Generation of red blood cells from human induced pluripotent stem cells

Abstract

Differentiation of human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) into the erythroid lineage of cells offers a novel opportunity to study erythroid development, regulation of globin switching, drug testing, and modeling of red blood cell (RBC) diseases in vitro. Here we describe an approach for the efficient generation of RBCs from hiPSC/hESCs using an OP9 coculture system to induce hematopoietic differentiation followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. We showed that fibroblast-derived transgenic hiPSCs generated using lentivirus-based vectors and transgene-free hiPSCs generated using episomal vectors can be differentiated into RBCs with an efficiency similar to that of H1 hESCs. Erythroid cultures established with this approach consisted of an essentially pure population of CD235a(+)CD45(-) leukocyte-free RBCs with robust expansion potential and long life span (up to 90 days). Similar to hESCs, hiPSC-derived RBCs expressed predominately fetal γ and embryonic ɛ globins, indicating complete reprogramming of β-globin locus following transition of fibroblasts to the pluripotent state. Although β-globin expression was detected in hiPSC/hESC-derived erythroid cells, its expression was substantially lower than the embryonic and fetal globins. Overall, these results demonstrate the feasibility of large-scale production of erythroid cells from fibroblast-derived hiPSCs, as has been described for hESCs. Since RBCs generated from transgene-free hiPSCs lack genomic integration and background expression of reprogramming genes, they would be a preferable cell source for modeling of diseases and for gene function studies.

FIG. 1.

Schematic diagram of the protocol used for production of red blood cells from hESCs/hIPSCs. To induce hematopoietic differentiation, hESC/hiPSCs were cocultured with OP9 for 7–8 days. Two methods were used to produce red blood cells from differentiated PSCs. In Method A, CD34⁺ or CD31⁺ hematopoietic progenitors were isolated from differentiated PSCs and expanded/differentiated into red blood cells in low adherent conditions. In Method B, erythroid progenitors were selectively expanded from bulk cultures and maintained on MS5 feeders. Red blood cells were induced to mature in coculture with MS5 without cytokines. hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; SCF, stem cell factor; EPO, erythropoietin; TPO, thrombopoietin; IL, interleukin. Color images available online at www.liebertonline.com/scd

FIG. 2.

Expansion profiles of individual erythroid cultures from hESCs (A), transgenic (B), and transgene-free iPSCs (C). The cell expansion number at each time point is calculated as a total number of cells generated per 1 hESC/hiPSC induced to differentiate in coculture with OP9. Day 0 indicates the day when erythroid cultures were initiated from OP9 differentiated hESC/hiPSCs. E1–E3 refers to individual experiments. CD31 and CD34 indicate experiments in which erythroid cells were generated using Method A by isolating corresponding cell population. Nonseparated (NS) erythroid cells generated using Method B without isolation of progenitors (NS cells). Erythroid cultures from transgene-free iPSCs depicted in (C) were terminated at day 37. (D). Red blood cell pellet (6×10⁸ cells) derived from 10⁵ hESCs using Method A after 30 days expansion. (E) Flow cytometric analysis of cells shown in (D) demonstrates that these CD235a⁺ erythroid cells are essentially free of CD45⁺ leukocytes.

FIG. 3.

Flow cytometric analysis of erythroid cultures at different time of expansion. Representative experiments demonstrating H1 hESCs and TiPSC1 hiPSCs are shown. TiPSC, transgenic iPSC.

FIG. 4.

Morphology of PSC-derived erythroid cells at different stages of expansion and maturation. Wright-stained cytospin of H1 hESC and TiPSC1 are shown. Bars are 40 μm (expansion) and 20 μm (maturation). Color images available online at www.liebertonline.com/scd

FIG. 5.

Characterization of hemoglobin expressed in erythroid cells obtained from PSCs by quantitative polymerase chain reaction (A) and flow cytometry (B). Expression of hemoglobin was analyzed by polymerase chain reaction on days 10 and 20 of expansion. Scale bars show individual experiments. E1 and E2 indicate experiments 1 and 2, respectively. Flow cytometric analysis shows representative analysis of hemoglobin expression in erythroid cultures from H1 hESC and TiPSC1 expanded for 20 days.