Temporal Activation of LRH-1 and RAR-γ in Human Pluripotent Stem Cells Induces a Functional Naïve-Like State (2020)
Figure 7

Figure 7. TGFβ-2iL converts primed hPSCs to the naïve state.

A qRT-PCR analysis of ligands associated with TGF-β signaling during conversion of primed to naïve-like pluripotency by 2a2iL induction protocol at days 1, 3, and 5. 2iL was selected as a control group. ***P<0.0001. One-way ANOVA. Data presented as mean ± SD (n=3).

B Inhibition of TGF-β pathway using SB43 during conversion of primed to naïve pluripotency. Scale bar: 100 µm.

C Morphology of TGFβ2iL-treated RH6 cells along with ALP activity. Scale bar: 200 µm.

D qRT-PCR analysis of naïve related genes in pRH6, 2a2iL-, and TGFβ2iL-RH6. **P<0.01, ***P<0.001. One-way ANOVA. Data presented as mean ± SD (n=3).

E Bar plot representing cell proliferation rates of 2a2iL- and TGFβ2iL-RH6 by cell counts. **P<0.01 (t-test). Data presented as mean ± SD (n=3).

F Bar plot of the cloning efficiency of 2a2iL and TGFβ2iL-RH6 based on the number of ALP positive colonies. ***P<0.001 (t-test). Data presented as mean ± SD (n=3).

G Immunofluorescence images show the expression of naïve pluripotency markers in TGF-β2iL-RH6. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.

Table 1. Primer sequences and reaction conditions of qRT-PCR or PCR.

Annealing

temperature (°C)

F: ctc att tcc tgg tat gac aac ga

R: ctt cct ctt gtg ctc ttg ct

F: ctg ggt tga tcc tcg gac ct

R: cac aga act cat acg gcg gg

F: aaa gaa tct tca cct atg cc

R: gaa gga aga gga gag aca gt

F: ggg aaa tgg aag ggg tgc aaa aga gg

R: ttg cgt gag tgt gga tgg gat tgg tg

F: att acc aag agc tca tgc ca

R:cct tga gat ggg aac tct ttg

F: ttt acg ttt ggg agg agg

R: gtg gtc agc tat tca gga g

F: aaa gct tcc gat aga ggg ga

R: tgc tca ccg aag aaa att cc

F: ttt gaa gac cat gga gcc cg

R: aca ttc gcc ttc ccg act tg

F: ggc aag acc tac acc aag ag

R: cac aga tgg cac tgg aat gg

F: cct ggt cca gac aag atg tga

R: gaa ctg gtc tac gac tga ggc

F: gtc gtg gtt tac ggc tcc tat

R: ggc aag ttt gag cat ccc tc

F: ttg ctg atg cca tcc ctt act

R: agctgacatatctggaggtgaa

F: gcc cag ttg cag agg act c

R: tct tgt tcc ata agc gca ttc t

F: cag ccc gag cac tac aac c

R: ctc cca gct tcc gat tct cc

F: cac aac tcg gag atc agc aa

R: ggt act tgt aat ccg ggt gc

F: gtc cat ctt tgc ttg gga aa

R: tag cca ggt tgc gaa gaa ct

F: gta tcc cga ccg cat cat

R: tct cat ccg tgt tct cca

F: tcc agg aac gga aaa tca ag

R: gcc tcc tca tcc cct act tc

F: cct gtc atc tca cta cgg

R: gct gtt cca aga gtc ctg

F: aat tgg tcc agc ctt gga at

R: cgt tgc tca cag acc aca

F: atg atg cat ttt ggg ggt ta

R: cag cac ctt cct cct ctc ag

F: aaa tgc gtt tct cgt tgc tt

R: gcc aca ggc caa tag ttt gt

F: gga gcg gtg aag atg gaa

R: tac gtg ttc atg ccg ttc at

F: ctc tgc ctc ctc cac gaa

R: cag aat cca gac ctg cac aa

F: tcc cag ctc tta cct tac ca

F: aaa ctc ctt ccc atc ctg ata ctc

F: cca acc tta ggg agt aac act g

R: ggg agt gct gct tct ttc tg

F: atg aag tca agg cta acc agc

R: cgt cat cgt cgt aca gga aga g

F: gtctttcttccgttctgatgag

R: ttcttcattcacctaaggacca

F: aactatctcctactactctttccc

R: cactcctcttcctatatgcct

F: agacaccagttctacctcct

R: ggtctctggaatattgctctg

F: gcg tac atg ctg agc ctc ta

R: ggt gac ctg gga caa agt g

F: ctttgtcccgtgtgtggagat

R: gtcggcccttacagcttcta

F: gaacgtacttctttcacccac

R: ttcctgaaccaaacctttactg

F: gacttgatgacacgtaccttcg

R: agcgtgacacttggagtcct

F: tgctacaggtgtcggtgcagagg

R: agaaacggacacttgaaggccagg

F: gggaattgggatacctggat

R: ctaaatatgcacgggcaagg

F: tagacatcgtactacacgacacg

R: tccaggtttatggagggttc

*BRA: Brachyury

Table 2. Primary and secondary antibodies used for immunofluorescence staining.

*HNA: Human nuclear antigen

Expanded View Figure legends