Low Glucose Dulbecco Research Articles

Effects of low-level laser on the expression of interleukin-6, tumor necrosis factor‑α, osteoprotegerin, and receptor activator of nuclear factor-κB ligand in human periodontal ligament cells.

This study aims to determine the effects of low-level laser (LLL) on the expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) in human periodontal ligament cells (HPDLCs) stimulated by high glucose; and identify the molecular mechanism of LLL therapy in the regulation of periodontal inflammation and bone remodeling during orthodontic treatment in diabetic patients. HPDLCs were cultured in vitro to simulate orthodontic after loading and irradiated with LLL therapy. The cultured cells were randomly divided into four groups: low glucose Dulbecco's modification of Eagle's medium (DMEM)+stress stimulation (group A), high glucose DMEM+stress stimulation (group B), hypoglycemic DMEM+LLL therapy+stress stimulation (group C), and hyperglycemic DMEM+LLL therapy+stress stimulation (group D). Groups C and D were further divided into C1 and D1 (energy density: 3.75 J/cm2) and C2 and D2 (energy density: 5.625 J/cm2). Cells in groups A, B, C, and D were irradiated by LLL before irradiation. At 0, 12, 24, 48, and 72 h, the supernatants of the cell cultures were extracted at regular intervals, and the protein expression levels of IL-6, TNF-α, OPG, and RANKL were detected by enzyme-linked immunosorbent assay. 1) The levels of IL-6 and TNF-α secreted by HPDLCs increased gradually with time under static pressure stimulation. After 12 h, the levels of IL-6 and TNF-α secreted by HPDLCs in group A were significantly higher than those in groups B, C1, and C2 (P<0.05), which in group B were significantly higher than those in groups D1, and D2 (P<0.01). 2) The OPG protein concentration showed an upward trend before 24 h and a downward trend thereafter. The RANKL protein concentration increased, whereas the OPG/RANKL ratio decreased with time. Significant differen-ces in OPG, RANKL, and OPG/RANKL ratio were found among group A and groups B, C1, C2 as well as group B and groups D1, D2 (P<0.05). 1) In the high glucose+stress stimulation environment, the concentrations of IL-6 and TNF-α secreted by HPDLCs increased with time, the expression of OPG decreased, the expression of RANKL increased, and the ratio of OPG/RANKL decreased. As such, high glucose environment can promote bone resorption. After LLL therapy, the levels of IL-6 and TNF-α decreased, indicating that LLL therapy could antagonize the increase in the levels of inflammatory factors induced by high glucose environment and upregulate the expression of OPG in human HPDLCs, downregulation of RANKL expression in HPDLCs resulted in the upregulation of the ratio of OPG/RANKL and reversed the imbalance of bone metabolism induced by high glucose levels. 2) The decrease in inflammatory factors and the regulation of bone metabolism in HPDLCs were enhanced with increasing laser energy density within 3.75-5.625 J/cm2. Hence, the ability of LLL therapy to modulate bone remodeling increases with increasing dose.

Safety Study of an Original Mesenchymal Stromal Cell Secretome-Based Medicinal Product for Spermatogenesis Restoration

SCIENTIFIC RELEVANCE. Currently, there are no effective and safe medicinal products for idiopathic male infertility. Previous studies in two animal models of infertility (short-term cryptorchidism in rats and doxorubicin-induced testicular injury in mice) have shown the effectiveness of an originator medicinal product based on the mesenchymal stromal cell (MSC) secretome.AIM. The aim of the study was to evaluate the toxicity profile of the MSC secretome-based medicinal product in rats after local intratesticular or intramuscular administration.MATERIALS AND METHODS. The MSC secretome is a combination of factors secreted by MSCs in low-glucose Dulbecco’s modified Eagle’s medium (DMEM-LG) for MSC conditioning. In the single-dose toxicity study, the MSC secretome-based medicinal product was injected under the testicular tunica albuginea of male Wistar rats (15 per group) at doses of 15 and 25 relative units (RU) per animal, which are 1.5 and 2.5 times higher than the therapeutic dose (10 RU). In the repeat-dose toxicity study, male Wistar rats (10 per group) received intramuscular thigh injections of the medicinal product on days 1, 6, and 12 at doses of 15 and 25 RU per animal. The local tolerance study involved histopathological examination of the testes and thighs at the injection site. All studies included control groups of intact animals and animals similarly injected with blank DMEM-LG. The early follow-up period was 14 days, and the late follow-up period was 42 days.RESULTS. The rats showed no changes in the general condition after single and repeated doses of the MSC secretome-based medicinal product. Single subtunical doses induced moderate irritation; its signs included pathological changes in individual seminiferous tubules: epithelial atrophy (70% of the animals on day 14; 55% at late follow-up) and sperm stasis (70% of the animals). Similar changes were observed in the blank DMEM-LG group (up to 80% of the animals). There were no pathological changes in the tissues after repeated injections. A transient increase in alkaline phosphatase activity was detected in animals after their third intramuscular injection at a dose of 25 RU; the other biochemical parameters were normal in all study groups.CONCLUSIONS. The MSC secretome-based medicinal product has a favourable safety profile following both intratesticular and intramuscular administration, as it does not cause any permanent changes in the studied organs and tissues.

The Role of Aloe vera in Inhibiting the P53 Protein Expression and Enhancing the HT29 Colon Cancer Cells Proliferation

Background: Aloe vera is one of the most widely recognized herbs considered a valid treatment option for its wound healing and anti-inflammatory properties. However, there is insufficient evidence regarding the effectiveness of its anti-tumor activity on colon cancer. Objectives: In this study, therefore, we investigated the effect of Aloe vera on the proliferation and survival of the HT29 colon cancer cell line. Methods: The effect of processed Aloe vera gel (PAG) on colon carcinogenesis was examined using low glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% serum and 1% penicillin-streptomycin in the HT29 cells. Then, we obtained the expression levels of P53 and BCL2 using the immunocytochemical (ICC) method. To determine the viability of the HT29 cells, we also performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The MTT assay showed that the HT29 cells were viable in 5%, 10%, 15%, and 20% of Aloe vera gel (AVG). However, these cells had a more obvious proliferation in the 10% Aloe vera group than in the control group. In the 10% concentration group, mitotic cells were evident in the center of the colonies. The P53 protein expression decreased, but the BCL2 protein expression remained unchanged in the 10% group compared to the control group. Conclusions: The results, therefore, indicated that in the 10% Aloe vera concentration, the expression levels of P53, known as a tumor suppressor protein in colon cancer, decreased in HT29. Our results also showed that the application of Aloe vera for its anti-tumor activity in colon cancer should be further evaluated.

Effect of Different Basal Culture Media and Sera Type Combinations on Primary Broiler Chicken Muscle Satellite Cell Heterogeneity during Proliferation and Differentiation.

Simple SummaryLittle consistency in the literature exists for optimal culture conditions for proliferating and differentiating primary broiler chicken muscle satellite cells regarding basal culture media, proliferation sera, and differentiation sera. This experiment assessed primary satellite cell proliferation and differentiation when cultured in different combinations of basal media and sera. Cells were cultured in different basal media: low glucose Dulbecco’s Modified Eagle’s medium, McCoy’s 5A, and high glucose Dulbecco’s Modified Eagle’s medium. Each media was supplemented with 15% chicken serum, or a combination of 5% horse serum + 10% chicken serum during proliferation while 3% horse serum or 3% chicken serum were supplemented during differentiation. Cultures were immunofluorescence stained for myogenic regulatory factors at different time points during proliferation and differentiation. During proliferation and differentiation, cells cultured in Dulbecco’s Modified Eagle’s medium tended to have higher proportions of myogenic cells expressing myogenic regulatory factors and promoted satellite cell fusion into myotubes compared with McCoy’s 5A. Low glucose media, glucose concentration similar to circulating glucose concentrations in broilers, combined with sera published in the literature may be the optimal culture media to promote satellite cell proliferation and differentiation.The objective of this experiment was to access primary satellite cell (SC) proliferation and differentiation when cultured in different combinations of basal media and sera due to little consistency being published on the optimal culture media for primary broiler chicken satellite cells. Cells were cultured in one of three different basal media: McCoy’s 5A, high glucose Dulbecco’s Modified Eagle’s medium (DMEM), and low glucose DMEM. Media were supplemented with 15% chicken serum (CS) or a combination of 5% horse serum (HS) + 10% CS during proliferation while 3% HS or 3% CS were added to the media during differentiation. Cultures were immunofluorescence stained for myogenic regulatory factors (MRF) at 48, 72, and 96 h post-plating for proliferation (Pax7, MyoD, and Myf-5) and 96 h post-proliferation during differentiation (Pax7 and MyoD), including MF20 to assess fusion. Cells cultured in Dulbecco’s Modified Eagle’s medium tended to have higher proportions of myogenic cells expressing MRF during proliferation and promoted fusion into myotubes compared with McCoy’s 5A during differentiation. Culturing primary SC in low glucose media, glucose concentrations similar to circulating glucose concentrations in broilers, HSCS during proliferation and CS during differentiation, appears to be optimal for promoting broiler chicken satellite cell proliferation and differentiation.

A Novel Strategy to Enhance Microfracture Treatment With Stromal Cell-Derived Factor-1 in a Rat Model.

BackgroundMicrofracture is one of the most widely used techniques for the repair of articular cartilage. However, microfracture often results in filling of the chondral defect with fibrocartilage, which exhibits poor durability and sub-optimal mechanical properties. Stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant for mesenchymal stem cells (MSCs) and is expressed at high levels in bone marrow adjacent to developing cartilage during endochondral bone formation. Integrating SDF-1 into an implantable collagen scaffold may provide a chondro-conductive and chondro-inductive milieu via chemotaxis of MSCs and promotion of chondrogenic differentiation, facilitating more robust hyaline cartilage formation following microfracture.ObjectiveThis work aimed to confirm the chemoattractive properties of SDF-1 in vitro and develop a one-step method for incorporating SDF-1 in vivo to enhance cartilage repair using a rat osteochondral defect model.MethodsBone marrow-derived MSCs (BMSCs) were harvested from the femurs of Sprague–Dawley rats and cultured in low-glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, with the medium changed every 3 days. Passage 1 MSCs were analyzed by flow cytometry with an S3 Cell Sorter (Bio-Rad). In vitro cell migration assays were performed on MSCs by labeling cells with carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE; Bio-Rad). For the microfracture model, a 1.6-mm-diameter osteochondral defect was created in the femoral trochleae of 20 Sprague–Dawley rats bilaterally until bone marrow spillage was seen under saline irrigation. One knee was chosen at random to receive implantation of the scaffold, and the contralateral knee was left unfilled as an empty control. Type I collagen scaffolds (Kensey Nash) were coated with either gelatin only or gelatin and SDF-1 using a dip coating process. The rats received implantation of either a gelatin-only scaffold (N = 10) or gelatin-and-SDF-1 scaffold (N = 10) at the site of the microfracture. Femurs were collected for histological analyses at 4- and 8-week time points post-operatively, and sections were stained with Safranin O/Fast Green. The samples were graded blindly by two observers using the Modified O’Driscoll score, a validated scoring system for chondral repair. A minimum of 10 separate grading scores were made per sample and averaged. Quantitative comparisons of cell migration in vitro were performed with one-way ANOVA. Cartilage repair in vivo was also compared among groups with one-way ANOVA, and the results were presented as mean ± standard deviation, with P-values < 0.05 considered as statistically significant.ResultsMSC migration showed a dose–response relationship with SDF-1, with an optimal dosage for chemotaxis between 10 and 100 ng/ml. After scaffold implantation, the SDF-1-treated group demonstrated complete filling of the cartilage defect with mature cartilage tissue, exhibiting strong proteoglycan content, smooth borders, and good incorporation into marginal cartilage. Modified O’Driscoll scores after 8 weeks showed a significant improvement of cartilage repair in the SDF-1 group relative to the empty control group (P < 0.01), with a trend toward improvement when compared with the gelatin-only-scaffold group (P < 0.1). No significant differences in scores were found between the empty defect group and gelatin-only group.ConclusionIn this study, we demonstrated a simple method for improving the quality of cartilage defect repair in a rat model of microfracture. We confirmed the chemotactic properties of SDF-1 on rat MSCs and found an optimized dosage range for chemotaxis between 10 and 100 ng/ml. Furthermore, we demonstrated a strategy to incorporate SDF-1 into gelatin–collagen I scaffolds in vivo at the site of an osteochondral defect. SDF-1-treated defects displayed robust hyaline cartilage resurfacing of the defect with minimal fibrous tissue, in contrast to the empty control group. The results of the in vitro and in vivo studies together suggest that SDF-1-mediated signaling may significantly improve the quality of cartilage regeneration in an osteochondral defect.

Bovine tongue epithelium-derived cells: A new source of bovine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) possess the ability to differentiate into multiple cell lineages, and thus, confer great potential for use in regenerative medicine and biotechnology. In the present study, we attempted to isolate and characterize bovine tongue tissue epithelium-derived MSCs (boT-MSCs) and investigate the culture conditions required for long-term culturing of boT-MSCs. boT-MSCs were successfully isolated by the collagenase digestion method and their proliferative capacity was maintained for up to 20 or more passages. We observed a significant increase in the proliferation of boT-MSCs during the 20 consecutive passages under low-glucose Dulbecco’s modified Eagle’s medium culture condition among the three culture conditions. These boT-MSCs presented pluripotency markers (octamer-binding transcription factor 3/4 (Oct3/4) and sex determining region Y-box2 (Sox2)) and cell surface markers, which included CD13, CD29, CD44, CD73, CD90, CD105, CD166, and major histocompatibility complex (MHC) class I (MHC-I) but not CD11b, CD14, CD31, CD34, CD45, CD80, CD86, CD106, CD117, and MHC-II at third passage. Moreover, these boT-MSCs could differentiate into mesodermal (adipocyte, osteocyte, and chondrocyte) cell lineages. Thus, the present study suggests that the tongue of bovines could be used as a source of bovine MSCs.

DNA Damage Levels and DNA Repair Capability of Bone Marrow‐Derived Mesenchymal Stem Cells from Sprague Dawley® SPF rats as a Quality Parameter Before Surgical Transfection for the Treatment of Gestational Diabetic Myopathy

Mesenchymal stem cells (MSCs) have an important role for regenerative medicine through their self‐renewal and differentiation capability; besides its involvement in tissue development, maintenance, and repair, being useful for skeletal muscle regeneration by the production of secretory factors with trophic and engraftment functions. We propose to use the alkaline comet assay to evaluate the DNA damage levels and DNA repair capability according to cell culture passages as one of the parameters to test the quality of MSCs before surgical transplantation to the periurethral and rectus abdominis muscle (RAM) for the treatment of gestational diabetic myopathy in female rats at postpartum. MSCs were collected from bone marrow of female Sprague Dawley® rats. The characterization of these cells was done according to the International Society for Stem Cell. After isolation using Ficoll‐Paque and washing in low glucose Dulbecco's Modified Eagle Medium (DMEM) enriched with L‐glutamine, the cells were seeded in 25 cm2 plastic flasks containing high glucose DMEM enriched with L‐glutamine plus 20% bovine fetal serum, penicillin, streptomycin and amphotericin B. The cells were harvested at 90% of confluence and subjected to comet assay analysis. The slides were incubated in alkaline electrophoresis solution for 20 minutes for DNA unwinding and the run was conducted at 25v, 0.3A and 8W for 30 minutes, at 4 °C. The slides were stained with SYBR GOLD® (1:10.000 dilution) and analyzed in a fluorescent microscope using COMET ASSAY VI software. The DNA damage levels were ranked according to the percentage of damage as follows: 1) 0 % – 5% of damage; 2) 6% – 20% of damage; 3) 21% – 40% of damage; 4) 41% –95% of damage; 5) &gt; 95% of damage. The percentage of nucleoids with such levels of damage was calculated and analyzed using the Chi‐Square test. The analysis was conducted using SPSS (21.0) and p &lt; 0.05 was considered significant. This study was approved by the local Animal Ethics Committee (1234/2017 – CEUA). The partial results demonstrated that the 5th passage has a high percentage (70.04%) of cells with high levels of DNA damage (41% – 95%) assessed by tail intensity. The proportion of cells with high percentage of DNA damage (41% – 95%) is statistically significant from the proportion of cells with low DNA damage levels (0% – 5%, 6% –20%, and 21% –40%; p &lt; 0.001). The preliminary results demonstrated that the 5th passage is not useful for surgical transplantation because of high DNA damage levels. Future experiments will be conducted to test the DNA damage of the other passages and also evaluated the effect of cell culture on DNA repair capability. Furthermore, such results will be compared with the functional evaluations of periurethral and RAM after the surgical transplantation of MSC.Support or Funding InformationSupported by São Paulo Research Foundation (2016/01743‐5)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In situ self-assembly of graphene oxide/polydopamine/Sr2+ nanosheets on titanium surfaces for enhanced osteogenic differentiation of mesenchymal stem cells

Bioinert surface is the greatest obstacle to the biomedical applications of titanium (Ti)-based materials. Herein, a bioactive coating as a carrier of strontium ions (Sr2+) on Ti surfaces was prepared by in situ self-assembly of graphene oxide/polydopamine/Sr2+ (GO/PDA/Sr2+) nanosheets. Surface physicochemical characteristics of the coating were evaluated by scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), contact angle (CA) and surface free energy (SFE) measurements. The results showed an ordered and Sr-incorporated nanosheets coating that may be beneficial to cell responses of mesenchymal stem cells (MSCs). Furthermore, 4.51 at.% Sr was introduced into the coating. During 30 days of immersion, GO/PDA/Sr2+ nanosheets coated Ti surfaces cumulatively released 20.36 ± 2.09 ppm Sr2+ in low glucose Dulbecco's Modified Eagle's Medium (L-DMEM), but only 16.62 ± 1.00 ppm in simulated body fluid (SBF). Moreover, the coating possessed outstanding capacity of protein adsorption. In vitro biological evaluation displayed that the synergies of PDA, GO, and released Sr2+ promoted the adhesion, spreading, and proliferation of MSCs grown on GO/PDA/Sr2+-coated Ti surfaces, subsequently enhancing the expressions of bone-specific genes and proteins, in turn accelerating extracellular matrix (ECM) mineralization. This work provided us an alternative for bio-functionalizing the surfaces of Ti-based implants.

Development of In Vitro NASH Model with Mechanically Compliant Substrate

Although a sedentary lifestyle is known to play a role in nonalcoholic steatohepatitis (NASH), understanding of the molecular mechanism of NASH development is incomplete and effective treatment is still greatly sought. Part of this challenging situation in tackling NASH is a lack of an appropriate in vitro hepatocyte culture that reproduces in vivo cellular functions ex vivo. Emerging evidence suggests that matching the stiffness of substrates with that of tissues helps reproducing in vivo functions of cells derived from them. Thus, we hypothesized that primary hepatocytes on 500 Pa polyacrylamide (PAA) gels, whose stiffness matches that of the liver, may exhibit steatosis and a pro-inflammatory response when treated with either fructose or palmitate to model over-nutrition. To this end, rat primary hepatocytes were seeded on either 500 Pa PAA gels or glass coverslips, which represent a conventional but unphysiological stiff culture device. Cells were cultured in low glucose Dulbecco’s Modified Eagle’s medium, whose glucose concentration matches the normal blood glucose level, with or without addition of 5.5 mM fructose or 0.5 mM palmitate. Addition of fructose or palmitate induced lipid accumulation in cells on 500 Pa gels and increased ROS (reactive oxygen species) accumulation in hepatocytes on 500 Pa PAA gels, but not in hepatocytes on glass. Fructose or palmitate treatment also caused HMGB1 (High Mobility Group Box 1) secretion by hepatocytes, implicating a proinflammatory response by these cells. These results suggest that exposure of hepatocytes on a mechanically compliant substrate to either fructose or palmitate may serve as an excellent ex vivo model of the initial phase of NASH development by showing ability to initiate an inflammatory process. Disclosure M. Morishima: None. K. Horikawa: None. M. Funaki: Research Support; Self; Otsuka Holdings Co., Ltd., The Knowledge Cluster from the Ministry of Education, Science, and Culture of Japan. Board Member; Self; Mechanogenic C.C..

Cartilage Repair with Mesenchymal Stem Cells (MSCs) Delivered in a Novel Chondroitin Sulfate / Polyethylene Glycol Hydrogel in a Rabbit Animal Model”

Objectives:To determine whether rabbit bone marrow-derived MSCs embedded in a chondroitin sulfate (ChS)/ poly (ethylene glycol) (PEG) biodegradable hydrogel display enhanced in vivo chondrogenesis as compared to ChS/PEG hydrogel alone, in a critical sized osteochondral defect in a rabbit animal model.Methods:Allogenic MSCs were harvested from bone marrow and expanded in specific media (20% fetal bovine serum, 50 U ml-1 penicillin, 50 mg ml-1 streptomycin, 20 mg ml-1 gentamicin, and 5 ng ml-1 bFGF (fibroblast growth factor) in low glucose Dulbecco’s modified Eagle media) under standard cell culture conditions (37o C with 5% CO2). Surgery was carried out in 10 mature New Zealand white rabbits (8 months old). A critical sized chondral defect (3mm) was performed bilaterally in the trochlear groove of the femoropatellar joint in all ten rabbits. Three treatment groups were established as follows: 1- hydrogel alone (5N), 2- hydrogel with MSCs (3 x 106 cell/ml) (5N), and 3- control defect with no treatment (10N). Animals were left to ambulate freely after surgery. At 6 months postoperative, euthanasia was performed. Macroscopic evaluation of defect repair was performed by four observers unaware of treatment groups using ICRS (International Cartilage Repair Society) scoring. Microscopic evaluation was performed using the O’Driscoll grading system. Using SigmaPlot 11.0 statistical software (Systat Software, San Jose, CA, USA), comparison between groups was performed with an ANOVA test to see if differences existed between treatment groups. Tukey’s correction was used to adjust for multiple group comparisons, and two independent t-tests: 1- between rabbits receiving hydrogel alone vs. their respective controls; 2- between rabbits receiving hydrogel / MSCs vs. their respective controls; for both ICRS and O’Driscoll scores, being a total of six statistical analyses.Results:At time of euthanasia, all hydrogels remained in place. There was no synovial reaction or macroscopic inflammation. Cartilage repair was evident in all groups. Macroscopically, no significant difference was evident according to ICRS scores. The average ICRS score for the groups was: 1 (hydrogel alone): 10 ± 1.73 SD, 2 (hydrogel with MSCs): 10 ± 1.41 SD and 3(control): 11.3 ± 1.06 SD (p= 0.088). Histological average scoring with O’Driscoll system for each group was highest for the hydrogel alone (1: 19.8 ± 5.5 SD, 2: 16 ± 3.54 SD, and 3: 18.5 ± 3.63 SD) (p= 0.227) (Figure 1). One way ANOVA, showed no statistical difference for both ICRS and O’Driscoll scores. The t-tests showed no statistical significance among groups (p>.05), except for ICRS score comparing control vs. hydrogel / MSCs which favored the control group (p=0.046).Conclusion:Hydrogels remained in place and induced chondrogenesis, especially when hydrogels were placed in the defect without MSCs. Adding MSCs to the hydrogel did not improve cartilage healing. Macroscopically, ICRS score was significantly worse for the MSCs/hydrogel group when compared to the control group. Limitations of this study include small animal model with natural good intrinsic cartilage regeneration. Still, all selected animals were mature, reducing the chances of natural healing. In addition, all animals had an established control defect. Future experiments will include adding transforming growth factor (TFGb) to the encapsulated MSCs to potentially improve chondrogenesis.

Isolation, Characterization and Expansion of Bone-Marrow derived Mesenchymal Stem cells (MSCs) from Chronic Kidney Disease (CKD) patients for Clinical Translation

Introduction: During the recent years, Mesenchymal Stem cell (MSC) has gained importance in tissue repair, tissue engineering and in immunosuppressive therapy. Autologous MSCs are being used in the treatment of various diseases, including cardiovascular diseases, acute renal failure, autoimmune diseases, neurodegenerative disorders and organ transplant. Recently, ex-vivo expanded MSC infusion in renal transplant cases is being explored as a possible intervention for induction phase. Past studies support the feasibility of lowering the dose or avoiding the use of immunosuppressive drugs after induction with MSCs. Several reports also demonstrate that MSCs lead to decreased acute rejection rates, better graft function and decreased infection rates. The main objective of the study was to isolate, expand and characterize BM-derived autologous MSCs from CKD (Chronic kidney disease) patients for clinical translation. Methods: Bone marrow aspirates of the patients collected aseptically from the posterior iliac crest. Samples were centrifuged and the cellular layer containing the MSCs was carefully removed, washed, counted and seeded in low glucose Dulbecco modified Eagle medium (DMEM) containing 10% serum albumin and 1%antibiotic-antimycotic agents. After three passages, quality assurance was done as per the International Society of Cell therapy and transplantation. The seeding density was ~5 X 106 cells in T75 flasks. MSCs were allowed to adhere to the surface of the flasks for 72 hours at 37°C and 5% CO2. The non-adherent cells in the media were removed, fresh media added, incubated till the cells became confluent. At 70-80% confluence, cells were detached and replated ~25,000 MSCs in T 75 flask. Results: Approximately 10X106 cells were obtained after three passages in 1 month’s time frame without any contamination. The viability of thawed cells was 85.5%, and they maintained the stemness even after 6 months of cryopreservation similar to fresh MSCs. They expressed CD44, CD73, and CD90 and were negative for CD19, CD34, CD45, and HLA DR. Cytogenetic analysis showed no chromosomal abnormalities. Conclusion: Optimization of the protocol for isolation, expansion of MSCs from bone marrow has been done successfully for implementing MSC-based therapy in kidney transplantation, and to promote its clinical translation.FIGURE 1: Microscopic Images of Mesenchymal Stem Cells.FIGURE 2: Enumerarion of MSC-specific CD markers (CD90, CD-73 and CD44) in cells from 3rd passage of patient No.10 in Panel B shows viablity of cells as stained by 7-AAD. Further Panel B and C shows MSC-negative markers for MSCs.FIGURE 3: Karyotype report showing no chromosomal abnormality.

Effects of kisspeptin-10 on in vitro proliferation and kisspeptin receptor expression in primary epithelial cell cultures isolated from bovine placental cotyledons of fetuses at the first trimester of pregnancy

Kisspeptin (Kp) and Kiss-1 receptor (Kiss-1R) expressions have been reported to be in the placenta, and a possible involvement of the Kiss-1R/Kps system in regulating trophoblast invasion and proliferation has been hypothesized. The aim of the present study was to investigate whether Kiss-1R activation by kisspeptin-10 (Kp-10) could modulate in vitro proliferation and progesterone (P4) secretion of bovine primary placental cell lines isolated from cotyledons of fetuses in the first trimester of pregnancy. The involvement of Kiss-1R in the cell responses observed was also analyzed. Uteri from cows at the first trimester of pregnancy were obtained from local abattoirs. Fetal cotyledon fragments were digested with collagenase in low glucose Dulbecco's Modified Eagle's Medium and cell lines were isolated. After being characterized for epithelial polygonal morphology, the presence of binucleate cells, male gender, and the expression of cytokeratin and zona occludens 2, cell lines were cultured in a low glucose Dulbecco's Modified Eagle's Medium–based expansion medium in the presence of 0.01, 0.1, 1, and 10 μM Kp-10. Control cells were cultured in the absence of Kp-10. Cell population doubling time was evaluated for each culture passage (P) from P1 to P10. Cells were tested for Kiss-1R mRNA expression analysis by real-time reverse transcription–polymerase chain reaction, and culture media were analyzed for P4 concentration by radioimmunoassay. Kisspeptin-10 modulated in vitro proliferation of epithelial cell lines isolated from cotyledons recovered from bovine fetuses in the first trimester of pregnancy. Inhibitory (line A) or stimulatory (line B) effects of Kp-10 on cell proliferation were found in different cell lines and observed cell responses were found to be related to Kiss-1R mRNA levels. Inhibition of cell proliferation matched with not significant variation of Kiss-1R expression, whereas stimulation of cell proliferation was found to be related to Kiss-1R upregulation. In both cell lines, no effect of Kp-10 on P4 secretion was found at any tested concentration. These results lead to the conclusion that the Kiss-1R/Kps system is involved in the regulation of cell proliferation of bovine placental cotyledon cell lines isolated at the first trimester of pregnancy but, at this gestational stage, it may not be involved in modulating placental P4 secretion.

Improvement of the Survival of Autologous Free-Fat Transplants in Rats Using Vascular Endothelial Growth Factor 165–Transfected Bone Mesenchymal Stem Cells

Autologous free-fat transplantation is limited by fat absorption and fibrosis due to fat necrosis. In this study, we explored the feasibility of using bone mesenchymal stem cells (BMSCs) transfected by vascular endothelial growth factor (VEGF) 165 gene to improve the survival of transplanted fat tissues in a rat model. Bone mesenchymal stem cells with (group A) and without (group B) VEGF165 gene transfection were each mixed with free transplanted fat tissue; then, they were injected subcutaneously at sites on the backs of 36 Sprague-Dawley rats. A control group (group C) was established by using low-glucose Dulbecco modified Eagle medium. The transplants from groups A, B, and C were gathered respectively at 30, 90, and 180 days after transplantation. Transplanted tissue volume and histology were evaluated, and revascularization was quantified by counting the number of capillaries. The survival rate of the A group was significantly higher than that of the B group (P < 0.05), which was significantly higher than that of the C group (P < 0.05). Histologic analysis revealed that both groups A and B had significantly less fat necrosis and fibrosis (P < 0.05). Group A had significantly higher capillary density than the other 2 groups (P < 0.05), and its chloromethyl-1-1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanineperchlorate-labeled BMSCs were also von Willebrand factor positive. When transfected by the VEGF165 gene, the BMSCs of a rat can better promote the regeneration of capillaries, which can improve the survival rate of transplanted free-fat tissue. This experiment combined correlative theory and techniques of stem cell research, genetic technology, and autologous free-fat transplantation. It may provide a new way to improve the survival of tissue undergoing autologous free-fat transplantation.

Enhanced adipogenic differentiation of bovine bone marrow-derived mesenchymal stem cells

Until now, the isolation and characterisation of bovine bone marrow-derived mesenchymal stem cells (bBM-MSCs) have not been established, which prompted us to optimise the differentiation protocol for bBM-MSCs. In this study, bBM-MSCs were freshly isolated from three six-month-old cattle and used for pluripotent or phenotypic characterisation by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometer. Initially, the cells were adherent to plastic surfaces and exhibited spindle-like morphology. The cells expressed pluripotent as well as typical MSC markers. In addition, bBM-MSCs were differentiated into chondrocytes and osteocytes, but less efficiently into adipocytes. Accordingly, we conducted this study to establish the optimal adipogenic differentiation protocol under specific culture conditions. For this purpose, we formulated the basal differentiation medium using low-glucose Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin (P/S), 1 µM dexamethasone, 0.5 mM indomethacin and 0.5 mM 3-isobutyl-l-methylxanthine, and added with three levels of insulin at 10, 15 and 20 µg/mL as insulin is the key adipogenesis inducer. The level of differentiation was evaluated by Oil Red O staining and by analysing the expression of adipocyte lineage-specific genes by a quantitative real time RT-PCR. In this study, we found that the bBM-MSCs were effectively differentiated into adipocytes as manifested by the presence of Oil Red O-stained lipid droplets. The mRNA levels of adipocyte-specific genes in the differentiated cells were highly expressed as compared with the non-differentiated bBM-MSCs. In conclusion, we successfully isolated and characterised bBM-MSCs with multipotent and differentiation potential. Additionally, enhanced in vitro adipogenic differentiation protocol for bBM-MSCs was established.

Isolation and characterization of equine amniotic membrane-derived mesenchymal stem cells

Recent studies have shown that mesenchymal stem cells (MSCs) are able to differentiate into multi-lineage cells such as adipocytes, chondroblasts, and osteoblasts. Amniotic membrane from whole placenta is a good source of stem cells in humans. This membrane can potentially be used for wound healing and corneal surface reconstruction. Moreover, it can be easily obtained after delivery and is usually discarded as classified waste. In the present study, we successfully isolated and characterized equine amniotic membrane-derived mesenchymal stem cells (eAM-MSCs) that were cultured and maintained in low glucose Dulbecco's modified Eagle's medium. The proliferation of eAM-MSCs was measured based on the cumulative population doubling level (CPDL). Immunophenotyping of eAM-MSCs by flow cytometry showed that the major population was of mesenchymal origin. To confirm differentiation potential, a multi-lineage differentiation assay was conducted. We found that under appropriate conditions, eAM-MSCs are capable of multi-lineage differentiation. Our results indicated that eAM-MSCs may be a good source of stem cells, making them potentially useful for veterinary regenerative medicine and cell-based therapy.

Generation of Insulin Producing Cells using Mesenchymal Stem Cells Derived from Bone Marrow of New-Zealand White Rabbits

Background: The utilization of stem cell trans-differentiation into insulin-producing cells (IPCs) would provide potential promising therapy for diabetes mellitus (DM). Objective: The study was aimed to investigate the differentiation potential of rabbit’s bone marrowderived cells into insulin producing cells. Methods: Bone marrow-derived mesenchymal stem cells (MSCs) were obtained from three New-Zealand male white rabbits and propagated in a primary culture for 14 days in low glucose Dulbecco's Modified Eagle's Medium (DMEM), harvested and subjected to another three passages encompassing 21 days. After that, MSCs were functionally defined by their ability to differentiate into osteoblasts. This was achieved by incubation with the DMEM containing 10 -7 M dexamethasone, 10 mM βglycerophosphate and 50 μl/ml of ascorbic acid. Osteogenic differentiation was followed up at days 2, 4, 7 and 9 by staining cells with alizarin red S and vonKossa. Results: It was found that the onset of differentiation was at day 7 and continued at day 9. On the other hand, another patch of MSCs were induced into insulin-producing cells by two step incubation. The first with high glucose serum-free DMEM containing 0.5 mmol/L β-mercaptoethanol for three days and the second follows by incubation with the same medium containing 10 mmol/L nicotinamideinstead of β-mercaptoethanolfor 18 days. Trans-differentiation was followed up at days 4, 9, 14 and 21. It was found that the cells have trans-differentiated into insulin-producing cells starting from day 14 and continued in the subsequent days as judged by their affinity to stain with diphenylthiocarbazone (dithizone or DTZ) at days 14 and 21. Conclusion: The results would provide some insights of using rabbit's bone marrow as a source of MSCs with their differentiation potentials. This might help for the development of a future stem cell therapy for diabetes as well as several other human diseases.

Improvement in Poor Graft Function After Allogeneic Hematopoietic Stem Cell Transplantation Upon Administration of Mesenchymal Stem Cells From Third-Party Donors: A Pilot Prospective Study

Abstract 1904 BackgroundPoor graft function (PGF) is a refractory complication that occurs in 5–27% patients, and is associated with considerable morbidity and mortality related to infections or hemorrhagic complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mesenchymal stem cells (MSCs) possess the capacity to differentiate into several types of mesenchymal tissues, to suppress immunological responses, and to support hematopoiesis. Clinical applications of MSCs are evolving rapidly with goals of improving hematopoietic engraftment, preventing and treating graft-versus-host disease (GVHD) after allo-HSCT and so on. In the present study, we prospectively evaluated the efficacy and safety of MSCs expanded from the bone marrow of a third-party donor to patients with PGF after allo-HSCT. MethodsTwenty patients, including primary PGF in 7 and secondary in 13, were enrolled in this prospective multicenter trial. Bone marrow-derived MSCs were obtained from HLA-unrelated third-party donors. MSCs were cultured and expanded with human MSCs growth medium (low-glucose Dulbecco’s modified Eagle’s medium [L-DMEM], Hyclone, Logan and UT) with 10% (v/v) fetal bovine serum. MSCs were harvested after 4–5 passages for clinical administration. MSCs were given at 4 weeks intervals at the dose of 1×106 cells/kg. If ANC counts did not achieve >1.5×109/L and PLT counts not >50×109/L within 28 d after MSCs treatment, a second course of MSCs treatment was given. Flow cytometry was used to examine the lymphocyte subsets in peripheral blood pre- and post-MSCs treatment. ResultsSeventeen patients were responsive and 3 (primary PGF in 2 and secondary in 1) were not to 1–3 cycles of MSCs treatment. Within the first 100 d after MSCs treatment, 13 patients developed 20 episodes of infections. Moreover, 5 patients experienced cytomegalovirus-DNA viremia and 7 experienced Epstein-Barr virus (EBV)-DNA viremia within the first 100 d after MSCs treatment. Within a median follow-up of 125 d (range 11–579 d) after MSCs treatment, 6 patients (primary PGF in 3 and secondary in 3) experienced CMV-DNA viremia, and 8 (primary PGF in 2 and secondary in 6) developed EBV-DNA viremia, including 3 with EBV-associated post-transplant lymphoproliferative disorders (PTLD). One patient developed gradeII acute GVHD and 2 developed local chronic GVHD after MSCs treatment; 2 developed acute GVHD and chronic GVHD, respectively, after donor lymphocyte infusion because of PTLD. The proportions of CD3+ T cells and CD3+CD4+ T cells at 56 d after MSCs treatment were higher than those prior to MSCs treatment (88.05 ± 1.26% vs 79.05 ± 3.23%, P=0.015 and 24.09 ± 1.95% vs 17.40 ± 1.57%, P=0.012, respectively). The proportion of CD3+CD8+ T cells at 56 d after MSCs treatment was lower than that prior to MSCs treatment (51.91 ± 2.86% vs 61.39 ± 3.43%, P=0.043), and the ratio of CD3+CD4+ to CD3+CD8+ T cells was higher after MSCs treatment (0.31 ± 0.04 vs 0.51 ± 0.06, P=0.01). The proportions of CD4+CD25+ FoxP3 regulatory T cells, CD19+ B cells, and NK cells did not change significantly after MSCs treatment. With a median follow-up time of 508 d (range, 166–904 d) post-transplantation, 9 patients were alive and 11 died. No short-term toxic side effects were observed after MSCs treatment. Two patients experienced leukemic relapse. With the exception of 3 patients with PTLD, no secondary tumors occurred. ConclusionsMSCs derived from the bone marrow of a third-party donor are effective to both primary and secondary PGF after allo-HSCT. However, whether such treatment might increase the risks of EBV infection and reactivation or the development of EBV-associated PTLD should be studied further. Disclosures:No relevant conflicts of interest to declare.

LKB1 Suppresses p21-activated Kinase-1 (PAK1) by Phosphorylation of Thr109 in the p21-binding Domain

The serine/threonine protein kinase LKB1 is a tumor suppressor gene mutated in Peutz-Jeghers syndrome patients. The mutations are found also in several types of sporadic cancer. Although LKB1 is implicated in suppression of cell growth and metastasis, the detailed mechanisms have not yet been elucidated. In this study, we investigated the effect of LKB1 on cell motility, whose acquisition occurs in early metastasis. The knockdown of LKB1 enhanced cell migration and PAK1 activity in human colon cancer HCT116 cells, whereas forced expression of LKB1 in Lkb1-null mouse embryonic fibroblasts suppressed PAK1 activity and PAK1-mediated cell migration simultaneously. Notably, LKB1 directly phosphorylated PAK1 at Thr(109) in the p21-binding domain in vitro. The phosphomimetic T109E mutant showed significantly lower protein kinase activity than wild-type PAK1, suggesting that the phosphorylation at Thr(109) by LKB1 was responsible for suppression of PAK1. Consistently, the nonphosphorylatable T109A mutant was resistant to suppression by LKB1. Furthermore, we found that PAK1 was activated in the hepatocellular carcinomas and the precancerous liver lesions of Lkb1(+/-) mice. Taken together, these results suggest that PAK1 is a direct downstream target of LKB1 and plays an essential role in LKB1-induced suppression of cell migration.

Effects of imatinib mesylate in osteoblastogenesis

Imatinib mesylate (IM), a tyrosine kinase inhibitor currently used in chronic myeloid leukemia (CML), may also affect the growth of other cellular systems besides CML cells. Because it has been reported that IM may affect bone tissue remodeling, we evaluated the effects of IM on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). After 21 days of culture, hBM-MSCs treated with IM (1 microM) alone or osteogenic medium (OM) + IM showed changes in morphology with evidence of extracellular mineralization and increased mRNA expression of osteogenic markers, such as RUNX2, osteocalcin (OCN), and bone morphogenetic protein (BMP-2). We also observed that levels of OCN and the osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL) ratio (OPG/RANKL ratio) were increased in the surnatant of the 21-day culture with IM or OM + IM compared to controls (p<0.005). In addition, we found that in 46 serum samples collected from CML patients treated with IM for 3 to 24 months, the OPG/RANKL ratio increased after 3 and 6 months (p<0.004) returning back to the basal level after 24 months of IM treatment. In these patients, OCN levels were low at diagnosis but they increased throughout the IM treatment, approaching normal levels at 24 months of IM therapy. In summary, our data show that IM increases mRNA expression of osteogenic markers in hBM-MSCs and increases the OPG/RANKL ratio and the OCN levels both in surnatant of hBM-MSCs cultured with IM and in serum of patients treated with IM, thus indicating that IM potentially favors osteoblastogenesis.

Influence of Culture Medium on Smooth Muscle Cell Differentiation from Human Bone Marrow–Derived Mesenchymal Stem Cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) represent an appealing source of smooth muscle cells (SMCs) for engineering small-diameter vascular grafts due to the limited availability and replicative capacity of somatic SMCs. However, lack of standardization of hMSC culture conditions has limited some progress in hMSC research. Because, at the moment, a chemically defined, serum-free medium without growth factors is not capable of amplifying hMSCs in vitro, the usage of serum (either human serum or fetal bovine serum [FBS]) continues in hMSC research. The emergence of commercial hMSCs and hMSC media opened a series of questions regarding the compatibility of commercial and homemade hMSCs and hMSC media. In this study, two types of commonly used FBS-containing hMSC media-MSCGM (containing 10% FBS) and MesenPro (containing 2% FBS), along with our homemade medium (low-glucose Dulbecco's modified Eagle's medium plus 10% selected lot FBS)-were compared in their ability to support SMC differentiation from hMSCs. The effects of FBS level, medium supplements (ascorbic acid, copper, etc.), and growth factors (transforming growth factor beta1) were also examined for their impact on SMC differentiation. It was discovered that MesenPro and transforming growth factor beta1 are the strongest SMC inducers from hMSCs. In contrast, hMSCs grown in homemade (10% Dulbecco's modified Eagle's medium) and commercial MSCGM media remained undifferentiated. FBS concentration did not affect SMC differentiation when 10% FBS was compared with 2%. Finally, the mechanism underlying SMC differentiation from hMSCs grown in FBS-containing medium was explored by following the expression changes of serum response factor during the establishment of hMSC culture.