Additional file 1: of Peri-foci adipose-derived stem cells promote chemoresistance in breast cancer

Supplementary material. (XLS 1016 kb

Microwave-Assisted One-Pot Synthesis of 1,6-Anhydrosugars and Orthogonally Protected Thioglycosides

Living organisms employ glycans as recognition elements because of their large structural information density. Well-defined sugar structures are needed to fully understand and take advantage of glycan functions, but sufficient quantities of these compounds cannot be readily obtained from natural sources and have to be synthesized. Among the bottlenecks in the chemical synthesis of complex glycans is the preparation of suitably protected monosaccharide building blocks. Thus, easy, rapid, and efficient methods for building-block acquisition are desirable. Herein, we describe routes directly starting from the free sugars toward notable monosaccharide derivatives through microwave-assisted one-pot synthesis. The procedure followed the in situ generation of per-<i>O</i>-trimethylsilylated monosaccharide intermediates, which provided 1,6-anhydrosugars or thioglycosides upon treatment with either trimethylsilyl trifluoromethanesulfonate or trimethyl­(4-methylphenylthio)­silane and ZnI₂, respectively, under microwave irradiation. We successfully extended the methodology to regioselective protecting group installation and manipulation toward a number of thioglucosides and the glycosylation of persilylated derivatives, all of which were conducted in a single vessel. These developed approaches open the possibility for generating arrays of suitably protected building blocks for oligosaccharide assembly in a short period with minimal number of purification stages

Ectopic Pregnancy-Derived Human Trophoblastic Stem Cells Regenerate Dopaminergic Nigrostriatal Pathway to Treat Parkinsonian Rats

<div><h3>Background</h3><p>Stem cell therapy is a potential strategy to treat patients with Parkinson’s disease (PD); however, several practical limitations remain. As such, finding the appropriate stem cell remains the primary issue in regenerative medicine today. We isolated a pre-placental pluripotent stem cell from the chorionic villi of women with early tubal ectopic pregnancies. Our objectives in this study were (i) to identify the characteristics of hTS cells as a potential cell source for therapy; and (ii) to test if hTS cells can be used as a potential therapeutic strategy for PD.</p> <h3>Methods and Findings</h3><p>hTS cells expressed gene markers of both the trophectoderm (TE) and the inner cell mass (ICM). hTS cells exhibited genetic and biological characteristics similar to that of hES cells, yet genetically distinct from placenta-derived mesenchymal stem cells. <em>All-trans</em> retinoic acid (RA) efficiently induced hTS cells into trophoblast neural stem cells (tNSCs) in 1-day. Overexpression of transcription factor Nanog was possibly achieved through a RA-induced non-genomic c-Src/Stat3/Nanog signaling pathway mediated by the subcellular c-Src mRNA localization for the maintenance of pluripotency in tNSCs. tNSC transplantation into the lesioned striatum of acute and chronic PD rats not only improved behavioral deficits but also regenerated dopaminergic neurons in the nigrostriatal pathway, evidenced by immunofluorescent and immunohistological analyses at 18-weeks. Furthermore, tNSCs showed immunological advantages for the application in regenerative medicine.</p> <h3>Conclusions</h3><p>We successfully isolated and characterized the unique ectopic pregnancy-derived hTS cells. hTS cells are pluripotent stem cells that can be efficiently induced to tNSCs with positive results in PD rat models. Our data suggest that the hTS cell is a dynamic stem cell platform that is potentially suitable for use in disease models, drug discovery, and cell therapy such as PD.</p> </div

TissueQuest Analysis of TH and CREB1 Expressions <i>in vivo</i>.

<p>(A) Left panel: Expression of immunoreactive CREB1 (red) and TH (green) in DA neurons in normal SNC (upper) and therapeutic SNC (lower). Right panel: Colocalization of TH and CREB1 in the regenerated DA neurons (arrow) of therapeutic SNC at 12-week postimplantation of the hTS cell-derived NSCs in PD rat brain. (B) A distributive correlation of CREB1 and TH intensity in DA neurons counted from the normal SNC and the therapeutic SNC after 12-week implantation. R² = 0.856. (C-D) Comparison of relative expression of TH and CREB1 between the normal DA neurons (left, n = 86 cells counted) and the regenerated DA neurons (right, n = 114 cells counted) in a brain section at postimplantation (C) and the mean intensity of TH to CREB1 ratio in DA neurons is significantly lower in therapeutic SNC than normal SNC (D). (E) RA increases levels of GIRK2, ALDH1, and Nurr1 mRNAs after one-day incubation by qPCR assay. (F) Immunofluorescence imaging reveals co-expression of TH with Foxa2 and Pitx3. n = 4. Data represent mean ± SD, ^★: p < 0.05, ^★★: p<0.01, ^★★★: p < 0.001.</p

Subcellular Localization of RARβ and RXRα.

<p>(A-B) Knockdown of TrkA and TrkB reduced Akt1 and Akt3 expressions at 4 hr (upper) and 24 hr (lower panel), respectively, evidenced by inhibitory GNF-5837 by immunoblotting assays. β-actin as loading control. (C) RA activated mTOR and eIF4E at 4 hr, but not 24 hr. (D) Knockdown of mTOR resulted in reduction of phosphorylation of 4EBP1, but not eIF4E, and knockdown of 4EBP1 did not reduce eIF4E. (E) RA reduced both RXRα mRNA (left) and RARβ mRNA (right) levels at 2 min by qPCR, but did not change by using eIF4E shRNA. Data represent mean ± SD, n = 6, ^★:p < 0.05. (F) RA-induced expressions of Gα_q/11, Gβ, RARβ and RXRα in a timeline and their responses by knockdown of eIF4E shRNA. shGFP as a positive control. (G) RA-reduced RXRα mRNA and RARβ mRNA levels were promoted by ACD (100 μg/ml), but not CHX (100 μg/ml) at 30 min by qPCR assay. Data represent mean ± SD, n = 4, ^★:p < 0.05. C: control. (H) RA induced different expression patterns of RARβ, RXRα, Gβ and Gα_q/11 by immunoblotting assay. (I) IP assay showed an interaction between Gβ and RARβ and also between Gα_q/11 and RXRα while (J) knockdown of RXRα inhibited the latter.</p

Imaging of RARβ/Gβ and RXRα/Gα_q/11 Complexes.

<p>(A) Representative micrographs of real-time confocal immunofluorescence microscopy showing RA at 0 min, 4.5 min, and 13 min. Bar scale: 30μm. (B) The distribution of Gβ (green) and RARβ (red) before RA stimulation (upper); while co-localization of them at the extended axonal membrane after RA (white arrow) (lower) and (C) a similar expression was observed in between Gα_q/11 (green) and RXRα (red). Bar scale: 30μm. (D) Representative electron micrographs showing: the subcellular large gold-tagged Gα_q/11 (20 μm, green arrow) and ER areas (blank arrow) before RA stimulation (upper). Co-localization of the small gold-tagged RXRα (6 μm, red arrow) and the large gold-tagged Gα_q/11 (green arrow) at the ER areas (blank arrow) (middle) and at the subcellular regions after RA stimulation (15 min; lower). N: nucleus, Bar scale: 1 μm.</p

CREB1 and MAPT as Central Core Regulators in Axon Growth.

<p>(A-B) Linkage of PKA/c-Src/c-Raf pathway (A) and c-Raf/MEK/Erk1/2/CREB1 pathway (B) in a timeline by immunoblotting assays supported by PKA inhibitor PP1 and knockdown of Gβ. (C) RA activated the molecular components of Gα_q/11/CaMKII/CREB1 pathway (upper), MAPT-centered complexes (middle) and Gα_q/11/CaN/NFAT1 pathway (lower) at 4 hr induction. (D) Responses of CaMKII and CaN to RA at different time induction by immunoblotting assay. β-actin: loading control. (E-J) ChIP-qPCR analysis at different site of the promoter of genes by RA induction showing: (E) CREB1 transcribed <i>MAPT</i> gene at 4 hr, but not 24 hr; (F) CREB1 transcribed <i>MEF2A</i> gene (left) and MEF2A performed a transcriptional autoregulation (right) at 4 hr; (G-H) MEF2A transcribed <i>SNCA</i> gene at 4 hr (G) and <i>PARK2</i> gene at 4 hr, but not 24 hr (H); (I) β-catenin transcribed <i>Pitx2</i> gene at both 2 hr and 24 hr (left panel) and MEF2A transcribed <i>Pitx2</i> gene at 2 hr and (J) Pitx2 transcribed <i>CDH2</i> gene prominently at 4 hr, but not 24 hr. Data represent mean ± SD of quadruplicates. (K) Immunofluorescence imaging (4 hr) revealed that RA induced co-expression of NFAT1 and MEF2A (arrow; upper) and β-catenin and Pitx2 (arrow; middle) in the nucleus, while β-catenin and N-cadherin (arrow; lower) at the subcellular regions. Bar scale: 20μm.</p

Characteristics of hTS Cells.

<p>(<b>A</b>) Histology of an ectopic pregnancy at fallopian tube by HE staining (left large panel). Bla: blastocyst, V: chorionic villi, FT: fallopian tube. Immunocytochemistry of SSEAs (red, arrow) showed SSEA-1 in the cytoplasm (left upper); SSEA-3 in the nucleus (middle upper); and SSEA-4 in both the cytoplasm and the membrane (right upper), corresponding to cytotrophoblasts in the ectopic chorionic villus (lower panel). (<b>B</b>) Immunofluorescent Oct4 and Cdx2 at passage 9 (P-9), Oct4 and Nanog expressed in the nuclei at passage 9 (P-9) and 17 (P-17) (left column). Scale bar: 20 µm. Expression of Cdx2, Oct4, Sox2, and Nanog in the nuclei in the amplified cells (middle and right columns). Scale bar: 10 µm. (<b>C</b>) Expression of specific genes of both trophectoderm (TE) and ICM by RT-PCR. (<b>D</b>) hTS cells expressed genes of three germ layers before induction (left column) and after appropriate induction (right column). (<b>E</b>) For cellular homogeneity, Oct4-positive cells in P-9 and P-17 with 100% and 97.3%, respectively, by TissueGnostics analysis. n = total cell number counted. (<b>F</b>) Flow cytometric analysis at passage 15, cells expressed positive Oct4, Nanog, Sox2, and Cdx2 were 97.9%, 98.7%, 95%, and 94%, respectively. (<b>G</b>) Southern blots showed no obvious change in telomere length at passages 3 (P-3) and 7 (P-7). (<b>H</b>) Distinction between hTS cells and PDMS cells in global gene expressions (total 54,675 genes; p<0.05 and fold change >2) with a homogenous histogram. (<b>I</b>) Different relative intensity values of genes (4,864 genes) between PDMS and hTS cells. Blue box indicating 50% of total genes and error bars indicating 25% and 75%. (<b>J</b>) Venn diagram illustrated the numbers of immue-related genes (upper panel) and mesenchymal genes (lower panel) in PDMS and hTS cells by microarray analysis. Overlap: common genes; Bilateral regions: unique genes.</p

Schematic Illustration of Molecular Networks of RA Induction at the Early Stage of Differentiation in hTS Cells.

<p>Schematic Illustration of Molecular Networks of RA Induction at the Early Stage of Differentiation in hTS Cells.</p

Schematic Illustration of a Variety of Distinct Transcriptional Pathways and Epigenetic Histone Modifications by RA Induction (24 hr) for <i>TH</i> Gene Expression in hTS Cells.

<p>Schematic Illustration of a Variety of Distinct Transcriptional Pathways and Epigenetic Histone Modifications by RA Induction (24 hr) for <i>TH</i> Gene Expression in hTS Cells.</p