Senescent Changes Research Articles

Abstract 53: Discrepancies in Senescence and Protein Expression from Cells in Nerve Autografts Compared to Injured Nerve

PURPOSE: Outcomes of nerve autografting are often merely adequate to poor, especially as the nerve gap increases in length. We hypothesize senescent cells accumulate in longer grafts and resultant changes in protein expression contribute to reduced regeneration. METHODS: A rat sciatic nerve transection model was used to compare short (2cm) and long (6cm) isografts (equivalent to autograft in syngeneic animals), while injured (no repair) and cut-repair nerves were used as controls.Grafts were analyzed after 4 and 8 weeks in vivo with histology, gene expression, histomorphometry, and retrograde labeling. RESULTS: Axon counts clearly demonstrated disproportionately reduced regeneration in the longer graft compared to the short graft when measuring at equivalent distances to the spinal cord: the number of regenerated axons in the long graft was 57% of those regenerated in the short graft. Additionally, retrograde labeling showed significantly fewer motoneurons were found to be regenerating axons to long grafts compared to the short graft repairs. Cell composition amongst the different grafts and injured nerve were surprisingly consistent: groups had similar total numbers of cells and similar proportions of fibroblasts, Schwann cells, and macrophages. Immunohistochemical analysis showed an increased percentage of cells with senescent markers in the long grafts and in the “normal” nerve proximal to the graft. While gene expression for senescent markers was increased in both long and short nerve grafts, these markers remained elevated over time in the long grafts. Interestingly, GDNF and IL-6 expression was elevated in the long grafts compared to the short. Finally, immunohistochemistry revealed increased NOTCH signalling in the long grafts. CONCLUSION: Comparison of short and long grafts, at points equidistant to the spinal cord, showed reduced axon regeneration and reduced number of motoneurons regenerating axons. Given this reduced regeneration in the presence of a long graft and the finding that cellular composition was unchanged, immunohistochemistry and gene expression were used to identify gene and protein expression differences in the grafts.The longer grafts were found to have a greater proportion of senescent cells and the nerve proximal to the graft even showed senescent changes. Gene expression associated with senescent was increased in the grafts but retained over time in the longer grafts. Prior work has linked increased NOTCH signalling to reduced neurite extension from dorsal root ganglia neurons and the current work links increasing graft length with increased NOTCH signalling. These protein and gene expression changes give insight as to the poor clinical outcomes associated with autografts and provide targets for improvement.

Senescent Changes and Topography of the Dark-Adapted Multifocal Electroretinogram.

PurposeTo investigate the topographic changes of the dark-adapted multifocal electroretinogram (mfERG) across adulthood in the central retina and compare the topography between macular versus extramacular, nasal versus temporal, and inferior versus superior retinal areas.MethodsSixty-five subjects (18–88 years) received a comprehensive dilated eye examination to ensure the health of their retina and were tested with a dark-adapted mfERG protocol using a 61-hexagon pattern. The lens absorption of each subject was also estimated using a heterochromatic flicker photometry (HFP) paradigm.ResultsThe response amplitude and latency of the dark-adapted mfERG showed a significant change with age, which was best described with a linear model. All the retinal areas examined demonstrated similar aging effects. The extramacular and temporal retina showed higher response amplitude and faster response latency when compared with the macular and nasal retinae, respectively. No difference was found in response amplitude and latency between the inferior and superior retina. The HFP results also showed a significant correlation with age, consistent with senescent increases in short wavelength absorption by the crystalline lens. However, the change in lens absorption did not exceed the magnitude of the change in response amplitude and latency.DiscussionOur results indicate that there is a decline in dark-adapted retinal activity as measured with the mfERG. These aging processes affect rods and rod-bipolar cells. Their decrease in response can be attributed to both optical and neural factors.

Variants of core histones and their roles in cell fate decisions, development and cancer.

Histone variants endow chromatin with unique properties and show a specific genomic distribution that is regulated by specific deposition and removal machineries. These variants - in particular, H2A.Z, macroH2A and H3.3 - have important roles in early embryonic development, and they regulate the lineage commitment of stem cells, as well as the converse process of somatic cell reprogramming to pluripotency. Recent progress has also shed light on how mutations, transcriptional deregulation and changes in the deposition machineries of histone variants affect the process of tumorigenesis. These alterations promote or even drive cancer development through mechanisms that involve changes in epigenetic plasticity, genomic stability and senescence, and by activating and sustaining cancer-promoting gene expression programmes.

Negative auto-regulators trap p53 in their web.

The transcriptional factor p53 activates the expression of a myriad of target genes involving a complicated signalling network, resulting in various cellular outcomes, such as growth arrest, senescence, apoptosis, and metabolic changes, and leading to consequent suppression of tumour growth and progression. Because of the profoundly adverse effect of p53 on growth and proliferation of cancer cells, several feedback mechanisms have been employed by the cells to constrain p53 activity. Two major antagonists MDM2 and MDMX (the long forms) are transcriptionally induced by p53, but in return block p53 activity, forming a negative feedback circuit and rendering chemoresistance of several cancer cells. However, they are not alone, as cancer cells also employ other proteins encoded by p53 target genes to inhibit p53 activity at transcriptional, translational, and posttranslational levels. This essay is thus composed to review a recent progress in understanding the mechanisms for how cancer cells hijack the p53 autoregulation by these proteins for their growth advantage and to discuss the clinical implications of these autoregulatory loops.

MicroRNAs, Aging, Cellular Senescence, and Alzheimer's Disease.

Aging is a normal process of living being. It has been reported that multiple cellular changes, including oxidative damage/mitochondrial dysfunction, telomere shortening, inflammation, may accelerate the aging process, leading to cellular senescence. These cellular changes induce age-related human diseases, including Alzheimer's, Parkinson's, multiple sclerosis, amyotrophic lateral sclerosis, cardiovascular, cancer, and skin diseases. Changes in somatic and germ-line DNA and epigenetics are reported to play large roles in accelerating the onset of human diseases. Cellular mechanisms of aging and age-related diseases are not completely understood. However, recent discoveries in molecular biology have revealed that microRNAs (miRNAs) are potential indicators of aging, cellular senescence, and Alzheimer's disease (AD). The purpose of our chapter is to highlight recent advancements in miRNAs and their involvement in cellular changes in aging, cellular senescence, and AD. This chapter also critically evaluates miRNA-based therapeutic drug targets for aging and age-related diseases, particularly Alzheimer's.

Mitotic catastrophe and cell cycle arrest are alternative cell death pathways executed by bortezomib in rituximab resistant B-cell lymphoma cells.

The ubiqutin-proteasome system (UPS) plays a role in rituximab-chemotherapy resistance and bortezomib (BTZ) possesses caspase-dependent (i.e. Bak stabilization) and a less characterized caspase–independent mechanism-of-action(s). Here, we define BTZ-induced caspase-independent cell death pathways. A panel of rituximab-sensitive (RSCL), rituximab-resistant cell lines (RRCL) and primary tumor cells derived from lymphoma patients (N = 13) were exposed to BTZ. Changes in cell viability, cell-cycle, senescence, and mitotic index were quantified. In resting conditions, RRCL exhibits a low-proliferation rate, accumulation of cells in S-phase and senescence. Exposure of RRCL to BTZ reduces cell senescence, induced G2-M phase cell-cycle arrest, and is associated with mitotic catastrophe. BTZ stabilized p21, CDC2, and cyclin B in RRCL and in primary tumor cells. Transient p21 knockdown alleviates BTZ-induced senescence inhibition, G2-M cell cycle blockade, and mitotic catastrophe. Our data suggest that BTZ can induce apoptosis or mitotic catastrophe and that p21 has a pivotal role in BTZ activity against RRCL.

The endocrine-brain-aging triad where many paths meet: female reproductive hormone changes at midlife and their influence on circuits important for learning and memory

Female mammals undergo natural fluctuations in sex steroid hormone levels throughout life. These fluctuations span from early development, to cyclic changes associated with the menstrual or estrous cycle and pregnancy, to marked hormone flux during perimenopause, and a final decline at reproductive senescence. While the transition to reproductive senescence is not yet fully understood, the vast majority of mammals experience this spontaneous, natural phenomenon with age, which has broad implications for long-lived species. Indeed, this post-reproductive life stage, and its transition, involves significant and enduring physiological changes, including considerably altered sex steroid hormone and gonadotropin profiles that impact multiple body systems, including the brain. The endocrine-brain-aging triad is especially noteworthy, as many paths meet and interact. Many of the brain regions affected by aging are also sensitive to changes in ovarian hormone levels, and aging and reproductive senescence are both associated with changes in memory performance. This review explores how menopause is related to cognitive aging, and discusses some of the key neural systems and molecular factors altered with age and reproductive hormone level changes, with an emphasis on brain regions important for learning and memory.

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products. Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions — namely, patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor's own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions — namely, patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor's own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Senescence-Like Phenotypes in Human Nevi.

Cellular senescence is an irreversible arrest of cell proliferation at the G1 stage of the cell cycle in which cells become refractory to growth stimuli. Senescence is a critical and potent defense mechanism that mammalian cells use to suppress tumors. While there are many ways to induce a senescence response, oncogene-induced senescence (OIS) remains the key to inhibiting progression of cells that have acquired oncogenic mutations. In primary cells in culture, OIS induces a set of measurable phenotypic and behavioral changes, in addition to cell cycle exit. Senescence-associated β-Galactosidase (SA-β-Gal) activity is a main hallmark of senescent cells, along with morphological changes that may depend on the oncogene that is activated, or on the primary cell type. Characteristic cellular changes of senescence include increased size, flattening, multinucleation, and extensive vacuolation. At the molecular level, tumor suppressor genes such as p53 and p16 INK4A may play a role in initiation or maintenance of OIS. Activation of a DNA damage response and a senescence-associated secretory phenotype could delineate the onset of senescence. Despite advances in our understanding of how OIS suppresses some tumor types, the in vivo role of OIS in melanocytic nevi and melanoma remains poorly understood and not validated. In an effort to stimulate research in this field, we review in this chapter the known markers of senescence and provide experimental protocols for their identification by immunofluorescent staining in melanocytic nevi and malignant melanoma.

The Impact of Age on the Female Reproductive System.

Evaluation of the female reproductive system in a general toxicity setting can be challenging for the toxicologic pathologist due to the cyclic nature of the estrous and menstrual cycles, timing of puberty and reproductive senescence, and species differences. Age in particular can have a significant impact on the histologic appearance of the female reproductive system and create challenges when trying to distinguish test article-related findings from normal developmental or senescent changes. This review describes the key physiologic and histologic features of immaturity, the transition through puberty, sexual maturity, and reproductive senescence in the female reproductive system, with an emphasis on practical applications for the toxicologic pathologist, and includes recommendations for distinguishing and documenting these developmental periods. Rats and cynomolgus monkeys are used as examples throughout with correlations to clinically observed end points to better aid the toxicologic pathologist in understanding how age may impact study interpretation.

Transplantation of osteoporotic bone marrow stromal cells rejuvenated by the overexpression of SATB2 prevents alveolar bone loss in ovariectomized rats

Estrogen-deficient osteoporosis is an aging-related disease with high morbidity that not only significantly increases a woman's risk of fragility fracture but is also associated with tooth and bone loss in the supporting alveolar bone of the jaw. Emerging evidence suggests that the aging of bone marrow stromal cells (BMSCs) contributes to the development of osteoporosis. In this study, we aimed to investigate the role of the special AT-rich sequence-binding protein 2 (SATB2), a stemness and senescence regulator of craniofacial BMSCs, in rat ovariectomy-induced alveolar osteoporosis. We also sought to determine whether transplantation of SATB2-modified BMSCs could ameliorate estrogen deficient alveolar bone loss. Our data revealed that BMSCs from ovariectomy-induced alveolar bone exhibited typical senescence phenotypes such as diminished stemness and osteogenic capacity, increased expression of senescence or osteoclastic markers and enhanced adipogenic potential. These phenotypic changes are a result of SATB2-mediated senescence dysregulation as evidenced by nuclear γH2AX foci formation. Moreover, overexpression of SATB2 significantly alleviated the senescence of osteoporotic BMSCs in vitro. Importantly, transplantation of SATB2-modified BMSCs significantly attenuated ovariectomy-induced alveolar bone loss in vivo. Together, our results revealed that SATB2 is a critical regulator of alveolar BMSC senescence, and its overexpression decreases these senescent changes both in vitro and in vivo. SATB2-modified BMSC delivery could be a viable and promising therapeutic strategy for alveolar bone loss induced by estrogen-deficient osteoporosis.

Monitoring Agronomic Parameters of Winter Wheat Crops with Low-Cost UAV Imagery

Monitoring the dynamics in wheat crops requires near-term observations with high spatial resolution due to the complex factors influencing wheat growth variability. We studied the prospects for monitoring the biophysical parameters and nitrogen status in wheat crops with low-cost imagery acquired from unmanned aerial vehicles (UAV) over an 11 ha field. Flight missions were conducted at approximately 50 m in altitude with a commercial copter and camera system—three missions were performed between booting and maturing of the wheat plants and one mission after tillage. Ultra-high resolution orthoimages of 1.2 cm·px−1 and surface models were generated for each mission from the standard red, green and blue (RGB) aerial images. The image variables were extracted from image tone and surface models, e.g., RGB ratios, crop coverage and plant height. During each mission, 20 plots within the wheat canopy with 1 × 1 m2 sample support were selected in the field, and the leaf area index, plant height, fresh and dry biomass and nitrogen concentrations were measured. From the generated UAV imagery, we were able to follow the changes in early senescence at the individual plant level in the wheat crops. Changes in the pattern of the wheat canopy varied drastically from one mission to the next, which supported the need for instantaneous observations, as delivered by UAV imagery. The correlations between the biophysical parameters and image variables were highly significant during each mission, and the regression models calculated with the principal components of the image variables yielded R2 values between 0.70 and 0.97. In contrast, the models of the nitrogen concentrations yielded low R2 values with the best model obtained at flowering (R2 = 0.65). The nitrogen nutrition index was calculated with an accuracy of 0.10 to 0.11 NNI for each mission. For all models, information about the surface models and image tone was important. We conclude that low-cost RGB UAV imagery will strongly aid farmers in observing biophysical characteristics, but it is limited for observing the nitrogen status within wheat crops.

Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability.

BackgroundHistone modification H4K20me3 and its methyltransferase SUV420H2 have been implicated in suppression of tumorigenesis. The underlying mechanism is unclear, although H4K20me3 abundance increases during cellular senescence, a stable proliferation arrest and tumor suppressor process, triggered by diverse molecular cues, including activated oncogenes. Here, we investigate the function of H4K20me3 in senescence and tumor suppression.ResultsUsing immunofluorescence and ChIP-seq we determine the distribution of H4K20me3 in proliferating and senescent human cells. Altered H4K20me3 in senescence is coupled to H4K16ac and DNA methylation changes in senescence. In senescent cells, H4K20me3 is especially enriched at DNA sequences contained within specialized domains of senescence-associated heterochromatin foci (SAHF), as well as specific families of non-genic and genic repeats. Altered H4K20me3 does not correlate strongly with changes in gene expression between proliferating and senescent cells; however, in senescent cells, but not proliferating cells, H4K20me3 enrichment at gene bodies correlates inversely with gene expression, reflecting de novo accumulation of H4K20me3 at repressed genes in senescent cells, including at genes also repressed in proliferating cells. Although elevated SUV420H2 upregulates H4K20me3, this does not accelerate senescence of primary human cells. However, elevated SUV420H2/H4K20me3 reinforces oncogene-induced senescence-associated proliferation arrest and slows tumorigenesis in vivo.ConclusionsThese results corroborate a role for chromatin in underpinning the senescence phenotype but do not support a major role for H4K20me3 in initiation of senescence. Rather, we speculate that H4K20me3 plays a role in heterochromatinization and stabilization of the epigenome and genome of pre-malignant, oncogene-expressing senescent cells, thereby suppressing epigenetic and genetic instability and contributing to long-term senescence-mediated tumor suppression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1017-x) contains supplementary material, which is available to authorized users.

Lower facial contouring: dermal filler approaches and techniques

With the onset of age, the lower face displays a number of senescent changes, manifested through skeletal remodelling and subcutaneous and deep facial fat descent, as well as textural changes in skin tone and texture. Anna Baker reviews the literature concerning the salient anatomy for lower facial rejuvenation and critically analyses treatment approaches with dermal fillers

Transcriptomic analyses reveal senescent and SnoRNA changes in B cells with age irrespective of vaccine-stimulation in mice.

Abstract Aging is associated with a decline in immune function that is not fully understood. Transcriptomic analysis is a powerful tool to evaluate age-related changes, but has not yet been reported for B-cells. To address this, we performed RNA-seq analyses on purified B-cells from naïve or vaccinated BALB/c mice of varying ages with the goal of expression profiling by age as opposed to any ongoing immunostimulation. Mouse age groups were Y (young, 15–20 wks), MA (middle-aged, 68–81 wks), and O (older, 103–111 wks) ± intramuscular Flulaval injection (n=5). Sequencing was performed using Ion Torrent/Ion Proton System. PCR and phenotype analyses were performed to validate selected results. In naïve groups, 230 genes were significantly differentially expressed by MA and O compared to Y. Nine of these were also significant comparing MA and O to Y in vaccinated groups. These included protein-coding genes: senescence-associated Cdkn2a; NFκB-mediator Edaradd; Klk4, a kallikrein implicated in cancer; and 4921504E06Rik, a gene region linked to inflammatory disease. Non-coding genes included: pseudogenes Gm2619 and RP24-374C7.3; antisense Gm16028; and box C/D small nuclear (sno) RNAs, Snord123 and Snord1a. The latter genes were particularly interesting as box C/D snoRNAs regulate rRNAs through methylation, and changes in their expression levels occur with neurodegenerative disorders, inflammatory diseases, and cancer. Altered DNA methylation or epigenetic drift is implicated in both healthy aging and age-related dysfunction but exact mechanisms are unclear. Our results suggest that Snord123 and Snord1a play a previously unappreciated role as regulators of altered methylation patterns contributing to age-related immune decline in B-cells.

In vitro lifespan and senescent behaviour of human periosteal derived stem cells

Periosteum derived progenitor cells (PDPCs) represent promising mesenchymal stem cells (MSCs) for skeletal regeneration and to test bone cell based tissue engineering strategies. Most of regenerative medicine approaches based on MSCs require a noteworthy amount of cells that must be expanded in vitro prior to their use. As culture expansion method may impact on cell behaviour, we assessed the replicative and metabolic capacity (nitric oxide production and glucose consumption), senescence hallmarks of PDPCs serially passaged as well as the expression of selected genes specifically related to early osteoblastic differentiation, bone remodelling and stemness during PDPC sequential passaging. We also scouted a Systems Biology approach to examine and elucidate the experimental results through mathematical modelling and in silico simulations. PDPC subculture led to a progressive proliferative decline but, despite this, PDPCs maintained almost constant their metabolic activity. In vitro, senescent PDPCs displayed the typical “replicative senescence” features, involving p16 and not p53 in the regulation of this phenomenon. Gene expression analysis evidenced the tendency of sub-cultured PDPCs to increase the expression of genes involved in bone resorption. The mathematical analysis of the experimental results showed a strict similarity between replicative senescence and age-related changes, enabling the definition of an in silico model mimicking PDPC behaviour in terms of nitric oxide (NO) production. The relationship between NO production and subculture passages could represent a cutting edge “replicative senescence index”. Overall, our findings suggest the possibility to use early-passage PDPCs for bone regenerative approaches based on the local recruitment of stem cells, whilst the later cell passages could be a suitable in vitro tool to validate scaffolds intended for bone regeneration in elderly subjects.

Monitoring Grassland Seasonal Carbon Dynamics, by Integrating MODIS NDVI, Proximal Optical Sampling, and Eddy Covariance Measurements

This study evaluated the seasonal productivity of a prairie grassland (Mattheis Ranch, in Alberta, Canada) using a combination of remote sensing, eddy covariance, and field sampling collected in 2012–2013. A primary objective was to evaluate different ways of parameterizing the light-use efficiency (LUE) model for assessing net ecosystem fluxes at two sites with contrasting productivity. Three variations on the NDVI (Normalized Difference Vegetation Index), differing by formula and footprint, were derived: (1) a narrow-band NDVI (NDVI680,800, derived from mobile field spectrometer readings); (2) a broad-band proxy NDVI (derived from an automated optical phenology station consisting of broad-band radiometers); and (3) a satellite NDVI (derived from MODIS AQUA and TERRA sensors). Harvested biomass, net CO2 flux, and NDVI values were compared to provide a basis for assessing seasonal ecosystem productivity and gap filling of tower flux data. All three NDVIs provided good estimates of dry green biomass and were able to clearly show seasonal changes in vegetation growth and senescence, confirming their utility as metrics of productivity. When relating fluxes and optical measurements, temporal aggregation periods were considered to determine the impact of aggregation on model accuracy. NDVI values from the different methods were also calibrated against fAPARgreen (the fraction of photosynthetically active radiation absorbed by green vegetation) values to parameterize the APARgreen (absorbed PAR) term of the LUE (light use efficiency) model for comparison with measured fluxes. While efficiency was assumed to be constant in the model, this analysis revealed hysteresis in the seasonal relationships between fluxes and optical measurements, suggesting a slight change in efficiency between the first and second half of the growing season. Consequently, the best results were obtained by splitting the data into two stages, a greening phase and a senescence phase, and applying separate fits to these two periods. By incorporating the dynamic irradiance regime, the model based on APARgreen rather than NDVI best captured the high variability of the fluxes and provided a more realistic depiction of missing fluxes. The strong correlations between these optical measurements and independently measured fluxes demonstrate the utility of integrating optical with flux measurements for gap filling, and provide a foundation for using remote sensing to extrapolate from the flux tower to larger regions (upscaling) for regional analysis of net carbon uptake by grassland ecosystems.

A Detailed Assessment of Varying Ejection Rate on Delivery Efficiency of Mesenchymal Stem Cells Using Narrow-Bore Needles

As the number of clinical trials exploring cell therapy rises, a thorough understanding of the limits of cell delivery is essential. We used an extensive toolset comprising various standard and multiplex assays for the assessment of cell delivery postejection. Primary human mesenchymal stem cell (hMSC) suspensions were drawn up into 100-µl Hamilton syringes with 30- and 34-gauge needles attached, before being ejected at rates ranging from 10 to 300 µl/minute. Effects of ejection rate, including changes in viability, apoptosis, senescence, and other key aspects of cellular health, were evaluated. Ejections at slower flow rates resulted in a lower percentage of the cell dose being delivered, and apoptosis measurements of samples ejected at 10 µl/minute were significantly higher than control samples. Immunophenotyping also revealed significant downregulation of CD105 expression in samples ejected at 10 µl/minute (p < .05). Differentiation of ejected hMSCs was investigated using qualitative markers of adipogenesis, osteogenesis, and chondrogenesis, which revealed that slower ejection rates exerted a considerable effect upon the differentiation capacity of ejected cells, thereby possibly influencing the success of cell-based therapies. The findings of this study demonstrate that ejection rate has substantial impact on the percentage of cell dose delivered and cellular health postejection.

Circadian rhythms of body temperature and locomotor activity in aging BALB/c mice: early and late life span predictors.

Impairment of one or more parameters of circadian rhythms (CR) of body temperature (BT) and locomotor activity (LMA) are considered among the hallmarks of mammalian aging. These alterations are frequently used as markers for imminent death in laboratory mice. However, there are still contradictory data for particular strains and it is also uncertain which changes might predict senescence changes later in life, including the force of mortality. In the present paper we use telemetry to study LMA and CR of BT during aging of BALB/c mice. At our knowledge this is the first time that CR of BT and LMA are investigated in this strain in a range of age covering the whole lifespan, from young adult up to very old age. CR of BT was analyzed with a cosine model using a cross sectional approach and follow-up measurements. The results show that BT, LMA, amplitude, goodness-of-fit (GoF) to circadian cycle of temperature decrease with different shapes during chronological age. Moreover, we found that the % change of amplitude and BT in early life (5-19months) can predict the remaining lifespan of the mice. Later in life (22-32months), best predictors are single measurements of LMA and GoF. The results of this study also offer potential measures to rapidly identifying freely unrestrained mice with the worst longitudinal outcome and against which existing or novel biomarkers and treatments may be assessed.