Generation of engraftable hematopoietic stem cells from induced pluripotent stem cells by way of teratoma formation

Abstract

In vitro generation of hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) has the potential to provide novel therapeutic approaches for replacing bone marrow (BM) transplantation without rejection or graft versus host disease. Hitherto, however, it has proved difficult to generate truly functional HSCs transplantable to adult host mice. Here, we demonstrate a unique in vivo differentiation system yielding engraftable HSCs from mouse and human iPSCs in teratoma-bearing animals in combination with a maneuver to facilitate hematopoiesis. In mice, we found that iPSC-derived HSCs migrate from teratomas into the BM and their intravenous injection into irradiated recipients resulted in multilineage and long-term reconstitution of the hematolymphopoietic system in serial transfers. Using this in vivo generation system, we could demonstrate that X-linked severe combined immunodeficiency (X-SCID) mice can be treated by HSCs derived from gene-corrected clonal iPSCs. It should also be noted that neither leukemia nor tumors were observed in recipients after transplantation of iPSC-derived HSCs. Taken our findings together, our system presented in this report should provide a useful tool not only for the study of HSCs, but also for practical application of iPSCs in the treatment of hematologic and immunologic diseases.

Figure 1

Generation of transplantable hematopoietic stem cells (HSCs) from LG-iPSCs through teratoma formation. (a) The hypothesized model for the homing of iPSC-derived HSCs from teratoma into bone marrow (BM). (b) Strategy to induce HSCs from iPSCs through teratoma formation. With or without OP9 cells, iPSCs were subcutaneously injected into nude mice. Cytokines were administered for 2 weeks using a micro-osmotic pump. BM cells, among which iPSC-derived HSCs were detectable, were transplanted into irradiated mice. (c) Flow cytometric analysis of peripheral blood (PB) in teratoma-bearing nude mice 12 weeks after iPSC injection. Numbers represent percentages of GFP⁺/CD45⁺ cells. (d) Time course change of GFP⁺/CD45⁺ cells in PB dependent on teratoma size. The X-axis represents teratoma size (***P < 0.001). (e) Percentages of GFP⁺/KSL cells in BM of nude teratoma-bearing mice. Analysis conducted 12 weeks after iPSC injection (n = 5 per group). Error bars represent SEM (***P < 0.001). (f) Hundred GFP⁺ CD34⁻ KSL cells in BM of teratoma-bearing mice were single-cell sorted and cultured for 10 days with cytokines for hematopoietic differentiation. (g) BM transplantation assay for chimerism of LG-iPSC–derived GFP⁺/CD45⁺ cells in PB of recipient mice. BM cells of teratoma-bearing mice were transplanted into irradiated mice. Secondary transplantation was also performed 12 weeks after primary transplantation. (Primary, n = 4; secondary, n = 10.) Error bars represent SEM. CFU-C, colony-forming unit in culture; GFP, green fluorescent protein; GM, granulocyte macrophage; GMM, granulocyte macrophage megakaryocyte; GME, granulocyte macrophage erythroid; GEMM, granulocyte erythroblast macrophage megakaryocyte multilineage; LG-iPSC, Lnk^−/− GFP transgenic mice-induced pluripotent stem cell; N.D., not detectable; SCF, stem cell factor; TPO, thrombopoietin.

Figure 2

Generation of transplantable hematopoietic stem cells (HSCs) from G-iPSCs through teratoma formation, with the gene therapy model of X-SCID. (a) Percentages of GFP⁺/CD45⁺ cells in peripheral blood (PB) of nude teratoma-bearing mice. Analysis was conducted 12 weeks after iPSC injection (n = 4 per group). Error bars represent SD (*P < 0.05, **P < 0.01). (b) Hundred GFP⁺ CD34⁻ KSL cells in bone marrow (BM) of teratoma-bearing mice were single-cell sorted and cultured with cytokines for hematopoietic differentiation. Numbers and types of CFC-GEMM were evaluated on day 10. (c) BM transplantation assay for chimerism of G-iPSC–derived GFP⁺ hematopoietic cells/CD45⁺ cells in PB of recipient mice. Whole BM cells of teratoma-bearing mice were transplanted into primary recipient mice. Forty FACS-sorted GFP⁺ CD34⁻ KSL cells in BM of primary recipient mouse were transplanted into secondary recipient mice. (Primary, n = 6; secondary, n = 5.). Error bars represent SD. (d) Flow chart explaining the generation of T cells from mγc-iPSCs in X-SCID mice. iPSCs were established from X-SCID mice and mγc was transduced, yielding mγc-iPSCs. mγc-iPSCs and OP9 cells were injected into X-SCID mice to generate teratomas. (e) Flow cytometric analysis of PB in teratoma-bearing mice 12 weeks after iPSC injection. Numbers represent percentages. CFC-GEMM, colony-forming cell-granulocyte erythroblast macrophage megakaryocyte multilineage; EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting; G-iPSC, GFP transgenic mice-induced pluripotent stem cell; IRES, internal ribosomal entry site; LTR, long terminal repeat; SCF, stem cell factor; TPO, thrombopoietin; X-SCID, X-linked severe combined immunodeficiency.

Figure 3

Induction of engraftable hematopoietic stem cells (HSCs) from human iPSCs through teratoma formation. (a) Strategy to induce HSCs from human induced pluripotent stem cells (iPSCs) through teratoma formation. iPSCs were injected with OP9 cells into testes of NOD/SCID mice. Cytokines were administered for 2 weeks via a micro-osmotic pump. BM cells of teratoma-bearing mice were transplanted into sublethally (3 Gy) irradiated NOD/SCID or NOD/SCID/JAK3^null mice. (b) Flow cytometric analysis of PB (left panel) and BM cells (right panels) in teratoma-bearing mice 12 weeks after human iPSC (hiPSC) injection. (c,d) Colony-forming assay with human iPSC-derived hCD45^dull hCD34⁺ HSCs isolated from BM of teratoma-bearing mice. (c) Wright-Giemsa staining of cytospin preparations of CFU-GEMM mixed colonies. (d) Reverse transcription-PCR analysis for the expression of embryonic, fetal, and adult globins in cells taken from CFU-E colonies derived from human iPSCs. Human (h) PB cDNA was used as positive control. CB-derived CFU-E cDNA was used as control. Water is the negative control. HBE1: ε chain, HBZ: ζ chain, HBA1: α chain, HBG1: γ chain, HBB: β chain, and HBD: δ chain. (e) mCD45⁻ hCD45⁺ hCD34⁺ 600 cells in BM of teratoma-bearing NOD/SCID mice were transplanted into irradiated NOD/SCID/JAK3^null mice with 2 × 10⁵ NOD/SCID/JAK3^null BM cells. PB chimerism in recipient mice 12 weeks after BM transplantation. APC, allophycocyanin; BM, bone marrow; CB, cord blood; CFU-E, colony-forming unit-erythroid; PB, peripheral blood; SCID, severe combined immunodeficiency.

Figure 4

Hematopoietic stem cell (HSC) niche environment formation in GFP-iPSC–derived teratomas. (a) Immunostaining of CD45⁺ cells in G-iPSC–derived teratomas. Bar, 75 μm. (b) Flow cytometric analysis of iPSC-derived GFP⁺ and GFP⁻ CD45⁺ cells and KSL cells in bone marrow (BM) and teratomas (TR). KSL cells in teratomas ranging from 93 to 620 cells. DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; G-iPSC, GFP transgenic mice-induced pluripotent stem cell.