Isolation and Differentiation of Erythroid Cells

Mononuclear cells were isolated from cord blood (CRB Saint- Louis Hospital, Paris, France) or adult bone marrow donors with written informed consent for research use, in accordance with the Declaration of Helsinki. The ethics evaluation committee of INSERM, the Institutional Review Board (IRB00003888), approved our research project (n. 16-319). Mononucleated cells were separated by Ficoll-Paque (Life Technologies) and purified with a human CD34 MicroBead Kit (cat. n. 130100453, Miltenyi Biotec). A two-step culture method for cell expansion and erythroid differentiation was used to obtain highly stage-enriched erythroblast populations.^{36 (link),37 (link)} Briefly, CD34⁺ cells (purity 94-98%) were cultured for 7 days with interleukin (IL)6, IL3 (10 ng/mL), and stem cell factor (SCF, 100 ng/mL) to expand hematopoietic progenitors.^{37 (link)} CD36⁺ erythroid progenitors were purified using magnetic microbeads (CD36 FA6.152 from Beckman Coulter and anti-mouse IgG1 MicroBeads from Miltenyi Biotec). Then, erythropoietin (EPO) was added for 10-18 days to allow erythroid differentiation. For assays of colony-forming cells (CFC, counted at 11-14 days), CD34⁺ hematopoietic stem cells were seeded in methylcellulose (H4034, StemCell Technologies). TGFbRI inhibitor SB431542 was from Selleckchem (cat. n. S1067)^{38 (link)} and TGFb was from Peprotech (cat. n. 100-21).

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Astori A., Matherat G., Munoz I., Gautier E.F., Surdez D., Zermati Y., Verdier F., Zaidi S., Feuillet V., Kadi A., Lauret E., Delattre O., Lefèvre C., Fontenay M., Ségal-Bendirdjian E., Dusanter-Fourt I., Bouscary D., Hermine O., Mayeux P, & Pendino F. (2020). The epigenetic regulator RINF (CXXC5) maintains SMAD7 expression in human immature erythroid cells and sustains red blood cell expansion. Haematologica, 107(1), 268-283.

Publication 2020

Ficoll-Paque anti-mouse IgG1 MicroBeads H4034 TGFb

Adult Assays Bone marrow Cell culture method Cells Committee ethics Cord blood Donors Erythroblast Erythropoietin Ficoll Hematopoietic Hematopoietic stem cells Human Igg1 Institutional review board Interleukin 6 Methylcellulose Microbeads Mouse Populations Sb431542 Stem cell factor Stemcell Tgfbri

Corresponding Organization :

Other organizations : Institut Cochin, Université Paris Cité, Inserm, Centre National de la Recherche Scientifique, La Ligue Contre le Cancer, Laboratory of Excellence GR-Ex, Institut Curie, Université Paris Sciences et Lettres, Assistance Publique – Hôpitaux de Paris, Hôpital Cochin

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Variable analysis

independent variables

TGFbRI inhibitor SB431542

dependent variables

Colony-forming cells (CFC) counted at 11-14 days
Erythroid differentiation

control variables

Mononuclear cells isolated from cord blood or adult bone marrow donors
Ficoll-Paque separation and CD34+ cell purification
Two-step culture method for cell expansion and erythroid differentiation
CD34+ cells cultured with IL6, IL3, and SCF for 7 days
CD36+ erythroid progenitors purified using magnetic microbeads
Erythropoietin (EPO) added for 10-18 days to allow erythroid differentiation

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