Sixth Passage Research Articles

PO-342 Establishment and molecular profile of a new cervical cancer cell line from brazilian patient biopsy

IntroductionIn vitro model using continuous tumour cell lines is very common in the screening of new drugs in the treatment of cancer. However, the low number of commercially available cervical cancer cell lines (CCCL) poses a challenge to generate an effective and representative means of screening. There is thus a need to establish new CCCL to improve this process. Therefore, we aimed to establish and to characterise a CCCL from a Brazilian patient.Material and methodsFollowing patient‘s consent (Ethics Committee HCB-985/2015), a biopsy sample was taken, fragmented and subjected to enzymatic digestion. The material was then cultured in a flask with keratinocyte medium and when cells reached sixth passage, they were submitted to immunocytochemistry to characterise its phenotype. To investigate the HPV status, PCR for GP5+/GP6 +sequences and the HPV test using COBAS were carried out. Short tandem repeat (STR) analysis from cell line and patient´s blood was done to confirm the patient origin. Following DNA isolation of cell line and patient‘s blood, whole-exome sequencing (WES) and copy number variation (CNV) assays were performed. Moreover, functional assays are being performed to evaluate its tumorigenic potential protein profile.Results and discussionsOverall, 35 tissues were collected and attempted to be established in vitro. Only one (2,9%) of the cultures survived for more than 3 months. The successful cell line, named HCB-514, was derived from a stage IIB squamous cell carcinoma of a 30 years old patient, with no previous treatment. It continued to grow after freeze-thaw cycles and showed positive expression for AE1/AE3 cytokeratin, confirming its epithelial phenotype. The STR analysis of HCB-514 compared with the patient’s blood confirmed its lineage. The HCB-514 was HPV 16 positive. The WES identified 93 non-silent mutations, among them 11 (HPS-3, IRX2, XPO5, CBWD1, TGFBR1, CUBN, TRHDE, BRCA2, THBS1, TGM5, CCDC22) have potentially pathogenic. CNV revealed 44 somatic alteration, including 30 regions with gains (NF1, CCND1, MYC, TP53, DCC, MAP2K4, AURKA, IGF1R, PIK3CA, GAB2, NKX2-1, BCL2L2, C8orf4, BCL2L1, ZNF217, EGFR, NCOA3, SKP2, FADD, ORAOV1, EEF1A2, REL, TERT, AKT3, PRKCI, MAPK7, DCUN1D1, BIRC2, YAP1, MYCN) and 14 losses (FHIT, GPC5, IRS2, BRCA2, RB1, PDGFRA, CDK6, SHH, APC, FOXO1, MET, MITF, KDR, KIT).ConclusionThe characterisation of a new cervical cancer cell line, HCB-514, will constitute an important in vitro tool for further biological and therapeutic studies of cervical cancer.

Molecular and biological characterization of the human-bovine rotavirus-based reassortant rotavirus

Rotaviruses (RV) are the leading cause of acute infantile gastroenteritis, associated with elevated mortality in low-income countries. Morbidity and mortality, length and rates of hospitalization due to RV gastroenteritis are dropping. Improving the quality of newborns life is an ongoing challenge for health-care providers. In this study, homemade reassortant human-bovine rotavirus was developed and biological activity and molecular characterization of candidate vaccine were evaluated for the vaccine stability. Virus titration and purification of reassortant rotavirus strains were evaluated by plaque assays, electropherotyping. The genetic stability after first, third and sixth passage was by sequencing.Due to WHO recommendation, developingment of national capacity for vaccine production in appropriate quantities and at affordable prices is the cornerstone of developing global vaccination policies. Such studies are critical to producing national vaccines and modeling herd protection.

The Dynamics of Cell Properties during Long-Term Cultivation of Two Lines of Mesenchymal Stem Cells Derived from Wharton’s Jelly of Human Umbilical Cord

The cell properties of two MSC lines (MSCWJ-1 and MSCWJ-2) established from Wharton’s jelly of human umbilical cord were analyzed at the 6th, 13th, and 28th passages. The following were discovered. (1) The cell morphology during long-term cultivation was changed in both lines, with the cells being larger and having more debris in the MSCWJ-2 line than in the MSCWJ-1 line. (2) As shown by analysis of growth characteristics, long-term cultivation was accompanied with a significant increase of the average population-doubling time in both cell lines. This was correlated with morphological changes and a gradual increase in β-galactosidase activity, indicating the occurrence of replicative senescence. The interline changes concerned the level of proliferative activity, the index of proliferation, the time of the entry into the phase of replicative senescence (30 population doublings in the MSCWJ-2 line and 60 population doublings in the MSCWJ-1 line), and the percentage of senescent cells (80% in MSCWJ-2 line compared to 58% in the MSCWJ-1 line). (3) Numerical karyotypic analysis showed that both cell lines at early and late passages had normal karyotype 46, XX (MSCWJ-1 cells), and 46, XY (MSCWJ-2 cells). Structural karyotypic analysis showed a cytogenetic heterogeneity in both lines: low frequency of random clonal and nonclonal chromosomal rearrangements disappeared during long-term cultivation. Unlike in the case of the MSCWJ-1 line, the clonal chromosomal rearrangement of the short arm of chromosome 7 with a frequency of 39% was detected at the sixth passage in the MSCWJ-2 line but disappeared at the 13th passage. (4) Surface marker analysis of these cell lines at the 6th, 13th, and 28th passages revealed that these cells expressed surface antigens characteristic of human MSC (CD44, CD73, CD90, CD105, HLA-ABC), as well as vimentin, and did not express CD34 and HLA-DR. (5) Analysis of nondifferentiated hESC markers in both lines showed a decrease in the level of expression of the surface marker SSEA-4 and a lack of expression of the transcription factor SOX-2 at the 13th passage in the MSCWJ-2 and at the 28th passage in MSCWJ-1 cells. (6) Cell adhesion in both lines was lower in late passages compared to the cells at the sixth passage. The cells retained an osteogenic and adipogenic differentiation potential at late passages. A lack of chondrogenic differentiation in MSCWJ-2 cells, but not in MSCWJ-1 cells, was registered at late passages. The MSCWJ-2 cell line exhibited a frequently occurring clonal chromosomal rearrangement at an early passage, early replicative senescence with a large proportion of senescent cells, a significantly decreased index of proliferation during long-term cultivation, a decrease or disappearance of the expression of nondifferentiated ESCs of markers, and decreased differentiation potential. Given the metabolic cooperation between the cells, we can cautiously suppose that the clonal chromosomal rearrangement of the short arm of chromosome 7 detected at an early passage with high frequency is coupled with activation of cellular processes that proceed in the absence of this chromosomal aberration.

The dynamics of a Chinese porcine G9P[23] rotavirus production in MA-104 cells and intestines of 3-day-old piglets.

Rotavirus A (RVA) G9 genotype is recognized as an emerging genotype which is spreading worldwide, however, our knowledge on pathogenicity of this virus is limited. In this study, porcine RVA strain HN03 was successfully isolated on MA-104 cells, and the isolate was propagated continuously for 7 passages after a virus cloning at passage 3. The virus titers from 4 to 10 passages ranged from 107.1 to 108.1 TCID50/ml. The growth curve of HN03 strain in cell culture was determined, and the virus production dynamics was confirmed by immunoperoxidase monolayer assay (IPMA). Sequence and phylogenetic analyses based on full-length VP7 and partial VP4 genes indicated that HN03 strain belongs to genotype G9P[23]. In addition, the sixth passage of strain HN03 in cell culture was subjected to 3-day-old piglets. All infected piglets developed severe watery diarrhea within 24 hr post-inoculation (hpi), but recovered from disease after 72 hpi. RVA antigen could be detected by IHC in the cytoplasm of villous enterocytes as early as 2 hr after appearance of clinical symptoms and virus antigen load kept increasing in the next 30 hr. The dynamics of RVA HN03 strain proliferation on cells and in pigs extended our understanding of rotavirus pathogenicity.

『仮名手本忠臣藏』六段目の二つの疑問(読む)

『仮名手本忠臣藏』六段目の二つの疑問(読む)

FBP1 is highly expressed in human hypertrophic scars and increases fibroblast proliferation, apoptosis, and collagen expression

ABSTRACTPurpose: FBP1, one of the far-upstream element binding proteins(FBPs), is a distal upstream binding protein of c-myc, which is highly expressed in tumor tissues. This study aimed to investigate FBP1 expression in human hypertrophic scars and to determine the effects of FBP1 on fibroblasts. Materials and methods: Human normal skin and scar specimens were collected during clinical surgery. One portion of each tissue specimen was embedded in paraffin and sliced to observe differences in histological features and FBP1 expression by immunohistochemistry and western blotting. The other portion of each tissue specimen was cultured to obtain fibroblasts. Fibroblasts from the second to the sixth passage were used for the experiments, which were divided into the following two groups: an experimental group, whose cells were transfected with an siRNA targeting FBP1, and a control group, whose cells where not transfected. MTT and TUNEL assays were performed, respectively, to assess fibroblast proliferation and apoptosis, and western blotting was performed to assess protein expression. Results: We obtained fibroblasts by primary tissue culture and found that FBP1 was highly expressed in hypertrophic scars. MTT assay showed that an siRNA targeting FBP1 significantly reduced fibroblast proliferation in siRNA-treated cells compared to control cells. TUNEL assay showed that there was no difference in apoptosis between the two groups; however, western blotting showed that collagen I, collagen III, c-myc, caspase-3, and caspase-9 expression levels were all decreased in the experimental group. Conclusion: FBP1 is highly expressed in human hypertrophic scars and increases fibroblast proliferation, apoptosis and collagen expression.

Порівняльна характеристика генетичної стабільності культур клітин жирової тканини та кісткового мозку щурів на ранніх пасажах

From literary sources, we received a number of controversial data regarding the risks of neoplastic transformation of stem cells in vitro. Inasmuch as today bone marrow has been sufficiently studied as a source of adult donor stem cells, and adipose tissue is an alternative source for obtaining cell material (from which it may be obtained using less invasive methods and in much greater quantity), determination of genetic stability of these particular cell cultures currently is the most relevant issue. Comparison of genetic stability of rat bone marrow and adipose cell cultures from the first to the sixth passages. Materials and methods: 3 non-linear 4 months old male rats and 9 non-linear 12 days old infant rats were used in the experiment. Adipose cell culture was obtained from subcutaneous fat using the standardized method in our own modification. Bone marrow cell culture was obtained from the rat femurs, tibiae, and humeri using the standardized method. Obtained cell mass was cultivated in a standard culture medium in a СО2 incubator at 37 ºС and with 5% СО2 concentration. Cytogenetic analysis was carried out on 30 metaphase plates from each passage. Cells from the first to the sixth passage we used in the research. A modification of the standard cytogenetic method was used to obtain chromosome preparations. In the preparations prepared in an aforementioned way, numerical chromosome abnormalities, like aneuploidy, polyploidy, were determined, as well as the number of binucleate cells, micronucleus cells, mitotic and apoptotic indexes. Here, we provide the comparison of genetic stability indicators for rat bone marrow and adipose cell cultures throughout the in vitro cultivation thereof. Changes in the genetic apparatus were identified in both bone marrow and adipose tissue cell cultures. Karyotype analysis of rat bone marrow and adipose cell cultures showed that, given the conditions that we used for the cultivation, the number of aneuploidies and polyploidies varies from one passage to another; however it does not go beyond the limits of spontaneous mutagenesis typical for mammals. Based on the results of cytogenetic evaluation of the culture, it was determined that the number of cells with micronuclei and the binucleate cell remains within the normal range from the first to the sixth passages. The number of cells with aneuploidy and micronuclei in adipose cell culture on all passages was lower than in bone marrow cell culture; this indicates its higher genetic stability in terms of these indicators. The highest mitotic index was determined on the first passage with its following decrease throughout the cultivation in inverse proportion to apoptotic index. The obtained data on the genetic stability of both adipose and bone marrow cell cultures are within normal range typical for mammals in terms of all studied indicators. This allows for the following studies relating to the transplantation with minimal risk of neoplastic transformation of the said cultures.

Expression of tumor necrosis factor-.ALPHA.-induced protein-8-like 2 of bone marrow in children with aplastic anemia and its significance

Objective To investigate the characteristics of the bone marrow mesenchymal stem cell(MSC) in children with aplastic anemia(AA)in vitro, and the expressions of tumor necrosis factor-α-induced protein-8-like 2(TIPE2) in the bone marrow, and the correlation between the level of TIPE2 mRNA with γ-interferon(IFN-γ) and IL-6 in AA patients. Methods Bone marrow samples were collected from 18 children with AA(AA group) and 8 children with bone injury (control group)who were hospitalized in Jinan Children′s Hospital from January 2012 to June 2015.MSC were isolated and cultured.The morphology of MSC was observed and immune phenotype was detected.The TIPE2 mRNA was detected by using real-time fluorescence quantitative PCR, and the levels of IFN-γ and IL-6 were detected by using enzyme linked immunosorbent assay. Results Different sizes had been presented in the primitive MSC of AA patients, but the third passage MSC until 80% confluence had manifested the uniform convergence with long spindle and swirl distribution.In the sixth passage, cells showed degenerative change.The primitive and first pa-ssage MSC in patients with AA was longer than that in the controls.CD73, CD105, CD44 and CD90 were expressed in MSC, while CD34, CD45, CD271 expressed rarely.The level of TIPE2 mRNA in AA patients (5.29±1.56) was obviously lower than that of the control group(8.68±2.00), and the difference was significant(t=-4.48, P 0.05). Conclusions The proliferation of MSC is significantly reduced in patients with AA.TIPE2, as an important role to stabilize the immune system, plays an important role in the occurrence of AA by its low expression and up-regulating the expression of inflammatory factors. Key words: Aplastic anemia; Tumor necrosis factor-α-induced protein-8-like 2; Mesenchymal stem cell; Inflammatory factor

Cytogenetic analysis of rat pancreatic cell cultures at early passages

Cell culture obtained from the pancreas can serve as a source of physiologically competent substitute for primary islets of Langerhans in the treatment of diabetes. However, it is possible to obtain the required number of cells only at long-term cultivation in vitro. Therefore, it is necessary to investigate the risks of neoplastic transformation of cells in vitro before transplantation.Materials and methods. Cell culture was obtained by explant method from pancreas of 12-day-old rats. Cell cultures of the first to sixth passages were used for the cytogenetic analysis. In this study the number of cells with altered karyotype, cells with micronuclei, binucleated cells and the cells in a state of apoptosis were considered, mitotic index was calculated.Results. Aneuploid cells were noted at all passages in an amount of 2.2 % (1st) to 16.6 % (4th). Polyploidy manifested in a population of cells from the second (1.1 %) to the sixth passage (4.4 %) with a maximum at passage four (7.8 %). A significant increase in their number was observed since the second passage (0.3 %). We have seen a significant increase in the number of binucleated cells from the first (0.1 %) to the sixth passage (0.8 %). During the study there was a decrease in mitotic index from the first (2.7 %), to the third passage (1.5 %) and its gradual increase in fourth (1.7 %) and sixth (2.0 %) passages. In addition, there was discovered a small percentage of cells in apoptosis, their number gradually increased to the 4th passage (0.5 %). The 5th-6th passages showed decrease in the number of apoptotic cells to 0.1 %.Conclusion. There have been revealed changes in the rat pancreatic cells culture as aneuploidies, polyploidies and micronuclei, the intensity of which varied depending on the passage. However, karyotype variability of mentioned cell did not exceed the level of spontaneous mutations characteristic of mammalians.

Cellular spheroids obtained from mesenchymal stem cells derived from bone marrow and limb muscle of early human embryo

Cellular spheroids were obtained from mesenchymal stem cells derived from different tissues of 5to 6-week-old embryos: bone marrow (FetMSC) and limb muscle (M-FetMSC). The properties of these cells maintained in monolayer (2D cultivation) or spheroids (3D cultivation) were compared. Cellular spheroids were obtained from monolayer cultures at the sixth passage after decryopreservation and assayed 48 h after their formation. Unlike monolayer cultures, spheroids are heterogeneous cell populations consisted of fibroblast-like and epithelioid cells. Two-day-old spheroids are actively proliferating structures. Cell surface markers were analyzed by flow cytometry. Monolayer culture and cellular spheroids expressed CD44, CD73, CD90, CD105, and HLA-ABC common for mesenchymal stem cells (MSCs) and lacked CD34 and HLA-DR. However, expression of CD90 and CD105 antigens was significantly lower in spheroids than in monolayer cultures. Expression of transcription factors and surface antigens typical for human embryonic stem cells were analyzed with immunofluorescence and flow cytometry. Expression of Sox-2 and SSEA-4 in 2D and 3D cultures, lack of Oct-4 in 2D cultures, and its increase in 3D cultures were found. Both monolayer cells and spheroids were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages, although monolayer cultures and spheroids differed. Adipogenic differentiation was more active in the cellular spheroids from M-FetMSC cells compared to corresponding monolayer cultures. Differences between the 2D and 3D cultures of both lines have been shown by the character of chondrogenic differentiation. As a whole, these findings confirm the MSC status for the cellular spheroids derived from FetMSC and M-FetMSC monolayer cultures and attest to their increased differentiation potential compared to monolayer cultures.

RAPD and ISSR based evaluation of genetic stability of micropropagated plantlets of Morus alba L. variety S-1

Plant regeneration through rapid in vitro clonal propagation of nodal explants of Morus alba L. variety S-1 was established along with genetic stability analysis of regenerates. Axillary shoot bud proliferation was achieved on Murashige and Skoog (MS) medium in various culture regimes. Highest number of shoots (5.62±0.01), with average length 4.19±0.01cm, was initially achieved with medium containing 0.5mg/lN6-benzyladenine (BA) and 3% sucrose. Repeated subculturing of newly formed nodal parts after each harvest up to sixth passage, yielded highest number of shoots (about 32.27) per explants was obtained after fourth passage. Rooting of shoots occurred on 1/2 MS medium supplemented with 1.0mg/1 Indole-3-butyric acid (IBA). About 90% (89.16) of the plantlets transferred to the mixture of sand:soil:organic manure (2:2:1) in small plastic pots acclimatized successfully. Genetic stability of the discussed protocol was confirmed by two DNA-based fingerprinting techniques i.e. RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat). This protocol can be used for commercial propagation and for future genetic improvement studies.

Evaluation of Immunomodulatory Effects of Mesenchymal Stem Cells Soluble Factors on miR-155 and miR-23b Expression in Mice Dendritic Cells

Mesenchymal stem cells (MSCs) can modulate dendritic cells (DCs) activation and induce tolerogenic characteristics in DCs. All mechanisms involved in MSCs-induced tolerogenic DCs are not fully understood. MicroRNAs (miRs) play important role in maturation and function of DCs. In this study, we investigated the effects of MSCs culture supernatant (C.S.) on expression of miR-155 and miR-23b in mice DCs. BALB/c mice spleens were used for DCs isolation. MSCs were isolated from the mice bone marrow and cultured in DMEM media. When MSCs expanded to sixth passage, C.S. was collected after 12, 24 and 48 h. Quantitative polymerase chain reaction (QPCR) was used to determine the expression of miR-155 and miR-23b in DCs treated with C.S. after 6 and 12 h. Secretion of IL-23 and TGF- β were detected in DCs treated with C.S. by ELISA after 24 h. miR-23b expression was significantly increased in DCs treated with 12 h C.S. for 12 h compared to negative controls. miR-155 expression did not change in DCs treated with C.S. after 6 and 12 h. miR-23b expression was significantly increased in DCs treated with 12 h C.S. for 12 h, compared to those treated with C.S. for 6 h. Similarly, miR-23b expression was increased in DCs treated with 24 h C.S. for 12 h when compared to those treated for 6 h. Production of TGF-β and IL-23 were not influenced by C.S. In conclusion, miR-23b is considered to be one of the mechanisms involved in tolerogenic DCs induction by C.S. in a time-dependent manner.

A comparative analysis of mesenchymal stem-cell lines derived from bone marrow and limb muscle of early human embryos

The properties of mesenchymal stem-cell (MSC) lines derived from bone marrow (FetMSC) and limb muscle (M-FetMSC) of 5- to 6-week human embryos were assayed. The main cell-line characteristics were obtained at the sixth passage. The average cell-population doubling time was 33.0 ± 1.4 h for FetMSC and 25.0 ± 0.1 h for M-FetMSC. Growth curves indicated active cell proliferation. Numerical and structural karyotypic analysis showed that these lines have normal karyotype, 46, XY. Cell surface markers were analyzed by flow cytometry. It was found that the cells express CD44, CD73, CD90, CD105, and HLA-ABC surface antigens and vimentin, which are common for human MSC beings, and lack CD34 and HLA-DR. The cells were capable of osteogenic, chondrogenic, and adipogenic differentiation. Immunofluorescence and flow-cytometry assays reveled a lack of surface antigen TRA-1-60, high expression of surface antigen SSEA-4, and low expression of transcription factor Oct-4 attributed to human embryonic stem cells. Immunofluorescence analysis showed the presence of early differentiation markers, three germ-layer derivatives for human embryonic stem cells. This makes MSCs useful for repair of damaged tissue in the corresponding microenvironment. Despite their shared MSC status, the FetMSC and M-FetMSC lines displayed some interlinear differences related to growth characteristics and differentiation potential. The MFetMSC line exhibited reduced potential for adipogenic differentiation compared to the FetMSC line. Immunofluorescence analysis revealed Z-disks in M-FetMSC, but not in FetMSC, during skeletal-muscle differentiation. These findings suggest that the different microenvironment has an influence when cells are in an organism before their transplantation in vitro.

Detection of bovine parainfluenza type 3 virus in Iraq

This study is considered the first study in our country to isolate bovine parainfluenzatype 3 virus (BPIV-3) from naturally infected calves. BPIV-3 virus was isolated from nasalswabs collected from calves with respiratory disease. The virus was isolated and titrated insecondary embryonic bovine kidney (SEBK) cell culture; other cell cultures (secondary sheeptestis and chicken embryos fibroblast cell cultures) were used for propagation of isolatedvirus. Examination of infected cell culture revealed giant cell formation and intracytoplasmicinclusions. The isolated virus agglutinated guinea pig’s erythrocytes and produces clearheamadsorption of guinea pig’s erythrocyte in infected SEBK cell culture. Virus titer was2×102TCID50/0.1ml in the sixth passage and became 2×104.5TCID 50/0.1ml in the ninthpassage. The new isolated virus was identified as BPIV-3 by heamagglutination inhibitiontest, serum neutralization test and indirect immunoflurescent technique, by using specificmonoclonal and polyclonal antibody. The virus was sensitive to ether. High titer of BPIV-3antibodies was detected in sera of infected animals during convalescent period by ELISA test

Evaluation of the Cell Viability of Human Wharton's Jelly Stem Cells for Use in Cell Therapy

Human umbilical cord Wharton's jelly stem cells (HWJSCs) are gaining attention as a possible clinical source of mesenchymal stem cells for cell therapy and tissue engineering due to their high accessibility, expansion potential, and plasticity. We employed a combination of highly sensitive techniques to determine the average cell viability levels and proliferation capabilities of 10 consecutive cell passages of cultured HWJSCs and then used RNA microarrays to identify genes associated with changes in cell viability levels. We found an initial decrease in cell viability from the first to the third cell passage followed by an increase until the sixth passage and a final decrease from the sixth to tenth cell passages. The highest cell viability levels corresponded to the fifth and sixth passages. The intracellular ionic contents of potassium, sodium, and chlorine suggest that the lower cell viability levels at passages 2, 3, and 8-10 may be associated with apoptotic cell death. In fact, gene expression analysis revealed that the average cell viability was significantly associated with genes with a function in apoptotic cell death, especially pro-apoptotic FASTKD2, BNIP3L genes and anti-apoptotic TNFAIP8 and BCL2L2 genes. This correlation with both pro-apoptotic and anti-apoptotic genes suggests that there may be a complex live-death equilibrium in cultured HWJSCs kept in culture for multiple cell passages. In this study, the highest cell viability levels corresponded to the fifth and sixth HWJSC passages, suggesting that these passages should be preferentially employed in cell therapy or tissue engineering protocols using this cell type.

Dissecting the Hematopoietic Niche to Improve Transplant: Hedgehog and Wnt Pathways Activation in Fetal Mesenchymal Cells (MSC) Are Associated with Better Support to Hematopoietic Progenitors and Higher Proliferative Capacity Compared to Adult MSC

Abstract 4809 Background.Mesenchymal Stem Cells (MSC) are among major players of the hematopoietic niche. In addition, they have shown to be useful in clinical studies both as immune-modulators as well as in regenerative medicine. However, a major obstacle for proving their usefulness is to obtain a clinically effective cell dose, since often less than 10^6 /kg of adult MSC have been used. Thus, their potential efficacy can be either disregarded or not formally proved. In addition, forcing in vitro the proliferative potential of adult BM derived MSC (BM-MSC) implies the risk of generating chromosomal abnormalities.The aim of the study is to investigate the potential use of fetal derived MSC (FT-MSC) which could provide means to overcome the above limits. In addition, we attempted to identify some molecular mechanisms regulating the proliferative potential of FT-MSC and their supportive capacity of hematopoietic progenitors. Methods:Specimens from 5 fetal samples were collected after mechanically induced abortion (median gestational age 11 weeks). FT-MSC were obtained from kidneys and femurs and adult MSC from normal BM. After 15 days of culture adherent cells were sub-cultured until 80% confluence. Immunophenotype and karyotype were evaluated. The telomere length was evaluated using the Telomere PNA system. FT-MSC were injected into NOD/SCID mice to rule out any tumorigenic potential. The immunosuppressive activity of FT-MSC and adult BM-MSC was evaluated by standard assays. To quantify the supportive capacity of FT-MSC we weekly measured the colony output using normal BM MNC layered over confluent FT-MSC compared to stromal cell line MS-5 and to BM-MSC. Protein expression of the key regulators of Hedgehog and Wnt pathways were investigated in FT-MSC and BM-MSC by immunofluorescence using monoclonal antibodies against Indian Hedghog (IHh), Sonic Hedgehog (SHh), Desert Hedgehog (DHh) and b-catenin. Fluorescent signal was detected by confocal microscope. Results.MSC successfully grew from 100% of fetal samples. FT-MSC maintained the typical MSC markers (CD73, CD105, CD44, CD106, CD166, HLA class I, negative for HLA class II) and possessed the same capacity to inhibit T cell proliferation as BM-MSC. FT-MSC compared to adult BM-MSC showed a higher proliferative potential: they reached the fifteenth generation and had a median cumulative population doubling of 26 (range 4–47) while BM-MSC grew until the sixth passage showing a median cumulative population doubling of 4 (range 2–5) (p<0.05 at the fourth, fifth and sixth passage). This might be ascribed to both Hedghog and Wnt pathways which were highly activated in FT-MSC compared to BM-MSC. The mean value of SHh protein expression was 11,5 RU (relative units) compared to 5,5 RU in BM-MSC and b-catenin was 17 RU in FT-MSC vs 3.7 RU in adult. Differently from BM-MSC, FT-MSC maintained a stable karyotype without any sign of tumorigenic potential in the mouse model and stable telomere length during culture. By contrast, 5 out of 32 adult MSC developed genetic abnormalities during in vitro expansion. Importantly, FT-MSC display a better support of hematopoiesis compared to BM-MSC or MS-5 cell line, in fact, during five weeks of culture normal bone marrow samples produced an overall colony number higher when FT-MSC were used as feeder layer compared to BM-MSC or MS-5 (p= 0.07 for both). IHh was significantly higher in FT-MSC compared to BM-MSC (121 RU vs 9 RU). This is in keeping with what already reported by Kobune et al. (Blood 2006) regarding the role of IHh in supporting hematopoiesis and provides the molecular basis for the high supportive capacity of FT-MSC.In conclusion, FT-MSC seem to be candidate for being used to support hematopoiesis in transplant settings. FT-MSC overcome the limitation imposed by adult MSC providing a remarkable yield in culture and they show a reassuring safety profile. FT-MSC show a peculiar profile of Hedgehog and Wnt activation possibly responsible for their biological properties described in this study. Disclosures:Saglio:Novartis Pharmaceutical: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Kinetics of anaerobic elemental sulfur oxidation by ferric iron in Acidithiobacillus ferrooxidans and protein identification by comparative 2-DE-MS/MS

Elemental sulfur oxidation by ferric iron in Acidithiobacillus ferrooxidans was investigated. The apparent Michaelis constant for ferric iron was 18.6mM. An absence of anaerobic ferric iron reduction ability was observed in bacteria maintained on elemental sulfur for an extended period of time. Upon transition from ferrous iron to elemental sulfur medium, the cells exhibited similar kinetic characteristics of ferric iron reduction under anaerobic conditions to those of cells that were originally maintained on ferrous iron. Nevertheless, a total loss of anaerobic ferric iron reduction ability after the sixth passage in elemental sulfur medium was demonstrated. The first proteomic screening of total cell lysates of anaerobically incubated bacteria resulted in the detection of 1599 protein spots in the master two-dimensional electrophoresis gel. A set of 59 more abundant and 49 less abundant protein spots that changed their protein abundances in an anaerobiosis-dependent manner was identified and compared to iron- and sulfur-grown cells, respectively. Proteomic analysis detected a significant increase in abundance under anoxic conditions of electron transporters, such as rusticyanin and cytochrome c(552), involved in the ferrous iron oxidation pathway. Therefore we suggest the incorporation of rus-operon encoded proteins in the anaerobic respiration pathway. Two sulfur metabolism proteins were identified, pyridine nucleotide-disulfide oxidoreductase and sulfide-quinone reductase. The important transcription regulator, ferric uptake regulation protein, was anaerobically more abundant. The anaerobic expression of several proteins involved in cell envelope formation indicated a gradual adaptation to elemental sulfur oxidation.

The effect of vascular endothelial growth factor and hypoxia on the secretion of opticin in retinal pigment epithelium cells

To evaluate the role of vascular endothelial growth factor or hypoxia on the secretion of opticin in retinal pigment epithelium cells. Human RPE cells were cultured, the third to sixth passage of retinal pigment epithelium (RPE) cells were placed in 6-well culture plates at a density of 4 × 10(4)/well. For hypoxia experiment, the cells were cultured under hypoxic condition for different times. For vascular endothelial growth factor (VEGF) experiment, the media was changed to DMEM containing different concentration VEGF (1, 10, 50, 100 µg/L) for 24 h respectively. VEGF mRNA levels were determined by RT-RCR method. The protein content of opticin in RPE cells or culture media was detected by Western blot. Matrix metalloproteinase activity in culture media was analysis by zymography. One way ANOVA was used to test the comparisons between experimental groups and control group. Western blot experiment showed the opticin expression was not changed in RPE cells after hypoxia treatment, however was significantly decreased in culture media. Compared with control group (0.21 ± 0.03). The relative density of VEGF mRNA levels (0.81 ± 0.04, 0.67 ± 0.07) in RPE cells were increased after 12 h or 24 h hypoxia treatment (F = 483.60, P < 0.05). Opticin expression in RPE cells was also remain unchanged after vary concentration VEGF addition treatment (F = 2.16, P > 0.05), the relative density of opticin expression in VEGF conditioned culture medium were 0.65 ± 0.02, 0.52 ± 0.04, 0.23 ± 0.03, 0.30 ± 0.03 respectively, and the difference in culture media was significant compared to control group (0.73 ± 0.04) (F = 141.38, P < 0.05). Zymography indicate a matrix metalloproteinases type 2 digest band, the activities were enhanced with VEGF increasing. The decrease of opticin in culture media after VEGF treatment could be inhibited by low condition of EDTA. VEGF and hypoxia have an effect on the on the secretion of opiticin in RPE cells, it may be contributed to the increasing levels of matrix metalloproteinases type 2.

Selective Isolation of Young Cells from Human Corneal Endothelium by the Sphere-Forming Assay

This study aimed to investigate whether a representative adult stem cell/precursor cell isolation method (the sphere-forming assay) could isolate cells with differences of telomere length, telomerase activity, and characteristics reflecting senescence. The sphere-forming assay was performed to obtain precursors from cultured sixth passage (P6) human corneal endothelial cells (CECs). P6 and P7 cultured CECs were used as the controls. Telomere length, telomerase activity, and senescence-associated factors were evaluated in precursors and controls. Precursors obtained from the spheres had longer telomeres and higher telomerase activity than cultured P6 cells. Strong positive staining for senescence-associated beta-galactosidase activity was detected in P6 and P7 cultured CECs, whereas little or no staining was detected in the precursors within spheres obtained from P6-cultured CECs or their progeny. The progeny of spheres derived from cultured CECs were small regular cells that grew at a higher density and contained more 5-bromo-2'-deoxyuridine-incorporating cells compared with the parental cultured cells. These findings indicate that the sphere-forming assay enriches precursors with longer telomeres, higher telomerase activity, and younger progeny than the original cells. Thus, the sphere-forming assay may contribute to obtaining the young cells needed for regenerative medicine.

The Antimicrobial Peptide LL37 Induces the Migration of Human Pulp Cells: A Possible Adjunct for Regenerative Endodontics

Introduction The antimicrobial peptide LL37 has multiple functions, such as the induction of angiogenesis and migration. Pulp cell migration is a key phenomenon in the early stage of pulp-dentin complex regeneration. In this study, we examined the effect of LL37 on the migration of human pulp (HP) cells. Methods HP cells at the sixth passage were exposed to LL37. The migration of HP cells was assessed by a wound-healing assay. The phosphorylation of epidermal growth factor receptor (EGFR) and c-Jun N-terminal kinase (JNK) was analyzed by immunoblotting. Results LL37 as well as heparin binding (HB)-EGF, which is an agonist of EGFR, induced HP cell migration. LL37 increased the level of phosphorylated EGFR. An anti-EGFR antibody, an EGFR tyrosine kinase inhibitor, and a JNK inhibitor abolished the migration induced by both LL37 and HB-EGF. Furthermore, the two peptides increased the levels of phosphorylated JNK. Conclusions LL37 activates EGFR and JNK to induce HP cell migration, and it may contribute to enhancing the regeneration of pulp-dentin complexes.