Lymphatic Endothelial Cell Differentiation from Human Pluripotent Stem Cells: A Novel Approach Using Transcription Factors and Hypoxia

Introduction: Generating new lymphatic vessels holds promise as a therapeutic strategy for various diseases, including neurodegenerative disorders, metabolic syndrome, cardiovascular disease, and lymphedema. However, efficient protocols to differentiate human induced pluripotent stem cells (hiPSCs) into lymphatic endothelial cells (LECs) are lacking. We describe a rapid and efficient method for generating LECs using the transcription factors ETS2 and ETV2, key regulators of embryonic vascular development and lymphatic specification. Our approach reproducibly differentiates four diverse hiPSC lines into LECs with exceptionally high efficiency.

Timely activation of ETS2 was critical for its interaction with Prox1, a master lymphatic regulator. The differentiated LECs express key lymphatic markers, VEGFR-3, LYVE-1, and Podoplanin, at levels comparable to mature LECs. Moreover, these LECs can assemble into stable lymphatic vascular networks in vitro and secrete the key lymphangiocrine factor, reelin. Our protocol has broad applications for the basic study of lymphatic biology and various approaches in lymphatic regeneration and personalized medicine.

Building upon this work, we investigated the role of hypoxia in the differentiation process. Hypoxia influences various aspects of stem cell biology and vascular development. We hypothesized that hypoxic conditions could enhance the efficiency and quality of LEC differentiation from hiPSCs. Our results demonstrate that exposure to hypoxia during key stages of differentiation significantly increases the expression of lymphatic markers, including VEGFR-3, LYVE-1, and Podoplanin, compared to normoxic conditions. This enhancement under hypoxic conditions suggests a synergistic role of hypoxia in promoting the functional and morphological maturation of LECs, underscoring its potential in optimizing stem cell-based regenerative therapies.

Materials and

Methods: Lentiviruses were produced in HEK293T cells by cotransfecting lentiviral vector DNA and helper plasmids. Viral titers were determined using qPCR. Human iPSCs were maintained in mTeSR1 medium on Matrigel-coated plates. For differentiation, hiPSCs were treated with CHIR99021 for 2 days, followed by lentiviral transduction of ETV2 and/or ETS2. The cells were then cultured in MV2 medium supplemented with VEGF-C and TGF-β inhibitor. Flow cytometry was used to analyze the expression of endothelial and lymphatic markers. Quantitative RT-PCR was performed to assess gene expression levels. ELISA was used to measure secreted proteins in cell culture supernatants. Tube formation assay was conducted by plating differentiated LECs on Matrigel and analyzing tube length and branch points. Statistical comparisons were made using Student's t-test for paired data and ANOVA for multiple comparisons with Tukey post hoc analysis.

Hypoxic experiments were conducted in the incubator at 5% oxygen level independent of ETS2 or ETV2 transduction. The differentiation process was driven by growth factors and small molecules. Different combinations of hypoxia/ normoxia were applied on S1/ S2 phase of the differentiation to check for better differentiation efficiency.

Results, Conclusions, and Discussions: We developed a feeder-free, chemically defined protocol that efficiently differentiates hiPSCs into lymphatic endothelial cells (LECs) using transcription factors ETV2 and ETS2. The three-stage protocol involves converting hiPSCs into mesodermal progenitor cells (MPCs), transducing MPCs with ETV2/ETS2 to generate endothelial cells (iECs), and treating iECs with VEGF-C and TGF-β inhibitor to produce lymphatic endothelial cells (iLECs). The protocol achieved up to 88.5% efficiency in generating CD144+/Prox1+ cells.

Transduction with ETS2 produced the highest efficiency of Prox1+ cells, which express high levels of lymphatic markers like LYVE-1 and Podoplanin, consistent with ETS2's interaction with Prox1, the master regulator of lymphatic genes. ETV2 transduction resulted in lower Prox1+ cell efficiency but higher LYVE-1 expression, corresponding to LECs in lymphatic capillaries. The differentiated LECs express key lymphatic markers comparable to primary LECs, form lymphatic networks in vitro, and secrete reelin, a key lymphangiocrine, at levels similar to primary LECs.

In the study investigating the effects of hypoxia on the differentiation of lymphatic endothelial cells (LECs), we observed that cells exposed to hypoxic conditions throughout the S1 and S2 phases of the differentiation exhibited poorer performance compared to cells maintained under normoxic conditions throughout the entire process. Interestingly, cells that were treated with normoxia during the S1 phase and subsequently exposed to hypoxia during the S2 phase demonstrated expression levels of VEGFR3, Podoplanin and Prox1 comparable to those of mature LECs. These findings suggest that the timing and duration of hypoxia exposure play a crucial role in the differentiation and maturation of LECs.

Overall, our findings demonstrate an efficient method for deriving LECs, which may be useful for modeling human diseases in vitro and understanding human lymphatic development. Future studies could investigate modulating ETV2 and ETS2 expression with modified mRNA to avoid introducing lentiviral components. The promising results of hypoxic treatment warrant further research into a stepwise, well-defined method for differentiating LECs from hiPSCs, with potential applications in lymphatic biology, regeneration, and personalized medicine.

Acknowledgements (Optional): We acknowledge support from the University of Notre Dame through “Advancing Our Vision” Initiative in Stem Cell Research, Harper Cancer Research Institute – American Cancer Society Institutional Research Grant (IRG-17-182-04), American Heart Association through Career Development Award (19-CDA-34630012 to D.H.-P.), National Science Foundation (2047903 to D.H.-P.) and from National Institutes of Health (1R35-GM-143055-01 to D.H.-P.).