Effect of passage number of genetically modified TGF-β3 expressing primary chondrocytes on the chondrogenesis of ATDC5 cells in a 3D coculture system

Due to the lack of blood vessels, nerves and lymphatics, articular cartilage is difficult to repair once damaged. Tissue engineering is considered to be a potential strategy for cartilage regeneration. Successful tissue engineering strategies depend on the effective combination of biomaterials, seed cells and biological factors. In our previous study, a genetically modified coculture system with chondrocytes and ATDC5 cells in an alginate hydrogel has exhibited a superior ability to enhance chondrogenesis. In this study, we further evaluated the influence of chondrocytes at various passages on chondrogenesis in the coculture system. The results demonstrated that transfection efficiency was hardly influenced by the passage of chondrocytes. The coculture system with passage 5 (P5) chondrocytes had a better effect on chondrogenesis of ATDC 5 cells, while chondrocytes in this coculture system presented higher levels of dedifferentiation than other groups with P1 or P3 chondrocytes. Therefore, P5 chondrocytes were shown to be more suitable for the coculture system, as they accumulated in sufficient cell numbers with more passages and had a higher level of dedifferentiation, which was prone to form a favorable niche for chondrogenesis of ATDC5 cells. This study may provide fresh insights for future cartilage tissue engineering strategies with a combination of a coculture system and advanced biomaterials.

Generation and characterization of human Fetal membrane and Decidual cell lines for reproductive biology experiments

Human fetal membrane and maternal decidua parietalis form one of the major feto-maternal interfaces during pregnancy. Studies on this feto-maternal interface is limited as several investigators have limited access to the placenta, and experience difficulties to isolate and maintain primary cells. Many cell lines that are currently available do not have the characteristics or properties of their primary cells of origin. Therefore, we created, characterized the immortalized cells from primary isolates from fetal membrane-derived amnion epithelial cells, amnion and chorion mesenchymal cells, chorion trophoblast cells and maternal decidua parietalis cells. Primary cells were isolated from a healthy full-term, not in labor placenta. Primary cells were immortalized using either a HPV16E6E7 retroviral or a SV40T lentiviral system. The immortalized cells were characterized for the morphology, cell type-specific markers, and cell signalling pathway activation. Genomic stability of these cells was tested using RNA seq, karyotyping, and short tandem repeats DNA analysis. Immortalized cells show their characteristic morphology, and express respective epithelial, mesenchymal and decidual markers similar to that of primary cells. Gene expression of immortalized and primary cells were highly correlated (R = 0.798 to R = 0.974). Short tandem repeats DNA analysis showed in the late passage number (>P30) of cell lines matched 84-100% to the early passage number (<P10) of the cell lines revealing there were no genetic drift over the passages. Karyotyping also revealed no chromosomal anomalies. Creation of these cell lines can standardize experimental approaches, eliminate subject to subject variabilities, and benefit the reproductive biological studies on pregnancies by using these cells.

Effect of Passage Number on Human Induced Pluripotent Stem Cell Derived Neurons Culture Contaminants with Non-Neuronal Cell Types

Background: The study of live human neurons has been hindered due to the complexity and potential irreversible damage to the patient during biopsy. However, reprogramming of adult human somatic cells into induced pluripotent stem cells (iPSCs) has proved to be a novel method in the study of the pathophysiology of disease and therapeutic targets of the human nervous system. There are several approaches, and the optimum time (i.e., passage number) to generate highly pure cultures is being studied. Therefore, our laboratory has investigated the effect of passage number on culture contaminants with non-neuronal cell types. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood from three cell lines and reprogrammed into iPSCs. Each cell line consisted of three samples that were analyzed after low (5-10), middle (20-26), and high (30-38) number of passages. Cells were maintained in an induction medium for eight days. On day nine, cells were dissociated and replated in a maintenance medium. On day 33, total RNA was extracted from cells. Normalized values for non-neuronal cell marker genes were compared using paired Student’s t-tests and two-way ANOVA, with the cell line and passage number as independent variables. P-values less than 0.05 were considered significant. Results: Our results showed that lower passage number was associated with decreased astrocyte and chondrocyte marker expression. High passage number was associated with decreased oligodendrocyte and glial precursor marker expression. Of the fibroblast markers evaluated, there were similar trends of expression between all three groups. There was no significant difference in microglial cell marker gene expression between all three groups. Conclusion and Potential Impact: Low gene expression suggests a purer culture. According to these results, as passage number increases, there is more contaminants with oligodendrocytes and glial precursor cells. Conversely, with low passage numbers, there are more contaminants with astrocytes and chondrocytes. Future studies will identify the impact of these non-neuronal contaminants and implications on research.

A DNAmCULTURE Epigenetic Fingerprint Recapitulates Physiological Aging

Aging elicits dramatic changes to DNA methylation (DNAm), however the causes and consequences of such alterations to the epigenome remain unclear. The utility of biomarkers of aging based on DNAm patterns would be greatly enhanced if in vitro models existed that recapitulated physiological phenotypes such that modulation could garnish mechanistic insights. Using DNAm from serially passaged mouse embryonic fibroblasts, we developed a marker of culture aging and asked if culture phenotypes, like exhaustive replication, are epigenetically analogous to physiological aging. Our measure, termed DNAmCULTURE, accurately estimated passage number and was shown to strongly increase with age when examined in multiple tissues. Furthermore, we observed epigenetic alterations indicative of early cultured cells in animals undergoing caloric restriction and in lung and kidney fibroblasts re-programmed to iPSCs. This study identifies culture-derived alterations to the methylome as physiologically relevant and implicates culture aging as an important feature in known epigenetic aging phenomena.

Impact of baseline culture conditions of mouse-derived cancer organoids when determining therapeutic response and tumor heterogeneity

Representative models are needed to screen new therapies for patients with cancer. Cancer organoids are a leap forward as a culture model that faithfully represents the disease. Mouse-derived cancer organoids (MDCOs) are becoming increasingly popular, however there has yet to be a standardized method to assess therapeutic response and identify subpopulation heterogeneity. There are multiple factors unique to organoid culture that could affect how therapeutic response and MDCO heterogeneity are assessed. Here we describe an analysis of nearly 3,500 individual MDCOs where individual organoid morphologic tracking was performed. Change in MDCO diameter was assessed in the presence of control media or targeted therapies. Individual organoid tracking was identified to be more sensitive to treatment response than well-level assessment. The impact of different generations of mice of the same genotype, different regions of the colon, and organoid specific characteristics including baseline size, passage number, plating density, and location within the matrix were examined. Only the starting size of the MDCO altered the subsequent growth. Here we establish organoid culture parameters for individual organoid morphologic tracking to determine therapeutic response and growth/response heterogeneity for translational studies using murine colorectal cancer organoids.

Molecular Profiling and Gene Banking of Rabbit EPCs Derived from Two Biological Sources

Endothelial progenitor cells (EPCs) have been broadly studied for several years due to their outstanding regenerative potential. Moreover, these cells might be a valuable source of genetic information for the preservation of endangered animal species. However, a controversy regarding their characterization still exists. The aim of this study was to isolate and compare the rabbit peripheral blood- and bone marrow-derived EPCs with human umbilical vein endothelial cells (HUVECs) in terms of their phenotype and morphology that could be affected by the passage number or cryopreservation as well as to assess their possible neuro-differentiation potential. Briefly, cells were isolated and cultured under standard endothelial conditions until passage 3. The morphological changes during the culture were monitored and each passage was analyzed for the typical phenotype using flow cytometry, quantitative real–time polymerase chain reaction (qPCR) and novel digital droplet PCR (ddPCR), and compared to HUVECs. The neurogenic differentiation was induced using a commercial kit. Rabbit cells were also cryopreserved for at least 3 months and then analyzed after thawing. According to the obtained results, both rabbit EPCs exhibit a spindle-shaped morphology and high proliferation rate. The both cell lines possess same stable phenotype: CD14-CD29+CD31-CD34-CD44+CD45-CD49f+CD73+CD90+CD105+CD133-CD146-CD166+VE-cadherin+VEGFR-2+SSEA-4+MSCA-1-vWF+eNOS+AcLDL+ALDH+vimentin+desmin+α-SMA+, slightly different from HUVECs. Moreover, both induced rabbit EPCs exhibit neuron-like morphological changes and expression of neuronal markers ENO2 and MAP2. In addition, cryopreserved rabbit cells maintained high viability (>85%) and endothelial phenotype after thawing. In conclusion, our findings suggest that cells expanded from the rabbit peripheral blood and bone marrow are of the endothelial origin with a stable marker expression and interesting proliferation and differentiation capacity.

Study on Pipetting Motion Optimization of Automatic Spheroid Culture System for Spheroid Formation

This research group has established a technology for producing a three-dimensional cell constructed using only the cell itself. This technology uses a property in which the spheroids fuse with each other. We developed a system that automates the spheroid production process to obtain reproducible spheroids and suppress variation factors that occur from human operation. However, it has become clear that the dispersion occurs in the diameter depending on the number of cells of the spheroid even if the cells are handled in the same manner. The purpose of this research is to examine an appropriate pipetting motion in accordance with the number of cells of the spheroid to be produced. Rabbit mesenchymal stem cells (rMSCs) are used as the objects. The number of cells was set to 2×104, 3×104, and 4×104 cells/well, and the passage number as 7. The appearance of spheroids cultured using the motion programmed in accordance with each number of cells was observed every 24 hours for 5 days after seeding. The results of the analysis indicate that the optimum motion in each number of cells has been successfully specified, and reproducible spheroids have been successfully produced.

Effect of low-passage number on dengue consensus genomes and intra-host variant frequencies

Intra-host single nucleotide variants (iSNVs) have been increasingly used in genomic epidemiology to increase phylogenetic resolution and reconstruct fine-scale outbreak dynamics. These analyses are preferably done on sequence data from direct clinical samples, but in many cases due to low viral loads, there might not be enough genetic material for deep sequencing and iSNV determination. Isolation of the virus from clinical samples with low-passage number increases viral load, but few studies have investigated how dengue virus (DENV) culture isolation from a clinical sample impacts the consensus sequence and the intra-host virus population frequencies. In this study, we investigate consensus and iSNV frequency differences between DENV sequenced directly from clinical samples and their corresponding low-passage isolates. Twenty five DENV1 and DENV2 positive sera and their corresponding viral isolates (T. splendens inoculation and C6/36 passage) were obtained from a prospective cohort study in the Philippines. These were sequenced on MiSeq with minimum nucleotide depth of coverage of 500×, and iSNVs were detected using LoFreq. For both DENV1 and DENV2, we found a maximum of one consensus nucleotide difference between clinical sample and isolate. Interestingly, we found that iSNVs with frequencies ≥5 % were often preserved between the samples, and that the number of iSNV positions, and sample diversity, at this frequency cutoff did not differ significantly between the sample pairs (clinical sample and isolate) in either DENV1 or DENV2 data. Our results show that low-passage DENV isolate consensus genomes are largely representative of their direct sample parental viruses, and that low-passage isolates often mirror high frequency within-host variants from direct samples.

A Study on Generating Fake Images of Podocyte Cells Based on Acgans with Restriction of Learning

In general, we need a lot of data for improving the accuracy of machine learning. However, the number of biological samples what we can obtain are not enough for machine learning. This problem exists in the classification of glomerular epithelial cells with the progress of disease, and its accuracy is contrary to our intuitive impression. Therefore, we would like to improve the accuracy by generating a lot of fake images using Generative Adversarial Nets (GANs). About podocyte cells, it was difficult to obtain an arbitrary disease by previous method. In this paper, we propose the model with restriction of learning by shapes information based on ACGANs, and we investigate how much fake images generated by our method are similar to real images. According to the results, the passage number of fake images by our method is 17% higher than conventional method.