Stem cell properties of human clonal salivary gland stem cells are enhanced by three-dimensional priming culture in nanofibrous microwells

BACKGROUND: Three-dimensional (3D) cultures recapitulate the microenvironment of tissue-resident stem cells and enable them to modulate their properties. We determined whether salivary gland-resident stem cells (SGSCs) are primed by a 3D spheroid culture prior to treating irradiation-induced salivary hypofunction using in-vitro coculture and in-vivo transplant models. METHODS: 3D spheroid-derived SGSCs (SGSCs(3D)) were obtained from 3D culture in microwells consisting of a nanofiber bottom and cell-repellent hydrogel walls, and were examined for salivary stem or epithelial gene/protein expression, differentiation potential, and paracrine secretory function compared with monolayer-cultured SGSCs (SGSCs(2D)) in vitro and in vivo. RESULTS: SGSCs(3D) expressed increased salivary stem cell markers (LGR5 and THY1) and pluripotency markers (POU5F1 and NANOG) compared with SGSCs(2D). Also, SGSCs(3D) exhibited enhanced potential to differentiate into salivary epithelial cells upon differentiation induction and increased paracrine secretion as compared to SGSCs(2D). Wnt signaling was activated by 3D spheroid formation in the microwells and suppression of the Wnt/beta-catenin pathway led to reduced stemness of SGSCs(3D). Enhanced radioprotective properties of SGSCs(3D) against radiation-induced salivary hypofunction was confirmed by an organotypic 3D coculture and in-vivo transplantation experiments. CONCLUSION: The 3D spheroid culture of SGSCs in nanofibrous microwells promotes stem cell properties via activation of Wnt signaling. This may contribute to SGSC priming prior to regenerative therapy to restore salivary hypofunction after radiotherapy.ope

Organotypic 3D Culture in Nanoscaffold Microwells Supports Salivary Gland Stem-Cell-Based Organization

The self-organizing properties of stem cells have been exploited to generate organoids, organ-specific, cell-containing, three-dimensional (3D) structures. The present study aimed to introduce a novel bioengineering technique for driving the effective organization of adult tissue stem cells via niche-independent 3D microwell culture. Microwells were fabricated by photopatterning poly(ethylene glycol) hydrogel in the presence of an electrospun polycaprolactone nanofibrous scaffold. Human single clonal salivary gland stem cells (SGSCs) were cultured in nanofibrous microwells through two simple steps, priming and differentiation. Before the induction of 3D organization, single clonal SGSCs were preconditioned to aggregate to form 3D spheroids in different matrices, such as Matrigel, floating dish, and microwells. Expression of salivary stem cell markers and pluripotency markers was greater in 3D spheroid cultures than in 2D plastic culture. Lobular structures were organized by changing media, and those in microwells exhibited higher salivary acinar, ductal, and tight junction marker levels and decreased stem-cell marker levels relative to other 3D cultures. Furthermore, higher α-amylase secretion and intracellular calcium levels were observed in the presence of adrenergic or cholinergic agonists, respectively, along with an increased intracellular pH, suggesting more functional salivary organoid formation. These microwell-driven organoids also engrafted successfully into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Our results showed that microwell-cultured SGSCs organize into salivary structures and that this biomimetic 3D culture technique can promote effective generation of niche-independent single stem-cell-based 3D organoids.ope

Comparative characteristics of laryngeal-resident mesenchymal stromal cell populations isolated from distinct sites in the rat larynx

BACKGROUND: Although tissue-resident mesenchymal stromal cells (MSCs) in the larynx have been described, their distinct characteristics and roles have not been thoroughly explored. Therefore, we investigated stem cell characteristics and regenerative potentials of single clonal populations isolated from rat epiglottic mucosa (EM), lamina propria (LP), and macula flava (MF) to determine whether they comprised laryngeal tissue-resident stem cells. METHODS: Single clonal laryngeal cells were isolated following microdissection of the EM, LP, and MF from the rat larynx. Several clonal populations from the three laryngeal subsites were selected and expanded in vitro. We compared the stem cell characteristics of self-renewal and differentiation potential, as well as the cell surface phenotypes and gene expression profiles, of laryngeal MSC-like cells to that of bone marrow MSCs (BM-MSCs). We also investigated the regenerative potential of the laryngeal cells in a radiation-induced laryngeal injury animal model. RESULTS: Self-renewing, clonal cell populations were obtained from rat EM, LP, and MF. EM-derived and LP-derived clonal cells had fibroblast-like features, while MF-resident clonal cells had stellate cell morphology and lipid droplets containing vitamin A. All laryngeal clonal cell populations had MSC-like cell surface marker expression (CD29, CD44, CD73, and CD90) and the potential to differentiate into bone and cartilage cell lineages; EM-derived and MF-derived cells, but not LP-derived cells, were also able to differentiate into adipocytes. Clonal cells isolated from the laryngeal subsites exhibited differential extracellular matrix-related gene expression. We found that the mesenchymal and stellate cell-related genes desmin and nestin were enriched in laryngeal MSC-like cells relative to BM-MSCs (P < 0.001). Growth differentiation factor 3 (GDF3) and glial fibrillary acidic protein (GFAP) transcript and protein levels were higher in MF-derived cells than in other laryngeal populations (P < 0.001). At 4 weeks after transplantation, laryngeal MF-derived and EM-derived cells contributed to laryngeal epithelial and/or glandular regeneration in response to radiation injury. CONCLUSIONS: These results suggest that cell populations with MSC characteristics reside in the EM, LP, and MF of the larynx. Laryngeal MSC-like cells contribute to regeneration of the larynx following injury; further investigation is needed to clarify the differential roles of the populations in laryngeal tissue regeneration, as well as the clinical implications for the treatment of laryngeal disease.ope

복막 중피세포의 상피-중간엽 세포이행과 세포사멸 억제의 새로운 치료 표적으로서 소포체 스트레스의 의의

Long-term peritoneal dialysis (PD) results in functional and structural alterations in the peritoneal membrane. Epithelial-to-Mesenchymal Transition (EMT) and apoptosis of peritoneal mesothelial cells are known to be the earliest mechanisms of peritoneal fibrosis. Endoplasmic reticulum (ER) stress with an unfolded protein response is regarded to play a role in the development of apoptosis and EMT. To investigate the potential role of ER stress as a target to prevent and/or delay the development of peritoneal fibrosis, the effect of modulation of ER stress on EMT or apoptosis of human peritoneal mesothelial cells (HPMCs) was examined with an elucidation of mechanisms of protective effect of ER stress preconditioning on TGF-β1-induced EMT. EMT was evaluated by morphological changes of HPMCs and the expressions of E-cadherin and α-smooth muscle actin (α-SMA) with ER stress inducers, tunicamycin (TM) or thapsigargin (TG). TM and TG induced EMT from the concentrations of 0.01 ng/ml and 0.01 nM, respectively with an enhanced phosphorylation of Smad2/3, an increased nuclear translocation of β-catenin and Snail expression. TM or TG at the concentrations inducing EMT in HPMCs did not induce apoptosis up to 48 hours, however high concentration of TM (&amp;gt;1 ng/ml) or TG (&amp;gt;1 nM) induced apoptosis at 12 hours with a persistent increase in the expression of C/EBP homologous protein (CHOP). TGF-β1 induced ER stress in HPMCs, which was expressed as an increase in the expression of GRP78/94 and ATF6 with XBP-1 splicing. TGF-β1 induced EMT of HPMCs at 24 and 48 hours, which was confirmed by a transition of cell morphology and altered expression of epithelial and mesenchymal cell markers. TGF-β1 also induced apoptosis of HPMCs. TUDCA, an endogenous bile acid known to attenuate ER stress, blocked TGF-β1-induced EMT and apoptosis in HPMCs. Pre-treatment with TM or TG for 4 hours protected the cells from TGF-β1-induced EMT and apoptosis in HPMCs, demonstrating the role of ER stress as an adaptive response that served to protect HPMCs from phenotype transition and apoptosis. Peritoneal mesothelial cells isolated from PD patients expressed an increase in GRP78/94 which was correlated with the degree of EMT. These findings suggest the modulation of ER stress in HPMCs could serve as a novel approach to ameliorate EMT and apoptosis in the pathogenesis of peritoneal fibrosis.;복막의 섬유화는 장기간 복막투석을 받는 환자에게 발생되는 합병증이다. 특히, 상피-중간엽 세포이행(Epithelial-to-Mesenchymal Transition, EMT)과 세포사멸(Apoptosis)은 복막손상의 발생이나 진행에 중요한 기전이다. 소포체 스트레스(Endoplasmic Reticulum Stress, ER stress)는 세포 항상성을 유지하기 위한 세포의 중요한 보상기전이지만, 강도가 과도거나 장기간 유지되는 소포체 스트레스는 세포사멸을 유도한다. 소포체 스트레스가 복막 중피세포의 EMT및 세포사멸에 미치는 영향에 관해서는 아직 연구된 바 없다. 본 연구에서는 복막 중피세포에서 소포체 스트레스의 조절이 EMT와 세포사멸에 미치는 영향과 관련 기전에 관해 연구하였다. 소포체 스트레스 유도제인 Tunicamycin (TM, 0.01ng/ml)과 Thapsigargin (TG, 0.01nM)을 처리하였을 때 복막 중피세포의 EMT (12시간)가 유도되었다. 또한, EMT에 중요한 메커니즘인 Smad2/3의 인산화, β-catenin의 핵 내 이동, Snail의 발현을 증가시켰다. TM과TG는 EMT를 유도하지만, 세포 사멸을 유도하지 않았다. 하지만, 고농도 TM (&amp;gt;1ng/ml)과TG (&amp;gt;1nM)를 처리하였을 때 복막 중피세포의 사멸을 유도하였고, C/EBP homologous protein (CHOP)의 발현을 증가 시켰다. 소포체 스트레스 차단제인 Tauroursodeoxycholic acid (TUDCA)는 TM과 TG에 의해 발생되는 EMT와 세포사멸을 차단하였다. TGF-β1에 의한 복막 중피세포의 EMT및 세포사멸은 TUDCA 및 TM과 TG의 전처치 (4시간)로 의미있게 완화되었다. 복막투석 환자에서 분리된 중피세포에서 GRP78/94 발현은 의미있게 증가되었으며E-cadehrin 및 α-SMA발현과 의미있는 상관을 보였다. 본 연구는 복막 중피세포에서 소포체 스트레스를 차단하거나 전처치 함으로써 중피세포의 EMT 및 세포사멸을 억제할 수 있다는 결과를 최초로 보고한 것으로 향후 복막섬유화 치료의 새로운 표적으로서의 가능성을 제시 하였다.I. Introduction 1 II. Materials and methods 3 A. Reagents 3 B. Isolation and maintenance of HPMCs 3 C. Cell proliferation and cytotoxicity assay 4 D. Cell morphology and immunofluorescence analysis of HPMC 4 E. Western blotting 5 F. Reverse transcriptase-PCR analysis for X-box-binding protein 1 splicing 6 G. Quantitative real time PCR analysis 6 H. Transfection of siRNA 7 I. Cell Apoptosis Assay: Flow Cytometric Analysis for Propidium Iodide Staining and Annexin V-FITC Binding 8 J. Effect of ER stress Modulation on TGF-β1-induced EMT in HPMCs 8 K. Isolation of Mesothelial Cells from Peritoneal Effluent in Patients on Peritoneal Dialysis (PD) 9 L. Peritoneal Equilibration Test (PET) 10 M. Statistical Analysis 10 III. Results 11 A. ER stress induced EMT of HPMCs 11 B. ER stress induced Snail expression by Smad 2/3 activation and nuclear translocation of β-catenin 16 C. ER stress induced apoptosis in a dose- and time-dependent manner 19 D. TGF-β1-induced a persistent ER stress which resulted in EMT and apoptosis of HPMCs 22 E. ER stress preconditioning protected HPMCs from TGF-β1-induced EMT and apoptosis with an amelioration of Smad 2/3 activation and Snail expression 25 F. Increased GRP expression in mesothelial cells isolated from peritoneal effluent was correlated with EMT in PD patients 28 IV. Discussion 30 References 37 국문초록 4