Reduced Proliferative Capacity and Defense against Staphylococcus aureus in Human Nasal Mucosal Epithelium Lacking ZNF365.

INTRODUCTION: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common chronic inflammatory disease of the nose characterized by barrier disruption and environmental susceptibility, and the deletion of ZNF365 may be a factor inducing these manifestations. However, there is no study on the mechanism of action between CRSwNP and ZNF365. Therefore, this study focuses on the effect of the zinc finger protein ZNF365 on the proliferation of nasal mucosal epithelial cells and their defense against Staphylococcus aureus (S. aureus). METHODS: Immunohistochemistry and Western blot were applied to verify the changes of ZNF365 expression in nasal polyp tissues and control tissues, as well as in primary epithelial cells. ZNF365 was knocked down in human nasal mucosa epithelial cell line (HNEpc), and the proliferation, migration, and transdifferentiation of epithelium were observed by immunofluorescence, QPCR, CCK8, and cell scratch assay. The changes of mesenchymal markers and TLR4-MAPK-NF-κB pathway were also observed after the addition of S. aureus. RESULTS: ZNF365 expression was reduced in NP tissues and primary nasal mucosal epithelial cells compared to controls. Knockdown of ZNF365 in HNEpc resulted in decreased proliferation and migration ability of epithelial cells and abnormal epithelial differentiation (decreased expression of tight junction proteins). S. aureus stimulation further inhibited epithelial cell proliferation and migration, while elevated markers of epithelial-mesenchymal transition and inflammatory responses occurred. CONCLUSION: ZNF365 is instrumental in maintaining the proliferative capacity of nasal mucosal epithelial cells and defending against the invasion of S. aureus. The findings suggest that ZNF365 may participate in the development of CRSwNP.

Exogenous Glutathione Promotes the Proliferation of Pinus koraiensis Embryonic Cells and the Synthesis of Glutathione and Ascorbic Acid.

Somatic embryogenesis (SE), which leads to the formation of embryonic callus (EC) tissue, is the most promising method for large-scale production and selective breeding of woody plants. However, in many species, SE suffers from low proliferation rates, hindering the production of improved plant materials. One way of improving proliferation rates is achieved by improving the redox status of the culture medium. In this study, we investigated the effects of exogenous glutathione (GSH) and L-buthionine sulfoximine (BSO, the inhibitor of glutathione synthase) on the EC proliferation rate in Korean pine (Pinus koraiensis), using cell lines with both high (F: 001#-001) and low (S: 001#-010) proliferation potential. We found that exogenous GSH promoted cell proliferation in both cell lines, while exogenous BSO inhibited proliferation in both cell lines. At 35 d with exogenous GSH treatment, the fresh weight of F and S cell lines increased by 35.48% and 48.39%, respectively, compared with the control. The exogenous application of GSH increased the intracellular levels of GSH, total GSH (T-GSH), oxidized glutathione (GSSG), ascorbic acid (ASA), total ASA (T-ASA), and the ratios of GSH:T-GSH and ASA:T-ASA in both F and S cell lines. Furthermore, exogenous GSH increased the activity of both glutathione reductase (GR) and dehydroascorbate reductase (DHAR) while decreasing the activity of ascorbate peroxidase (APX) in both cell lines. It appears that the application of exogenous GSH promotes a reducing cultural environment, which is conducive to EC proliferation in Korean pine. By helping to reveal the mechanism whereby GSH regulates redox homeostasis in Korean pine EC cells, we have laid the foundation for a large-scale breeding of Korean pine somatic embryogenesis technology system.

Marrow changes and reduced proliferative capacity of mesenchymal stromal cells from patients with "no-option" critical limb ischemia; observations on feasibility of the autologous approach from a clinical trial.

BACKGROUND AIMS: Approximately 1 in 3 patients with critical limb ischemia (CLI) are not suitable for surgical or endovascular revascularization. Those "no-option" patients are at high risk of amputation and death. Autologous bone marrow mesenchymal stromal cells (MSCs) may provide a limb salvage option. In this study, bone marrow characteristics and expansion potentials of CLI-derived MSCs produced during a phase 1b clinical trial were compared with young healthy donor MSCs to determine the feasibility of an autologous approach. Cells were produced under Good Manufacturing Practice conditions and underwent appropriate release testing. METHODS: Five bone marrow aspirates derived from patients with CLI were compared with six young healthy donor marrows in terms of number of colony-forming units-fibroblast (CFUF) and mononuclear cells. The mean population doubling times and final cell yields were used to evaluate expansion potential. The effect of increasing the volume of marrow on the CFUF count and final cell yield was evaluated by comparing 5 CLI-derived MSCs batches produced from a targeted 30 mL of marrow aspirate to five batches produced from a targeted 100 mL of marrow. RESULTS: CLI-derived marrow aspirate showed significantly lower numbers of mononuclear cells with no difference in the number of CFUFs when compared with healthy donors' marrow aspirate. CLI-derived MSCs showed a significantly longer population doubling time and reduced final cell yield compared with young healthy donors' MSCs. The poor growth kinetics of CLI MSCs were not mitigated by increasing the bone marrow aspirate from 30 to 100 mL. CONCLUSIONS: In addition to the previously reported karyotype abnormalities in MSCs isolated from patients with CLI, but not in cells from healthy donors, the feasibility of autologous transplantation of bone marrow MSCs for patients with no-option CLI is further limited by the increased expansion time and the reduced cell yield.

Downregulation of TCF1 in HIV Infection Impairs T-cell Proliferative Capacity by Disrupting Mitochondrial Function.

Background: Despite the benefits of antiretroviral therapy (ART) for people with HIV, T-cell dysfunction cannot be fully restored. Metabolic dysregulation is associated with dysfunction of HIV-1-specific T-cells. Exploration of the factors regulating metabolic fitness can help reverse T-cell dysfunction and provide new insights into the underlying mechanism. Methods: In this study, HIV-infected individuals and HIV-negative control individuals (NCs) were enrolled. T-cell factor (TCF)1 expression in cells was determined by quantitative reverse-transcriptase polymerase chain reaction and flow cytometry. Relevant microarray data from the GEO database were analyzed to explore the underlying mechanism. The effects of TCF1 on T-cell function and metabolic function were assessed in vitro. Results: TCF7 mRNA expression in peripheral blood mononuclear cells was downregulated in rapid progressors compared with long-term non-progressors individuals and NCs. TCF1 expression on CD4+ and CD8+ T-cells was downregulated in treatment-naïve HIV-infected individuals compared with NCs. Interleukin (IL)2 production and proliferative capacity were impaired in TCF1 knockdown T-cells. Moreover, glycolytic capacity and mitochondrial respiratory function were decreased in TCF1 knockdown T-cells, and depolarized mitochondria were increased in TCF1 knockdown T-cells. Conclusion: Downregulation of TCF1 in HIV infection impairs T-cell proliferative capacity by disrupting mitochondrial function. These findings highlight the metabolic regulation as a pivotal mechanism of TCF1 in the regulation of T-cell dysfunction.

Quantitative Evaluation of the Reduced Capacity of Skeletal Muscle Hypertrophy after Total Body Irradiation in Relation to Stem/Progenitor Cells.

The effects of total body irradiation (TBI) to the capacity of skeletal muscle hypertrophy were quantified using the compensatory muscle hypertrophy model. We additionally assessed the responses of stem and/or progenitor cells in the muscles. A single TBI of 9.0, 5.0 and 2.5 Gy was delivered to C57BL/6 mice. Bone marrow stromal cells were obtained from GFP-Tg mice, and were injected into the tail vein of the recipient mice (1 × 106 cells/mouse), for bone marrow transplantation (BMT). Five weeks after TBI, the mean GFP-chimerism in the blood was 96 ± 0.8% in the 9 Gy, 83 ± 3.9% in the 5 Gy, and 8.4 ± 3.4% in the 2.5 Gy groups. This implied that the impact of 2.5 Gy is quite low and unavailable as the BMT treatment. Six weeks after the TBI/BMT procedure, muscle hypertrophy was induced in the right plantaris muscle by surgical ablation (SA) of the synergist muscles (gastrocnemius and soleus), and the contralateral left side was preserved as a control. The muscle hypertrophy capacity significantly decreased by 95% in the 9 Gy, 48% in the 5 Gy, and 36% in the 2.5 Gy groups. Furthermore, stem/progenitor cells in the muscle were enzymatically isolated and fractionated into non-sorted bulk cells, CD45-/34-/29+ (Sk-DN), and CD45-/34+ (Sk-34) cells, and myogenic capacity was confirmed by the presence of Pax7+ and MyoD+ cells in culture. Myogenic capacity also declined significantly in the Bulk and Sk-DN cell groups in all three TBI conditions, possibly implying that skeletal muscles are more susceptible to TBI than bone marrow. However, interstitial Sk-34 cells were insusceptible to TBI, retaining their myogenic/proliferative capacity.

Comparison of the rabbit and human corneal endothelial proteomes regarding proliferative capacity.

The shortage of human donor corneas has raised important concerns about engineering of corneal endothelial cells (CECs) for clinical use. However, due to the limited proliferative capacity of human CECs, driving them into proliferation and regeneration may be difficult. Unlike human CECs, rabbit CECs have a marked proliferative capacity. To clarify the potential reason for this difference, we analysed the proteomes of four human corneal endothelium samples and four rabbit corneal endothelium samples with quantitative label-free proteomics and downstream analysis. We discovered that vitamin and selenocompound metabolism and some signaling pathways such as NF-kappa B signaling pathway differed between the samples. Moreover, TGFß, PITX2 and keratocan were distinctively expressed in rabbit samples, which might be associated with active proliferation in rabbit CECs. This study illustrates the proteomic differences between human and rabbit CECs and might promote CEC engineering strategies.

α-Tocopherol Attenuates Oxidative Phosphorylation of CD34+ Cells, Enhances Their G0 Phase Fraction and Promotes Hematopoietic Stem and Primitive Progenitor Cell Maintenance.

Alpha tocopherol acetate (αTOA) is an analogue of alpha tocopherol (αTOC) that exists in the form of an injectable drug. In the context of the metabolic hypothesis of stem cells, we studied the impact of αTOA on the metabolic energetic profile and functional properties of hematopoietic stem and progenitor cells. In ex vivo experiments performed on cord blood CD34+ cells, we found that αTOA effectively attenuates oxidative phosphorylation without affecting the glycolysis rate. This effect concerns complex I and complex II of the mitochondrial respiratory chain and is related to the relatively late increase (3 days) in ROS (Reactive Oxygen Species). The most interesting effect was the inhibition of Hypoxia-Inducible Factor (HIF)-2α (Hexpression, which is a determinant of the most pronounced biological effect-the accumulation of CD34+ cells in the G0 phase of the cell cycle. In parallel, better maintenance of the primitive stem cell activity was revealed by the expansion seen in secondary cultures (higher production of colony forming cells (CFC) and Severe Combined Immunodeficiency-mice (scid)-repopulating cells (SRC)). While the presence of αTOA enhanced the maintenance of Hematopoietic Stem Cells (HSC) and contained their proliferation ex vivo, whether it could play the same role in vivo remained unknown. Creating αTOC deficiency via a vitamin E-free diet in mice, we found an accelerated proliferation of CFC and an expanded compartment of LSK (lineagenegative Sca-1+cKit+) and SLAM (cells expressing Signaling Lymphocytic Activation Molecule family receptors) bone marrow cell populations whose in vivo repopulating capacity was decreased. These in vivo data are in favor of our hypothesis that αTOC may have a physiological role in the maintenance of stem cells. Taking into account that αTOC also exhibits an effect on proliferative capacity, it may also be relevant for the ex vivo manipulation of hematopoietic stem cells. For this purpose, low non-toxic doses of αTOA should be used.

Early Development of Resident Macrophages in the Mouse Cochlea Depends on Yolk Sac Hematopoiesis.

Resident macrophages reside in all tissues throughout the body and play a central role in both tissue homeostasis and inflammation. Although the inner ear was once believed to be "immune-privileged," recent studies have shown that macrophages are distributed in the cochlea and may play important roles in the immune system thereof. Resident macrophages have heterogeneous origins among tissues and throughout developmental stages. However, the origins of embryonic cochlear macrophages remain unknown. Here, we show that the early development of resident macrophages in the mouse cochlea depends on yolk sac hematopoiesis. Accordingly, our results found that macrophages emerging around the developing otocyst at E10.5 exhibited dynamic changes in distribution and in situ proliferative capacity during embryonic and neonatal stages. Cochlear examination in Csf1r-null mice revealed a substantial decrease in the number of Iba1-positive macrophages in the spiral ganglion and spiral ligament, whereas they were still observed in the cochlear mesenchyme or on the intraluminal surface of the perilymphatic space. Our results demonstrated that two subtypes of resident macrophages are present in the embryonic cochlea, one being Csf1r-dependent macrophages that originate from the yolk sac and the other being Csf1r-independent macrophages that appear to be derived from the fetal liver via systemic circulation. We consider the present study to be a starting point for elucidating the roles of embryonic cochlear resident macrophages. Furthermore, resident macrophages in the embryonic cochlea could be a novel target for the treatment of various inner ear disorders.

Timing the spinal cord development with neural progenitor cells losing their proliferative capacity: a theoretical analysis.

In the developing neural tube in chicken and mammals, neural stem cells proliferate and differentiate according to a stereotyped spatiotemporal pattern. Several actors have been identified in the control of this process, from tissue-scale morphogens patterning to intrinsic determinants in neural progenitor cells. In a previous study (Bonnet et al. eLife 7, 2018), we have shown that the CDC25B phosphatase promotes the transition from proliferation to differentiation by stimulating neurogenic divisions, suggesting that it acts as a maturating factor for neural progenitors. In this previous study, we set up a mathematical model linking fixed progenitor modes of division to the dynamics of progenitors and differentiated populations. Here, we extend this model over time to propose a complete dynamical picture of this process. We start from the standard paradigm that progenitors are homogeneous and can perform any type of divisions (proliferative division yielding two progenitors, asymmetric neurogenic divisions yielding one progenitor and one neuron, and terminal symmetric divisions yielding two neurons). We calibrate this model using data published by Saade et al. (Cell Reports 4, 2013) about mode of divisions and population dynamics of progenitors/neurons at different developmental stages. Next, we explore the scenarios in which the progenitor population is actually split into two different pools, one of which is composed of cells that have lost the capacity to perform proliferative divisions. The scenario in which asymmetric neurogenic division would induce such a loss of proliferative capacity appears very relevant.

Effect of an Exercise Program on Lymphocyte Proliferative Responses of COPD Patients.

Exercise training has been shown to reduce symptoms and exacerbations in COPD patients; however, the exercise effect on patients' immune response is poorly known. We thus verified if an exercise program (EP) impacted on proliferative T cell response of COPD patients. Fourteen non-O2 dependent COPD patients on standard treatment were studied. EP consisted in 24 sessions of aerobic and muscular training. Peripheral blood mononuclear cells were stimulated with the mitogen phytohemagglutinin and antigens from Haemophilus influenzae and cytomegalovirus, and the lymphocyte proliferative response (LPR) was assessed through the expression of Ki67 before and after the EP. The Quality of life [COPD assessment test (CAT)], dyspnea [(modified Medical Research Council scale (mMRC)], and 6-min walk distance were also assessed. The EP program increased significantly the LPR of TCD4+ lymphocytes to phytohemagglutinin and cytomegalovirus and H. influenzae antigens, but with TCD8+ lymphocytes the increase was less marked. Consistent with this, a higher proportion of TCD8+ than TCD4+ cells did not express the costimulatory molecule CD28. The EP also resulted in improvement of the quality of life, dyspnea, and physical capacity. The improvement in TCD4+ cell function may represent an additional mechanism through which the EP results in less exacerbations and hospitalizations.

Human mesenchymal stromal cell therapy for damaged cochlea repair in nod-scid mice deafened with kanamycin.

BACKGROUND: Kanamycin, mainly used in the treatment of drug-resistant-tuberculosis, is known to cause irreversible hearing loss. Using the xeno-transplant model, we compared both in vitro and in vivo characteristics of human mesenchymal stromal cells (MSCs) derived from adult tissues, bone marrow (BM-MSCs) and adipose tissue (ADSCs). These tissues were selected for their availability, in vitro multipotency and regenerative potential in vivo in kanamycin-deafened nod-scid mice. METHODS: MSCs were isolated from informed donors and expanded ex vivo. We evaluated their proliferation capacity in vitro using the hexosaminidase assay, the phenotypic profile using flow-cytometry of a panel of surface antigens, the osteogenic potential using alkaline phosphatase activity and the adipogenic potential using oil-red-O staining. MSCs were intravenously injected in deafened mice and cochleae, liver, spleen and kidney were sampled 7 and 30 days after transplantation. The dissected organs were analyzed using lectin histochemistry, immunohistochemistry, polymerase chain reaction (PCR) and dual color fluorescence in situ hybridization (DC-FISH). RESULTS: MSCs showed similar in vitro characteristics, but ADSCs appeared to be more efficient after prolonged expansion. Both cell types engrafted in the cochlea of damaged mice, inducing regeneration of the damaged sensory structures. Several hybrid cells were detected in engrafted tissues. DISCUSSION: BM-MSCs and ADSCs showed in vitro characteristics suitable for tissue regeneration and fused with resident cells in engrafted tissues. The data suggest that paracrine effect is the prevalent mechanism inducing tissue recovery. Overall, BM-MSCs and ADSCs appear to be valuable tools in regenerative medicine for hearing loss recovery.

Stem cell evolutionary paradigm and cell engineering.

Studying hematopoietic and mesenchymal stem cells for almost three decades revealed some similarities between the stem cell entity and the single-celled eukaryotes exhibiting the anaerobic/facultative aerobic metabolic features. A careful analysis of nowadays knowledge concerning the early eukaryotic evolution allowed us to reveal some analogies between stem cells in the metazoan tissues and the single-celled eukaryotes which existed during the first phase of eukaryotes evolution in mid-Proterozoic era. In fact, it is possible to trace the principle of the self-renewal back to the first eukaryotic common ancestor, the first undifferentiated nucleated cell possessing the primitive, mostly anaerobically-respiring mitochondria and a capacity to reproduction by a simple cell division "à l'identique". Similarly, the diversification of these single-cell eukaryotes and acquiring of complex life cycle allowed/conditioned by the increase of O2 in atmosphere (and consequently in the water environment) represents a prototype for the phenomenon of commitment/differentiation. This point of view allowed to predict the ex-vivo behavior of stem cells with respect to the O2 availability and metabolic profile which enabled to conceive the successful protocols of stem cell expansion and ex vivo conditioning based on "respecting" this relationship between the anaerobiosis and stemness. In this review, the basic elements of this paradigm and a possible application in cell engineering were discussed.

Phenotype and multipotency of rabbit (Oryctolagus cuniculus) amniotic stem cells.

BACKGROUND: Stem cells are capable of unlimited self-renewal and are able to remain undifferentiated for extended periods of time prior to their differentiation into specific cell lineages. Because of the issues (ethical and religious) involved in the use of embryonic stem cells and the limited plasticity of adult stem cells, an alternative cell source could be foetal stem cells derived from extra-embryonic tissue, which are highly proliferative, grow in vitro and possess interesting immunogenic characteristics. As a result, the amniotic membrane of several species has been studied as an important new source of stem cells. METHODS: Here, we cultured and characterized mesenchymal progenitor cells derived from the rabbit amniotic membrane, and investigated their differentiation potential. In total, amniotic membranes were collected from eight rabbit foetuses and were isolated by the explant technique. The obtained cells were cultured in DMEM-HIGH glucose and incubated at 37 °C in a humidified atmosphere with 5% CO2. RESULTS: The cells adhered to the culture plates and showed a high proliferative capacity with fibroblast-like morphologies. The cells showed a positive response for markers for the cytoskeleton, mesenchymal stem cells and proliferation, pluripotency and haematopoietic precursor stem cells. However, the cells were negative for CD45, a marker of haematopoietic cells. Furthermore, the cells had the capacity to be induced to differentiate into osteogenic, adipogenic and chondrogenic lineages. In addition, when the cells were injected into nude mice, we did not observe the formation of tumours. CONCLUSIONS: In summary, our results demonstrate that multipotent mesenchymal stem cells can be obtained from the rabbit amniotic membrane for possible use in future cell therapy applications.

A biological study establishing the endotoxin limit for in vitro proliferation of human mesenchymal stem cells.

INTRODUCTION: Multipotent mesenchymal stem cells (MSCs) are widespread in adult organisms and are implicated in tissue maintenance and repair, regulation of hematopoiesis, and immunologic responses. Human (h)MSCs have applications in tissue engineering, cell-based therapy, and medical devices but it is unclear how they respond to unfavorable conditions such as hypoxia or inflammation after in vivo transplantation. Although endotoxin testing is a requirement for evaluating the quality and safety of transplanted MSCs, there have been no reports on the dose response to endotoxins to establish limits for in vitro MSC culture systems. The present study aimed to accurately quantify the risk of endotoxin contamination in cell culture systems in order to establish the acceptable endotoxin limit for hMSC proliferation. METHODS: Three types of bone marrow-derived hMSC (hMSC-1: 21 years, M/B; hMSC-2: 36 years, M/B; hMSC-3: 43 years, M/C) and adipose-derived stem cells (ADSCs; StemPro Human) were cultured in medium from commercial kits containing various concentrations of endotoxin (0.1-1000 ng/ml). The proliferative capacity of cells was estimated by cell counts using a hemocytometer. To clarify the molecular mechanism underlying the effect of endotoxin on hMSCs proliferation, cellular proteins were extracted from cultured cells and subjected to liquid chromatograph-tandem mass spectrometry shotgun proteomics analysis. The expression of Cu/Zn-type superoxide dismutase (SOD1) and Fe/Mn-type superoxide dismutase (SOD2) induced in hMSCs by endotoxin stimulation were evaluated by enzyme-linked immunosorbent assay (ELISA), and the effect of SOD2 on hMSC proliferation was also estimated. RESULTS: Although there was no change in cell morphology during the culture period, proliferative capacity increased with endotoxin concentration to over 0.1 ng/ml for ADSCs, 1 ng/ml for hMSC-1, and 100 ng/ml for hMSC-2; hMSC-3 proliferation was unaffected by the presence of endotoxin. A proteomic analysis of hMSC-1 revealed that various proteins related to the cell cycle, apoptosis, and host defense against infection were altered by endotoxin stimulation, whereas SOD2 expression was significantly and consistently upregulated during the culture period. The latter was also confirmed by ELISA. Moreover, recombinant SOD2 increased proliferative capacity in hMSC-1 cells in a manner similar to endotoxin. These results suggest that endotoxin protects MSCs from oxidative stress via upregulation of SOD2 to improve cell survival. CONCLUSIONS: Since endotoxins can affect various cellular functions, an endotoxin limit should be set for in vitro MSC cultures. The lowest observed adverse effect level was determined to be 0.1 ng/ml based on the effect on MSC proliferation.

Rotation is the primary motion of paired human epidermal keratinocytes.

BACKGROUND: Collective motion of keratinocytes is involved in morphogenesis, homeostasis, and wound healing of the epidermis. Yet how the collective motion of keratinocytes emerges from the behavior of individual cells is still largely unknown. OBJECTIVE: The aim of this study was to find the cellular behavior that links single and collective motion of keratinocytes. METHODS: We investigated the behavior of two-cell colonies of HaCaT keratinocytes by a combination of time-lapse imaging and image processing. RESULTS: The two-cell colonies of HaCaT cells were formed as a contacted pair of keratinocyte clones. Image analysis and cell culture experiments revealed that the rotational speed of two-cell colonies was positively associated with their proliferative capacity. α6 integrin was required for the rotational motion of two-cell keratinocyte colonies. We also confirmed that two-cell colonies of keratinocytes predominantly exhibited the rotational, but not translational, motion, two modes of motion in a contact pair of rotating objects. CONCLUSION: The rotational motion is the primary motion of two-cell keratinocyte colonies and its speed is positively associated with their proliferative capacity. This study suggests that the assembly of rotating keratinocytes generates the collective motion of proliferative keratinocytes during morphogenesis and wound healing of the epidermis.

microRNA-103/107 Family Regulates Multiple Epithelial Stem Cell Characteristics.

The stem cell niche is thought to affect cell cycle quiescence, proliferative capacity, and communication between stem cells and their neighbors. How these activities are controlled is not completely understood. Here we define a microRNA family (miRs-103/107) preferentially expressed in the stem cell-enriched limbal epithelium that regulates and integrates these stem cell characteristics. miRs-103/107 target the ribosomal kinase p90RSK2, thereby arresting cells in G0/G1 and contributing to a slow-cycling phenotype. Furthermore, miRs-103/107 increase the proliferative capacity of keratinocytes by targeting Wnt3a, which enhances Sox9 and YAP1 levels and thus promotes a stem cell phenotype. This miRNA family also regulates keratinocyte cell-cell communication by targeting: (a) the scaffolding protein NEDD9, preserving E-cadherin-mediated cell adhesion; and (b) the tyrosine phosphatase PTPRM, which negatively regulates connexin 43-based gap junctions. We propose that such regulation of cell communication and adhesion molecules maintains the integrity of the stem cell niche ultimately preserving self-renewal, a hallmark of epithelial stem cells.

Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells.

Telomere extension has been proposed as a means to improve cell culture and tissue engineering and to treat disease. However, telomere extension by nonviral, nonintegrating methods remains inefficient. Here we report that delivery of modified mRNA encoding TERT to human fibroblasts and myoblasts increases telomerase activity transiently (24-48 h) and rapidly extends telomeres, after which telomeres resume shortening. Three successive transfections over a 4 d period extended telomeres up to 0.9 kb in a cell type-specific manner in fibroblasts and myoblasts and conferred an additional 28 ± 1.5 and 3.4 ± 0.4 population doublings (PDs), respectively. Proliferative capacity increased in a dose-dependent manner. The second and third transfections had less effect on proliferative capacity than the first, revealing a refractory period. However, the refractory period was transient as a later fourth transfection increased fibroblast proliferative capacity by an additional 15.2 ± 1.1 PDs, similar to the first transfection. Overall, these treatments led to an increase in absolute cell number of more than 10(12)-fold. Notably, unlike immortalized cells, all treated cell populations eventually stopped increasing in number and expressed senescence markers to the same extent as untreated cells. This rapid method of extending telomeres and increasing cell proliferative capacity without risk of insertional mutagenesis should have broad utility in disease modeling, drug screening, and regenerative medicine.

SENIEUR status of the originating cell donor negates certain 'anti-immunosenescence' effects of ebselen and N-acetyl cysteine in human T cell clone cultures.

BACKGROUND: Damage to T cells of the immune system by reactive oxygen species may result in altered cell function or cell death and thereby potentially impact upon the efficacy of a subsequent immune response. Here, we assess the impact of the antioxidants Ebselen and N-acetyl cysteine on a range of biological markers in human T cells derived from a SENIEUR status donor. In addition, the impact of these antioxidants on different MAP kinase pathways in T cells from donors of different ages was also examined. METHODS: T cell clones were derived from healthy 26, 45 and SENIEUR status 80 year old people and the impact of titrated concentrations of Ebselen or N-acetyl cysteine on their proliferation and in vitro lifespan, GSH:GSSG ratio as well as levels of oxidative DNA damage and on MAP kinase signaling pathways was examined. RESULTS: In this investigation neither Ebselen nor N-acetyl cysteine supplementation had any impact on the biological endpoints examined in the T cells derived from the SENIEUR status 80 year old donor. This is in contrast to the anti-immunosenescent effects of these antioxidants on T cells from donors of 26 or 45 years of age. The analysis of MAP kinases showed that pro-apoptotic pathways become activated in T cells with increasing in vitro age and that Ebselen or N-acetyl cysteine could decrease activation (phosphorylation) in T cells from 26 or 45 year old donors, but not from the SENIEUR status 80 year old donor. CONCLUSIONS: The results of this investigation demonstrate that the biological phenotype of SENIEUR status derived human T cells negates the anti-immunosenescence effects of Ebselen and also N-acetyl cysteine. The results highlight the importance of pre-antioxidant intervention evaluation to determine risk-benefit.

Chemical characteristics combined with bioactivity for comprehensive evaluation of Panax ginseng C.A. Meyer in different ages and seasons based on HPLC-DAD and chemometric methods.

Panax ginseng C.A. Meyer has been known as a valuable traditional Chinese medicines for thousands years of history. Ginsenosides, the main active constituents, exhibit prominent immunoregulation effect. The present study first describes a holistic method based on chemical characteristic and lymphocyte proliferative capacity to evaluate systematically the quality of P. ginseng in thirty samples from different seasons during 2-6 years. The HPLC fingerprints were evaluated using principle component analysis (PCA) and hierarchical clustering analysis (HCA). The spectrum-efficacy model between HPLC fingerprints and T-lymphocyte proliferative activities was investigated by principal component regression (PCR) and partial least squares (PLS). The results indicated that the growth of the ginsenosides could be grouped into three periods and from August of the fifth year, P. ginseng appeared significant lymphocyte proliferative capacity. Close correlation existed between the spectrum-efficacy relationship and ginsenosides Rb1, Ro, Rc, Rb2 and Re were the main contributive components to the lymphocyte proliferative capacity. This comprehensive strategy, providing reliable and adequate scientific evidence, could be applied to other TCMs to ameliorate their quality control.