Specific complexes derived from extracellular matrix facilitate generation of structural and drug-responsive human salivary gland microtissues through maintenance stem cell homeostasis
Siqi Zhang
Department of Oral and Maxillofacial Surgery/Central Laboratory, School and Hospital of Stomatology, Peking University, Beijing, China
Laboratory of Biomaterials and Regenerative Medicine, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
Search for more papers by this authorYi Sui
Department of Oral and Maxillofacial Surgery/Central Laboratory, School and Hospital of Stomatology, Peking University, Beijing, China
Search for more papers by this authorXiaoming Fu
Department of Oral and Maxillofacial Surgery/Central Laboratory, School and Hospital of Stomatology, Peking University, Beijing, China
Search for more papers by this authorYanrui Feng
Department of Oral and Maxillofacial Surgery/Central Laboratory, School and Hospital of Stomatology, Peking University, Beijing, China
Search for more papers by this authorZuyuan Luo
Laboratory of Biomaterials and Regenerative Medicine, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
Search for more papers by this authorCorresponding Author
Yuanyuan Zhang
Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC
Correspondence
Shicheng Wei, Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Peking University, No. 22 Zhong-Guan-Cun South Road, Hai-Dian District, Beijing 100081, China.
Email: [email protected]
Yuanyuan Zhang, Wake Forest Institute for Regenerative Medicine, 391 Technology Way, Winston-Salem 27157, NC.
Email: [email protected]
Search for more papers by this authorCorresponding Author
Shicheng Wei
Department of Oral and Maxillofacial Surgery/Central Laboratory, School and Hospital of Stomatology, Peking University, Beijing, China
Laboratory of Biomaterials and Regenerative Medicine, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
Correspondence
Shicheng Wei, Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Peking University, No. 22 Zhong-Guan-Cun South Road, Hai-Dian District, Beijing 100081, China.
Email: [email protected]
Yuanyuan Zhang, Wake Forest Institute for Regenerative Medicine, 391 Technology Way, Winston-Salem 27157, NC.
Email: [email protected]
Search for more papers by this authorSiqi Zhang
Department of Oral and Maxillofacial Surgery/Central Laboratory, School and Hospital of Stomatology, Peking University, Beijing, China
Laboratory of Biomaterials and Regenerative Medicine, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
Search for more papers by this authorYi Sui
Department of Oral and Maxillofacial Surgery/Central Laboratory, School and Hospital of Stomatology, Peking University, Beijing, China
Search for more papers by this authorXiaoming Fu
Department of Oral and Maxillofacial Surgery/Central Laboratory, School and Hospital of Stomatology, Peking University, Beijing, China
Search for more papers by this authorYanrui Feng
Department of Oral and Maxillofacial Surgery/Central Laboratory, School and Hospital of Stomatology, Peking University, Beijing, China
Search for more papers by this authorZuyuan Luo
Laboratory of Biomaterials and Regenerative Medicine, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
Search for more papers by this authorCorresponding Author
Yuanyuan Zhang
Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC
Correspondence
Shicheng Wei, Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Peking University, No. 22 Zhong-Guan-Cun South Road, Hai-Dian District, Beijing 100081, China.
Email: [email protected]
Yuanyuan Zhang, Wake Forest Institute for Regenerative Medicine, 391 Technology Way, Winston-Salem 27157, NC.
Email: [email protected]
Search for more papers by this authorCorresponding Author
Shicheng Wei
Department of Oral and Maxillofacial Surgery/Central Laboratory, School and Hospital of Stomatology, Peking University, Beijing, China
Laboratory of Biomaterials and Regenerative Medicine, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
Correspondence
Shicheng Wei, Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Peking University, No. 22 Zhong-Guan-Cun South Road, Hai-Dian District, Beijing 100081, China.
Email: [email protected]
Yuanyuan Zhang, Wake Forest Institute for Regenerative Medicine, 391 Technology Way, Winston-Salem 27157, NC.
Email: [email protected]
Search for more papers by this authorAbstract
Three-dimensional cultured salivary glands (SGs) microtissues hold great potentials for clinical research. However, most SGs microtissues still lack convincing structure and function due to poor supplementation of factors to maintain stem cell homeostasis. Extracellular matrix (ECM) plays a crucial role in regulating stem cell behavior. Thus, it is necessary to model stem cell microenvironment in vitro by supplementing culture medium with proteins derived from ECM. We prepared specific complexes from human SG ECM (s-Ecx) and analyzed the components of the s-Ecx. Human SG epithelial and mesenchymal cells were used to generate microtissues, and the optimum seeding cell number and ratio of two cell types were determined. Then, the s-Ecx was introduced to the culture medium to assess its effect on stem cell behavior. Multiple specific factors were presented in s-Ecx. s-Ecx promoted maintenance of the stem cell and formation of specific structures resembling that of salivary glands and containing mucins, which suggested stem cell differentiation potential. Moreover, treatment of the microtissues with s-Ecx increased their sensitivity to neurotransmitters. On the basis of the analysis of components, we believed that the presented growth factors are able to interact with stem cell they encountered in vivo, which promote the capacity to maintain stem cell homeostasis. This work provided foundations to study molecular mechanism of stem cell homeostasis in SGs and develop novel therapies for dry mouth through new drug discovery and disease modeling.
CONFLICT OF INTEREST
The authors have declared that no competing interest exists.
Supporting Information
| Filename | Description |
|---|---|
| term2992-sup-0001-Supplementary_Material.docxWord 2007 document , 3.9 MB |
Figure S1. Optimization of SG microtissues culture conditions. a) schematic diagram of a novel hanging drop 3D culture platform and uniform microtissues were generated; b) growth factor protein array map corresponding to figure 1c. POS: positive control of protein array assay. Figure S2. Optimization of SG microtissues culture conditions. a) Structural aspects of SG microtissues (5000-cells) with a low frequency of apoptosis, as assessed by TUNEL and DAPI staining. b) Number of apoptotic cells according to the epithelial: mesenchymal cell ratio compared to the ratio of 9:1. c) Disrupted structure of microtissues with a mesenchymal:epithelial cell ratio of 1:9 at day 21. Scale bar indicates 100 μm. Data are means ± standard deviations (SD) of three independent experiments. *, p < 0.05; ***, p < 0.01. Figure S3. Optimization of SG microtissues culture conditions. a-f) Immunofluorescence staining for ki67 of microtissues with different staring number and the ratio of two cell types. g) Immunofluorescence staining for CD73 of microtissues with cell ratio of 9:1. Scale bar indicate 10 μm (a'), 25 μm (b' and c'), 50 μm (a, d', e', f') and 100 μm (b-g) Figure S4. Optimization of the s-Ecx concentration. a) Morphology of microtissues treated with 0% (control) or 1% s-Ecx at day 7. b) 10% s-Ecx did not generate SG microtissues. c) Morphology and histological staining of microtissues treated with 0% (control) and 1% s-Ecx at day 14. Scale bar indicates 100 μm. Figure S5. Identify of microtissues with or without s-Ecx. a) Immunofluorescence staining of AQP5 following treatment with s-Ecx. b) Immunofluorescence staining of the SG progenitor cell markers K5 and K19 treatment for 24 h and 48 h. c-e) Immunofluorescence staining of ki67 treatment for 24 h and 48 h. Scale bar indicate 25 μm (c'-e') and 50 μm (a-e). |
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