In a funded NSF/CASIS grant, we are employing engineered skeletal muscle system as a tissue chip for studying the effects of microgravity in mimicking salient aspects of sarcopenia.  Sarcopenia is an age-related muscle wasting syndrome that takes decades to progress.  In part because of its slow progression, there is no current FDA-approved drugs.  Through this grant, we developed a tissue chip and bioreactor system composed of 3D engineered skeletal muscle.  With our recent launch of these bioreactors to the International Space Station in Aug 2021, we will be able to study the potential effects of microgravity to accelerate the process of sarcopenia.  We will compare the effect of microgravity vs gravity in modulating the formation of muscle myotubes, as well as in modulating their proteomic/transcriptomic signature.  We hope this microgravity platform may become a novel approach for screening drugs that combat sarcopenia.

Read more: Tissue Engineering in Space Could Treat Age-Related Muscle Loss on Earth

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 Hypoxia. Acta Biomater. 44: 199-199, 2016.
 

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 vivo.  Front 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

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 NF.  Nanoscale Patterning of Extracellular Matrix Alters Endothelial Function under Shear Stress.  Nano 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 Arteriogenesis.  ACS Nano 9: 6900–6908, 2015.
 

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.   

Hu C, Ayan B, Chiang G, Chang AHP, Rando TA, Huang NF. Comparative Effects of Basic Fibroblast Growth Factor Delivery or Voluntary Exercise on Muscle Regeneration after Volumetric Muscle Loss. Bioengineering 9: 37 2022.  From the Special Issue, Extracellular Matrix in Musculoskeletal Regeneration.

Mulorz J, Shayan M, Hu C, Alcazar C, Chan AHP, Briggs M, Wen Y, Walvedar AP, Ramasubramanian A, Spin JM, Chen B, Tsao PS, Huang NF. Peri-Adventitial Delivery of Smooth Muscle Cells in Porous Collagen Scaffolds For Treatment of Experimental Abdominal Aortic Aneurysm. Biomater Sci 9: 6903–6914, 2021.

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. 8:5376-5389. doi: 10.1039/d0bm00990c. (^# co-corresponding author)

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 mRNA. Tissue 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.

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 NF. Near-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 NF.  Nanoscale 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 NF.  Near-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.