Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors.

Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in...

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Autores principales:	Pilar Muñoz, Miguel G Toscano, Pedro J Real, Karim Benabdellah, Marién Cobo, Clara Bueno, Verónica Ramos-Mejía, Pablo Menendez, Per Anderson, Francisco Martín
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2cc09e86081a40df98800d1d0a3ee8b4
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spelling	oai:doaj.org-article:2cc09e86081a40df98800d1d0a3ee8b42021-11-18T07:15:28ZSpecific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors.1932-620310.1371/journal.pone.0039091https://doaj.org/article/2cc09e86081a40df98800d1d0a3ee8b42012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22720040/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.Pilar MuñozMiguel G ToscanoPedro J RealKarim BenabdellahMarién CoboClara BuenoVerónica Ramos-MejíaPablo MenendezPer AndersonFrancisco MartínPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 6, p e39091 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Pilar Muñoz Miguel G Toscano Pedro J Real Karim Benabdellah Marién Cobo Clara Bueno Verónica Ramos-Mejía Pablo Menendez Per Anderson Francisco Martín Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors.
description	Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.
format	article
author	Pilar Muñoz Miguel G Toscano Pedro J Real Karim Benabdellah Marién Cobo Clara Bueno Verónica Ramos-Mejía Pablo Menendez Per Anderson Francisco Martín
author_facet	Pilar Muñoz Miguel G Toscano Pedro J Real Karim Benabdellah Marién Cobo Clara Bueno Verónica Ramos-Mejía Pablo Menendez Per Anderson Francisco Martín
author_sort	Pilar Muñoz
title	Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors.
title_short	Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors.
title_full	Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors.
title_fullStr	Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors.
title_full_unstemmed	Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors.
title_sort	specific marking of hescs-derived hematopoietic lineage by was-promoter driven lentiviral vectors.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/2cc09e86081a40df98800d1d0a3ee8b4
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Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors.

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