Extracellular Matrix Microenvironments that Modulate Angiogenesis or Cell Fate Commitment

We are broadly interested in manipulating the extracellular matrix (ECM) microenvironment to induce cell fate commitment or to modulate other cellular functions.  Some of the ECM properties of interest include the ECM chemical composition, topographical patterning, and rigidity.  We are developing oriented nano-scale and micro-scale biomaterials that guide cell organization and modulates cell function. We hypothesize that spatially patterned biomaterials can alter cytoskeletal tension and chromatin remodeling to influence cell fate.  We are also engineering high-throughput approaches for testing combinatorial ECM compositions and rigidities that favor cell fate commitment of induced pluripotent stem cells towards cardiovascular lineages. Our recent R01-funded project focuses on the use of engineered ECM mimetics with controllable chemical ligands, stiffness, and stress relaxation properties.

1.    Hou L, Coller J, Natu V, Hastie TJ, Huang NF. Combinatorial Extracellular Matrix Microenvironments Promote Survival and Phenotype of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells in Hypoxia. Acta Biomater.  Epub.

2.    Hou L, Kim JJ, Wanjare M, Patlolla B, Coller J, Natu V, Hastie TJ, Huang NF. Combinatorial Extracellular Matrix Microenvironments for Probing Endothelial Differentiation of Human Pluripotent Stem Cells. Sci Rep 7, 6551, 2017.

Microscale combinatorial extracellular matrix microarrays are fabricated consisting of multi-factorial compositions of collagen IV (C), fibronectin (F), gelatin (G), heparan sulfate (H), laminin (L), and matrigel (M).  Human induced pluripotent stem cell-derived endothelial cells seeded on the arrays are used for high-throughput quantitative assessment of viability, maintenance of phenotype, and nitric oxide production. Source: Hou et al., Acta Biomater, 2016

Mechanotransduction Pathways that Induce Cell Fate Commitment

Using customizable ECM microenvironments, we are interested in studying mechanotransduction pathways that induce cell fate commitment or function.  Some of the mechanotransduction pathways of interest include those that are activated by integrins, focal adhesions, and actin-binding proteins. Current projects include mechanotransduction pathways that are involved cardiovascular differentiation of induced pluripotent stem cells, direct transdifferentiation of fibroblasts into cardiovascular lineages, effects of stiffness on endothelial-to-mesenchymal transition, and reprogramming of fibroblasts into induced pluripotent stem cells.  

1.    Nakayama KH, Hong G, Lee JC, Patel J, Edwards B, Zaitseva TS, Paukshto MV, Dai H, Cooke JP, Woo YJ, Huang NF. Aligned-Braided Nanofibrillar Scaffold with Endothelial Cells Enhances ArteriogenesisACS Nano 9: 6900–6908, 2015.

2.    Nakayama KH, Surya VN, Gole M, Walker TW, Yang W, Lai ES, Ostrowski MA, Fuller GG, Dunn AR, Huang NF.  Nanoscale Patterning of Extracellular Matrix Alters Endothelial Function under Shear StressNano Lett, 16:410-9, 2016.

Endothelial outgrowth from aligned nanofibrillar scaffolds.HumanECsseededon fibronectin-precoated control or aligned scaffold were encapsulated into a 3D hydrogel for tracking cellular outgrowth. (A)Fluorescently labeled endothelial cells are shown migrating from scaffold into the surrounding hydrogel after 3 days. Dotted line denotes border of scaffold. (B) Quantification of cellular outgrowth from control or aligned scaffold after 3 days (n = 3, * P < 0.01). (C) qPCR analysis of integrin subunit gene expression (n=3,*P<0.05). (D) Cellular outgrowth from aligned nanofibrillar scaffolds in the presence of integrin α1 inhibition antibody or IgG control (n = 3, *P < 0.001). Scale bar: 200 μm. Source: Nakayama et al., ACS Nano, 2015.

Tissue Engineering and Regenerative Medicine

By gaining fundamental insights in the role of ECM-mediated mechanotransduction pathways on cell fate commitment, we will engineer three-dimensional vascular conduits, cardiac patches, and skeletal muscle grafts with physiologically relevant cellular and ECM compositions.  We are also working with industry partners to engineer nanofibrillar scaffolds that can induce angiogenesis or lymphangiogenesis.   

1.    Mulyasasmita W, Cai L, Dewi RE, Jha A, Ullmann SD, Luong RH, Huang NF, Heilshorn SC. Avidity-controlled hydrogels for injectable co-delivery of induced pluripotent stem cell-derived endothelial cells and growth factors. J Control Release 191:71-81, 2014.

2.    Nakayama KH, Joshi PA, Lai ES, Gujar P, Joubert L-M, Chen B, Huang NF. Bi-layered vascular graft derived from human induced pluripotent stem cells with biomimetic structure and function. Regen Med 10:745-55, 2015. 

3.    Hadamitzky C, Zaitseva TS, Bazalova-Carter M, Paukshto MV, Hou L, Strassberg Z, Ferguson J, Matsuura Y, Dash R, Yang PC, Kretchetov S, Vogt PM, Rockson SG, Cooke JP, Huang NF. Aligned nanofibrillar collagen scaffolds - Guiding lymphangiogenesis for treatment of acquired lymphedema. Biomaterials 102:259-67, 2016.

4.     Foster AA, Dewi RE, Cai L, Hou L, Strassberg Z, Alcazar CA, Heilshorn SC, Huang NF. Protein-engineered hydrogels enhance the survival of induced pluripotent stem cell-derived endothelial cells for treatment of peripheral arterial disease. Biomater Sci. 6:614-622, 2018.

5.     Zaitseva T, Alcazar C, Zamani M, Hou L, Sawamura S, Yakubov E, Hopkins M, Woo YJ, Paukshto M, Huang NF. Aligned Nanofibrillar Scaffolds for Controlled Delivery of Modified mRNA. Tissue Eng Part A. 2018 (epub).

Production of a bilayered aligned nanofibrillar collagen graft. Schematic of the bilayered graft fabrication showing extrusion of collagen using concentric mandrels to aligned collagen fibrils in the longitudinal direction to create the inner hollow graft. The longitudinally aligned inner layer is circumferentially wrapped with an outer layer of aligned collagen. (A) The cellularized vascular graft has an inner layer of longitudinally oriented endothelial cells and an outer layer of circumferentially oriented smooth muscle cells. (B) The inner longitudinal layer alone (bottom) and the complete bilayered graft are supported by Nylon tubing before drying.  Scanning electron microscopy image shows the organization of aligned collagen fibrils. (C) Arrow depicts direction of fibrils. Scanning electron microscopy depicts transverse section of the bilayered graft. (D) Arrow depicts direction of fibrils, and dotted line marks the interface between the two layers. (E) Fluorescently labeled inner layer (green) and outer layer (red) of the bilayered graft provide increased visualization of the two layers. EC: Endothelial cell; iPSC: Induced pluripotent stem cell; SMC: Smooth muscle cell. Source: Nakayama et al., Regen Med, 2015

Imaging and Devices Technology

In order to study the function of cells and/or engineered constructs in vivo, we have engineered devices or platforms to overcome existing technological limitations. We are engineering microscale high-throughput arrayed platforms for studying combinatorial extracellular matrix proteins on stem cell fate in a facile manner. To study the role of hemodynamic shear stress gradients on cell behavior, we developed a novel fluid flow device that recapitulates shear stress gradients and disturbed flow. In another example, we have tested the utility of near infrared fluorophores in the second window (~1400nm emission) as angiographic contrast dyes to visualize blood flow, blood perfusion, and microvasculature at high architectural resolution in mice. These technological developments together enable us to study cell biology and pathophysiology of limb ischemia in unprecedented ways.

1.    Hong G, Lee JC, Robinson JT, Raaz U, Xie L, Huang NF, Cooke JP, Dai H. Multi-Functional In Vivo Vascular Imaging Using Near-Infrared II Fluorescence. Nat Med, 18:1841-6, 2012.

2.    Hong G, Lee JC, Jha A, Diao S, Nakayama KH, Hou L, Doyle TC, Robinson JT, Antaris AL, Dai H, Cooke JP, Huang NFNear-Infrared II Fluorescence for Imaging Hindlimb Vessel Regeneration with Dynamic Tissue Perfusion Measurement. Circ Cardiovasc Imaging 7:517-525, 2014.

3.    Nakayama KH, Surya VN, Gole M, Walker TW, Yang W, Lai ES, Ostrowski MA, Fuller GG, Dunn AR, Huang NF.  Nanoscale Patterning of Extracellular Matrix Alters Endothelial Function under Shear Stress.  Nano Lett, 16:410-9, 2016.

4.    Ma Z, Zhang M, Yue J, Alcazar C, Zhong Y, Doyle TC, Dai H, Huang NF. Near-Infrared IIb Fluorescence Imaging of Vascular Regeneration with Dynamic Tissue Perfusion Measurement and High Spatial Resolution. Adv Funct Mater, 2018 (in press).