Cellular Ontogeny and Hierarchy Influence the Reprogramming Efficiency of Human B Cells into Induced Pluripotent Stem Cells

Abstract

Although B cells have been shown to be refractory to reprogramming into pluripotency, induced pluripotent stem cells (iPSCs) have been very recently generated, at very low efficiency, from human cord blood (CB)-and peripheral blood (PB)-derived CD191CD20 1 B cells using nonintegrative tetracistronic OSKM-expressing Sendai Virus (SeV). Here, we addressed whether cell ontogeny and hierarchy influence the reprogramming efficiency of the B-cell compartment. We demonstrate that human fetal liver (FL)-derived CD19 1 B cells are 110-fold easier to reprogram into iPSCs than those from CB/PB. Similarly, FL-derived CD341CD19 1 B progenitors are reprogrammed much easier than mature B cells (0.46% vs. 0.11%). All FL B-cell iPSCs carry complete VDJH rearrangements while 55% and 45% of the FL B-progenitor iPSCs carry incomplete and complete VDJH rearrangements, respectively, reflecting the reprogramming of developmentally different B progenitors (pro-B vs. pre-B) within a continuous differentiation process. Finally, our data suggest that successful B-cell reprogramming relies on active cell proliferation, and it is MYC-dependent as identical nonintegrative polycistronic SeV lacking MYC (OSKL or OSKLN) fail to reprogram B cells. The ability to efficiently reprogram human fetal primary B cells and B precursors offers an unprecedented opportunity for studying developmental B-lymphopoiesis and modeling B-cell malignances. STEM CELLS 2016;34:581-587 SIGNIFICANCE STATEMENTUsing tetracistronic OSKM-expressing nonintegrative Sendai vectors, we show that the developmental ontogeny and cellular hierarchy largely influences the reprogramming efficiency of human B cells into iPSCs. We also show that Ig/VDJH rearrangements do not constitute a reprogramming barrier and that B-cell reprogramming seems to rely on cell proliferation and it is cMYC-dependent. The ability to efficiently reprogram human fetal primary B cells and B precursors offers an unprecedented opportunity for studying developmental B-lymphopoiesis and modeling B-cell malignances.

Generation and characterization of a human iPSC cell line expressing inducible Cas9 in the “safe harbor” AAVS1 locus

“…iPSCs were generated using OKSM polycistronic SeV vector (Munoz-Lopez et al, 2016a, Munoz-Lopez et al, 2016b) and were fully characterized before and after gene edition as previously described (Bueno et al, 2016). …”

“…Human tissue was provided by The Vrelinghuis abortion clinic (Utrecht, The Netherlands) upon signed informed consent and approval by our local Ethics and Biozahard Board Committees (CMRB-CEIC-26/2013) through a formal collaboration with the Erasmus-Medical Centre, Rotterdam, The Netherlands). Mononuclear cells (MNCs) were isolated using Ficoll-Hypaque and CD34 + CD19 + B-cell progenitors (BCP) were FACS-purified and reprogrammed by infection with non-integrative tetracistronic SeV vectors encoding the transcription factors OCT4, SOX2, KLF4, and MYC (OSKM) (Munoz-Lopez et al, 2016a, Munoz-Lopez et al, 2016b). Resulting FL CD34 + CD19 + − iPSC lines (FL-BCP-hiPSC) were established as previously reported (Munoz-Lopez et al, 2016a, Munoz-Lopez et al, 2016b, Bueno et al, 2016).…”

“…Mononuclear cells (MNCs) were isolated using Ficoll-Hypaque and CD34 + CD19 + B-cell progenitors (BCP) were FACS-purified and reprogrammed by infection with non-integrative tetracistronic SeV vectors encoding the transcription factors OCT4, SOX2, KLF4, and MYC (OSKM) (Munoz-Lopez et al, 2016a, Munoz-Lopez et al, 2016b). Resulting FL CD34 + CD19 + − iPSC lines (FL-BCP-hiPSC) were established as previously reported (Munoz-Lopez et al, 2016a, Munoz-Lopez et al, 2016b, Bueno et al, 2016). This FL-BCP-hiPSC was genome-edited to harbor the Cas9 coding sequence controlled by a doxycycline-inducible cassette in the genomic “safe harbor” AAVS1 (iCas9-FL-BCP-hiPSC).…”

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

“…Because leukemia manifests as a developmental cell blockage, the generation and differentiation of leukemia-specific iPSCs offers a promising in vitro model to study the earliest events leading to the specification of both normal and malignant hematopoietic tissue, and to unravel the molecular and cellular mechanisms underlying the pathogenesis of human leukemia. iPSCs are easily generated from healthy and diseased, patient-specific, cells at different developmental stages (7). However, reprogramming human primary cancer cells remains challenging, and only a few reports have demonstrated successful reprogramming of malignant cells.…”

“…A stumbling block for advancing iPSC-based leukemia disease models is the extremely low reprogramming frequency of primary leukemic cells and cell lines (7), which prevents prospective genotype-phenotype studies comparing a large cohort of cancer-iPSCs from adult and pediatric human cancers carrying different genetic composites. Consistent with the developmental refractoriness of human B-cell ALL to reprogramming to pluripotency (7), the work by Chao et al reports the generation of AML-iPSCs from just two patients, further emphasizing the notion that reprogramming of primary leukemias is not a trivial task.…”