01Abnormal protein accumulation and aggregate formation are involved in the pathogenesis of various neurodegenerative diseases

Pathological analyses have shown abnormal protein accumulation in neural tissues of patients with various neurodegenerative diseases. These abnormal proteins are thought to form aggregates upon aging and various stresses, resulting in neurodegeneration. Therefore, many attempts have been made to develop therapeutics targeting the accumulation of these abnormal proteins. However, various findings have suggested that the accumulation of abnormal proteins alone is insufficient to explain the pathogenesis of neurodegeneration. To overcome neurodegenerative diseases, we believe it is necessary to clarify pathogenic events not dependent on the accumulation of abnormal proteins (early-stage pathology). Post-mortem pathology specimens obtained from autopsies are extremely important for investigating the cause of disease but only allow for observations of neurodegeneration after it has occurred; therefore, most human specimens are unsuitable for analyses of disease onset or processes of neurodegeneration. Accordingly, we believe that it is important to use ‘disease models that can reproduce the exact state of the disease’.

02Elucidating the mechanism of neurodegeneration by analyzing differentiation and maturation of neurons generated from patient-derived iPSCs (disease-specific iPSCs)

We believe induced pluripotent stem cells (iPSCs) derived from patients with neurological diseases can be turned into neuronal cells as a ‘disease model that can reproduce the exact state of the disease’. To elucidate the pathogenesis of neurological diseases and explore new therapeutic approaches, we are generating iPSCs from somatic cells obtained from patients with neurodegenerative diseases (disease-specific iPSCs) and differentiating ‘patients’ neurons’ from these iPSCs. In particular, we are focusing on processes to differentiate and mature patient-derived iPSCs into neurons that reproduce the onset and progression of diseases (e.g., accumulation or aggregate-formation of abnormal proteins during neuronal degeneration on a culture dish). By employing such techniques, we are creating new disease models that use patient-derived neural cells to elucidate mechanisms of neurodegeneration.

03Development of methods for neural differentiation of human iPSCs

To advance research on neurological diseases using human iPSCs, it is first necessary to induce neural stem cells and neuronal cells from human iPSCs. To this end, we have developed culture methods that induce highly efficient differentiation of neural stem cells and neuronal cells from human iPSCs.

These differentiation methods can recapitulate human neural development in a dish and are useful for elucidating its underlying mechanisms. Therefore, we are using human iPSC-derived neural cells not only to study neurological diseases but also elucidate mechanisms underlying human nervous system development. In addition, we are working on quality assessments of human iPSC-derived neural stem cells (NSCs) and neural cells intended for use in disease analysis and regenerative medicine.

04Recapitulation of various neurological diseases on a culture dish using patient-derived iPSCs

So far, we have generated neural cells from disease-specific iPSCs from patients with various neurological diseases to perform disease modeling and pathophysiological analysis. Patient iPSC-derived neural cells reproduce the patient’s disease condition well and can be used for both detailed pathological analysis and drug evaluations.

In our laboratory, we have primarily been working on iPSCs derived from patients with motor neuron diseases, such as spinal and bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS). Recently, we have expanded our studies to include iPSCs derived from patients with other neurological diseases.

05Establishment of iPSCs from patients with neurodegenerative diseases

We are establishing iPSCs by introducing reprogramming factors such as OCT4, SOX2, KLF4, c-MYC (also known as the four Yamanaka factors) or a combination of OCT4, SOX2, KLF4, LIN28, L-MYC, shP53, and other pluripotency-related genes into fibroblasts or cells in peripheral blood obtained from patients. We are then evaluating established iPSCs for quality and using them for analysis. The quality of iPSCs is typically evaluated based on their expression of pluripotency markers, ability to differentiate into various cell types (teratoma formation and in vitro differentiation potentials), the presence of chromosomal abnormalities, and preservation of patient genetic mutations.

06Rapid and efficient differentiation of human iPSCs into motor neurons

In our laboratory, we developed a rapid and efficient method to induce differentiation of human iPSCs into motor neurons. We are able to induce motor neurons with an efficiency of approximately 40%–60% in about two weeks. These motor neurons can generate synaptic connections, also called neuromuscular junctions (NMJs), with skeletal muscle cells when co-cultured. We are currently working on creating a variety of cells besides motor neurons.

07Development of novel therapeutics for neurodegeneration

We are investigating the molecular mechanisms underlying neurodegeneration using patient-derived neurons generated from patient-derived iPSCs. Our aim is to develop new therapeutics targeting the molecules and signals involved in neurodegeneration for treatment of neurodegenerative disorders.

08Disease modeling and pathophysiological analysis focusing on interactions between motor neurons and skeletal muscle

Interactions between motor neurons and skeletal muscle play important roles in the pathogenesis of various neuromuscular diseases. In other words, analysis of motor neurons or skeletal muscle alone may not allow for fundamental pathogenesis to be elucidated. Amyotrophic lateral sclerosis (ALS), spinal-bulbar muscular atrophy (SBMA), and spinal muscular atrophy (SMA) are neurodegenerative diseases in which motor neurons are selectively degenerated. Traditionally, motor neurons have been thought to be the cause of neurodegeneration. In recent years, however, the involvement of neuromuscular junctions (NMJs) and skeletal muscles in the pathogenesis of these diseases has been suggested, making them important targets for pathological analysis and therapeutic development. In our laboratory, we are generating iPSCs from ALS and SBMA patients, differentiating them into motor neurons and skeletal muscles, and co-culturing these cells to reproduce neuromuscular interactions and synaptic pathology to elucidate the pathogenesis of neuromuscular diseases from a new perspective.

Development of a rapid and efficient method to induce differentiation of iPSCs into skeletal muscle

iPSC-derived skeletal muscle forms neuromuscular junctions (NMJs) with iPSC-derived motor neurons.

09Neuronal regeneration in damaged peripheral nerves using human iPSC-derived motor neurons

Because there are no effective treatments for nerve injuries or intractable neurological diseases such as amyotrophic lateral sclerosis (ALS), there is an urgent need for effective therapies that reconstruct motor functions. For the treatment of spinal cord injury and Parkinson’s disease, attempts have been made to reconstruct impaired neural functions using neural cells generated from pluripotent stem cells such as ESCs and iPSCs. However, it remains difficult to reconstruct the complex neural networks of the central nervous system. In contrast, regeneration of the peripheral nervous system is expected to be relatively feasible because it is relatively simple and the number of cells required is small. Our collaborator, a group at Nagoya University Department of Hand Surgery, has successfully regenerated and reconstructed injured peripheral nerves using fetal-derived motor neurons in a rat model. By combining this technology with iPSC technology and ‘optogenetics’ technology to stimulate nerve cells using light, we are now conducting research to regenerate neuronal cells in the peripheral nervous system using human iPSC-derived motor neurons and reconstruct motor functions.