ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n . logical role in a range of hematologic diseases, such as inherited dyserythropoiesis, sideroblastic anemias and low-grade myelodysplastic symdromes. 8,12 In addition, transcriptome, epigenetic and proteomic studies in stem cell systems have indicated that specific metabolic/mitochondrial properties are essential for regulating the balance between self-renewal and differentiation. 13,14 Although recent work has begun to shed light on the mitochondrial response during murine stem cell differentiation, 7,10,15,16 how and to what extent the mitochondrial mass/function contributes to human hematopoietic stem and progenitor function remains poorly understood. Here, we found that mitochondrial mass correlates strongly with mitochondrial membrane potential (ΔΨm Design and Methods Cord blood collection and CD34 + cell isolation and cultureFresh umbilical cord blood units from healthy neonates were obtained from local hospitals following approval from our local Ethics and Biohazard Board Committee. The cord blood samples were pooled to reduce variability among individual units. Mononuclear cells were isolated using Ficoll-Hypaque and after lysing the red cells (Cytognos, Salamanca, Spain), CD34 + cells were purified by magnetic bead separation using the human CD34 MicroBead kit and the AutoMACS Pro separator (Miltenyi Biotec) as instructed by the manufacturer. [17][18][19] The purity of the CD34 + fraction was assessed by flow cytometry using an anti-CD34-PE antibody (Miltenyi Biotec), and only CD34 + fractions showing purity higher than 90% were used. [17][18][19] The CD34 -fraction was irradiated (15 Gy) and used as accessory cells for co-transplantation with CD34 + cells. MitoTracker staining and cell sortingCD34 + cells were stained with MitoTracker Red (CMXRos) and MitoTracker Green FM dye (Molecular Probes) for 10 min and 20 min, respectively, according to the manufacturer's guidelines and analyzed by wide confocal cytometry. 6 For functional assays, CD34 + cells were FACS-sorted (FACSAria-II, BD Biosciences) based on MitoTracker Green levels into CD34 + Mito High and CD34 + Mito Low (n=10). Measurements of ATP and reactive oxygen speciesATP levels were measured using a Cell-Titer-Glo ® Luminescent Cell Viability Assay (Promega) according to the manufacturer's guidelines. Briefly, equal numbers of cells (5x10 4 /100 mL) were seeded in a 96-well plate and 100 mL of the reaction reagent were added to each well. After 10 min of shaking, the luminescence signal was detected using the GloMax ® -Multi Detection System (Promega) and compared against the ATP Standard Curve using ATP disodium salt (Promega). 6 Reactive oxygen species were measured using the mitochondrial superoxide indicator MitoSOX Red, as previously described. 20 Briefly, CD34 + Mito Low and CD34 + Mito High cells were treated with 3 mM MitoSOX for 20 min and were then washed twice in Hanks balanced salt solution and analyzed by flow cytometry (Online Supplementary Figure S1A). Gene expression by q...

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.

SummaryInduced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.