Design, Synthesis, and Characterization of Novel Styryl Dyes as Fluorescent Probes for Tau Aggregate Detection in Vitro and in Cells.

A series of novel styryl dye derivatives incorporating indolium and quinolinium core structures were successfully synthesized to explore their interacting and binding capabilities with tau aggregates in vitro and in cells. The synthesized dyes exhibited enhanced fluorescence emission in viscous environments due to the rotatable bond confinement in the core structure. Dye 4, containing a quinolinium moeity and featuring two cationic sites, demonstrated a 28-fold increase in fluorescence emission upon binding to tau aggregates. This dye could also stain tau aggregates in living cells, confirmed by cell imaging using confocal fluorescence microscopy. A molecular docking study was conducted to provide additional visualization and support for binding interactions. This work offers novel and non-cytotoxic fluorescent probes with desirable photophysical properties, which could potentially be used for studying tau aggregates in living cells, prompting further development of new fluorescent probes for early Alzheimer's disease detection.

Label-free tumor cells classification using deep learning and high-content imaging.

Many studies have shown that cellular morphology can be used to distinguish spiked-in tumor cells in blood sample background. However, most validation experiments included only homogeneous cell lines and inadequately captured the broad morphological heterogeneity of cancer cells. Furthermore, normal, non-blood cells could be erroneously classified as cancer because their morphology differ from blood cells. Here, we constructed a dataset of microscopic images of organoid-derived cancer and normal cell with diverse morphology and developed a proof-of-concept deep learning model that can distinguish cancer cells from normal cells within an unlabeled microscopy image. In total, more than 75,000 organoid-drived cells from 3 cholangiocarcinoma patients were collected. The model achieved an area under the receiver operating characteristics curve (AUROC) of 0.78 and can generalize to cell images from an unseen patient. These resources serve as a foundation for an automated, robust platform for circulating tumor cell detection.

A germline STAT6 gain-of-function variant is associated with early-onset allergies.

BACKGROUND: The signal transducer and activator of transcription 6 (STAT6) signaling pathway plays a central role in allergic inflammation. To date, however, there have been no descriptions of STAT6 gain-of-function variants leading to allergies in humans. OBJECTIVE: We report a STAT6 gain-of-function variant associated with early-onset multiorgan allergies in a family with 3 affected members. METHODS: Exome sequencing and immunophenotyping of T-helper cell subsets were conducted. The function of the STAT6 protein was analyzed by Western blot, immunofluorescence, electrophoretic mobility shift assays, and luciferase assays. Gastric organoids obtained from the index patient were used to study downstream effector cytokines. RESULTS: We identified a heterozygous missense variant (c.1129G>A;p.Glu377Lys) in the DNA binding domain of STAT6 that was de novo in the index patient's father and was inherited by 2 of his 3 children. Severe atopic dermatitis and food allergy were key presentations. Clinical heterogeneity was observed among the affected individuals. Higher levels of peripheral blood TH2 lymphocytes were detected. The mutant STAT6 displayed a strong preference for nuclear localization, increased DNA binding affinity, and spontaneous transcriptional activity. Moreover, gastric organoids showed constitutive activation of STAT6 downstream signaling molecules. CONCLUSIONS: A germline STAT6 gain-of-function variant results in spontaneous activation of the STAT6 signaling pathway and is associated with an early-onset and severe allergic phenotype in humans. These observations enhance our knowledge of the molecular mechanisms underlying allergic diseases and will potentially contribute to novel therapeutic interventions.

Circulating tumor cells as a prognostic biomarker in patients with hepatocellular carcinoma.

Circulating tumor cells (CTCs) have been shown as a surrogate for cancer progression and prognostication. We aimed to determine an association between CTCs and survival of hepatocellular carcinoma (HCC) patients. Peripheral blood was obtained from 73 HCC patients to enumerate for epithelial CTCs/8 mL blood. CTCs were detected by immunoaffinity-based method using epithelial cell adhesion molecule (EpCAM) and mucin1 (MUC1). The CTCs detection rates of BCLC stages A, B, and C patients were 65.4% (17/26), 77.3% (17/22), and 96% (24/25), respectively, p = 0.018. Patients with CTCs < 5 cells/8 mL had significantly longer survival than those with CTCs ≥ 5 cells/8 mL (>36 vs. 4.6 months, p < 0.001). In multivariate analysis, CTP B, BCLC B, BCLC C, AFP ≥ 400 ng/mL, and CTC ≥ 5 cells/8 mL were independently associated with survival, with adjusted HRs (95%CI) of 4.1 (2.0-8.4), 3.5 (1.1-11.4), 4.7 (1.4-15.4), 2.4 (1.1-5.0), and 2.6 (1.2-8.4); p < 0.001, 0.036, 0.011, 0.025 and 0.012, respectively. The combination of CTCs ≥ 5 cells/8 mL and AFP ≥ 400 ng/mL provided additively increased HR to 5.3 (2.5-11.1), compared to HRs of 4.0 (2.0-8.0) and 3.5 (1.8-6.7) for CTCs ≥ 5 cells/8 mL and AFP ≥ 400 ng/mL, p < 0.001, respectively. The larger number of peripheral CTCs is correlated with higher tumor aggressive features and poorer survival of HCC patients. CTCs can potentially become novel prognostic biomarker in HCC.

Generation and characterization of HLA-universal platelets derived from induced pluripotent stem cells.

Platelet demand has increased around the world. However, the inadequacy of donors, the risk of transfusion-transmitted infections and associated reactions, and the refractory nature of platelet transfusions are among the limitations of allogeneic platelet transfusions. To alleviate these problems, we propose generating platelets in a laboratory that do not induce alloimmunity to human leukocyte antigen (HLA) class I, which is a major cause of immune reaction in platelet transfusion refractoriness. Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) of a healthy Thai woman. We then knocked out the ß2-microglobulin (ß2m) gene in the cells using paired CRISPR/Cas9 nickases and sequentially differentiated the cells into haematopoietic stem cells (HSCs), megakaryocytes (MKs) and platelets. Silencing of HLA class I expression was observed on the cell surface of ß2m-knockout iPSCs, iPSC-derived HSCs, MKs and platelets. The HLA-universal iPSC-derived platelets were shown to be activated, and they aggregated after stimulation. In addition, our in vivo platelet survival experiments demonstrated that human platelets were detectable at 2 and 24 hours after injecting the ß2m-KO MKs. In summary, we successfully generated functional iPSC-derived platelets in vitro without HLA class I expression by knocking out the ß2m gene using paired CRISPR/Cas9 nickases.

Study of Bernard-Soulier Syndrome Megakaryocytes and Platelets Using Patient-Derived Induced Pluripotent Stem Cells.

Bernard-Soulier syndrome (BSS) is a hereditary macrothrombocytopenia caused by defects in the glycoprotein (GP) Ib-IX-V complex. The mechanism of giant platelet formation remains undefined. Currently, megakaryocytes (MKs) can be generated from induced pluripotent stem cells (iPSCs) to study platelet production under pharmacological or genetic manipulations. Here, we generated iPSC lines from two BSS patients with mutations in different genes (GP1BA and GP1BB: termed BSS-A and BSS-B, respectively). The iPSC-derived MKs and platelets were examined under electron microscopy and stained by immunofluorescence to observe proplatelet formation and measure platelet diameters which were defined by circumferential tubulin. BSS-iPSCs produced abnormal proplatelets with thick shafts and tips. In addition, compared with the normal iPSCs, the diameters were larger in platelets derived from BSS-A and BSS-B with the means ± standard deviations of 4.34 ± 0.043 and 3.88 ± 0.045 µm, respectively (wild-type iPSCs 2.61 ± 0.025 µm, p < 0.001). Electron microscopy revealed giant platelets with the abnormal demarcation membrane system. Correction of BSS-A and BSS-B-iPSCs using lentiviral vectors containing respective GP1BA and GP1BB genes improved proplatelet structures and platelet ultrastructures as well as reduced platelets sizes. In conclusion, the iPSC model can be used to explore molecular mechanisms and potential therapy for BSS.

Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells.

DNA methylation of specific genome locations contributes to the distinct functions of multicellular organisms. DNA methylation can be governed by RNA-dependent DNA methylation (RdDM). RdDM is carried out by endogenous small-RNA-guided epigenomic editing complexes that add a methyl group to a precise DNA location. In plants, the Argonaute 4 (AGO4) protein is one of the main catalytic components involved in RdDM. Although small interfering RNA or short hairpin RNA has been shown to be able to guide DNA methylation in human cells, AGO protein-regulated RdDM in humans has not yet been evaluated. This study aimed to identify a key regulatory AGO protein involved in human RdDM by bioinformatics and to explore its function in RdDM by a combination of AGO4 knockdown, Alu small interfering RNA transfection, AGO4-expressing plasmid transfection, chromatin immunoprecipitation, cell-penetrating peptide-tagged AGO4 combined Alu single-guide RNA transfection, and methylation analyses. We found that first, human AGO4 showed stronger genome-wide association with DNA methylation than AGO1-AGO3. Second, endogenous AGO4 depletion demethylated DNA of known AGO4 bound loci. Finally, exogenous AGO4 de novo methylated the bound DNA sequences. Therefore, we discovered that AGO4 plays a role in human RdDM.

Rabbit induced pluripotent stem cells retain capability of in vitro cardiac differentiation.

Stem cells are promising cell source for treatment of multiple diseases as well as myocardial infarction. Rabbit model has essentially used for cardiovascular diseases and regeneration but information on establishment of induced pluripotent stem cells (iPSCs) and differentiation potential is fairly limited. In addition, there is no report of cardiac differentiation from iPSCs in the rabbit model. In this study, we generated rabbit iPSCs by reprogramming rabbit fibroblasts using the 4 transcription factors (OCT3/4, SOX2, KLF4, and c-Myc). Three iPSC lines were established. The iPSCs from all cell lines expressed genes (OCT3/4, SOX2, KLF4 and NANOG) and proteins (alkaline phosphatase, OCT-3/4 and SSEA-4) essentially described for pluripotency (in vivo and in vitro differentiation). Furthermore, they also had ability to form embryoid body (EB) resulting in three-germ layer differentiation. However, ability of particular cell lines and cell numbers at seeding markedly influenced on EB formation and also their diameters. The cell density at 20,000 cells per EB was selected for cardiac differentiation. After plating, the EBs attached and cardiac-like beating areas were seen as soon as 11 days of culture. The differentiated cells expressed cardiac progenitor marker FLK1 (51 ± 1.48%) on day 5 and cardiac troponin-T protein (10.29 ± 1.37%) on day 14. Other cardiac marker genes (cardiac ryanodine receptors (RYR2), α-actinin and PECAM1) were also expressed. This study concluded that rabbit iPSCs remained their in vitro pluripotency with capability of differentiation into mature-phenotype cardiomyocytes. However, the efficiency of cardiac differentiation is still restricted.

Human Umbilical Cord Blood-Derived Serum for Culturing the Supportive Feeder Cells of Human Pluripotent Stem Cell Lines.

Although human pluripotent stem cells (hPSCs) can proliferate robustly on the feeder-free culture system, genetic instability of hPSCs has been reported in such environment. Alternatively, feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS) prior to coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. Recently, human cord blood-derived serum (hUCS) showed a positive effect on culture of mesenchymal stem cells. It is interesting to test whether hUCS can be used for culture of feeder cells of hPSCs. This study was aimed to replace FBS with hUCS for culturing the human foreskin fibroblasts (HFFs) prior to feeder cell preparation. The results showed that HFFs cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and TGFß1, which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore, the results demonstrated the benefit of hUCS for hPSCs culture system.

Wiskott-Aldrich syndrome iPS cells produce megakaryocytes with defects in cytoskeletal rearrangement and proplatelet formation.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterised by microthrombocytopenia, complex immunodeficiency, autoimmunity, and haematologic malignancies. It is caused by mutations in the gene encoding WAS protein (WASP), a regulator of actin cytoskeleton and chromatin structure in various blood cell lineages. The molecular mechanisms underlying microthrombocytopenia caused by WASP mutations remain elusive. Murine models of WASP deficiency exhibited only mild thrombocytopenia with normal-sized platelets. Here we report on the successful generation of induced pluripotent stem cell (iPSC) lines from two patients with different mutations in WASP (c.1507T>A and c.55C>T). When differentiated into early CD34+ haematopoietic and megakaryocyte progenitors, the WAS-iPSC lines were indistinguishable from the wild-type iPSCs. However, all WAS-iPSC lines exhibited defects in platelet productionin vitro. WAS-iPSCs produced platelets with more irregular shapes and smaller sizes. Immunofluorescence and electron micrograph showed defects in cytoskeletal rearrangement, F-actin distribution, and proplatelet formation. Proplatelet defects were more pronounced when using culture systems with stromal feeders comparing to feeder-free culture condition. Overexpression of WASP in the WAS-iPSCs using a lentiviral vector improved proplatelet structures and increased the platelet size. Our findings substantiate the use of iPSC technology to elucidate the disease mechanisms of WAS in thrombopoiesis.