Pluripotent lines and viral infections
Mouse embryonic fibroblasts (MEFs) used to derive primary induced pluripotent stem cell (iPSC) lines by infections with inducible lentiviruses were harvested at 13.5 days post coitum from F1 mating between ROSA26-M2rtTA and Nanog-GFP mice9
. Lentiviral preparation and infection with Doxycycline (DOX)-inducible lentiviruses encoding Oct4, Klf4, c-Myc and Sox2 cDNAs driven by the TetO/CMV promoter, were previously described21
. The NGFP1 iPSC line used in this study was derived after culturing the infected MEFs with DOX, and grew stably in culture independent of DOX. To generate NGFP1-p53KD and p21KD clonal cell lines, 50,000 NGFP1 cells were infected with the pSicoR-PGK-puro vector encoding a hairpin for mouse p21
(targeting sequence: GCAGATTGGTCTTCTGCAA) and a previously described specific hairpin for the mouse p53
tumor suppressor gene28
. Hairpin against the CD8 sequence was used as control where indicated. 10 µg of lentiviral vector and packaging vectors were co-transfected in 293T cells by using the FuGENE 6 reagent (Roche Diagnostics). Supernatants were collected 36–48 hours after transfection, filtered through a 0.45-µm filter, and used to infect the iPSCs for 48 hours. Afterwards, the cells were trypsinized, plated at low densities and subcloned to test for knockdown specificity (NGFP1 already contains a puromycin selection cassette in the ROSA26 locus restricting our ability to use puromycin as a marker for infected cells). To verify integration of PsicoR lentiviral vectors the following oligos were used: Forward, CCCGGTTAATTTGCATATAATATTTC; and Reverse, CATGATACAAAGGCATTAAAGCAG. cDNAs encoding Nanog and Lin28 transcripts where cloned into EcoRI site of TetO-FUW lentiviral vectors and were used to infect NGFP1 iPSC. Integration for TetO-Nanog and TetO-Lin28 lentiviral vectors was verified by PCR and southern analysis on genomic DNA using the EcoRI digested insert as a probe (data not shown). A NGFP1 subclone that demonstrated >95% constitutive knockdown of p53
) or p21
genes products, termed NGFP1-p53KD or NGFP1-p21KD, were used in the subsequent experiments. iPSCs were cultured in DME containing 15% fetal calf serum (FCS), leukemia inhibiting factor (LIF), penicillin/streptomycin, L-glutamine, beta-mercaptoethanol and nonessential amino acids. Chromosomal karyotyping of iPSC lines was performed by Cell Line Genetics on 20 G-banded metaphase cells from each line tested.
Chimera and teratoma formation
iPS cells were injected in C57B6 X 129Sv F1 Rag2−/− or BDF2 diploid blastocysts that were initially harvested 94–98 hours after hormone injection and placed in a drop of DMEM with 15% FCS under mineral oil. A flat-tip microinjection pipette with an internal diameter of 1.2–1.5 mm was used for iPSC injection (using a Piezo micromanipulator). A controlled number of cells was injected into the blastocyst cavity. After injection, blastocysts were returned to KSOM media (Invitrogen) and placed at 37°C until transferred to recipient females. Ten to fifteen injected blastocysts were transferred to each uterine horn of 2.5 days post coitum pseudo-pregnant females. To recover full-term pups, recipient mothers were sacrificed at 19.5 days post coitum. For teratoma generation, 2×106 iPSCs were injected subcutaneously into both flanks of recipient SCID mice, and tumors were harvested for sectioning 3–6 weeks after initial injection.
Reprogramming into iPSCs
Transgenic Pre-BCR+ (Igµ+ IgK− IgL−) early Pre-B cells, which are not generated in Rag2−/−
mice due to their inability to undergo heavy and light chain rearrangements, were isolated from bone marrow of 3–5 week old Rag2−/−
NGFP1 chimeras and single cell sorted into 96 well plates. This choice of host ensured that any isolated B cells were derived from the injected iPSCs, and not from the host blastocysts. In certain experiments, we labeled the NGFP1-iPS cell clone with a lentivirus constitutively expressing dTomato (a kind gift from K. Hochedlinger) and injected cells into BDF2 blastocysts to produce chimeric mice, and utilized the dTomato as a marker of transgenic cells. One of the experimental replicates was performed on B220+CD25+ Pro-B cells isolated from reprogrammable mice carry the same set of transgenes11
. Cells were plated in gelatinized and irradiated (3000 Rad) OP9 coated wells in ES medium containing DOX (4 µg/ml). IL-7 and SCF (10 ng/ml each, Peprotech) were added to the medium for the first 1–2 weeks to optimize cloning efficiency in the presence of DOX (the plating efficiency of Pre-B cells to grow as single cells was > 80%). Cells on DOX did not exhibit contact inhibition and grew both on gelatin and in suspension. After 2 weeks of DOX induction, cultures were trypsinized every week and passaged on gelatinized plates in ES medium + DOX. Populations were defined as being positive for iPSC generation when >0.5% of the adherent/semi-adherent screened cells were GFP+. Upon detection of a GFP+ fraction, further flow cytometric (FACS) assaying of the clonal population was discontinued and, in all cases, stable DOX independent iPSC lines were derived by growing the cells in the absence of DOX (which allows the iPSCs to overgrow transgene-dependent, partially reprogrammed GFP- cell in the population). A fraction of ~250,000 cells, including a quarter of all fully-adherent cells, was replated at the end of each week (beginning at week 2) for further follow-up analysis. By including adherent cells in the culture plate upon passaging, we consistently retained all iPSCs generated during a given culture period, as no iPSCs were detected by FACS in the non-adherent fraction (Supplementary Fig. 1
). NGFP1-p53KD and NGFP1-p21KD derived cells were passaged and replated twice a week (versus once a week at the time of GFP assays for other cells) to avoid over-confluence in the culture wells due to their higher proliferation rate. Population-averaged doubling time for reprogramming and iPSC populations was determined by plating 105
cells, and counting total cell number after 24 and 48 hours in duplicates. Numbers of cells as a function of time were fit using exponential growth regression in Excel (R2
ranging from 0.97 to 1.00). The CD11b+ myeloid fraction was sorted from spleen and cells were plated in gelatinized and irradiated OP9 coated 96 wells with ES medium supplemented with DOX (4 µg/ml). M-CSF, Flt3L, LPS and SCF (10 ng/ml, Peprotech) was added to the medium for the first 2 weeks to boost plating efficiency. Wild-type CD11b+ cells were obtained from “reprogrammable” transgenic mice carrying identical DOX inducible copies of the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, Rosa26-M2rtTA and a Nanog-GFP knock-in reporter. Unlike iPSC-chimeras, all cells in these “reprogrammable” mice carrying the same set of transgenes11
. However, unlike for Pre-B cells which are not found in Rag2−/−
hosts and thus all isolated cells were of transgenic origin, CD11b+ cells were isolated from reprogrammable” mice carry the same set of transgenes11
or from dTomato labeled chimeric donor mice. V(D)J-IgH rearrangements were amplified from genomic DNA samples by PCR using degenerate primer sets as described9
Cells were fixed in 4% paraformaldehyde for 20 minutes at 25 °C, washed 3 times with PBS and blocked for 15 min with 5% FBS in PBS containing 0.1% Triton-X. After incubation with primary antibodies against Oct4 (Santa Cruz), Nanog (polyclonal rabbit, Bethyl) and SSEA1 (monoclonal mouse, Developmental Studies Hybridoma Bank) for 1 h in 1% FBS in PBS containing 0.1% Triton-X, cells were washed 3 times with PBS and incubated with fluorophore-labeled appropriate secondary antibodies purchased from Jackson Immunoresearch. Specimens were analyzed on an Olympus Fluorescence microscope, and images were acquired with a Zeiss Axiocam camera.
Total RNA was isolated using Rneasy Kit (Qiagen). Three micrograms of total RNA was treated with DNase I to remove potential contamination of genomic DNA using a DNA Free RNA kit (Zymo Research, Orange, CA). One microgram of DNase I-treated RNA was reverse transcribed using a First Strand Synthesis kit (Invitrogen) and ultimately resuspended in 100 ul of water. Quantitative PCR analysis was performed in triplicate using 1/50 of the reverse transcription reaction in an ABI Prism 7000 (Applied Biosystems, Foster City, CA) with Platinum SYBR green qPCR SuperMix-UDG with ROX (Invitrogen). Error bars indicate standard deviation of triplicate measurements for each clone. Primers used for transgene encoded amplification were as follows: c-Myc: Forward, 5’-ACCTAACTCGAGGAGGAGCTGG-3’ and Reverse, 5’-TCCACATAGCGTAAAAGGAGC-3’; Klf4: Forward, 5’-ACACTGTCTTCCCACGAGGG-3’ and Reverse, 5’-GGCATTAAAGCAGCGTATCCA-3’; Sox2: Forward, 5’-CATTAACGGCACACTGCCC-3’ and Reverse, 5’-GGCATTAAAGCAGCGTATCCA-3’; Oct4: Forward, 5’-AGCCTGGCCTGTCTGTCACTC-3’ and Reverse, 5’-GGCATTAAAGCAGCGTATCCA-3’. Nanog: Forward 5’-ACATGCAACCTGAAGACGTG-3’ and Reverse; 5’-CACATAGCGTAAAAGGAGCAA-3’. To ensure equal loading of cDNA into RT reactions, GAPDH mRNA was amplified using the following primers: Forward, 5’-TTCACCACCATGGAGAAGGC-3’; and Reverse, 5’-CCCTTTTGGCTCCACCCT-3’. Data were extracted from the linear range of amplification.
Antibodies and apoptosis measurement assays
Fluorescently conjugated antibodies (PE, FITC, Cy-Chrome or APC labeled) were used for flow cytometric analysis, and for cell sorting: anti-CD11b+, Pre-BCR, IgK, IgL, CD19, B220, CD45.2 and IL7R antibodies (BD-Biosciences) were used. Enrichment for CD11bhigh cells by using CD11b magnetic bead isolation kit was use prior to sorting. Cell sorting was performed by using FACS-Aria (BD-Biosciences), and consistently achieved cell sorting purity of >99%. Antibodies for western blot analysis: anti-mouse p53 (BAF1355, R&D systems), anti-β actin (ab8226, Abcam) and anti-p21cip (clone C-19, Santa Cruz Biotechnology). To determine the rate of apoptosis, samples were stained with annexin V staining kit (BD Biosciences) and propidium iodide (PI) according to the manufacturer's recommendations and analyzed by flow cytometer. In addition, samples were subjected to the Tdt-mediated dUTP-biotin nick end labeling (TUNEL) test for apoptotic cells by using flow cytometry based MEBSTAIN apoptosis kit (MBL).
Non-parametric statistical analysis
The latency of iPSC appearance of each well was used to generate a survival curve for each experiment. Latency was considered either as a function of absolute time (i.e., the weeks on DOX until GFP was detected) or as a function of the population-averaged number of cell divisions during latency (i.e., latency in time divided by doubling time). Any wells that failed to generate Nanog-GFP+ cells at the end of each experiment were considered to be censored, and are shown with solid geometric shapes in –. Histograms of the fraction of cells at initial GFP detection were performed using Prism 5 (version 5.0b; Graphpad Software, Inc.). Using the censored latency data sets, the logrank (Mantel-Cox) test, a nonparametric statistical test appropriate for right-censored data, was used to test the null-hypothesis that survival functions do not differ across groups. In contrast to parametric analyses, this test does not require knowledge about the shape of the survival curve or the distribution of survival times. Analysis was performed using Prism 5 (version 5.0b; Graphpad Software, Inc.), and two-tailed p-values. p-values above 0.05 indicate that the latencies between the two groups were similar (i.e., the treatment did not change survival; accept the null hypothesis at a 95% confidence level), while p-values less than 0.05 indicate that latencies between groups were not similar (i.e., survival functions differ; reject the null hypothesis at a 95% confidence level).
Parametric statistical analysis
The censored latency data sets were also fit to several univariate probability distributions using maximum likelihood estimation via the ‘dfitool’ in Matlab (The Mathworks, Inc.). For optimal univariate distribution fits, the chi-squared goodness-of-fit statistic, χ2
, was used to assess the quality of each fit. Gamma distributions had the lowest χ2
of any fit distribution (see Supplementary Fig. 12
Simulations of the proposed model were generated using a hybrid scheme in which the size of each subpopulation (either B-cells or iPSC) was evolved by considering (a) stochastic Gillespie-like evolution of Poissonian growth dynamics for small population sizes, (b) deterministic evolution of population sizes using a time step of Δt = 0.001 weeks whenever the probability of generating a new cell in the time Δt exceeded 0.1, (c) Gillespie-like evolution of the reprogramming transition, (d) periodic replating, selection of iPSCs and observation of the fraction of reprogrammed cells according to the details of the experimental protocol followed in each experiment.