Endogenous Wnt signalling in human embryonic stem cells generates an equilibrium of distinct lineage-specified progenitors

Abstract

The pluripotent nature of human embryonic stem cells (hESCs) makes them convenient for deriving therapeutically relevant cells. Here we show using Wnt reporter hESC lines that the cells are heterogeneous with respect to endogenous Wnt signalling activity. Moreover, the level of Wnt signalling activity in individual cells correlates with differences in clonogenic potential and lineage-specific differentiation propensity. The addition of Wnt protein or, conversely, a small-molecule Wnt inhibitor (IWP2) reduces heterogeneity, allowing stable expansion of Wnt(high) or Wnt(low) hESC populations, respectively. On differentiation, the Wnt(high) hESCs predominantly form endodermal and cardiac cells, whereas the Wnt(low) hESCs generate primarily neuroectodermal cells. Thus, heterogeneity with respect to endogenous Wnt signalling underlies much of the inefficiency in directing hESCs towards specific cell types. The relatively uniform differentiation potential of the Wnt(high) and Wnt(low) hESCs leads to faster and more efficient derivation of targeted cell types from these populations.

Figure 1. Endogenous Wnt signalling reveals distinct subpopulations of hESCs.

Analysis of transgenic 7xTCF-GFP hESCs in standard self-renewing conditions. (a) TCF-GFP expression pattern in live hESC colonies. Scale bar, 200 μm. (b) FACS analysis of TCF-GFP expression in live cells after 4 days of IWP2, Wnt3a or CHIR99021 treatment. Pre-gated for live, non-mouse cells. (c) Quantitative Clonogenic Potential Assay. Double FACS-sorted populations of TCF-GFP^high and TCF-GFP^low cells were plated as single cells in standard hESC. Clonogenic potential was determined by counting the number of alkaline phosphatase (AP)-positive (left) and OCT4-positive (right) colonies 7 days after plating. Error bars show the range of two biological replicates and are representative of three independent experiments. (d) Gene expression analysis in double FACS-sorted populations of >99.5% purity. The ratio of glyceraldehyde 3-phosphate dehydrogenase-normalized gene expression level in TCF-GFP^high hESCs divided by TCF-GFP^low hESCs is shown. ND, not detectable. ±s.d of three biological replicates and are representative of two independent experiments. (e) GFP expression pattern of live colonies derived from plating single cells of >99.5% pure populations at clonal density (500 cells per cm²) in standard self-renewing conditions. The starting populations are indicated on the x axis, examples of the three GFP expression patterns counted are shown in a. (f) FACS analysis of GFP expression in hESC cultures 9 days after plating single cells from the indicated population of sorted cells. %GFP^pos is based on live, non-mouse cells. (g) FACS analysis of GFP expression in hESC passage 2 cultures initiated from single colonies that were entirely GFP^low or GFP^high. %GFP^pos is based on live, non-mouse cells.

Figure 2. Role of Wnt signalling in proliferation and survival.

(a) FACS analysis of TCF-GFP hESCs grown in standard hESC conditions showing the number of proliferating (EdU-positive) cells in log-phase cultures (boxed cells). Cells were pre-gated for OCT4-positive non-mouse cells single cells, and separated into either TCF^low (left panel) or TCF^high (right panel) populations. (b) The percentage of proliferating OCT4-positive cells in TCF^low and TCF^high populations in a was averaged across two independent experiments±range. (c) The percentage of proliferating OCT4-positive hESCs after 3 days of treatment with Wnt3a or vehicle was measured as in a and b. The average from two independent experiments is shown. (d) Log-phase hESCs were treated with IWP2 or Wnt3a for 24 h, digested to single cells, counted and plated at 200,000 cells per cm². The number of viable attached cells still present 24 h after passage was measured. The average number of cells in four replicates of two independent experiments is shown. The P-value is calculated from a two-tailed equal variance T-test. (e) Identical numbers of H9 or H9-BCL2 hESCs were plated in standard hESC media on Matrigel in the presence of dimethyl sulfoxide (DMSO; control for IWP2), IWP2, vehicle (control for Wnt3a) or Wnt3a. The number of AP-positive colonies was counted 6 days later. The average of three replicates in a single experiment are shown, ±s.d.

Figure 3. Serum replacement blocks Wnt-mediated differentiation.

(a) Alkaline phosphatase (AP) staining of cells grown in 'standard (std)', 'N2B27' and 'mTeSR1' media for three passages in the continuous presence of Wnt3a protein or control buffer (vehicle). Scale bar, 2 mm. (b) Quantification of Wnt-mediated changes in self-renewal during passage as clumps. The number of AP+ colonies was determined after each passage. The ratio of cumulative number of AP+ colonies in Wnt3a-treated cultures divided by AP+ colonies in vehicle-treated cultures is shown for each passage in each media. 100% represents an equal number of AP+ colonies in Wnt3a- and vehicle (Veh)-treated cultures. The average ±s.d. of three biological replicates is shown, and is representative of two independent experiments. (c) Quantification of Wnt-mediated changes in total cell number during passage as clumps. The ratio of cumulative cell number in Wnt3a-treated cultures divided vehicle-treated cultures is shown on a log scale. The average ±s.d. of three biological replicates is shown, and is representative of two independent experiments. (d) Phase-contrast and OCT4, Brachyury and GATA4 immunofluorescence images of cells treated with Wnt3a for 7 days in standard hESC media, N2B27 or mTeSR1. Scale bar, 200 μm. (e) Phase-contrast images of hESCs treated for 4 days with Wnt3a in N2B27 media alone, or the same supplemented with 20% PBS or 20% knockout serum replacement (KOSR). Scale bar, 200 μm. (f) AP staining of hESC treated for 3 days with Wnt3a in standard hESC media, or the same media in which the KOSR concentration was reduced to 2%. Scale bar, 200 μm. (g) Phase contrast and GFP fluorescence in live colonies after 5 days of growth on N2B27 in the presence of vehicle, 20% PBS or 20% KOSR. Scale bar, 200 μm. (h,i) Gene expression analysis in cells grown in N2B27 plus the indicated combination of vehicle, Wnt3a and KOSR for four passages. Values were normalized to glyceraldehyde 3-phosphate dehydrogenase, and are expressed relative to vehicle-treated cells at passage 1. The average and range of two biological replicates is shown.

Figure 4. Lysophosphatidic acid (LPA) accounts for much of KOSR activity in blocking Wnt-mediated differentiation.

(a) KOSR was filtered through different molecular-weight cutoff (MWCO) filters. The flow-through containing proteins smaller than the MWCO at their original concentration was added to 20% (v/v) in N2B27 media. The ability of Wnt3a to induce morphological changes after 3 days was used to determine which fraction contained the factor(s) that prevent Wnt-mediated differentiation. The >100 kDa is enriched for this size, but still contains all smaller proteins at ¼ of the original concentration. Phase-contrast images of hESCs on N2B27 treated with vehicle (Veh) or Wnt3a plus the indicated fraction of KOSR for 3 days are shown. Scale bar, 200 μm. (b) TCF-GFP hESCs were grown on N2B27 plus 20% PBS, 1.5% fatty acid-free BSA (FAF-BSA), 1.6% AlbuMaxII (Invitrogen) or 10 μM LPA supplemented with 1% BSA. Phase-contrast and GFP fluorescence images are shown for cells treated with Veh or Wnt3a for 3 days. Scale bar, 200 μm. (c) FACS analysis of cells in b showing the fraction of Wnt^high hESCs. (d) hESCs were grown in N2B27 media, except that irradiated feeders were used in lieu of Matrigel, for three passages in the presence of 1% FAF-BSA, 1 μM LPA or 10 μM LPA. Veh or Wnt3a was added fresh daily throughout. Alkaline phosphatase (AP) staining after three passages is shown. Scale bar, 500 μm. (e–g) The cumulative number of AP-positive colonies present after each of three passages in the indicated conditions are shown. The average ±s.d. of three biological replicates is shown.

Figure 5. Distinct lineage-specific differentiation propensities of Wnt^low and Wnt^high hESCs.

(a,b) hESCs maintained with various levels of Wnt pathway activation were subjected to the neural differentiation protocol for 5 days. Neuralectodermal differentiation was detected by immunostaining for Pax6 (a) and Otx2 (b). Scale bar, 200 μm. (c) FACS-sorted populations of >95% pure TCF-GFP^high or TCF-GFP^low were subjected to the neural differentiation protocol for 5 days. Neural differentiation was detected by PAX6 staining, and compared with the number of DAPI-positive nuclei using an automated counting algorithm. Average ±s.d. from five fields of view, representing three independent experiments. (d) Immunofluorescent staining for endodermal marker FoxA2 in hESCs subjected to the neural differentiation protocol for 5 days. Scale bar, 200 μm. (e) Immunofluorescent staining for endodermal marker Gata4 on day 5 of the endodermal differentiation protocol. Scale bar, 200 μm. (f) Schematic description of cardiogenic differentiation protocol. (g) IWP2- and Wnt3a-treated hESCs in standard hESC media were subjected to the cardiogenic differentiation protocol, and shortened versions thereof. The number of spontaneously beating sectors observed on days 15–18 of the full-length protocol (days 12–15 of the shortest protocol) were averaged. Error bars ±s.d., n=4. (h–m) Hematoxylin and eosin staining of tumours arising 8 weeks after injecting hESCs maintained in N2B27 with Wnt3a and KOSR for five passages under the kidney capsule of nude mice. (h) Neural rosette, (i) pigmented epithelium, (j,k) glandular structures, (l) cartilage and (m) bone. Scale bar, 50 μm.