Fu et al.

Clin Proteom (2020) 17:15

https://doi.org/10.1186/s12014-020-09279-6 Clinical Proteomics

RESEARCH Open Access

Effects of cryopreservation and long‑term

culture on biological characteristics
and proteomic profiles of human umbilical
cord‑derived mesenchymal stem cells
Xufeng Fu1,2*† , Bo Xu1†, Jiang Jiang3†, Xing Du1, Xiaoli Yu1, Yaping Yan2, Shanshan Li2, Briauna Marie Inglis2,
Huiming Ma1, Hongyan Wang1, Xiuying Pei1* and Wei Si2*

Abstract
Background: Human umbilical cord-derived MSCs (hUC-MSCs) have been identified as promising seeding cells in
tissue engineering and clinical applications of regenerative medicine due to their advantages of simple acquisition
procedure and the capability to come from a young tissue donor over the other MSCs sources. In clinical applications,
large scale production is required and optimal cryopreservation and culture conditions are essential to autologous
and allogeneic transplantation in the future. However, the influence of cryopreserved post-thaw and long-term cul-
ture on hUC-MSCs remains unknown, especially in terms of specific protein expression. Therefore, biological charac-
teristics and proteomic profiles of hUC-MSCs after cryopreserving and long-term culturing were investigated.
Methods: Firstly, hUC-MSCs were isolated from human umbilical cord tissues and identified through morphology,
surface markers and tri-lineage differentiation potential at passage 3, and then the biological characteristics and prot-
eomic profiles were detected and compared after cryopreserving and long-term culturing at passage 4 and continu-
ously cultured to passage 10 with detection occurring here as well. The proteomic profiles were tested by using the
isobaric tags for relative and absolute quantification (iTRAQ) labeling technique and differential protein were con-
firmed by mass spectrometry.
Results: The results showed no significant differences in phenotypes including morphology, surface marker and
tri-lineage differentiation potential but have obvious changes in translation level, which is involved in metabolism, cell
cycle and other pathways.
Conclusion: This suggests that protein expression may be used as an indicator of hUC-MSCs security testing before
applying in clinical settings, and it is also expected to provide the foundation or standardization guide of hUC-MSCs
applications in regenerative medicine.
Keywords: Human umbilical cord, Mesenchymal stem cells, Cryopreservation, Long-term culture, Proteomic analysis

Background
*Correspondence: fuxufeng100@163.com; peixiuying@163.com; siw@lpbr.cn
†
Xufeng Fu, Bo Xu and Jiang Jiang contributed equally
Mesenchymal stem cells (MSCs) have been regarded as
1
Key Laboratory of Fertility Preservation and Maintenance of Ministry one of the most promising adult stem cells for clinical
of Education, Ningxia Medical University, Yinchuan 750004, China
2
applications in cell therapy and regenerative medicine
Yunnan Key Laboratory of Primate Biomedical Research, Institute
of Primate Translational Medicine, Kunming University of Science
due to the capabilities of self-renewal, immunomodula-
and Technology, Kunming 650500, China tion, multi-lineage differentiation and paracrine function
Full list of author information is available at the end of the article [1]. Moreover, insignificant ethical issues cause MSCs

© The Author(s) 2020. 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 the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other 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
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zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Fu et al. Clin Proteom (2020) 17:15 Page 2 of 18

to be seen as more advantageous in clinical applications influence of cryopreservation and cultivation on protein
compared to embryonic stem cells [2]. Since the discov- expression, and help facilitate the applications of hUC-
ery of MSCs in bone marrow in 1966, various tissues MSCs in cell therapeutic medicine.
have been reported as the sources of MSCs [3]. The iso-
lation of MSCs from human umbilical cord (hUC) has Materials and methods
been recognized as a major alternative source. Normally, Ethics statement
postnatal tissues after childbirth are discarded as medical The ethical approval was obtained in advance by the
waste, and the harvest and utilization of human umbili- Ethics Review Board of Ningxia Medical University and
cal cords is noninvasive and causes negligible bioethics General Hospital of Ningxia Medical University, and
concerns [4]. The hUC-MSCs originate from newborns, informed patient consent for participation was obtained
while the range of bone marrow derived MSC (BM-MSC) from all subjects.
donors’ ages is wide and the harvest process of bone
marrow is invasive [5]. A positive correlation between Isolation and culture of hUCs derived MSCs
donor ages and the accumulation of mutations in MSCs Three hUCs collected from full-term births were used
has been observed in previous studies [6–8]. Moreover, and evaluated separately for this study. The hUC tissues
hUC-MSCs show lower immunogenicity after cell trans- were sanitized with 75% alcohol for 5 min and trans-
plantation compared to other sources derived MSCs [9]. ferred to the lab within 1 h in Hanks balanced salt solu-
Therefore, hUC-MSCs show better superiority than BM- tion (HBSS, Sangon biotech, Shanghai, China). The hUCs
MSCs in terms of source and their unique characteristics were cut into 0.5 × 0.5 cm pieces with sterile forceps
make hUC-MSCs an extremely valuable candidate for and curved scissors. The pieces were cultured in sterile
cell therapeutic medicine [5]. 10 mm plastic Petri dishes containing 10 ml of low glu-
Conventionally, the dosages for MSCs transplantations cose Dulbecco’s modified Eagle’s medium (DMEM, Gibco
is ­106 cells/kg body weight and the total amount of MSCs BRL, Grand Island, NY, USA) supplemented with 10%
for one patient is about 1 ­ 08 per cell therapy in clinical (v/v) fetal bovine serum (FBS, Gibco) and 1% (v/v) peni-
trials [10]. Usually, the number of MSCs derived from cillin/streptomycin (Gibco) at 37 °C in an incubator with
either autologous or allogeneic tissues is limited, and a humidified atmosphere of 5% C ­ O2 and the medium
it is necessary to expand MSCs in vitro before therapy. was refreshed every 48 h. A large amount of fibroblast-
However, the long-term cultivation of MSCs can result like cells around the hUCs tissue pieces appeared 1 week
in differentiation-related gene expression and mitochon- later. The remained hUCs tissues were removed and these
drial morphology change, reactive oxygen species (ROS) primary fibroblast-like cells (passage 0) were passaged at
generation and cell senescence, which may deteriorate 80% confluency by using 0.25% trypsin (Gibco). The cells
MSCs features [11]. Therefore, the development of an were resuspended in culture medium at a dilution ratio of
ideal technique is essential to large-scale MSCs produc- 1:3 and expanded on a new plastic Petri dish to passage
tion and storage and it also requires minimal impact on 1 [14]. The morphology, surface markers and differentia-
MSCs. tion potency of MSCs were identified at passage 3.
Cell cryopreservation is a widely used technology for
long-term storage of cells by cooling the cells to cryogenic Morphological and immunophenotypic characterization
temperatures (− 196 °C in liquid nitrogen, for example) of hUC‑MSCs
[12]. In our previous study, we found that BM-MSCs of The morphological characteristics of hUC-MSCs were
a nonhuman primate vitrified with a high level (5.6 M) assessed under a light microscope (Nikon DIAPHOT
of the penetrating cryoprotectant either DMSO or ethyl- 300, Japan) at primary culture and upon passaging in all
ene glycol (EG) resulted in changes of a large number of the experimental groups. The morphological images in
transcripts [13]. Currently, the most widely used method this present study were taken at 20 × magnification. The
for MSCs cryopreservation is the slow-freezing approach expression of cell surface markers were evaluated using
with using a low level of DMSO (1.5 M) as the penetrat- a Human MSC Analysis Kit (BD Biosciences, San Jose,
ing cryoprotectant. However, the effects of slow-freezing CA) with a C6 flow cytometer (BD Biosciences, San Jose,
with a low level of DMSO on the global gene transcripts CA) at 3rd, 4th and 10th passages. Briefly, hUC-MSCs
and proteomics profiles of MSCs have not been studied were collected and washed with 500 μL of PBS (contain-
(Additional file 1: Table S1). ing 3% FBS, PBSF) and the concentration was adjusted at
In the present study, we aimed to comprehend the 1 × 106 cells/mL by using a hemacytometer. Then a total
effects of conventional slow-freezing cryopreservation of 100 μL of the cell suspension (approximately 5 × 105
and long-term cultivation on the proteomic profiles cells) was distributed in a 1.5 mL centrifugal tube and
of hUC-MSCs. The study will provide a basis for the incubated with 5 μL (10 μg/μL) of human monoclonal
Fu et al. Clin Proteom (2020) 17:15 Page 3 of 18

antibodies against a positive (CD44, CD73, CD90 and cooled at approximately 1 °C/min from 25 to − 80 °C in a
CD105) and negative cocktail (including CD34, CD45, freezing container (Nalgene, Rochester, NY) for 12 h and
CD14, CD19, and HLA-DR) at room temperature for then the cryovials were plunged directly into liquid nitro-
30 min according to the manufacturer’s instructions. gen for storage. This is the most commonly used method
Unbound antibodies were washed off with PBS and sub- and equipment for MSCs cryopreservation in laborato-
sequently the cells were resuspended in 500 μL of PBSF ries all over the world [16, 17]. After being stored in liq-
mixture before flow cytometric testing [13]. uid nitrogen for 24 h, the cells were rapidly warmed by
immersing the cryovial in a 37 °C water bath for 5 min.
Evaluation of the differentiation potential of hUC‑MSCs Post-thawed cells were cultured for 24 h for recovery
For adipogenic differentiation, hUC-MSCs were seeded and subsequently evaluated as described in the follow-
into 24-well plates and cultured for 12 h at a density of ing assays. The cryopreserved MSCs (abbreviated as “C”
8 × 104 cells per well. Subsequently, the medium was from now on) were subcultured for 24 and 48 h at P4 and
substituted with the adipogenic differentiation medium P10, respectively, and non-cryopreserved MSCs (abbre-
(Biological Industries, Israel) for 21 days, and the viated as “N” from now on) cultured for 24 and 48 h at
medium was refreshed every 3 days. The induced cells same passages were used as controls. The schematic illus-
were stained with Oil Red O in a MSCs Adipo-Staining tration of the procedure was shown in Fig. 1. A total of 8
Kit (XP Biomed Ltd., Shanghai, China) according the groups of hUC-MSCs were involved in this study as fol-
instructions. lows: non-cryopreserved and sub-cultured for 24 h at P4
For osteogenic differentiation, hUC-MSCs were seeded (P4N24), cryopreserved and sub-cultured for 24 h at P4
into 24-well plates and cultured for 12 h at a density of (P4C24), non-cryopreserved and sub-cultured for 48 h
4 × 104 cells per well. Subsequently, the medium was sub- at P4 (P4N48), cryopreserved and sub-cultured for 48 h
stituted with the osteogenic differentiation medium (Bio- at P4 (P4C48), non-cryopreserved and sub-cultured for
logical Industries, Israel) for 21 days, and the medium 24 h at P10 (P10N24), cryopreserved and sub-cultured
was refreshed every 3 days. The induced cells were for 24 h at P10 (P10C24), non-cryopreserved and sub-
stained with alizarin red solution in a MSCs Osteo-Stain- cultured for 48 h at P10 (P10N48) and cryopreserved and
ing Kit (XP Biomed Ltd., Shanghai, China) according the sub-cultured for 48 h at P10 (P10C48).
instructions.
For chondrogenic differentiation, 2 × 105 hUC-MSCs Measurement of cell viability
were pelleted in 15-mL centrifuge tubes and cultured The viability of cells from P4N24, P4C24, P4N48, P4C48,
with the chondrogenic differentiation medium (Bio- P10N24, P10C24, P10N48 and P10C48 groups were
logical Industries, Israel) for 21 days and the medium measured by trypan blue dye (Solarbio, Beijing, China)
was refreshed every 3 days. The chondroid pellets were exclusion assay. Ten μL of cell suspension was mixed 10
sectioned by a freezing microtome and the slices were μL 0.4% w/v trypan blue solution for 5 min, and the dead
stained with toluidine blue in a MSCs Chondro-Staining cells were stained and counted with a haemocytometer
Kit (XP Biomed Ltd., Shanghai, China) according the under a light microscope.
instructions [15].
All differentiation evaluations were repeated 3 times. Proteomics analysis and targeted quantitative detection
of hUC‑MSCs
Cryopreservation of hUC‑MSCs The cells from non-cryopreserved groups (P4N24,
The hUC-MSCs from the three donors were harvested P4N48, P10N24, P10C24, and P10N48) and cryopre-
at passage 4 and 10 for the cryopreservation assay when served groups (P4C24, P4C48, P10C24 and P10C48)
the cells reached 80% confluency. The cell suspension groups were collected for proteomic profile detection.
was divided into two equal aliquots at a density of 2 × 106 The proteomics procedures were performed by PTM
cells/mL. One of the aliquots without cryopreservation Biolabs Lnc. (Hangzhou, Zhejiang, China). Briefly, a cell
was sub-cultured in fresh medium for 24 h, and cell via- sample was sonicated by high intensity ultrasonic proces-
bility, immunophenotype surface markers, proliferation sor in lysis buffer of urea and protease inhibitor cocktail,
and metabolic activity were subsequently examined as a and the remaining cell debris was removed by centrifu-
non-frozen control. The other cells were cryopreserved gation. The protein concentration of the supernatant was
by the conventional cell freezing method with the freez- collected and quantified with BCA kit (Thermo Fisher,
ing medium composed of DMEM medium supplemented USA), and prokaryotic standard protein was added for
with 10% FBS and 10% DMSO. The mixture of freezing detecting quality control [18]. Then, the protein solu-
medium and hUC-MSC suspension (1 mL) in a 1.8 mL tion was reduced with dithiothreitol and alkylated with
cryovial containing a density of 1 × 106 cells/mL was iodoacetamide, and the urea concentration was diluted
Fu et al. Clin Proteom (2020) 17:15 Page 4 of 18

Fig. 1 A schematic illustration of the procedure for hUC-MSCs cryopreservation and long-term culture. L­ N2: liquid nitrogen. P4N24:
non-cryopreserved and sub-cultured for 24 h at P4. P4C24: cryopreserved and sub-cultured for 24 h at P4, P4N48: non-cryopreserved and
sub-cultured for 48 h at P4, P4C48: cryopreserved and sub-cultured for 48 h at P4, P10N24: non-cryopreserved and sub-cultured for 24 h at P10,
P10C24: cryopreserved and sub-cultured for 24 h at P10, P10N48: non-cryopreserved and sub-cultured for 48 h at P10, P10C48: cryopreserved and
sub-cultured for 48 h at P10

by adding tetraethylammonium bromide, and then the proteins was also performed depending on Parallel Reac-
protein samples were digested by trypsin. After trypsin tion Monitoring (PRM) technology by PTM Biolabs Lnc.
digestion, the peptide was desalted and processed accord- according to their commercial manufacturer’s instruc-
ing to the manufacturer’s protocol for TMT/iTRAQ kit. tions. The pre-processing of samples as well as proteom-
The tryptic peptides were fractionated into fractions by ics analysis, besides, quantitative analysis was used as a
high pH reverse-phase HPLC using Agilent 300 Extend standard to quantify special protein from samples.
C18 column, and the peptides were dissolved by acetoni-
trile and analyzed by tandem mass spectrometry in Q Statistical analysis
ExactiveTM Plus (Thermo) coupled online to the EASY- The data from viability and markers expression were
nLC 1000UPLC. The data of tandem mass spectrometry significantly analyzed statistically using Graphpad
were processed using Maxquant search engine (v.1.5.2.8) software (GraphPad Prism; Graphpad Software, Inc.,
and annotation results from database were collected for San Diego, CA) and presented as the mean ± SD. Com-
analysis. Quantitative analysis of differentially expressed parative assessment of mean value among various
Fu et al. Clin Proteom (2020) 17:15 Page 5 of 18

factors was performed using ANOVA and unpaired t Effect of long‑term culture and cryopreservation
test and a P-value < 0.05 was considered statistically on the biological characteristics of hUC‑MSCs
significant. As shown in Fig. 3a, the viability of hUC-MSCs were
Differential protein screening was based on a 1.3- significantly decreased after instant freezing and thaw-
fold change, and the ratio between the samples at more ing (abbreviated as “C” groups from now on) com-
than 1.3-fold change or less than 1/1.3-fold change pared to non-cryopreserved control (abbreviated as “N”
were considered up-regulated or down-regulated groups from now on) either at passage 4 (P4, N vs. C,
trend P-value < 0.05. For further study of the hier- 99.61 ± 0.22% vs. 94.42 ± 1.53%) or passage 10 (P10, N
archical clustering, all the categories were obtained vs. C, 99.44 ± 0.51 vs. 93.82 ± 2.13%). After a sub-culture
and enriched in clusters depending on P-value < 0.05, for 24 h or 48 h post thawing, the hUC-MSCs either at
and the cluster membership were visualized by a heat P4 or P10 remained to possess a high level expression
map using the “heatmap.2” function from the “gplots” of positive surface markers (CD44, CD73, CD90 and
R-package. Proteins were classified by Gene Ontology CD105) and barely expressed negative markers of MSCs,
(GO) annotation, which was derived from the UniProt- and no significant differences were observed compared
GOA database (www. http://www.ebi.ac.uk/GOA/). The to non-cryopreserved controls. The results suggested
pathways of different proteins were classified accord- that the expression of surface markers was not affected
ing to the Kyoto Encyclopedia of Genes and Genomes by cryopreservation and long-term culture (Fig. 3b). The
(KEGG) database website. morphology of cells from non-frozen control and cryo-
Identified proteins domain functional description preserved groups following a 24 h and 48 h sub-culture
was annotated by InterProScan based on InterPro post thawing are shown in Fig. 3c. No obvious morpho-
(http://www.ebi.ac.uk/inter​pro/) domain database. logical changes were observed among the eight groups.
These enrichment analyses were tested according Similar to the cells from control groups, the differen-
to the database of identified proteins and employed tiation potency of hUCs from N24, N48, C24 and C48
two-tailed Fisher’s exact test, all terms with corrected groups at P4 and P10 showed no obvious difference eval-
P-values < 0.05 were considered significantly enriched uated by adipogenic (Fig. 4a), osteogenic (Fig. 4b) and
differentially expressed proteins. chondrogenic differentiation (Fig. 4c).

Results Effect of cryopreservation and long‑term culture

Basic characterization of hUC‑MSCs on proteome profiles of hUC‑MSCs
During primary culture, the spindle-shaped and The number of significantly modulated proteins of hUC-
fibroblast-like cells were dispersed around the shred- MSCs among the 8 groups are summarized in Fig. 5a.
ded umbilical cord tissues. These cells grew adhe- These results indicated that the proteome profiles of
sively in plastic dishes in a scattered manner, formed hUC-MSCs were affected by either long-term culture
colonies and appeared heterogeneously regarded as or cryopreservation. The functional enrichment analy-
hUC-MSCs of passage 0 (P0, Fig. 2a). The hUC-MSCs sis according to Gene Ontology (GO) of differentially
colonies at passage 0 were extended to passage 3 (P3) expressed proteins among hUC-MSCs from the 8 groups
with subsequent subculture, and the P3 hUC-MSCs was summarized in Fig. 5b–d. The heatmap graphs of the
also showed a spindle-shaped and fibroblast-like mor- GO display the distribution of the biological terms pre-
phology (Fig. 2b). The surface marker profiles of the sented in molecular function (Fig. 5b), biological process
hUC-MSCs were analyzed at P3 by flow cytometry. (Fig. 5c) and cellular component (Fig. 5d). In molecular
The percentage of positively expressed surface mark- function, protein kinase activity and microtubule motor
ers was 100.0 ± 0.0% of CD44, 99.3 ± 0.2% of CD73 activity were affected by continuous culture from 24 h to
and 85.0 ± 1.4% of CD105, and the percentage of nega- 48 h at P4 without cryopreservation (P4N24 vs. P4N48).
tive expressed cocktail surface markers was 0.2 ± 0.1% Retinoid, isoprenoid binding and cytokine activity were
(Fig. 2c–h). After adipogenic, osteogenic and chondro- affected by long-term culture from P4 to P10 with-
genic differentiation, the P3 hUC-MSCs formed numer- out cryopreservation (P4N48 vs. P10N48). Oxidore-
ous neutral lipid droplets in the cytoplasm identified ductase, hydrolase and peptidyl-proline dioxygenase
by Oil Red O staining (Fig. 2i), mineral accumulation were affected by cryopreservation and sub-culture for
and bone nodules formation was identified by alizarin 48 h at P10 compared to P4 (P4C48 vs. P10C48), which
red staining (Fig. 2j) and proteoglycan and hyaluronic were shown in Fig. 5b. In biological process as shown
acid accumulation was identified by alcian blue staining in Fig. 5c, regulation of nuclear division was affected by
(Fig. 2k). continuous culturing from 24 h to 48 h at P4 without
Fu et al. Clin Proteom (2020) 17:15 Page 6 of 18

Fig. 2 Fibroblast-like morphology of MSCs at passage 0 (a) and passage 3 (b). Scale bars: 100 μm. c–g Surface markers expression on human
umbilical cord-derived MSCs at passage 3 analyzed using flow cytometry. Black lines represent isotype control. h Quantitative profile of surface
markers expression (n = 3). i–k Differentiation potency of MSCs at passage 3. i Adipogenic differentiation (oil red staining, × 200); j Osteogenic
differentiation (alizarin red staining, × 100); k Chondrogenic differentiation (alcian blue staining, × 50). Scale bars: i was 50 μm, j was 100 μm and k
was 500 μm
Fu et al. Clin Proteom (2020) 17:15 Page 7 of 18

Fig. 3 Comparison of the cell viability (n = 3) (a), surface markers expression (n = 3) (b) and morphology (c) between non-cryopreserved (N) and
cryopreserved (C) groups after being sub-cultured for 24 h or 48 h at P4 and P10, respectively

cryopreservation (P4N24 vs. P4N48), protein activation enriched in biological processes of GO classification hav-
cascade was affected by cryopreservation and sub-cul- ing known identities in MSCs functions. The functions
ture for 24 h at P4 compared to non-cryopreserved and of these differentially hUC-MSCs proteins are listed in
sub-cultured for 24 h (P4N24 vs. P4C24), regulation of Table 1, which are associated with differentiation, immu-
smooth muscle cell proliferation was affected by long- noregulation, wound healing and regeneration, apoptotic
term culture from P4 to P10 without cryopreservation signaling pathway, oxidation resistance, cartilage devel-
(P4N48 vs. P10N48), cell proliferation and programmed opment, regulation of cytokine production, cell migra-
cell death were affected by continuous culturing from tion and others. Specific protein information and the fold
24 h to 48 h at P10 (P10N24 vs. P10N48), and cell com- of change in different groups were shown in Table 2.
munication and signal transduction were affected by Protein domain was analyzed after cryopreserva-
post-thawing and sub-culturing for 24 h at P4 compared tion and sub-culture for 24 h and 48 h at P4 and P10,
to non-cryopreservation at P10 (P10N24 vs. P10C24). In respectively, compared with non-cryopreserved groups
cellular component as shown in Fig. 5d, cytoskeleton and as shown in Fig. 5e. The results showed that immuno-
chromosome passenger complex were affected by contin- globulin-like fold domain was affected by the continuous
uous culture from 24 h to 48 h at P4 (P4N24 vs. P4N48), culture from 24 h to 48 h at P4 without cryopreservation
extracellular region and lysosome were affected by long- (P4N24 vs. P4N48). BRICHOS domain and galactose-
term culture from P4 to P10 without cryopreservation binding domain-like were affected by long-term culture
(P4N48 vs. P10N48), nuclear replication fork, lysosomal from P4 to P10 without cryopreservation and sub-cul-
and endoplasmic reticulum lumen were affected by cryo- ture for 24 h (P4N24 vs. P10N24). Chemokine domain
preservation and sub-culture for 48 h at P10 compared to was affected by continuous culturing from by long-term
P4 (P4C48 vs. P10C48). culture from P4 to P10 without cryopreservation and
In order to further analyze the effect of cryopreser- sub-culture for 48 h (P4N48 vs. P10N48). Hydroxy-
vation on hUC-MSCs function, differential proteins lase, iron-dependent dioxygenase, glycoside hydrolase
Fu et al. Clin Proteom (2020) 17:15 Page 8 of 18

Fig. 4 Comparison of adipogenic (a), osteogenic (b) and chondrogenic (c) differentiation potency between non-cryopreserved (N) control and
cryopreserved (C) groups after being sub-cultured for 24 h or 48 h at P4 and P10, respectively

superfamily and thioredoxin-like fold domain were and Genomes) to show the network of pathway interac-
affected by cryopreservation and sub-culturing 48 h at tions (The raw data of differentially expressed proteins
P10 compared to P4 (P4C48 vs. P10C48). enriched in KEGG database as shown in Additional file 1:
In addition, differentially expressed proteins were Table S1). The results as shown in Fig. 6a indicated that
also analyzed by KEGG (Kyoto Encyclopedia of Genes progesterone mediated oocyte maturation, complement
Fu et al. Clin Proteom (2020) 17:15 Page 9 of 18

Fig. 5 The number and the GO heatmaps of differentially expressed proteins. a The histogram of differentially expressed proteins. The heatmap of
differential proteins enriched pathways in molecular function (b), biological process (c), cellular component (d) and protein domain (e)
Fu et al. Clin Proteom (2020) 17:15 Page 10 of 18

Table 1 Biological processes classification of differential identified proteins in MSCs

Biological processes Gene name

Differentiation GATA6, DKK1, STC1, PDGFRB, COL5A2, FST, CCNB1, AURKA, TOP2A, INHBA, COL1A1, ANLN, JUN,
SEMA7A, NCAM1, COL12A1, NRP2, FBN2, HGF
Immune system regulation process TNFAIP3, KIF2C, PTX3, TMBIM1, IGHG1, JUN, NDRG1, NCAM1, MYO10, KIF22, COL1A1, RACGAP1,
SEMA7A, KIF11, INHBA, MT2A, FST, C3, GEM, TOP2A, SERPINE1, KIF23, ANLN, PDCD1LG2, CRISPLD2,
JUN
Wound healing and regeneration TNFAIP3, SERPINB2, GATA6, MKI67, SERPINE1, F3, FOSL1, AURKA, COL1A1, DCN, NRP2, HGF, JUN, C3,
PDGFRB, CCNB1, TFPI2, HIST2H3A, CCNA2
Apoptotic signaling pathway BIRC5, STK17B, F3, TNFAIP3, SERPINE1, INHBA, TMBIM1, TIMP3, TOP2A, CHEK1, HGF, PDGFRB, TPX2,
GATA6, SERPINB2, AURKA, CCNB1, TNFAIP3, NUAK1, FOSL1, AURKB, CPEB4, PLK1, JUN, ARAF, AMIGO2
Myeloid cell differentiation and ossification INHBA, FBN2, STC1, COL5A2, JUN, HGF, COL1A1, SEMA7A
Oxidation resistance TNFAIP3, PTX3, NDRG1, TIMP3, SERPINE1, COL1A1, NDRG1, STK17B,AURKA, PLK1, PDGFRB, CPEB4,
AURKB, AMIGO2, TMBIM1, JUN, STC1, ARAF, GATA6, HGF, FOSL1, CCNB1, TOP2A, F3, CPEB4, BIRC5,
SERPINB2, INHBA, CCNA2
Adaptive immune response TNFAIP3, IGHG1, MYO10, DCN, C3, HGF, PDCD1LG2, JUN, SEMA7A, FST, INHBA, PTX3, TOP2A, SERPINE1,
NCAM1, ANLN, MT2A
Inflammatory response TNFAIP3, PTX3, SERPINE1, PDCD1LG2, SEMA7A, F3, HGF, C3
Interferon-gamma-mediated signaling pathway PDCD1LG2, NCAM1, INHBA, MT2A
Cartilage development STC1, BNC2, LUM, COL1A1, STC1, COL1A1, MEX3C
Regulation of cytokine production GATA6, LUM, TNFAIP3, SERPINE1, PDCD1LG2, SEMA7A, INHBA, HGF, C3
Angiogenesis SERPINE1, GATA6, PDGFRB, F3, JUN, HGF, NRP2, C3
Antigen processing and presentation RACGAP1, TNFAIP3, CCNA2, KIF22, INHBA, KIF11, KIF2C, KIF23
Cell migration F3, STC1, SERPINE1, SMURF2, DCN, COL1A1, PDGFRB, HGF, NRP2, JUN
Transforming growth factor beta1 production GATA6, COL1A1, C3, LUM, JUN, SERPINE1, TNFAIP3, PDCD1LG2, SEMA7A, HGF, INHBA
Response to growth factor PDGFRB, GATA6, NRP2, DKK1, SHCBP1, SHCBP1, CCNA2, SMURF2, COL1A1, SMURF2, JUN, FBN2, HGF,
DCN, LUM
Aging CHEK1, SERPINE1, AURKB, PDGFRB, DCN, JUN
Regulation of endothelial cell proliferation SERPINE1, GATA6, DCN, TNFAIP3, THBS2, HGF, F3, C3, JUN, NRP2

and coagulation cascades and protein digestion related by cryopreservation as well as long-term culturing at P4
pathways were affected by continuous culturing from and P10.
24 h to 48 h at P4 (P4N24 vs. P4N48) without cryo-
preservation. Retinol metabolism and vitamin absorp- Verification of cryopreservation and long‑term culture
tion related pathways were affected by long-term culture induced candidate proteins by PRM
from P4 to P10 without cryopreservation and sub-culture The differentially expressed proteins were separated into
for 24 h (P4N24 vs. P10N24). Steroid hormone biosyn- several categories according to their functions by GO
thesis related pathways was effected by post-thawing and and KEGG enrichment analysis, to validate the results of
sub-culturing for 48 h compared to non-cryopreserva- MS and to compare the influence mechanisms of cryo-
tion and sub-culture for 48 h at P4 (P4N48 vs. P4C48). preservation and long-term culture on hUC-MSCs, we
Nicotinamide metabolism, antifolate resistance and used PRM analysis to assess the abundance of 14 candi-
staphylococcus aureus infection related pathways (Not date proteins whole abundance changes in response to
contaminated) were affected by long-term culture from hUC-MSCs cryopreservation and long-term culture as
P4 to P10 without cryopreservation and sub-culture for determined by TMT. The 14 differentially expressed pro-
48 h (P4N48 vs. P10N48). Glycosaminoglycan degrada- teins as well as enriched various pathways were selected
tion and DNA replication related pathways were affected from 4 groups (P4N24 vs. P4C24, P4N24 vs. P10N24,
by cryopreservation and sub-culture for 48 h at P10 com- P4C24 vs. P10C24, P10N24 vs. P10C24) and involved
pared to P4 (P4C48 vs. P10C48). In addition, the differen- in tdioxygenase activity, cell development, extracellular
tially expressed proteins of the enriched KEGG pathway matrix, oxidoreductase activity, reproductive process,
were listed as Fig. 6b, the red and blue present up-reg- hydrolase activity, ATP binding, protein kinase activity,
ulaed and down-regulated proteins, respectively. These immune process, cell growth and division. As shown in
results indicated that the expression of hUC-MSCs pro- Table 3, 14 proteins in PRM analysis was consistent with
teins which are involved in many pathways were changed the results of TMT-based quantitation results. Although
Fu et al. Clin Proteom (2020) 17:15 Page 11 of 18

Table 2 Differential proteins associated with MSCs function

P4N24 P4N24 P4N24 P4N48 P4N48 P4C24 P4C24 P4C48 P10N24 P10N24 P10N48 P10C24
Protein
Gene name vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs.
accession
P4N48 P4C24 P10N24 P4C48 P10N48 P10C48 P10C24 P10C48 P10N48 P10C24 P10C48 P10C48
Q86SJ2 AMIGO2 0.671 NS NS 1.422 1.377 0.749 NS NS NS NS NS NS
Q9NQW6 ANLN 1.861 NS 1.363 0.741 NS NS 1.711 2.167 1.620 1.335 1.354 1.642
P10398 ARAF 0.760 NS 0.766 NS NS NS NS 0.741 NS NS NS NS
O14965 AURKA 1.930 NS NS NS NS NS NS NS 1.725 1.429 NS NS
Q96GD4 AURKB 1.592 NS 1.308 NS NS NS NS NS 1.398 NS NS NS
O15392 BIRC5 1.612 NS NS NS NS NS 1.622 2.275 1.803 NS NS 1.791
P01024 C3 3.316 3.729 4.611 1.347 1.409 NS NS 0.724 NS NS 0.692 NS
P20248 CCNA2 1.845 NS 1.324 NS NS 1.369 1.408 1.689 1.545 NS NS NS
P14635 CCNB1 1.751 NS 1.312 0.734 NS 1.488 1.864 2.181 1.631 NS NS NS
O14757 CHEK1 1.331 NS NS NS NS NS 1.390 1.928 NS NS 1.367 1.613
Q99715 COL12A1 0.723 NS 0.706 1.459 NS NS 0.714 0.508 NS NS 0.766 0.585
P02452 COL1A1 0.707 NS NS NS 1.302 NS 0.725 0.630 NS NS 0.603 0.681
P05997 COL5A2 0.768 NS NS NS NS NS 0.742 0.629 NS NS 0.691 0.683
Q17RY0 CPEB4 1.372 NS NS NS NS NS NS NS NS NS NS NS
Q9H0B8 CRISPLD2 0.557 NS NS 1.662 NS NS NS 0.537 0.608 NS NS 0.638
P07585 DCN 0.760 NS NS NS NS 0.744 0.619 0.520 NS NS 0.715 0.626
O94907 DKK1 1.584 NS 1.651 0.703 1.387 NS 1.806 2.744 1.331 1.387 1.391 NS
P13726 F3 1.414 1.408 0.754 NS NS NS NS 1.790 1.852 1.903 NS 1.369
P35556 FBN2 0.762 NS NS NS NS NS NS NS NS NS NS NS
P15407 FOSL1 1.775 NS NS 0.753 NS NS 1.736 1.619 1.494 1.643 NS NS
P19883 FST 2.036 NS NS 0.581 NS NS 1.528 1.894 1.880 1.719 NS 1.452
Q92908 GATA6 0.746 NS NS NS 0.728 NS NS NS NS NS NS
P55040 GEM 1.308 NS NS 0.663 NS NS 1.321 1.675 1.320 NS NS NS
P14210 HGF 0.767 NS NS 1.453 1.448 NS NS NS NS NS NS NS
Q71DI3 HIST2H3A 1.445 NS 1.572 NS 1.333 1.339 1.500 1.809 NS NS NS 1.614
P01857 IGHG1 2.704 2.998 4.018 1.524 NS 1.374 1.663 NS NS NS NS NS
P08476 INHBA 1.478 NS NS NS NS NS NS NS 1.373 1.592 NS NS
P05412 JUN 1.350 NS NS NS 1.337 NS 1.718 1.584 1.467 NS NS NS
P52732 KIF11 1.417 NS 1.313 NS NS NS 1.587 2.325 1.307 NS 1.496 1.794
Q14807 KIF22 1.413 NS NS NS NS NS NS 1.433 1.311 NS NS NS
Q02241 KIF23 1.417 NS NS NS NS NS 1.590 2.307 NS NS 1.753 NS
Q99661 KIF2C 1.435 NS NS NS NS NS 1.449 2.158 NS NS 1.499 1.828
P51884 LUM 0.627 NS NS NS NS 0.572 0.538 NS NS NS NS 0.722
Q5U5Q3 MEX3C 0.769 NS NS NS NS NS NS NS 0.768 0.746 NS NS
P46013 MKI67 1.458 NS 1.473 0.760 NS NS 1.694 2.337 NS NS 1.452 1.715
P02795 MT2A 1.383 0.732 NS 0.701 NS 1.324 NS 2.449 1.650 NS 1.693 2.624
Q9HD67 MYO10 1.300 NS NS NS NS NS NS NS 1.402 NS NS NS
P13591 NCAM1 0.747 NS NS NS 1.424 NS NS NS NS NS NS NS
Fu et al. Clin Proteom (2020) 17:15 Page 12 of 18

Table 2 (continued)
Q92597 NDRG1 1.450 NS NS NS NS 1.462 NS NS NS NS NS NS
O60462 NRP2 0.655 NS 0.632 1.464 NS NS NS 0.638 NS NS NS NS
O60285 NUAK1 0.730 NS NS NS NS NS NS 0.751 NS NS NS NS
Q9BQ51 PDCD1LG2 1.534 1.350 NS NS NS NS NS NS 1.339 NS 1.534 NS
P09619 PDGFRB 0.700 NS NS NS NS NS NS NS NS NS NS NS
P53350 PLK1 1.617 NS NS NS NS NS 1.677 1.819 1.450 NS NS 1.407
P26022 PTX3 1.354 1.313 NS 0.672 NS 0.693 NS 1.699 1.357 1.432 NS NS
Q9H0H5 RACGAP1 1.392 NS NS NS NS NS 1.501 2.203 NS NS 1.626 1.747
O75326 SEMA7A 1.647 NS NS 0.740 NS NS 1.701 1.792 1.618 1.638 NS NS
P05120 SERPINB2 1.505 NS NS 0.736 NS NS NS NS 1.309 NS NS NS
P05121 SERPINE1 1.686 NS NS NS NS NS NS NS 1.595 1.730 NS NS
P52823 STC1 1.794 NS 0.556 NS 0.647 1.734 NS NS 2.088 2.108 NS NS
O94768 STK17B 1.343 NS NS NS NS NS NS NS 1.582 1.571 NS NS
P48307 TFPI2 1.731 NS 0.655 NS 0.526 1.491 NS NS 1.391 1.981 1.780 NS
P35442 THBS2 0.731 NS NS NS NS NS 0.768 0.694 NS NS NS 0.706
P35625 TIMP3 1.529 NS 0.730 NS NS NS 1.428 1.336 1.730 1.850 NS NS
Q969X1 TMBIM1 1.602 NS NS NS NS NS NS NS NS NS NS NS
P21580 TNFAIP3 1.494 NS NS NS NS 1.357 1.327 NS NS NS NS NS
P11388 TOP2A 1.782 NS NS 0.733 NS 1.439 1.584 2.334 1.679 NS 1.407 2.119
Q9ULW0 TPX2 1.663 NS NS 0.758 NS NS 1.393 1.778 1.517 NS NS 1.495

Red is up-regulation and blue is down-regulation, NS is no significant difference

the fold changes of SMTN in P4C24/P10C24, SEMA7A ethylene glycol (EG) have been used for vitrification of
in P4C24/P10C24 and P10N24/P10C24 analyzed by PRM MSCs, and the results showed that the viability of cells
more than TMT, whereas the TMT and PRM results all vitrified by DMSO is less than those by EG. However,
showed a rising trend. Our PRM results were in consist- the transcripts of larger numbers of genes affected by
ent with the data from TMT analysis (Table 3), which EG are much more than those by DMSO [13]. There-
further confirmed the credibility of the proteomics data. fore, the method of conventional slow freezing method
by using 10% DMSO was selected in the present study
Discussion and it is still the most widely used method at present
Human umbilical cord-derived MSCs are promising [16, 17]. In regard to the store period (24 h) of MSCs in
seeding cells in cell therapy and regenerative medicine liquid nitrogen, whether long-term storage more than
due to their unique advantages. Cryopreservation plays 24 h will have more profound effects remains need to
an important role in the maintenance of MSCs function be further studied [13].
and avoids adverse effects caused by long-term culture In this present study, the conventional slow freez-
[19]. DMSO is a widely used penetrating cryoprotect- ing method using 10% DMSO was used for MSC cryo-
ant for MSCs cryopreservation when using the conven- preservation. The freezing and thawing process decrease
tional slow freezing protocol. Although efforts for the the viability of cells either at P4 (94.42 ± 1.53%) or P10
reduction of DMSO concentrations have been made to (93.82 ± 2.13%). In previous studies, Fong et al. reported
alleviate the adverse reactions of DMSO and decreased that hUC-MSCs viability was 85–90% after thawing by
DMSO concentration (as low as 2% combined with using the same slow cooling method [21], and Woods
other cryoprotectants) has been successfully employed et al. reported the post-thaw viability of human MSCs
[20], the viability of MSCs cannot be guaranteed. In was about 91% by using 1.0 M (about 7.1%, w/v) and
addition, the combination of multiple penetrating cryo- 1.5 M (about 10.65%, w/v) DMSO freezing with this
protectants is not conducive to understand the adverse method [22]. Our results showed similar viabilities com-
mechanisms of each cryoprotectant on cell recovery pared to the previous studies. Although the conventional
or engraftment. In our previous study, DMSO and slow freezing method has been widely used and can also
Fu et al. Clin Proteom (2020) 17:15 Page 13 of 18

Fig. 6 Heatmap of pathways (a) and differentially expressed proteins (b) enriched according to KEGG database among the eight groups
Fu et al. Clin Proteom (2020) 17:15 Page 14 of 18

Table 3 Comparison of the quantification results between TMT and PRM of the 14 candidate different expression
proteins
Protein accession Proteins Signature peptides P4N24/ P4N24/ P4C24/ P10N24/
P4C24 P10N24 P10C24 P10C24
TMT PRM TMT PRM TMT PRM TMT PRM

Q8IVL6 P3H3 DLETPPHWAAYDTGLELLGR 1.03 1.17 0.96 1.06 0.91 0.86 0.98 0.96
P53814 SMTN AQEIEAATLAGRLQDGTPQAALSPLTPAR 1.04 1.04 1.23 1.27 1.92 3.29 1.63 2.68
Q96CG8 CTHRC1 QCSWSSLNYGIDLGKVLFSGSLR 1.47 2.08 0.89 0.81 0.61 0.39 1.01 1.00
O75326 SEMA7A DPYCGWDQGR 1.06 1.09 1.10 1.08 1.70 3.03 1.64 3.08
P35354 PTGS2 SHLIDSPPTYNADYGYKSGLDDINPTVLLK 1.40 2.79 0.57 0.50 0.81 0.53 2.00 2.99
Q9NQW6 ANLN LLLIATGKGFLTIFEDVSGFGAWHR 1.06 1.08 1.36 1.32 1.71 2.44 1.34 1.99
P58335 ANTXR2 VSPVGETYIHEGLKLDALWALLR 0.87 0.97 0.75 0.70 1.18 1.17 1.36 1.63
P48307 TFPI2 LQVSVDDQCEGSTEKTCDAFTYT​GCG​GNDNNFVSR 1.08 0.96 0.66 0.47 1.20 1.23 1.98 2.51
Q13642 FHL1 FWHDTCFR 1.56 1.45 2.06 2.00 1.69 2.53 1.28 1.84
Q02241 KIF23 ALLQEFDNAVLSK 0.96 1.05 1.29 1.49 1.59 1.82 1.18 1.28
Q9H0H5 RACGAP1 SIGSAVDQGNESIVAK 0.97 0.87 1.25 1.56 1.50 2.47 1.17 1.38
P53350 PLK1 LILYNDGDSLQYIER 0.96 1.01 1.27 1.89 1.68 2.70 1.27 1.44
P00749 PLAU FEVENLILHK 0.62 0.42 0.43 0.27 1.23 1.50 1.76 2.30
O00762 UBE2C GISAFPESDNLFKLSLEFPSGYPYNAPTVK 1.02 1.05 1.27 1.52 1.63 2.06 1.31 1.42

obtain better viability, profound influence of the freezing the traditional identification standards based on qualita-
process and cryoprotectant on the transcript and protein tive detection (post thaw viability, morphology, surface
function of hUC-MSCs remains unknown. markers and tri-lineage differentiation potency) may be
Conventionally, the morphology, surface marker insufficient for the evaluation of the change of biological
expression and tri-lineage differentiation potency are characteristics after cryopreservation or environmental
regarded as a “gold standard” for identifying MSCs stimulus during long-term culture. Therefore, it is nec-
according to the International Society for Cellular Ther- essary to explore quantitative methods for MSCs qual-
apy. In this study, there are no significant differences ity evaluation such as a protein targeting quantification
between non-cryopreserved and post-thaw following method in preclinical or clinical application.
sub-culture 24 or 48 h in morphology, surface markers Previous studies have reported that cryopreservation
and tri-lineage differentiation potency at P4 and P10. can affect the immunomodulatory properties of MSCs,
Hence, these results concluded that cryopreservation and the levels of heat shock proteins increased and the
and long-term culture did not affect the characteristics inflammatory response was impaired within 24 h after
of hUC-MSCs, which are consistent with previous stud- thawing. However, these studies considered that the
ies [13, 23]. To our knowledge, almost all of the studies function of MSCs would be completely recovered after
have shown that cryopreservation does not affect the 24 h of culturing [25–27]. The protein expression recov-
morphology, surface markers and differentiation potency ery of cryopreserved MSCs is essential to maintain their
as description in a review [24] and proven by our previ- properties after transplantation in vivo. In this present
ous [13] and present study. However, our previous study study, the proteomics profile showed that the 47 and 81
revealed that though the morphology, surface markers proteins of hUC-MSCs were affected by freeze–thawing
and tri-lineage differentiation potency of MSCs were and a 24 h sub-culture at P4 and P10, respectively. In this
not affected by cryopreservation, the global gene expres- study, two time points (24 and 48 h) were chose in this
sion was affected either vitrified with DMSO or EG as study because over-time culture can induce over-conflu-
a cryoprotectant [13]. In the present study, many pro- ency of hUC-MSCs that is not conducive to evaluate the
tein’s expression was affected by cryopreservation and status of cells, and hUC-MSC passage with fresh culture
long-term culture revealed by the proteomics analysis. medium contains serum can affect many proteins expres-
A total of 47 and 81 proteins expressed were affected by sion, which may not reflect the true status of cells after
freezing and thawing at P4 (P4N24 vs. P4C24) and P10 thawing [28]. In P4, the different proteins were enriched
(P10N24 vs. P10C24), respectively, as well as cell com- in microRNA in cancer, small cell lung cancer, hyper-
munication and signal transduction were obviously trophic cardiomyopathy and dilated cardiomyopathy
affected though GO analysis. Therefore, in our opinion, due to the proteins such as TIMP3 (Metalloproteinase
Fu et al. Clin Proteom (2020) 17:15 Page 15 of 18

inhibitor 3), ITGA6 (Integrin alpha-6) and TPMs (Tro- 48 h at P10, it maybe that serum or nutritional compo-
pomyosins) were affected by culturing from 24 h to 48 h nents for hUC-MSCs growth was less with consumption,
(P4N24 vs. P4N48) and freeze–thawing for culturing and this would cause interference in the expression of
24 h (P4N24 vs. P4C24), and these gene were clustered a variety of proteins [28, 36]. These results suggest that
in pathway of those disease. TIMP3, ITGA6 and TPM properly prolonging the time of continuous culture after
are involved in the extracellular matrix, cytoskeleton and freeze-thawing can alleviate the effect of cryopreser-
cell adhesion that directly related to the cellular regu- vation on the change of proteins expression. In addi-
lar function, and these genes change may be caused by tion, rare studies have reported that cryopreservation
cryopreservation or cryoprotectant, and cryopreserva- reduces the homing/engraftment potential of MSCs by
tion could affect surface adhesion molecules had been poor binding to the extracellular matrix such as fibronec-
reported [29]. It is indicated that TIMP3, ITGA6 and tin and the immunosuppression ability of MSCs play an
TPM may be good markers to detecting impairment important role in MSCs homing/engraftment. However,
of cell function which is still need to be further stud- the knowledge about the recovery status of the main
ied. Many studies have shown that extracellular matrix, immunoregulation proteins of MSCs after cryopreserva-
cytoskeleton and cell adhesion are connected with lung tion and sub-culture is poor [27, 37].Therefore, it is nec-
cancer and cardiomyopathy. TIMP-3 inhibits the activity essary to sub-culture and recover the functional proteins
of metalloproteinases that play important roles in devel- of hUC-MSCs after cryopreservation and before trans-
opment and progression of lung tumors [30]. TIMP-3 is plantation, and the optimal recovery methods for MSCs
up-expressed in cardiac fibroblasts and cardiomyocytes are still need to be further explored.
but down-expressed in the failing heart [31]. Early stud- The proliferation of MSCs is limited during long-term
ies have reported that ITGA6 is involved in the occur- culture and the MSCs exhibit a aberrant phenotype of
rence and development of lung cancer [32]. It is reported irregular flattened geometry and enlarged size [38]. Yang
that ITGA6 corresponds to the activation of regeneration et al. found human bone marrow-derived MSCs undergo
involving an epithelial-mesenchymal transition in adult senescence during extensive passage and result in mor-
heart [33]. TPM is a potential marker in lung cancer phological, phenotypic and genetic changes from P4 to
diagnosis [34], and the latest study showed TPM pseudo- P8 [38]. De Witte et al. reported that long-term expan-
phosphorylation results in dilated cardiomyopathy [35]. sion induced aging of hUC-MSCs exhibiting stable phe-
However, the relationship between cryopreservation of notype but reduced immunosuppressive properties from
hUC-MSCs after long-term culture and diseases includ- P4 to P12 [39]. Facchin et al. reported that umbilical cord
ing cancer and cardiomyopathy remains unknown and Wharton’s Jelly-derived MSCs showed higher antioxidant
need to be further studied. ability to senescence than human adipose tissue-derived
The complement and coagulation cascades were alle- MSCs at high subculture passages, and they considered
viated by sub-culturing from 24 h to 48 h after freeze– that the age of tissue donors is likely to be the main cause
thawing compared with the non-cryopreserved group of senescence [40]. Moreover, recently, studies found
with a sub-culture for 24 h or 48 h parallelly. Meanwhile, that transcriptome and epigenetic regulations changes
the proteins of fat digestion and absorption, steroid of hUC-MSCs occurred during long-term expansion [41,
hormone biosynthesis, and hematopoietic cell lineage 42]. These studies not only indicated that long-term cul-
pathways were affected (P4N24 vs. P4C24 and P4N48 ture and expansion induces aging of hUC-MSCs as well
vs. P4C48). In P10, many pathways including cytokine– as genes expression changed, but also suggested that the
cytokine receptor interaction, hippo signaling pathway, antioxidant ability of hUC-MSC is superior to others that
wnt signaling pathway, microRNA in cancer, small cell were derived from human adult such as bone marrow
lung cancer, NF-kappa B signaling pathway and others and adipose tissue. In this present study, the morphol-
were significantly alleviated by sub-culturing from 24 h ogy, surface markers expression, tri-lineage differentia-
to 48 h after freeze-thawing (P10N24 vs. P10C24 and tion potency and proteomic analysis of hUC-MSCs were
P10N48 vs. P10C48). These results indicated that the evaluated after long-term culturing and expanding from
effect of cryopreservation on the protein expression of P4 to P10, and the results showed that the morphol-
MSCs at P10 was greater than those at P4. For example, ogy, surface markers and differentiation potency were
related proteins of complement and coagulation cascades not affected but large scale of proteins were changed
including CLU (Clustering), PLAU(Urokinase-type plas- from P4 to P10, which involve in proteins related to cell
minogen activator), C3 (Complement C3) and F3(Tissue cycle and P53 pathways including CCNB1(G2/mitotic-
factor) were not recovered until a sub-culture to 48 h at specific cyclin-B1), CCND1(G1/S-specific cyclin-
P4, and related proteins of Th17 cell differentiation IL-1B D1), CHEK1 (Serine/threonine-protein kinase Chk1),
and SMAD3 were not recovered until a sub-culture to RRM2(Ribonucleoside-diphosphate reductase subunit
Fu et al. Clin Proteom (2020) 17:15 Page 16 of 18

M2), SERPINE1(Plasminogen activator inhibitor 1) MSCs function, and CHEK1, SERPINE1, PDGFRB and
and P53 pathway has been reported to relate to aging JUN were also enriched in aging pathway of MSCs bio-
of MSCs in previous studies [6, 43, 44]. Superoxide dis- logical process. Therefore, these proteins may be used as
mutase 2 (SOD2) has been reported to participate in the indicators for the detection of MSCs after cryopreserva-
aging of MSCs [45, 46]. In the present study, superoxide tion and long-term culturing. However, whether these
dismutase 2 (SOD2) is up-regulated in MSCs at P10 com- proteins can be used as markers in clinical detection
pare to those at P4, which indicated that oxidative stress remains to be further studied.
may be activated.
The identified differential proteins of hUC-MSCs cry-
opreserved and thawed at P4 and P10 were enriched in Conclusion
the biological processes pathways of GO classification The morphology, surface markers and tri-lineage differ-
including differentiation, immunoregulation, wound entiation potential of P4 and P10 hUC-MSCs were tested
healing and regeneration, apoptotic signaling pathway, after cryopreservation and a sub-culturing for 24 h and
oxidation resistance, cartilage development, regulation 48 h which was compared with non-cryopreservation
of cytokine production, cell migration, aging and others and sub-culturing 24 h and 48 h, and the results showed
as shown in Table 1, and some proteins were enriched no obvious differences among these groups. However, the
and appeared multiple times in various signaling path- proteomics analysis found that cryopreservation leads to
ways of hUC-MSCs biological processes including STC1 changes in a large number of proteins expression com-
(Stanniocalcin-1), TNFAIP3 (Tumor necrosis factor pared to those of the controls. This report is the first to
alpha-induced protein 3), SERPINE1, COL1A1 (Col- show the different effects of freeze-thaw and long-term
lagen alpha-1(I)), PDGFR (Platelet-derived growth fac- culture on the proteome of hUC-MSCs. These results
tor receptor), NCAM1 (Neural cell adhesion molecule will be beneficial to understand the biological process
1), C3, JUN (Transcription factor AP-1), GATA6 (Tran- involved in the cryopreservation and long-term culture
scription factor GATA-6), HGF (Hepatocyte growth fac- of hUC-MSCs and contribute to improved cryopreser-
tor), F3 and other proteins likely be used as markers to vation protocols that maintain proteomic identity for
evaluate hUC-MSCs after cryopreserving and long-term clinical research, and promote scientists’ attention to
culturing. MSCs can secrete STC1 to protect cancer the recovery of main proteins and MSCs function after
cells from apoptosis by reducing reactive oxygen radi- cryopreservation. This will also provide a foundation for
cal (ROS), it suggests that STC1 play an important role safety detection and standardization guide of hUC-MSCs
in antioxidant activity of MSCs [47]. The deficiency of applications in clinical.
TNFAIP3 in MSCs can induce immune thrombocytope-
nia and influence megakaryocytic differentiation through Supplementary information
Supplementary information accompanies this paper at https​://doi.
terminating the NF-κB pathway that suggests TNFAIP3 org/10.1186/s1201​4-020-09279​-6.
play a critical role in the process of MSCs alleviate s
autoimmune disease [48]. The mutation of COL1A1 and
Additional file 1: Table S1. The raw data of pathways and differentially
COL1A2 in MSCs could cause osteogenesis imperfecta, it expressed proteins enriched in KEGG database among the eight groups.
likely that COL1A1 and COL1A2 play an important role
in osteogenesis differentiation from MSCs [49]. PDGFR Abbreviations
signaling is emerging as a critical regulatory mechanism hUC-MSCs: Human umbilical cord-derived mesenchymal stem cells; KEGG:
and important therapeutic target that critically directs Kyoto Encyclopedia of Genes and Genomes; DMSO: Dimethyl sulfoxide; GO:
Gene Ontology; iTRAQ: Isobaric tags for relative and absolute quantification;
the fate of mesenchymal stem cells during postnatal neo- TMT: Tandem mass tags; PRM: Parallel reaction monitoring; P4N24: Non-
vascularization [50]. It is reported that JUN not only can cryopreserved and sub-cultured for 24 h at P4; P4C24: Cryopreserved and sub-
regulate human bone marrow MSCs differentiates into cultured for 24 h at P4; P4N48: Non-cryopreserved and sub-cultured for 48 h
at P4; P4C48: Cryopreserved and sub-cultured for 48 h at P4; P10N24: Non-
neuron-like cells and acilitates neurite outgrowth, but cryopreserved and sub-cultured for 24 h at P10; P10C24: Cryopreserved and
also play a key role in human MSCs aging and therapeu- sub-cultured for 24 h at P10; P10N48: Non-cryopreserved and sub-cultured for
tic potency maintaining [51, 52]. C3 was secreted from 48 h at P10; P10C48: Cryopreserved and sub-cultured for 48 h at P10.
MSCs that has an important role in the immunomodu- Acknowledgements
latory and liver regeneration [53, 54]. HGF may have an The authors would like to thank Open Foundation of State Key Laboratory of
important role in MSC recruitment sites of tissue regen- Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environ-
mental Sciences, Chinese Academy of Sciences.
eration, and may be beneficial in tissue engineering and
cell therapy employing hMSCs [55]. These proteins such Authors’ contributions
as STC1, TNFAIP3, SERPINE1, COL1A1, PDGFR, C3, XF, XP and WS wrote the manuscript. XF, BMI and WS revised the manuscript.
BX, XD, XY and YY analysed the data. XF and WS designed the experiment. BX,
JUN and HGF present important roles in maintaining
Fu et al. Clin Proteom (2020) 17:15 Page 17 of 18

SL, HM and HW operated experiment. All authors read and approved the final 10. Si YL, Zhao YL, Hao HJ, Fu XB, Han WD. MSCs: biological characteristics,
manuscript. clinical applications and their outstanding concerns. Ageing Res Rev.
2011;10(1):93–103.
Funding 11. Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I. Study
This work was supported by grants from the National Natural Science of telomere length reveals rapid aging of human marrow stromal cells
Foundation of China (Grant Nos. 81960270, 31872973, 31560329), Major following in vitro expansion. Stem Cells. 2004;22(5):675–82.
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