Dynamic and scalable assessment of the senescence-associated secretory phenotype (SASP).

Dual-faced cellular senescence is responsible for beneficial biological processes and for age-related pathologies. Senescent cells under stable proliferation arrest develop numerous senescence-associated phenotypes such as the potent pro-inflammatory secretome called the senescence-associated secretory phenotype (SASP). The SASP shapes the senescent microenvironment and influences the biology of adjacent cells, including the modulation of proliferation and migration/invasion, reinforcement/induction of peripheral senescence, and immune cell activity or recruitment. The SASP is a dynamic process with multiple waves of secreted factors described to interlace over a period of many days. Whether the senescence phenotype reaches a mature stable state remains controversial. Overall, the complexity of the context-dependent and timely SASP compositions and its varied microenvironmental impact demonstrate the importance of properly assessing SASP over time. In this chapter, we focus on scalable and dynamic experimental procedures to prepare SASP conditioned medium over time from cells receiving senescence-inducing stimuli. This SASP-containing conditioned medium can be used to assess the composition of the SASP, study SASP-related signaling pathways or evaluate the paracrine microenvironmental impact of senescent cells.

Comparison of the Cost and Effect of Combined Conditioned Medium and Conventional Medium for Fallopian Tube Organoid Cultures.

Fallopian tube epithelial cells (FTEC) are thought to be the cell of origin of high-grade serous ovarian carcinoma. FTEC organoids can be used as research models for the disease. Nevertheless, culturing organoids requires a medium supplemented with several expensive growth factors. We proposed that a combined conditioned medium based on the composition of the fallopian tubes, including epithelial, stromal, and endothelial cells could enhance FTEC organoid formation. We derived two primary culture cell lines from the fimbria portion of the fallopian tubes. The organoids were split into conventional or combined medium groups based on what medium they were grown in and compared. The number and size of the organoids were evaluated. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC) were used to evaluate gene and protein expression (PAX8, FOXJ1, beta-catenin, and stemness genes). Enzyme-linked immunosorbent assay was used to measure Wnt3a and RSPO1 in both mediums. DKK1 and LiCl were added to the mediums to evaluate their influence on beta-catenin signaling. The growth factor in the combined medium was evaluated by the growth factor array. We found that the conventional medium was better for organoids regarding proliferation (number and size). In addition, WNT3A and RSPO1 concentrations were too low in the combined medium and needed to be added making the cost equivalent to the conventional medium. However, the organoid formation rate was 100% in both groups. Furthermore, the combined medium group had higher PAX8 and stemness gene expression (OLFM4, SSEA4, LGR5, B3GALT5) when compared with the conventional medium group. Wnt signaling was evident in the organoids grown in the conventional medium but not in the combined medium. PLGF, IGFBP6, VEGF, bFGF, and SCFR were found to be enriched in the combined medium. In conclusion, the combined medium could successfully culture organoids and enhance PAX8 and stemness gene expression. However, the conventional medium was a better medium for organoid proliferation. The expense of both mediums was comparable. The benefit of using a combined medium requires further exploration.

Primary hepatocyte urea assessment in the sodium-alginate patterned hydrogel by electrochemical procedure containing umbilical cord conditioned media.

Limitations in liver transplantation and advances in cell therapy methods motivated us to study primary hepatocytes. The main challenge in using primary hepatocytes for liver regeneration is that they lose their functionalities. We aimed to develop a controlled-shape hydrogel and apply the conditioned-media of mesenchymal stromal cells (CM-MSCs) to improve in vitro hepatocyte functions. In this experimental study, following rat hepatocyte isolation by collagenase perfusion and collection of human umbilical cord CM-MSCs, a simple and precise system called electrodeposition was used to produce the patterned alginate hydrogel. To reduce the cytopathic effects, we used an indirect electrodeposition method. For characterizing this structure, mechanical properties, Fourier-transform infrared spectroscopy (FTIR), water uptake, in-vitro degradation, and hydrogel stability were studied. Urea synthesis as a basic function of hepatocytes was assessed in five different groups. Scanning electron microscope (SEM) was utilized to evaluate the primary hepatocyte morphology and their dispersion in the fabricated structure. We observed a significant increase in urea synthesis in the presence of CM-MSCs in patterned hydrogel alginate compared to 2D culture on day 3 (p<0.05). However, there was no significant difference in simple and patterned hydrogel on day 2. We found that the electrodeposition method is appropriate for the rapid fabricating of hydrogel structures with arbitrary patterns for 3D cell culture.

Assessment of Small Cellular Particles from Four Different Natural Sources and Liposomes by Interferometric Light Microscopy.

Small particles in natural sources are a subject of interest for their potential role in intercellular, inter-organism, and inter-species interactions, but their harvesting and assessment present a challenge due to their small size and transient identity. We applied a recently developed interferometric light microscopy (ILM) to assess the number density and hydrodynamic radius (Rh) of isolated small cellular particles (SCPs) from blood preparations (plasma and washed erythrocytes) (B), spruce needle homogenate (S), suspension of flagellae of microalgae Tetraselmis chuii (T), conditioned culture media of microalgae Phaeodactylum tricornutum (P), and liposomes (L). The aliquots were also assessed by flow cytometry (FCM), dynamic light scattering (DLS), ultraviolet-visible spectrometry (UV-vis), and imaging by cryogenic transmission electron microscopy (cryo-TEM). In Rh, ILM showed agreement with DLS within the measurement error in 10 out of 13 samples and was the only method used here that yielded particle density. Cryo-TEM revealed that representative SCPs from Tetraselmis chuii flagella (T) did not have a globular shape, so the interpretation by Rh of the batch methods was biased. Cryo-TEM showed the presence of thin filaments in isolates from Phaeodactylum tricornutum conditioned culture media (P), which provides an explanation for the considerably larger Rh obtained by batch methods than the sizes of particles observed by cryo-TEM images. ILM proved convenient for assessment of number density and Rh of SCPs in blood preparations (e.g., plasma); therefore, its use in population and clinical studies is indicated.

Virus matrix interference on assessment of virucidal activity of high-touch surfaces designed to prevent hospital-acquired infections.

OBJECTIVES/PURPOSE: High-touch surfaces are a critical reservoir in the spread of nosocomial infections. Although disinfection and infection control protocols are well developed, they lack the ability to passively reduce the pathogenic load of high-touch surfaces. Copper and its alloys have been suggested as a surface that exhibit continuous biocidal effects. Antimicrobial studies on these surfaces are prevalent, while virucidal studies are not as well explored. The goal of this study was to first determine the virucidal activity of a copper-nickel-zinc alloy and to then examine the effect of soiling and virus preparation on virucidal activity. METHODS: A baculovirus vector was used as an easily quantifiable model of an infectious enveloped animal cell virus. Droplets containing virus were deposited on surfaces and allowed to stay wet using humidity control or were dried onto the surface. Virus was then recovered from the surface and assayed for infectivity. To examine how the composition of the droplet affected the survival of the virus, 3 different soiling conditions were tested. The first two were recommended by the United States Environmental Protection Agency and the third consisted of cell debris resulting from virus amplification. RESULTS: A copper-nickel-zinc alloy was shown to have strong virucidal effects for an enveloped virus. Copper, nickel, and zinc ions were all shown to leach from the alloy surface and are the likely cause of virucidal activity by this surface. Virucidal activity was achieved under moderate soiling but lost under high soiling generated by routine virus amplification procedures. The surface was able to repeatably inactivate dried virus droplets under moderate soiling conditions, but unable to do so for virus droplets kept wet using high humidity. CONCLUSION: Ion leaching was associated with virucidal activity in both wet and dried virus conditions. Soiling protected the virus by quenching metal ions, and not by inhibiting leaching. The composition of the solution containing virus plays a critical role in evaluating the virucidal activity of surfaces and surface coatings.

Harnessing the Secretome of Mesenchymal Stromal Cells for Traumatic Spinal Cord Injury: Multicell Comparison and Assessment of In Vivo Efficacy.

Cell therapy offers significant promise for traumatic spinal cord injury (SCI), which despite many medical advances, has limited treatment strategies. Able to address the multifactorial and dynamic pathophysiology of SCI, cells present various advantages over standard pharmacological approaches. However, the use of live cells is also severely hampered by logistical and practical considerations. These include specialized equipment and expertise, standardization of cell stocks, sustained cell viability post-thawing, and cryopreservation-induced delayed-onset cell death. For this reason, we suggest a novel and clinically translatable alternative to live-cell systemic infusion, which retains the efficacy of the latter while overcoming many of its limitations. This strategy involves the administration of concentrated cell secretome and exploits the trophic mechanism by which stromal cells function. In this study, we compare the efficacy of intravenously delivered concentrated conditioned media (CM) from human umbilical cord matrix cells (HUCMCs), bone marrow mesenchymal stromal cells, as well as newborn and adult fibroblasts in a rat model of moderately severe cervical clip compression/contusion injury (C7--T1, 35 g). This is further paired with a thorough profile of the CM cytokines, chemokines, and angiogenic factors. The HUCMC-derived CM was most effective at limiting acute (48 h post-SCI) vascular pathology, specifically lesion volume, and functional vascularity. Principle component analysis (PCA), hierarchical clustering, and interaction analysis of proteins highly expressed in the HUCMC secretome suggest involvement of the MAPK/ERK, JAK/STAT, and immune cell migratory pathways. This "secretotherapeutic" strategy represents a novel and minimally invasive method to target multiple organ systems and several pathologies shortly after traumatic SCI.

Non-invasive molecular assessment of human embryo development and implantation potential.

In vitro fertilization (IVF) is the most common assisted reproductive technology used to treat infertility. Embryo selection for transfer in IVF cycles relies on the morphological evaluation by embryologists, either by conventional microscopic assessment or more recently by time-lapse imaging systems. Despite the introduction of time-lapse imaging improvements in IVF success rates have failed to materialize, therefore alternative approaches are needed. Recent studies have shown that embryos resulting in successful pregnancy differ in their secretome and metabolism compared to embryos that fail to implant, suggesting that molecular analysis of embryo culture medium could assist in non-invasive single embryo selection. However, this approach has yet to be adopted clinically due to the lack of appropriate highly sensitive screening technologies needed to assess volume-limited samples. Here we report the detection of hCGß, IL-8 and TNFα from conditioned culture media of single human embryos using electrochemical impedance spectroscopy. The impedimetric immunosensors revealed that morphologically non-viable embryos produce higher levels of IL-8 and TNFα, associated with abnormal cell division and cell death, respectively. More importantly, hCGß detection was able to discriminate apparently morphologically identical viable embryos. This work brings an objective dimension to embryo selection, which could overcome the major limitations of morphology-based embryo selection for implantation. Future work should include the validation of these biomarkers in a large patient cohort.

Sequential Scalp Assessment in Hair Regeneration Therapy Using an Adipose-Derived Stem Cell-Conditioned Medium.

BACKGROUND: An adipose-derived stem cell-conditioned medium (ADSC-CM) reportedly exerts skin-rejuvenating and hair growth-promoting effects. In the therapeutic application of ADSC-CM for alopecia, changes to the interfollicular scalp remain unclear although some evidence has indicated hair growth-promoting effects. OBJECTIVE: To evaluate the effects of ADSC-CM not only on hair follicles, but also on the interfollicular scalp. METHODS: Forty patients (21 men, 19 women; age range, 23-74 years) with alopecia were treated by intradermal injection of ADSC-CM every month for 6 months. Eighty fixed sites on patients were investigated by trichograms, physiological examinations, and ultrasonographic examinations at 4 time points (before treatment and 2, 4, and 6 months after the initial treatment). RESULTS: Hair density and anagen hair rate increased significantly. As physiological parameters, transepidermal water loss value gradually increased, with significant differences at 4 and 6 months after the initial treatment, but hydration state of the stratum corneum and skin surface lipid level showed no obvious changes. As ultrasonographic parameters, dermal thickness and dermal echogenicity were increased significantly. CONCLUSION: Intradermal administration of ADSC-CM on the scalp has strong potential to provide regenerative effects for hair follicles and the interfollicular scalp. An adipose-derived stem cell-conditioned medium offers a promising prospect as an alternative treatment for alopecia.

Assessment of biological functions for C3A cells interacting with adverse environments of liver failure plasma.

BACKGROUND: For its better differentiated hepatocyte phenotype, C3A cell line has been utilized in bioartificial liver system. However, up to now, there are only a few of studies working at the metabolic alternations of C3A cells under the culture conditions with liver failure plasma, which mainly focus on carbohydrate metabolism, total protein synthesis and ureagenesis. In this study, we investigated the effects of acute liver failure plasma on the growth and biological functions of C3A cells, especially on CYP450 enzymes. METHODS: C3A cells were treated with fresh DMEM medium containing 10% FBS, fresh DMEM medium containing 10% normal plasma and acute liver failure plasma, respectively. After incubation, the C3A cells were assessed for cell viabilities, lactate dehydrogenase leakage, gene transcription, protein levels, albumin secretion, ammonia metabolism and CYP450 enzyme activities. RESULTS: Cell viabilities decreased 15%, and lactate dehydrogenase leakage had 1.3-fold elevation in acute liver failure plasma group. Gene transcription exhibited up-regulation, down-regulation or stability for different hepatic genes. In contrast, protein expression levels for several CYP450 enzymes kept constant, while the CYP450 enzyme activities decreased or remained stable. Albumin secretion reduced about 48%, and ammonia accumulation increased approximately 41%. CONCLUSIONS: C3A cells cultured with acute liver failure plasma showed mild inhibition of cell viabilities, reduction of albumin secretion, and increase of ammonia accumulation. Furthermore, CYP450 enzymes demonstrated various alterations on gene transcription, protein expression and enzyme activities.

Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles.

RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles.

Human Corneal Endothelial Cell Assessment From Tissues Preserved in Serum-Based and Synthetic Storage Media.

PURPOSE: To assess the difference between endothelial cells from tissues preserved in media supplemented with fetal bovine serum (FBS) and recombinant human serum albumin (rHSA). METHODS: In a donor-matched study, 48 tissues were preserved for 28 days at 31°C in Cornea Max and Cornea Syn supplemented with FBS and rHSA, respectively. Endothelial cells were visualized by 2 masked observers before and after preservation. Endothelial cell density (ECD) and the number of iatrogenic folds were counted manually. Alizarin red staining and tight junction protein (Zonula Occludens-1) were used to assess cell morphology (hexagonality and polymorphism). Intraobserver and interobserver cell counts were recorded and analyzed. Wilcoxon and one-way analysis of variance tests were used, where P < 0.05 was deemed statistically significantly different. RESULTS: Significant amount of iatrogenic folds were observed in the tissues supplemented with FBS compared with rHSA postpreservation (P = 0.0007). Approximately 69% and 71% hexagonal cells (P = 0.0303) and 29% and 26% polymorphic cells (P = 0.0234) were observed in the FBS and rHSA groups, respectively. Postpreservation, operator 1 counted 1766 cells/mm in FBS and 1864 cells/mm in rHSA. Operator 2 counted 1702 cells/mm in FBS and 1858 cells/mm in rHSA. ECD counts from FBS (interoperator) were statistically significant (P = 0.0429). However, significance was not observed in the ECD counts (interoperator) from the rHSA-preserved tissues (P = 0.8738). CONCLUSIONS: rHSA-supplemented media allow better visualization of the corneal endothelial cells. This reduces the rate of discard observed due to counting errors. Use of rHSA improves the current standard of care and reduces the use of animal-derived products.

Assessment of tumor promoting effects of amniotic and umbilical cord mesenchymal stem cells in vitro and in vivo.

PURPOSE: Human mesenchymal stem cells (hMSCs) have been applied in a variety of therapies recently. However, the role of MSCs in tumor progression remains largely elusive. Some studies demonstrated that MSCs can promote tumor growth, while others had opposite results. Therefore, the lack of evidence about the effect of MSCs on tumor cells impedes its further use. METHODS: In the current study, hMSCs from amniotic membrane (hAMSCs) and umbilical cord (hUCMSCs) were used to evaluate the effects of MSCs on tumor development in vitro and in vivo. Two different animal models based on subcutaneous xenograft bearing nude mice and a murine experimental metastatic model were established for in vivo study. Moreover, cytokines regulated by MSCs co-cultured with cancer cells SPC-A-1 were also analyzed by cytokine array. RESULTS: Our results indicated that hUCMSCs not only did not promote proliferation in cancer cells, but also inhibited migration. In addition, they inhibited tube formation in human umbilical vein endothelial cells (HUVECs). Although hAMSCs also showed inhibitory effects on cancer cell motility, the proliferation of cancer cells was indeed enhanced. The in vivo data revealed that hUCMSCs did not promote tumor progression in lung adenocarcinoma and gastric carcinoma xenografts. Nevertheless, hAMSCs could do. The results from murine experimental metastatic model also demonstrated that neither hUCMSCs nor hAMSCs significantly enhanced the lung metastasis. The data from cytokine array showed that 11 inflammatory factors, 8 growth factors and 11 chemokines were remarkably secreted and changed. CONCLUSIONS: In view of the data from in vitro and in vivo studies, the exploitation of hUCMSCs in new therapeutic strategies should be safe compared to hAMSCs under malignant conditions. Moreover, this is the first report to systematically elucidate the possible molecular mechanisms involved in UCMSC- and AMSC-affected tumor growth and metastasis.

Assessment of HBEC-5i endothelial cell line cultivated in astrocyte conditioned medium as a human blood-brain barrier model for ABC drug transport studies.

Endothelial cells are main components of the Blood-Brain Barrier (BBB) and form a tight monolayer that regulates the passage of molecules, with the ATP-Binding Cassette (ABC) transporters efflux pumps. We have developed a human in vitro model of HBEC-5i endothelial cells cultivated alone or with human astrocytes conditioned medium on insert. HBEC-5i cells showed a tight monolayer within 14 days, expressing ZO-1 and claudin 5, a low apparent permeability to small molecules, with a TEER stability during five days. The P-gp, BCRP, MRPs transporters were well expressed and functional. Accumulation and efflux ratio measurement with different ABC transporters substrates (Rhodamine 123, BCECF AM, Hoechst 33342) and inhibitors (verapamil, Ko143, probenecid and cyclosporin A) were conducted. At barrier level, the functionality of ABC transporters was three-fold enhanced in astrocyte conditioned medium. We validated our model by the transport of pharmacological substrates: caffeine, rivaroxaban, and methotrexate. The rivaroxaban and methotrexate were released with an efflux ratio >3 and were decreased by more than half with inhibitors. HBEC-5i model could be used as relevant tool in preclinical studies for assessing the permeability of therapeutic molecules to cross human BBB.

Methods for the Assessment of Active Transforming Growth Factor-ß in Cells and Tissues.

The potent and pluripotent cytokine TGFß has important roles in normal homeostasis and disease pathogenesis. Once released from cells, TGFß exists in both latent and functionally active forms. Large amounts of latent TGFß are secreted from cells and sequestered in extracellular matrix, only a small proportion of which is activated at any given time. Accurate assessment of TGFß activity levels is an important measurement in biological research and requires methods distinct from measuring total levels of TGFß expression as small changes in TGFß activity levels could be masked by the large amounts of latent TGFß available to be measured. In this chapter, we describe detailed experimental methods for assessing levels of active TGFß in cells and tissues. This chapter includes methods for the assessment of TGFß activity in cells in vitro, in ex vivo precision cut tissue, and in vivo.

In-house made nucleofection buffer for efficient and cost effective transfection of RAW 264.7 macrophages.

Electroporation is the most widely employed method of gene transfer into macrophages which are hard to transfect. RAW 264.7 is a widely used cell line for studying macrophage responses. Electroporation of RAW 264.7 cells with commercial reagents although very efficient is expensive necessitating the development of cost effective alternatives. In this study, we have formulated an economical electroporation buffer for electroporation of RAW 264.7 cells compatible with commercial nucleofector apparatus. We observed that supplementation of membrane fusogenic agents such as Ficoll, PEG and membrane resealing agent, poloxamer P188, enhanced the transfection efficiency of macrophages to a level comparable to the commercially available solutions thereby providing us a cost effective solution for genetic manipulation of macrophages especially in large numbers.

In vitro assessment of the growth and plasma membrane H+ -ATPase inhibitory activity of ebselen and structurally related selenium- and sulfur-containing compounds in Candida albicans.

Ebselen (EB, compound 1) is an investigational organoselenium compound that reduces fungal growth, in part, through inhibition of the fungal plasma membrane H+ -ATPase (Pma1p). In the present study, the growth inhibitory activity of EB and of five structural analogs was assessed in a fluconazole (FLU)-resistant strain of Candida albicans (S2). While none of the compounds were more effective than EB at inhibiting fungal growth (IC50  âˆ¼ 18 µM), two compounds, compounds 5 and 6, were similar in potency. Medium acidification assays performed with S2 yeast cells revealed that compounds 4 and 6, but not compounds 2, 3, or 5, exerted an inhibitory activity comparable to EB (IC50  âˆ¼ 14 µM). Using a partially purified Pma1p preparation obtained from S2 yeast cells, EB and all the analogs demonstrated a similar inhibitory activity. Taken together, these results indicate that EB analogs are worth exploring further for use as growth inhibitors of FLU-resistant fungi.

Assessment of MiRNA Regulation of Endothelial Progenitor Cell Mediated Angiogenesis.

Organ outgrowth, embryonic development, wound healing, and many such processes require the process of angiogenesis, whereby new blood vessels are developed from the preexisting vessels. microRNAs (miRs) are 18-24 nucleotide-containing endogenous RNAs that, via a posttranscriptional mechanism, exert substantial gene regulatory effects. It was discovered by recent advances that, through direct targeting of certain critical secretory factors and transcription factors, miRs exert potent angiogenic control in a cell autonomous and non-cell autonomous manner. This chapter comprehensively summarizes step-by-step protocols for the (1) transfection of miRNA in EPCs (2) advantages and limitations of the principal tubule formation assays in use.

Assessment of Antigen-Specific Cellular Immunogenicity Using Intracellular Cytokine Staining, ELISpot, and Culture Supernatants.

Quantification of cytokine production by CD4 and CD8 T cells after in vitro recall stimulation with the immunizing antigen is a powerful approach to characterize the cellular immune responses to immunization. Here we describe three complementary methods for such quantification including flow cytometric analysis of cytokine production by intracellular staining, ELISpot determination of the numbers of cytokine-producing cells, and generation of secreted cytokines and chemokines in culture supernatants for analysis by ELISA and/or cytometric bead arrays.

Identification of a Novel Human E-Cadherin Splice Variant and Assessment of Its Effects Upon EMT-Related Events.

Epithelial Cadherin (E-cadherin) is involved in calcium-dependent cell-cell adhesion and signal transduction. The E-cadherin decrease/loss is a hallmark of Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. The underlying molecular mechanisms that trigger E-cadherin loss and consequent EMT have not been completely elucidated. This study reports the identification of a novel human E-cadherin variant mRNA produced by alternative splicing. A bioinformatics evaluation of the novel mRNA sequence and biochemical verifications suggest its regulation by Nonsense-Mediated mRNA Decay (NMD). The novel E-cadherin variant was detected in 29/42 (69%) human tumor cell lines, expressed at variable levels (E-cadherin variant expression relative to the wild type mRNA = 0.05-11.6%). Stable transfection of the novel E-cadherin variant in MCF-7 cells (MCF7Ecadvar) resulted in downregulation of wild type E-cadherin expression (transcript/protein) and EMT-related changes, among them acquisition of a fibroblastic-like cell phenotype, increased expression of Twist, Snail, Zeb1, and Slug transcriptional repressors and decreased expression of ESRP1 and ESRP2 RNA binding proteins. Moreover, loss of cytokeratins and gain of vimentin, N-cadherin and Dysadherin/FXYD5 proteins was observed. Dramatic changes in cell behavior were found in MCF7Ecadvar, as judged by the decreased cell-cell adhesion (Hanging-drop assay), increased cell motility (Wound Healing) and increased cell migration (Transwell) and invasion (Transwell w/Matrigel). Some changes were found in MCF-7 cells incubated with culture medium supplemented with conditioned medium from HEK-293 cells transfected with the E-cadherin variant mRNA. Further characterization of the novel E-cadherin variant will help understanding the molecular basis of tumor progression and improve cancer diagnosis. J. Cell. Physiol. 232: 1368-1386, 2017. © 2016 Wiley Periodicals, Inc.

Canine mesenchymal stem cells are neurotrophic and angiogenic: An in vitro assessment of their paracrine activity.

Mesenchymal stem cells (MSCs) have been used in cell replacement therapies for connective tissue damage, but also can stimulate wound healing through paracrine activity. In order to further understand the potential use of MSCs to treat dogs with neurological disorders, this study examined the paracrine action of adipose-derived canine MSCs on neuronal and endothelial cell models. The culture-expanded MSCs exhibited a MSC phenotype according to plastic adherence, cell morphology, CD profiling and differentiation potential along mesenchymal lineages. Treating the SH-SY5Y neuronal cell line with serum-free MSC culture-conditioned medium (MSC CM) significantly increased SH-SY5Y cell proliferation (P <0.01), neurite outgrowth (P = 0.0055) and immunopositivity for the neuronal marker ßIII-tubulin (P = 0.0002). Treatment of the EA.hy926 endothelial cell line with MSC CM significantly increased the rate of wound closure in endothelial cell scratch wound assays (P = 0.0409), which was associated with significantly increased endothelial cell proliferation (P <0.05) and migration (P = 0.0001). Furthermore, canine MSC CM induced endothelial tubule formation in EA.hy926 cells in a soluble basement membrane matrix. Hence, this study has demonstrated that adipose-derived canine MSC CM stimulated neuronal and endothelial cells probably through the paracrine activity of MSC-secreted factors. This supports the use of canine MSC transplants or their secreted products in the clinical treatment of dogs with neurological disorders and provides some insight into possible mechanisms of action.