iPS Cell Technology Research Articles

Pharmacological rescue of mitochondrial and neuronal defects in SPG7 hereditary spastic paraplegia patient neurons using high throughput assays.

SPG7 is the most common form of autosomal recessive hereditary spastic paraplegia (HSP). There is a lack of HSP-SPG7 human neuronal models to understand the disease mechanism and identify new drug treatments. We generated a human neuronal model of HSP-SPG7 using induced pluripotent stem (iPS) cell technology. We first generated iPS cells from three HSP-SPG7 patients carrying different disease-causing variants and three healthy controls. The iPS cells were differentiated to form neural progenitor cells (NPCs) and then from NPCs to mature cortical neurons. Mitochondrial and neuronal defects were measured using a high throughout imaging and analysis-based assay in live cells. Our results show that compared to control NPCs, patient NPCs had aberrant mitochondrial morphology with increased mitochondrial size and reduced membrane potential. Patient NPCs develop to form mature cortical neurons with amplified mitochondrial morphology and functional defects along with defects in neuron morphology - reduced neurite complexity and length, reduced synaptic gene, protein expression and activity, reduced viability and increased axonal degeneration. Treatment of patient neurons with Bz-423, a mitochondria permeability pore regulator, restored the mitochondrial and neurite morphological defects and mitochondrial membrane potential back to control neuron levels and rescued the low viability and increased degeneration in patient neurons. This study establishes a direct link between mitochondrial and neuronal defects in HSP-SPG7 patient neurons. We present a strategy for testing mitochondrial targeting drugs to rescue neuronal defects in HSP-SPG7 patient neurons.

Recent Clinical Treatment and Basic Research on the Alveolar Bone.

The periodontal ligament is located between the bone (alveolar bone) and the cementum of the tooth, and it is connected by tough fibers called Sharpey's fibers. To maintain healthy teeth, the foundation supporting the teeth must be healthy. Periodontal diseases, also known as tooth loss, cause the alveolar bone to dissolve. The alveolar bone, similar to the bones in other body parts, is repeatedly resorbed by osteoclasts and renewed by osteogenic cells. This means that an old bone is constantly being resorbed and replaced by a new bone. In periodontal diseases, the alveolar bone around the teeth is absorbed, and as the disease progresses, the alveolar bone shrinks gradually. In most cases, the resorbed alveolar bone does not return to its original form even after periodontal disease is cured. Gum covers the tooth surface so that it matches the shape of the resorbed alveolar bone, exposing more of the tooth surface than before, making the teeth look longer, leaving gaps between the teeth, and in some cases causing teeth to sting. Previously, the only treatment for periodontal diseases was to stop the disease from progressing further before the teeth fell out, and restoration to the original condition was almost impossible. However, a treatment method that can help in the regeneration of the supporting tissues of the teeth destroyed by periodontal diseases and the restoration of the teeth to their original healthy state as much as possible is introduced. Recently, with improvements in implant material properties, implant therapy has become an indispensable treatment method in dentistry and an important prosthetic option. Treatment methods and techniques, which are mainly based on experience, have gradually accumulated scientific evidence, and the number of indications for treatment has increased. The development of bone augmentation methods has contributed remarkably to the expansion of indications, and this has been made possible by various advances in materials science. The induced pluripotent stem cell (iPS) cell technology for regenerating periodontal tissues, including alveolar bone, is expected to be applied in the treatment of diseases, such as tooth loss and periodontitis. This review focuses on the alveolar bone and describes clinical practice, techniques, and the latest basic research.

IPS cell technologies toward overcoming neurological diseases

Neurological diseases are often life threatening, with severely affecting an individual's quality of life. However, the disease mechanisms are still less understood, mainly because of lacking good disease models. Over the past decades, researchers developed many models using cell lines or animals, but most of them did not faithfully recapitulate the disease phenotypes. In particular, it is almost impossible to create animal models for multifactorial diseases or sporadic cases of unknown etiology. In these circumstances, it has come to be expected that induced pluripotent stem cells (iPSCs) can revolutionize neurological disease research as they retain patient's genetic information and provide an expandable source of disease-relevant neurons and glial cells. iPSC technologies are now widely used for disease modeling, and further for drug discovery and regenerative medicine. They are also enabling previously infeasible studies such as those uncovering how disease-associated single nucleotide polymorphism (SNP) and genetic variants increase the disease risk. This review describes a variety of iPSC technologies to produce various types of neurons and brain-like tissues (brain organoids) and summarize recent trends in iPSC technology-based neurological disease research. We also discuss the remaining challenges for understanding and overcoming brain disorders.

Elucidation of pathology and drug discovery in mental disorders using genomic information and iPS cell technology

Elucidation of pathology and drug discovery in mental disorders using genomic information and iPS cell technology

Development of COVID-19 drugs using human iPS cell technology

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which results in a rapid increase in the number of patients and deaths. In addition, various mutant strains have emerged and to be considered to accelerate the number of infected persons. To overcome this situation, effective strategies against COVID-19 include the development of vaccines to prevent SARS-CoV-2 infection and therapeutic agents that suppress the severity after infection. The drug repositioning approach, which search existing drugs that are effective against COVID-19, are expected to develop anti-COVID-19 drugs. In addition, various methods using human iPSC-derived differentiated cells has been developed to evaluate the efficacy and safety of drugs, and are also used for searching for therapeutic drugs for COVID-19. Here, we would like to describe the recent research and future perspectives for COVID-19 therapeutic drugs from the viewpoint of human iPS cell technology.

Human iPSC-based models to evaluate COVID-19 drugs

New approaches methodologies (NAMs) have been developed to predict adverse events in humans more accurately. In vitro methods, such as human iPS cell technology, are expected to accelerate the efficiency of drug development and ensure the patients' safety and speed-up of the review process. We have evaluated the iPSC-based tools to assess safety and effectiveness of chemicals from the viewpoint of regulatory science. Based on the current COVID-19 pandemic situation and animal alternative methods, we have made various iPSC models, such as cardiomyocytes, blood-brain barrier, and intestine, to evaluate the effects of COVID-19 drugs. Here we would like to summarize the current status and future perspectives of iPSC-based system for COVID-19 in drug development.

Current status and future directions of clinical applications using iPS cells-focus on Japan.

Regenerative medicine using iPS cell technologies has progressed remarkably in recent years. In this review, we summarize these technologies and their clinical application. First, we discuss progress in the establishment of iPS cells, including the HLA-homo iPS cell stock project in Japan and the advancement of low antigenic iPS cells using genome-editing technology. Then, we describe iPS cell-based therapies in or approaching clinical application, including those for ophthalmological, neurological, cardiac, hematological, cartilage, and metabolic diseases. Next, we introduce disease models generated from patient iPS cells and successfully used to identify therapeutic agents for intractable diseases. Clinical medicine using iPS cells has advanced safely and effectively by making full use of current scientific standards, but tests on cell safety need to be further developed and validated. The next decades will see the further spread of iPS cell technology-based regenerative medicine.

Electrophysiological Quality Control of Human Dopaminergic Neurons: Are We Doing Enough?

The breakthrough of induced pluripotent stem (iPS) cell technology (Takahashi and Yamanaka, 2006) has allowed scientists to design protocols allowing the differentiation of these cells into the main cell types of our organism. This possibility is particularly exciting in the field of Parkinson's disease (PD), since early experiments using heterologous dopaminergic (DA) or chromaffin cells have demonstrated the ability of such cells, when injected in adequate sites, to improve symptoms of the disease in a sustained manner (Kefalopoulou et al., 2014). The possibility of reproducing this effect using autologous cells, thereby free of ethical concerns, is of course fascinating (Barker et al., 2017; Parmar et al., 2020). It has clearly been demonstrated that symptoms of experimental parkinsonism can be relieved in several species by such grafts (Kikuchi et al., 2017; Song et al., 2020), with a first human case report study yielding encouraging results (Schweitzer et al., 2020). Protocols are being refined to improve the yield of DA neurons and minimize the survival of undifferentiated cells (e.g., Piao et al., 2021). Immunohistochemical methods allow to clearly define the presence of DA neurons, the absence of other monoaminergic neurons (e.g., serotoninergic neurons) and the molecular subtype of these DA neurons (by examining the expression of a variety of transcription factors such as NURR1, PITX3, etc…). Many experiments have confirmed the possibility of obtaining preparations devoid of oncogenic potential. In the published papers, some efforts have been made to characterize the physiological properties of the DA neurons, but I argue that, in many cases, the data is not completely convincing and, more importantly, that the reader has no idea of the percentage of neurons that display the specific properties of these neurons. I believe that more standardization of these experiments is important to assess the functional quality of the future grafts. Although I reckon that safety is of paramount importance, I also believe that suboptimal functionality may impair the clinical effectiveness of future procedures.

Genome-wide CRISPR/Cas9 screening in human iPS derived cardiomyocytes uncovers novel mediators of doxorubicin cardiotoxicity

Human induced pluripotent stem (iPS) cell technologies coupled with genetic engineering now facilitate the study of the molecular underpinnings of disease in relevant human cell types. Application of CRISPR/Cas9-based approaches for genome-scale functional screening in iPS-derived cells, however, has been limited by technical constraints, including inefficient transduction in pooled format, loss of library representation, and poor cellular differentiation. Herein, we present optimized approaches for whole-genome CRISPR/Cas9 based screening in human iPS derived cardiomyocytes with near genome-wide representation at both the iPS and differentiated cell stages. As proof-of-concept, we perform a screen to investigate mechanisms underlying doxorubicin mediated cell death in iPS derived cardiomyocytes. We identified two poorly characterized, human-specific transporters (SLCO1A2, SLCO1B3) whose loss of function protects against doxorubicin-cardiotoxicity, but does not affect cell death in cancer cells. This study provides a technical framework for genome-wide functional screening in iPS derived cells and identifies new targets to mitigate doxorubicin-cardiotoxicity in humans.

MDA5 Governs the Innate Immune Response to SARS-CoV-2 in Lung Epithelial Cells

Recent studies profiling the innate immune signatures in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suggest that cellular responses to viral challenge impact disease severity. Yet, the molecular events that underlie cellular recognition and response to SARS-CoV-2 infection remains to be elucidated. Here, we find that SARS-CoV-2 replication induces a delayed interferon (IFN) response in lung epithelial cells. Through a survey of putative sensors involved in detection of RNA virus infection, we found that MDA5 and LGP2 primarily regulate IFN induction in response to SARS-CoV-2 infection. Additionally, we find that IRF-3, -5, and NF-kB/p65 are the key transcription factors regulating the IFN response during SARS-CoV-2 infection. In summary, these findings provide critical insights into the molecular basis of the innate immune recognition and signaling response to SARS-CoV-2.Funding: This work was supported by the following grants to the Sanford Burnham Prebys Medical Discovery Institute: DoD: W81XWH-20-1-0270; DHIPC: U19 AI118610; Fluomics/NOSI: U19 AI135972, as well as generous philanthropic donations from Dinah Ruch and Susan & James Blair. This work was additionally supported by the following grants to Northwestern University Feinberg School of Medicine: a CTSA supplement to NCATS: UL1 TR002389; a CTSA supplement to NUCATS with the generous support of the Dixon family: UL1 TR001422; and a Cancer Center supplement: P30 CA060553. Development and implementation of iPS cell technology for production of airway epithelial cells was supported by Incubation Program from Office of Society Academia Collaboration for Innovation, Kyoto University. Conflict of Interest: The authors declare no competing interests.

Current challenges and future perspectives of iPSC-based neurotoxicity testing

Predicting drug-induced side effects in central nervous system is important because they can lead to the discontinuation of new drugs/candidates or the withdrawal of marketed drugs. Although many efforts are made, evaluation system using animals have not been highly predictive in humans. In addition, animal experiments are time-consuming and costly. To address these issues, in vitro evaluation methods, such as the use of New Approach Methodologies (NAM) have been explored. Human iPS cell technology has already been applied to assess drug-induced cardiotoxicity. In addition, the use of human iPS cell technology and in silico has been promoted for neurotoxicity assessment during the developmental neurotoxicity in terms of chemical safety issues. Organization for Economic Cooperation and Development (OECD) guidance regarding developmental neurotoxicity is under preparation. In this review, we will review the current trends in safety assessment methods for the central nervous system in light of these international trends.

Integrated safety evaluation of benzodiazepines using human iPS cell technology

Integrated safety evaluation of benzodiazepines using human iPS cell technology

Development of high efficient method in human iPSinduction from patient derived primary cells.

Abstract: The greatest advantage of iPS cell technology is that undifferentiatedcells can be obtained from the patient's own cells. Although iPS celldifferentiation is easy using the established cell line, its efficiency isextremely poor using patient-derived primary cultured cells because ofpathological aging. The purpose of this study was to establish a highlyefficient iPS induction method that can be applied to poor quality somatic cellssuch as pathogenic cells and senescent cells. Method and material: Dermalfibroblasts were isolated and cultured from three elderly patients who hadundergone cardiac surgery. As next step, episomatic vectors containing the fourYamanaka factors were transformed into these cells to induce iPS cells. Since itis difficult to induce iPS from pathogenic and senescent cells using thestandard iPS cell protocol, we had developed the optimal induction method. Then,we would verify whether the established iPS cell induction method usingfour-chamber myocardial tissue and fibroblasts derived from dermal tissue ofpatients with end-stage heart failure isolated by heart transplantation aspathological somatic cells. Results and conclusion: Pathological fibroblasts aremainly myofibroblasts that highly express alpha-smooth muscle actin and theywere factors that attenuated iPS induction efficiency. Therefore, by controllingthe factors in the culture medium, we succeeded in inducing iPS cells frompathological senescent cells obtained from the heart tissue of patient withend-stage heart failure.

Drug-induced neurotoxicity assessment using human iPS cell technology

Drug-induced neurotoxicity assessment using human iPS cell technology

Unveiling the impact of epigenetic regulation in disease pathogenesis with iPS cell technology

Unveiling the impact of epigenetic regulation in disease pathogenesis with iPS cell technology

Development of human intestinal organoids from iPS cell technology

Development of human intestinal organoids from iPS cell technology

Platelets using iPS cell technology; large scale manufacturing

Induced pluripotent stem cell (iPSC) derived-platelet like particle product (iPS-platelets) is aimed to complement the current blood donor-dependent system, which is expecting the shortage of blood donors in the younger population due to the aging societies in developed countries and platelet transfusion refractoriness due to alloimmune responses. One of the strategies is to establish expandable megakaryocyte lines as a source of manufacturing cGMP grade platelets. Additionally, by scaling up of the bioreactor with novel physical parameters in optimal range, more than 100 billion iPS-platelets were produced in a 8L newly developed reactor tank towards supply of an one unit platelets concentrate (300 billion of platelets, USA). In vitro and in vivo evaluation of iPS-platelets showed the functionality comparable with donor-derived platelets. We further plan to establish the proof-of-concept of the universal HLA class-I knocked out platelets towards clinical application and the further industrial production.

Cytoplasmic fusion between an enlarged embryonic stem cell and a somatic cell using a microtunnel device

Based on a previous finding that fusion of a somatic cell with an embryonic stem (ES) cell reprogrammed the somatic cell, genes for reprogramming transcription factors were selected and induced pluripotent stem (iPS) cell technology was developed. The cell fusion itself produced a tetraploid cell. To avoid nuclear fusion, a method for cytoplasmic fusion using a microtunnel device was developed. However, the ES cell was too small for cell pairing at the device. Therefore, in the present study, ES cell enlargement was carried out with the colchicine derivative demecolcine (DC). DC induced enlargement of ES cells without loss of their stemness. When an enlarged ES cell was paired with a somatic cell in the microtunnel device, cytoplasmic fusion was observed. The present method may be useful for further development of reprogramming techniques for iPS cell preparation without gene transfection.

Abnormal nuclear aggregation and myotube degeneration in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is caused by CTG nucleotide repeat expansions in the 3'-untranslated region (3'-UTR) of the dystrophia myotonica protein kinase (DMPK) gene. The expanded CTG repeats encode toxic CUG RNAs that cause disease, largely through RNA gain-of-function. DM1 is a fatal disease characterized by progressive muscle wasting, which has no cure. Regenerative medicine has emerged as a promising therapeutic modality for DM1, especially with the advancement of induced pluripotent stem (iPS) cell technology and therapeutic genome editing. However, there is an unmet need to identify in vitro outcome measures to demonstrate the therapeutic effects prior to in vivo clinical trials. In this study, we examined the muscle regeneration (myotube formation) in normal and DM1 myoblasts in vitro to establish outcome measures for therapeutic monitoring. We found normal proliferation of DM1 myoblasts, but abnormal nuclear aggregation during the early stage myotube formation, as well as myotube degeneration during the late stage of myotube formation. We concluded that early abnormal nuclear aggregation and late myotube degeneration offer easy and sensitive outcome measures to monitor therapeutic effects in vitro.

IPS cell technology for periodontal tissue regeneration

iPS cell technology for periodontal tissue regeneration