Serum- and stromal cell-free hypoxic generation of embryonic stem cell-derived hematopoietic cells in vitro, capable of multilineage repopulation of immunocompetent mice

Abstract

Induced pluripotent stem cells (iPSCs) may become a promising source for the generation of patient-specific hematopoietic stem cells (HSCs) in vitro. A crucial prerequisite will be the availability of reliable protocols for the directed and efficient differentiation toward HSCs. So far, the most robust strategy for generating HSCs from pluripotent cells in vitro has been established in the mouse model involving ectopic expression of the human transcription factor HOXB4. However, most differentiation protocols include coculture on a xenogenic stroma cell line and the use of animal serum. Involvement of any of both would pose a major barrier to the translation of those protocols to human autologous iPSCs intended for clinical use. Therefore, we asked whether long-term repopulating HSCs can, in principle, be generated from embryonic stem cells without stroma cells or serum. Here, we showed that long-term multilineage engraftment could be accomplished in immunocompetent mice when HSCs were generated in serum-free medium without stroma cell support and when hypoxic conditions were used. Under those conditions, HOXB4(+) embryonic stem cell-derived hematopoietic stem and progenitor cells were immunophenotypically similar to definitive bone marrow resident E-SLAM(+) (CD150(+)CD48(-)CD45(+)CD201(+)) HSCs. Thus, our findings may ease the development of definitive, adult-type HSCs from pluripotent stem cells, entirely in vitro.

Figure 1.

Characterization of hematopoietic differentiation of murine embryonic stem cells during EB development under serum and serum-free conditions. (A): Expression of CD41 and CD45 during EB development of embryonic stem cells differentiated with or without serum support. A representative result is shown. (B): In vitro proliferation of HOXB4-expressing ESC-HCs after dissociation of EBs at day 6 (EBd6) and continuation of suspension cultures in serum-free medium containing stem cell factor, Flt3-ligand, interleukin (IL)-3, IL-6, insulin-like growth factor-I, and dexamethasone (SFI36D). (C): Flow cytometry analysis of hematopoietic surface markers on ESC-HCs after 10 days of cultivation of dissociated EBd6 cells. Abbreviations: d, day; EB, embryoid body; ESC-HC, embryonic stem cell-derived hematopoietic cell; S, cytokine-supplemented serum; SF, serum-free.

Figure 2.

Influence of stromal cell and serum support on hematopoietic differentiation of HOXB4 expressing ESC-HCs under normoxic (20% pO₂) and reduced (5% pO₂) oxygen levels. Dissociated embryoid body day 6 cells generated under serum-free conditions were cocultivated with different stromal cell lines (AFT024, OP9 cells) or only in the presence of cytokines with and without serum supplementation. After 10 days of culture under normoxic (A, B) or reduced O₂ (C, D) levels, the contents of colony-forming cells and the hematopoietic immunophenotype were evaluated. Data are the average number of colony-forming cells performed in triplicate from two independent experiments. p values were determined by unpaired, two-tailed Student's t test. *, p < .05; **, p < .01; ***, p < .001. Abbreviations: CFU, colony-forming unit; ESC-HC, embryonic stem cell-derived hematopoietic cell; S, cytokine-supplemented serum; SF, serum-free; STFV, stem cell factor, thrombopoietin, Flt3-ligand, and vascular endothelial growth factor.

Figure 3.

Donor cell chimerism in irradiated primary immunocompetent recipient mice (129S6/SvEvTac). (A): Donor chimerism (percentage of GFP⁺ cells) in the peripheral blood of mice transplanted with 3 × 10⁶ hematopoietic cells differentiated from HOXB4-expressing embryonic stem cells and expanded for 10 days under hypoxic conditions with the support of AFT024 or OP9 stromal cells with cytokines. Analysis was performed 4 and 25 weeks after transplantation. Arithmetic means of the donor cell engraftments are shown. (B): Kaplan-Meier survival analyses of transplanted recipient mice. Abbreviations: BM, freshly isolated bone marrow cells from 129S6/SvEvTac mice (3 × 10⁶ cells per mouse); d, day; ESC-HC, embryonic stem cell-derived hematopoietic cell; GFP, green fluorescence protein; PB, peripheral blood; pTx, post-transplantation; w, week.

Figure 4.

Donor cell chimerism in immunocompetent recipient mice (129S6/SvEvTac) after limiting dilution of HOXB4⁺ embryonic stem cell-derived hematopoietic stem and progenitor cells (ES-HSPCs) cocultured on OP9 stroma. (A): Donor chimerism (percentage of GFP⁺ cells) in the peripheral blood of mice transplanted with HOXB4-expressing ES-HSPCs that had been expanded for 10 days under hypoxic conditions on OP9 stromal cells. Analysis was performed 7, 18, and 24 weeks after transplantation. The arithmetic means of the donor cell engraftments are shown. (B): Chimerism 18 weeks post-transplantation was predominantly myeloid (GFP⁺CD11b⁺). Lymphoid engraftment above 5% was observed only at a transplanted cell dose of 3 × 10⁶ cells. Arithmetic means of the assigned animal numbers are shown. Error bars represent ± SD. (C): Kaplan-Meier survival analyses of transplanted recipient mice. *, Ocular bacterial infection unrelated to any aplasia but necessitating euthanization. Abbreviations: BM, freshly isolated bone marrow cells from 129S6/SvEvTac mice (3 × 10⁶ cells per mouse); GFP, green fluorescence protein; PB, peripheral blood; pTx, post-transplantation; w, week.

Figure 5.

Donor cell chimerism in immunocompetent recipient mice (129S6/SvEvTac) of HOXB4⁺ embryonic stem cell-derived hematopoietic stem and progenitor cells (ES-HSPCs) with and without prior coculture on stroma. (A): Donor chimerism (percentage of GFP⁺ cells) in the peripheral blood of individual mice transplanted with 3 × 10⁶ HOXB4-expressing ES-HSPCs that had been expanded for 10 days under hypoxic conditions, either stroma-free with serum supplement (STFV+S) or on AFT024 or OP9 stromal cells. Analysis was performed 7, 18, and 24 weeks after transplantation. (B): Under all three conditions, chimerism 18 weeks post-transplantation was predominantly myeloid (GFP⁺CD11b⁺). Lymphoid engraftment (GFP⁺CD3⁺) above 5% was only observed after coculture on OP9 stroma cells. Arithmetic means of the donor cell engraftments are shown. Error bars represent ± SD. (C): Kaplan-Meier plots representing survival rates of transplanted recipient mice. *, Ocular bacterial infections unrelated to any aplasia but necessitating euthanization. Abbreviations: BM, freshly isolated bone marrow cells from 129S6/SvEvTac mice (3 × 10⁶ cells per mouse); GFP, green fluorescence protein; PB, peripheral blood; S, cytokine-supplemented serum; SF, serum-free; STFV, stem cell factor, thrombopoietin, Flt3-ligand, and vascular endothelial growth factor; pTx, post-transplantation; w, week.

Figure 6.

Donor cell chimerism in immunocompetent recipient mice (129S6/SvEvTac) of HOXB4⁺ embryonic stem cell-derived hematopoietic stem and progenitor cells (ES-HSPCs) grown under stroma-free conditions. (A): Donor chimerism (percentage of GFP⁺ cells) in the peripheral blood of individual mice transplanted with 3 × 10⁶ HOXB4-expressing ES-HSPCs that had been expanded for 23 days under hypoxic conditions, stroma-free and with or without serum supplement (STFV+S or STFV-SF, respectively). Analysis was performed 12 weeks post-transplantation. Median chimerism rates are shown. (B): Chimerism 12 weeks post-transplantation was predominantly myeloid (GFP⁺CD11b⁺) with a lymphoid engraftment (GFP⁺CD3⁺) <1%. Arithmetic means of the assigned animal numbers are shown. Error bars represent SD. (C): Flow-cytometric analysis for CD150, CD48, CD4, and CD201 expression after 23 days of cultivation. An E-SLAM⁺ cell population, being CD150⁺CD48⁻CD45⁺CD201⁺, was unambiguously detectable only in serum-free cultures, with an average frequency of approximately 0.8% of all nucleated cells. Abbreviations: ESC-HC, embryonic stem cell-derived hematopoietic cell; GFP, green fluorescence protein; PB, peripheral blood; pTx, post-transplantation; S, cytokine-supplemented serum; SF, serum-free; STFV, stem cell factor, thrombopoietin, Flt3-ligand, and vascular endothelial growth factor; w, week.