Even though HPB-AML-I was established from the PBMCs of an AML-M1 case , this cell line presents distinctive morphological features from AML. In terms of cell-surface antigen expression, multilineage differentiation, and CD3+ T-cell suppression, the characteristics of HPB-AML-I were found to be similar to those of MSCs. Our findings presented here suggest that HPB-AML-I may be a neoplastic cell line with MSC properties. Few reports have dealt with the establishment of human neoplastic MSC lines. A previous study established F6, a human neoplastic MSC line, from embryonic bone marrow MSCs. Transplantation of F6 cells into the SCID-nude mice resulted in fibrosarcoma formation and tissue metastasis [21, 24]. To the best of our knowledge, however, HPB-AML-I is the first neoplastic MSC line derived from a leukemic case.
The appearance of HPB-AML-I cells in suspension phase with their round-polygonal morphology intrigued us. We observed that an increase in the population of HPB-AML-I cells with such morphological patterns occurs in conjunction with the increased confluence of cultured cells. Morphological changes during culturing have previously been described in the case of bone marrow MSCs. Choi et al. reported that the morphology of bone marrow MSCs changed from small spindle-like in the first passage to large polygonal in the later passages. In contrast to many other adherent cell lines, HPB-AML-I cells with their round-polygonal morphology were viable and capable of proliferating and adhering to plastic surfaces following cell passage. Similar findings have been reported for the F6 cell line . While the exact mechanisms remain to be elucidated, we speculate that the loss of adherent capacity after confluent condition may be a pivotal property to neoplasms originated from mesenchymal stem cells.
Flow cytometric analysis of HPB-AML-I disclosed that, based on ISCT criteria, the cell-surface antigen expression patterns of this cell line were similar to those of human MSCs (reviewed by ) with positive CD73 and negative CD14, CD19, CD34, CD45 and HLA-DR expression. However, contrary to those criteria (reviewed by ), HPB-AML-I did not express CD90 and CD105. Absence of CD90 expression has also been observed in UCBTERT-21  and in human MSCs obtained from umbilical cord blood [15, 26]. MSCs lacking CD105 expression have been reported by Jiang et al. and Ishimura et al., who isolated MSCs from the subcutaneous adipose tissue, and by Lopez-Villar et al., who extracted MSCs from the bone marrow of a myelodysplastic syndrome case. These reports suggested that the absence of CD90 and CD105 expression in HPB-AML-I does not necessarily exclude the possibility that this cell line is derived from MSCs. The differentiation capability of MSCs with a negative CD105 expression has been investigated by Jiang et al. and Ishimura et al.. They found that this population of MSCs, while showing adipogenic differentiation, lacked chondrogenic and osteogenic differentiation. It is interesting that HPB-AML-I could differentiate into three lineages despite of CD105 negativity. In addition, a subpopulation of HPB-AML-I expressed CD45, even though most of HPB-AML-I cells were negative for CD45. Generally, CD45 is negative in MSCs, but CD45 expression has been detected in bone marrow MSCs from cases with multiple myeloma [30, 31]. It is therefore not surprising that neoplastic MSC line, such as HPB-AML-I, shows the aberrant expression of this antigen. Interestingly, CD45 expression in HPB-AML-I cells is likely to be transient, as the expression levels of CD45 increased in round-polygonal cells in the confluent cell culture and they decreased after passage of round-polygonal cells. Normal cells are known to have the property of contact inhibition, which is lost in transformed cells. Therefore, cell-to-cell contact might induce the aberrant expression of CD45 with an unknown reason in HPB-AML-I cells.
By using inverted microscopic examination and cytochemical staining, we demonstrated that HPB-AML-I cells are able to acquire the properties of adipocytes, chondrocytes, and osteocytes. The capability of MSCs to differentiate toward mesenchymal lineage cells reportedly correlates with their morphological and cell-surface antigen expression patterns. Chang et al. demonstrated that MSCs isolated from human umbilical cord blood consisted of cells with a flattened or spindle-like morphology and that the capability of differentiating toward adipocytes of the spindle-like MSCs was superior than that of the flattened cells. Since such heterogeneous morphology is shared by HPB-AML-I, further analyses are needed to characterize the difference between the round-polygonal and spindle-like cells.
As also reported by previous studies of the immunomodulatory effects on MSCs [18, 32], we demonstrated that HPB-AML-I cells are capable of suppressing CD3+ T-cell proliferation. Similar studies have been performed on MSCs isolated from cases with various hematopoietic neoplasms, such as ALL, Hodgkin's disease, non-Hodgkin's lymphoma, myelodysplastic syndrome, AML , and chronic myeloid leukemia (CML) . In contrast to our results, Zhi-Gang et al. reported that bone marrow MSCs isolated from AML cases did not inhibit the proliferation of CD3+ T-cells . These findings suggest that bone marrow MSCs from cases with hematopoietic neoplasms may or may not be capable of inhibiting CD3+ T-cell proliferation as a consequence of the secretion of humoral factors by neoplastic cells or the direct interaction with them. It is therefore very interesting that HPB-AML-I, regardless of its HSC or MSC origin, maintains the capability of inhibiting T-cell proliferation even after neoplastic transformation.
The cytogenetic analysis revealed the presence of complex chromosomal abnormalities in HPB-AML-I, although these were not the same as the frequently observed chromosomal alterations in AML cases. While it is not fully understood whether MSCs isolated from leukemic cases carry the cytogenetic characteristics common to leukemic cells, previous studies reported the absence of t(9;22)(q34;q11) chromosomal translocation or BCR-ABL rearrangement in bone marrow MSCs obtained from cases with Philadelphia (Ph) chromosome-positive CML [35, 36]. On the other hand, a recent study demonstrated the presence of leukemic reciprocal translocation and fusion gene expression in bone marrow MSCs of MLL-AF4-positive B-ALL cases . However, monoclonal Ig gene rearrangements, uncontrolled cell proliferation, diminished cell apoptosis, and cell-cycle arrest characteristic of leukemic cells were not observed in the bone marrow MSCs of those cases . Unfortunately, we could not obtain the karyotype of the original leukemic cells. Therefore, the complex karyotype in HPB-AML-I may not correspond to the cytogenetic status of the primary cells. It is possible that the complex karyotype of HPB-AML-I may include the additional genetic changes, which occurred in vitro during and after the establishment of the cell line. Nevertheless, the MSC-like properties of HPB-AML-I, as shown in this study, suggest the possibility that the first genetic event might have occurred at the stage of MSC.