Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPα

Abstract

B cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphoblastoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus. Co-expression of C/EBPα with OSKM facilitates iPSC generation from both CB- and PB-derived B cells. We also demonstrate that myeloid cells are much easier to reprogram than B and T lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B cells because biased reprogramming was avoided by using freshly isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B cells offers an unprecedented opportunity for studying developmental B lymphopoiesis and modeling B-cell malignancies.

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

“…Furthermore, these procedure were repeated three times. The reprogramming efficiency (iPSC clones/ infected cells 15 ) of our method appears to be close to the efficiency reported by Bueno. The purpose of establishing BiPSCs from normal B cells was to find or make an abnormal B cell that could be considered as an origin of B lymphoid tumors.…”

“…Recently, Stefano et al reported that the overexpression of Tet2 induced by the transfection of C/EBPα enhances Yamanaka factor-induced B-cell reprograming 14 . Subsequently, BiPSCs were established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors and C/EBPα using a Sendai virus vector 15 .…”

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

“…It is possible that, compared with the method of Bueno et al . who used a Sendai virus vector 15 , the transfection efficiency of genes using the retroviral vector of our study might be lower. Ultimately, we succeeded in the establishment of BiPSCs from B cells using Inaki’s method (unpublished method; supplemental Materials and Methods).…”

“…reported that the overexpression of Tet2 induced by the transfection of C/EBPα enhances Yamanaka factor-induced B-cell reprograming 14 . Subsequently, BiPSCs were established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors and C/EBPα using a Sendai virus vector 15 .…”

Human acute leukemia induced pluripotent stem cells: a unique model for investigating disease development and pathogenesis

“…Indeed, iPSCs from primary leukemic cells have been generated only from chronic hematological malignances, including Philadelphia+ CML, PMF, JAK2-V617F+ PV and JMML (8)(9)(10). The generation of iPSCs from acute myeloid (AML) or acute lymphoid leukemias (ALL) has not been reported to date, whereas iPSCs have been generated from normal human myeloid, T-cells and B-cells (11). Our laboratory has recently demonstrated that despite multiple in vitro and ex vivo combinations of reprogramming factors using diverse delivery strategies together with transcriptomic and epigenetic "booster" reprogramming strategies, neither primary B-ALL blasts nor B-ALL cell lines could be reprogrammed to pluripotency (12).…”