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Reprogramming mouse embryonic fibroblasts with transcription factors to induce a hemogenic program

Daniel, Michael G.; Pereira, Carlos-Filipe LU ; Bernitz, Jeffrey M.; Lemischka, Ihor R. and Moore, Kateri (2016) In Journal of Visualized Experiments
Abstract

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of... (More)

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis “in a dish,” allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

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author
publishing date
type
Contribution to journal
publication status
published
keywords
Cell fate conversion, Developmental Biology, Fibroblasts Hematopoiesis, Hematopoietic stem cell, Hemogenesis, Issue 118, Reprogramming, Transcription factors
in
Journal of Visualized Experiments
issue
118
publisher
JoVE
external identifiers
  • scopus:85015946426
ISSN
1940-087X
DOI
10.3791/54372
language
English
LU publication?
no
id
2b45bd28-ed41-4ba0-9fb9-407d32211a46
date added to LUP
2017-10-02 17:23:14
date last changed
2017-10-12 08:51:50
@article{2b45bd28-ed41-4ba0-9fb9-407d32211a46,
  abstract     = {<p>This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34<sup>+</sup> cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP<sup>+</sup> cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP<sup>+</sup> cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis “in a dish,” allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.</p>},
  articleno    = {e54372},
  author       = {Daniel, Michael G. and Pereira, Carlos-Filipe and Bernitz, Jeffrey M. and Lemischka, Ihor R. and Moore, Kateri},
  issn         = {1940-087X},
  keyword      = {Cell fate conversion,Developmental Biology,Fibroblasts Hematopoiesis,Hematopoietic stem cell,Hemogenesis,Issue 118,Reprogramming,Transcription factors},
  language     = {eng},
  month        = {12},
  number       = {118},
  publisher    = {JoVE},
  series       = {Journal of Visualized Experiments},
  title        = {Reprogramming mouse embryonic fibroblasts with transcription factors to induce a hemogenic program},
  url          = {http://dx.doi.org/10.3791/54372},
  year         = {2016},
}