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Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

Silvério-Alves, Rita LU ; Gomes, Andreia M. ; Kurochkin, Ilia LU ; Moore, Kateri A. and Pereira, Carlos Filipe LU (2019) In Journal of visualized experiments : JoVE
Abstract

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced... (More)

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of visualized experiments : JoVE
issue
153
publisher
JoVE
external identifiers
  • pmid:31736500
  • scopus:85075191300
ISSN
1940-087X
DOI
10.3791/60112
language
English
LU publication?
yes
id
4155b441-2f7e-42b7-aca9-335ee36630cd
date added to LUP
2019-12-06 08:49:30
date last changed
2020-01-13 02:34:46
@article{4155b441-2f7e-42b7-aca9-335ee36630cd,
  abstract     = {<p>The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.</p>},
  author       = {Silvério-Alves, Rita and Gomes, Andreia M. and Kurochkin, Ilia and Moore, Kateri A. and Pereira, Carlos Filipe},
  issn         = {1940-087X},
  language     = {eng},
  month        = {11},
  number       = {153},
  publisher    = {JoVE},
  series       = {Journal of visualized experiments : JoVE},
  title        = {Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors},
  url          = {http://dx.doi.org/10.3791/60112},
  doi          = {10.3791/60112},
  year         = {2019},
}