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sc-12-0256_sm_SupplFigure1.pdf113KFIGURE S1: SMAD7-induction promotes neural conversion of hESCs via inhibition of Activin/Nodal and BMP signaling in hESCs. A. SMAD7-induction prevents BMP4-mediated trophoblast differentiation of hESCs. Cultures were preinduced with DOX for 24hrs and subsequently challenged with 100ng/mL BMP4 in CM for 48hrs. Cells were then fixed and stained for CDX2, an early marker of trophectoderm induction, and NANOG. While both CDX2 and NANOG cells are found in uninduced cultures, a complete absence of CDX2 and NANOG is observed in EGFP-positive cells in induced cultures. Scale bars represent 200μm. B. Schematic of the neural induction protocol for gene expression and immunofluorescence analysis.
sc-12-0256_sm_SupplFigure2.pdf2305KFIGURE S2: Schematics of expression of markers mentioned in the manuscript and IF analysis of the neural crest marker SOX10 A. Schematics of expression of markers mentioned in the manuscript. Note that the schematics show a more advanced stage of embryonic development compared to the early neuroepithelia we are generating here for purposes of clarity and should only be used as a guide. B. Immunofluorescence analysis of the neural crest progenitor marker SOX10. Only rare nuclear BRA+ cells are observed while SOX10 is never observed. Scale bars represent 200μm.
sc-12-0256_sm_SupplFigure3.pdf1610KFIGURE S3: Gene expression analysis of SMAD7-induced cultures DAVID Gene ontology analysis of significantly expressed genes shows that SMAD7-induced hESCs are enriched for neural, forebrain, and developmental genes
sc-12-0256_sm_SupplFigure4.pdf1775KFIGURE S4: Heatmaps of developmentally relevant signaling pathways dynamically changing over time in SMAD7-induced hESCs: A) NOTCH, B) MAPK, C) SHH, D) WNT, E) TGFβ, and F) JAK-STAT. The complete list of genes in the order presented can be found in Table S3.
sc-12-0256_sm_SupplFigure5.pdf3868KFIGURE S5: Time-course comparison of lineage-specific genes expressed in A) MEK-I/SMAD7 and B) SMAD7-only combinations. Observe the decrease in pluripotency transcripts (OCT4, NANOG) in both conditions, and comparable expression profile of trophectodermal (CDX2), ectodermal (KRT14), endodermal (SOX17), neural crest (SOX10), or mesodermal (BRA, MIXL1) genes. There is a selective increase in the neural determinant PAX6 in both conditions. qRT-PCR data is presented as LOG2-fold change over uninduced hESCs. ATP5O was used for normalization at each time point. Bars represent n = 3 -4 ± SEM.
sc-12-0256_sm_SupplFigure6.pdf1588KFIGURE S6: SMAD7 imposes an anterior neural fate in hESCs via a default pathway. A. SMAD7-induction results in a neural fate in hESCs. DOX induction of the transgenic RUES1- SMAD7/EGFP line in pluripotency conditions (CM) resulted in down-regulation of the pluripotency determinant OCT4, expression of the PAX6, and the neuroepithelial gene SOX1 by Day 7. Coexpression of PAX6 and SOX1 was seen in most areas, though patches of PAX6+ only and SOX1+ only cells were also observed. The morphology of the cells mostly resembled that of pre-rosette neuroepithelium; however neural rosettes were also observed. The majority of cells are EGFP+ at this stage, suggesting presence of SMAD7 expression. B. SMAD7 mediated neural conversion is cell-intrinsic and does not require secondary induction centers. The induced cultures were largely devoid of mesoderm (BRA), endoderm (SOX17), visceral endoderm (HNF3β) on Day 7. Rare BRA+ cells (10-15/96-well) were observed in both induced and uninduced cultures, but never in clusters. C. Induced neural cells are of anterior character. Expression of the forebrain-midbrain specific transcription factor OTX2 is homogeneously observed on Day 7 in SMAD7 cultures. Almost complete co-localization of OTX2 is observed with PAX6 in many areas of the culture (also see Figure 1). We did not observe expression of the midbrain marker EN1/2 (Engrailed) or the hindbrain marker HOXB4. Scale bars represent 200μm.
sc-12-0256_sm_SupplFigure7.pdf2910KFIGURE S7: A. SMAD7 induced neuroepithelium can differentiate into GFAP +ve astroglia after 8 weeks. RUES1- SMAD7/EGFP hESCs were induced with DOX for 11 days and then allowed to differentiate on laminin/poly-ornithine coated surfaces for 8 weeks. CNTF was added between weeks 6-8. Observe the typical glial morphology of the differentiated cultures. B. Small molecule inhibitors of Activin-Nodal and BMP receptors also induce anterior neuroectoderm in hESCs. The two compounds used here were: the Activin/Nodal receptor inhibitor SB431542, which targets ALKs 4, 5, and 7, and the BMP receptor inhibitor LDN193189, which inhibits ALKs 2 and 3. The small molecules induced neural fate with similar efficiency to SMAD7, as determined by IF of RUES2 cultures for PAX6, OTX2, and SOX1 on Day 11. C. SMAD7 induces neural fate in different hESC lines. Two additional DOX-inducible SMAD7/EGFP lines were established: RUES2-SMAD7/EGFP, and a new clone of RUES1-SMAD7/EGFP. Both lines show identical expression of several neural markers at day 11 such as PAX6, OTX2 and DCX on DOX induction. Flow cytometry analysis of both lines for PAX6 showed acquisition of neural fate by the majority of EGFP+ cells in both these lines on Day 11. Scale bars represent 100μm. Insets show representative higher magnification views. The nuclear counterstain (DAPI) is pseudocolored blue.
sc-12-0256_sm_SupplInfor.pdf69KSupplementary Data
sc-12-0256_sm_SupplTable1.xls28KSupplementary Table 1
sc-12-0256_sm_SupplTable2.xls29KSupplementary Table 2
sc-12-0256_sm_SupplTable3.xls44KSupplementary Table 3

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