Microarray analysis of sorted hUCB VSEL cells and comparison to other stem and mature cell types (listed in Table S2).

<p>A+B) A genome-wide unbiased principal component analysis including 12621 genes classifies VSEL cells as remote to both pluripotent and adult stem cell populations as well as mature blood cell lineages. C) Hierarchical clustering represented by an un-rooted tree diagram confirms the results of the PCA. D) A heatmap comparison of 371 selected pluripotency associated genes shows substantial differences between VSEL cells and pluripotent stem cells, but a high degree of similarity between ES and iPS cells. E) Normalized transcription level of the core pluripotency genes Oct4 (POU5F1), Nanog and Sox2 as part of D. Note that the Affymetrix microarray probes for POU5F1 do not comply with up to date quality standards as they are known to detect pseudogenes.</p

Immunophenotype of CD45-Lin-Hoechst 33342+PI- cells.

<p>hUCB cells were subjected to multiparameter FACS analysis, and gated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034899#pone-0034899-g001" target="_blank"><b>Figure 1</b></a> D (either Lin⁻ or CD235a⁻). Among 22 markers tested, three were found positive on CD45⁻Lin⁻ cells: CXCR4, CD31 and CD84. However, in contrast to previous reports, CD45⁻Lin⁻ cells contain very low amounts of either CD34⁺ (1,2%+/−0,6%; n = 5) or CD133⁺ (1,8%+/−2,3%; n = 5) cells (B middle & right). A) Representative single analysis of markers on CD45⁻Lin⁻ cells. B+C) Representative replicate analysis of the expression of CXCR4, CD34 and CD133 on CD45⁻Lin⁻ cells and on CD34⁺CD45^dimcells.</p

Immunophenotyping and transcriptional profiling of Lin-CD45- and VSEL cells.

<p>#)surface antigen expression measured by FACS analysis;</p><p>*)mRNA expression level measured by microarray analysis.</p

Cytometric and karyotypic analysis of CD34+CD45dim cells and VSEL cells.

<p>A) Comparison of the median Hoechst 33342 fluorescence, FSC-H and SSC-H of CD34⁺CD45^dim cells and VSEL cells, respectively (n = 15); B) Cell diameter of freshly FACS sorted CD34⁺CD45^dim cells and VSEL cells (n≥110; p<0.001); C) May-Grünwald/Giemsa stained cytospin preparations of CD34⁺CD45^dim cells (top) and VSEL cells (bottom). D–E) Karyotypic analysis of cytospin preparations of CD34⁺CD45^dim cells (top) and VSEL cells (bottom) stained with probes against D) centromere 7 (red) and centromere 8 (green), and E) p53 (red) and DLeu (green). scales = 10 µm.</p

Gating of VSEL cells and CD34+CD45dim cells in hUCB.

<p>hUCB TNC were isolated by NH₄Cl lysis and stained with fluorescent antibodies, Hoechst 33342, and PI. A) Gating of living (PI⁻) nucleated (Hoechst 33342⁺) cells; B) pulse processing: exclusion of doublets (Hoechst-W^high) and granulocytes (SSC-A^high). C) Gating of CD34⁺CD45^dim Hoechst 33342⁺PI⁻. D) Gating of CD45⁻Lin⁻ cells. E) Gating of CD45⁻Lin⁻ CXCR4⁺Hoechst 33342⁺PI⁻ VSEL cells. F) Expression of CD133 in CD34⁺CD45^dim cells (blue) and VSEL cells (black).</p

Separation of CD34+CD45dim cells and VSEL cells by ficoll density gradient centrifugation.

<p>Cells recovered from interphase and pellet were lysed with NH₄Cl and analyzed for the frequency of CD34⁺CD45^dim cells and CD45⁻Lin⁻CXCR4⁺ VSEL cells, respectively. A) Recovery of TNC in interphase and pellet (n = 15). B) Recovery of CD34⁺CD45^dim cells and VSEL cells in the interphase (n = 15). C) Frequency of CD34⁺CD45^dim cells and VSEL cells in interphase and pellet, respectively (n = 15).</p

FACS sorting of VSEL cells from Lin− hUCB cells derived from MACS lineage depletion of TNC.

<p>A+B) Two extreme UCB samples with many (A) and very few (B) CD34⁺VSEL cells, respectively. C) Frequency of CD34⁺CD45^dim cells (upper middle gate in A+B), CD34⁻VSEL (CD45⁻Lin⁻CXCR4⁺CD34⁻, lower left gate in A+B), and CD34⁺VSEL (CD45⁻Lin⁻CXCR4⁺CD34⁺, upper left gate in A+B) cells within PI⁻ lymphocytes (n = 14).</p

Humanized mice generated functional human B and T cells.

<p>(A, B) The concentration of human IgM and IgG in the plasma of humanized mice was determined by cytometric bead array 19–21 weeks after intrahepatic transplantation of CD34⁺ cells from CCB or FCB. (C) Spleen cells were labeled with CFDA-SE and cultured for 5 d in the presence (dark gray bars) or absence (gray bars) of an anti-CD3 antibody. Proliferation of cells was determined by flow cytometry. Box plots depict median and 5–95% percentile. Level of significance is given.</p

The purity of CD34⁺ separation decreases with an increasing number of MNC in CCB.

<p>MNC were isolated from CCB or FCB by washing with DNAse buffer or Ficoll-paque density gradient centrifugation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046772#s2" target="_blank">materials and methods</a>. CD34⁺ stem cells were isolated from MNC by a positive magnetic separation of CD34⁺ cells and the purity was determined by flow cytometry. The correlations between the number of MNC and the purity of the CD34⁺ separation for CCB (A; no significance) and FCB (B) are shown. Correlations were evaluated using GraphPad Prism software and the Spearman rank correlation coefficient method.</p

Differences and parallelism in the purity of CD34⁺ -cell-purifications from FCB and blood bank CCB.

<p>(A) MNC were isolated from FCB and blood bank CCB by washing with DNAse buffer or Ficoll-paque density gradient centrifugation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046772#s2" target="_blank">materials and methods</a>. CD34⁺ stem cells were isolated from MNC by a positive magnetic separation of CD34+ cells and the purity was analyzed by flow cytometry. Dot plots depict the percentage of CD34⁺ cells of one representative separation from CCB and FCB (lower panels). The quadrant was set according to the isotype controls (upper panels). (B) The bar chart depicts the percentage of CD34⁺ cells after isolation of CD34⁺ cells from 26 CCB and 37 FCB samples. Box plots depict median and 5–95% percentile. Level of significance is given. n. s. = no significance.</p