Human Fetal Fibroblasts (IMR90 Cell Line, ATCC, Catalogue Number CCL-186) Were Grown To

Supplementary methods

Fibroblast reprogramming and iPSC validation

Human fetal fibroblasts (IMR90 cell line, ATCC, catalogue number CCL-186) were grown to confluence in 60-mm culture dishes in DMEM supplemented with 20% (vol/vol) fetal bovine serum (FBS). These cells were infected twice (once every 24 hours) with retroviruses expressing the transcription factors OCT4, SOX2 and KLF4. Retroviral production was performed as described before[1]. The day following the second infection, cells were plated on mitotically inactivated murine embryonic fibroblasts (MEFs) layer on Matrigel (BD)-coated dishes. Approximately after 6 weeks of culture in DMEM-F12, supplemented with 20% Knockout serum replacement, non-essential amino acids, sodium pyruvate, 1% penicillin-streptomycin, 1% L-glutamine, 0.1 mM β-mercaptoethanol (Gibco) and 10 ng/ml basic Fibroblast Growth Factor (bFGF, Peprotech), iPSCs colonies started to appear and were manually picked using a stereomicroscope (Leica). Then, they were cultured in human embryonic stem cell conditions on MEFs and split in 1:3 ratio by Collagenase IV (1 mg ml-1, Life Technologies) digestion once a week. To verify in vitro the ability to differentiate towards the three germ layers iPSCs were cultured in ESCs medium without bFGF supplemented with 20% fetal bovine serum on 1 mg/ml gelatine (Sigma)-coated dishes for up to 20 days and then tested by immunofluorescence with corresponding markers: Sox17 (clone 245013, R&D) for endoderm, Smooth Muscle Actin (SMA, clone 1A4 Abcam) for mesoderm and Neuronal Class III Beta Tubulin (clone Tuj1, Covance) and Nestin (clone MAB353, Millipore) for ectoderm. To corroborate their pluripotency, 1*106 hiPSCs (2 clones for each reprogrammed line) were subcutaneously injected into immunodeficient mice (6 week old SCID female mice) to test their ability to generate teratomas. After 2-3 months mice displayed subcutaneous masses that were dissected and paraffin-embedded. An histological evaluation of the explanted tissue by hematoxylin and eosin staining was performed to confirm the presence of teratoma. Differentiation experiments were performed on two clones (number 1 and 5) of reprogrammed fetal fibroblasts and on one clone of reprogrammed adult fibroblasts.

Differentiation of iPSCs into pancreatic cells

The differentiation schedule consists of 6 stages with different culture conditions: (i) stage 1, mesendoderm formation: RPMI 1640 (Lonza) supplemented with 100 ng/ml Activin A and 25 ng/ml Wnt3a (R&D Systems) for 2 days; (ii) stage 2, definitive endoderm formation: RPMI supplemented with 0.2% FBS (Lonza) and 100 ng/ml Activin A for 2 days; (iii) stage 3, primitive gut tube formation: RPMI supplemented with 2% FBS, 50ng/ml of Fibroblast Growth Factor-10 (FGF-10, R&D Systems) and 0.2 μM KAAD-cyclopamine (CYC, Calbiochem) for 2 days; (iv) stage 4, posterior foregut formation: DMEM (Lonza) supplemented with 1% B27 (Invitrogen), 50 ng/ml FGF-10, 0.2 μM CYC and 2 μM Retinoic Acid (Sigma Aldrich) for 4 days, replacing with fresh medium on the second day; (v) stage 5, pancreatic endoderm formation: DMEM supplemented with 1% B27, 1 μM N-[N-(3,5-Difluorophenacetyl) L-alanyl]-S-phenylglycine t-butyl ester (DAPT, Sigma Aldrich) and 50 ng/ml Exendin-4 (AnaSpec) for 3 days; (vi) stage 6, hormone expressing endocrine cells formation: CMRL-1066 (Connaught Medical Research Laboratories, Mediatech) supplemented with 1% B27, 50 ng/ml Exendin-4 , 50 ng/ml Insulin Growth Factor- 1 (IGF-1, Sigma Aldrich) and 50 ng/ml Hepatocyte Growth Factor (HGF, Peprotech) for 5 days, replacing with fresh medium on the second day. Starting from this outline, two modifications were introduced: (i) 300 nM Indolactam V[2] (Alexis Biochemicals) was added to the culture during stage 4 and (ii) cells were detached with 4 mg/ml collagenase IV (Gibco) and re-seeded in Ultra Low Attachment Plates (Corning) between stage 4 and 5.

Molecular analysis by Droplet Digital PCR (ddPCR) of iPSC differentiation

A total of 50ng were used to set-up 3 replicate ddPCR reactions; these were emulsified in a QX100 droplet generator (Bio-Rad), transferred to 96 well plates and subjected to thermal cycling on a T100 instrument (Bio-Rad), according to the manufacturer’s instructions. After amplification, plates were read and individual sample droplets analyzed on a Bio-Rad QX100 droplet reader. The number of gene copies/ng of equivalent RNA was determined using the QuantaSoft v1.2.10 software, applying a correction based on the Poisson distribution to the counted number of droplets positive for the different time points.

MicroRNAs expression analysis

RT reactions containing 10ng of RNA, specific stem-loop primers for each miRNA, 1X buffer, dNTPs, reverse transcriptase and RNAse inhibitor were incubated in a DOPPIO™ Thermalcycler (VWR) for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C and then held at 4°C. Real Time PCR runs were performed by using miRNA specific TaqMan MGB probe and TaqMan Universal Master Mix II in duplicate in a VIIA7 Real Time PCR instrument (Applied Biosystem). MiRNAs expression levels were normalized by using two different endogenous control smallRNAs: RNU6 and RNU48.

1. Takahashi K, Tanabe K, Ohnuki M, et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–72. doi: 10.1016/j.cell.2007.11.019

2. Chen S, Borowiak M, Fox JL, et al. (2009) A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nat Chem Biol 5:258–65. doi: 10.1038/nchembio.154