Figures and Tables

Figure 1: Comparative analysis of cKit^pos Sca-1^pos Lin^neg Rhodamine^lo BM (Rho^lo) stem cell activity before and after LTC on AFT024

CFC content in 100 fresh, Day 0, (n=6) or the equivalent of 100 Rho^lo cells after 35 days of culture (n=8). The frequency of LT-CRSC of freshly purified Rho^lo cells, 1 in 14, compared to the frequency of CAFC, 1 in 13±1.7 (n=11), after 28-35 days culture on AFT024. Limiting-dilution CRSC and CAFC frequency was determined by Poisson statistics at 37% negative mice or wells. Rho^lo cells from the same purification were used to determine both CRSC and CAFC frequencies. %Ly5.2 peripheral blood cells from 25 Day 0 Rho^lo cells (n=5) compared to the 35 day cultured equivalent of 25 Rho^lo cells (n=5) from the same purification; both transplanted in CRSC assay.

Figure 2: Construction of an annotated database that profiles gene expression in a hematopoietic stem cell-supporting microenvironment

1. Flow diagram depicting the subtracted cDNA library strategy and essential elements comprising Stromal Cell Database (StroCDB).
2. Categorization of informative sequences by homology (left) or by putative or known protein type (right).

Figure 3: Novel cell surface or secreted proteins contain peptide motifs and/or sequence homology to interesting developmental regulators.

1. One novel and 2 known cadherin domain-containing proteins are aligned with a Cadherin. The novel LL6in11084 protein is related to the Drosophila Fat morphogenic protein.
2. The novel protein LL6in12391 is homologous to a KIAA1402 cDNA that contains a fringe motif.
3. Two novel leucine rich repeat-containing proteins are shown, LL6in12065 and LL6in12362. LL6in12362 is homologous to a human gene product p37NB. These proteins are most related to proteins that show homology to the chemokinetic protein Slit.
4. Keilin is a secreted, Xenopus laevis singaling protein that mediates inductive activities of the embryonic midline. E25 is the first mouse homologue.

Figure 4: A gene expression profile may predict stem cell-supporting phenotype in stromal cell lines.

Expression arrays developed from the AFT024-subtracted library were interrogated with probes derived from stromal cell lines. A clustering algorithm was used to profile the data and informative clusters were selected. The contents of the clusters revealed in the cDNA microarray (StroChip) analysis were compared to those selected in the custom filter array analysis. The overlapping gene products are displated as their relative signal to background ratios in red to green coloration for both the StroChip (left panel) and filter arrays (right panel). The Pearson correlation coefficient (r) of this subset of gene products for each line compared to AFT024 is; 2012 (0.32), 2058 (0.42), and 2018 (0.04). Tables of correlation coefficients of all array elements and comparisons are shown below the gene expression images.

Figure 5: Correlating gene expression and stem cell support.

The ability of stromal cell lines to support enriched stem cell populations was studied. A. Limiting-dilution CAFC assay on each stroma at 4 weeks. B. Stem cells were cultured on each stroma and then after 4 weeks replated into limiting-dilution CAFC assay onto fresh AFT024 monolayers. This assay determines the ability of each stroma to maintain stem cells with a capacity to generate secondary CAFC. Limiting-dilution frequency was determined at 37% negative wells according to Poisson statistics and is shown in parentheses for both graphs. The ability of each line to maintain stem cells in the secondary CAFC assay relative to AFT024 is; 2012 (32%), 2058 (31%), and 2018 (1%). These data correlate well with the results from the gene expression analysis in Figure 4.

Table 1 (Web-enhanced version): Novel candidate stem cell regulatory molecules

Table 2 (Web-only): Known secreted, cell surface, matrix, and cytoskeletal proteins in StroCDB

Table 3 (Web-only): Neuronal-related proteins