Figure 4.

Selected Four Transcription Factors Induce Human PAX3-Positive Muscle Stem Cells Derived from the Mesodermal Lineage

(A) Procedure for the induction of PAX3-GFP cells derived from mesodermal lineage.

(B) GFP or mCherry expression in transient MYOD-induced populations after expression of PAX3, HEYL, and KLF4 transcription factors (+3F), and doxycycline for 72 h (+DOX).

(C) The contrasting proportion of induced PAX3-GFP cells with transient single doxycycline treatment (+DOX) and three transcription factors and transient/continuous doxycycline (+3F+DOX/+3F++DOX).

(D) The FACS profile of CD56 and CD82 expression, human myogenic cell-surface markers, in induced cells. More than 60% of PAX3-GFP cells were merged with CD56⁺CD82⁺ as double positives.

(E) Immunostaining with anti-PAX7 (left panel, labeled with Alexa 488, light blue) and anti-MYOGENIN (MYOG; right panel, labeled with Alexa 647, purple) antibodies in CD56⁺CD82⁺GFP⁺ sorted cells. GFP signals were not detected after immunostaining.

(F and G) In vitro myogenic differentiation assay with induced PAX3-GFP-positive (CD56⁺GFP⁺) or -negative (CD56⁺GFP⁻) cells of CD56-positive cells in 2% of horse serum medium for 7 days. MYOG, purple; MYHC, light blue (labeled with Alexa 647); DAPI, blue.

(H) Percentage of differentiated myogenic cells presented as the ratio of MYOG or MYHC/DAPI fluorescence. n = 3 independent replicates; p values are determined by t test from a two-tailed distribution. ^∗p < 0.01.

(I) Ratio of transcripts of PAX3 relative to RPL13A in myogenic differentiated cells from PAX3-GFP-positive (+) or -negative cells (−) of the CD56⁺ population. n = 3 independent replicates; p values are determined by t test from a two-tailed distribution. ^∗p < 0.01.

(J) Fetal (NFIX) and adult (NAP1L5) transcriptional analyses with PAX3-GFP-positive or -negative cells of CD56-positive cells induced by transient DOX treatment plus three transcription factors (+3F+DOX). Mesodermal GFP-expressing cells, marked by DMRT2 (dermomyotome), were used as control (GFP⁺ mesoderm). n = 3 independent replicates; p values are determined by Dunnett's multiple-comparisons test. ^∗p < 0.01.

(K) Transcriptional analyses with GFP-positive cells induced by transient DOX treatment plus three transcription factors (+3F+DOX). GFP-expressing cells with or without DOX were used as control (GFP⁺ −DOX/+DOX). n = 3 independent replicates; p values are determined by Dunnett's multiple-comparisons test. ^∗p < 0.01.

Error bars indicate ±SEM. Scale bars, 50 μm.