Original Article

Hypoxic ucMSC-secreted exosomal miR-125b

promotes endothelial cell survival and migration
during wound healing by targeting TP53INP1
Xiao-Fei Zhang,1 Ting Wang,2 Zi-Xuan Wang,3 Kun-Peng Huang,3 Yun-Wei Zhang,4 Guo-Liang Wang,5
Hong-Ji Zhang,3 Zi-Han Chen,2 Chang-Yan Wang,2 Jin-Xiang Zhang,3 and Hui Wang2
1Center for Translational Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; 2Department of
Medical Genetics, Basic school of Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China; 3Department
of Emergency Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; 4Department of Emergency,
Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; 5Department of Hepatobiliary Surgery, Union
Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China

A hypoxic microenvironment is a common feature of skin tissue, and umbilical cords (UCs).4 Among the types of MSCs,
wounds. Our previous study demonstrated that three-dimen- umbilical cord MSCs (ucMSCs) are advantageous because their
sional coculture of umbilical cord-derived mesenchymal stem collection causes no risk or discomfort to the donor, and they
cells (ucMSCs) and endothelial cells facilitates cell communica- have a highly similar gene expression pattern to that of skin
tion and host integration in skin tissue engineering. Here, we fibroblasts;5,6 thus, ucMSCs have a substantial potential in skin
aimed to identify the mechanism by which ucMSCs affect endo- wound repair or tissue engineering. However, the clinical applica-
thelial cells under hypoxic conditions after skin injury. We tion of ucMSCs still faces several problems, such as the low cell
demonstrate that hypoxia enhances the exosome-mediated viability of transplanted cells and the risk of immune responses
paracrine function of ucMSCs, which increases endothelial in recipients.7
cell proliferation and migration. In a mouse full-thickness
skin injury model, ucMSC-derived exosomes can be taken up Exosomes (Exos) derived from MSCs exhibit similar functions to
by endothelial cells and accelerate wound healing. Hypoxic exo- MSCs, but have their own set of advantages, such as their anti-inflam-
somes lead to a better outcome than normoxic exosomes by matory, anti-apoptotic, and angiogenesis-promoting effects.8,9
promoting proliferation and inhibiting apoptosis. Mechanisti- Exosomes are lipid bilayer vesicles containing mRNAs, microRNAs
cally, microRNA-125b (miR-125b) transcription is induced by (miRs), lipids, and proteins.10,11 By delivering various RNAs and pro-
hypoxia in ucMSCs. After being packaged into hypoxic exo- teins to neighboring or distant cells, exosomes play an important role
somes and transported to endothelial cells, miR-125b targets in cell-cell communication and disease progression.10,11 The admin-
and suppresses the expression of tumor protein p53 inducible istration of exosomes is considered an attractive cell-free approach to
nuclear protein 1 (TP53INP1) and alleviates hypoxia-induced tissue repair and organ regeneration.12–14 However, the composition
cell apoptosis. Inhibition of miR-125b-TP53INP1 interaction of exosomes varies widely according to the parent cells from which
attenuates the protective effect of hypoxic exosomes. Moreover, they are derived and is profoundly affected by physiological and
artificial agomiR-125b can accelerate wound healing in vivo. pathological conditions, such as mechanical or metabolic stresses,
Our findings reveal communication between ucMSCs and hypoxia, or abnormal pH.15–17 During skin injury, a condition of
endothelial cells via exosomal miR-125b/TP53INP1 signaling hypoxia arises. The exosomes secreted by ucMSCs under hypoxic
in the hypoxic microenvironment and present hypoxic exo- conditions are significantly different from those secreted under nor-
somes as a promising therapeutic strategy to enhance cuta- moxic conditions, including the enrichment of specific types of
neous repair. miR-21 and miR-23a.18,19 However, whether hypoxic ucMSC-derived
exosomes have a special function in skin wound healing remains to be
further elucidated.
INTRODUCTION
Skin, an effective barrier that prevents body dehydration and
external microorganism penetration, is extremely vulnerable to Received 29 January 2021; accepted 17 July 2021;
https://doi.org/10.1016/j.omtn.2021.07.014.
different types of lesions, such as burns, ulcers, and wounds.
Correspondence: Hui Wang, Department of Medical Genetics, Basic school of
Stem cell-based skin tissue engineering has shown great promise
Tongji Medical College, Huazhong University of Science and Technology, 13
for wound healing.1–3 Mesenchymal stem cells (MSCs) exist Hangkong Road, Wuhan 430030, China.
in many adult tissues, such as the bone marrow, adipose E-mail: [email protected]

Molecular Therapy: Nucleic Acids Vol. 26 December 2021 ª 2021 The Authors. 347
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Endothelial cells play a vital role in skin repair. The locally hypoxic with GW4869 (10 mM, an inhibitor of exosome biogenesis and
environment in wounds enhances the proliferation and migration release) for 48 h. The culture media were replaced by fresh complete
of endothelial cells to induce revascularization.20,21 We previously co- media, and the cells were exposed to hypoxic conditions for 12 h.
cultured ucMSCs and endothelial cells in a three-dimensional gelatin Then, the supernatants were harvested to treat HUVECs. The
methacryloyl hydrogel to construct engineered skin analogs. We CCK-8 assay showed that GW4869 pretreatment significantly
found that three-dimensional coculture of the two types of cells mark- blocked the promoting effect of ucMSCs on the viabilities of HUVECs
edly enhanced the gene expression levels of CHD1, FGF2, and VEGFA (Figure 1D), demonstrating that exosomes are an essential mediator
in vitro and promoted host integration in vivo when compared with of ucMSC functions. Then, ucMSCs were cultured for 72 h under nor-
culture of ucMSCs or endothelial cells alone; these results suggested moxic and hypoxic conditions, and normoxic and hypoxic exosomes
that cell-cell communication might be beneficial to angiogenesis were isolated from the culture media by ultracentrifugation. The par-
and tissue regeneration.22 Given that hydrogels also create a hypoxic ticle size and zeta potential of the normoxic and hypoxic exosomes
environment for cells, we postulated that the communication between were assessed by a particle detector. In good agreement with the range
ucMSCs and endothelial cells under hypoxic conditions might be a of standard values of exosomes, the average diameters of the two types
favorable factor for skin repair. of exosomes were within 104.2~118.5 nm (Figure 1E), and the average
zeta potential value ranged from
3.9~
6.9 mV (Figure 1F). Under
Here, we found that hypoxia enhanced the paracrine effect of ucMSCs transmission electron microscopy, the two types of exosomes dis-
on endothelial cell proliferation and migration in vitro, and the played representative cup-shaped morphology (Figure 1G), which
impact of ucMSCs on endothelial cells was mediated by exosomes. was consistent with previous reports.25,26 Known exosomal markers,
Mechanistically, miR-125b enriched in hypoxic exosomes was a key including CD9, CD63, CD81, and HSP70, were also expressed in both
molecule that promoted the survival and migration of endothelial types of exosomes (Figure 1H). Then, exosomes labeled with 3,30 -di-
cells. Our study further reveals that exosomal-miR-125b/TP53INP1 octadecyloxacarbocyanine perchlorate (DIOC18(3), a green fluores-
signaling is crucial for the communication of ucMSCs and endothelial cent lipophilic tracer) were used to treat HUVECs. The images
cells, which provides a therapeutic target for exosome-based cell-free showed that HUVECs indeed took up the exosomes, which were
skin tissue engineering. mainly distributed in the perinuclear region of HUVECs (Figure 1I).
Then, exosomes were added to the culture media of HUVECs
RESULTS to determine their effect on the function of endothelial cells. As Fig-
ucMSC-derived exosomes mediate the paracrine effect of ures 1J and 1K show, hypoxic exosomes induced higher endothelial
ucMSCs on the cell proliferation and migration of endothelial cell viabilities and migratory activities than normoxic exosomes.
cells under hypoxic conditions These data indicated that exosomes derived from ucMSCs mediated
To investigate the role of hypoxia in the interaction of ucMSCs and the paracrine effect of ucMSCs on HUVECs, and the hypoxic micro-
endothelial cells, we treated human umbilical vein endothelial cells environment in local wounds might create a favorable context for the
(HUVECs) with the supernatants from ucMSCs cultured under function of ucMSCs.
normoxic or hypoxic conditions. The results from the Cell Counting
Kit-8 (CCK-8) assay showed that both the normoxic and hypoxic su- Hypoxic exosomes lead to a better outcome of regenerated skin
pernatants of ucMSCs increased the viability of HUVECs, and the by enhancing cell proliferation and inhibiting cell apoptosis
hypoxic supernatants resulted in higher cell viability than the nor- compared to normoxic exosomes
moxic supernatants (Figure 1A). The migration of endothelial cells A mouse full-thickness cutaneous injury model was established to
plays an important role in tissue repair. To study the effect of the hyp- verify the effect of normoxic or hypoxic exosomes on wound healing.
oxic supernatants of ucMSCs on HUVEC migration, we performed The wounds were photographed, and wound healing curves were
wound healing and Transwell migration assays. The wound-healing generated. As shown in Figure 2A, the wound areas in both normoxic
assay demonstrated that the hypoxic supernatants of ucMSCs and hypoxic exosome-treated groups were smaller than those in the
increased cell migration, as indicated by the significantly smaller PBS-treated group at day 3 and day 8, and the wounds of the three
wound area in the hypoxic group than in the normoxic group at groups almost healed at day 12. Statistical analysis showed the faster
16 h (Figure 1B). Then, an indirect coculture model using a Transwell healing rates of the normoxic and hypoxic exosome-treated groups
system was established (Figure 1C). The HUVECs exposed to the than of the PBS group, while the healing rate of the hypoxic exo-
hypoxic supernatants of ucMSCs displayed the greatest migratory some-treated group was very close to that of the normoxic exo-
ability among the three groups of cells (Figure 1C). These data indi- some-treated group (Figure 2B). With an in vivo tracking assay, we
cated that hypoxia promoted the paracrine effect of ucMSCs on the observed a remarkable colocalization of exosomes (DIOC18(3),
proliferation and migration of endothelial cells. green) and endothelial cells (von Willebrand factor, red) at the areas
adjacent to the injury sites, which confirmed the uptake of exosomes
Exosomes, as major paracrine components of mesenchymal stem by endothelial cells (Figure 2C). Hematoxylin and eosin (H&E) stain-
cells, mediate intercellular communication in various physiological ing also showed that there were no statistically significant differences
or pathological processes.23–25 To further explore the role of exo- in the thickness of the epidermis among normoxic exosome-, hypoxic
somes in the paracrine function of ucMSCs, we pretreated ucMSCs exosome-, and PBS-treated groups; however, the epidermis in both

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Figure 1. ucMSC-derived exosomes induce proliferation and migration of endothelial cells under hypoxic conditions
HUVECs were treated with NC (complete media without serum), N-S (the supernatants of ucMSCs cultured under normoxic conditions without serum), and H-S (the
supernatants of ucMSCs cultured under hypoxic conditions without serum) for the indicated times. (A) CCK-8 analysis of cell proliferation of HUVECs. *p < 0.05, N-S group
versus H-S group. (B and C) Wound-healing assay (B) and Transwell assay (C) of the migratory activities of HUVECs. Scale bar, 200 mm; *p < 0.05. (D) ucMSCs were
pretreated with GW4869 (10 mM) for 48 h. The culture media were replaced with fresh complete media, and the cells were exposed to hypoxia for 12 h. Then, the
supernatants were used to treat HUVECs. CCK-8 analysis of cell proliferation of HUVECs. *p < 0.05, **p < 0.01. (E and F) Particle size distribution (E) and zeta potential (F) of
normoxic exosomes (N-Exos) or hypoxic exosomes (H-Exos). (G) Morphology of normoxic and hypoxic exosomes photographed by transmission electron microscopy. Scale
bar, 200 nm. (H) Western blotting of the expression of the surface markers CD9, CD63, CD81, and HSP70 in equal amounts of normoxic and hypoxic exosomes. (I) The
internalization of DIOC18(3)-labeled exosomes by HUVECs. Scale bar, 50 mm. (J) CCK-8 assay of HUVECs treated with normoxic or hypoxic exosomes. *p < 0.05 versus
PBS treated group, #p < 0.05 versus normoxic exosome group. (K) Transwell migration assay of HUVECs treated with normoxic or hypoxic exosomes. Scale bar, 200 mm;
*p < 0.05. The data are presented as the mean ± SD.

the normoxic (26.4 ± 2.9 mm) and hypoxic (27.3 ± 9.8 mm) exosome- evaluate cell proliferation and apoptosis (Figure 2D). The exosome in-
treated groups was slightly thinner than that in the PBS group (30.4 ± jection groups had significantly more Ki67-positive cells and fewer
4.5 mm) (Figure 2D). Ki67 and TUNEL staining were performed to TUNEL-positive cells than the PBS control group. Interestingly, we

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Figure 2. Hypoxic exosomes improve the outcome of wound healing in a mouse full-thickness skin injury model
(A) Gross view of wound areas of mice injected with normoxic or hypoxic exosomes. Scale bar, 5 mm. Line segments with arrows represent the diameters of wound areas.
The wound areas at D0, D3, and D8 are outlined with a circular dotted box, and with a square dotted box at D12 due to the difficulty in defining the border. (B) Quantification of
wound closure rates. n = 5. *p < 0.05, normoxic exosome-treated group versus PBS-treated group; #p < 0.05, hypoxic exosome-treated group versus PBS-treated group.
(C) In vivo uptake of DIOC18(3)-labeled exosomes (GFP) by HUVECs (von Willebrand factor, red). Scale bar, 10 mm. (D) Representative images of H&E staining, Ki67 staining,
and TUNEL staining, and quantitative analysis of the thickness of newly formed epidermis, the number of Ki67-positive cells, and the number of TUNEL-positive cells at the
end point of wound repair (day 12). Scale bar, 400 mm in H&E staining and 50 mm in Ki67 and TUNEL staining. ns, no significance. *p < 0.05. The data are presented as the
mean ± SD.

observed a significantly increased number of Ki67-positive cells and a the abundance of microRNAs in exosomes was important for their
decreased number of TUNEL-positive cells in the hypoxic exosome- functions, so we confirmed the expression of these microRNAs and
treated group compared to the normoxic exosome-treated group analyzed their differential expression between hypoxic exosomes
(Figure 2D), suggesting a better outcome of hypoxic exosome and normoxic exosomes from ucMSCs by quantitative PCR. The
treatment. expression of miR-125b was found to be most notably increased in
hypoxic exosomes compared with normoxic exosomes (Figure 3A).
ucMSC-derived exosomal miR-125b, transcriptionally induced To explore the correlation of the miR-125b levels in exosomes and
by hypoxia, modulates the behaviors of endothelial cells their source ucMSCs, we assessed the expression of miR-125b in
Exosomes always carry and deliver mRNAs, microRNAs, and pro- ucMSCs exposed to hypoxic conditions, including a tri-gas culture
teins to target cells, and the levels of exosome contents are strongly for 3 h and 6 h or stimulation with cobalt chloride (CoCl2) to create
correlated with their levels in the cells of origin.27,28 Hypoxic condi- a chemical hypoxia model. We observed an upregulation of miR-125b
tions have been implicated in modulating the expression profile of expression in ucMSCs cultured under hypoxic conditions (Figures 3B
microRNAs in different types of cells as well as in the exosomes and 3C). A luciferase assay also confirmed increased reporter
derived from these cells.29–31 A previous study investigated the exoso- activity of the miR-125b promoter under hypoxic conditions (Fig-
mal microRNA expression profile in ucMSCs and screened several ure 3D). These data suggested that the accumulation of miR-125b
abundant microRNAs, including miR-21-5p, miR-125b-5p, miR- in exosomes was largely due to its upregulation in ucMSCs under
23a-3p, let-7f-5p, let-7a-5p, and miR-145-5p.18 We speculated that hypoxic conditions.

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Figure 3. Hypoxic exosomal miR-125b promotes cell proliferation, migration, and survival of endothelial cells under hypoxic conditions
(A) The RNA levels of let-7f-5p, miR-21-5p, miR-23a-3p, miR-125b-5p, miR-145-5p, and let-7a-5p in H-Exos or N-Exos from ucMSCs. **p < 0.01, ****p < 0.0001. ns, no
significance. (B and C) miR-125b levels in ucMSCs placed into three gas incubators and exposed to hypoxia for 1 h and 3 h (B) or 50 mM CoCl2 treatment for 8 h (C). **p <
0.01, ***p < 0.001. (D) Luciferase assay to analyze the promoter activity of miR-125b in ucMSCs exposed to 50 mM CoCl2 for 8 h. *p < 0.05. (E) The expression of mature miR-
125b in HUVECs treated with DRB (20 mM, 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole, an RNA polymerase II inhibitor) for 48 h and H-Exos or N-Exos for another 48 h.
**p < 0.01, ***p < 0.001. (F) CCK-8 analysis of HUVEC proliferation following the indicated treatment. *p < 0.05. (G) Representative images (left panel) and quantitative analysis
(right panel) of Transwell migration assays of HUVECs after different treatments. Scale bar, 200 mm; *p < 0.05. (H) Flow cytometry analysis of the apoptosis of HUVECs treated
with the indicated exosomes or oligos (upper panel). Quantitative analysis of flow cytometry data (lower panel). #p < 0.05. The data are presented as the mean ± SD.

To further confirm whether exosomal miR-125b could be taken up the HUVECs. DRB treatment markedly decreased the endogenous
by endothelial cells, we cocultured normoxic exosomes or hypoxic expression of mature miR-125b in HUVECs; exosome addition
exosomes with HUVECs for 48 h, and the HUVECs were obviously improved the level of mature miR-125b, indicating
pretreated with 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole that the increase in miR-125b expression in HUVECs was due
(DRB, an RNA polymerase II inhibitor, 20 mM) for the previous to their uptake from exosomes rather than the enhanced transcrip-
48 h. Then, we detected the expression of mature miR-125b in tion of miR-125b in HUVECs. Consistent with the dramatic

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Figure 4. Exosomal miR-125b targets TP53INP1 in endothelial cells

(A) The predicted targets of miR-125b in four databases. (B) DAVID analysis of 30 common targets of miR-125b. (C) The binding site between hsa-miR-125b and TP53INP1.
(D) The reporter activity of the 30 untranslated region of TP53INP1 in HUVECs transfected with miR-125b mimics or control oligos. **p < 0.01. (E and F) The mRNA and protein

(legend continued on next page)

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increase in miR-125b expression in hypoxic exosomes, as shown in To further study the interaction of miR-125b and TP53INP1, we con-
Figure 3A, the level of mature miR-125b expression in hypoxic structed stable TP53INP1-overexpressing or TP53INP1-knockdown
exosome-treated HUVECs was much higher than that in normoxic HUVECs using a lentivirus delivery system (Figure 4G). Then, we
exosome-treated HUVECs. The above data, together with the cyto- verified the role of the interaction of miR-125b and TP53INP1 in
plasmic localization of exosomes in endothelial cells (Figure 2C), the process of endothelial cell apoptosis by determining the protein
further demonstrated much more uptake by HUVECs of miR- levels of TP53INP1 and cell apoptotic markers, including caspase-3,
125b from hypoxic exosomes than of miR-125b from normoxic B cell lymphoma 2-associated X protein (Bax), and B cell lymphoma
exosomes (Figure 3E). 2 (Bcl2). As Figure 4H demonstrates, overexpression of miR-125b
decreased the levels of cleaved caspase-3 and Bax and increased the
To further address whether the function of exosomes depends on level of Bcl2 in HUVECs treated with high concentrations of CoCl2.
miR-125b, we treated HUVECs with hypoxic exosomes with or Cotransfection of TP53INP1 without the 30 untranslated region
without miR-125b inhibitors. As CCK-8 (Figure 3F) and migration reversed the effect of miR-125b overexpression on the above protein
assays (Figure 3G) showed, inhibition of miR-125b blocked the pro- levels. Moreover, inhibition of miR-125b upregulated cleaved
moting effect of hypoxic exosomes on cell growth and migration. caspase 3, and Bax expression and downregulated Bcl2 expression,
Consistent with the in vivo study results (Figures 2F and 2G), hypoxic whereas cotransfection of shTP53INP1 impaired the promotion effect
exosome treatment significantly decreased the cell apoptosis induced of miR-125b inhibitors on cell apoptosis, indicating that the function
by high concentrations of CoCl2, while inhibition of miR-125b of miR-125b during hypoxia was due, at least in part, to the downre-
abrogated the protective effect of hypoxic exosomes on cell apoptosis gulation of TP53INP1 expression (Figure 4H).
(Figure 3H). Taken together, we demonstrated that ucMSC-derived
exosomal miR-125b, whose transcription was induced by hypoxic To identify whether the protective effect of hypoxic exosomes on
conditions, was necessary for cell migration and survival of endothe- endothelial cells was dependent on TP53INP1 downregulation, we
lial cells. analyzed the mRNA and protein levels of TP53INP1 following exo-
some treatment. Hypoxic exosome treatment reduced the level of
The interaction of miR-125b and TP53INP1 is crucial for the TP53INP1, while inhibition of miR-125b abrogated this suppressive
function of hypoxic exosomes effect under hypoxic conditions (Figure 4I). Then, cell growth, migra-
MicroRNAs participate in diverse cellular signaling pathways by tion, and survival were assessed under both normoxic and hypoxic
regulating their targets. To examine the functional mechanism of exo- conditions. As CCK-8 assays showed (Figure 4J), TP53INP1 overex-
somal miR-125b in endothelial cells, we analyzed the targets of miR- pression led to lower cell viability than the corresponding control.
125b using online databases, including TargetScan (http://www. Hypoxic exosome treatment improved cell viability, whereas the pro-
targetscan.org/vert_72/), miRTarBase (https://mirtarbase.cuhk.edu. tective effect was reversed by TP53INP1 overexpression (Figure 4J). A
cn/miRTarBase/miRTarBase_2019/php/download.php), miRDB similar phenomenon was observed in the migration assay; hypoxic
(http://mirdb.org/), and PicTar (https://pictar.mdc-berlin.de/), and exosomes increased the number of migrated cells, whereas TP53INP1
screened 30 common targets (Figure 4A). DAVID analysis showed overexpression inhibited the promoting effect of hypoxic exosomes
that these targets were enriched in the cell apoptotic process (p = following hypoxia stimulation (Figure 4K). Apoptosis of TP53INP1-
0.0018), and tumor protein p53 inducible nuclear protein 1 overexpressing endothelial cells exposed to hypoxia was also detected.
(TP53INP1) was an interesting target mRNA of them (Figures 4B As expected, we observed a higher cell apoptotic rate in hypoxic
and 4C). Luciferase reporter assay confirmed the direct binding of exosome-treated TP53INP1-overexpressing cells than in hypoxic exo-
miR-125b to the 30 untranslated region of wild-type TP53INP1 (Fig- some-treated control cells (Figure 4L). The data described above
ure 4D). Then the expression of TP53INP1 was detected by quantita- further illustrated that exosomal miR-125b targeted TP53INP1
tive PCR and western blotting. The mRNA and protein levels of mRNA in endothelial cells and that the interaction of miR-125b
TP53INP1 were obviously downregulated in endothelial cells by and TP53INP1 was crucial for the function of exosomes.
exposure to hypoxia (Figures 4E and 4F), indicating the involvement
of TP53INP1 in hypoxia-induced cell damage. miR-125b overexpres- Artificial miR-125b promotes cutaneous wound healing
sion suppressed the level of TP53INP1 under both normoxic and To further determine the function of miR-125b in skin wound
hypoxic conditions, and vice versa, suggesting that miR-125b repair in vivo, we injected agomiR-125b or agomiR control oligos
increased the mRNA degradation of TP53INP1 (Figures 4E and 4F). into the areas adjacent to skin injury sites by subcutaneous injection.

levels of TP53INP1 in HUVECs transfected with the indicated oligos under normoxic or hypoxic (200 mM CoCl2 for 48 h) conditions. *p < 0.05, **p < 0.01, ***p < 0.001. (G) The
mRNA and protein levels of TP53INP1 in HUVECs stably transfected with the indicated plasmids. **p < 0.01, ***p < 0.001. (H) Western blot analysis of the protein levels of
TP53INP1, total caspase 3, cleaved caspase-3, Bax, and Bcl2 in HUVECs stably transfected with the indicated plasmids. (I) The mRNA and protein levels of TP53INP1 in
HUVECs transfected with the indicated exosomes or oligos under normoxic or hypoxic (100 mM CoCl2 for 48 h) conditions. *p < 0.05, **p < 0.01. (J) CCK-8 analysis of HUVEC
proliferation following the indicated treatments. *p < 0.05, **p < 0.01. (K) Representative images (left panel) and quantitative analysis (right panel) of Transwell migration assays
of HUVECs after different treatments. Scale bar, 200 mm; *p < 0.05. (L) Flow cytometry analysis of the apoptosis of HUVECs after different treatments (upper panel).
Quantitative analysis of flow cytometry data (lower panel). *p < 0.05, **p < 0.01. The data are presented as the mean ± SD.

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Figure 5. AgomiR-125b enhances cutaneous wound healing

(A) Gross view of wound areas of mice injected with agomiR control or agomiR-125b. Scale bar, 5 mm. Line segments with arrows represent the diameters of wound areas.
The wound areas at D0, D3, and D8 are outlined with a circular dotted box, and with a square dotted box at D12 due to the difficulty in defining the border. (B) Quantification of
wound-closure rates. *p < 0.05, **p < 0.01 versus agomiR control group. n = 5. (C) Representative images of H&E staining, Ki67 staining, TUNEL staining, and TP53INP1
staining of wound sections at day 12 post wounding (left panel). The right panel shows, from top to bottom, the thickness of the newly formed epidermis, the number of Ki67-
positive cells, the number of TUNEL-positive cells, and the number of TP53INP1-positive cells. Scale bar, 400 mm in H&E staining and 50 mm in Ki67 and TUNEL staining.
ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001. (D) The RNA levels of miR-125b and Tp53inp1 in wound areas of mice injected with the indicated exosomes or oligos at
day 12 post wounding. *p < 0.05. The data are presented as the mean ± SD.

Compared with the agomiR control group, agomirR-125b led to an Tp53inp1 expression was lower in the exosome- or agomiR-125b-in-
apparent decrease in wounds at day 3 and the following days (Fig- jected groups (Figure 5D, right). All these data further defined the
ure 5A). The wound closure rate of the agomiR-125b group was interaction of miR-125b and TP53INP1 during exosome-mediated
higher than that of the agomiR control group, as shown in Figure 5B. skin tissue repair.
AgomiR-125b injection led to a slight increase in the thickness of
the newly formed epidermis of 49.8 ± 12.3 mm compared with DISCUSSION
that of the agomiR control group of 40.7 ± 9.9 mm, but the differ- Numerous studies have revealed the beneficial effect of MSCs in the
ence was not statistically significant (Figure 5C). The number of repair of various tissues or organs; however, clinical trials of the uti-
Ki67-positive cells increased and the number of TUNEL-positive lization of MSCs in patients remain cautious due to the low survival
cells decreased following agomiR-125b treatment (Figure 5C). The and possible inflammatory responses elicited by MSCs.32–34 Exo-
expression of TP53INP1 was also tested, and we observed a reduc- somes secreted by MSCs play a significant role in both intercellular
tion in TP53INP1 levels in the agomiR-125b group (Figure 5C). communications and interactions with cellular microenvironments.
Then, the RNA levels of miR-125b and Tp53inp1 in the wound sites Evidence has also accumulated that exosomes can be used as effective
of exosome- or oligo-treated animals at the end point of injury nanocarriers in cell-free-based tissue regeneration; they could modu-
repair were detected by quantitative PCR, and the results showed late inflammation and promote proliferation and angiogenesis,
that the expression of miR-125b was increased following exosome similar to their source cells.35–38 Nevertheless, although the current
injection (Figure 5D, left). Although there was no significant differ- results of exosomes are encouraging, additional research is necessary
ence in the mRNA levels of Tp53inp1 among the five groups, to optimize the cell source of exosomes, to optimize the culture

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condition of MSCs, and to understand the mechanism by which of chemicals should be addressed. Although the mechanisms of the
exosomes function in various microenvironments. two hypoxic conditions were different, we observed a significant in-
duction of miR-125b expression under both conditions. The tri-gas
In the present study, ucMSCs were chosen due to their similar gene incubator sustainably increased miR-125b expression over time,
expression to that of skin fibroblasts and their pro-keratinocyte differ- while a low concentration of CoCl2 (50 mM) induced higher expres-
entiation potential.5,6 Both normoxic and hypoxic exosomes derived sion of miR-125b than a high concentration of CoCl2 (100 mM).
from ucMSCs increased the behaviors of endothelial cells and facilitated The effect of hypoxic exosomes under different hypoxic conditions
wound healing, suggesting that ucMSCs could serve as a good cell source was not explored in our present study. CoCl2 might be more suited
for exosomes in skin regeneration. We have reported that ucMSCs could for large-scale cell culture to produce more exosomes due to its
communicate with endothelial cells and enhance the function of gelatin advantages of low cost, ease of use, and better control of the variable
methacryloyl hydrogel-based engineered skin.22 Here, we found that the degrees of hypoxia. Collectively, further research is needed to opti-
protective paracrine effect of ucMSCs on endothelial cells was mainly mize the hypoxic conditions and compare the functions of hypoxic
mediated by exosomes, as indicated by the loss of function of ucMSCs exosomes under different hypoxic conditions.
after GW4869 pretreatment. In vivo experiments also demonstrated
that exosomes derived from ucMSCs indeed facilitated wound healing, Multiple studies have shown that exosomes communicate with target
which is consistent with previous reports.18,19 cells by delivering certain microRNAs. An elegant study previously
analyzed the microRNA expression profiles of exosomes derived
Several reports have demonstrated that hypoxic exosomes enhance from ucMSCs or human embryonic kidney 293T cells, characterizing
cell proliferation and angiogenesis during myocardial infarction or miR-21, miR-125b-5p, miR-23a-3p, miR-100-5p, let-7f-5p, let-7a-5p,
tumor development.31,40,41 In the present study, we found that hypox- and miR-145-5p as the first seven microRNAs with high abundances
ic exosomes enhanced endothelial cell proliferation and migration of 12.5%, 10.2%, 4.9%, 3.4%, 3.2%, 2.5%, and 2%, respectively.18 We
in vitro, improving the quality of regenerated skin and conferring a speculated that the abundance of microRNAs in exosomes was
higher proliferation rate and lower apoptosis rate, although their ef- important for their functions, and therefore we chose the most highly
fect on the wound closure rate was comparable to that of normoxic abundant microRNAs and analyzed their levels in exosomes from
exosomes. However, there are several limitations to the study. First, ucMSCs cultured under normoxic and hypoxic conditions. We found
our present study only focused on the communication of exosomes a marked increase in the miR-125b levels in hypoxic exosomes from
with endothelial cells. In fact, Ki67 or TUNEL staining differences ucMSCs. miR-125b has also been reported to be increased in bone-
in other skin cell types, including basal cells and fibroblasts, were pre- marrow derived mesenchymal stem cell-derived exosomes after expo-
sent in the PBS-treated group and exosome-treated groups, suggest- sure to hypoxia,31 which suggests that hypoxia-induced levels of miR-
ing a direct or indirect interaction of these cells with exosomes, which 125b in exosomes might be a common phenomenon in MSCs.
should be addressed in our further study. In addition, we observed
that there was no statistical difference in the epidermal thicknesses The levels of microRNAs in exosomes were intensively correlated
among normoxic exosome-, hypoxic exosome-, or PBS-treated with their expression in their cells of origin.27,28 We compared the
groups, which suggested that the impact of exosomes on epidermal expression of miR-125b in ucMSCs and 293T cells and observed
remodeling is not prominent. Indeed, the thickness of the newly that the miR-125b level was at least 10 times greater in ucMSCs
formed epidermis is the comprehensive effect of exosomes on the spe- than that in 293T cells in a quiescent state; exposure to hypoxia
cific epidermal cells and proteins, especially keratinocytes and kera- further induced miR-125b expression in both ucMSCs and 293T cells
tins, which were arranged in an orderly fashion to form the structure (data not shown). These results support the above conclusion, but the
of epidermis. Our study is limited to the protective effect of exosomes primary factors regulating the sorting of miR-125b or other micro-
on endothelial cells. In future studies, the impact of exosomes on the RNAs to exosomes following hypoxia stimulus require further study.
organization of keratinocytes and keratins should be further clarified. It is also necessary to characterize the function of exosomes from
In addition, it is possible that day 12 is not an optimal time to observe different cell sources under various physiological conditions, as well
the thickness of regenerated epidermis in our study. Second, we only as the underlying mechanisms, to provide a basis for their clinical
evaluated the effect of the two types of exosomes at day 12, after heal- application.
ing was almost complete, and their effects on the phases of inflamma-
tion and reepithelialization occurring at the early stages of wound TP53INP1 is a protein with multiple functions and is involved in cell
repair require further study. It is noteworthy that wound healing in cycle arrest and apoptosis. In the present study, we showed that hyp-
a mouse is fundamentally different from that of humans, as it primar- oxia profoundly suppressed TP53INP1 expression, suggesting the
ily occurs via contraction.42 A splint incorporated around the wounds involvement of TP53INP1 in hypoxia-induced cell death. TP53INP1
of mice or a rat full-thickness skin injury model could better simulate is a known target of miR-125b in various diseases or cell processes,
the conditions of human skin repair.42 such as ischemia-reperfusion-induced neuroinflammation and endo-
metrial cancer cell migration.43,44 However, whether this interaction
Physical (tri-gas incubator) and chemical (CoCl2) hypoxia models exists between exosomal miR-125b and TP53INP1 in targeted cells
were used in our study. The duration of stimulation or concentration remains to be determined. We verified that the induction of cell

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 Molecular Therapy: Nucleic Acids

proliferation and migration and the inhibition of cell apoptosis by with H&E. For each mouse, epidermal thickness of regenerated skin
exosomal miR-125b were dependent on its interaction with flanked by the normal skin with hair follicles was measured from stra-
TP53INP1, further indicating that TP53INP1 was an important target tum basal to stratum corneum at six equidistant sites. Immunofluo-
of miR-125b. The RNA level of miR-125b was increased at exosome- rescence staining for Ki67 (Servicebio, #GB111141, Wuhan, China)
or agomiR-125b-injected wound sites in mice, whereas Tp53inp1 and TP53INP1 (ABclonal, #A8274) were performed to evaluate cell
expression was reduced in these groups, further indicating an interac- proliferation and the level of TP53INP1, respectively. TUNEL (termi-
tion of miR-125b and TP53INP1. Overall, the miR-125b-TP53INP1 nal deoxynucleotidyl transferase dUTP nick end labeling) assays were
interaction was crucial for the function of endothelial cells. performed to detect cell apoptosis according to the manufacturer’s in-
structions (Servicebio, #G1507, Wuhan, China).
Notably, we observed that inhibition of miR-125b expression did not
entirely eliminate the protective effect of hypoxic exosomes in in vitro Exosome isolation and identification
experiments. Our in vivo study also showed that the level of miR-125b ucMSCs (Cyagen, Guangdong, China) were cultured to a density of
in wound sites injected with exosomes was considerably higher than 2  107 cells/75 mm flask. Culture media were removed, and cells
in those injected with agomiR-125b at day 12, which might be caused were washed three times with PBS and then cultured in serum-free
by factors such as (1) the inequality of miR-125b contents in media for 48 h. The media were collected, and cell debris was
exosomes (100 ng) and agomiR-125b (2 nM); (2) other protective discarded by gradient centrifugation at 300  g for 10 min and
components of exosomes, such as miR-21 and miR-23a;18 or (3) bet- 10,000  g for 30 min at 4 C. Further centrifugation (100,000  g,
ter stability of exosomes in vivo. The results suggested a better effect of 4 C, 70 min) was performed to obtain exosomes. The size distribution
exosomes than agomiR oligos. In our further research, exosomes or and zeta potential of exosomes was measured by Zetasizer Nano ZS90
oligos encapsulated in hydrogels will be used in a cutaneous repair (Malvern Panalytical, Westborough, MA, USA). The morphologies of
model to compare their different therapeutic effects. exosomes were photographed with transmission electron microscopy
(Hitachi HT 7800, Tokyo, Japan). The exosomes were measured for
In conclusion, we demonstrated that hypoxic exosomes from ucMSCs their protein content using the BCA protein assay kit (Pierce Protein
enhanced skin wound repair by promoting cell growth and migration Biology, Thermo Fisher Scientific Life Sciences). For labeling, exo-
and decreasing cell apoptosis. Mechanistically, miR-125b, whose somes were incubated with DIOC18(3), a green fluorescent lipophilic
transcription was induced by hypoxia, was sorted into exosomes in tracer (Beyotime), at room temperature for 30 min and centrifuged at
ucMSCs. After internalization by endothelial cells, miR-125b targeted 100,000  g, 4 C, for 70 min, incubated with HUVECs in normoxic
the 30 untranslated region of TP53INP1 mRNA and suppressed and hypoxic conditions for 3 h, and then photographed.
TP53INP1-mediated cell apoptosis, which finally led to cell survival
and subsequent wound healing.
In vivo exosome tracking assay
MATERIALS AND METHODS To assess exosome uptake by skin endothelial cells, 20 mL
Animals DIOC18(3)-labeled exosomes (1 mg/mL) were injected into the
All experimental procedures involving animals were approved by the around areas of wounded skin. Mice were sacrificed at 5 h after
Animal Care and Use Committee of Wuhan Union Hospital. All mice injection. The skin was dehydrated and frozen, then sliced into
were housed in an environment with controlled light (12 h light/12 h 10-mm-thick cryosections. The sections were incubated with primary
dark), temperature (23 C ± 2 C), and humidity. 8-week-old male antibody against von Willebrand factor (Servicebio, #GB11020,
BALB/C mice were used. For the full-thickness skin wound model, Wuhan, China) overnight at 4 C and then dyed with DAPI. The
mice were anesthetized by intraperitoneal (i.p.) administration of internalization of exosomes into skin endothelial cells was observed
50 mg/kg pentobarbital sodium. A skin wound 8 mm in diameter by confocal microscope.
was created on the dorsum. In total, 25 mice were randomly divided
into control group (PBS), normoxic or hypoxic exosome groups Cell culture
(200 mg in 100 mL PBS), and agomiR-125b or agomiR-control groups, ucMSCs and HUVECs were cultured in specific complete media
with 5 mice in each group. The above exosomes or oligos were subcu- (Cyagen, Guangdong, China). 293T were cultured in DMEM (Gibco)
taneously injected at four points around the wound sites. The wounds supplemented with 10% fetal bovine serum (FBS) and 100 U/mL
were photographed every day and the wound size was analyzed by penicillin-streptomycin (Gibco). For hypoxic treatment, cells were
ImageJ software. 12 days after injection, mice were sacrificed, and exposed to 1% O2, 95% N2, and 5% CO2 for 3–6 h in a tri-gas incu-
skin samples were harvested for further analysis. The wound-healing bator. When required, cells were treated with a high concentration
rate on a specific day (day X) was the ratio of the wound area differ- of CoCl2 (200 mM) for 24 h to induce cell growth arrest and apoptosis,
ences between day 0 and day X to wound area at day 0. while low concentration of CoCl2 with 50 mM was used to induce the
production of exosomes The optimal concentration of CoCl2 (50 mM)
Histology staining and TUNEL staining was determined by dose-dependent (using 25–200 mM) experiments
Skin samples were fixed in 4% paraformaldehyde, dehydrated, to produce maximal induction of HIF1a and miR-125b at 12 h,
embedded in paraffin, sectioned into 5-mm-thick pieces, and stained without affecting cell viabilities (data not shown). For coculture of

356 Molecular Therapy: Nucleic Acids Vol. 26 December 2021

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exosomes and cells, 50 mg/mL of exosomes were incubated with For stable cell line construction, HEK293T cells were transfected
HUVECs at 37 C for 24 h. with overexpression or knockdown plasmids with packaging plas-
mids pSPAX2 and pMD2G for 60 h. The supernatants were har-
Cell viability assay vested, filtered with 0.22 mm membrane, and added into pre-seeded
CCK-8 assays were performed to analyze cellular proliferation and HUVECs in a 6-well plate. Puromycin (2 mg/mL) was utilized to
activity. HUVECs were seeded at a density of 2  103 cells/well in screen stable expression clones.
96-well plates. At 24, 48, 72, 96, and 120 h, 10 mL of CCK-8 solution
(Dojindo Laboratories, Kumamoto, Japan) was added to each well of Luciferase reporter assay
the plate and incubated for 1 h. Cell viability was then determined us- 293T cells were seeded in 24-well plates at a density of 2  105
ing a spectrophotometer set (ELx800, BioTek, Winooski, VT, USA) at cells/well and transfected with the indicated plasmids for 24 h. Lucif-
a wavelength of 450 nm. erase assays were performed using a dual-luciferase assay kit (#E1960,
Promega, Madison, WI, USA) according to the manufacturer’s
Wound-healing assay instructions.
HUVEC migrations were performed in 12-well plates by wound-
healing assays in vitro. Cells were seeded at a density of 5  105 cells Quantitative PCR
per well and starved for 24 h. Wounds were made by scratching with Total RNA was extracted using TRIzol reagent (#9109, TaKaRa,
a 200 mL pipette tip (3 wounds per well). The cell debris was Japan). cDNA was obtained by using an oligo (dT) primer and reverse
removed with PBS washing. Then cells were incubated with super- transcriptase (Thermo Fisher Scientific, Waltham, MA, USA)
natants from ucMSCs treated with or without hypoxic treatment. following standard protocols. MicroRNA was reverse transcribed by
The scratch of each group was observed using a microscope at 0 h using specific Bulge-Loop RT primers (RiboBio, Guangzhou, China).
and 24 h. The area of the scratch was measured using ImageJ The relative expression levels of mRNA and microRNA were normal-
software. ized to GAPDH or U6, respectively.

Transwell migration assay Western blot

For Transwell assay, 3  104 cells were suspended in serum-free me- Cells were lysed in RIPA lysis buffer (#P00138, Beyotime Biotech-
dium and seeded into the upper chamber of Transwell 24-well plates nology, Shanghai, China). Equal amounts of protein were separated
(8 mm pore filters, Corning, Corning, NY, USA). Complete medium by SDS-PAGE gels and transferred to polyvinylidene fluoride
with or without exosomes was added to the lower chamber. After (PVDF) membrane. Antibodies against CD9 (#A19027), CD63
16 h, the cells on the upper surface of the inserts were wiped. Migrated (#A5271), CD81 (#A5270), and HSP70 (#A0284) were obtained
cells of the lower surface were stained with crystal violet and photo- from ABclonal Technology (ABclonal, Wuhan, China). The anti-
graphed with a microscope. bodies against HIF1a (#36169, Cell Signaling Technology [CST],
Danvers, MA, USA), Bax (#5023, CST, Danvers, MA, USA), Bcl-2
Flow cytometry analysis (#15071, CST, Danvers, MA, USA), caspase3 (#9665, CST, Danvers,
Cells treated with indicated exosomes or oligos were exposed to MA, USA), cleved-caspase3 (#9661, CST, Danvers, MA, USA), p53
200 mM CoCl2 for 24 h, and cell apoptosis was tested using (#9282, CST, Danvers, MA, USA), and b-tubulin (#2128, CST, Dan-
PI/Annexin V-FITC staining (#556547, BD Biosciences, St. Louis, vers, MA, USA) were purchased from Cell Signaling Technology. A
MO, USA) according to the manufacturer’s protocol for another ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA) was
10 min. After incubation, the cells were subjected to apoptosis anal- used for signal detection.
ysis using BD Accuri C6 Plus cytometer (Beckman Coulter, Fullerton,
CA, USA), and the results were analyzed with BD Accuri C6 Plus Statistical analysis
software. All the data in this study are expressed as means ± SD. Two treatment
groups were compared by Student’s t test. Multiple groups were
Plasmid and stable transfected cell lines analyzed by one-way ANOVA analysis. GraphPad Prism version
The mimics, inhibitors, and corresponding negative control oligos for 8.0 was used for statistical analyses. Statistical significance was consid-
hsa-miR-125b-5p were purchased from Guangzhou RiboBio ered when p < 0.05.
(Guangzhou, China). Oligo transfection was performed as per man-
ufacturer’s instructions. Human TP53INP1 cDNA was amplified ACKNOWLEDGMENTS
following the primers: 50 -ATGGACAATATGTCTATTAC-30 (for- This study was supported by grants from the National Natural Sci-
ward) and 50 -TCAGTCTAAAGGTTGTGGG-30 (reverse). The short ence Foundation of China (no. 81801923, no. 81700558, no.
hairpin RNA (shRNA) sequences against TP53INP1 were as follows: 81570570, no. 81670575, and no. 81070355), the Program of HUST
(1) 50 -CGAGTTGTATCACCTGGAATT-30 , (2) 50 -GTACTTCATA Academic Frontier Youth Team (2018QYTD02), and the Pre-
CCATGCCGATT-30 , and (3) the scramble sequence, 50 -AATT Research Fund for Free Innovation of Union Hospital, Huazhong
CTCCGAACGTGTCACGT-30 . The cDNAs were ligated into University of Science and Technology (no. 02.03.2017-312, no.
pHAGE-flag vector, and shRNAs were ligated into pLKO.1 vector. 02.03.2017-59, and no. 02.03.2018-126). We would like to thank

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 Molecular Therapy: Nucleic Acids

Ying Liu and the Core Facility of Medical Research Institute at Wu- 16. Xu, R., Rai, A., Chen, M., Suwakulsiri, W., Greening, D.W., and Simpson, R.J. (2018).
Extracellular vesicles in cancer - implications for future improvements in cancer care.
han University for flow cytometry and histological analysis.
Nat. Rev. Clin. Oncol. 15, 617–638.
17. Xue, M., Chen, W., Xiang, A., Wang, R., Chen, H., Pan, J., Pang, H., An, H., Wang, X.,
AUTHOR CONTRIBUTIONS Hou, H., and Li, X. (2017). Hypoxic exosomes facilitate bladder tumor growth
X.-F.Z. and H.W. designed the topic and performed statistical and development through transferring long non-coding RNA-UCA1. Mol. Cancer
analyses. X.-F.Z., T.W., Z.-X.W., and K.-P.H. performed animal ex- 16, 143.

periments. X.-F.Z., Z.-X.W., and T.W. performed function studies. 18. Fang, S., Xu, C., Zhang, Y., Xue, C., Yang, C., Bi, H., Qian, X., Wu, M., Ji, K., Zhao, Y.,
et al. (2016). Umbilical Cord-Derived Mesenchymal Stem Cell-Derived
Z.-X.W., T.W., Y.-W.Z., G.-L.W., H.-J.Z., Z.-H.C., and C.-Y.W. per-
Exosomal MicroRNAs Suppress Myofibroblast Differentiation by Inhibiting the
formed mechanism studies. X.-F.Z., H.W., and J.-X.Z. wrote the Transforming Growth Factor-b/SMAD2 Pathway During Wound Healing. Stem
manuscript. All authors discussed the results and reviewed and Cells Transl. Med. 5, 1425–1439.
approved the final manuscript. 19. Hu, Y., Rao, S.S., Wang, Z.X., Cao, J., Tan, Y.J., Luo, J., Li, H.M., Zhang, W.S., Chen,
C.Y., and Xie, H. (2018). Exosomes from human umbilical cord blood accelerate cuta-
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DECLARATION OF INTERESTS fibroblast function. Theranostics 8, 169–184.
The authors declare no competing interests. 20. Niinikoski, J., Grislis, G., and Hunt, T.K. (1972). Respiratory gas tensions and
collagen in infected wounds. Ann. Surg. 175, 588–593.
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