A novel human pluripotent stem cell-based gene activation system identifies IGFBP2 as a mediator in the production of hematopoietic progenitors in vitro

Reviewer #1 (Public Review):

Summary:

The work from Petazzi et al. aimed at identifying novel factors supporting the differentiation of human hematopoietic progenitors from induced pluripotent stem cells (iPSCs). The authors developed an inducible CRISPR-mediated activation strategy (iCRISPRa) to test the impact of newly identified candidate factors on the generation of hematopoietic progenitors in vitro. They first compared previously published transcriptomic data of iPSC-derived hemato-endothelial populations with cells isolated ex vivo from the aorta-gonad-mesonephros (AGM) region of the human embryo and they identified 9 transcription factors expressed in the aortic hemogenic endothelium that were poorly expressed in the in vitro differentiated cells. They then tested the activation of these candidate factors in an iPSC-based culture system supporting the differentiation of hematopoietic progenitors in vitro. They found that the IGF binding protein 2 (IGFBP2) was the most upregulated gene in arterial endothelium after activation and they demonstrated that IGFBP2 promotes the generation of functional hematopoietic progenitors in vitro.

Strengths:

The authors developed an extremely useful doxycycline-inducible system to activate the expression of specific candidate genes in human iPSC. This approach allows us to simultaneously test the impact of 9 different transcription factors on in vitro differentiation of hematopoietic cells, and the system appears to be very versatile and applicable to a broad variety of studies.

The system was extensively validated for the expression of 1 transcription factor (RUNX1) in both HeLa cells and human iPSC, and a detailed characterization of this test experiment was provided.

The authors exhaustively demonstrated the role of IGFBP2 in promoting the generation of functional hematopoietic progenitors in vitro from iPSCs. Even though the use of IGFBP2-interacting proteins IGF1 and IGF2 have been previously reported in human iPSC-derived hematopoietic differentiation in vitro (Ditadi and Sturgeon, Methods 2016; Ng et al., Nature Biotechnology 2016), and IGFBP-2 itself has been shown to promote adult HSC expansion ex vivo (Zhang et al., Blood 2008), its role on supporting in vitro hematopoiesis was demonstrated here for the first time.

Weaknesses:

Although the authors performed a very thorough characterization of the system in proof-of-principle experiments activating a single transcription factor, the data provided when 9 independent factors were used is not sufficient to fully validate the experimental strategy. Indeed, in the current version of the manuscript, it is not clear whether the results presented in both the scRNAseq analysis and the functional assays are the consequence of the simultaneous activation of all 9 TF or just a subset of them. This is essential to establish whether all the proposed factors play a role during embryonic hematopoiesis, and a more complete analysis of the scRNAseq dataset could help clarify this aspect.

Similarly, the data presented in the manuscript are not sufficient to clarify at what stage of the endothelial-to-hematopoietic transition (EHT) the TF activation has an impact. Indeed, even though the overall increase of functional hematopoietic progenitors is fully demonstrated, the assays proposed in the manuscript do not clarify whether this is due to a specific effect at the endothelial level or to an increased proliferation rate of the generated hematopoietic progenitors. Similar conclusions can be applied to the functional validation of IGFBP2 in vitro.

The overall conclusions are sometimes vague and not always supported by the data. For instance, the authors state that the CRISPR activation strategy resulted in transcriptional remodeling and a steer in cell identity, but they do not specify which cell types are involved and at what level of the EHT process this is happening. In the discussion, the authors also claim that they provided evidence to support that RUNX1T1 could regulate IGFBP2 expression. However, this is exclusively based on the enrichment of RUNX1T1 gRNA in cells expressing higher levels of IGFBP2 and it does not demonstrate any direct or indirect association of the two factors.

Reviewer #2 (Public Review):

To enable robust production of hematopoietic progenitors in-vitro, Petazzi et al examined the role of transcription factors in the arterial hemogenic endothelium. They use IGFBP2 as a candidate gene to increase the directed differentiation of iPSCs into hematopoietic progenitors. They have established a novel induced-CRISPR mediated activation strategy to drive the expression of multiple endogenous transcription factors and show enhanced production of hematopoietic progenitors through expansion of the arterial endothelial cells. Further, upregulation of IGFBP2 in the arterial cells facilitates the metabolic switch from glycolysis to oxidative phosphorylation, inducing hematopoietic differentiation. While the overall study and resources generated are good, assertions in the manuscript are not entirely supported by the experimental data and some claims need further experimental validation.

Author response:

We thank both reviewers for their feedback and for underlying the potential of our new tool and experimental approach to identify signalling molecules that can improve the in vitro derivation of specific cell types from human pluripotent stem cells. To address the reviewers' points we plan to carry out further analysis that should solidify our conclusions. We will also edit the text to temper conclusions where appropriate.