Robust Differentiation of Human Pluripotent Stem Cells into Lymphatic Endothelial Cells Using Transcription Factors

Abstract

Introduction: Generating new lymphatic vessels has been postulated as an innovative therapeutic strategy for various disease phenotypes, including neurodegenerative diseases, metabolic syndrome, cardiovascular disease, and lymphedema. Yet, compared to the blood vascular system, protocols to differentiate human induced pluripotent stem cells (hiPSCs) into lymphatic endothelial cells (LECs) are still lacking.

Methods: Transcription factors, ETS2 and ETV2 are key regulators of embryonic vascular development, including lymphatic specification. While ETV2 has been shown to efficiently generate blood endothelial cells, little is known about ETS2 and its role in lymphatic differentiation. Here, we describe a method for rapid and efficient generation of LECs using transcription factors, ETS2 and ETV2.

Results: This approach reproducibly differentiates four diverse hiPSCs into LECs with exceedingly high efficiency. Timely activation of ETS2 was critical, to enable its interaction with Prox1, a master lymphatic regulator. Differentiated LECs express key lymphatic markers, VEGFR3, LYVE-1, and Podoplanin, in comparable levels to mature LECs. The differentiated LECs are able to assemble into stable lymphatic vascular networks in vitro, and secrete key lymphangiocrine, reelin.

Conclusion: Overall, our protocol has broad applications for basic study of lymphatic biology, as well as toward various approaches in lymphatic regeneration and personalized medicine.

Keywords: Biomedical engineering; Lymphatic biology; Stem cell differentiation; Tissue engineering; Transcription factors; Vascular biology.

Fig. 1.

Schematic of deriving LECs from hiPSCs. The cultured hiPSCs were transduced with viral vector containing specific transcription factor and seeded onto matrigel-coated plate. Next, the transcription factors were activated, and cells were cultured in differentiation media. Then differentiated ECs were seeded onto fibronectin-coated plates and treated with VEGF-C for 3 more days to make them committed to lymphatic endothelial lineage. The second row represents bright-field images of the corresponding cell states.

Fig. 2.

Characterization of differentiated LEC by flow cytometry. a–c Prox1 shift compared to Isotype control; d–f LYVE-1 and Podoplanin expression of three transduced group. h-iLECs were characterized by quantitative protein expression of Podoplanin, LYVE-1, and Prox1. Among three transduced group, ETS2 showed more Podoplanin expression leading up to over 88%, followed by ETV2+ETS2 group (44.2%) and ETV2 group. Similarly, Prox1 expression was higher in ETS2 group as well, rising up to 95% whereas the ETV2 and combination group showed 69.5% and 81% expression, respectively.

Fig. 3.

Immunofluorescent data show presence of key endothelial and lymphatic marker in h-iLECs. a–i represents ETV2, ETS2, and ETV2+ETS2 transduced group respectively. Blue, green, and red are indicator of DAPI, ETS-related gene and Prox1 in that order. All three-cohort shows nuclear presence of ERG and PROX1.

Fig. 4.

Genotypic characterization of iLECs. a–d qRT-PCR data of LYVE-1, PDPN, Prox1, and VEGRR3 of three transduced group, respectively, in each case growth factor derived LECs (endogenous activation of ETV2/ETS2) were used as control. The data show that all three transduced group showed higher expression of the genes of interest compared to control. Especially cohort having ETS2 showed very significant expression of all the LEC markers. Data represent mean ± standard deviation, n = 4 per condition. Significance levels were set at: *p < 0.5 and **p < 0.01. Three biological replicates (n = 3) were collected per condition and analyzed with real-time qRT-PCR.

Fig. 5.

Functional properties of iLECs. a–c Capillary-like network formation of ETV2, ETS2, and ETV2+ETS2 group where Live cells are stained by cell tracker green. d, e Quantitative analysis of tube formation assay by measuring the tube length and no. of branches respectively. f, g Quantification of Reelin and VEGFC secreted by the iLECs obtained through ELISA assay. Significance level was set at: **p < 0.01.