A Cylindrical Molding Method for the Biofabrication of Plane-Shaped Skeletal Muscle Tissue.

Muscle tissues can be fabricated in vitro by culturing myoblast-populated hydrogels. To counter the shrinkage of the myoblast-populated hydrogels during culture, a pair of anchors are generally utilized to fix the two ends of the hydrogel. Here, we propose an alternative method to counter the shrinkage of the hydrogel and fabricate plane-shaped skeletal muscle tissues. The method forms myoblast-populated hydrogel in a cylindrical cavity with a central pillar, which can prevent tissue shrinkage along the circumferential direction. By eliminating the usages of the anchor pairs, our proposed method can produce plane-shaped skeletal muscle tissues with uniform width and thickness. In experiments, we demonstrate the fabrication of plane-shaped (length: ca. 10 mm, width: 5~15 mm) skeletal muscle tissue with submillimeter thickness. The tissues have uniform shapes and are populated with differentiated muscle cells stained positive for myogenic differentiation markers (i.e., myosin heavy chains). In addition, we show the assembly of subcentimeter-order tissue blocks by stacking the plane-shaped skeletal muscle tissues. The proposed method can be further optimized and scaled up to produce cultured animal products such as cultured meat.

Formation of contractile 3D bovine muscle tissue for construction of millimetre-thick cultured steak.

Owing to the increase in the global demand of meat, cultured meat technology is being developed to circumvent a shortage of meat in the future. However, methods for construction of millimetre-thick bovine muscle tissues with highly aligned myotubes have not yet been established. Here, we propose a culture method for constructing 3D-cultured bovine muscle tissue containing myotubes aligned along its long-axial direction, which contracted in response to electrical stimulation. First, we optimised the composition of biomaterials used in the construction and the electrical stimulation applied to the tissue during culture. Subsequently, we fabricated millimetre-thick bovine muscle tissues containing highly aligned myotubes by accumulating bovine myoblast-laden hydrogel modules. The microbial content of the bovine muscle tissue cultured for 14 days was below the detection limit, indicating that the muscle tissues were sterile, unlike commercial meat. Therefore, the proposed construction method for bovine muscle tissues will be useful for the production of clean cultured steak meat simulating real meat.

Three-dimensional co-culture of blood-brain barrier-composing cells in a culture insert with a collagen vitrigel membrane.

The blood-brain barrier (BBB) is a structure located in brain capillaries that protects the brain from toxic substances in blood due to its high barrier function. The brain capillaries form a layered structure with pericytes, neurons, glial cells, and extracellular matrix proteins that is called neurovascular unit, and the structure is important to express the high barrier function of BBB. Here, we propose a method to construct a three-dimensional BBB tissue using three human BBB-composing cells, including brain endothelial cells, pericytes, and astrocytes, that mimics the in vivo BBB-like layered structure. Primary human brain endothelial cells were plated on the back side (outside) of the collagen vitrigel membrane of a culture insert, pericytes were plated on the upper side (inside), and astrocytes mixed in Matrigel were plated on the pericyte layer. The layered structure was maintained for at least 2 wk. The BBB tissue-loaded collagen vitrigel membrane can be detached from the insert frame using acetone with the tissue fixed intact and used for vertical cryosectioning to analyze the tissue interior. We also measured transendothelial electrical resistance (TEER) in the three-dimensional BBB co-culture to investigate barrier function of the brain endothelial cells. We believe that our co-culture method is useful to study engineered BBB tissues and develop reliable in vitro human BBB models in the future.

Cell-laden microfibers fabricated using µl cell-suspension.

Current microfluidic methods for cell-laden microfiber fabrication generally require larger than 100 µl of cell-suspensions. Since some 'rare' cells can be only acquired in small amounts, the preparation of >100 µl cell-suspensions with high-cell density can be both expensive and time consuming. Here, we present a facile method capable of fabricating cell-laden microfibers using small-volume cell-suspensions. The method utilizes a 3D-printed coaxial microfluidic device featured with a 'luer-lock inlet' to effectively load cell-suspensions in a deterministic volume (down to 5 µl) with a low sample-loss. In experiments, we demonstrate the formation of fibrous tissues consisting of various kinds of cells. Investigations on the morphology and function of the encapsulated cells show the viability of the cells is not significantly affected by the fabrication process, and also indicate the potential of using our method to perform quantitative assays on fiber-shaped tissues, while reducing the overall material and time consumption.

Cell fibers promote proliferation of co-cultured cells on a dish.

This paper describes a co-culture method using cell fiber technology. Cell fibers are cell-laden hydrogel microfibers, in which cells are cultured three-dimensionally and allowed to reach more mature state than the conventional two-dimensional cell culture. Cells in the cell fibers are encapsulated by alginate shell. Only cellular secretome is released into the surrounding environment through the shell while the cells were retained by the fiber. With their high handleability and retrievability, we propose to use the cell fibers for co-culture to ensure steady supply of cellular secretome. We cultured mouse C2C12 myoblasts with mouse 3T3 fibroblasts encapsulated in the cell fibers for two days. The number of C2C12 cells increased proportionally to the number of co-cultured 3T3 fibers, suggesting that the secretome of 3T3 fibers promoted survival and proliferation of C2C12 cells. We believe that cell fiber technology is a useful tool for co-culturing cells, and it will contribute to both basic cell biology and tissue engineering with its unique features.

Three-dimensional contractile muscle tissue consisting of human skeletal myocyte cell line.

This paper describes a method to construct three-dimensional (3D) contractile human skeletal muscle tissues from a cell line. The 3D tissue was fabricated as a fiber-based structure and cultured for two weeks under tension by anchoring its both ends. While myotubes from the immortalized human skeletal myocytes used in this study never contracted in the conventional two-dimensional (2D) monolayer culture, myotubes in the 3D tissue showed spontaneous contraction at a high frequency and also reacted to the electrical stimulation. Immunofluorescence revealed that the myotubes in the 3D tissues had sarcomeres and expressed ryanodine receptor (RyR) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). In addition, intracellular calcium oscillations in the myotubes in the 3D tissue were observed. These results indicated that the 3D culture enabled the myocyte cell line to reach a more highly matured state compared to 2D culture. Since contraction is the most significant feature of skeletal muscle, we believe that our 3D human muscle tissue with the contractile ability would be a useful tool for both basic biology research and drug discovery as one of the muscle-on-chips.

Gender Effects on the Clinical Phenotype in Japanese Patients with Spinal Muscular Atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is a neuromuscular disease caused by a mutation in SMN1. SMA is classified into three subtypes (types 1, 2, 3) based on achieved motor milestones. Although NAIP and SMN2 are widely accepted as SMA-modifying factors, gender-related modifying factors or gender effects on the clinical phenotype are still controversial. METHODS: A total of 122 Japanese patients with SMA, of which SMN1 was homozygously deleted, were analyzed from the perspective of the achieved motor milestone, NAIP status and SMN2 copy number. RESULTS: A predominance of male patients was observed in SMA type 3 (the walker group) without NAIP-deletion or with high SMN2 copy number (3 or 4 copies). CONCLUSION: We suggest the presence of gender-related modifiers on disease severity in SMA patients. The modifiers may contribute only in the presence of NAIP and a high copy number of SMN2.

Alternative splicing of a cryptic exon embedded in intron 6 of SMN1 and SMN2.

Both survival of motor neuron (SMN) genes are associated with spinal muscular atrophy; mutations in SMN1 cause the disease, and SMN2 modulates its severity. It is established that different alternative splicing of exon 7 occurs for SMN1 and SMN2, and a cryptic exon was recently found in intron 6 of both genes. Here, we characterize this cryptic exon and clarify its alternative splicing pattern in control and spinal muscular atrophy cells.

Tadalafil Treatment Delays the Onset of Cardiomyopathy in Dystrophin-Deficient Hearts.

BACKGROUND: Cardiomyopathy is a leading cause of mortality among Duchenne muscular dystrophy patients and lacks effective therapies. Phosphodiesterase type 5 is implicated in dystrophic pathology, and the phosphodiesterase type 5 inhibitor tadalafil has recently been studied in a clinical trial for Duchenne muscular dystrophy. METHODS AND RESULTS: Tadalafil was evaluated for the prevention of cardiomyopathy in the mdx mouse and golden retriever muscular dystrophy dog models of Duchenne muscular dystrophy. Tadalafil blunted the adrenergic response in mdx hearts during a 30-minute dobutamine challenge, which coincided with cardioprotective signaling, reduced induction of µ-calpain levels, and decreased sarcomeric protein proteolysis. Dogs with golden retriever muscular dystrophy began daily tadalafil treatment prior to detectable cardiomyopathy and demonstrated preserved cardiac function, as assessed by echocardiography and magnetic resonance imaging at ages 18, 21, and 25 months. Tadalafil treatment improved golden retriever muscular dystrophy histopathological features, decreased levels of the cation channel TRPC6, increased total threonine phosphorylation status of TRPC6, decreased m-calpain levels and indicators of calpain target proteolysis, and elevated levels of utrophin. In addition, we showed that Duchenne muscular dystrophy patient myocardium exhibited increased TRPC6, m-calpain, and calpain cleavage products compared with control human myocardium. CONCLUSIONS: Prophylactic use of tadalafil delays the onset of dystrophic cardiomyopathy, which is likely attributed to modulation of TRPC6 levels and permeability and inhibition of protease content and activity. Consequently, phosphodiesterase type 5 inhibition is a candidate therapy for slowing the development of cardiomyopathy in Duchenne muscular dystrophy patients.

Telomeric Region of the Spinal Muscular Atrophy Locus Is Susceptible to Structural Variations.

BACKGROUND: Most patients with spinal muscular atrophy lack the survival motor neuron 1 gene (SMN1) in the telomeric region of the spinal muscular atrophy locus on chromosome 5q13. On the other hand, the copy number of SMN2, a centromeric homolog of SMN1, is increased in many of these patients. This study aimed to clarify the mechanism underlying these structural variations. METHODS: We determined the copy numbers of telomeric and centromeric genes in the spinal muscular atrophy locus of 86 patients and 22 control subjects using multiplex ligation-dependent probe amplification analysis. Then, we chose 74 patients lacking SMN1 exons 7 and 8, and compared their dataset with that of 22 control subjects retaining SMN1 exons 7 and 8. RESULTS: The SMN2 copy number was shown to vary widely and to correlate with the disease severity of the patients. Interestingly, telomeric NAIP and telomeric GTF2H2 showed similar tendencies. We also noted positive correlations among the copy number of SMN2 and the telomeric genes of the spinal muscular atrophy locus. However, the copy numbers of centromeric NAIP and centromeric GTF2H2 were stable among the patients, with both approximating a value of two. CONCLUSION: Our findings suggested that the telomeric region of the spinal muscular atrophy locus appears to be susceptible to structural variation, whereas the centromeric region is stable. Moreover, according to our results, new SMN2 copies may be generated in the telomeric region of the spinal muscular atrophy locus, supporting the SMN1-to-SMN2 gene conversion theory.

Low culture temperature inhibits myogenic differentiation through mitochondrial activity.

A previous study by our group reported that mouse and human myoblasts fail to express myogenin and to fuse into multi-nucleate myotubes when cultured at low temperature, such as 30°C, but that this activity is rescued by adding IGF-I and vitamin C to the culture medium. In the present study, we examined mitochondrial activity as a target of the inhibitory effects of the low culture temperature. It has been suggested that mitochondria regulate myogenesis. By using a mouse myoblast cell line C2C12, we demonstrate that the expression of cytochrome c oxidase subunit I (COX I), which is encoded in mitochondrial genome, increases during myogenic differentiation at the normal culture temperature (38°C), but that this up-regulation is inhibited at 30°C. The mitochondrial membrane potential also decreased at 30°C compared to the culture at 38°C. However, IGF-I and vitamin C rescued both COX I expression and mitochondrial membrane potential at 30°C as promoting muscle differentiation. We also find that the rescue of mitochondrial activity by IGF-I and vitamin C at 30°C occurred after the myogenin expression, which suggests that myogenin regulates mitochondrial function during myogenesis. We suggest that our low temperature-culture system may be suitable for use in studying the detailed mechanism of myogenin-related phenomena during myogenesis.

Myosin VI deafness mutation prevents the initiation of processive runs on actin.

Mutations in the reverse-direction myosin, myosin VI, are associated with deafness in humans and mice. A myosin VI deafness mutation, D179Y, which is in the transducer of the motor, uncoupled the release of the ATP hydrolysis product, inorganic phosphate (Pi), from dependency on actin binding and destroyed the ability of single dimeric molecules to move processively on actin filaments. We observed that processive movement is rescued if ATP is added to the mutant dimer following binding of both heads to actin in the absence of ATP, demonstrating that the mutation selectively destroys the initiation of processive runs at physiological ATP levels. A drug (omecamtiv) that accelerates the actin-activated activity of cardiac myosin was able to rescue processivity of the D179Y mutant dimers at physiological ATP concentrations by slowing the actin-independent release of Pi. Thus, it may be possible to create myosin VI-specific drugs that rescue the function of deafness-causing mutations.

Salbutamol inhibits ubiquitin-mediated survival motor neuron protein degradation in spinal muscular atrophy cells.

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is currently incurable. SMA is caused by decreased levels of the survival motor neuron protein (SMN), as a result of loss or mutation of SMN1. Although the SMN1 homolog SMN2 also produces some SMN protein, it does not fully compensate for the loss or dysfunction of SMN1. Salbutamol, a ß2-adrenergic receptor agonist and well-known bronchodilator used in asthma patients, has recently been shown to ameliorate symptoms in SMA patients. However, the precise mechanism of salbutamol action is unclear. We treated SMA fibroblast cells lacking SMN1 and HeLa cells with salbutamol and analyzed SMN2 mRNA and SMN protein levels in SMA fibroblasts, and changes in SMN protein ubiquitination in HeLa cells. Salbutamol increased SMN protein levels in a dose-dependent manner in SMA fibroblast cells lacking SMN1, though no significant changes in SMN2 mRNA levels were observed. Notably, the salbutamol-induced increase in SMN was blocked by a protein kinase A (PKA) inhibitor and deubiquitinase inhibitor, respectively. Co-immunoprecipitation assay using HeLa cells showed that ubiquitinated SMN levels decreased in the presence of salbutamol, suggesting that salbutamol inhibited ubiquitination. The results of this study suggest that salbutamol may increase SMN protein levels in SMA by inhibiting ubiquitin-mediated SMN degradation via activating ß2-adrenergic receptor-PKA pathways.

IGF-I and vitamin C promote myogenic differentiation of mouse and human skeletal muscle cells at low temperatures.

In a previous study investigating the effects of low temperature on skeletal muscle differentiation, we demonstrated that C2C12 mouse myoblasts cultured at 30°C do not express myogenin, a myogenic regulatory factor (MRF), or fuse into multinucleated myotubes. At this low temperature, the myoblasts continuously express Id3, a negative regulator of MRFs, and do not upregulate muscle-specific microRNAs. In this study, we examined if insulin-like growth factor-I (IGF-I) and a stable form of vitamin C (L-ascorbic acid phosphate) could alleviate the low temperature-induced inhibition of myogenic differentiation in C2C12 cells. Although the addition of either IGF-I or vitamin C alone could promote myogenin expression in C2C12 cells at 30°C, elongated multinucleated myotubes were not formed unless both IGF-I and vitamin C were continuously administered. In human skeletal muscle cells, low temperature-induced blockage of myogenic differentiation was also ameliorated by exogenous IGF-I and vitamin C. In addition, we demonstrated that satellite cells of IGF-I overexpressing transgenic mice in single-fiber culture expressed myogenin at a higher level than those of wild-type mice at 30°C. This study suggests that body temperature plays an important role in myogenic differentiation of endotherms, but the sensitivity to low temperature could be buffered by certain factors in vivo, such as IGF-I and vitamin C.

The expression of myogenin, but not of MyoD, is temperature-sensitive in mouse skeletal muscle cells.

Homothermal animals need to keep their body temperature within a narrow range. Only a few degrees Celsius change in temperature has a dynamic influence on many physiological processes. To investigate the effect of the body temperature on muscle cell differentiation, we cultured the mouse myoblast cell lines C2C12 and Sol8 at lower temperatures than mouse body temperature. At 38 degrees C, the cells fused into multinucleated myotubes within 4 days after the induction of differentiation. However, myotube formation was blocked at 30 degrees C, whereas it was delayed but relatively normal at 35 degrees C. The myoblasts expressed MyoD, but not myogenin, at 30 degrees C. Id3, which acts as a negative regulator of myogenic regulatory factors (MRFs), was expressed at a higher level at 30 degrees C than at 38 degrees C, whereas the expression level of E2A, which acts as a positive regulator of MRF expression, exhibited no difference between these temperatures. We also found that the expression of muscle-enriched microRNAs decreased at 30 degrees C. In addition, we investigated the expressions of MyoD and myogenin during mouse satellite-cell activation in single-fiber culture as an in-vivo model, and found that the expression of myogenin, but not of MyoD, was inhibited. These results suggest that skeletal muscle formation can be regulated by temperature, and that the physiological body temperature plays a crucial role in the myogenesis of homothermal animals.