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In this study, hematopoietic precursors were shown to acquire long-term multilineage hematopoietic …
Biology Articles » Methods & Techniques » 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 » Figures
Figure 1 Determination of the development stages enriched in immature precursors. Upper panel: Temporal evolution of lmo2 expression from the OB to LHF stages: initially expressed by most extra-embryonic mesodermal cells (A), lmo2 expression rapidly restricts to a smaller cell number at the EB stage (B). Lmo2 expression subsequently expands with the development of blood islands endothelial and hematopoietic cells from LB (C) to LHF (D), and subsequent stages. Middle panel: Transcripts of the embryonic globin β-H1 are present in a minute cell number at the OB/EB stages (E, F). During the following stages (LB: G to LHF: H, and subsequent stages), β-H1 expressing erythroid cells rapidly expand. The arrows in the upper and middle panels point to equivalent zone of the blood islands. Lower panel: The comparative evolution of lmo2 and β-H1 expressions points to embryos at the OB/EB stages as enriched in immature precursors. Scale bar: 100 μm.
Figure 2 Plasmid injection protocol. Abbreviations: am: amnion; EPC: Ectoplacental cone; YSC: YS cavity OB-EB stage embryos are dissected from the decidua (A). The Reichert membrane is removed, while the EPC is kept in place (B). The plasmid solution is injected into the YSC, from the node region through the amnion (C). Plasmid filling of the YSC is visualised with fast green (D). Prior to pulse application, the electrodes are positioned parallel to the prospective blood islands ring (E). After electroporation, the YS is dissected from the embryo and placed in organ culture (D). Scale bar: 100 μm.
Figure 3 In situ electroporation parameters. Left: Scheme of a square wave pulse delivered during in situ electroporation. Right: Optimal parameters used for YS in situ electroporation at the OB-EB stages.
Figure 4 Cytological analysis of electroporated OrgD3 YS-explants. Abbreviations: AFP: α-foeto-protein; Control non-electroporated YS-explants (A), maintained in organ culture, organise into a "bubble-like" structure that contains a compacted part, at the site adhering to the culture dish (asterisk). Erythroid cells (arrow), differentiated from explanted YS mesoderm, are located close to this adhering site (A). The "bubble-like" part harbours clusters similar to YS-blood islands (B: Enlargement of the square in A), which also contains erythroid cells (arrow). CD31+ cells are present in the both in the "bubble-like" structure and adhering site (asterisk) (C). Whereas in the adhering site, the nature of labelled cells (arrows) is unclear, CD31+ endothelial cells (D) are clearly present cells in the bubble. In electroporated YS explants, both without (E, F) or with (G, H) plasmid, the endoderm, revealed by AFP wholemount in situ hybridization, remains external, but does not cover the whole mesoderm, as shown on the sections of wholemount embryos (F, H) made along the axis shown in E, G. β-H1+ erythroid cells (I, control YS and J, electroporated YS) are mostly located at the adhering site (asterisk), while lmo2-expressing cells (K, control YS) are distributed either in the adhering site (asterisk) or in close contact with the endoderm (arrow). At the end of the organ culture step, both the blood islands-like clusters and the adhering sites (L, N) display GFP+ cells (M, O: same explants as L, N), often in area containing erythroid cells (arrows). Scale bars: 100 μm, except D: 20 μm.
Figure 5 A: Hematopoietic progeny obtained from OrgD3 electroporated YS. GFP+ cells display hematopoietic markers indicating that pre-hematopoietic mesodermal cells were targeted. B: GFP+ cells sorted from electroporated OrgD3-YS give rise, after a 5 day culture on OP9 stroma, to the hematopoietic progeny typical of the YS, i.e. few precursors (left panel), myeloid (middle panel) and erythroid (right panel) cells. C: Frequency of hematopoietic precursors within OrgD3 control YS (see also Table 1) obtained by limiting dilution assay.
Figure 6A: 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).
Figure 7Hematopoietic development from transduced cells. A: Comparison of the clonogenic potential of GFP+ cells sorted at day 4 post-infection from YS infected upon explantation or after one day in organ culture. B: Phenotype analyses of OrgD1-YS infected cells cultured for 1 day on OP9 stromal cells, show that erythroid (Ter199+) and myeloid (CD45+) cells are identically distributed within GFP+ and GFP- subsets. C: At day 4 post infection, the distribution of erythroid (Ter199+) and myeloid (CD45+) cells within the GFP+ population is similar to that obtained from normal YS (never exposed to viral supernatant). D: Clonogenic assays, performed at day 1 (left panel) and 4 (right panel) post-infection, indicate that the transduction procedure does not modify the type and numbers of precursors recovered.
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