Enhancement of human adipose-derived stem cell expansion and stability for clinical use

Co-culture techniques associating both dermal fibroblasts and epidermal keratinocytes have shown to have better clinical outcome than keratinocyte culture alone for the treatment of severe burns. Since fat grafting has been shown to improve scar remodelling, new techniques such as cell-therapy-assisted surgical reconstruction with isolated and expanded autologous adipose-derived stem cells (ASCs) would be of benefit to increase graft acceptation. Therefore, integrating ASCs into standardized procedures for cultured skin grafting could be of benefit for the patient if cell quality and quantity could be maintained. The purpose of this study was to evaluate ASC processing from adult tissue with simple isolation (without enzymatic steps), expansion (low density of 325-3,000 cells/cm2) and storage conditions to assure methods to enhance the cellular resistance when transferred back to the patient. Co-culture with cell-banked skin progenitor cells (FE002-SK2) showed an increase of 40-50% ASCs yield at high passages alongside with a better preservation of morphology, proper adipogenic and osteogenic differentiation and efficient biocompatibility with 3D collagen scaffolds. ASCs can be considered as a valuable additional cell source to be delivered in biological bandages to the patient in a need of tissue reconstruction such as burn patients.

Fibroblast growth factor-23 (FGF-23) levels differ across populations by degree of industrialization.

CONTEXT: Compensatory increases in FGF23 with increasing phosphate intake may adversely impact health. However, population and clinical studies examining the link between phosphate intake and FGF23 levels have focused mainly on populations living in highly industrialized societies in which phosphate exposure may be homogenous. OBJECTIVE: Contrast dietary phosphate intake, urinary measures of phosphate excretion and FGF23 levels across populations that differ by level of industrialization. DESIGN: Cross-sectional analysis of three populations Setting: Maywood, IL, U.S., Mah|fe Island, Seychelles, and Kumasi, Ghana Participants: Adults with African ancestry aged 25-45 years Main Outcome: Fibroblast growth factor 23 (FGF23) levels Results: The mean age was 35.1 (6.3) years and 47.9% were male. Mean phosphate intake and fractional excretion of phosphate were significantly higher in the U.S. vs. Ghana while no significant difference in phosphate intake or fractional excretion of phosphate was noted between U.S. and Seychelles for men or women. Overall, median FGF23 values were 57.41 RU/ml (IQR 43.42, 75.09) in U.S., 42.49 RU/ml (IQR 33.06, 55.39) in Seychelles and 33.32 RU/ml (IQR 24.83, 47.36) in Ghana. In the pooled sample, FGF23 levels were significantly and positively correlated with dietary phosphate intake (r=0.11; P < 0.001), and the fractional excretion of phosphate (r=0.13; P < 0.001) but not with plasma phosphate levels (-0.001; P = 0.8). Dietary phosphate intake was significantly and positively associated with the fractional excretion of phosphate (r=0.23; P < 0.001). CONCLUSION: The distribution of FGF23 levels in a given population may be influenced by the level of industrialization, likely due to differences in access to foods preserved with phosphate additives.

Porcine follicular fluid concentration of free insulin-like growth factor-I collected from different diameter ovarian follicles

The study aimed to quantify the concentrations of free IGF-I in serum and fluid of ovarian follicles in pre-pubertal gilts and describe the ovarian morphology by measuring the size of the ovaries and counting the number of surface follicles. Ovaries (n=1,000) from pre-pubertal gilts were obtained immediately after slaughter. A total of 10 samplings were performed, with ovaries obtained from 50 females for each collection. The follicles situated on the surface of each ovary were classified as small (SFs, 2 to 5mm in diameter) or large (LFs 6 to 10mm in diameter) and the follicular fluid was obtained by follicle aspiration. The collection of serum samples was performed after the gilts exsanguination using sterile tubes. From the pool of serum and follicular fluid obtained from 50 females, the concentration of free IGF-I was determined in each sample using an enzyme immunoassay kit (ELISA). The description of ovarian morphometry was performed in 100 ovaries from randomly selected gilts. The larger and smaller lengths of ovaries were measured, and the total number of SFs and LFs present on the surface of each ovary were also counted. The IGF-I concentration was greater (P<0.05) in LFs (170.92±88.29 ng/mL) compared with SFs (67.39±49.90ng/mL) and serum (73.48±34.63ng/mL). The largest and smallest length of the ovaries was 26.0±3.0 and 19.0mm ±2.0mm, respectively. The number of SFs (70.86±25.76) was greater (P<0.01) than LFs (6.54±5.26). The study concluded that LFs present greater levels of IGF-I when compared with SFs and blood, which is related to increased activity of the LFs and its differentiation to ovulation. In addition, ovaries of pre-pubertal gilts have a higher number of SFs compared to LFs. Therefore, our study demonstrated unique data regarding the physiological concentration of free IGF-I in ovarian follicles, that can be used in future research to evaluate the addition of this hormone in the in vitro production media of porcine embryos with the goal to improve the technique efficiency.

Influence of Insulin-like Growth Factor I (IGF-I) on the survival and the in vitro development of caprine preantral follicles

The aim of this study was to investigate the effects of the insulin-like growth factor -I (IGF-I) on survival, activation (transition from primordial to primary follicles) and growth of caprine preantral follicles cultured in vitro. Fragments of ovarian cortex were cultured for one and seven days in the absence or presence of IGF-I (0, 50 and 100ng/ml). The non-cultured and cultured tissues were processed and analyzed by histology and transmission electron microscopy. The culture for one day in a medium with 100ng/ml of IGF-I showed 86.7% of morphologically normal follicles. These results were similar (P>0.05) to the percentage of normal follicles found in the control (96.7%). It was also found that this medium increased the percentage of follicular activation (developing follicles) with one day of culture. The oocyte and follicular diameters remained similar to the control by culturing for one day in a medium containing 100ng/ml of IGF-I. The ultrastructural analysis did not confirm the integrity of the follicular fragments in a medium containing IGF-I (100ng/ml) after one and seven days of culture. In conclusion, this study demonstrated that the addition of 100 ng/ml of IGF-I in the culture medium enables the development of preantral follicles of goats with one day of culture. However, it is not sufficient to maintain the follicular integrity and the follicular survival rate after seven days of culture.

The Effects of Fibroblast Growth Factor 8b on Reproductive Organs and Prostate Tumorigenesis

Fibroblast growth factors (FGFs) are involved in the development and homeostasis of the prostate and other reproductive organs. FGF signaling is altered in prostate cancer. Fibroblast growth factor 8 (FGF8) is a mitogenic growth factor and its expression is elevated in prostate cancer and in premalignant prostatic intraepithelial neoplasia (PIN) lesions. FGF8b is the most transforming isoform of FGF8. Experimental models show that FGF8b promotes several phases of prostate tumorigenesis - including cancer initiation, tumor growth, angiogenesis, invasion and development of bone metastasis. The mechanisms activated by FGF8b in the prostate are unclear. In the present study, to examine the tumorigenic effects of FGF8b on the prostate and other FGF8b expressing organs, an FGF8b transgenic (TG) mouse model was generated. The effect of estrogen receptor beta (ERβ) deficiency on FGF8binduced prostate tumorigenesis was studied by breeding FGF8b-TG mice with ERβ knockout mice (BERKOFVB). Overexpression of FGF8b caused progressive histological and morphological changes in the prostate, epididymis and testis of FGF8b-TG-mice. In the prostate, hyperplastic, preneoplastic and neoplastic changes, including mouse PIN (mPIN) lesions, adenocarcinomas, sarcomas and carcinosarcomas were present in the epithelium and stroma. In the epididymis, a highly cancer-resistant tissue, the epithelium contained dysplasias and the stroma had neoplasias and hyperplasias with atypical cells. Besides similar histological changes in the prostate and epididymis, overexpression of FGF8b induced similar changes in the expression of genes such as osteopontin (Spp1), connective tissue growth factor (Ctgf) and FGF receptors (Fgfrs) in these two tissues. In the testes of the FGF8b-TG mice, the seminiferous epithelium was frequently degenerative and the number of spermatids was decreased. A portion of the FGF8b-TG male mice was infertile. Deficiency of ERβ did not accelerate prostate tumorigenesis in the FGF8b-TG mice, but increased significantly the frequency of mucinous metaplasia and slightly the frequency of inflammation in the prostate. This suggests putative differentiation promoting and anti-inflammatory roles for ERβ. In summary, these results underscore the importance of FGF signaling in male reproductive organs and provide novel evidence for a role of FGF8b in stromal activation and prostate tumorigenesis.

Analysis of trophic responses in lesioned brain: focus on basic fibroblast growth factor mechanisms

The actions of fibroblast growth factors (FGFs), particularly the basic form (bFGF), have been described in a large number of cells and include mitogenicity, angiogenicity and wound repair. The present review discusses the presence of the bFGF protein and messenger RNA as well as the presence of the FGF receptor messenger RNA in the rodent brain by means of semiquantitative radioactive in situ hybridization in combination with immunohistochemistry. Chemical and mechanical injuries to the brain trigger a reduction in neurotransmitter synthesis and neuronal death which are accompanied by astroglial reaction. The altered synthesis of bFGF following brain lesions or stimulation was analyzed. Lesions of the central nervous system trigger bFGF gene expression by neurons and/or activated astrocytes, depending on the type of lesion and time post-manipulation. The changes in bFGF messenger RNA are frequently accompanied by a subsequent increase of bFGF immunoreactivity in astrocytes in the lesioned pathway. The reactive astrocytes and injured neurons synthesize increased amount of bFGF, which may act as a paracrine/autocrine factor, protecting neurons from death and also stimulating neuronal plasticity and tissue repair

Transforming growth factor beta activity in urine of patients with type 2 diabetes and diabetic nephropathy

Diabetic nephropathy (DN) is characterized structurally by progressive mesangial deposition of extracellular matrix (ECM). Transforming growth factor-ß (TGF-ß) is considered to be one of the major cytokines involved in the regulation of ECM synthesis and degradation. Several studies suggest that an increase in urinary TGF-ß levels may reflect an enhanced production of this polypeptide by the kidney cells. We evaluated TGF-ß in occasional urine samples from 14 normal individuals and 23 patients with type 2 diabetes (13 with persistent proteinuria >500 mg/24 h, DN, 6 with microalbuminuria, DMMA, and 4 with normal urinary albumin excretion, DMN) by enzyme immunoassay. An increase in the rate of urinary TGF-ß excretion (pg/mg UCreat.) was observed in patients with DN (296.07 ± 330.77) (P<0.001) compared to normal individuals (17.04 ± 18.56) (Kruskal-Wallis nonparametric analysis of variance); however, this increase was not observed in patients with DMMA (25.13 ± 11.30) or in DMN (18.16 ± 11.82). There was a positive correlation between the rate of urinary TGF-ß excretion and proteinuria (r = 0.70, a = 0.05) (Pearson's analysis), one of the parameters of disease progression.

Differential role of entorhinal and hippocampal nerve growth factor in short- and long-term memory modulation

We studied the effects of infusion of nerve growth factor (NGF) into the hippocampus and entorhinal cortex of male Wistar rats (250-300 g, N = 11-13 per group) on inhibitory avoidance retention. In order to evaluate the modulation of entorhinal and hippocampal NGF in short- and long-term memory, animals were implanted with cannulae in the CA1 area of the dorsal hippocampus or entorhinal cortex and trained in one-trial step-down inhibitory avoidance (foot shock, 0.4 mA). Retention tests were carried out 1.5 h or 24 h after training to measure short- and long-term memory, respectively. Immediately after training, rats received 5 µl NGF (0.05, 0.5 or 5.0 ng) or saline per side into the CA1 area and entorhinal cortex. The correct position of the cannulae was confirmed by histological analysis. The highest dose of NGF (5.0 ng) into the hippocampus blocked short-term memory (P < 0.05), whereas the doses of 0.5 (P < 0.05) and 5.0 ng (P < 0.01) NGF enhanced long-term memory. NGF administration into the entorhinal cortex improved long-term memory at the dose of 5.0 ng (P < 0.05) and did not alter short-term memory. Taken as a whole, our results suggest a differential modulation by entorhinal and hippocampal NGF of short- and long-term memory.

Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells

Gamma-irradiation (gamma-IR) is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD), specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P < 0.001) antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control) to 49% (IR cells), with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.

Interaction between growth differentiation factor 9, insulin-like growth factor I and growth hormone on the in vitro development and survival of goat preantral follicles

The objective of this study was to determine the effects of GDF-9, IGF-I, and GH alone or combined on preantral follicle survival, activation and development after 1 and 7 days of in vitro culture. Either fresh (non-cultured) or cultured ovarian tissue was processed for histological and fluorescence analysis. For all media tested, the percent of normal follicles was greater when compared to minimum essential medium supplemented (MEM+) alone, except when ovarian tissue was cultured with GDF-9/IGF-I or GDF-9/GH (P < 0.05). Fluorescence analysis showed that the percent of viable follicles after 7 days of culture was similar for non-cultured tissue and for all treatments tested. The percent of primordial follicles was reduced (P < 0.05) and there was a significant and concomitant increase in the percent of intermediate and primary follicles in all treatments tested after 7 days of culture when compared to non-cultured tissue. After 7 days of culture, the highest percent of intermediate follicles was observed with IGF-I/GH (61.3%), and the highest percent of primary follicles was achieved with IGF-I (57.7%). After 7 days of culture in MEM+ containing GDF-9, IGF-I and GH alone or in all associations, a significant increase in follicular diameter was observed when compared to MEM+ alone and non-cultured tissue. In conclusion, GDF-9, IGF-I and GH alone or in combination maintain preantral follicle survival and promote primordial follicle activation. Nevertheless, the data showed that IGF-I/GH and IGF-I alone are efficient in promoting the transition from primordial to intermediate follicles and from intermediate to primary follicles, respectively.

Stimulated mast cells promote maturation of myocardial microvascular endothelial cell neovessels by modulating the angiopoietin-Tie-2 signaling pathway

Angiopoietin (Ang)-1 and Ang-2 interact in angiogenesis to activate the Tie-2 receptor, which may be involved in new vessel maturation and regression. Mast cells (MCs) are also involved in formation of new blood vessels and angiogenesis. The present study was designed to test whether MCs can mediate angiogenesis in myocardial microvascular endothelial cells (MMVECs). Using a rat MMVEC and MC co-culture system, we observed that Ang-1 protein levels were very low even though its mRNA levels were increased by MCs. Interestingly, MCs were able to enhance migration, proliferation, and capillary-like tube formation, which were associated with suppressed Ang-2 protein expression, but not Tie-2 expression levels. These MCs induced effects that could be reversed by either tryptase inhibitor [N-tosyl-L-lysine chloromethyl ketone (TLCK)] or chymase inhibitor (N-tosyl-L-phenylalanyl chloromethyl ketone), with TLCK showing greater effects. In conclusion, our data indicated that MCs can interrupt neovessel maturation via suppression of the Ang-2/Tie-2 signaling pathway.

PPARγ induces growth inhibition and apoptosis through upregulation of insulin-like growth factor-binding protein-3 in gastric cancer cells

Peroxisome proliferator activator receptor-gamma (PPARγ) is a ligand-activated transcriptional factor involved in the carcinogenesis of various cancers. Insulin-like growth factor-binding protein-3 (IGFBP-3) is a tumor suppressor gene that has anti-apoptotic activity. The purpose of this study was to investigate the anticancer mechanism of PPARγ with respect to IGFBP-3. PPARγ was overexpressed in SNU-668 gastric cancer cells using an adenovirus gene transfer system. The cells in which PPARγ was overexpressed exhibited growth inhibition, induction of apoptosis, and a significant increase in IGFBP-3 expression. We investigated the underlying molecular mechanisms of PPARγ in SNU-668 cells using an IGFBP-3 promoter/luciferase reporter system. Luciferase activity was increased up to 15-fold in PPARγ transfected cells, suggesting that PPARγ may directly interact with IGFBP-3 promoter to induce its expression. Deletion analysis of the IGFBP-3 promoter showed that luciferase activity was markedly reduced in cells without putative p53-binding sites (-Δ1755, -Δ1795). This suggests that the critical PPARγ-response region is located within the p53-binding region of the IGFBP-3 promoter. We further demonstrated an increase in PPARγ-induced luciferase activity even in cells treated with siRNA to silence p53 expression. Taken together, these data suggest that PPARγ exhibits its anticancer effect by increasing IGFBP-3 expression, and that IGFBP-3 is a significant tumor suppressor.

Análisis de asociación de los genes receptor de rianodina (RYR-1) e insulin-like growth factor-2 (IGF-2) con caracteres productivos en una población porcina de tipo comercial en el estado de Nuevo León, México

Tesis (Maestría en Ciencias Veterinarias) U.A.N.L., 2006.

Analysis of HIV-1 Vpr determinants responsible for cell growth arrest in Saccharomyces cerevisiae

Affiliation: Département de microbiologie et immunologie, Faculté de médecine, Université de Montréal

Exploring TERRA (TElomeric Repeat-containing RNA) Expression and Regulation During Cell Growth in Saccharomyces cerevisiae

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