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Nerve-derived factors have attracted attention in bone regeneration therapy due to their ability to promote bone regeneration and nerve innervation. Mesenchymal stem cells transported to target sites promote osteogenesis. However, there are few reports on the effects of neural stem cells on bone regeneration. Therefore, the aim of this study was to investigate the role of neural stem cells in osteogenesis. Here, embryoid bodies (EB) or primary neurospheres (1NS) were generated using mouse induced pluripotent stem cells (iPS cells), which were then seeded onto gelatin (Gel) sponges. The seeded Gel sponges were then transplanted into mouse calvarial bone defects. We noted that 1NS-seeded Gel promoted bone regeneration and the presence of tartrate-resistant acid phosphatase (TRAP)-positive cells, whereas the EB-seeded Gel did not. RNA-sequencing of the 1NS-seeded and EB seeded Gels showed an upregulation of the transforming growth factor (TGF)-β signaling pathway in the 1NS-seeded Gel group. Immunostaining confirmed the presence of Id3 positive cells in mice with bone defects treated with the 1NS-seeded Gel. These findings suggest that the transplantation of neural stem cells may contribute to the promotion of bone regeneration. This study aimed to investigate whether neural stem cells, when seeded in Gel sponges, promoted bone regeneration. It has been well documented that bone is tightly linked with the nervous systems. Bioscaffolds comprising factors that promote innervation and bone regeneration have been investigated for use in bone therapy. However, there is limited research on the use of neural stem cells for promoting bone formation. To assess this relationship, we conducted both in vivo and in vitro assays to determine whether neural stem cells promoted bone formation. We noted that 1NS-seeded Gel sponges promoted bone formation significantly in mice with calvarial defects after 4 weeks. This study provides a novel approach of neural stem cells for bone therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

Xeroderma pigmentosum (XP) is characterized by photosensitivity that causes pigmentary disorder and predisposition to skin cancers on sunlight-exposed areas due to DNA repair deficiency. Patients with XP group A (XP-A) develop freckle-like pigmented maculae and depigmented maculae within a year unless strict sun-protection is enforced. Although it is crucial to study pigment cells (melanocytes: MCs) as disease target cells, establishing MCs in primary cultures is challenging. Elucidation of the disease pathogenesis by comparison between MCs differentiated from XP-A induced pluripotent stem cells (iPSCs) and healthy control iPSCs on the response to UV irradiation. iPSCs were established from a XP-A fibroblasts and differentiated into MCs. Differences in gene expression profiles between XP-A-iPSC-derived melanocytes (XP-A-iMCs) and Healthy control iPSC-derived MCs (HC-iMCs) were analyzed 4 and 12 h after irradiation with 30 or 150 J/m2 of UV-B using microarray analysis. XP-A-iMCs expressed SOX10, MITF, and TYR, and showed melanin synthesis. Further, XP-A-iMCs showed reduced DNA repair ability. Gene expression profile between XP-A-iMCs and HC-iMCs revealed that, numerous gene probes that were specifically upregulated or downregulated in XP-A-iMCs after 150-J/m2 of UV-B irradiation did not return to basal levels. Of note that apoptotic pathways were highly upregulated at 150 J/m2 UV exposure in XP-A-iMCs, and cytokine-related pathways were upregulated even at 30 J/m2 UV exposure. We revealed for the first time that cytokine-related pathways were upregulated even at low-dose UV exposure in XP-A-iMCs. Disease-specific iPSCs are useful to elucidate the disease pathogenesis and develop treatment strategies of XP. • Induced pluripotent stem cells (iPSCs) were established from a XP-A patient. • Melanocytes(MCs) derived from XP-A-iPSCs and healthy control-iPSCs were analyzed. • Apoptotic pathways were highly upregulated at high-dose UV exposure in XP-A-iMCs. • Cytokine-related pathways were upregulated at low-dose UV exposure in XP-A-iMCs. • Disease-specific iPSCs are useful to elucidate the disease pathogenesis. [ABSTRACT FROM AUTHOR]

The high mortality in the global population due to chronic diseases highlights the urgency to identify effective alternative therapies. Regenerative medicine provides promising new approaches for this purpose, particularly in the use of induced pluripotent stem cells (iPSCs). The aim of the work is to establish a new pluripotency cell line obtained for the first time by reprogramming human gingival mesenchymal stem cells (hGMSCs) by a non-integrating method. The hGMSC-derived iPS line characterization is performed through morphological analysis with optical and electron scanning microscopy and through the pluripotency markers expression evaluation in cytofluorimetry, immunofluorescence, and RT-PCR. To confirm the pluripotency of new hGMSC-derived iPS, the formation of embryoid bodies (EBs), as an alternative to the teratoma formation test, is studied in morphological analysis and through three germ layers' markers' expression in immunofluorescence and RT-PCR. At the end, a comparative study between parental hGMSCs and derived iPS cells is performed also for the extracellular vesicles (EVs) and their miRNA content. The new hGMSC-derived iPS line demonstrated to be pluripotent in all aspects, thus representing an innovative dynamic platform for personalized tissue regeneration. [ABSTRACT FROM AUTHOR]

The thymus is where T cells, among the most important immune cells involved in biological defense and homeostasis, are produced and developed. The thymus plays an important role in the defense against infection and cancer as well as the prevention of autoimmune diseases. However, the thymus gland atrophies with age, which might have pathological functions, and in some circumstances, there is a congenital defect in the thymus. These can be the cause of many diseases related to the dysregulation of T cell functions. Thus, the enhancement and/or normalization of thymic function may lead to protection against and treatment of a wide variety of diseases. Therefore, thymus transplantation is considered a strong candidate for permanent treatment. The status and issues related to thymus transplantation for possible immunotherapy are discussed although it is still at an early stage of development. [ABSTRACT FROM AUTHOR]

Juvenile nephronophthisis is an inherited renal ciliopathy with cystic kidney disease, renal fibrosis, and end-stage renal failure in children and young adults. Mutations in the NPHP1 gene encoding nephrocystin-1 protein have been identified as the most frequently responsible gene and cause the formation of cysts in the renal medulla. The molecular pathogenesis of juvenile nephronophthisis remains elusive, and no effective medicines to prevent end-stage renal failure exist even today. No human cellular models have been available yet. Here, we report a first disease model of juvenile nephronophthisis using patient-derived and gene-edited human induced pluripotent stem cells (hiPSCs) and kidney organoids derived from these hiPSCs. We established NPHP1-overexpressing hiPSCs from patient-derived hiPSCs and NPHP1-deficient hiPSCs from healthy donor hiPSCs. Comparing these series of hiPSCs, we found abnormalities in primary cilia associated with NPHP1 deficiency in hiPSCs. Kidney organoids generated from the hiPSCs lacking NPHP1 formed renal cysts frequently in suspension culture with constant rotation. This cyst formation in patient-derived kidney organoids was rescued by overexpression of NPHP1. Transcriptome analysis on these kidney organoids revealed that loss of NPHP1 caused lower expression of genes related to primary cilia in epithelial cells and higher expression of genes related to the cell cycle. These findings suggested the relationship between abnormality in primary cilia induced by NPHP1 loss and abnormal proliferative characteristics in the formation of renal cysts. These findings demonstrated that hiPSC-based systematic disease modeling of juvenile nephronophthisis contributed to elucidating the molecular pathogenesis and developing new therapies. [ABSTRACT FROM AUTHOR]

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, continues to spread around the world with serious cases and deaths. It has also been suggested that different genetic variants in the human genome affect both the susceptibility to infection and severity of disease in COVID-19 patients. Angiotensin-converting enzyme 2 (ACE2) has been identified as a cell surface receptor for SARS-CoV and SARS-CoV-2 entry into cells. The construction of an experimental model system using human iPS cells would enable further studies of the association between viral characteristics and genetic variants. Airway and alveolar epithelial cells are cell types of the lung that express high levels of ACE2 and are suitable for in vitro infection experiments. Here, we show that human iPS cell-derived airway and alveolar epithelial cells are highly susceptible to viral infection of SARS-CoV-2. Using gene knockout with CRISPR-Cas9 in human iPS cells we demonstrate that ACE2 plays an essential role in the airway and alveolar epithelial cell entry of SARS-CoV-2 in vitro. Replication of SARS-CoV-2 was strongly suppressed in ACE2 knockout (KO) lung cells. Our model system based on human iPS cell-derived lung cells may be applied to understand the molecular biology regulating viral respiratory infection leading to potential therapeutic developments for COVID-19 and the prevention of future pandemics. [ABSTRACT FROM AUTHOR]

Preclinical transplantations using human neuroepithelial stem (NES) cells in spinal cord injury models have exhibited promising results and demonstrated cell integration and functional improvement in transplanted animals. Previous studies have relied on the generation of research grade cell lines in continuous culture. Using fresh cells presents logistic hurdles for clinical transition regarding time and resources for maintaining high quality standards. In this study, we generated a good manufacturing practice (GMP) compliant human iPS cell line in GMP clean rooms alongside a research grade iPS cell line which was produced using standardized protocols with GMP compliant chemicals. These two iPS cell lines were differentiated into human NES cells, from which six batches of cell therapy doses were produced. The doses were cryopreserved, thawed on demand and grafted in a rat spinal cord injury model. Our findings demonstrate that NES cells can be directly grafted post-thaw with high cell viability, maintaining their cell identity and differentiation capacity. This opens the possibility of manufacturing off-the-shelf cell therapy products. Moreover, our manufacturing process yields stable cell doses with minimal batch-to-batch variability, characterized by consistent expression of identity markers as well as similar viability of cells across the two iPS cell lines. These cryopreserved cell doses exhibit sustained viability, functionality, and quality for at least 2 years. Our results provide proof of concept that cryopreserved NES cells present a viable alternative to transplanting freshly cultured cells in future cell therapies and exemplify a platform from which cell formulation can be optimized and facilitate the transition to clinical trials. [ABSTRACT FROM AUTHOR]

Primary ciliary dyskinesia (PCD) is a relatively rare genetic disorder that affects approximately 1 in 20,000 people. Approximately 50 genes are currently known to cause PCD. In light of differences in causative genes and the medical system in Japan compared with other countries, a practical guide was needed for the diagnosis and management of Japanese PCD patients. An ad hoc academic committee was organized under the Japanese Rhinologic Society to produce a practical guide, with participation by committee members from several academic societies in Japan. The practical guide including diagnostic criteria for PCD was approved by the Japanese Rhinologic Society, Japanese Society of Otolaryngology–Head and Neck Surgery, Japanese Respiratory Society, and Japanese Society of Pediatric Pulmonology. The diagnostic criteria for PCD consist of six clinical features, six laboratory findings, differential diagnosis, and genetic testing. The diagnosis of PCD is categorized as definite, probable, or possible PCD based on a combination of the four items above. Diagnosis of definite PCD requires exclusion of cystic fibrosis and primary immunodeficiency, at least one of the six clinical features, and a positive result for at least one of the following: (1) Class 1 defect on electron microscopy of cilia, (2) pathogenic or likely pathogenic variants in a PCD-related gene, or (3) impairment of ciliary motility that can be repaired by correcting the causative gene variants in iPS cells established from the patient's peripheral blood cells. This practical guide provides clinicians with useful information for the diagnosis and management of PCD in Japan. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 genome editing is a rapidly advancing technology that has the potential to accelerate research and development in a variety of fields. However, manual genome editing processes suffer from limitations in scalability, efficiency, and standardization. The implementation of automated systems for genome editing addresses these challenges, allowing researchers to cover the increasing need and perform large-scale studies for disease modeling, drug development, and personalized medicine. In this study, we developed an automated CRISPR/Cas9-based genome editing process on the StemCellFactory platform. We implemented a 4D-Nucleofector with a 96-well shuttle device into the StemCellFactory, optimized several parameters for single cell culturing and established an automated workflow for CRISPR/Cas9-based genome editing. When validated with a variety of genetic backgrounds and target genes, the automated workflow showed genome editing efficiencies similar to manual methods, with indel rates of up to 98%. Monoclonal colony growth was achieved and monitored using the StemCellFactory-integrated CellCelector, which allowed the exclusion of colonies derived from multiple cells or growing too close to neighbouring colonies. In summary, we demonstrate the successful establishment of an automated CRISPR/Cas9-based genome editing process on the StemCellFactory platform. The development of such a standardized and scalable automated CRISPR/Cas9 system represents an exciting new tool in genome editing, enhancing our ability to address a wide range of scientific questions in disease modeling, drug development and personalized medicine.

The ex vivo production of platelets from induced pluripotent cells (iPSCs) may offer a safer and sustainable alternative for transfusions and drug delivery systems (DDS). However, the mass production of the clinically required number of iPSC-derived platelets (iPSC-PLTs) is challenging. Here, we introduce recent technologies for mass production and the first-in-human clinical trial using ex vivo iPSC-PLTs. To this end, we established immortalized megakaryocyte progenitor cell lines (imMKCLs) as an expandable master cell bank (MCB) through the overexpression of c-MYC, BMI1 and BCL-XL, which modulated megakaryopoiesis and thrombopoiesis. We also optimized a culture cocktail for maturation of the imMKCLs by mixing an aryl hydrocarbon receptor (AhR) antagonist, SR1/GNF-316; a Rho-associated protein kinase (ROCK) inhibitor, Y-27632/Y-39983; and a small-molecule compound replacing recombinant thrombopoietin (TPO), TA-316. Finally, we discovered the importance of turbulence on the manufacturing of intact iPSC-PLTs, allowing us to develop a turbulence-based bioreactor, VerMES. Combination of the MCB and VerMES enabled us to produce more than 100 billion iPSC-PLTs, leading to the first-in-human clinical trial. Despite these advancements, many challenges remain before expanding the clinical implementation of this strategy.

Periodontal tissue regeneration is important for preserving teeth. Periodontal ligament stem cells (PDLSCs) are useful in periodontal tissue regeneration; however, tooth extraction is required to obtain these cells. Therefore, we focused on induced pluripotent stem (iPS) cells and established a method to obtain PDLSC-like cells from iPS cells. Specifically, we first differentiated iPS cells into neural crest–like cells (iNCs). Next, we obtained PDLSC-like cells (iPDLSCs) by culturing iNCs on extracellular matrix (ECM) derived from human primary periodontal ligament cells (HPDLCs). This differentiation method suggested that ECM derived from HPDLCs is important for iPDLSC differentiation. Thus, we aimed to identify the PDLSC-inducing factor present in HPDLC-derived ECM in this study. We first performed comprehensive analyses of HPDLC genes and identified fibrillin-2 (FBN2), an ECM-related factor. Furthermore, to clarify the effect of FBN2 on iPDLSC differentiation, we cultured iNCs using ECM derived from HPDLCs with FBN2 knocked down. As a result, expression of PDL-related markers was reduced in iNCs cultured on ECM derived from HPDLCs transfected with FBN2 siRNA (iNC-siFBN2) compared with iPDLSCs. Furthermore, the expression of CD105 (a mesenchymal stem cell marker), proliferation ability, and multipotency of iNC-siFBN2 were lower compared with iPDLSCs. Next, we cultured iNCs on FBN2 recombinant protein; however, expression of PDL-related markers did not increase compared with iPDLSC. The present results suggest the critical involvement of FBN2 in inducing iPDLSCs from iNCs when in fact it does not promote iPDLSC differantiation. Therefore, we need to elucidate the entire HPDLC-ECMs, responsible for iPDLSCs induction. [ABSTRACT FROM AUTHOR]

A patient with the PSEN1 E280A mutation and homozygous for APOE3 Christchurch (APOE3Ch) displayed extreme resistance to Alzheimer's disease (AD) cognitive decline and tauopathy, despite having a high amyloid burden. To further investigate the differences in biological processes attributed to APOE3Ch, we generated induced pluripotent stem (iPS) cell-derived cerebral organoids from this resistant case and a non-protected control, using CRISPR/Cas9 gene editing to modulate APOE3Ch expression. In the APOE3Ch cerebral organoids, we observed a protective pattern from early tau phosphorylation. ScRNA sequencing revealed regulation of Cadherin and Wnt signaling pathways by APOE3Ch, with immunostaining indicating elevated ß-catenin protein levels. Further in vitro reporter assays unexpectedly demonstrated that ApoE3Ch functions as a Wnt3a signaling enhancer. This work uncovered a neomorphic molecular mechanism of protection of ApoE3 Christchurch, which may serve as the foundation for the future development of protected case-inspired therapeutics targeting AD and tauopathies. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSCs) and their derivatives have been described to display epigenetic memory of their founder cells, as well as de novo reprogramming-associated alterations. In order to selectively explore changes due to the reprogramming process and not to heterologous somatic memory, we devised a circular reprogramming approach where somatic stem cells are used to generate iPSCs, which are subsequently re-differentiated into their original fate. As somatic founder cells, we employed human embryonic stem cell-derived neural stem cells (NSCs) and compared them to iPSC-derived NSCs derived thereof. Global transcription profiling of this isogenic circular system revealed remarkably similar transcriptomes of both NSC populations, with the exception of 36 transcripts. Amongst these we detected a disproportionately large fraction of X chromosomal genes, all of which were upregulated in iPSC-NSCs. Concurrently, we detected differential methylation of X chromosomal sites spatially coinciding with regions harboring differentially expressed genes. While our data point to a pronounced overall reinstallation of autosomal transcriptomic and methylation signatures when a defined somatic lineage is propagated through pluripotency, they also indicate that X chromosomal genes may partially escape this reinstallation process. Considering the broad application of iPSCs in disease modeling and regenerative approaches, such reprogramming-associated alterations in X chromosomal gene expression and DNA methylation deserve particular attention. [ABSTRACT FROM AUTHOR]

Cytomegalovirus (CMV), a type of herpes virus, is the predominant cause of congenital anomalies due to intrauterine infections in humans. Adverse outcomes related to intrauterine infections with human cytomegalovirus (HCMV) vary widely, depending on factors such as fetal infection timing, infection route, and viral virulence. The precise mechanism underlying HCMV susceptibility remains unclear. In this study, we compared the susceptibility of neonatal human dermal fibroblast cells (NHDFCs) and human induced pluripotent stem cells (hiPSCs) derived from NHDFCs, which are genetically identical to HCMV, using immunostaining, microarray, in situ hybridization, quantitative PCR, and scanning electron microscopy. These cells were previously used to compare CMV susceptibility, but the underlying mechanisms were not fully elucidated. HCMV susceptibility of hiPSCs was significantly lower in the earliest phase. No shared gene ontologies were observed immediately post-infection between the two cell types using microarray analysis. Early-stage expression of HCMV antigens and the HCMV genome was minimal in immunostaining and in in situ hybridization in hiPSCs. This strongly suggests that HCMV does not readily bind to hiPSC surfaces. Scanning electron microscopy performed using the NanoSuit method confirmed the scarcity of HCMV particles on hiPSC surfaces. The zeta potential and charge mapping of the charged surface in NHDFCs and hiPSCs exhibited minimal differences when assessed using zeta potential analyzer and scanning ion conductance microscopy; however, the expression of heparan sulfate (HS) was significantly lower in hiPSCs compared with that in NHDFCs. Thus, HS expression could be a primary determinant of HCMV resistance in hiPSCs at the attachment level. [ABSTRACT FROM AUTHOR]

In currently ongoing adoptive T-cell therapies, T cells collected from patients are given back to them after ex vivo activation and expansion. In some cases, T cells are transduced with chimeric antigen receptor (CAR) or T-cell receptor (TCR) genes during the ex vivo culture period in order to endow T cells with the desired antigen specificity. Although such strategies are effective in some types of cancer, there remain issues to be solved: (i) the limited number of cells, (ii) it is time-consuming, (iii) it is costly, and (iv) the quality can be unstable. Points (ii) and (iv) can be solved by preparing allogeneic T cells and cryopreserving them in advance and methods are being developed using healthy donor-derived T cells or pluripotent stem cells as materials. Whereas it is difficult to solve (i) and (iii) in the former case, all the issues can be cleared in the latter case. However, in either case, a new problem arises: rejection by the patient's immune system. Deletion of human leukocyte antigen (HLA) avoids rejection by recipient T cells, but causes rejection by NK cells, which can recognize loss of HLA class I. Various countermeasures have been developed, but no definitive solution is yet available. Therefore, further research and development are necessary. [ABSTRACT FROM AUTHOR]

Simple Summary: Axons are highly elongated extensions of neurons that transmit electrical impulses to their connecting targets and are integral for neuronal function. In neurodegenerative diseases such as Parkinson's disease, axons degenerate early in the disease course, breaking essential connections, leading to the development of clinical phenotypes over time. Thus, developing a better understanding of axonal pathology is crucial. For this reason, we used patient-derived induced pluripotent stem cells to generate neurons which were then used to develop assays to characterize how neurodegenerative diseases such as Parkinson's disease might affect axons. We show that LRRK2 G2019S, which is one of the most common known mutations causing Parkinson's disease, subtly affects axonal function and the injury response. Our assays could be used in the future to better understand axonal degeneration and test potential therapeutics for their ability to protect axons against degeneration. The degeneration of axon terminals before the soma, referred to as "dying back", is a feature of Parkinson's disease (PD). Axonal assays are needed to model early PD pathogenesis as well as identify protective therapeutics. We hypothesized that defects in axon lysosomal trafficking as well as injury repair might be important contributing factors to "dying back" pathology in PD. Since primary human PD neurons are inaccessible, we developed assays to quantify axonal trafficking and injury repair using induced pluripotent stem cell (iPSC)-derived neurons with LRRK2 G2019S, which is one of the most common known PD mutations, and isogenic controls. We observed a subtle axonal trafficking phenotype that was partially rescued by a LRRK2 inhibitor. Mutant LRRK2 neurons showed increased phosphorylated Rab10-positive lysosomes, and lysosomal membrane damage increased LRRK2-dependent Rab10 phosphorylation. Neurons with mutant LRRK2 showed a transient increase in lysosomes at axotomy injury sites. This was a pilot study that used two patient-derived lines to develop its methodology; we observed subtle phenotypes that might correlate with heterogeneity in LRRK2-PD patients. Further analysis using additional iPSC lines is needed. Therefore, our axonal lysosomal assays can potentially be used to characterize early PD pathogenesis and test possible therapeutics. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) and induced pluripotent stem (iPS) cells have great potential as cell sources for tissue engineering and regenerative medicine. This study aimed to investigate whether iPS cells can be differentiated into MSCs using MSCGM, a commercially available MSC culture system. The cells were characterized by flow cytometry, immunostaining, and gene expression analyses. We also examined their potential to differentiate into osteoblasts and chondrocytes. Our results showed that iPS cells cultured in MSCGM (iPS-MSCGM) exhibited a fibroblast-like morphology and expressed CD73 and CD90 genes, as well as positive markers for CD73, CD90, and CD105. Moreover, iPS-MSCGM cells demonstrated the ability to differentiate into osteoblasts and chondrocytes in vitro. This study demonstrates a new and simple method for inducing the differentiation of iPS cells to MSCs using MSCGM. [ABSTRACT FROM AUTHOR]

The function of mucosal-associated invariant T (MAIT) cells, a burgeoning member of innate-like T cells abundant in humans and implicated in many diseases, remains obscure. To explore this, mice with a rearranged T cell receptor (TCR) α or β locus, specific for MAIT cells, were generated via induced pluripotent stem cells derived from MAIT cells and were designated Vα19 and Vβ8 mice, respectively. Both groups of mice expressed large numbers of MAIT cells. The MAIT cells from these mice were activated by cytokines and an agonist to produce IFN-γ and IL-17. While Vβ8 mice showed resistance in a cancer metastasis model, Vα19 mice did not. Adoptive transfer of MAIT cells from the latter into the control mice, however, recapitulated the resistance. These mice present an implication for understanding the role of MAIT cells in health and disease and in developing treatments for the plethora of diseases in which MAIT cells are implicated. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that extracellular vesicles (EVs) derived from an anaplastic mouse melanoma cell line made using Nanog overexpression of F10 (Nanog+F10) suppressed the metastasis of Nanog+F10. Here, an induced pluripotent stem (iPS) cell line was focused as a more anaplastic cell line, potentially producing EVs with higher metastasis-suppressive effects. The EVs were introduced into the tail vein nine times before introducing Nanog+F10 cells. Two weeks later, the liver and lung were resected and metastatic colonies were quantified. The involvement of macrophages (invasion inhibiting ability, phagocytic activity) and cytotoxic T cells (cytotoxicity) was evaluated using J774.1 and CTLL-2 cell lines. iPS EVs showed similar level effects to Nanog+F10 EVs in every item relevant to metastasis suppression. Differential expression analysis of miRNAs in EVs and functional network database analysis revealed that dominant regulatory miRNAs were predicted. The candidate hub genes most highly associated with the metastasis suppression mechanism were predicted as six genes, including Trp53 and Hif1a, for Nanog+F10 EVs and ten genes, including Ins1 and Kitl, for iPS EVs. Regarding the mechanism, Nanog+F10 EVs and iPS EVs were very different. This suggests synergistic effect when used together as metastasis preventive vaccine. [ABSTRACT FROM AUTHOR]

Preclinical transplantations using human neuroepithelial stem (NES) cells in spinal cord injury models have exhibited promising results and demonstrated cell integration and functional improvement in transplanted animals. Previous studies have relied on the generation of research grade cell lines in continuous culture. Using fresh cells presents logistic hurdles for clinical transition regarding time and resources for maintaining high quality standards. In this study, we generated a good manufacturing practice (GMP) compliant human iPS cell line in GMP clean rooms alongside a research grade iPS cell line which was produced using standardized protocols with GMP compliant chemicals. These two iPS cell lines were differentiated into human NES cells, from which six batches of cell therapy doses were produced. The doses were cryopreserved, thawed on demand and grafted in a rat spinal cord injury model. Our findings demonstrate that NES cells can be directly grafted post-thaw with high cell viability, maintaining their cell identity and differentiation capacity. This opens the possibility of manufacturing off-the-shelf cell therapy products. Moreover, our manufacturing process yields stable cell doses with minimal batch-to-batch variability, characterized by consistent expression of identity markers as well as similar viability of cells across the two iPS cell lines. These cryopreserved cell doses exhibit sustained viability, functionality, and quality for at least 2 years. Our results provide proof of concept that cryopreserved NES cells present a viable alternative to transplanting freshly cultured cells in future cell therapies and exemplify a platform from which cell formulation can be optimized and facilitate the transition to clinical trials.

A patient with the PSEN1 E280A mutation and homozygous for APOE3 Christchurch (APOE3Ch) displayed extreme resistance to Alzheimer’s disease (AD) cognitive decline and tauopathy, despite having a high amyloid burden. To further investigate the differences in biological processes attributed to APOE3Ch, we generated induced pluripotent stem (iPS) cell-derived cerebral organoids from this resistant case and a non-protected control, using CRISPR/Cas9 gene editing to modulate APOE3Ch expression. In the APOE3Ch cerebral organoids, we observed a protective pattern from early tau phosphorylation. ScRNA sequencing revealed regulation of Cadherin and Wnt signaling pathways by APOE3Ch, with immunostaining indicating elevated β-catenin protein levels. Further in vitro reporter assays unexpectedly demonstrated that ApoE3Ch functions as a Wnt3a signaling enhancer. This work uncovered a neomorphic molecular mechanism of protection of ApoE3 Christchurch, which may serve as the foundation for the future development of protected case-inspired therapeutics targeting AD and tauopathies.

Abstract Background Human induced pluripotent stem cell-derived neural stem/progenitor cell (hiPSC-NS/PC)-based cell transplantation has emerged as a groundbreaking method for replacing damaged neural cells and stimulating functional recovery, but its efficacy is strongly influenced by the state of the injured spinal microenvironment. This study evaluates the impact of a dual therapeutic intervention utilizing hepatocyte growth factor (HGF) and hiPSC-NS/PC transplantation on motor function restoration following spinal cord injury (SCI). Methods Severe contusive SCI was induced in immunocompromised rats, followed by continuous administration of recombinant human HGF protein into the subarachnoid space immediately after SCI for two weeks. Acute-phase histological and RNA sequencing analyses were conducted. Nine days after the injury, hiPSC-NS/PCs were transplanted into the lesion epicenter of the injured spinal cord, and the functional and histological outcomes were determined. Results The acute-phase HGF-treated group exhibited vascularization, diverse anti-inflammatory effects, and activation of endogenous neural stem cells after SCI, which collectively contributed to tissue preservation. Following cell transplantation into a favorable environment, the transplanted NS/PCs survived well, facilitating remyelination and neuronal regeneration in host tissues. These comprehensive effects led to substantial enhancements in motor function in the dual-therapy group compared to the single-treatment groups. Conclusions We demonstrate that the combined therapeutic approach of HGF preconditioning and hiPSC-NS/PC transplantation enhances locomotor functional recovery post-SCI, highlighting a highly promising therapeutic strategy for acute to subacute SCI.

Abstract Background Disease-specific induced pluripotent stem cells (iPSCs) are useful tools for pathological analysis and diagnosis of rare diseases. Given the limited available resources, banking such disease-derived iPSCs and promoting their widespread use would be a promising approach for untangling the mysteries of rare diseases. Herein, we comprehensively established iPSCs from patients with designated intractable diseases in Japan and evaluated their properties to enrich rare disease iPSC resources. Methods Patients with designated intractable diseases were recruited for the study and blood samples were collected after written informed consent was obtained from the patients or their guardians. From the obtained samples, iPSCs were established using the episomal method. The established iPSCs were deposited in a cell bank. Results We established 1,532 iPSC clones from 259 patients with 139 designated intractable diseases. The efficiency of iPSC establishment did not vary based on age and sex. Most iPSC clones originated from non-T and non-B hematopoietic cells. All iPSC clones expressed key transcription factors, OCT3/4 (range 0.27–1.51; mean 0.79) and NANOG (range 0.15–3.03; mean 1.00), relative to the reference 201B7 iPSC clone. Conclusions These newly established iPSCs are readily available to the researchers and can prove to be a useful resource for research on rare intractable diseases.

The high mortality in the global population due to chronic diseases highlights the urgency to identify effective alternative therapies. Regenerative medicine provides promising new approaches for this purpose, particularly in the use of induced pluripotent stem cells (iPSCs). The aim of the work is to establish a new pluripotency cell line obtained for the first time by reprogramming human gingival mesenchymal stem cells (hGMSCs) by a non-integrating method. The hGMSC-derived iPS line characterization is performed through morphological analysis with optical and electron scanning microscopy and through the pluripotency markers expression evaluation in cytofluorimetry, immunofluorescence, and RT-PCR. To confirm the pluripotency of new hGMSC-derived iPS, the formation of embryoid bodies (EBs), as an alternative to the teratoma formation test, is studied in morphological analysis and through three germ layers’ markers’ expression in immunofluorescence and RT-PCR. At the end, a comparative study between parental hGMSCs and derived iPS cells is performed also for the extracellular vesicles (EVs) and their miRNA content. The new hGMSC-derived iPS line demonstrated to be pluripotent in all aspects, thus representing an innovative dynamic platform for personalized tissue regeneration.

Functional human tissues engineered from patient-specific induced pluripotent stem cells (hiPSCs) hold great promise for investigating the progression, mechanisms, and treatment of musculoskeletal diseases in a controlled and systematic manner. For example, bioengineered models of innervated human skeletal muscle could be used to identify novel therapeutic targets and treatments for patients with complex central and peripheral nervous system disorders. There is a need to develop standardized and objective quantitative methods for engineering and using these complex tissues, in order increase their robustness, reproducibility, and predictiveness across users. Here we describe a standardized method for engineering an isogenic, patient specific human neuromuscular junction (NMJ) that allows for automated quantification of NMJ function to diagnose disease using a small sample of blood serum and evaluate new therapeutic modalities. By combining tissue engineering, optogenetics, microfabrication, optoelectronics and video processing, we created a novel platform for the precise investigation of the development and degeneration of human NMJ. We demonstrate the utility of this platform for the detection and diagnosis of myasthenia gravis, an antibody-mediated autoimmune disease that disrupts the NMJ function.

Objective: Heterozygous coding sequence mutations of the INS gene are a cause of permanent neonatal diabetes (PNDM), requiring insulin therapy similar to T1D. While the negative effects on insulin processing and secretion are known, how dominant insulin mutations result in a continued decline of beta cell function after birth is not well understood. Methods: We explored the causes of beta cell failure in two PNDM patients with two distinct INS mutations using patient-derived iPSCs and mutated hESCs. Results: we detected accumulation of misfolded proinsulin and impaired proinsulin processing in vitro, and a dominant-negative effect of these mutations on beta-cell mass and function after transplantation into mice. In addition to anticipated ER stress, we found evidence of beta-cell dedifferentiation, characterized by an increase of cells expressing both Nkx6.1 and ALDH1A3, but negative for insulin and glucagon. Conclusions: These results highlight a novel mechanism, the loss of beta cell identity, contributing to the loss and functional failure of human beta cells with specific insulin gene mutations.

Hereditary transthyretin (TTR) amyloidosis (ATTRv amyloidosis) is autosomal dominant and caused by mutation of TTR gene. Heterozygous ATTR Tyr114Cys (p.Tyr134Cys) amyloidosis is a lethal disease with a life expectancy of about 10 years after onset of the disease. However, the molecular pathogenesis of ATTR Tyr114Cys amyloidosis is still largely unknown. In this study, we took advantage of disease-specific induced pluripotent stem (iPS) cells and generated & characterized the heterozygous ATTR Tyr114Cys amyloidosis-specific iPS cells (Y114C iPS cells), to determine whether Y114C iPS cells could be useful for elucidating the pathogenesis of ATTR Tyr114Cys amyloidosis. We successfully differentiated heterozygous Y114C iPS cells into hepatocyte like cells (HLCs) mainly producing TTR protein. On day 27 after differentiation, the expression of hepatocyte maker albumin was detected, and TTR expression was significantly increased in HLCs differentiated from Y114C iPS cells. LC–MS/MS analysis showed that both WT TTR & ATTR Y114C protein were indeed expressed in the HLCs differentiated from Y114C iPS cells. Notably, the number of detected peptides derived from ATTR Y114C protein was lower than that of WT TTR protein, indeed indicating the clinical phenotype of ATTR Tyr114Cys amyloidosis. Taken together, we first reported the heterozygous Y114C iPS cells generated from patient with ATTR Tyr114Cys amyloidosis, and suggested that Y114C iPS cells could be a potential pathological tool, which may contribute to elucidating the molecular pathogenesis of heterozygous ATTR Tyr114Cys amyloidosis.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, continues to spread around the world with serious cases and deaths. It has also been suggested that different genetic variants in the human genome affect both the susceptibility to infection and severity of disease in COVID-19 patients. Angiotensin-converting enzyme 2 (ACE2) has been identified as a cell surface receptor for SARS-CoV and SARS-CoV-2 entry into cells. The construction of an experimental model system using human iPS cells would enable further studies of the association between viral characteristics and genetic variants. Airway and alveolar epithelial cells are cell types of the lung that express high levels of ACE2 and are suitable for in vitro infection experiments. Here, we show that human iPS cell-derived airway and alveolar epithelial cells are highly susceptible to viral infection of SARS-CoV-2. Using gene knockout with CRISPR-Cas9 in human iPS cells we demonstrate that ACE2 plays an essential role in the airway and alveolar epithelial cell entry of SARS-CoV-2 in vitro. Replication of SARS-CoV-2 was strongly suppressed in ACE2 knockout (KO) lung cells. Our model system based on human iPS cell-derived lung cells may be applied to understand the molecular biology regulating viral respiratory infection leading to potential therapeutic developments for COVID-19 and the prevention of future pandemics.

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia characterized by ineffective erythropoiesis. DBA is majorly caused by mutations in the ribosomal protein (RP) genes (Gadhiya and Wills in Diamond-Blackfan Anemia, https://www.statpearls.com/; 2023). A suitable disease model that yields a continuous supply of erythroid cells is required to study disease pathogenesis and drug discovery. Toward this, we reprogrammed dermal fibroblasts from a DBA patient with a heterozygous mutation c.22-23delAG in the RPS19 gene identified through exome sequencing. To generate induced pluripotent stem cells (iPSCs), we induced episomal expression of the reprogramming factors OTC3/4, L-MYC, LIN28, SOX2, and KLF4, and a p53 shRNA2. The DBA-iPSC line CSCRi006-A generated during this study was extensively characterized for its pluripotency and genome stability. The clone retained normal karyotype and showed high expression levels of pluripotency markers, OCT4, NANOG, SOX2, TRA-I-60, TRA-I-81, and SSEA4. It could differentiate into cells originating from all three germ cell layers, as identified by immunostaining for SOX17 (endoderm), Brachyury (mesoderm), and PAX6 (ectoderm). IPSCs provide a renewable source of cells for in vitro disease modeling. CSCRi006-A, a thoroughly characterized iPSC line carrying heterozygous RPS19 c.22-23delAG mutation, is a valuable cell line for the disease modeling of DBA. This iPSC line can be differentiated into different blood cell types to study the mechanisms of disease development and identify potential treatments. [ABSTRACT FROM AUTHOR]

Cardiac arrhythmias are a major cause of cardiovascular mortality worldwide. Many arrhythmias are caused by reentry, a phenomenon where excitation waves circulate in the heart. Optical mapping techniques have revealed the role of reentry in arrhythmia initiation and fibrillation transition, but the underlying biophysical mechanisms are still difficult to investigate in intact hearts. Tissue engineering models of cardiac tissue can mimic the structure and function of native cardiac tissue and enable interactive observation of reentry formation and wave propagation. This review will present various approaches to constructing cardiac tissue models for reentry studies, using the authors' work as examples. The review will highlight the evolution of tissue engineering designs based on different substrates, cell types, and structural parameters. A new approach using polymer materials and cellular reprogramming to create biomimetic cardiac tissues will be introduced. The review will also show how computational modeling of cardiac tissue can complement experimental data and how such models can be applied in the biomimetics of cardiac tissue. [ABSTRACT FROM AUTHOR]

The cornea is a pioneering area of regenerative medicine, and Japanese researchers have led the world in this field. In Japan, 3 different epithelial sheet regenerative medicine products have been approved for corneal epithelial stem cell deficiency, and the first-in-human studies of cultured corneal endothelial cell suspension transplants, induced pluripotent stem cell (iPS cell)-derived corneal epithelial sheet transplants, and iPS cell-derived corneal endothelial substitute cell transplants were all conducted and reported globally for the first time by Japanese researchers. In the field of corneal epithelial regenerative medicine, Pellegrini et al. (Lancet 349:990–3, 1997) performed the first in-human transplant of autologous cultured corneal epithelial sheets. More than 20 years later, autologous cultivated corneal epithelium and autologous cultivated oral mucosal epithelium products were launched in Japan. In addition, clinical studies of iPS cell-derived corneal epithelial cell sheet transplant have begun, which may solve the issues with conventional autologous epithelial sheets. In corneal endothelium regenerative medicine, a clinical study of transplant of allogenic cultured corneal endothelial cell suspension for bullous keratopathy was reported, in which corneal endothelial cells derived from donor corneas were grown in culture and then injected into the anterior chamber with a ROCK inhibitor (Kinoshita et al. in N Engl J Med 378:995–1003, 2018). Our research group is also developing iPS-cell-derived corneal endothelium-like cells, termed corneal endothelial cell substitute from iPS cells (CECSi cells), and we are conducting a clinical study to treat bullous keratopathy with these cells (Hatou et al. in Stem Cell Res 55:102497, 2021). This review describes the progress and challenges of corneal epithelial and endothelial regenerative medicine and the promising future of corneal regenerative medicine with iPS cells. [ABSTRACT FROM AUTHOR]

Optical mapping is a powerful imaging technique widely adopted to measure membrane potential changes and intracellular Ca2+ variations in excitable tissues using voltage-sensitive dyes and Ca2+ indicators, respectively. This powerful tool has rapidly become indispensable in the field of cardiac electrophysiology for studying depolarization wave propagation, estimating the conduction velocity of electrical impulses, and measuring Ca2+ dynamics in cardiac cells and tissues. In addition, mapping these electrophysiological parameters is important for understanding cardiac arrhythmia mechanisms. In this review, we delve into the fundamentals of cardiac optical mapping technology and its applications when applied to hiPSC-derived cardiomyocytes and discuss related advantages and challenges. We also provide a detailed description of the processing and analysis of optical mapping data, which is a crucial step in the study of cardiac diseases and arrhythmia mechanisms for extracting and comparing relevant electrophysiological parameters. [ABSTRACT FROM AUTHOR]

Genetic editing of induced pluripotent stem (iPS) cells represents a promising avenue for an HIV cure. However, certain challenges remain before bringing this approach to the clinic. Among them, in vivo engraftment of cells genetically edited in vitro needs to be achieved. In this study, CD34+ cells derived in vitro from iPS cells genetically modified to carry the CCR5Δ32 mutant alleles did not engraft in humanized immunodeficient mice. However, the CD34+ cells isolated from teratomas generated in vivo from these genetically edited iPS cells engrafted in all experiments. These CD34+ cells also gave rise to peripheral blood mononuclear cells in the mice that, when inoculated with HIV in cell culture, were resistant to HIV R5-tropic isolates. This study indicates that teratomas can provide an environment that can help evaluate the engraftment potential of CD34+ cells derived from the genetically modified iPS cells in vitro. The results further confirm the possibility of using genetically engineered iPS cells to derive engraftable hematopoietic stem cells resistant to HIV as an approach toward an HIV cure.

Alzheimer’s Disease (AD) is characterized by synapse and neuronal loss and the accumulation of neurofibrillary tangles and Amyloid β plaques. Despite significant research efforts to understand the late stages of the disease, its etiology remains largely unknown. This is in part because of the imprecise AD models in current use. In addition, little attention has been paid to neural stem cells (NSC), which are the cells responsible for the development and maintenance of brain tissue during an individual’s lifespan. Thus, an in vitro 3D human brain tissue model using induced pluripotent stem (iPS) cell-derived neural cells in human physiological conditions may be an excellent alternative to standard models to investigate AD pathology. Following the differentiation process mimicking development, iPS cells can be turned into NSCs and, ultimately, neural cells. During differentiation, the traditionally used xenogeneic products may alter the cells’ physiology and prevent accurate disease pathology modeling. Hence, establishing a xenogeneic material-free cell culture and differentiation protocol is essential. This study investigated the differentiation of iPS cells to neural cells using a novel extracellular matrix derived from human platelet lysates (PL Matrix). We compared the stemness properties and differentiation efficacies of iPS cells in a PL matrix against those in a conventional 3D scaffold made of an oncogenic murine-matrix. Using well-defined conditions without xenogeneic material, we successfully expanded and differentiated iPS cells into NSCs via dual-SMAD inhibition, which regulates the BMP and TGF signaling cascades in a manner closer to human conditions. This in vitro, 3D, xenogeneic-free scaffold will enhance the quality of disease modeling for neurodegenerative disease research, and the knowledge produced could be used in developing more effective translational medicine.

Division Session "Soft-nanotechnology for brain science and biological systems" was organized at JVSS 2023 by the Division of Soft-Nanotechnology at the Japan Society of Vacuum and Surface Science. The current Special Issue gathers review articles authored by the five invited speakers of the Session. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSCs) and their derivatives have been described to display epigenetic memory of their founder cells, as well as de novo reprogramming-associated alterations. In order to selectively explore changes due to the reprogramming process and not to heterologous somatic memory, we devised a circular reprogramming approach where somatic stem cells are used to generate iPSCs, which are subsequently re-differentiated into their original fate. As somatic founder cells, we employed human embryonic stem cell-derived neural stem cells (NSCs) and compared them to iPSC-derived NSCs derived thereof. Global transcription profiling of this isogenic circular system revealed remarkably similar transcriptomes of both NSC populations, with the exception of 36 transcripts. Amongst these we detected a disproportionately large fraction of X chromosomal genes, all of which were upregulated in iPSC-NSCs. Concurrently, we detected differential methylation of X chromosomal sites spatially coinciding with regions harboring differentially expressed genes. While our data point to a pronounced overall reinstallation of autosomal transcriptomic and methylation signatures when a defined somatic lineage is propagated through pluripotency, they also indicate that X chromosomal genes may partially escape this reinstallation process. Considering the broad application of iPSCs in disease modeling and regenerative approaches, such reprogramming-associated alterations in X chromosomal gene expression and DNA methylation deserve particular attention.

In vitro culture systems that structurally model human myogenesis and promote PAX7+ myogenic progenitor maturation have not been established. Here we report that human skeletal muscle organoids can be differentiated from induced pluripotent stem cell lines to contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures reassembling neural plate border and dermomyotome. Culture conditions instigate neural lineage arrest and promote fetal hypaxial myogenesis toward limb axial anatomical identity, with generation of sustainable uncommitted PAX7 myogenic progenitors and fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from the second trimester of human gestation. Single-cell comparison to human fetal and adult myogenic progenitor /satellite cells reveals distinct molecular signatures for non-dividing myogenic progenitors in activated (CD44High/CD98+/MYOD1+) and dormant (PAX7High/FBN1High/SPRY1High) states. Our approach provides a robust 3D in vitro developmental system for investigating muscle tissue morphogenesis and homeostasis.

IntroductionWith the expected increase in patients with heart failure and ischemic 15 cardiomyopathy, the development of myocardial regenerative medicine using cell transplantation as a novel treatment method is progressing. This first-in-human clinical trial aimed to confirm the safety of cardiomyocyte patch transplantation derived from allogeneic induced pluripotent stem (iPS) cells based on the results of several preclinical studies.Study designThe inclusion criteria were left ventricular ejection fraction of 35% or less; heart failure symptoms of New York Heart Association class III or higher despite existing therapies such as revascularization; and a 1-year observation period that included a 3-month immunosuppressive drug administration period after transplantation of iPS cell-derived cardiomyocyte patches to evaluate adverse events, cardiac function, myocardial blood flow, heart failure symptoms, and immune response.ResultsIn the first three cases of this trial, no transplanted cell-related adverse events were observed during the 1-year observation period, and improvement in heart failure symptoms was observed. In addition, improvements in left ventricular contractility and myocardial blood flow were observed in two of the three patients. Regarding immune response, an increase in transplant cell-specific antibody titer was observed in all three patients after immunosuppressive drug administration. In one patient with poor improvement in cardiac function and myocardial blood flow, an increase in antibody titer against HLA-DQ was observed even before cell transplantation.ConclusionsOur case findings demonstrate that the transplantation of iPS cell-derived cardiomyocyte patches for ischemic cardiomyopathy can be safely performed; however, further investigation of the therapeutic effect and its relationship with an immune response is needed by accumulating the number of patients through continued clinical trials.

Airway–liquid interface cultures of primary epithelial cells and of induced pluripotent stem-cell-derived airway epithelial cells (ALI and iALI, respectively) are physiologically relevant models for respiratory virus infection studies because they can mimic the in vivo human bronchial epithelium. Here, we investigated gene expression profiles in human airway cultures (ALI and iALI models), infected or not with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), using our own and publicly available bulk and single-cell transcriptome datasets. SARS-CoV-2 infection significantly increased the expression of interferon-stimulated genes (IFI44, IFIT1, IFIT3, IFI35, IRF9, MX1, OAS1, OAS3 and ISG15) and inflammatory genes (NFKBIA, CSF1, FOSL1, IL32 and CXCL10) by day 4 post-infection, indicating activation of the interferon and immune responses to the virus. Extracellular matrix genes (ITGB6, ITGB1 and GJA1) were also altered in infected cells. Single-cell RNA sequencing data revealed that SARS-CoV-2 infection damaged the respiratory epithelium, particularly mature ciliated cells. The expression of genes encoding intercellular communication and adhesion proteins was also deregulated, suggesting a mechanism to promote shedding of infected epithelial cells. These data demonstrate that ALI/iALI models help to explain the airway epithelium response to SARS-CoV-2 infection and are a key tool for developing COVID-19 treatments. [ABSTRACT FROM AUTHOR]

Background: Parkinson’s disease (PD) is a progressive neurodegenerative condition that primarily affects motor functions; it is caused by the loss of midbrain dopaminergic (mDA) neurons. The therapeutic effects of transplanting humaninduced pluripotent stem cell (iPSC)-derived mDA neural progenitor cells in animal PD models are known and are being evaluated in an ongoing clinical trial. However, However, improvements in the safety and efficiency of differentiation-inducing methods are crucial for providing a larger scale of cell therapy studies. This study aimed to investigate the usefulness of dopaminergic progenitor cells derived from human iPSCs by our previously reported method, which promotes differentiation and neuronal maturation by treating iPSCs with three inhibitors at the start of induction. Methods: Healthy subject-derived iPS cells were induced into mDA progenitor cells by the CTraS-mediated method we previously reported, and their proprieties and dopaminergic differentiation efficiency were examined in vitro. Then, the induced mDA progenitors were transplanted into 6-hydroxydopamine-lesioned PD model mice, and their efficacy in improving motor function, cell viability, and differentiation ability in vivo was evaluated for 16 weeks. Results: Approximately ≥80% of cells induced by this method without sorting expressed mDA progenitor markers and differentiated primarily into A9 dopaminergic neurons in vitro. After transplantation in 6-hydroxydopamine-lesioned PD model mice, more than 90% of the engrafted cells differentiated into the lineage of mDA neurons, and approximately 15% developed into mature mDA neurons without tumour formation. The grafted PD model mice also demonstrated significantly improved motor functions. Conclusion: This study suggests that the differentiation protocol for the preparation of mDA progenitors is a promising option for cell therapy in patients with PD. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas system has a broad range of possible medical applications, but its clinical translation has been hampered, particularly by the lack of safe and efficient vector systems mediating the short-term expression of its components. Recently, different virus-like particles (VLPs) have been introduced as promising vectors for the delivery of CRISPR/Cas genome editing components. Here, we characterized and directly compared three different types of retrovirus-based (R) VLPs, two derived from the γ-retrovirus murine leukemia virus (gRVLPs and "enhanced" egRVLPs) and one from the lentivirus human immunodeficiency virus, HIV (LVLPs). First, we unified and optimized the production of the different RVLPs. To ensure maximal comparability of the produced RVLPs, we adapted several assays, including nanoparticle tracking analysis (NTA), multi-parametric imaging flow cytometry (IFC), and Cas9-ELISA, to analyze their morphology, surface composition, size, and concentration. Next, we comparatively tested the three RVLPs targeting different genes in 293T model cells. Using identical gRNAs, we found egRVLPs to mediate the most efficient editing. Functional analyses indicated better cargo (i.e., Cas9) transfer and/or release as the underlying reason for their superior performance. Finally, we compared on- and off-target activities of the three RVLPs in human-induced pluripotent stem cells (hiPSC) exploiting the clinically relevant C-C motif chemokine receptor 5 (CCR5) as the target. Again, egRVLPs facilitated the highest, almost 100% knockout rates, importantly with minimal off-target activity. In conclusion, in direct comparison, egRVLPs were the most efficient RVLPs. Moreover, we established methods for in-depth characterization of VLPs, facilitating their validation and thus more predictable and safe application. [ABSTRACT FROM AUTHOR]

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Islet transplantation, including pancreatic beta cells, has become an approved treatment for type I diabetes. To date, the number of donors limits the availability of treatment. Induction of pancreatic endocrine cells from pluripotent stem cells including iPSCs in vitro offers promise as a solution, but continues to face problems including high reagent costs and cumbersome differentiation procedures. In a previous study, we developed a low-cost, simplified differentiation method, but its efficiency for inducing pancreatic endocrine cells was not sufficient: induction of endocrine cells is non-uniform, resulting in colonies containing relatively high ratio of non-pancreatic-related cells. Here, we applied cyclin-dependent kinase inhibitors (CDKi) within a specific time window, which improved the efficiency of pancreatic endocrine cell induction. CDKi treatment reduced the prevalence of multi-layered regions and enhanced expression of the endocrine progenitor–related marker genes PDX1 and NGN3 resulting in enhanced production of both INSULIN and GLUCAGON. These findings support a step forward in the field of regenerative medicine of pancreatic endocrine cells. [ABSTRACT FROM AUTHOR]

Abstract Background Ischemic stroke in white matter of the brain induces not only demyelination, but also neuroinflammation. Peripheral T lymphocytes, especially regulatory T cells (Tregs), are known to infiltrate into ischemic brain and play a crucial role in modulation of inflammatory response there. We previously reported that transplantation of vascular endothelial cells generated from human induced pluripotent stem cells (iVECs) ameliorated white matter infarct. The aim of this study is to investigate contribution of the immune system, especially Tregs, to the mechanism whereby iVEC transplantation ameliorates white matter infarct. Methods iVECs and human Tregs were transplanted into the site of white matter lesion seven days after induction of ischemia. The egress of T lymphocytes from lymph nodes was sequestered by treating the animals with fingolimod (FTY720). The infarct size was evaluated by magnetic resonance imaging. Immunohistochemistry was performed to detect the activated microglia and macrophages, T cells, Tregs, and oligodendrocyte lineage cells. Remyelination was examined by Luxol fast blue staining. Results iVEC transplantation reduced ED-1+ inflammatory cells and CD4+ T cells, while increased Tregs in the white matter infarct. Treatment of the animals with FTY720 suppressed neuroinflammation and reduced the number of both CD4+ T cells and Tregs in the lesion, suggesting the importance of infiltration of these peripheral immune cells into the lesion in aggravation of neuroinflammation. Suppression of neuroinflammation by FTY720 per se, however, did not promote remyelination in the infarct. FTY720 treatment negated the increase in the number of Tregs by iVEC transplantation in the infarct, and attenuated remyelination promoted by transplanted iVECs, while it did not affect the number of oligodendrocyte lineage cells increased by iVEC transplantation. Transplantation of Tregs together with iVECs into FTY720-treated ischemic white matter did not affect the number of oligodendrocyte lineage cells, while it remarkably promoted myelin regeneration. Conclusions iVEC transplantation suppresses neuroinflammation, but suppression of neuroinflammation per se does not promote remyelination. Recruitment of Tregs by transplanted iVECs contributes significantly to promotion of remyelination in the injured white matter.

Introduction: In a diploid organism, two alleles from a single genetic locus are expressed to generate a normal phenotype. Heterozygous deleterious mutation causes a reduction of functional proteins to a half dose and insufficient amounts of functional proteins can occur to generate an in–normal phenotype, namely haploinsufficiency. Heterozygous deleterious mutation of microRNAs (miRs), non-coding RNAs that regulate the expression level of target transcripts, is still not well understood. The hsa-miR-302/367 cluster is the most abundant and specifically up-regulated miR cluster in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) and plays an important role in the maintenance of pluripotency. Methods: We targeted the hsa-miR-302/367 region via a Cas9 nuclease complex with guide RNA and replaced that region with green fluorescent protein (GFP). Using a homologous donor, consisting of left and right arms and GFP, we confirmed deletion of the hsa-miR-302/367 cluster by homologous recombination without cellular destruction by microscopy. We sub-cloned GFP-positive colonies and checked the genotype of each sub-clone by genomic PCR. We then analyzed the pluripotency of heterozygous knockout cells with a hsa-miR-302/367 cluster by assessing cell proliferation ratio, morphology, and undifferentiated marker gene expression. We also used an embryoid body formation assay and transplanted wild-type and heterozygous knockout cells into immune-deficient mice. Furthermore, to analyze the lineage-specific differentiation potential of heterozygous knockout cells, we differentiated both wild-type and heterozygous knockout cells into neural stem cells. Results: Here, we show that the half dose of mature miRs from the hsa-miR-302/367 cluster loci was sufficient for the continued self-renewal of hiPSCs. All GFP-positive clones were revealed to be heterozygous knockout cells, suggesting hsa-miR-302/367 cluster homozygous knockout cells were not maintained. The cell proliferation ratio, morphology, and expression of undifferentiated marker genes were comparable between wild-type and heterozygous knockout of undifferentiated human iPSCs. In addition, we found that heterozygous knockout human iPSCs have the capacity to differentiate into three germ layers, including neural stem cells. Conclusions: Taken together, a single allele of the hsa-miR-302/367 cluster expresses a sufficient amount of miRs to maintain the pluripotent properties of human stem cells.

Introduction: Human induced pluripotent stem cells (hiPSCs) are generated through the reprogramming of somatic cells expressing a defined set of transcription factors. The advent of autologous iPSCs has enabled the generation of patient-specific iPSC lines and is expected to contribute to the exploration of cures and causes of diseases, drug screening, and tailor-made regenerative medicines. Efficient control of hiPSC derivation is beneficial for industrial applications. However, the mechanisms underlying somatic cell reprogramming remain unknown, while reprogramming efficiency remains extremely low, especially in human cells. Methods and results: We previously reported that chemical inhibition of the NOTCH signaling pathway and DOT1L promoted the generation of hiPSCs from keratinocytes, but the mechanisms and effect of this double inhibition on other types of cells remain to be investigated. Here, we found that the NOTCH/DOT1L inhibition markedly increased iPSC colony generation from human fibroblast cells via mRNA reprogramming, and mesenchymal to epithelial transition (MET)-related genes are significantly expressed in the early phase of the reprogramming. We successfully derived hiPSC lines using a single-cell sorting system under efficient reprogramming conditions. Conclusions: This user-friendly reprogramming approach paves the way for the development of hiPSC derivations in industrial applications of disease modeling and drug screening.

Background: Recently, various regenerative therapies have been developed based on induced pluripotent stem (iPS) cells. However, hygienic control strategies have not been established at the manufacturing facilities. We aimed to evaluate the safety and effects of continuous exposure to low-dose chlorine dioxide (ClO2) gas on cell fates, and to determine the optimum dose for safe usage of this disinfectant. Methods: We cultured an iPS cell line in the absence or presence of various doses of ClO2 gas. We evaluated cell proliferation, cell death, the maintenance of undifferentiated state, and cell senescence. Results: We found that iPS cell proliferation was not affected by 0.05 or 0.1 ppmv ClO2 gas in the atmosphere. Although 0.1 ppmv ClO2 slightly affected apoptosis, it was not a significant effect. Moreover, neither at 0.05 nor 0.1 ppmv ClO2 gas significantly affected the characteristics of iPS cells. Discussion and conclusion: Continuous exposure to 0.05 or 0.1 ppmv ClO2 gas did not affect the fate of iPS cells. These results may contribute to the development of new strategies for hygiene control in cell processing facilities.