The superiority of conditioned medium derived from rapidly expanded mesenchymal stem cells for neural repair

Abstracts Background Spinal cord injury (SCI) is a complex and severe neurological condition. Mesenchymal stem cells (MSCs) and their secreted factors show promising potential for regenerative medicine. Many studies have investigated MSC expansion efficacy of all kinds of culture medium formulations, such as growth factor-supplemented or xeno-free medium. However, very few studies have focused on the potential of human MSC (hMSC) culture medium formulations for injured spinal cord repair. In this study, we investigated the effect of hMSC-conditioned medium supplemented with bFGF, EGF, and patient plasma, namely, neural regeneration laboratory medium (NRLM), on SCI in vitro and in vivo. Methods Commercial and patient bone marrow hMSCs were obtained for cultivation in standard medium and NRLM separately. Several characteristics, including CD marker expression, differentiation, and growth curves, were compared between MSCs cultured in standard medium and NRLM. Additionally, we investigated the effect of the conditioned medium (referred to as NRLM-CM) on neural repair, including inflammation inhibition, neurite regeneration, and spinal cord injury (SCI), and used a coculture system to detect the neural repair function of NRLM-MSCs. Results Compared to standard culture medium, NRLM-CM had superior in inflammation reduction and neurite regeneration effects in vitro and improved functional restoration in SCI rats in vivo. In comparison with standard culture medium MSCs, NRLM-MSCs proliferated faster regardless of the age of the donor. NRLM-MSCs also showed increased adipose differentiative potential and reduced CD90 expression. Both types of hMSC CM effectively enhanced injured neurite outgrowth and protected against H2O2 toxicity in spinal cord neuron cultures. Cytokine arrays performed in hMSC-CM further revealed the presence of at least 120 proteins. Among these proteins, 6 demonstrated significantly increased expression in NRLM-CM: adiponectin (Acrp30), angiogenin (ANG), HGF, NAP-2, uPAR, and IGFBP2. Conclusions The NRLM culture system provides rapid expansion effects and functional hMSCs. The superiority of the derived conditioned medium on neural repair shows potential for future clinical applications.

The effect of Malaysian stingless bee, Trigona spp. honey in promoting proliferation of the undifferentiated stem cell

Stem cells provide various potential applications in regenerative medicine through its ability of self-renewal and differentiation. Among the various stem cells, dental pulp stem cells (DPSCs) have shown encouraging results in their ability to regenerate. Honey has been used in traditional culture as a natural medicine in supporting wound healing. Yet, very few studies on honey were conducted for its potential as a proliferative agent for stem cells. The aim of this study is to evaluate the stability of two Trigona spp. honeys (1 and 2) added in culture media and its proliferative effect on DPSCs. Both honeys were diluted with standard culture medium through dilution process to prepare the concentrations of 0.01%, 0.04%, 0.10% and 0.25%. DPSCs were treated with the diluted honeys for 24 hours. The proliferative activity was determined through the images taken using an inverted microscope for every six hours. In addition, the MTT assay was conducted to determine the cell viability of DPSCs when treated with both honey 1 and 2 at various concentrations. The results showed a stable culture media added with honey for three days and a dose-dependent proliferative effect of both Trigona spp. honey samples on DPSCs. Optimum proliferative effects were observed at 24 hours for both Trigona spp. honey 1 and 2 on DPSCs. The optimum concentration of Trigona spp. honey 1 was from 0.04% to 0.10% and Trigona spp. honey 2 was below 0.01%. It is concluded that Trigona spp. honey has a promising proliferative effect on DPSCs.

73 Fatty Acid Supplementation in Culture Medium with Reduced Nutrient Concentrations Improves Bovine Blastocyst Development Compared with Standard Culture Medium

Lipids are a potent source of cellular energy and are metabolized within mitochondria via fatty acid β-oxidation, a process that also requires carnitine. Embryos metabolize lipids during pre-implantation development, but relatively little is known about the effect of fatty acid supplementation for early bovine embryogenesis in culture. The objective of this study was to evaluate the effect of lipid supplementation (via albumin) and l-carnitine (C; 5 mM) during embryo culture in a novel medium with reduced concentrations of nutrients, compared with our standard culture medium (control). Following in vitro maturation and IVF, zygotes were cultured using a serum-free sequential media system (0-72 h step 1; 72-168 h step 2). Concentrations of salts, bicarbonate, and protein [2.5 mg mL−1 fatty acid-free (FAF) or fraction V (FrV) BSA] were the same in all treatments to maintain consistent osmolarity and pH. Nutrients (glucose/fructose, citrate, lactate, pyruvate, amino acids, vitamins, and EDTA) were diluted to 6.25% of control. In addition to the control medium (100%+FAF; n = 587), experimental treatments included 6.25%+FAF+C (essentially lipid free; n = 573) and 6.25%+FrV+C (lipid rich; n = 585). Following in vitro culture (7 reps), hatching blastocysts were stained to determine inner cell mass (ICM; SOX2+) and trophectoderm (TE; CDX2+) cell numbers. Lipid content of single expanded blastocysts was determined using gas chromatography coupled to an ISQ-LT MS/MS (GC-MS). Data (mean ± SEM) were analysed by ANOVA. Embryo cleavage did not differ between treatments. Blastocyst development (per cleaved embryo) was higher (P < 0.05) after culture in lipid rich (38.3 ± 1.5%) compared with control (29.6 ± 2.2%) and lipid free (28.1 ± 3.6%). Blastocyst hatching was reduced (P < 0.05) in lipid free (1.4 ± 0.7%) but not in lipid rich (5.2 ± 1.7) compared with control (9.8 ± 2.1). However, blastocysts developed in lipid rich and lipid free had reduced cell numbers compared with control: TE, 98.7 ± 5.9 and 98.8 ± 9.1 v. 160.3 ± 9.0; ICM, 19.2 ± 2.9 and 25.2 ± 6.1 v. 43.3 ± 4.0; and total cell number, 117.9 ± 7.3 and 124.0 ± 8.7 v. 203.6 ± 10.2, respectively. Analysis by GC-MS identified 40 annotated lipids (i.e. triacylglycerols and phosphatidyl cholines) that were significantly reduced in blastocysts cultured in lipid rich compared with control. In summary, blastocyst development was significantly improved after supplementation of fatty acids and l-carnitine to a medium with reduced nutrient concentrations. The mechanism underlying this phenomenon may be related to increased lipid metabolism in the low nutrient environment. Although more embryos developed in this novel medium, these blastocysts had reduced cell numbers even though blastocyst expansion and hatching were not affected. This reduced nutrient medium may provide an experimental model in which to independently study pathways controlling cell proliferation and blastocyst development. Future studies will investigate whether embryo cell number can be rescued while maintaining improved blastocyst development.

Producción de ácido giberélico a partir de Gibberella fujikuroi utilizando lodo residual municipal como sustrato

<p><strong></strong><strong>Objective. </strong>To use municipal sewage sludge (LRM) from a wastewater treatment plant located in Toluca, State of Mexico, to grow the fungus <em>Gibberella fujikuroi</em> in submerged fermentation and to produce gibberellic acid (AG₃). <strong>Materials and methods. </strong>We used<em> Gibberella fujikuroi</em> (CDBB: 268). To obtain AG3, production was verified using as a substrate the standard culture medium (MCE). Gibberellic acid determination was done with high performance liquid chromatography (HPLC) with a Varian 9050.9012 equipment. We obtained 6 samples of sludge from a wastewater treatment plant in Toluca, State of Mexico, that were then characterized. Finally, both substrates (LRM and MCE) were used in submerged fermentation, and GA3 was obtained by extraction and quantified using HPLC.<strong> Results. </strong>The LRM characterization showed that the organic matter content (MO) is of 5.20% (w/v) and the total nitrogen content (N_T) is of 0.25% (w/v). Such composition is within the range as a substrate for the production of AG₃ by <em>Gibberella fujikuroi</em>. The fungus was cultivated for 3, 8, 13 and 30 days in sterile sewage sludge with a moisture content of 95.6% (w/v) and in standard culture medium (MCE). Samples were processed and analyzed by high performance liquid chromatography (HPLC). Production of AG₃ in the LRM was of 460.06 mg/L after 30 days in submerged fermentation at pH 4.0, and of 1,014.46 mg/L in the control.<strong> Conclusion</strong>. The nutrient content of LRM is suitable for the growth of the fungus <em>Gibberella fujikuroi</em> and for the production of GA₃ when used as a substrate.</p> <p><strong>Key words</strong>: gibberellic acid,<em> Gibberella fujikuroi</em>, sewage sludge</p><br />