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M. Ackermann et al.
sorted populations confirmed the high expression of SOX17 in HEP, whereas the hematopoietic TF GATA2 and RUNX1 as well as the protein tyrosine phosphatase CD45 were particularly expressed in HP. We furthermore evalu- ated the expression of the TF GFI1, which has been shown to play a crucial role in the intra-embryonic EHT.32 In the hemanoids, GFI1 expression was first observed in KDR+ mesoderm and increased during further differentia- tion stages from HEP to HP. Quantitative PCR as well as whole transcriptome analysis confirmed increased expression of the IL3Ra already at the HEP stage (Figure 6A). In order to verify this early expression of the IL-3 receptor, we performed RNA in situ hybridization of E9.5 murine embryos, a developmental stage where Sox17+ hemogenic endothelium has already developed in the AGM, but no hematopoietic cells characterized by expression of Gata2 are yet detectable. Also in this in vivo scenario we observed partial co-expression of the IL3ra and Sox17 in cells lining the dorsal aorta before the EHT, although IL3ra appeared to be more widely expressed in the AGM domain (Figure 7).
Expression analysis of stage-specific gene ontologies in whole transcriptome data demonstrated characteristic expression profiles of all defined cell populations. Transcripts associated with pluripotency (black) such as NANOG, DNMT3B, and POU5F1 were highly expressed in iPSC and rapidly downregulated upon induction of hematopoietic differentiation. In contrast, genes associat- ed with primitive streak and mesoderm (dark blue) for- mation such as PDGFRA, HAND1 or CDH11 were highly enriched in the Tra-1-60-/KDR+ early mesoderm popula- tion. Emerging HEP showed characteristic expression of transcripts associated with blood vessel endothelium (green) such as genes involved in transforming growth factor (TGF) (e.g., ACVRL1) and Notch (NOTCH1, DLL4) signaling. When analyzing gene ontologies associated with hematopoiesis (red) we found the important regula- tor of EHT HOXA9 highly expressed in our HEP, further confirming their hematopoietic potential. Classical genes involved in hematopoiesis such as GFI1B, GATA2, GATA1, EVI2B, SPI1 or ANGPT1 were highly expressed in HP (Figure 6B). Gene set enrichment analysis con- firmed the endothelial phenotype of HEP as well as the upregulation of genes specific for HSC in HP (Figure 6C). Further analysis of HEP revealed an upregulation of genes associated with an arterial endothelium such as DLL4, EFNB2 and NOTCH1, whereas markers of venous endothelial fate did not reveal gene upregulation, except from EPHB4 (Online Supplementary Figure S4A and B, respectively). Next, we analyzed the signaling pathways important for the development of iPSC towards HP. Signaling pathways at the defined differentiation stages revealed the glycine, serine and threonine metabolism pathways as well as hedgehog and WNT signaling path- ways were highly enriched in the KDR+ early mesoderm population sorted from EB (EnrichR, KEGG2018). In con- trast, developing HEP showed highest enrichment scores for hematopoietic stem cell differentiation, angiogenesis, and dorsal aorta development (EnrichR, GO Biological Processes), whereas in HP, especially genes associated with IL5, IL-3 or B-cell receptor signaling were upregulat- ed when compared to HEP (EnrichR, WikiPathways, Online Supplementary Figure S4C).
Taken together, our hemanoid system recapitulates early human hematopoietic development and shows the
temporally regulated emergence of KDR+/CD34high/ CD144+/CD43–CD45– HEP with endothelial and hematopoietic potential and a characteristic transcription- al fingerprint. Also the emergence of clonogenic CD34low/CD144–/CD43+CD45+ HP expressing important TF characteristic for hematopoietic stem and progenitor cells was observed at later stages of differentiation.
Influence of IL-3 on the early hematopoietic specification of human induced pluripotent stem cells
After defining the spatiotemporal emergence of human HEP and HP in our hemanoid system, we next evaluated the effect of IL-3 on the emergence of early HP. We disso- ciated whole hemanoids cultured in different cytokine conditions at specific days of differentiation and analyzed the frequency of HEP and HP populations. No significant difference in the overall frequency of emerging CD34+ cells irrespective of the addition of IL-3 could be observed. Similarly, cultures treated with an anti-IL-3 antibody to block any effect by autocrine or paracrine secreted IL-3 within the hemanoids, revealed a similar frequency of total CD34+ cells (Figure 8A-B). However, when further analyzing the fate of CD34+ cells within the hemanoids by scoring CD144 (HEP) or CD45 (HP) expression, we noted strong differences in cultures treated with IL-3, compared to cultures in which IL-3 was omitted (w/o) or blocked by the addition of an IL-3 blocking antibody (w/o +IL-3 block). In IL-3 treated cultures or cultures w/o cytokines analyzed at day 8 of differentiation CD34+ con- tained CD34high/CD144+ and CD34low/CD45+ positive sub- populations (Online Supplementary Figure S5A). In contrast, nearly all CD34+ cells remained CD144+ and were unable to undergo the EHT process in cultures treated with an IL- 3 blocking antibody (Figure 8B). Moreover, CD34+ cells in hemanoids supplemented with IL-3 showed a decline in CD34high/CD144+ /CD45– HEP cells and an increasing pro- portion of CD45 expressing cells over time, suggesting successful EHT and the progression towards a CD34low/CD144–/CD45+ HP phenotype (Figure 8C). Interestingly, also without the exogenous addition of IL-3 we observed the emergence of 18.18±11,16% (mean± SD, n=6) CD34low/CD144–/ CD45+ HP in hemanoid day 8, an effect that could be completely blocked upon addition of an anti-IL-3 antibody to the differentiation cultures (1.85±1,90% HP, mean±SD, n=5). This data suggests local production of IL-3 by the hemanoid complex, which is sufficient to induce the EHT of some HEP (Figure 8C). In order to further specify at which stage the EHT is arrested upon IL-3 inhibition, we analyzed the expression of CD43, one of the earliest surface markers indicating the hematopoietic fate of human embryonic endothelium.33 Also the emergence of CD34+/CD144–/CD43+ was strong- ly reduced in cultures without addition of IL-3 and this effect was even more pronounced in cultures where autocrine/paracrine IL-3 signaling was blocked (Online Supplementary Figure S5B). In order to further assess the important role of IL-3 in the progression of HEP to HP, we subjected FACS-purified HEP from the hemanoid system to the OP-9 based in vitro EHT culture assay. Similar to our 3D hemanoid culture, we observed significantly more (>5 fold) CFU when IL-3 was added to the EHT culture medium compared to culture conditions without IL-3 (Figure 8D).
Of note, the stimulating effect of IL-3 on the EHT could not be recapitulated by the addition of Oncostatin M or
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haematologica | 2021; 106(5)