Fig 1: 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 α‐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.
Fig 2: Reprogramming of HEKs into SwG cells by combining the stimulation of β 2‐AR, forced transgenic expression of EDA and SGM culture. a) Scheme of ISO‐based reprogramming procedure. ISO‐treated HEKs were transduced with EDA and plated in Epilife. Then cells were transferred into SGM supplemented with 5 µm ISO (Day 0) and cultured for indicated days; b) Phase contrast images showing the morphological changes of iSwGCs in optimized SGM containing ISO. Scale bar = 100 µm. Insets, higher magnification of the boxed areas; c) qPCR analysis of transcriptional expression of CK5, CK18, AQP5, α‐SMA, and hair follicle‐specific genes LHX2, CDH3 in HEKs, and iSwGCs after 8 days of induction. Primarily isolated SwG cells from human skin samples (hSwGCs) were used as positive controls. The genes showing significant change in PCR array assay are presented; d) Representative immunofluorescence of CK5, CK10, AQP5, CK18, and α‐SMA in HEKs, and iSwGCs at day 20 after SGM treatment with ISO. Scale bar = 50 µm; e) Percentages of CK5+, CK10+, AQP5+, CK18+, and α‐SMA+ cells in HEKs and iSwGCs calculated according to the immunostaining. Quantification was done with 5 randomly selected microscopy fields from each of the 3 independent experiments; f) FACS analysis showing the cell fractions labeled with antibodies against CK5, CK10, AQP5, CK18, and α‐SMA in HEKs, iSwGCs and native hSwGCs. g) Proportions and absolute numbers of the CK5+/CK10+, AQP5+/CK18+, and CK5+/α‐SMA+ cell population in HEKs, iSwGCs, and hSwGCs. n = 3. Data are mean ± SD and analyzed by two‐tailed t‐tests, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.
Fig 3: 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 α‐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+/α‐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 α‐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 α‐SMA in tubular structures generated from iSwGOs in response to bFGF gradients. Scale bar = 25 µm.
Fig 4: Functional analysis of iSGC. a Representative immunofluorescence of CK18+ and AQP5+ in HDF and iSGC. Scale bar = 50 μm. b Percentages of CK18+ and AQP5+ cells in HDF and iSGC calculated according to the immunostaining. Quantification was done with 5 randomly selected microscopy fields from each of the 3 independent experiments. c Calcium activity analysis was used to assess the reactivity to acetylcholine. d The data presented the intracellular free Ca2+ intensity of iSGCs was higher than HF-EDA in SGM and similar to that of the pSGC, (60.79 ± 7.71)%, (12.65 ± 2.07)% and (70.59 ± 0.34)%, respectively. n = 3. Data were expressed as mean ± SD and analyzed by two-tailed t-tests, **P < 0.01, ***P < 0.001, ns not significant, HDF human dermal fibroblasts, iSGC induced sweat gland-like cell, CK18 cytokeratin 18, AQP5 aquaporin 5, EDA ectodermal dysplasia antigen, SGM sweat gland culture medium, pSGC primary sweat gland cell, ns not significant
Fig 5: Negr1 expressed in mouse and human salivary glands(A) Messenger RNA expression levels of mouse Negr1 genes were monitored in the mouse submandibular glands, cerebral cortex, hippocampus, and stomach of wildtype (WT) and Negr1 knockout (KO) mice. Gapdh: glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene used as a PCR positive control.(B) Messenger RNA expression levels of human Negr1 genes were monitored in a human submandibular gland (HSG) cell line and human submandibular gland tissue. Gapdh: a housekeeping gene used as a PCR positive control.(C) Quantitative RT-PCR analysis of Negr1 messenger RNA in the mouse submandibular glands, cerebral cortex, hippocampus, and stomach of WT (n = 5) and Negr1 KO mice (n = 5) normalized to a reference gene (Gapdh). Fold change in Negr1 mRNA expression is relative to WT mice and averaged from independent experiments.(D) The gross morphology of the submandibular gland tissue from WT and Negr1 KO mice was visualized by hematoxylin and eosin staining. Scale bars, 50 μm.(E) The submandibular gland size was monitored in WT (n = 8) and Negr1 KO mice (n = 8).(F) The expression levels of aquaporin-5 (AQP5) and E-cadherin (E-cad) were monitored in the submandibular gland of WT (n = 4) and Negr1 KO mice (n = 4).(G) Representative immunofluorescence images of mouse submandibular gland tissues were immunostained with anti-AQP5 (green) and anti-E-cadherin (red), and merged with DAPI image (blue). Scale bar, 10 μm. Data are represented as mean ± SEM. ∗p < 0.05.
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