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Single-cell transcriptome analysis of embryonic and adult endothelial cells allows to rank the hemogenic potential of post-natal endothelium

Artem Adamov, Yasmin Natalia Serina Secanechia, Christophe Lancrin

Preprint posted on June 16, 2021 https://www.biorxiv.org/content/10.1101/2021.06.16.447688v1

Adamov et al. use scRNA-seq to rank the potential of adult mouse tissues to be reprogrammed into HSCs – providing insight into a potential alternative for transplant patients.

Selected by Bobby Ranjan

Background

Every year, thousands of patients require haematopoietic stem cell (HSC) transplantations to cure non-malignant diseases (e.g. thalassemia) and blood cancers like acute lymphocytic leukaemia (Passweg et al., 2019). Unfortunately, these transplantations require a suitable donor, which can be a major limitation. Cellular reprogramming of patient cells into HSCs has therefore been explored as an alternative to circumvent this problem.

In our body, HSCs are produced from endothelial cells, the building blocks of blood vessels, through a process called Endothelial to Haematopoietic Transition (EHT). EHT occurs in the embryo at major haematopoietic sites such as the Aorta-Gonad-Mesonephros (AGM) region and the Yolk Sac (YS). Multiple recent studies in non-human species have shown that non-embryonic/adult endothelial cells could also undergo EHT if exposed to the right conditions.

To explore the EHT potential of adult endothelial cells at single-cell resolution, Adamov et al. selected EHT-related single-cell clusters from three different datasets of the AGM region between E9.5 and E11 in the mouse (Embryo_dataset_1: Hou et al., 2020; Embryo_dataset_2: Vink et al., 2020; and Embryo_dataset_3: Zhu et al., 2020). These cells were integrated with data from 12 adult tissues from Tabula Muris, a consortium of murine single-cell data (Tabula Muris Consortium, 2018).

The authors compared the transcriptomes of adult and embryonic endothelial cells based on gene expression patterns and gene regulatory networks. Based on this analysis, they ranked adult tissues based on the potential of their endothelial cells to undergo EHT.

Key Findings

Single-cell gene and TF expression patterns do not provide direct evidence for EHT in adult mice

To find evidence of EHT in adult tissues, the authors clustered cells in the integrated single-cell dataset and identified fourteen distinct clusters. Of these, only one cluster (of 100% embryonic origin) specifically expressed hemogenic markers such as Runx1 (Sroczynska et al., 2009), Itga4 (Li et al., 2018) and Neurl3 (Hou et al., 2020). No such signature was observed in the adult cells.

Since there was no evidence of EHT in the overall gene expression patterns, the authors specifically looked for the expression of key transcription factors (TFs) crucial to the EHT process and haematopoiesis: Cbfa2t3, Cbfb, Erg, Fli1, Gata1, Gata2, Ldb1, Lmo2, Lyl1, Runx1 and Tal1 (aka “seed TFs”). In particular, the co-expression of Cbfb, Erg, Fli1, Gata2, Lmo2, Lyl1, Runx1 and Tal1 at the single-cell level was characteristic of the endothelial cells initiating the expression of haematopoietic genes (Bergiers et al., 2018). As Runx1 is the main regulator of EHT, (Lancrin et al., 2009; Sroczynska et al., 2009; LieA-Ling et al., 2018) endothelial cells expressing this transcription factor were of particular interest for further investigation.

The abovementioned TFs were highly expressed (detected in nearly all cells) and co-expressed (over half the cells expressed nine out of ten TFs simultaneously) in two out of the three embryonic datasets used. The authors attribute the anomalous TF expression in the third dataset to limitations in the 10X Genomics technology. In adult tissues, TF expression tended to be moderate (detected in roughly 25-50% of cells). The aorta, brain, lung and pancreas tissues had about half of the endothelial cells co-expressing five-to-seven TFs.

Both these results were consistent with the conventional belief in the field that EHT only occurs in embryonic tissues.

 

While many adult tissues had high overlap of target genes between seed TFs, overlap with Runx1 target genes was rare

The systematic difference in TF expression and co-expression between embryonic and adult endothelial cells could have an effect on their respective regulatory programs. To interrogate these differences in more detail, the authors constructed Gene Regulatory Networks (GRN) using the scTarNet R package (Bergiers et al., 2018). A GRN describes how TFs and their target genes interact with each other to influence gene expression.

The authors used seed TFs to construct a GRN for each tissue, and observed a strong positive association between TFs and their target genes in nearly all adult tissues. In the network constructed from embryonic endothelial cells, Runx1 target genes overlapped with those of Erg and Fli1, which could be indicative of EHT. In adult endothelial cell GRNs, despite the fact that target genes of many seed TFs had strong overlaps, an overlap of Runx1 target genes was rarely observed.

Runx1 gene expression is not sufficient to trigger EHT in adult endothelial cells

Clustering and differential expression analysis of Runx1+ endothelial cells versus the rest of the endothelial cells revealed 13 common marker genes across the adult tissues. Of these, only Cd44, Notch2 and Cd63 have been linked to haematopoiesis (Oatley et al., 2020). Intriguingly, adult Runx1+ endothelial cells showed no evidence of haematopoietic cell fate and expressed none of the key Runx1 targets in EHT (Lacrin et al., 2012), indicating that expression of Runx1 is not sufficient for EHT in adult endothelial cells.

Pancreas, brain, kidney and liver are the most suitable for reprogramming

Using the analyses conducted above, the authors computed a normalized aggregate score to rank adult tissues based on their likelihood to undergo EHT based on the following criteria:

  1. Expression of seed TFs,
  2. Co-expression of seed TFs, and
  3. Overlap between target genes of seed TFs in endothelial cells.

Figure 1 shows the relative aggregate scores of the adult tissues. In summary, the pancreas, brain, kidney and liver are the four most promising candidate tissues for undergoing EHT.

Figure 1. Identification of the most promising tissues for reprogramming. Ranking of the tissues according to the expression of the key transcription factors, their co-expression and the results of the network analysis. The top 4 organs are highlighted with their corresponding drawing.

Why I chose this preprint

As described above, there is significant clinical value in reprogramming cells from adult tissues into HSCs for patients requiring transplantations. In this preprint, the authors attempt to identify adult tissues having the highest potential for cellular reprogramming into HSCs using a computational single-cell-based strategy. In the process, they also make novel biological discoveries regarding the role of Runx1 in EHT. This resource could be a stepping-stone for attempting in vivo reprogramming by targeting the most promising endothelial cells in the adult organism.

References

  • Passweg JR, Baldomero H, Basak GW, et al. The EBMT activity survey report 2017: a focus on allogeneic HCT for nonmalignant indications and on the use of non-HCT cell therapies. Bone Marrow Transplant. 2019 Oct;54(10):1575-1585. doi: 10.1038/s41409-019- 0465-9.
  • Hou S, Li Z, Zheng X, et al. Embryonic endothelial evolution towards first hematopoietic stem cells revealed by single-cell transcriptomic and functional analyses. Cell Res. 2020 May;30(5):376-392. doi: 10.1038/s41422-020-0300-2.
  • Lancrin C, Mazan M, Stefanska M, et al. GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment. Blood. 2012 Jul 12;120(2):314-22. doi: 10.1182/blood-2011-10-386094.
  • Tabula Muris Consortium; Overall coordination; Logistical coordination; Organ collection and processing; Library preparation and sequencing; Computational data analysis; Cell type annotation; Writing group; Supplemental text writing group; Principal investigators. Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature. 2018 Oct;562(7727):367-372. doi: 10.1038/s41586-018-0590-4.
  • Vink CS, Calero-Nieto FJ, Wang X, et al. Iterative Single-Cell Analyses Define the Transcriptome of the First Functional Hematopoietic Stem Cells. Cell Rep. 2020 May 12;31(6):107627. doi: 10.1016/j.celrep.2020.107627.
  • Zhu Q, Gao P, Tober J, et al. Developmental trajectory of prehematopoietic stem cell formation from endothelium. Blood. 2020 Aug 13;136(7):845-856. doi: 10.1182/blood.2020004801.
  • Oatley M, Vargel Bölükbası Ö, Svensson V, et al. Single-cell transcriptomics identifies CD44 as a marker and regulator of endothelial to haematopoietic transition. Nat Commun. 2020 Jan 29;11(1):586. doi: 10.1038/s41467-019-14171-5.
  • Bergiers I, Andrews T, Vargel Bölükbaşı Ö, et al. Single-cell transcriptomics reveals a new dynamical function of transcription factors during embryonic hematopoiesis. Elife. 2018 Mar 20;7:e29312. doi: 10.7554/eLife.29312.
  • Li D, Xue W, Li M et al. VCAM-1+ macrophages guide the homing of HSPCs to a vascular niche. Nature. 2018 Dec;564(7734):119-124. doi: 10.1038/s41586-018-0709-7.
  • Sroczynska P, Lancrin C, Kouskoff V, Lacaud G. The differential activities of Runx1 promoters define milestones during embryonic hematopoiesis. Blood. 2009 Dec 17;114(26):5279-89. doi: 10.1182/blood-2009-05-222307.
  • Lie-A-Ling M, Marinopoulou E, Lilly AJ, et al. Regulation of RUNX1 dosage is crucial for efficient blood formation from hemogenic endothelium. Development. 2018 Mar 12;145(5):dev149419. doi: 10.1242/dev.149419.
  • Lancrin C, Sroczynska P, Stephenson C, Allen T, Kouskoff V, Lacaud G. The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage. Nature. 2009 Feb 12;457(7231):892-5. doi: 10.1038/nature07679.

 

Posted on: 15th July 2021

doi: https://doi.org/10.1242/prelights.30062

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Author's response

Christophe Lancrin and Yasmin Natalia Serina Secanechia shared

Have you tried to perform RNA velocity analysis to see whether adding information of unspliced reads can help identify adult endothelial cells that may have EHT tendencies?

CL:  We have not tried RNA velocity on these datasets. Using this tool may be helpful to highlight differences between endothelial cells but we are not certain that this could be readily applicable to determine EHT “tendencies”. The results of this analysis could be quite difficult to interpret.

Just as EHT only happens in a narrow time window during embryonic development, could the age of adult mice affect the potential of endothelial cells to be reprogrammed into HSCs?

CL: Actually, it has been shown in a study by Yvernogeau et al (2019, PMID: 31685991) that EHT could occur for a few weeks after birth in the mouse and chicken species. Then the process would stop. In short, it would seem that the age of an animal has a negative impact of the hemogenic capacity of endothelial cells. At the moment, we do not know why it is the case but discovering the reasons behind this block of EHT in adult animals would have a big impact to our reprogramming efforts.

YNSS: The age of the animal seems indeed to have an impact on the ability of its cells to be reprogrammed into haematopoietic progenitors. In 2014, Batta and co-workers (PMID: 25466247) showed that the ectopic expression of five of the “seed TFs” (Tal1, Lmo2, Runx1, Gata2 and Erg) could transdifferentiate mouse embryonic fibroblasts (MEFs) into multipotent haematopoietic progenitors more efficiently compared to mouse adult fibroblasts (MAFs), while the ectopic expression of only two “seed TFs” (Tal1 and Lmo2) could transdifferentiate MEFs but not MAFs. This suggests that adult fibroblasts might be less plastic compared to embryonic ones, and the same might also be true for endothelial cells. It is therefore possible that the age of the adult mouse could affect the potential of endothelial cells to be reprogrammed into HSCs. Nevertheless, thinking of future personalised medicine applications, it would be important to identify conditions that allow to generate HSCs from the endothelium or other tissues of adult animals of a wide range of ages.

Are the seed TFs and their corresponding target genes also crucial to the EHT process and haematopoiesis in humans?

CL: Yes. The seed TFs that we have highlighted in the preprint have the same roles in the human EHT. We therefore believe that our findings in the mouse could be applicable to the humans. Of course, in the longer term, studies with human endothelium single-cell transcriptomes would be better. However, the number of good qualities human datasets is not sufficient yet.

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