In view of these features, we further investigated the possibility of HPB-AML-I being a neoplasm of MSC origin. Recently, some human MSC lines have been established from the bone marrow [13, 14] and umbilical cord blood  cells of
healthy donors. To establish a stable cell line, genes encoding the human Linsitinib supplier telomerase reverse transcriptase (hTERT), bmi-1, E6, and E7 proteins were transduced to prolong the life span XMU-MP-1 of the healthy donor-originated MSCs [13–15]. However, there have been no reports of the establishment of MSC lines from human bone marrow cells without in vitro gene transduction. Since a number of the characteristics of HPB-AML-I are not typically observed in leukemic cells, we wondered whether HPB-AML-I cells are neoplastic cells originating from the non-hematopoietic compartments of bone marrow, such
as MSCs. Methods Cell lines and cell culture HPB-AML-I cells were kindly provided by Dr. K. Morikawa (Sagami Women’s University, Sagamihara, Japan) and 5 × 105 of these cells were cultured in 10 ml of RPMI-1640 medium supplemented with L-glutamine (Gibco, Carlsbad, CA), 10% fetal bovine serum (FBS) (Clontech, Mountain C59 wnt price View, CA), 50 U/ml of penicillin (Lonza, Walkersville, MD), and 50 μg/ml of streptomycin (Lonza). Cell culture was performed in a T-25 flask and was maintained in a 37°C incubator humidified with 5% CO2. Cell passage was performed twice a week. UCBTERT-21, the hTERT -transduced umbilical cord blood mesenchymal stem cell (MSC) line
, was obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan) and propagated in a T-75 flask in a total number of 1.5 × 105 cells. Cell culture was maintained in 15 GBA3 ml of Plusoid-M medium (Med Shirotori, Tokyo, Japan) containing 5 μg/ml of gentamicin (Gibco). The culture medium was replaced twice a week and cell passage was performed when the cultured cells reached 80-90% of confluence. Cytochemical analysis The following cytochemical staining was performed according to the manufacturer’s instructions: May Grünwald-Giemsa (Sysmex, Kobe, Japan), myeloperoxidase-Giemsa, toluidine blue, alkaline phosphatase-Safranin O (Muto, Tokyo, Japan), Sudan Black B-hematoxylin, oil red O-hematoxylin (Sigma-Aldrich, St. Louis, MO), and von Kossa-nuclear fast red (Diagnostic BioSystems, Pleasanton, CA). Cytogenetic analysis Cytogenetic analysis was performed according to the standard protocols. The karyotype was determined by G-banding using trypsin and Giemsa (GTG)  to examine 50 cells. The best metaphase was then photographed to determine the karyotype. The specimen was also submitted to spectral karyotyping (SKY)-fluorescence in situ hybridization (FISH) assay according to Ried’s method using whole chromosome painting (WCP) libraries (cytocell for WCP) and α-satellite DNA probes .