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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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Specific expression of WAS-promoter driven LVs in hESCs-derived hematopoietic cells.(A) Representation of the SIN LVs used to transduce hESCs. WE vector [31] contains a 500-bp fragment of the human WAS proximal promoter driving the expression of the reporter gene eGFP. The AWE vector [33] contains an aditional 387-bp fragment of the WAS alternative promoter. The CE and pLVTHM are control lentiviral vectors expressing constitutively eGFP through the CMV and the EF1α promoters, respectively. (B) Constitutive LVs (CE and pLVTHM) express eGFP in hESC undifferentiated cells (AND-1) while the hematopoietic-specific AWE and WE LVs are silent. AND-1 cells were transduced with the LVs in order to obtain 0.5–3 vg/c (see materials and methods). (C) Hematopoietic differentiation induces eGFP expression in AWE and WE-transduced hESCs. AWE-, WE- and pLVTHM- transduced H9 cells were prompt to differentiate and 22 days later analyzed for eGFP expression (Middle plots). eGFP+ (right) and eGFP− (left) populations were analyzed for expression of mature hematopoietic markers CD45 and CD33. Note that WE and AWE-transduced cells (top and middle plots) mark specifically CD45+CD33+ cells while pLVTHM (bottom plots) express eGFP equally in hematopoietic and non-hematopoietic cells. (D) Graph showing the percentage of CD45+ cells within the eGFP+ population from AWE-, WE- and pLVTHM-transduced hESCs at day 22. AWE and WE- transduced cells did not show statistics differences. pLVTHM-transduced hESCs were used as controls. Data are average of at least three independent experiments +/− SEM *** P = 0.0002; ** P = 0.0023 (E) Neuronal and endothelial differentiation of AWE-transduced hESCs does not induce eGFP expression. AWE-transduced H9 cells (AWE) were differentiated into neural progenitors (Top plots) and endothelium (middle plots) and analyzed for eGFP, A2B5 (an early neuroectodermal marker) and VE-Cadherine (an endothelial marker) expression. The AWE-transduced H9 cells were also used for EB-mediated hematopoietic differentiation and analyzed after 22 days for eGFP and CD45 (Right plot). Untransduced (NT) H9 were used as a negative control for eGFP expression.
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pone-0039091-g002: Specific expression of WAS-promoter driven LVs in hESCs-derived hematopoietic cells.(A) Representation of the SIN LVs used to transduce hESCs. WE vector [31] contains a 500-bp fragment of the human WAS proximal promoter driving the expression of the reporter gene eGFP. The AWE vector [33] contains an aditional 387-bp fragment of the WAS alternative promoter. The CE and pLVTHM are control lentiviral vectors expressing constitutively eGFP through the CMV and the EF1α promoters, respectively. (B) Constitutive LVs (CE and pLVTHM) express eGFP in hESC undifferentiated cells (AND-1) while the hematopoietic-specific AWE and WE LVs are silent. AND-1 cells were transduced with the LVs in order to obtain 0.5–3 vg/c (see materials and methods). (C) Hematopoietic differentiation induces eGFP expression in AWE and WE-transduced hESCs. AWE-, WE- and pLVTHM- transduced H9 cells were prompt to differentiate and 22 days later analyzed for eGFP expression (Middle plots). eGFP+ (right) and eGFP− (left) populations were analyzed for expression of mature hematopoietic markers CD45 and CD33. Note that WE and AWE-transduced cells (top and middle plots) mark specifically CD45+CD33+ cells while pLVTHM (bottom plots) express eGFP equally in hematopoietic and non-hematopoietic cells. (D) Graph showing the percentage of CD45+ cells within the eGFP+ population from AWE-, WE- and pLVTHM-transduced hESCs at day 22. AWE and WE- transduced cells did not show statistics differences. pLVTHM-transduced hESCs were used as controls. Data are average of at least three independent experiments +/− SEM *** P = 0.0002; ** P = 0.0023 (E) Neuronal and endothelial differentiation of AWE-transduced hESCs does not induce eGFP expression. AWE-transduced H9 cells (AWE) were differentiated into neural progenitors (Top plots) and endothelium (middle plots) and analyzed for eGFP, A2B5 (an early neuroectodermal marker) and VE-Cadherine (an endothelial marker) expression. The AWE-transduced H9 cells were also used for EB-mediated hematopoietic differentiation and analyzed after 22 days for eGFP and CD45 (Right plot). Untransduced (NT) H9 were used as a negative control for eGFP expression.

Mentions: We have previously described the development of two hematopoietic-specific WAS-promoter-driven LVs: WE [31] an AWE [33] (Figure 2A). The WE LV harbours a 500 bp fragment from the WAS proximal promoter driving the expression of eGFP and the AWE LV contains an additional 387 pb from the WAS alternative promoter upstream of the proximal promoter. In order to determine the ability of WE and AWE LVs to transduce hESCs, we incubated the AND-1 cell line with the LV-containing supernatants. Two other constitutive vectors, CE [40] and pLVTHM [39], driving eGFP expression through the CMV and EF1-α promoters respectively, were used as controls. All LVs efficiently integrated into the hESCs (as determined by Q-PCR) achieving 0.6–1.4 vector genome per cell (vg/c). However, only the constitutive vectors CE and pLVTHM expressed the transgene in the undifferentiated hESCs (Figure 2B). The transduction protocol did not affect the pluripotency of the hESCs as demonstrated by the expression of pluripotency markers and their ability to form teratomas in NOD-SCID mice (data not shown).


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)

Specific expression of WAS-promoter driven LVs in hESCs-derived hematopoietic cells.(A) Representation of the SIN LVs used to transduce hESCs. WE vector [31] contains a 500-bp fragment of the human WAS proximal promoter driving the expression of the reporter gene eGFP. The AWE vector [33] contains an aditional 387-bp fragment of the WAS alternative promoter. The CE and pLVTHM are control lentiviral vectors expressing constitutively eGFP through the CMV and the EF1α promoters, respectively. (B) Constitutive LVs (CE and pLVTHM) express eGFP in hESC undifferentiated cells (AND-1) while the hematopoietic-specific AWE and WE LVs are silent. AND-1 cells were transduced with the LVs in order to obtain 0.5–3 vg/c (see materials and methods). (C) Hematopoietic differentiation induces eGFP expression in AWE and WE-transduced hESCs. AWE-, WE- and pLVTHM- transduced H9 cells were prompt to differentiate and 22 days later analyzed for eGFP expression (Middle plots). eGFP+ (right) and eGFP− (left) populations were analyzed for expression of mature hematopoietic markers CD45 and CD33. Note that WE and AWE-transduced cells (top and middle plots) mark specifically CD45+CD33+ cells while pLVTHM (bottom plots) express eGFP equally in hematopoietic and non-hematopoietic cells. (D) Graph showing the percentage of CD45+ cells within the eGFP+ population from AWE-, WE- and pLVTHM-transduced hESCs at day 22. AWE and WE- transduced cells did not show statistics differences. pLVTHM-transduced hESCs were used as controls. Data are average of at least three independent experiments +/− SEM *** P = 0.0002; ** P = 0.0023 (E) Neuronal and endothelial differentiation of AWE-transduced hESCs does not induce eGFP expression. AWE-transduced H9 cells (AWE) were differentiated into neural progenitors (Top plots) and endothelium (middle plots) and analyzed for eGFP, A2B5 (an early neuroectodermal marker) and VE-Cadherine (an endothelial marker) expression. The AWE-transduced H9 cells were also used for EB-mediated hematopoietic differentiation and analyzed after 22 days for eGFP and CD45 (Right plot). Untransduced (NT) H9 were used as a negative control for eGFP expression.
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Related In: Results  -  Collection

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

pone-0039091-g002: Specific expression of WAS-promoter driven LVs in hESCs-derived hematopoietic cells.(A) Representation of the SIN LVs used to transduce hESCs. WE vector [31] contains a 500-bp fragment of the human WAS proximal promoter driving the expression of the reporter gene eGFP. The AWE vector [33] contains an aditional 387-bp fragment of the WAS alternative promoter. The CE and pLVTHM are control lentiviral vectors expressing constitutively eGFP through the CMV and the EF1α promoters, respectively. (B) Constitutive LVs (CE and pLVTHM) express eGFP in hESC undifferentiated cells (AND-1) while the hematopoietic-specific AWE and WE LVs are silent. AND-1 cells were transduced with the LVs in order to obtain 0.5–3 vg/c (see materials and methods). (C) Hematopoietic differentiation induces eGFP expression in AWE and WE-transduced hESCs. AWE-, WE- and pLVTHM- transduced H9 cells were prompt to differentiate and 22 days later analyzed for eGFP expression (Middle plots). eGFP+ (right) and eGFP− (left) populations were analyzed for expression of mature hematopoietic markers CD45 and CD33. Note that WE and AWE-transduced cells (top and middle plots) mark specifically CD45+CD33+ cells while pLVTHM (bottom plots) express eGFP equally in hematopoietic and non-hematopoietic cells. (D) Graph showing the percentage of CD45+ cells within the eGFP+ population from AWE-, WE- and pLVTHM-transduced hESCs at day 22. AWE and WE- transduced cells did not show statistics differences. pLVTHM-transduced hESCs were used as controls. Data are average of at least three independent experiments +/− SEM *** P = 0.0002; ** P = 0.0023 (E) Neuronal and endothelial differentiation of AWE-transduced hESCs does not induce eGFP expression. AWE-transduced H9 cells (AWE) were differentiated into neural progenitors (Top plots) and endothelium (middle plots) and analyzed for eGFP, A2B5 (an early neuroectodermal marker) and VE-Cadherine (an endothelial marker) expression. The AWE-transduced H9 cells were also used for EB-mediated hematopoietic differentiation and analyzed after 22 days for eGFP and CD45 (Right plot). Untransduced (NT) H9 were used as a negative control for eGFP expression.
Mentions: We have previously described the development of two hematopoietic-specific WAS-promoter-driven LVs: WE [31] an AWE [33] (Figure 2A). The WE LV harbours a 500 bp fragment from the WAS proximal promoter driving the expression of eGFP and the AWE LV contains an additional 387 pb from the WAS alternative promoter upstream of the proximal promoter. In order to determine the ability of WE and AWE LVs to transduce hESCs, we incubated the AND-1 cell line with the LV-containing supernatants. Two other constitutive vectors, CE [40] and pLVTHM [39], driving eGFP expression through the CMV and EF1-α promoters respectively, were used as controls. All LVs efficiently integrated into the hESCs (as determined by Q-PCR) achieving 0.6–1.4 vector genome per cell (vg/c). However, only the constitutive vectors CE and pLVTHM expressed the transgene in the undifferentiated hESCs (Figure 2B). The transduction protocol did not affect the pluripotency of the hESCs as demonstrated by the expression of pluripotency markers and their ability to form teratomas in NOD-SCID mice (data not shown).

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