Ocular instillation of conditioned medium from mesenchymal stem cells is effective for dry eye syndrome by improving corneal barrier function.

Dry eye syndrome (DES) is a chronic ocular disease that induces epithelial damage to the cornea by decreasing tear production and quality. Adequate treatment options have not been established for severe DES such as Sjogren's syndrome due to complicated pathological conditions. To solve this problem, we focused on the conditioned medium of human adipose-derived mesenchymal stem cells (hAdMSC-CM), which have multiple therapeutic properties. Here, we showed that hAdMSC-CM suppressed Benzalkonium Chloride (BAC)-induced cytotoxicity and inflammation in human corneal epithelial cells (hCECs). In addition, hAdMSC-CM increased the expression level and regulated the localisation of barrier function-related components, and improved the BAC-induced barrier dysfunction in hCECs. RNA-seq analysis and pharmacological inhibition experiments revealed that the effects of hAdMSC-CM were associated with the TGFß and JAK-STAT signalling pathways. Moreover, in DES model rats with exorbital and intraorbital lacrimal gland excision, ocular instillation of hAdMSC-CM suppressed corneal epithelial damage by improving barrier dysfunction of the cornea. Thus, we demonstrated that hAdMSC-CM has multiple therapeutic properties associated with TGFß and JAK-STAT signalling pathways, and ocular instillation of hAdMSC-CM may serve as an innovative therapeutic agent for DES by improving corneal barrier function.

Generation of 3D lacrimal gland organoids from human pluripotent stem cells.

Lacrimal glands are the main exocrine glands of the eyes. Situated within the orbit, behind the upper eyelid and towards the temporal side of each eye, they secrete lacrimal fluid as a major component of the tear film. Here we identify cells with characteristics of lacrimal gland primordia that emerge in two-dimensional eye-like organoids cultured from human pluripotent stem cells1. When isolated by cell sorting and grown under defined conditions, the cells form a three-dimensional lacrimal-gland-like tissue organoid with ducts and acini, enabled by budding and branching. Clonal colony analyses indicate that the organoids originate from multipotent ocular surface epithelial stem cells. The organoids exhibit notable similarities to native lacrimal glands on the basis of their morphology, immunolabelling characteristics and gene expression patterns, and undergo functional maturation when transplanted adjacent to the eyes of recipient rats, developing lumina and producing tear-film proteins.

Role of CD99 in regulating homeostasis and differentiation in normal human epidermal keratinocytes.

CD99 is a glycoprotein primarily expressed in immune cells. Physiologically, it is involved in the adhesion, migration, and development of immune cells. The presence of CD99 in the skin was first reported in 2016 and its function is yet to be determined. In this study, we aimed to understand the role of CD99 in the skin using normal human epidermal keratinocytes (NHEK). CD99 expression increased with the confluency of NHEK, while the CD99-high expressing NHEK lost their stem cell properties and played a role in barrier function. We characterized CD99-expressing NHEK as cells committed to early differentiation because they expressed early differentiation markers. However, the deficiency of CD99 in NHEK disrupted homeostasis and caused aberrant differentiation, as evidenced by larger cells with lesser Ki67 staining and higher expression of terminal differentiation markers. Hence, we propose that CD99 is involved in maintaining homeostasis and initiating early differentiation in the skin.

Study on the Umbilical Cord-Mesenchymal Stem Cell Manufacturing Using Clinical-Grade Culture Medium.

Mesenchymal stem/stromal cell (MSC)-based therapies have been gaining increasing attention owing to their application in various diseases and conditions. In this study, we aimed to identify the optimal condition for industrial-scale MSC manufacturing. MSCs were isolated from umbilical cord (UC) tissues by implementing the explant method (Exp) or a collagenase based-enzymatic digestion method (Col), using a good manufacturing practice-compatible serum-free medium developed in-house. Microarray analysis demonstrated that the gene expression profiles of Exp-MSCs and Col-MSCs did not significantly differ according to the method of isolation or the culture conditions used. The isolated UC-MSCs were then subjected to expansion using conventional static culture (ST) or microcarrier-based culture in stirred-tank bioreactors (MC). Metabolomic and cytokine array analyses were conducted to evaluate the biochemical status of the MSCs. However, no remarkable differences in the metabolic profile and cytokine secretome between ST-MSCs and MC-MSCs were observed. On the contrary, we observed for the first time that the hydrophobic components of ST-MSCs and MC-MSCs were different, which suggested that the cell membrane distribution of fatty acids and lipids was altered in the process of adaptation to shear stress in MC-MSCs. These results establish the flexibility of the isolation and expansion method for UC-MSCs during the manufacturing processes and provide new insights into the minor differences between expansion methods that may exert remarkable effects on MSCs. In conclusion, we demonstrated the feasibility of both Exp-MSCs and Col-MSCs and MC and ST culture methods for scale-up and scale-out of MSC production, as well as the equivalence of these cells. As for the industrialized mass production of MSCs, enzyme-based methods for isolation and cell expansion in a bioreactor were considered to be more suitable. The methods developed, which underwent comprehensive evaluation in this study, may contribute toward the provision of sufficient MSC sources and the establishment of cost-effective MSC therapies. Impact statement Our in-house-developed good manufacturing practice-grade serum-free medium could be used for both isolation (Exp and Col) and expansion (ST and MC) of umbilical cord (UC)-mesenchymal stem/stromal cells (MSCs). Characteristics of the obtained UC-MSCs were widely assessed with regard to gene expression, metabolome, and secretome. Cellular characteristics and efficacy were observed to be equivalently maintained among whichever technique was applied. In addition, our research presents the first evidence that bioreactor and microcarrier-based MSC cultures alter the fatty acid and phospholipid composition of MSCs. These results provide new insights into the differences between expansion methods that may exert remarkable effects on MSCs.

Generation of knockout rabbits with X-linked severe combined immunodeficiency (X-SCID) using CRISPR/Cas9.

Severe immunodeficient mice are widely used to examine human and animal cells behaviour in vivo. However, mice are short-lived and small in size; while large animals require specific large-scale equipment. Rabbits are also commonly employed as experimental models and are larger than mice or rats, easy to handle, and suitable for long-term observational and pre-clinical studies. Herein, we sought to develop and maintain stable strains of rabbits with X-linked severe combined immunodeficiency (X-SCID) via the CRISPR/Cas9 system targeting Il2rg. Consequently, X-SCID rabbits presented immunodeficient phenotypes including the loss of T and B cells and hypoplasia of the thymus. Further, these rabbits exhibited a higher success rate with engraftments upon allogeneic transplantation of skin tissue than did wild type controls. X-SCID rabbits could be stably maintained for a minimum of four generations. These results indicate that X-SCID rabbits are effective animals for use in a non-rodent model of severe immunodeficiency.

Cell-Type-Specific Adhesiveness and Proliferation Propensity on Laminin Isoforms Enable Purification of iPSC-Derived Corneal Epithelium.

A treatment for intractable diseases is expected to be the replacement of damaged tissues with products from human induced pluripotent stem cells (hiPSCs). Target cell purification is a critical step for realizing hiPSC-based therapy. Here, we found that hiPSC-derived ocular cell types exhibited unique adhesion specificities and growth characteristics on distinct E8 fragments of laminin isoforms (LNE8s): hiPSC-derived corneal epithelial cells (iCECs) and other non-CECs rapidly adhered preferentially to LN332/411/511E8 and LN211E8, respectively, through differential expression of laminin-binding integrins. Furthermore, LN332E8 promoted epithelial cell proliferation but not that of the other eye-related cells, leading to non-CEC elimination by cell competition. Combining these features with magnetic sorting, highly pure iCEC sheets were fabricated. Thus, we established a simple method for isolating iCECs from various hiPSC-derived cells without using fluorescence-activated cell sorting. This study will facilitate efficient manufacture of iCEC sheets for corneal disease treatment and provide insights into target cell-specific scaffold selection.

Generation and validation of a PITX2-EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells.

PITX2 (Paired-like homeodomain transcription factor 2) plays important roles in asymmetric development of the internal organs and symmetric development of eye tissues. During eye development, cranial neural crest cells migrate from the neural tube and form the periocular mesenchyme (POM). POM cells differentiate into several ocular cell types, such as corneal endothelial cells, keratocytes, and some ocular mesenchymal cells. In this study, we used transcription activator-like effector nuclease technology to establish a human induced pluripotent stem cell (hiPSC) line expressing a fluorescent reporter gene from the PITX2 promoter. Using homologous recombination, we heterozygously inserted a PITX2-IRES2-EGFP sequence downstream of the stop codon in exon 8 of PITX2 Cellular pluripotency was monitored with alkaline phosphatase and immunofluorescence staining of pluripotency markers, and the hiPSC line formed normal self-formed ectodermal autonomous multizones. Using a combination of previously reported methods, we induced PITX2 in the hiPSC line and observed simultaneous EGFP and PITX2 expression, as indicated by immunoblotting and immunofluorescence staining. PITX2 mRNA levels were increased in EGFP-positive cells, which were collected by cell sorting, and marker gene expression analysis of EGFP-positive cells induced in self-formed ectodermal autonomous multizones revealed that they were genuine POM cells. Moreover, after 2 days of culture, EGFP-positive cells expressed the PITX2 protein, which co-localized with forkhead box C1 (FOXC1) protein in the nucleus. We anticipate that the PITX2-EGFP hiPSC reporter cell line established and validated here can be utilized to isolate POM cells and to analyze PITX2 expression during POM cell induction.

The secretome of adipose-derived mesenchymal stem cells attenuates epithelial-mesenchymal transition in human corneal epithelium.

INTRODUCTION: Epithelial-mesenchymal transition (EMT) induces the loss of cell-cell interactions in polarized epithelial cells and converts these cells to invasive mesenchymal-like cells. It is also involved in tissue fibrosis including that occurring in some ocular surface diseases such as pterygium and in subepithelial corneal fibrosis in limbal stem cell deficiency. Here, we examined the effects of the secretome of human adipose-derived mesenchymal stem cells (AdMSCs) on EMT in human corneal epithelial cells (CECs). METHODS: EMT was induced with transforming growth factor-ß (TGF-ß) in primary human CECs isolated from the human corneal limbus. The effects of the AdMSC secretome on EMT in these cells or stratified CEC sheets were analyzed by co-cultivation experiments with the addition of AdMSC conditioned-medium. The expression of EMT-related genes and proteins in CECs was analyzed. The superstructure of CECs was observed by scanning electron microscopy. Furthermore, the barrier function of CEC sheets was analyzed by measuring transepithelial electrical resistance (TER). RESULTS: The AdMSC secretome was found to suppress EMT-related gene expression and attenuate TGF-ß-induced corneal epithelial dysfunction including the dissociation of cell-cell interactions and decreases in TER in constructed CEC sheets. CONCLUSIONS: The secretome of AdMSCs can inhibit TGF-ß-induced EMT in CECs. These findings suggest that this could be a useful source for the treatment for EMT-related ocular surface diseases.

Selective Laminin-Directed Differentiation of Human Induced Pluripotent Stem Cells into Distinct Ocular Lineages.

The extracellular matrix plays a key role in stem cell maintenance, expansion, and differentiation. Laminin, a basement membrane protein, is a widely used substrate for cell culture including the growth of human induced pluripotent stem cells (hiPSCs). Here, we show that different isoforms of laminin lead to the selective differentiation of hiPSCs into different eye-like tissues. Specifically, the 211 isoform of the E8 fragment of laminin (LN211E8) promotes differentiation into neural crest cells via Wnt activation, whereas LN332E8 promotes differentiation into corneal epithelial cells. The immunohistochemical distributions of these laminin isoforms in the developing mouse eye mirrors the hiPSC type that was induced in vitro. Moreover, LN511E8 enables generation of dense hiPSC colonies due to actomyosin contraction, which in turn led to cell density-dependent YAP inactivation and subsequent retinal differentiation in colony centers. Thus, distinct laminin isoforms determine the fate of expanded hiPSCs into eye-like tissues.

Use of homeobox gene expression patterns to determine anatomical regions of origin for body surface tissues derived from adult mice.

Anatomical regions of the skin have distinct functions and anatomical characteristics, including thicker or thinner epidermis, more or fewer hair follicles, and lighter or darker skin. For a better therapeutic outcome of skin transplantation, site-specific characteristics of grafted tissues need to be taken into account in terms of their functionality and beauty. However, there is no method for evaluating positional information of epidermal cells. Homeobox genes are expressed along the anterior-posterior axis and direct the body plan in the animal development process. Although the expression of several HOX genes is known to be retained as the positional information in adult tissue, their expression patterns in the body surface tissues in adult mammals are still incompletely understood. In this study, we investigated the expression patterns of 40 homeobox genes, including 39 Hox genes and the paired box 6 (Pax6) gene, in body surface tissues of adult mice. On the basis of the results obtained, we proposed, for the first time, a method for determining anatomical regions of origin for body surface tissues derived from adult mice using Hox genes and Pax6. Evaluation of expression levels of at least 7 Hox genes and Pax6 should be sufficient to distinguish 11 anatomical body surface tissues derived from the adult mouse body. The proposed method may be useful not only for determining the origin of surface tissues from specific anatomical regions of the mammalian body but also for predicting positional information of epithelial cells generated from pluripotent stem cells.

Age-Associated Increase in Skin Fibroblast-Derived Prostaglandin E2 Contributes to Reduced Collagen Levels in Elderly Human Skin.

Production of type I collagen declines during aging, leading to skin thinning and impaired function. Prostaglandin E2 (PGE2) is a pleiotropic lipid mediator that is synthesized from arachidonic acid by the sequential actions of cyclooxygenases (COX) and PGE synthases (PTGES). PGE2 inhibits collagen production by fibroblasts in vitro. We report that PTGES1 and COX2 progressively increase with aging in sun-protected human skin. PTGES1 and COX2 mRNA were increased 3.4-fold and 2.7-fold, respectively, in the dermis of elderly (>80 years) versus young (21-30 years) individuals. Fibroblasts were the major cell source of both enzymes. PGE2 levels were increased 70% in elderly skin. Fibroblasts in aged skin display reduced spreading due to collagen fibril fragmentation. To investigate the relationship between spreading and PGE2 synthesis, fibroblasts were cultured on micropost arrays or hydrogels of varying mechanical compliance. Reduced spreading/mechanical force resulted in increased expression of both PTGES1 and COX2 and elevated levels of PGE2. Inhibition of PGE2 synthesis by diclofenac enhanced collagen production in skin organ cultures. These data suggest that reduced spreading/mechanical force of fibroblasts in aged skin elevates PGE2 production, contributing to reduced collagen production. Inhibition of PGE2 production may be therapeutically beneficial for combating age-associated collagen deficit in human skin.

Elevated cysteine-rich protein 61 (CCN1) promotes skin aging via upregulation of IL-1ß in chronically sun-exposed human skin.

Chronic exposure of human skin to solar ultraviolet (UV) irradiation causes premature skin aging, which is characterized by reduced type I collagen production and increased fragmentation of the dermal collagenous extracellular matrix. This imbalance of collagen homeostasis is mediated, in part, by elevated expression of the matricellular protein cysteine-rich protein 61 (CCN1), in dermal fibroblasts, the primary collagen producing cell type in human skin. Here, we report that the actions of CCN1 are mediated by induction of interleukin 1ß (IL-1ß). CCN1 and IL-1ß are strikingly induced by acute UV irradiation, and constitutively elevated in sun-exposed prematurely aged human skin. Elevated CCN1 rapidly induces IL-1ß, inhibits type I collagen production, and upregulates matrix metalloproteinase-1, which degrades collagen fibrils. Blockade of IL-1ß actions by IL-1 receptor antagonist largely prevents the deleterious effects of CCN1 on collagen homeostasis. Furthermore, knockdown of CCN1 significantly reduces induction of IL-1ß by UV irradiation, and thereby partially prevents collagen loss. These data demonstrate that elevated CCN1promotes inflammaging and collagen loss via induction of IL-1ß and thereby contributes to the pathophysiology of premature aging in chronically sun-exposed human skin.

Phenethyl isothiocyanate suppresses nitric oxide production via inhibition of phosphoinositide 3-kinase/Akt-induced IFN-gamma secretion in LPS-activated peritoneal macrophages.

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, is well known to have versatile physiological activities, including chemopreventive effects. On the other hand, its anti-inflammatory effects are poorly reported. Nitric oxide (NO) is associated with a wide variety of inflammatory diseases. In this study, we investigated the effects of PEITC on NO production in LPS-activated peritoneal macrophages from ICR mice. The signaling pathway of LPS-induced NO production was examined using neutralizing antibodies [anti-interferon (IFN)-gamma and anti-interleukin (IL-12)] and specific protein kinase inhibitors, as well as others. The activity of PEITC toward NOx production was assessed in mice that received LPS via intraperitoneal administration. The neutralizing antibody of anti-IFN-gamma, but not anti-IL-12, suppressed LPS-induced NO production by 90%. LY294002, a specific inhibitor of phosphoinositide-3-kinase, suppressed Akt and IFN-gamma mRNA expression up-regulated by LPS, whereas PEITC exhibited a similar inhibition profile. Furthermore, oral administration of PEITC significantly suppressed the serum concentration of NOx in ICR mice. Our results suggest that PEITC suppresses LPS-induced NO production via inhibition of Akt activation and the resultant decrease in expression of IFN-gamma. This is one of the first reports to demonstrate a marked anti-inflammatory effect of PEITC following its oral administration.