SUPPLEMENTARY INFORMATION

Supplementary Materials and Methods

Virus Production

To generate lentiviruses, 293T cells (Open Biosystems) were transfected at 80% confluence using calcium phosphate-mediated transient transfection.

Constructs for the four Yamanaka’s factors were a gift from Konrad Hochedlinger: FU-tet-o-hc-MYC (Addgene plasmid #19775), FU-tet-o-hKLF4 (Addgene plasmid #19777), FU-tet-o-hOct4 (Addgene plasmid #19778), FU-tet-o-hSox2 (Addgene plasmid #19779) and FUdeltaGW-rtTA (Addgene plasmid #19780) [1]. A mix of packaging construct (pMDL/pRRE, pRSV.Rev, pMD2.G) were used. Viruses were harvested over 3 days and concentrated 500-fold.

Cell Culture and hiPSC generation

Fibroblasts derived from dermis of 40-year-old healthy female were grown in DMEM with 10% Fetal Bovine Serum (FBS; EuroClone), 1% nonessential amino acids (NEAA; EuroClone), 2 mM L-glutamine (EuroClone), and 1% penicillin streptomycin (P/S; EuroClone).

For a standard reprogramming, in a 35 mm dish, fibroblast (105 cells, P2) were infected with doxycycline (dox)-inducible lentiviruses encoding for the Yamanaka’s factors: 10 µl rtTA + 5 µl factors (OCT4, SOX2, KLF4) + 2 µl cMYC was used in an overnight infection supplemented with 6 μg/ml polybrene (Sigma Aldrich). Two days after transduction, fibroblast were harvested by trypsinization and replated on a feeder layer of irridadiated mouse embryonic fibroblast (MEFs; GlobalStem, Rockville, MD). The next day medium was switched to human embryonic stem cell (hES)-medium including knock-out DMEM (KO-DMEM) (Gibco), 10% knock-out serum replacement (KSR) (GIBCO), 0,055 mM 2-mercaptoethanol (Gibco), 1% NEAA, 1 % GlutaMAX (Gibco), 1% P/S and supplemented with 10 ng/ml bFGF (R&D Systems).

Human iPS cells (hiPSCs) were generated as described by Maherali et al. [1]. hiPSC colonies were picked manually based on morphology between 4 to 6 weeks after dox induction and manually maintained and passaged according to hES protocols in the absence of dox. hiPSCs phenotypic characterization was performed using the Fluorescent Human ES/iPS cell Characterization Kit (Millipore, Billerica, MA, USA) following manufacturer directions.

In vitro differentiation

Embryoid bodies (EBs) were generated from large colonies fragments maintained in non-adherent dishes for 3-4 days in hES medium. EBs were than plated in 0,1% gelatin-coated dishes and cultured in differentiation medium (DMEM, 10% FBS, 1% GlutaMAX, 2-mercaptoethanol, 1% NEAA and 1% P/S) for 2-3 weeks.

The cells were stained with specific markers for the three germ layers: anti α-smooth muscle actin (α-SMA) monoclonal antibody (Sigma Aldrich), anti α-fetoprotein (AFP) polyclonal antibody (Dako), anti βIII tubulin polyclonal antibody (Millipore), anti glial fibrillary acidic protein (GFAP) polyclonal antibody (Dako, Denmark), anti fibronectin polyclonal antibody (Sigma Aldrich), anti desmin polyclonal antibody (Millipore) (Supplementary Table S2).

Karyotype analysis

Karyotyping was performed by incubating colonies for 3h in Colcemid (Invitrogen). 10/20 colonies from each samples were picked up. Metaphase suitable for analysis is sequentially Q banded according to the routine methods. To confirm analysis, fluorescence in situ hybridization (FISH) technique with commercial probe SEX(DXZ1)/Y(DYZ3) was used.

Quantitative RT-PCR (qPCR) and Scorecard analysis

Total RNA was extracted from hiPSC and EBs pellets using Tri Reagent (Sigma). Eight cDNA reactions were set up from 1 µg of total RNA per sample using a High-Capacity cDNA RT kit (Life Technologies) and following manufacturer instructions. qPCR was performed on TaqMan® hPSC Scorecard™ Panel, 96-well Fast (Life Technologies) plates using Viia7 RUO software and a ViiA7 instrument (Applied Biosystems).

Data were analyzed through TaqMan® hPSC Scorecard™ Analysis Software through Life Technologies website.

Supplementary Results

hiPSC Generation and Characterization

Human fibroblasts derived from dermis of 40-year-old healthy female were established and used for reprogramming at passages 2 using the Yamanaka’s factors [2]. Briefly, fibroblasts were infected with doxycycline (dox) -inducible lentiviral vectors carrying the human cDNAs encoding the four transcription factors OCT4, SOX2, cMYC, KLF4. Fibroblasts were simultaneously infected with a constitutively active lentivirus transducing the reverse tetracycline transactivator (rtTA). Human iPS cells (hiPSCs) were generated as described by Maherali et al. [1]. In particular, two days after transduction, cells were plated onto irridadiated mouse embryonic fibroblast (MEF) feeder cells and cultured in human embryonic stem cell (hES) medium in the presence of dox (Fig. S1A , panel a). Following 4 days, the morphology of fibroblasts changed (Fig. S1A, panel b) leading to the acquisition at day 20 of the typical hES features, such as the well-defined phase-bright borders, an high nucleus-to-cytoplasm ratio and prominent nucleoli (Fig. S1A, panel c). These colonies were manually picked at day 30 and expanded on MEF feeders in hES medium containing bFGF in the absence of dox (Fig.S1A, panel d). By using immunocytochemistry, we found that hiPSC clones uniformly expressed OCT4, SOX2 and NANOG, transcription factors strongly associated with the acquisition of pluripotency (Fig. S1A panels e, f, g). In addition, the expression of the surface markers tumor-related (TRA)-1-60, TRA-1-81 and TRA-2-49/6E (alkaline phosphatase, AP) were also detected (Fig.S1A, panels h, i, j).

The differentiation ability of hiPSCs in vitro was next tested. To this aim, cells were allowed to grow in suspension for 7 days to form Embryoid Bodies (EBs) (Fig.S1B, panel k and l) and successively plated and cultured for 8 more days. The presence of various type of cells derived from the three main embryo germ layers were detected by using immunofluorescence analysis. In particular, we clearly detected βIII tubulin (Tuj1) and glial fibrillary acidic protein (GFAP), used as a marker for ectoderm (Fig. S1B, panel m-n), α-smooth muscle actin (α-SMA) and desmin (Fig.S1B, panel o, p), as marker for mesoderm and α-fetoprotein (AFP) and fibronectin (Fig. S1B, panel q, r), marker for endoderm thus indicating the pluripotency of the hiPSC.

To further assess pluripotency and three-germ-layer differentiation potential of hiPSCs generated, the TaqMan® hPSC Scorecard Assay (Life Technologies) was performed. The assay quantitatively confirms trilineage differentiation potential on the basis of 94 predefined TaqMan® Gene Expression assays (Fig. S1C). Total RNA was isolated from hiPSCs and from EBs. Increased expression profiles of markers of all three-germ-layers could be detected in EBs, with highest up-regulation of genes associated with the mesodermal lineage (Fig. S1D).

Finally, genome integrity was assessed by karotyping analysis showing a normal karyotype [46; XX] of the hiPSC (Fig.S1E).

Altogether, these results indicate that the hiPSCs generated by our protocol possesses the morphological, molecular and functional properties typically associated with hES.

Supplementary Figures

Fig. S1 Induction of iPSC from human dermal fibroblast. A) Phenotypic characterization of human iPSC (hiPSC) (a) Morphology of human fibroblasts. (b) Morphology of human fibroblast at day 4 from lentiviral infection. (c) Typical image of hES cell-like colony at day 30. (d) Morphology of established hiPSC line at day 60. (e-j) Immunofluorescence staining of hiPSCs colonies for pluripotency markers performed after 5 days in culture. Representative microphotographs of hiPSCs stained for (e) Oct4, (f) SOX2, (g), Nanog (h) TRA-1-60, (i) TRA-1-81 and (j) alkaline phosphatase. Nuclei were stained with DAPI (blue). Scale bars=50 μM. B) Pluripotency of iPSCs derived from human fibroblasts. Left column shows floating culture of hiPSCs at day 8 (panel k-l). Immunocytochemistry analysis of the in vitro differentiation into all three germ layers: ectoderm (m) βIII-tubulin (TuJ1) and (n) GFAP; mesoderm (o) α-SMA and (p) Desmin; endoderm (q) AFP and (r) Fibronectin. Nuclei were stained with DAPI (blue). Scale barr=50 μm. C) Transcriptome analysis shows down-regulation of genes involved in self-renewal in embryoid bodies (EBs) compared with hiPSC, whereas some of these genes were up-regulated in hiPSC. D) Summary analysis of the transcriptome profile confirms down-regulation of self-renewal genes and up-regulation of markers of all three-germ-layers in EBs. E) Karyotype analysis of hiPSC [46;XX]

Supplementary Table

GENE / Forward 5'-3' / Reverse 5'-3' / Product size / TA (°C)
D2R / GAAAGCCACTCAGATGCTCG / GCAGTGGAGGATCTTCAGGA / 223 / 60
D3R / GTCTGGGTACTGGCCTTTGC / AGTCACTCCAAAGGGCAGGT / 144 / 60
D4R / ccttcttcgtggtgcacat / aactcggcgttgaagacagt / 139 / 60
DAT / TCACCACCTCCATCAACTCC / GGGTGAGCAGCATGATGAAG / 210 / 59
DDC / ACATGGAAGGCATTGAGGGA / GGGGAAGTAGGCGAAGAAGT / 185 / 59
FOXA2 / ACAACCTCATGTCCTCGGAG / TCATAATGGGCCGGGAGTAC / 228 / 59
GAD67 / TCTTGCAAAGGACCAACAGC / ATGTCCACCACTTCCAGGAG / 220 / 59
GAPDH / AGGTCGGAGTCAACGGATTT / ATCTCGCTCCTGGAAGATGG / 230 / 60
GIRK2 / GGTCTTTTCCACCCATGCAG / ACACCAGAAACAGACGGTCA / 215 / 59
HTR2C / GTGATTGGACTGAGGGACGA / CCGTATTCCTCTTGCAGCAC / 244 / 59
LMX1A / CTCAACAGAGGCGAGCATTC / GGCCAGCTTCTTCATCTTCG / 152 / 59
MAP2 / CCCCTTGCTTCCCTGTAGAA / ATTTCCTCCTGGCAACCTCA / 231 / 59
NESTIN / TCTTTGCTCCCAGTCCTGAG / GGGCTCTGATCTCTGCATCT / 187 / 55
NET / TTGCTCTGTCTGATGGTCGT / GAGTTGCGGCATCAATCCAT / 229 / 59
NURR1 / CACTTCTCTCCCCAGCTTCA / CGGCATCATCTCCTCAGACT / 184 / 59
OCT4 / CTCTGAGGTGTGGGGGATT / AGCTAAGCTGCAGAGCCTCA / 419 / 64
PAX6 / TTGCCCGAGAAAGACTAGCA / TGGAGCCAGATGTGAAGGAG / 228 / 59
PTX3 / CAGGCCGAGCTATGCAAAG / GACCGAGTTGAAGGCGAATG / 159 / 59
SERT / TCATCTTCACAGTGCTCGGT / ACGAGGGATCCAAAGAAGCA / 304 / 59
SOX2 / ACTTTTGTCGGAGACGGAGA / TATAATCCGGGTGCTCCTTC / 86 / 64
TH / CCGAGCTGTGAAGGTGTTTG / ACACTTGTCCAGCTCTGACA / 238 / 59
VGAT / CTACACCGGCAAGATCCTCA / CCGGGAAGCTGTTGTACATG / 224 / 59
VGLUT2 / CATGGTCAACAACAGCACCA / ACTCTGGCTGCTGATGGAAT / 234 / 59
VMAT2 / CCGTACATCCTCATTGCTGC / CCACCTCCCCATTTTGTGTG / 201 / 59
α4*nAChR / CGAACGTATGGGTGAAGCAG / GCATGTTCACCAGGTCGATC / 330 / 59
α6*nAChR / CGTGGTGACTGTGTTTGTGT / CTCCGAATTTTCCACCACCC / 319 / 59
β2*nAChR / TCACCTGGAAGCCTGAAGAG / TGTCCACGTACGTAGAGTCG / 307 / 59

Table S1 List of primers used in RT-PCR analysis

ANTIBODY / SOURCE / COMPANY / CODE / APPLICATION
AADC / Rabbit / Sigma Aldrich / #HPA017742 / IF/WB
α-SYN (211) / Mouse / Santa Cruz / #sc-12767 / IF
DAT / Rat / Santa Cruz / #sc-32258 / IF/WB
FoxA2 / Goat / Santa Cruz / #sc-9187 / IF
Lmx1α / Rabbit / Sigma Aldrich / #HPA030088 / IF
MAP2 / Rabbit / Merck Millipore / #AB5622 / IF
Nestin / Mouse / Chemicon / #MAB353 / IF
NET / Rabbit / Santa Cruz / #sc-67216 / IF
Pax6 / Rabbit / Covance / #PRB2789 / IF
p-ERK1/2 / Mouse / Santa Cruz / #sc-7383 / WB
TH / Mouse / Merck Millipore / #MAB318 / IF
TH / Rabbit / Santa Cruz / #sc-14007 / IF/ICC
TH / Rabbit / Merck Millipore / #AB152 / WB
α-tubulin / Mouse / Sigma Aldrich / #T5168 / WB
α-SMA / Mouse / Sigma Aldrich / #A2547 / IF
α-fetoprotein (AFP) / Rabbit / Dako / #A0008 / IF
βIII-tubulin / Rabbit / Merck Millipore / IF
GFAP / Rabbit / Dako / #Z0334 / IF
Fibronectin / Rabbit / Sigma Aldrich / #F3648 / IF
Desmin / Rabbit / Merck Millipore / IF

Table S2 List of antibodies used in immunofluorescence (IF), immunocytochemistry (ICC) and western blot (WB) analysis


Supplementary References

1.  Maherali N, Ahfeldt T, Rigamonti A, Utikal J, Cowan C, Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell 3(3): 340-5

2.  Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861-872

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