Identification of New Rat Bone Marrow-Derived Population of Very Small Stem Cell with Oct-4A and Nanog Expression by Flow Cytometric Platforms

Figure 1

Expression of CD54 (ICAM-I) and CD106 (VCAM-I) on rat BM cells. (a) Gating strategy for identification of CD54⁺ and CD106⁺ populations by classical flow cytometry. Total nucleated cells (TNCs) derived from WT rat BM were stained for CD45 (PE-Cy7), hematopoietic lineages markers (Lin: TCRαβ, CD3, CD11b, CD45RA; FITC), and CD54 (PE) or CD106 (PE) and further analyzed by LSR II (Becton Dickinson). BM cells are visualized on dot-plot showing FSC versus SSC signals, which are related to the size and granularity/complexity of the cell, respectively. 2% of total of analyzed TNCs is only displayed in this dot-plot to visualize the population distribution. Objects from region P1 (lymphgate including / objects) were further analyzed for Lin markers expression and only Lin⁻ events are included into region P2. Upper, middle dot-plot cells stained for CD54, derived from gate P2, and analyzed for CD54 versus CD45 expression. Two fractions of cells potentially enriched in stem/progenitor cells are gated: rHSCs, CD45⁺/Lin⁻/CD54⁺ (region P3), and rVSELs, CD45⁻/Lin⁻/CD54⁺ (region P4). Both populations are further “back-gated” on FSC versus SSC dot-plot to visualize cell size distribution. Lower, middle dot-plot cells stained for CD106, derived from gate P2, and analyzed for CD106 versus CD45 expression; rHSC, CD45⁺/Lin⁻/CD106⁺ (region P3), and rVSELs, CD45⁻/Lin⁻/CD106⁺ (region P4). Both populations are further “back-gated” on FSC versus SSC dot-plot to visualize cell size distribution. Percentages show content of each subpopulation among TNCs in representative sample. Total of TNCs was typically collected for each sample to identify the SC populations. (b) Representative images of nonhematopoietic cells expressing CD54 and CD106 by ImageStream X system (ISS-X). TNCs were stained for Lin markers (FITC, green), CD45 (Alexa Fluor 647, blue), CD54 (PE, orange), or CD106 (PE, orange). Cells were fixed and nuclei were stained with Hoechst 33342 prior to analysis by ISS. Extracellular expression of CD106 and CD54 is shown on combo as well as on magnified images (right panels). The scale bars indicate 10 μm.

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