Fig 2: SALL4 is expressed in undifferentiated spermatogonia in adult mouse seminiferous tubules.Double immunohistochemistry of tissue sections was used to examine the localization of SALL4 in comparison to PLZF (A–D) and LIN28 (E–H), two known markers for undifferentiated spermatogonia. (D, H) Data are expressed as the average number of single or double stained cells per tubular cross section ± SEM. (I–K) SALL4 was not expressed in GATA4-positive Sertoli cells. (L) SALL4, PLZF and LIN28-positive cells were normalized to 1000 Sertoli cells to express the relative size of the respective spermatogonial populations. The basement membrane of each tubule is marked by a dashed line. Scale bars = 10 µm. For quantitative analysis, 100 circular tubule cross sections in testes from at least 3 adult male mice were scored.

Fig 3: Construction of LIN28A-responsive devices. (A) Predicted secondary structures of the LIN28A aptamers used in this study. Small letters represent the nucleotides changed from the original preE-let7d sequence. Purple shadows represent the LIN28A recognition sequences. Color scale represents base pairing probabilities calculated by CentroidFold software. (B) Overlaid dot plots of the flow cytometry analysis. Red, cells co-transfected with EGFP reporter plasmid and trigger plasmid encoding LIN28A and tagRFP. Blue, cells co-transfected with EGFP reporter plasmid and control plasmid without LIN28A. (C) Translational efficiency of LIN28A-responsive devices. All values were normalized by cells transfected with a plasmid lacking aptamers (No aptamer). Transfection was performed in a 24-well format. Error bars represent mean ± SD (n = 3 independent experiments, each performed in triplicate). (D) Translational efficiency of defective LIN28A-responsive devices (sequences are described in Supplementary Figure S2). All values were normalized by cells transfected with No aptamer. Transfection was performed in a 96-well format. (E) Translational efficiency using mutant LIN28A (W46A, LIN28Amut), which weakens RNA binding. All values were normalized by cells co-transfected with No aptamer and trigger plasmid coding for wild-type LIN28A. Transfection was performed in a 24-well format. Error bars represent mean ± SD (n = 3 independent experiments, each performed in triplicate). (F) Translational efficiency of LIN28A-responsive devices in HeLa cells. Transfection was performed in a 24-well format. Error bars represent mean ± SD (n = 3 independent experiments, each performed in triplicate). All values were normalized as described in D. *P < 0.05, **P < 0.005, ***P < 0.0005 (Welch's t-test).

Fig 4: Summary of spermatogonial marker expression in mouse spermatogonia.Graphical summary of quantitative data from Figures 6–8 were used to calculate the ratio of cells expressing each marker within the populations of As, Apr, and Aal spermatogonia. Each bar represents 10% GFRa1-, SALL4-, PLZF-, LIN28- or cKIT- positive cells, respectively, and half bars represent approximately 5% positive cells. For example, co-staining revealed that 71% of As spermatogonia expressed GFRa1, therefore 7 bars (each representing 10%), are shown in the diagram. The heterogeneity of the molecular phenotype of spermatogonia clone sizes is represented by the extent of overlap among bars across marker profiles.

Fig 5: Distinction of living cell types using LIN28A-responsive mRNA devices. (A) Distinction of human iPSCs with differentiated cells by detecting endogenous LIN28A expression level. LIN28A is highly expressed in iPSCs, and its expression level is decreased after differentiation. LIN28A in iPSCs reduces the translation level from LIN28A-responsive hmAG1 mRNA. In differentiated cells, LIN28A expression level is low, resulting in normal translation (left and middle panels). After co-transfection of the LIN28A-responsive hmAG1 mRNA and reference mRNA encoding mKO2, the cells typically show belt-like distribution in a two-dimensional plot by flow cytometry analysis (right panel). A shift of the population is observed as the result of translational repression of hmAG1 in iPSCs but not in differentiated cells, resulting in the separation of iPSCs from differentiated cells based on LIN28A detection. (B) Relative translational efficiency of transfected mRNAs in iPSCs and D14. Relative translational efficiency = (N.I. of iPSCs)/(N.I. of D14) (See ‘Flow cytometry data analysis’ in ‘Materials and Methods’ section). Error bars represent mean ± SD (n = 3 independent experiments, each performed in duplicate). ***P < 0.0005 (Welch's t-test). (C) Representative overlaid dot plots of iPSCs (red) and differentiated cells (D14, blue). (D) Representative histograms related to Figure 7C. Red, hiPSCs; blue, D14. (E) Western blot analysis of the LIN28A expression. A total of 0 ng Dox, 10 ng Dox and 293FT were applied as controls, and GAPDH was used as internal control. The samples used in these experiments were also used for the data in Figure 5C. iPSCs and D14 samples were collected in different experiments. LIN28A was detected by anti-LIN28A. (F) Endogenous miRNA expression levels by LIN28A-responsive mRNA transfection. Mature let7d, let7g and miR-98 levels were analyzed by qRT-PCR. We used differentiated cells (D14, black bar) as a control, because the expression levels of these miRNAs increased in differentiated cells compared with iPSCs. Thus, the expression levels of miRNAs in untransfected (white), mRNA (without aptamer)-transfected (blue) and stbC-containing mRNA-transfected (red) iPSCs were normalized by the expression level of the miRNAs in D14. Error bars indicate SD for three independent experiments. Significant differences were not observed among untransfected, no aptamer and stbC.