A molecular profile of a hematopoietic stem cell niche

Abstract

The hematopoietic microenvironment provides a complex molecular milieu that regulates the self-renewal and differentiation activities of stem cells. We have characterized a stem cell supportive stromal cell line, AFT024, that was derived from murine fetal liver. Highly purified in vivo transplantable mouse stem cells are maintained in AFT024 cultures at input levels, whereas other primitive progenitors are expanded. In addition, human stem cells are very effectively supported by AFT024. We suggest that the AFT024 cell line represents a component of an in vivo stem cell niche. To determine the molecular signals elaborated in this niche, we undertook a functional genomics approach that combines extensive sequence mining of a subtracted cDNA library, high-density array hybridization and in-depth bioinformatic analyses. The data have been assembled into a biological process oriented database, and represent a molecular profile of a candidate stem cell niche.

Figure 1

Comparative analysis of cKit^pos Sca-1^pos Lin^neg Rhodamine^lo BM (Rho^lo) stem cell activity before (day 0) 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 with 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 with 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. (A) Flow diagram depicting the subtracted cDNA library strategy and essential elements comprising StroCDB. (B) Categorization of informative sequences by homology (Left) or by putative or known protein type and function (Right).

Figure 3

Previously undescribed cell surface or secreted proteins contain peptide motifs and/or sequence homology to interesting developmental regulators. (A) One previously undescribed and 2 known cadherin domain-containing proteins are aligned with Cadherin. The LL6in11084 protein is related to the Drosophila Fat morphogenic protein. (B) LL6in12391 is homologous to KIAA1402 which contains a Fringe motif. (C) Two leucine-rich repeat-containing proteins are shown, LL6in120165 and LL6in12362. LL6in12362 is homologous to a human gene product, p37NB. These proteins are most related to proteins homologous to the chemokinetic protein Slit. (D) Keilin is a secreted, Xenopus laevis, signaling protein that mediates inductive activities in the embryonic midline. E25 represents the mouse homolog.

Figure 4

A gene expression profile may correlate with a 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 with those selected in the custom filter array analysis. The overlapping gene products are displayed as their relative signal to background ratios in red to green coloration for the filter arrays. The Pearson correlation coefficient (r) of this subset of gene products for each line compared with AFT024 is; 2012 (0.32), 2058 (0.42), and 2018 (0.04). An expanded version of the figure and the correlation coefficients of all array elements and comparisons is available in StroCDB: Figures and Microarray Analysis. StroCDB identifier (LL6inXXXXX) and the protein identities are listed.

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, were 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 Fig. 4.