culture dishes which were left uncoated (CON) or coated with collagen (COL, 2ug/cm2), fibronectin (FBN (100ng/cm2) or Poly-l-lysine (PLL, 100ng/cm2) for 48–72 hours. (A) Doubling time was calculated by cell counting of MSC grown on different matrices. (B) Cell proliferation on different matrices were analysed by MTT assay. *p<0.05 represents cell proliferation significantly higher in cells grown on COL compared to CON, FBN and PLL surfaces. (C) MSC cultured on COL, FBN and PLL and TC surface (CON) was collected by trypsinization and stained with propidium iodide and the cell cycle profile was analysed flow cytometrically. Values are Mean±SD, n = 5–7 samples. (D) Equal numbers of MSC were seeded on TC (CON), COL, FBN and PLL surfaces and non-adherent cells were removed after 2hr or 12hr and the adherent cells were counted. Five different high power fields were counted microscopically for each sample. Values are Mean ± SD, n = 3 samples (E) MSC were treated with H 2 O 2 (400μm) for 2–3 hours under low serum conditions (0.1% FBS) and the percentage of cell death was analysed by counting the live and dead cells in each condition. *p<0.05 represents that cell death in cells grown on COL was significantly lower compared to other conditions. (F, G) MSC grown on different matrices were serum starved for 48 hours and mitosox superoxide indicator was added to cells for 30 minutes and mitosox red fluorescence was analysed by flow cytometry and the percentage positive cells were determined. (G) Representative flow cytometric plot showing mitosox fluorescence analysis and gating in control (i), COL (ii), FBN (iii), PLL(iv). Values are Mean±SD, n = 3 independent experiments. (H) Total RNA was extracted from 5 samples cultured on uncoated TC (CON), COL, FBN and PLL coated tissue culture dishes. Semi-quantitative real-time PCR was done to analyse the mRNA expression levels of IL6 and the values were normalized to GAPDH expression levels in the respective samples. *p<0.05 represents that IL6 levels were significantly higher in control (CON) compared to other conditions. Values are Mean±SD, n = 5 samples. *p<0.05, **p<0.005, ***p<0.0005
The ADSC secrete a number of cytokines and growth factors with paracrine and autocrine activities, such as macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor-macrophage (GM-CSF), macrophage inflammatory protein (MIP-1α/CCL3), as demonstrated by Sibille [ 56 ] and Cerqueira [ 55 ]. These secreted factors produce a series of responses in the local immune system, enhance angiogenesis and induce the proliferationand differentiation of tissue stemcells. Moreover, the ADSC induce the expression of junction proteins, increase microvascular integrity and NO production. The paracrine effect of ADSC was observed in co-culture with activated macrophages, with the production of NO. The macrophages are essential for effective tissue regeneration since they regulate the recruitment, proliferationand differentiation of the target cells, such as cardiomyoblasts, fibroblasts andmesenchymalstemcells. ( Figure 11 ) shows the intense production of NO in the presence of Ch-Ma, Ch-Co-Ge-Ma, Ch-Ma-ADSC, Ch-Co- Ma-ADSC and Ch-Co-Ge-Ma-ADSC revealing the importance of the presence of chitosan, collagen, ADSC genipin and in the process of tissue regeneration, because these scaffolds strongly stimulated NO production that may be responsible, in vivo, by vasodilation present in physiological angiogenesis, in addition to function as a mediator in wound healing, and as an inhibitor of leukocyte adhesion in post capillary microvessels. This was also demonstrated by Chiono [ 10 ]. Nitric oxide(NO) is an important antimicrobial agent, plays an important role in the decontamination site of implantation of scaffolds, and regulates the secretion ofcollagen by fibroblasts, influencing the mechanical strength of the new tissue formation.
Chondrocytes and bone marrow mesenchymalstemcells (BMSCs) are frequently used as seed cells in cartilage tissue en- gineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagenand aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagenand aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagenand aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.
of the most important components of liver ECM may lead to the activation of a large number of integrins on the cell surface (9). Integrins are a family of cell adhesion receptors, linking the ECM to stimulate and control several intracellular signaling pathways (10- 12). Thus, it seems that laminin coating improves survivalof hBM-MSCs during differentiation period. Takayama et al (25) showed that the type III laminin had positive effects on the maintenance and expansion of hepatoblast-like cells derived from ES/iPS cells, which confirmes our results of cell survival improvement due to laminin treatment. However, to the best of our knowledge, no data was available to address the effects of laminin on improving hepatogenic differentiation of MSCs. Considering the fact that during the initiation of the regenerative process in damaged liver, the hepatic progenitors acquire an ability to attach to laminin (24), prompted us to examine the possibility that laminin as a liver specific ECM component might
This study was undertaken to clarify the role and mechanism of pyruvate dehydrogenase kinase isoform 2 (PDK2) in chondrogenic differentiation ofmesenchymalstemcells (MSCs). MSCs were isolated from femurs and tibias of Sprague- Dawley rats, weighing 300–400 g (5 females and 5 males). Overexpression and knockdown of PDK2 were transfected into MSCs and then cell viability, adhesionand migration were assessed. Additionally, the roles of aberrant PDK2 in chondrogenesis markers SRY-related high mobility group-box 6 (Sox6), type II procollagen gene (COL2A1), cartilage oligomeric matrix protein (COMP), aggrecan (AGC1), type IX procollagen gene (COL9A2) andcollagen type 1 alpha 1 (COL1A1) were measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The expressions of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and extracellular regulated protein kinase (ERK) were measured. Overexpressing PDK2 promoted cell viability, adhesionand inhibited cell migration in MSCs (all Po0.05). qRT-PCR assay showed a potent increase in the mRNA expressions of all chondrogenesis markers in response to overexpressing PDK2 (Po0.01 or Po0.05). PDK2 overexpression also induced a signiﬁcant accumulation in mRNA and protein expressions of JNK, p38MAPK and ERK in MSCs compared to the control (Po0.01 or Po0.05). Meanwhile, silencing PDK2 exerted the opposite effects on MSCs. This study shows a preliminary positive role and potential mechanisms of PDK2 in chondrogenic differentiation of MSCs. It lays the theoretical groundwork for uncovering the functions of PDK2 and provides a promising basis for repairing cartilage lesions in osteoarthritis. Key words: Pyruvate dehydrogenase kinase isoform 2; Chondrogenic differentiation; Mesenchymalstem cell; SRY-related high mobility group-box 6; c-Jun N-terminal kinase (JNK)
In this study, ESCs were cultured on mouse embry- onic fibroblasts. These cells can be cultured, passaged and cryopreserved, and retain their proliferative capacity after continuous passage. Several totipotency molecular markers with an important role in maintaining the capacity for self-renewal (Eiges et al., 2001; Richards et al., 2002) oc- cur in these cells, including stage-specific embryonic anti- gens (SSEA-1, SSEA-3, SSEA-4), transcriptional regulation antigens (TRA-1-60, TRA-1-81), homologous protein transcription factor (Nanog) and transcription fac- tors OCT3\4 and SOX-2. As shown here, three germ layer teratomas were also generated. These results indicate these cells are totipotent and that the method for establishing them is feasible. MSCs were generated from ESCs. With continuous passage, the cells began to form fibroblast colo- nies. MSCs can proliferate indefinitely while retaining their potential for differentiation, as shown by the identification of totipotency factors. Indeed, MSCs were totipotent and could differentiate into osteoblasts, chondrocytes and adipocytes after stimulation with a specific inducer. MSCs can therefore be regarded as seed cells for tissue engineer- ing. The relatively controllable differentiation may provide a new approach for adipose tissue engineering.
In April 2017, 1091 CTC/7,5mL were collected and analysed from the patient’s blood. Using CellSearch, CTC have been detected in nearly 60% of metastatic BC, with a median value of 2 CTC/7.5mL [38, 39]. Higher numbers have been found in patients with positive ER, multiple bone or both bone and visceral metastasis [39, 40]. Indeed, this is concordant with the high CTC level seen in this case report, making feasible their extraction and molecular/genomic characterization. Also, at this time, the patient had already been submitted to three lines of ET and four regimens of chemotherapy, thus >5 CTC/7.5mL of blood represented a significantly worse clinical benefit rate, PFS and OS, and previous treatments’ inefficiency . However, the lack of serial CTC evaluations makes it impossible to make greater conclusions as a patient’s CTC history and dynamics (throughout treatments) would better reflect the overall aggressiveness and prognosis of that BC than a current CTC status alone .
60. Adewumi O, Aflatoonian B, Ahrlund-Richter L, Amit M, Andrews PW, Beighton G, Bello PA, Benvenisty N, Berry LS, Bevan S, Blum B, Brooking J, Chen KG, Choo AB, Churchill GA, Corbel M, Damjanov I, Draper JS, Dvorak P, Emanuelsson K, Fleck RA, Ford A, Gertow K, Gertsenstein M, Gokhale PJ, Hamilton RS, Hampl A, Healy LE, Hovatta O, Hyllner J, Imreh MP,Itskovitz-Eldor J, Jackson J, Johnson JL, Jones M, Kee K, King BL, Knowles BB, Lako M, Lebrin F, Mallon BS, Manning D, Mayshar Y, McKay RD, Michalska AE, Mikkola M, Mileikovsky M, Minger SL, Moore HD, Mummery CL, Nagy A, Nakatsuji N, O'Brien CM, Oh SK, Olsson C, Otonkoski T, Park KY, Passier R, Patel H, Patel M, Pedersen R, Pera MF, Piekarczyk MS, Pera RA, Reubinoff BE, Robins AJ, Rossant J, Rugg-Gunn P, Schulz TC, Semb H, Sherrer ES, Siemen H, Stacey GN, Stojkovic M, Suemori H, Szatkiewicz J, Turetsky T, Tuuri T, van den Brink S, Vintersten K, Vuoristo S, Ward D, Weaver TA, Young LA, Zhang W. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol. 2007 Jul;25(7):803- 16.
E. coli CD (EC18.104.22.168) converts 5-FC to 5-fluorouridine (5-FU). 5-FU, a pyrimidine analogue, is metabolized in vivo by mammalian enzymes to active forms that inhibit DNA and RNA synthesis. We have used HB1.F3 cells transduced to express CD. This CD-expressing cell line was generated using an amphotropic replication- deficient retrovirus in which CD expression is regulated by the retrovirus LTR. A clonal cell population was isolated following selection of transduced cells with puromycin. The established clonal cell line expresses functional CD. Single copies of the therapeutic gene (CD) andof the immortalizing gene (v-myc) inserted into ‘relatively barren’ regions of the host cell genome (KS Aboody and MK Danks, unpublished observations). The karyotype of the isolated cell line is stable for at least 26 passages in vitro. Systemically administered HB1.F3 cellsand also 5-FC cross the blood-brain barrier, making this approach uniquely well suited for the treatment of CNS tumors. Further, CD/5-FC produces a significant by- stander effect. 123 Using this approach, we have observed
Flow cytometry was performed using a FacsCalibur instrument (Becton Dickinson, USA) equipped with an argon-ion laser tuned at 488 nm and the data were ana- lyzed using the CELLQuest software (Becton Dickinson). The cells were incubated with antibodies for 30 min at 4°C and were then washed with PBS. 7-Aminoactinomycin D (7AAD; Molecular Probes, Inc., USA) at a final concentration of 1 µg/mL was used to identify dead cells. The following specific antibodies for the human antigens were purchased from PharMingen/Becton Dickinson, USA): CD29/PE, CD34/PE, CD44/FITC, CD45/FITC, CD90/FITC, CD117/ PE, CD133/PE, CD146/FITC, CD184/PE, HLA-DR/FITC, and Stro-1/FITC, as well as the isotype control antibodies mouse IgG1,k/FITC and IgG1,k/PE. The gating strategy was used in order to acquire 10,000 events corresponding to a live MNC cluster. In this gate, the analysis was performed to observe double labeling in this population using a com- bination of the other antibodies.
Bone marrow contains a population ofstemcells that can support hematopoiesis and can differentiate into different cell lines including adipocytes, osteocytes, chondrocytes, myocytes, astrocytes, and tenocytes. These cells have been denoted mesenchymalstemcells. In the present study we isolated a cell population derived from the endothelium and subendothelium of the umbilical cord vein which possesses morphological, immunophenotypical and cell differentia- tion characteristics similar to those ofmesenchymalstemcells isolated from bone marrow. The cells were isolated from three umbilical cords after treatment of the umbilical vein lumen with collagenase. The cell population isolated consisted of adherent cells with fibroblastoid morphology which, when properly stimulated, gave origin to adipo- cytes and osteocytes in culture. Immunophenotypically, this cell popu- lation was found to be positive for the CD29, CD13, CD44, CD49e, CD54, CD90 and HLA-class 1 markers and negative for CD45, CD14, glycophorin A, HLA-DR, CD51/61, CD106, and CD49d. The charac- teristics described are the same as those presented by bone marrow mesenchymalstemcells. Taken together, these findings indicate that the umbilical cord obtained from term deliveries is an important source ofmesenchymalstemcells that could be used in cell therapy protocols.
Introduction: Stemcells (SCs) are capable of inducing tissue regeneration and are, there- fore, potentially therapeutic. Similarly to bone marrow and umbilical cords, dental pulp is one of the available sources of SCs. The fact that these cells are easily accessible and that deciduous teeth are not vital organs, and are normally discarded after exfoliation, make them particularly attractive for use in safety and viability tests. Objective: To de- scribe the collection, isolation and culture of SCs obtained from the pulp of deciduous teeth as well as their characterization by flow cytometry, and the induction of differen- tiation into osteogenic and adipogenic lineages. Methods: SCs were obtained in a rela- tively straightforward manner and showed good proliferative capacity, even from a small amount of pulp tissue. Results: Analysis by flow cytometry confirmed the characteristics ofmesenchymal SCs with low expression of CD34 and CD45 antigens, which are mark- ers for hematopoietic cells, and high levels of expression of CD105, CD166, CD90 and CD73 antigens, which are markers for mesenchymal SCs. Cell plasticity was confirmed by identifying calcium deposits in cultures that received osteogenic medium, and in- tracellular lipid accumulation in adipogenic cultures that received adipogenic medium. Conclusions: SCs collected from deciduous teeth show promising potential for applica- tion in tissue regeneration. Therefore, it is important that knowledge about the existence and characteristics of this source ofstemcells be disseminated among dentists and that the technique, its limitations and possible indications are highlighted and discussed. Abstract
Culture of mouse Mesenchymal Stromal Cells (mMSCs) All animals (C57BL/6J mice) were handled in strict accordance with institutional guidelines for animal care approved by the Animal Care and Ethics Committees (ACEC), University of Sydney and UNSW, Sydney, NSW, Australia (approval number N08/32). Mouse MSCs were prepared from bone marrow as previously described [29,57]. Briefly, C57BL/6J (B6) mice were anesthetized with Avertin (2, 2, 2-Tribromoethanol) and killed by cervical dislocation. Mouse femurs and tibias were aseptically dissected and bone marrow was extruded by flushing media used for the culture using a 10-mL syringe and a 21-gauge needle. The cell suspension obtained was centrifuged at 500 g for 10 minutes and washed with fresh medium: a-MEM or DMEM/F12 (Gibco, Invitrogen) containing 10% non-inactivated ESFBS (Gibco, Invitrogen), specially tested for the ability to sustain the growth of MSCs, 2 mmol/L L-glutamine (JRH Bioscience), 100 units/mL penicillin and 100 mg/mL streptomycin (penicillin-streptomycin, GSL) . The cell suspension was passed through a 70 m m nylon filter and centrifuged at 500 g for 10 minutes, washed one more time and centrifuged at 500 g for 10 minutes. In order to remove the erythrocytes, the cell suspension was treated with lysis buffer (0.144 M ammonium chloride, 17 mM TrisHCl). Viable cells were counted after a trypan blue stain (Sigma) using a Neubauer hemocytometer. Cell suspension at a density of 20610 6 whole marrow cells per mL media was either: i) sorted using MACS microbeads; ii) plated in chamber slides (SlideFlask, 10 cm 2 , Nunc) for immunostaining; iii) plated in 6-well plates for selection of mMSCs by adhesion to plastic dishes and subcultures, for RNA isolation and differentiation assessments. When the mMSCs became confluent, they were resuspended with 0.25% trypsin (JRH Biosciences, Brooklyn, Australia) and then subcultured. The media was changed twice a week and the cells cultured in humidified 5% CO 2 /95% air (37 uC). Custom made tryptophan-
Proteins from DVSMCs were extracted using RIPA lysis buffer with the protease inhibitor phenylmethanesulfonyl fluoride. Proteins were separated on 8% or 10% Tris-glycine gels by sodium dodecyl sulfate polyacrylamide gel electro- phoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% milk for 2 h at room temperature, the membranes were incubated with primary antibodies, includ- ing ERK, phospho-ERK, p38, phospho-p38, JNK, phospho- JNK and GAPDH, overnight at 4˚C. The membranes were washed and incubated with appropriate horseradish perox- idase (HRP)-conjugated secondary antibodies for 1.5 h at room temperature. The proteins were detected with ECL detection reagents.
It was reported that increased plasma levels of miR- let-7b can be an indicator ofsurvival. Therefore, decreases in the plasma expression of let-7b were associated with worse prognosis and poorer survival. The reduction in serum miR-223 expression was also associated with poor survival outcomes in TNM stage I patients. These findings showed that LC patients with epigenetic alterations were predisposed to more aggressive disease . Considering that Let-7 can clinically increase the postoperative survivalof patients with LC by suppressing tumor proliferationandsurvival through the mediation of oncogenes and other cell functions, it has been one of the main potential therapeutic targets studied in cancer therapy. Inflammation is one of the mechanism clinically affected by Let-7 expression in cancer. Molecules related to inflammation, such as NFκB, have are involved in the regulatory feedback loop controlling Let-7 expression in inflammation and cancer. Positive feedback occurs when NFκB reduces let-7 levels, inhibiting IL-6 expression and consequently activating NFκB .
Corneal chemical burns are common ophthalmic injuries that may result in permanent visual impairment. Although significant advances have been achieved on the treatment of such cases, the structural and functional restoration of a chemical burn-injured cornea remains challenging. The applications of polysaccharide hydrogel and subconjunctival injection ofmesenchymalstemcells (MSCs) have been reported to promote the healing of corneal wounds. In this study, polysaccharide was extracted from Hardy Orchid andmesenchymalstemcells (MSCs) were derived from Sprague-Dawley rats. Supplementation of the polysac- charide significantly enhanced the migration rate of primarily cultured rat corneal epithelial cells. We examined the therapeutic effects of polysaccharide in conjunction with MSCs appli- cation on the healing of corneal alkali burns in rats. Compared with either treatment alone, the combination strategy resulted in significantly better recovery of corneal epithelium and reduc- tion in inflammation, neovascularization and opacity of healed cornea. Polysaccharide and MSCs acted additively to increase the expression of anti-inflammatory cytokine (TGF-β), anti- angiogenic cytokine (TSP-1) and decrease those promoting inflammation (TNF-α), chemo- taxis (MIP-1α and MCP-1) and angiogenesis (VEGF and MMP-2). This study provided evidence that Hardy Orchid derived polysaccharide and MSCs are safe and effective treat- ments for corneal alkali burns and that their benefits are additive when used in combination. We concluded that combination therapy with polysaccharide and MSCs is a promising clinical treatment for corneal alkali burns and may be applicable for other types of corneal disorder.
comparing both collected areas directs the ADSC extraction from the inguinal region, ensuring greater concentration ofcells collected in comparision to the peritoneal region, which in turn can be useful in designing future studies aimed at testing the properties of ADSC, as done in this work, which envisaged its closest application to clinical practice.
by using an ELISA Kit to detect early and late markers of differentiation. Results: The level of osteonectin (ON, early osteogenic marker) decreased, which indicated that the osteogenic differentiation may be accelerated with addition of extracts. However, the level of osteocalcin (OCN, late osteogenic marker and sign of calcium granulation) differed among the extracts, in which S. ar om at icum presented the highest value, followed by S. t r iloba and C. zeylanicum . Surprisingly, the determined calcium granules were reduced in S. ar om at icum and S. t r iloba. In response to tumor necrosis factor alpha (TNF- D), S. t r iloba-treated DPSCs showed the most reduced level of IL-6 cytokine level. We suggest C. zeylanicum as a promising osteogenic inducer and S. t riloba
(trans) diferenciação em células aptas a adotar fenótipo endócrino pancreático expressando insulina 1 in vitro. Os mecanismos que levam à diferenciação de células adultas em células produtoras de insulina ainda são mal compreendidos, já os indutores utilizados neste estudo são reconhecidamente importantes neste processo. A exendina-4, descoberta na saliva do lagarto Heloderma suspectum, é um peptídio sintético 53% homólogo ao hormônio GLP-1(do inglês, Glucagon like- peptide-1) (Eng et al.,1992) o que lhe confere características importantes no que diz respeito à diferenciação, aumento de massa celular e neogênese de ilhotas pancreáticas. Logo, é um potente secretagogo de insulina (Xu et al., 1999; Hui et al. 2001; Abraham et al. 2002 Bai; Meredith G and Tuch, 2005). A nicotinamida é um inibidor da síntese da poli ADP-ribose e possui efeito estimulatório das células progenitoras do pâncreas em células produtoras de insulina (Tang et al., 2004). Na necessidade de recriar in vitro um ambiente similar à fase embriogênica, as células mesenquimais foram induzidas a formar a endoderme pancreática enriquecendo o meio com ativina A, uma proteína da família do TGF-β (do inglês, transforming growth factor β) (D’Amour et al., 2005).
ABSTRACT - The possibility for isolating bovine mesenchymal multipotent stromal cells (MSCs) from fetal adnexa is an interesting prospect due to the potential use of these cells in biotechnological applications. However, little is known about the properties of these progenitor cells in the bovine species. Wharton‟s jelly (WJ) MSC cells were obtained from the umbilical cord of bovine fetuses at three different stages of pregnancy and divided into groups 1, 2 and 3 according to gestational trimester. Cell morphology, from three stages of pregnancy, typically appeared fibroblast-like spindle shape, presenting the same viability and number. Moreover, the proliferative ability of T cells in response to a mitogenic stimulus was suppressed when WJMSC cells were added to the culture. Multilineage properties were confirmed by their ability to undergo adipogenic, osteogenic/chondrogenic and neurogenic differentiation. Mesenchymal phenotyping, CD105+, CD29+, CD73+, CD90+ cell markers were detected in all three cell groups, yet these markers were considered more expressed in MSCs of Group 2 (p<0.005). Expression of cytokines IL2, IL6RR, INFAC, INFB1, IFNG, TNF and LTBR were downregulated; whereas IL1F10 expression was upregulated in all tested WJMSCs. The present study demonstrated that WJMSCs harvested from bovine umbilical cord at different gestational stages showed proliferative capacity, immune privileged and stemness potential.