Enhanced reconstruction of rat calvarial defects achieved by plasma-treated electrospun scaffolds and induced pluripotent stem cells.

Tissue engineering with a combination of stem cells and nanofibrous scaffolds has attracted interest with regard to bone regeneration applications. In the present study, human induced pluripotent stem cells (iPSCs) were cultured on polymeric nanofibrous polyethersulfone (PES) with and without plasma treatment. The capacity of PES and plasma-treated PES (Plasma-PES) scaffolds to support the proliferation and osteogenic differentiation of iPSCs was investigated by MTT assay and for common osteogenic markers such as alkaline phosphatase activity, calcium mineral deposition and bone-related genes. Plasma-PES scaffolds with or without iPSCs were subsequently used to evaluate bone regeneration of critical-size defects in the rat by digital mammography, multislice spiral-computed tomography imaging and histological analysis. The results of in vitro analysis showed that plasma treatment significantly enhanced iPSC proliferation and osteogenesis. After 8 weeks of iPSC-loaded Plasma-PES implantation, no mortality or complication was observed in animals or at the site of surgery. Imaging analysis revealed more extensive bone reconstruction in rats receiving nanofibers compared with untreated control groups. Moreover, Plasma-PES seeded with iPSCs induced the highest regeneration of bone defects among all groups. These findings were confirmed by histological staining. Affective osseointegration was observed in implanted scaffolds. Thus, plasma-treated nanofibrous scaffolds are suitable tissue-engineered matrices for supporting the proliferation and osteogenic differentiation of iPSCs and might also be appropriate for the reconstruction of bone defects.

A comparison of pluripotency and differentiation status of four mesenchymal adult stem cells.

The self-renewal and differentiation status of a stem cell is very important in the applications concerning regenerative medicine. Proliferation capacity, differentiation potentials and epigenetic properties of stem cells differ between sources. Studies have shown the high potentials of stem cells in iPS reprogramming. To examine this; we have compared the stem-ness and differential potential of four adult stem cells from common sources. We show a correlation between pluripotency and differentiation status of each stem cell with available data on the reprogramming efficiency. Four human adult stem cells including, adipose tissue-mesenchymal stem cells (AT-MSC), bone marrow mesenchymal stem cells (BM-MSCs), nasal septum derived multipotent progenitors (NSP) and umbilical cord blood stem cells (USSCs) were isolated and characterized. The self- renewal and differentiation potentials of each stem cell were assessed. Stem-ness transcription factors and the propagation potentials of all cells were analyzed. Furthermore the differentiation potentials were evaluated using treatment with induction factors and specific MicroRNA profile. Real-time PCR results showed that our stem cells express innate differentiation factors, miR145 and Let7g, which regulate the stem-ness and also the reprogramming potentials of each stem cell. To complete our view, we compared the propagation and differentiation potentials by correlating the stem-ness gene expression with differentiation MicroRNAs, also the direct effect of these factors on reprogramming. Our results suggest that the potentials of adipose tissue stem cells for GMP (Good Manufacturing Practice) compliant starting material are adequate for clinical applications. Our results indicate a low risk potential for AT-MSCs as starting material for iPS production. Although let7g and mir145 are well known for their differentiation promoting effects, but function more of a fine tuning system between self-renewal and differentiation status.

MicroRNA 17-92 expressed by a transposone-based vector changes expression level of cell-cycle-related genes.

The miR-17-92 cluster is composed of seven miRNAs (microRNAs; miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1). Previous studies have indicated that this cluster is involved in cell proliferation and their overexpression has been seen in several types of cancer. We have assessed the overexpression effects of miR-17-92 on the expression of several genes associated with cell-cycle regulation. The human miR-17-92 gene was cloned into a transposone-based vector, piggyBac and transfected into HEK-293T [HEK-293 cells (human embryonic kidney cells) expressing the large T-antigen of SV40 (simian virus 40)] cell line. Gene expression analysis indicated that up-regulation of this cluster causes significant changes in the expression of several cell-cycle related genes, including CDK2 (cyclin-dependent kinase 2), cyclin-D2, c-Myc and CREB (cAMP-response-element-binding protein). Other methods of transcripts assessment confirmed miR-17-92 overexpression enhances cell proliferation.

miR-424 induces apoptosis in glioblastoma cells and targets AKT1 and RAF1 oncogenes from the ERBB signaling pathway.

Glioblastoma is a lethal and incurable cancer. Tumor suppressor miRNAs are promising gene therapy tools for cancer treatment. In silico, we predicted miR-424 as a tumor suppressor. It had several target genes from the epidermal growth factor receptor (ERBB) signaling pathway that are overactive in most glioblastoma cases. We overexpressed miR-424 by lentiviral transduction of U-251 and U-87 glioblastoma cells confirmed with fluorescent microscopy and real-time quantitative PCR (qRT-PCR). Then the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) proliferation assay and scratch wound migration assay were performed to investigate the miR-424 tumor suppressor effect in glioblastoma. miR-424's effect on glioblastoma apoptosis and cell-cycle arrest was verified using Annexin V- phosphatidylethanolamine (PE) and 7-minoactinomycin D (7-AAD) apoptosis assay and cell-cycle assay. miR-424 predicted target genes mRNA and protein level were measured after miR-424 overexpression in comparison to the control group by qRT-PCR and western blotting, respectively. We confirmed miR-424 direct target genes by dual-luciferase reporter assay. miR-424 overexpression significantly suppressed cell proliferation and migration rate in glioblastoma cells based on the MTT and scratch assays. Flow cytometry results confirmed that miR-424 promotes apoptosis and cell-cycle arrest in glioblastoma cells. Predicted target genes of miR-424 from the ERBB pathway were downregulated by miR-424 overexpression. qRT-PCR and western blotting showed that KRAS, RAF1, MAP2K1, EGFR, PDGFRA, AKT1, and mTOR mRNA expression levels and KRAS, RAF1, MAP2K1, EGFR, and AKT1 protein level, respectively, had significantly decreased as a result of miR-424 overexpression in comparison to the control group. Dual-luciferase reporter assay confirmed that miR-424 directly targets RAF1 and AKT1 oncogenes. Overall, miR-424 acts as tumor suppressor miRNA in glioblastoma cells.