Therapeutic efficacy of thrombin-preconditioned mesenchymal stromal cell-derived extracellular vesicles on Escherichia coli-induced acute lung injury in mice.

BACKGROUND: Acute lung injury (ALI) following pneumonia involves uncontrolled inflammation and tissue injury, leading to high mortality. We previously confirmed the significantly increased cargo content and extracellular vesicle (EV) production in thrombin-preconditioned human mesenchymal stromal cells (thMSCs) compared to those in naïve and other preconditioning methods. This study aimed to investigate the therapeutic efficacy of EVs derived from thMSCs in protecting against inflammation and tissue injury in an Escherichia coli (E. coli)-induced ALI mouse model. METHODS: In vitro, RAW 264.7 cells were stimulated with 0.1 µg/mL liposaccharides (LPS) for 1 h, then were treated with either PBS (LPS Ctrl) or 5 × 107 particles of thMSC-EVs (LPS + thMSC-EVs) for 24 h. Cells and media were harvested for flow cytometry and ELISA. In vivo, ICR mice were anesthetized, intubated, administered 2 × 107 CFU/100 µl of E. coli. 50 min after, mice were then either administered 50 µL saline (ECS) or 1 × 109 particles/50 µL of thMSC-EVs (EME). Three days later, the therapeutic efficacy of thMSC-EVs was assessed using extracted lung tissue, bronchoalveolar lavage fluid (BALF), and in vivo computed tomography scans. One-way analysis of variance with post-hoc TUKEY test was used to compare the experimental groups statistically. RESULTS: In vitro, IL-1ß, CCL-2, and MMP-9 levels were significantly lower in the LPS + thMSC-EVs group than in the LPS Ctrl group. The percentages of M1 macrophages in the normal control, LPS Ctrl, and LPS + thMSC-EV groups were 12.5, 98.4, and 65.9%, respectively. In vivo, the EME group exhibited significantly lower histological scores for alveolar congestion, hemorrhage, wall thickening, and leukocyte infiltration than the ECS group. The wet-dry ratio for the lungs was significantly lower in the EME group than in the ECS group. The BALF levels of CCL2, TNF-a, and IL-6 were significantly lower in the EME group than in the ECS group. In vivo CT analysis revealed a significantly lower percentage of damaged lungs in the EME group than in the ECS group. CONCLUSION: Intratracheal thMSC-EVs administration significantly reduced E. coli-induced inflammation and lung tissue damage. Overall, these results suggest therapeutically enhanced thMSC-EVs as a novel promising therapeutic option for ARDS/ALI.

A Brief Review of Anatomy Education in Korea, Encompassing Its Past, Present, and Future Direction.

Anatomy is a foundational subject in medicine and serves as its language. Hippocrates highlighted its importance, while Herophilus pioneered human dissection, earning him the title of the founder of anatomy. Vesalius later established modern anatomy, which has since evolved historically. In Korea, formal anatomy education for medical training began with the introduction of Western medicine during the late Joseon Dynasty. Before and after the Japanese occupation, anatomy education was conducted in the German style, and after liberation, it was maintained and developed by a small number of domestic anatomists. Medicine in Korea has grown alongside the country's rapid economic and social development. Today, 40 medical colleges produce world-class doctors to provide the best medical care service in the country. However, the societal demand for more doctors is growing in order to proactively address to challenges such as public healthcare issues, essential healthcare provision, regional medical service disparities, and an aging population. This study examines the history, current state, and challenges of anatomy education in Korea, emphasizing the availability of medical educators, support staff, and cadavers for gross anatomy instruction. While variations exist between Seoul and provincial medical colleges, each manages to deliver adequate education under challenging conditions. However, the rapid increase in medical student enrollment threatens to strain existing anatomy education resources, potentially compromising educational quality. To address these concerns, we propose strategies for training qualified gross anatomy educators, ensuring a sustainable cadaver supply, and enhancing infrastructure.

Simplified in vitro 3D co-culture-based blood-brain barrier model using transwell.

The blood-brain barrier (BBB) is a major hurdle for treatment of brain diseases. To overcome this, precise and reproducible BBB model is one of the key factors for successful evaluation of BBB-penetrating efficacy of developmental drugs. Thus, in vitro BBB model recapitulating the physiological structure of the BBB is a valuable tool for drug discovery and development for brain diseases. Here, we develop a simplified 3D co-culture-based BBB model using immortalized human brain endothelial cells and immortalized human astrocytes mixed with Matrigel allowing model preparation within 30 min. We directly compare our 3D BBB model to a 2D BBB model comprised solely of immortalized brain endothelial cells, to demonstrate that our 3D BBB model blocks penetration of Dextran molecules with various molecular weights, remain durable and impermeable even in a BBB-degrading condition, and rapidly form tight junctions while the 2D BBB model do not. In conclusion, this establishes our simplified 3D BBB model as a valuable tool for high throughput screening of drug candidates for brain diseases.

Functional validation of the simplified in vitro 3D Co-culture based BBB model.

Various methods of generating 2D and 3D in vitro blood-brain barrier (BBB) models have previously been published with the objective of developing therapeutics for brain diseases. In general, published methods including our published method demonstrate that in vivo-like semi-permeable barrier can be generated. To further verify that an in vitro BBB model closely represents BBB, functional validation is required. Here, we functionally validate our in vitro 3D BBB model using rituximab as a representative therapeutic antibody and previously published anti-TfR (transferrin receptor) antibodies as representative BBB-penetrating antibodies. We demonstrate that our BBB model can efficiently block rituximab while allowing receptor-mediated transcytosis (RMT) of anti-TfR antibodies. In addition, we showed that RMT efficacy of anti-TfR antibodies with different binding affinity can be displayed using our BBB model. In conclusion, this demonstrates that our BBB model functionally mimics the BBB as well as having BBB-like physical properties, further establishing our BBB model as a screening tool for discovery and development of therapeutics for brain diseases.

Significant Therapeutic Effects of Adult Human Neural Stem Cells for Spinal Cord Injury Are Mediated by Monocyte Chemoattractant Protein-1 (MCP-1).

The limited capability of regeneration in the human central nervous system leads to severe and permanent disabilities following spinal cord injury (SCI) while patients suffer from no viable treatment option. Adult human neural stem cells (ahNSCs) are unique cells derived from the adult human brain, which have the essential characteristics of NSCs. The objective of this study was to characterize the therapeutic effects of ahNSCs isolated from the temporal lobes of focal cortical dysplasia type IIIa for SCI and to elucidate their treatment mechanisms. Results showed that the recovery of motor functions was significantly improved in groups transplanted with ahNSCs, where, in damaged regions of spinal cords, the numbers of both spread and regenerated nerve fibers were observed to be higher than the vehicle group. In addition, the distance between neuronal nuclei in damaged spinal cord tissue was significantly closer in treatment groups than the vehicle group. Based on an immunohistochemistry analysis, those neuroprotective effects of ahNSCs in SCI were found to be mediated by inhibiting apoptosis of spinal cord neurons. Moreover, the analysis of the conditioned medium (CM) of ahNSCs revealed that such neuroprotective effects were mediated by paracrine effects with various types of cytokines released from ahNSCs, where monocyte chemoattractant protein-1 (MCP-1, also known as CCL2) was identified as a key paracrine mediator. These results of ahNSCs could be utilized further in the preclinical and clinical development of effective and safe cell therapeutics for SCI, with no available therapeutic options at present.

Thrombin Preconditioning Improves the Therapeutic Efficacy of Mesenchymal Stem Cells in Severe Intraventricular Hemorrhage Induced Neonatal Rats.

Severe intraventricular hemorrhage (IVH) remains a major cause of high mortality and morbidity in extremely preterm infants. Mesenchymal stem cell (MSC) transplantation is a possible therapeutic option, and development of therapeutics with enhanced efficacy is necessary. This study investigated whether thrombin preconditioning improves the therapeutic efficacy of human Wharton's jelly-derived MSC transplantation for severe neonatal IVH, using a rat model. Severe neonatal IVH was induced by injecting 150 µL blood into each lateral ventricle on postnatal day (P) 4 in Sprague-Dawley rats. After 2 days (P6), naïve MSCs or thrombin-preconditioned MSCs (1 × 105/10 µL) were transplanted intraventricularly. After behavioral tests, brain tissues and cerebrospinal fluid of P35 rats were obtained for histological and biochemical analyses, respectively. Thrombin-preconditioned MSC transplantation significantly reduced IVH-induced ventricular dilatation on in vivo magnetic resonance imaging, which was coincident with attenuations of reactive gliosis, cell death, and the number of activated microglia and levels of inflammatory cytokines after IVH induction, compared to naïve MSC transplantation. In the behavioral tests, the sensorimotor and memory functions significantly improved after transplantation of thrombin-preconditioned MSCs, compared to naïve MSCs. Overall, thrombin preconditioning significantly improves the therapeutic potential and more effectively attenuates brain injury, including progressive ventricular dilatation, gliosis, cell death, inflammation, and neurobehavioral functional impairment, in newborn rats with induced severe IVH than does naïve MSC transplantation.

Optimal Preclinical Conditions for Using Adult Human Multipotent Neural Cells in the Treatment of Spinal Cord Injury.

Stem cell-based therapeutics are amongst the most promising next-generation therapeutic approaches for the treatment of spinal cord injury (SCI), as they may promote the repair or regeneration of damaged spinal cord tissues. However, preclinical optimization should be performed before clinical application to guarantee safety and therapeutic effect. Here, we investigated the optimal injection route and dose for adult human multipotent neural cells (ahMNCs) from patients with hemorrhagic stroke using an SCI animal model. ahMNCs demonstrate several characteristics associated with neural stem cells (NSCs), including the expression of NSC-specific markers, self-renewal, and multi neural cell lineage differentiation potential. When ahMNCs were transplanted into the lateral ventricle of the SCI animal model, they specifically migrated within 24 h of injection to the damaged spinal cord, where they survived for at least 5 weeks after injection. Although ahMNC transplantation promoted significant locomotor recovery, the injection dose was shown to influence treatment outcomes, with a 1 × 106 (medium) dose of ahMNCs producing significantly better functional recovery than a 3 × 105 (low) dose. There was no significant gain in effect with the 3 × 106 ahMNCs dose. Histological analysis suggested that ahMNCs exert their effects by modulating glial scar formation, neuroprotection, and/or angiogenesis. These data indicate that ahMNCs from patients with hemorrhagic stroke could be used to develop stem cell therapies for SCI and that the indirect injection route could be clinically relevant. Moreover, the optimal transplantation dose of ahMNCs defined in this preclinical study might be helpful in calculating its optimal injection dose for patients with SCI in the future.

Elucidation of Novel Therapeutic Targets for Acute Myeloid Leukemias with RUNX1-RUNX1T1 Fusion.

The RUNX1-RUNX1T1 fusion is a frequent chromosomal alteration in acute myeloid leukemias (AMLs). Although RUNX1-RUNX1T1 fusion protein has pivotal roles in the development of AMLs with the fusion, RUNX1-RUNX1T1, fusion protein is difficult to target, as it lacks kinase activities. Here, we used bioinformatic tools to elucidate targetable signaling pathways in AMLs with RUNX1-RUNX1T1 fusion. After analysis of 93 AML cases from The Cancer Genome Atlas (TCGA) database, we found expression of 293 genes that correlated to the expression of the RUNX1-RUNX1T1 fusion gene. Based on these 293 genes, the cyclooxygenase (COX), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR) pathways were predicted to be specifically activated in AMLs with RUNX1-RUNX1T1 fusion. Moreover, the in vitro proliferation of AML cells with RUNX1-RUNX1T1 fusion decreased significantly more than that of AML cells without the fusion, when the pathways were inhibited pharmacologically. The results indicate that novel targetable signaling pathways could be identified by the analysis of the gene expression features of AMLs with non-targetable genetic alterations. The elucidation of specific molecular targets for AMLs that have a specific genetic alteration would promote personalized treatment of AMLs and improve clinical outcomes.

Optimized Clump Culture Methods for Adult Human Multipotent Neural Cells.

Adult human multipotent neural cell (ahMNC) is a candidate for regeneration therapy for neurodegenerative diseases. Here, we developed a primary clump culture method for ahMNCs to increase the efficiency of isolation and in vitro expansion. The same amount of human temporal lobe (1 g) was partially digested and then filtered through strainers with various pore sizes, resulting in four types of clumps: Clump I > 100 µm, 70 µm < Clump II < 100 µm, 40 µm < Clump III < 70 µm, and Clump IV < 40 µm. At 3 and 6 days after culture, Clump II showed significantly higher number of colonies than the other Clumps. Moreover, ahMNCs derived from Clump II (ahMNCs-Clump II) showed stable proliferation, and shortened the time to first passage from 19 to 15 days, and the time to 1 × 108 cells from 42 to 34 days compared with the previous single-cell method. ahMNCs-Clump II had neural differentiation and pro-angiogenic potentials, which are the characteristics of ahMNCs. In conclusion, the novel clump culture method for ahMNCs has significantly higher efficiency than previous techniques. Considering the small amount of available human brain tissue, the clump culture method would promote further clinical applications of ahMNCs.

Tpl2 induces castration resistant prostate cancer progression and metastasis.

Progression to metastatic castration resistant prostate cancer (CRPC) is the major lethal pathway of prostate cancer (PC). Herein, we demonstrated that tumor progression locus 2 (Tpl2) kinase is the fundamental molecule provoking progression and metastasis of CRPC. Tpl2 upregulates CXCR4 and focal adhesion kinase (FAK) to activate CXCL12/CXCR4 and FAK/Akt signalling pathway. Consequently, epithelial-mesenchymal transition (EMT) and stemness of androgen depletion independent (ADI) PC cells are induced, which is dependent on the kinase activity of Tpl2. In vitro, proliferation, clonogenicity, migration, invasion and chemoresistance of ADI PC cells were enhanced by Tpl2. In vivo, Tpl2 overexpression and downregulation showed significant stimulatory and inhibitory effects on tumorigenic and metastatic potential of ADI PC cells, respectively. Moreover, the prognostic effects of Tpl2 and expressional correlation between Tpl2 and EMT-related molecules/CXCR4 were validated in clinical PC databases. Since Tpl2 exerts metastatic progression promoting activities in CRPC, Tpl2 could serve as a novel therapeutic target for metastatic CRPC.

Natural killer (NK) cells inhibit systemic metastasis of glioblastoma cells and have therapeutic effects against glioblastomas in the brain.

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by extensive local invasion, which is in contrast with extremely rare systemic metastasis of GBM. Molecular mechanisms inhibiting systemic metastasis of GBM would be a novel therapeutic candidate for GBM in the brain. METHODS: Patient-derived GBM cells were primarily cultured from surgical samples of GBM patients and were inoculated into the brains of immune deficient BALB/c-nude or NOD-SCID IL2Rgamma(null) (NSG) mice. Human NK cells were isolated from peripheral blood mononucleated cells and expanded in vitro. RESULTS: Patient-derived GBM cells in the brains of NSG mice unexpectedly induced spontaneous lung metastasis although no metastasis was detected in BALB/c-nude mice. Based on the difference of the innate immunity between two mouse strains, NK cell activities of orthotopic GBM xenograft models based on BALB/c-nude mice were inhibited. NK cell inactivation induced spontaneous lung metastasis of GBM cells, which indicated that NK cells inhibit the systemic metastasis. In vitro cytotoxic activities of human NK cells against GBM cells indicated that cytotoxic activity of NK cells against GBM cells prevents systemic metastasis of GBM and that NK cells could be effective cell therapeutics against GBM. Accordingly, NK cells transplanted into orthotopic GBM xenograft models intravenously or intratumorally induced apoptosis of GBM cells in the brain and showed significant therapeutic effects. CONCLUSIONS: Our results suggest that innate NK immunity is responsible for rare systemic metastasis of GBM and that sufficient supplementation of NK cells could be a promising immunotherapeutic strategy for GBM in the brain.

Tumor progression locus 2 (Tpl2) kinase as a novel therapeutic target for cancer: double-sided effects of Tpl2 on cancer.

Tumor progression locus 2 (Tpl2) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that conveys various intra- and extra-cellular stimuli to effector proteins of cells provoking adequate adoptive responses. Recent studies have elucidated that Tpl2 is an indispensable signal transducer as an MAP3K family member in diverse signaling pathways that regulate cell proliferation, survival, and death. Since tumorigenesis results from dysregulation of cellular proliferation, differentiation, and apoptosis, Tpl2 participates in many decisive molecular processes of tumor development and progression. Moreover, Tpl2 is closely associated with cytokine release of inflammatory cells, which has crucial effects on not only tumor cells but also tumor microenvironments. These critical roles of Tpl2 in human cancers make it an attractive anti-cancer therapeutic target. However, Tpl2 contradictorily works as a tumor suppressor in some cancers. The double-sided effects of Tpl2 originate from the specific upstream and downstream signaling environment of each tumor, since Tpl2 interacts with various signaling components. This review summarizes recent studies concerning the possible roles of Tpl2 in human cancers and considers its possibility as a therapeutic target, against which novel anti-cancer agents could be developed.

Colorectal cancer patient-derived xenografted tumors maintain characteristic features of the original tumors.

BACKGROUND: Despite significant improvements in colon cancer outcomes over the past few decades, preclinical development of more effective therapeutic strategies is still limited by the availability of clinically relevant animal models. To meet those clinical unmet needs, we generated a well-characterized in vivo preclinical platform for colorectal cancer using fresh surgical samples. METHODS: Primary and metastatic colorectal tumor tissues (1-2 mm(3)) that originate from surgery were implanted into the subcutaneous space of nude mice and serially passaged in vivo. Mutation status, hematoxylin and eosin staining, short tandem repeat profiling, and array comparative genomic hybridization were used to validate the similarity of molecular characteristics between the patient tumors and tumors obtained from xenografts. RESULTS: From surgical specimens of 143 patients, 97 xenograft models were obtained in immunodeficient mice (establish rate = 67%). Thirty-nine xenograft models were serially expanded further in mice with a mean time to reach a size of 1000-1500 mm(3) of 90 ± 20 d. Histologic and immunohistochemical analyses revealed a high degree of pathologic similarity including histologic architecture and expression of CEA, CK7, and CD20 between the patient and xenograft tumors. Molecular analysis showed that genetic mutations, genomic alterations, and gene expression patterns of each patient tumor were also well conserved in the corresponding xenograft tumor. CONCLUSIONS: Xenograft animal models derived from fresh surgical sample maintained the key characteristic features of the original tumors, suggesting that this in vivo platform can be useful for preclinical development of novel therapeutic approaches to colorectal cancers.

Drosophila HCN mediates gustatory homeostasis by preserving sensillar transepithelial potential in sweet environments.

Establishing transepithelial ion disparities is crucial for sensory functions in animals. In insect sensory organs called sensilla, a transepithelial potential, known as the sensillum potential (SP), arises through active ion transport across accessory cells, sensitizing receptor neurons such as mechanoreceptors and chemoreceptors. Because multiple receptor neurons are often co-housed in a sensillum and share SP, niche-prevalent overstimulation of single sensory neurons can compromise neighboring receptors by depleting SP. However, how such potential depletion is prevented to maintain sensory homeostasis remains unknown. Here, we find that the Ih-encoded hyperpolarization-activated cyclic nucleotide-gated (HCN) channel bolsters the activity of bitter-sensing gustatory receptor neurons (bGRNs), albeit acting in sweet-sensing GRNs (sGRNs). For this task, HCN maintains SP despite prolonged sGRN stimulation induced by the diet mimicking their sweet feeding niche, such as overripe fruit. We present evidence that Ih-dependent demarcation of sGRN excitability is implemented to throttle SP consumption, which may have facilitated adaptation to a sweetness-dominated environment. Thus, HCN expressed in sGRNs serves as a key component of a simple yet versatile peripheral coding that regulates bitterness for optimal food intake in two contrasting ways: sweet-resilient preservation of bitter aversion and the previously reported sweet-dependent suppression of bitter taste.

Thrombin Priming Promotes the Neuroprotective Effects of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Via the HGF/AKT/STAT3 Signaling Pathway.

Mesenchymal stem cells (MSCs) directly differentiate into neurons and endothelial cells after transplantation, and their secretome has considerable potential for treating brain injuries. Previous studies have suggested that the effects of MSCs priming with exposure to hypoxia, cytokines, growth factors, or chemical agents could optimize the paracrine potency and therapeutic potential of MSCs. Studies have suggested that thrombin-primed Wharton's Jelly-derived mesenchymal stem cells (Th.WJ-MSCs) significantly enhance the neuroprotective beneficial effects of naive MSCs in brain injury such as hypoxic-ischemic brain injury (HIE) and intraventricular hemorrhage (IVH). This study aimed to characterize WJ-MSCs in terms of stem cell markers, differentiation, cell proliferation, and paracrine factors by comparing naive and Th.WJ-MSCs. We demonstrated that compared with naive MSCs, Th.MSCs significantly enhanced the neuroprotective effects in vitro. Moreover, we identified differentially expressed proteins in the conditioned media of naive and Th.WJ-MSCs by liquid chromatography-tandem mass spectrometry analysis. Secretome analysis of the conditioned medium of WJ-MSCs revealed that such neuroprotective effects were mediated by paracrine effects with secretomes of Th.WJ-MSCs, and hepatocyte growth factor was identified as a key paracrine mediator. These results can be applied further in the preclinical and clinical development of effective and safe cell therapeutics for brain injuries such as HIE and IVH.

Radiosensitization of brain metastasis by targeting c-MET.

Radiotherapy is the most widely used therapeutic modality in brain metastasis; however, it only provides palliation due to inevitable tumor recurrence. Resistance of tumor cells to ionizing radiation is a major cause of treatment failure. A critical unmet need in oncology is to develop rationale driven approaches that can enhance the efficacy of radiotherapy against metastatic tumor. Utilizing in vivo orthotopic primary tumor and brain metastasis models that recapitulate clinical situation of the patients with metastatic breast cancer, we investigated a molecular mechanism through which metastatic tumor cells acquire resistance to radiation. Recent studies have demonstrated that the hepatocyte growth factor (HGF)-c-Met pathway is essential for the pathologic development and progression of many human cancers such as proliferation, invasion and resistance to anticancer therapies. In this study, c-Met signaling activity as well as total c-Met expression was significantly upregulated in both breast cancer cell lines irradiated in vitro and ex vivo radio-resistant cells derived from breast cancer brain metastatic xenografts. To interrogate the role of c-Met signaling in radioresistance of brain metastasis, we evaluated the effects on tumor cell viability, clonogenicity, sensitivity to radiation, and in vitro/in vivo tumor growth after targeting c-Met by small-hairpin RNA (shRNA) or small-molecule kinase inhibitor (PF-2341066). Although c-Met silencing or radiation alone demonstrated a modest decrease in clonogenic growth of parental breast cancers and brain metastatic derivatives, combination of two modalities showed synergistic antitumor effects resulting in significant prolongation of overall survival in tumor-bearing mice. Taken together, optimizing c-Met targeting in combination with radiation is critical to enhance the effectiveness of radiotherapy in the treatments of brain metastasis.

Effects of ethanol washing and storage duration on primary culture of stem cells from human exfoliated deciduous teeth.

Purpose: Since the oral environment harbors various microorganisms, the removal of contaminants during the primary culture process of stem cells from human exfoliated deciduous teeth (SHEDs) is very important. We investigated optimal methods for primary culture of SHEDs with minimal contamination rates. Materials and methods: Three different storage conditions for deciduous teeth were utilized:1) storing teeth in Hank's Balanced Salt Solution (HBSS) with 3% penicillin and streptomycin (P/S), 2) storing teeth in HBSS with 3% antibiotics and antimycotics (A-A), and 3) storing teeth in HBSS with A-A, and additional washing with 70% ethanol just before primary culture of dental pulp. In addition, the storage time from the extraction of teeth to the primary culture was measured. Results: The contamination rates were about 70% for HBSS with P/S, 40% for HBSS with A-A, and less than 10% for HBSS with A-A and additional washing with 70% ethanol. When the primary culture was conducted within 12 h after teeth extraction, the contamination rate was the lowest in all conditions. Furthermore, when the teeth were delivered in HBSS with A-A and an additional 70% ethanol washing was performed, the contamination rate was 0% until 48 h after teeth extraction. Ethanol washing had little effect on the cellular characteristics and stemness of SHEDs, including their morphology, growth rate, expression of surface markers, and differentiation potential. Conclusions: We suggested that both delivering teeth in HBSS with A-A and additional 70% ethanol washing are critical considerations for the successful culture of SHEDs without contamination.

Application of RapidHIT™ ID for cell authentication by fast and convenient STR profiling.

BACKGROUND: As cell therapies are injected directly into the body, cell authentication is essential. Short tandem repeat (STR) profiling is used for human identification in forensics as well as for cell authentication. The standard methodology (DNA extraction, quantification, polymerase chain reaction, and capillary electrophoresis) takes at least 6 h and requires several instruments to obtain an STR profile. RapidHIT™ ID is a single automated instrument that provides an STR profile in 90 min. OBJECTIVE: In this study, we aimed to propose a method to use RapidHIT™ ID for cell authentication. METHODS: Four types of cells which are used for cell therapy or in the production process were used. The sensitivity of STR profiling was compared by the cell type and cell count using RapidHIT™ ID. Moreover, the effect of preservation solutions, pre-treatment with cell lysis solution, proteinase K, Flinders Technology Associates (FTA) cards, and dried or wet cotton swabs (with a single cell type or a mixture of two) were examined. The results were compared to those obtained by the standard methodology using genetic analyzer ThermoFisher SeqStudio. RESULTS: We accomplished a high sensitivity through our proposed method that can benefit cytology laboratories. Although the pre-treatment process affected the quality of the STR profile, other variables did not significantly affect STR profiling. CONCLUSION: As a result of the experiment, RapidHIT™ ID can be used as a faster and simpler instrument for cell authentication.

Dopamine receptor D2 regulates glioblastoma survival and death through MET and death receptor 4/5.

Recent studies indicate that signaling molecules traditionally associated with central nervous system function play critical roles in cancer. Dopamine receptor signaling is implicated in various cancers including glioblastoma (GBM) and it is a recognized therapeutic target, as evidenced by recent clinical trials with a selective dopamine receptor D2 (DRD2) inhibitor ONC201. Understanding the molecular mechanism(s) of the dopamine receptor signaling will be critical for development of potent therapeutic options. Using the human GBM patient-derived tumors treated with dopamine receptor agonists and antagonists, we identified the proteins that interact with DRD2. DRD2 signaling promotes glioblastoma (GBM) stem-like cells and GBM growth by activating MET. In contrast, pharmacological inhibition of DRD2 induces DRD2-TRAIL receptor interaction and subsequent cell death. Thus, our findings demonstrate a molecular circuitry of oncogenic DRD2 signaling in which MET and TRAIL receptors, critical factors for tumor cell survival and cell death, respectively, govern GBM survival and death. Finally, tumor-derived dopamine and expression of dopamine biosynthesis enzymes in a subset of GBM may guide patient stratification for DRD2 targeting therapy.