Engineering Functional Biomaterials with Stem Cells for Therapeutic Lymphangiogenesis

Introduction::
Controlling the formation of new lymphatic vessels has been suggested as an innovative therapeutic strategy for various disease phenotypes, including metabolic syndrome, Alzheimer’s, and cardiovascular diseases. Yet, little is known about the molecular regulation that controls stem cell differentiation and morphogenesis into lymphatic networks in a synthetic matrix.¹ Here, we hypothesize that strategies to derive lymphatic endothelial cells (LECs) from human pluripotent stem cells (hPSCs) can be used as a clinically relevant cell source for therapeutic lymphangiogenesis in a synthetic and controllable matrix environment.




Materials and Methods:: Stepwise differentiation protocols were used to induce hPSCs into mesodermal cell lineage and eventually LECs. Employing hyaluronic acid modified with norbornene (NorHA), hydrogels were designed with the ability to spatially and temporally pattern specific peptides and viscoelastic properties. Matrix metalloproteinase (MMP)-sensitive crosslinker allowed cells to degrade and remodel their matrix, and the incorporation of RGD-binding motif provided sites for cell attachment.


Results, Conclusions, and Discussions::
We observed that treatment with sodium acetate, an acetyl-CoA precursor, increases the expression of lymphatic master regulator gene Prox1, which indicates the activation of positive feedback loop between VEGFR3 and Prox1 (Fig A-D). Moreover, hPSC-LECs express LYVE-1, a specific receptor for HA, and provide a unique advantage for engineered matrices containing HA (Fig E).² We show that matrix stiffness primes hPSC-LECs to undergo lymphatic tube formation directed by high level of VEGF-C (Fig F).³ Viscoelastic property further enhances lymphatic morphogenesis (Fig G). By three days post-encapsulation, we observe mature vessels with open lumens. Upon transplantation into mammary fat pads, the engineered lymphatic networks integrated with the murine lymphatic system and continued to mature, as evidenced by fluorescent staining at two weeks post-implantation (Fig H-I).

Collectively, we show that the signaling pathways of stem cell differentiation and morphogenesis into lymphatic tube networks can be precisely regulated in a synthetic matrix, resulting in lymphatic networks which may be useful as therapeutics for a range of lymphatic disorders and approaches in tissue regeneration.




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.), National Institutes of Health (1R35-GM-143055-01 to D.H.-P.), and from the Walther Cancer Foundation (0180.01 to Pre-doctoral Fellowship to L.A.).




References (Optional): :
1. Alderfer, L., et al., JBE 12, 32 (2018).




2. Jeong, D. P. et al., CMBE (2022).




3. Alderfer, L., et al. FASEB Journal (2021).