Prognostic Analysis of Human Pluripotent Stem Cells Based on Their Morphological Portrait and Expression of Pluripotent Markers

The ability of human pluripotent stem cells for unlimited proliferation and self-renewal promotes their application in the fields of regenerative medicine. The morphological assessment of growing colonies and cells, as a non-invasive method, allows the best clones for further clinical applications to be safely selected. For this purpose, we analyzed seven morphological parameters of both colonies and cells extracted from the phase-contrast images of human embryonic stem cell line H9, control human induced pluripotent stem cell (hiPSC) line AD3, and hiPSC line HPCASRi002-A (CaSR) in various passages during their growth for 120 h. The morphological phenotype of each colony was classified using a visual analysis and associated with its potential for pluripotency and clonality maintenance, thus defining the colony phenotype as the control parameter. Using the analysis of variance for the morphological parameters of each line, we showed that selected parameters carried information about different cell lines and different phenotypes within each line. We demonstrated that a model of classification of colonies and cells by phenotype, built on the selected parameters as predictors, recognized the phenotype with an accuracy of 70–75%. In addition, we performed a qRT-PCR analysis of eleven pluripotency markers genes. By analyzing the variance of their expression in samples from different lines and with different phenotypes, we identified group-specific sets of genes that could be used as the most informative ones for the separation of the best clones. Our results indicated the fundamental possibility of constructing a morphological portrait of a colony informative for the automatic identification of the phenotype and for linking this portrait to the expression of pluripotency markers.     

Bufalin Inhibits Tumorigenesis,Stemness, and Epithelial–Mesenchymal Transition in Colorectal Cancer through a C-Kit/Slug Signaling Axis

Colorectal cancer (CRC) is a major source of morbidity and mortality, characterized by intratumoral heterogeneity and the presence of cancer stem cells (CSCs). Bufalin has potent activity against many tumors, but studies of its effect on CRC stemness are limited. We explored bufalin’s function and mechanism using CRC patient-derived organoids (PDOs) and cell lines. In CRC cells, bufalin prevented nuclear translocation of β-catenin and down-regulated CSC markers (CD44, CD133, LGR5), pluripotency factors, and epithelial–mesenchymal transition (EMT) markers (N-Cadherin, Slug, ZEB1). Functionally, bufalin inhibited CRC spheroid formation, aldehyde dehydrogenase activity, migration, and invasion. Network analysis identified a C-Kit/Slug signaling axis accounting for bufalin’s anti-stemness activity. Bufalin treatment significantly downregulated C-Kit, as predicted. Furthermore, overexpression of C-Kit induced Slug expression, spheroid formation, and bufalin resistance. Similarly, overexpression of Slug resulted in increased expression of C-Kit and identical functional effects, demonstrating a pro-stemness feedback loop. For further study, we established PDOs from diagnostic colonoscopy. Bufalin differentially inhibited PDO growth and proliferation, induced apoptosis, restored E-cadherin, and downregulated CSC markers CD133 and C-Myc, dependent on C-Kit/Slug. These findings suggest that the C-Kit/Slug axis plays a pivotal role in regulating CRC stemness, and reveal that targeting this axis can inhibit CRC growth and progression.     

What Are the Reasons for Continuing Failures in Cancer Therapy? Are Misleading/Inappropriate Preclinical Assays to Be Blamed? Might Some Modern Therapies Cause More Harm than Benefit?

Over 50 years of cancer research has resulted in the generation of massive amounts of information, but relatively little progress has been made in the treatment of patients with solid tumors, except for extending their survival for a few months at best. Here, we will briefly discuss some of the reasons for this failure, focusing on the limitations and sometimes misunderstanding of the clinical relevance of preclinical assays that are widely used to identify novel anticancer drugs and treatment strategies (e.g., “synthetic lethality”). These include colony formation, apoptosis (e.g., caspase-3 activation), immunoblotting, and high-content multiwell plate cell-based assays, as well as tumor growth studies in animal models. A major limitation is that such assays are rarely designed to recapitulate the tumor repopulating properties associated with therapy-induced cancer cell dormancy (durable proliferation arrest) reflecting, for example, premature senescence, polyploidy and/or multinucleation. Furthermore, pro-survival properties of apoptotic cancer cells through phoenix rising, failed apoptosis, and/or anastasis (return from the brink of death), as well as cancer immunoediting and the impact of therapeutic agents on interactions between cancer and immune cells are often overlooked in preclinical studies. A brief review of the history of cancer research makes one wonder if modern strategies for treating patients with solid tumors may sometimes cause more harm than benefit.     

From 2D to 3D Co-Culture Systems: A Review of Co-Culture Models to Study the Neural Cells Interaction

The central nervous system (CNS) controls and regulates the functional activities of the organ systems and maintains the unity between the body and the external environment. The advent of co-culture systems has made it possible to elucidate the interactions between neural cells in vitro and to reproduce complex neural circuits. Here, we classified the co-culture system as a two-dimensional (2D) co-culture system, a cell-based three-dimensional (3D) co-culture system, a tissue slice-based 3D co-culture system, an organoid-based 3D co-culture system, and a microfluidic platform-based 3D co-culture system. We provide an overview of these different co-culture models and their applications in the study of neural cell interaction. The application of co-culture systems in virus-infected CNS disease models is also discussed here. Finally, the direction of the co-culture system in future research is prospected.     

MicroRNA-381 Regulates Proliferation and Differentiation of Caprine Skeletal Muscle Satellite Cells by Targeting PTEN and JAG2

In our previous study, microRNA (miR)-381 was found to be the most down-regulated miRNA in skeletal muscle of Liaoning cashmere goats with higher skeletal muscle mass, but the molecular mechanism involved remains unclear. In this study, primary caprine skeletal muscle satellite cells (SMSCs) were isolated and identified. We investigated the effect of miR-381 on the viability, proliferation and differentiation of caprine SMSCs, and the target relationships of miR-381 with jagged canonical Notch ligand 2 (JAG2) and phosphatase and tensin homolog (PTEN). Cells isolated were positive for SMSC-specific marker protein Pax7. This suggests that purified SMSCs were obtained. The expression level of miR-381 achieved a peak value on day 4 after SMSC differentiation, and miR-381 also significantly increased the expression levels of myogenic differentiation marker genes: myosin heavy chain (MyHC), myogenin (MyoG) and myocyte enhancer factor 2C (MEF2C) in differentiated SMSCs, the area of MyHC-positive myotubes and the myogenic index. These findings suggest that miR-381 promoted myogenic differentiation of caprine SMSCs. The CCK8 assay and EDU staining analysis showed that miR-381 mimic both inhibited the viability of SMSCs and decreased the percentage of EDU-labeled positive SMSCs. In contrast, miR-381 inhibitor had the opposite effect with miR-381 mimic. A dual luciferase reporter assay verified that miR-381 can target JAG2 and PTEN by binding to the 3′-untranslated regions (3′-UTR) of the genes. The transfection of miR-381 mimic into caprine SMSCs resulted in decreases in expression levels of JAG2 and PTEN, while miR-381 inhibitor increased the two target genes in expression. This is the first study to reveal the biological mechanisms by which miR-381 regulates caprine SMSC activities.     

Circulating miR-200 Family and CTCs in Metastatic Breast Cancer before,during, and after a New Line of Systemic Treatment

The extracellular circulating microRNA (miR)-200 regulates epithelial-mesenchymal transition and, thus, plays an essential role in the metastatic cascade and has shown itself to be a promising prognostic and predictive biomarker in metastatic breast cancer (MBC). Expression levels of the plasma miR-200 family were analyzed in relationship to systemic treatment, circulating tumor cells (CTC) count, progression-free survival (PFS), and overall survival (OS). Expression of miR-200a, miR-200b, miR-200c, miR-141, and miR-429, and CTC status (CTC-positive ≥ 5 CTC/7.5 mL) was assessed in 47 patients at baseline (BL), after the first completed cycle of a new line of systemic therapy (1C), and upon the progression of disease (PD). MiR-200a, miR-200b, and miR-141 expression was reduced at 1C compared to BL. Upon PD, all miR-200s were upregulated compared to 1C. At all timepoints, the levels of miR-200s were elevated in CTC-positive versus CTC-negative patients. Further, heightened miR-200s expression and positive CTC status were associated with poorer OS at BL and 1C. In MBC patients, circulating miR-200 family members decreased after one cycle of a new line of systemic therapy, were elevated during PD, and were indicative of CTC status. Notably, increased levels of miR-200s and elevated CTC count correlated with poorer OS and PFS. As such, both are promising biomarkers for optimizing the clinical management of MBC.     

The Neuroprotective Effects of Exosomes Derived from TSG101-Overexpressing Human Neural Stem Cells in a Stroke Model

Although tissue-type plasminogen activator was approved by the FDA for early reperfusion of occluded vessels, there is a need for an effective neuroprotective drug for stroke patients. In this study, we established tumor susceptibility gene (TSG)101-overexpressing human neural stem cells (F3.TSG) and investigated whether they showed enhanced secretion of exosomes and whether treatment with exosomes during reperfusion alleviated ischemia-reperfusion-mediated brain damage. F3.TSG cells secreted higher amounts of exosomes than the parental F3 cells. In N2A cells subjected to oxygen–glucose deprivation (OGD), treatment with exosomes or coculture with F3.TSG cells significantly attenuated lactate dehydrogenase release, the mRNA expression of proinflammatory factors, and the protein expression of DNA-damage-related proteins. In a middle cerebral artery occlusion (MCAO) rat model, treatment with exosomes, F3 cells, or F3.TSG cells after 2 h of occlusion followed by reperfusion reduced the infarction volume and suppressed inflammatory cytokines, DNA-damage-related proteins, and glial fibrillary acidic protein, and upregulated several neurotrophic factors. Thus, TSG101-overexpressing neural stem cells showed enhanced exosome secretion; exosome treatment protected against MCAO-induced brain damage via anti-inflammatory activities, DNA damage pathway inhibition, and growth/trophic factor induction. Therefore, exosomes and F3.TSG cells can affect neuroprotection and functional recovery in acute stroke patients.     

Compound C Inhibits Renca Renal Epithelial Carcinoma Growth in Syngeneic Mouse Models by Blocking Cell Cycle Progression,Adhesion and Invasion

Compound C (CompC), an inhibitor of AMP-activated protein kinase, reduces the viability of various renal carcinoma cells. The molecular mechanism underlying anti-proliferative effect was investigated by flow cytometry and western blot analysis in Renca cells. Its effect on the growth of Renca xenografts was also examined in a syngeneic BALB/c mouse model. Subsequent results demonstrated that CompC reduced platelet-derived growth factor receptor signaling pathways and increased ERK1/2 activation as well as reactive oxygen species (ROS) production. CompC also increased the level of active Wee1 tyrosine kinase (P-Ser⁶⁴²-Wee1) and the inactive form of Cdk1 (P-Tyr¹⁵-Cdk1) while reducing the level of active histone H3 (P-Ser¹⁰-H3). ROS-dependent ERK1/2 activation and sequential alterations in Wee1, Cdk1, and histone H3 might be responsible for the CompC-induced G2/M cell cycle arrest and cell viability reduction. In addition, CompC reduced the adhesion, migration, and invasion of Renca cells in the in vitro cell systems, and growth of Renca xenografts in the BALB/c mouse model. Taken together, the inhibition of in vivo tumor growth by CompC may be attributed to the blockage of cell cycle progression, adhesion, migration, and invasion of tumor cells. These findings suggest the therapeutic potential of CompC against tumor development and progression.     

Unilateral Cervical Vagotomy Modulates Immune Cell Profiles and the Response to a Traumatic Brain Injury

TBI induces splenic B and T cell expansion that contributes to neuroinflammation and neurodegeneration. The vagus nerve, the longest of the cranial nerves, is the predominant parasympathetic pathway allowing the central nervous system (CNS) control over peripheral organs, including regulation of inflammatory responses. One way this is accomplished is by vagus innervation of the celiac ganglion, from which the splenic nerve innervates the spleen. This splenic innervation enables modulation of the splenic immune response, including splenocyte selection, activation, and downstream signaling. Considering that the left and right vagus nerves have distinct courses, it is possible that they differentially influence the splenic immune response following a CNS injury. To test this possibility, immune cell subsets were profiled and quantified following either a left or a right unilateral vagotomy. Both unilateral vagotomies caused similar effects with respect to the percentage of B cells and in the decreased percentage of macrophages and T cells following vagotomy. We next tested the hypothesis that a left unilateral vagotomy would modulate the splenic immune response to a traumatic brain injury (TBI). Mice received a left cervical vagotomy or a sham vagotomy 3 days prior to a fluid percussion injury (FPI), a well-characterized mouse model of TBI that consistently elicits an immune and neuroimmune response. Flow cytometric analysis showed that vagotomy prior to FPI resulted in fewer CLIP+ B cells, and CD4+, CD25+, and CD8+ T cells. Vagotomy followed by FPI also resulted in an altered distribution of CD11b^high and CD11b^low macrophages. Thus, transduction of immune signals from the CNS to the periphery via the vagus nerve can be targeted to modulate the immune response following TBI.     

Asymmetric Non-Fullerene Small Molecule Acceptor with Unidirectional Non-Fused π-Bridge and Extended Terminal Group for High-Efficiency Organic Solar Cells

We designed and synthesized an asymmetric non-fullerene small molecule acceptor (NF-SMA) IDT-TNIC with an A–D–π–A structure, based on an indacenodithiophene (IDT) central core, with a unidirectional non-fused alkylthio-thiophene (T) π-bridge, and 2-(3-oxo-2,3-dihydro-1H-cyclopenta[b]naphthalen-1-ylidene)malononitrile (NIC) extended terminal groups. IDT-TNIC molecules still maintain a good coplanar structure, which benefits from the non-covalent conformational locks (NCL) between O···S and S···S. The asymmetric structure increases the molecular dipole moment, and the extended terminal group broadens the absorption of the material, resulting in an excellent photovoltaic performance of IDT-TNIC. The photovoltaic device, based on PBDB-T:IDT-TNIC, exhibits an energetic PCE of 11.32% with a high V_oc of 0.87 V, high J_sc of 19.85 mA cm⁻², and a low energy loss of 0.57 eV. More importantly, IDT-TNICs with asymmetric structures show a superior property compared to symmetric IDT-Ns. The results demonstrate that it is an effectual strategy to enhance the properties of asymmetric A–D–π–A-based NF-SMAs with non-fused NCL π-bridges and extended terminal groups.