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

Muñoz P, Toscano MG, Real PJ, Benabdellah K, Cobo M, Bueno C, Ramos-Mejía V, Menendez P, Anderson P, Martín F - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Human DNA Variability Department, Pfizer-Universidad de Granada-Junta de Andalucía Centre for Genomics and Oncological Research, GENYO, Granada, Spain.

ABSTRACT
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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WAS gene expression is restricted to hematopoietic cells and hemogenic progenitors.(A) Analysis of WAS gene expression in different cell lines. mRNA was obtained from hematopoietic cell lines (AlloT and Raji), hESCs-derived myeloid CFUs (And-1 CFUs), undifferentiated hESCs (AND-1 and SHEF-2), a human fibroblastic cell line (293T) and human endothelial cells (HUVEC) and analyzed by RT-PCR for WAS expression (see M&M for details). (B) Time-course analysis of WAS gene expression during hematopoietic differentiation of AND-1 and H9 hESCs (see M&M and Figure S1 for details). mRNA was extracted at different days during differentiation as indicated and WAS expression analyzed by RT-PCR. (C) Analysis of WAS gene expression in hemogenic progenitors and hematopoietic cells. CD45−CD31+ (containing hemogenic progenitors) and CD45+CD31+ (containing hematopoietic cells) populations were sorter from AND-1 (left) and H9 (right) hESCs after 15 days of hematopoietic differentiation. WAS mRNA relative levels were determined in sorted cells by RT-PCR. ΔCt value for WAS expression was obtained using GAPDH as reference gene in all experiments. CFUs: Colony Forming Units.
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pone-0039091-g001: WAS gene expression is restricted to hematopoietic cells and hemogenic progenitors.(A) Analysis of WAS gene expression in different cell lines. mRNA was obtained from hematopoietic cell lines (AlloT and Raji), hESCs-derived myeloid CFUs (And-1 CFUs), undifferentiated hESCs (AND-1 and SHEF-2), a human fibroblastic cell line (293T) and human endothelial cells (HUVEC) and analyzed by RT-PCR for WAS expression (see M&M for details). (B) Time-course analysis of WAS gene expression during hematopoietic differentiation of AND-1 and H9 hESCs (see M&M and Figure S1 for details). mRNA was extracted at different days during differentiation as indicated and WAS expression analyzed by RT-PCR. (C) Analysis of WAS gene expression in hemogenic progenitors and hematopoietic cells. CD45−CD31+ (containing hemogenic progenitors) and CD45+CD31+ (containing hematopoietic cells) populations were sorter from AND-1 (left) and H9 (right) hESCs after 15 days of hematopoietic differentiation. WAS mRNA relative levels were determined in sorted cells by RT-PCR. ΔCt value for WAS expression was obtained using GAPDH as reference gene in all experiments. CFUs: Colony Forming Units.

Mentions: Although the hematopoietic-specific expression of WAS gene is well established [45], [46], [47], its expression pattern during early hematopoietic development remains unknown. We therefore studied WAS gene expression in hESCs during hematopoietic differentiation (Figure 1). We first analyzed by RT-PCR the relative endogenous WAS expression in non-hematopoietic cells (293T and HUVEC) and hematopoietic (AlloT and Raji) cell lines as well as in undifferentiated hESCs (AND-1 and SHEF-2) and hESCs-derivedCFUs from AND-1 (Figure 1A). We confirmed that only hematopoietic cell lines (AlloT and Raji) or hESCs-derived hematopoietic cells (AND-1 CFUs) expressed WAS over background levels (Figure 1A). We could not detect any significant expression of WAS in endothelial cells (HUVEC), nor in undifferentiated hESCs (AND-1 and SHEF-2). However, upon hematopoietic differentiation (see Figure S1 for details), WAS expression appears early (day 3–5) and increased over time (Figure 1B), paralleling hematopoietic commitment of hESCs. In order to further characterize WAS expression during hematopoietic commitment, we sorted CD45− CD31− non-hematopoietic cells, CD45−CD31+ hemogenic progenitors and CD45+CD31+ hematopoietic cells at day 15 of differentiation and analyzed for WAS expression (Figure 1C). In line with the expression kinetics shown in Figure 1B, CD45+ hematopoietic cells contained the highest levels of WAS mRNA followed by CD31+CD45− hemogenic progenitors. Together, this data indicates that WAS expression parallels hematopoietic commitment from hESCs.


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

Muñoz P, Toscano MG, Real PJ, Benabdellah K, Cobo M, Bueno C, Ramos-Mejía V, Menendez P, Anderson P, Martín F - PLoS ONE (2012)

WAS gene expression is restricted to hematopoietic cells and hemogenic progenitors.(A) Analysis of WAS gene expression in different cell lines. mRNA was obtained from hematopoietic cell lines (AlloT and Raji), hESCs-derived myeloid CFUs (And-1 CFUs), undifferentiated hESCs (AND-1 and SHEF-2), a human fibroblastic cell line (293T) and human endothelial cells (HUVEC) and analyzed by RT-PCR for WAS expression (see M&M for details). (B) Time-course analysis of WAS gene expression during hematopoietic differentiation of AND-1 and H9 hESCs (see M&M and Figure S1 for details). mRNA was extracted at different days during differentiation as indicated and WAS expression analyzed by RT-PCR. (C) Analysis of WAS gene expression in hemogenic progenitors and hematopoietic cells. CD45−CD31+ (containing hemogenic progenitors) and CD45+CD31+ (containing hematopoietic cells) populations were sorter from AND-1 (left) and H9 (right) hESCs after 15 days of hematopoietic differentiation. WAS mRNA relative levels were determined in sorted cells by RT-PCR. ΔCt value for WAS expression was obtained using GAPDH as reference gene in all experiments. CFUs: Colony Forming Units.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375235&req=5

pone-0039091-g001: WAS gene expression is restricted to hematopoietic cells and hemogenic progenitors.(A) Analysis of WAS gene expression in different cell lines. mRNA was obtained from hematopoietic cell lines (AlloT and Raji), hESCs-derived myeloid CFUs (And-1 CFUs), undifferentiated hESCs (AND-1 and SHEF-2), a human fibroblastic cell line (293T) and human endothelial cells (HUVEC) and analyzed by RT-PCR for WAS expression (see M&M for details). (B) Time-course analysis of WAS gene expression during hematopoietic differentiation of AND-1 and H9 hESCs (see M&M and Figure S1 for details). mRNA was extracted at different days during differentiation as indicated and WAS expression analyzed by RT-PCR. (C) Analysis of WAS gene expression in hemogenic progenitors and hematopoietic cells. CD45−CD31+ (containing hemogenic progenitors) and CD45+CD31+ (containing hematopoietic cells) populations were sorter from AND-1 (left) and H9 (right) hESCs after 15 days of hematopoietic differentiation. WAS mRNA relative levels were determined in sorted cells by RT-PCR. ΔCt value for WAS expression was obtained using GAPDH as reference gene in all experiments. CFUs: Colony Forming Units.
Mentions: Although the hematopoietic-specific expression of WAS gene is well established [45], [46], [47], its expression pattern during early hematopoietic development remains unknown. We therefore studied WAS gene expression in hESCs during hematopoietic differentiation (Figure 1). We first analyzed by RT-PCR the relative endogenous WAS expression in non-hematopoietic cells (293T and HUVEC) and hematopoietic (AlloT and Raji) cell lines as well as in undifferentiated hESCs (AND-1 and SHEF-2) and hESCs-derivedCFUs from AND-1 (Figure 1A). We confirmed that only hematopoietic cell lines (AlloT and Raji) or hESCs-derived hematopoietic cells (AND-1 CFUs) expressed WAS over background levels (Figure 1A). We could not detect any significant expression of WAS in endothelial cells (HUVEC), nor in undifferentiated hESCs (AND-1 and SHEF-2). However, upon hematopoietic differentiation (see Figure S1 for details), WAS expression appears early (day 3–5) and increased over time (Figure 1B), paralleling hematopoietic commitment of hESCs. In order to further characterize WAS expression during hematopoietic commitment, we sorted CD45− CD31− non-hematopoietic cells, CD45−CD31+ hemogenic progenitors and CD45+CD31+ hematopoietic cells at day 15 of differentiation and analyzed for WAS expression (Figure 1C). In line with the expression kinetics shown in Figure 1B, CD45+ hematopoietic cells contained the highest levels of WAS mRNA followed by CD31+CD45− hemogenic progenitors. Together, this data indicates that WAS expression parallels hematopoietic commitment from hESCs.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Human DNA Variability Department, Pfizer-Universidad de Granada-Junta de Andalucía Centre for Genomics and Oncological Research, GENYO, Granada, Spain.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus