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Timed-pregnant mice and 5-7 weeks old females (C57Bl/6J, Ly5.2) were purchased from the Jackson Laboratory (Bar Harbor, ME). Congenic C57Bl/6, Ly5.1 female mice were purchased from the National Cancer Institute (Frederick, MD). All mice were housed in the Princeton University Barrier Animal Facility, in autoclaved micro-isolator cages on ventilated cage racks. The animals received sterile, irradiated food, and acidified, autoclaved water ad libitum.
Stromal cell lines were routinely cultured in Dulbecco's modified Eagles's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5X10-5 mol/L b-mercaptoethanol (2-ME), at 32o, 5% CO2, 100% humidity. Sera were obtained from Hyclone, Logan, UT. Other biochemical reagents were obtained from Sigma, St. Louis, MO. The fetal liver stromal cell lines used in this study were derived as previously described (Wineman, et al. 1996). Briefly, day 14 fetal livers were dissected from fetuses and disassociated by gentle pipeting. Cells were suspended in a modified Dexter media containing 12.5% horse serum, 12.5% fetal bovine serum, 5x10-5 mol/L 2-ME, 1X10-7 mol/L hydrocortisone cultured at 37o, 5% CO2, 100% humidity. After adherent cells became 50% confluent, they were immortalized by infection with viral supernatants from a retroviral vector encoding a temperature sensitive (TSa-58) SV40 T antigen and G418 resistance. 3 days later the cells were placed in selection with media containing 500 ug/ml G418. After 2-3 weeks of selection, individual clones of adherent cells were isolated and expanded. The lines used in this study have been demonstrated to be clonal by Southern blot analysis for the retroviral integration site. The AFT024 and 2012 cell lines have been described as supporters of highly enriched hematopoietic stem cells while the 2018 line does not support repopulating stem cell activity (Moore, et al. 1997). An additional line, BFC012 has also been described as a non-supporting line (Moore, et al. 1997). Additional biological data pertaining to the stromal cell lines can be found here.
Stem cells were purified from day 14-14.5 fetal livers essentially as described (Moore, et al. 1997). The AA4.1pos fraction has been shown to contain all repopulating stem cell activity present in day 14 fetal liver (Jordan, et al. 1990). Initial studies used an immuno-panning technique to remove AA4.1neg cells, while later studies identified AA4.1pos by sorting after labeling with phycoerythrin (PE) congugated AA4.1 mAb. Lineage positive cells were removed either by staining with a cocktail of fluorescein isothiocyanate (FITC) labeled rat monoclonal antibodies and sorting or by 2X depletion with anti-Rat immunomagnetic beads (Dynal , Oslo, Norway) of lineage cocktail stained cells. The lineage cocktail consisted of CD3, CD4, CD5, CD8, B220, Gr-1, and TER-119. The AA4.1pos cells were sorted for c-kit allophycocyanin (APC) and Sca-1 FITC or PE positive cells depending on the lineage depletion method. The lineage mAb hybridomas were obtained from Dr. Gerald Spangrude (University of Utah), AA4.1 hybridoma was obtained from Dr. J. McKearn, (Monsanto, St. Louis, MO) all other antibodies purchased from BD PharMingen, San Diego, CA. Propoidium Iodideneg (PI) Lineageneg/lo AA4.1pos c-kitpos, and Sca-1pos cells were sorted on a FACS Vantage with Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, CA). A second sort of these cells for the same parameters has enriched the stem cell frequency in this population to one in sixteen.
Bone marrow was purified essentially as described with modifications (Spangrude, et al. 1995). Cells from the femora and tibiae of 2-4 month old C57Bl/6J mice were extracted, red cells lysed in Ammonium Chloride Potassium Phosphate Buffer (ACK), and 2X lineage-depleted as above after staining with a lineage cocktail where CD4 was replaced by Mac-1. Lineageneg cells were threshold sorted for Sca-1(PE) positive cells that were further stained with c-kit (APC) and resorted for PIneg Lineageneg Sca-1pos c-kitpos cells. These cells were then stained with Rhodamine-123 (Rho), effluxed and sorted again into Rholo and Rhohi populations. The stem cell frequency in the quiescent Rholo population is one in 12-14 while the Rhohi fraction does not contain long-term (>4 months) repopulating stem cells.
Stromal cell lines were seeded onto tissue culture dishes that had been coated with 0.1% gelatin (Specialty Media, Lavallette, NJ) and were grown at 320C, 5% CO2, 100% humidity. Confluent monolayers were irradiated (20 Gy, 137Cesium source, Gammacell 40, Nordion International Inc. Ontario, Canada) and cultured in modified Dexter media (Dexter, et al. 1977) (DMEM, 10% FBS, 10% horse serum, 5X10-5 mol/L 2-ME, 1X10-7 mol/L hydrocortisone). For Dexter-LTC, enriched hematopoietic stem cells were added and the cultures were maintained at 37o C, 5% CO2, 100% humidity. Irradiated (20 Gy) 2018 monolayers in 96-well trays were used in limiting-dilution Whitlock-Witte (LD-WW) assays (Whitlock, et al. 1984) (DMEM, 10% FBS, 10% horse serum, 5X10-5 mol/L 2-ME, 1X10-7 mol/L hydrocortisone) to assess stromal-dependent B-lymphopoiesis content of both freshly purified and cultured stem cells. These cultures were established in RPMI media with 5% FBS, 2 mmol/L glutamine, 1 mmol/L Na pyruvate, and 5X10-5 mol/L 2-ME at 37o C, 5% CO2, 100% humidity.
The progenitor content of freshly purified hematopoietic stem cell populations and AFT024/stem cell cocultures was assessed using a variety of in vitro assays. To determine the time course of Cobblestone Area (CA) development, enriched stem cells were seeded onto irradiated AFT024 monolayers in bulk co-cultures or in limiting-dilution in 96-well trays. CA development was followed over time and characteristic clusters were quantitated as described (Ploemacher, et al. 1989). At different time points of the stem cell/AFT024 cultures, individual wells were harvested and replated into cytokine-supplemented semisolid clonogenic progenitor assays (CFU-C). The cytokine-enriched (rmIL-3 10 ng/mL, rhIL-6 10 ng/mL, rmSL 50 ng/mL, Epo 3 U/mL) methyl-cellulose mixture was purchased from Stem Cell Technologies Inc., Vancouver BC, Canada. Colonies were scored after 8-14 days of culture at 37o, 5% CO2 , 100% humidity according to established criteria. Colonies that reached >1mm in size after 8 days and which contained erythroid bursts and multiple myeloid cell lineages including megakaryocytes were scored as high-proliferative potential-mixed lineage colonies (CFU-HPP-Mix). Lineage content of typical colonies was determined by Wright's/Giemsa staining of cytospin slide preparation from individual colonies. To determine the ability of the AFT024 cell line to maintain primitive lymphoid progenitors, 4 week cocultures were plated into LD-WW assay as described above. Resulting pro-B cell colonies were scored after 7 days. In a similar manner, the frequency of secondary CA-initiating cells in long-term (> 4 weeks) stem cell cocultures was also determined by replating them in limiting-dilution onto fresh, irradiated AFT024 monolayers in 96-well trays. For all limiting dilution assays the progenitor frequency was predicted according to Poisson statistics, where the cell number at 37% negative wells (as calculated from the line of best fit) is the frequency.
Competitive repopulation was used to measure stem cell activity present in both freshly isolated and cultured stem cell populations (Harrison 1980). This assay was performed using the congenic Ly5.1/5.2 mouse system. Freshly purified stem cells (Ly5.2) were to transplant mice without culture and a portion was seeded onto irradiated stromal monolayers and maintained in Dexter-LTC conditions. At the end of the LTC period, the cultures were harvested by vigorous trituration. Single cell suspensions were prepared by passage through 22-gauge needles, mixed with fresh congenic BM (Ly5.1) and transplanted into lethally irradiated congenic mice (10 Gy, split dose 3 hours apart, 1 Gy/min, Gammacell 40). In order to assess reconstitution, mice were periodically bled by capillary puncture of the orbital venous plexus. After red cell lysis with ACK, nucleated cells were stained with lineage antibodies (T-cells; CD4-PE, CD8-PE, B-cells; B220-APC, Myeloid Cells; Biotinylated Mac- 1/Gr-1 developed with streptavidin Texas Red), Ly5.2-FITC and propidium iodide. Five color analysis of peripheral blood cells from mice transplanted with stem cell/stromal cell cultures was performed on the Becton Dickinson FACS Vantage.
We performed subtractive hybridization using two directionally cloned cDNA libraries. This procedure is based on the hybridization of a single stranded (ssDNA) phagemid cDNA library (target, AFT024) to biotinylated RNA (driver, 2018) transcribed from a double stranded (dsDNA) phagemid cDNA library with cDNA inserts cloned in the opposite (complementary) orientation. To make these libraries we isolated total cellular RNA by guanidine isothiocyanate solubilization followed by CsCl gradient centrifugation, acid phenol-choloroform extraction and ethanol precipitation. The polyA-plus fraction (mRNA) was purified on oligo-dT cellulose columns. Prior to first-strand cDNA synthesis mRNA was treated with RNase-free DNase I. First and second strand cDNA were synthesized using reverse transcriptase SuperScript II, DNA polymeraseI, RNAseH and E.coli ligase. The first-strand primer was oligo-dT with a 5' NotI site. Double-stranded cDNA was blunt-ended with T4-DNA polymerase and ligated to a SalI adapter with T4-DNA ligase.
The vectors for the library construction, pSport1 and pSport2 are commercially available from Life Technologies and were used as described (Li, et al. 1994). Accordingly, two cDNA libraries were constructed according to recommended protocols. AFT024 Sport1 contained 25x106 clones and 2018-Sport2 contained 17x106 clones. The average insert size for both libraries was 1000 bp and ranged from 500-4000 bp. To perform the subtractive hybridization, ssDNA was generated by infection of AFT024-Sport1 transformants with helper phage. Biotinylated driver 2018-RNA was likewise prepared from the Sal I linearized 2018-Sport2 library by in vitro transcription with T7 RNA polymerase. In a typical round of subtractive hybridization 600 ng of AFT024 target ssDNA was hybridized to 80 mg of biotinylated 2018 driver RNA. The hybridization proceeded at 42o C for 48 hours to obtain a Cot value of 1000. The hybridized biotinylated product and remaining driver were removed with streptavidin and phenol-chloroform extraction as recommended. The remaining ssDNA was converted to dsDNA with Taq polymerase. This product was then electroporated into bacteria. The resulting clones, 4.2x105 compared to control, (same reaction conditions without driver) 3.7x107 constituted an almost 2 log reduction in transformants {Librarystats.txt}. In order to eliminate the plasmids which did not contain inserts that are enriched following subtraction, the DNA from the subtracted library was linearized with NotI and subjected to four successive agarose gel electrophoresis fractionations. After each electrophoresis gel the DNA smear corresponding in size to vector plus 1 kb. and greater was excised and eluted using the sodium iodide/glass bead procedure. Four such fractionations eliminated essentially all empty plasmids. The resultant DNA population was introduced into bacteria. Portions of the transformed bacteria were plated and random individual clones were selected for manual sequencing and expression analysis. A portion of the remaining transformants were robotically picked into 384-well plates for automated sequencing. A separate aliquot of the library was amplified as a population and used to prepare DNA. A direct measure of subtraction efficiency was obtained by hybridizing pre and post-subtraction cDNA Southern blots with a beta-actin probe. This showed a complete depletion of this very abundant gene product from the subtracted cDNA library. In addition, the randomly selected clones readily revealed both gene specific or enriched expression in AFT024 compared to 2018.
Initial sequences were obtained by chain termination using the Sequenase Version 2.0 kit (U.S. Biochemicals). The majority of randomly selected sequences were generated by single-pass automated sequencers by Commonwealth Biotechnologies, Inc. (Richmond, VA), cDNAs A01-F89; or by Incyte Pharmaceuticals, Inc. (Palo Alto, CA), all LL6inxxxxx cDNAs.
DNA sequences and conceptual translations were compared in-house with known nucleotide and protein sequences using the BLAST (Basic Local Alignment Search Tool) algorithm (blastn for nucleotide and blastx for protein databases). Six publicly-accessible databases were searched: SwissProt, Genbank non-redundant (nr) protein, Genbank nr nucleotide, dbEST expressed sequence tags, and the murine and human DoTS databases of transcribed sequences. The DoTS databases have been coalesced into Allgenes. Sequences were also compared with those in StroCDB itself as a measure of internal redundancy. Potential open reading frames (ORFs) were located using ORF Finder. Protein motif searches were performed using four different motif identification programs: Prosite, Pfam, Prodom, and SMART. Transmembrane helices were detected using the TMPred server and potential signal peptides were detected with SignalP. Categorization of sequence homology was based on the following criteria: 1) Exact match, identity to a published mouse protein; 2) Homolog: near-identity to a published protein from a species other than mouse; 3) Family Member: homology indicating relatedness to a described protein family; 4) EST only: no extensive homology to any published or characterized protein, but identity to expressed sequence tags from mouse or another species; 5) No Match: no extensive homology to any nucleotide or protein sequence in any of the public databases.
The StroCDB database is a relational database with MySQL as the database manager. It contains all primary sequence data, results of bioinformatic analyses, and array expression results. The database architecture allows incorporation of new expression data or results of other experiments as they become available.
Multi sequence format (MSF) display of protein sequences was generated through (Multalin) and used with MacBoxShade v.2.15 (Kay Hoffman: Boxshade; Michael D Baron: MacBoxshade derivative)to generate alignments.
The StroCDB database is a relational database with MySQL as the database manager. It contains all primary sequence data, results of bioinformatic analyses, and array expression results. The database architecture allows incorporation of new expression data or results of other experiments as they become available.
Total and poly A+ RNA were prepared from stromal cell monolayers using commercial reagents (Life Technologies and Ambion). The quantity and quality of the mRNA was evaluated by Northern blotting (data not shown). The Stromal Cell cDNA Microarray (StroChip) was developed and printed by Incyte Genomics Inc. from non-redundant cDNAs in the AFT024-subtracted library. To determine the non-redundant clone set, individual sequences were compared by BLAST (basic local alignment sequence tool, (Altschul, et al. 1997)) against the mouse and human UniGene databases (http://www.ncbi.nlm.nih.gov/UniGene). Highly significant matches (e < 10-15) were assembled into the same cluster. For entries with multiple representatives, the one containing the largest sequence was selected. 3600 PCR-amplified DNAs were arrayed on glass slides and correspond to an annotated entry in StroCDB. Controls on the cDNA arrays included housekeeping genes (alpha tubulin, 23 kD HBP, and ribosomal subunit S9), a complex target to measure probe complexity, as well as sensitivity controls and fluorescence intensity controls (doped spots), spotted in triplicate on each chip. Cy3 and Cy5 fluorescent labels were incorporated into cDNA transcribed from RNA templates, hybridized to microarrays, and quantitated. Probe labeling, microarray hybridizations and image quantitation were performed by Incyte Genomics Inc. The stromal cell lines, 2012, 2018, 2058, and BFC012 were each compared to AFT024. Each comparison was performed in two labeling orientations, allowing compensation for different dye incorporation efficiencies and an experimental replicate. The resulting data were processed as follows. Cy5 signal was normalized relative to the Cy3 signal by whole chip balancing to differences in the intensity of each fluorochrome. In order to assess dataset quality, each type of control was grouped and asked, by t-test, if the means of each signal (Cy3 and Cy5, after balancing) were significantly different. In no case was there a significant difference. The data were filtered as log2 > 1 across all four comparisons and 381 cDNAs passed this filter. log2 transformed, balanced signals were expressed as a ratio (AFT024 vs. the compared cell line) and averaged. These results were grouped into 18 clusters using a k-means clustering algorithm (adapted by JAH from the cclust R package written by E. Dimitriadou), using Euclidean distance as the similarity metric. Clusters were chosen with patterns of expression that correlated with a biological phenotype, i.e. higher expression in AFT024 compared to known non-supporting stromal cell lines.
Approximately 2000 clones from the subtracted library were arrayed on nylon membranes where each duplicate spot corresponds to an entry in StroCDB. The membranes were hybridized with 33P-dCTP-labeled subtracted probes produced using the PCR Select technology (Clontech). Probes were generated from AFT024, 2012, 2018, and 2058 where each test line was subtracted with BFC012. We have previously shown that BFC012 cells do not support stem cells in culture (Moore, et al. 1997). Image data were collected by a Molecular Probes Phosphoimager and quantitated by Incyte Genomics Inc. using Arrayvision software. Raw signal/background intensities were log2 transformed and values for a given probe were averaged. These values were also subjected to the same k-means clustering algorithm. Representative clusters that showed higher expression in AFT024 than 2018 were selected. The log-transformed, averaged data were used to generate red to green pseudo-colored images of the signal/background intensity.
