Determination of dynamic changes in gene expression and chromatin accessibility during cellular reprogramming

Abstract

To deconstruct the heterogeneity, reprogramming cells were collected at various time points after induction and subjected for single-cell next-generation sequencing (NGS) library preparation. Reprogramming was induced from BJ fibroblast with polycistronic O2S, K2M vectors delivered using lentivirus. BJ, D2, D8, D16 TRA-1-60+, and D16 TRA-1-60− were harvested for scRNA-Seq and scATAC-Seq library preparation on the Fluidigm C1 platform. The same series of time points, with the addition of D12, was collected for scRNA-Seq library preparation using the droplet-based 10X Genomics. scRNA-Seq library construction (Fluidigm)

The scRNA-Seq libraries were prepared following the published protocol with minor modifications (11). Briefly, cells undergoing reprogramming were trypsinized into single cells at each time point. Cells were washed three times with C1 DNA-seq Cell Wash Buffer (Fluidigm). Cells at a concentration of 300 to 600 cells/μl were mixed with C1 Cell Suspension Reagent at a ratio of 3:2. Next, the cell mixture was loaded into C1 Single-Cell Auto Prep IFC (integrated fluidic circuits) microfluidic chips according to the “STRT seq, 1862× (10-17 μm diameter cells)” protocol. Cells were captured at the 96 capture sites in the microfluidic chip and stained using a green fluorescent calcein-AM dye (LIVE/DEAD cell viability assay, Life Technologies) and imaged by a Leica CTR 6000 microscope. mRNA-Seq libraries were prepared following the Generate complementary DNA (cDNA) libraries with the C1 Single-Cell mRNA Seq HT IFC and Reagent Kit V2 manual. scRNA-Seq library construction (10X Genomics)

The 10X Genomics scRNA-Seq libraries were prepared following the Single-cell 3′ Reagents Kits v2 User Guide. Briefly, cell suspensions (~6000 cells) were loaded in a C1 Chromium Instrument (10X Genomics) to generate single-cell gel beads in emulsion (GEMs). scRNA-Seq libraries were prepared using the Chromium Single Cell 3′ Library, Gel Bead Kit v2, and Chromium i7 Multiplex Kit (10X Genomics). Sequencing was performed on Illumina HiSeq 4000 platform using 98 base pairs (bp) pair-end sequencing parameter scATAC-Seq library construction (Fluidigm)

The scATAC-Seq libraries were prepared following the published protocol with minor modifications (10). Briefly, cells undergoing reprogramming at different time points were trypsinized into single cells and washed three times with the C1 DNA-seq Cell Wash Buffer (Fluidigm). Cells at a concentration of 300 to 600 cells/μl were combined mixed with the C1 Cell Suspension Reagent at a ratio of 3:2. The mixture of cells was then loaded into the C1 Single-Cell Auto Prep IFC microfluidic chips according to the “ATACseq: Cell Load and Stain (1862×)” protocol. Cells were captured at the 96 capture sites in the chip and stained using the green fluorescent calcein-AM dye (LIVE/DEAD cell viability assay, Life Technologies) and imaged by the Leica CTR 6000 microscope. On the IFC, the lysis and transposition reaction took place at 37°C for 30 min, followed by inactivation of Tn5 using EDTA at 50°C for 30 min. Next, excess EDTA was quenched by MgCl2 at room temperature. The digested accessible regions were then amplified for 8 cycles. The 96 scATAC-Seq libraries were harvested from microfluidic chips and transferred to a 96-well plate for incorporating cell-specific indexes by additional 14 cycles of polymerase chain reaction (PCR). Following that, the 96 scATAC-Seq libraries were pooled and purified by a Qiagen PCR purification kit, and the quality of the libraries was assessed by an Agilent 2100 bioanalyzer. Cell lines and reagents

The human neonatal fibroblast cell line BJ (Stemgent, Cambridge, MA) and the human lung fibroblast cell line MRC-5 (American Type Culture Collection (ATCC) CCL-171) were maintained in the medium, which is composed of Dulbecco’s Modified Eagles Medium (DMEM) with High Glucose (4500 mg/l) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), minimum essential medium (MEM) nonessential amino acid solution (100X), 200 mM L-glutamine (100X), and penicillin-streptomycin (10,000 U/ml) (100X). hESCs and iPSCs were grown in mTeSR medium (STEMCELL Technologies). The mentioned reagents were purchased from Life Technologies, unless otherwise specified. Cellular reprogramming (lentivirus)

At one day prior to the induction, BJ was seeded at a density of 25,000 cells/ml onto a 12-well plate. At D0, BJ cells were infected with polycistronic O2S, K2M lentivirus (Addgene) in the presence of polybrene (4 mg/ml; Sigma-Aldrich). These cells were maintained with BJ medium until D6. At D5, cells were trypsinized, replated at a density of 50,000 cells/ml onto a Matrigel (Corning)–coated 12-well plate. From D6 to D12, reprogramming cells were cultured with hESC medium, composed of 20% knockout serum replacement, MEM nonessential amino acid solution (100X), 200 mM L-glutamine (100X), penicillin-streptomycin (10,000 U/ml) (100X), basic fibroblast growth factor (8 ng/ml), and 0.1 mM β-mercaptoethanol in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12). From day 12 onward, medium was changed to a mixture of mouse embryonic fibroblast (MEF)–conditioned hESC medium and mTeSR (STEMCELL Technologies) in a 1:1 ratio. Production of lentiviral supernatants

293T cells were plated at a density of 1.0 × 107 cells per 15-cm dish. The next day, cells were transfected with 9 μg of overexpression vector, 9 μg of psPAX2, and 0.9 μg of pMD2.G plasmid diluted with OptiMEM at a total of 225ul by mixing with 54 μl of TransIT LT1 (Mirus Bio) diluted with 696 μl of OptiMEM (Invitrogen) per plate. The supernatant was collected at 48 and 72 hours after transfection, filtered through 0.45-μm pore size filters, and concentrated using ultracentrifugation for 1.5 hours at 23,000 rpm. Cellular reprogramming (Sendai virus)

At two days prior to the induction, 25,000 BJ or MSC cells were plated onto a 12-well plate. At D0, cells were infected with KOS, MYC, and KLF4 Sendai virus (CytoTune-iPS 2.0 Sendai Reprogramming Kit) at multiplicities of infection of 10:10:6. On the next day, medium was changed to reduce the toxicity effects of the Sendai virus. Cells were cultured in BJ and MSC medium for the first 4 to 5 days. At D4 or D5, cells were passaged and transferred onto Matrigel (Corning)–coated plates. Reprogramming cells were then cultured with medium, composed of BJ/MSC medium and mTeSR medium (STEMCELL Technologies) in a 1:1 ratio. A83-01 (STEMCELL Technologies) was supplemented to the reprogramming medium to increase the reprogramming efficiency. Magnetic-activated cell sorting

Enrichment of cells by magnetic-activated cell sorting (MACS) was performed according to the manufacturer’s instructions. Briefly, cells were trypsinized to single cells and resuspended in 1% FBS (Sigma-Aldrich) at a concentration of 20 million/ml. Single-cell suspensions were incubated with anti-human TRA-1-60-PE (phycoerythrin) antibody (Miltenyi Biotec), anti-human CD13-FITC (fluorescein isothiocyanate) antibody (Miltenyi Biotec), and anti-human CD201-PE antibody (Miltenyi Biotec) in a 1:11 dilution, in the fridge for 10 min. Cells were then incubated with anti-PE MicroBeads (Miltenyi Biotec) in a 1:5 dilution, in the fridge for 15 min. Next, cells were separated into TRA-1-60+ and TRA-1-60− (flow-through) cells using the magnetic sorter. Staining and imaging in 96-well plates (fluorescent probes screen)

A preprepared library of fluorescent probes was used for screen. For staining, cells were washed with phosphate-buffered saline (PBS) and incubated in cell culture medium supplemented with an optimal concentration of fluorescent probes for 1 hour at 37°C. It was followed by three washes with PBS and destaining with culture medium for 3 hours at 37°C. Hoechst 3342 (1:20,000; Invitrogen) was added to each well and stained for 30 min. The cells were then washed once with PBS. Cells were imaged with an IXU ultra plate-scanning confocal microscope (Molecular Devices) at ×10 magnification, and nine pictures were taken per well. Granule area, integrated fluorescent intensity, and nuclei number were quantified using MetaXpress Image Acquisition and Analysis software V2. BDD2-C8 dye live staining and FACS sorting

D8 reprogramming cells were incubated with hESC medium containing BDD2-C8 at a final concentration of 0.05 μM at 37°C for 1 hour. It was followed by three washes with PBS and destaining with hESC medium for 3 hours at 37°C. During destaining steps, fresh hESC medium was changed every 1 hour. BDD2-C8–stained cells were trypsinized and subjected for sorting with flow cytometry (MoFlo XDP Cell Sorter, Beckman Coulter). According to the staining intensity of BDD2-C8, the top 10% and bottom 10% of D8 reprogramming cells were enriched for single-cell real-time quantitative reverse transcription PCR (qRT-PCR), scRNA-Seq library preparation, and replating onto a 12-well plate for TRA-1-60+ colony counting assay. Organelle probe and fluorescent probe costaining

Cells were incubated with organelle-specific probes (diluted with culture media) at 37°C for 30 min and washed three times with PBS. Cells were then stained with fluorescent probes at 37°C for 1 hour, followed by destaining at 37°C for 3 hours. The information of organelle-specific probes is as follows: ER-specific probe: ER-Tracker Red, 500 nM; Golgi-specific probe: BODIPY FL C5-ceramide, 5 μM; mitochondria-specific probe: MitoTracker Green FM, 250 nM. Costaining of cell surface markers and BDD2-C8

Cells were stained with BDD2-C8 for 1 hour, followed by 3 hours of destaining. Next, cells were trypsinized and resuspended with 1% FBS (Sigma-Aldrich) at a concentration of 1.5 million/ml. Single-cell suspensions were incubated with anti-human CD13-FITC, anti-human CD201(EPCR)-APC-Vio770 antibody (at a dilution of 1:11), and anti-human CD44-VioBlue (at a dilution of 1:51) (Miltenyi Biotec), in the fridge for 10 min.