Fig 2: Expression of precursor cell markers in mouse brDFAT and wDFAT cells. (A) Both brDFAT and wDFAT cells showed high expression of Sca-1 as determined by FACS after one week of culture. Low expression of CD90, CD105, and SSEA1 was found in both cell types. However, only the brDFAT cells expressed the endothelial progenitor marker CD34. Representative of 3 experiments. (B) Time course expression of the neurogenic markers Neurogenic Differentiation 1 (NeuroD1), glial fibrillary acidic protein (GFAP), Nestin, ß tubulin III (ßTubIII), Oligodendrocyte Transcription Factor 2 (OLIG2), and S100 Calcium Binding Protein B (S100ß) in brDFAT and wDFAT cells, as determined by qPCR. The cells were treated with neural induction medium; wDFAT cells in basic control medium were used for comparison (day 3 was set to 1) (n = 3 cell preparations. P-values for statistically significant differences are indicated (two-way ANOVA). * < 0.05, ** < 0.01, *** < 0.001.

Fig 3: Alkbh5 depletion induces G2/M cell cycle arrest and impairs the MET process. (A) Cell proliferation of reprogrammed MEFs was assessed by FACS measured by BrdU incorporation on day 3 of reprogramming using either scrambled shRNA or two different shRNAs targeting Alkbh5. (B) Quantification of the mean percentage of each of the populations G1, S, and G2/M from FACS data shown in A. (C) Cell proliferation was assessed by counting Alkbh5f/f cells with or without the addition of 1 µM 4-OH Tam for Alkbh5 depletion. (D) Expression of mitotic checkpoint complex (MCC) factors as assessed by qPCR on day 3 of reprogramming using either scrambled shRNA (SCR) or two different shRNAs targeting Alkbh5 (KD1 and KD2). The data were normalized to the level of the housekeeping gene Gapdh. (E) Immunoblot analysis of the protein levels of several cell cycle regulators on day 3 of reprogramming using either scrambled shRNA or two different shRNAs targeting Alkbh5. a-tubulin (A-TUB) was used as a loading control. (F) Expression of mesenchymal and epithelial genes as assessed by qPCR on day 6 of reprogramming after infection either with scrambled shRNA or two different shRNAs targeting Alkbh5. The data were normalized to the housekeeping gene Gapdh. (G) Estimation of E-cadherin (E-cad) and Thy-1-positive populations by FACS in WT and Alkbh5 KO cells on day 6 of reprogramming. (H) Estimation of E-cad- and PDGFRß-positive populations by FACS in WT and KO Alkbh5 MEFs on day 6 of reprogramming. (I) Estimation of E-cad- and SSEA1-positive populations by FACS in WT and Alkbh5 KO MEFs or MEFs infected with either empty vector (EV) or ALKBH5–HA on day 7 of reprogramming. Images in A, E, G–I are representative of three repeats. Quantitative data are shown as the mean±s.d.; n=3. *P<0.05, **P<0.01, ***P<0.001 (paired Student's t-test).

Fig 4: Alkbh5 depletion impairs somatic cell reprogramming efficiency. (A) Relative expression of Alkbh5 during somatic cell reprogramming detected by qPCR. MEFs and mouse ESCs cultured in serum plus leukemia inhibitory factor (LIF), described as S/L, were used as negative and positive controls of pluripotency, respectively. Data are normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh). (B) Immunoblot analysis of ALKBH5 protein levels during reprogramming. D, day. a-tubulin (A-TUB) was used as a loading control. (C) Immunoblot analysis of ALKBH5 protein levels in MEFs after lentiviral infection with either scrambled or two different shRNAs targeting Alkbh5 (KD1 and KD2). Scrambled (SCR) shRNA used as a negative control. A-TUB was used as a loading control. (D) Experimental design showing the timing of Alkbh5 knockdown, onset of reprogramming, counting of GFP-positive colonies and FACS analysis of the ?-PE-Oct4-GFP-positive population. (E) Fraction of ?-PE-Oct4-GFP-positive cells determined by FACS analysis after Alkbh5 knockdown during the early phase of reprogramming. (F) Number of ?-PE-Oct4-GFP-positive colonies on day 14 of reprogramming. (G) Fraction of SSEA1-positive cells determined by FACS analysis after Alkbh5 knockdown during the early phase of reprogramming. Negative control is unreprogrammed MEFs. (H) Reprogramming efficiency was measured by counting the number of ALP-positive colonies. (I) Fraction of SSEA1-positive cells determined by FACS for reprogrammed wild-type (WT) and knockout (KO) Alkbh5 MEFs assessed at day 7 of reprogramming. (J) Fraction of SSEA1-positive cells determined by FACS analysis of reprogrammed homozygous floxed Alkbh5 (Alkbh5f/f) treated with ethanol as a control or 1 µM 4-OH Tam for depletion of Alkbh5 at either day 2 or day 4. Images in B, C, E, G, I and J are representative of three repeats. Quantitative data are shown as the mean±s.d.; n=3. *P<0.05, ***P<0.001 (paired Student's t-test).

Fig 5: Cellular Pluripotency Marker Expression Levels in EpiSCs Derived by the IWP-2 Method(A–J) Immunofluorescence images for SSEA1 (A and B), PECAM1 (red) and OCT4 (green, C and D), NANOG (E and F), and histone H3K27 trimethylation (G and H) in mESCs (A, C, E, and G) and EpiSCs (B, D, F, and H). Nuclear staining is shown using TO-PRO3 (blue). Cytograms of EpiSCs and mESCs are shown using anti-SSEA1 (I) and anti-PECAM1 (J) antibodies.(K) Relative expression levels of marker genes in EpiSCs detected by qRT-PCR compared with J1 mESCs. For each gene, technical triplicate assays and two independent experiments were performed. Error bars represent the standard SEM.(L) Growth curves of EpiSCs. At the indicated time points, 2.0 × 104 cells were plated and counted. Averaged data from three independent experiments were plotted.Scale bars, 20 µm (A, B, C, D, G, and H) and 50 µm (E and F).