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1、Mauritz Flk-1 progenitor cells from murine iPSCsSUPPLEMENTARY MATERIALSupplementary methodsCultivation of iPSCsThe iPSC clone B4 was routinely cultivated and expanded on mitotically inactivated murine embryonic fibroblasts (MEFs). The culture medium was composed of Dulbeccos Modified Eagles Medium (
2、Invitrogen, Karlsruhe, Germany) supplemented with 15% FCS (Thermo Scientific, Bonn, Germany), 0.2mM L-glutamine (Invitrogen), 0.1mM bmercaptoethanol (Invitrogen), 0.1mM non-essential amino acid stock (Invitrogen), and 0.1% huLIF (human leukemia inhibitory factor) conditioned medium. The latter had b
3、een produced by transient transfection of a huLIF expression plasmid into human embryonic kidney 293 T cells. Every three to four days colonies were detached with 0.2% collagenase IV (Invitrogen), dissociated into single cells with 0.025% trypsin (Sigma-Aldrich, Taufkirchen, Germany) and 0.1% chicke
4、n serum (Invitrogen) in phosphate-buffered saline (PBS) and plated again onto MEFs.Feeder-free iPSC culture for initiation of differentiation and for colony assays was performed on 6-well culture dishes (Thermo Scientific) coated with 0.1% gelatin (Figure S1). The medium was supplemented with 1M inh
5、ibitor of glycogen synthase kinase-3 (Merck, Darmstadt, Germany)1. Teratoma assay of iPSCs2.5x105 viable iPSCs from the B4 clone were injected into lower limb skeletal muscle of SCID beige mice (n=4). Four weeks later tumours were dissected and paraffin sections were stained with haematoxylin and eo
6、sin (H&E). All sections were analysed by an experienced pathologist with regard to teratoma formation.In vitro differentiation of iPSCsBefore starting differentiation iPSCs were cultivated for several passages without feeder cells to eliminate contaminating MEFs. Colonies were detached with 0.2% col
7、lagenase IV and dissociated into single cells with 0.025% trypsin and 0.1% chicken serum. To initiate embryoid body (EB) formation in suspension iPSCs were cultured at 0.5x105 cells/ml in differentiation medium consisting of Iscoves Modified Dulbeccos Medium (Invitrogen) supplemented with 15% FCS, 0
8、.2 mM L-glutamine, 0.1mM bmercaptoethanol and 0.1mM non-essential amino acid stock and kept on an orbital shaker. For the formation of “hanging drop”-EBs 600 cells were aggregated per 20l of differentiation medium.On day 5 of differentiation EBs were either plated onto 0.1% gelatin-coated 6-well cul
9、ture dishes or were processed for fluorescent activated cell sorting (FACS). To study the potential of the iPSC clone B4 to differentiate into cardiovascular lineages, plated EBs were examined for the appearance of spontaneously beating areas. Cells were immunostained with antibodies against cardiac
10、 troponin T (cTnT), connexin 43 (Cx43), Cd31 and -smooth muscle actinin (-SMA). Flow cytometry and cell sorting of iPSC-derived Flk-1 cellsOn days 3 to 6 of differentiation EBs were dissociated into single cells by incubation for 4min with 0.025% trypsin and 0.1% chicken serum. Cells were stained wi
11、th rat anti-mouse Flk-1-Allophycocyanin (APC) or with the corresponding rat IgG2a-APC isotype control (BD Biosciences, Heidelberg, Germany) and the percentage of living Flk-1pos cells excluding propidium iodide (BD Biosciences) was analysed on a FACSCalibur (BD). On day 5 of differentiation cells we
12、re sorted on a FACSAria IIu cell sorter (BD) into nucVenuspos/Oct4-GFPneg/Flk-1-APCneg (Flk-1neg) and nucVenuspos/Oct4-GFPneg/Flk-1-APCpos (Flk-1pos) populations. Those populations were either directly used for quantitative real-time RT-PCR (qRT-PCR) analyses or for intramyocardial injections or the
13、y were further cultivated as reaggregates. For the formation of reaggregates “hanging drops” composed of 6x103 viable cells in 20l differentiation medium were generated. Two days later reaggregates were plated onto gelatin-coated 6-well culture dishes.Analysis of mRNA expression by qRT-PCRTotal RNA
14、was isolated using TriZol (Invitrogen), according to the manufacturers instructions. Integrity of total RNA was controlled using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Potentially contaminating genomic DNA was digested by DNAse I (Stratagene, La Jolla, CA, USA) for 15m
15、in at 37C followed by phenol/chloroform-extraction. After precipitation, 300ng RNA was used for Oligo(dT)18-primed cDNA synthesis with RevertAidTM H Minus First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany). qRT-PCR was performed using a Mastercycler ep realplex (Eppendorf, Hamburg, G
16、ermany) and the ABsolute QPCR SYBR Green Mix (Thermo Scientific). PCR reaction mixtures contained cDNA template, primers and 1x ABsolute QPCR SYBR Green Mix in a final volume of 25l. Real-time PCR conditions included enzyme activation at 95C for 15min, followed by 40 cycles (denaturation at 95C for 15sec, annealing at TA (for annealing temperatures of individual primer pairs (see Table S1) for 60sec and extension at 72C for 60sec,
