News Article | May 24, 2017
A step-by step protocol describing the HSPC conversion of human PSCs can be found at Protocol Exchange48. All experiments were performed with H9 hESC (NIHhESC-10-0062), PB34 iPS49, MSC-iPS150, 1045-iPSC, and 1157-iPSC established by the hES Core Facility at Boston Children’s Hospital. Human ESCs and iPSCs were maintained on hESC-qualified Matrigel (BD) in mTeSR1 media (Stem Cell Technologies) or mouse embryonic fibroblasts (GlobalStem) feeders in DMEM/F12 + 20% KnockOutSerum Replacement (Invitrogen), 1 mM l-glutamine, 1 mM NEAA, 0.1 mM β-mercaptoethanol, and 10 ng ml−1 bFGF on 10 cm gelatinized culture dishes. Medium was changed daily, and cells were passaged 1:4 onto fresh feeders every 7 days using standard clump passaging with dispase. Morphology of PSCs was checked by microscopy daily. As a quality control, only dishes with more than 70% of typical PSC colonies were processed for embryoid body formation. Cell lines were tested for mycoplasma routinely. Embryoid body differentiation was performed as previously described19. Briefly, hPSC colonies were dissociated with 0.05% trypsin for 5 min at 37 °C, pipetted thoroughly with p1000 to form small aggregates, washed twice with PBS + 2% FBS, and resuspended in StemPro-34 (Invitrogen, 10639-011) supplemented with l-glutamine (2 mM), penicillin/streptomycin (10 ng ml−1), ascorbic acid (1 mM), human holo-Transferrin (150 μg ml−1, Sigma T0665), monothioglycerol (MTG, 0.4 mM) (referred to as ‘supplemented StemPro-34’), BMP4 (10 ng ml−1), and Y-27632 (10 μM). Cells were then seeded into non-adherent spheroid formation 10 cm plates (Ezsphere, Asahi Glass; well size diameter 400–500 μm, depth 100–200 μm; number of wells 14,000 per dish) at a density of 5 million per dish. Twenty-four hours later, bFGF (5 ng ml−1) and BMP4 (10 ng ml−1) were added to the medium. On day 2, the developing embryoid bodies were collected and resuspended in supplemented StemPro-34 with SB431542 (6 μM), CHIR99021 (3 μM), bFGF (5 ng ml−1), and BMP4 (10 ng ml−1). The formation of embryoid bodies was checked by microscopy on day 4 and the decision was made to continue embryoid body formation on the basis of the size and morphology of aggregations (quality control; >100 μM, compaction-like tight contact of cells). On day 4, medium was replaced by supplemented StemPro-34 with VEGF (15 ng ml−1) and bFGF (10 ng ml−1). At day 6, medium was replaced by supplemented StemPro-34 with bFGF (10 ng ml−1), VEGF (15 ng ml−1), interleukin (IL)-6 (10 ng ml−1), IGF-1 (25 ng ml−1), IL-11 (5 ng ml−1), and SCF (50 ng ml−1). Cultures were maintained in a 5% CO /5% O /90% N environment. All recombinant factors were human and purchased from Peprotech. To avoid potential damage resulting from hydrodynamic pressure and contamination through fluorescence-activated cell sorting (FACS), for functional assay, isolation of haemogenic endothelium was performed by magnetic cell isolation. Freshly dissociated embryoid body cells (at the day 8 time point) by 0.05% trypsin were filtered through a 70 μm filter and stained with CD34 microbeads (Miltenyi) for 30 min at 4 °C. CD34+ cells were isolated with LS columns (Miltenyi). Around 0.3 × 105 to 1.0 × 105 cells were obtained per 10 cm dish of embryoid body formation. A sample from each batch was analysed by FACS to validate its purity of haemogenic endothelium with the panel CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), CD43 PE (1G10; BD), and 4′,6-diamidino-2-phenylindole (DAPI). For expression profiling by microarray and qRT–PCR, isolation of haemogenic endothelium was performed by FACS. Dissociated embryoid bodies (at the day 8 time point) were resuspended at 1 × 106 to 3 × 106 per 100 μl of staining buffer (PBS + 2% FBS). Cells were stained with a 1:50 dilution of CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), CD43 PE (1G10; BD), and DAPI for 30 min at 4 °C in the dark. All FACS sorting was performed on a BD FACS Aria II cell sorter using an 85 μm nozzle to avoid potential damage to haemogenic endothelium. All the samples used for microarray analysis were FACS-sorted. Haemogenic endothelium panel: CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), and CD43 PE (1G10; BD). Fetal-liver HSCs were purchased from StemCell Technologies and stained with HSC panel: CD38 PE-Cy5 (LS198-4-3; Clontech), CD34 PE-Cy7 (8G12; BD), and CD45 PE (HI30; BD). Between 10,000 and 50,000 cells were sorted for each cell type with two or three biological replicates. An RNAeasy Microkit (Qiagen) was used to collect and prepare total RNA for microarray analysis. The Ovation Picokit (Nugen) was used for preamplification, where required. Gene expression profiling was performed on Affymetrix 430 2.0 gene chips according to standard protocol. Microarray data were analysed according to standard protocol using R/Bioconductor. Embryoid bodies were dissociated on day 8 by digestion with 0.05% trypsin for 5 min at 37 °C, pipetted thoroughly with p1000 to generate a single-cell suspension and washed with PBS + 2%FBS. Dissociated embryoid bodies were immediately processed for isolation of haemogenic endothelium. Cells were resuspended in 1mL PBS+2%FBS and incubated with human CD34 MicroBead kit for 1 h (Miltenyl Biotec, 130-046-702). After incubation, cells were washed with PBS+2%FBS and isolated by magnetic cell isolation using LS columns (Miltenyl Biotec, 130-042-401). Sorted CD34+ cells were resuspended in supplemented StemPro-34 medium, containing Y-27632 (10 μM), TPO (30 ng ml−1), IL-3 (10 ng ml−1), SCF (50 ng ml−1), IL-6 (10 ng ml−1), IL-11 (5 ng ml−1), IGF-1 (25 ng ml−1), VEGF (5 ng ml−1), bFGF (5 ng ml−1), BMP4 (10 ng ml−1), and FLT3 (10 ng ml−1) as reported20 and seeded at a density of 25 × 103 to 50 × 103 cells per well onto thin-layer Matrigel-coated 24-well plates. All recombinant factors were human and most were purchased from Peprotech. Plasmids for the transcription factor library were obtained as Gateway plasmids (Harvard Plasmid Service; GeneCopoeia). Open reading frames were cloned into lentiviral vectors using LR Clonase (Invitrogen). Two vectors were used, pSMAL-GFP (constitutive) and pINDUCER-21 (ORF-EG)51. pINDUCER21 (ORF-EG) was a gift from S. Elledge and T. Westbrook (Addgene plasmid 46948). Lentiviral particles were produced by transfecting 293T-17 cells (ATCC) with the second-generation packaging plasmids (pMD2.G and psPAX2 from Addgene). Virus were harvested 36 and 60 h after transfection and concentrated by ultracentrifugation at 23,000 r.p.m. for 2 h 15 min at 4 °C. Viruses were reconstituted with 50 μl of EHT culture medium. Constructs were titred by serial dilution on 293T cells using GFP as an indicator. Polycistronic vectors were made as follows: LCOR-P2A-HOXA9-T2A-HOXA5 and RUNX1-P2A-ERG DNA fragments were synthesized and cloned into pENTR-D/TOPO cloning vector by GenScript, then Gateway-recombined with pINDUCER-21 (ORF-EG). At day 3 of EHT culture, haemogenic endothelium cells were beginning to produce potentially haematopoietic ‘round’ cells; the occurrence of this phenomenon was used as quality control of haemogenic endothelium induction and transition to haematopoietic cells for each batch of experiments. The infection medium was EHT culture medium supplemented with Polybrene (8 μg ml−1, Sigma). Lentiviral infections were performed in a total volume of 250 μl (24-well plate). The multiplicity of infection for the factors was as follows: Library 3.0 for each, ERG 5.0, HOXA5 5.0, HOXA9 5.0, HOXA10 5.0, LCOR 5.0, RUNX1 5.0, SPI1 5.0, LCOR–HOXA9–HOXA5 2.0, and RUNX1–ERG 2.0. Haemogenic endothelium was vulnerable to damage during spinoculation, thus infections were performed static for 12 h, then 250 μl of fresh EHT medium was supplemented to dilute Polybrene. Parallel wells were cultured for an additional 3 days to measure infection efficiency by the percentage of GFP+ DAPI cells by FACS, achieving 30–70% of infection efficiency. Followed by lentiviral gene transfer, cells were maintained for 5 days in EHT culture medium supplemented with doxycycline (2 μg ml−1, Sigma) to induce transgene expression in vitro. Fifty thousand cells were plated into 3 ml complete methylcellulose (H4434; StemCell Technologies). Additional cytokines added were 10 ng ml−1 FLT3, 10 ng ml−1 IL6, and 50 ng ml−1 TPO (R&D Systems). The mixture was distributed into two 60 mm dishes and maintained in a humidified chamber at 37 °C for 14 days. Colonies were scored manually or using a BD Pathway 855 fluorescent imager. At 14 days, granulocyte, erythrocyte, monocyte, megakaryocyte (GEMM) colonies were picked up by P20 pipette. Between 10 and 20 GEMM colonies were picked with 2 or 3 biological replicates. A QIAamp DNA Micro Kit (Qiagen) was used to collect and prepare total genomic DNA for PCR detection of transgenes. Nested PCR reaction was as follows: first round with LNCX forward primer (5′-AGC TCG TTT AGT GAA CCG TCA GAT C-3′) and EGFP N reverse primer (5′-CGT CGC CGT CCA GCT CGA CCA G-3′), 95 °C 5 min, 36 cycles of (95 °C for 30 s, 60 °C for 30 s, 72 °C for 5 min), 72 °C for 5 min, 4 °C hold; second round with forward primer for each gene and HA reverse primer (5′-TCT GGG ACG TCG TAT GGG TA-3′), 95 °C 5 min, 36 cycles of (95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s), 72 °C for 5 min, 4 °C hold. Twelve hours after lentiviral gene transfer, cells were recovered by dispase for 5 min at 37 °C, and washed by PBS three times to ensure no carry-over of virus. Cells were resuspended at 0.3 × 105 to 3.0 × 105 cells per 25 μl buffer (PBS + 2% FBS from StemCell Technologies) and kept on ice until injection. Thirty thousand to 3.0 × 105 cells were intrafemorally injected in to NOD/LtSz-scidIL2Rgnull (NSG) mice and treated with doxycycline as described below (see section on ‘Mouse transplantation’). Up to 100 μl peripheral blood was collected every 2–4 weeks, to 14 weeks. Mice were euthanized and bone marrow and thymus removed at 8–14 weeks. For transgene detection in engrafted cells, each lineage of cells was FACS-sorted from bone marrow. Myeloid cells: CD33 APC (P67.6; BD), CD45 PE-Cy5 (J33; Coulter). B cells: CD19 PE (HIB19; BD), CD45 PE-Cy5 (J33; Coulter). T cells: CD3 PE-Cy7 (SK7; BD), CD45 PE-Cy5 (J33; Coulter). Between 10,000 and 50,000 cells were isolated with 2 or 3 biological replicates for multiple cell lines (iPSCs and ESCs). The QIAamp DNA Micro kit (Qiagen) was used to collect and prepare total genomic DNA for PCR detection of transgenes. Nested PCR reaction was performed similarly to the in vitro screening described in the above section. NOD/LtSz-scidIL2Rgnull (NSG) mice (The Jackson Laboratory) were bred and housed at the Boston Children’s Hospital animal care facility. Animal experiments were performed in accordance with institutional guidelines approved by Boston Children’s Hospital Animal Care Committee. Intrafemoral transplantations were conducted with 6- to 10-week-old female mice irradiated (250 rad) 12 h before transplantation. Before transplantation, mice were temporarily sedated with isoflurane. A 26-half-gauge needle was used to drill the femur and a 0.3 × 105 to 3.0 × 105 range of cells was transplanted in a 25 μl volume using a 28.5-gauge insulin needle. Sulfatrim was administered in drinking water to prevent infections after irradiation. Doxycycline Rodent Diet (Envigo-Teklad Diets; 625 p.p.m.) and doxycycline (1.0 mg ml−1) were added to the drinking water to maintain transgene expression in vivo for 2 weeks (ref. 2). Secondary transplantation was performed with 1,000–3,000 human CD34+ cells (isolated from bone marrow by magnetic cell isolation with CD34 microbeads) at 8 weeks. Isolated cells were resuspended at 1,000–3,000 cells per 25 μl buffer (PBS + 2% FBS from StemCell Technologies) and kept on ice until injection. Cells were intrafemorally injected in to NSG mice. Sorted CD34+CD43+CD45+ (25,000 cells) or CD34+CD43−CD45− (25,000 cells) HE-7TF cells were either intrafemorally or intravenously injected. For non-irradiated c-Kit-deficient immune-deficient recipients, the NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ model was used (The Jackson Laboratory). Investigators were blinded for the analysis of mice. The experiments were not randomized. No statistical methods were used to predetermine sample size. For this analysis, multi-lineage engraftment was defined as chimaerism of human CD45+ cells in bone marrow encompassing four distinct lineages (myeloid, erythroid, B- and T-lymphoid, each comprising more than 1% of engrafted human CD45+ cells) in this study. Cells grown in EHT culture or harvested animal tissues were stained with the following antibody panels. Haemogenic endothelium panel: CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), and CD43 PE (1G10; BD). HSPC panel: CD38 PE-Cy5 (LS198-4-3; Clontech), CD34 PE-Cy7, and CD45 PE (HI30; BD). Lineage panel: CD235a/glycophorin (GLY)-A PE-Cy7 or FITC (11E4B-7-6; Coulter), CD33 APC (P67.6; BD), CD19 PE (HIB19; BD), IgM BV510 (G20-127; BD), CD4 PE-Cy5 (13B8.2; Coulter), CD3 PE-Cy7 (SK7; BD), CD8 V450 (RPA-T8; BD), TCRαβ BV510 (T10B9; BD), TCRγδ APC (B1; BD), CD45 PE-Cy5 (J33; Coulter), CD15 APC (HI98; BD), and CD31/PECAM PE (WM59; BD). All stains were performed with fewer than 1 × 106 cells per 100 μl staining buffer (PBS + 2% FBS) with 1:100 dilution of each antibody, for 30 min at 4 °C in the dark. Compensation was performed by automated compensation with anti-mouse Igk negative beads (BD) and cord blood MNC stained with individual antibodies. All acquisitions were performed on a BD Fortessa cytometer. For detection of engraftment, human cord-blood-engrafted mouse marrow was used as a control to set gating; sorting was performed on a BD FACS Aria II cell sorter. Five thousand to 10,000 FACS-sorted erythroid cells (CD235a/glycophorin (GLY)-A PE-Cy7 or FITC (11E4B-7-6; Coulter)), plasmacytoid lymphocytes (CD19 PE (HIB19; BD), IgM BV510 (G20-127; BD), CD38 PE-Cy5 (LS198-4-3; Clontech)), neutrophils (CD15 APC (HI98; BD), CD31/PECAM PE (WM59; BD) and CD45 PE-Cy5 (J33; Coulter)) were cytospun onto slides (500 r.p.m. for 10 min), air dried, and stained with May-Grunwald and Giemsa stains (both from Sigma), washed with water, air dried, and mounted, followed by examination by light microscopy. RNA extraction was performed using an RNAeasy Microkit (Qiagen). Reverse transcription was performed using Superscript III (>5,000 cells) or VILO reagent (<5,000 cells) (Invitrogen). Quantitative PCR was performed in triplicate with SYBR Green (Applied Biosystems). Transcript abundance was calculated using the standard curve method. Primers used for globin genes were as follows52: huHbB F (5′-CTG AGG AGA AGT CTG CCG TTA-3′), huHbB R (5′-AGC ATC AGG AGT GGA CAG AT-3′), huHbG F (5′-TGG ATG ATC TCA AGG GCA C-3′), huHbG R (5′-TCA GTG GTA TCT GGA GGA CA-3′), huHbE F (5′-GCA AGA AGG TGC TGA CTT CC-3′), and huHbE R (5′-ACC ATC ACG TTA CCC AGG AG-3′). FACS-isolated neutrophils (CD15+PECAM+CD45+) and T cells (CD3+CD45+) were cultured in IMDM + 10%FBS overnight in 96-well plates (flat-bottom), seeding 5,000–20,000 cells per well obtained from mice engrafted over 10% in primary recipients, or pooled mice engrafted less than 5% in primary recipients. Then supernatant was taken and analysed by MPO- or IFN-γ-ELISA –Ready-SET-Go! Kit (eBioscience) according to the manufacturer’s protocol. The amount of IFN-γ was normalized per 1,000 cells. PMA (20 ng ml−1) and ionomycin (1 μg ml−1) were added to either neutrophils or T cells, then cells were cultured overnight (6–18 h). Human Ig production was measured from 50 μl of serum from NSG mice at 8 weeks (IgM) and 14 weeks after engraftment (IgG). Immunization of mice was done with OVA (F5503, Sigma) with Freund’s complete adjuvant (F5881, Sigma), followed by booster doses of Freund’s incomplete adjuvant (F5506, Sigma) according to the manufacturer’s instructions. Six to 14 weeks after engraftment, mice were injected with antigen OVA (0.1%) emulsified in complete adjuvant subcutaneously at two sites on the back, injecting 100 μl at each site. A booster injection of antigen OVA (0.1%) emulsified in incomplete adjuvant was administered 14 days after immunization. The booster was given as a single subcutaneous injection with 100 μl at one site on the back. A serum sample was isolated from mice 7 days after the first booster dose, and human ova-specific antibody concentration was tested with an ovalbumin-specific IgG, OVA sIgG, ELISA Kit (Mybiosource, MB S700766) for human Ova-specific IgG and a Human Anti-Ovalbumin (Gal d 2) IgM ELISA Kit (Alpha Diagnostic, 670-145-OVM) to detect human Ova-specific IgG and IgM, respectively. The technical replicates were done with three measurements of the same experimental setup. Human CD3+ T cells were FACS-isolated from thymus of engrafted NSG mice. Purified DNA was subjected to next-generation sequencing of CDR3 using immunoSEQ (Adaptive Biotechnology, Seattle, Washington, USA) and analysed with immunoSEQ Analyzer software (Adaptive Biotechnology). Aliquots (250 ng) of genomic DNA from human CD45+ bone marrow cells (CD45 PE-Cy5 (J33; Coulter)) from engrafted NSG mice and original PSCs (two biological replicates) were digested with either Nsp1 or Sty1. A universal adaptor oligonucleotide was then ligated to the digested DNAs. The ligated DNAs were diluted with water and three 10 μl aliquots from each well of the Sty 1 plate and four 10 μl aliquots from each well of the Nsp 1 plate were transferred to fresh 96-well plates. PCR master mix was added to each well and the reactions cycled as follows: 94 °C for 3 min; 30 cycles of 94 °C for 30 s, 60 °C for 45 s, 68 °C for 15 s; 68 °C for 7 min; 4 °C hold. After PCR, the seven reactions for each sample were combined and purified by precipitation from 2-propanol/7.5 M ammonium acetate. The ultraviolet absorbance of the purified PCR products was measured to ensure a yield ≥4 μg μl−1. Forty-five microlitres (≥180 μg) of each PCR product were fragmented with DNase 1 so the largest fragments were <185 base pairs. The fragmented PCR products were then end-labelled with a biotinylated nucleotide using terminal deoxynucleotidyl transferase. For hybridization, the end-labelled PCR products were combined with hybridization cocktail, denatured at 95 °C for 10 min and incubated at 49 °C. Two hundred microlitres of each mixture was loaded on a GeneChip and hybridized overnight at 50 °C and 60 r.p.m. After 16–18 h of hybridization, the chips were washed and stained using the GenomeWideSNP6_450 fluidics protocol with the appropriate buffers and stains. After washing and staining, the GeneChips were scanned on a GeneChip Scanner 3000 using AGCC software. Genotype calls (chp files) were generated in Affymetrix Genotyping Console using the default parameters. The resulting chp files were analysed for familial relationships using the identity by state algorithm implemented in Partek Genomics Suite. Engrafted human CD34+CD38−CD45+ HSCs were isolated from bone marrow from either iPS-derived haemogenic endothelium- or cord-blood-injected NSG mice, then RNA was purified with an RNeasy Micro kit (Qiagen). Quality control of RNA was done by Bioanalyzer and qubit analysis. Samples that passed quality control were converted into libraries and sequenced by a Nextseq PE75 kit. Raw reads were aligned to the human genome/transcriptome using TopHat2 software53. Gene expression levels and reads per kilobase per million (RPKM) values were estimated using a htseq-count tool54 and the edgeR package55. For a legitimate transcriptome-wide comparison, we retrieved raw RNA-seq data of two published from the Gene Expression Omnibus database (Long non-coding RNA profiling of human lymphoid progenitors reveals transcriptional divergence of B-cell and T-cell lineages, accession number GSE69239; Distinct routes of lineage development reshape the human blood hierarchy across ontogeny, accession number GSE76234) and calculated RPKM values using a same analysis pipeline. Engrafted human CD34+CD38−CD45+ HSCs were isolated from bone marrow from either iPS-derived haemogenic endothelium- or cord-blood-injected NSG mice, then processed for in-droplet barcoding according to a previous report29. The library was QCed with Bioanalyzer and sequenced by Nextseq PE 75 kit. The t-SNE algorithm was used to visualize transcriptome similarities and population heterogeneity of cord blood HSCs and iPSC-derived HSCs. The t-SNE algorithm performs a dimensionality reduction of multidimensional single-cell RNA-seq data into a low-dimensional space, preserving pairwise distances between data points as well as possible, allowing a global visualization of subpopulation structure and cell–cell similarities. We used the R package tsne in our analyses. The t-SNE map was initialized with point-to-point distances computed by classical multidimensional scaling, and the R plot function was used to visualize t-SNE maps annotated by cord blood or iPSC-derived HSCs. Plots showing t-SNE maps coloured by expression of selected genes were created using the ggplot2 package. For subpopulation identification, we used the top 500 genes with the highest variance to elucidate global differences among single cells. To assess transcriptome similarities in terms of induction of haematopoietic genes in iPSC-derived HSCs, we used 62 haematopoietic genes for t-SNE analysis in Supplementary Table 2. Gene set enrichment analysis was performed with the desktop client version (javaGSEA, http://software.broadinstitute.org/gsea/downloads.jsp) with default parameters. RPKM values from the 7F-HSPC were obtained from the RNA-seq (described previously). These values were normalized to a terminally differentiated cell and the normalized values were used to rank the most differentially expressed genes. These differentially expressed genes were used to run gene set enrichment analysis with gene sets obtained from mSigDB (KEGG, Hallmark, immunological, transcription factors, and chemical and genetic perturbations gene sets were used). In addition, gene sets specific to progenitors, cord blood, or fetal-liver HSC were obtained from previous reports16, 56. FDR < 0.25 with P < 0.05 was considered significant. CD33+ myeloid cells, CD19+ B cells, and CD3+ T cells were isolated from bone marrow from haemogenic-endothelium-injected NSG mice. Genomic DNA was purified with a QIAamp DNA Micro kit (Qiagen). Ligation-mediated PCR-based detection of lentiviral integration sites was done with a Lenti-X Integration Site Analysis Kit (Clontech) according to the manufacturer’s instructions. Sequencing-based detection (integration sequencing) was done as previously described57. RNA-seq from this study have been deposited in the Gene Expression Omnibus under accession number GSE85112. We retrieved raw RNA-seq data of two published from the Gene Expression Omnibus database (Long non-coding RNA profiling of human lymphoid progenitors reveals transcriptional divergence of B-cell and T-cell lineages, accession number GSE69239; Distinct routes of lineage development reshape the human blood hierarchy across ontogeny, accession number GSE76234). The data are all in the paper, or are available from the corresponding author upon reasonable request if not.
News Article | May 24, 2017
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. To construct the 35S:uORFs –LUC reporter, the 35S promoter and the TBF1 exon1 (including the R-motif, uORF1-uORF2, and the coding sequence of the first 73 amino acids of TBF1) were amplified from p35S:uORF1-uORF2-GUS1 using Reporter-F/R primers, and ligated into pGWB235 (ref. 22) via gateway recombination. The 35S:ccdB cassette–LUC-NOS construct was generated by fusing PCR fragments of the 35S promoter from pMDC140 (ref. 23), the ccdB cassette, and the NOS terminator from pRNAi-LIC24 and LUC from pGWB235 (ref. 22). The 35S:ccdB cassette–LUC-NOS was then inserted into pCAMBIA1300 via PstI and EcoRI and designated as pGX301 for cloning 5′ leader sequences through replacement of the ApaI-flanked ccdB cassette24. Similarly, the 35S:RLUC-HA-rbs terminator construct was made through fusion of PCR fragments of 35S from pMDC140 (ref. 23), RLUC from pmirGLO (Promega, E1330), and rbs terminator from pCRG3301 (ref. 25). The 35S:RLUC-HA-rbs fragment flanked with EcoRI was inserted into pTZ-57rt (Thermo Fisher, K1213) via TA cloning to generate pGX125. The 5′ leader sequences were amplified from the Arabidopsis (Col-0) genomic DNA or synthesized by Bio Basics (New York, USA) and inserted into pGX301 followed by transferring 35S:RLUC-HA-rbs from pGX125 via EcoRI. EFR, PAB2, PAB4, and PAB8 were amplified from U21686, C104970, U10212, and U15101 (from the Arabidopsis Biological Resource Center), respectively, and fused with the amino (N) terminus of enhanced green fluorescent protein (EGFP) by PCR. Fusion fragments were then inserted between the 35S promoter and the rbs terminator to generate 35S:EFR–EGFP (pGX664), 35S:EFR (pGX665), and 35S:PAB2–EGFP (pGX694). Information on all plasmids and primers in this study can be found in Supplementary Table 6. Plants were grown on soil (Metro Mix 360) at 22 °C under 12/12-h light/dark cycles with 55% relative humidity. Mutants efr-1 (ref. 6), ers1-10 (a weak gain-of-function mutant; ERS, ethylene receptor-related gene family member)26, ein4-1 (a gain-of-function mutant; EIN4, ethylene receptor-related gene family member)27, wei7-4 (a loss-of-function mutant; WEI7, involved in ethylene-mediated auxin increase)28, eicbp.b (camta 1-3; SALK_108806; EICBP.B, an ethylene-induced calmodulin-binding protein)29, and pab2/4 (ref. 18) and pab2/8 (ref. 18) were previously described; erf7 (SALK_205018; ERF7, a homologue of the ethylene responsive transcription factor gene ERF1) and gcn2 (GABI_862B02) were from the Arabidopsis Biological Resource Center. Transgenic plants were generated using the floral dip method30. Leaves from ~24 3-week-old plants (two leaves per plant; ~1.0 g) were collected. Tissue was fast frozen and ground in liquid nitrogen. Five millilitres of cold polysome extraction buffer (PEB; 200 mM Tris pH 9.0, 200 mM KCl, 35 mM MgCl , 25 mM EGTA, 5 mM DTT, 1 mM phenylmethanesulfonylfluoride (PMSF), 50 μg ml−1 cycloheximide, 50 μg ml−1 chloramphenicol, 1% (v/v) Brij-35, 1% (v/v) Igepal CA630, 1% (v/v) Tween 20, 1% (v/v) Triton X-100, 1% sodium deoxycholate (DOC), 1% (v/v) polyoxyethylene 10 tridecyl ether (PTE)) was added. After thawing on ice for 10 min, lysate was centrifuged at 4 °C/16,000g for 2 min. Supernatant was transferred to 40 μm filter falcon tube and centrifuged at 4 °C/7,000g for 1 min. Supernatant was then transferred into a 2-ml tube and centrifuged at 4 °C/16,000g for 15 min and this step was repeated once. Lysate (0.25 ml) was saved for total RNA extraction for making the RNA-seq library. Another 1 ml of lysate was layered on top of 0.9 ml sucrose cushion (400 mM Tris·HCl pH 9.0, 200 mM KCl, 35 mM MgCl , 1.75 M sucrose, 5 mM DTT, 50 μg ml−1 chloramphenicol, 50 μg ml−1 cycloheximide) in an ultracentrifuge tube (349623, Beckman). The samples were then centrifuged at 4 °C/70,000 r.p.m. for 4 h in a TLA100.1 rotor. The pellet was washed twice with cold water, resuspended in 300 μl RNase I digestion buffer (20 mM Tris·HCl pH 7.4, 140 mM KCl, 35 mM MgCl , 50 μg ml−1 cycloheximide, 50 μg ml−1 chloramphenicol)10 and then transferred to a new tube for brief centrifugation. The supernatant was then transferred to another new tube where 10 μl RNase I (100 U μl−1) was added before 60 min incubation at 25 °C. 15 μl SUPERase-In (20 U μl−1) was then added to stop the reaction. The subsequent steps including ribosome recovery, footprint fragment purification, PNK treatment, and linker ligation were performed as previously reported31. Two and a half microlitres of 5′ deadenylase (NEB) were then added to the ligation system and incubated at 30 °C for 1 h. Two and a half microlitres of RecJ exonuclease (NEB) was subsequently added for 1 h incubation at 37 °C. The enzymes were inactivated at 70 °C for 20 min and 10 μl of the samples were taken as template for reverse transcription (Extended Data Fig. 2). The rest of the steps for the library construction were performed as in the reported protocol31, with the exception of using biotinylated oligos, rRNA1 and rRNA2, for Arabidopsis according to another reported method10. TRIzol LS (0.75 ml; Ambion) was added to the 0.25 ml lysate saved from the Ribo-seq library construction, from which total RNA was extracted, quantified, and qualified using Nanodrop (Thermo Fisher Scientific). Total RNA (50-75 μg) was used for mRNA purification with Dynabeads Oligo (dT) (Invitrogen). Twenty microlitres of the purified poly (A) mRNA was mixed with 20 μl 2× fragmentation buffer (2 mM EDTA, 10 mM Na CO , 90 mM NaHCO ) and incubated for 40 min at 95 °C before cooling on ice. Five hundred microlitres of cold water, 1.5 μl of GlycoBlue, and 60 μl of cold 3 M sodium acetate were then added to the samples and mixed. Subsequently, 600 μl isopropanol was added before precipitation at −80 °C for at least 30 min. Samples were then centrifuged at 4°C/15,000g for 30 min to remove all liquid and air dried for 10 min before resuspension in 5 μl of 10 mM Tris pH 8. The rest of the steps were the same as Ribo-seq library preparation with quality control data shown in Extended Data Fig. 3. To record the 35S:uORFs –LUC reporter activity, 3-week-old Arabidopsis plants were sprayed with 1 mM luciferin 12 h before infiltration with either 10 μM elf18 (synthesized by GenScript) or 10 mM MgCl as Mock. Luciferase activity was recorded in a CCD (charge-coupled device) camera-equipped box (Lightshade Company) with each exposure time of 20 min. For the dual-luciferase assay, Nicotiana benthamiana plants were grown at 22 °C under 12/12-h light/dark cycles. Dual-luciferase constructs were transformed into the Agrobacterium strain GV3101, which was cultured overnight at 28 °C in Luria-Bertani broth supplied with kanamycin (50 mg l−1), gentamycin (50 mg l−1), and rifampicin (25 mg l−1). Cells were then spun down at 2,600g for 5 min, resuspended in infiltration buffer (10 mM 2-(N-morpholino) ethanesulfonic acid (MES), 10 mM MgCl , 200 μM acetosyringone), adjusted to an opitcal density at 600 nm (OD ) = 0.1, and incubated at room temperature for an additional 4 h before infiltration using 1 ml needleless syringes. For elf18 induction, 10 mM MgCl (Mock) solution or 10 μM elf18 were infiltrated 20 h after the dual-luciferase construct and EFR–EGFP had been co-infiltrated at the ratio of 1:1, and samples were collected 2 h after treatment. For the PAB2–EGFP co-expression assay, Agrobacterium containing a dual-luciferase construct was mixed with Agrobacterium containing the PAB2–EGFP construct at a ratio of 1:5. Leaf discs were collected, ground in liquid nitrogen, and lysed with the PLB buffer (Promega, E1910). Lysate was spun down at 15,000g for 1 min, from which 10 μl was used for measuring LUC and RLUC activity using a Victor3 plate reader (PerkinElmer). At 25 °C, substrates for LUC and RLUC were added using the automatic injector and after 3 s shaking and 3 s delay, the signals were captured for 3 s and recorded as counts per second. For elf18-induced growth inhibition assay, seeds were sterilized in a 2% PPM solution (Plant Cell Technology) at 4 °C for 3 days and sown on MS media (1/2 MS basal salts, 1% sucrose, and 0.8% agar) with or without 100 nM elf18. Ten-day-old seedlings were weighed with ten seedlings per sample. For elf18-induced resistance to Psm ES4326, 1 μM elf18 or Mock (10 mM MgCl ) was infiltrated into 3-week-old soil-grown plants 1 day before Psm ES4326 (OD = 0.001) infection of the same leaf. Bacterial growth was scored 3 days after infection. For elf18-induced resistance to Psm ES4326 in primary transformants overexpressing PAB2 in the pab2/8 mutant (OE-PAB2), transgenic plants expressing yellow fluorescent protein (YFP) in the WT background were used as control, and both control and OE-PAB2 were selected for basta-resistance and further confirmed by PCR. For MAPK activation, 12-day-old seedlings grown on MS media were flooded with 1 μM elf18 solution and 25 seedlings were collected at the indicated time points. Protein was extracted with co-IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% (v/v) Triton X-100, 0.2% (v/v) Nonidet P-40, protease inhibitor cocktail (Roche), phos-stop phosphatase inhibitor cocktail (Roche)). Antibody information and conditions can be found in Supplementary Table 6. For callose deposition, 3-week-old soil-grown plants were infiltrated with 1 μM elf18. After 20 h of incubation, leaves were collected, decolorized in 100% ethanol with gentle shaking for 4 h, and rehydrated in water for 30 min before stained in 0.01% (w/v) aniline blue in 0.01 M K PO pH 12 covered with aluminium foil for 24 h with gentle shaking. Callose deposition was observed with a Zeiss-510 inverted confocal microscope using a 405 nm laser for excitation and 420–480 nm filter for emission. PAB2–EGFP was amplified from pGX694. GA, G(A) , and G(A) were synthesized using Bio Basics (New York, USA) while poly(A) and G(A) were synthesized by IDT (https://www.idtdna.com/site). The sequences used for in vitro biotin-RNA synthesis can be found in Supplementary Table 6. In vitro transcription and translation were performed using the wheat germ translation system according to the manufacturer’s instructions (BioSieg, Japan). To make biotin-labelled RNA probes, 2 μl of 10 mM biotin-16-UTP (11388908910, Roche) was added into the transcription system. DNase I was then used to remove the DNA template. Biotin-labelled RNA (0.2 nmol) was conjugated to 50 μl streptavidin magnetic beads (65001, Thermo Fisher) according to the manufacturer’s instructions. In vitro synthesized PAB2–EGFP was incubated with biotin-labelled RNA in the glycerol-co-IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2.5 mM EDTA, 10% (v/v) glycerol, 1 mM PMSF, 20 U ml−1 Super-In RNase inhibitor, protease inhibitor cocktail (Roche)). To perform in vivo pull-down experiment, PAB2–EGFP was co-expressed with the elf18 receptor EFR (pGX665) for 40 h in N. benthamiana, which was then treated with Mock or elf18 for 2 h. Protein was extracted with glycerol-co-IP buffer and used in the pull-down assay at 4 °C for 4 h. YFP was expressed as a control. Antibody information and assay conditions can be found in Supplementary Table 6. Arabidopsis tissue (0.6 g) was ground in liquid nitrogen with 2 ml cold PEB buffer. One millilitre of crude lysate was loaded to 10.8 ml 15–60% sucrose gradient and centrifuged at 4 °C for 10 h (35,000 r.p.m., SW 41 Ti rotor). A absorbance recording and fractionation were performed as described previously32. Polysomal RNA was isolated by pelleting polysomes, and polysomal/total mRNA ratio was calculated as described previously8. About 50 mg of leaf tissue was used for total RNA extraction using TRIzol following the manufacturer’s instructions (Ambion). After DNase I (Ambion) treatment, reverse transcription was performed following the instruction of SuperScript III Reverse Transcriptase (Invitrogen) using oligo (dT). Real-time reverse-transcription polymerase chain reaction (RT–PCR) was done using FastStart Universal SYBR Green Master (Roche). Primer sequences can be found in Supplementary Table 6. Read processing and statistical methods were conducted following the criteria illustrated in Extended Data Fig. 4. Generally, Bowtie2 (ref. 33) was used to align reads to the Arabidopsis TAIR10 genome. Read assignment was achieved using HT-Seq34. Transcriptome and translatome changes were calculated using DESeq2 (ref. 35). Transcriptome fold changes (RS ) for protein-coding genes were determined using reads assigned to exon by gene. Translatome fold changes (RF ) for protein-coding genes were measured using reads assigned to CDS by gene. Translational efficiency was calculated by combining reads for all genes that passed reads per kilobase of transcript per million mapped reads (RPKM) ≥ 1 in CDS threshold in two biological replicates and normalizing Ribo-seq RPKM to RNA-seq RPKM as reported12. The criteria used for uORF prediction are shown in Extended Data Fig. 6 and were performed using systemPipeR (https://github.com/tgirke/systemPipeR). The MEME online tool36 was used to search strand-specific 5′ leader sequences for enriched consensuses compared with whole-genome 5′ leader sequences with default parameters. The density plot was presented using IGB37. The nucleotide resolution of the coverage around start and stop codons was performed using the 15th nucleotide of 30-nucleotide reads of Ribo-seq, similar as reported previously10, 38. Whole-transcriptome R-motif search was performed using the FIMO tool in the MEME suite36. LUC/RLUC ratio was first tested for normal distribution using a Shapiro–Wilk test. A two-sided Student’s t-test was used for comparison between two samples. Two-sided one-way or two-way analysis of variance was used for more than two samples, and Tukey’s test was used for multiple comparisons. GraphPad Prism 6 was used for all the statistical analyses. Unless specifically stated, sample size n means the biological replicate and experiment was performed three times with similar results. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate significant increases; NS, no significance; †††P < 0.001 indicates a significant decrease. The authors declare that the main data supporting the findings of this study are available within the article and its Source Data files. Extra data are available from the corresponding author upon request. The RNA-seq and Ribo-seq data have been deposited in Gene Expression Omnibus under accession number GSE86581.
News Article | May 2, 2017
Last May a seemingly commonplace meeting kicked off a firestorm of controversy. More than 100 experts in genetics and bioengineering convened at Harvard Medical School for a meeting that was closed to the public — attendees were asked not to contact news media or to post about the meeting on social media. The same group is getting back together in New York City next week. To the meeting organizers, last year's secretive measures were, counterintuitively, to make sure as many people heard about the project as possible. They were submitting a paper about the project to a scientific journal and were discouraged from sharing the information publicly before it was published. But there's another reason why this group of scientists, while encouraging debate and public involvement, would be wary of attracting too much attention. Their project is an effort to synthesize DNA, including human DNA. Researchers will start with simpler organisms, such as microbes and plants, but hope to ultimately create strands of human genetic code. One of the group's organizers, Jef Boeke, director of the Institute for Systems Genetics at NYU School of Medicine, told CNBC that incorporating synthesized DNA into mammalian (or even human) cells could happen in four to five years. This project follows in the footsteps of the Human Genome Project (HGP), the 13-year, $2.7 billion project that enabled scientists to first decode the human genome. "HGP allowed us to read the genome, but we still don't completely understand it," said Nancy Kelley, the coordinator of the new effort, dubbed GP-write. High school biology covers the basic building blocks for DNA, called nucleotides — adenine (A), cytosine (C), guanine (G) and thymine (T). Humans' 3 billion pairs provide the blueprints for how to build our cells. The intention of GP-write is to provide a better fundamental understanding of how these pieces work together. Using synthesized genomes has both pragmatic and theoretical implications — it could lead to lower cost and higher quality of DNA synthesis, discoveries about DNA assembly in cells and the ability to test many DNA variations. "If you do that, you gain a much deeper understanding of how a complicated apparatus goes," Boeke said. Boeke likens the genome to a bicycle — you can only fully understand something once you take it apart and put it back together. "Really, a synthetic genome is an engine for learning new information." More from Modern Medicine: Medical breakthroughs are way behind for the hard of hearing In the land of Vikings, an ambitious effort to find a cancer cure New guidelines for prostate cancer screening Boeke is particularly excited about what he calls an "ultrasafe cell line." Certain types of mammalian cells intended to produce certain types of large molecule drugs, called biologics. "[Cell lines] have been cultured in dishes in labs for decades. But you can't engineer the genomes — the tools for doing that are quite crude, relatively speaking," Boeke said. Sometimes these cells get infected with a virus, and it completely shuts down drug production. A synthetic cell that lacked unnecessary genetic material could, evidence suggests, be virus-resistant, consistently producing useful drugs to treat disease. The results of GP-write could also lead to stem cell therapy that doesn't run the risk of infecting the patient with another disease, which appears to be what happened to one patient who received stem cell treatment in Mexico. Or they could create a line of microorganisms that could help humans generate some of their own amino acids — nutrients we usually get from food. These outcomes, of course, won't happen overnight. Boeke, who has spent years synthesizing yeast DNA, knows there will be plenty of technical hurdles. "Getting big pieces of DNA efficiently into mammalian cells, engineering them rapidly, these will be major challenges," he said. Scientists will also have to do that without breaking the bank. Right now, Kelley estimates that it costs 10 cents to synthesize every base pair, the bonded molecules that make up the double helix of DNA (start-up GenScript advertises even higher prices, at 23 cents for "economy"). Considering that humans have 3 billion base pairs. "If we can get that [cost] down to one cent per base pair, it would really make a difference," Kelley said. Since last May's meeting, Kelley, Boeke and their collaborators have published an article in Science about the project, as well as a white paper outlining its timeline. Close to 200 researchers and collaborators around the world have expressed interest in participating, Kelley says, ranging from institutional researchers to corporate scientists. Preliminary experiments are already underway, and the project organizers are discussing the project with companies as well as federal and state agencies that might help them reach their goal of raising $100 million this year. They estimate GP-write should cost less, in total, than the $3 billion Human Genome Project, though they have not provided more specific cost projections. It might not be so bad if these advances took some time. After news broke of the May meeting, some criticized the way the rollout was handled. "Given that human genome synthesis is a technology that can completely redefine the core of what now joins all of humanity together as a species, we argue that discussions of making such capacities real ... should not take place without open and advance consideration of whether it is morally right to proceed," read one op-ed, published in Cosmos. Boeke says a public and scientific discussion is exactly what the GP-write organizers intend to have. "I think articulation of our plan not to start right off synthesizing a full human genome tomorrow was helpful. We have a four- to five-year period where there can be plenty of time for debate about the wisdom of that, whether resources should be put in that direction or in another. Whenever it's human, everyone has an opinion and wants their voice to be heard. We want to hear what people have to say," Boeke said. 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