Jing Liao, Chun Cui, Siye Chen, Jiangtao Ren, Jijun Chen, Yuan Gao, Hui Li, Nannan Jia, Lu Cheng, Huasheng Xiao, and Lei Xiao

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1 Cell Stem Cell, Volume 4 Supplemental Data Generation of Induced Pluripotent Stem Cell Lines from Adult Rat Cells Jing Liao, Chun Cui, Siye Chen, Jiangtao Ren, Jijun Chen, Yuan Gao, Hui Li, Nannan Jia, Lu Cheng, Huasheng Xiao, and Lei Xiao Supplemental Experimental Procedures Cell culture. Rat bone marrow cells (BMC) were cultured in αdmem culture medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum. Rat primary ear fibroblasts (PEF) were cultured in DMEM culture medium (Invitrogen) supplemented with 10% fetal bovine serum. Rat ips cells were maintained on irradiated mouse embryonic fibroblasts (MEF) in Knockout DMEM supplemented with 10% ES cell qualified fetal bovine serum, 10% KnockOut serum replacer, 0.1 mm non-essential amino acids, 1 mm L-glutamine, and 0.1 mm ß-mercaptoethanol (ES media) (all from Invitrogen, Carlsbad, CA). Rat ips cells were trypsinized into single cells with TTL trypsin and split at a ratio of 1:10 every 3 days. To form embryoid bodies, rat ips cells were trypsinized into single cells and transferred to a Petri dish in differentiation medium consisting of DMEM (Invitrogen) supplemented with 10% fetal bovine serum. Lentiviral transduction and reprogramming culture. The cdna of human gene were inserted downstream of EF-1 alpha promoter and upstream of IRES-EFGP of lenti-vector(liao et al., 2008). Half a million rat PEF or BMC from SD rat were transduced with a lentivirus carrying GFP (negative control) or a cocktail of lentivirus carrying reprogramming factors at day 0. Two days later, cells were dissociated with trypsin and transferred to 6-well plates coated with an MEF feeder and cultured in ES media. On day 10, the ips colonies were picked, dissociated by trypsin digestion, and plated into new culture dishes. Immunostaining. Immunostaining was carried out similarly as described (Xiao et al., 2006). The primary antibodies used were anti-nanog (R&D Systems), anti-ssea1 (Developmental Studies Hybridoma Bank), anti-ssea3 (Developmental Studies Hybridoma Bank) and anti-ssea4 (Developmental Studies Hybridoma Bank), anti-alpha-fetal protein (AFP) (Abcam, ab46799). AFP was used as a marker of gut-like epithelia in teratoma(aleckovic and Simon, 2008). The photo of SSEA1 staining was acquired by confocal microscopy. 1

2 Real-time reverse transcription-polymerase chain reaction. Total RNA was prepared using the RNAeasy kit (Qiagen) and used as a template for reverse transcription-polymerase chain reaction (RT-PCR). Real-time PCR was performed in an Eppendorf Mastercycler ep realplex real-time PCR system using a SYBR Green-based PCR Master mix (TOBOYO). PCR primers are listed in Supplemental Table S2. A standard curve for both the gene of interest and the endogenous control (GAPDH) were acquired. The unit defined by standard curve is copy number. The CT data of the gene of interest and the endogenous control (GAPDH) obtained from the real time PCR were transform into copy number by the standard curve. The expression value of each gene was normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase cdna in order to calculate the relative amount of RNA present in each sample. The copy number of the target gene was defined as copies per 10 6 copies of GAPDH. Microarray Total RNA was extracted with TRIZOL reagent (Invitrogen) and further purified using an RNeasy column (Qiagen, Hilden, Germany, The labeling procedure was carried out by using a RNA Fluorescent Linear Amplification Kit (Agilent Technologies, Palo Alto, CA, agilent.com). The sample and control were labeled with cy-3 and cy-5, respectively. Fragmentation was carried out by incubating at 60 C for 30 minutes in fragmentation buffer (Agilent Technologies) and stopped by adding equal volume of hybridization buffer (Agilent Technologies). Fragmented target was applied to Whole Genome Oligo Microarray of rat, mouse and human, respectively (Agilent Technologies). Hybridization proceeded at 60 C for 17 hours in a hybridization oven (Robbins Scientific, Sunnyvale, CA). The hybridized array was scanned with Agilent microarray scanner. The TIFF image generated was loaded into Feature Extraction Software (Agilent Technologies) for feature data extraction. The data analysis was performed with GeneSpring To do a cross-species comparison, we have to set a baseline for each species. Then we can compare sample versus the baseline to get relative data (a ratio of change). At the end, we can compare the relative data in different samples to get the information of the similarity of the samples from different species. MEFs are a mixture of many cell types. A mixed cell sample for the rat or human should be more similar to MEF than one cell type. To identify the enriched genes, mouse cells were compared with murine embryonic fibroblast (MEF), human cells were compared with a mixture (1:1) of Human newborn foreskin fibroblasts (ATCC, catalog number CRL-2097) and IMR90 fetal fibroblasts (ATCC, catalog number CCL-186), and rat cells were compared with a mixture (1:1) of BMC and PEF. MEF, human foreskin fibroblast cells and human fetal lung cell were routinely used to produce mouse and human ips cells, respectively(park et al., 2008; Takahashi and Yamanaka, 2006; Yu et al., 2007). BMC and PEF were the cells used to produce rat ips cells in our study. The significantly enriched genes were selected according to the following criteria: P 0.01 and fold change 3. The ortholog search was performed with GeneSprings The signal ratios of orthologous genes were used to perform hierarchical clustering (conditioned tree) with GeneSpring The signal ratio is log ratio unless specifically defined. The microarray data have been submitted to GEO. Accession number is GSE

3 Note: 1. The probes on the Agilent Oligo Microarray were designed to locate in the 3 -UTR region of each gene. Rat Nanog(ACCESSION NO. AB275459)doesn t contain 3 -UTR. Therefore, there is no probe for Nanog in Agilent Oligo Microarray. The exogenous genes don t contain 3 -UTR neither. 2. The probe on rat microarray, A_44_P506024, represents rat sox2 (IMAGE clone: ). Bisulfite genomic sequencing Bisulfite treatment was performed using the CpGenome modification kit (Chemicon) according to the manufacturer s recommendations. PCR primers are listed in Table S3. Amplified products were cloned into T-vector, and at least ten randomly selected clones were sequenced. Telomerase activity of ips cells The telomerase activity of the ips cells and ES cells was determined with the TRAPEZE telomerase detection kit (Chemicon) according to the manufacturer s recommendations. The lysates were heated at 85 C for 10 minutes and used as negative controls. Reactions were separated with non-denaturing TBE-based 12% polyacrylamide gel electrophoresis and visualized with SYBR Gold staining. Teratoma formation Cells were injected intramuscularly into non-obese diabetic/severe combined immune deficient (NOD/SCID) mice (~ cells per site). After 4 6 weeks, tumors were processed for hematoxylin-eosin staining. All animal experiments were conducted in accordance with the Guide for the Care and Use of Animals for Research Purposes and were approved by the SIBS Animal Care Committee. Supplemental References Aleckovic, M., and Simon, C. (2008). Is teratoma formation in stem cell research a characterization tool or a window to developmental biology? Reprod Biomed Online 17, Liao, J., Wu, Z., Wang, Y., Cheng, L., Cui, C., Gao, Y., Chen, T., Rao, L., Chen, S., Jia, N., et al. (2008). Enhanced efficiency of generating induced pluripotent stem (ips) cells from human somatic cells by a combination of six transcription factors. Cell Res 18, Park, I. H., Zhao, R., West, J. A., Yabuuchi, A., Huo, H., Ince, T. A., Lerou, P. H., Lensch, M. W., and Daley, G. Q. (2008). Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 3

4 Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, Xiao, L., Yuan, X., and Sharkis, S. J. (2006). Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells. Stem Cells 24, Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318,

5 Table S1. Characterization of rat ips cell lines. Cell name AP SSEA1 SSEA3 SSEA4 NANOG Teratoma Karyotyping Telomerase PEF ND 42XY - BMC ND 42XY - M11 + ND ND ND ND - ND ND M ND ND + M XY + F ND + F ND ND ND ND F XY + F ND + F ND ND ND F XY + F ND ND ND 5

6 Table S2. Primers for RT-PCR Gene Name Oct4 endo-f Oct4 endo-r Oct4 exo-f Oct4 exo-r Sox2 endo-f Sox2 endo-r Sox2 exo-f Sox2 exo-r Nanog endo-f Nanog endo-r Nanog exo-f Nanog exo-r Nodal-f Nodal -r Fgf4-f Fgf4-r Gal-f GalL-r Sequence CGAGGCCTTTCCCTCTGTTCCT TCTCTTTGTCTACCTCCCTTCCTTGC AGAAGGATGTGGTCCGAGTGTG CAGAGTGGTGACAGAGACAGGG GGCCATTAACGGCACACTGCC TTACTCTCCTCTTTTGCACCCCTCC TCTTGGCTCCATGGGTTCGG AGTGCTGGGACATGTGAAGTCTG ACCTACCTCTTCAAGATAGCCCTG ACCTTTGCCTCTGAAACCTATCCT GCTGAGATGCCTCACACGGA GGTCTTCACCTGTTTGTAGCTGAG GAGCGTGTTTGGATGGAGAGG ATGCCAACACTTTCCTGCTTGAC GTGTGCCTTTCTTTACCGACGAGTG GGAAGTGGGTTACCTTCATGGTCG TGGAAGTGGAGGAAGGGAGACTAGG GGGATGCCAGGCAGGCTGTC 6

7 Leftyb-f Leftyb-r Lin28 3'UTR-f Lin28 3'UTR-r Gabrb-f Gabrb-r Sox17-f Sox17-r AFP-f AFP-r NCAM-f NCAM-r PAX6-f PAX6-r SM22-a-f SM22-a-r Myod-f Myod-r TGACCATCAGGTGGCCATTTCTG TGTTGGGCAGGCTGACCACTTG CGGGAGGAGGAAGAAGAGATCCAC CCACTCTGCGGATTGATGCCTC AATCAACCGGGTGGATGCTC GTGCGGGATGCTTCTGTCTC GGCACGGAACCCAACCAGC CAGTCGTGTCCCTGGTAGGGAAGAC TCTGAAACGCCATCGAAATGCC AATGTAAATGTCGGCCAGTCCCT TGCTCAAGTCCCTAGACTGGAACG CTTCTCGGGCTCTGTCAGTGGTGTGG TGCTCAAGTCCCTAGACTGGAACG GGACGGGAACTGACACTCCAGG GCTGAAGAATGGCGTGATTCTGAG CCTTCAAAGAGGTCAACAGTCTGG GCTCAGGAAGATTGCTGTGTCCA CCGCAACACTCCATGCATATCTCC 7

8 Table S3. PCR primers for Bisulfite genomic sequencing Name Sequence Region (ATG +1) roct4-of-1 TTATAGTGAATGTATAGATATTGGGAG -1794bp~-1202bp roct4-or-1 CCTCCAAAATCCCATTACTAAC -1794bp~-1202bp roct4-of-1 TTATAGTGAATGTATAGATATTGGGAG -1794bp~-1202bp roct4-ir-1 ACTAACCCAATACTTAATTTATCTTTAC -1794bp~-1202bp roct4-of-2 TTTAAGATTTAGGAGGTAAGAAGTCG -2357bp~-1932bp roct4-or-2 AAAACTAAAACCACCCAATTC -2357bp~-1932bp roct4-if-2 TTCGTGGTTATCGGGTTAATATTAG -2357bp~-1932bp roct4-ir-2 CTACCCCCAAAACAAAACTATC -2357bp~-1932bp 8

9 Figure. S1. The expressions of endogenous Oct4 and Nanog in rat ips cells were not maintained by LIF. Figure. S2. The relative expressions of Tert were determined by real-time PCR. Figure. S3. PEF and BMC don t express SSEA1. 9