Generation of Induced Pluripotent Stem Cells From Primary Chronic Myelogenous Leukemia Patient Sample.

Induced pluripotent stem cells (iPSCs) can be generated from various cell types by the expression of defined transcription factors. In addition to the regenerative medicine, iPSCs have been used for the study of the pathogenesis of inherited genetic disease. Recently, it was reported that iPSCs were generated not only from normal tissue, but also from malignant cells. In those cases, cancer cells themselves must be the starting material from which iPSCs are derived. However, in almost all the cases, they used the established cell lines (chronic myelogenous leukemia (CML), gastrointestinal cancers, and melanoma) except for the JAK2-V617F mutation (+) polycythemia vera (PV) patient. In this study, we established the iPSCs from primary CML patient sample.

After obtaining informed consent, bone marrow cells from CML patient were reprogrammed by introducing the transcription factors Oct3/4, Sox2, KLF4, and c-myc. To improve the efficiency of the development of iPSCs, we added valproic acid (VPA), a histone deacetylase inhibitor, to the culture. Two CML derived iPSCs (CML-iPSCs) were generated. CML-iPSCs expressed the pluripotency markers such as SSEA-4 and Tra-1-60, and the endogenous expression of embryonic stem cell (ESC) characteristic transcripts (Oct3/4, Sox2, KLF4, Nanog, LIN28, REX1) was confirmed by RT-PCR. Oct4 and Nanog promoter regions were demetylated in the CML-iPSCs. Although CML-iPSCs expressed bcr-abl, they were resistant to the imatinib. Then we differentiated them into hematopoietic progenitors within the ‘unique sac-like structures’ (iPS-sacs). This method was reported to be able to produce the hematopoietic progenitors with higher efficiency than the usual embryoid body formation method using human ESCs (Takayama et al., Blood, 111, 5298–306, 2008). The hematopoietic progenitors showed the hematopoietic marker CD45 and immature marker CD34, and recovered the sensitivity to the imatinib, which recapitulated the feature of initial CML disease. Then we investigated the mechanism of the resistance to the imatinib in CML-iPSCs. The phosphorylation state of ERK1/2, AKT, and STAT5, which are the essential for the survival of bcr-abl (+) hematopoietic progenitors, were evaluated after imatinib treatment in CML-iPSCs. The phosphorylation of ERK1/2 and AKT, which were also essential for the maintenance of iPSCs, were unchanged after treatment, although STAT5 was not activated both before and after treatment. These results showed that the signaling for iPSCs maintenance compensated for the inhibition of bcr-abl in CML-iPSCs and that the oncogene addiction was lost in CML-iPSCs.

We generated the iPSCs from primary CML patient samples, re-differentiated them into hematopoietic lineage and showed the recapitulation of the features of initial disease. Primary samples of hematological malignancy are usually difficult to be expanded. However, if once they are reprogrammed to iPSCs, they can expand unlimitedly. As a result, we can obtain the genetically abnormal hematopoietic cells continuously by re-differentiating them into hematopoietic cells and use them for the studies which require the large number of living cells such as the analysis for leukemia stem cells or drug screening. Thus iPSCs technology would be useful for the study of hematological malignancy, especially for which animal model was not established such as myelodysplastic syndrome and be applicable for other cancers than hematological malignancies. We are now trying to establish the iPSCs derived from other hematological malignancies.

No relevant conflicts of interest to declare.