Browsing by Author "National University of Singapore, Faculty of Dentistry"

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    Engineering innervated secretory epithelial organoids by magnetic three-dimensional bioprinting for stimulating epithelial growth in salivary glands
    (2018-10-01) Christabella Adine; Kiaw K. Ng; Sasitorn Rungarunlert; Glauco R. Souza; João N. Ferreira; National University of Singapore, Faculty of Dentistry; Chulalongkorn University; University of Texas Health Science Center at Houston; National Institute of Dental and Craniofacial Research; Mahidol University; Nano3D Biosciences
    © 2018 Elsevier Ltd Current saliva-based stimulation therapies for radiotherapy-induced xerostomia are not fully effective due to the presence of damaged secretory epithelia and nerves in the salivary gland (SG). Hence, three-dimensional bio-engineered organoids are essential to regenerate the damaged SG. Herein, a recently validated three-dimensional (3D) biofabrication system, the magnetic 3D bioprinting (M3DB), is tested to generate innervated secretory epithelial organoids from a neural crest-derived mesenchymal stem cell, the human dental pulp stem cell (hDPSC). Cells are tagged with magnetic nanoparticles (MNP) and spatially arranged with magnet dots to generate 3D spheroids. Next, a SG epithelial differentiation stage was completed with fibroblast growth factor 10 (4–400 ng/ml) to recapitulate SG epithelial morphogenesis and neurogenesis. The SG organoids were then transplanted into ex vivo model to evaluate their epithelial growth and innervation. M3DB-formed spheroids exhibited both high cell viability rate (>90%) and stable ATP intracellular activity compared to MNP-free spheroids. After differentiation, spheroids expressed SG epithelial compartments including secretory epithelial, ductal, myoepithelial, and neuronal. Fabricated organoids also produced salivary α-amylase upon FGF10 stimulation, and intracellular calcium mobilization and trans-epithelial resistance was elicited upon neurostimulation with different neurotransmitters. After transplantation, the SG-like organoids significantly stimulated epithelial and neuronal growth in damaged SG. It is the first time bio-functional innervated SG-like organoids are bioprinted. Thus, this is an important step towards SG regeneration and the treatment of radiotherapy-induced xerostomia.
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    Generation of a pig induced pluripotent stem cell (piPSC) line from embryonic fibroblasts by incorporating LIN28 to the four transcriptional factor-mediated reprogramming: VSMUi001-D
    (2017-10-01) Warunya Chakritbudsabong; Ladawan Sariya; Sirikron Pamonsupornvichit; Rassmeepen Pronarkngver; Somjit Chaiwattanarungruengpaisan; Joao N. Ferreira; Piyathip Setthawong; Praopilas Phakdeedindan; Mongkol Techakumphu; Theerawat Tharasanit; Sasitorn Rungarunlert; Mahidol University; National University of Singapore, Faculty of Dentistry; Chulalongkorn University
    © 2017 The Authors Pig induced pluripotent stem cell (piPSC) line was generated from embryonic fibroblast cells using retroviral transduction approaches carrying human transcriptional factors: OCT4, SOX2, KLF4, c-MYC and LIN28. The generated piPSC line, VSMUi001-D, was positive for alkaline phosphatase activity and expressed the pluripotency associated transcription factors including OCT4, SOX2, NANOG and surface markers SSEA-1, all iPSC hallmarks of authenticity. Furthermore, VSMUi001-D exhibited a normal karyotype and formed embryoid bodies in vitro and teratomas in vivo. Upon cardiac differentiation, VSMUi001-D displayed spontaneous beating and expressed cardiomyocyte markers, like cardiac Troponin T.
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    A magnetic three-dimensional levitated primary cell culture system for the development of secretory salivary gland-like organoids
    (2019-03-01) Joao N. Ferreira; Riasat Hasan; Ganokon Urkasemsin; Kiaw K. Ng; Christabella Adine; Sujatha Muthumariappan; Glauco R. Souza; National University of Singapore, Faculty of Dentistry; Chulalongkorn University; University of Texas Health Science Center at Houston; Mahidol University; Nano3D Biosciences
    © 2019 John Wiley & Sons, Ltd. Salivary gland (SG) hypofunction and oral dryness can be induced by radiotherapy for head and neck cancers or autoimmune disorders. These are common clinical conditions that involve loss of saliva-secreting epithelial cells. Several oral complications arise with SG hypofunction that interfere with routine daily activities such as chewing, swallowing, and speaking. Hence, there is a need for replacing these saliva-secreting cells. Recently, researchers have proposed to repair SG hypofunction via various cell-based approaches in three-dimensional (3D) scaffold-based systems. However, majority of the scaffolds used cannot be translated clinically due to the presence of non-human-based substrates. Herein, saliva-secreting organoids/mini-glands were developed using a new scaffold/substrate-free culture system named magnetic 3D levitation (M3DL), which assembles and levitates magnetized primary SG-derived cells (SGDCs), allowing them to produce their own extracellular matrices. Primary SGDCs were assembled in M3DL to generate SG-like organoids in well-established SG epithelial differentiation conditions for 7 days. After such culture time, these organoids consistently presented uniform spheres with greater cell viability and pro-mitotic cells, when compared with conventional salisphere cultures. Additionally, organoids formed by M3DL expressed SG-specific markers from different cellular compartments: acinar epithelial including adherens junctions (NKCC1, cholinergic muscarinic receptor type 3, E-cadherin, and EpCAM); ductal epithelial and myoepithelial (cytokeratin 14 and α-smooth muscle actin); and neuronal (β3-tubulin and vesicular acetylcholine transferase). Lastly, intracellular calcium and α-amylase activity assays showed functional organoids with SG-specific secretory activity upon cholinergic stimulation. Thus, the functional organoid produced herein indicate that this M3DL system can be a promising tool to generate SG-like mini-glands for SG secretory repair.
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    Novel bioreactor platform for scalable cardiomyogenic differentiation from pluripotent stem cell-derived embryoid bodies
    (2016-01-01) Sasitorn Rungarunlert; Joao N. Ferreira; Andras Dinnyes; Mahidol University; National University of Singapore, Faculty of Dentistry; BioTalentum Ltd.; Szent Istvan Egyetem
    © Springer Science+Business Media New York 2016. Generation of cardiomyocytes from pluripotent stem cells (PSCs) is a common and valuable approach to produce large amount of cells for various applications, including assays and models for drug development, cell-based therapies, and tissue engineering. All these applications would benefit from a reliable bioreactorbased methodology to consistently generate homogenous PSC-derived embryoid bodies (EBs) at a large scale, which can further undergo cardiomyogenic differentiation. The goal of this chapter is to describe a scalable method to consistently generate large amount of homogeneous and synchronized EBs from PSCs. This method utilizes a slow-turning lateral vessel bioreactor to direct the EB formation and their subsequent cardiomyogenic lineage differentiation.
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    Occlusal dysesthesia and dysfunction
    (2017-07-06) Somsak Mitrirattanakul; Tan Hee Hon; João N.A.R. Ferreira; Mahidol University; National University of Singapore, Faculty of Dentistry
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    Strategies for developing functional secretory epithelia from porcine salivary gland explant outgrowth culture models
    (2019-11-01) Ganokon Urkasemsin; Phoebe Castillo; Sasitorn Rungarunlert; Nuttha Klincumhom; Joao N. Ferreira; National University of Singapore, Faculty of Dentistry; Chulalongkorn University; Mahidol University
    © 2019 by the authors. Licensee MDPI, Basel, Switzerland. Research efforts have been made to develop human salivary gland (SG) secretory epithelia for transplantation in patients with SG hypofunction and dry mouth (xerostomia). However, the limited availability of human biopsies hinders the generation of sufficient cell numbers for epithelia formation and regeneration. Porcine SG have several similarities to their human counterparts, hence could replace human cells in SG modelling studies in vitro. Our study aims to establish porcine SG explant outgrowth models to generate functional secretory epithelia for regeneration purposes to rescue hyposalivation. Cells were isolated and expanded from porcine submandibular and parotid gland explants. Flow cytometry, immunocytochemistry, and gene arrays were performed to assess proliferation, standard mesenchymal stem cell, and putative SG epithelial stem/progenitor cell markers. Epithelial differentiation was induced and different SG-specific markers investigated. Functional assays upon neurostimulation determined α-amylase activity, trans-epithelial electrical resistance, and calcium influx. Primary cells exhibited SG epithelial progenitors and proliferation markers. After differentiation, SG markers were abundantly expressed resembling epithelial lineages (E-cadherin, Krt5, Krt14), and myoepithelial (α-smooth muscle actin) and neuronal (β3-tubulin, Chrm3) compartments. Differentiated cells from submandibular gland explant models displayed significantly greater proliferation, number of epithelial progenitors, amylase activity, and epithelial barrier function when compared to parotid gland models. Intracellular calcium was mobilized upon cholinergic and adrenergic neurostimulation. In summary, this study highlights new strategies to develop secretory epithelia from porcine SG explants, suitable for future proof-of-concept SG regeneration studies, as well as for testing novel muscarinic agonists and other biomolecules for dry mouth.
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    Three-Dimensional Bioprinting Nanotechnologies towards Clinical Application of Stem Cells and Their Secretome in Salivary Gland Regeneration
    (2016-01-01) Joao N. Ferreira; Sasitorn Rungarunlert; Ganokon Urkasemsin; Christabella Adine; Glauco R. Souza; National University of Singapore, Faculty of Dentistry; National Institute of Dental and Craniofacial Research; Mahidol University; University of Texas Health Science Center at Houston; Nano3D Biosciences
    © 2016 Joao N. Ferreira et al. Salivary gland (SG) functional damage and severe dry mouth (or xerostomia) are commonly observed in a wide range of medical conditions from autoimmune to metabolic disorders as well as after radiotherapy to treat specific head and neck cancers. No effective therapy has been developed to completely restore the SG functional damage on the long-term and reverse the poor quality of life of xerostomia patients. Cell- and secretome-based strategies are currently being tested in vitro and in vivo for the repair and/or regeneration of the damaged SG using (1) epithelial SG stem/progenitor cells from salispheres or explant cultures as well as (2) nonepithelial stem cell types and/or their bioactive secretome. These strategies will be the focus of our review. Herein, innovative 3D bioprinting nanotechnologies for the generation of organotypic cultures and SG organoids/mini-glands will also be discussed. These bioprinting technologies will allow researchers to analyze the secretome components and extracellular matrix production, as well as their biofunctional effects in 3D mini-glands ex vivo. Improving our understanding of the SG secretome is critical to develop effective secretome-based therapies towards the regeneration and/or repair of all SG compartments for proper restoration of saliva secretion and flow into the oral cavity.