Immortalization of erythroblasts by c-MYC and BCL-XL enables large-scale erythrocyte production from human pluripotent stem cells

Abstract

The lack of knowledge about the mechanism of erythrocyte biogenesis through self-replication makes the in vitro generation of large quantities of cells difficult. We show that transduction of c-MYC and BCL-XL into multipotent hematopoietic progenitor cells derived from pluripotent stem cells and gene overexpression enable sustained exponential self-replication of glycophorin A(+) erythroblasts, which we term immortalized erythrocyte progenitor cells (imERYPCs). In an inducible expression system, turning off the overexpression of c-MYC and BCL-XL enabled imERYPCs to mature with chromatin condensation and reduced cell size, hemoglobin synthesis, downregulation of GCN5, upregulation of GATA1, and endogenous BCL-XL and RAF1, all of which appeared to recapitulate normal erythropoiesis. imERYPCs mostly displayed fetal-type hemoglobin and normal oxygen dissociation in vitro and circulation in immunodeficient mice following transfusion. Using critical factors to induce imERYPCs provides a model of erythrocyte biogenesis that could potentially contribute to a stable supply of erythrocytes for donor-independent transfusion.

Figure 1

c-MYC and BCL-XL Are Self-Replication Factors for Erythrocyte Progenitors Derived from Human PSCs (A) Numbers of glycophorin A (GPA)⁺ cells transduced with c-MYC on days 0, 14, and 21. (B) Expression of BCL-XL in cells transduced with vehicle (mock) or a c-MYC overexpression vector. (C) Numbers of GPA⁺ cells transduced with c-MYC alone, BCL-XL alone, or a combination of c-MYC plus BCL-XL on days 12, 18, and 28. (D and E) Expression of BCL-XL (D) and the annexin⁺ cell fraction (E) among cells transduced with c-MYC alone or c-MYC plus BCL-XL. Data in (A)–(E) represent the mean ± SE from three independent experiments. (F) Schematic diagram of immortalized erythrocyte progenitor cell (imERYPC) induction from human PSCs. Hematopoietic progenitor cells (HPCs) within ES/iPS sacs were collected and transduced with c-MYC and BCL-XL using doxycycline (DOX)-inducible lentiviral vectors. The cells were then expanded for 12 days (induction phase). For clonal selection, on day 12 of culture, the cells were transferred to semisolid culture medium containing DOX and cultivated for an additional 12 days (clonal selection phase). The hematopoietic clones were then picked up and cultured separately for several months (clonal expansion phase). (G) Numbers of GPA⁺ cells derived from two highly proliferative imERYPC clones (clones 8 and 16) from day 50 to day 80 of culture. These clones grew exponentially for several months. (H) Dot plots showing imERYPC clone 8 derived from human ESCs. Left panel: x axis, GFP; y axis, GPA. Right panel: x axis, CD71; y axis, GPA.

Figure 2

Immortalized Erythrocyte Progenitor Cells Can Be Differentiated into Functional Erythroblasts Exhibiting Hemoglobin Synthesis and Chromatin Condensation after Genes Are Turned Off (A) Numbers of the cells obtained from clones 8 and 16 cultured for 7 days in the presence or absence of DOX. The cells stopped growing when DOX was absent. Results are expressed as means ± SE from four independent experiments. (B) Photomicrographs showing representative Giemsa staining of imERYPC clone 8 in the presence of DOX or 2, 4, and 7 days after DOX off. (C) Transmission electron micrographs of imERYPC with DOX on (i) or 5 days after DOX off (ii and iii). Arrows indicate a mitochondrion. Arrowheads indicate chromatin condensation. Asterisks indicate endosomal vacuoles. (D) Pictures showing representative imERYPC clone 8 pellets obtained in the presence or absence of DOX. In the absence of DOX, cells generate heme and show the characteristic red color. (E) Percent heme-positive cells obtained 7 days after DOX withdrawal. Results are expressed as means ± SE from four independent experiments. (F) Chromatograms showing CE-HPLC analyses of hemoglobin generated in imERYPC clones 8 and 16. (G) Oxygen dissociation curves (ODCs) for adult erythrocytes derived from peripheral blood (PB-erythrocyte) and fetal erythrocytes differentiated from cord blood CD34⁺ cells (CB-erythrocyte) in imERYPC clone 8 and clone 16. (F) and (G) show representative data from three independent experiments.

Figure 3

Quantitative PCR Analysis during the Course of imERYPC and CB-Derived Erythroblast Maturation Time course of changes in the expression of total c-MYC, total BCL-XL, GATA1, RAF1, and GCN5 in imERYPCs after genes were turned off (A–E) or CB-erythroblasts (F–J). Black and blue bars represent clones 8 and 16, respectively, in (A)–(E). Results are expressed as means ± SE from four independent experiments.

Figure 4

In Vivo Circulation and Enucleation of Immortalized Erythrocyte Progenitor Cells in NOG Mice (A) Representative dot plots showing the chimerism of human PB-RBCs in NOD/SCID/IL-2Rg null (NOG) mice. A total of 1 × 10⁹ RBCs were transfused intraperitoneally, after which PB samples were collected on days 1, 2, and 3. (B) Schematic diagram of imERYPC transfusion into NOG mice. NOG mice irradiated with 2 Gy were intravenously administered clodronate-liposomes (1 mg/body, n = 6 from three independent experiments) or PBS (n = 6 from three independent experiments) and transfused intraperitoneally with 2 × 10⁹ human peripheral RBCs 13 days after irradiation. Then 24 hr later they received 1 × 10⁹ imERYPCs with 5 days of DOX off (clone 8) labeled with CFSE. Peripheral blood samples were then collected on days 1, 3, 5, and 7 after imERYPC transfusion. (C) Representative dot plots showing the circulation chimerism of imERYPCs (CSFE⁺) in mice treated with or without clodronate-liposomes. (D) Peripheral blood chimerism of imERYPCs on days 1, 3, 5, and 7 after transfusion. Black and blue bars represent the circulation rates in mice treated with (blue bars) or without (black bars) clodronate-liposomes. Results are expressed as means ± SE. (E) Representative Giemsa staining of sorted imERYPCs from mice treated with clodronate-liposomes. (F) Percent enucleation of circulating imERYPCs in mice. The enucleation ratios were calculated as Hoechst-negative cells per total CSFE⁺ imERYPCs. Results are expressed as means ± SE.