the tsc lines used in this study were b6ts4 and egfp - ts3.5 , which were derived from a blastocyst 
 of the c57bl/6 and icr mouse strain , respectively . tscs were cultured as described previously . in brief , cells were cultured on mitomycin - c ( sigma - aldrich , st . louis , 
 mo , usa)-treated primary mouse embryonic fibroblasts in rpmi1640 medium ( thermo fisher scientific , san jose , 
 ca , usa ) with 20% fetal bovine serum ( thermo fisher scientific ) , 25 ng / ml human recombinant fgf4 ( wako pure 
 chemicals , osaka , japan ) , 1 g / ml heparin ( sigma - aldrich ) , 100 m 2-mercaptoethanol ( sigma - aldrich ) , 1% 
 glutamax ( thermo fisher scientific ) , and 1 mm sodium pyruvate ( thermo fisher scientific ) . representative colony 
 morphologies are shown in fig . 1 , together with descriptions of 
 their morphological characters . there was an additional type ( type 5 ) , but this was not analyzed here because 
 it rarely emerged under normal tsc culture conditions . at 96 h after passaging , colonies were picked up using 
 a glass capillary pipette under a dissecting microscope and used for rt - qpcr analysis . total rna for rt - qpcr was extracted with rneasy micro kits ( qiagen , venlo , netherlands ) from tsc colonies . following extraction , the first strand cdna was synthesized with a superscript iii reverse transcriptase 
 reagent set ( thermo fisher scientific ) . gene expression was assessed by qpcr on a steponeplus instrument 
 ( thermo fisher scientific ) using quantitect sybr green pcr kits ( qiagen ) according to the manufacturer s 
 instructions . samples were denatured at 95c for 10 min , followed by 40 amplification cycles consisting of 
 denaturation at 95c for 15 sec , and an annealing and extension step at 60c for 1 min . raw cq ( ct ) values 
 ( pcr cycles at which the fluorescence signal crosses threshold ) were calculated using stepone software ( v. 
 2.1 ; thermo fisher scientific ) setting baseline and appropriate threshold values . all runs were performed in 
 triplicate and an identical sample was used in each different runs as an inter - run calibration sample to 
 correct for the technical variance between the runs and thus compare results from different plates [ 4 , 6 ] . the primer sets used for tsc 
 marker genes were the following : cdx2 , 5gcagtccctaggaagccaag3 and 
 5gcagccagctcacttttcct3 ; elf5 , 5gtggcatcctggaatgggaa3 and 5cactaacctccggtcaaccc3. 
 for quantification of reference genes ( atp5b , canx , cyc1 , 
 gapdh , ubc , rn18s ) , the primer sets from the mouse genorm 
 kit ( primerdesign ltd . , southampton , uk ) were used . actb was analyzed as an additional 
 reference gene using the primer set 5ctgtcgagtcgcgtcca3 and 5acccattcccaccatcacac3. the baseline cycles and cycle thresholds were established manually for each gene . to assess the stability of 
 the reference genes , the raw cq values of each reference gene were analyzed with the genorm algorithmic tool 
 using qbase software ( biogazelle ) . these values were converted into normalized relative 
 quantities ( nrqs ) with multiple reference genes based on the modified classic --ct method   or a single reference gene . nrqs were scaled in relation to the mean 
 across all samples for each target or to reference genes . we ranked the stability of the reference genes based 
 on the mean expression stability ( genorm m value ) provided by the genorm program . to determine the optimal 
 number of reference genes across different colony types , qbase was also used to calculate 
 pairwise variation ( v value ) . normalization of the expression levels of tsc marker genes 
 ( cdx2 and elf5 ) was performed by qbase software using 
 reference gene(s ) ranked as described above . to compare the gene expression levels between colony types within the tsc lines , normalized relative mrna 
 levels were analyzed with kruskal - wallis tests followed by dunnett s multiple comparison tests ; p < 0.05 
 was considered statistically significant . the tsc lines used in this study were b6ts4 and egfp - ts3.5 , which were derived from a blastocyst 
 of the c57bl/6 and icr mouse strain , respectively . tscs were cultured as described previously . in brief , cells were cultured on mitomycin - c ( sigma - aldrich , st . louis , 
 mo , usa)-treated primary mouse embryonic fibroblasts in rpmi1640 medium ( thermo fisher scientific , san jose , 
 ca , usa ) with 20% fetal bovine serum ( thermo fisher scientific ) , 25 ng / ml human recombinant fgf4 ( wako pure 
 chemicals , osaka , japan ) , 1 g / ml heparin ( sigma - aldrich ) , 100 m 2-mercaptoethanol ( sigma - aldrich ) , 1% 
 glutamax ( thermo fisher scientific ) , and 1 mm sodium pyruvate ( thermo fisher scientific ) . representative colony 
 morphologies are shown in fig . 1 , together with descriptions of 
 their morphological characters . there was an additional type ( type 5 ) , but this was not analyzed here because 
 it rarely emerged under normal tsc culture conditions . at 96 h after passaging , colonies were picked up using 
 a glass capillary pipette under a dissecting microscope and used for rt - qpcr analysis . total rna for rt - qpcr was extracted with rneasy micro kits ( qiagen , venlo , netherlands ) from tsc colonies . following extraction , the first strand cdna was synthesized with a superscript iii reverse transcriptase 
 reagent set ( thermo fisher scientific ) . gene expression was assessed by qpcr on a steponeplus instrument 
 ( thermo fisher scientific ) using quantitect sybr green pcr kits ( qiagen ) according to the manufacturer s 
 instructions . samples were denatured at 95c for 10 min , followed by 40 amplification cycles consisting of 
 denaturation at 95c for 15 sec , and an annealing and extension step at 60c for 1 min . raw cq ( ct ) values 
 ( pcr cycles at which the fluorescence signal crosses threshold ) were calculated using stepone software ( v. 
 2.1 ; thermo fisher scientific ) setting baseline and appropriate threshold values . all runs were performed in 
 triplicate and an identical sample was used in each different runs as an inter - run calibration sample to 
 correct for the technical variance between the runs and thus compare results from different plates [ 4 , 6 ] . the primer sets used for tsc 
 marker genes were the following : cdx2 , 5gcagtccctaggaagccaag3 and 
 5gcagccagctcacttttcct3 ; elf5 , 5gtggcatcctggaatgggaa3 and 5cactaacctccggtcaaccc3. 
 for quantification of reference genes ( atp5b , canx , cyc1 , 
 gapdh , ubc , rn18s ) , the primer sets from the mouse genorm 
 kit ( primerdesign ltd . , southampton , uk ) were used . actb was analyzed as an additional 
 reference gene using the primer set 5ctgtcgagtcgcgtcca3 and 5acccattcccaccatcacac3. the baseline cycles and cycle thresholds were established manually for each gene . to assess the stability of 
 the reference genes , the raw cq values of each reference gene were analyzed with the genorm algorithmic tool 
 using qbase software ( biogazelle ) . these values were converted into normalized relative 
 quantities ( nrqs ) with multiple reference genes based on the modified classic --ct method   or a single reference gene . nrqs were scaled in relation to the mean 
 across all samples for each target or to reference genes . we ranked the stability of the reference genes based 
 on the mean expression stability ( genorm m value ) provided by the genorm program . to determine the optimal 
 number of reference genes across different colony types , qbase was also used to calculate 
 pairwise variation ( v value ) . normalization of the expression levels of tsc marker genes 
 ( cdx2 and elf5 ) was performed by qbase software using 
 reference gene(s ) ranked as described above . to compare the gene expression levels between colony types within the tsc lines , normalized relative mrna 
 levels were analyzed with kruskal - wallis tests followed by dunnett s multiple comparison tests ; p < 0.05 
 was considered statistically significant .