engineering-characterization-an-optogenetic-model-human-neuromuscular

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

Columbia University

Gladstone Institutes

Columbia College

Bioreactor systems allow safe and reproducible production of tissue constructs and functional analysis of cell behavior in biomaterials. However, current procedures for the analysis of tissue generated in biomaterials are destructive. We describe a transparent perfusion system that allows real-time bioluminescence imaging of luciferase expressing cells seeded in scaffolds for the study of cell-biomaterial interactions and bioreactor performance. A prototype provided with a poly(lactic) acid scaffold was used for "proof of principle" studies to monitor cell survival in the scaffold (up to 22 days). Moreover, using cells expressing a luciferase reporter under the control of inducible tissue-specific promoters, it was possible to monitor changes in gene expression resulting from hypoxic state and endothelial cell differentiation. This system should be useful in numerous tissue engineering applications, the optimization of bioreactor operation conditions, and the analysis of cell behavior in three-dimensional scaffolds.

Real-Time Bioluminescence Imaging of Cell Distribution, Growth, and Differentiation in a Three-Dimensional Scaffold Under Interstitial Perfusion for Tissue Engineering.

A preclinical model of glioblastoma (GB) bystander cell therapy using human adipose mesenchymal stromal cells (hAMSCs) is used to address the issues of cell availability, quality, and feasibility of tumor cure. We show that a fast proliferating variety of hAMSCs expressing thymidine kinase (TK) has therapeutic capacity equivalent to that of TK-expressing hAMSCs and can be used in a multiple-inoculation procedure to reduce GB tumors to a chronically inhibited state. We also show that up to 25% of unmodified hAMSCs can be tolerated in the therapeutic procedure without reducing efficacy. Moreover, mimicking a clinical situation, tumor debulking previous to cell therapy inhibits GB tumor growth. To understand these striking results at a cellular level, we used a bioluminescence imaging strategy and showed that tumor-implanted therapeutic cells do not proliferate, are unaffected by GCV, and spontaneously decrease to a stable level. Moreover, using the CLARITY procedure for tridimensional visualization of fluorescent cells in transparent brains, we find therapeutic cells forming vascular-like structures that often associate with tumor cells.  experiments show that therapeutic cells exposed to GCV produce cytotoxic extracellular vesicles and suggest that a similar mechanism may be responsible for the  therapeutic effectiveness of TK-expressing hAMSCs.

Glioblastoma Bystander Cell Therapy: Improvements in Treatment and Insights into the Therapy Mechanisms.

The study of human neuromuscular diseases has traditionally been performed in animal models, due to the difficulty of performing studies in human subjects. Despite the unquestioned value of animal models, inter-species differences hamper the translation of these findings to clinical trials. Tissue-engineered models of the neuromuscular junction (NMJ) allow for the recapitulation of the human physiology in tightly controlled  settings. : Here we report the first human patient-specific tissue-engineered model of the neuromuscular junction (NMJ) that combines stem cell technology with tissue engineering, optogenetics, microfabrication and image processing. The combination of custom-made hardware and software allows for repeated, quantitative measurements of NMJ function in a user-independent manner. : We demonstrate the utility of this model for basic and translational research by characterizing in real time the functional changes during physiological and pathological processes. : This system holds great potential for the study of neuromuscular diseases and drug screening, allowing for the extraction of quantitative functional data from a human, patient-specific system.

Quantification of human neuromuscular function through optogenetics.

: Neuromuscular Junctions (NMJs) are the synapses between motor neurons and skeletal muscle fibers, and they are responsible for voluntary motor function. NMJs are affected at early stages of numerous neurodegenerative and neuroimmunological diseases. Due to the difficulty of systematically studying and manipulating NMJs in live subjects,  systems with human tissue models would provide a powerful complement to simple cell cultures and animal models for mechanistic and drug development studies.: The authors review the latest advances in  models of NMJs, from traditional cell co-culture systems to novel tissue culture approaches, with focus on disease modeling and drug testing.: In recent years, more sophisticated  models of human NMJs have been established. The combination of human stem cell technology with advanced tissue culture systems has resulted in systems that better recapitulate the human NMJ structure and function, and thereby allow for high-throughput quantitative functional measurements under both healthy and diseased conditions. Although they still have limitations, these advanced systems are increasingly demonstrating their utility for evaluating new therapies for motoneuron and autoimmune neuromuscular diseases, and we expect them to become an integral part of the drug discovery process in the near future.

In vitro models of neuromuscular junctions and their potential for novel drug discovery and development.

Joint disorders can be detrimental to quality of life. There is an unmet need for precise functional reconstruction of native-like cartilage and bone tissues in the craniofacial space and particularly for the temporomandibular joint (TMJ). Current surgical methods suffer from lack of precision and comorbidities and frequently involve multiple operations. Studies have sought to improve craniofacial bone grafts without addressing the cartilage, which is essential to TMJ function. For the human-sized TMJ in the Yucatan minipig model, we engineered autologous, biologically, and anatomically matched cartilage-bone grafts for repairing the ramus-condyle unit (RCU), a geometrically intricate structure subjected to complex loading forces. Using image-guided micromilling, anatomically precise scaffolds were created from decellularized bone matrix and infused with autologous adipose-derived chondrogenic and osteogenic progenitor cells. The resulting constructs were cultured in a dual perfusion bioreactor for 5 weeks before implantation. Six months after implantation, the bioengineered RCUs maintained their predefined anatomical structure and regenerated full-thickness, stratified, and mechanically robust cartilage over the underlying bone, to a greater extent than either autologous bone-only engineered grafts or acellular scaffolds. Tracking of implanted cells and parallel bioreactor studies enabled additional insights into the progression of cartilage and bone regeneration. This study demonstrates the feasibility of TMJ regeneration using anatomically precise, autologous, living cartilage-bone grafts for functional, personalized total joint replacement. Inclusion of the adjacent tissues such as soft connective tissues and the TMJ disc could further extend the functional integration of engineered RCUs with the host.

Tissue engineered autologous cartilage-bone grafts for temporomandibular joint regeneration.

Functional human tissues engineered from patient-specific induced pluripotent stem cells (hiPSCs) hold great promise for investigating the progression, mechanisms, and treatment of musculoskeletal diseases in a controlled and systematic manner. For example, bioengineered models of innervated human skeletal muscle could be used to identify novel therapeutic targets and treatments for patients with complex central and peripheral nervous system disorders. There is a need to develop standardized and objective quantitative methods for engineering and using these complex tissues, in order increase their robustness, reproducibility, and predictiveness across users. Here we describe a standardized method for engineering an isogenic, patient specific human neuromuscular junction (NMJ) that allows for automated quantification of NMJ function to diagnose disease using a small sample of blood serum and evaluate new therapeutic modalities. By combining tissue engineering, optogenetics, microfabrication, optoelectronics and video processing, we created a novel platform for the precise investigation of the development and degeneration of human NMJ. We demonstrate the utility of this platform for the detection and diagnosis of myasthenia gravis, an antibody-mediated autoimmune disease that disrupts the NMJ function.

Article	Year
Real-Time Bioluminescence Imaging of Cell Distribution, Growth, and Differentiation in a Three-Dimensional Scaffold Under Interstitial Perfusion for Tissue Engineering. Tissue engineering. Part C, Methods\| PubMed ID: 27339005	2016
Glioblastoma Bystander Cell Therapy: Improvements in Treatment and Insights into the Therapy Mechanisms. Molecular therapy oncolytics\| PubMed ID: 30364660	2018
Quantification of human neuromuscular function through optogenetics. Theranostics\| PubMed ID: 30867827	2019
In vitro models of neuromuscular junctions and their potential for novel drug discovery and development. Expert opinion on drug discovery\| PubMed ID: 31846349	2020
Tissue engineered autologous cartilage-bone grafts for temporomandibular joint regeneration. Science translational medicine\| PubMed ID: 33055244	2020
Bioengineered optogenetic model of human neuromuscular junction. Biomaterials\| PubMed ID: 34403849	2021

Olaia F. Vila

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