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Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse yolk sac: a comparative study between in situ electroporation and retroviral transduction.

Giroux SJ, Alves-Leiva C, Lécluse Y, Martin P, Albagli O, Godin I - BMC Dev. Biol. (2007)

Bottom Line: Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated.We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U790, Institut Gustave Roussy-PR1, Villejuif, France. Sebastien.Giroux@u-bourgogne.fr <Sebastien.Giroux@u-bourgogne.fr>

ABSTRACT

Background: Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.

Results: One transduction protocol involves transient modification of gene expression through in situ electroporation of the prospective blood islands, which allows the evolution of transfected mesodermal cells in their "normal" environment, upon organ culture. Following in situ electroporation of a GFP reporter construct into the YS cavity of embryos at post-streak (mesodermal/pre-hematopoietic precursors) or early somite (hematopoietic precursors) stages, high GFP expression levels as well as a good preservation of cell viability is observed in YS explants. Moreover, the erythro-myeloid progeny typical of the YS arises from GFP+ mesodermal cells or hematopoietic precursors, even if the number of targeted precursors is low. The second approach, based on retroviral transduction allows a very efficient transduction of large precursor numbers, but may only be used to target 8 dpc YS hematopoietic precursors. Again, transduced cells generate a progeny quantitatively and qualitatively similar to that of control YS.

Conclusion: We thus provide two protocols whose combination may allow a thorough study of both early and late events of hematopoietic development in the murine YS. In situ electroporation constitutes the only possible gene transfer method to transduce mesodermal/pre-hematopoietic precursors and analyze the earliest steps of hematopoietic development. Both in situ electroporation and retroviral transduction may be used to target early hematopoietic precursors, but the latter appears more convenient if a large pool of stably transduced cells is required. We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.

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A: Compared evolution of cell production obtained from GFP+ cell sorted from 8 dpc YS infected upon explantation (between bracket: number of cells recovered at day 8), or after one day in organ culture. B: Evolution of GFP expression in the whole population or in GFP- cells sorted from transduced 8 dpc OrgD1-YS after culture on OP9 stromal cells. A steady state GFP expression is attained at day 4 post-infection. Moreover, GFP expression is acquired, during culture, by a subset of GFP- sorted cells. C: GFP expression in transduced OrgD1-YS cells (top panel left) persists in sorted GFP+ cells cultured on OP9 stromal cells (top panel right). The colonies generated by transduced cells also remain entirely GFP+ (bottom panel).
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Figure 6: A: Compared evolution of cell production obtained from GFP+ cell sorted from 8 dpc YS infected upon explantation (between bracket: number of cells recovered at day 8), or after one day in organ culture. B: Evolution of GFP expression in the whole population or in GFP- cells sorted from transduced 8 dpc OrgD1-YS after culture on OP9 stromal cells. A steady state GFP expression is attained at day 4 post-infection. Moreover, GFP expression is acquired, during culture, by a subset of GFP- sorted cells. C: GFP expression in transduced OrgD1-YS cells (top panel left) persists in sorted GFP+ cells cultured on OP9 stromal cells (top panel right). The colonies generated by transduced cells also remain entirely GFP+ (bottom panel).

Mentions: In our first tests, we used the MPI retroviral vector to transduce 8 dpc YS directly upon dissection. After dissociation, YS cells are seeded in a 48-well plate in 1 ml medium supplemented with cytokines and 4 μg polybrene (see Materials and Methods). After addition of the retroviral supernatant (MOI = 1), the culture is maintained at 37°C, 5% CO2 for 12 hours and re-plated in fresh medium and either seeded on OP9 stromal cells or analysed for clonogenic potential in methylcellulose assay. In these conditions, the initial efficiency of transduction is reproducible and quantitatively high (60–70% GFP+ cells). However, the GFP+ population rapidly decreases in culture to completely vanish after 4–5 days (Fig. 6A). The 8 dpc YS is enriched in terminally differentiated hematopoietic cells and immature erythro-myeloid precursors only appear at the 2–5 somite-stage [25,26]. Accordingly, we suspect that the rapid exhaustion of the GFP+ population during the 4 days culture arises as a consequence of the relative maturity of the transduced cells, which therefore rapidly complete differentiation and disappear.


Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse yolk sac: a comparative study between in situ electroporation and retroviral transduction.

Giroux SJ, Alves-Leiva C, Lécluse Y, Martin P, Albagli O, Godin I - BMC Dev. Biol. (2007)

A: Compared evolution of cell production obtained from GFP+ cell sorted from 8 dpc YS infected upon explantation (between bracket: number of cells recovered at day 8), or after one day in organ culture. B: Evolution of GFP expression in the whole population or in GFP- cells sorted from transduced 8 dpc OrgD1-YS after culture on OP9 stromal cells. A steady state GFP expression is attained at day 4 post-infection. Moreover, GFP expression is acquired, during culture, by a subset of GFP- sorted cells. C: GFP expression in transduced OrgD1-YS cells (top panel left) persists in sorted GFP+ cells cultured on OP9 stromal cells (top panel right). The colonies generated by transduced cells also remain entirely GFP+ (bottom panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1941737&req=5

Figure 6: A: Compared evolution of cell production obtained from GFP+ cell sorted from 8 dpc YS infected upon explantation (between bracket: number of cells recovered at day 8), or after one day in organ culture. B: Evolution of GFP expression in the whole population or in GFP- cells sorted from transduced 8 dpc OrgD1-YS after culture on OP9 stromal cells. A steady state GFP expression is attained at day 4 post-infection. Moreover, GFP expression is acquired, during culture, by a subset of GFP- sorted cells. C: GFP expression in transduced OrgD1-YS cells (top panel left) persists in sorted GFP+ cells cultured on OP9 stromal cells (top panel right). The colonies generated by transduced cells also remain entirely GFP+ (bottom panel).
Mentions: In our first tests, we used the MPI retroviral vector to transduce 8 dpc YS directly upon dissection. After dissociation, YS cells are seeded in a 48-well plate in 1 ml medium supplemented with cytokines and 4 μg polybrene (see Materials and Methods). After addition of the retroviral supernatant (MOI = 1), the culture is maintained at 37°C, 5% CO2 for 12 hours and re-plated in fresh medium and either seeded on OP9 stromal cells or analysed for clonogenic potential in methylcellulose assay. In these conditions, the initial efficiency of transduction is reproducible and quantitatively high (60–70% GFP+ cells). However, the GFP+ population rapidly decreases in culture to completely vanish after 4–5 days (Fig. 6A). The 8 dpc YS is enriched in terminally differentiated hematopoietic cells and immature erythro-myeloid precursors only appear at the 2–5 somite-stage [25,26]. Accordingly, we suspect that the rapid exhaustion of the GFP+ population during the 4 days culture arises as a consequence of the relative maturity of the transduced cells, which therefore rapidly complete differentiation and disappear.

Bottom Line: Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated.We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U790, Institut Gustave Roussy-PR1, Villejuif, France. Sebastien.Giroux@u-bourgogne.fr <Sebastien.Giroux@u-bourgogne.fr>

ABSTRACT

Background: Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.

Results: One transduction protocol involves transient modification of gene expression through in situ electroporation of the prospective blood islands, which allows the evolution of transfected mesodermal cells in their "normal" environment, upon organ culture. Following in situ electroporation of a GFP reporter construct into the YS cavity of embryos at post-streak (mesodermal/pre-hematopoietic precursors) or early somite (hematopoietic precursors) stages, high GFP expression levels as well as a good preservation of cell viability is observed in YS explants. Moreover, the erythro-myeloid progeny typical of the YS arises from GFP+ mesodermal cells or hematopoietic precursors, even if the number of targeted precursors is low. The second approach, based on retroviral transduction allows a very efficient transduction of large precursor numbers, but may only be used to target 8 dpc YS hematopoietic precursors. Again, transduced cells generate a progeny quantitatively and qualitatively similar to that of control YS.

Conclusion: We thus provide two protocols whose combination may allow a thorough study of both early and late events of hematopoietic development in the murine YS. In situ electroporation constitutes the only possible gene transfer method to transduce mesodermal/pre-hematopoietic precursors and analyze the earliest steps of hematopoietic development. Both in situ electroporation and retroviral transduction may be used to target early hematopoietic precursors, but the latter appears more convenient if a large pool of stably transduced cells is required. We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.

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