Notch inhibition allows oncogene-independent generation of iPS cells

Abstract

The reprogramming of somatic cells to pluripotency using defined transcription factors holds great promise for biomedicine. However, human reprogramming remains inefficient and relies either on the use of the potentially dangerous oncogenes KLF4 and CMYC or the genetic inhibition of the tumor suppressor gene p53. We hypothesized that inhibition of signal transduction pathways that promote differentiation of the target somatic cells during development might relieve the requirement for non-core pluripotency factors during induced pluripotent stem cell (iPSC) reprogramming. Here, we show that inhibition of Notch greatly improves the efficiency of iPSC generation from mouse and human keratinocytes by suppressing p21 in a p53-independent manner and thereby enriching for undifferentiated cells capable of long-term self-renewal. Pharmacological inhibition of Notch enabled routine production of human iPSCs without KLF4 and CMYC while leaving p53 activity intact. Thus, restricting the development of somatic cells by altering intercellular communication enables the production of safer human iPSCs.

Figure 1. DAPT treatment promotes mouse and human keratinocyte reprogramming

a, Chemical structure of DAPT. b, The efficiency of iPSC generation from mouse and human keratinocytes transduced with Oct4, Sox2, Klf4, and cMyc with DMSO or DAPT treatment (DAPT used at 10 μM in mouse experiment). c, The efficiency of iPSC generation from mouse and human keratinocytes transduced with all combinations of 2 reprogramming factors with DMSO or 2.5 μM DAPT treatment from days 1-18 post-transduction. d, A P0 mouse and human iPSC colony generated using OCT4, SOX2, and DAPT, scale bars = 100 μm. g, Teratoma generated by iPSCs derived from human neonatal kerationcytes using OCT4, SOX2, and DAPT, scale bar = 50 μm. j, NANOG+/TRA-1-81+ iPSC line generated from human adult keratinocytes using OCT4, SOX2 + DAPT, scale bars = 100 μm. For all experiments, error bars represent the standard deviation between two or three biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test.

Figure 2. γ-secretase inhibition promotes reprogramming by blocking Notch signaling

a, Chemical structure of DBZ. b, The efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4, KLF4, SOX2, and CMYC and treated with different concentrations of DBZ from days 1-18 post-transduction. c, The efficiency of NANOG+/ TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT, SOX2, KLF4, and CMYC and GFP or NOTCH ICD and treated with DMSO or 10 μM DAPT from days 1-18 post-transduction. Cells were transduced with NOTCH ICD or GFP lentivirus 1 day after transduction with the reprogramming factors. d, qPCR analysis of expression levels of NOTCH-dependent gene HES1 in human neonatal keratinocytes transduced with dominant-negative MASTERMIND-LIKE-1 (dnMAML) or RFP. e, The efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT, SOX2, KLF4, and CMYC and RFP or dnMAML and treated with DMSO or 10 μM DAPT from days 1-18 post-transduction. For all experiments, error bars represent the standard deviation between two or three biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test.

Figure 3. Notch inhibition promotes keratinocyte reprogramming by suppressing p21

a, Schematic of the DAPT treatment time course on human neonatal keratinocytes. b, Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4, SOX2, KLF4, and CMYC and treated with intervals of 10 μM DAPT or c, 2 μM DBZ. d, Western blot for p21 in human neonatal keratinocytes transduced with OCT4 and SOX2 and treated with DMSO or 10 μM DAPT. Full blot shown in Supplementary Figure 7c. e, Western blot for INVOLUCRIN in human neonatal keratinocytes treated with DMSO, 10 μM DAPT, or 1.2 mM calcium chloride for 6 days. Calcium was used as a positive control to induce keratinocyte differentiation. Full blot shown in Supplementary Figure 7d. f, Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4, KLF4, SOX2, and CMYC and a scrambled shRNA or a p21 shRNA at day 0 of reprogramming. DAPT was added at 10 μM. g, Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4 and SOX2 and a scrambled shRNA control or a p21 shRNA at day 0 of reprogramming. DAPT was added at 2.5 μM. h, Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT, SOX2, KLF4, and CMYC and GFP or p21 and treated with DMSO or 10 μM DAPT from days 1-18 post-transduction. For all experiments, error bars represent the standard deviation between two-three biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test.

Figure 4. Highly efficient reprogramming with NOTCH and DOT1L inhibition

a, Comparison of NANOG+/TRA-1-81+ iPSC generation from OCT4, SOX2, KLF4, and CMYC-transduced human neonatal keratinocytes using 10 μM DAPT versus other published reprogramming chemicals. “A83” = A8301 (.5 μM), “PD” = PD0325901 (.5 μM), “All from ref (13)” = A8301 (.5 μM), PD0325901 (.5 μM), PS48 (5 μM), sodium butyrate (.25 mM), Parnate (2 μM), CHIR99021 (3 μM), “AZA” = 5-aza-cytidine (.5 μM), “VPA” = valproic acid (.5 mM), “iDOT1L” = EPZ004777 (3 μM). b, Comparison of NANOG+/TRA-1-81+ iPSC generation from OCT4- and SOX2-transduced human neonatal keratinocytes using 2.5 μM DAPT versus other published reprogramming chemicals. c, iPSC line generated from human neonatal keratinocytes using OCT4, SOX2, DAPT, and iDOT1L. scale bars = 100 μm. For all experiments, error bars represent the standard deviation between two-three biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test.

Figure 5. NOTCH inhibition suppresses p21 without reducing p53 activity

a, qPCR analysis of p53-dependent genes in human neonatal keratinocytes 3 days after transduction of GFP or OCT4 and SOX2. b, Western blot of p53 levels in human neonatal keratinocytes with DMSO or 10 μM DAPT treatment for 3 days. Full blot shown in Supplementary Figure 7g. c, qPCR analysis of p53-dependent genes after 10 μM DAPT or 2 μM DBZ treatment for 3 days in OCT4, SOX2-transduced human keratinocytes. d, The efficiency of NANOG+/TRA-1-81+ iPSC generation in OCT4, SOX2, KLF4, and CMYC-transduced human neonatal keratinocytes transduced with p53DD or GFP with or without exposure to UV irradiation. e, γH2AX immunostaining in human neonatal keratinocytes 10 days after transduction with OCT4, SOX2, KLF4, and CMYC and treatment with DAPT, p53DD, or p53 shRNA. Scale bars = 50 μm. f, The percentage of TUNEL-positive cells in human neonatal keratinocyte reprogramming cultures with active or inactive p53 (p53DD expression) 10 days after transduction with OCT4, SOX2, KLF4, and CMYC. g, The number of insertions or deletions (indels) per iPSC line derived under normal, DAPT, or p53DD conditions, as determined by array CGH. For all experiments, error bars represent the standard deviation between two biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test. * denotes significance p-value < .05.

Figure 6. Model of iPSC generation from human keratinocytes

Notch inhibition allows the production of safer oncogene-free iPSCs by suppressing p21 in a p53-independent manner.