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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: Muñoz, Pilar, Toscano, Miguel G., Real, Pedro J., Benabdellah, Karim, Cobo, Marién, Bueno, Clara, Ramos-Mejía, Verónica, Menendez, Pablo, Anderson, Per, Martín, Francisco
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3375235/
https://www.ncbi.nlm.nih.gov/pubmed/22720040
http://dx.doi.org/10.1371/journal.pone.0039091
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author Muñoz, Pilar
Toscano, Miguel G.
Real, Pedro J.
Benabdellah, Karim
Cobo, Marién
Bueno, Clara
Ramos-Mejía, Verónica
Menendez, Pablo
Anderson, Per
Martín, Francisco
author_facet Muñoz, Pilar
Toscano, Miguel G.
Real, Pedro J.
Benabdellah, Karim
Cobo, Marién
Bueno, Clara
Ramos-Mejía, Verónica
Menendez, Pablo
Anderson, Per
Martín, Francisco
author_sort Muñoz, Pilar
collection PubMed
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.
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spelling pubmed-33752352012-06-20 Specific Marking of hESCs-Derived Hematopoietic Lineage by WAS-Promoter Driven Lentiviral Vectors Muñoz, Pilar Toscano, Miguel G. Real, Pedro J. Benabdellah, Karim Cobo, Marién Bueno, Clara Ramos-Mejía, Verónica Menendez, Pablo Anderson, Per Martín, Francisco PLoS One Research Article 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. Public Library of Science 2012-06-14 /pmc/articles/PMC3375235/ /pubmed/22720040 http://dx.doi.org/10.1371/journal.pone.0039091 Text en Muñoz et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Muñoz, Pilar
Toscano, Miguel G.
Real, Pedro J.
Benabdellah, Karim
Cobo, Marién
Bueno, Clara
Ramos-Mejía, Verónica
Menendez, Pablo
Anderson, Per
Martín, Francisco
Specific Marking of hESCs-Derived Hematopoietic Lineage by WAS-Promoter Driven Lentiviral Vectors
title 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_short 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
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3375235/
https://www.ncbi.nlm.nih.gov/pubmed/22720040
http://dx.doi.org/10.1371/journal.pone.0039091
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