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STEM_211_sm_suppinfofig1.tif602KSupplementary Figure 1 - Presence of studied surface antigens on various cell sources (A) Representative flow plots of examples for positive versus negative single marker analysis on various human and mouse cell types for CD15, CD24, CD29 antigens. Species-specific antibodies were used for CD24 and CD29, as appropriate (see Suppl. Table 1). (B) Heat map, summarizing a profiling of FORSE-1, CD15, CD24, CD29 and CD56 surface antigens on various cell sources obtained by flow cytometric analysis. Cells stained for the respective surface antigens are presented as percent of positive cells within the overall live cell population. Abbreviations: VM= mouse E13 ventral midbrain; SC= mouse E13 spinal cord; STRO= mouse E13 stromal tissue; iPS NP= induced pluripotent stem cells (clone OS9) of neural precursor stage; msES= immature mouse ES cells (J1); ms NP= neurally induced msES cell cultures (J1; stage 4:2); ms ND= neuronally differentiated msES cell cultures (J1; stage 5:10); hESC H9 ES= immature hESC (H9); hESC div21= neurally induced hESC (H9) at the neuroepithelial precursor stage (21 div); hESC div30= hESC at the neural precursor stage (30 div); hESC div40= hESC at the neuronal differentiation stage (40 div); BE-M17= human neuroblastoma cell line.
STEM_211_sm_suppinfofig2A-D.tif869KSupplementary Figure 2 - Characterization of CD24 antigen surface expression (A) Immunoreactivity for CD24 antigen (red) on neural precursor cells, genetically labeled by Sox1-GFP (mouse ES-cell derived neural cells (19)). Compare to flow cytometric analysis in Fig. 2 A, and to immunofluorescence in Fig. 3 B. (B) Specificity of CD24 antigen staining on human ES cell derived mature neurons, as detected by co-staining of neuronal processes with CD24 (green) and MAP-2 (red). Right panel depicts orthogonal reconstruction of the z-axis images obtained by confocal microscopy. (C) Temporal changes of CD24 surface antigen expression as detected by flow cytometry. Neuronal maturation in vitro is paralleled by changes in CD24 antigen surface expression (here obtained by subsequent analysis of hES ND samples from div 43 to 48). Occasionally, an increasingly clear, two-peak separation into CD24LO versus CD24HI populations can be detected already in single marker analysis (lower histogram). (D) Characterization of hES neural differentiation by combined intracellular and surface antigen flow cytometric analysis. Pax3 expression is restricted to the CD24-/LO fraction, as detected by combined surface and intracellular antigen flow cytometric analysis (panel 3). Neuronal maturation monitored by CD24HI expression is accompanied by downregulation of nestin and ki-67 immunoreactivity (panels 4 and 5), and upregulation of neuroblast and mature neuronal markers (doublecortin, tau, TuJ1; panels 6 to 8). Representative dot-plots display CD24 surface antigen staining versus the respective intracellular antigens as labeled. Dcx= doublecortin. Note: Left panels show unstained/secondary antibody only control. In staining for intracellular antigens, varying levels of background fluorescence were observed, depending on the primary antibody, making this a qualitative assay for relative comparison, rather than an absolute numerical quantification of cells in culture. Dashed lines in panel 1 depict gates set for CD24- negative, low and high populations.
STEM_211_sm_suppinfofig2E-F.tif2530K(E) Relative abundance of CD24 expression on neuronally differentiating populations in primary neural tissue culture (here mouse embryonic day 13 forebrain). CD24 strongly colabels with nestin and MAP2 (1st and 4th columns), but is absent/ expressed in low levels on GFAP-positive astroglial cells (2nd column, arrowhead) or on neural cells of nonneuronal morphology (cells of “flat” morphology; 3rd column, arrowhead). Arrows indicate strongly CD24-expressing cell types. Div=days in vitro; NSE= neuron-specific enolase; Hoe= Hoechst. (F) Cell sorting of primary neural tissue (here mouse embryonic day 13 ventral midbrain) enables the derivation of FACS-purified neuronal cell cultures. Image depicts typical appearance at 2 to 3 days post-FACS, using sorting parameters optimized for neural cell types according to Pruszak et al., 2007 (15). Scale bars= 50 μm.
STEM_211_sm_suppinfofig3.tif1558KSupplementary Figure 3 - Characterization of CD29 antigen surface expression (A) Pax6-positive proliferative proliferative clusters strongly coexpressed CD29 antigen, while doublecortin (dcx)-positve cells emerging from such immature neural clusters were CD29-negative (B). Scale bars= 50μm. (C) Gating strategy for combined CD29 surface and intracellular antigen (TuJ1; TH) detection. In a typical flow cytometric analysis of hES-derived neuronal differentiation stage cells, the CD29LO population was characterized by 67.5% cells positive for TuJ1, and a mean intensity of fluorescence (MFI) of 7335. In contrast, CD29HI cells contained 25.6% of TuJ1+ cells (MFI of 2550) (D). Similarly, positivity for the catecholaminergic marker tyrosine hydroxylase (TH) was restricted to the CD29LO fraction (lower panels), identifying CD29LO as a marker criterion for the isolation of mature neuronal cell types. (E) In addition, the CD29LO population also contained a higher fraction of doublecortin-positive neuroblasts.
STEM_211_sm_suppinfofig4.tif1735KSupplementary Figure 4 - Combinatorial application of CD15/CD24/CD29 surface antigens (A) Visual cell counts showed that populations sorted according to CD15/CD24/CD29 expression segregated into proliferative versus neuronally differentiated cells types, substantiating FACS-based analysis for intracellular antigens (see Suppl. Fig. 2 D). HES-derived neural cell suspensions were FACS-enriched for NS, NC and ND cell populations to only ca. 80% purity prior to replating in vitro and fluorescence microscopic quantification by blinded investigators. (B,C) Distribution of cell populations defined by CD15/CD24/CD29 expression changed over time during in vitro maturation, consistent with the putative NS, NC and ND phenotypes defined by this combinatorial profile. Without a sorting step, all three subpopulations remained present, also after prolonged culture (up to 70 div, i.e. 30 div under differentiation conditions analyzed). (D) Graft analysis of CD15-/CD24HI/CD29LO grafts at 5 weeks shows neuronal composition without tumor formation and sparse ki67-positivity. HES-derived neural cell suspensions were enriched for ND cell populations to ca. 80% purity prior to transplantation. hNCAM= human-specific NCAM (Eric-1). (E) A neurosphere formation assay confirmed reduced proliferative capacity in the ND population. 5,000 cells of each population were cultured in round bottom 96-wells. Formation of spheres and/or proliferative clusters was closely observed and quantified at five weeks post-FACS using Hoechst stain and fluorescence microscopy. Spheres per well are shown; vertical dashed lines indicate NS, NC ND groups. Cells sorted for CD15-/CD24HI/CD29LO contained significantly less spheres or clusters than the NS and NC populations (P<0.05; single experiment; five wells per group). An intermediate population, CD15-/CD24HI/CD29″MID″, was included as an additional control, as indicated. Scale bars= 100μm.
STEM_211_sm_suppinfotable1.tif329KSupplementary Table 1 - Antibodies used for flow cytometric analysis of surface antigens
STEM_211_sm_suppinfotable2.tif388KSupplementary Table 2 - Antibodies used for immunocytochemical and immunofluorescence analysis
STEM_211_sm_suppinfotable3.tif26KSupplementary Table 3 - List of primers used in qRT-PCR

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