Extended Data Fig. 6: The impact of vitamin B12 administration on in vitro reprogramming.

(a) Changes in gene expression of the vitamin B12 uptake receptor Cd320 and Mtr (encoding MS) during a previously published time course of iPSC generation by OSKM expression. iPSCs were derived from MEFs of two different genetic backgrounds (Gatad2a-/- and Mbd3-/) that both yield high-efficiency reprogramming. Each row is an independent embryo of the indicated genotype. Data source: 40 (b) Vitamin B12 supplementation does not accelerate iPSC formation. OSKM MEFs were treated with doxycycline with or without vitamin B12 supplementation as indicated, and iPSC colony formation was assessed by alkaline phosphatase staining after 7 and 10 days of treatment. Representative images are shown of MEFs derived from a single embryo; n = 3 independent embryos were tested. (c) Vitamin B12 enhances doxycycline-free retroviral reprogramming. Wild type MEFs were transduced with retroviral vectors encoding Oct4, Sox2, Klf4, and cMyc. Samples were collected on day 5 post-transduction for histone extraction and immunoblot (lower). iPSC colony formation was evaluated by alkaline phosphatase (AP) staining on day 14 (upper). Immunoblot and AP images are representative from n = 3 independent MEFs. (d-e) Stable isotope labeling (SIL) with fully labeled 13C-serine during in vitro reprogramming. (d) Schematic of 1 C metabolism indicating how fully labeled 13C-serine contributes to methyl m + 1 labeling of SAM. Orange points indicate 13 C atoms. Figure adapted from ref. ³³, Springer Nature Limited. (e) SIL does not affect reprogramming efficiency. Doxycycline-inducible OSKM MEFs were cultured in normal iPS media with doxycycline without or without vitamin B12 supplementation as indicated. 13C-serine labeled media was administered as descried in Methods and reprogramming was quantified by iPS colonies evaluated by AP staining on day 10. Quantification of colonies is shown for the indicated treatments. Each point represents an individual MEF clone. P-values represent comparison of the OSKM vs OSKM + B12 condition for each media type. (f) Changes in histone methylation during in vitro reprogramming with vitamin B12 supplementation. Histone extracts were prepared from OSKM MEFs treated with doxycycline for 3 and 10 days, with or without vitamin B12 supplementation. Levels of the indicated histone methylation modifications were measured by colorimetric multiplex assay and the relative abundance (fold change, FC) in cells reprogrammed in the presence as compared to absence of vitamin B12 is shown. Note that MEFs derived from independent embryos were used for day 3 and day 10 analyses. (g) Expression of Setd2, which encodes the H3K36 trimethyl-transferase in the colon (left) and stomach (right) from WT and OSKM mice after 7 days of doxycycline treatment. Mice are from Fig. 1b n = 4 mice (WT), n = 8 (OSKM colon), n = 6 (OSKM stomach). (h) H3K36me3 during in vivo reprogramming in the colon and stomach of OSKM mice with and without B12 supplementation. Representative images are shown (left) and quantified (right) in a subset (n = 5 for each group) of mice from Fig. 1e. Scale bar is 100 µm. Mice that received folate in addition to B12 are represented by open points. Graphs represent average ± SD. P-values by two-tailed Student’s t-test. used for day 3 and day 10 analyses. (g) Expression of Setd2, which encodes the H3K36 trimethyl-transferase in the colon (left) and stomach (right) from WT and OSKM mice after 7 days of doxycycline treatment. Mice are from Fig. 1b n = 4 mice (WT), n = 8 (OSKM colon), n = 6 (OSKM stomach). (h) H3K36me3 during in vivo reprogramming in the colon and stomach of OSKM mice with and without B12 supplementation. Representative images are shown (left) and quantified (right) in a subset (n = 5 for each group) of mice from Fig. 1e. Scale bar is 100 µm. Mice that received folate in addition to B12 are represented by open points. Graphs represent average ± SD. P-values by two-tailed Student’s t-test.

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