Role of mir-15a/16-1 in early B cell development in a mouse model of chronic lymphocytic leukemia

In both human chronic lymphocytic leukemia (CLL) and the New Zealand Black (NZB) murine model of CLL, decreased levels of microRNAs miR-15a/16 play an important role in the disease. Here we investigate the effects of this microRNA on early steps of B cell development and the capacity of miR-15a-deficient hematopoietic stem cells (HSC) and B1 progenitor cells (B1P) to reproduce CLL-like phenotype both in vitro and in vivo. Our results demonstrate that both miR-15a deficient HSC and B1P cells are capable of repopulating irradiated recipients and produce higher numbers of B1 cells than sources with normal miR-15a/16 levels. Furthermore, induced pluripotent stem (iPS) cells derived for the first time from NZB mice, provided insights into the B cell differentiation roadblock inherent in this strain. In addition, exogenously delivered miR-15a into the NZB derived B cell line provided valuable clues into novel targets such as Mmp10 and Mt2. Our data supports the hypothesis that miR-15a/16 deficient stem cells and B1Ps experience a maturation blockage, which contributes to B1 cells bias in development. This work will help understand the role of miR-15a in early events of CLL and points to B1P cells as potential cells of origin for this incurable disease.

HEK293 cell line was used for lentiviral production. This was maintained in DMEM supplemented with 10% FBS, 1% sodium pyruvate, 1% penicillin-streptomycin at 37°C and 5% CO2. For in vitro differentiation of primitive hematopoietic stem cells with the phenotype (lineage -, Sca1 + c-Kit + ) or LSK progenitors, the OP9 (ATCC® CRL-2749) stromal cell line was used. Alpha Minimum Essential Medium without ribonucleosides and deoxyribonucleosides and with 2.2 g/L sodium bicarbonate with fetal bovine serum to a final concentration of 20% was used for OP9 system co-culture. NZB ES cells were a kind gift from Dr. Ken-Ichi Yagami Mice: NZB/BlNJ (stock #000684) and DBA/2J (wild type control strain; stock #000671) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and housed under standard pathogen-free conditions at the research animal facility at Rutgers University-New Jersey Medical School, Newark, NJ, USA. DBA congenic strain bearing NZB mir-15a/16-1 loci (DBA -/-) were generated by systematic back-crossing as described previously 20,43 . The immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice were purchased from Jackson Laboratory (www.jax.org) and housed under standard pathogen free conditions. Mouse femurs and tibias were flushed to harvest cells from the bone marrow as previously described 44 . Twenty thousand HCS or B1P were sorted from the bone marrow source and used for retro-orbital intravenous adoptive transfer experiments (in 200 uL of sterile PBS). NSG recipients were sub-lethally irradiated by 275 Gr prior to injection of sorted cells. Mice were sacrificed 48 days post injection followed by harvesting of cells and flow cytometry analysis. All studies were in compliance with principles of laboratory animal care guidelines and were IACUC approved.

iPS cells generation and lentiviral constructs:
For iPS production, the packaging (psPAX2, Addgene #12260) and envelope (pMD2.G, Addgene, #12259) plasmids along with polycistronic lentiviral construct (pKP332, Addgene #21627) were mixed in 4:3.5:7.5 ratio and transfected using Lipofectamine-2000 (Invitrogen, Carlsbad, CA) following manufacturer's instructions into 293T. The pseudoviral particles were concentrated from supernatant by centrifugation at 26,000 rpm for 90 minutes at 8°C in an SW-28 rotor using a Beckman XL-100 ultracentrifuge and viral titer was confirmed using LentiX GoStix assay (Clontech). For iPS cell induction, 3 × 10 5 NZB mouse splenic stromal cells (NSF) were seeded onto one well of a six-well plate. The next day, 2.5 uL of the concentrated virus was mixed with 2 ml of ES cell medium containing 8 μg/ml polybrene and added to the target cells. Forty-eight hours later, the NSFs were trypsinized and transferred to a 100-mm dish without MEFs and continuously cultured on the same dish for 3 weeks with daily media changes. ES media with LIF and differentiation inhibitors (2i) was used.
Potential iPS cell colonies started to appear after 2-3 weeks. These colonies were individually picked and expanded on MEFs for analysis and subsequent experiment. One million iPS cells in 100 uL of PBS were injected via a 21-G needle into the dorsal flanks of immunodeficient (NOD/SCID) mice subcutaneously. Teratomas were recovered 4-5 weeks post-injection and processed for histological H&E staining and analysis. Photographs were taken and analyzed by an expert pathologist for the presence of the tissues derived from three different germinal layers ectoderm, mesoderm and endoderm. For alkaline phosphatase staining, 100 to 200 iPS cells were seeded onto one well of a six-well plate with pre-seeded mouse embryonic fibroblasts (MEF) and cultured for one week with daily ES media changes. iPS cells were then stained using the Vector Blue Alkaline Phosphatase Substrate Kit III according to the manufacturer's instructions.
For LNC transduction, the miR-15a-lenti-GFP (SBI, Mountain View, CA) lentiviral aliquot was thawed on ice and added dropwise to the pre-seeded (24 h) culture plates (MOI 10). Polybrene was added at a final concentration of 8 ug/mL to increase transduction efficiency. The plates were incubated at 32°C for 24 hours. Lenti-GFP lentivirus was used as a negative control. For NZB cell line transduction LNC cells were plated 24 hours before at a concentration of 0.3x10 6 /mL in a 6 well plate. Concentrated lentiviral aliquot was thawed on ice and added dropwise to the culture plates (MOI 10). Polybrene was added at a final concentration of 8 ug/mL to increase transduction efficiency. The plates were incubated at 32°C for 24 hours instead of 37°C to assure the virus stability. The transduction efficiency was tested qualitatively using fluorescence microscopy followed by quantitative assessment by flow cytometry. GFP lentivirus (with no miR-15a/16-1) was used as a negative control for miR-15a overexpression.
Quantitative PCR: Total RNA was isolated from cells with Trizol reagent (Invitrogen). RNA was pretreated with RQ1 RNase-free DNase (Promega, Madison, WI) and reverse transcribed with SuperScript First-Strand Synthesis System (Invitrogen) using oligo d(T)n. Gene expression levels were measured using Power SYBR Green Kit (Ambion Inc., Austin, TX, USA) or SensiMix SYBR Low-ROX Kit (Bioline, London, UK) and custom designed primers (Supplemental Table   2). MicroRNA-specific cDNA was prepared using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. The qPCR reaction was performed on 7500 Real-Time PCR System for 40 cycles at 60°C. The standard 2 -∆∆CT method was used for relative quantitation of microRNA levels using AB7500 software (v. 2.0.6, Applied Biosystems). The following pre-made TaqMan Assays (Life Technologies) were used for realtime quantitation-mmu-miR-15a (assay ID 000389), mmu-miR-150 (assay ID002637) and U6 (Assay ID001973, housekeeping control).
RNA FISH: Dleu2, PU.1, IL10 and Pax5 RNAs were imaged using single-molecule RNA FISH custom probes as previously described 22 . Briefly, a set of 35 probes was designed to hybridize to each target RNA and was synthesized with a 3′ amino modification from Biosearch Technologies (Novato, CA, USA). The individual probes for a given target were pooled in equimolar amounts and then coupled with succinimidyl ester of TMR (for Dleu2 and IL10), Alexa594 (for Pax5) or Cy5 (for PU.1). The coupled fraction was purified using highperformance liquid chromatography and the concentration was determined using Nanodrop.
The coverslips were washed with 1 × phosphate-buffered saline, fixed in 4% formaldehyde, permeabilized with 70% ethanol and hybridized with the chosen probes. Hybridization was carried out overnight at 37 °C. The coverslips were washed (with 10% formamide in 2 × SSC) to remove unbound probes and imaged using Zeiss wide field fluorescence microscope (Carl Zeiss, Thornwood, NY, USA). For each image, z-stacks were obtained and merged to get the final image. The image acquisition was carried out by Openlab software (Perkin-Elmer, Waltham, MA, USA) and numbers of mRNAs were counted using custom written algorithms in MATLAB (MathWorks, Natick, MA, USA). LNC cell line was analyzed using individual probes separately whereas primary splenocytes were probed for three target mRNAs simultaneously to count Pax5 + B cells only.

In vitro Differentiation: OP9 cells were used for iPS or ES in vitro differentiation according to
published protocols with slight modifications 23 . To initiate co-culture, ES or iPS cells were harvested as a single cell suspension by trypsin-mediated disaggregation and added to OP9 monolayer, with changes of media with culture. The non-adherent cells were reseeded on fresh OP9 and 5 ng/mL of recombinant human Flt-3L was added and cultured with media changes.
The non-adherent cells were seeded in 3 mL per well of a 6-well plate containing OP9 cells to generate B-lineage and myelo-erythroid cells. The cytokines were added to each well at final concentrations of 5 ng/mL for Flt-3L and 1 ng/mL for IL7. On day 12 the cells were replated on fresh OP9 with 5 ng/mL Flt-3L and 1 ng/mL IL-7 and the media changed at interval. At day 38 lymphocyte-like non-adherent cells were harvested and analyzed by flow cytometry.
2 × 10 4 sorted LSK cells were plated with OP9 cells with 5 ng/mL Flt-3L and 1 ng/mL IL-7 and feed and reseeded. At day 11 the cells were analyzed by flow cytometry using B cell specific antibodies.     (G) Additional teratoma formation assays on NZB iPS cells. 2x10 6 NZB iPS #1-1 cells were injected into dorsal flanks of NOD/SCID mice subcutaneously. Four to five weeks later the mice were sacrificed and teratomas were harvested, minced and processed for H&E staining. Tissues derived from ectoderm (keratinized epithelium), mesoderm (striated muscles) and endoderm (ciliated epithelium) are shown (indicated by arrows).   (Mt2) following exogenous delivery of miR-15a; q<0.05 (FDR adjusted p value) was considered significant. (C) Western blot analysis of LNC cell line with and without transduction with lentiviral miR-15a construct. The A20 cell line is a control non-NZB B cell line. β-actin was used as a housekeeping control.