Increased aldehyde dehydrogenase (ALDH) activity has been found in murine and human hematopoietic stem cells and human multiple myeloma stem cells (MMSCs). 1–3 ALDHs are a group of NAD (P)-dependent enzymes involved in drug resistance and retinoic acid metabolism, which are crucial for the protection of stem cells against toxic endogenous and exogenous aldehydes. Recently, the isolation and functional analysis of ALDH1-positive (ALDH1þ) cells have been greatly facilitated by the development of the Aldefluor assay. 4 Matsui et al. 3 reported that ALDH1 activity is increased in MMSCs. 5 In the present study, we have addressed the question of whether ALDH1þ MM cells can initiate MM tumor formation and have evaluated the functional role of ALDH1 in MM cell growth, clonogenic ability and cell signaling. To determine whether MM cell lines contain an ALDH1 stem cell-like population, we evaluated ALDH1 activity using the Aldefluor assay in 11 human and one mouse MM cell lines. Flow cytometry demonstrated that all MM cell lines contained a very small subset of cells that were positive for ALDH1 (ALDH1þ) with a range from 0.1 to 4.85% of total cells (Figures 1a and b). Human MM cell lines (KMS12BM, XG6, U266 and ARP1) and the mouse MM cell line (5TGM1) had the highest fraction of ALDH1þ cells with 4.85%, 3.6%, 2.1%, 1.7%, and 1.9%, respectively (Figure 1b), whereas the remainder of the human MM cell lines (OPM1, JJN3, H929, KMS28, 8226, XG1, OCI-MY5 and OPM2) showed a very low percentage of ALDH1þ cells (o1. 5%; Figure 1b). We also analyzed ALDH1þ cells in primary MM samples by combing enzyme activity measurements with flow cytometric determinations of monotypic cytoplasmic immunoglobulin light-chain expression: k in one case and l in another. We found that the kþ tumor contained 0.69% ALDH1þ/kþ cells (Figure 1c, right panel), whereas the lþ tumor contained only 0.08% ALDH1þ/lþ cells (not shown). To determine whether ALDH1þ cells have increased clonogenic potential, the ALDH1þ fraction was selected from the ARP1 and KMS12BM cell lines. A total of 10000 ALDH1þ and ALDH1–cells per well were seeded in triplicates in a 12-well plate on methylcellulose. 6 After 2 weeks at 37 1C, the number of colonies from ALDH1þ and ALDH1–cells was compared. The colonyforming capacity of ALDH1þ cells in both ARP1 and KMS12BM cells showed greater colony formation capacity than ALDH–cells (5.68% vs 1.15% in APR1 and 2.97% vs 0.83% in KMS12BM, Figure 1d). Tumorigenicity of ALDH1þ MM cells was evaluated using an in vivo mouse model. A total of B10 000 ALDH1þ and 10 000 ALDH1–cells from ARP1 and KMS12BM cells were injected into the NOD. Cg-Rag1 mice (n ¼ 5 for each group), respectively. After 8 weeks, ARP1-ALDH1þ and KMS12BM-ALDH1þ cells showed significantly greater capacity of tumor formation compared with their ALDH1–cells correspondingly (Figures 1e and f). To explore ALDH1þ-related cell signaling, we performed gene expression profiling (GEP) analyses to identify ALDH1-associated transcriptional signatures from three myeloma cell lines. ALDH1þ cells were selected from XG6, KMS12BM and ARP1 by flow cytometry. GEPs were compared between ALDH1þ and ALDH1–cells using a paired Student’s t-test. A total of 20 genes identified were significantly differentially expressed between the two fractions, which had a P-value of o0. 001 and had greater than twofold change in absolute value. Of those, 17 genes were upregulated and three were downregulated in ALDH1þ cells. Interestingly, the largest functional category of the ALDH1-associated genes …