Hepatogenic Differentiation of Human Induced Pluripotent Stem cells on Collagen-Coated Polyethersulfone Nanofibers.

Many scientists have been fascinated with induced pluripotent stem cells (iPSCs) for cell replacement therapies. Nanofibrous biocompatible scaffolds have been shown to foster better cell adhesion and improve stem cell differentiation. In the current study, after fabrication using electrospinning technique and surface modifications, the characteristics of polyethersulfone (PES) nanofibers were determined by scanning electron microscopy, attenuated total reflection Fourier transform infrared spectroscopy, and 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide (MTT) assay. Then, the hepatogenic potential of iPSCs was evaluated using real-time reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC) after culture on collagen-coated polyethersulfone (PES/COL) scaffolds. After scaffolds characterization, analysis of two important definitive endoderm specific markers (Sox17 and Foxa2) using real-time RT-PCR and ICC indicated increase in their mRNA and protein levels after 5 days of hepatogenic induction. In addition, to determine hepatic differentiation of iPSCs cultured on PES/COL, the expression of albumin and α-fetoprotein was evaluated by ICC after 20 days. Real-time RT-PCR analysis showed increased expression of albumin, TAT, cytokeratin 19, and Cyp7A1 genes during the course of differentiation program. Finally, enzyme-linked immunosorbent assay analysis demonstrated an increased expression of albumin in the protein level after 28 days of differentiation. In conclusion, our results demonstrated that PES/COL nanofibrous scaffolds could be a proper substrate to significantly increase the hepatogenic differentiation potential of iPSCs and could also be introduced as a promising candidate for liver tissue engineering applications.

MicroRNA-129-1 acts as tumour suppressor and induces cell cycle arrest of GBM cancer cells through targeting IGF2BP3 and MAPK1.

BACKGROUND: MicroRNA-129-1 (miR-129-1) seems to behave as a tumour suppressor since its decreased expression is associated with different tumours such as glioblastoma multiforme (GBM). GBM is the most common form of brain tumours originating from glial cells. The impact of miR-129-1 downregulation on GBM pathogenesis has yet to be elucidated. METHODS: MiR-129-1 was overexpressed in GBM cells, and its effect on proliferation was investigated by cell cycle assay. MiR-129-1 predicted targets (CDK6, IGF1, HDAC2, IGF2BP3 and MAPK1) were also evaluated by western blot and luciferase assay. RESULTS: Restoration of miR-129-1 reduced cell proliferation and induced G1 accumulation, significantly. Several functional assays confirmed IGF2BP3, MAPK1 and CDK6 as targets of miR-129-1. Despite the fact that IGF1 expression can be suppressed by miR-129-1, through 3'-untranslated region complementary sequence, we could not find any association between IGF1 expression and GBM. MiR-129-1 expression inversely correlates with CDK6, IGF2BP3 and MAPK1 in primary clinical samples. CONCLUSION: This is the first study to propose miR129-1 as a negative regulator of IGF2BP3 and MAPK1 and also a cell cycle arrest inducer in GBM cells. Our data suggests miR-129-1 as a potential tumour suppressor and presents a rationale for the use of miR-129-1 as a novel strategy to improve treatment response in GBM.

Controlled release of rhEGF and rhbFGF from electrospun scaffolds for skin regeneration.

Controlled delivery of multiple therapeutic agents can be considered as an effective approach in skin tissue engineering. In this study, recombinant human epidermal growth factor (rhEGF) and recombinant human basic fibroblast growth factor (rhbFGF) encapsulated in PLGA microspheres were loaded in hybrid scaffolds of PLGA and PEO. The scaffolds with various formulations were fabricated through electrospinning in order to maintain dual, individual or different release rate of rhEGF and rhbFGF. Morphological, physical and mechanical properties of the scaffold were investigated. The scaffold possessed uniform morphology with an average diameter of 280 nm for PLGA and 760 nm for PEO nanofibers. Furthermore, the mechanical properties of the scaffolds were shown to be akin to those of human skin. Bioactivity of the scaffolds for human skin fibroblasts was evaluated. The HSF acquired significant proliferation and well-spread morphology on the scaffolds particularly in the case of different release rate of rhEGF and rhbFGF which implies the synergistic effect of the growth factors. Additionally, collagen and elastin gene expression was significantly up-regulated in the HSF seeded on the scaffolds in the case of individual delivery of rhEGF and dual delivery of rhEGF and rhbFGF. In conclusion, the prepared scaffolds as a suitable supportive substrate and multiple growth factor delivery system can find extensive utilization in skin tissue engineering.

Association of glucose-6-phosphate dehydrogenase activity with oocyte cytoplasmic lipid content, developmental competence, and expression of candidate genes in a sheep model.

PURPOSE: To evaluate associations of glucose-6-phosphate dehydrogenase (G6PDH) activity in sheep oocytes with cytoplasmic lipid content, maturational competence, developmental competence to the blastocyst stage, and gene expression of certain molecular markers. METHODS: Before brilliant cresyl blue (BCB) staining test, oocytes were classified as high, middle, and low cytoplasmic lipid content (HCLC, MCLC, and LCLC) and after the test as having low or high G6PDH-activity (BCB(+) and BCB(-), respectively). After maturation in vitro, a group of oocytes were subjected to IVF followed by in vitro embryo culture and another group was used for evaluation of expression of candidate genes. RESULTS: The cleavage and blastosyst rates were lowest (P < 0.05) in LCLC group, intermediate (P < 0.05) in MCLC group and highest (P < 0.05) in HCLC group. More (P < 0.05) oocytes in HCLC group were BCB(+), and higher (P < 0.05) maturation, cleavage, and blastocyst rates were seen for BCB(+) oocytes than the BCB(-) oocytes. Our gene expression data indicated that mRNA transcript abundance of ITGB2, pZP3, BMP15, and GDF9 genes was similar between BCB oocytes groups. However, the expression of ATP1A1 was higher (P < 0.05) for BCB(+) oocytes compared to BCB(-) oocytes. In addition, BAX transcript abundance was similar (P > 0.05) among BCB(+), BCB(-), and control groups, before and after maturation in vitro. CONCLUSION: Activity of G6PDH in sheep oocytes is highly associated with lipid content, and compared with the morphological parameters might be a more precise and objective predictor for subsequent developmental competence in vitro.

Efficient programming of human eye conjunctiva-derived induced pluripotent stem (ECiPS) cells into definitive endoderm-like cells.

Due to pluripotency of induced pluripotent stem (iPS) cells, and the lack of immunological incompatibility and ethical issues, iPS cells have been considered as an invaluable cell source for future cell replacement therapy. This study was aimed first at establishment of novel iPS cells, ECiPS, which directly reprogrammed from human Eye Conjunctiva-derived Mesenchymal Stem Cells (EC-MSCs); second, comparing the inductive effects of Wnt3a/Activin A biomolecules to IDE1 small molecule in derivation of definitive endoderm (DE) from the ECiPS cells. To that end, first, the EC-MSCs were transduced by SOKM-expressing lentiviruses and characterized for endogenous expression of embryonic markers Then the established ECiPS cells were induced to DE formation by Wnt3a/Activin A or IDE1. Quantification of GSC, Sox17 and Foxa2 expression, as DE-specific markers, in both mRNA and protein levels revealed that induction of ECiPS cells by either Wnt3a/Activin A or IDE1 could enhance the expression level of the genes; however the levels of increase were higher in Wnt3a/Activin A induced ECiPS-EBs than IDE1 induced cells. Furthermore, the flow cytometry analyses showed no synergistic effect between Activin A and Wnt3a to derive DE-like cells from ECiPS cells. The comparative findings suggest that although both Wnt3a/Activin A signaling and IDE1 molecule could be used for differentiation of iPS into DE cells, the DE-inducing effect of Wnt3a/Activin A was statistically higher than IDE1.

Human unrestricted somatic stem cell administration fails to protect nude mice from cisplatin-induced acute kidney injury.

BACKGROUND: Kidney failure is a debilitating disorder with limited treatment options. The kidney-protective effects of stem cells have been vastly investigated and promising results have been achieved with various sources of stem cells. However, in spite of beneficial effects on other disease models, the renoprotective potential of human cord blood-derived unrestricted somatic stem cells (USSC) has not been examined so far. METHODS: In the present study, acute kidney failure was induced in female nude mice and the effect of USSC transplantation on kidney function and structure was assessed. Furthermore, the expression of some cytokine genes was examined by real-time PCR. Homing of the transplanted cells into kidneys was assessed by flow cytometry, immunohistochemistry, and real-time PCR. RESULTS: USSC-conditioned medium did not attenuate the in vitro nephrotoxic effects of cisplatin. Transplantation of USSC to nude mice did not protect kidney function and was associated with worsened kidney structural damage. USSC transplantation was also associated with a decline in the renal expression of VEGF-A gene. In spite of these effects, the transplanted cells could not be detected in the kidneys by any of the exploited methods and they were mainly entrapped in the lungs. CONCLUSION: These data indicate that USSC are not suitable for cell therapy in the setting of acute kidney injury. Also, this study shows that these stem cells are able to affect damaged kidneys even if they are not homed there.

In vitro differentiation of human cord blood-derived unrestricted somatic stem cells into hepatocyte-like cells on poly(epsilon-caprolactone) nanofiber scaffolds.

Tissue engineering of implantable cellular constructs is an emerging cellular therapy for hepatic disease. In this study, we tested the ability of poly(epsilon-caprolactone) (PCL) nanofiber scaffold to support and maintain hepatic differentiation of human cord blood-derived unrestricted somatic stem cells (USSCs) in vitro. USSCs, self-renewing pluripotent cells, were isolated from human cord blood. The electrospun PCL nanofiber porous scaffold was constructed of uniform, randomly oriented nanofibers. USSCs were seeded onto PCL nanofiber scaffolds, and were induced to differentiate into hepatogenic lineages by culturing with differentiation factors for 6 weeks. RT-PCR analysis of endoderm and hepatic-specific gene expression, immunohistochemical detection of cytokeratin 18 (CK-18), alpha-fetoprotein, albumin, glycogen storage and indocyanine green uptake confirmed the differentiation of USSCs into endoderm and hepatocyte-like cells. In the present study, we show that hepatocyte-like cells differentiated from USSCs on the PCL nanofiber scaffold can be candidate for tissue engineering and cell therapy of hepatic tissues.

Characterization of fibroblast-like cells from the rat olfactory bulb.

The isolation and characterization of stem cells from an alternative tissue is a subject of intensive investigation. In the present study, we have focused on the characterization of fibroblastic cells in olfactory bulb tissue of the rat. To this end, 4-6 week old rats were killed and their olfactory bulb tissue was dissected out. Olfactory bulb derived fibroblast-like cells were recovered by adhesion to cell culture plastic. The plastic adherent cultivated cells were induced to differentiate along osteoblastic, adipogenic and chondrogenic lineages. Furthermore, the expression of some surface antigens was investigated. We obtained purified cells with spindle shaped morphology in primary culture, which differentiated into mesenchymal lineages. These cells expressed CD29 and CD90 (Thy1.1) surface antigens, but not CD31, CD34 and CD45. Our results indicate that fibroblast-like cells from the olfactory bulb are mesenchymal stem cells in nature. Taken together, our data suggest that olfactory bulb tissue may constitute a new source of mesenchymal stem cells and could be used for the treatment of injury.