Modeling RET-Rearranged Non-Small Cell Lung Cancer (NSCLC): Generation of Lung Progenitor Cells (LPCs) from Patient-Derived Induced Pluripotent Stem Cells (iPSCs).

REarranged during Transfection (RET) oncogenic rearrangements can occur in 1-2% of lung adenocarcinomas. While RET-driven NSCLC models have been developed using various approaches, no model based on patient-derived induced pluripotent stem cells (iPSCs) has yet been described. Patient-derived iPSCs hold great promise for disease modeling and drug screening. However, generating iPSCs with specific oncogenic drivers, like RET rearrangements, presents challenges due to reprogramming efficiency and genotypic variability within tumors. To address this issue, we aimed to generate lung progenitor cells (LPCs) from patient-derived iPSCs carrying the mutation RETC634Y, commonly associated with medullary thyroid carcinoma. Additionally, we established a RETC634Y knock-in iPSC model to validate the effect of this oncogenic mutation during LPC differentiation. We successfully generated LPCs from RETC634Y iPSCs using a 16-day protocol and detected an overexpression of cancer-associated markers as compared to control iPSCs. Transcriptomic analysis revealed a distinct signature of NSCLC tumor repression, suggesting a lung multilineage lung dedifferentiation, along with an upregulated signature associated with RETC634Y mutation, potentially linked to poor NSCLC prognosis. These findings were validated using the RETC634Y knock-in iPSC model, highlighting key cancerous targets such as PROM2 and C1QTNF6, known to be associated with poor prognostic outcomes. Furthermore, the LPCs derived from RETC634Y iPSCs exhibited a positive response to the RET inhibitor pralsetinib, evidenced by the downregulation of the cancer markers. This study provides a novel patient-derived off-the-shelf iPSC model of RET-driven NSCLC, paving the way for exploring the molecular mechanisms involved in RET-driven NSCLC to study disease progression and to uncover potential therapeutic targets.

A novel berberine derivative targeting adipocyte differentiation to alleviate TNF-α-induced inflammatory effects and insulin resistance in OP9 cells.

Inflammation and insulin resistance play important roles in the development and progression of type 2 diabetes mellitus. The enhancement of adipocyte differentiation can improve insulin sensitivity by increasing glucose uptake, improving insulin signaling, and reducing inflammation. However, only a few adipogenic agents have shown clinical success in patients with type 2 diabetes mellitus. The therapeutic potential of berberine in type 2 diabetes mellitus was confirmed in terms of the target gene-disease relationship using a network pharmacology database prior to synthesizing the derivatives. Novel berberine derivatives were synthesized, and compound 3b promoted adipocyte differentiation and improvement of insulin resistance in OP9 cells. Compound 3b significantly increased the expression of key adipogenic markers including peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein ß (C/EBPß) and promoted lipid accumulation without cytotoxicity. Furthermore, tumor necrosis factor α (TNF-α)-induced inhibition of adipocyte differentiation and the elevation of inflammatory responses were reversed by compound 3b. Subsequently glucose uptake level through insulin sensitivity improvement was enhanced by compound 3b. Mechanistically, TNF-α activated mitogen-activated protein kinases (MAPKs): ERK, JNK, and p38, whereas compound 3b attenuated phosphorylation of three MAPKs. Finally, in silico molecular docking suggested the possible binding sites of compound 3b on PPARγ. Collectively, the adipogenic and glucose uptake effects of compound 3b were associated with its anti-inflammatory effects and reduced phosphorylation of MAPKs. These findings suggest that the berberine derivative compound 3b may be a potent antidiabetic agent.

Novel bone Morphogenetic Protein (BMP)-2/4 Consensus Peptide (BCP) for the Osteogenic Differentiation of C2C12 Cells.

BACKGROUND: Despite the promising clinical potential of bone morphogenetic protein (BMP)-related therapies for bone formation, their side effects warrant the need for alternative therapeutic peptides. BMP family members can aid in bone repair; however, peptides derived from BMP2/ 4 have not yet been investigated. METHODS: In this study, three candidates BMP2/4 consensus peptide (BCP) 1, 2, and 3 were identified and their ability to induce osteogenesis in C2C12 cells was analyzed. First, an alkaline phosphatase (ALP) staining assay was performed to evaluate the osteogenic effects of BCPs. Next, the effects of BCPs on RNA expression levels and protein abundances of osteogenic markers were explored. Furthermore, the transcriptional activity of ALP by BCP1 and in silico molecular docking model on BMP type IA receptor (BRIA) were performed. RESULTS: BCP1-3 induced higher RUNX2 expression than BMP2. Interestingly, among them, BCP1 significantly promoted osteoblast differentiation more than BMP2 in ALP staining with no cytotoxicity. BCP1 significantly induced the osteoblast markers, and the highest RUNX2 expression was observed at 100 ng/mL compared to other concentrations. In transfection experiments, BCP1 stimulated osteoblast differentiation via RUNX2 activation and the Smad signaling pathway. Finally, in silico molecular docking suggested the possible binding sites of BCP1 on BRIA. CONCLUSION: These results show that BCP1 promotes osteogenicity in C2C12 cells. This study suggests that BCP1 is the most promising candidate peptide to replace BMP2 for osteoblast differentiation.

Single cell transcriptomics reveals reduced stress response in stem cells manipulated using localized electric fields.

Membrane disruption using Bulk Electroporation (BEP) is a widely used non-viral method for delivering biomolecules into cells. Recently, its microfluidic counterpart, Localized Electroporation (LEP), has been successfully used for several applications ranging from reprogramming and engineering cells for therapeutic purposes to non-destructive sampling from live cells for temporal analysis. However, the side effects of these processes on gene expression, that can affect the physiology of sensitive stem cells are not well understood. Here, we use single cell RNA sequencing (scRNA-seq) to investigate the effects of BEP and LEP on murine neural stem cell (NSC) gene expression. Our results indicate that unlike BEP, LEP does not lead to extensive cell death or activation of cell stress response pathways that may affect their long-term physiology. Additionally, our demonstrations show that LEP is suitable for multi-day delivery protocols as it enables better preservation of cell viability and integrity as compared to BEP.

Ca2+ entry through reverse Na+/Ca2+ exchanger in NCI-H716, glucagon-like peptide-1 secreting cells.

Glucagon like peptide-1 (GLP-1) released from enteroendocine L-cells in the intestine has incretin effects due to its ability to amplify glucose-dependent insulin secretion. Promotion of an endogenous release of GLP-1 is one of therapeutic targets for type 2 diabetes mellitus. Although the secretion of GLP-1 in response to nutrient or neural stimuli can be triggered by cytosolic Ca2+ elevation, the stimulus-secretion pathway is not completely understood yet. Therefore, the aim of this study was to investigate the role of reverse Na+/Ca2+ exchanger (rNCX) in Ca2+ entry induced by muscarinic stimulation in NCI-H716 cells, a human enteroendocrine GLP-1 secreting cell line. Intracellular Ca2+ was repetitively oscillated by the perfusion of carbamylcholine (CCh), a muscarinic agonist. The oscillation of cytosolic Ca2+ was ceased by substituting extracellular Na+ with Li+ or NMG+. KB-R7943, a specific rNCX blocker, completely diminished CCh-induced cytosolic Ca2+ oscillation. Type 1 Na+/Ca2+ exchanger (NCX1) proteins were expressed in NCI-H716 cells. These results suggest that rNCX might play a crucial role in Ca2+ entry induced by cholinergic stimulation in NCI-H716 cells, a GLP-1 secreting cell line.

Heme oxygenase-1 gene delivery for altering high mobility group box-1 protein in pancreatic islet.

Pancreatic islet transplantation is a promising strategy for the treatment of type I diabetes. High-mobility group box-1 (HMGB1), highly expressed in islet cells, is a potent immune stimulator in immune rejection. Heme oxygenase-1 (HO1) gene therapy can modulate the release of HMGB1 by altering intracellular molecules for successful cell transplantation. After delivery of the heme oxygenase-1 (HO1) gene to islet cells using an adeno-associated viral vector (AAV), it was evaluated the changes in cytoplasmic Ca2+ ions and calcineurin activity as well as histone acetyltransferase (HAT) and Poly(ADP) ribose polymerase-1 (PARP-1). Inhibition of HMGB1 release was evaluated through altering these intracellular molecules. Then, after transplantation of HO1-transduced islets, the therapeutic effect of them was evaluated through measuring blood glucose level to diabetic mice and through immunohistochemical analysis. The transduced HO1 gene significantly inhibited HMGB1 release in islets that was under the cell damage by hypoxia exposure. It was confirmed that this result was initially due to the decrease in cytoplasmic Ca2+ ion concentration and calcineurin activity. In addition, the delivered HO1 gene simultaneously reduced the activity of HAT and PARP-1, which are involved in the translocation of HMGB1 from the nucleus to the cytoplasm. As a result, when the HO1 gene-transduced islets were transplanted into diabetic mice, the treatment efficiency of diabetes was effectively improved by increasing the survival rate of the islets. Collectively, these results suggest that HO1 gene transfer can be used for successful islet transplantation by altering the activity of intracellular signal molecules and reducing HMGB1 release.

Effects of solvent osmolarity and viscosity on cartilage energy dissipation under high-frequency loading.

Articular cartilage is a spatially heterogeneous, dissipative biological hydrogel with a high fluid volume fraction. Although energy dissipation is important in the context of delaying cartilage damage, the dynamic behavior of articular cartilage equilibrated in media of varied osmolarity and viscosity is not widely understood. This study investigated the mechanical behaviors of cartilage when equilibrated to media of varying osmolarity and viscosity. Dynamic moduli and phase shift were measured at both low (1 Hz) and high (75-300 Hz) frequency, with cartilage samples compressed to varied offset strain levels. Increasing solution osmolarity and viscosity both independently resulted in larger energy dissipation and decreased dynamic modulus of cartilage at both low and high frequency. Mechanical property alterations induced by varying osmolarity are likely due to the change in permeability and fluid volume fraction within the tissue. The effects of solution viscosity are likely due to frictional interactions at the solid-fluid interface, affecting energy dissipation. These findings highlight the significance of interstitial fluid on the energy dissipation capabilities of the tissue, which can influence the onset of cartilage damage.

Detection of Hematopoietic Stem Cell Transcriptome in Human Fetal Kidneys and Kidney Organoids Derived From Human Induced Pluripotent Stem Cells.

BACKGROUND: In mammalians, hematopoietic stem cells (HSCs) arise in the dorsal aorta from the hemogenic endothelium, followed by their migration to the fetal liver and to the bone marrow. In zebrafish, the kidney is the site of primary hematopoiesis. In humans, the presence of HSCs in the fetal or adult kidney has not been established. METHODS: We analyzed the presence of HSC markers in the human fetal kidneys by analysis of single-cell datasets. We then analyzed in kidney organoids derived from induced pluripotent stem cells (iPSCs) the presence of hematopoietic markers using transcriptome analyses. RESULTS: Twelve clusters were identified as stromal, endothelial, and nephron cell type-specific markers in the two fetal stage (17 weeks) kidney datasets. Among these, the expression of hematopoietic cells in cluster 9 showed an expression of primitive markers. Moreover, whole transcriptome analysis of our iPSC-derived kidney organoids revealed induction of the primitive hematopoietic transcription factor RUNX1 as found in the human fetal kidney cortex. CONCLUSION: These finding support the presence of cells expressing HSC transcriptome in the human kidney. The mechanisms of the appearance of the cells with the same transcriptional features during iPSC-derived kidney organoid generation require further investigation.

Asarylaldehyde enhances osteogenic differentiation of human periodontal ligament stem cells through the ERK/p38 MAPK signaling pathway.

Periodontitis is an inflammatory disease that affects tooth-supporting tissues. Chronic inflammation can progress to periodontitis, which results in loss of alveolar bone. Asarylaldehyde is a potential substance for bone metabolism present in natural compounds. Here, we propose the application of asarylaldehyde in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) to prevent bone loss. We investigated the effect of asarylaldehyde on hPDLSCs together with bone differentiation media in vitro. The osteogenic differentiation effect was observed after treatment of hPDLSCs with several concentrations of asarylaldehyde. After 21 days, osteogenic cells were identified by mineralization. We also observed that asarylaldehyde increased the mRNA expression of osteoblast-specific markers in hPDLSCs. Interestingly, asarylaldehyde regulated the levels of alkaline phosphatase (ALP) transcriptional activity through the p38/extracellular-signal-regulated kinase (ERK) signaling pathway. Notably, asarylaldehyde induced hPDLSCs to promote osteogenic differentiation. These results suggest that asarylaldehyde plays a key role in the osteogenic differentiation of hPDLSCs. Asarylaldehyde may be a good candidate for the application of natural compounds in future in periodontal regeneration.

Embryonic Program Activated during Blast Crisis of Chronic Myelogenous Leukemia (CML) Implicates a TCF7L2 and MYC Cooperative Chromatin Binding.

Chronic myeloid leukemia (CML) is characterized by an inherent genetic instability, which contributes to the progression of the disease towards an accelerated phase (AP) and blast crisis (BC). Several cytogenetic and genomic alterations have been reported in the progression towards BC, but the precise molecular mechanisms of this event are undetermined. Transcription Factor 7 like 2 (TFC7L2) is a member of the TCF family of proteins that are known to activate WNT target genes such as Cyclin D1. TCF7L2 has been shown to be overexpressed in acute myeloid leukemia (AML) and represents a druggable target. We report here that TCF7L2 transcription factor expression was found to be correlated to blast cell numbers during the progression of the disease. In these cells, TCF7L2 CHIP-sequencing highlighted distal cis active enhancer, such as elements in SMAD3, ATF5, and PRMT1 genomic regions and a proximal active transcriptional program of 144 genes. The analysis of CHIP-sequencing of MYC revealed a significant overlapping of TCF7L2 epigenetic program with MYC. The ß-catenin activator lithium chloride and the MYC-MAX dimerization inhibitor 10058-F4 significantly modified the expression of three epigenetic targets in the BC cell line K562. These results suggest for the first time the cooperative role of TCF7L2 and MYC during CML-BC and they strengthen previous data showing a possible involvement of embryonic genes in this process.

Synergistic stimulating effect of 2-hydroxymelatonin and BMP-4 on osteogenic differentiation in vitro.

2-Hydroxymelatonin is a metabolite produced when melatonin 2-hydroxylase catalyzes melatonin. Recent studies have reported the important roles of melatonin in bone metabolism. However, the roles of 2-hydroxymelatonin in bone metabolism remains poorly understood. The purpose of this study is to present evidence of the effect of 2-hydroxymelatonin on osteogenic differentiation in C2C12 cells. In this study, we demonstrated the synergistic stimulating effect of 2-hydroxymelatonin and bone morphogenetic protein (BMP)-4 on osteogenic differentiation in vitro, using alkaline phosphatase (ALP) staining, Alizarin red S (ARS) staining, qPCR, and luciferase reporter assay. The combination of 2-hydroxymelatonin and BMP-4 revealed a synergistic effect on osteogenic differentiation in vitro. This finding provides evidence that optimal concentrations of both 2-hydroxymelatonin and BMP-4 are beneficial for anabolic effects on bone in vitro.

A novel neuronal organoid model mimicking glioblastoma (GBM) features from induced pluripotent stem cells (iPSC).

BACKGROUND: Current experimental models using either human or mouse cell lines, are not representative of the complex features of GBM. In particular, there is no model to study patient-derived iPSCs to generate a GBM model. Overexpression of c-met gene is one of the molecular features of GBM leading to increased signaling via STAT3 phosphorylation. We generated an iPSC line from a patient with c-met mutation and we asked whether we could use it to generate neuronal-like organoids mimicking features of GBM. METHODS: We have generated iPSC-aggregates differentiating towards organoids. We analyzed them by gene expression profiling, immunostaining and transmission electronic microscopy analyses (TEM). RESULTS: Herein we describe that c-met-mutated iPSC aggregates spontaneously differentiate into dopaminergic neurons more rapidly than control iPSC aggregates in culture. Gene expression profiling of c-met-mutated iPSC aggregates at day +90 showed neuronal- and GBM-related genes, reproducing a genomic network described in primary human GBM. Comparative TEM analyses confirmed the enrichment of these structures in intermediate filaments and abnormal cilia, a feature described in human GBM. The c-met-mutated iPSC-derived organoids, as compared to controls expressed high levels of glial fibrillary acidic protein (GFAP), which is a typical marker of human GBM, as well as high levels of phospho-MET and phospho-STAT3. The use of temozolomide (TMZ) showed a preferential cytotoxicity of this drug in c-met-mutated neuronal-like organoids. GENERAL SIGNIFICANCE: This study shows the feasibility of generating "off-the shelf" neuronal-like organoid model mimicking GBM using c-met-mutated iPSC aggregates and its potential future use in research.

iPSC-Derived Embryoid Bodies as Models of c-Met-Mutated Hereditary Papillary Renal Cell Carcinoma.

Hereditary cancers with cancer-predisposing mutations represent unique models of human oncogenesis, as a driving oncogenic event is present in germline. Currently, there are no satisfactory models to study these malignancies. We report the generation of IPSC from the somatic cells of a patient with hereditary c-met-mutated papillary renal cell carcinoma (PRCC). From these cells we have generated spontaneous aggregates organizing in structures which expressed kidney markers such as PODXL and Six2. These structures expressed PRCC markers both in vitro and in vivo in NSG mice. Gene-expression profiling showed striking molecular similarities with signatures found in a large cohort of PRCC tumor samples. This analysis, applied to primary cancers with and without c-met mutation, showed overexpression of the BHLHE40 and KDM4C only in the c-met-mutated PRCC tumors, as predicted by c-met-mutated embryoid bodies transcriptome. These data therefore represent the first proof of concept of "hereditary renal cancer in a dish" model using c-met-mutated iPSC-derived embryoid bodies, opening new perspectives for discovery of novel predictive progression markers and for drug-screening for future precision-medicine strategies.

Modeling malignancies using induced pluripotent stem cells: from chronic myeloid leukemia to hereditary cancers.

Over the last decade, the possibility of reprogramming malignant cells to a pluripotent state has been achieved in several hematological malignancies, including myeloproliferative neoplasms, myelodysplastic syndromes, and chronic myeloid leukemia (CML). It has been shown that it is readily possible to generate induced pluripotent stem cells (iPSCs) from several types of primary CML cells and to generate progenitors and differentiated cells with variable efficiency. Although these experiments have brought some new insights in the understanding of CML pathophysiology, the ultimate goal of generating induced leukemic stem cells (LSCs) with long-term multilineage potential has not yet been demonstrated. Experiments under way will determine whether additional signaling events are required to induce the emergence of bona fide LSCs. However, iPSC modeling offers the unique possibility to generate pluripotent cells harboring cancer-predisposing mutations using patient-derived noncancerous cells, as has been shown in Li-Fraumeni syndrome, BRCA-1 associated breast carcinomas, or RET-mutated medullary thyroid carcinomas. In these conditions, mutated iPSCs can then be used to study the mutational history that precedes the appearance of the malignant transformation and to develop novel drug-screening strategies. The ability to induce a successful differentiation program toward the tissue in which a given cancer develops or to generate tissue-specific cancer organoids in which the full oncogenic potential can be revealed remains a major challenge in the field. Similarly, in hematological malignancies, a significant hurdle remains due to the lack of adequate technology to induce the emergence of leukemic cells that resemble LSCs, which hinders our ability to study the mechanisms of therapy resistance.

Effects of the fibrous topography-mediated macrophage phenotype transition on the recruitment of mesenchymal stem cells: An in vivo study.

Host responses to a biomaterial critically influence its in vivo performance. Biomaterial architectures that can recruit endogenous host stem cells could be beneficial in tissue regeneration or integration. Here, we report that the fibrous topography of biomaterials promotes the recruitment of host mesenchymal stem cells (MSCs) by facilitating the macrophage phenotype transition from M1-to-M2. Electrospun poly (ε-caprolactone) fiber (PCL-fiber) films were implanted into the subcutaneous tissues of rats, and the response of host cells to the PCL-fiber was evaluated and compared with those of solid ones (PCL-solid). During the initial post-implantation period, greater numbers of cells were recruited and adhered to the PCL-fiber compared to the PCL-solid, and the cells exhibited the M1 phenotype, which was supported by the enhanced adsorption of complement C3a to the implanted PCL-fiber. Subsequently, the PCL-fiber supported the macrophage phenotype transition from M1-to-M2, which was confirmed by the ratio of M2/M1 marker (CD163/CCR7)-positive cells and by the expression of M2/M1 markers (arginase-1/iNOS). The PCL-fiber also reduced the formation of foreign body giant cells. MSC marker (CD29, CD44, and CD90)-positive cells began to appear as early as day 4 on the PCL-fiber, while few MSCs were observed on the PCL-solid. The MSCs migration ex vivo assay showed that MSCs substantially migrated across the trans-wells toward the implanted PCL-fiber. The cells on the implanted PCL-fiber expressed and secreted substantial levels of SDF-1 (CXCL-12), while anti-SDF-1 neutralizing antibody abrogated the MSCs migration. Taken together, these results provide evidence that the fibrous topography of biomaterials enhances the recruitment of MSCs by promoting macrophage recruitment, facilitating M1-to-M2 transition, and enhancing SDF-1 secretion.

Comparative, randomized, double-blind clinical study of alveolar ridge preservation using an extracellular matrix-based dental resorbable membrane in the extraction socket.

PURPOSE: The aim of this study was to radiographically and clinically compare the effect of extracellular matrix (ECM) membranes on dimensional alterations following a ridge preservation procedure. METHODS: One of 2 different ECM membranes was applied during a ridge preservation procedure. A widely used ECM membrane (WEM; Bio-Gide, Geistlich Biomaterials, Wolhusen, Switzerland) was applied in the treatment group and a newly developed ECM membrane (NEM; Lyso-Gide, Oscotec Inc., Seongnam, Korea) was applied in the control group. Cone-beam computed tomography (CBCT) scans and alginate impressions were obtained 1 week and 6 months after the ridge preservation procedure. Results were analyzed using the independent t-test and the nonparametric Mann-Whitney U test. RESULTS: There were no significant differences between the ECM membranes in the changes in the dimension, width, and height of the extraction socket or the quantity of bone tissue. CONCLUSIONS: The NEM showed comparable clinical and radiographic results to the WEM following the ridge preservation procedure.

A randomized controlled clinical study of periodontal tissue regeneration using an extracellular matrix-based resorbable membrane in combination with a collagenated bovine bone graft in intrabony defects.

PURPOSE: The purpose of this study was to investigate the feasibility of regenerative therapy with a collagenated bone graft and resorbable membrane in intrabony defects, and to evaluate the effects of the novel extracellular matrix (ECM)-based membrane clinically and radiologically. METHODS: Periodontal tissue regeneration procedure was performed using an ECM-based resorbable membrane in combination with a collagenated bovine bone graft in intrabony defects around the teeth and implants. A novel extracellular matrix membrane (NEM) and a widely-used membrane (WEM) were randomly applied to the test group and the control group, respectively. Cone-beam computed tomography images were obtained on the day of surgery and 6 months after the procedure. Alginate impressions were taken and plaster models were made 1 week and 6 months postoperatively. RESULTS: The quantity of bone tissue, the dimensional changes of the surgically treated intrabony defects, and the changes in width and height below the grafted bone substitutes showed no significant difference between the test and control groups at the 6-month examination. CONCLUSIONS: The use of NEM for periodontal regeneration with a collagenated bovine bone graft showed similar clinical and radiologic results to those obtained using WEM.

Effect of Extracellular Matrix Membrane on Bone Formation in a Rabbit Tibial Defect Model.

Absorbable extracellular matrix (ECM) membrane has recently been used as a barrier membrane (BM) in guided tissue regeneration (GTR) and guided bone regeneration (GBR). Absorbable BMs are mostly based on collagen, which is more biocompatible than synthetic materials. However, implanted absorbable BMs can be rapidly degraded by enzymes in vivo. In a previous study, to delay degradation time, collagen fibers were treated with cross-linking agents. These compounds prevented the enzymatic degradation of BMs. However, cross-linked BMs can exhibit delayed tissue integration. In addition, the remaining cross-linker could induce inflammation. Here, we attempted to overcome these problems using a natural ECM membrane. The membrane consisted of freshly harvested porcine pericardium that was stripped from cells and immunoreagents by a cleaning process. Acellular porcine pericardium (APP) showed a bilayer structure with a smooth upper surface and a significantly coarser bottom layer. APP is an ECM with a thin layer (0.18-0.35 mm) but with excellent mechanical properties. Tensile strength of APP was 14.15 ± 2.24 MPa. In in vivo experiments, APP was transplanted into rabbit tibia. The biocompatible material was retained for up to 3 months without the need for cross-linking. Therefore, we conclude that APP could support osteogenesis as a BM for up to 3 months.

Endometrial stromal sarcoma presented as an incidental lung mass with multiple pulmonary nodules.

Low-grade endometrial stromal sarcoma (ESS) is an uncommon gynecologic malignancy of mesodermal origin. Pulmonary metastasis of low-grade ESS can occur years and decades after the treatment of the primary disease. Low-grade ESS is frequently mistaken as benign uterine neoplasm like uterine leiomyoma, which can potentially lead to a misdiagnosis. We present a case of a 42-year-old woman with low-grade ESS, that initially presented as an incidental lung mass with multiple pulmonary nodules, seven years after an uterine myomectomy. A 6.9×5.8 cm-sized intrapelvic mass suspected of uterine origin was discovered while searching for potential extrathoracic primary origin. A pelviscopy and simultaneous thoracoscopic lung biopsy were conducted for pathologic diagnosis. Finally, the diagnosis was confirmed as low-grade ESS with lung metastasis based on the histopathologic examination with immunohistochemical stain, which was showed positive for CD10 and hormone receptor markers (estrogen and progesterone receptors) in both pelvic and lung specimens.

In situ formation and collagen-alginate composite encapsulation of pancreatic islet spheroids.

In this study, we suggest in situ islet spheroid formation and encapsulation on a single platform without replating as a method for producing mono-disperse spheroids and minimizing damage to spheroids during encapsulation. Using this approach, the size of spheroid can be controlled by modulating the size of the concave well. Here, we used 300 µm concave wells to reduce spheroid size and thereby eliminating the central necrosis caused by large volume. As the encapsulation material, we used alginate and collagen-alginate composite (CAC), and evaluated their suitability through diverse in vitro tests, including measurements of viability, oxygen consumption rate (OCR), hypoxic damage to encapsulated spheroids, and insulin secretion. For in situ encapsulation, alginate or CAC was spread over a concave microwell array containing spheroids, and CaCl(2) solution was diffused through a nano-porous dialysis membrane to achieve uniform polymerization, forming convex structures. By this process, the formation of uniform-size islet spheroids and their encapsulation without an intervening replating step was successfully performed. As a control, intact islets were evaluated concurrently. The in vitro test demonstrated excellent performance of CAC-encapsulated spheroids, and on the basis of these results, we transplanted the islet spheroids-encapsulated with CAC into the intraperitoneal cavity of mice with induced diabetes for 4 weeks, and evaluated subsequent glucose control. Intact islets were also transplanted as control to investigate the effect of encapsulation. Transplanted CAC-encapsulated islet spheroids maintained glucose levels below 200 mg/dL for 4 weeks, at which they were still active. At the end of the implantation experiment, we carried out intraperitoneal glucose tolerance test (IPGTT) in mice to investigate whether the implanted islets remained responsive to glucose. The glucose level in mice with CAC-encapsulated islet spheroids dropped below 200 mg/dL 60 min after glucose injection and was stably maintained. In conclusion, the proposed encapsulation method enhances the viability and function of islet spheroids, and protects these spheroids from immune attack.