Employment of diverse in vitro systems for analyzing multiple aspects of disease, hereditary hemorrhagic telangiectasia (HHT).

BACKGROUND: In vitro disease modeling enables translational research by providing insight into disease pathophysiology and molecular mechanisms, leading to the development of novel therapeutics. Nevertheless, in vitro systems have limitations for recapitulating the complexity of tissues, and a single model system is insufficient to gain a comprehensive understanding of a disease. RESULTS: Here we explored the potential of using several models in combination to provide mechanistic insight into hereditary hemorrhagic telangiectasia (HHT), a genetic vascular disorder. Genome editing was performed to establish hPSCs (H9) with ENG haploinsufficiency and several in vitro models were used to recapitulate the functional aspects of the cells that constitute blood vessels. In a 2D culture system, endothelial cells showed early senescence, reduced viability, and heightened susceptibility to apoptotic insults, and smooth muscle cells (SMCs) exhibited similar behavior to their wild-type counterparts. Features of HHT were evident in 3D blood-vessel organoid systems, including thickening of capillary structures, decreased interaction between ECs and surrounding SMCs, and reduced cell viability. Features of ENG haploinsufficiency were observed in arterial and venous EC subtypes, with arterial ECs showing significant impairments. Molecular biological approaches confirmed the significant downregulation of Notch signaling in HHT-ECs. CONCLUSIONS: Overall, we demonstrated refined research strategies to enhance our comprehension of HHT, providing valuable insights for pathogenic analysis and the exploration of innovative therapeutic interventions. Additionally, these results underscore the importance of employing diverse in vitro systems to assess multiple aspects of disease, which is challenging using a single in vitro system.

Ferric citrate and apo-transferrin enable erythroblast maturation with ß-globin from hemogenic endothelium.

Red blood cell (RBC) generation from human pluripotent stem cells (PSCs) offers potential for innovative cell therapy in regenerative medicine as well as developmental studies. Ex vivo erythropoiesis from PSCs is currently limited by the low efficiency of functional RBCs with ß-globin expression in culture systems. During induction of ß-globin expression, the absence of a physiological microenvironment, such as a bone marrow niche, may impair cell maturation and lineage specification. Here, we describe a simple and reproducible culture system that can be used to generate erythroblasts with ß-globin expression. We prepared a two-dimensional defined culture with ferric citrate treatment based on definitive hemogenic endothelium (HE). Floating erythroblasts derived from HE cells were primarily CD45+CD71+CD235a+ cells, and their number increased remarkably upon Fe treatment. Upon maturation, the erythroblasts cultured in the presence of ferric citrate showed high transcriptional levels of ß-globin and enrichment of genes associated with heme synthesis and cell cycle regulation, indicating functionality. The rapid maturation of these erythroblasts into RBCs was observed when injected in vivo, suggesting the development of RBCs that were ready to grow. Hence, induction of ß-globin expression may be explained by the effects of ferric citrate that promote cell maturation by binding with soluble transferrin and entering the cells.Taken together, upon treatment with Fe, erythroblasts showed advanced maturity with a high transcription of ß-globin. These findings can help devise a stable protocol for the generation of clinically applicable RBCs.

Maintenance of Hypoimmunogenic Features via Regulation of Endogenous Antigen Processing and Presentation Machinery.

Universally acceptable donor cells have been developed to address the unmet need for immunotypically matched materials for regenerative medicine. Since forced expression of hypoimmunogenic genes represses the immune response, we established universal pluripotent stem cells (PSCs) by replacing endogenous ß2-microglobulin (ß2m) with ß2m directly conjugated to human leukocyte antigen (HLA)-G, thereby simultaneously suppressing HLA-I expression and the natural killer (NK) cell-mediated immune response. These modified human PSCs retained their pluripotency and differentiation capacity; however, surface presentation of HLA-G was absent from subsequently differentiated cells, particularly cells of neural lineages, due to the downregulation of antigen processing and presentation machinery (APM) genes. Induction of APM genes by overexpression of NLR-family CARD domain-containing 5 (NLRC5) or activator subunit of nuclear factor kappa B (NF-κB) heterodimer (RelA) recovered the surface expression of HLA-G and the hypoimmunogenicity of neural cells. Our findings enhance the utility of hypoimmunogenic cells as universal donors and will contribute to the development of off-the-shelf stem-cell therapeutics.

Generation and characterization of human umbilical cord blood-derived induced pluripotent stem cells (KRIBBi005-A).

Induced pluripotent stem cells (iPSCs) hold great promise as a potential source for regenerative medicine. Umbilical cord blood (UCB) cells possess a high level of stem cells, exhibit less immune rejection, and have fewer DNA mutations and, thus, are an easily accessible and valuable cell source for iPSC generation for basic research and clinical applications. Here, we reprogrammed CD34+-UCB cells with the group O/D-negative (RhD-) blood type as a universal source for red blood cell (RBC) generation. The resulting iPSCs, with features typical of pluripotent stem cells, will further advance the development of various therapeutics, including blood transfusion.

Generation of induced pluripotent stem cell line (KRIBBi004-A) from adult bone marrow CD34+ cells from a patient carrying 46,XX,t(1;5)(p31.1;35.1) karyotype.

Induced pluripotent stem cell (iPS) technology may be advantageous for the study of genetic aberrations in terms of recapitulating the full manifestation of pathological features in vitro, identifying underlying pathways, and developing personalized therapeutics rather than procuring somatic cells from patients. Here, we derived an iPSC line from a patient with reciprocal chromosome translocation, t(1;5)(p31.1;35.1), as a novel alternative model to identify clinical phenotypes induced by genetic instability. The resulting iPSC line generated from somatic cells with an existing instability showed representative characteristics of PSCs, and might serve as an unparalleled cellular resource for the development of a custom remedy.

Human Blood Vessel Organoids Penetrate Human Cerebral Organoids and Form a Vessel-Like System.

Vascularization of tissues, organoids and organ-on-chip models has been attempted using endothelial cells. However, the cultured endothelial cells lack the capacity to interact with other somatic cell types, which is distinct from developing vascular cells in vivo. Recently, it was demonstrated that blood vessel organoids (BVOs) recreate the structure and functions of developing human blood vessels. However, the tissue-specific adaptability of BVOs had not been assessed in somatic tissues. Herein, we investigated whether BVOs infiltrate human cerebral organoids and form a blood-brain barrier. As a result, vascular cells arising from BVOs penetrated the cerebral organoids and developed a vessel-like architecture composed of CD31+ endothelial tubes coated with SMA+ or PDGFR+ mural cells. Molecular markers of the blood-brain barrier were detected in the vascularized cerebral organoids. We revealed that BVOs can form neural-specific blood-vessel networks that can be maintained for over 50 days.

Establishment of iPSC (KRIBBi001-A) from CD34+ group O D-negative bone marrow blood.

Human induced pluripotent stem cells with indefinite propagation in vitro provide a potential donor source of cells including erythroid cells for human therapy. Since group O D-negative (RhD-) blood cells are considered as universal donors for transfusion, it is compelling to derive iPSC line from group O/RhD- sample as a new cellular source to generate universal RBCs. The resulting iPSC line derived from group O/RhD- somatic source showed typical features of pluripotent stem cells and could provide an unprecedented cellular tool to develop universal therapeutics for blood transfusion.

BRM270 targets cancer stem cells and augments chemo-sensitivity in cancer.

Over the past decade, a number of studies have demonstrated the resistance of cancer cells to conventional drugs and have recognized this as a major challenge in cancer therapy. While attempting to understand the underlying mechanisms of chemoresistance, several studies have suggested that the presence of cancer stem cells (CSCs) in tumors is one of the major pathways contributing toward resistance. Chemoresistance leads to cancer treatment failure and worsens the prognosis of patients. Natural herbal compounds are gaining attention as an alternative treatment strategy for cancer. These compounds may be effective against chemoresistant cells either alone or synergistically alongside conventional drugs, sensitizing cancer cells and enhancing the therapeutic efficacy. BRM270 is a natural compound made from seven herbal plant (Saururus chinensis, Citrus unshiu Markovich, Aloe vera, Arnebia euchroma, Portulaca oleracea, Prunella vulgaris var. lilacina and Scutellaria bacicalensis) extracts used in Asian traditional medicine and has the potential to target CSCs. Several studies have demonstrated the positive effects of BRM270 against chemoresistant cancer and its synergy alongside existing cancer drugs, including paclitaxel and gefitinib. These effects have been observed against various cancer types, including resistant non-small cell lung cancer (NSCLC), glioblastoma, multi-drug resistant osteosarcoma, cervical cancer, pancreatic cancer and hepatocarcinoma. The present review discusses the effects of BRM270 treatment against CSC-associated chemoresistance in common types of cancer.

Wogonin Influences Osteosarcoma Stem Cell Stemness Through ROS-dependent Signaling.

Backgorund/Aim: Wogonin, a flavonoid-like compound extracted from the root of Scutellaria baicalensis Georgi, has been shown to have anticancer effects against cancer cells. Osteosarcoma is the most malignant type of bone cancer and can appear in any bone, with a high propensity for relapse and metastasis. The present study aimed to assess the anticancer effects of wogonin on osteosarcoma stem cells. MATERIALS AND METHODS: The cytotoxic effects of wogonin on CD133+ Cal72 osteosarcoma stem cells were assessed through in vitro experiments by MTT assay, transwell assay, sphere-formation assay, flow cytometry, immunocytochemistry and western blotting. RESULTS: Wogonin suppressed stem cell characteristics and the expression of stem cell-related genes by regulating reactive oxygen species (ROS) levels and ROS-related signaling of CD133+ Cal72 cells, effects which were reversed by ROS scavenger N-acetylcysteine. CONCLUSION: Wogonin may be a promising candidate for successful clinical management of osteosarcoma by regulating ROS-related mechanisms and stem cell-related genes.

BRM270 Inhibits the Proliferation of CD44 Positive Pancreatic Ductal Adenocarcinoma Cells via Downregulation of Sonic Hedgehog Signaling.

Pancreatic cancer has a poor survival rate as compared to other types of cancer. Surface marker CD44 plays important role in epithelial-mesenchymal transition and cancer stem cell phenotype. Therefore, targeting CD44 positive pancreatic cancer cells might enhance therapies effectiveness. Our previous studies indicated the antitumorigenesis effect of BRM270 in osteosarcoma, lung cancer, and glioblastoma; however there is no evidence on BRM270 impacts on pancreatic cancer growth. In this study, we investigated the effect of BRM270 on the isolated CD44 positive pancreatic ductal adenocarcinoma cells (CD44+ PDAC). Results showed that CD44 positive cells undergo apoptosis induced by BRM270. Moreover, BRM270 also inhibits stemness and metastasis traits in CD44+ PDAC via Sonic hedgehog signaling pathway and SALL4 expression. In vivo study indicated that tumor growth derived from CD44+ PDAC was suppressed as daily uptake by BRM270 5 mg/kg. These data suggest the alternative approach in antipancreatic tumorigenesis via herbal plants extract and selectively targeting CD44+ PDAC cells in tumor.

Synthesis and evaluation of the antiproliferative efficacy of BRM270 phytocomposite nanoparticles against human hepatoma cancer cell lines.

BRM270 is the most leading phytochemical extract that possesses potent anticancer properties. A major challenge associated with this drug is its low bioavailability and thus requires high dosages for cancer treatment. Here, we report the novel nano-synthesis of phyto-composite, BRM270 for the first time by mechanical milling method with specific modifications for enhanced cytotoxicity against HepG2 human hepatoma cancer cells. Unlike free BRM270 and other phytomedicines, BRM270 nanoparticles (BRM270 NPs) are well-dispersed and small sized (23 to 70 nm) which is believed to greatly enhanced cellular uptake. Furthermore, the acidic tumor microenvironment attracts BRM270 NPs enhancing targeted therapy while leaving normal cells less affected. The comparative cytotoxicity analysis using MTT assay among the three treatment groups, such as free BRM270, BRM270 NPs, and doxorubicin demonstrated that BRM270 NPs induced greater cytotoxicity against HepG2 cells with an effective drug concentration of 12 µg/ml. From FACS analysis, we observed an apoptotic cell death of 44.4% at BRM270 NPs treated cells while only 12.5% found in the free BRM270 treated cells. Further, the comparative relative expression profiling of the candidate genes were showed significant (p < 0.05) down-regulation of IL6, BCL2, p53, and MMP9 in the BRM270 NPs treated cells, compared to the free BRM270 and doxorubicin. Indeed, the genes, CASPASE 9 and BAX have shown significant (p < 0.05) upregulation in cells treated with BRM270 NPs as compared to counter treatment groups. The investigation of the signal pathways and protein-protein network associations were also carried out to elucidate the functional insights underlying anti-cancer potential of BRM270 NPs in HepG2 cells. Taken together, our findings demonstrated that these uniquely engineered BRM270 NPs effectively enter into the cancer cells due to its acidic microenvironment thereby inducing apoptosis and regulate the cell-proliferation in-vitro at extremely low dosages.

Specific Chicken Egg Yolk Antibody Improves the Protective Response against Gallibacterium anatis Infection.

Gallibacterium anatis is a pathogen associated with peritonitis and salpingitis in chickens and other avian species. Novel safety prevention strategies are urgently needed because of widespread multidrug resistance and antigenic diversity. The objective of this study was to produce a specific chicken egg yolk antibody and evaluate its protective response against a G. anatis infection model in 4-week-old chicks. Enzyme-linked immunosorbent assays showed that hens immunized with the recombinant N terminus of Gallibacterium toxin A (GtxA-N) had significantly increased antibody titers against GtxA-N in serum and egg yolk IgY. Western blotting showed that IgY antibody had specificity against GtxA-N in the egg yolks of immunized hens. The growth of G. anatis in brain heart infusion (BHI) broth and agar was significantly inhibited by the GtxA-N-specific IgY antibody. The protective effects of the specific IgY antibody were evaluated in G. anatis-infected chicks after intramuscular injection (10 mg/ml). The anti-GtxA-N antibody titers in the sera of G. anatis-challenged chicks following an injection of specific IgY antibody were significantly higher than those of the control and the nonspecific IgY groups, but lower lesion scores for the peritoneum, liver, and duodenum were found after specific IgY antibody treatment. The results from this study suggest that the GtxA-N-specific IgY antibody could potentially improve the protective response against G. anatis infection in chicks.

MicroRNA-128 suppresses paclitaxel-resistant lung cancer by inhibiting MUC1-C and BMI-1 in cancer stem cells.

The existence of cancer stem cells (CSCs) is the main reason for failure of cancer treatment caused by drug resistance. Therefore, eradicating cancers by targeting CSCs remains a significant challenge. In the present study, because of the important role of BMI-1 proto-oncogene, polycomb ring finger (BMI-1) and C-terminal Mucin1 (MUC1-C) in tumor growth and maintenance of CSCs, we aimed to confirm that microRNA miR-128, as an inhibitor of BMI-1 and MUC1-C, could effectively suppress paclitaxel (PTX)-resistant lung cancer stem cells. We showed that CSCs have significantly higher expression levels of BMI-1, MUC1-C, stemness proteins, signaling factors, and higher malignancy compared with normal tumor cells. After transfection with miR-128, the BMI-1 and MUC1-C levels in CSCs were suppressed. When miR-128 was stably expressed in PTX-resistant lung cancer stem cells, the cells showed decreased proliferation, metastasis, self-renewal, migration, invasive ability, clonogenicity, and tumorigenicity in vitro and in vivo and increased apoptosis compared with miR-NC (negative control) CSCs. Furthermore, miR-128 effectively decreased the levels of ß-catenin and intracellular signaling pathway-related factors in CSCs. MiR-128 also decreased the luciferase activity of MUC1 reporter constructs and reduced the levels of transmembrane MUC1-C and BMI-1. These results suggested miR-128 as an attractive therapeutic strategy for PTX-resistant lung cancer via inhibition of BMI-1 and MUC1-C.