Introduction 1 2 3 8 9 16 17 18 c-kit Materials and methods Mice C57BL/6 (B6-Ly5.2) mice were purchased from Charles River Laboratories Japan (Yokohama, Japan). C57BL/6 mice congenic for the Ly5 locus (B6-Ly5.1) were obtained from RIKEN BRC. B6-Ly5.1/Ly5.2 F1 mice were obtained from mating pairs of B6-Ly5.1 and B6-Ly5.2 mice. All animal experiments were approved by the Animal Experiment Committee of the RIKEN Tsukuba Institute.  Stromal cell culture and lentiviral transduction 2 19 − −/low + + − − 20 + − 4 Colony-forming cell assay −  Competitive repopulation assay 8 − 5 + Microarray analysis Total RNA was isolated from stromal cells using the ISOGEN reagent (Nippon Gene, Tokyo, Japan) and purified with the RNeasy MinElute cleanup kit (Qiagen, Hilden, Germany). Biotin-labeled cRNA was prepared from 1 μg of the purified total RNA with a one-cycle cDNA synthesis kit and 3′-amplification reagents for IVT labeling (Affymetrix, Santa Clara, CA) and was hybridized to an Affymetrix Gene Chip Mouse Genome 430 2.0 array (Affymetrix), which contains approximately 45,000 probe sets for analyzing the expression levels of more than 34,000 mouse genes. After washing and staining with the antibody amplification procedure, the microarrays were scanned with an Affymetrix GeneChip Scanner 3000 7G. All these procedures were carried out according to the manufacturer’s instructions. The expression value (Signal) and detection call (Present (P), Absent (A), or Marginal (M)) for each probe set were calculated using GeneChip Operating Software version 1.4 (Affymetrix). The Signal values were normalized so that their mean in each experiment was 100 in order to adjust for minor differences between the experiments. The change value (Signal Log Ratio) and change call (Increase, Marginal Increase, No Change, Marginal Decrease, or Decrease) for each probe set were calculated by Comparison Analysis of the software. All experiments were performed in duplicate using two independent cell samples. To identify differentially expressed genes, we selected probe sets that showed a change call of Decrease and a Signal Log Ratio value of ≤–1 (more than twofold down-regulation) or a change call of Increase and a Signal Log Ratio value of ≥1 (more than twofold up-regulation) in each of the two independent experiments. Quantitative real-time polymerase chain reaction 1 Table 1 Primers used in quantitative real-time PCR Gene Primer sequence Product size (bp) 1110007F12Rik Forward 5′-GCCCTGTGCCTGATGTTCTAC-3′ 111  Reverse 5′-GCCCATGTCCTCCTTCCAC-3′ 2900064A13Rik Forward 5′-GTTTGACCCTGTCCGAGTCG-3′ 205  Reverse 5′-CGGGAGAACCATCATCATAACC-3′ Ccl2 Forward 5′-TTAAAAACCTGGATCGGAACCAA-3′ 121  Reverse 5′-GCATTAGCTTCAGATTTACGGGT-3′ Ccl9 Forward 5′-TCAGATTGCTGCCTGTCCTAT-3′ 117  Reverse 5′-GAACCCCCTCTTGCTGATAAAG-3′ Cxcl5 Forward 5′-TGCGTTGTGTTTGCTTAACCG-3′ 107  Reverse 5′-AGCTATGACTTCCACCGTAGG-3′ IL-1rn Forward 5′-GCTCATTGCTGGGTACTTACAA-3′ 132  Reverse 5′-CCAGACTTGGCACAAGACAGG-3′ IL-6 Forward 5′-TAGTCCTTCCTACCCCAATTTCC-3′ 76  Reverse 5′-TTGGTCCTTAGCCACTCCTTC-3′ Results and discussion Subclones of the PA6 cell line are defective in supporting HSCs 18 − 20 13 2 Table 2 CFC frequency and competitive repopulation capacity Stromal cells CFC frequency No. of reconstituted mice (%) % Chimerism PA6 10.8 ± 7.51 6/9 (66.7) 13.2 ± 19.9 PA6 S-2 0.60 ± 0.97* 4/10 (40.0) 4.5 ± 11.2** PA6 S-12 3.2 ± 3.05* 3/8 (37.5) 3.4 ± 5.9** OP9 28.2 ± 6.61 5/9 (55.6) 8.6 ± 11.5 − − − P P t Identification of genes that were specifically down-regulated in PA6 subclone cells 3 Table 3 List of candidate genes down-regulated in PA6 subclone cells compared with PA6 cells Gene symbol Gene title Refseq ID Microarray qPCR vs. PA6 vs. OP9 vs. PA6 vs. OP9 PA6 S-2 PA6 S-12 PA6 S-2 PA6 S-12 PA6 S-2 PA6 S-12 PA6 S-2 PA6 S-12 1110007F12Rik RIKEN cDNA 1110007F12 gene NM_197986 0.30 0.46 0.11 0.19 0.29 0.23 0.16 0.13 1200009O22Rik RIKEN cDNA 1200009O22 gene NM_025817 0.30 0.21 2.79 1.48 ND ND ND ND 2900064A13Rik RIKEN cDNA 2900064A13 gene NM_133749 0.41 0.37 1.23 1.16 0.29 0.33 0.48 0.55 Ccl2 Chemokine (C-C motif) ligand 2 NM_011333 0.09 0.13 0.04 0.06 0.16 0.07 0.09 0.04 Ccl9 Chemokine (C-C motif) ligand 9 NM_011338 0.14 0.19 0.03 0.04 0.10 0.09 0.04 0.03 Cd53 CD53 antigen NM_007651 0.23 0.15 0.28 0.14 ND ND ND ND Cxcl5 Chemokine (C-X-C motif) ligand 5 NM_009141 0.33 0.23 0.43 0.38 0.20 0.13 0.90 0.60 Hgf Hepatocyte growth factor NM_010427 0.41 0.41 0.85 0.88 ND ND ND ND Il1rn(IL-1rn) Interleukin 1 receptor antagonist NM_031167 0.09 0.12 0.26 0.31 <0.03 0.04 <1.25 1.45 Il6(IL-6) Interleukin 6 NM_031168 0.38 0.44 2.07 2.62 0.30 0.16 2.42 1.31 Ppap2b Phosphatidic acid phosphatase type 2B NM_080555 0.22 0.22 0.09 0.09 ND ND ND ND Relative expression levels in PA6 subclone cells (S-2 and S-14) compared with PA6 and OP9 cells were calculated using data from microarray and quantitative real-time PCR (qPCR) analyses. Data represent the average of fold changes from two independent experiments ND Characterization of candidate genes − 1 Hgf Ccl2 and Ccl9 IL-6 Fig. 1 −  Ccl9 IL-6 Ppap2b Cxcl5 4 1200009O22Rik IL-6 1200009O22Rik IL-6 IL-6 3 − 8 21 Table 4 Effect of overexpression of candidate genes on the competitive repopulation capacity  No. of reconstituted mice (%) % Chimerism Total Myeloid B-lymphoid T-lymphoid PA6 9/9 (100) 22.1 ± 25.1 30.9 ± 30.9 25.1 ± 26.5 17.4 ± 23.0 PA6 S-2 4/10 (40) 7.3 ± 14.0 4.0 ± 7.4 10.8 ± 18.8 9.2 ± 17.8 1110007F12Rik 7/10 (70) 16.2 ± 14.8* 21.8 ± 26.3 18.7 ± 16.9 15.5 ± 16.7 1200009O22Rik 8/8 (100) 8.2 ± 10.2 14.5 ± 12.2 9.6 ± 13.4 8.3 ± 11.3 2900064A13Rik 2/5 (40) 3.3 ± 4.6 10.5 ± 20.6 3.6 ± 5.4 1.0 ± 2.2 Ccl9 2/5 (40) 11.1 ± 15.0 14.2 ± 19.5 12.7 ± 17.6 11.4 ± 15.7 IL-6 9/9 (100) 8.5 ± 13.5 8.1 ± 7.1 11.6 ± 16.9 9.2 ± 16.3 Ppap2b 8/10 (80) 9.7 ± 10.4 20.5 ± 26.7 9.2 ± 9.7 9.6 ± 14.6 Cxcl5 3/5 (60) 3.0 ± 4.0 12.8 ± 23.6 2.5 ± 3.5 1.9 ± 1.8 − P t 2900064A13Rik CCl9 1110007F12Rik Ppap2b Cxcl5 1200009O22Rik IL- 1110007F12Rik 4 1110007F12Rik 1110007F12Rik 3 1110007F12Rik 1110007F12Rik 1 1110007F12Rik 1110007F12Rik Tmem140 1110007F12Rik 22 1110007F12Rik Electronic supplementary material Below is the link to the Supplemental data.
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