 1940-087X
IS - 121
M1 - e55456
ER -