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.
