In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

Kyoto University

Human CO respiration requires rapid conversion between CO and HCO Carbonic anhydrase II facilitates this reversible reaction inside red blood cells, and band 3 [anion exchanger 1 (AE1)] provides a passage for HCO flux across the cell membrane. These 2 proteins are core components of the CO transport metabolon. Intracellular HO is necessary for CO/HCO conversion. However, abundantly expressed aquaporin 1 (AQP1) in erythrocytes is thought not to be part of band 3 complexes or the CO transport metabolon. To solve this conundrum, we used Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging (FLIM-FRET) and identified interaction between aquaporin-1 and band 3 at a distance of 8 nm, within the range of dipole-dipole interaction. Notably, their interaction was adaptable to membrane tonicity changes. This suggests that the function of AQP1 in tonicity response could be coupled or correlated to its function in band 3-mediated CO/HCO exchange. By demonstrating AQP1 as a mobile component of the CO transport metabolon, our results uncover a potential role of water channel in blood CO transport and respiration.-Hsu, K., Lee, T.-Y., Periasamy, A., Kao, F.-J., Li, L.-T., Lin, C.-Y., Lin, H.-J., Lin, M. Adaptable interaction between aquaporin-1 and band 3 reveals a potential role of water channel in blood CO transport.

Adaptable interaction between aquaporin-1 and band 3 reveals a potential role of water channel in blood CO transport.

Satellite cells, a population of skeletal muscular stem cells, are generally recognized as the main and, possibly, the sole source of postnatal muscle regeneration. Previous studies have revealed the potential of low-level laser (LLL) irradiation in promoting satellite cell proliferation, which, thereby, boosts the recovery of skeletal muscle from atrophy. The purpose of this study is to investigate the beneficial effect of LLL on disuse-induced atrophy. The optimal irradiation condition of LLL (808 nm) enhancing the proliferation of Pax7 cells, isolated from tibialis anterior (TA) muscle, was examined and applied on TA muscle of disuse-induced atrophy model of the rats accordingly. Healthy rats were used as the control. On one hand, transiently, LLL was able to postpone the progression of atrophy for 1 week through a reduction of apoptosis in Pax7MyoD (myocyte) population. Simultaneously, a significant enhancement was observed in Pax7MyoD population; however, most of the increased cells underwent apoptosis since the second week, which suggested an impaired maturation of the population. On the other hand, in normal control rats with LLL irradiation, a significant increase in Pax7MyoD cells and a significant decrease of apoptosis were observed. As a result, a strengthened muscle contraction was observed. Our data showed the capability of LLL in postponing the progression of disuse-induced atrophy for the first time. Furthermore, the result of normal rats with LLL irradiation showed the effectiveness of LLL to strengthen muscle contraction in healthy control.

A transient protective effect of low-level laser irradiation against disuse-induced atrophy of rats.

The control of voluntary skeletal muscle contraction relies on action potentials, which send signals from the motor neuron through the neuromuscular junction (NMJ). Although dysfunction of the NMJ causes various neuromuscular diseases, a reliable in vitro system for disease modeling is currently unavailable. Here, we present a potentially novel 2-step, self-organizing approach for generating in vitro human NMJs from human induced pluripotent stem cells. Our simple and robust approach results in a complex NMJ structure that includes functional connectivity, recapitulating in vivo synapse formation. We used these in vitro NMJs to model the pathological features of spinal muscular atrophy, revealing the developmental and functional defects of NMJ formation and NMJ-dependent muscular contraction. Our differentiation system is therefore useful for investigating and understanding the physiology and pathology of human NMJs.

iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases.

The aim of this study was to evaluate the response of satellite cells to muscular atrophies which possess different pathological characteristics and which were induced by distinct damages. Right lower limbs of rats were exposed to denervation or disuse and later its tibialis anterior (TA) or soleus (SOL) muscles were analyzed. After confirming their functional impairments indicated by common but distinct pathological and electrophysiological characteristics, the quantitative polymerase chain reaction analysis of Pax7 and Pax3 expressions and the number of Pax7 and Pax3 cells were analyzed sequentially at day 0, day 7, and day 14. TA muscles of both denervation- and disuse-induced atrophy models showed persisted low level of Pax7 expression from day 7 (0.91 ± 0.23 and 0.31 ± 0.07, P = 0.06, n = 6) through day 14 (1.09 ± 0.15 and 0.4 ± 0.09 [P < 0.05]). On the other hand, significant elevations were observed in Pax3 expression in both atrophy models (2.73 ± 0.46 and 2.75 ± 0.26 [P < 0.05]) at day 7. Similar to TA muscle, resembled pattern of Pax7 and Pax3 expression changes were observed between the SOL muscles of denervation- and disused-atrophy models. These trends were further confirmed by the changes in Pax7 and Pax3 cell numbers of TA and SOL muscles in both atrophy models. Despite the distinct pathological findings, similar patterns in the changes of Pax3 and Pax7 expressions and the changes of Pax7 and Pax3 cell numbers were observed between the denervation- and disuse-induced atrophy models and this commonality was admitted among the muscle type. Therefore, we claim that the muscle regeneration orchestrated by satellite cells was governed by the muscle type in which satellite cells reside.

Muscle type from which satellite cells are derived plays a role in their damage response.

 sp., such as  (GT), play an important role in traditional Chinese medicine.  sp. contains several constituents, including Sacacchin, which has recently drawn attention because it can not only enhance the repair of muscle damage but also strengthen the muscle enforcement. Although  sp. have a therapeutic effect for neuromuscular disorders, the underlying mechanism remains unclear. This study investigated the effect and underlying molecular mechanism of micronized sacchachitin (mSC) on satellite cells (SCs), which are known as the muscle stem cells.

Micronized sacchachitin promotes satellite cell proliferation through TAK1-JNK-AP-1 signaling pathway predominantly by TLR2 activation.

Despite remarkable advancements in modern medicine, muscular atrophy remains as an unsolved problem. It is well known that pathological characteristics of different atrophy types could vary according to the pathophysiological causes. In fact, the lesion of atrophy is not always homogenously distributed but often predominantly evident in either fast or slow myofibers. As the focalization of the atrophic lesions, the existence and the functional impairment of each fast and slow progenitor/satellite cell (SC) are suspected though there are still controversies about this hypothesis. In this study, we isolated Pax7 positive (Pax7) SCs from the tibia anterior (fast) and soleus (slow) muscles respectively and successfully demonstrated, for the first time, the difference between optimal exposure durations of photobiomodulation (PBM) which was known as low level laser irradiation (LLLI) in promoting proliferation of Pax7 SC which were acquired from fast and slow muscles respectively. Moreover, a hypertrophy-accompanied bidirectional change in myofiber composition with neuromuscular junction alteration, either from slow to fast or fast to slow, were achieved by applying different PBM durations. Simultaneously, PBM exhibited a synergistic effect with muscle exercise on the increase in myofiber size. Our data suggested the existence of at least two different populations of Pax7 SC which possess distinct sensitivities towards PBM. As our data revealed the capability of PBM in bidirectional changes of skeletal muscle composition and neuromuscular junction constitution thereby strengthen its contractility through altering the irradiation condition, we believe PBM showed the potential to be as a promising clinical treatment for muscular atrophy.

Chuang-Yu Lin

Department of Clinical Application,
Center for iPS Cell Research and Application (CiRA),
Department of Clinical Application, Center for iPS Cell Research and Application (CiRA)

Kyoto University

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

Chuang-Yu Lin

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Clinical Application,Center for iPS Cell Research and Application (CiRA),Department of Clinical Application, Center for iPS Cell Research and Application (CiRA)

Kyoto University

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

Department of Clinical Application,
Center for iPS Cell Research and Application (CiRA),
Department of Clinical Application, Center for iPS Cell Research and Application (CiRA)