Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells

Giarratana, Marie‐Catherine; Kobari, Ladan; Lapillonne, Hélène; Chalmers, David; Kiger, Laurent; Cynober, Thérèse; Marden, Michael C.; Wajcman, Henri; Douay, Luc

doi:10.1038/nbt1047

Cited by 518 publications

(622 citation statements)

References 42 publications

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“…22 The 3 culture steps produced similar results in our hands: at the end of the first step (day 10), the cell population was composed of 53% proerythroblasts (pro-EBs) and 36% basophilic erythroblasts (baso-EBs). At the end of the second step (day 13), cultured cells were composed of 50% polychromatophilic erythroblasts (poly-EBs) and 40% orthochromatophilic erythroblasts (ortho-EBs).…”

Section: Expression Of Endogenous Ikaros During Erythroid Differentiasupporting

confidence: 68%

“…16,20,21 ␥-globin genes are first expressed in fetal liver and cord blood (CB) erythropoiesis. 19,22 Shortly after birth, a switch between fetal and adult globins allows the expression of ␦-and ␤-globin genes, and the decrease of ␥-globin gene expression. 21,23,24 Adult erythropoiesis recapitulates fetal-to-adult hemoglobin switching in the so-called "compressed switch."…”

Section: Introductionmentioning

confidence: 99%

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The role of Ikaros in human erythroid differentiation

Dijon

Bardin

Murati

et al. 2008

Blood

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Ikaros-a factor that positively or negatively controls gene transcription-is active in murine adult erythroid cells, and involved in fetal to adult globin switching. Mice with Ikaros mutations have defects in erythropoiesis and anemia. In this paper, we have studied the role of Ikaros in human erythroid development for the first time. Using a gene-transfer strategy, we expressed Ikaros 6 (Ik6)-a known dominant-negative protein that interferes with normal Ikaros activity-in cord blood or apheresis CD34 ؉ cells that were induced to differentiate along the erythroid pathway. Lentivirally induced Ik6-forced expression resulted in increased cell death, decreased cell proliferation, and decreased expression of erythroid-specific genes, including GATA1 and fetal and adult globins. In contrast, we observed the maintenance of a residual myeloid population that can be detected in this culture system, with a relative increase of myeloid gene expression, including PU1. IntroductionThe transcription factor Ikaros (also known as LyF-1) was first described to interact with Cd3␦ and TdT promoters in murine thymocyte cells. 1,2 Its sequence is highly conserved between mice and humans, [3][4][5] and its expression is high in the developing and adult hematopoietic systems. 1,6 Ikaros proteins are characterized by the presence of 2 Krüppel-like zinc finger domains. The N-terminal domain is involved in DNA binding, 2,7 while the C-terminal domain is required for homo-or heterodimerization with family members and is composed of 2 zinc fingers. 8 The Ikaros gene contains 7 translated exons and encodes 9 isoforms by means of alternative splicing that alters expression of exons 3 to 6. 2,7 In these different proteins, the number of N-terminal zinc fingers varies from 0 to 4; this combination determines DNA binding affinity. At least 3 zinc fingers in this domain are necessary to efficiently bind to DNA. Ik1, Ik2, Ik3, and IKX are considered as functional isoforms. However, Ik4 can only bind DNA on palindromic sequences. 7 Ik5, Ik6, Ik7, and Ik8 are considered dominantnegative (DN) isoforms because of their capacity to bind other isoforms and their inefficiency to bind DNA. 8 Gene-targeting studies evidenced the fundamental role of Ikaros in hematopoiesis, in particular in T and B lymphocytes, natural killer (NK) and dendritic cells, and stem cells. [9][10][11][12] In addition, analysis of Ikaros L/L mice (insertion of the ␤-Gal gene in exon-2 of the Ikaros gene) revealed that Ikaros is expressed at low levels in a majority of Ter-119 ϩ erythroid cells. 10 Other studies of Ikaros DN (deletion of exon 7) and null (deletion of dimerization domain) mice show that Ikaros is important for erythroid differentiation. 13 These mice display a decrease of erythroid precursor numbers, and a drop in hematocrit levels 2 to 3 weeks after birth. In MEL (murine erythroleukemia) cells, Ikaros is associated with the chromatin remodelling PYR complex which binds to an intergenic domain between the ␥-globin and ␤-globin genes, and facilitates the switch be...

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Section: Expression Of Endogenous Ikaros During Erythroid Differentiasupporting

confidence: 68%

Section: Introductionmentioning

confidence: 99%

The role of Ikaros in human erythroid differentiation

Dijon

Bardin

Murati

et al. 2008

Blood

View full text Add to dashboard Cite

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“…Then the erythroid protocol (D0 to 25) was adapted from our previous procedures. 3 From day 0 to day 8 dissociated hEB were plated at a density of 10 6 cells/mL in liquid culture medium containing 10% human plasma, insulin 10 mg/mL (Cellgenix, France) and 3 U/mL heparin, in the presence of SCF (100 ng/mL), IL-3 (5 ng/mL) and Epo (3 U/mL). From day 8 to day 11 the cells were suspended at a density of 3¥10 5 cells/mL and cultured in fresh medium supplemented with SCF (100 ng/mL) and Epo (3 U/mL).…”

Section: Erythroid Induction and Differentiationmentioning

confidence: 99%

“…1 This is the concept of cultured RBC produced ex vivo from hematopoietic stem cells originating from bone marrow, peripheral blood or cord blood. 2,3 Research is already focused on the feasibility of this approach at the industrial level [US Defence Advanced Research Programs Agency (USDARPA)'s 'Blood Pharming' program (2007) (http://www.darpa.mil/dso/solicitations/baa07-21mod6.html)]. On this background, the identification of an unlimited source of stem cells is a major objective to free the production of cultured RBC from the constraints of the supply of hematopoietic stem cells.…”

Section: Introductionmentioning

confidence: 99%

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

Lapillonne¹,

Kobari²,

Mazurier³

et al. 2010

Haematologica

213

256

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The online version of this article has a a Supplementary Appendix. BackgroundEx 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 MethodsWe 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. ResultsWe 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. ConclusionsRed 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. © F e r r a t a S t o r t i F o u n d a t i o n

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“…Several teams of investigators have already taken up this challenge [7,8], seeking to generate immortalized erythroid cell lines able to produce transfusable RBCs in vitro, which, were they to be established, would be an immensely valuable resource. Establishment of embryonic stem cell-derived erythroid progenitor cell lines represents one promising approach to the production of functional red blood cells [9].…”

Section: Yield (Of Red Cells)mentioning

confidence: 99%

Guest editorial: the seven wonders of erythropoiesis

Sawada

2011

Int J Hematol

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Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells

Cited by 518 publications

References 42 publications

The role of Ikaros in human erythroid differentiation

The role of Ikaros in human erythroid differentiation

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

Guest editorial: the seven wonders of erythropoiesis

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