3D osteogenic differentiation of human iPSCs reveals the role of TGFß signal in the transition from progenitors to osteoblasts and osteoblasts to osteocytes.

Although the formation of bone-like nodules is regarded as the differentiation process from stem cells to osteogenic cells, including osteoblasts and osteocytes, the precise biological events during nodule formation are unknown. Here we performed the osteogenic induction of human induced pluripotent stem cells using a three-dimensional (3D) culture system using type I collagen gel and a rapid induction method with retinoic acid. Confocal and time-lapse imaging revealed the osteogenic differentiation was initiated with vigorous focal proliferation followed by aggregation, from which cells invaded the gel. Invading cells changed their morphology and expressed osteocyte marker genes, suggesting the transition from osteoblasts to osteocytes. Single-cell RNA sequencing analysis revealed that 3D culture-induced cells with features of periosteal skeletal stem cells, some of which expressed TGFß-regulated osteoblast-related molecules. The role of TGFß signal was further analyzed in the transition from osteoblasts to osteocytes, which revealed that modulation of the TGFß signal changed the morphology and motility of cells isolated from the 3D culture, suggesting that the TGFß signal maintains the osteoblastic phenotype and the transition into osteocytes requires down-regulation of the TGFß signal.

Optimization of the proliferation and persistency of CAR T cells derived from human induced pluripotent stem cells.

The effectiveness of chimaeric antigen receptor (CAR) T-cell immunotherapies against solid tumours relies on the accumulation, proliferation and persistency of T cells at the tumour site. Here we show that the proliferation of CD8αß cytotoxic CAR T cells in solid tumours can be enhanced by deriving and expanding them from a single human induced-pluripotent-stem-cell clone bearing a CAR selected for efficient differentiation. We also show that the proliferation and persistency of the effector cells in the tumours can be further enhanced by genetically knocking out diacylglycerol kinase, which inhibits antigen-receptor signalling, and by transducing the cells with genes encoding for membrane-bound interleukin-15 (IL-15) and its receptor subunit IL-15Rα. In multiple tumour-bearing animal models, the engineered hiPSC-derived CAR T cells led to therapeutic outcomes similar to those of primary CD8 T cells bearing the same CAR. The optimization of effector CAR T cells derived from pluripotent stem cells may aid the development of long-lasting antigen-specific T-cell immunotherapies for the treatment of solid tumours.

Inhibition of dopamine receptor D1 signaling promotes human bile duct cancer progression via WNT signaling.

Bile duct cancer (BDC) frequently invades the nerve fibers, making complete surgical resection difficult. A single tumor mass contains cells of variable malignancy and cell-differentiation states, with cancer stem cells (CSCs) considered responsible for poor clinical outcomes. This study aimed to investigate the contribution of autosynthesized dopamine to CSC-related properties in BDC. Sphere formation assays using 13 commercially available BDC cell lines demonstrated that blocking dopamine receptor D1 (DRD1) signaling promoted CSC-related anchorage-independent growth. Additionally, we newly established four new BDC patient-derived organoids (PDOs) and found that blocking DRD1 increased resistance to chemotherapy and enabled xenotransplantation in vivo. Single-cell analysis revealed that the BDC PDO cells varied in their cell-differentiation states and responses to dopamine signaling. Further, DRD1 inhibition increased WNT7B expression in cells with bile duct-like phenotype, and it induced proliferation of other cell types expressing Wnt receptors and stem cell-like signatures. Reagents that inhibited Wnt function canceled the effect of DRD1 inhibition and reduced cell proliferation in BDC PDOs. In summary, in BDCs, DRD1 is a crucial protein involved in autonomous CSC proliferation through the regulation of endogenous WNT7B. As such, inhibition of the DRD1 feedback signaling may be a potential treatment strategy for BDC.

[Short-Term Outcomes of Neoadjuvant Chemoradiotherapy for Locally Advanced Rectal Cancer].

In recent years, an increasing number of reports have demonstrated the usefulness of neoadjuvant chemoradiotherapy (NACRT). In our department, we consider cT3-4 and/or cN-positive locally advanced rectal cancer as an indication for NACRT. We have retrospectively evaluated the efficacy and safety of NACRT in 11 patients who underwent NACRT from November 2018 to July 2022. All patients were male, with a median age of 69 years, and cStage was Ⅱa: 1, Ⅱc: 1, Ⅲb: 5, Ⅲc: 3, and Ⅳa: 1. All patients completed NACRT, and there were no cases of CTCAE Grade 3 or higher adverse events or treatment interruptions. The response rate was 72.7%, and histological response grade were Grade 3: 1(9.1%), 2: 4 (36.4%), 1b: 6(54.5%), and surgical margin was negative in all cases. Pathological down stage was obtained in 45.5% of cases, and pCR was obtained in 1 case(9.1%). The median observation period was 17 months, and during the period, 2 cases(18.2%)developed recurrence, both of which were pulmonary metastases, and no local recurrence including pelvic lymph node recurrence was observed. NACRT for locally advanced rectal cancer is considered a relatively safe and highly locally controllable preoperative treatment.

[A Case of BRAF V600E-Mutant Colorectal Cancer Treated Effectively by Encorafenib, Binimetinib, and Cetuximab Triple Therapy].

A 76-year-old woman was diagnosed with left-sided transverse colon cancer invading the pancreatic tail with multiple liver metastases and peritoneal dissemination. Preoperative diagnosis was cT4b(SI)N2aM1c(H3, P1), cStage Ⅳc, harboring BRAF V600E mutation. Transverse colostomy was performed, and FOLFOXIRI plus bevacizumab(BEV)was administered. After 12 chemotherapy cycles, the primary tumor and metastatic lesions showed partial response. Because of CEA elevating after 5-FU plus LV plus BEV as maintenance therapy was changed, the regimen was switched to encorafenib plus binimetinib plus cetuximab as the second-line chemotherapy. The patient developed dermatitis around the colostomy after the start of the second-line chemotherapy, resulting in temporally cetuximab monotherapy. After improvement of dermatitis, the patient resumed encorafenib plus binimetinib, improving liver metastases. Eight months after the start of the second- line, the patient has been administered with triple therapy and had stable disease status.

Oxidative Stress as a Biomarker for Predicting the Prognosis of Patients with Colorectal Cancer.

INTRODUCTION: Excess oxidative stress is generated by inflammation and cancer, and it is involved in the development and metastasis of colorectal cancer. However, there are few reports on the relationship between blood oxidative stress and prognosis. This study examined the usefulness of derivatives of reactive oxygen metabolites (d-ROMs), a measure of oxidative stress, and the neutrophil-to-lymphocyte ratio (NLR), an inflammatory marker, as prognostic markers in colorectal cancer. METHODS: The study enrolled 163 patients who underwent colorectal cancer resection at our institution between 2013 and 2018. Blood samples were taken preoperatively to measure d-ROMs and NLR. Spearman's correlation analysis was used to examine the relationships between d-ROMs and NLR, and Cox regression analysis was performed to identify factors associated with d-ROMs and NLR. The Kaplan-Meier method was used to calculate disease-specific survival (DSS). RESULTS: There was no correlation between d-ROMs and NLR. Tumor size was significantly associated with d-ROMs and NLR. DSS was significantly worse among patients with high d-ROMs or high NLR, although patients with high d-ROMs and high NLR had the worst DSS. In the multivariate analysis, distant metastasis and the high d-ROM/NLR combination were significant factors associated with DSS (p < 0.001, hazard ratio [HR] = 22.880 and p = 0.049, HR = 2.391, respectively). CONCLUSION: Preoperative d-ROMs and NLR reflect the tumor size among patients with colorectal cancer. The combination of d-ROMs and NLR may effectively predict prognosis in patients with colorectal cancer.

Single-cell RNA sequencing identifies a migratory keratinocyte subpopulation expressing THBS1 in epidermal wound healing.

Keratinocyte differentiation is an intricate process that is regulated by multiple mediators. Using cultured human keratinocytes, we found that lysophosphatidic acid (LPA) induced the differentiation of a previously unsuspected keratinocyte subpopulation expressing the extracellular matrix protein, thrombospondin-1 (THBS1). This action of LPA was mediated by the RHO/ROCK-SRF signaling downstream of LPA1 and LPA5 receptors and required ERK activity. Suppression of THBS1 in vitro suggested a migratory role of THBS1+ keratinocytes. Moreover, we analyzed publicly deposited single-cell RNA sequencing dataset and identified Thbs1-expressing keratinocytes in the mouse wound skin. Immunohistochemistry analysis revealed that Thbs1+ keratinocytes were apparently differentiated from basal keratinocytes upon wounding, subsequently polarized and migrated suprabasally toward the wound front, and eventually underwent terminal differentiation in the neo-epidermis. Importantly, inhibition of Erk activity suppressed Thbs1+ keratinocyte differentiation in wound healing. Based on these findings, we suggest that THBS1+ keratinocyte is a migratory keratinocyte subpopulation that facilitates epidermal wound healing.

Induction of inverted morphology in brain organoids by vertical-mixing bioreactors.

Organoid technology provides an opportunity to generate brain-like structures by recapitulating developmental steps in the manner of self-organization. Here we examined the vertical-mixing effect on brain organoid structures using bioreactors and established inverted brain organoids. The organoids generated by vertical mixing showed neurons that migrated from the outer periphery to the inner core of organoids, in contrast to orbital mixing. Computational analysis of flow dynamics clarified that, by comparison with orbital mixing, vertical mixing maintained the high turbulent energy around organoids, and continuously kept inter-organoid distances by dispersing and adding uniform rheological force on organoids. To uncover the mechanisms of the inverted structure, we investigated the direction of primary cilia, a cellular mechanosensor. Primary cilia of neural progenitors by vertical mixing were aligned in a multidirectional manner, and those by orbital mixing in a bidirectional manner. Single-cell RNA sequencing revealed that neurons of inverted brain organoids presented a GABAergic character of the ventral forebrain. These results suggest that controlling fluid dynamics by biomechanical engineering can direct stem cell differentiation of brain organoids, and that inverted brain organoids will be applicable for studying human brain development and disorders in the future.

Single-Cell Transcriptome Analysis Dissects the Replicating Process of Pancreatic Beta Cells in Partial Pancreatectomy Model.

Heterogeneity of gene expression and rarity of replication hamper molecular analysis of ß-cell mass restoration in adult pancreas. Here, we show transcriptional dynamics in ß-cell replication process by single-cell RNA sequencing of murine pancreas with or without partial pancreatectomy. We observed heterogeneity of Ins1-expressing ß-cells and identified the one cluster as replicating ß-cells with high expression of cell proliferation markers Pcna and Mki67. We also recapitulated cell cycle transition accompanied with switching expression of cyclins and E2F transcription factors. Both transient activation of endoplasmic reticulum stress responders like Atf6 and Hspa5 and elevated expression of tumor suppressors like Trp53, Rb1, and Brca1 and DNA damage responders like Atm, Atr, Rad51, Chek1, and Chek2 during the transition to replication associated fine balance of cell cycle progression and protection from DNA damage. Taken together, these results provide a high-resolution map depicting a sophisticated genetic circuit for replication of the ß-cells.

Initiation of Supporting Cell Activation for Hair Cell Regeneration in the Avian Auditory Epithelium: An Explant Culture Model.

Sensorineural hearing loss is a common disability often caused by the loss of sensory hair cells in the cochlea. Hair cell (HCs) regeneration has long been the main target for the development of novel therapeutics for sensorineural hearing loss. In the mammalian cochlea, hair cell regeneration is limited, but the auditory epithelia of non-mammalian organisms retain the capacity for hair cell regeneration. In the avian basilar papilla (BP), supporting cells (SCs), which give rise to regenerated hair cells, are usually quiescent. Hair cell loss induces both direct transdifferentiation and mitotic division of supporting cells. Here, we established an explant culture model for hair cell regeneration in chick basilar papillae and validated it for investigating the initial phase of hair cell regeneration. The histological assessment demonstrated hair cell regeneration via direct transdifferentiation of supporting cells. Labeling with 5-ethynyl-2'-deoxyuridine (EdU) revealed the occurrence of mitotic division in the supporting cells at specific locations in the basilar papillae, while no EdU labeling was observed in newly generated hair cells. RNA sequencing indicated alterations in known signaling pathways associated with hair cell regeneration, consistent with previous findings. Also, unbiased analyses of RNA sequencing data revealed novel genes and signaling pathways that may be related to the induction of supporting cell activation in the chick basilar papillae. These results indicate the advantages of our explant culture model of the chick basilar papillae for exploring the molecular mechanisms of hair cell regeneration.

Expansion of Human iPSC-Derived Ureteric Bud Organoids with Repeated Branching Potential.

Ureteric bud (UB) is the embryonic kidney progenitor tissue that gives rise to the collecting duct and lower urinary tract. UB-like structures generated from human pluripotent stem cells by previously reported methods show limited developmental ability and limited branching. Here we report a method to generate UB organoids that possess epithelial polarity and tubular lumen and repeat branching morphogenesis. We also succeed in monitoring UB tip cells by utilizing the ability of tip cells to uptake very-low-density lipoprotein, cryopreserving UB progenitor cells, and expanding UB tip cells that can reconstitute the organoids and differentiate into collecting duct progenitors. Moreover, we successfully reproduce some phenotypes of multicystic dysplastic kidney (MCDK) using the UB organoids. These methods will help elucidate the developmental mechanisms of UB branching and develop a selective differentiation method for collecting duct cells, contributing to the creation of disease models for congenital renal abnormalities.

A Modular Differentiation System Maps Multiple Human Kidney Lineages from Pluripotent Stem Cells.

Recent studies using human pluripotent stem cells (hPSCs) have developed protocols to induce kidney-lineage cells and reconstruct kidney organoids. However, the separate generation of metanephric nephron progenitors (NPs), mesonephric NPs, and ureteric bud (UB) cells, which constitute embryonic kidneys, in in vitro differentiation culture systems has not been fully investigated. Here, we create a culture system in which these mesoderm-like cell types and paraxial and lateral plate mesoderm-like cells are separately generated from hPSCs. We recapitulate nephrogenic niches from separately induced metanephric NP-like and UB-like cells, which are subsequently differentiated into glomeruli, renal tubules, and collecting ducts in vitro and further vascularized in vivo. Our selective differentiation protocols should contribute to understanding the mechanisms underlying human kidney development and disease and also supply cell sources for regenerative therapies.

[Dissecting early development of the kidney by single cell transcriptomics].

Each of the billions of the cells in our body exhibits their identity with unique gene expression profile. Recent advances in single cell transcriptomics enable to conduct cell taxonomy identifying new cell types and to re-arrange cells in order of pseudo-time course describing differentiation status of each cell. Even though the cost is still high, the single cell transcriptomics now becomes one of the conventional assays. We have applied the single cell gene expression analysis to dissect human development. In this article, we show our recent progress on a study describing early development of the kidney using human iPS cells by the single cell transcriptomics.

mDia1/3 generate cortical F-actin meshwork in Sertoli cells that is continuous with contractile F-actin bundles and indispensable for spermatogenesis and male fertility.

Formin is one of the two major classes of actin binding proteins (ABPs) with nucleation and polymerization activity. However, despite advances in our understanding of its biochemical activity, whether and how formins generate specific architecture of the actin cytoskeleton and function in a physiological context in vivo remain largely obscure. It is also unknown how actin filaments generated by formins interact with other ABPs in the cell. Here, we combine genetic manipulation of formins mammalian diaphanous homolog1 (mDia1) and 3 (mDia3) with superresolution microscopy and single-molecule imaging, and show that the formins mDia1 and mDia3 are dominantly expressed in Sertoli cells of mouse seminiferous tubule and together generate a highly dynamic cortical filamentous actin (F-actin) meshwork that is continuous with the contractile actomyosin bundles. Loss of mDia1/3 impaired these F-actin architectures, induced ectopic noncontractile espin1-containing F-actin bundles, and disrupted Sertoli cell-germ cell interaction, resulting in impaired spermatogenesis. These results together demonstrate the previously unsuspected mDia-dependent regulatory mechanism of cortical F-actin that is indispensable for mammalian sperm development and male fertility.

Diffusion-weighted imaging of local recurrent prostate cancer after radiation therapy: comparison with 22-core three-dimensional prostate mapping biopsy.

Accurate localization of local recurrence within the prostate gland is important to perform focal salvage therapy effectively with minimal complications. The purpose of this study was to evaluate the usefulness of diffusion-weighted imaging (DWI) in the detection and localization of prostate cancer recurrence in patients with biochemical failure after definitive radiation therapy using 22-core three-dimensional prostate mapping biopsy (3D-PMB) as a standard reference. Ten patients who underwent magnetic resonance imaging and 22-core 3D-PMB were retrospectively analyzed. For visual assessment of DWI, the prostate was divided into 22 regions corresponding to 3D-PMB. Two diagnostic radiologists determined the presence of abnormal high signal intensity in each region on DWI, and the results of DWI were compared with those of 3D-PMB. Of the 220 regions, 16 regions in six patients were positive for cancer on 3D-PMB, and 30 regions in six patients were judged as positive on DWI. On a patient-by-patient basis, sensitivity and specificity were 100% (6/6) and 100% (4/4), respectively. On a region-by-region basis, sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 69% (11/16), 91% (185/204), 37% (11/30), 97% (185/190) and 89% (196/220), respectively. For discrepant localization between DWI and pathology, DWI-positive and pathology-positive regions tended to be adjacent to each other. In conclusion, DWI is a useful tool for the detection and localization of recurrent prostate cancer in patients with biochemical failure after radiation therapy and may be helpful in the planning of focal salvage therapy.

A new role of multi scaffold protein Liprin-α: Liprin-α suppresses Rho-mDia mediated stress fiber formation.

Regulation of the actin cytoskeleton is crucial for cell morphology and migration. One of the key molecules that regulates actin remodeling is the small GTPase Rho. Rho shuttles between the inactive GDP-bound form and the active GTP-bound form, and works as a molecular switch in actin remodeling in response to both extra- and intra-cellular stimuli. Mammalian homolog of Diaphanous (mDia) is one of the Rho effectors and produces unbranched actin filaments. While Rho GTPases activate mDia, the mechanisms of how the activity of mDia is downregulated in cells remains largely unknown. In our recent paper, we identified Liprin-α as an mDia interacting protein and found that Liprin-α negatively regulates the activity of mDia in the cell by displacing it from the plasma membrane through binding to the DID-DD region of mDia. Here, we review these findings and discuss how Liprin-α regulates the Rho-mDia pathway and how the mDia-Liprin-α complex functions in vivo.

Liprin-α controls stress fiber formation by binding to mDia and regulating its membrane localization.

Regulation of the actin cytoskeleton is crucial for cell morphology and migration. mDia is an actin nucleator that produces unbranched actin filaments downstream of Rho. However, the mechanisms by which mDia activity is regulated in the cell remain unknown. We pulled down Liprin-α as an mDia-binding protein. The binding is mediated through the central region of Liprin-α and through the N-terminal Dia-inhibitory domain (DID) and dimerization domain (DD) of mDia. Liprin-α competes with Dia autoregulatory domain (DAD) for binding to DID, and binds preferably to the open form of mDia. Overexpression of a Liprin-α fragment containing the mDia-binding region decreases localization of mDia to the plasma membrane and attenuates the Rho-mDia-mediated formation of stress fibers in cultured cells. Conversely, depletion of Liprin-α by RNA interference (RNAi) increases the amount of mDia in the membrane fraction and enhances formation of actin stress fibers. Thus, Liprin-α negatively regulates the activity of mDia in the cell by displacing it from the plasma membrane through binding to the DID-DD region.

Diffusion-weighted imaging of the breast: principles and clinical applications.

Diffusion-weighted imaging provides a novel contrast mechanism in magnetic resonance (MR) imaging and has a high sensitivity in the detection of changes in the local biologic environment. A significant advantage of diffusion-weighted MR imaging over conventional contrast material-enhanced MR imaging is its high sensitivity to change in the microscopic cellular environment without the need for intravenous contrast material injection. Approaches to the assessment of diffusion-weighted breast imaging findings include assessment of these data alone and interpretation of the data in conjunction with T2-weighted imaging findings. In addition, the analysis of apparent diffusion coefficient (ADC) value can be undertaken either in isolation or in combination with diffusion-weighted and T2-weighted imaging. Most previous studies have evaluated ADC value alone; however, overlap in the ADC values of malignant and benign disease has been observed. This overlap may be partly due to selection of b value, which can influence the concomitant effect of perfusion and emphasize the contribution of multicomponent model influences. The simultaneous assessment of diffusion-weighted and T2-weighted imaging data and ADC value has the potential to improve specificity. In addition, the use of diffusion-weighted imaging in a standard breast MR imaging protocol may heighten sensitivity and thereby improve diagnostic accuracy. Standardization of diffusion-weighted imaging parameters is needed to allow comparison of multicenter studies and assessment of the clinical utility of diffusion-weighted imaging and ADC values in breast evaluation.

Rho and anillin-dependent control of mDia2 localization and function in cytokinesis.

Diaphanous-related formin, mDia, is an actin nucleation/polymerization factor functioning downstream of the small GTPase Rho. Although Rho is critically involved in cytokinesis, it remains elusive how Rho effectors and other regulators of cytoskeletons work together to accomplish this process. Here we focused on mDia2, an mDia isoform involved in cytokinesis of NIH 3T3 cells, and analyzed mechanisms of its localization in cytokinesis. We found that targeting of mDia2 to the cleavage furrow requires not only its binding to RhoA but also its diaphanous-inhibitory domain (DID). We then performed pulldown assays using a fragment containing the latter domain as a bait and identified anillin as a novel mDia2 interaction partner. The anillin-binding is competitive with the diaphanous autoregulatory domain (DAD) of mDia2 in its autoinhibitory interaction. A series of RNA interference and functional rescue experiments has revealed that, in addition to the Rho GTPase-mediated activation, the interaction between mDia2 and anillin is required for the localization and function of mDia2 in cytokinesis.

Effect of autogenic training on cardiac autonomic nervous activity in high-risk fire service workers for posttraumatic stress disorder.

OBJECTIVE: We investigated the effect of autogenic training (AT) on cardiac autonomic nervous activity in fire services workers with the use of the questionnaire of the Japanese-language version of Impact of Event Scale-Revised (IES-R-J) and indexes of heart rate variability. METHODS: We studied 22 male fire services workers who were divided into posttraumatic stress disorder (PTSD)-related stress group (n=10) and control group (n=12). They underwent AT twice or three times a week for 2 months. RESULTS: Posttraumatic stress disorder-related stress group showed a significantly higher cardiac sympathetic nervous activity and a significantly lower cardiac parasympathetic nervous activity than control group at baseline. Autogenic training significantly decreased cardiac sympathetic nervous activity and significantly increased cardiac parasympathetic nervous activity in both groups. These changes were accompanied by a significant decrease in the total points of IES-R-J. CONCLUSION: Autogenic training is effective for ameliorating the disturbance of cardiac autonomic nervous activity and psychological issues secondary to PTSD.