Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells

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.

Figure 1

Cell surface antigen analysis by two-color flow cytometry. (a) Phenotype analysis of lymphocytes of the lymph node by flow cytometric analysis with a combination of anti-CD3/-CD20 and anti-CD27/-CD20 antibodies. B cells are surrounded by a dotted line. (b) Phenotype analysis of B cells purified using CD19-microbeads by flow cytomeric analysis with a combination of anti-CD3/-CD20 antibodies. Purified B cells are surrounded by a dotted line.

Figure 2

Generation of B cell derived iPSC (BiPSC). (a) Time schedule and protocol of BiPSC generation. (b) Colony formation of a representative BiPSC13 (x200). (c) Teratoma derived from human BiPSC13 (x4). Nine weeks after the injection of BiPSC13, teratomas were dissected from bulging lower abdomen. Hematoxylin and eosin staining of the teratoma. (A) Nerve tissue (ectoderm), (B) Cartilage tissue (mesoderm), (C) Bone tissue (mesoderm), (D) Connective tissue (mesoderm) and endodermal epithelial tissues with a lumen structure (Arrow head), and ectodermal epithelial tissue consisting of ependymal cell-like cells (Double arrow heads). (d) Monoclonal VDJ rearrangements of the IgH gene (Arrows) in BiPSC13 detected using PCR. (e) Normal G-banding karyotype of BiPSC13.

Figure 3

Characterization of the BiPSCs. (a) Immunofluorescence staining of BiPSC13 and MIB2-6 for expression of the pluripotent markers Oct3/4, Nanog, SSEA4, TRA-1-60, and TRA-1-81. (b) Expression of endogenous Oct3/4, Sox2, Klf4, cMyc, Pax5, AID, and GAPDH in BiPSCs (BiPSC13, MIB2-6) and normal B cells (CD19) from the lymph node analyzed using RT-PCR. (c) RT-PCR analysis of the expression of retrovirus-derived Oct3/4, Sox2, Klf4, and cMyc in BiPSCs (BiPSC13, MIB2-6). HUC-Fm, human umbilical cord fibroblast cells, (male; RIKEN, Tsukuba, Japan) infected with a retrovirus containing Oct3/4, Sox2, Klf4, c-Myc, and GAPDH for 5 days were used as the positive control.

Figure 4

Hematopoietic progenitor cells differentiation of BiPSCs. (a) Flow cytometric analysis of the cell phenotype after differentiation of BiPSCs into HPCs. (A) BiPSC13, (B) MIB2-6. The population of CD34-positive cells is surrounded by a dotted line. (b) Representative morphology (x1000) and Wright stain of BiPSC13 (A) and MIB2-6 (B) cells before and after differentiation into CD34⁺ cells. (c) Morphology of formed colonies (x50) and Wright staining of cytospins picked up from a colony (x1000). (A) BiPSC13, (B) MIB2-6.

Figure 5

AID expression in BiPSCs induced with the doxycycline-controlled (Tet-off) system. (a) Induction of AID expression in the absence of doxycycline was confirmed in two clones (#1 and #2) derived from BiPSC13 by western blotting, and (b) qRT-PCR. In (b), the numbers on the Y axis are the relative ratio comparing the expression of AID mRNA of each sample standardized by the expression of GAPDH mRNA with that of CD19⁺ normal B cells purified from normal LN using CD19-microbeads. Raji, Burkitt lymphoma cell line. Data were analyzed in triplicates and normalized to glyceraldehyde 3-phosphate dehydrogenase. (c) Immunofluorescence analysis of the expression of Oct3/4 and Nanog in BiPSC13-AID (#1 and #2) after the induction of AID expression in the absence of doxycycline. (d) Induction of AID expression in the absence of doxycycline in two clones (#16 and #17) derived from MIB2-6 by western blotting, and (e) qRT-PCR as described in the legend to (b). (f) Immunofluorescence analysis of the expression of Oct3/4 and Nanog in MIB-AID (#16 and #17) after the induction of AID expression in the absence of doxycycline.

Figure 6

Hematopoietic differentiation from BiPSC13 with AID expression induced using the doxycycline-controlled (Tet-off) system. (a) Flow cytometric analysis of the cell phenotype after differentiation of BiPSC13#1-AID into hematopoietic progenitors. The population of CD34-positive cells is surrounded by a dotted line. (A) BiPSC13#1-AID were cultured in the presence of doxycycline, (B) Originally, BiPSC13#1-AID were cultured in the presence of doxycycline to inhibit expression of AID, and subsequently, doxycycline was withdrawn 10 days before sorting of the CD34-positive cells, and (C) BiPSC13#1-AID were cultured in the absence of doxycycline to express AID constitutively. (b) Immunofluorescence analysis of the expression of AID in the sorted CD34-positive cells. AID expressed cells were detected in (B) and (C) partially. Arrow heads and arrows indicate AID-positive or –negative cells, respectively. Culture condition of (A), (B), and (C) are as described in (a). (c) Wright staining of cytospins picked up from a colony (x400). Culture condition of (A), (B), and (C) are as described in (a).

Figure 7

Abnormal B cells as the origin of myeloma cells (hypothesis). When expression of Yamanaka factors is induced for any reason in mature B cells, the cells are reprogrammed and B cell derived iPS cells (BiPSCs) are established (a). During the process of redifferentiation of these BiPSCs into hematopoietic stem cells and further into B cells, cleavages occur on chromosome 14 and another chromosome due to AID expression, resulting in the formation of abnormal B cells (a) with reciprocal translocation of these chromosomes (b). One of the alleles of the IgH gene of these B cells is a productive (functional) allele that had performed VDJ rearrangement as well as class switch recombination and produces the so-called M protein. The other allele, however, is a nonproductive (nonfunctional) allele (c), in which case binding to oncogenes, etc. (b) due to reciprocal translocation with another chromosome following cleavage between the J and C segments due to AID expression results in myelomagenesis.