iPS Cell-derived Cells Research Articles

Induced pluripotent stem cell-derived macrophages as a platform for modelling human disease.

Macrophages are innate immune cells that are present in essentially all tissues, where they have vital roles in tissue development, homeostasis and pathogenesis. The importance of macrophages in tissue function is reflected by their association with various human diseases, and studying macrophage functions in both homeostasis and pathological tissue settings is a promising avenue for new targeted therapies that will improve human health. The ability to generate macrophages from induced pluripotent stem(iPS) cells has revolutionized macrophage biology, with the generation of iPScell-derived macrophages (iMacs) providing unlimited access to genotype-specific cells that can be used to model various human diseases involving macrophage dysregulation. Such disease modelling is achieved by generating iPScells from patient-derived cells carrying disease-related mutations or by introducing mutations into iPScells from healthy donors using CRISPR-Cas9 technology. These iMacs that carry disease-related mutations can be used to study the aetiology of the particular disease in vitro. To achieve more physiological relevance, iMacs can be co-cultured in 2D systems with iPScell-derived cells or in 3D systems with iPScell-derived organoids. Here, we discuss the studies that have attempted to model various human diseases using iMacs, highlighting how these have advanced our knowledge about the role of macrophages in health and disease.

Two-year safety outcomes of iPS cell-derived mesenchymal stromal cells in acute steroid-resistant graft-versus-host disease

The first completed clinical trial of induced pluripotent stem cell (iPS cell)-derived cells was conducted in 15 participants with steroid-resistant acute graft-versus-host disease. After intravenous infusion of mesenchymal stromal cells (CYP-001 derived from a clone of human iPS cells), we reported the safety, tolerability and efficacy within the primary evaluation period at day 100. We now report results at the 2-year follow-up: 9 of 15 (60%) participants survived, which compares favorably with previously reported outcomes in studies of steroid-resistant acute graft-versus-host disease. Causes of death were complications commonly observed in recipients of allogeneic hematopoietic stem cell transplantation, and not considered by the investigators to be related to CYP-001 treatment. There were no serious adverse events, tumors or other safety concerns related to CYP-001. In conclusion, systemic delivery of iPS cell-derived cells was safe and well tolerated over 2 years of follow-up, with sustained outcomes up to 2 years after the first infusion. ClinicalTrials.gov registration: NCT02923375.

Functional intestinal monolayers from organoids derived from human iPS cells for drug discovery research

BackgroundHuman induced pluripotent stem (iPS) cell-derived enterocyte-like cells (ELCs) are expected to be useful for evaluating the intestinal absorption and metabolism of orally administered drugs. However, it is difficult to generate large amounts of ELCs with high quality because they cannot proliferate and be passaged.MethodsTo solve the issue above, we have established intestinal organoids from ELCs generated using our protocol. Furthermore, monolayers were produced from the organoids. We evaluated the usefulness of the monolayers by comparing their functions with those of the original ELCs and the organoids.ResultsWe established organoids from ELCs (ELC-org) that could be passaged and maintained for more than a year. When ELC-org were dissociated into single cells and seeded on cell culture inserts (ELC-org-mono), they formed a tight monolayer in 3 days. Both ELC-org and ELC-org-mono were composed exclusively of epithelial cells. Gene expressions of many drug-metabolizing enzymes and drug transporters in ELC-org-mono were enhanced, as compared with those in ELC-org, to a level comparable to those in adult human small intestine. The CYP3A4 activity level in ELC-org-mono was comparable or higher than that in primary cryopreserved human small intestinal cells. ELC-org-mono had the efflux activities of P-gp and BCRP. Importantly, ELC-org-mono maintained high intestinal functions without any negative effects even after long-term culture (for more than a year) or cryopreservation. RNA-seq analysis showed that ELC-org-mono were more mature as intestinal epithelial cells than ELCs or ELC-org.ConclusionsWe have successfully improved the function and convenience of ELCs by utilizing organoid technology.

ACE2 knockout hinders SARS-CoV-2 propagation in iPS cell-derived airway and alveolar epithelial cells.

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.

Development of a Perfusing Small Intestine–Liver Microphysiological System Device

There is an increasing need to develop alternatives to animal modeling and testing for pre-clinical studies as researchers face major challenges, such as the study of dynamic systems in laboratory settings. Microphysiological system (MPS) technology has recently shown great potential for addressing such limitations. We developed a perfusing small intestine–liver-connected device that is easy to operate and highly reproducible. In non-clinical pharmacokinetics and safety studies, the use of human-derived materials is necessary. We used human iPS cell-derived small intestinal epithelial cells (HiEs) and cryopreserved human primary hepatocytes. Hepatocytes in 3D culture were co-cultured with swiss-albino 3T3 cells as feeder cells. We evaluated the effects of co-culturing hepatocytes and HiEs using our small intestine–liver device. The mRNA expression levels of CYP1A2 and CYP3A4 in hepatocytes were significantly increased in the 3D culture. The TEER values were increased in HiEs co-cultured with hepatocytes in the 3D culture. We evaluated the differential proliferation and function characteristics of the hepatocytes and HiEs following perfusion and verified the utility of our proposed small intestine–liver device for evaluating multiple cell populations. The perfusion culture system of our small intestine–liver device can be used to investigate distinct effects on co-cultured hepatocytes and HiEs.

Neural Differentiation of Induced Pluripotent Stem Cells for a Xenogeneic Material-Free 3D Neurological Disease Model Neurulation from Pluripotent Cells Using a Human Hydrogel.

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.

Cardiac cell sheet engineering for regenerative medicine and tissue modeling.

Stem cell biology and tissue engineering are essential techniques for cardiac tissue construction. We have succeeded in fabricating human cardiac tissue using the mass production technology of human iPS cell-derived cardiomyocytes and cell sheet engineering, and we are developing regenerative medicine and tissue models to apply this tissue to heart disease research. Cardiac tissue fabrication and tissue functional evaluation technologies for contractile and electrophysiological function are indispensable, which lead to the functional improvement of bioengineered human cardiac tissue.

Cancer Immunotherapy Utilizing iPS Cell-Derived Dendritic Cells and Macrophages

Cancer Immunotherapy Utilizing iPS Cell-Derived Dendritic Cells and Macrophages

Development of HLA-modified induced pluripotent stem cell-derived dendritic cells for a novel cancer immunotherapy

We previously established a method to generate a human iPS cell-derived myeloid cells (iPS-MLs) from a human leukocyte antigen (HLA)-A *24:02 donor, which could differentiate into dendritic cells (DCs) and had a high potential in stimulating T cells. However, for clinical applications, the histoincompatibility between iPS cells and cells in cancer patients has remained as a key challenge. One solution is to construct the iPS cell library covering all HLA haplotypes, but it is excessively laborious and impractical. In order to overcome this problem, we generated HLA-modified iPS cells to avoid immune rejection. We disrupted HLA-A , -B and DRA genes by using a CRISPR-Cas9 system and subsequently introduced HLA-A *02:01 into iPS-MLs. We examined the immunogenic capacities of these HLA-deficient iPS-MLs for inducing antigen-specific CD8 + T cells derived from an allogeneic donor. HLA-deficient iPS-MLs could avoid recognition by alloreactive CD8 + T cells. HLA-deficient iPS-MLs with introduced HLA-A *02:01 differentiated into functional DCs upon stimulation with IL-4, and induced HLA-A2-restricted melanoma antigen recognized by T cells-1 (MART)−1-specific CD8 + T cells derived from an allogeneic donor. Furthermore, HLA-deficient iPS-MLs with introduced HLA-A *02:01 and MART-1 could induce HLA-A2-restricted MART-1-specific CD8 + T cells derived from an allogeneic donor. We developed a method that can exchange HLA alleles from HLA-A *24:02 to* 02:01 in iPS-MLs, and our findings may overcome the obstacle of histoincompatibility in DC vaccination therapy, regardless of HLA allele.

Exposure to a Pathological Condition May Be Required for the Cells to Secrete Exosomes Containing mtDNA Aberration.

Exosomes, nanovesicles secreted by all cells, carry out intercellular communication by transmitting biologically active cargo comprising DNA, RNA, and proteins. These biomolecules reflect the status of their parent cells and can be altered by pathological conditions. Therefore, the researchers have been investigating differential sequences and quantities of DNA associated with exosomes as valuable biomarkers of diseases. Exosomes carry different types of DNA molecules, including genomic, cytoplasmic, and mitochondrial (mtDNA). The mtDNA aberrations are reported to be a hallmark of diseases involving oxidative stress, such as cancer and neurodegenerative diseases. Establishing robust in vitro models comprising appropriate cell lineages is the first step towards investigating disease-specific anomalies and testing therapeutics. Induced pluripotent stem (iPS) cells from patients with diseases have been used for this purpose since they can differentiate into various cells. The current study investigated mtDNA aberrations in exosomes secreted by primary cancer cells and neural stem cells (NSCs) differentiated from iPS cells. The primary cancer cells were isolated from surgically removed glioblastoma multiforme (GBM) tissue, and the iPS cells were produced from control and Alzheimer's disease (AD) subjects' B lymphocytes. We detected aberrations in mtDNA associated with exosomes secreted from GBM cells but not from the NSCs. This result indicates that the cells may not secrete exosomes carrying mtDNA aberration without exposure to a pathological condition. Thus, we may need to consider this fact when we use iPS cell-derived cells as an in vitro disease model.

Development of theoretical model expressing self-beating deformation behaviour of human iPS cardiomyocytes tube

In recent years, a regenerative therapy method using human iPS cell-derived myocardial cells (hiPS-CM) has attracted attention. In the future, large-scale myocardial three-dimensional structures that apply tissue engineering technology are expected. In this study, we developed a three-dimensional structure with a pump function by the self-beating power of hiPS-CM. Then, the deformation behavior of the 3D hiPS-CM structure associated with self-pulsation was examined. The pulsatile behavior of hiPS-CM was quantitatively evaluated by using a high-speed optical measurement system with the digital image correlation method. Then, the experimental strain data was used to construct the stress-strain response with use of a theoretical active stress model originally developed by Guccione et al. A theoretical constitutive model was also developed by combining the Guccione's active stress model and the viscoelastic Maxwell model. As a result, this theoretical model stress-strain behavior showed a good agreement with the combination model of experimental strain and theoretical stress.

ヒトiPS細胞由来の近位尿細管細胞を用いたMPSにおけるP-gpとSGLT2の輸送能評価

Proximal tubule plays an important role in reabsorption of nutrients and excretion of drugs. Such reabsorption and excretion processes are mediated by proteins known as transporters in the proximal tubule cells. To evaluate the transport of nutrients and xenobiotics in vitro, proximal tubule microphysiological systems (MPS) have been developed. However, conventional proximal tubule MPS employ cells derived from experimental animals or cells immortalized by genetic modification. Apparently, the expression levels of transporters are insufficient in these cells. In this study, we prepared MPS employing proximal tubular cells isolated from kidney organoids generated from iPS cells. In this MPS, we quantitatively evaluated the transport of SGLT2, which is responsible for glucose reabsorption, and P-gp, which is responsible for drug excretion. As a result, compared to MPS using immortalized cells, MPS using iPS cell-derived cells showed an approximately 1.6-fold improvement in the transport capacity. This indicates that we were able to produce an MPS that better reproduced reabsorption and excretion rates in vivo.

Improvement of contractile function of human iPS cell-derived cardiac cell sheets by electric stimulation for several days

Improvement of contractile function of human iPS cell-derived cardiac cell sheets by electric stimulation for several days

In vitro drug evaluation model of human iPS cell-derived neural cell by using novel gelatin fiber scaffold

In vitro drug evaluation model of human iPS cell-derived neural cell by using novel gelatin fiber scaffold

A Personalized Glomerulus Chip Engineered from Stem Cell-Derived Epithelium and Vascular Endothelium.

Progress in understanding kidney disease mechanisms and the development of targeted therapeutics have been limited by the lack of functional in vitro models that can closely recapitulate human physiological responses. Organ Chip (or organ-on-a-chip) microfluidic devices provide unique opportunities to overcome some of these challenges given their ability to model the structure and function of tissues and organs in vitro. Previously established organ chip models typically consist of heterogenous cell populations sourced from multiple donors, limiting their applications in patient-specific disease modeling and personalized medicine. In this study, we engineered a personalized glomerulus chip system reconstituted from human induced pluripotent stem (iPS) cell-derived vascular endothelial cells (ECs) and podocytes from a single patient. Our stem cell-derived kidney glomerulus chip successfully mimics the structure and some essential functions of the glomerular filtration barrier. We further modeled glomerular injury in our tissue chips by administering a clinically relevant dose of the chemotherapy drug Adriamycin. The drug disrupts the structural integrity of the endothelium and the podocyte tissue layers, leading to significant albuminuria as observed in patients with glomerulopathies. We anticipate that the personalized glomerulus chip model established in this report could help advance future studies of kidney disease mechanisms and the discovery of personalized therapies. Given the remarkable ability of human iPS cells to differentiate into almost any cell type, this work also provides a blueprint for the establishment of more personalized organ chip and ‘body-on-a-chip’ models in the future.

Human iPS Cell-Derived Cell Aggregates Exhibited Dermal Papilla Cell Properties in in vitro Three-Dimensional Assemblage Mimicking Hair Follicle Structures.

Current approaches for human hair follicle (HF) regeneration mostly adopt cell-autonomous tissue reassembly in a permissive murine intracorporeal environment. This, together with the limitation in human-derived trichogenic starting materials, potentially hinders the bioengineering of human HF structures, especially for the drug discovery and treatment of hair loss disorders. In this study, we attempted to reproduce the anatomical relationship between an epithelial main body and the dermal papilla (DP) within HF in vitro by three-dimensionally assembling columnarly molded human keratinocytes (KCs) and the aggregates of DP cells and evaluated how HF characteristics were reproduced in the constructs. The replaceability of human-induced pluripotent stem cell (hiPSC)-derived DP substitutes was assessed using the aforementioned reconstruction assay. Human DP cell aggregates were embedded into Matrigel as a cluster. Subsequently, highly condensed human KCs were cylindrically injected onto DP spheroids. After 2-week culture, the structures visually mimicking HFs were obtained. KC-DP constructs partially reproduced HF microanatomy and demonstrated differential keratin (KRT) expression pattern in HFs: KRT14 in the outermost part and KRT13, KRT17, and KRT40, respectively, in the inner portion of the main body. KC-DP constructs tended to upregulate HF-related genes, KRT25, KRT33A, KRT82, WNT5A, and LEF1. Next, DP substitutes were prepared by exposing hiPSC-derived mesenchymal cells to retinoic acid and subsequently to WNT, BMP, and FGF signal activators, followed by cell aggregation. The resultant hiPSC-derived DP substitutes (iDPs) were combined with KCs in the invented assay. KC-iDP constructs morphologically resemble KC-DP constructs and analogously mimicked KRT expression pattern in HF. iDP in the constructs expressed DP-related markers, such as vimentin and versican. Intriguingly, KC-iDP constructs more intensely expressed KRT33A, KRT82, and LEF1, which were stepwisely upregulated by the addition of WNT ligand and the mixture of WNT, SHH, and EDA signaling activators, supporting the idea that iDP exhibited biological properties analogous to DP cell aggregates in the constructs in vitro. These preliminary findings suggested the possibility of regenerating DP equivalents with in vitro hair-inductive capacity using hiPSC-derived cell composites, which potentially reduce the necessity of human tissue-derived trichogenic cell subset and eventually allow xeno-free bioengineering of human HFs.

SARS-CoV-2 research using human pluripotent stem cells and organoids.

Experimental cell models are indispensable for clarifying the pathophysiology of coronavirus disease 2019 (COVID‐19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, and for developing therapeutic agents. To recapitulate the symptoms and drug response of COVID‐19 patients in vitro, SARS‐CoV‐2 studies using physiologically relevant human embryonic stem (ES)/induced pluripotent stem (iPS) cell‐derived somatic cells and organoids are ongoing. These cells and organoids have been used to show that SARS‐CoV‐2 can infect and damage various organs including the lung, heart, brain, intestinal tract, kidney, and pancreas. They are also being used to develop COVID‐19 therapeutic agents, including evaluation of their antiviral efficacy and safety. The relationship between COVID‐19 aggravation and human genetic backgrounds has been investigated using genetically modified ES/iPS cells and patient‐derived iPS cells. This review summarizes the latest results and issues of SARS‐CoV‐2 research using human ES/iPS cell‐derived somatic cells and organoids.

Macrophage-like iPS-derived Suppressor Cells Reduce Th1-mediated Immune Response to a Retinal Antigen

ABSTRACT Purpose To investigate the immunotherapeutic effects of macrophage-like induced pluripotent stem (iPS) cell-derived suppressor cells (SCs) in ocular immune response and experimental autoimmune uveoretinitis (EAU). Methods The genes of Oct3/4, Sox2, Klf4, and c-Myc were transferred to B cells enriched from the spleen cells of C57BL/6 mice by using retrovirus vectors. Transferred B cells were cultured for 17 days to obtain colonies of iPS cells. Through additional steps, iPS-SCs were induced. An antigen-specific T cell proliferation assay was performed with CD4+ T cells collected from draining lymph nodes of the mice immunized with human interphotoreceptor retinoid-binding protein (hIRBP) peptide and co-cultured with iPS-SCs. Cytokine concentrations in the culture supernatant were examined. Mice were immunized with hIRBP peptide to induce EAU. The iPS-SCs were administered into the mice one day before the induction of EAU. Results The iPS-SCs decreased hIRBP-specific T cell proliferation depending on the number of cells. Productions of tumor necrosis factor-α and interferon-γ were significantly decreased; however, transforming growth factor-β1, nitric oxide, interleukin (IL)-13, IL-17A, and IL-17 F levels were elevated in the supernatant when the collected T cells were co-cultured with iPS-SCs. The iPS-SCs had immunosuppressant effects even without cell-to-cell contact, and their effects were non-specific to the antigen preloaded on iPS-SCs. EAU was significantly milder in the mice administered iPS-SCs prior to immunization. Conclusions Macrophage-like iPS-SCs reduced Th1 immune response to a retinal antigen and Th1-mediated EAU in mice. These results showed the possibility of the application of iPS technology to the treatment of noninfectious ocular inflammation, endogenous uveitis, in the future.

Application of human iPS cell-derived vascular cell differentiation induction method to elucidation of vascular pathology

Cardiovascular diseases such as arteriosclerosis are a leading cause of death in developed countries. Various factors, including diet, exercise and genetics, can play a role in the onset of such conditions and the development of treatment options is complex and often involves the use of animal models. However, there are limitations with the use of animal models due to their inherent differences to humans, meaning that research results aren't always translatable. Associate Professor Daisuke Taura, Department of Diabetes, Endocrinology and Nutrition, Kyoto University, Japan, is interested in the use of in vitro techniques for investigating cardiovascular conditions. He is conducting vascular cell differentiation induction research using human embryonic stem cells (ES) cells and induced pluripotent cells (iPS), which can be induced to differentiate into any type of human cell, provided the right culturing conditions are present. In a world first, Taura successfully induced vascular constituent cells from human iPS cells, which led to important results in the establishment of vascular cells with autosomal dominant polycystic kidney disease (ADPKD) and moyamoya disease. Ultimately, Taura and his team are working towards a radical treatment for atherosclerotic diseases, and are also seeking to induce differentiation of vascular constituent cells using human ES/ iPS cells and explore their development and differentiation processes. Taura has been successful in improving on culture methods and, in doing so, achieved the differentiation of human ES cells into vascular endothelial cells and mural cells, and the differentiation of human iPS cells into vascular endothelial cells and mural cells.

Sulfate Conjugates of Isoflavone as Potential Physiological Substrates of BCRP for Estimation of in vivo Inhibition

Breast cancer resistant protein (BCRP/ABCG2) is expressed throughout the body including brain, liver, kidney, and small intestine and extrudes its substrates from cells. Although several drugs potentially inhibit BCRP in vitro, limited information is available on its in vivo inhibition. Therefore, potential assessment of the change in BCRP function in vivo using its physiological substrates as a biomarker is desirable for clarifying the DDI potential via this transporter. The purpose of this study is to find and characterize physiological BCRP substrates based on metabolomics approach. Lapatinib was orally administered in mice to inhibit BCRP in vivo, and plasma samples were comprehensively measured by LC-TOFMS with all-ion fragmentation acquisition and quantified by LC-MS/MS. The untargeted metabolomics has revealed change in sulfate conjugates of isoflavone, such as daidzein sulfate (DS) and genistein sulfate (GS), in plasma. Oral administration of lapatinib, significantly increased AUC0-7h for DS, GS, and equal sulfate (ES) by 3.6-, 5.6-, and 1.6-fold, respectively. Another BCRP inhibitor febuxostat also increased AUC0-7h of DS, GS, and ES. To evaluate the possible inhibition of BCRP by lapatinib in human small intestinal epithelial cells, transcellular transport in iPS cell-derived small intestinal epithelial-like cells (F-hiSIEC) was examined. After addition of daidzein, genistein, and equal to apical chamber, the basal excretion of DS, GS, and ES was significantly increased with lapatinib treatment, suggesting lapatinib may inhibit intestinal BCRP. ATP-dependent uptake of DS and GS was observed in BCRP expressing membrane vesicles, and this uptake was inhibited by lapatinib. These results suggest that lapatinib affects disposition of DS, GS, and ES by inhibition of the intestinal BCRP. Further studies are needed to evaluate these sulfate conjugates as potential biomarker for the estimation of BCRP inhibition in vivo.