Assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts

Antonio Fernandez-Perez, Nikhil V. Munshi

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 languageEnglish (US)
Article numbere55456
JournalJournal of Visualized Experiments
Volume2017
Issue number121
DOIs
StatePublished - Mar 22 2017

Fingerprint

Transcription factors
Fibroblasts
Cardiac Myocytes
Transcription Factors
Muscle Cells
Pacemakers
Confocal microscopy
Antibodies
Regenerative Medicine
Confocal Microscopy
Experiments
Specifications
Immunohistochemistry
Research Personnel
Infection

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 journalArticle

@article{558a84da30ac435294435d3b9a770317,
title = "Assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts",
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.",
keywords = "Cardiomyocyte, Developmental biology, Direct reprogramming, Gata4, Hand2, Hcn4, iAM, iPM, Issue 121, iVM, Mef2c, Myl2, Nppa, Tbx5",
author = "Antonio Fernandez-Perez and Munshi, {Nikhil V.}",
year = "2017",
month = "3",
day = "22",
doi = "10.3791/55456",
language = "English (US)",
volume = "2017",
journal = "Journal of Visualized Experiments",
issn = "1940-087X",
publisher = "MYJoVE Corporation",
number = "121",

}

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 -