Fig. 8: menRNA stimulates TM cell rejuvenation and proliferation through enhancing paraspeckle assembly.

a Left panel: Quantification of pre-stained pTM cellularity following co-culture with iPSCs at low and high densities (n = 3 technical repeats), iPSC-TM after differentiation for 4 (n = 3 technical repeats) or 15–18 days (n = 9 technical repeats over 3 independent experiments), and pTM (n = 15 technical repeats over 5 independent experiments). The result indicates that iPSC-TM has a significantly greater pro-proliferative capacity compared to iPSCs and pTM (Supplementary Data 5). Right panel: quantification analysis (Supplementary Data 5) of paraspeckle showing that iPSC-TM (5 days: n = 65 cells; 16 days: n = 51 cells) has a significantly higher amount than both iPSCs (n = 73 cells) and pTM (n = 29 cells). RT-PCR results (Supplementary Data 5) showing a higher level of NEAT1_2 (b) but a lower expression of menRNA (c) in pTM compared to those in iPSC-TM. n = 3. d RT-PCR results (Supplementary Data 5) confirming increased expression of menRNA in pTM after menRNA transfection for 12 and 48 h. n = 3. e Immunohistochemical staining of NONO (red; left panel), along with quantification of paraspeckle amount (right panel; Supplementary Data 5), indicating a significantly higher amount of paraspeckle in pTM after overexpressing menRNA for 12 and 48 h. Scale bar, 10 µm. n = 58, 153, 165, and 216 cells before or after menRNA treatment for 12 and 48 h. f A representative co-culture image of pTM pre-stained with CellTracker Red and pTM overexpressing menRNA pre-labeled with green fluorescence indicates the transfer of menRNA between cells. Scale bar, 50 µm. g Upper panel: a Log (Fold change) analysis using RT-PCR results showing induced expressions of neural crest cell (NCC) biomarkers (NANOG, FOXC1, NES), stem cell biomarkers (ZFP42, TBX3, TFAP2C, DUS6), and Ki-67 in pTM after overexpressing menRNA. Lower panel: FPKM values of these genes are shown in pTM before (U) and after (T) co-culture with iPSC-TM. h Quantification of pTM after menRNA transfection for 48 h revealing menRNA overexpression significantly increased pTM cellularity (Supplementary Data 5). Cells transfected with LipoFiter^TM transfection reagent were used as a control. n = 22 and 24 regions of pTM before and after menRNA transfection across 3 technical repeats. **P < 0.01, ****P < 0.0001 by two-tailed Student’s t test in (d) and (h), while *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Tukey’s post-hoc test in other panels.