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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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The AWE vectors identify different sub-populations at different times of hematopoietic differentiations.(A) AWE-transduced H9 cells were incubated in hematopoietic differentiation media and analyzed for CD45, CD31 and CD34 expression at days 10, 15 and 22. eGFP+ (right) and eGFP− (left) populations were first analyzed for expression of CD45 and CD31. hESCs CD31+CD45+ and CD31+CD45− were further analyzed for expression of CD34 (top and bottom plots respectively). (B) A similar experiment as in A) was performed using pLVTHM -transduced H9 cells. Note that using the pLVTHM constitutive vectors there are no differences in eGFP expression from day 10 to day 22. (C) AWE- marked cells are enriched in CD34dim hemogenic progenitors and hematopoietic cells. Graph representing fold enrichment of eGFP+ versus eGFP− population in hemogenic progenitors (CD45−CD31+), hematopoietic progenitors (CD45+CD34+) and myeloid cells (CD45+CD33+). CD34dim and CD34bright progenitor cells were analyzed independently. Data were obtained by dividing the percentages of the different cell types in the eGFP+ fraction of the AWE-transduced hESCs (from Figure 4A, right plots) with the values obtained in the eGFP− population (Figure 4A, left plots) and plotted as fold enrichment. Data represent average of at least five separate experiments (+/− SEM) using H9 and AND-1cells at medium stage of hematopoietic development (day 10 for H9 and day 15 for AND-1). ** P = 0.008; * P = 0.032.
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pone-0039091-g004: The AWE vectors identify different sub-populations at different times of hematopoietic differentiations.(A) AWE-transduced H9 cells were incubated in hematopoietic differentiation media and analyzed for CD45, CD31 and CD34 expression at days 10, 15 and 22. eGFP+ (right) and eGFP− (left) populations were first analyzed for expression of CD45 and CD31. hESCs CD31+CD45+ and CD31+CD45− were further analyzed for expression of CD34 (top and bottom plots respectively). (B) A similar experiment as in A) was performed using pLVTHM -transduced H9 cells. Note that using the pLVTHM constitutive vectors there are no differences in eGFP expression from day 10 to day 22. (C) AWE- marked cells are enriched in CD34dim hemogenic progenitors and hematopoietic cells. Graph representing fold enrichment of eGFP+ versus eGFP− population in hemogenic progenitors (CD45−CD31+), hematopoietic progenitors (CD45+CD34+) and myeloid cells (CD45+CD33+). CD34dim and CD34bright progenitor cells were analyzed independently. Data were obtained by dividing the percentages of the different cell types in the eGFP+ fraction of the AWE-transduced hESCs (from Figure 4A, right plots) with the values obtained in the eGFP− population (Figure 4A, left plots) and plotted as fold enrichment. Data represent average of at least five separate experiments (+/− SEM) using H9 and AND-1cells at medium stage of hematopoietic development (day 10 for H9 and day 15 for AND-1). ** P = 0.008; * P = 0.032.

Mentions: We further investigated the eGFP expression kinetics through hematopoietic differentiation of AWE- and pLVTHM-transduced-hESCs. Transduced hESCs were allowed to differentiate towards the hematopoietic lineage and analysed at different time points (days 10, 15 and 22). eGFP expression driven by the AWE LV was manifested at day 10 and increased progressively at days 15 and 22 (Figure 4A, black plots) paralleling CD45 expression (Figure 4A and Figure S4). The phenotypic analysis of the eGFP+ cells showed specific marking of different subpopulations at different days of differentiation. At day 10, around 33% of the eGFP+ cells were CD45−CD31+CD34+, markers characteristic of hemogenic progenitors (Figure 4A, Day 10, right plots, highlighted in red). 12% were CD45+CD31+ hematopoietic cells of which 60% were CD34+, markers that identify hematopoietic precursors (Figure 4A, Day 10, right plots, highlighted in blue). The remaining 55% eGFP+ cells were CD45−CD31low/− (Figure 4A, Day 10, right plots, highlighted in green). Note that the CD31 expression levels of eGFP+ cells is slightly higher than the observed in eGFP− cells (Figure 4A, right plots, green population, versus left plots black population; day 10 and day 15). Further analysis of this population showed that they were also negative for CD34 (Figure S5). We therefore annotate this population as CD45−CD31low/−CD34−. As differentiation continued to days 15 and 22, most of the eGFP+CD45− populations disappeared (Figure 4A, middle-right plots, highlighted in green and red) and over 93% of the eGFP+ cells became CD45+CD31+. The phenotype of pLVTHM-transduced hESCs was identical in the eGFP+ and eGFP− regions (Figure 4B), indicating equal expression of this vector in all populations.


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)

The AWE vectors identify different sub-populations at different times of hematopoietic differentiations.(A) AWE-transduced H9 cells were incubated in hematopoietic differentiation media and analyzed for CD45, CD31 and CD34 expression at days 10, 15 and 22. eGFP+ (right) and eGFP− (left) populations were first analyzed for expression of CD45 and CD31. hESCs CD31+CD45+ and CD31+CD45− were further analyzed for expression of CD34 (top and bottom plots respectively). (B) A similar experiment as in A) was performed using pLVTHM -transduced H9 cells. Note that using the pLVTHM constitutive vectors there are no differences in eGFP expression from day 10 to day 22. (C) AWE- marked cells are enriched in CD34dim hemogenic progenitors and hematopoietic cells. Graph representing fold enrichment of eGFP+ versus eGFP− population in hemogenic progenitors (CD45−CD31+), hematopoietic progenitors (CD45+CD34+) and myeloid cells (CD45+CD33+). CD34dim and CD34bright progenitor cells were analyzed independently. Data were obtained by dividing the percentages of the different cell types in the eGFP+ fraction of the AWE-transduced hESCs (from Figure 4A, right plots) with the values obtained in the eGFP− population (Figure 4A, left plots) and plotted as fold enrichment. Data represent average of at least five separate experiments (+/− SEM) using H9 and AND-1cells at medium stage of hematopoietic development (day 10 for H9 and day 15 for AND-1). ** P = 0.008; * P = 0.032.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375235&req=5

pone-0039091-g004: The AWE vectors identify different sub-populations at different times of hematopoietic differentiations.(A) AWE-transduced H9 cells were incubated in hematopoietic differentiation media and analyzed for CD45, CD31 and CD34 expression at days 10, 15 and 22. eGFP+ (right) and eGFP− (left) populations were first analyzed for expression of CD45 and CD31. hESCs CD31+CD45+ and CD31+CD45− were further analyzed for expression of CD34 (top and bottom plots respectively). (B) A similar experiment as in A) was performed using pLVTHM -transduced H9 cells. Note that using the pLVTHM constitutive vectors there are no differences in eGFP expression from day 10 to day 22. (C) AWE- marked cells are enriched in CD34dim hemogenic progenitors and hematopoietic cells. Graph representing fold enrichment of eGFP+ versus eGFP− population in hemogenic progenitors (CD45−CD31+), hematopoietic progenitors (CD45+CD34+) and myeloid cells (CD45+CD33+). CD34dim and CD34bright progenitor cells were analyzed independently. Data were obtained by dividing the percentages of the different cell types in the eGFP+ fraction of the AWE-transduced hESCs (from Figure 4A, right plots) with the values obtained in the eGFP− population (Figure 4A, left plots) and plotted as fold enrichment. Data represent average of at least five separate experiments (+/− SEM) using H9 and AND-1cells at medium stage of hematopoietic development (day 10 for H9 and day 15 for AND-1). ** P = 0.008; * P = 0.032.
Mentions: We further investigated the eGFP expression kinetics through hematopoietic differentiation of AWE- and pLVTHM-transduced-hESCs. Transduced hESCs were allowed to differentiate towards the hematopoietic lineage and analysed at different time points (days 10, 15 and 22). eGFP expression driven by the AWE LV was manifested at day 10 and increased progressively at days 15 and 22 (Figure 4A, black plots) paralleling CD45 expression (Figure 4A and Figure S4). The phenotypic analysis of the eGFP+ cells showed specific marking of different subpopulations at different days of differentiation. At day 10, around 33% of the eGFP+ cells were CD45−CD31+CD34+, markers characteristic of hemogenic progenitors (Figure 4A, Day 10, right plots, highlighted in red). 12% were CD45+CD31+ hematopoietic cells of which 60% were CD34+, markers that identify hematopoietic precursors (Figure 4A, Day 10, right plots, highlighted in blue). The remaining 55% eGFP+ cells were CD45−CD31low/− (Figure 4A, Day 10, right plots, highlighted in green). Note that the CD31 expression levels of eGFP+ cells is slightly higher than the observed in eGFP− cells (Figure 4A, right plots, green population, versus left plots black population; day 10 and day 15). Further analysis of this population showed that they were also negative for CD34 (Figure S5). We therefore annotate this population as CD45−CD31low/−CD34−. As differentiation continued to days 15 and 22, most of the eGFP+CD45− populations disappeared (Figure 4A, middle-right plots, highlighted in green and red) and over 93% of the eGFP+ cells became CD45+CD31+. The phenotype of pLVTHM-transduced hESCs was identical in the eGFP+ and eGFP− regions (Figure 4B), indicating equal expression of this vector in all populations.

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