In vitro culture of primary human fibroblasts and lentivirus reprogramming

Human fibroblasts for iPSCs derivation were obtained from Coriell Institute (Camden, NJ) and reprogrammed using a single polycistronic vector using four-factor 2A (4F2A) doxycycline (DOX)-inducible lentivirus encoding mouse cDNAs for Oct4, Sox2, Klf4, and c-Myc separated by three different 2A peptides (P2A, T2A, and E2A, respectively). The lentiviral plasmids are p20321 (TetO-FUW-OSKM) and p20342 (FUW-M2rtTA) (Addgene, Cambridge, MA) originally developed by Carey et al.9. Lentiviral particles (4F2A and M2rtTA) were packaged in HEK 293T cells. The primary fibroblast cells were co-transfected using the lentivirus construct, psPAX and pCMV-VSVG vectors by calcium phosphate co-precipitation. Viral supernatants from cultures packaging each of the two viruses were pooled, filtered through a 0.45 m filter and concentrated by ultracentrifugation and stored at 80C.

The 5 human fibroblast lines were transduced by viral particles in xenofree human fibroblast culture medium10 in the presence of polybrene (8g/mL). Forty-eight hours after infection, less than 15% of fibroblasts tested immunopositive for viral-derived OCT4. The procedure was carried out in 1 well of a 6-well plate with cells at 70% confluence to allow for cell growth after viral infection an appearance of stem cell colonies. The medium was replaced two days after infection, and then daily, with xenofree hES medium plus doxycycline (1g/ml) formulated to maintain stem cell pluripotency10,11,12. After 35 days of culture, small cell clumps distinguishable from the fibroblast morphology appeared. Those that formed cell colonies with hESC-like morphology were mechanically isolated and passed on to mitotically inactivated xenofree human foreskin feeder cells (ATCC PCS-201010). Overall reprogramming efficiency by this method was calculated to be 0.002 ~ 0.004%. The iPSC colonies were expanded for several passages under xenofree conditions without doxycycline and evaluated for expression of markers of pluripotency by quantitative RT-PCR (qRT-PCR) and immunocytology.

Quantitative PCR analysis was done by isolation of total RNA from the hESC or iPSC lines and parental fibroblast lines and purification using the NucleoSpin RNA XS Total RNA isolation kit (Clontech). Reverse transcription (RT) was performed in a 20ul reaction volume using Superscript II (Invitrogen) and the cDNA reaction was diluted to a 300ul working stock volume. Primers for use in qPCR were first validated by maximally amplifying cDNA from a range of samples to confirm that a single PCR reaction product was produced and that the amplicon was of the predicted length. For validation, 10ul of cDNA from H9 hESCs (WA09, Wicell, Madison, WI), control fibroblasts (line A-2), and two of iPSC lines (A-2.2.1 & A-2.2.2) for each primer set was amplified for 36 cycles (95C 30s, 55C 30s, 72C 30s). For endogenous and transgene expression, 5ul of cDNA from each iPSC lines for each primer set was amplified for 32 cycles and resolved on a 3% nusieve agarose gel and visualized by ethidium bromide staining. Quantitative PCRs contained 10ng of cDNA, 400nM of each primer, and SYBR Green PCR Master Mix (AppliedBiosystems). Each sample was analyzed by triplicate by an ABI PRISM 7000 sequence detection system. Data was analyzed using the systems software. The expression of gene of interest was normalized to GAPDH in all cases and compared with hESCs.

We used the MycoAlertTM PLUS Assay mycoplasma detection kit (Lonza, Allendale, NJ) essentially as manufacturers instructions. Briefly, after centrifugation (1500rpm, 5min) of cell supernatant during passage of suspension iPSC cultures, the supernatants were transferred into luminescence compatible tubes (Corning Inc., Corning, NJ). The viable mycoplasma was lysed to allow enzymes to react with MycoAlertTM PLUS substrate, catalyzing the conversion of ADP to ATP. The level of ATP in the sample both before (reading A; ATP background) and after (reading B) the addition of MycoAlertTM PLUS substrate was assessed using a luminometer (Victor3, Perkin-Elmer, Waltham, Massachusetts, USA), so that a ratio B/A was obtained. Reading B assesses the conversion of ADP to ATP and is a monitor of contaminated samples. If the ratio of B/A is greater than 1 the cell culture was considered to be contaminated by mycoplasma. For control samples, the MycoAlert TM assay positive and negative control set was used.

Ethnically diverse-induced pluripotent stem cell (ED-iPSC) lines maintained on human foreskin fibroblast feeders were transferred to feeder-free conditions in non-tissue culture treated dishes coated with xenofree vitronectin (StemCell Technologies, Vancouver, Canada) or 1:100 Matrigel (10mg/ml; BD Biosciences, San Jose, CA) diluted into Hanks Buffered Saline Solution (Gibco HBSS; Life Technologies, Grand Island, NY). Cells were maintained in mTeSR2 complete media (StemCell Technologies, Vancouver, Canada) and mechanically passaged between days 5 and 7. Media was replaced on day 1 after the first passage of the series and cells grown overnight. On day 2, slow release Stem Beads FGF2 (20 microliters of PLGA beads loaded with hFGF2; StemBeads; Stem Culture Inc., Rensselaer, NY) were added with fresh mTeSR2 media. Media changes were done every 3 days with Stem Beads FGF2 and mTeSR2. Preparation of uniform sized EBs from iPSCs colonies was done in custom lithography template microarrays (LTA) generated in-house. Chemical dissociation of the stem cell colonies into single cell suspension was done before and loading of the cells into LTA- polydimethylsiloxane (PDMS) grids in mTeSR2 media in the presence of 10M Rock inhibitor (Sigma-Aldrich, St. Louis, MO) at day 0. Stem cells were maintained in grids for five days with media changes every two days. For directed multi-lineage early differentiation we used the Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN).

For immunocytology of biomarkers in iPSC colonies, cells were prepared by two methods. Cells were fixed using 4% paraformaldehyde in PBS for 15min at room temperature and blocked by incubating cells for 90min in a solution containing 3% normal donkey serum and permeabilized by 0.1% Triton-X 100 for 10min before antibody addition. Incubations with the primary antibodies of anti-Nanog (Santa Cruz Biotechnology, Dallas, TX) and anti-SSEA4 (Santa Cruz Biotechnology, Dallas, TX) were done at 4C overnight, followed by incubation with a secondary antibody conjugated with Alexa 647 or Alexa 488 (Abcam, Cambridge, MA). After rinsing with phosphate buffered saline (PBS), the DNA was stained with bisBenzimide H 33258 (Sigma-Aldrich, St. Louis, MO) and cells imaged using a digital camera connected to a Nikon TE-2000 inverted microscope.

Phase imaging for in vitro differentiated samples was done on a Nikon 80i epifluorescence microscope using a PLAN 100.30 NA DL objective and images captured with a cooled QICam CCD camera. Fluorescent images were obtained on a Leica SP5 Laser Scanning Confocal Microscope using either HC PL FLUOTAR 100.30 NA or HCX PL APO CS 20X .70 NA objectives and also on a Zeiss AxioObserver Z1 Inverted Microscope with Colibri LED illumination, using a 100X oil 1.45 NA PlanFLUAR or 63X Plan-Apochromat 1.4 NA oil DIC objectives. Images were captured with a Hamamatsu ORCA ER CCD camera and Zeiss Axiovision Rel 4.8 acquisition software. Figures were compiled using Adobe Photoshop (Adobe Systems Inc., San Jose, CA) and Microsoft PowerPoint (Microsoft Corp., Redmond, WA) software.

The immunocytology of 2D cell cultures or three dimensional EBs was done by first fixing cells for 10 minutes at room temperature in 4% paraformaldehyde and stored overnight in PBS+0.1% Tween20 at 4C. Immediately before incubation with antibodies, the cells were permeabilized with PBS+0.5% Triton X-100 for 1 hour at 4C. Nonspecific binding was blocked by 20 minute incubation in 1% BSA in HBSS and followed by a single HBSS wash. Antibodies used for gauging pluripotency recognized Oct4A C-10 (Santa Cruz Biotechnology, Dallas, TX) and anti-SSEA4 (Millipore, Billerica, MA) (1:1000 each). Analysis of lineage commitment to differentiation was done using antibodies to OTX2 (ectoderm), SOX17 (endoderm), and Brachyury (mesoderm; 1:100 each) provided in the Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN). Secondary antibodies were either AlexaFluor 488 or AlexaFluor 594 (A-11001, A-11037, Invitrogen, Carlsbad, CA). Nuclei were stained with bisBenzimide H 33258 (Sigma-Aldrich, St. Louis, MO) at 4C overnight and followed by washing one hour in HBSS at 4C. Samples were mounted in ProLong Gold antifade reagent (Life Technologies, Grand Island, NY) at 20C overnight in the dark before imaging immediately or storing at 4C.

Approximately 2 million ED-iPSCs were injected subcutaneously in the flank region of NOD scid gamma (NSG) mice (The Jackson Lab, Bar harbor, ME). After 1224 weeks, teratomas were formed from 10 iPSC lines, and tumors were excised & fixed in 10% normal buffered formalin (NBF) overnight. The samples were processed for histology by the Division of Human Pathology at MSU. Hematoxylin- and eosin (H&E)-stained sections were examined under a microscope.

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