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.


Zaitseva TS, Yang G, Dionyssiou D, Zamani M, Sawamura S, Yakubov E, Ferguson J, Hallet RL, Fleischmann D, Paukshto MV, Huang NF. Delivery of hepatocyte growth factor mRNA from nanofibrillar scaffolds in a pig model of peripheral arterial disease. Regen Med 2020, epub

Nakayama KH, Alcazar C, Yang G, Quarta M, Paine P, Doan L, Davis A, Rando TA, Huang NF. Rehabilitative Exercise and Spatially Patterned Nanofibrillar Scaffolds Enhance Vascularization and Innervation Following Volumetric Muscle Loss. npj Regen Med, 3:16, 2018.

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.

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 HypoxiaActa Biomater. 44: 199-199, 2016.
 

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

Stem Cell Therapeutics for Treatment of Ischemic Cardiovascular Diseases

To identify promising stem cell candidate for tissue engineering and regenerative medicine applications, we have made substantial progress in evaluating the efficacy of adult and pluripotent stem cells for cardiovascular remodeling and for restoring function and revascularization to ischemic cardiovascular tissues, such as the ischemic limb or ischemic myocardium. These cells include those derived from embryonic stem cells, induced pluripotent stem cells (iPSCs), and bone marrow-derived mesenchymal stem cells. We have focused on iPSCs, owing to their theoretically infinite expansion potential and autologous nature, for the treatment of peripheral arterial disease. Our early studies of injecting iPSC-derived endothelial cells in saline alone demonstrate therapeutic benefit in enhancing blood perfusion and angiogenesis. But a critical limitation of saline-injected cells was poor survival due to needle shear and the harsh ischemic environment. We then developed shear-thinning protein hydrogels that protect the iPSC-ECs during injection, leading to improved cell survival, leading to improved angiogenesis. The hydrogels could be further functionalized with pro-angiogenic factors like vascular endothelial growth factor for added potency for therapeutic revascularization. These studies highlight the importance of instructive biomaterial interactions to enhance cell survival.


Hu C, Zaitseva TS, Alcazar C, Tabada P, Sawamura S, Yang G, Borrelli MR, Wan DC, Nguyen DH, Paukshto MV, Huang NF. Delivery of Human Stromal Vascular Fraction Cells on Nanofibrillar Scaffolds for Treatment of Peripheral Arterial Disease. Front Bioeng Biotechnol 8:689, 2020. eCollection 2020.

Wanjare M, Kawamura M, Hu C, Alcazar C, Wang H, Woo YJ, Huang NF. Vascularization of engineered spatially patterned myocardial tissue derived from human pluripotent stem cells in vivoFront Bioeng Biotechnol 7:208, 2019.

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 

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.

Abdul Jalil R, Huang NF*, Jame S, Lee J, Nguyen HN, Byers B, De A, Okogbaa J, Rollins MD, Reijo-Pera R, Gambhir SS, Cooke JP. Endothelial cells derived from human iPSCs increase capillary density and improve perfusion in a mouse model of peripheral arterial disease. Arterioscler Thromb Vasc Biol, 31:e72-79, 2011

Mean blood perfusion recovery after implantation of aligned nanofibrillar scaffolds seeded with SVF. (A) Representative laser Doppler spectroscopy images from day 0 and day 14. (B) Quantification of mean perfusion ratio (ischemic/control) over the course of 14 days (n = 5–9 per group). *denotes statistically significantly higher mean perfusion ratio in the SVF+Scaffold group, relative to the PBS group (p < 0.05). Arrow denotes ischemic limb.  Source: Hu et al., Front Bioeng Biotechnol 2020

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.   


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

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.
 

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. For example, by employing spatially patterned and/or three-dimensional biomaterials, we have built prototypes of engineered skeletal muscle or cardiac tissue constructs. Some of these constructs employ nanoscale or microscale spatially patterning cues derived from the extracellular matrix to induce cellular reorganization, resulting in engineered tissues with physiologically relevant morphology. Our published works demonstrate the ability of these tissue constructs to mimic physiological structure and/or function of native muscle structure. Additionally, we are also collaborating with industry partners to engineer spatially patterned scaffolds that can induce angiogenesis or lymphangiogenesis.   


Alcazar C, Hu C, Rando TA, Huang NF#, Nakayama KH. Transplantation of Insulin-like Growth Factor-1 Laden Scaffolds Combined with Exercise Promotes Neurovascular Regeneration and Angiogenesis in a Muscle Injury Preclinical Model. Biomater Sci 2020 (epub)

Nakayama KH, Quarta M, Paine P, Alcazar C, Karakikes I, Garcia V, Abilez O, Calvo NS, Simmons CS, Rando TA, Huang NF. Treatment of Volumetric Muscle Loss Using Spatially Patterned Scaffolds Enhances Vascular Organization and Functional Integration. Commun Biol  2:170, 2019. eCollection 2019.

Wanjare M, Kawamura M, Hu C, Alcazar C, Wang H, Woo YJ, Huang NF. Vascularization of engineered spatially patterned myocardial tissue derived from human pluripotent stem cells in vivo. Front Bioeng Biotechnol 7:208, 2019.  doi: 10.3389/fbioe.2019.00208.

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 mRNATissue Eng Part A. 2019 25:121-130.

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.

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.

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.

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.

Implantation of murine engineered skeletal muscle in a mouse model of volumetric muscle loss. Longitudinal sections of endothelialized engineered muscle formed randomly oriented or aligned skeletal muscle constructs. A. Donor transplanted myofibers are denoted by the co-expression of green fluorescence protein (GFP, green) and myosin heavy chain (MHC, red). Native myofibers are denoted by MHC+/GFP- expression. Insets show higher magnification images of the GFP+ region depicted by white boxes in the center of the engineered tissues. B. Representative image of a tissue cross-section of muscle transplanted with an endothelialized engineered skeletal muscle construct at 21 days after induction of traumatic muscle injury. MHC (red) indicates mature myofibers, GFP (green) indicates the transplanted myoblast population, and nuclei are visualized in blue using Hoechst 33342 dye. C. The density of MHC+/GFP+ transplanted myofibers in the region of regeneration is shown (n ≥ 3, **p ≤ 0.01). Error bars represent standard deviation. Arrow designates orientation of aligned nanofibrillar scaffold. Scale bars denote 50 μm (inset) or 500 μm (a); 500 μm (b).  Source: Nakayama et al., Commun Biol, 2019

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.


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, 28: 1803417, 2018.  https://doi.org/10.1002/adfm.201803417.

Ma Z, Zhang M, Yue J, Alcazar C, Zhong Y, Doyle TC, Dai H, Huang NFNear-Infrared IIb Fluorescence Imaging of Vascular Regeneration with Dynamic Tissue Perfusion Measurement and High Spatial Resolution. Adv Funct Mater 28: 1803417, 2018.

Zhong Y, Ma Z, Zhu S, Yue J, Zhang M, Antaris AL, Yuan J, Cui R, Wan H, Zhou Y, Wang W, Huang NF, Luo J, Hu Z, Dai H. Boosting the down-shifting luminescence of rare-earth nanocrystals for biological imaging beyond 1500 nm. Nat Commun. 2017 8:737.

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

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.

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.
 

Comparison of vascular imaging in normal mice using lead sulfide/cadmium sulfide (PbS/CdS) and single-walled nanotubes (SWNT) as vascular contrast dyes. A,B) Whole body fluorescence imaging of mouse hindlimb vasculature after systemic injection of SWNTs by NIR-II imaging (A), in comparison to NIR-IIb imaging of systemically injected PbS/CdS contrast agent (B). E,F) High magnification imaging by NIR-II (E) and NIR-IIb (F). Adv Funct Mater, 2018.