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3D iPSC-differentiation model
stem cell factor (SCF), both described as regulators of early hematopoietic development. While we did not see a change in the frequency of emerging HEP in the hemanoids, we observed a significant increase of HP only in cultures treated with IL-3, but not upon addition of Oncostatin M (Online Supplementary Figure S5C). Similarly, also the addition of SCF, independent of a simultaneous block of endogenous IL-3 signaling, did not lead to an increase in the frequency of HP compared to cultures without cytokine supplementation. Also, the combined supplementation of differentiation cultures with IL-3 and SCF did not lead to an increase in HP compared to cultures with only IL-3 supplementation (Online Supplementary Figure S5D).
Given the heterogeneity between different iPSC lines, we confirmed our key findings in a second iPSC line, which was derived from fetal fibroblasts (F134iPSC).34 Similar to our previous finding, the addition of only IL-3 resulted in the efficient production of hematopoietic cells from the hemanoids (Online Supplementary Figure S6A). In addition, the transition of HEP towards HP was again sig- nificantly improved by the addition of IL-3, highlighting the regulatory role of IL-3 signaling in our hemanoid sys- tem (Online Supplementary Figure S6B to D). Of note, in this iPSC line, we did not observe an effect of the IL-3-block- ing antibody, indicating that endogenous expression of IL- 3 is not a uniform feature of the hemanoids and might dif- fer between different iPSC lines, regardless of their hematopoietic differentiation potential upon cytokine supplementation.
In summary, we here highlight the potential use of a hemanoid-based iPSC model to study human hematopoi- etic development and demonstrate the important role for IL-3 in early human hematopoiesis by supporting the EHT of hemato-endothelial progenitor cells (Figure 8E).
Discussion
Although well studied in adult hematopoiesis, the role of cytokines in general and IL-3 in particular remains large- ly unexplored during human hematopoietic development. Utilizing an iPSC-based differentiation system, which recapitulates key stages of embryonic hematopoiesis, we here demonstrate an important role for IL-3 in the endothelial to EHT of KDR+/CD144+/CD34high/CD43–/ CD45– HEP.
We used a hematopoietic differentiation protocol, which maintains the 3D organization of iPSC-derived cells developing within an EB and introduced “hemanoids” as a model system to study early human hematopoietic specification. Notably, this system does not rely on excessive cytokine priming and might follow a more physiological differentiation process, as only low frequencies of CD34+ cells are detected. HEP developing within the hemanoids started to form endothelial tube-like structures already during the first days of differentiation and formed an organized/arborizing vasculature-like network within 8 to 16 days. A similar effect was recently observed by using a SOX17/RUNX1C transgenic hESC reporter-line.35 The authors demonstrated the development of HOXA+ hemogenic vasculature which resembles the AGM within an EB-based protocol. Interestingly, a simultaneous modulation of activin and WNT signaling by SB/CHIR during mesoderm patterning
was necessary to induce the development of an organized vasculature. In contrast, in our hemanoid system we observed the formation of vascular structures without fur- ther exogenous modification of these two pathways. Moreover, transcriptome profiling of developing HEP con- firmed the upregulation of genes associated with Notch signaling, an important regulator of EHT in hemogenic endothelium and indispensable for lympho-myeloid hematopoiesis and HSC-specification.36,37 Further pathway analysis revealed high scores for dorsal aorta develop- ment/regulation in HEP compared to HP, indicating the hemogenic capacity of this cell population. Likewise, analysis of specific genes involved in arterial and venous development indicate an arterial fate of the developing endothelium, an important prerequisite for the develop- ment of definitive hemogenic endothelium.38,39
In fact, the idea of maintaining the 3D organization of iPSC-derived cells during the differentiation process has been applied in a variety of different tissues such as kid- ney, liver, brain or gut in organoid-based differentiation systems.19 Those organoids are characterized by the self- organization of cells within a 3D complex, have been shown to recapitulate human organ development remark- ably similar to in vivo development and especially support the often problematic final maturation of iPSC-derived progeny as e.g., demonstrated for iPSC-derived hepato- cytes.40,41 Thus, it is likely that the recapitulation of the spatial organization and structures formed within the AGM might also support the maturation and definitive hematopoietic development of human iPSC, which remains hampered without genetic modification. However, it is worth mentioning that Ng and colleagues did not achieve long-term engraftment with the iPSC- derived cells from 3D cultures either,35 suggesting that important cues are still missing. Considering the onset of blood flow as an important instructive developmental sig- nal in the early embryo,42,43 maybe this or other important determinants have to be considered in more detail and implemented into the organoid-like differentiation sys- tems.
After we confirmed the suitability of our hemanoid- based differentiation protocol to recapitulate human hematopoietic development, we aimed to investigate the role of IL-3 on the hematopoietic specification of human iPSC. Although intensively studied during adult hematopoiesis, the role of IL-3 in early human hematopoi- etic development remains as of yet elusive. We demon- strate by flow cytometry and gene expression analysis, that HEP already express IL3RA before the EHT, indicating a responsiveness to IL-3 signaling. We also confirmed the physiological relevance of this finding in murine embryos and demonstrate the expression of the IL-3R in SOX17+, GATA2– E9.5 AGM cells. This observation is in line with previous reports, showing expression of IL-3R at very early stages of murine embryogenesis on E10.5 and E7.5 hemangioblastcells.12
Withdrawal of IL-3 or inhibition of IL-3 signaling arrest- ed the hematopoietic specification at the hemogenic endothelium stage. This observation concurs with previ- ous reports studying the role of IL-3 in early murine hematopoietic development. As an example, Robin and colleagues proved an IL-3 dependent amplification effect on the HSC pool in midgestation mouse embryos and showed, that IL-3 functions either as a proliferation and/or survival factor for the earliest HSC in the embryo.
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