Additional file 1: of Self-templated Synthesis of Nickel Silicate Hydroxide/Reduced Graphene Oxide Composite Hollow Microspheres as Highly Stable Supercapacitor Electrode Material

The experimental details for synthesis of SiO2 microspheres and SiO2/GO composite microspheres. Figure S1 (a, b) FESEM images of pristine SiO2 microspheres at low and high magnifications, respectively. (c, d) FESEM images of SiO2/GO composite microspheres at low and high magnifications, respectively. Figure S2 FESEM images of Ni3Si2O5(OH)4/RGO hybrid material synthesized in the absence of PVP. Figure S3 (a) GCD curves of bare Ni3Si2O5(OH)4 hollow microspheres measured at a set of varied current densities. (b) Specific capacitance of bare Ni3Si2O5(OH)4 hollow microsphere electrode deduced from the GCD curves depicted in (a) as a function of current density. (DOCX 5399 kb

Efficient Reprogramming of Naïve-Like Induced Pluripotent Stem Cells from Porcine Adipose-Derived Stem Cells with a Feeder-Independent and Serum-Free System

<div><p>Induced pluripotent stem cells (iPSCs) are somatic cells reprogrammed by ectopic expression of transcription factors or small molecule treatment, which resemble embryonic stem cells (ESCs). They hold great promise for improving the generation of genetically modified large animals. However, few porcine iPSCs (piPSCs) lines obtained currently can support development of cloned embryos. Here, we generated iPSCs from porcine adipose-derived stem cells (pADSCs), using drug-inducible expression of defined human factors (Oct4, Sox2, c-Myc and Klf4). Reprogramming of iPSCs from pADSCs was more efficient than from fibroblasts, regardless of using feeder-independent or feeder-dependent manners. By addition of Lif-2i medium containing mouse Lif, CHIR99021 and PD0325901 (Lif-2i), naïve-like piPSCs were obtained under feeder-independent and serum-free conditions. These successfully reprogrammed piPSCs were characterized by short cell cycle intervals, alkaline phosphatase (AP) staining, expression of Oct4, Sox2, Nanog, SSEA3 and SSEA4, and normal karyotypes. The resemblance of piPSCs to naïve ESCs was confirmed by their packed dome morphology, growth after single-cell dissociation, Lif-dependency, up-regulation of Stella and Eras, low expression levels of TRA-1-60, TRA-1-81 and MHC I and activation of both X chromosomes. Full reprogramming of naïve-like piPSCs was evaluated by the significant up-regulation of Lin28, Esrrb, Utf1 and Dppa5, differentiating into cell types of all three germ layers <i>in vitro</i> and <i>in vivo</i>. Furthermore, nuclear transfer embryos from naïve-like piPSCs could develop to blastocysts with improved quality. Thus, we provided an efficient protocol for generating naïve-like piPSCs from pADSCs in a feeder-independent and serum-free system with controlled regulation of exogenous genes, which may facilitate optimization of culture media and the production of transgenic pigs.</p></div

Evidences of naïve-like state of piPSCs.

<p>(A) Morphology of naïve-like piPSCs at phase contrast (a, scale bar = 50 µm) and immunofluorescence (GFP) imaging (b, scale bar = 50 µm). (B) AP staining of C4-6 NpiPSCs, when Lif was present (a, scale bar = 500 µm) and withdrawn (b, scale bar = 500 µm). (C) mRNA levels of MHC I in pADSCs, C4-6 NpiPSCs and C4-30 NpiPSCs. (D) mRNA levels of stella and Eras in pADSCs, C4-6 NpiPSCs and C4-30 NpiPSCs. (E) Immunofluorescence staining of H3K27me3 in pADSCs and naïve-like piPSCs (C4-30 NpiPSCs), nuclei surrounded by squares were magnified (right panels), arrows indicate H3K27me3-positive areas, scale bar = 50 µm.</p

Characterization of fully reprogrammed porcine naïve-like iPSCs.

<p>(A) Real-time PCR analysis of expression levels of genes associated with fully reprogramming in pADSCs, C4-6 NpiPSCs and C4-30 NpiPSCs. (B) Reverse transcription PCR analysis of differentiation markers for the three germ layers in the EBs. (C) Hematoxylin and eosin staining of naïve-like piPSCs-derived teratoma (C4-30 NpiPSCs), the tumor was differentiated into the tissues of three germ layers, including cuticulated epithelium (a, ectoderm), adipose tissue (b, mesoderm) and gut-like epithelium (c, endoderm). Black arrows and square denote the specific structure of ectoderm mesoderm and endoderm, scale bar = 100 µm.</p

Generation of piPSCs from pADSCs.

<p>(A) AP staining of colonies which were reprogrammed from pADSCs and pEFs in the presence of feeders and feeder-independent condition. The rate of AP positive colonies was compared between the groups, different superscripts above the bars denote significant difference (<i>P</i><0.05). (B) Schematic of the reprogramming strategy and the, morphology of cells at day 3, 6, 8 and 10. (C) Expression levels of genes associated with reprogramming, including Oct4, Sox2, c-Myc, Klf4, Lin28 and Nanog were evaluated in pADSCs and pEFs. (D) Expression levels of 5-mC and 5-hmC were analyzed in pADSCs and pEFs by immunofluorescence staining, *<i>P</i><0.05, **<i>P</i><0.01.</p

Characterization of naïve-like piPSCs produced by DOX-inducible system.

<p>(A) AP staining of naïve-like piPSCs, scale bar = 50 µm. (B) Pluripotency of naïve-like piPSCs was demonstrated by immunofluorescence staining of Sox2, Nanog, SSEA1, SSEA3, SSEA4, TRA-1-60 and TRA-1-81, scale bar = 100 µm. (C) Real-time PCR analysis of expression level of pluripotency genes in pADSCs, C4-6 NpiPSCs and C4-30 NpiPSCs. (D) DNA methylation analysis of the Nanog promoter in C4-6 NpiPSCs and C4-30 NpiPSCs. (E) Karyotype analysis of naïve-like piPSCs.</p

NpiPSCs for nuclear transfer.

<p>(A) Blastocyst rate of nuclear transfer embryos from NpiPSCs and pADSCs. Blastocyst rate I  =  No. blastocyst/No. cultured embryos (a); Blastocyst rate II  =  No. blastocyst/No. cleaved embryos (b). Different superscripts above the bars denote significant difference (P<0.05). (B) Total cell number of blastocyst from NpiPSCs and pADSCs. (C) Spindle-like morphology of cells 24h after the withdrawal of DOX. (D) Reverse transcription PCR analysis of expression of exogenous genes 6 days after the withdrawal of DOX.</p

Identification of pADSCs.

<p>(A) Morphology of pADSCs at passage 3, scale bar = 100 µm. (B) Multi-lineage differentiation of pADSCs, mature adipocytes were detected by Oil Red O staining (a), scale bar = 50 µm; osteogenesis was analysis by Alizarin Red S staining (c), scale bar = 200 µm. Cells cultured in the corresponding proliferation medium served as negative controls, respectively (b, scale bar = 50 µm; d, scale bar = 200 µm). (C) Expression of cell surface markers in pADSCs at passage 3 including CD29, CD44, CD90, CD105,CD31, CD45 and HLA-DR. Positive cells were gated based on staining with isotype antibody controls.</p