More Efficient Generation of β-Globin-Expressing Erythroid Cells Using Stromal Cell-Derived Induced Pluripotent Stem Cells
Authors: Uchida Naoya , Atsushi Fujita , Juan Haro-Mora J , Winkler Thomas , Tisdale John F 
Published: October 2019

Abstract
Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. Using traditional embryoid body methods, iPS cell-derived erythroid cells predominantly produce ε-globin and γ-globin without β-globin expression. We recently demonstrated that ES cell-derived sacs (ES sacs), known to express hemangioblast markers, allow for efficient erythroid cell generation with β-globin production, which is associated with emergence of CD34+ hematopoietic stem/progenitor cells (HSPCs) (2014 ASH). In the current study, we extend this work to evaluate erythroid cell generation using iPS cell lines generated from various sources including patients with sickle cell disease (SCD).
To test our two hypotheses; (1) erythroid progenitor (EP)-derived iPS cells more efficiently differentiate to erythroid cells, and (2) stromal cell (ST)-derived iPS cells more efficiently emerge hemangioblast-like immature HSPCs which results in greater erythroid cell generation, we generated several clones of iPS cells which were derived from (1) EPs (6 clones) which were differentiated from peripheral blood mononuclear cells and (2) bone marrow STs (5 clones) in SCD patients. Transgene-free iPS cells were generated and characterized according to Merling et al. (Blood. 2013). These iPS cells and controls (2 clones of fibroblast (FB)-derived iPS cells and H1 ES cells) were used to generate ES/iPS sacs for 15 days. After a 2 day culture of ES/iPS sac-derived spherical cells on OP9 feeder cells, the suspension cells were differentiated into erythroid cells using human erythroid massive amplification culture for 13 days (Blood cells Mol Dis. 2002).
Following ES/iPS sac generation, 3.5-4.8 fold greater amounts of CD34+CD45+ HSPCs emerged in both EP- and ST-derived iPS sacs, compared to FB-derived iPS sacs (p<0.01). After an additional 2 weeks of erythroid differentiation, we observed 4.5-8.7 fold greater amounts of GPA+ erythroid cells from both EP- and SC-derived iPS sacs, compared to FB-derived iPS sacs (p<0.01). Interestingly, ST-derived iPS sacs resulted in 1.4-2.0 fold greater amounts of CD34+CD45+ HSPCs and GPA+ erythroid cells (p<0.01), compared to EP-derived iPS sacs. Higher β-globin expression (21.5±4.3%) was observed by RT-qPCR in erythroid cells from ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (4.4±2.5% and 8.3±4.2%, respectively, p<0.01), which was comparable to ES sacs (23.3%). Sickle hemoglobin was detected by hemoglobin electrophoresis. The ES/iPS sac-derived erythroid cell generation was more strongly affected by cell sources (5-6 fold larger SD) than variations among iPS cell clones. These data demonstrate that ST-derived iPS sacs allow more efficient erythroid cell generation with higher β-globin production, compared to EP- and FB-derived iPS sacs.
We hypothesized that ST-derived iPS sacs contain greater amounts of immature HSPCs (including hemogenic endothelium) and immature EPs (including megakaryoerythroid progenitors), since more expansion of ST-derived cells was observed during the late phase of erythroid differentiation, compared to EP- and FB-derived cells. We evaluated hemogenic endothelium markers at day 15, and observed 7.7 fold greater amounts of VEGFR+GPA- cells (p<0.01) and 1.3-1.4 fold greater amounts of CD31+CD34+ cells in ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (not detectable VEGFR+GPA- cells in EP-derived iPS sacs). Before erythroid differentiation, 3.2-16.4 fold greater amounts of GPA+CD41a+ megakaryoerythroid progenitors were observed in ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (p<0.05). In colony forming unit assays, 1.8-5.0 fold greater amounts of myeloid and erythroid colonies were observed in ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (p<0.01). These data suggest that ST-derived iPS sacs more efficiently produce immature HSPCs and immature EPs, which may result in more efficient generation of erythroid cells with β-globin production.
In summary, we demonstrated that human ST-derived iPS sacs allow for more efficient erythroid cell generation with higher β-globin production, which could be caused by heightened emergence of hemogenic endothelium in ST-derived iPS sacs. Our findings should be important for in in vitro iPS cell-derived erythroid cell generation with high β-globin expression.

Disclosures
Winkler: Novartis: Research Funding; GSK: Research Funding.