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M. Ackermann et al.
Likewise, IL-3 promotes HSC activity in E10 AGM and YS (at a time when normally no HSC are yet detected), sug- gesting an even earlier role of IL-3 during murine hematopoietic development. Furthermore, IL-3 has been demonstrated to elevate numbers as well as intrinsic hematopoietic commitment of the hemangioblast cells by inducing the downregulation of Flk-1 and upregulation of hematopoietic genes in single cell suspension derived from E7.5 embryos.12 These results are in line with our observations, showing that also in the human system, IL-3 can support the EHT and the emergence of CD144–/CD34low/CD43+/CD45+ HP with clonogenic potential.
Previous studies revealed IL-3 expressing cells within the lumen of the aorta in E11 murine embryos, indicating a physiological role of paracrine IL-3 in HSC regulation. In our hemanoid system, we also observed a more pro- nounced differentiation arrest when an IL-3 blocking anti- body was applied, suggesting the endogenous production of IL-3 within our hemanoids. This observation favors the concept, that within the hemanoids specific developmen- tal stages of embryonic development are recapitulated and also a specific microenvironment supporting the early hematopoiesis develops. However, endogenous IL-3 pro- duction was only observed in one of the employed iPSC lines, indicating that there is some heterogeneity in the differentiation. Here, further studies are needed to evalu- ate the differences of endogenous IL-3 production and to determine the source of IL-3 within the hemanoid microenvironment.
Despite the similarities of our hemanoid system to the murine in vivo scenario, a contradictory role of IL-3 has been reported. Rybtsov et al. propose that IL-3 only acts on E11 Type II pre-HSC (CD144+CD41low CD43+D45–), whereas SCF is the driving factor to induce engraftment potential and the emergence of definitive HSC from murine E9 pro-HSC (CD144+CD41+RUNX1+CD43–D45–). In our human model, however, we did not observe an effect of SCF on the EHT of HEP (CD144+CD34+CD43–CD45–) and subsequent emergence of either CD43+ or CD45+ cells, which only emerge when IL-3 signaling is active. However, the different subtypes are not that specifically defined in the human system and more studies are needed to shed further light onto the
exact mechanism of human hematopoietic development. In fact, our model would be of high value to study the effect of other cytokines known to promote the genera- tion of HSC in the adult system. Given the minimal cytokine administration schedule, it would be of great interest to study the effect of e.g., FLT3-L,TPO or other cytokines to improve robust HSC production in the future.
Our hemanoid system provides proof-of-evidence, that the spatial organization of developing cells in vitro has an effect on early human hematopoietic specification and that organoid-like structures generated from hPSC may be used to unravel new determinants in the generation of de novo HSC in the near future.
Disclosures
MA, HK, and NL are authors od a pending patent applica- tion: "Stem-cell derived myeloid cells, generation and use thereof, EP 17 169 454.0". All authors declare no conflict of interest.
Contributions
MA designed the study, performed research, analyzed and interpreted data, and wrote the manuscript; NL designed the study, interpreted data and wrote the manuscript; KH, HK, PK, ACW, DH, MA, MPK, SM and AHNN performed research and analyzed data; AK, MM, DJ and TT designed research, all authors reviewed the manuscript.
Acknowledgments
The authors thank Theresa Buchegger and Doreen Lüttge for excellent technical support. We are also grateful to Prof. Dr. T. Moritz for critical comments and support. We are grateful to M. Ballmaier (Cell Sorting Facility, Hannover Medical School). We moreover thank O. Papp and Prof. Dr. T Cantz for providing the F134iPSC.
Funding
This work was supported by grants from Hannover Medical School (Hochschulinterne Leistungsförderung [HiLF] to MA), the Joachim Herz Stiftung (Add-on Fellowship to MA), the Deutsche Forschungsgemeinschaft (Cluster of Excellence REBIRTH; Exc 62/3 to NL as well as DFG LA 3680/2-1), the European Union (ERC XHaLe to DJ) and the Else Kröner-Fresenius-Stiftung (EKFS; 2015_A92 to NL and EKFS; 2016_A146 to MA).
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