Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons

Abstract

Amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease involving loss of motor neurons (MNs) and muscle atrophy, still has no effective treatment, despite much research effort. To provide a platform for testing drug candidates and investigating the pathogenesis of ALS, we developed an ALS-on-a-chip technology (i.e., an ALS motor unit) using three-dimensional skeletal muscle bundles along with induced pluripotent stem cell (iPSC)-derived and light-sensitive channelrhodopsin-2-induced MN spheroids from a patient with sporadic ALS. Each tissue was cultured in a different compartment of a microfluidic device. Axon outgrowth formed neuromuscular junctions on the muscle fiber bundles. Light was used to activate muscle contraction, which was measured on the basis of pillar deflections. Compared to a non-ALS motor unit, the ALS motor unit generated fewer muscle contractions, there was MN degradation, and apoptosis increased in the muscle. Furthermore, the muscle contractions were recovered by single treatments and cotreatment with rapamycin (a mechanistic target of rapamycin inhibitor) and bosutinib (an Src/c-Abl inhibitor). This recovery was associated with up-regulation of autophagy and degradation of TAR DNA binding protein-43 in the MNs. Moreover, administering the drugs via an endothelial cell barrier decreased the expression of P-glycoprotein (an efflux pump that transports bosutinib) in the endothelial cells, indicating that rapamycin and bosutinib cotreatment has considerable potential for ALS treatment. This ALS-on-a-chip and optogenetics technology could help to elucidate the pathogenesis of ALS and to screen for drug candidates.

Fig. 1. Compartmentalized design of a human motor unit on a chip microfluidic device.

(A) The micro fabricated motor unit mimic device uses polydimethylsiloxane (PDMS) microchannels to form four identical sites on a single chip, each composed of a muscle fiber bundle attaching pillar structures and culture MN spheroids. Each site has two medium reservoirs, two gel injection ports, and three compartments. (B) Photos of the microfluidic device. Each device had three distinct culture regions: for MN spheroids (left), muscle tissues (right), and neurite elongation (middle). The distance between two pillars is 1500 μm. (C) iPS-derived skeletal myoblasts were injected into the right compartment with the collagen/Matrigel mixture from gel injection port 1. Within 1 day, a skeletal muscle fiber bundle was formed on pillar structures. After 13 days of differentiation, an MN spheroid with collagen gel was injected into the left compartment from gel injection port 2. Neural outgrowth occurs by 14 days, resulting in the formation of a human motor unit along with NMJ. (D) An MN spheroid and a skeletal muscle fiber bundle in a microfluidic chip on day 0 (D0). A differentiated MN spheroid and a muscle fiber bundle were embedded in collagen gel. Scale bars, 200 μm. (E) Preparation and differentiation of skeletal muscle cell and MN cells. hESC-derived NSC spheroids were formed and differentiated into mature MNs by treatment with appropriate growth factors. Meanwhile, hiPS-derived skeletal muscle fiber bundles were formed in the microfluidic device and differentiated into mature myotubes. Then, an MN spheroid was injected for coculture of the two tissues. Timeline A indicates 0 d = initial day of coculture; timeline B indicates 0 d = initial day of generating neurospheorid; and timeline C indicates 0 d = initial day of seeding skeletal muscle cells into the device. KSR, knockout serum replacement; IGF-1, insulin-like growth factor 1; SHH, sonic hedgehog; bFGF, basic fibroblast growth factor; BDNF, brain-derived neurotrophic factor; GDNF, glial cell–derived neurotrophic factor. (F) Muscle contraction force driven by electrical stimulation and chemical stimulation via MN is estimated by pillar displacement.

Fig. 2. Characterization of an iPS-derived skeletal muscle fiber bundle and an MN spheroid.

(A) Fabricated skeletal muscle fiber bundle approximately 1500 μm in length attaching the pillars at D7 and D21. Scale bars, 200 μm. (B) The population of myogenin-positive cells on muscle fiber bundle. n = 5. (C) Fusion index for skeletal muscle cells in a 3D micro device and a 2D monolayer. n = 8. (D) Regular pattern of a sarcomere structure stained by sarcomeric α-actinin and F-actin. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (E) Gene expression change of MyoD, myogenin, and FHL1 (F) at D7, D14, and D21 against D0 or D7. n = 5. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. N.D., not determined. (G) Muscle contraction powered by electrical stimulation at different frequencies (0.5 to 4 Hz). (H) Formation of an NSC spheroid and differentiation characterized by the immunostaining of HB9 and 3D reconstruction stained by Tuj1. Scale bars, 200 μm. (I) mRNA expression level of an MN spheroid at D14, D28, and D42. MNP, motor neuron progenitor. (J) Determination of necrotic cell death and cell cycle determined by HIF-1α expression and Ki67. N.S., not significant. (K) Histological immunostaining of PDK1 and DAPI. The MN spheroid is divided into three regions inside the spheroid: (i) normoxia region, (ii) hypoxia region, and (iii) necrotic death region. Scale bars, 200 μm. (L) NSC-MN spheroid culture (green, Tuj1; red, islet1) on a laminin-coated dish. After the formation of NSC spheroids on D20 and D40, the spheroids were seeded into a laminin-coated dish and cultured for an additional 5 days to visualize neurite elongation. Thick nerve fiber can be seen connecting two MN spheroids on D45. Scale bars, 200 μm. *P < 0.05; **P < 0.01, Student’s t test and one-way analysis of variance (ANOVA). Error bars ± SD.

Fig. 3. Neural outgrowth and formation of a human motor unit along with NMJ.

(A) Injection of an MN spheroid into the left compartment of the microfluidic device with collagen gel on D0. (B) MNs started neural outgrowth toward the muscle fiber bundle on D4. (C) After 7 days of coculture, many axons reached the muscle fiber bundle and end feet of neurons attached to myotubes, resulting in the formation of NMJs. (D) By D4 of coculture, thick neural fibers were observed. Soma of MNs also migrated from the original position. (E) Characterization of mature MNs stained by ChAT and islet1 (F) and a mature myotube (G) after D14. Scale bars, 100 μm (A to G). (H) Localization of nAChR on the muscle fiber bundle on D7 and D14. Scale bars, 10 μm. (I) The number of clusters of nAChRs on single muscle fiber bundles increased over time. n = 4. (J) Muscle contraction force estimation by pillar displacement on D14 of coculture. (K) Frequency of spontaneous muscle contraction and spontaneous muscle contraction force (L) on D0 (before NMJ formation, without NMJ) and D7 (after NMJ formation, with NMJ) without glutamic acid stimulation. n = 6. (M) Measurement of muscle contraction force by adding glutamic acid on D4, D7, and D14. **P < 0.05; *P < 0.01, Student’s t test and one-way ANOVA. Error bars ± SD.

Fig. 4. Excitotoxicity of the 3D human motor unit model induced by excess glutamic acid treatment.

(A) Time scale of glutamic acid treatment. After formation of the motor unit with NMJ by D7, glutamic acid (5 mM) treatment was started alongside the control. Muscle contraction was measured on D7, D10, and D14 by applying glutamic acid (0.1 mM). (B and C) Representative images of the 3D human motor unit on D14 with and without continuous treatment of glutamic acid. Glutamic acid treatment caused loss of thick neural fibers. Scale bars, 100 μm. (D) Number of motor neural fascicles with glutamic acid treatment at D14 is less than that of control. n = 2. (E) The average force of muscle contraction with treatment slightly decreased on D10 and significantly decreased after D14 although contraction force of control consistently increased over time. n = 2. (F) Average frequency of muscle contractions also decreased with treatment over time. Average contraction force fell from ~1.3 to 0.5 μN, and frequency was also reduced from 1.8 to 0.7 Hz. n = 2. (G) Difference of muscle contraction force between chemical and electrical stimulation. Electrical stimulation produced higher muscle contractility compared to treatment of glutamic acid via MN activity. n = 2. Error bars ± SD.

Fig. 5. ALS patient–derived motor unit and drug treatment.

(A and B) Morphology and immunostaining of SMI-32 (red) and DAPI (blue) of ALS patient–derived iPS MNs on D35 after differentiation. The expression of neurofilament heavy chain indicates maturation of MNs. Scale bars, 100 μm. (C) Percentage of islet1-positive cells of iALS-MN is lower than that of control (ESC-derived MNs) (counting 100 cells, n = 5). (D) TDP-43 expression is higher than that of control, whereas expression of NEFL and NEFM is lower than that of control. n = 3. Scale bars, 20 μm. a.u., arbitrary units. (E) Immunostaining of TDP-43 (red) indicates abnormal aggregation and inclusion in iALS-MN G298S compared with control (hES-MN). (F) Transfection of channelrhodopsin-2[H134R] in ALS patient–derived NSCs. Scale bars, 20 μm. (G) Representative images of iALS-MN spheroids on D14 and D28. Scale bars, 200 μm. (H) Comparison of mRNA expression on D45 related to MN differentiation between iALS-MN spheroids and ESC-derived MN spheroids revealed that no significant changes were observed in OLIG2, HB9, and GFAP. In contrast, the decrease in islet1, ChAT, SMI-32, and Synapsin I indicated an immature function of neural activity. n = 8. (I) Representative images of the ALS motor unit and control D14 of coculture stained by Tuj1 (green), F-actin (purple), and DAPI (blue). Fewer thick neural fibers and less NMJ formation were seen on the ALS motor unit model compared with the ES-derived motor unit. Scale bars, 100 μm. (J) Average neurite elongation speed in the ALS motor unit was slower than that in control. (K) Treatment of potential drugs (bosutinib and rapamycin) to the ALS motor unit model. n = 4. Muscle contraction of the ALS motor unit was weaker than that of the ES-derived motor unit without drug treatment on D7 and D14. No significant effect of the drug treatments on D7. However, significant neuroprotection by treatments can be seen on D14. (L and M) After D14 of culture in the microfluidic system with the MN spheroid, muscle fiber was live stained for caspase3/7 and then stained for α-actinin and DAPI. In the normal motor unit (ESC-derived MN and iPS-derived skeletal muscle), few caspase3/7-positive cells can be observed. The number of caspase3/7-positive cells increased in the ALS motor unit (iALS-MN and iPS-derived skeletal muscle). Treatment with rapamycin and rapamycin/bosutinib significantly decreased the number of caspase3/7-positive cells, indicating that the treatment of drug reversed muscle apoptosis. n = 2. Scale bars, 100 μm. *P < 0.05; **P < 0.01, Student’s t test and two-way ANOVA. Error bars ± SD.

Fig. 6. Muscle contraction by optical stimulation with rapamycin and bosutinib and expression of autophagy.

(A to C) Muscle contraction force by optical stimulation without drugs and with rapamycin and cotreatment of rapamycin and bosutinib. Red arrows indicate the absence of muscle contraction with light stimulation. Blue dashed lines indicate times of light stimulation. (D) Successful rate of muscle contraction by light stimulation in three cases. The ALS motor unit often misses muscle contraction (~56%), whereas drug treatments returned the success rate of muscle to nearly normal; n = 6. ALS, ALS^+Rap, ALS^{+Rap/+bosutinib} ; n = 4. (E) To measure gene expression after coculture in the device, two tissues were collected separately and then total RNA was purified. (F) Gene expression change of the MN spheroid after D14 of culture. ATG5, ATG7, ATG16L2, BECN1, ULK1, ULK2, and LC3 regulating autophagy increased by the addition of rapamycin and bosutinib, while expression of TDP-43 decreased. n = 4. (G) Changes in gene expression related to the myogenesis and apoptosis of muscle tissue after D14 of culture with drug treatment. n = 4. (H) TDP-43 turnover in MN in the microfluidic devices after 14 days of culture. In the presence of drugs, the aggregation of TDP-43 was somewhat suppressed. Scale bars, 20 μm. *P < 0.05; **P < 0.01, two-way ANOVA. Error bars ± SD.