Tracking the Differentiation Status of Human Neural Stem Cells through Label-Free Raman Spectroscopy and Machine Learning-Based Analysis.

The ability to noninvasively monitor stem cells' differentiation is important to stem cell studies. Raman spectroscopy is a non-harmful imaging approach that acquires the cellular biochemical signatures. Herein, we report the first use of label-free Raman spectroscopy to characterize the gradual change during the differentiation process of live human neural stem cells (NSCs) in the in vitro cultures. Raman spectra of 600-1800 cm-1 were measured with human NSC cultures from the undifferentiated stage (NSC-predominant) to the highly differentiated one (neuron-predominant) and subsequently analyzed using various mathematical methods. Hierarchical cluster analysis distinguished two cell types (NSCs and neurons) through the spectra. The subsequently derived differentiation rate matched that measured by immunocytochemistry. The key spectral biomarkers were identified by time-dependent trend analysis and principal component analysis. Furthermore, through machine learning-based analysis, a set of eight spectral data points were found to be highly accurate in classifying cell types and predicting the differentiation rate. The predictive accuracy was the highest using the artificial neural network (ANN) and slightly lowered using the logistic regression model and linear discriminant analysis. In conclusion, label-free Raman spectroscopy with the aid of machine learning analysis can provide the noninvasive classification of cell types at the single-cell level and thus accurately track the human NSC differentiation. A set of eight spectral data points combined with the ANN method were found to be the most efficient and accurate. Establishing this non-harmful and efficient strategy will shed light on the in vivo and clinical studies of NSCs.

Functional analysis of HSPA1A and HSPA8 in parturition.

Many factors are involved in parturition, such as apoptosis, inflammatory mediators, and hormones. Previous studies indicated that HSP70 directly or indirectly regulates apoptosis, inflammatory immune response and hormone stimulus. To gain new insights into molecular mechanism underlying HSP70 for regulating parturition, we overexpressed and knocked down two representative members of HSP70 (HSPA1A and HSPA8) through transfection of their recombinant plasmid and si-RNA separately in WISH (human amniotic epithelial) cells. The expression changes of several pathways' marker genes were investigated by Western blotting and quantitative real-time PCR (qRT-PCR). Results showed extreme expression changes in the genes of IL-8 and ESR2. HSP70 was found to stimulate estrogen response by regulating ESR2 through ERK1/2 after treating WISH cells with the special phosphorylation inhibitor of ERK1/2 and analyzing the changes of E2 concentration by ELISA. HSP70 was also observed to contribute to preterm birth after administering the special inhibitor of HSP70-PFT-µ with LPS-induced preterm birth mouse model. Overall, HSP70 induces parturition through stimulating immune inflammatory and estrogen response. The balanced HSP70 expression could ensure a smooth parturition, while the imbalanced expression may cause a pathological state like preterm.

MicroRNAs in regulation of osteogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs), also referred to as multipotent stromal cells, have been isolated from various adult tissue sources because of their capabilities of differentiating into multiple cell lineages including osteoblasts, thus providing a novel approach for treating bone diseases and metabolic disorders. Despite extensive potential in cell therapy and widespread interest in clinical applications of MSCs, the molecular mechanisms with regard to the regulation of their therapeutic properties and osteoblast differentiation remain to be fully elucidated. MicroRNAs (miRNAs), a novel class of endogenous small noncoding RNAs, regulate gene expressions by translational repression or degradation of their targets. Recently, emerging evidence has shown that miRNAs are closely involved in controlling the key steps of osteoblast differentiation in MSCs. This review focuses on miRNAs and their roles in regulating osteogenic differentiation of MSCs.

Roles and regulation of the matrix metalloproteinase system in parturition.

Significant tissue destruction, repair, and remodeling are involved in parturition, which involves fetal membrane rupture, cervical ripening, and uterine contraction and its subsequent involution. Extracellular matrix degradation and remodeling by proteolytic enzymes, such as matrix metalloproteinases (MMPs), are required for the final steps of parturition. MMPs participate in physiological degradation and remodeling through their proteolytic activities on specific substrates, and are balanced by the action of their inhibitors. Disruption to this balance can result in pathological stress that ends with preterm or post-term birth or pre-eclampsia. In this review, we examine the roles and regulation of the MMP system in physiological and pathological labor, and propose a model that illustrates the mechanisms by which the MMP system contributes to these processes.

Versatile activity and morphological effects of zinc oxide submicron particles as anticancer agents.

Background: Submicron particles (SMPs), as novel bionanomaterials, offer complementary benefits to their conventional nano-counterparts. Aim: To explore zinc oxide (ZnO) SMPs' bioimaging and anticancer potentials. Materials & methods: ZnO SMPs were synthesized into two shapes. Fluorescent spectrum and microscopy were studied for the bioimaging property. Wound healing and Live/Dead assays of glioblastoma cells were characterized for anticancer activities. Results: ZnO SMPs exhibited a high quantum yield (49%) with stable orange fluorescence emission. Both morphologies (most significant in the rod shape) showed tumor-selective properties in cytotoxicity, inhibition to cell migration and attenuating the cancer-upregulated genes. The tumor selectivity was attributed to particle degradation and surface properties on pH dependency. Conclusion: The authors propose that ZnO SMPs could be a promising anticancer drug with tunable, morphology-dependent properties for bioimaging and controlled release.

Submicron particles (SMPs) are a novel nanomaterial whose total size is microscale (around one-millionth of a meter), but at least one dimension is nanoscale (around one-billionth of a meter). Their combined micro- and nanoscale properties are complementary, which can be an improvement on their conventional nano-counterparts. The aim of this study was to explore the bioimaging and anticancer properties of zinc oxide (ZnO) SMPs. ZnO SMPs were synthesized in two shapes: rod-shaped and flower-shaped. The fluorescence spectra and microscopy images were studied to investigate their potential for imaging applications, and wound healing and viability assays of glioblastoma cells were used to characterize anticancer activity. ZnO SMPs exhibited strong and stable orange fluorescence emission. Both shapes of ZnO SMPs showed tumor-selective toxicity, inhibition to cell migration and attenuating the cancer-upregulated genes; however, these effects were more significant for the rod-shaped particles. The tumor selectivity was attributed to pH-dependent particle degradation related to surface properties. The authors therefore propose that ZnO SMPs could be a promising anticancer drug with tunable, morphology-dependent properties for bioimaging and controlled release.

Fabrication of Tapered 3D Microstructure Arrays Using Dual-Exposure Lithography (DEL).

Three-dimensional (3D) microstructure arrays (MSAs) have been widely used in material science and biomedical applications by providing superhydrophobic surfaces, cell-interactive topography, and optical diffraction. These properties are tunable through the engineering of microstructure shapes, dimensions, tapering, and aspect ratios. However, the current fabrication methods are often too complex, expensive, or low-throughput. Here, we present a cost-effective approach to fabricating tapered 3D MSAs using dual-exposure lithography (DEL) and soft lithography. DEL used a strip-patterned film mask to expose the SU-8 photoresist twice. The mask was re-oriented between exposures (90° or 45°), forming an array of dual-exposed areas. The intensity distribution from both exposures overlapped and created an array of 3D overcut micro-pockets in the unexposed regions. These micro-pockets were replicated to DEL-MSAs in polydimethylsiloxane (PDMS). The shape and dimension of DEL-MSAs were tuned by varying the DEL parameters (e.g., exposure energy, inter-exposure wait time, and the photomask re-orientation angle). Further, we characterized various properties of our DEL-MSAs and studied the impact of their shape and dimension. All DEL-MSAs showed optical diffraction capability and increased hydrophobicity compared to plain PDMS surface. The hydrophobicity and diffraction angles were tunable based on the MSA shape and aspect ratio. Among the five MSAs fabricated, the two tallest DEL-MSAs demonstrated superhydrophobicity (contact angles >150°). Further, these tallest structures also demonstrated patterning proteins (with ~6-7 µm resolution), and mammalian cells, through microcontact printing and direct culturing, respectively. Our DEL method is simple, scalable, and cost-effective to fabricate structure-tunable microstructures for anti-wetting, optical-, and bio-applications.

MiR-144-3p regulates osteogenic differentiation and proliferation of murine mesenchymal stem cells by specifically targeting Smad4.

Despite extensive research on osteoblast differentiation and proliferation in mesenchymal stem cells (MSCs), the accurate mechanism remains to be further elucidated. MicroRNAs have been reported to be key regulators of osteoblast differentiation and proliferation. Here, we found that miR-144-3p is down-regulated during osteoblast differentiation of C3H10T1/2 cells. Overexpression of miR-144-3p inhibited osteogenic differentiation, whereas inhibition of miR-144-3p reversed this process. Furthermore, miR-144-3p inhibited the proliferation of C3H10T1/2 cells by arresting cells at the G0/G1 phase. Results from bioinformatics analysis, luciferase assay and western blotting demonstrated that miR-144-3p directly targeted Smad4. Additionally, Smad4 knockdown blocks the effects of miR-144-3p inhibitor. Therefore, we conclude that miR-144-3p negatively regulates osteogenic differentiation and proliferation of C3H10T1/2 cells by targeting Smad4.

miR-144 and targets, c-fos and cyclooxygenase-2 (COX2), modulate synthesis of PGE2 in the amnion during pregnancy and labor.

Labor is initiated as a result of hormonal changes that are induced by the activation of the inflammatory response and a series of biochemical events. The amnion, which is the primary source of prostaglandin E2 (PGE2), plays an important role in the process of labor. In the present study, we uncovered a pathway in which c-fos, cyclooxygenase-2 (COX2) and miR-144 function as hormonal modulators in the amnions of pregnant mice and humans. miR-144 down-regulated the synthesis of PGE2 during pregnancy by directly and indirectly inhibiting COX2 expression and by directly inhibiting the expression of c-fos, a transcriptional activator of COX2 and miR-144. Estrogen (E2) activated c-fos, thus promoting the expression of miR-144 and COX2 during labor. However, the increase in COX2 resulted in the partial inhibition of COX2 expression by miR-144, thereby slightly reducing the secretion of PGE2. These observations suggest that miR-144 inhibits PGE2 secretion by section to prevent the initiation of premature labor. Up-regulated expression of miR-144, c-fos and COX2 was also observed both in preterm mice and in mice undergoing normal labor. In summary, miR-144, c-fos and COX2 play important roles in regulating PGE2 secretion in the amnion during pregnancy and labor.

Integrated analysis of miRNA/mRNA network in placenta identifies key factors associated with labor onset of Large White and Qingping sows.

Labour onset is a very complex physiological process, and its mechanism is poorly understood. Here, we obtained the mRNA and miRNA expression profiles from the placentas of four groups of sows: Qingping sows 112 days after insemination with signs of labour onset (QS), Qingping sows 114 days after insemination with signs of labour onset (QL), Large White sows 114 days after insemination with signs of labour onset (LL) and Large White sows 112 days after insemination without signs of labour onset (LN). A set of differentially expressed genes, including 2164 mRNAs and 39 miRNAs, were found. A DAVID analysis of these differentially expressed genes revealed their critical roles in response to hormone stimulus, immune response. Cytoscape Network analysis of the functional genes found node mRNAs and that the regulatory network between the node mRNAs and miRNAs was established. A comparison of the sequencing data from the shorter gestation period (QS) and the normal gestation period (QL) indicated that these genes were responsible for the quicker and more sensitive reaction to the regulation of labour onset. This research not only detected the key factors that were involved in labour onset but also provided useful information for the research of gynaecological diseases.