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. 2010 Oct;95(10):1651-9.
doi: 10.3324/haematol.2010.023556. Epub 2010 May 21.

Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine

Hélène Lapillonne  1 , Ladan Kobari, Christelle Mazurier, Philippe Tropel, Marie-Catherine Giarratana, Isabelle Zanella-Cleon, Laurent Kiger, Marie Wattenhofer-Donzé, Hélène Puccio, Nicolas Hebert, Alain Francina, Georges Andreu, Stéphane Viville, Luc Douay
Affiliations

Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine

Hélène Lapillonne et al. Haematologica. 2010 Oct.

Abstract

Background: Ex vivo manufacture of red blood cells from stem cells is a potential means to ensure an adequate and safe supply of blood cell products. Advances in somatic cell reprogramming of human induced pluripotent stem cells have opened the door to generating specific cells for cell therapy. Human induced pluripotent stem cells represent a potentially unlimited source of stem cells for erythroid generation for transfusion medicine.

Design and methods: We characterized the erythroid differentiation and maturation of human induced pluripotent stem cell lines obtained from human fetal (IMR90) and adult fibroblasts (FD-136) compared to those of a human embryonic stem cell line (H1). Our protocol comprises two steps: (i) differentiation of human induced pluripotent stem cells by formation of embryoid bodies with indispensable conditioning in the presence of cytokines and human plasma to obtain early erythroid commitment, and (ii) differentiation/maturation to the stage of cultured red blood cells in the presence of cytokines. The protocol dispenses with major constraints such as an obligatory passage through a hematopoietic progenitor, co-culture on a cellular stroma and use of proteins of animal origin.

Results: We report for the first time the complete differentiation of human induced pluripotent stem cells into definitive erythrocytes capable of maturation up to enucleated red blood cells containing fetal hemoglobin in a functional tetrameric form.

Conclusions: Red blood cells generated from human induced pluripotent stem cells pave the way for future development of allogeneic transfusion products. This could be done by banking a very limited number of red cell phenotype combinations enabling the safe transfusion of a great number of immunized patients.

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Figures

Figure 1.
Figure 1.
Schematic representation of the successive culture steps for production of cultured red blood cells (cRBC) from pluripotent stem cells. First step: clumps of undifferentiated hiPSC and hESC were cultured in “erythroid body (EB) medium” for 20 days. Second step: dissociated D20-EB were then cultured in a liquid medium for up to 25 days in the presence of sequential cocktails of cytokines (see Design and Methods section).
Figure 2.
Figure 2.
Phenotypic analyses of hiPSC-IMR90-16 (A, B, C) and hESC-H1-derived cells (D, E, F) during EB differentiation from day 0 to day 20. (A) and (D): percentage expression of undifferentiated cell markers (SSEA-4, Tra 1–60, Tra 1–81) and representative dot plots. (B) and (E): percentage expression of the hematopoietic markers (CD45, CD34) and representative dot plots. (C) and (F): percentage expression of erythroid markers (CD71, CD36, CD235a) and representative dot plots as determined by flow cytometry in one representative experiment.
Figure 3.
Figure 3.
Morphology of the erythroid cells generated from hiPSC-IMR90-16 (A) and hESC-H1 (B). During the second step of the protocol (differentiation and maturation to mature cultured RBC), aliquots of cells were taken at the indicated times for morphological analysis of the cells by May-Grünwald-Giemsa staining. Photographs show each stage of erythroid maturation on days 0, 8, 11, 15, 20, and 25 (magnification x 630). One representative experiment.
Figure 4.
Figure 4.
Analyses of cultured RBC maturation. (A) Kinetics of expression of antigens during erythroid differentiation from hiPSC-IMR90-16 and hESC-H1 by flow cytometry. (B) The size of mature cultured RBC (cRBC) generated from D25-hiPSC and D25 -hESC was compared to that of mature cRBC derived from CD34+ hematopoietic stem cells from leukapheresis (Lk) and control adult reticulocytes from peripheral blood (PB reticulocytes). Measures were performed in 100 cells with an optical micrometer and P values calculated by the Mann-Whitney test. One representative experiment.
Figure 5.
Figure 5.
Hemoglobin analyses and functionality of erythroid cells generated from hiPSC-IMR90-16 (A, B, C) and hESC-H1. (D, E, F). (AD) Representative RP-LC profiles of globin chains identified by mass spectrometry for D16 mature cultured RBC. (B, and E) CE-HPLC analysis of the hemoglobin for mature cultured RBC at D16. [*acetylated HbF, **non-acetylated HbF]. (C, and F) CO rebinding kinetics after flash photolysis of cultured RBC hemoglobin (black curves with triangles) and hemoglobin from control native cord blood RBC (black curves with circles). The two samples show similar binding properties, including the allosteric transition. The fraction of T-state tetramers is similar for hemoglobin from cultured RBC control, hiPSC and hESC at different intensities of laser photodissociation, The increase in allosteric transition to the low affinity T-state tetramers upon addition of 0.1 mM inositol hexaphosphate (IHP) is larger for the HbA than that for HbF and hemoglobin from cultured RBC, hiPSC and hESC with regard to the photodissociation level (about 30 % higher T-state transition). This is explained by a lower allosteric response of HbF to the IHP binding as already reported for 2,3 DPG.25
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