Decellularized amniotic membrane Scaffolds improve differentiation of iPSCs to functional hepatocyte-like cells

Human-induced pluripotent stem cells-derived hepatocyte-like cells (hiPSCs-HLCs) holds considerable promise for future clinical personalized therapy of liver disease. However, the low engraftment of these cells in the damaged liver microenvironment is still an obstacle for potential application. In this study, we explored the effectiveness of decellularized amniotic membrane (dAM) matrices for culturing of iPSCs and promoting their differentiation into HLCs. The DNA content assay and histological evaluation indicated that cellular and nuclear residues were efficiently eliminated and the AM extracellular matrix component was maintained during decelluarization. DAM matrices were developed as three-dimensional scaffolds and hiPSCs were seeded into these scaffolds in defined induction media. In dAM scaffolds, hiPSCs-HLCs gradually took a typical shape of hepatocytes (polygonal morphology). HiPSCs-HLCs that were cultured into dAM scaffolds showed a higher level of hepatic markers than those cultured in tissue culture plates (TCPs). Moreover, functional activities in term of albumin and urea synthesis and CYP3A activity were significantly higher in dAM scaffolds than TCPs over the same differentiation period. Thus, based on our results, dAM scaffold might have a considerable potential in liver tissue engineering, because it can improve hepatic differentiation of hiPSCs which exhibited higher level of the hepatic marker and more stable metabolic functions.     

Mn(II) complex of a vitamin B6 Schiff base as an exclusive apoptosis inducer in human MCF7 and HepG2 cancer cells: Synthesis,characterization, and biological studies

Herein, a Mn(II) complex of the N,N′-dipyridoxyl(1,4-butanediamine) (═H₂L) Schiff base has been newly synthesized. The synthesized complex was characterized by several experimental methods. In addition, the density functional theory approaches were used for theoretical identification of the complex. A good agreement between the computed and experimental infrared frequencies demonstrates validity of the optimized geometry for the synthesized complex. In a N₂O₂ manner, two azomethine nitrogens and two phenolate oxygens of the L²⁻ ligand are coordinated to the Mn²⁺ metal ion. The biological studies indicate an efficient apoptotic and antioxidant activities of the synthesized [MnL(CH₃OH)₂] complex on both of the HepG2 and MCF7 cancer cells. Since it has been suggested that the complex is an exclusive potent antitumor for treatment of the human breast and liver cancers.     

Incorporation of SPION-casein core-shells into silk-fibroin nanofibers for cardiac tissue engineering

Mimicking the structure of extracellular matrix (ECM) of myocardium is necessary for fabrication of functional cardiac tissue. The superparamagnetic iron oxide nanoparticles (SPIONs, Fe₃O₄), as new generation of magnetic nanoparticles (NPs), are highly intended in biomedical studies. Here, SPION NPs (1 wt%) were synthesized and incorporated into silk-fibroin (SF) electrospun nanofibers to enhance mechanical properties and topography of the scaffolds. Then, the mouse embryonic cardiac cells (ECCs) were seeded on the scaffolds for in vitro studies. The SPION NPs were studied by scanning electron microscope (SEM), X-ray diffraction (XRD), and transmission electron microscope (TEM). SF nanofibers were characterized after incorporation of SPIONs by SEM, TEM, water contact angle measurement, and tensile test. Furthermore, cytocompatibility of scaffolds was confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. SEM images showed that ECCs attached to the scaffolds with elongated morphologies. Also, the real-time PCR and immunostaining studies approved upregulation of cardiac functional genes in ECCs seeded on the SF/SPION-casein scaffolds including GATA-4, cardiac troponin T, Nkx 2.5, and alpha-myosin heavy chain, compared with the ones in SF. In conclusion, incorporation of core-shells in SF supports cardiac differentiation, while has no negative impact on ECCs' proliferation and self-renewal capacity.     

Reaction of sunflower varieties to Sclerotinia sclerotiorum under various inoculation methods,time of field evaluation and phylogenic stages of host

Sunflower cultivation is affected seriously by Sclerotinia sclerotiorum (Lib.) de Bary in Iran, particularly north-western areas. Because of economic and environmental harms by chemical control, it is necessary to develop cultivars with adequate genetic resistance for reduction of yield losses. The purpose of this study was to find an effective method of inoculation with S. sclerotiorum under field evaluations. Three stem-inoculation techniques including: 1 – mycelium plug, 2 – oxalic acid solution (OAS) and 3 – infested wheat seeds with Sclerotinia mycelium were employed under field conditions. Four genotypes including Ghalami (local variety in market), Confeta, Allstar and Master were used in this study. The lesion length, lesion width and lesion as up and down leading on the stem from inoculation site were measured after 3, 7, 10 and 14 days of inoculation. The analysis of variance showed significant difference between all employed techniques and incubation days after inoculation. Mycelial plug (MP) inoculation technique produced significantly more developed lesions on the treated stems. In spite of this effect, Master variety demonstrated reasonable resistance reaction against the disease. The progress of disease in wounded treatments was also faster than the non-wounded ones. And, the shortest time to obtain significant differences between varieties was 10?days after inoculation. By comparison of results of lesion length at flowering and seed-filling stages, the more obvious effectiveness of the disease was observed at the second stage. Finally, there were negative correlations between mean temperature and mean lesion length in all three inoculation methods.     

Combination of oocyte and zygote selection by brilliant cresyl blue (BCB) test enhanced prediction of developmental potential to the blastocyst in cattle

The cumulus oocyte complexes (COCs) were obtained from local abattoir. After aspiration, the COCs were allotted into four treatments to evaluation of brilliant cresyl blue (BCB) test. Control treatment (C): oocytes were cultured directly (without exposure to BCB) after recovery in in vitro production (IVP) process. Oocyte treatment (OBCB): immediately after aspiration, COCs were incubated in modified Dulbecco's phosphate-buffered saline (mDPBS) supplemented with 26 μM of BCB for 90 min and classified into two classes: oocytes with blue cytoplasm coloration (OBCB+: more competent oocytes) and oocytes without blue cytoplasm coloration (OBCB−: low competent oocytes). Directly after classification, the oocytes were maintained undisrupted in the IVP process. Zygote treatment (ZBCB): After oocyte collection, maturation and fertilization, zygotes were stained with BCB for 10 min and categorized into three ways, according to whether they were highly stained (ZBCB++: low competent zygotes), moderately stained (ZBCB+: moderate competent zygotes) and unstained (ZBCB−: more competent zygotes). Directly after classification, the zygotes were maintained undisrupted in the culture process. Oocyte and zygote treatments (OBCB/ZBCB): COCs were stained with BCB after recovery and classified into two classes (OBCB+ and OBCB−). After fertilization, the zygotes produced from OBCB+ and OBCB− oocytes were further stained with BCB for 10 min and categorized six ways (OBCB+/ZBCB++, OBCB+/ZBCB+, OBCB+/ZBCB−, OBCB−/ZBCB++, OBCB−/ZBCB+ and OBCB−/ZBCB−). Directly after classification, the zygotes were maintained undisrupted in the culture process. The selection rate produced from OBCB treatment (OBCB+; 54.3%) was greater (P < 0.05) than ZBCB treatment (ZBCB−; 44.3%). In addition, the selection rate produced from double application (combination of oocyte and zygote selection) of BCB test (OBCB+/ZBCB−: 28.8%) was less (P < 0.01) than single application of BCB test (ZBCB−: 44.3%or OBCB+: 54.3%). The percentage of blastocyst production from OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%) were greater (P < 0.05) than that from C oocytes (25.7%), OBCB− oocytes (16.5%), ZBCB++ (13.5%) and ZBCB+ zygotes (21.3%). However, there were no significant differences (P > 0.05) in the percentages of blastocyst production between OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%). The proportion of blastocyst production from double application of BCB test (OBCB+/ZBCB−: 48.0%) was greater (P < 0.05) than that from single application of BCB test (OBCB+: 35.7% or ZBCB−: 36.6%). In conclusion, current results confirmed that combination of oocyte and zygote selection by BCB test enhanced the efficiency of selecting for high quality embryos, compared to the single BCB test.     

Multiple spectroscopic studies of the interaction between a quaternary ammonium-based cationic Gemini surfactant (as a carrier) and human erythropoietin

Erythropoietin (EPO) is a hematopoietic growth factor. This substance, as a strong cell protector, can increase cell maintenance during different damages of central nervous system. Since the brain-blood barrier prevents the entrance of large proteins similar to EPO into the brain, its systemic delivery gets limited. The aim of this study was to find an alternative approach for EPO delivery into the brain to skip the blood-brain barrier prevention. So, a new quaternary ammonium-based cationic Gemini surfactant has been used to study the interaction of the cationic Gemini surfactant (as a carrier) with EPO using various spectroscopic techniques of (fluorescence and circular dichroism (CD)) and thermal denaturation. Fluorescence spectroscopy studies show the formation of Gemini-EPO complex and also static quenching of protein upon this interaction. The binding parameters of number of binding sites, binding affinity, Gibbs free energy, enthalpy, and entropy were calculated according to fluorescence quenching studies. Also, CD results have further represented that the content of regular secondary structure of EPO did not show any significant alterations by increasing the Gemini concentration. Finally, thermal denaturation behavior of EPO results indicates decreasing the thermal stability of protein in the presence of Gemini. In conclusion, the obtained results proposed that Gemini as a cationic surfactant can bind to EPO without any significant diverse effects on the structure of this drug (EPO) which can be considered as a candidate for EPO delivery in future.     

Comparative immunomodulatory properties of adipose‐derived mesenchymal stem cells conditioned media from BALB/c,C57BL/6, and DBA mouse strains

Adipose tissue‐derived mesenchymal stem cells (AD‐MSCs) have been shown to be capable of differentiating into multiple cell type and exert immunomodulatory effects. Since the selection of ideal stem cell is apparently crucial for the outcome of experimental stem cell therapies, therefore, in this study we compared AD‐MSCs conditioned media (CM) from BALB/c, C57BL/6, and DBA mouse strains. No significant difference was found in the morphology, cell surface markers, in vitro differentiation and proliferation potentials of AD‐MSCs isolated from C57BL/6, BALB/c, and DBA mice. The immunological assays showed some variation among the strains in the cytokines, nitric oxide (NO), and indoleamine 2,3‐dioxygenase (IDO) production and immunomodulatory effects on splenocytes functions. Our results indicated a suppression of splenocytes proliferation in the presence of AD‐MSC CM from the three inbred mouse strains. However, BALB/c CM exerted a higher suppression of splenocytes proliferation. AD‐MSCs isolated from C57BL/6 and BALB/c mice produced higher levels of TGF‐β than those from DBA mice. Furthermore, IL‐17 and IDO production was higher in AD‐MSCs isolated from BALB/c mice. Our results indicated an increased production of TGF‐β, IL‐4, IL‐10, NO, and IDO by splenocytes in response to CM from BALB/c AD‐MSCs. In conclusion, our results showed that the immunomodulatory properties of mouse AD‐MSCs is strain‐dependent and this variation should be considered during selection of appropriate stem cell source for in vivo experiments and stem cell therapy strategies. J. Cell. Biochem. 114: 955–965, 2013. © 2012 Wiley Periodicals, Inc.