Periodic Reporting for period 1 - MusclePlate (Human skeletal muscle platform for disease modelling and high-throughput drug screening)
Okres sprawozdawczy: 2023-05-01 do 2024-10-31
Neuromuscular disorders (NMDs) are a group of diseases affecting skeletal muscle and motor nerves, either individually or simultaneously. Although rare, NMDs impose a significant socioeconomic burden and severely impact patients' quality of life. Most NMDs are currently incurable, with existing therapies focusing solely on symptom management.
Drug development (DD) is a lengthy, costly, and inefficient process, often delaying the introduction of new therapies to the market. A major challenge is the poor translation of preclinical findings to humans, largely due to the limitations of current models in accurately reflecting human biology, physiology, and disease mechanisms. For NMDs specifically, the lack of human in vitro skeletal muscle models for drug screening and validation has significantly hindered the discovery of new treatments. Developing a relevant preclinical NMD model requires overcoming several challenges:
• Creation of a mature, biomimetic, and functional human tissue
• Ensuring sample reproducibility and handling convenience
• Achieving robust assay readouts
• Compatibility with high-throughput screening
This project aims to address these challenges by developing an in vitro human skeletal muscle platform for NMD modeling and preclinical drug testing. This technology offers a valuable research tool for pharmaceutical development, enabling accurate predictions of drug activity in humans, identifying promising candidates, and discarding ineffective ones early in the process.
The project has significant scientific, societal, and economic implications, including:
• Discovery and validation of novel or repurposed drugs for NMDs
• Identification of new molecular targets in NMDs
• Reduction in animal use, costs, and time associated with DD
• Enhanced understanding of muscle biology and physiology in healthy and pathological states
• Accelerated "time to market" for NMD drugs
• Availability of novel and more effective therapies for NMD patients
• Decreased healthcare and R&D costs related to NMDs
• Potential applications in other research areas, such as studying muscle development and repair mechanisms
Drug development (DD) is a lengthy, costly, and inefficient process, often delaying the introduction of new therapies to the market. A major challenge is the poor translation of preclinical findings to humans, largely due to the limitations of current models in accurately reflecting human biology, physiology, and disease mechanisms. For NMDs specifically, the lack of human in vitro skeletal muscle models for drug screening and validation has significantly hindered the discovery of new treatments. Developing a relevant preclinical NMD model requires overcoming several challenges:
• Creation of a mature, biomimetic, and functional human tissue
• Ensuring sample reproducibility and handling convenience
• Achieving robust assay readouts
• Compatibility with high-throughput screening
This project aims to address these challenges by developing an in vitro human skeletal muscle platform for NMD modeling and preclinical drug testing. This technology offers a valuable research tool for pharmaceutical development, enabling accurate predictions of drug activity in humans, identifying promising candidates, and discarding ineffective ones early in the process.
The project has significant scientific, societal, and economic implications, including:
• Discovery and validation of novel or repurposed drugs for NMDs
• Identification of new molecular targets in NMDs
• Reduction in animal use, costs, and time associated with DD
• Enhanced understanding of muscle biology and physiology in healthy and pathological states
• Accelerated "time to market" for NMD drugs
• Availability of novel and more effective therapies for NMD patients
• Decreased healthcare and R&D costs related to NMDs
• Potential applications in other research areas, such as studying muscle development and repair mechanisms
The technical part of this project had three main objectives:
1. Platform development and validation
2. Cell culture protocol optimization
3. Proof-of-concept for NMD modeling and drug testing
Objective 1: Platform Development and Validation
To achieve this, we designed a CAD model for the platform, dividing it into a bottom and top component. The bottom component is a glass substrate with an array of thin, aligned grooves (Fig. 1) to induce muscle cell alignment. The top component is a PDMS chip containing compartments for muscle cell seeding and anchoring channels.
The bottom substrate was fabricated by depositing SiO2 onto the glass substrate, coating it with a positive photoresist, and applying UV photolithography (using a custom-made hard mask with the intended pattern). This was followed by reactive ion etching and dicing into the desired shape. For the top component, a master mold in SU-8 was produced using UV photolithography, which served to create multiple PDMS replicas. These replicas were aligned and bonded to the glass substrate via oxygen plasma bonding. Quality control was performed using scanning electron microscopy and a profilometer (Fig. 1).
Objective 2: Cell Culture Protocol Optimization
To develop human skeletal muscle tissue, we tested protocols using hiPSCs previously differentiated into myogenic progenitors (cryopreserved as such). Various cell seeding densities and hydrogel formulations (composed of collagen, fibrin, and Matrigel blends) were evaluated. The optimal conditions were found to be a density of 50k/cm² and a hydrogel comprising fibrin (4 mg/ml), collagen (2.5 mg/ml), and 10% Matrigel.
Muscle fiber characterization was conducted via immunostaining for muscle markers, such as α-actinin, desmin, myosin heavy chain, and bin-1 (Fig. 2). Spontaneous muscle contractions were observed using a bright-field microscope and analyzed with a PIV algorithm in MATLAB. After generating a skeletal muscle model, we added human motor neurospheres (with optogenetic probes) to develop a neuromuscular model. The established protocol for neurosphere creation enabled efficient co-culture integration.
After optimizing the culture conditions, we characterized neuromuscular junction (NMJ) morphology in 14DIV co-cultures via immunostaining and evaluated function by analyzing muscle contractions after light-induced neuron firing. An optogenetic training protocol (blue light stimulation at specific frequencies and intensities) was implemented to strengthen NMJs. Optogenetically trained co-cultures exhibited stronger contractions (Fig. 4), suggesting increased neuron firing enhances NMJ formation and strength.
Objective 3: Proof-of-Concept for NMD Modeling and Drug Testing
An ALS model was implemented using hiPSC-derived motor neurons from ALS patients with familial mutations (SOD1) and sporadic ALS. After validating the ALS phenotype (Fig. 5), co-cultures were created following the same process. ALS cultures demonstrated lower cell viability and slower muscle contractions compared to healthy controls (Fig. 6).
With the platform established, we are prepared to conduct proof-of-concept drug testing for ALS. Two drugs, tofersen (for SOD1 mutants) and ropinirole (for sporadic ALS), were selected for initial validation. Following this, a larger drug library will be screened to identify potential therapeutics.
In summary, most technical objectives were achieved, and the platform is now ready for drug testing applications.
1. Platform development and validation
2. Cell culture protocol optimization
3. Proof-of-concept for NMD modeling and drug testing
Objective 1: Platform Development and Validation
To achieve this, we designed a CAD model for the platform, dividing it into a bottom and top component. The bottom component is a glass substrate with an array of thin, aligned grooves (Fig. 1) to induce muscle cell alignment. The top component is a PDMS chip containing compartments for muscle cell seeding and anchoring channels.
The bottom substrate was fabricated by depositing SiO2 onto the glass substrate, coating it with a positive photoresist, and applying UV photolithography (using a custom-made hard mask with the intended pattern). This was followed by reactive ion etching and dicing into the desired shape. For the top component, a master mold in SU-8 was produced using UV photolithography, which served to create multiple PDMS replicas. These replicas were aligned and bonded to the glass substrate via oxygen plasma bonding. Quality control was performed using scanning electron microscopy and a profilometer (Fig. 1).
Objective 2: Cell Culture Protocol Optimization
To develop human skeletal muscle tissue, we tested protocols using hiPSCs previously differentiated into myogenic progenitors (cryopreserved as such). Various cell seeding densities and hydrogel formulations (composed of collagen, fibrin, and Matrigel blends) were evaluated. The optimal conditions were found to be a density of 50k/cm² and a hydrogel comprising fibrin (4 mg/ml), collagen (2.5 mg/ml), and 10% Matrigel.
Muscle fiber characterization was conducted via immunostaining for muscle markers, such as α-actinin, desmin, myosin heavy chain, and bin-1 (Fig. 2). Spontaneous muscle contractions were observed using a bright-field microscope and analyzed with a PIV algorithm in MATLAB. After generating a skeletal muscle model, we added human motor neurospheres (with optogenetic probes) to develop a neuromuscular model. The established protocol for neurosphere creation enabled efficient co-culture integration.
After optimizing the culture conditions, we characterized neuromuscular junction (NMJ) morphology in 14DIV co-cultures via immunostaining and evaluated function by analyzing muscle contractions after light-induced neuron firing. An optogenetic training protocol (blue light stimulation at specific frequencies and intensities) was implemented to strengthen NMJs. Optogenetically trained co-cultures exhibited stronger contractions (Fig. 4), suggesting increased neuron firing enhances NMJ formation and strength.
Objective 3: Proof-of-Concept for NMD Modeling and Drug Testing
An ALS model was implemented using hiPSC-derived motor neurons from ALS patients with familial mutations (SOD1) and sporadic ALS. After validating the ALS phenotype (Fig. 5), co-cultures were created following the same process. ALS cultures demonstrated lower cell viability and slower muscle contractions compared to healthy controls (Fig. 6).
With the platform established, we are prepared to conduct proof-of-concept drug testing for ALS. Two drugs, tofersen (for SOD1 mutants) and ropinirole (for sporadic ALS), were selected for initial validation. Following this, a larger drug library will be screened to identify potential therapeutics.
In summary, most technical objectives were achieved, and the platform is now ready for drug testing applications.
In this project, we were able to engineer a cell culture platform containing multiple and reproducible replicas of functional human skeletal muscle and neuromuscular tissue. These models evidenced high morphological/phenotypical maturity as well as stable contractions, that could be externally controlled via blue light stimulation (for NMJ cultures). Using cells derived from ALS patients, we were able to create an ALS model within our platform that shows hallmarks of the disease, such as pathological markers presence and decreased cell viability/muscle function. Next steps will be the validation of drug testing ability with field drugs, followed by a large compound screening. These results constitute a foundational step in creating novel tools for drug development that can increase testing accuracy and accelerate the discovery of novel/repurposed drug candidates.
To achieve this and bring our technology to a commercial stage, we started taking important steps such as submitting a provisional IP request, conducting a freedom-to-operate analysis and designing a business plan that contains our financial roadmap and go-to-market strategy.
In sum, this project completion resulted in the development of a novel tool that is superior to current alternatives and has the potential to aid the biomedical field and pharmaceutical industry in fighting NMDs
To achieve this and bring our technology to a commercial stage, we started taking important steps such as submitting a provisional IP request, conducting a freedom-to-operate analysis and designing a business plan that contains our financial roadmap and go-to-market strategy.
In sum, this project completion resulted in the development of a novel tool that is superior to current alternatives and has the potential to aid the biomedical field and pharmaceutical industry in fighting NMDs