A rare human syndrome provides genetic evidence that WNT signaling is required for reprogramming of fibroblasts to induced pluripotent stem cells

Abstract

WNT signaling promotes the reprogramming of somatic cells to an induced pluripotent state. We provide genetic evidence that WNT signaling is a requisite step during the induction of pluripotency. Fibroblasts from individuals with focal dermal hypoplasia (FDH), a rare genetic syndrome caused by mutations in the essential WNT processing enzyme PORCN, fail to reprogram with standard methods. This blockade in reprogramming is overcome by ectopic WNT signaling and PORCN overexpression, thus demonstrating that WNT signaling is essential for reprogramming. The rescue of reprogramming is critically dependent on the level of WNT signaling: steady baseline activation of the WNT pathway yields karyotypically normal iPSCs, whereas daily stimulation with Wnt3a produces FDH-iPSCs with severely abnormal karyotypes. Therefore, although WNT signaling is required for cellular reprogramming, inappropriate activation of WNT signaling induces chromosomal instability, highlighting the precarious nature of ectopic WNT activation and its tight relationship with oncogenic transformation.

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Ectopic WNT signaling and PORCN overexpression restores reprogramming of PORCN mutant fibroblasts. A. Flow chart of reprogramming experiments. Fibroblast cultures established from unaffected and affected FDH individuals were transduced with reprogramming factors (KLF4, MYC, OCT4, SOX2 [KMOS] or KLF4, OCT4, SOX2 [KOS]) via viral infections with either retro- (RV), lenti- (LV) or Sendai-viral (SV) vectors. After passaging to hESC culture conditions, culture media were supplemented with compounds that perturb WNT signaling. Once colonies were visible by eye, cultures were fixed and stained for Alkaline Phosphatase (AP) to quantify colony numbers per condition, or individual colonies were isolated and expanded as iPS cell lines. B.PORCN mutant fibroblasts (FDH2 and 3) fail to reprogram in the absence of exogenous WNT activation. Addition of Wnt3a during reprogramming restores generation of AP+ colonies. Buffer = WNT storage buffer. Shown are representative images of AP stained cultures. Scale bar = 10 mm. C. Quantitation of reprogramming experiments in FDH fibroblasts. (mean ± SD of four biological replicates for WT and of three biological replicates for FDH2 and 3). D. Overexpression of PORCN in FDH3 fibroblasts, as well as treatment with the GSK3 inhibitor CHIR98014 (CHIR), rescues the reprogramming defect of PORCN mutant cells. Scale bar = 10 mm. E. Quantitation of reprogramming experiments in FDH fibroblasts. (mean ± SD of three biological replicates for FDH3). F. A PORCN inhibitor blocks reprogramming. Fibroblasts undergoing reprogramming in the presence of the PORCN inhibitor IWP fail to yield AP positive (AP+) colonies. This inhibitory effect is overcome by addition of Wnt3a protein. (mean ± SD of three biological replicates. **P < 0.005; ***P < 0.001). See also Figure S1 and Table S1.

Figure 2

WNT signaling is required to maintain the pluripotent state. A. WT iPS or FDH iPS (Clone 3.C.1) cells were cultured for 5 days with increasing doses of CHIR98014 (CHIR) and expression of POU5F1/OCT4 and NANOG was determined by qRT-PCR. See also Figure S2. B. Clone 3.C.1 requires low levels of WNT stimulation to maintain an optimal hPSC morphology. In the absence of CHIR, WT iPSCs exhibit a normal hPSC morphology whereas FDH-iPSCs fail to grow with the characteristic morphology associated with hPSCs. At CHIR concentrations of 20nM, FDH iPS cell colonies most closely resemble WT iPS cells. At concentrations of 200nM, both cell populations assume a cell morphology indicative of differentiation. Scale bar = 500μM. C. FDH iPS (Clone 3.C.4) cells were cultured for 5 days in the presence (left) or absence (right) of CHIR (20nM), stained for the cell surface markers SSEA4 and TRA1-81 and analyzed by flow cytometry. Significantly higher cell surface staining for these two pluripotency-associated markers is observed in the presence of CHIR (70%) than in the absence of CHIR (21%).

Figure 3

Characterization of FDH-iPS cell clone. A. A phase contrast image of a FDH-iPS cell clone (3.C.1) derived in the presence of CHIR exhibits characteristics of human pluripotent stem cells. Note: as shown in Figure 4D, these cells have a normal number of chromosomes. Scale bar = 500 μm. B. Quantitative reverse transcription PCR (qRT-PCR) confirms expression of the pluripotency markers NANOG and OCT4/POU5F1 at levels comparable to a human embryonic stem cell line (H1/WA01). C. Flow cytometry confirms presence of the cell surface pluripotency markers TRA1-81 at levels comparable to that found on H1 cells. D–F. Upon differentiation via embryoid body (EB) formation, cells express markers associated with endodermal (D), mesodermal (E), and ectodermal (F) lineages. See also Figure S3.

Figure 4

Ectopic Wnt3a stimulation yields karyotypically abnormal iPS cells. A. A FDH3-iPS cell derived in the presence of exogenous Wn3a (Clone 3.W.1) exhibits an abnormal chromosome number. Additional images of abnormal chromosome spreads highlighting chromosomal anomalies, such as insertions, deletions, translocations and inversions, are provided in the Supplemental Information (Figure S4). B. FDH3 fibroblasts exhibit a normal karyotype. C. An iPS cell derived from unaffected fibroblasts (BJ, ATCC CRL-2522) in the presence of Wnt3a is euploid. Additional characterization of this iPS cell clone is provided in Supplemental Information (Figure S5). D. A FDH-iPS cell (Clone 3.C.1) derived in the presence of the GSK3 inhibitor CHIR98014 (CHIR) is euploid. E. Quantitation of chromosome numbers in FDH-iPS cells. Chromosome numbers were obtained by counting condensed chromosomes of metaphase-arrested cells (for representative images see Supplemental Information Figure S4C). The dashed line marks 46 chromosomes. Like HEK-293T (293T) cells, FDH-iPS cells derived in the presence of exogenous Wnt3a carried significantly increased numbers of chromosomes. The hESC line H1, BJ-iPS cells derived in the presence of Wnt3a (K.W.1 and K.W.2) and FDH-iPS cells derived in the presence of CHIR (clones 3.C.1 and 3.C.2) have 46 chromosomes. Note: This method to quantify chromosome numbers is only reliable in determining the maximum number of chromosomes; metaphase spreads with less than 46 are most likely due to loss of chromosomes during the preparation of the samples. See also Figure S4.

Figure 5

Modulation of WNT signaling influences reprogramming efficiencies. Fibroblasts were reprogrammed with factors KOS or KMOS in the presence of the indicated compounds or transgenes. A. Wnt3a and the GSK3 inhibitor BIO increase KOS reprogramming of wild-type fibroblasts. (Quantification represents mean number of AP+ colonies ± SD, normalized to untreated condition [CTL]; n=7 for CTL, n=6 for Wnt3a, and n=3 for BIO. * P ≤ 0.05, **** P ≤ 0.0001). B. Wnt3a increases KMOS reprogramming of wild-type fibroblasts. (Quantification represents mean number of AP+ colonies ± SD, normalized to untreated condition [CTL]; n=23 for CTL and n=6 for Wnt3a. ** P ≤ 0.01). C. Overexpression (OE) of constitutively active β-catenin (β-catenin 4A) increases reprogramming efficiencies. (Quantification represents mean number of AP+ colonies ± SD, normalized to untreated condition [no Wnt3a and no RSPO1]; n=13 for KOS, n=3 for all other conditions. * P ≤ 0.05). D. RSPO1 sensitizes cells to low levels of Wnt3a. (Quantification represents mean number of AP+ colonies ± SD, normalized to untreated condition [no Wnt3a and no RSPO1]; n=13 for KOS, n=3 for all other conditions. P-values are between corresponding Wnt3a concentrations ± RSPO1, ns = not significant [p-value for 2.5nM Wnt3a data points = 0.061], * P ≤ 0.05 [p-value for 0nM Wnt3a data points = 0.022], ** P ≤ 0.01). E. Addition of Wnt3a increases AP+ colony numbers when added early during the reprogramming process, whereas addition of IWP reduces AP+ colony numbers when added early or late. (Quantification represents mean number of AP+ colonies, normalized to untreated condition [CTL]; n=3 for all conditions). CONT. = continuous treatment with Wnt3a or IWP. F. The Tankyrase inhibitor IWR, which induces AXIN stabilization, reduces numbers of AP+ colonies. (Quantification represents mean number of AP+ colonies ± SD, normalized to untreated KOS condition; n=7 for KOS, n=5 for KOS+IWR, n=13 for KMOS, and n=3 for KMOS+IWR; ** P ≤ 0.01). G.AXIN overexpression (OE) by lenti-viral gene transduction reduces numbers of AP+ colonies. (Quantification represents mean number of AP+ colonies ± SD, normalized to KMOS condition [CTL]; n=23 for CTL and n=9 for AXIN; ** P ≤ 0.01). H. Treatment of reprogramming cultures with Wnt5a, which antagonizes WNT/β-catenin signaling, reduces numbers of AP+ colonies. (Quantification represents mean number of AP+ colonies ± SD, normalized to untreated KOS condition; n=7 for KOS, n=4 for KOS+Wnt5a, n=23 for KMOS, and n=4 for KMOS+Wnt5a; * P ≤ 0.05, *** P ≤ 0.001). I. FZD7 knockdown (KD) by lenti-viral transduction of a FZD7-specific shRNA reduces numbers of AP+ colonies. (Quantification represents mean number of AP+ colonies ± SD, normalized to standard condition [CTL]; n=13 for CTL and n=8 for FZD7 knockdown, ** P ≤ 0.01). See also Figure S5.

Figure 6

A model summarizing the role of WNT signaling during the reprogramming process. An endogenous WNT signaling loop is required for acquisition of the pluripotent state and for the expansion of iPS cells.