Inter-cellular mRNA Transfer Alters Human Pluripotent Stem Cell State.

Inter-cellular transmission of mRNA is being explored in mammalian species using immortal cell lines (1-3). Here, we uncover an inter-cellular mRNA transfer phenomenon that allows for the adaptation and reprogramming of human primed pluripotent stem cells (hPSCs). This process is induced by the direct cell contact-mediated coculture with mouse embryonic stem cells (mESCs) under the condition impermissible for human primed PSC culture. Mouse-derived mRNA contents are transmitted into adapted hPSCs only in the coculture. Transfer-specific mRNA analysis show the enrichment for divergent biological pathways involving transcription/translational machinery and stress-coping mechanisms, wherein such transfer is diminished when direct cell contacts are lost. After 5 days of mESC culture, surface marker analysis, and global gene profiling confirmed that mRNA transfer-prone hPSC efficiently gains a naïve-like state. Furthermore, transfer-specific knockdown experiments targeting mouse-specific transcription factor-coding mRNAs in hPSC show that mouse-derived Tfcp2l1, Tfap2c, and Klf4 are indispensable for human naïve-like conversion. Thus, inter-species mRNA transfer triggers cellular reprogramming in mammalian cells. Our results support that episodic mRNA transfer can occur in cell cooperative and competitive processes(4), which provides a fresh perspective on understanding the roles of mRNA mobility for intra- and inter-species cellular communications.

Cellotype-phenotype associations using 'organoid villages'.

En masse phenotyping technology, using massively mosaic donor-derived cells and organoids, can offer enriched insights for cellotype-phenotype association in a cell-type-specific regulatory context. This emerging approach will help to discover biomarkers, inform genetic-epigenetic interactions and identify personalized therapeutic targets, offering hope for precision medicine against highly heterogeneous metabolic diseases.

Giant bullous emphysema successfully treated with percutaneous drainage followed by resection: A case complicated by lung cancer diagnosed by intraoperative biopsy.

We present a case of bilateral giant bullous emphysema (GBE) with rapidly progressive dyspnea. The dyspnea was thought to be due to tension bullae caused by the check valve mechanism in COVID-19 bronchitis. Multiple nodules were also detected on both sides of the lung. As the patient had poor pulmonary reserve for surgical bullectomy, we first performed percutaneous intracavitary drainage. Prior to this procedure, we placed a chest tube in the thoracic cavity to avoid tension pneumothorax. As a result, the patient's remaining lung expanded and respiratory status improved, allowing him to undergo surgical bullectomy. Intraoperatively, needle biopsy of the lung nodule was directly performed, which led to a diagnosis of adenocarcinoma. Despite multiple distant metastases, the patient's general condition improved postoperatively, and chemotherapy was successfully initiated.

A Pillar and Perfusion Plate Platform for Robust Human Organoid Culture and Analysis.

Human organoids have the potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo. This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by the lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, these challenges are overcome by engineering "microarray three-dimensional (3D) bioprinting" technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing, and encapsulation techniques are demonstrated on a pillar plate, which is coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels are differentiated into liver and intestine organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

Preparation of mechanically patterned hydrogels for controlling the self-condensation of cells.

Synthetic protocols providing mechanical patterns to culture substrate are essential to control the self-condensation of cells for organoid engineering. Here, we present a protocol for preparing hydrogels with mechanical patterns. We describe steps for hydrogel synthesis, mechanical evaluation of the substrate, and time-lapse imaging of cell self-organization. This protocol will facilitate the rational design of culture substrates with mechanical patterns for the engineering of various functional organoids. For complete details on the use and execution of this protocol, please refer to Takebe et al. (2015) and Matsuzaki et al. (2014, 2022).1,2,3.

Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells.

Although the differentiation of human induced pluripotent stem cells (hiPSCs) into various types of blood cells has been well established, approaches for clinical-scale production of multipotent hematopoietic progenitor cells (HPCs) remain challenging. We found that hiPSCs cocultured with stromal cells as spheroids (hematopoietic spheroids [Hp-spheroids]) can grow in a stirred bioreactor and develop into yolk sac-like organoids without the addition of exogenous factors. Hp-spheroid-induced organoids recapitulated a yolk sac-characteristic cellular complement and structures as well as the functional ability to generate HPCs with lympho-myeloid potential. Moreover, sequential hemato-vascular ontogenesis could also be observed during organoid formation. We demonstrated that organoid-induced HPCs can be differentiated into erythroid cells, macrophages, and T lymphocytes with current maturation protocols. Notably, the Hp-spheroid system can be performed in an autologous and xeno-free manner, thereby improving the feasibility of bulk production of hiPSC-derived HPCs in clinical, therapeutic contexts.

Extra-Anatomical Bypass for Chronic Mesenteric Ischemia with Saphenous Vein Grafts from a Previous Axillofemoral Artery Bypass Graft: A Case Report.

A 74-year-old woman who was diagnosed with chronic mesenteric ischemia was under hemodialysis maintenance and had previously undergone axillobifemoral bypass surgery because of abdominal aortoiliac occlusion. Endovascular and antegrade or retrograde surgical revascularizations from the aortoiliac artery were contraindicated because of a severely calcified arteriosclerotic lesion, which included aortoiliac occlusion. During median laparotomy, revascularization consisting of bypass grafting from a previous prosthetic graft to the mesenteric arteries was performed using saphenous vein grafts. Although extra-anatomical bypass for chronic mesenteric ischemia is challenging, it provides a feasible option in cases where conventional endovascular or surgical revascularization is contraindicated.

A Pillar and Perfusion Plate Platform for Robust Human Organoid Culture and Analysis.

Human organoids have potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo . This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, we overcome these challenges by engineering "microarray three-dimensional (3D) bioprinting" technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing and encapsulation techniques were demonstrated on a pillar plate, which was coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels were differentiated into liver and intestine organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

An unusual case of hepatosplenic T-cell lymphoma-like unclassifiable T/NK-cell lymphoma accompanied by acute myeloid leukemia.

We describe a case of unclassifiable T/NK-cell lymphoma with concomitant acute myeloid leukemia (AML). A 73-year-old Japanese man was diagnosed with AML by bone marrow smear, but the presence of splenomegaly and liver tumor was incompatible with AML. Splenectomy and hepatic resection were performed to resolve the thrombocytopenia and define the diagnosis. The pathological findings showed sinusoidal involvement of abnormal lymphoid cells that were CD3-positive but negative for T-cell receptor (TCR) rearrangement. Our case could not be categorized as hepatosplenic T-cell lymphoma because of the lack of immunohistological expression of TCR, despite the clinical similarity.

Mechanical guidance of self-condensation patterns of differentiating progeny.

Spatially controlled self-organization represents a major challenge for organoid engineering. We have developed a mechanically patterned hydrogel for controlling self-condensation process to generate multi-cellular organoids. We first found that local stiffening with intrinsic mechanical gradient (IG > 0.008) induced single condensates of mesenchymal myoblasts, whereas the local softening led to stochastic aggregation. Besides, we revealed the cellular mechanism of two-step self-condensation: (1) cellular adhesion and migration at the mechanical boundary and (2) cell-cell contraction driven by intercellular actin-myosin networks. Finally, human pluripotent stem cell-derived hepatic progenitors with mesenchymal/endothelial cells (i.e., liver bud organoids) experienced collective migration toward locally stiffened regions generating condensates of the concave to spherical shapes. The underlying mechanism can be explained by force competition of cell-cell and cell-hydrogel biomechanical interactions between stiff and soft regions. These insights will facilitate the rational design of culture substrates inducing symmetry breaking in self-condensation of differentiating progeny toward future organoid engineering.

En masse organoid phenotyping informs metabolic-associated genetic susceptibility to NASH.

Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T. Analysis of NASH clinical cohorts revealed that GCKR-rs1260326-T allele elevates disease severity only under diabetic state but protects from fibrosis under non-diabetic states. Transcriptomic, metabolomic, and pharmacological analyses indicate significant mitochondrial dysfunction incurred by GCKR-rs1260326, which was not reversed with metformin. Uncoupling oxidative mechanisms mitigated mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress, forming a basis for future therapeutic approaches for diabetic NASH. Thus, "in-a-dish" genotype-phenotype association strategies disentangle the opposing roles of metabolic-associated gene variant functions and offer a rich mechanistic, diagnostic, and therapeutic inference toolbox toward precision hepatology. VIDEO ABSTRACT.

Incidence of genitourinary injuries in pelvic fractures: A 12-year single-center retrospective study.

OBJECTIVE: This study aimed to determine the epidemiology of genitourinary injuries in pelvic fractures and elucidate the clinical outcomes of patients with pelvic fractures with and without genitourinary injuries at a tertiary trauma center in Japan. METHODS: Patients with pelvic fractures in our tertiary trauma center between May 2009 and April 2021 were retrospectively assessed. The patients' demographics, mechanism of injury, and hospital course details were collected. The outcomes of patients with pelvic fractures with and without genitourinary injuries were compared. RESULTS: Of 402 patients with pelvic fractures, 18 (4.5%) had genitourinary injuries. Falls were the most common mechanisms of injury for all pelvic fractures The incidence of bladder, kidney, urethral, and testis injuries were 2.0%, 1.2%, 1.2%, and 0.5%, respectively. Patients with genitourinary injuries were significantly younger (median age, 26 vs. 51 years; p < 0.001), had a higher rate of intensive care unit admission (94% vs. 58%; p = 0.002), remained hospitalized longer (median duration, 82 vs. 45 days; p < 0.001), and had a longer intensive care unit stay (median duration, 6 vs. 2 days; p < 0.001) when compared to patients without genitourinary injuries. Genitourinary injuries were not associated with in-hospital mortality. CONCLUSIONS: The incidence of genitourinary injuries with pelvic fractures was 4.5%. The presence of genitourinary injuries was associated with a higher rate of intensive care unit admission, longer hospital stay, and longer intensive care unit stay, but it was not associated with in-hospital mortality.

Correction of a Factor VIII genomic inversion with designer-recombinases.

Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient-derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, results in 12% correction of the inversion and restores Factor VIII mRNA expression. In this work, we present designer-recombinases as an efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.

Two-Stage Portal Vein Ligation Facilitates Liver Regeneration Safely in Rats with Liver Cirrhosis.

BACKGROUND: Portal vein (PV) embolization is performed prior to extended hepatectomy for the damaged liver to increase future remnant liver volume and prevent postoperative liver failure. This study examined whether two-stage PV ligation (PVL) increased regeneration and hypertrophy of the future remnant liver compared to conventional PVL, and whether two-stage PVL was safe for damaged liver. METHOD: We produced a cirrhotic liver rat model with perioperatively maintained fibrosis. Rats were divided into: Group A (70%PVL), ligation of left branch of PV; Group B (90%PVL), ligation of right and left branches of PV; and Group C (two-stage 90%PVL), two-stage PVL with left branch ligation of PV followed by right branch ligation 7 days later. To evaluate liver regeneration, liver weight ratios, proliferating cell nuclear antigen (PCNA) labeling index (LI), mitotic index (MI), and TdT-mediated dUTP-biotin nick end labeling (TUNEL) LI in the non-ligated caudate lobe were measured. RESULTS: Fourteen-day survival rate was 20% in Group B but 100% in Group C. TUNEL LI differed significantly between Groups A and B at 2 and 7 days postoperatively. Weight ratios were significantly higher in Group C than in Groups A and B at 14 days postoperatively. PCNA LI and MI in the non-ligated caudate lobe decreased to preoperative levels by 7 days postoperatively in Groups A and B, but remained elevated until 14 days postoperatively in Group C. CONCLUSION: In cirrhotic liver rats, two-stage PVL avoided the lethal liver failure seen with one-stage PVL, and significantly facilitated liver regeneration more than one-stage PVL.

Higher serum alkaline phosphatase value indicates the need for bone mineral density testing in non-metastatic prostate cancer patients undergoing androgen deprivation therapy.

PURPOSE: Osteoporosis is a well-known adverse effect of androgen deprivation therapy for prostate cancer. This study aimed to reveal the factors associated with the diagnosis of osteoporosis in prostate cancer patients undergoing androgen deprivation therapy. METHODS: This retrospective cross-sectional study included 106 prostate cancer patients treated with androgen deprivation therapy. Patients with bone metastasis at the initiation of androgen deprivation therapy and those with castration-resistant prostate cancer were excluded. Bone mineral density was measured at the lumbar spine and femoral neck using dual-energy X-ray absorptiometry. Osteoporosis was defined as bone mineral density equal to or below either -2.5 SD or 70% of the mean in young adults. The association between clinicopathological variables and bone mineral density or diagnosis of osteoporosis was investigated. RESULTS: Thirty-six (34%) patients were found to have osteoporosis. The incidence of osteoporosis increased in a stepwise manner depending on the duration of androgen deprivation therapy. Multivariate logistic regression analysis identified a longer duration of androgen deprivation therapy (months, odd's ratio = 1.017, P = 0.006), lower body mass index (kg/m2, odd's ratio = 0.801, P = 0.005) and higher serum alkaline phosphatase value (U/l, odd's ratio 1.007, P = 0.014) as the factors independently associated with the diagnosis of osteoporosis. Eleven out of 50 (22%), 14 out of 35 (40%) and 11 out of 20 patients (55%) were osteoporotic in the patients with serum alkaline phosphatase values <238 U/l, 238-322 U/l and >322 U/l, respectively (P = 0.022). CONCLUSIONS: Osteoporosis is common in prostate cancer patients undergoing androgen deprivation therapy; furthermore, its incidence increases depending on the duration of androgen deprivation therapy. Bone mineral density testing should be considered for all patients on androgen deprivation therapy, especially for those with a lower body mass index and higher serum alkaline phosphatase value.

Four- and two-armed hetero porphyrin dimers: their specific recognition and self-sorting behaviours.

In this study we self-assembled the four-armed porphyrin hetero dimer capsule Cap4, stabilized through amidinium-carboxylate salt bridges, in CH2Cl2 and CHCl3. The dimer capsule Cap4 was kinetically and thermodynamically more stable than the corresponding two-armed dimer Cap2. The number of arms strongly influenced their recognition behaviour; guests possessing small aromatic faces (e.g., 1,3,5-trinitrobenzene) preferred residing in the cavity of the two-armed capsule Cap2, rather than in Cap4, both thermodynamically and kinetically; in contrast, large aromatic guests (e.g., 9,10-dibromoanthracene) were encapsulated predominantly by Cap4 because of favourable entropic effects. The number of arms enabled self-sorting behaviour of the dimer formation; complexation studies using an equimolar mixture of the four porphyrin constituents of the two capsules revealed the quantitative formation of the corresponding dimers Cap2 and Cap4. Furthermore, we examined the specific molecular recognition of Cap2 and Cap4; NMR experiments of mixtures of Cap2 and Cap4 in the presence of favourable guests for Cap2 and Cap4 revealed that these guest molecules were encapsulated selectively by their preferred hosts.

Synthesis and application of POLYseq for profiling human liver organoids.

Detailed herein is the protocol for synthesis, characterization, and application of POLYseq for cell pooling in single-cell sequencing runs. POLYseq is synthesized through commercially available reagents and is highly tailorable. Synthesis is easily performed in a two-step protocol, utilizing Michael addition only requiring a temperature-stable hot bath capable of holding 90°C. However, care must be taken when mixing reagents for synthesis, as the final product is sensitive to initial mixing ratios of POLYseq reagents. For complete details on the use and execution of this protocol, please refer to Dunn et al. (2021).

POLYseq: A poly(ß-amino ester)-based vector for multifunctional cellular barcoding.

Despite evolving biological application of next-generation sequencing (NGS) at single-cell level, current techniques in NGS library preparation restrict multiplexing, necessitating the costly preparation of distinct libraries for each sample. Here, we report the development of a novel poly(ß-amino) ester labeling system synthesized with inexpensive, common reagents, termed POLYseq, capable of efficiently delivering fluorescent molecules or sample-distinguishing DNA barcodes through non-covalent binding enabling rapid creation of custom sample pools. Chemical formulation was found to determine cellular labeling propensity. Live image-based tracking of fluorescent conjugated POLYseq vectors demonstrated lysosomal compartmentalization. Barcode labeling was uniformly detected across 90% of cells by single-cell RNA sequencing, allowing for the successful identification of human and mouse cultured cell lines from a single pool. These findings highlight the multifunctional applications of POLYseq in live cell imaging and NGS in a scalable and cost-effective manner.

Penetrating penile injury due to the splintering of the floorboards in the gymnasium.

INTRODUCTION: We present the case of a patient with penetrating penile injury caused by splintering floorboards in a gymnasium. CASE PRESENTATION: A 24-year-old man was brought to the emergency department of our hospital because of an unintentional penetrating penile injury sustained while playing volleyball at a gymnasium. He dove into the wooden floor to fly-receive the ball. When sliding with his abdomen on the floor, a wooden splinter from the floorboard stuck from the base of his penis to near the glans penis. The splinter was gently removed without bleeding under local anesthesia. CONCLUSIONS: Splintering floorboards in gymnasiums can cause serious trauma, including penile injuries. Health-care workers and users of public facilities, such as gymnasiums, should be aware of the accident risk associated with wooden floors.

A unique case of splenic tumor exhibiting a serous carcinoma phenotype.

The spleen has no epithelial element; thus, primary carcinoma of the spleen is quite rare. We present the case of a patient with serous carcinoma of the spleen. A 76-year-old woman with no significant medical history presented with a huge lesion in the spleen. Except this lesion, clinical examination, including imaging examination, revealed no remarkable findings. She underwent excision of the spleen for treatment and diagnosis. Postoperative pathological examination revealed neoplastic cells with pleomorphic and hyperchromatic nuclei, prominent nucleoli, and frequent mitotic activity. The neoplastic cells exhibited a papillary pattern with psammoma bodies. Immunohistochemistry showed positivity for cytokeratin 7, PAX-8, WT-1, p16, p53, and Ber-EP4 and negativity for cytokeratin 20, thyroid transcription factor-1, carcinoembryonic antigen, CD10, estrogen receptor, calretinin, D2-40, intelectin-1, and sialylated HEG1. We inferred that this tumor was a primary splenic serous carcinoma. Serous tubal intraepithelial carcinoma is the plausible origin of most pelvic serous carcinomas. However, the origin of serous carcinoma of the spleen remains unknown. We speculated that endosalpingiosis might be the origin of the tumor.