FBS Medium Research Articles

Biodegradation study in FBS media of polycaprolactone patch as a potential prenatal treatment for myelomeningocele.

Myelomeningocele (MMC) is a congenital defect of the spine characterized by meningeal and spinal cord protrusion through open vertebral archs, and its exposure to the amniotic fluid. Given that the progression of neuronal loss begins early in fetal life, an early coverage of the defect is required to improve the neurological outcomes. Several studies have proposed patches as an alternative to full surgical repair, to achieve an early protection of the spine and possibly reduce the rate of complications of current prenatal surgical procedures. In our previous work, we developed a biocompatible, watertight and biodegradable patch to improve in utero MMC repair. This patch offers an anti-adhesive internal surface to prevent adhesion to spinal cord tissue, and a bioactive external surface to promote tissue coverage. The aim of this study is to assess the patch's in vitro degradation in an amniotic-fluid-like medium and investigate the surface functionalization effect, to understand its mechanism and predict the patch's behavior over time. The study was carried out for 24weeks in FBS medium and after each period the samples were characterized by differential scanning calorimetry, scanning electron microscopy, steric exclusion chromatography, toluidine blue assay and contact angle measurement. The results revealed a progression of PCL hydrolysis over time, characterized by a decrease in molar mass and evidence of erosion as observed by SEM. Furthermore, this process appears to be accelerated by ozonation, compared to surface functionalization without ozonation. The latter can be considered as the most suitable technique to preserve the patch structure over time, while benefiting from the grafting polymers properties during the first weeks of implantation.

Fluorinated polyethyleneimine vectors with serum resistance and adjuvant effect to deliver LMP2 mRNA vaccine for nasopharyngeal carcinoma therapy.

Latent membrane protein 2 (LMP2), which is an important protein of Epstein-Barr virus (EBV) in the latent phase to mediate metastasis and recurrence, has shown great potential as a targeting antigen in mRNA vaccine for nasopharyngeal carcinoma (NPC) therapy. In this study, an LMP2 mRNA vaccine was developed based on a serum-resistant fluorinated polyethyleneimine (TKPF) with the self-adjuvant effect for achieving a strong anti-tumor immunity in NPC treatment. Specifically, the proposed vaccine PEG[TKPF/mLMP2] was comprised of a TKPF/mLMP2 core formed by the cationic TKPF and LMP2 mRNA, together with a dialdehyde poly (ethyl glycol) (OHC-PEG-CHO) coating. PEG[TKPF/mLMP2] showed less protein absorption to enable serum resistance to maintain ∼50 % transfection efficiency under 50 % FBS media. In addition, PEG[TKPF/mLMP2] could render enhanced internalization and lysosomal escape of mRNA by DC cells via positive charge and fluorine groups, followed by efficient transfection and expression, eventually triggering DC maturation and antigen presentation to T cells as demonstrated by in vitro studies. The activated antigen-specific T cells would attack tumor cells expressing LMP2 and release pro-inflammatory cytokines including IFN-γ, IL-6, and TNF-α. Furthermore, in vivo studies manifested effective spleen transfection and activated T cells by PEG[TKPF/mLMP2] to prevent tumor cell growth and prolong mouse survival in both prophylactical and therapeutical models. Notably, PEG[TKPF] revealed self-adjuvant effect to induce a strong immune response for boosting the anti-tumor potency of LMP2 mRNA. In summary, the fabricated LMP2 mRNA vaccine facilitated by the efficient and self-adjuvant vector induced robust immunotherapeutic efficacy, providing a possible solution for NPC therapy. STATEMENT OF SIGNIFICANCE: Latent membrane protein 2 (LMP2), which is a key Epstein-Barr virus (EBV) protein for metastasis and recurrence, can be targeted as an antigen for mRNA vaccine development to treat nasopharyngeal carcinoma (NPC). However, the current LMP2 vaccine is still inefficient in inducing potent anti-NPC immunity. Although mRNA has emerged as an effective tool to rejuvenate LMP2 vaccine development, it still suffers from vulnerability to serum conditions and weak immune response. In this study, we developed an LMP2 mRNA vaccine based on a serum-resistant fluorinated polyethyleneimine (TKPF) with self-adjuvant effects to achieve strong anti-tumor immunity in NPC treatment. The proposed PEG[TKPF/mLMP2] vaccine efficiently delivers to dendritic cells (DCs) for activating T cell maturation, ultimately suppressing the growth of LMP2-expressing tumors in both prophylactic and therapeutic mouse models.

Development of a Magnetic Bead-Based Electrochemical Sandwich Assay to Investigate Inflammatory Cytokines in an Instrumented Fetal Membrane on-a-Chip

The instrumented fetal membrane on-a-chip (IFMOC) is a 3D microfluidic device that mimics the human fetal membranes and functions as a physiologically relevant model to investigate bacterial infections and the preterm premature rupture of the fetal membranes (PPROM) in-vitro. The magnetic bead-based electrochemical sandwich assay (MBESA) serves as an alternative to traditional enzyme-linked immunosorbent assays (ELISAs) to quantify inflammatory cytokines elevated during preterm birth (PTB), such as interleukin-1β (IL-1β). In this work, a MBESA is developed to detect IL-1β levels present within IFMOC cell cultures infected with lipopolysaccharides (LPS) and Group B Streptococcus (GBS). We present the design and application of the MBESA, highlighting the high sensitivity of an electrochemical technique in a biological platform. We discuss the fabrication and implementation of the IFMOC with the MBESA in reproductive research. The electrochemical assay was able to reproducibly detect IL-1β levels up to 600 pg/mL in RPMI 1640 with 10% FBS media. The MBESA is a promising technique to detect multiple inflammatory cytokines present in infected fetal membrane organ on-a-chip cultures. Future work includes optimizing the electrochemical performance of the MBESA, examining the inflammatory cytokine TNF-α to further elucidate PPROM mechanisms, and subsequently investigating therapeutic methods to prevent the onset of PTB. Figure 1

Interactions Between Silver Nanoparticles and Culture Medium Biomolecules with Dose and Time Dependencies.

The interaction between silver nanoparticles (AgNPs) and molecules producing coronas plays a key role in cytotoxicity mechanisms. Once adsorbed coronas determine the destiny of nanomaterials in vivo, their effective deployment in the biomedical field requires a comprehensive understanding of the dynamic interactions of biomolecules with nanoparticles. In this work, we characterized 40nm AgNPs in three different nutritional cell media at different molar concentrations and incubation times to study the binding mechanism of molecules on surface nanoparticles. In addition, their cytotoxic effects have been studied in three cell lineages used as tissue regeneration models: FN1, HUV-EC-C, RAW 264.7. According to the data, when biomolecules from DMEM medium were in contact with AgNPs, agglomeration and precipitation occurred. However, FBS medium proteins indicated the formation of coronas over the nanoparticles. Nonetheless, little adsorption of molecules around the nanoparticles was observed when compared to DMEM supplemented with 10% FBS. These findings indicate that when nanoparticles and bioproteins from supplemented media interact, inorganic salts from DMEM contribute to produce large bio-coronas, the size of which varies with the concentration and time. The static quenching mechanism was shown to be responsible for the fluorescence quenching of the bioprotein aggregates on the AgNPs surface. The calculated bioprotein-nanoparticle surface binding constants were on the order of 105M-1 at 37°C, with hydrophobic interactions driven by enthalpy and entropy playing a role, as confirmed by thermodynamic analysis. Cytotoxicity data showed a systematic degrowth in the viable cell population as the number of nanoparticles increased and the diameter of coronas decreased. Cytotoxic intervals associated with half decrease of cell population were established for AgNPs molar concentration of 75µM for 24h and 50µM for 48h. In summary, through the cytotoxicity mechanism of bio-coronas we are able to manipulate cells' expansion rates to promote specific processes, such inflammatory mechanisms, at different time instants.

248 Divergent Satellite Cell Profiles of Bovine Trapezius, Longissimus, and Bicep Femoris Muscles in Utero

Abstract As we look to better understand whole body growth and development, there are still many mechanisms of individual muscle growth that we have yet to comprehend. Therefore, the objective of this study was to compare proliferative capacity and satellite cell number within the trapezius (TP), longissimus (LM), and bicep femoris (BF) muscles of fetal bovine throughout mid and late gestation. We hypothesized a difference in satellite cell number across these three muscles may further explain the differential growth pattern of each of these muscles in utero. Muscle samples were harvested from bovine fetuses at a commercial abattoir and aged using crown rump length (~150-220d in utero). These samples were then dissected into single fibers and seeded in a 3D hydrogel culture with 20% FBS media. Cells were fixed using 4% paraformaldehyde and stained for DAPI, Pax-7, Myf-5 (Invitrogen, Bedford, MA) at 72, 96, and 120h. Wells were imaged and analyzed for proliferation rate using the Cytation 5 (Agilent; La Jolla, CA) Prime Gen 5 software. Data were analyzed as a two-way ANOVA with repeated measures using GraphPad Prism (Version 9.5). For Myf-5, Pax-7, and dual positive cells (P ≤ 0.05) there was a difference across muscle type with BF having an increased number of positive cells compared with the TP muscle and LM being intermediate (Myf-5 mean = 51, 23, and 40 respectively; Pax-7 mean = 45, 30, and 20 respectively; Dual mean = 39, 20, 16 respectively). This variation was further supported by a similar divergence in proliferation rate across muscle type (P = 0.04), with BF muscle having increased proliferative capacity over TP muscle and LM muscle being intermediary. These findings suggest contrasting growth patterns across muscle types in utero that may influence postnatal growth patterns. Further research is needed to understand how the variability in satellite cell number contributes to differences in individual muscle growth.

Platelet-Rich Fibrin-Conditioned Medium as an Alternative to Fetal Bovine Serum Promotes Osteogenesis of Human Dental Pulp Stem Cells.

Human dental pulp stem cells (DPSCs) exhibit multilineage differentiation capabilities and superior clonogenic and proliferative properties. However, the use of animal-derived components such as FBS raises concerns regarding the clinical application of stem-cell-based therapies. Platelet-rich fibrin (PRF) derived from human blood is rich in fibrin, platelets, and growth factors and acts as a bioactive scaffold for grafting with biomaterials. In this study, we assessed the efficacy of PRF-conditioned medium (CM) in promoting DPSCs proliferation and osteogenic differentiation compared with the standard culture medium supplemented with FBS. A comparison of DPSCs cultured in FBS and PRF-CM revealed no differences in characteristics or morphology. However, cells cultured with PRF-CM exhibited inferior proliferation rates and cell numbers during passage in comparison with those cultured with FBS. In contrast, DPSCs cultured in PRF-CM showed significantly higher levels of calcification, and RT-PCR confirmed that the gene expression levels of markers associated with osteoblast differentiation were significantly increased. The PRF-CM approach offers a convenient, straightforward, and advantageous method for culturing DPSCs, without relying on animal-derived components. In summary, this study introduces a novel application of PRF-CM for enhancing the osteogenesis of DPSCs, which provides an alternative to FBS culture medium and addresses concerns associated with the use of animal-derived components in clinical settings.

Continuous ultraviolet irradiation induces the development of irreversible resistance of melanoma cells to anticancer drugs

Introduction. Melanoma belongs to the group of the most malignant tumors characterized by aggressive growth and active metastasis. At the same time, the effectiveness of therapy, primarily targeted therapy, is largely limited by the rapid development of drug resistance.Aim. To study the effect of chronic ultraviolet (UV) irradiation on the formation of a population of radiation-resistant melanoma cells; to study the features of cell signaling and the sensitivity of UV-resistant melanoma cells to the antitumor drugs.Materials and methods. The experiments were carried out on in vitro cultured A375 melanoma cells. Cells were cultured in a standard DMEM + 10 % FBS medium; cell growth rate was analyzed using the MTT assay; cell survival after irradiation was analyzed using a colony-forming test. Determination of the transcriptional activity of the estrogen receptor (ER) was performed by reporter analysis upon transfection into cells of a plasmid containing the luciferase reporter gene controlled by estrogen responsive element. The immunoblotting method was used to analyze the expression of cellular proteins; comparative analysis of ERα and ERβ expression was performed by immunofluorescent method.Results. Long-term UV irradiation leads to the formation of a UV-resistant subpopulation of A375 melanoma cells, which is characterized by decreased sensitivity to targeted (vemurafenib) and hormonal (tamoxifen) drugs, increased expression of Snail, an activator of the epithelial-mesenchymal transition, and in the absence of noticeable changes in the expression of PI3K / mTOR signaling. Metformin reduces Snail expression in both parental and UV-resistant A375 cells and enhances the cytostatic effect in combination with vemurafenib or tamoxifen.Conclusion. The data obtained demonstrate a decrease in the sensitivity of melanoma cells to targeted drugs under the long-term exposure to UV. The ability of metformin to potentiate the action of targeted drugs and inhibit Snail allows us to consider metformin not only as an antitumor agent, but also as a potential inhibitor of the epithelial-mesenchymal transition.

BIOL-24. USING NEEDLE WASH SPECIMEN AFTER STEREOTACTIC BIOPSY TO DEVELOP PDOX MODELS OF DIPG AND DIFFUSE MIDLINE GLIOMAS

Abstract Obtaining viable tissue remains a major barrier for lab-based studies, especially in brain tumors deemed surgically unresectable such as diffuse midline gliomas. Surgical biopsy is performed for those tumors invested in deep and vital brain structures however, biopsied specimens are usually of small volume without additional tissue for research after clinical testing. To overcome the barrier of limited surgical specimen volume, we partnered with neurosurgeons to develop a protocol which procures viable cells from discarded biopsy needle after stereotactic biopsies to develop patient-derived orthotopic xenograft (PDOX) models. After procurement of surgical specimen and prior to disposal of biopsy needle, each stereotactic biopsy needle was washed with FBS media twice. Wash solution was collected and immediately transported on ice to the research lab. Cell number was counted for in vitro culture and for orthotropic in vivo tumor implantation. To date, 29 samples of biopsy needle washes have been obtained including 12 diffuse midline gliomas of the brain stem, 8 supratentorial diffuse midline gliomas, and 3 pineal region tumors with harvested cell number from no visible cells to 5.3 million cells (median 1 million). 14 tumors have been orthotopically implanted into matched locations of immunodeficient mouse brains. Tumor formation was confirmed in 4 out of 15 (28.5%), including 2 brain stem tumors, 1 infiltrating astrocytoma, and 1 midline glioma, after implantation of cell numbers as low as 12,500. 3 models were successfully sub-transplanted to passages II and III. Histological examination confirmed the replication of key pathological features of the originating patient tumors. In conclusion, we have developed a novel protocol to procure clinical specimen by using wash solution of stereotactic brain tumor biopsy needles for surgically unresectable tumors. Our work demonstrates a new possibility for clinical sample acquisition and tumor modeling, opening new frontiers in the field of pediatric brain tumor research.

TGF-BETA STIMULATES PERIOSTEAL OSTEOGENIC ACTIVITY

Osteoprogenitors on the inner layer of periosteum are the major cellular contributors to appositional bone growth and bone repair by callus formation. Previous work showed that periosteal-derived cells have little or no osteogenic activity under standard in vitro osteogenic culture conditions. This study was conducted to determine what growth factor(s) can activate periosteal osteogenic capacity.This study was conducted with IACUC approval. Periosteum from five equine donors was digested in collagenase for 3-4 hours at 37C. Isolated periosteal cells were maintained in DMEM/10% FBS medium and exposed to PDGF, Prostaglandin E2, BMP-2 and TGF-b3 at a range of concentrations for 72 hours. Changes in osteogenic gene expression (Runx2, OSX and ALP) were measured by qPCR. Periosteal cells were pre-treated with TGF-b3 or maintained in control medium were transferred into basal or osteogenic medium. Osteogenic status was assessed by Alizarin Red staining for mineralized matrix, ALP enzymatic activity and induction of osteogenic genes.PDGF, PgE2 and BMP-2 had little impact on expression of osteogenic markers by periosteal cells. In contrast, TGF-b3 stimulated significant increases in Osterix (over 100-fold) ALP expression (over 70-fold). Pre-treating periosteal cells with TGF-b3 for 72 hours stimulated rapid cell aggregation and aggregate mineralization once cells were transferred to osteogenic medium, while cells not exposed to TGF-b3 exhibited minimal evidence of osteogenic activity.This study indicate that TGF-b signaling is vital for periosteal osteogenic activity. Transient ‘priming’ of periosteal cells through TGF-b exposure was sufficient to activate subsequent osteogenesis without requiring ongoing growth factor stimulation. TGF beta ligands are secreted by many cell types, including periosteal progenitors and osteocytes, providing opportunities for both autocrine and paracrine pathways to regulate periosteal bone formation under homeostatic and reparative conditions.

Room-temperature synthesis of nanometric and luminescent silver-MOFs.

Three silver-MOFs were prepared using an optimized, room-temperature methodology starting from AgNO₃ and dicarboxylate ligands in water/ethanol yielding Ag 2 BDC, Ag 2 NDC (UAM-1), and Ag 2 TDC (UAM-2) at 38%-48% (BDC, benzenedicarboxylate; NDC, 1,8-naphthalene-dicarboxylate; TDC, p-terphenyl-4,4″-dicarboxylate). They were characterized by PXRD/FT-IR/TGA/photoluminescence spectroscopy, and the former two by SEM. These materials started decomposing at 330°C, while showing stability. The crystal structure of UAM-1 was determined by PXRD, DFT calculations, and Rietveld refinement. In general, the structure was 3D, with the largest Ag-O bond interlinking 2D layers. The FT-IR spectra revealed 1450 and 1680 bands (cm-1) of asymmetrically stretching aniso-/iso-bidentate -COO in coordination with 2/3-Ag atoms, accompanied by Ag-O bands at 780-740 cm-1, all demonstrating the network formation. XRD and SEM showed nanometric-scale crystals in Ag₂BDC, and UAM-1 developed micrometric single-stranded/agglomerated fibrillar particles of varying nanometric widths. Luminescence spectroscopy showed emission by Ag₂BDC, which was attributed to ligand-to-metal or ligand-to-metal-metal transitions, suggesting energy transfer due to the short distance between adjacent BDC molecules. UAM-1 and UAM-2 did not show luminescence emission attributable to ligand-to-metal transition; rather, they presented only UV emission. The stabilities of Ag₂BDC and UAM-1 were evaluated in PBS/DMEM/DMEM+FBS media by XRD, which showed that they lost their crystallinity, resulting in AgCl due to soft-soft (Pearson's principle) affinity.

Abstract 11789: Exosomes Released by Human Cardiac Fibroblasts Promote the Maturation of Human Induced-Pluripotent Stem Cells Derived Cardiomyocyte via AMPK Signal Pathway

Background: Human induced-pluripotent stem cell derived cardiomyocytes (hiPS-CMs) is phenotypically less matured than adult cardiomyocytes. The aim of this study was to explore the potential mechanism by which human cardiac fibroblast-derived exosomes (hc-FB-EXOs) enhance the maturation of hiPS-CMs. Method: Exosomes were extracted from human cardiac fibroblasts (hc-FBs) which were cultured with exosome-free FBS medium. The cultured hiPS-CMs were randomly devided into two groups: co-culture with (EXO) and without (NC) hc-FB-EXOs. The morphology of hiPS-CMs was assessed via immunostaining. Mitochondrial functions assay were measured by Seahorse XF. Potential mechanism was assessed via mRNA-Seq, real-time PCR and western blot. Results: Morphologically, the cells treated in EXO group was significantly larger than the in NC group in terms of cell area and sarcomere length (Panel A). In cellular metabolism, the mitochondrial oxygen consumption rate of EXO group was significantly higher than that of NC group, including basal respiration, proton leak, maximal respiration and spare respiratory capacity (Panel B). The expression of cardiac Troponin I (cTnI), Myosin Heavy Chain 7 (MYH7), Cyclin Dependent Kinase Inhibitor 2C (CDKN2C or p18), phospho-WEE1 and CAV3 (caveolin3) were significant higher, while the expression of of Myosin Heavy Chain 6 (MYH6) was significant lower in EXO group than in NC group (Panel C). Furthermore, qPCR showed that TNNI1, TNNT2, TNNI3/TNNI1, total titin, N2B, MYH7, LPL, ND1, N2BA, and MYH6 were significantly upregulated in EXO group compared with the NC group. Furthermore, western blot validation of RNA-Seq data confirmed that significantly upregulated of phospho-AMPK and downregulated of ACC1 and SCD1 were detected in EXO group compared with the NC group (Panel D). Conclusion: hc-FB-EXOs could significantly facilitate maturation of hiPS-CMs via the phosphorylation of AMPK.

Abstract 3983: Dysregulated SerpinA6 levels and progression of prostate cancer

Abstract SerpinA6 is a major cortisol binding transport protein, which helps in cortisol tissue delivery particularly at inflammatory sites. Moreover, SerpinA6 has been reported to participate in stress response by releasing cortisol specifically at inflammatory sites upon cleavage by human neutrophil elastase between residues 344 and 345. In castrate resistant prostate cancer (CRPC) patients, reports suggest castration induces inflammation in the prostate, which increases glucocorticoids (GCs) levels and elevates pro-inflammatory cytokine IL-6 levels. We propose declining SerpinA6 levels in the prostate may play a role in prostate cancer disease progression. This could be due to increasing IL-6 levels after castration, which may inhibit or degrade SerpinA6. Deficient SerpinA6 may not be sufficient to keep steroids inactive, due to the increasing GCs. Our data suggests IL-6 treatment of androgen independent prostate cancer (PC-3) cells decreases SerpinA6 level and conversely increases glucocorticoid receptor (GR) expression in a dose (1-10ng/ml) and time (30-180 minutes) dependent fashion. Moreover, IL-6 dose dependent treatment in PC-3 cells decreases FOXO3a expression, a major apoptotic protein dysregulated in prostate cancer. Next, we determine the role of SerpinA6 (30µg/ml) and IL-6(5ng/ml) independently and in combination to PC-3 cells. We observed IL-6 and SerpinA6 co-treatment to PC-3 cells (charcoal stripped FBS media) for 6 hours reduced Neutrophil elastase levels significantly. Additionally we observed increased FOXO3a and SerpinA6 expression, whereas decreased GR and HNF3α levels. However, silencing IL-6 in PC-3 cells increased SerpinA6 expression. To further support our findings, silencing SerpinA6 in metastatic variant of PC-3 cells increased phosphor-Akt-(ser-473), glucocorticoid receptor and increased apoptotic protein FOXO3a expressions. Using dexamethasone (15µM), which is known to suppress PC-3 cell migration, we found increased expression of SerpinA6. Conversely, Mifepristone (15µM), which inhibits PC-3 cell growth, deceases SerpinA6 expression after 24 hours of treatment. In humans the normal range of circulatory SerpinA6 level are 19-45ug/ml. We treated normal prostate epithelial cells (RWPE1) and prostate cancer (PC-3) cells with human recombinant protein 18 and 36ug/ml of doses for 24 hours; RWPE1 cells represented a SerpinA6 dose dependent increase in cellular viability, whereas PC-3 cells demonstrated no change in the viability after a dose response treatment with SerpinA6 for 24 hours. These findings suggest the SerpinA6 context dependent role. We suggest that decreasing levels of SerpinA6 and FOXO3a expressions in the prostate cancer patients may have potential role in disease progression. Moreover, we need to have an effective agent, which could prevent the decreasing levels of SerpinA6 and FOXO3a in prostate to inhibit the progression of prostate cancer. Citation Format: Carlos Riveros, Victor Chalfant, Jatinder Kumar, Sanjeev Shukla. Dysregulated SerpinA6 levels and progression of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3983.

Senescence State in Mesenchymal Stem Cells at Low Passages: Implications in Clinical Use.

Mesenchymal stem cells (MSCs) are multipotent cells found in various tissues and are easily cultivated. For use in clinical protocols, MSCs must be expanded to obtain an adequate number of cells, but a senescence state may be instituted after some passages, reducing their replicative potential. In this study, we report a case where MSC derived from an elderly donor acquired a senescence state after three passages. The bone marrow was aspirated from a female patient submitted to a cell therapy for the incontinency urinary protocol; MSCs were cultivated with DMEM low glucose, supplemented with 10% autologous serum (AS) plus 1% L-glutamine and 1% antibiotic/antimycotic. Senescence analysis was performed by β-galactosidase staining after 24 and 48 h. Controls were established using BM-MSC from healthy donors and used for senescence and gene expression assays. Gene expression was performed using RT-PCR for pluripotency genes, such as SOX2, POU5F1, NANOG, and KLF4. MSC telomere length was measured by the Southern blotting technique, and MSCs were also analyzed for their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. The patient’s MSC expansion using AS displayed an early senescence state. In order to understand the role of AS in senescence, MSCs were then submitted to two different culture conditions: 1) with AS or 2) with FBS supplementation. Senescence state was assessed after 24 h, and no statistical differences were observed between the two conditions. However, patients’ cells cultured with AS displayed a higher number of senescence cells than FBS medium after 48 h (p = 0.0018). Gene expression was performed in both conditions; increased expression of KLF4 was observed in the patient’s cells in comparison to healthy controls (p = 0.0016); reduced gene expression was observed for NANOG (p = 0.0016) and SOX2 (p = 0.0014) genes. Telomere length of the patient’s cells was shorter than that of a healthy donor and that of a patient of similar age. Osteocyte differentiation seemed to be more diffuse than that of the healthy donor and that of the patient of similar age. MSCs could enter a senescence state during expansion in early passages and can impact MSC quality for clinical applications, reducing their efficacy when administered.

The fluorination effect on the transfection efficacy of cell penetrating peptide complexes

Cell penetrating peptides (CPPs) have been used as alternative delivery vectors to translocate therapeutic cargo molecules across cell membranes. One example of CPPs is the dTAT peptide, which has shown great promise in the design of highly efficient and low-cytotoxic gene vectors when condensed via “soft” calcium cross links. Here, we investigated the effect of fluorination on the formulation of dTAT complexes and explored their potential for pDNA delivery to cells. Fluorinated dTAT complexes achieve excellent gene transfection efficacy compared to fluorinated PEI polyplexes in A549, HeLa, and MCF-7 cell lines. Furthermore, the fluorinated dTAT complexes exhibit excellent serum resistance, high gene transfection efficacy even in 10% FBS medium, and no detectable cytotoxicity on transfected cells. The optimum NaF concentration (14 mM) resulted in an over 1000-fold enhancement in dTAT complexes (N/P 33) transfection efficiency. According to these findings, fluorination seems to be a potential strategy for creating gene vectors without requiring complex syntheses.

Comparison of the Confluence-Initiated Neurogenic Differentiation Tendency of Adipose-Derived and Bone Marrow-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ADSCs), which tended to neurogenically differentiate spontaneously after achieving high confluence, were observed. Human ADSCs reaching 80% confluence were cultured in DMEM without an inducing factor for 24 h and then maintained in DMEM plus 1% FBS medium for 7 days. The neurogenic, adipogenic, and osteogenic genes of the factor-induced and confluence-initiated differentiation of the ADSCs and bone marrow-derived mesenchymal stem cells (BMSCs) at passages 3 to 5 were determined and compared using RT-qPCR, and the neurogenic differentiation was confirmed using immunofluorescent staining. In vitro tests revealed that the RNA and protein expression of neuronal markers, including class III β-tubulin (TUBB3), microtubule-associated protein 2 (MAP2), neurofilament medium polypeptide (NEFM), neurofilament heavy polypeptide (NEFH), and neurofilament light polypeptide (NEFL), had been enhanced in the confluence-initiated differentiation of the ADSCs. In addition, the expressions of neurotrophins, such as the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial cell-derived neurotrophic factor (GDNF), were also elevated in the confluence-initiated differentiation of the ADSCs. However, the confluent ADSCs did not show a tendency toward spontaneous adipogenic and osteogenic differentiation. Moreover, compared with the confluent ADSCs, the tendency of spontaneous neurogenic, adipogenic, and osteogenic differentiation of the confluent human bone marrow mesenchymal stem cells (BMSCs) was not observed. The results indicated that ADSCs had the potential to spontaneously differentiate into neuron-like cells during the confluent culture period; however, this tendency was not observed in BMSCs.

PDE-5 Inhibition Decreases Cancer Tumor Burden through Immunomodulation

Myeloid-derived suppressor cells (MDSC) are CD14-/CD11b+/Gr1+ cells that have been shown to suppress T-cell anti-tumor activity. They do so namely by using arginase and iNOS to deplete the microenvironment of L-arginine and producing unopposed NO, respectively. Sildenafil, a PDE5 inhibitor, has been shown to decrease tumor burden and increase T-cell tumor infiltration, though its mechanism is poorly understood. This study aimed at determining the mechanisms by which Sildenafil affected MDSC suppression mechanisms on cytotoxic T-cells and investigate the effects on invasive 4T1 cancer cells both in vitro and in vivo.In our in vitro model, lymphoid cells were obtained from 4T1 tumor bearing BalbC mice 14 days after exposure and extracted using a magnetic bead separation technique, then expanded for 6 days using ionomicin, bryostatin and IL-2 pulsed for 24 hours followed by IL-7 and IL-15 in RPMI and FBS media. MDSCs were obtained from the spleens of 4T1 exposed mice. The sildenafil-exposed MDSCs showed a significant decrease in arginase activity. Additionally, when cultured in sildenafil and media, 4T1 cells showed a 50% reduction in growth. In this same model, T-cells had a 13% increase in growth with a therapeutic dose of sildenafil.In vivo studies were performed using BalbC mice injected with 4T1 cells and treated with sildenafil injections as well as PBS control injections. Spleens were harvested at day 14 and purified using flow cytometry to GR1 and CD11b antigens. Sildenafil-treated mice showed a 43% decrease in tumor burden and a significant decrease in arginase activity, though iNOS levels showed no significant difference between groups. The MDSC pathway remains a promising area of study in allowing activated T-cells to decrease tumor and the use of sildenafil has proven effective in reducing MDSC activity through decreases in arginase activity. DisclosuresNo relevant conflicts of interest to declare.

369-P: Wound Repair by P53 Silenced Human Endothelial Cell-Derived Conditioned Media, In Vitro

Introduction: Diabetes is associated with impaired wound healing. Studies have demonstrated that endothelial progenitor cells (EPCs) and mature human endothelial cells (EC) are susceptible to apoptosis in hyperglycemic environment and p53 gene silencing prevents cellular senescence and helps vascular regeneration. Our goal was to test whether cell conditioned media obtained from p53 silenced human cells can also improve cellular regeneration. Methods: Ad-human-P53 (TP53)-shRNA was used to silence P53 and Ad-scrambled-null-shRNA was used as control. To collect condition media, different human cells such as HEK, human CD14+ve cells and HUVEC (human umbilical vein endothelial cells) were cultured in exosome free FBS media for 5 days, following transduction with Ad-p53sh or Ad-null, as the case may be. After transduction, cells were grown in media containing 2% BSA (with no FBS and growth factors) for only 24 h and the collected supernatant was concentrated 10-fold to obtain the conditioned media (CM). Endothelial regeneration or wound healing was tested using endothelial wound healing assay kit (from Cell Biolabs # CBA-120-T). Results: We compared CM obtained from null and p53sh in HEK, CD14+ve and HUVEC cells on human endothelial cells. Proliferation capacity of the endothelial cells was greater with p53sh CM compared to null and non-transduced CM. The CM obtained from the untreated and null samples did not show increased proliferation in any of the 3 kinds of cells tested. The CM from p53sh HUVEC was more effective than CM from other cells. We are analyzing the CM (exosome and non-exosome fraction) for identification of specific secretory proteins. Conclusion: P53 silencing appears to improve HUVEC paracrine factors and this particular modality of cell free therapy may have therapeutic role in treating diabetes complications and even aging. Disclosure D. K. Rivera: None. S. Nandula: None. S. Sen: None. Funding American Heart Association

The Effect of Ethynil Estradiol and Desogestrel on Proliferation and Apoptosis Hydatidiform Mole Trophoblast Cell

Background: To explore the effect of ethynil estradiol and desogestrel on proliferation and apoptosis hydatidiform mole trophoblast cell.
 Methods: From April 2008 until March 2009, we collected 15 samples of hydatidiform mole tissue. Trophoblast cells was isolated and culture at RPMI 20% FBS medium. Only 7 samples (46%) shown good growth. Cell was identified using cytology and β HCG test. Trophoblast cells good quality than devided into three groups observation. First group get ethynil estradiol 10 nmol/mL, second group get desogestrel 100 nmol/mL, Third group get DMSO 1%. Cells incubated and observe at 24, 48, 72, 96 hours. Cell cycle, apoptosis and β HCG was evaluated at each time observation. Cell cycle evaluation using BD cycle test plus DNA reagent, apoptosis evaluation using FITC-Annexin V. Analyze using FACSCalibur flowcytometer. β HCG evaluation using Abbott AxSym total β-HCG reagent pack.
 Results: The group of cell that get ethynil estradiol in concentration 10 nmol/mL had cell proliferation index, amount cells and β HCG level higher than control after 72 hours observation. The group of cell that get desogestrel in concentration 100 nmol/mL have cell proliferation index, amount cells and β HCG level lower than control after 48 hours observation. There are no differences of apoptosis between the two group and control.
 Conclusion: Ethynil estradiol will increase proliferation of hydatidiform mole trophoblast cell, while desogestrel will decrease proliferation of hydatidiform mole trophoblast cell. There are no effect of ethynil estradiol and desogestrel on apoptosis of hydatidiform mole trophoblast cell.

The Effect of Ethynil Estradiol and Desogestrel on Proliferation and Apoptosis Hydatidiform Mole Trophoblast Cell

A B S T R A C TIntroduction To explore the effect of ethynil estradiol and desogestrel onproliferation and apoptosis hydatidiform mole trophoblast cell. Methods: From April2008 until March 2009, we collected 15 samples of hydatidiform mole tissue.Trophoblast cells was isolated and culture at RPMI 20% FBS medium. Only 7samples (46%)shown good growth. Cell was identified using cytology and β HCG test.Trophoblast cells good quality than devided into three groups observation. Firstgroup get ethynil estradiol 10 nmol/mL, second group get desogestrel 100 nmol/mL,Third group get DMSO 1%. Cells incubated and observe at 24, 48, 72, 96 hours. Cellcycle, apoptosis and β HCG was evaluated at each time observation. Cell cycleevaluation using BD cycle test plus DNA reagent, apoptosis evaluation using FITC-Annexin V. Analyze using FACSCalibur flowcytometer. β HCG evaluation usingAbbott AxSym total β-HCG reagent pack. Results: The group of cell that get ethynilestradiol in concentration 10 nmol/mL had cell proliferation index, amount cells andβ HCG level higher than control after 72 hours observation. The group of cell thatget desogestrel in concentration 100 nmol/mL have cell proliferation index, amountcells and β HCG level lower than control after 48 hours observation. There are nodifferences of apoptosis between the two group and control. Conclusion: Ethynilestradiol will increase proliferation of hydatidiform mole trophoblast cell, whiledesogestrel will decrease proliferation of hydatidiform mole trophoblast cell. Thereare no effect of ethynil estradiol and desogestrel on apoptosis of hydatidiform moletrophoblast cell.

Long-Term Human Hematopoietic Stem Cell Culture in Microdroplets.

We previously reported a new approach for micromanipulation and encapsulation of human stem cells using a droplet-based microfluidic device. This approach demonstrated the possibility of encapsulating and culturing difficult-to-preserve primary human hematopoietic stem cells using an engineered double-layered bead composed by an inner layer of alginate and an outer layer of Puramatrix. We also demonstrated the maintenance and expansion of Multiple Myeloma cells in this construction. Here, the presented microfluidic technique is applied to construct a 3D biomimetic model to recapitulate the human hematopoietic stem cell niche using double-layered hydrogel beads cultured in 10% FBS culture medium. In this model, the long-term maintenance of the number of cells and expansion of hHSCS encapsulated in the proposed structures was observed. Additionally, a phenotypic characterization of the human hematopoietic stem cells generated in the presented biomimetic model was performed in order to assess their long-term stemness maintenance. Results indicate that the ex vivo cultured human CD34+ cells from bone marrow were viable, maintained, and expanded over a time span of eight weeks. This novel long-term stem cell culture methodology could represent a novel breakthrough to improve Hematopoietic Progenitor cell Transplant (HPT) as well as a novel tool for further study of the biochemical and biophysical factors influencing stem cell behavior. This technology opens a myriad of new applications as a universal stem cell niche model potentially able to expand other types of cells.