(2023) 24:8
Dan et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-023-00467-4

BMC Molecular andCell Biology

Open Access

RESEARCH

Urine‑derived mesenchymal stem
cells‑derived exosomes enhances survival
and proliferation of aging retinal ganglion cells
Qi‑Qin Dan1, Li Chen1, Lan‑Lan shi2, Xiu Zhou3, Ting‑Hua Wang1* and Hua Liu4*

Abstract
Objectives This study was designed to investigate to test the effect of exosomes from urine-derived mesenchymal
stem cells (USCs) on the survival and viability of aging retinal ganglion cells (RGCs), and explored the preliminary
related mechanisms.
Methods Primary USCs were cultured and identified by immunofluorescence staining. Aging RGCs models were
established by D-galactose treatment and identified by β-Galactosidase staining. After treatment with USCs condi‑
tioned medium (with USCs removal), flow cytometry was performed to examine the apoptosis and cell cycle of RGCs.
Cell viability of RGCs was detected by Cell-counting Kit 8 (CCK8) assay. Moreover, gene sequencing and bioinformatics
analysis were applied to analyze the genetic variation after medium treatment in RGCs along with the biological func‑
tions of differentially expressed genes (DEGs).
Results The number of apoptotic aging RGCs was significantly reduced in USCs medium-treated RGCs. Besides,
USCs-derived exosomes exert significant promotion on the cell viability and proliferation of aging RGCs. Further,sequencing data analyzed and identified DEGs expressed in aging RGCs and aging RGCs treated with USCs condi‑
tioned medium. The sequencing outcomes demonstrated 117 upregulated genes and 186 downregulated genes innormal RGCs group vs aging RGCs group, 137 upregulated ones and 517 downregulated ones in aging RGCs groupvs aging RGCs + USCs medium group. These DEGs involves in numerous positive molecular activities to promote therecovery of RGCs function.
Conclusions Collectively, the therapeutic potentials of USCs-derived exosomes include suppression on cell apop‑
tosis, enhancement on cell viability and proliferation of aging RGCs. The underlying mechanism involves multiple
genetic variation and changes of transduction signaling pathways.
Keywords Retinal ganglion cells, Urine-derived stem cells-derived exosomes, Aging-related RGCs function loss, Cell
viability and proliferation*Correspondence:
Ting‑Hua Wang
Wangth_email@163.com
Hua Liu
1604571378@qq.com
Full list of author information is available at the end of the article© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Dan et al. BMC Molecular and Cell Biology(2023) 24:8

Introduction
Retinal ganglion cells (RGCs) are nerve cells located in
the final segment of retina. RGCs axons are optic nerve
fibers in the eyeball, which are distributed on the surfaceof omentum and collected in the papilla of optic tract(optic nerve) [1, 2]. After coming out of the eyeball, they
pass through the intersection of optic tract and end in
the lateral geniculate body. In the retina, photoreceptors
transmit the visual information to interneurons which
process and pass on the information to RGCs, axons ofwhich then travel through the optic nerve, informing therest of the brain of all it will ever know about the visual
world [3, 4]. It has been reported that there are appropriately 30 types of RGCs, and distinct RGC types exhibit
different abilities to survive diseases like glaucoma or surgical trauma like axotomy [5]. Considerable efforts havebeen dedicated in previous studies to identify the cascade of structural and functional alterations in RGCs thateventually lead to their death [6]. Previous studies demonstrated that RGCs function is compromised within ashort period after intraocular pressure (IOP) increase [7,8], followed by notable morphological variation in dendrites. The subsequently progressive loss and death of
RGCs result in vision loss. Accumulating evidences fromexperimental and clinical studies suggest that RGCs arecapable of recovering themselves after periods of functional loss at early onset of glaucoma, however, the abilities to recover functions may be negatively impacted withaging [9]. With aging, the function loss of RGCs wasaggravated gradually. It’s of vital importance to find theeffective therapy to prevent aging-related RGCs function
loss and boost functional recovery of RGCs in the progression of aging.
Among a variety of approaches applied to ameliorate
vision loss, lowering IOP is the most widely used [10].
Nevertheless, it’s quite tough work to hinder the aging
process of RGCs as human get older. Stem cells are a kindof pluripotent cells with strong self-replication abilities
which enable them differentiate into a variety of functional cells and have the potential function of regenerating various tissues and organs. Currently, it’s quite
familiar to us with various types of stem cells, such asneural stem cells, embryonic stem cells, bone marrowmesenchymal stem cells, umbilical cord mesenchymal
stem cells, etc., which have been implicated in a great
many disorders and diseases. Urine-derived mesenchymal stem cells (USCs) can be obtained from patients’urine samples in a non-invasive way, and these cells inurine can differentiate into bladder-like cells, such assmooth muscle and urinary tract epithelial cell lines, evenembryonic stem cells [11, 12]. At the same time, USCs
are revealed to be involved in the formation of bone,
soft bone, skeletal muscle and other tissues [13]. The

Page 2 of 11

pluripotency of USCs reveals potency that they might
be used in the treatment of numerous diseases. Suchfunctions are mediated through the secretion of particles from USCs and resident cells known as extracellular
vesicles (EVs) within a microenvironment. EVs are a heterogeneous population of membranous vesicles released
from cells that transfer many types of active biomolecules
to recipient cells and induce physiologic and phenotypicalterations in the tissue environment [14]. Exosomes, asubtype of EVs mediate intercellular communication,
target cells inducing cellular development through different signaling pathways. EVs like exosomes originating from the endosomal pathway carry different types of
biomolecules such as nucleic acids, proteins, and lipids;
participate in cell-to-cell communication [15]. Targeting exosomes represents novel approaches for the treatment of cancers through using them as cell-free therapy
and drug-delivery system and inhibiting their biogenesis
and distribution [16]. Mesenchymal stem cells (MSCs)
derived exosomes have great potentials to improve ageing and age-related diseases [17]. Exosomes-therapy may
serve as a new approach to combat aging.
In this study, we investigated the effect of USCs-derived
exosomes on preventing the functional loss of aging
RGCs and preliminarily analyzed the underlying mechanisms. Our finding revealed that treatment with the conditioned USCs medium reversed the aging progression of
RGCs cells, which provide novel insights into the prevention and treatment of vision disorders.

Materials and methods
Cell culture

RGCs used in this study were purchased from Qingqi
Biotechnology Development Co., Ltd. (Shanghai, China).
RGCs were cultured with high glucose medium containing 10% inactivated fetal bovine serum (FBS) in an
incubator with 5% ­CO2 at 37 ℃. When RGCs reached
80%—90% confluence and grew well, they were digested
with trypsin and subcultured.
Urine was collected from healthy adult females, and
USCs were separated and cultured in special medium of
urine stem cell which were purchased from Xihan Biotechnology Co., Ltd. (Shanghai, China), incubated with
5% ­CO2 at 37℃. When the cell confluence reached about
85%, they were generally subcultured every 3–5 days.
If the cells grew well with 85% cell confluence, it is recommended to subculture at ratio of 1:3. Otherwise, thecells with lower density can be subcultured at ratio of
1:2. There’re some precautions in the process of operation. Before passage, a warm bath at 37 ℃ is necessary
for trypsin, and digestion should be carried out in a 37
℃ incubator. The duration of digestion shall be controlled within about 1 min to avoid affecting the cell state.

Dan et al. BMC Molecular and Cell Biology(2023) 24:8

The third-generation cells were used in the following
experiments.
Identification of USCs by immunofluorescence (IF) staining

USCs, primarily cultured in our laboratory, were identified by flow cytometry in Yunnan Key Laboratory ofStem Cell Translation. The biological characteristics of
USCs resemble to those of MSCs, and it has been shownthat USCs highly express four markers, CD44, CD73,
CD90 and CD105. Identification of MSCs was performed
as previously described [18, 19]. The cells were preparedinto single cell suspension, 5 μl of CD44 and CD90 antibodies were added and incubated for 30 min at room
temperature in dark. Afterwards, they were washed twicewith phosphate buffer (PBS, pH 7.2) and centrifuged at
1,500 r/min for 5 min. Then, the 200 μl of PBS was added,
and the cells were reconstituted into cell suspension for
detection on florescence intensity of cells by flow cytometry [20].
Cell counting kit (CCK8) assay

RGCs in logarithmic growth stage were inoculated in 96
well plate (200 μL/well) at a density of 1.6 × ­105/ml. There
were blank group, normal group and aging groups, with
6 replicated wells in each group. The blank group did notcontain cells. The cells in the normal group were cultured
in complete medium composed of high glucose medium,
10% FBS, 1% penicillin–streptomycin double antibodies.
The cells in the aging group were cultured in complete
culture medium with D-galactose at concentrations of
5, 10, 20, 30 and 40 mg/ml for 24 h in an incubator with
5% ­CO2 at 37 ℃, and then the complete culture medium
was changed for another 24-h incubation. Subsequently,
10 μL CCK-8 solution per 100 μL medium was added
into each well. After continuous culture for half hour,
the absorbance value (A) of each well at the wavelengthof 450 nm was detected using a microplate reader andthe cell survival rate was calculate as: cell survival rate(%) = (A value of test group—A value of blank group) / (Avalue of normal group—A value of blank group) × 100%.
The test was repeated 3 times.

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Afterwards, the cell fixative was removed, and followedby 3 washes with PBS for 3 min each time. An appropriate amount of cell staining working solution was subsequently added into cells which were incubated overnightat 37 ℃. The aging cells were stained light blue to dark
blue under the optical microscope.
Flow cytometry detection

RGCs in logarithmic growth stage were inoculated into
6-well plates (2 ml/well) at a density of 4 × ­108 pieces/L.
After culturing for 48 h, trypsin was added into eachwell for digestion. Being centrifuged at 1000 r / min for
5 min, cell precipitates were collected and rinsed twicewith PBS. Later, according to the instructions of FITCAnnexin V apoptosis detection cell kit, flow cytometry
was used to detect the apoptosis at the excitation wavelength of 488 nm and the apoptosis rate was calculated
as: apoptosis rate = early apoptosis rate + late apoptosis
rate [21].
The cells treated according to the above-method steps
until twice washes with PBS, were supplemented with
75% ethanol for fixation overnight at 4 ℃. Subsequently,
they were washed twice with 4 ℃ PBS and then centrifuged for 5 min at 1000 R / min. Propidium iodide [5]staining reagent was prepared according to the instructions of cell cycle kit, followed by a warm bath in dark at
37 ℃. Cell cycles were analyzed by flow cytometry [22].
The test was repeated 3 times.
Gene sequencing

Gene sequencing was employed to elucidate variation of
DEGs among normal, aging and USCs medium treatedgroups, 3 replicated wells for each group. The detailed
steps were similar to our previously published article[23]. Then, the volcano plot analysis, enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of
Genes and Genomes (KEGG) were performed for the
biological function determination of DEGs.

β‑Galactosidase staining of aging cells

The Protein–Protein Interactions (PPIs) Network Analysis
of Disease Intersection Targets

RGCs in logarithmic growth stage were inoculated into
6-well plates at a density of 1 × ­104 cells. The cells werearranged into normal group, aging group and USCsconditioned medium (with USCs removal) group.
After receiving respective treatment, they were incubated for 48 h at 37 ℃ with 5% ­CO2. As instructed in
β-Galactosidase detection kit, we sucked out the cell culture medium, and washed cells with PBS once. An appropriate amount of SA-β-Gal staining fixative for fixation
was added into cells for 15 min at room temperature.

The PPIs network of disease targets was established andanalyzed by STRING database (https://​www.​string-​db.​org/) [24]. The top 100 upregulated genes were imported
into String for analysis. Those PPIs with high confidence
score (> 0.90) should be selected for network construction and analysis to ensure the accuracy of the results.
All the network can be constructed by employing the
network visualization software Cytoscape 3.7.2 (https://​cytos​cape.​org/). The connected genes from PPI network
were imported into Cytoscpe for analysis of hub genes.

Dan et al. BMC Molecular and Cell Biology(2023) 24:8

Page 4 of 11

GO and KEGG pathway enrichment analysis

GO and KEGG analysis was employed on all DEGs. GO
enrichment analysis provides all GO terms that are significantly enriched in targets compared to the genome
background and filters the targets that correspond tobiological functions [25]. Pathway-based analysis isemployed to characterize the biological functions oftargets [26]. KEGG pathway enrichment analysis identified significantly signal transduction pathways in targets.
In the present study, top 100 upregulated genes were
mapped into Metascape database (http://​metas​cape.​org/​
gp/​index.​html#/​main/​step1), and gene numbers were
calculated for every term.
Statistical analysis

Graphpad prism 8.0 software was used for statisticalanalysis. Data were expressed as mean ± standard deviation (SD), and one-way analysis of variance (ANOVA)
was used for comparison among more than 3 groups. P
value less than 0.05 is considered statistically significant.

Results
Effects of D‑galactose on survival rate of RGC cells

The cell viability of RGCs treated with different concentrations of D-galactose was detected by CCK8 assay. As
demonstrated in Table 1, the cell survival rate was negatively correlated with the concentrations of D-galactose.
With the concentration of D-galactose increasing, thesurvival rate gradually decreased, reaching about 58%
when treated with 30 mg/mL D-galactose. The survival
rate at 30 mg/mL was significantly lower than that of control group (p < 0.001, Fig. 1). Thus, 30 mg/mL D-galactose
was determined to be the final concentration of inducing
aging RGCs.
Identification of USCs

Immunostaining results demonstrated that over 99%
positive expression of CD44 and CD90 identified thepurity of cultured USCs (Fig. 2). The high percentage ofTable 1 Survival rate of RGCs
concentrations of D-galactose
Groupswithdifferent

Concentrations

Survival rate

5 mg/ml

83% ± 0.017

Control group
Aging grouptreated100%
10 mg/ml
20 mg/ml
30 mg/ml
40 mg/ml

73.68% ± 0.036

67.52% ± 0.02

58.17% ± 0.0196

48.27% ± 0.028

Fig. 1 Survival rate of RGCs treated with different concentrations ofD-galactose. N = 8 /group, ***p < 0.001

cells expressing CD44 and CD90 indicates MSCs characteristics of USCs.
Apoptosis of aging RGCs was inhibited after USCsconditioned medium treatment

Flow cytometry was employed to detect the apoptosis
rate of RGCs. The results revealed that apoptotic RGCs
were significantly increased in the aging group, while thenumber of apoptotic cells got smaller in the aging RGCs
cultured in USCs medium (Fig. 3A, B). Correspondingly,
the apoptosis rate in aging group was much higher thanthat of normal group (p < 0.01, Fig. 3C), but it was markedly diminished in RGCs treated with USCs medium(p < 0.01, Fig. 3C). The results revealed that USCs-derived
exosomes could inhibit the apoptosis of aging RGCs. Further, the cell count of RGCs in aging group at Freq G1
was largely increased, but obviously decreased at Freq S
compared with that of the normal group (Fig. 3D, E). No
obvious variation was revealed in the number of cells at
Freq G2 among normal, aging and USCs medium groups(Fig. 3D, E). Relative to normal group, there’s a prominent increase of the cell number observed at Freq G1 in
aging group, while a significant reduction at Freq G1 in
USCs medium group (Fig. 3D, E).
USCs conditioned medium promoted survival
and proliferation of aging RGCs

Three kinds of medium (DMEM; USCs conditioned
medium; DMEM to USCs conditioned medium at1:1) were used to culture RGCs in normal and aging
groups. Normal RGCs in DMEM are irregular hexagon-shaped with uniform size, growing in goodstate (Fig. 4A). When cultured in USCs conditioned

Dan et al. BMC Molecular and Cell Biology(2023) 24:8

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Fig. 2 Immunostaining of CD44 and CD90 for USCs identification. Green fluorescence indicates CD44 positive cells, and red fluorescence indicates
CD90 positive cells. Scale bar = 100 μm, n = 6 /group

medium, the number of normal RGCs was increased
and arranged more closely. In the combined medium
with DMEM and USCs conditioned medium, variationin cell morphology was observed and cell number was
reduced (Fig. 4A). When aging RGCs were cultured
in DMEM, the cell count was significantly decreased
relative to that of normal RGCs in DMEM (p = 0.00,
Fig. 4B). However, aging cells in USCs conditioned
medium were augmented markedly compared to cells
in DMEM medium (p = 0.00, Fig. 4A, B) and combined
medium. Cell viability was detected using CCK8 testat 24 h and 48 h, which demonstrated that in DMEM
medium, the OD value of aging RGCs significantly
reduced compared to that of normal RGCs at 24 h and48 h (p = 0.00), while aging RGCs in USCs conditioned
medium showed obvious augmented OD value in comparison to aging RGCs in DMEM at 24 h and 48 h
(p = 0.00, Fig. 4C, D). The results of β-Galactosidase
staining uncovered that there were more aging cells
in DMEM medium, decreased number of aging RGCs
and increased number of normal cells when cultured in
USCs conditioned medium (Fig. 4E).

Identification of DEGs and analysis on their biological
functions

In order to further investigate the underlying mechanisms related to the effect of USCs-derived exosomeson aging RGCs, the gene sequencing was performed toanalyze the potential genetic variation. The sequencing outcomes demonstrated 117 upregulated genes
and 186 downregulated genes in normal RGCs groupvs aging RGCs group, 137 upregulated ones and
517 downregulated ones in aging RGCs group vsaging RGCs + USCs medium group (Fig. 5A). These
DEGs underwent subsequent GO and KEGG analysis. As revealed, the top 20 enriched KEGG pathways
involved by DEGs in aging RGCs vs normal RGCs
were presented: Cholesterol metabolism Biosynthesis of amino acids, Complement and coagulation cascades, ABC transporters Metabolic pathways, Seleno
compound metabolism, Steroid biosynthesis Osteoclast differentiation, Prion diseases, Alanine, aspartate
and glutamate metabolism, Ovarian Steroidogenesis
p53 signaling pathway, Glycine, Serine and threonine
metabolism, Chemical carcinogenesis, Synaptic vesicle

Dan et al. BMC Molecular and Cell Biology(2023) 24:8

Page 6 of 11

Fig. 3 Apoptosis and cell cycle of RGCs among normal, aging and USCs medium groups. A Light field images of RGCs in normal group, aging
group and aging + USCs medium group, scale bar = 100 μm. B The flow cytometry detection and C the apoptosis rate of RGCs among normal,
aging and aging + USCs medium groups. D The cell cycle and E cycle rate of RGCs among normal, aging and aging + USCs medium groups. Alldata are shown as the mean ± SD, n = 6 /group. **p < 0.01

cycle, Pertussis, One carbon pool by folate, Ether lipid
metabolism Arginine biosynthesis, Dorso-ventral axis
formation (Fig. 5B). Meanwhile, the top 20 enriched
KEGG pathways related to DEGs in aging RGCs vsaging cells cultured in USCs conditioned medium were
Cytokine-cytokine receptor interaction (map04060),
Jak-STAT signaling pathway (map04630), Circadianentrainment (map04713), Proximal tubule bicarbonate reclamation (map04964), MicroRNAs in cancer(map05206), Viral protein interaction with cytokine
and cytokine receptor (map04061), Chemokine
signaling pathway (map04062), Mineral absorption (map04978), Protein digestion and absorption(map04974), Apelin signaling pathway (map04371),
TNF signaling pathway (map04668), Aldosteroneregulated sodium reabsorption (map04960), Estrogensignaling pathway (map04915), Glycine, serine andthreonine metabolism (map00260), Axon guidance(map04360), PPAR signaling pathway (map03320),
Other types of O-glycan biosynthesis (map00514),
Insulin resistance (map04931), IL-17 signaling pathway(map04657), Relaxin signaling pathway (map04926). In

addition, the GO terms DEGs involved are also displayed (Fig. 5C). Top 10 biological processes in which
DEGs in aging RGCs vs normal cells participatedincluded cellular process, biological regulation, regulation of biological process, metabolic process, responseto stimulus, multicellular organismal process, negative regulation of biological process, developmental
process, localization, positive regulation of biological process (Fig. 5D). Cell components of these DEGs
include cell, cell part, organelle, membrane, organellepart, membrane part, extracellular region. Molecular functions of these DEGs involved mainly include
binding, catalytic activity, molecular function regulator, transporter activity (Fig. 5D). Comparatively, top
10 biological processes in which DEGs in aging cells vsaging RGCs + USCs medium participated involved cellular process, biological regulation, metabolic process,regulation of biological process, response to stimulus,
multicellular organismal process, developmental process, negative regulation of biological process, signaling, positive regulation of biological process (Fig. 5E).

Dan et al. BMC Molecular and Cell Biology(2023) 24:8

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Fig. 4 The effect of USCs conditioned medium on ameliorating aging RGCs. A Light field images of RGCs in normal and aging groups cultured inthree kinds of culture medium, respectively, scale bar = 100 μm. B The cell number of RGCs in three kinds of culture medium between normal andaging groups, n = 12/group. OD value of RGCs in normal + DMEM medium, normal + USCs medium, aging + DMEM medium and aging + USCs
medium groups at (C) 24 h and (D) 48 h, n = 6/group. E β-Galactosidase staining images of RGCs in normal + DMEM medium, aging + DMEM
medium and aging + USCs medium groups, scale bar = 100 μm, n = 6/group

Cell components of these DEGs included cell, cellpart, organelle, membrane, membrane part, organellepart, extracellular region, membrane-enclosed lumen.
Molecular functions of these DEGs involved binding,catalytic activity, molecular function regulator, transporter activity (Fig. 5E).

Discussion
Vision loss resulting from damage and death of RGCs is
usually irreversible as human get older and older, whichcauses extremely miserable life experience to those suffering vision disorders. MSCs are advantageous cells inregenerative medicine, exerting pleiotropic functions byproducing soluble factors, such as exosomes [17]. Here,
we established aging RGCs models and carried out cultures of aging RGCs in USCs conditioned medium,
aiming to reveal the potential efficacy of USCs-derived
exosomes in reversing the aging process of RGCs andboost functional recovery of aging RGCs. The results
demonstrated positive therapeutic effect of USCs-derived
exosomes on the cell viability and proliferation of RGCs.
Further, sequencing data identified differential genes
expressed in aging RGCs and aging RGCs treated with
USCs medium and analyzed their biological functions.
Damage or death of RGCs is usually accompanied byfunctional alterations. A previous study reported thatfunctional alterations to RGCs in inner retinal neurons

Dan et al. BMC Molecular and Cell Biology(2023) 24:8

Page 8 of 11

Fig. 5 Bioinformatics analysis of DEGs involved in the effect of USCs-derived exosomes on aging RGCs. A The number of DEGs and volcano maps
in normal RGCs group vs aging RGCs group, and aging RGCs group vs aging RGCs + USCs medium group, n = 3/group. B, C The enriched KEGG
pathways involved by DEGs. D, E GO terms enriched by DEGs

Dan et al. BMC Molecular and Cell Biology(2023) 24:8

already appear before the following detected evidenceof cell degeneration [27]. Besides, early signs of damage
have been detected at the level of RGCs’ axons [28, 29],subsequently, RGCs cell bodies shrink and their dendrites are pruned before cell death [6, 30. Thus, it’s necessary for us to focus on the morphologies of RGCs prior
to the detection of RGCs functions since defects in RGCs
function appear later than its variation in morphology.
In this study, we applied morphological detection andobserved the morphological variation and cell countchange of aging RGCs. Cell bodies of aging RGCs exhibited obvious shrinkage and pruned dendrites, along withsharp reduction of cell number. Comparatively, the morphology and amount aging cells cultured in USCs conditioned medium nearly approached the RGCs at normal
level. CCK8 assay and flow cytometry detection further
proved the cell viability of aging RGCs was enhanced in
USCs conditioned medium, and the number of apoptotic
aging RGCs was also significantly reduced, which suggested that USCs-derived exosomes might have the capabilities of inhibiting apoptosis of aging RGCs and help
recover the cell viability of aging RGCs.
Being derived from the convoluted tubules of nephron,renal pelvis, ureters, bladder and urethra, USCs have asimilar phenotype to MSCs and can be reprogrammed
into induced pluripotent stem cells [31]. The therapeutic potential of USCs in urologic surgeries and dysfunctions has been shown in previous publications [13]. USCs
were induced to differentiate into urothelial and smoothmuscle cells in vitro and then seeded on small intestinal
submucosa scaffolds which were then implanted into
nude mice. Then, multilayered tissue-like structures composed of urothelium and smooth muscle were visualized,
which proved the mighty feasibility to apply USCs as asource of cells for tissues-engineered strategies [31]. In
addition, it has been demonstrated the abilities of USCs
differentiating into neurogenic cells. After transplantation of USCs into rat brains, they survived in the lesion
site, migrated to other brain areas, and expressed neurogenic markers such as GFAP, β-III tubulin and nestin[32]. These outcomes revealed the abilities of USCs differentiating into neuron-like cells in the rat brain andtheir possibilities for regenerative medicine, which correspondingly provide evidence for the efficacy of USCs in
rescuing the viability of aging RGCs. Strikingly, in addition to MSCs themselves, their exosomes are capable ofmediating intercellular communication, targeting cells
inducing cellular development through different signaling pathways. Exosomes produced by endothelial cells
exhibit distinct cargoes that may be useful as a biomarker
of age-related vascular disease [33]. Also, MSCs-derived
exosomes can improve ageing and age-related diseases[17]. Consistently, our work revealed that exosomes fromPage 9 of 11

USCs-derived MSCs inhibited the apoptosis of aging
RGCs and enhanced the cell viability of aging RGCs.
Consequently, USCs-derived exosomes might present
a potential therapeutic method for RGCs loss-induced
vision disorders.
Nevertheless, the detailed mechanisms underlying
the efficacy of USCs-derived exosomes in hampering
age-related disorders remain yet elusive. It’s critical toinform the understanding of molecular characterizationof aging RGCs and specify the stereotyped connections
underlying their physiological function. Therefore, we’ve
analyzed the genetic variation of normal RGCs vs aging
RGCs and aging RGCs vs aging RGCs with USCs conditioned medium by employing gene sequencing. Oursequencing data displayed 117 upregulated genes and
186 downregulated genes in normal RGCs group vs aging
RGCs group, 137 upregulated ones and 517 downregulated ones in aging RGCs group vs aging RGCs + USCs
medium group, suggesting the aging RGCs and the effectof USCs-derived exosomes involved massive genetic
variation inside. It has been reported that early signs ofdamage have been detected at the level of RGCs’ axons
involving metabolic changes, impairment of axonaltransport, and downregulation of specific genes [28, 29].
Similarly, the 5 primary biological processes in which
DEGs in aging RGCs and aging RGCs in USCs medium
participated in are Cellular process, Biological regulation, Regulation of biological process, Metabolic process,Response to stimulus. Among the enriched KEGG pathways involved by DEGs from aging RGCs, the top five
are Cholesterol metabolism Biosynthesis of amino acids,
Complement and coagulation cascades, ABC transporters metabolic pathways, Selenocompound metabolism,
Steroid biosynthesis Osteoclast differentiation. Comparatively, of the enriched KEGG pathways involved by DEGs
from aging RGCs in USCs medium, the top 10 included
Cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, Circadian entrainment, Proximal tubulebicarbonate reclamation, MicroRNAs in cancer Viral,
protein interaction with cytokine and cytokine receptor
Chemokine signaling pathway, Mineral absorption, Protein digestion and absorption, Apelin signaling pathway
TNF signaling pathway, Aldosterone-regulated sodium
reabsorption. JAK/STAT signaling pathway is known asa universally expressed intracellular signal transduction
pathway and took active action in many crucial biological processes, including cell proliferation, differentiation,
apoptosis, and immune regulation [34]. These revealed
that the effect of USCs-derived exosomes activates many
positive molecular activities to promote the recovery of
RGCs function. A separate study suggested an induction in the exosomal secretory pathway coincided witha block in autophagy progress, accelerating senescence,

Dan et al. BMC Molecular and Cell Biology(2023) 24:8

which might be targeted for the treatment of age-relateddiseases [35]. Targeting exosomes represents novel
approaches as cell-free therapy and drug-delivery system for the treatment of age-related diseases. Although
exosomes-therapy may serve as a new approach to combat ageing, the translation of preclinical results to clinic
needs more extensive investigation on exosomes both on
their biology and related techniques. Overall, scrutiny
on the effect of ageing on USCs and vice versa is vital for
designing novel therapy using USCs with focus on the
management of older individuals.

Conclusions
On the whole, the therapeutic potentials of USCs-derived
exosomes for aging RGCs were presented in this study,
including suppression on cell apoptosis, enhancement
on cell viability and proliferation of RGCs. The underlying mechanism involves multiple genetic variation andchanges of transduction signaling pathways, which provide referential basis for the later in-depth mechanistic
investigations on the efficacy of USCs-derived exosomesin the age-related disease.
Acknowledgements
Not applicable.
Authors’ contributions
Study concept: Ting-Hua Wang, Hua Liu. Data collection: Lan-Lan Shi, Xiu
Zhou. Data analysis and interpretation: Qi-Qin Dan, Lan-Lan Shi, Xiu Zhou.
Paper writing: Qi-Qin Dan, Li Chen. The author(s) read and approved the final
manuscript.
Funding
Not applicable.
Availability of data and materials
The raw sequence data reported in this paper have been deposited in theGene Expression Ominibus of National Center for Biotechnology Information,
under accession number GSE224109 and are publicly accessible at https://​www.​ncbi.​nlm.​nih.​gov/​geo/​query/​acc.​cgi?​acc=​GSE22​4109. The token num‑ber (edszuwosnzevxit) is provided for review only.

Declarations
Ethics approval and consent to participate
All experiments were performed in accordance with relevant guidelines andregulations. Urine collection from healthy individuals have been performed inaccordance with the guidelines and regulations in Declaration of Helsinki andthe study have been approved by the ethics committee of Kunming Medi‑
cal University. The informed consent to participate in the study have beenobtained from all the participants.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Present Address: Institute of Neurological Disease, West China Hospital,
Sichuan University, No. 88 Keyuan South Road, Chengdu 610041, China.
2
Department of Human Anatomy and Tissue Embryology, Kunming Medical
University, Kunming 650500, China. 3 Laboratory Animal Department, InstitutePage 10 of 11

of Neuroscience, Kunming Medical University, Kunming 650500, China.
Department of Anatomy, Institute of Eyes, Jinzhou Medical University, No.40,
Section 3, Songpo Road, Linghe District, JinzhouLiaoning 121001, China.

4

Received: 25 March 2022 Accepted: 6 February 2023

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