Ivor J Lim's research while affiliated with National University of Singapore and other places

Significant improvement in skin tone was reported after topical application of a facial cream (CALECIM® Professional Multi-Action Cream, CALECIM Cosmeceuticals, Singapore) containing conditioned media (CM) derived from Red Deer Umbilical Cord Lining Mesenchymal Stem Cell (RD-CLMSC) culture. This study investigates the paracrine effects of RD-CLMSC-CM on human dermal fibroblasts (HDF) to understand how it may increase skin turgor and elasticity. Skin aging is associated with lower levels of extracellular matrix components such as hyaluronic acid (HA) and elastin, resulting in poor skin turgor and elasticity. Histochemical staining followed by photocolorimetry demonstrated that RD-CLMSC-CM upregulated HDF expression of elastin by 56% and HA by 83% compared with DMEM/10% Fetal Calf Serum (FCS).To further quantify the effects of CM, a proliferation assay was used to assess HDF response to RD-CLMSC-CM exposure. Exposure to RD-CLMSC-CM resulted in the highest increase in HDF proliferation over DMEM/10% FCS (113%) followed by Human (H)-CLMSC-CM (112%), then Human Foreskin Fibroblast (FSF)-CM (16%).These experimental results demonstrate both the cross-species efficacy and lack of toxicity of RD-CLMSC-CM on HDF. These pre-clinical studies also suggest the clinical effects of RD-CLMSC-CM on skin turgor may be related to increased HA and elastin production by HDF, as well as enhanced proliferation. J Drugs Dermatol. 2023;21(1):82-89. doi:10.36849/JDD.6906.

To determine the therapeutic efficacy of human umbilical cord lining mesenchymal stromal cells (CL-MSCs) (US Patent number 9,737,568) in lupus-prone MRL/lpr (Faslpr) mice and elucidate its working mechanisms. A total of 4 doses of (20–25) × 106 cells/kg of CL-MSCs was given to 16-week-old female Faslpr mice by intraperitoneal injection. Three subsequent doses were given on 17 weeks, 18 weeks, and 22 weeks, respectively. Six-week-old Faslpr mice were used as disease pre-onset controls. Mice were monitored for 10 weeks. Mouse kidney function was evaluated by examining complement component 3 (C3) deposition, urinary albumin-to-creatinine ratio (ACR), and lupus nephritis (LN) activity and chronicity. Working mechanisms were elucidated by flow cytometry, Luminex/ELISA (detection of anti-dsDNA and isotype antibodies), and RNA sequencing. CL-MSCs improved mice survival and kidney function by reducing LN activity and chronicity and lymphocyte infiltration over 10 weeks. CL-MSCs also reduced urinary ACR, renal complement C3 deposition, anti-dsDNA, and isotype antibodies that include IgA, IgG1, IgG2a, IgG2b, and IgM. Immune and cytokine profiling demonstrated that CL-MSCs dampened inflammation by suppressing splenic neutrophils and monocytes/macrophages, reducing plasma IL-6, IL-12, and CXCL1 and stabilizing plasma interferon-γ and TNF-α. RNA sequencing further showed that CL-MSCs mediated immunomodulation via concerted action of pro-proinflammatory cytokine-induced chemokines and production of nitric oxide in macrophages. CL-MSCs may provide a novel myeloid (neutrophils and monocytes/macrophages)-targeting therapy for SLE.

Antimicrobial polymers with a broad spectrum of action and high selectivity towards pathogens (versus mammalian cells) provide the opportunity to combat infections with only a limited chance of resistance development. To this end, functional antimicrobial biodegradable materials derived from inexpensive renewable resources such as polysaccharides would pave the way for broader applications. Here we report a facile one-pot approach to access potent functional antimicrobial polymers from soluble starch, a renewable plant-based starting material and other readily available reagents. In solution-state, these polymers with optimal composition demonstrate excellent antibacterial activity, but limited antifungal properties. The reactive allyl groups installed onto this polymeric platform enabled seamless translation of these polymers into antimicrobial hydrogels and coatings via photo-mediated thiol-ene reactions. Both the hydrogels and hydrogel-coated cotton-pads exhibited high killing efficiencies across multiple strains of pathogens ( 100 % for S. aureus, E. coli and P. aeruginosa). Installation of reactive groups onto antimicrobial platforms allows for rapid development of formulations and prototypes in different forms such as solutions, gels and surface coatings by minimizing or completely eliminating the need to optimize the polymer to suit different applications. These results demonstrate the potential utility of cationic starch-based biodegradable polymers as antimicrobial materials and coatings for the prevention of infections.

Objective: Nanofibers for tissue scaffolding and wound dressings hold great potential in realizing enhanced healing of wounds in comparison with conventional counterparts. Previously, we demonstrated good fibroblast adherence and growth on a newly developed scaffold, Tegaderm™-Nanofiber (TG-NF), made from poly ɛ-caprolactone (PCL)/gelatin nanofibers electrospun onto Tegaderm (TG). The purpose of this study is to evaluate the performance and safety of TG-NF dressings in partial-thickness wound in a pig healing model. Approach: To evaluate the rate of reepithelialization, control TG, human dermal fibroblast-seeded TG-NF(+) and -unseeded TG-NF(-) were randomly dressed onto 80 partial-thickness burns created on four female and four male pigs. Wound inspections and dressings were done after burns on day 7, 14, 21, and 28. On day 28, full-thickness biopsies were taken for histopathological evaluation by Masson-Trichrome staining for collagen and hematoxylin-eosin staining for cell counting. Results: No infection and severe inflammation were recorded. Wounds treated with TG-NF(+) reepithelialized significantly faster than TG-NF(-) and control. Wound site inflammatory responses to study groups were similar as total cell counts on granulation tissues show no significant differences. Most of the wounds completely reepithelialized by day 28, except for two wounds in control and TG-NF(-). A higher collagen coverage was also recorded in the granulation tissues treated with TG-NF(+). Innovation and Conclusion: With better reepithelialization achieved by TG-NF(+) and similar rates of wound closure by TG-NF(-) and control, and the absence of elevated inflammatory responses to TG-NF constructs, TG-NF constructs are safe and demonstrated good healing potentials that are comparable to Tegaderm.

Intense scientific research over the past two decades has yielded much knowledge about embryonic stem cells, mesenchymal stem cells from bone marrow, as well as epithelial stem cells from the skin and cornea. However, the billions of dollars spent in this research have not overcome the fundamental difficulties intrinsic to these stem cell strains related to ethics (embryonic stem cells), as well as to technical issues such as accessibility, ease of cell selection and cultivation, and expansion/mass production whilst maintaining consistency of cell stemness (all of the stem cell strains already mentioned). Overcoming these technical hurdles have made stem cell technology expensive, and any potential translational products unaffordable for most patients. Commercialization efforts have been rendered unfeasible by this high cost. Advanced biomedical research is on the rise in Asia and new innovations have started to overcome these challenges. The Nobel prize winning Japanese development of iPS cells has effectively introduced a possible replacement for embryonic stem cells. For non-embryonic stem cells, cord lining stem cells (CLSCs) have overcome the pre-existing difficulties inherent to mesenchymal stem cells from the bone marrow as well as epithelial stem cells from the skin and cornea, offering a realistic, practical and affordable alternative for tissue repair and regeneration. This novel CLSC technology was developed in Singapore in 2004, and has 18 international patents granted to date, including those from the USA and UK. CLSCs are derived from the umbilical cord outer lining membrane (usually regarded as medical waste), and is therefore free from ethical dilemmas related to its collection. The large quantity of umbilical cord lining membrane that can be collected translates to billions of stem cells that can be grown in primary stem cell culture, and therefore very rapid and inexpensive cell cultivation and expansion for clinical translational therapies. Both mesenchymal and epithelial stem cells can be isolated from the umbilical cord lining membrane, usefully regenerating not only mesenchymal tissue as bone, cartilage, cardiac and striated muscle, but also epithelial tissue as skin, cornea and liver. Both mesenchymal and epithelial CLSCs are immune-privileged and resist rejection. Clinically, CLSCs have proved effective in the treatment of difficult-to-heal human wounds such as diabetic ulcers, recalcitrant chronic wounds, and even persistent epithelial defects of the cornea. Heart and liver regeneration have been shown to be successful in animal studies and await human trials. CLSCs have also been shown to be an effective feeder layer for cord blood haematopoietic stem cells, and more recently, has been recognised as an abundant and high-quality source of cells for iPS cell production. Banking of CLSCs by cord blood banks in both private and public settings is now available in many countries, so that individuals may have their personal stores of CLSCs for future translational applications for both themselves and their families. Cord Lining Stem Cells are strongly positioned to be the future of cell therapy and regenerative medicine.

Keloid scarring is a dermal fibroproliferative disorder characterized by increased fibroblast proliferation and excessive production of collagen and extracellular matrix (ECM) components. To date, the role of cytokines in keloid pathogenesis has not been completely unravelled. Interleukin (IL)-18 is a pro-inflammatory cytokine that plays important roles in wound healing, fibrogenesis and carcinogenesis. Our aim was to study the role of the IL-18 system in keloid pathogenesis. Expression and localization of IL-18 and its receptor (IL-18R) were investigated in normal skin and keloid tissues using Western blot and immunohistochemistry. We further studied the expression of the IL-18 system in normal and keloid-derived cell lines in a coculture model. Results from Western blot and immunohistochemistry revealed that IL-18, IL-18Rα and IL-18Rβ expression was elevated in keloid tissue compared with normal skin tissue. Studies on the expression of IL-18 and its antagonist, IL-18 binding protein (IL-18BP), using a coculture model demonstrated severe IL-18/IL-18BP imbalance in keloid keratinocyte/keloid fibroblast (KK/KF) cocultures with significant elevation of bioactive IL-18 whereas IL-18BP levels remained the same. This overproduction of bioactive IL-18 in keloid cocultures could be due to increased caspase-1 and decreased caspase-3 expression in keloid tissue, as well as decreased soluble IL-10 levels observed in keloid cocultures. The important inductive effects of IL-18 on KFs were further underscored by the observation that exposure of KF to IL-18 resulted in increased collagen and ECM component synthesis, and increased secretion of profibrotic cytokines such as IL-6 and IL-8. Finally, the addition of phosphatidylinositol 3-kinase (PI3K), mitogen activation protein kinase (MAPK), specificity protein 1 (Sp1) and mammalian target of rapamycin (mTOR) inhibitors inhibited IL-18 secretion in keloid cocultures. The present study has proven that the IL-18 system plays an important role in keloid pathogenesis via epithelial-mesenchymal interactions. It also suggests a therapeutic potential of PI3K, MAPK, Sp1 and mTOR inhibitors in the treatment of keloid scarring.

Keloids are fibroproliferative disorders characterized by increased deposition of extracellular matrix components. Stem cell factor (SCF) and its receptor c-KIT are expressed in a wide variety of cells and have also been demonstrated to be important modulators of the wound healing process. To examine the role of the SCF/c-KIT system in keloid pathogenesis. Immunohistochemical staining and Western blot analyses were used to examine localization and expression of SCF and c-KIT in keloid and normal skin tissue. This was followed by the detection of SCF and c-KIT expression in fibroblasts cultured in vitro and fibroblasts exposed to serum. To investigate the effect of epithelial-mesenchymal interactions, a two-chamber system was employed in which keratinocytes on membrane inserts were cocultured with the fibroblasts. SCF and c-KIT expression levels in all cell extracts and conditioned media were assayed by Western blotting. In another set of experiments, the effect of imatinib (Glivec(®), Gleevec(®); Novartis Pharma AG, Basel, Switzerland) on keloid fibroblasts was examined. SCF and c-KIT were upregulated in keloid scar tissue and in cultured fibroblasts stimulated with serum, highlighting their importance in the initial phase of wound healing. We further demonstrated that epithelial-mesenchymal interactions, mimicked by coculture of keratinocytes and fibroblasts in vitro, not only stimulated secretion of the soluble form of SCF in keloid cocultures but also brought about shedding of the extracellular domain of c-KIT perhaps by upregulation of tumour necrosis factor-α converting enzyme which was also upregulated in keloid scars in vivo and keloid cocultures in vitro. In addition keloid cocultures expressed increased levels of phosphorylated c-KIT highlighting an activation of the SCF/c-KIT system. Finally, we demonstrated that imatinib, a tyrosine kinase inhibitor, may be a possible therapeutic agent for keloids. These data indicate that the SCF/c-KIT system plays an important role in scar pathogenesis, and underscore the role of imatinib as a key therapeutic agent in keloid scars.

Keloid scar is a fibroproliferative disorder characterized by increased deposition of extracellular matrix components. Hepatocyte growth factor (HGF), also known as the "scatter factor," and its receptor, a product of the Met oncogene, play multiple roles in regulating cell behavior. However, the role of this system in pathogenic fibrosis is still unclear. Our aim was to investigate and to clarify the role of HGF and its receptor c-Met in pathogenesis of keloid scars. This study investigated the expression profile of HGF and c-Met in keloid and normal skin tissue. In addition, the role of normal and keloid keratinocytes in modulating the expression of fibroblast HGF (epithelial-mesenchymal interactions) was examined using a two-chamber serum-free coculture model. The effect of serum stimulation on fibroblast expression of HGF and c-Met was also studied. Increased levels of HGF and c-Met were observed in tissue extracts obtained from keloid tissue. Increased levels of HGF and c-Met localization were observed in the basal epidermis and in the dermis of keloid tissue compared with normal skin. Serum stimulation seemed to upregulate the expression of both HGF and c-Met in fibroblasts. Finally, coculture of keloid keratinocytes with keloid fibroblasts upregulated levels of both HGF and c-Met in keratinocyte cell lysates and conditioned media obtained from fibroblast culture. These findings emphasize the importance of the HGF/c-Met system in keloid biology and pathogenesis and suggest a possible target for therapeutic intervention in the prevention and treatment of keloids.

Keloids are pathological scars and, despite numerous available treatment modalities, continue to plague physicians and patients. Identification of molecular mediators that contribute to this fibrotic phenotype. Two-dimensional gel electrophoresis, MALDI-TOF, Mascot online database searching algorithm and Melanie 5 gel analysis software were employed for comparative proteomic analysis between normal skin (NS) and keloid scar (KS) tissue extracts. Seventy-nine protein spots corresponding to 23 and 32 differentially expressed proteins were identified in NS and KS, respectively. Isoforms of heat shock proteins, gelsolin, carbonic anhydrase and notably keratin 10 were strongly expressed in NS along with manganese superoxide dismutase, immune components, antitrypsin, prostatic binding protein and crystalline. Various classes of proteins were found either to be present or to be upregulated in keloid tissue: (i) inflammatory/differentiated keratinocyte markers: S100 proteins, peroxiredoxin I; (ii) wound healing proteins: gelsolin-like capping protein; (iii) fibrogenetic proteins: mast cell β-tryptase, macrophage migration inhibitory factor (MIF); (iv) antifibrotic proteins: asporin; (v) tumour suppressor proteins: stratifin, galectin-1, maspin; and (vi) antiangiogenic proteins: pigment epithelium-derived factor. Significant increases in expression of asporin, stratifin, galectin-1 and MIF were observed by Western blot analysis in KS. This work has identified differentially expressed proteins specific to KS tissue extracts which can potentially be used as specific targets for therapeutic intervention.

Growth factors and cytokines involved in the wound healing process seem to be immobilized at the cell surface and extracellular matrix via binding with proteoglycans, making them important modulators of cell dynamics. Our aim was to investigate the expression of two proteoglycans, namely syndecan-2 and decorin, and to elucidate their role in the pathogenesis of an aberrant wound healing process leading to keloid scar. Intrinsic expression of syndecan-2, fibroblast growth factor (FGF)-2, and decorin in keloid tissue was investigated using Western blotting and immunohistochemistry. Normal and keloid fibroblasts were treated with serum to see the effects of serum growth factors on the expression of syndecan-2 and decorin. The role of epithelial-mesenchymal interactions in modulating syndecan-2, FGF-2, and decorin expression was investigated using an established two-chamber serum-free coculture model. Finally, the antifibrotic effect of decorin was investigated by studying its effect on the expression of extracellular matrix components. Syndecan-2 and FGF-2 were upregulated in keloid tissue; decorin was downregulated. Normal and keloid fibroblasts treated with serum led to increase in syndecan-2 and decrease in decorin expression. Under coculture conditions, syndecan-2 was shed in the conditioned media. FGF-2 was also upregulated under coculture conditions and, when added to fibroblast monocultures, increased shedding of syndecan-2. Decorin levels were upregulated under coculture conditions only in normal cocultures. Decorin was also able to decrease extracellular matrix proteins, highlighting its importance as an antifibrotic agent. Syndecan-2 and FGF-2 are not only overexpressed in keloid tissues but may interact with each other resulting in the shedding of syndecan-2, which in turn might activate a whole cascade of events responsible for a keloidic phenotype. In addition, decorin had an antifibrotic effect and could well be used as a potential therapeutic agent for keloids.