Fig 2: Ascorbate-induced changes in cell surface antigen expression. (A) Changes in cell surface markers associated with monocytic differentiation. Common granulocyte/monocyte precursor cells express immature cell antigens such as CD34 and CD117 which persist up until the monocyte precursor/monoblast differentiation stage. Once these immature cells mature into promonocytes and mature monocytes, CD117 and CD34 expression is lost and the cells begin to express CD15, CD33, CD13 and CD11b along with other characteristic cell surface markers (40). These changes are mirrored by SKM-1 cells following growth in ascorbate-replete media with notable increases in CD15, CD13, CD11b and a decrease in CD177. (B) Representative histograms showing shifts in the fluorescence intensity signal for each cell surface marker after treatment with phosphoascorbate, relative to the control. (C) Relative changes in cell surface antigen expression following treatment with phosphoascorbate and ascorbate. Measurements are n = 3 except for CD45RA expression, which was measured at n = 1 (at 12 days) and n = 2 (at 22 days). For all other measurements the 13-day timepoint includes one measurement taken at 12 days and two measurements at 13 days. For CD117, when two distinct subpopulations were present, the change in MFI was calculated by gating to select the CD117+ subpopulation and calculating the MFI change for this subpopulation. Statistically significant changes are marked as: *p < 0.05, **p < 0.01. Parts of this figure were made using BioRender.com.

Fig 3: Transiently reprogrammed cells reacquire their initial cellular identity.(A) Mean DNA methylation age (calculated using the multi-tissue clock; Horvath, 2013) throughout the reprogramming process where cells were transduced with our tetO-GFP-hOKMS vector and treated continuously with 2 µg/ml of doxycycline. Reprogramming is divided in three distinct phases: initiation phase (IP), maturation phase (MP), and stabilization phase (SP). DNA methylation age decreased substantially during the MP of reprogramming in cells that were successfully reprogramming (magenta line) but not in control cells (yellow and orange lines represent non-transduced cells and cells expressing hOKMS but failing to reprogram as indicated by cell surface markers, respectively). Points represent the mean and error bars the standard deviation. N=3 biological replicates per condition, where fibroblasts were derived from different donors. N=2 biological replicates for the iPSC time point (day 51). (B) Experimental scheme for maturation phase transient reprogramming (MPTR). The tetO-GFP-hOKMS reprogramming construct was introduced into fibroblasts from older donors by lentiviral transduction. Alternatively, cells were ‘mock infected’ as a negative control. Following this, cells were grown in the presence of 2 µg/ml doxycycline to initiate reprogramming. At several time points during the MP, cells were flow sorted and successfully reprogramming cells (CD13− SSEA4+) and cells that were failing to reprogram (CD13+ SSEA4−) were collected for analysis. These were termed ‘transient reprogramming intermediate’ and ‘failing to transiently reprogram intermediate,’ respectively. Sorted cells were also further cultured, and grown in the absence of doxycycline for at least 4 weeks—these were termed ‘transiently reprogrammed’ (CD13− SSEA4+) or ‘failed to transiently reprogram’ (CD13+ SSEA4−). (C) Phase-contrast microscope images of cells after doxycycline treatment (transient reprogramming intermediate) and after withdrawal of doxycycline (transiently reprogrammed) as described in (B). The morphology of some cells changed after doxycycline treatment. These cells appeared to form colonies, which became larger with longer exposure to doxycycline. After sorting, these cells were cultured in medium no longer containing doxycycline, and appeared to return to their initial fibroblast morphology. (D) Roundness ratio of cells before, during, and after MPTR (with 13 days of reprogramming). Roundness ratio was calculated by dividing maximum length by perpendicular width. Fibroblasts became significantly rounder during MPTR and returned to a more elongated state upon the completion of MPTR. Values from individual cells have been represented as violin plots. Points represent mean values and are connected with lines. Significance was calculated with a Tukey’s range test. Representative 3D renderings of cells (generated using Volocity) before, during, and after successful transient reprogramming are included below the plot. CD13 is colored in green, SSEA4 is colored in red, and DAPI staining is colored in blue. White scale bars represent a distance of 20 µm. (E) Principal component analysis of transient reprogramming and reference reprogramming sample transcriptomes (light blue to dark blue and black crosses, data from Banovich et al., 2018, Fleischer et al., 2018 and our novel Sendai reprogramming data set). Reference samples form a reprogramming trajectory along PC1. In the Sendai reprogramming reference data set, cells that were not reprogramming (CD13+ SSEA4−) were also profiled and clustered midway along PC1 suggesting some transcriptional changes had still occurred in these cells. Transient reprogramming samples moved along this trajectory with continued exposure to doxycycline (light magenta points) and returned to the beginning of the trajectory after withdrawal of doxycycline (magenta points). Control samples (yellow and orange points) remained at the beginning of the trajectory throughout the experiment. (F) Mean gene expression levels for the fibroblast specific gene FSP1 and the iPSC specific gene NANOG. Transiently reprogrammed samples expressed these genes at levels similar to control fibroblasts. Bars represent the mean and error bars the standard deviation. Samples transiently reprogrammed for 10, 13, 15, or 17 days were pooled. The number of distinct samples in each group is indicated in brackets. (G) Principal component analysis of transient reprogramming (magenta points) and reference reprogramming sample methylomes (light blue to dark blue and black crosses, data from Banovich et al., 2018, Ohnuki et al., 2014 and our novel Sendai reprogramming data set). Reference samples formed a reprogramming trajectory along PC1. Transient reprogramming samples moved along this trajectory with continued exposure to doxycycline (light magenta points) and returned to the beginning of the trajectory after withdrawal of doxycycline (magenta points). Control samples (yellow and orange points) remained at the beginning of the trajectory throughout the experiment. (H) Mean DNA methylation levels across the fibroblast-specific gene FSP1 and the iPSC-specific gene POU5F1 (encoding OCT4). Transiently reprogrammed samples had methylation profiles across these genes that resemble those found in fibroblasts. Gray bars and black bars indicate the locations of Ensembl annotated promoters and genes, respectively. Samples transiently reprogrammed for 10, 13, 15, or 17 days were pooled for visualization purposes. The number of distinct samples in each group is indicated in brackets. iPSC, induced pluripotent stem cell; MPTR, maturation phase transient reprogramming.

Fig 4: The EGFRC329R mutant impairs erythroid differentiation in the human TF1.8 erythroleukemia cell line. (a) Dot plots representative of CD235a (glycophorin A, erythroid marker) and CD13 (myeloid marker) surface expression on TF1.8/MIG, TF1.8/EGFR and TF1.8/C329R cells on day 5 of culture with GM-CSF (5 ng/mL), EPO (5 U/mL) or rhEGF (0.5 ng/mL or 10 ng/mL). (b) Percentage of cells in gates (a) or (c) (identified in (Fig. 3a) in GM-CSF culture (top panel), and plotted over a 7 day time-course for EPO (middle panels) or EGF (bottom panel) containing cultures. Points represent mean ± SE of 3 independent experiments. T-test *P < 0.05, **P < 0.01 (MIG vs. C329R). (c) Schematics of temporal acquisition of mutations (originating from a WT stem/progenitor cell) as determined from genotyping of individual BFU-E colonies plated from PBMNC. The percentage of each clone in PBMNC is shown.

Fig 5: Immunolabeling and RNA ISH reveals progressive emergence of theca lineage markers.a Immunostaining for ACTA2, ENG and ANPEP in primary (i), secondary (ii) and early antral (iii) follicles. Red, blue, and red/blue arrows point to cells that are positive for ENG, ACTA2 or both markers, respectively; in (iii) green arrows indicate ANPEP+ cell layer, while red arrows indicate an ENG+ area that is negative for both ANPEP and ACTA2. b–c Staining of early antral follicles for ANPEP and CYP17A1 (b), or ANPEP and THBD (c); red arrows indicate THBD+ cells and blue arrows show ANPEP+ cells. d Representative Immunolabeling for TCF21, ACTA2 and ANPEP in (i) primordial, (ii) transitioning primary, (iii) primary, (iv) secondary and (v) early antral follicles. Blue arrows indicate ACTA2+ cells and green arrows indicate TCF21+ cells; white boxes indicate areas magnified in bottom panels. e RNA ISH labeling for TCF21 RNA in a primary follicle. f Staining for ACTA2 and GLI1 in a secondary follicle; representative cells expressing both markers are indicated with red and blue arrow. g RNAScope labeling for PTCH1 RNA in primary and secondary follicles. Blue channel shows transmitted light in (e and g). Scale bars – 100 μm. a–e Nuclear counterstain (DAPI) is shown in white in (a–d and f). as = anti-sense RNA probe in (e and g).