Fig 2: DDR1 is required for breast tissue regeneration.a Percentage of patient-derived single primary breast epithelial cells able to give rise to a branched stricture in 3D hydrogel. Data for patients 1–3 was derived from n = 7 gels each, and n = 8 gels for patient 4. Data are presented as mean values ± SD. b Development of single primary breast cells in hydrogels with or without DDR1i treatment. Inset in D18 top panel depicts image directly below (DDR1i, day 18) scaled to match top image (Ctrl, day 18). Results are representative of two independent repeats of this experiment. Scale bar = 100 µm. c Development of breast tissue organoids in hydrogels with or without DDR1i treatment (left) and quantification of lobules (right). Red arrowheads indicate lobules, blue arrows indicate ducts that terminated without a lobule. P = 0.0167 (two-sided Student’s t-test). Data were derived from n = 11 and n = 14 gels for the Ctrl and DDR1i-treated groups, respectively. Data are presented as mean values ± SEM. Scale bars = 100 µm. d Schematic of DDR1-inhibition and withdrawal experiments (left) and representative bright-field images after withdrawing DDR1i (right). Scale bars = 100 µm. e Schematic of stem/progenitor assay in primary breast organoids with or without DDR1i treatment. Representative image of vehicle-treated development after 14 days is shown below. Scale bar = 100 µm. f Clone formation by cells isolated from primary breast organoids with or without DDR1i treatment, and 2 days following release from DDR1 inhibition. Data were derived from n = 12 gels for each treatment group. P(Ctrl vs. DDR1i) = 0.0003; P(Ctrl vs. DDR1i-Rel) = 0.02 (ordinary one-way ANOVA with Tukey’s multiple comparisons test). Data are presented as mean values ± SD. g Percentage of clones composed exclusively of cells stained with either of the lineage markers CK18 and p63, or mixed. Data was derived from n = 12 gels for each treatment group. Data are presented as mean values ± SD. h Mammosphere formation by cells isolated from primary breast organoids cultured in hydrogel with or without DDR1i treatment. P = 0.0006 (two-sided Student’s t-test). Data were derived from n = 24 gels for each treatment group. Data are presented as mean values ± SD. i Mammosphere formation by primary breast cells cultured in suspension culture with or without DDR1i treatment. P = 8.8 × 10-5, 0.001 and 3 × 10-6 comparing Ctrl and DDR1i groups, for patients A, B, and C, respectively (ordinary two-way ANOVA with Sidak’s multiple comparisons test). Data were derived from n = 3 gels per patient for each treatment group. Data are presented as mean values ± SD. j Flow-cytometry dot-plot analysis of EpCAM and CD49f expression on cells from primary breast organoids grown in hydrogels for 14 days without or with DDR1i treatment, or released from DDR1 inhibition for the last 2 days. k Graph depicts cell population percentages under each condition. Data were derived from n = 2, n = 4, and n = 3 gels for the Ctrl, DDR1i and DDR1i-Rel groups, respectively. Data are presented as mean values ± SD. * Indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001. Source data are provided as a Source data file.

Fig 3: Ovalbumin is able to downregulate a-SMA biosynthesis in MDA-MB-231 cells.Phase contrast microscopy. (A): confluent MCF-7 cells in OV condition (day 7). (B): confluent MDA-MB-231 cells in OV condition (day 7): OV-treated cells did not form acini or duct-like structures in both cell lines. (C) and (D): representative western blot showing the levels of the protein ß-casein in MCF-7 and MDA-MB-231 cells respectively, after 5 days. (E) and (F): representative western blot showing the levels of the proteins E-cadherin, CK18, and a-SMA in MDA-MB-231 cells, after 5 days; a-tubulin was used as a loading control. Ctrl (control), EW (egg white), and OV (ovalbumin).

Fig 4: Establishment of human SwG organoids from iSwGCs. a) Representative phase contrast images showing iSwGO cultures. Scale bar = 100 µm; b) Representative images of lumen-containing organoids derived from iSwGCs. d and l represent the diameter (lumen width) and long axis of the lumen, respectively; c) Signal distribution acquired by confocal microscopy showing the quantification of luminal-containing organoids derived from reprogrammed HEKs (n = 23). Organoids with similar size were analyzed independently from 3 biological replicates; d,e) Scatter plots representing the features of iSwGOs. HEKs cultured under the same 3D condition were controls; f) Immunofluorescence assay of ductal markers CK5 and CK10, luminal markers AQP5 and CK18, and myoepithelial markers CK5 and a-SMA in iSwGOs. The iSwGOs were obtained at passages 2–4 after the initiation of 3D culture. Scale bar = 50 µm; g–i) Percentages of CK5+/CK10+-, AQP5+/CK18+-, and CK5+/a-SMA+-expressing organoids in each GFP-positive population were shown. Quantifications involved > 100 organoids from 3 independent experiments. Data are mean ± SD; j) Immunofluorescence co-staining of a-SMA with CK18 or CK19 in iSwGOs generated from CD49fhiCD29hi cells. Scale bar = 75 µm; k) Fluorescence live cell imaging of intracellular Ca2+ activity in iSwGOs after ACh addition. Scale bar = 25 µm, n = 3; l) Immunofluorescence analysis of expression of ductal markers CK5 and CK10, luminal markers AQP5 and CK18 and myoepithelial markers CK5 and a-SMA in tubular structures generated from iSwGOs in response to bFGF gradients. Scale bar = 25 µm.

Fig 5: Engraftment of iSwGOs functionally repopulated damaged skin. a) Schematic diagram representing the experimental procedure; b) Starch–iodine sweat tests on paw skin of thermal-injured mice showed that only paws of iSwGO-treated mice responded by displaying indigo-black dots at day 21 after transplantation; c) Starch–iodine assessment of newly formed SwGs in the defect regions of thermally injured mice at day 30 after SGM, iSwGC and iSwGO treatment. The number of mice positive for starch-iodine reaction increased in a time-dependent manner. After 30 days treatment with iSwGOs, 34.4 ± 13.0% of the recipient mice (n = 60 per group) exhibited sweat production as compared with those treated with iSwGCs (13.3 ± 10.6%, n = 60), SGM (0, n = 60) and vehicle control (0, n = 60). Data are mean ± SD of 6 independent experiments; d) H&E staining was conducted to visualize SGM-, iSwGC-, iSwGO-treated wounds at day 21 post-injury. Emerging glandular structures were seen in the dermis of iSwGO-treated mice. Dotted line represents the ridges of epidermis where the sweat pores open. Note that the rete ridges in iSwGO-engrafted paw skin were elongated and intertwined with underlying dermal tissues comparable to those derived from normal skin. Scale bar = 100 µm; e) Immunofluorescence and quantification of SwG marker expression in nascent glands at day 21 after iSwGO transplantation (left). EDA-GFP+ cells were seen in the newly generated glandular structures. Newly emerged glands showed typical SwG morphological features and expressed SwG specific markers, for example, ductal markers CK5 and CK10, luminal markers AQP5, CK18, and myoepithelial markers CK5 and a-SMA. Scale bar = 25 µm. Quantification (right) involved > 150 cells from 3 independent experiments, and GFP+ cells within the duct, luminal, and myoepithelial domain were measured, respectively; f) Immunofluorescence of human-specific histone protein expression in nascent SwGs at day 21 after iSwGO transplantation. Scale bar = 25 µm; g) Wound healing curves for quantification of the wound coverage at different times in SGM-, iSwGC-, iSwGO-treated mice (n = 15, 3 independent experiments). Mice treated with DMEM/F-12 were vehicle controls. Data are mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.