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. 2024 Mar 22;21(Supplemental):e211015.
doi: 10.2142/biophysico.bppb-v21.s015. eCollection 2024.

Real-time imaging of human endothelial-to-hematopoietic transition in vitro using pluripotent stem cell derived hemogenic endothelium

Yuriko Yoneda  1 , Hisaya Kato  1 , Yoshiro Maezawa  1 , Koutaro Yokote  1 , Mio Nakanishi  1
Affiliations

Real-time imaging of human endothelial-to-hematopoietic transition in vitro using pluripotent stem cell derived hemogenic endothelium

Yuriko Yoneda et al. Biophys Physicobiol. .

Abstract

During embryogenesis, human hematopoietic stem cells (HSCs) first emerge in the aorta-gonad-mesonephros (AGM) region via transformation of specialized hemogenic endothelial (HE) cells into premature HSC precursors. This process is termed endothelial-to-hematopoietic transition (EHT), in which the HE cells undergo drastic functional and morphological changes from flat, anchorage-dependent endothelial cells to free-floating round hematopoietic cells. Despite its essential role in human HSC development, molecular mechanisms underlying the EHT are largely unknown. This is due to lack of methods to visualize the emergence of human HSC precursors in real time in contrast to mouse and other model organisms. In this study, by inducing HE from human pluripotent stem cells in feeder-free monolayer cultures, we achieved real-time observation of the human EHT in vitro. By continuous observation and single-cell tracking in the culture, it was possible to visualize a process that a single endothelial cell gives rise to a hematopoietic cell and subsequently form a hematopoietic-cell cluster. The EHT was also confirmed by a drastic HE-to-HSC switching in molecular marker expressions. Notably, HSC precursor emergence was not linked to asymmetric cell division, whereas the hematopoietic cell cluster was formed through proliferation and assembling of the floating cells after the EHT. These results reveal unappreciated dynamics in the human EHT, and we anticipate that our human EHT model in vitro will provide an opportunity to improve our understanding of the human HSC development.

Keywords: aorta-gonad-mesonephros region; hematopoietic stem and progenitor cell; live cell imaging.

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Figures

Figure 1
Figure 1
(A) Schematic representation of the two protocols to induce differentiation of human PSCs into HE based on two previous studies by Uenishi et al. and Shen et al. Wnt represents chemical Wnt activators. (B) Flow cytometric analysis of the cultures containing CD43–CD34+CD31+CD184+ HE-like cells induced from human PSCs with the protocols based on Uenishi et al. (upper panels) and Shen et al. (lower panels) represented in (A).
Figure 2
Figure 2
(A) Phase contrast images of the PSC-derived HE cultures on day 6 (1 day after re-seeding the induced HE), and on days 8 and 11 (3- and 6 days after re-seeding, respectively) with nascent blood-like cells. Scale bars=50 μm. (B) Flow cytometric analysis of the blood-like cells emerged from the PSC-derived HE harvested on day 12. Of note, CD31+ cell was not found among the 20,000 blood-like cells analyzed in the experiment.
Figure 3
Figure 3
Live-cell imaging of emergence of the blood-like cells from the PSC-derived HE starting at day 6 (1 day after re-seeding the induced HE). A single adherent epithelial cell indicated with arrowheads gradually transformed into a round blood-like cell (min 0–min 100), started to float (min 120–min 180), and then proliferated (min 200–min 240). Scale bar=10 μm.
See this image and copyright information in PMC

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