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Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells

Maria Teresa Mitjavila-Garcia1,*,{dagger}, Michel Cailleret1, Isabelle Godin2, Maria Manuela Nogueira1, Karine Cohen-Solal1, Valérie Schiavon1, Yann Lecluse1, Françoise Le Pesteur1, Anne Hélène Lagrue1 and William Vainchenker1

1 INSERM U.362, Institut Gustave Roussy, Villejuif 94805, Cedex, France
2 CNRS FRE 2160, Institut d’Embryologie Cellulaire et Moléculaire, Nogent sur Marne, 94736, France
* Present address: INSERM U.421, Faculté de Médecine, 8 rue du Général Sarrail, 94010 Créteil Cedex, France



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  		Fig. 1. Immunophenotyping of EB6 cells. Flow cytometric analysis was performed as described in Materials and Methods with MoAbs recognizing murine cell surface hematopoietic antigens. The results of one representative experiment are shown. At least three experiments were carried out for each marker; for CD41, 15 experiments were performed. Control immunoglobulins, broken lines; specific monoclonal antibodies, unbroken lines.

 


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  		Fig. 2. Cell sorting of different CD34 and CD41 populations from EB6 cells. Double staining was performed with a FITC-conjugated anti-CD41 MoAb and a biotinylated anti-CD34 MoAb, using a streptavidin-R-PE as the secondary reagent. Four cell subsets (CD34+CD41+, CD34–CD41+, CD34+CD41– and CD34–CD41–) were sorted by flow cytometry in R1, R2, R3 and R4, respectively. The percentages are shown for one representative experiment (n=4).

 


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  		Fig. 3. Hematopoietic colony formation in EB6 cells from the different CD34 and CD41 populations. Various EB6 cell fractions were sorted by flow cytometry according to CD34 and CD41 expression and cells were plated in methylcellulose for colony assays. Control 1 (C1) is the unfractionated cell population. Control 2 (C2) corresponds to EB6 cells sorted with the side scatter and forward scatter gating used for CD41 purification (n=6). Results are expressed as percentages (number of progenitors for 100 plated cells).

 


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  		Fig. 4. Identification of myeloid progenitors present in the different CD34 and CD41 populations purified from EB6, yolk sac, aorta-gonad-mesonephros (AGM), fetal liver and bone marrow cells. The initial population (unfractionated) was subdivided into CD34+CD41+, CD34+CD41– and CD34–CD41+ cell populations for EB6, yolk sac (YS), fetal liver (FL) and bone marrow (BM) cells. AGM cells, were subdivided into the entire CD41+, the CD34+CD41– and CD34–CD41– cell fractions. Myeloid colony formation from each population was studied in methylcellulose. Results are expressed as a distribution (expressed in percentage) of various myeloid colonies including mixed colonies in each population.

 


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  		Fig. 5. Identification of primitive erythropoiesis in the different CD34 and CD41 populations purified from EB6 and yolk sac (YS) cells. Erythroid colonies derived from primitive erythropoiesis (PE) were identified on an inverted microscope. They were observed in both the CD41+ and CD41– cell fractions, but essentially in CD34–CD41+ cells. Results are expressed in percentages (ratio of the number of primitive erythroid colonies in each fraction to the total number of primitive erythroid colonies).

 


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  		Fig. 6. CD41 and CD61 are associated in EB6 cells. (A) Western blot analysis was performed in reducing conditions with 30 µg of protein from unfractionated EB6 (U), CD41+ (+) and CD41– (–) cells obtained after immunomagnetic purification and 5 µg of proteins from murine platelets (Pts) used as a control. The anti-CD41 MoAb (anti-GPIIb) (the same MoAb used for purification and cell sorting) detected a doublet at 125 kDa. This doublet was at the threshold of detection in the CD41– cell fraction and unfractionated EB6 cells. These data were confirmed with a polyclonal antibody directed against human CD41, which crossreacts with the murine protein. CD61 (GPIIIa) was detected as a 90 kDa protein, with a rabbit anti-CD61 human antibody used to probe the CD41+ cell fraction and mouse platelets. This band was less intense in the CD41– cell fraction and in the unfractionated EB6 cells. The expression of GPIIb and GPIIIa was drastically increased in the CD41+ versus CD41– cell fractions, when results are normalized to actin expression. (B) The anti-CD41 MoAb was used to immunoprecipitate CD41 protein from unfractionated EB6 cells (EB6) (300 µg of protein) and murine platelets (Pts) (30 µg of protein). Bone marrow cells from CD41 knockout mice (BM) and Baf3 cells (Baf3) (300 µg of proteins), were used as a negative control in these experiments. The immunoprecipitate was probed with rabbit anti-human CD41 and anti-human CD61 antibodies. Two major bands were detected: one at 125 kDa, corresponding to CD41, and another at 90 kDa, corresponding to CD61 in EB6 and platelets (but not in the negative controls). These bands were not detected when the immunoprecipitate was probed with a pre-immune rabbit serum (data not shown).

 


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  		Fig. 7. Cell sorting of CD41+ and CD41– cells from EB6, yolk sac, AGM, fetal liver and bone marrow cells. (A) Cells from yolk sac, AGM, fetal liver and bone marrow were sorted by flow cytometry according to CD41 antigen expression. The percentages of CD41+ and CD41– cells in one representative experiment are shown. The CD41– and CD41+ gates were slightly different in these various tissues. This is especially true for AGM, where a high autofluorescence is found in CD41– cells. (B) Content in progenitor cells in the CD41+ and CD41– cell fractions. An immunomagnetic technique was used to separate EB6 into CD41+ and CD41– cell fractions. The results for EB6 cells are the mean of the ten experiments. 9.5 dpc yolk sac (YS) (n=3), 10.5 dpc AGM (n=3), 13.5 dpc fetal liver (FL) (n=5) and bone marrow (BM) (n=5) were purified by flow cytometry according to CD41 expression. The number of progenitors (yield) in each fraction (expressed as percentage) was calculated as the ratio between the numbers of progenitors contained in each cell fractions to the number of progenitors contained in the starting population.

 


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  		Fig. 8. Cell sorting of different CD34 and CD41 populations from yolk sac (YS), AGM, fetal liver (FL) and bone marrow (BM) cells. Double staining was performed with an FITC-conjugated anti-CD41 MoAb and a biotinylated anti-CD34 MoAb with streptavidin-R-PE used as the secondary reagent. For yolk sac, fetal liver and bone marrow cells, four cell subsets represent CD34+CD41+, CD34–CD41+, CD34+CD41– and CD34–CD41– were sorted in the R1, R2, R3 and R4 gates, respectively. For AGM, three cell subsets, the entire CD41+ cell population, the CD34+CD41– and CD34–CD41– cell fractions were sorted, corresponding to the R1, R3 and R4 gates, respectively.

 




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