Population Doubling Research Articles

Comparison of natural prey Tetranychus turkestani, date palm pollen, and bee pollen diets on development, reproduction, and life table parameters of the predator Amblyseius swirskii

The predatory mite Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae) is produced commercially for biological control of insects and mites on crop plants around the world. The aim of this study was to compare the effects of a diet of natural prey, Tetranychus turkestani Ugarov and Nikolskii (Acari: Tetranychidae), date palm pollen, or bee-collected pollen on A. swirskii development, reproduction, and life table parameters. Completely randomized, no-choice experiments were conducted in replicated experimental units, i.e., 3 x 3 cm plastic sheets inside Petri dishes. A life table analysis was also conducted. Results indicated that diet type did not affect A. swirskii preimaginal survival; it ranged from 97-100% on T. turkestani and both pollen diets. However, total development time was significantly shorter for A. swirskii females fed date palm pollen than T. turkestani or bee pollen. Adult females fed T. turkestani lived longer, had longer oviposition periods, and produced more eggs. The intrinsic rate of natural increase (0.396 d<sup>-1</sup>) was higher for A. swirskii fed date palm pollen than T. turkestani or bee pollen. The finite rate of increase, net reproductive rate, and gross reproductive rate did not differ significantly between A. swirskii fed date palm pollen or T. turkestani. Mean generation time (7.56 d) and population doubling time (1.043 d) were shorter for A. swirskii fed date palm pollen than T. turkestani or bee pollen. This study suggests that the T. turkestani diet or date palm pollen diet is suitable for A. swirskii. Date palm pollen has great potential as a cost effective diet to mass produce A. swirskii in the absence of natural prey. Future research could determine whether long-term rearing on date palm pollen reduces the ability of A. swirskii to locate, capture, and consume T. turkestani or other Tetranychus species on crop plants.

Oxidative stress and its impact on skin, scalp and hair.

Oxidative stress is an unbalanced condition in which the tissues of the body are not sufficiently able to counteract either exogenous or endogenous sources of reactive oxygen species. Oxidative stress is strongly associated with ageing, both local and systemic, as well as a wide range of local health conditions. This review focuses on the oxidative stress data known for skin, scalp and hair. This oxidative stress may be the 'currency' by which an unhealthy scalp leads to deleterious consequences to the hair. The ramifications of this scalp oxidative stress to normal hair elongation, retention and replacement are discussed.

Comparative study on the biological characteristics of menstrual blood- and endometrium-derived endometrial cells.

During a woman's reproductive period, the endometrial tissue is shed and regenerated every month to prepare for pregnancy or for the next cycle. The aim of the present study was to isolate, culture and characterize human endometrial cells (ECs) derived from menstrual blood (MB) and the endometrium (E). MB-derived ECs (MB-ECs) were isolated from women's MB. E-derived ECs (E-ECs) were isolated from women's endometrial tissues. The present study investigated the epithelial cell marker cytokeratin 18 (CK18) in MB-ECs and E-ECs. Cell proliferation analyses indicated that E-ECs (population doubling time, 20.85 h) grew faster than MB-ECs (population doubling time, 22.05 h; P<0.05). Cell migration ability was found to be significantly greater for MB-ECs than for E-ECs at 48 h (P<0.01). MB-ECs incubated with TGF-β1 (3 ng/ml) exhibited significantly decreased CK18 mRNA expression (P<0.01), and significantly increased vimentin (Vim) mRNA (P<0.05) and protein (P<0.01) expression at 6 and 12 h, respectively. E-EC incubation with TGF-β1 (3 ng/ml) significantly decreased CK18 mRNA expression (P<0.01) at 12 h and significantly increased Vim mRNA (P<0.01) and protein expression (P<0.05) at 6 h. The present results indicated that MB-ECs and E-ECs were biologically different, and that epithelial-mesenchymal transdifferentiation could be induced by TGF-β1 treatment.

Neurovascular dysfunction and neuroinflammation in a Cockayne syndrome mouse model.

Cockayne syndrome (CS) is a rare, autosomal genetic disorder characterized by premature aging-like features, such as cachectic dwarfism, retinal atrophy, and progressive neurodegeneration. The molecular defect in CS lies in genes associated with the transcription-coupled branch of the nucleotide excision DNA repair (NER) pathway, though it is not yet clear how DNA repair deficiency leads to the multiorgan dysfunction symptoms of CS. In this work, we used a mouse model of severe CS with complete loss of NER (Csa−/−/Xpa−/−), which recapitulates several CS-related phenotypes, resulting in premature death of these mice at approximately 20 weeks of age. Although this CS model exhibits a severe progeroid phenotype, we found no evidence of in vitro endothelial cell dysfunction, as assessed by measuring population doubling time, migration capacity, and ICAM-1 expression. Furthermore, aortas from CX mice did not exhibit early senescence nor reduced angiogenesis capacity. Despite these observations, CX mice presented blood brain barrier disruption and increased senescence of brain endothelial cells. This was accompanied by an upregulation of inflammatory markers in the brains of CX mice, such as ICAM-1, TNFα, p-p65, and glial cell activation. Inhibition of neovascularization did not exacerbate neither astro- nor microgliosis, suggesting that the pro-inflammatory phenotype is independent of the neurovascular dysfunction present in CX mice. These findings have implications for the etiology of this disease and could contribute to the study of novel therapeutic targets for treating Cockayne syndrome patients.

Impact of donor nutritional balance on the growth and development of mesenchymal stem cells from caprine umbilical cord Wharton´s jelly.

Mesenchymal stem cells (MSCs) from the umbilical cord (UC) have aroused considerable interest. However, little is known about the maternal effect on these cells. The aim of this study was to verify the impact of the nutritional status of donor goats on the growth and differentiation of MSCs from the UC. At parturition, 19 goats were grouped based on their low or high body mass index (low BMI, LBMI, n = 9; and high BMI, HBMI, n = 10). UCs were collected during delivery and Wharton's jelly (WJ) fragments cultured. WJ-MSCs were differentiated into osteocytes, adipocytes, chondrocytes, and the population doubling time (PDT) was determined. Samples of WJ-MSCs were also used to verify the expression of the CD90, CD73, CD34, CD45, and CD105 genes. Media used for WJ-MSC primary cultures were analyzed using near-infrared spectroscopy. The lag phase was 7.5 ± 0.6days and the entire culture took 26.7 ± 1.3days, with a cell proliferation rate of 8.500 cells/day. The mean PDT from subculture was 30.0 ± 0.7h. The CD105 gene was sub-expressed in LBMI, and the spectra of the spent media from the second to fourth day of WJ-MSC primary culture were segregated into negative scores by multivariate analysis. We conclude that, in goats, the nutritional balance of the donor did not affect the in vitro growth of MSCs derived from the UC. However, the molecular profile observed in the low BMI group suggests that the use of MSCs for therapeutic purposes should be considered more carefully.

337 DermaCult™, a Serum- and BPE-free Medium Supporting Long-term Feeder-free Expansion of Primary Human Epidermal Keratinocytes that Retain Their Differentiation Potential at Air Liquid Interface

Primary human epidermal keratinocytes (HEKs) typically undergo growth arrest at approximately 15 - 30 population doublings (PDs) when maintained in traditional feeder-free culture medium formulations. To circumvent this early senescence, we developed DermaCult™, a serum- and BPE-free medium that promotes long-term growth (> 30 PDs) of HEKs while maintaining their air liquid interface (ALI) differentiation potential. To measure cell expansion over the entire lifespan of the culture, HEKs derived from either neonatal or adult skin were thawed and seeded onto uncoated tissue cultureware at a density of 1 - 2.5 x 103 cells/cm2, and passaged when cultures reached approximately 50 - 70% confluence. At various time points, keratinocytes were analyzed by qPCR and flow cytometry to detect expression of basal and differentiated cell markers. In separate cultures, keratinocytes were also differentiated by increasing the calcium concentration in feeder- free monolayer culture or establishing ALI culture. Keratinocytes derived from adult or neonatal skin and cultured in DermaCult™ proliferated between 50 - 70 or >70 PDs before undergoing senescence, respectively (n=4). In monolayer cultures, keratinocytes predominantly maintained a cobblestone morphology, expressed basal cell markers (TP63, KRT14, ITGA6), and were devoid of suprabasal marker KRT10 (n=4). Induction of differentiation resulted in the expression of KRT10, LOR, and formation of ZO-1 tight junctions, with concomitant down-regulation of basal markers TP63 and KRT14. In ALI culture, HEKs generated a stratified epithelium with the appropriate cellular architecture and expression of markers similar to their in vivo counterparts. In summary, we have developed an improved and first BPE-free medium for primary HEKs culture that promotes greater expansion of HEKs and maintenance of their differentiation potential at later passages.

Expression of Runt-related Transcription Factor 3 in Human Colon Cancer Cell Line HCT-116 Resistant to 5-Fluorouracil and the Mechanism of Drug Resistance

Objective To establish a human colon cancer cell line HCT-116/5-FU resistant to 5-fluorouracil(5-FU)and explore the relationship between runt-related transcription factor 3(RUNX3)and drug resistance of colorectal cancer.Methods The human colon cancer cell line HCT-116/5-FU with resistance to 5-FU was established by low concentration gradient increment combined with high-dose intermittent shock.CCK-8 method was used to determine the half maximal inhibitory concentration(IC50)of 5-FU on the parent line HCT-116 and drug-resistant line HCT-116/5-FU.The cell growth curve was established for the calculation of population doubling time(TD).The mRNA levels and protein levels of RUNX3,P-glycoprotein(P-gp),multidrug resistance-associated protein 1(MRP1),and lung resistance-related protein(LRP)in HCT-116 and HCT-116/5-FU cells were determined by qRT-PCR and Western blotting,respectively.The RUNX3 expression in HCT-116 cells was knocked down by siRNA technique,and the cells were divided into RUNX3 knockdown groups(si-RUNX3-1 group and si-RUNX3-2 group)and negative control group(si-NC group).The knockdown efficiency was verified by qRT-PCR at the mRNA level and Western blotting at the protein level.The IC50 in si-RUNX3 groups and si-NC group was determined with CCK-8 method,and the expression of P-gp,MRP1,and LRP in the two groups was detected by Western blotting.Results A stable human colon cancer drug-resistant cell line HCT-116/5-FU was successfully constructed.HCT-116/5-FU showed the TD 1.38 times as long as that of HCT-116(P=0.002)and changed morphology.The mRNA level of RUNX3 in HCT-116/5-FU cells was significantly lower than that in HCT-116 cells(P=0.048),and those of P-gp(P=0.008),MRP1(P=0.001),and LRP(P=0.001)showed the opposite trend.The protein level of RUNX3 in HCT-116/5-FU cells was significantly lower than that in HCT-116(P<0.001),and those of P-gp,MRP1,and LRP presented the opposite trend(all P<0.001).The HCT-116 cell model with low expression of RUNX3 was successfully established.The mRNA level of RUNX3 had no significant difference between si-RUNX3-1 group and si-NC group(P=0.064),while the level in si-RUNX3-2 group was significantly lower than that in si-NC group(P=0.034).The protein levels of RUNX3 in si-RUNX3-1 group and si-RUNX3-2 group were lower than that in si-NC group(both P<0.001).The results demonstrated higher knocking efficiency in si-RUNX3-2 group,which was thus selected to complete the follow-up test.The IC50 of si-RUNX3 group was significantly higher than that of si-NC group(P<0.001),which indicated that the down-regulated expression of RUNX3 could reduce the sensitivity of HCT-116 cells to 5-FU.The relative protein levels of P-gp,MRP1,and LRP in si-RUNX3 group were significantly higher than those in si-NC group(all P<0.001).Conclusion The down-regulation of RUNX3 expression can reduce the sensitivity of HCT-116 cells to 5-FU,which is considered to be related to the up-regulated expression of P-gp,MRP1,and LRP.

Establishment of Down\u2019s syndrome periodontal ligament cells by transfection with SV40T-Ag and hTERT

Down’s syndrome is one of the most common human congenital genetic diseases and affected patients have increased risk of periodontal disease. To examine involvement of the disease with periodontal disease development, we established immortalized periodontal ligament cells obtained from a Down’s syndrome patient by use of SV40T-Ag and hTERT gene transfection. Expressions of SV40T-Ag and hTERT were observed in periodontal ligament cell-derived immortalized cells established from healthy (STPDL) and Down’s syndrome patient (STPDLDS) samples. Primary cultured periodontal ligament cells obtained from a healthy subject (pPDL) had a limited number of population doublings (< 40), while STPDL and STPDLDS cells continued to grow with more than 80 population doublings. Primary cultured periodontal ligament cells obtained from the patient showed a chromosome pattern characteristic of Down’s syndrome with trisomy 21, whereas STPDLDS samples showed a large number of abnormal chromosomes in those results. Gene expression analysis revealed that expression of DSCR-1 in STPDLDS is greater than that in STPDL. These results suggest that the newly established STPDLDS cell line may be a useful tool for study of periodontal disease in Down’s syndrome patients.

Evaluating Artificial Diets for the Fall Armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) through Nutritional Indices and an Age-Stage, Two-Sex Life Table Approach

Spodoptera frugiperda (J.E. Smith) is an invasive and key pest of maize, which has been recently introduced to South India. There are two strains reported, viz. rice strain and maize strain. Though several diets were tried in different parts of the world, none of them contained rice leaf powder as a component from natural host. Hence a study was conducted to evaluate the biology, nutritional indices, age-stage and two-sex life table to assess the suitability of the different artificial diets for S. frugiperda larval development. Six differently modified artificial diets were tested to evaluate the following biological parameters: duration of larva and pupae, survival of larvae and pupae, pupal weight, sex ratio, fecundity, adult pre-oviposition period, total pre-oviposition period, adult longevity and total lifespan. The following nutritional indices were determined: growth rate, relative growth rate, consumption rate, consumption index, approximate digestibility, efficiency of conversion of ingested food, efficiency of conversion of digested food and metabolic cost. Larval development showed a significant difference among the diets, in relation to the life table parameters. The results indicated that optimal growth, development and food utilisation by S. frugiperda occurred in the mixture of rice leaf powder and chickpea flour-based diet followed by the mixture of maize leaf powder and common bean powder-based diet. Evaluated diets also had a significant effect on the net reproductive rate, gross reproductive rate, intrinsic rate of increase, finite rate of increase, mean generation time and population doubling time. The values of these parameters were higher in the mixture of rice leaf powder and chickpea flour-based diet followed by mixture of maize leaf powder and common bean powder-based diet. Based on the results, the most adequate diet for rearing in the laboratory was the mixture of rice leaf powder and chickpea flour-based diet.

Cultural Properties of Cryopreserved Thymic Multipotent Stromal Cells and Fetal Skin and Muscle-Derived Cells

The paper provides a comparison of properties of cryopreserved fetal murine multipotent stromal cells (MSCs) of skin-muscular origin and those derived from adult thymus in culture in vitro. Fetal MSCs showed a 30% higher number of average population doublings within 24 hrs, and 41% lower average population doubling time. It was found that the fetal MSCs of the 4th passage had a 39% higher clonogenic activity than the adult thymus-derived ones. Fetal MSCs and those derived from adult thymus differentiated in osteogenic and adipogenic lineages with equal efficiency in special culture media. Fetal and thymus-derived MSCs were characterized by almost the same high ability of contact interaction with thymocytes, and the fibroblast-lymphocyte rosette (FLR) formation. They were far less active in FLR formation with lymph node cells. This indicated the presence of membrane affinity for immature lymphoid cells in both MSC subpopulations. The results showed the fetal MSCs to be significantly different from the adult thymus-derived MSCs by more active kinetics of growth and clonogenic potential. However, both cell subpopulations had virtually the same ability for linear differentiation and showed high activity during contact with immature lymphoid cells. Linear differentiation and the ability to interact with lymphocytes were found to be quite stable properties of MSCs, but a proliferative activity and in vitro colony formation distinguished significantly in different types of MSCs. This can be taken into account when choosing the cells for therapy, research and results assessment.

A critical appraisal of humanized alternatives to fetal bovine serum for clinical applications of umbilical cord derived mesenchymal stromal cells.

The study is aimed to verify the possibility of using humanized alternatives to fetal bovine serum (FBS) such as umbilical cord blood plasma (CBP) and AB+ plasma to support the long-term growth of mesenchymal stromal cells (MSCs) derived from the umbilical cord. We hypothesized that umbilical CBP would be a potential substitute to FBS, especially for small scale autologous clinical transplantations. The MSCs were cultured for six consecutive passages to evaluate xeno-free media's ability to support long-term growth. Cell proliferation rates, colony-forming-unit (CFU) efficiency and population doublings of expanded MSCs, were investigated. Ex vivo expanded MSCs were further characterized using flow cytometry and quantitative PCR. The impact of cryopreservation and composition of cryomedium on phenotype, viability of MSC was also assessed. Our results on cell proliferation, colony-forming unit efficiency suggested that the expansion of the cells was successfully carried out in media supplemented with humanized alternatives. MSCs showed lower CFU counts in FBS (~ 25) than humanized alternatives (~ 35). The gene expression analysis revealed that transcripts showed significant differential expression by two to three folds in the FBS group compared with MSCs grown in medium with humanized alternatives (p < 0.05). In addition, MSCs grown in a medium with FBS had more osteogenic activity, a signatureof unwanted differentiation. The majority of ex vivo expanded MSCs at early and late passages expressed CD44+, CD73+, CD105+, CD90+, and CD166+ in all the experimental groups tested (~ 90%). In contrast to the other MSC surface markers, expression levels of STRO-1+ (~ 21-10%) and TNAP+ (~ 29-11%) decreased with the increase in passage number for MSCs cultured in a FBS-supplemented medium (p < 0.05). Our results established that CBP supported culture of umbilical cord tissue-derived MSCs and is a safer Xeno free replacement to FBS. The use of CBP also enables the storage of umbilical cord tissue derived MSCs in patient-specific conditions to minimize adverse events if cells are delivered directly to the patient.

Multifarious Functions of Butyrylcholinesterase in Neuroblastoma: Impact of BCHE Deletion on the Neuroblastoma Growth In Vitro and In Vivo.

The physiological functions of butyrylcholinesterase (BChE) and its role in malignancy remain unexplained. Our studies in children newly diagnosed with neuroblastoma indicated that BChE expressions is proportional to MYCN amplification suggesting that pathogenesis of high-risk disease may be related to the persistent expression of abnormally high levels of tumor-associated BChE. BChE-deficient neuroblastoma cells (KO [knockout]) were produced from MYCN -amplified BE(2)-C cells (WT [wild-type]) by the CRISPR-Cas9 targeted disruption of the BCHE locus. KO cells have no detectable BChE activity. The compensatory acetylcholinesterase activity was not detected. The average population doubling time of KO cells is 47.0±2.4 hours, >2× longer than WT cells. Reduced proliferation rates of KO cells were accompanied by the loss of N-Myc protein and a significant deactivation of tyrosine kinase receptors associated with the aggressive neuroblastoma phenotype including Ros1, TrkB, and Ltk. Tumorigenicity of WT and KO cells in male mice was essentially identical. In contrast, KO xenografts in female mice were very small (0.37±0.10 g), ~3× smaller compared with WT xenografts (1.11±0.30 g). Unexpectedly, KO xenografts produced changes in plasma BChE similarly to WT tumors but lesser in magnitude. The disruption of BCHE locus in MYCN -amplified neuroblastoma cells decelerates proliferation and produces neuroblastoma cells that are less aggressive in female mice.

Parámetros reproductivos de Tecia solanivora (Povolný, 1973) (Lepidoptera: Gelechiidae) en genotipos de papa (Solanum tuberosum L.)

The Guatemalan potato moth Tecia solanivora is the main pest of potato crop in Colombia. In 2003, Agrosavia began the identification and selection of potato genotypes from the Central Colombian Collection (CCC) for T. solanivora resistance. In 2013 reciprocal diallelic crosses were carried out between commercial varieties and the Guatemalan moth resistant native genotypes. The objective of this study was to determine the effect of potato hybrids obtained from crosses, on T. solanivora demographic parameters under laboratory conditions. Five genotypes and two commercial varieties were evaluated in a non-choice trial, through the development of a life table with two consecutive cohorts. Genotypes 4-69, 23-31 and Perla Negra (PN) were found to cause the highest larval mortality (90, 98 and 98%); 23-31 and PN did not allow adults development. In genotypes 4-69 and 23-31, the net reproductive rate (Ro) (3.92 and 4.38) and the intrinsic rate of natural growth (rm) (0.030 and 0.035) decreased, and the initial population doubling time (TD) increased significantly (18,14 and 19,35). These results suggest that three of the evaluated genotypes could have attributes of resistance to T. solanivora due to the antibiosis mechanism by affecting its biology. This study contributes to the search for materials with resistance to the Guatemalan moth, a line of research for potato breeding in Colombia. Additional Keywords: Guatemalan potato moth, life table, antibiosis, antixenosis, pest resistance.

Environmentally Relevant Levels of Depleted Uranium Impacts Dermal Fibroblast Proliferation, Viability, Metabolic Activity, and Scratch Closure.

Uranium (U) is a heavy metal used in military and industrial settings, with a large portion being mined from the Southwest region of the United States. Uranium has uses in energy and military weaponry, but the mining process has released U into soil and surface waters that may pose threats to human and environmental health. The majority of literature regarding U’s human health concern focuses on outcomes based on unintentional ingestion or inhalation, and limited data are available about its influence via cutaneous contact. Utilizing skin dermis cells, we evaluated U’s topical chemotoxicity. Employing soluble depleted uranium (DU) in the form of uranyl nitrate (UN), we hypothesized that in vitro exposure of UN will have cytotoxic effects on primary dermal fibroblasts by affecting cell viability and metabolic activity and, further, may delay wound healing aspects via altering cell proliferation and migration. Using environmentally relevant levels of U found in water (0.1 M to 100 M [UN]; 23.8–23,800 ppb [U]), we quantified cellular mitosis and migration through growth curves and in vitro scratch assays. Cells were exposed from 24 h to 144 h for a time-course evaluation of UN chemical toxicity. The effects of UN were observed at concentrations above and below the Environmental Protection Agency threshold for safe exposure limits. UN exposure resulted in a dose-dependent decrease in the viable cell count; however, it produced an increase in metabolism when corrected for the viable cells present. Furthermore, cellular proliferation, population doubling, and percent closure was hindered at levels ≥10 M UN. Therefore, inadvertent exposure may exacerbate pre-existing skin diseases in at-risk demographics, and additionally, it may substantially interfere in cutaneous tissue repair processes.

Abstract P325: The Role Of Runx1 In Cardiomyocyte Cell Cycle And Ploidy

Adult mammalian cardiomyocytes (CMs) are thought to be post-mitotic and therefore unable to regenerate the myocardium after injury. In recent years various studies have shown that the adult mammalian CM is capable of a small amount of proliferation, potentially restricted to a subset of CMs. One such study demonstrated that having greater percentages of the rare mononuclear diploid cardiomyocyte (MNDCM) is associated with improved outcomes after myocardial infarction (MI). An accompanying genome-wide association analysis identified genetic loci associated with the frequency of the MNDCM population. One candidate to come out of this screen was Runx1. Concurrently, RUNX1 captured the attention of cardiac regeneration researchers due to its increased presence in disease states, with some suggesting it may be a marker for dedifferentiation (fetal gene induction). One recent study demonstrated improved calcium handling and decreased eccentric hypertrophy following RUNX1 ablation after injury, perhaps corroborating the idea that RUNX1 is involved with CM dedifferentiation. We hypothesize that Runx1 influences dedifferentiation in CMs, impacting ploidy, as well as CM cell cycle activity and post-MI outcomes. We found that CM-specific overexpression (OE) of Runx1 results in a doubling of the MNDCM population, thereby validating its influence on the population. Via multiple contexts including postnatal development and adult injury, knocking out Runx1 decreases DNA synthesis while Runx1 OE increases DNA synthesis. Furthermore, an initial analysis of RNAseq data demonstrates that RUNX1 OE in a neonatal mouse hearts demonstrated differential expression in genes related to cardiac conduction, contraction, heart development, regeneration, and regulation of cell differentiation . After MI in the adult mouse heart, the effects of Runx1 OE resulted in transient benefits which included increased cell cycle activity and preservation of function. These data suggest that Runx1 is not simply a marker of CM dedifferentiation, but also a regulator of the process including cell cycle activation. Ongoing work will tease apart this role in more detail and could establish RUNX1 as a prominent therapeutic target for mitigating effects of cardiac injury.

A study of the transformation of umbilical cord mesenchymal stem cells by interferon-gamma.

Objective(s):Mesenchymal stem cells (MSCs) exist in almost all tissues. Their unique nature is completed by their immunomodulatory functions, holding promise for the treatment of many diseases. An inflammatory environment precedes the immunosuppressive abilities of MSCs and this study was intended to better understand how umbilical cord MSCs (UCMSCs) react to the process of inflammation, regarding their basic characteristics and behavior when primed with the key pro-inflammatory cytokine, Interferon-γ (IFNγ).Materials and Methods:Human MSCs from the umbilical cord were isolated, expanded, and treated with IFNγ. Primed cells were analyzed to define their ability to form colonies, their morphology, differentiation potential, proliferation, and apoptosis rate. Results:UCMSCs treated with IFNγ changed their fibroblast-like morphology and retained the expression of typical MSCs markers. IFNγ treated UCMSCs had significantly higher MFI levels regarding the expression of HLA-I (980.43 ± 556.64) and PD-L1 (598.04 ± 416.90) compared with the control cells (144.97 ± 78.5 and 122.05 ± 103.83, respectively; P<0.01). Under the influence of IFNγ, the cells had a lower population doubling time compared with the control cultures (50.345 ± 9.155 versus 61.135 ± 21.110, respectively; P<0.01) and higher numbers of colony-forming unit-fibroblasts (26.0 ± 12.2 versus 10.2 ± 8.0, respectively; P<0.05). The primed MSCs could not undergo osteogenic and adipogenic differentiation. IFNγ increased the percentage of cells in the apoptotic state on day eight (29.470 ± 6.59 versus 15.708 ± 6.190, respectively; P<0.01).Conclusion:The properties of UCMSCs can be influenced by the pro-inflammatory cytokine IFNγ.

Hot Water Extract from Saccharomyces cerevisiae Scavenges DPPH and Reduces Senescence Associated b-galactosidase (SA-b-Gal) in Human Dermal Fibroblasts

Extracts from Saccharomyces cerevisiae are incorporated in a lot of cosmetic products on the market, but the benefits of the extracts lack scientific reports. We tested the toxicity and anti-senescent activity of an extract from S. cerevisiae on an in vitro model, the human dermal fibroblast (HDF) cell culture. We chronicled the development of the extraction method and the subsequent biochemical assays. We used two extraction methods which were hot water extraction and rapid spin. The optimum duration and growth phase to harvest S. cerevisiae were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, which also proved that the extracts exhibited antioxidant activity. Hot water extract showed a higher antioxidant activity, and not toxic to HDF. When subjected to senescence-associated β-galactosidase (SA-β-Gal) assay, the hot water extract significantly reduced the expression of SA-β-Gal in pre-senescent (passage 20, 30 &lt; population doubling &lt; 40) and senescent (passage 30, population doubling &gt; 50) HDF. In conclusion, S. cerevisiae hot water extract possessed antioxidant activity by scavenging DPPH, and anti-senescent activity by reducing the expression of SA-β-Gal in pre-senescent and senescent HDF.

Expired Platelet Concentrate as a Source of Human Platelet Lysate for Xenogeneic-Free Culture of Human Dermal Fibroblasts

Dermal fibroblasts have been used clinically to promote wound healing and to reduce wrinkles. Most of the time, fetal bovine serum (FBS) is used for the expansion of fibroblasts. In addition, chemically defined medium can also be used for fibroblast expansion. Nonetheless, both FBS and chemically defined medium are not ideal to culture cells that will be used clinically as FBS has the risk of pathogen transmission and induction of xenogeneic immune response whilst chemically defined medium is extremely expensive. In this study, we examine the potential of using human platelet lysate (hPL) prepared from expired platelet concentrates to culture human dermal fibroblasts. For the experiments, fibroblasts were cultured with 5 and 10% hPL, with 10% FBS as the control group to compare the cell morphology, viability, growth rate, extracellular matrix gene expression and wound healing. Results showed that fibroblasts cultured with hPL were more elongated and smaller in size. The cell viability was higher than 90% for all groups. Expansion with 10% hPL significantly shorten the population doubling time compared to the 5% hPL and 10% FBS groups. However, fibroblasts cultured with hPL have lower expression of type I collagen, type III collagen and fibronection as well as slower wound closure. In summary, hPL has the potential to be used as a serum substitute for FBS to expand fibroblasts as it significantly increases the cell proliferation. However, further studies are required to determine if the changes in the ECM gene expression and migration of the hPL-expanded fibroblasts will affect the efficacy of the cells in promoting in vivo wound healing.

In vitro Comparison of Adipogenic Differentiation in Human Adipose-Derived Stem Cells Cultured with Collagen Gel and Platelet-Rich Fibrin.

Background: Adipose-derived stem cells (ADSCs) are the most preferred cell type, based on their phenotypic characteristics, plasticity, and favorable immunological properties for applications in soft-tissue augmentation. Hence, the present in vitro study was aimed to evaluate the adipogenic differentiation potential of human ADSCs upon culturing individually with collagen gel and platelet-rich fibrin (PRF). Materials and methods: The collected lipoaspirate was used for establishing ADSCs using enzymatic digestion method. Then, the cells were analyzed for their morphology, viability, proliferation rate, population doubling time (PDT), colony-forming ability, cell surface markers expression, and osteogenic differentiation as biological properties. Further, ADSCs were evaluated for their adipogenicity using induction media alone, and by culturing with collagen gel and PRF individually for prospective tissue augmentation. Results: ADSCs were successfully established in vitro and exhibited a fibroblast-like morphology throughout the culture period. Cells had higher viability, proliferation potential and showed their ability to form colonies. The positive expression of cell surface markers and osteogenic ability confirmed the potency of ADSCs. The ADSCs cultured on collagen gel and PRF, individually, showed higher number of differentiated adipocytes than ADSCs grown with adipogenic induction medium alone. Conclusion: The extent of lipid accumulation by ADSCs was slightly higher when cultured on collagen gel than on PRF. Additional experiments are required to confirm better suitability of scaffold materials for soft-tissue regeneration.

Restoration of keratinocytic phenotypes in autonomous trisomy-rescued cells

BackgroundAn extra copy of chromosome 21 in humans can alter cellular phenotypes as well as immune and metabolic systems. Down syndrome is associated with many health-related problems and age-related disorders including dermatological abnormalities. However, few studies have focused on the impact of trisomy 21 (T21) on epidermal stem cells and progenitor cell dysfunction. Here, we investigated the differences in keratinocytic characteristics between Down syndrome and euploid cells by differentiating cells from trisomy 21-induced pluripotent stem cells (T21-iPSCs) and autonomous rescued disomy 21-iPSCs (D21-iPSCs).MethodsOur protocol for keratinocytic differentiation of T21-iPSCs and D21-iPSCs was employed. For propagation of T21- and D21-iPSC-derived keratinocytes and cell sheet formation, the culture medium supplemented with Rho kinase inhibitor on mouse feeder cells was introduced as growth rate decreased. Before passaging, selection of a keratinocytic population with differential dispase reactivity was performed. Three-dimensional (3D) air-liquid interface was performed in order to evaluate the ability of iPSC-derived keratinocytes to differentiate and form stratified squamous epithelium.ResultsTrisomy-rescued disomy 21-iPSCs were capable of epidermal differentiation and expressed keratinocytic markers such as KRT14 and TP63 upon differentiation compared to trisomy 21-iPSCs. The lifespan of iPSC-derived keratinocytes could successfully be extended on mouse feeder cells in media containing Rho kinase inhibitor, to more than 34 population doublings over a period of 160 days. Dispase-based purification of disomy iPSC-derived keratinocytes contributed epidermal sheet formation. The trisomy-rescued disomy 21-iPSC-derived keratinocytes with an expanded lifespan generated 3D skin in combination with a dermal fibroblast component.ConclusionsKeratinocytes derived from autonomous trisomy-rescued iPSC have the ability of stratification for manufacturing 3D skin with restoration of keratinocytic functions.