We evaluated the characteristics of bone marrow stromal cells (BMSCs) and hematopoietic cells (HCs) from patients of myelodysplastic syndrome (MDS, n = 21) and acute myeloid leukemia (AML, n = 58), and compared the results with control BMSCs derived from healthy donors (n = 8). The patient BMSCs had lower proliferative activity than that of the controls due to increased senescence. This retarded proliferation induced failure to obtain enough metaphase cells for karyotyping in patient BMSCs (10%). Patient BMSCs were genetically altered which was demonstrated by chromosome abnormalities in 5% of the patients (one MDS and three AML), whereas no clonal abnormalities were detected in the controls. The most common abnormality of the BMSCs was an extra chromosome 5, followed by an extra chromosome 7 and balanced translocations. The proportion of the abnormal metaphase cells was low (17.8%). We also analyzed the epigenetic changes of long interspersed nucleotide element 1 (LINE-1) repetitive element and CDKN2B using pyrosequencing. The quantitative measurement of global LINE-1 methylation demonstrated that patient BMSCs revealed global hypomethylation (68.2 ± 3.8) compared with controls (72.9 ± 3.4, P < 0.001) and that the global hypomethylation of BMSCs were more significant in AML than in MDS patients (67.9 ± 3.8, 69.4 ± 4.2, respectively). These findings seem worthy of further evaluation of their association with ineffective hematopoiesis and leukemogenesis.