Cell Culture of Pluripotent Stem Cells
Prior to FF adaptation, conventionally cultured hiPSCs were routinely maintained on feeder cells, mitomycin C treated MEF cells (Millipore), and cultured with conventional hESC medium (referred to as conventional medium in the text) containing DMEM/F12 (Mediatech), 20% v/v knockout serum replacement (Invitrogen), 1% v/v non-essential amino acids (Mediatech), 2 mM L-glutamine (Mediatech), 100 µM β-mercaptoethanol (Invitrogen) and 10 ng/mL bFGF (Invitrogen). Upon confluency, conventionally cultured hiPSCs were enzymatically dissociated using 1 mg/mL collagenase IV (Invitrogen) for 7 min at 37°C followed by mechanical dissociation into small pieces (termed as clump passaging), collected and dilute passaged 1:3–1:4 onto freshly seeded mitomycin C treated MEF cells every 5–7 days with daily addition of hESC medium. Cell cultures were maintained in a humidified incubator set at 37°C and 5% CO2
. For adaptation to FF and single cell culture, upon confluency conventionally cultured hiPSCs were clump dissociated and resuspended in MEF cell conditioned medium (hESC medium conditioned on MEF cells for 24 hrs and supplemented with 10 ng/mL bFGF prior to use) and transferred to FF tissue culture plates (BD Biosciences) that were previously coated with Matrigel™ (1
25 dilution; BD Biosciences) for 1–2 hrs in 37°C. MEF conditioned medium was changed daily. Upon confluency of greater than 90%, conditioned medium was switched to SMC4 supplemented medium [Supplementary Table 1
, hESC medium supplemented with 0.4 µM PD0325901 (Biovision), 1 µM CHIR99021 (Biovision), 5 µM Thiazovivin (internally synthesized at Fate Therapeutics Inc. and purchased from Biovision) and 2 µM SB431542 (Biovision)] two hours prior to single cell dissociation, see below. Small molecules were maintained in −20°C prior to the addition to medium at a stock concentration of 5–25 mM in DMSO. All working media were maintained in 4°C for the duration of usage. In studies that included Y27632 (Ascent), 10 μM was used.
For single cell dissociation, hiPSCs were washed once with phosphate buffered saline (PBS) (Mediatech) and treated with Accutase (Millipore) for 3–5 min at 37°C followed with pipetting to ensure single cell dissociation. The single cell suspension was then mixed in equal volume with conventional medium, centrifuged at 250 g for 5 min and resuspened in SMC4 supplemented medium (Supplementary Table S1
After resuspension in SMC4 supplemented medium, the single cells were transferred to FF tissue culture plates that were previously coated with Matrigel™ for 1–2 hrs in 37°C. In this format, cells routinely received fresh medium every other day and were passaged when confluency had reach 65–75%, which normally occurred 4–5 days post passage. With each passage cells were re-dissociated into single cells and transferred to a new tissue culture plate coated with Matrigel™ at a dilution passage of 1:8–1:15.
Induction of Reprogramming
To initiate the reprogramming process, ectopic expression of reprogramming factors were induced by lentiviral transduction. Most typically OKSM or OKS were used as indicated per each study. In most cases, the starting cells were plated at 10% confluency (i.e. 1x105
cells per well of a 6-well plate) on gelatin (Mediatech) coated surface. For the method of viral infection, freshly collected lentivirus was added to the starting cells at a dilution of 1
2, supplemented with 4 µg/mL polybrene (Millipore), and transferred to 37°C and 5% CO2
for 8–12 hrs. After the completion of the incubation, the cells were washed three times with PBS and fed with fibroblast medium: DMEM (Mediatech), 10% FBS (Invitrogen), 1x glutamax (Invitrogen), 1x non-essential amino acids (Mediatech). Upon confluency (usually between days 4–6) the cells were dissociated with trypsin (Invitrogen), mixed with equal part fibroblast medium, centrifuged at 250 g for 5 min, resuspended in SMC4 supplemented medium and expanded 1:10 into a larger culture plate. Cultures are maintained in SMC4 until the next application.
Unique Population Enrichment
Approximately 8–12 days after the induction of reprogramming, cells were dissociated into single cells with Accutase (Millipore) and stained with various surface markers of pluripotency, markers of somatic cells and/or markers of incomplete reprogramming. Briefly, dissociated cells were resuspended in staining solution containing Hanks' Balanced Salt Solution (Invitrogen), 4% fetal bovine serum (Invitrogen) and 10 mM Hepes (Invitrogen) and kept on ice. Per recommended manufacturers' dilution, conjugated primary antibodies were added to the cell solution and incubated on ice for 15 min. The cell solution was washed and resuspended in staining buffer and maintained on ice. At this point various enrichment/depletion strategies were taken; including Fluorescent Activated Cell Sorting (BD Biosciences, see below) and Magnetic Cell Sorting (Miltenyi Biotec, see below).
Flow cytometry sorting was performed on FACS Aria II (BD Biosciences). Primary antibodies used include SSEA4-Alexa Fluor-488/555 (BD Biosciences), Tra181-Alexa Fluor-488/647 (BD Biosciences), Tra161-Alexa Fluor-488/647 (BD Biosciences). The cells were collected in SMC4 supplemented medium. The sorted cells were then centrifuged and resuspended in SMC4 supplemented medium and transferred to Matrigel™ coated tissue culture plates. For additional improvement in seeding, 5 μg/mL Fibronectin (BD Biosciences) can be added to SMC4 for the first two days. When sorted into microwells, i.e. 96 well plates, each well is prefilled with SMC4 and upon completion of the sort the plates were centrifuged for 2 min at 300 g prior to incubation. The SMC4 supplemented medium was replaced every other day except in case of 96-well plate sorting where it was replaced after 3–4 days in culture. Colony formation was typically seen 2–4 days post sort. Flow cytometry analysis was performed on Guava EasyCyte 8 HT (Millipore).
MACS Microbeads (Miltenyi Biotec) separation was performed according to protocol. Briefly, cells were dissociated into single cells and stained with appropriate FITC-conjugated primary antibodies, including SSEA4-FITC (BD Biosciences) and Tra181-FITC (BD Biosciences). Cells were then magnetically labeled with Anti-FITC Microbeads (Miltenyi Biotec). The labeled cell suspension was then loaded onto a LS MACS Column (Miltenyi Biotec). The collected cells from either positively or negatively selected fractions were centrifuged at 250 g for 5 min and resuspended in SMC4 supplemented medium and transferred to Matrigel™ (BD Biosciences) coated tissue culture plates. The following day, fresh medium is added to the culture and subsequently replaced every other day. The appearance of colonies is typically seen 2–4 days post sort.
Isolation and culture of patient consented human neonatal foreskin fibroblast cells
Protocol and donor consent form were approved by the independent Institutional Review Board of Chesapeake Research Review (Columbia, MD). The parents of 2-day old baby boy provided their written informed consent for the use of their son's foreskin biopsy for the generation of iPSCs. The tissue was collected in Hanks' balanced salt solution (HBSS), cut into small pieces (3–4 mm), and incubated with dispase (BD Biosciences) overnight at 4°C. The dermis was separated from the epidermis and cut further into 0.5 mm-pieces and 10 to 15 pieces placed in 100-mm dish. Autoclaved cover-slips were laid on top of the dermal pieces to hold them down and 6 ml of fibroblast media slowly added to the edge of the dish. Fresh medium was added every 3–4 days and floating tissue pieces were removed. After two weeks, cells were ready to collect using trypsin. Additional patient consented fibroblast lines were obtained from ZenBio Inc.
Alkaline Phosphatase Staining
Cells were fixed in 4% v/v paraformaldehyde (Alfa Aesar), washed three times with PBS and stained with Alkaline Phosphatase Staining Kit (Sigma-Aldrich). Briefly, 1 mL Sodium Nitrite Solution was added to 1 mL FRV-Alkaline Solution, mixed and incubated at 25°C for 2 min. The solution was then mixed with 45 mL of H2O followed by the addition of 1 mL Naphthol AS-BI Alkaline Solution. The alkaline-dye mixture was added to the fixed cells and incubated at 25°C for 15 min followed by a PBS wash. The cells were then scored for the presence of alkaline phosphatase.
Cells were fixed using 4% v/v paraformaldehyde (Alfa Aesar), washed three times with PBS containing 0.2% v/v Tween (PBST) (Fisher Scientific) and permeablized using 0.15% v/v TritonX-100 (Sigma-Aldrich) in PBS for 1 hr at 25°C. After permeabilization, cells were blocked with 1% v/v BSA (Invitrogen) in PBST (PBSTB) (Fisher Scientific) for 30 min at 25°C. After gentle removal of PBSTB, cells were incubated with primary antibody in PBSTB overnight at 4°C. Primary antibodies used in this study include Nanog (Abcam), Tra160 (BD Biosciences), Tra181 (BD Biosciences), SSEA4 (BD Biosciences), β-III Tubulin (R&D Systems), α-Smooth Muscle Actin (Sigma), FoxA2 (R&D Systems) and Sox17 (R&D Systems). After the overnight incubation, cells were washed three times with PBST and stained with secondary antibody (Alexa 488 or 555; Invitrogen) diluted 1:500 in PBSTB for 1 hr at 25°C. The cells were washed three times in PBST and stained with Hoechst dye (Invitrogen). Images of the stained cells were captured using the Zeiss fluorescence microscope and CCD camera.
Induction of Differentiation
hiPSC were differentiated as EBs or monolayers in differentiation medium containing DMEM/F12 (Mediatech), 20% fetal bovine serum (Invitrogen), 1% non-essential amino acids (Mediatech), 2 mM L-glutamine (Mediatech) and 100 µM β-mercaptoethanol. Briefly, for EB formation hiPSCs were single cell dissociated with Accutase (Millipore) and resuspended in differentiation medium to a final concentration of 75,000 cells/mL and 5 uM Thiazovivin was added. Cells were seeded in 100 µL/well in V-bottom 96-well non-tissue culture plate (Nunc) and centrifuged at 950 g for 5 min. The following day compact “ball-like clumps” were transfer to ultra-low binding 6-well plate (Corning) using P1000 at approximately 30–40 EBs/well. After 7 days, EBs were transferred at 1:1 to Matrigel coated 6-well plate. After 3 weeks in culture, cells were fixed and stained. For monolayer differentiation, hiPSCs were seeded in SMC4 and switched to differentiation medium the next day. Once the medium had been switched, the hiPSCs were allowed to differentiate for 14–21 days. Medium was changed every 2–3 days.
RNA was isolated using the PicoPure RNA Isolation kit (Life Technologies), and 0.5 µg RNA was used to generate first strand cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). Relative gene expression levels were determined using the TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and the FAM-labeled TaqMan probes listed in Supplementary Table 3
Gene Expression Analysis
Total RNA was isolated from cells using Pico Pure RNA Isolation Kit (Life Technologies). In brief, biotinylated aRNA was prepared using the standard protocol for MessageAmp II aRNA Amplification Kit (Applied Biosystems/Ambion, Austin, TX) utilizing the optional Second Round Amplification and then transcribed into biotin labeled aRNA using MessageAmp II Biotin Enhanced Kit (Applied Biosystems/Ambion, Austin, TX) using the standard protocol. Biotin labeled aRNA was purified and fragmented according to Affymetrix recommendations. 20 µg of fragmented aRNA were used to hybridize to the Human Genome U133 Plus 2.0 chips (Affymetrix Inc. Santa Clara, CA) for 16 hrs at 45°C. The arrays were washed and stained in the Affymetrix Fluidics Station 450 and scanned using the Affymetrix GeneChip Scanner 3000 7G. The image data were analyzed using Affymetrix Expression Console software using default analysis settings. Arrays were normalized by log scale robust multi-array analysis (RMA) and visualized in Spotfire for Genomics 3.1 (Tibco Spotfire, Palo Alto, CA).
Cytogenetic analysis was performed on twenty G-banded metaphase cells by Cell Line Genetics (Madison, WI) or WiCell (Madison, WI).
Comparative Genomic Hybridization
High resolution comparative genomic hybridization (NimbleGen 12x135 k array; HG18 WG CGH v3.1 HX12) and subsequent copy number variation analysis was conducted by WiCell (Madison, WI). Briefly, relative copy number is determined by comparative differential hybridization of labeled genomic DNA to the 135,000 oligonucleotide whole genome tiling array.
Student's t test was used for statistical evaluations pertaining to standard deviation. StepOne Software v2.2 (Life Technologies) was used to determine RQ minimum and maximum values (error bars).
Teratoma grafting and analyses was conducted by Applied Stem Cells (Menlo Park, CA). Briefly, 1–5 million single cell dissociated hiPSCs were mixed in 100 uL SMC4 supplemented medium and 100 uL Matrigel and introduced to the renal capsule and testis of Beige SCID mice. The developed teratomas were harvested, sectioned and analyzed for various differentiated cell types and structures.
The GEO accession number for the Affymetrix profiling reported in this paper is GSE28815. The GEO accession numbers for CGH array reported in this paper are GSM828394, 828395 and 828396.