Abstract
B cell derived induced pluripotent stem cells (BiPSCs) were recently established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) and C/EBPα using a Sendai virus vector. Here, using a different method, we established BiPSCs with immunoglobulin heavy chain (IgH) gene rearrangement from normal B cells purified from lymph nodes. The critical points of our method are pre-stimulation of B cells with IL-21 and CD40-ligand (CD40L), followed by consecutive transfection of highly concentrated Yamanaka factors using a retroviral vector. Following each transfection the cells were centrifuged onto a retronectin coated plate and the activated by IL-4, IL-2, and CD40L. Furthermore, we established BiPSCs (BiPSC-A) in which activation-induced cytidine deaminase (AID) could be induced using the doxycycline-controlled. Both the parental BiPSC and BiPSC-A showed the capability of differentiating into hematopoietic progenitor cells (HPCs) based on confirmation of CD34 expression and colony-formation from CD34-positive cells. The findings that BiPSC-A can differentiate into HPCs suggest that there is a possibility that induction of AID expression would result in chromosomal translocations in the process of differentiation from BiPSCs, and therefore that these BiPSCs could be useful in elucidating the tumor origin of abnormal B cells in myelomagenesis.
Highlights
Somatic cells can be reprogrammed into induced pluripotent stem cells[1] by exogenous expression of reprogramming factors (Yamanaka factors) such as Oct[4], Sox[2], Klf[4] and c-Myc
The critical points of these processes are that, after pre-stimulation of the B cells with IL-21 and CD40L, three successive transfections and stimulations were performed, in each of which, after the transfection of highly concentrated Yamanaka factors into the B cells, the cells were centrifuged for 2 h and were stimulated with IL-4, IL-2, and CD40L for 22 h
We tried to establish induced pluripotent stem cells (iPSCs) from B cells purified from lymph nodes using a retroviral vector for the transfection of C/EBPα followed by transfection of Yamanaka factors; no colony formation indicating the generation of iPSCs was observed
Summary
Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs)[1] by exogenous expression of reprogramming factors (Yamanaka factors) such as Oct[4], Sox[2], Klf[4] and c-Myc. BiPSCs that had a B cell receptor (BCR) that was identical to that of B cells isolated from the chimeric mice were established by reactivation of Yamanaka factors together with either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-α (C/EBPα) or specific knockdown of the B cell transcription factor Pax[512]. BiPSCs were established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors and C/EBPα using a Sendai virus vector[15]. Since BiPSCs established from this mouse have IgH gene rearrangement, we subsequently established BiPSCs in which AID expression could be induced by using a doxycycline-controlled system (tet-off system), in order to determine whether those BiPSCs would produce B cells with chromosomal translocation, which is thought to be one origin of B cell malignancies
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