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 .
   mouse trophoblast stem cells ( tscs ) form colonies of different sizes and morphologies , which might reflect 
 their degrees of differentiation . 
 therefore , each colony type can have a characteristic gene expression 
 profile ; however , the expression levels of internal reference genes may also change , causing fluctuations in 
 their estimated gene expression levels . in this study , we validated seven housekeeping genes by using a 
 geometric averaging method and identified gapdh as the most stable gene across different 
 colony types . 
 indeed , when gapdh was used as the reference , expression levels of 
 elf5 , a tsc marker gene , stringently classified tsc colonies into two groups : a high 
 expression groups consisting of type 1 and 2 colonies , and a lower expression group consisting of type 3 and 4 
 colonies . 
 this clustering was consistent with our putative classification of undifferentiated / differentiated 
 colonies based on their time - dependent colony transitions . by contrast , use of an unstable reference gene 
 ( rn18s ) allowed no such clear classification . 
 cdx2 , another tsc marker , 
 did not show any significant colony type - specific expression pattern irrespective of the reference gene . 
 
 selection of stable reference genes for quantitative gene expression analysis might be critical , especially 
 when cell lines consisting of heterogeneous cell populations are used . 
Materials and Methods
Cell lines
Colony classification and sample collection
RT-qPCR
Data analysis
Statistical Analysis