R11 - Application of iPSC-derived lymphatic and blood endothelial cells in tissue engineering

Introduction: : Induced pluripotent stem cell (iPSC)-derived endothelial cells (dEC) are crucial for developing engineered vascularized tissues for personalized therapeutics and drug screening applications. In particular, we focus on the lymphatic endothelial cells (LEC) due to their importance in immune functions, fluid homeostasis, and cytokine production which are crucial for generating mature, physiologically relevant engineered tissues. However, currently there are two major challenges to incorporating LECs into tissue engineering. First, there is a lack of efficient, xeno-free protocol to generate iPSC-derived LECs (dLEC) that retains both the transcriptomic and functional characteristics of native LECs. Second, there is a lack of mechanism to precisely control angiogenesis at the microvasculature level, as most approaches to including microvasculature relies on spontaneous angiogenesis. One potential approach to developing a highly efficient differentiation protocol for dLEC is through leveraging the unique metabolic needs of LECs. LECs rely heavily on fatty acid oxidation for its energy and for generation of acetyl-CoA used for activation of VEGFR3, an LEC-specific marker with a positive feedback loop to a master lymphatic regulator gene, Prox1. Additionally, in angiogenesis, ECs respond to growth factors to migrate and proliferate, which uses intracellular calcium as a signal transduction mechanism. This suggests that the endothelial cells can respond to external electrical current which affects the calcium signaling and therefore allow precise spatiotemporal angiogenesis control. In this study, we propose a differentiation protocol that yields functional dLECs that can be incorporated into in vitro and in vivo models which can respond to electrical stimulus to direct angiogenesis.

Materials and

Methods: : Based on the dEC protocol published by Harding and colleagues, we developed the following modified protocol (Fig 1A). We induce mesodermal identity through 2 days of treatment with 6μM of CHIR99021, then we induce endothelial identity through 2 days of treatment with 50ng/mL of VEGF-A, FGF2, and BMP4. Cells are replated to density of 50,000 cells/cm2. Lastly, we treat the cells with 100ng/mL of VEGF-C and up to 50mM of sodium acetate (NaAc) for 4 days to induce LEC markers.

The cells were embedded into various hydrogels and allowed to vascularize, then stained, imaged, and analyzed for functional lumens in vessels and compared to native LECs. To demonstrate the capability of our dLEC to integrate into in vivo models, 50μL of norbornene-modified hyaluronic acid (NorHA) gels containing dLECs were implanted into the mammary fat pads and allowed to integrate into the host’s lymphatic system. Furthermore, to demonstrate the therapeutic effect of our dLECs, we performed ligation of the two collecting lymphatic vessels in the tail and injected Matrigel-dLEC mixture into the wound site to promote lymphatic regeneration.
Lastly, we analyzed the calcium signaling pattern of native and differentiated ECs in response to VEGF stimulation by staining the cells with a calcium reporter dye. We optimized the electrical current and voltage values that mimics the calcium signaling pattern caused by VEGF, then analyzed the F-actin structure of the cells subject to VEGF treatment and current.

Results, Conclusions, and Discussions:: After 8 days of our differentiation protocol, we performed qPCR and flow cytometry for various LEC markers and CD31, a mature endothelial marker and found a significant increase in lymphatic markers PDPN and Prox1 when treated with 50mM NaAc (Fig 1B-C). We replicated the method across three iPSC cell lines and saw an increase in PDPN+/CD31+ LECs with NaAc treatment (Fig 1D). We also confirm that our cells express Prox1, VEGFR3, and PDPN at a significantly higher level compared to the current standard method of LEC differentiation using OP9 co-culture (Fig 1E-G).

We further found that our dLECs retain many of the key functionalities of native LECs, including cytokine production, transcriptomic profile, and angiogenic potential. dLECs expressed reelin, a key lymphatic cytokine, at a comparable level to native LECs (Fig 2A). Furthermore, dLECs formed networks in Matrigel- and fibrin-based angiogenesis assays (Fig 2B-G). The dLECs also formed functional 3D networks in NorHA gels, forming junctions and lumens similar to native lymphatic vasculature (Fig 2H-I). We also found that our dLECs can incorporate into the mouse vasculature and significantly reduce swelling of the mouse tail following lymphatic ligation (Fig 2J-K).

We also demonstrated our ability to mimic the endothelial response to VEGF using electrical current. We show that our dLECs have similar calcium signaling dynamic compared to native endothelial cells when fully matured (Fig 3A-C). Furthermore, we demonstrate that electrical stimulation can mimic both the calcium signaling pattern and actin remodeling phenomena that we observe with VEGF stimulation (Fig 3D).

Our results suggest that our proposed protocol results in a functional dLEC that retains the gene expression characteristics and the functionality of native LECs. We demonstrate the angiogenic potential of our dLECs by demonstrating that they can be successfully incorporated into engineered tissues and form functional vessels. We also show their therapeutic potential by demonstrating that our dLECs can reduce inflammation in mouse tail with damaged lymphatics. Lastly, our dLECs are potentially compatible with our proposed mechanism of using precise electrical stimulation to spatially control angiogenesis through selective actin remodeling, further demonstrating their value in the field of lymphatic tissue engineering.

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