Protective effects of low-molecular-weight components of adipose stem cell-derived conditioned medium on dry eye syndrome in mice

The present study demonstrated the protective effects of low-molecular-weight adipose-derived stem cell-conditioned medium (LADSC-CM) in a mouse model of dry eye syndrome. Mice subjected to desiccating stress and benzalkonium chloride had decreased tear secretion, impaired corneal epithelial tight junction with microvilli, and decreased conjunctival goblet cells. Topical application of adipose-derived stem cell-conditioned medium (ADSC-CM) stimulated lacrimal tear secretion, preserved tight junction and microvilli of the corneal epithelium, and increased the density of goblet cells and MUC16 expression in the conjunctiva. The low-molecular-weight fractions (< 10 kDa and < 3 kDa) of ADSC-CM (LADSC-CM) provided better protections than the > 10 kDa or > 3 kDa fractions of ADSC-CM. In the in vitro study, desiccation for 10 min or hyperosmolarity (490 osmols) for 24 h caused decreased viability of human corneal epithelial cells, which were reversed by LADSC-CM. The active ingredients in the LADSC-CM were lipophobic and stable after heating and lyophilization. Our study demonstrated that LADSC-CM had beneficial effects on experimental dry eye. It is worthy of further exploration for the active ingredient(s) and the mechanism.


Results
In a controlled-environment chamber (CEC) [48][49][50] combined with benzalkonium chloride (BAC) [51][52][53] BALB/c mice dry eye model, topical application of ADSC-CM showed better protection than Refresh Plus lubricant eye drops (Allergan, Westport, Ireland), Iscove's modified Dulbecco's medium supplemented with glutamine, 10% fetal bovine serum, and mesenchymal stem cells culture adjuvant (abbreviated as IMMCA, which was the same medium as ADSC-CM but not conditioned by ADSCs). The confocal microscopy study of corneal epithelium revealed a decreased expression of zonula occludens-1 (ZO-1), occludin, and keratin 12 (K12) in the CEC-induced dry eye. The impaired expression was partially reversed by Refresh Plus and IMMCA, but the best expression was noted in the ADSC-CM group (Fig. 1A,B). The scanning electron microscopy (SEM) study demonstrated that the microvilli of corneal epithelium were lost in the CEC-induced dry eye mice. There were some preserved microvilli but also with bare area in the group treated with Refresh Plus and IMMCA, while most microvilli were well maintained in the group treated with ADSC-CM (Fig. 1C). Periodic acid-Schiff (PAS) staining showed that conjunctival goblet cells were reduced in the CEC-induced dry eye. Refresh Plus and IMMCA partially reversed the reduction, while ADSC-CM best preserved the density of goblet cells ( Fig. 2A,B). The immunohistochemical study of MUC16 also showed that the ADSC-CM group had the best expression of MUC16 (Fig. 2C).
Further characterization of the active components in the LADSC-CM showed that heating to 56 °C for 30 min or 100 °C for 3 min did not reduce the defensive effects of LADSC-CM on HCECs against desiccating stress or hyperosmolarity stress. The protective capabilities did not lose after lyophilization, storage, and reconstitution. Neither did 1:1 hexane extraction for three times change the protective effects of LADSC-CM (Fig. 7).

Discussion
Aging is one of the most critical factors in dry eye 59,60 , and antiaging approaches have been suggested for dry eye treatment 61,62 . Stem cells play essential roles against aging, but the application of stem cells might provoke the concerns of tumorigenesis or cellular rejection. In contrast, trophic factors in the conditioned medium secreted by stem cells also help tissue repair but minimize rejection problems as the conditioned medium is devoid of cells [63][64][65][66][67] . Besides, trophic factors in the conditioned medium might be freeze-dried or manufactured, packaged, and transported more easily. Therefore, stem cell-derived condition medium is promising as a pharmaceutical for regenerative medicine.
BAC has been used to induce dry eye in animals 51,52 , and the BAC-induced dry eye model was proved stable and widely used for research 31,53,[68][69][70][71][72][73][74][75] . CEC-induced dry eye is another widely used model 48,76 . Disruption of tight junction, loss of conjunctival goblet cell, and impairment of membrane-associated mucin have been described in dry syndrome [77][78][79] . Previous studies described a similar finding in the BAC-or CEC-dry eye model 48,[80][81][82][83][84][85][86] . In our study, the mice in the CEC treated with BAC concomitantly had significantly decreased expression of ZO-1 and occludin in the corneal epithelium. Topical application of ADSC-CM reversed or even increased the reduced expression. The corneal epithelium in the ADSC-CM group also showed the best presentation of K12, which meant preserving the characteristics of corneal epithelium 87 . The preservation of tight junction by ADSC-CM might help the corneal epithelium carry out housekeeping functions that border the external environment, including providing a barrier to fluid loss, toxin irritation, and pathogen entrance. Microvilli injury indicated by the surface covered by microvilli was suggested as the best determining indicator of progressive corneal exposure to dry eye conditions 88  ADSC-CM protected not only the tight junction of corneal epithelial cells but also the conjunctival goblet cells and the membrane-associated mucin Muc16 expression. Conjunctival goblet cell density was an ocular biomarker of dry eye 89 . Muc16 was one of the major membrane-associated mucins expressed on the ocular surface epithelium 90 . Although Muc16 was expressed only in the conjunctival epithelia in mice 91 , in contrast to both the corneal and conjunctival epithelia in humans 92 , the loss of Muc16 in the conjunctiva affected the homeostasis of the corneal epithelium and stroma and upregulated the inflammatory signaling cascade 93 . In our study, the goblet cell density was decreased in the CEC mice, which was alleviated by Refresh Plus lubricant eye drops, IMMCA, or ADSC-CM. ADSC-CM protected the goblet cells density best to the level similar to that of non-dry mice. The Muc16 expression was continuous in the non-dry group and interrupted in the dry control group. ADSC-CM preserved the integrity of Muc16 expression from dry injury.
HCEC desiccation stress and hyperosmolarity stress are two widely used in vitro models to simulate dry eye conditions 55,94-100 . In the HCEC desiccation stress study, ADSC-CM showed better protective or regenerative effect than the commercial corneal epithelial basal medium CEM, with the fractions of < 30 kD, < 10 kD, and < 3 www.nature.com/scientificreports/ kD providing better cell viabilities than those of > 30 kD, > 10 kD, and > 3 kD. From above, we believe something in LADSC-CM (< 3 kD) beneficial for dry eye and are exploring the possible underlying mechanism. Topical application of human ADSC-derived exosomes (size about 100 nM) has recently been reported to alleviate ocular surface damage in a mouse model of dry eye disease 47 . In contrast, our study showed better protective effects of the low-molecule-weight (< 3 kD, less than 0.6 nM; < 10 kD, less than 1.5 nM ) fractions of ADSC-CM (LADSC-CM). Mice treated with LADSC-CM had more tear secretion, well-preserved tight junction and MUC16 expression of the cornea, higher conjunctival goblet cell density, and less damaged microvilli of corneal epithelium. LADSC-CM contained only molecules smaller than the known smallest virus that is in the size of about 15 nM, making its clinical application free from infection risk. The protective capabilities remain similar after heating to 56 °C for 30 min or 100 °C for 3 min, or lyophilization and reconstitution, which meant the active ingredients were relatively stable and might be easier for transportation. The active components in LADSC-CM were resistant to hexane extractions and were probably polar in characters.
In conclusion, our study demonstrated the beneficial effects of LADSC-CM in both the in vitro HCEC desiccation stress study and in vivo mice dry-induced ocular surface injury. The active ingredients might be stable and polar molecules. Further investigation of the exact active ingredient(s) and the underlying mechanism is needed.

Isolation of ADSCs and preparation of ADSC-CM. This study was approved by the Buddhist Tzu Chi
General Hospital Internal Review Board (IRB102-130). All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained from all participants. Human adipose tissue was harvested during cosmetic liposuction from abdominal subcutaneous fat of three women (age: 23, 28, and 30). Stromal-vascular fraction (SVF) cells were isolated using a modified method described by Griesche and colleagues 101 . Collagenase type I (final concentration: 0.4 mg/mL; Sigma) was added for enzymatic digestion in a hybridization oven (37 °C, 30° angle, 15 rpm, 45 min). Digested adipose tissue was centrifuged at 400 × g for 10 min to generate the SVF pellets for subsequent ADSCs culture. The stemness of the ADSCs was confirmed by their osteogenesis, chondrogenesis, and adipogenesis after induction. ADSCs at passages 2 to 5 were cultured in non-Phenol Red Iscove's modified Dulbecco's medium (Gibco™) supplemented with glutamine (200 mM; Gibco™), 10% FBS (HyClone™) and mesenchymal stem cells culture adjuvant (FGF2, 10 ng/ml, R&D Systems; N-acetyl-L-cysteine, 2 mM, Sigma; L-ascorbic acid-2-phosphate, 0.2 mM, Sigma) (The medium was abbreviated

Characterization of active components in the LADSC-CM. For the heating test, LADSC-CM was
incubated at either 56 °C for 30 min or 100 °C for 3 min and was tested for activity. For the lyophilization test, aliquots of the dialyzed LADSC-CM samples (1 ml) were prepared in 5 ml lyophilized vials followed by lyophilization in a programmable freeze dryer. The lyophilized products were stored at 4 °C for one week and were then reconstituted with water for injection for activity test. For the lipophilicity test, equal volumes of LADSC-CM and hexane (3 mL for each) were mixed and vortexed for 20 min and centrifuged at 2000 rpm for 5 min, and the hexane fraction was discarded. The extraction was repeated three times, the lower layer (aqueous phase) was collected for test. HCECs treated with fractions of molecular size < 30 kD, < 10 kD, < 3kD after desiccation showed better viability than thosed treated with > 30 kD, > 10 kD, and > 3 kD. (N = 3) *p < 0.05, **p < 0.01, ***p < 0.001 compared with the non-dry control. #p < 0.05, ##p < 0.01, ### p < 0.001 compared with the corneal epithelial cell basal medium (CEM). (N = 5) (B) In the CEC-induced dry eye study, mice reated with fractions ADSC-CM with molecular size < 10 kD or < 3kD had more tear secretion than thosed treated with > 10 kD or > 3 kD. (N = 4). *p < 0.05, **p < 0.01, *** p < 0.001, compared with Non-dry control. # p < 0.05, # # p < 0.01, # # # p < 0.001, compared with Dry control. The values were expressed as the means ± SD. (N = 5). Desiccating stress. A modified in vitro desiccation stress on HCECs was used in our study 55,57,96 . Briefly, 2 × 10 4 HCECs were seeded in 96-well dishes and cultivated for 24 h to attach to the dishes (about 80% confluence). The medium was aspirated, and the dishes were left dry for 10 min at 37 °C. After desiccation, the testing culture mediums were replenished to the respective culture dishes. The HCECs that did not undergo the desiccation stress were deemed as control. After incubation for four hours, the cells were counted using the Cell Counting Kit-8 (CCK-8 assay).
Hyperosmolarity stress. 1.5 × 10 4 HCECs were seeded in 96 well-dishes and cultivated overnight to attach to the dishes (approximately 60% confluence) and were then treated for 24 h with fresh medium (311 mOsm/ kg, normal control) or the medium containing another 90 mM NaCl (490 mOsm/kg, hypertonic groups). After Induced murine dry eye model. The study is reported in accordance with ARRIVE guidelines (https:// arriv eguid elines. org). All experimental procedures were approved by the Laboratory Animal Care and Use Committee at Tzu Chi University. All methods were performed in accordance with the relevant guidelines and regulations. Dry eye-related ocular surface signs of BALB/c mice were induced in a controlled-environment chamber (CEC) [48][49][50] combined with topical BAC [51][52][53] . Briefly, 12-week-old female BALB/c mice were housed in CEC with a relative humidity of 10 ± 3%, temperatures of 21-25℃, and airflows of 10-15 L/min. Each experimental and control group consisted of 5 mice. Mice of dry control and experimental groups were housed in the CECs and received topical 0.2% BAC daily. Mice of the non-dry control group were in a chamber of humidity of 75 ± 3%. The experimental groups received the respective testing eye drops twice a day for 28 days. In the first experiment, the testing eye drops were Refresh Plus lubricant eye drops, IMMCA, and ADSC-CM. In the second part experiment, the testing eye drops were the original ADSC-CM, ADSC-CM with a molecular weight > 10 Negative control studies were also performed without using the primary antibodies. After dehydration in graded ethanol and xylene, sections were mounted in Histokit (Hecht Assistent, Sondheim, Germany) and analyzed.
Scanning electron microscopy analysis. Fresh corneas were first fixed in 2% paraformaldehyde for 24 h, and then in 2.5% glutaraldehyde solution in 0.2 M cacodylate buffer and 1% tannic acid at pH 7.0-7.3 for another 24 h, followed by postfixation with 1% osmium tetroxide solution in 0.2 M cacodylate buffer solution for 1 h. Samples were then dehydrated by a critical point dryer (Hitachi Ltd., Japan) and coated with platinum in an ion sputter coater (Hitachi Ltd., Japan). Finally, the samples were observed and photographed with the scanning electron microscope (Hitachi Ltd., Japan). www.nature.com/scientificreports/ Statistical analysis. Data were expressed as means ± SD. Only one sample from each mouse was used for the analysis of each examination result. In the tear secretion assay, the average of estimates from both eyes was used. For goblet cell density, only the left eye of each mouse was sectioned for Periodic acid-Schiff stain and calculation. For CCK viability assay, each sample in the same group was from different rounds of experiments. One-way ANOVA and two-sample t-test were used to compare CCK assay, tear secretion assay, and conjunctival goblet cell density. A p < 0.05 was considered statistically significant.