Research ArticleHEALTH AND MEDICINE

Biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation for functional sweat gland regeneration

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Science Advances  04 Mar 2020:
Vol. 6, no. 10, eaaz1094
DOI: 10.1126/sciadv.aaz1094
  • Fig. 1 Schematic illustration of 3D-bioprinted MSC-loaded constructs and cellular survival, proliferation, and morphology of printed construct.

    (A) Schematic description of the approach. (B) Full view of the cellular construct and representative microscopic and fluorescent images and the quantitative parameters of 3D-printed construct (scale bars, 200 μm). Photo credit: Bin Yao, Wound Healing and Cell Biology Laboratory, Institute of Basic Medical Sciences, General Hospital of PLA. (C) Representative microscopy images of cell aggregates and tissue morphology at 3, 7, and 14 days of culture (scale bars, 50 μm) and scanning electron microscopy (sem) images of 3D structure (scale bars, 20 μm). PD+/PD, 3D construct with and without PD. (D) DNA contents, collagen, and GAGs of native tissue and PD. (E) Proliferating cells were detected through Ki67 stain at 3, 7, and 14 days of culture. (F) Live/dead assay show cell viability at days 3, 7, and 14. *P < 0.05.

  • Fig. 2 Transcriptional and translational level of SG-specific and secretion-related markers in 3D-bioprinted cells with or without PD.

    (A) Transcriptional expression of K8, K18, Fxyd2, Aqp5, and ATP1a1 in 3D-bioprinted cells with and without PD in days 3, 7, and 14 culture by quantitative real-time polymerase chain reaction (qRT-PCR). Data are means ± SEM. (B) Comparison of SG-specific markers K8 and K18 in 3D-bioprinted cells with and without PD (K8 and K18, red; DAPI, blue; scale bars, 50 μm). (C and D) Comparison of SG secretion-related markers ATP1a1 (C) and Ca2+ (D) in 3D-bioprinted cells with and without PD [ATP1a1 and Ca2+, red; 4′,6-diamidino-2-phenylindole (DAPI), blue; scale bars, 50 μm].

  • Fig. 3 Expression of CTHRC1 in different ECM.

    (A and B) Differential expression of CTHRC1 and TSP1in PD and back dermis (DD) ECM of mice by proteomics analysis (A) and Western blotting (B). (C) CTHRC1 and TSP1 expression in back and plantar skin of mice at different developmental times. (Cthrc1/TSP1, red; DAPI, blue; scale bars, 50 μm).

  • Fig. 4 The transcriptional analysis of four groups of cells revealed the specific gene response to 3D structure.

    (A) Gene expression file of four groups of cells (R2DC, MSCs; R2DT, MSC with PD treatment; R3DC, MSC cultured in 3D construct; and R3DT, MSC treated with 3D/PD). (B) Up-regulated genes after treatment (2DC, MSCs; 2DT, MSC with PD treatment; 3DC, MSC cultured in 3D construct; and 3DT, MSC treated with 3D/PD). (C) Differentially expressed genes were further validated by RT-PCR analysis. [For all RT-PCR analyses, gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with 40 cycles, data are represented as the means ± SEM, and n = 3].

  • Fig. 5 CTHRC1 and HMOX1 synergistically boost SG fate of MSC.

    (A and B) Transcriptional analysis (A) and translational analysis (PD, MSCs; PD+, MSCs with 3D/PD treatment; CAPE, MSCs treated with 3D/PD and Hmox1 activator; Snpp, MSCs treated with 3D/PD and Hmox1 inhibitor; Cthrc1, MSCs treated with 3D/PD and recombinant CTHRC1; anti, MSCs treated with 3D/PD and CTHRC1 antibody: +/+, MSCs treated with 3D/PD and Hmox1 activator and recombinant CTHRC1; and −/−, MSCs treated with 3D/PD and Hmox1 inhibitor and CTHRC1 antibody. Data are represented as the means ± SEM and n = 3) (B) of bmp2 with regulation of CTHRC1 and Hmox1. (C) The graphic illustration of 3D-bioprinted matrix directed MSC differentiation. CTHRC1 is the main biochemical cues during SG development, and structural cues up-regulated the expression of Hmox1 synergistically initiated branching morphogenesis of SG. *P < 0.05.

  • Fig. 6 Directed regeneration of SG in thermal-injured mouse model after transplantation of iSGCs.

    (A) Schematic illustration of approaches for engineering iSGCs and transplantation. (B) Sweat test of mice treated with different cells. Photo credit: Bin Yao, Wound Healing and Cell Biology Laboratory, Institute of Basic Medical Sciences, General Hospital of PLA. (C) Histology of plantar region without treatment and transplantation of MSCs and iSGCs (scale bars, 200 μm). (D) Involvement of GFP-labeled iSGCs in directed regeneration of SG tissue in thermal-injured mouse model (K14, red; GFP, green; DAPI, blue; scale bar, 200 μm). (E) SG-specific markers K14, K19, K8, and K18 detected in regenerated SG tissue (arrows). (K14, K19, K8, and K18, red; GFP, green; scale bars, 50 μm).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/10/eaaz1094/DC1

    Fig. S1. Biocompatibility of 3D-bioprinted construct and cellular morphology in 2D monolayer culture.

    Fig. S2. Expression of SG-specific and secretion-related markers in MSCs and SG cells in vitro.

    Fig. S3. Transcriptional and translational expression of epithelial and mesenchymal markers in 3D-bioprinted cells with and without PD.

    Fig. S4. Expression of N- and E-cadherin in MSCs and SG cells in 2D monolayer culture.

    Fig. S5. Proteomic microarray assay of differential gene expression between PD and DD ECM in postnatal mice.

    Fig. S6. GO term analysis of differentially expressed pathways.

    Fig. S7. Heat maps illustrating differential expression of genes implicated in ECM organization, cell division, and gland and branch morphogenesis.

    Fig. S8. The expression of Hmox1 and the concentration of CTHRC1 on treatment and the related effects on cell proliferation.

    Fig. S9. The expression of K8 and K18 with Hmox1 and CTHRC1 regulation.

    Table S1. Primers for qRT-PCR of all the genes.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Biocompatibility of 3D-bioprinted construct and cellular morphology in 2D monolayer culture.
    • Fig. S2. Expression of SG-specific and secretion-related markers in MSCs and SG cells in vitro.
    • Fig. S3. Transcriptional and translational expression of epithelial and mesenchymal markers in 3D-bioprinted cells with and without PD.
    • Fig. S4. Expression of N- and E-cadherin in MSCs and SG cells in 2D monolayer culture.
    • Fig. S5. Proteomic microarray assay of differential gene expression between PD and DD ECM in postnatal mice.
    • Fig. S6. GO term analysis of differentially expressed pathways.
    • Fig. S7. Heat maps illustrating differential expression of genes implicated in ECM organization, cell division, and gland and branch morphogenesis.
    • Fig. S8. The expression of Hmox1 and the concentration of CTHRC1 on treatment and the related effects on cell proliferation.
    • Fig. S9. The expression of K8 and K18 with Hmox1 and CTHRC1 regulation.
    • Table S1. Primers for qRT-PCR of all the genes.

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