Two human stromal cell lines, HS-5 and HS-27a, represent functionally distinct components of the bone marrow microenvironment.1 2 HS-27a supports cobblestone area formation by early hematopoietic progenitors, whereas HS-5 secretes multiple cytokines that support the proliferation of committed progenitors. These cell lines, which are available from the American Type Culture Collection (ATCC, Manassas, VA), have been distributed to research groups worldwide for use as a tool to understand interactions between hematopoietic cells and their microenvironment. We have recently used DNA microarray technology to characterize and compare the expression of over 17 000 genes in these cell lines.
Briefly, microarray construction and hybridization protocols were modified from Marton et al.3 The microarrays were constructed using a set of more than 17 000 sequence-verified clones from Research Genetics (Huntsville, AL). Of the 17 761 features (spots) on the microarray, 186 are control nonexpressed or nonhuman sequences or housekeeping genes. UniGene cluster IDs could be assigned by GenBank to 16 592 of the features as of June 1, 2001 (UniGene Build 133), indicating that they are representatives of nonredundant unique genes. Many have been functionally characterized, and chromosomal location and tissue expression patterns are known for others. Total RNA was isolated from semiconfluent cultures and reverse-transcribed into cDNA in a nucleotide mix containing amino-allyl deoxyuracil triphosphate (dUTP). The cDNA from stromal cells and Universal RNA (Stratagene, La Jolla, CA) was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively. The Universal RNA consists of a mixture of RNA from 10 different human cell lines with a broad expression coverage of over 80% of the sequences on the array, allowing comparison of expression patterns of multiple different samples of different origin. The Cy5- and Cy3-labeled cDNAs were combined for hybridization to the microarray. Fluorescent array images were collected for both Cy3 and Cy5, and image-intensity data were extracted and analyzed to obtain expression ratios to Universal RNA for each stromal cell line. From these the expression in the 2 lines could be compared.
Here, we present tables identifying the genes with greater than 10-fold and significant differences (Student t test,P < .05) in expression between the 2 cell lines (Tables1 and2). The interleukin-7 receptor, among the genes with much higher expression in HS-27a, is the subject of a separate manuscript appearing in this issue (Iwata et al, page 1318).4 A summary table presenting the complete expression profile for each cell line, as well as a comparison between them, for all 17 000 sequences, is available athttp://parma.fhcrc.org/MIwata. This site also presents technical details of array spotting, hybridization, cDNA synthesis, and fluor-coupling procedures. The entire raw data set, consisting of 4 microarrays per cell line, is publicly available athttp://www.ncbi.nlm.nih.gov/geo/.
Gene expression differences in cytokines/chemokines, G-protein signaling molecules, and multiple extracellular matrix proteins add to the known protein and functional characterization of the lines, leading to new insight into the differences in their support function for hematopoietic progenitors.
Supported in part by grants HL62923, CA15704, and DK56465 from the National Institutes of Health, Bethesda, MD.
