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.
(Less)
- author
- Silvério-Alves, Rita
LU
; Gomes, Andreia M.
; Kurochkin, Ilia
LU
; Moore, Kateri A.
and Pereira, Carlos Filipe
LU
- organization
- publishing date
- 2019-11-04
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of visualized experiments : JoVE
- issue
- 153
- publisher
- JoVE
- external identifiers
-
- scopus:85075191300
- pmid:31736500
- ISSN
- 1940-087X
- DOI
- 10.3791/60112
- project
- Elucidating the Mechanisms of Hematopoietic Reprogramming
- language
- English
- LU publication?
- yes
- id
- 4155b441-2f7e-42b7-aca9-335ee36630cd
- date added to LUP
- 2019-12-06 08:49:30
- date last changed
- 2024-03-04 09:32:36
@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}},
}