Genomic stability is critical for the clinical use of human embryonic and induced pluripotent stem cells. We performed high-resolution SNP (single-nucleotide polymorphism) analysis on 186 pluripotent ...and 119 nonpluripotent samples. We report a higher frequency of subchromosomal copy number variations in pluripotent samples compared to nonpluripotent samples, with variations enriched in specific genomic regions. The distribution of these variations differed between hESCs and hiPSCs, characterized by large numbers of duplications found in a few hESC samples and moderate numbers of deletions distributed across many hiPSC samples. For hiPSCs, the reprogramming process was associated with deletions of tumor-suppressor genes, whereas time in culture was associated with duplications of oncogenic genes. We also observed duplications that arose during a differentiation protocol. Our results illustrate the dynamic nature of genomic abnormalities in pluripotent stem cells and the need for frequent genomic monitoring to assure phenotypic stability and clinical safety.
► hESCs and hiPSCs show more gene copy number variation than somatic cells ► Degree of abnormality differs more between hESC lines than hiPSC lines ► Deletions common in hiPSCs after reprogramming, duplications appear over time ► Recurrent duplications occur at specific genomic loci in pluripotent cells
Human pluripotent stem cells (hPSCs) are potential sources of cells for modeling disease and development, drug discovery, and regenerative medicine. However, it is important to identify factors that ...may impact the utility of hPSCs for these applications. In an unbiased analysis of 205 hPSC and 130 somatic samples, we identified hPSC-specific epigenetic and transcriptional aberrations in genes subject to X chromosome inactivation (XCI) and genomic imprinting, which were not corrected during directed differentiation. We also found that specific tissue types were distinguished by unique patterns of DNA hypomethylation, which were recapitulated by DNA demethylation during in vitro directed differentiation. Our results suggest that verification of baseline epigenetic status is critical for hPSC-based disease models in which the observed phenotype depends on proper XCI or imprinting and that tissue-specific DNA methylation patterns can be accurately modeled during directed differentiation of hPSCs, even in the presence of variations in XCI or imprinting.
► Global analysis of DNA methylation differences between somatic and pluripotent cells ► Tissue-specific DNA demethylation occurs during differentiation ► X chromosome inactivation is unstable in pluripotent cells over time in culture ► Aberrations in X inactivation and imprinting are maintained during differentiation
The nanoscale spatiotemporal resolution of single-particle tracking (SPT) renders it a powerful method for exploring single-molecule dynamics in living cells or tissues, despite the disadvantages of ...using traditional organic fluorescence probes, such as the weak fluorescent signal against the strong cellular autofluorescence background coupled with a fast-photobleaching rate. Quantum dots (QDs), which enable tracking targets in multiple colors, have been proposed as an alternative to traditional organic fluorescence dyes; however, they are not ideally suitable for applying SPT due to their hydrophobicity, cytotoxicity, and blinking problems. This study reports an improved SPT method using silica-coated QD-embedded silica nanoparticles (QD
), which represent brighter fluorescence and are less toxic than single QDs. After treatment of QD
in 10 μg/mL, the label was retained for 96 h with 83.76% of labeling efficiency, without impaired cell function such as angiogenesis. The improved stability of QD
facilitates the visualization of in situ endothelial vessel formation without real-time staining. Cells retain QD
fluorescence signal for 15 days at 4 °C without significant photobleaching, indicating that QD
has overcome the limitations of SPT enabling long-term intracellular tracking. These results proved that QD
could be used for SPT as a substitute for traditional organic fluorophores or single quantum dots, with its photostability, biocompatibility, and superior brightness.
Aim
Insulin resistance is a metabolic state where insulin sensitivity is lower than normal condition and strongly related to type 2 diabetes. However, an in vitro model mimicking insulin resistance ...is rare and thus screening drugs for insulin resistance severely depends on an in vivo model. Here, to increase anti‐diabetic drug selectivity for humans, 3D ADMSCs and macrophages were co‐cultured with in‐house fabricated co‐culture plates.
Material and methods
3D co‐culture plates were designed to load ADMSCs and RAW264.7 cells containing hydrogels in separate wells while allowing cell‐cell interaction with co‐culturing media. Hydrogels were constructed using a 3D cell‐printing system containing 20 mg/ml alginate, 0.5 mg/ml gelatin and 0.5 mg/ml type I collagen. Cells containing hydrogels in 3D co‐culture plates were incubated for 10 min to allow stabilization before the experiment. 3D co‐culture plates were incubated with the CaCl2 solution for 5 min to complete the cross linking of alginate hydrogel. Cells in 3D co‐culture plates were cultured for up to 12 days depending on the experiment and wells containing adipocytes and macrophages were separated and used for assays.
Results
KR‐1, KR‐2 and KR‐3 compounds were applied during differentiation (12 days) in 3D co‐cultured mouse 3T3‐L1 adipocytes and 3D co‐cultured human ADMSCs. Glucose uptake assay using 2‐DG6P and 2‐NBDG and western blot analysis were performed to investigate changes of insulin resistance in the 3D co‐cultured model for interspecies selectivity of drug screening. KR‐1 (mouse potent enantiomer) and KR‐3 (racemic mixture) showed improvement of 2‐DG and 2‐NBDG uptake compared with KR‐2 (human potent enantiomer) in 3D co‐cultured 3T3‐L1 adipocytes. In connection with insulin resistance in a 3D 3T3‐L1 co‐cultured model, KR‐1 and KR‐3 showed improvement of insulin sensitivity compared to KR‐2 by markedly increasing GLUT4 expression. In contrast to the result of 3D co‐cultured 3T3‐L1 adipocytes, KR‐1 failed to significantly improve 2‐DG and 2‐NBDG uptake in 3D co‐cultured ADMSC adipocytes. Results of 2‐NBDG accumulation and western blot analysis also showed that KR‐2 and KR‐3 improved insulin sensitivity relatively better than KR‐1.
Conclusions
Our 3D co‐culture model with/without 3D co‐culture plates can successfully mimic insulin resistance while allowing investigation of the effects of anti‐obesity or anti‐diabetic drugs on human or mouse co‐culturing cell type. This 3D co‐culture system may accelerate screening of drugs for insulin resistance depending on species.
This study was performed to evaluate the anticancer effects of tolerable doses of metformin with or without medroxyprogesterone (MPA) in endometrial cancer cells. Cell viability, cell invasion, and ...levels of matrix metallopeptidase (MMP) and transforming growth factor (TGF)-β1 were analyzed using three human endometrial adenocarcinoma cell lines (Ishikawa, KLE, and uterine serous papillary cancer (USPC)) after treatment with different dose combinations of MPA and metformin. Combining metformin (0, 100, 1000 µM) and 10 µM MPA induced significantly decreased cell viability in a time- and dose-dependent manner in Ishikawa cells, but not in KLE and USPC cells. In KLE cells, metformin treatment alone significantly inhibited cell invasion in a dose-dependent manner. The inhibitory effect of metformin was reversed when 10 µM MPA was combined, which was significantly inhibited again after treatment of MMP-2/9 inhibitor and/or TGF-β inhibitor. Changes of MMP-9 and TGF-β1 according to combinations of MPA and metformin were similar to those of invasion in KLE cells. In conclusion, the anticancer effects of tolerable doses of metformin varied according to cell type and combinations with MPA. Anti-invasive effect of metformin in KLE cells was completely reversed by the addition of MPA; this might be associated with MMP-9 and TGF-β1.
The complex process of bone regeneration is influenced by factors such as inflammatory responses, tissue interactions, and progenitor cells. Currently, multiple traumas can interfere with fracture ...healing, causing the prolonging or failure of healing. In these cases, bone grafting is the most effective treatment. However, there are several drawbacks, such as morbidity at the donor site and availability of suitable materials. Advantages have been provided in this field by a variety of stem cell types. Adipose-derived stem cells (ASCs) show promise. In the radiological examination of this study, it was confirmed that the C/S group showed faster regeneration than the other groups, and Micro-CT also showed that the degree of bone formation in the defect area was highest in the C/S group. Compared to the control group, the change in cortical bone area in the defect area decreased in the sham group (0.874), while it slightly increased in the C/S group (1.027). An increase in relative vascularity indicates a decrease in overall bone density, but a weak depression filled with fibrous tissue was observed outside the compact bone. It was confirmed that newly formed cortical bone showed a slight difference in bone density compared to surrounding normal bone tissue due to increased distribution of cortical bone. In this study, we investigated the effect of bone regeneration by ADMSCs measured by radiation and pathological effects. These data can ultimately be applied to humans with important clinical applications in various bone diseases, regenerative, and early stages of formative differentiation.
Abstract The physical factors of cell-culture environment have received little attention despite their anticipated significant role in human embryonic stem cell (hESC) culture optimization. Here we ...show that hESC culture conditions can be optimized by utilizing polyethylene terephthalate (PET) membranes whose defined pore densities (PDs) determine membrane surface hardness. The PET membranes with 1–4 × 106 pores/cm2 (0.291–0.345 GPa) supported the adherence and survival of hESCs without matrix coating. Furthermore, PET membrane with 4 × 106 pores/cm2 (0.345 GPa) supported optimal hESC self renewal as well as by the increase in cell proliferation. The expression level and activity of Rho-associated kinase (ROCK) were specifically down-regulated in hESCs cultured on the optimal PET membrane. We suggest that PET membranes of a defined PD/hardness provide an excellent culture substrate for the maintenance of uniform and undifferentiated hESCs.
Background
High‐functional cosmetic products combined with the concept of “treatment” cosmetics are being introduced to the market. Cosmetic products containing a skin‐derived microbiome, a ...three‐dimensional (3D) stem cell culture medium, and low‐molecular‐weight collagen are being introduced, and these products are leading the cosmeceutical market. We aimed to confirm the potential of a 3D stem cell culture medium‐containing cream as a skin‐whitening and moisturizing product.
Aim
To determine the enhancing effects of a cream containing 3D adipose tissue‐derived mesenchymal stem cell‐conditioned media (3D ADMSC‐CM) on whitening and moisturization.
Methods
The inhibitory activities of tyrosinase (TYR) and melanin were confirmed using 3D ADMSC‐CM. Furthermore, hyaluronic acid expression in 3D ADMSC‐CM was verified. The clinical efficacy of the cream containing 3D ADMSC‐CM was established by evaluating its antioxidant properties and effects on skin tone, radiance, freckles, and moisturization.
Results
The use of 3D ADMSC‐CM suppressed the inhibitory effects of TYR and melanin by approximately 24% and 33%, respectively, and increased the expression of hyaluronic acid synthase. A significant difference was observed after 4 weeks of using 3D ADMSC‐CM in the skin antioxidant evaluation. After 2 and 4 weeks of use, skin tone and radiance increased and skin freckles decreased significantly. Under extremely cold and dry weather conditions, the use of the cream increased skin moisturization.
Conclusions
The 3D ADMSC‐CM cream evaluated in an environment similar to the human body was found to enhance skin whitening and moisturization and can therefore be used in the skin care and cosmetic industries as a biocosmetic product.
Although biologiocal ancillay materials (AMs) have specific risk associated with their derivations, it plays key role to manufature cell and gene therapy (CGT) products. It is important to understand ...the regulation relevant to AMs for developers.
The authors investigated the guidelines and pharmacopeia (hereinafter referred to as “guidelines”) for biological AMs used for the manufacture of CGT products in Asia (China, India, Japan, Korea and Taiwan). In addition, the authors benchmarked the relevant guidelines in the United States (US) and European Union (EU).
The guidelines could be classified into two types based on whether specific AMs are scoped: (i) general guidelines for risk assessment of AMs and (ii) guidelines for specific AMs. The authors compared the risk categories for each type of AM provided in the general guidelines between the US and China and the specific requirements for bovine serum and trypsin in the guidelines of China, Japan, Taiwan, US and EU. The authors further compiled in-depth descriptions of the respective regulations in China, India, Japan, Korea and Taiwan. There is limited availability of some guidelines for specific AMs. Moreover, there are no common requirements established across the surveyed countries and regions. Therefore, the authors suggest a risk assessment approach for AMs with consideration of their biological origin and traceability, production steps applied and ability to control or remove AMs from the final medicinal product over the CGT manufacturing process.
In previous work, we established an equine induced pluripotent stem cell line (E-iPSCs) from equine adipose-derived stem cells (ASCs) using a lentiviral vector encoding four transcription factors: ...Oct4, Sox2, Klf4, and c-Myc. In the current study, we attempted to differentiate these established E-iPSCs into mesenchymal stem cells (MSCs) by serial passaging using MSC-defined media for stem cell expansion. Differentiation of the MSCs was confirmed by analyzing expression levels of the MSC surface markers CD44 and CD29, and the pluripotency markers Nanog and Oct4. Results indicated that the E-iPSC-derived MSCs (E-iPSC-MSCs) retained the characteristics of MSCs, including the ability to differentiate into chondrogenic, osteogenic, or myogenic lineages. E-iPSC-MSCs were rendered suitable for therapeutic use by inhibiting immune rejection through exposure to transforming growth factor beta 2 (TGF-β2) in culture, which down-regulated the expression of major histocompatibility complex class I (MHC class I) proteins that cause immune rejection if they are incompatible with the MHC antigen of the recipient. We reported 16 cases of E-iPSC-MSC transplantations into injured horses with generally positive effects, such as reduced lameness and fraction lines. Our findings indicate that E-iPSC-MSCs can demonstrate MSC characteristics and be safely and practically used in the treatment of musculoskeletal injuries in horses.
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