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Stemness properties of SSEA-4+
subpopulation isolated from
heterogenous Wharton’s jelly
mesenchymal stem/stromal cells
Agnieszka Smolinska
1
, Magdalena Chodkowska
1
,
Agata Kominek
2
, Jakub Janiec
2
, Katarzyna Piwocka
2
,
Dorota Sulejczak
3
and Anna Sarnowska
1
*
1
Translational Platform for Regenerative Medicine, Mossakowski Medical Research Institute, Polish
Academy of Sciences, Warsaw, Poland,
2
Laboratory of Cytometry, Nencki Institute of Experimental
Biology, Polish Academy of Sciences, Warsaw, Poland,
3
Department of Experimental Pharmacology,
Mossakowski Medical Research Institute, Polish Academy of Sciences, Warsaw, Poland
Background: High heterogeneity of mesenchymal stem/stromal cells (MSCs) due
to different degrees of differentiation of cell subpopulations poses a considerable
challenge in preclinical studies. The cells at a pluripotent-like stage represent a
stem cell population of interest for many researchers worldwide, which is worthy
of identification, isolation, and functional characterization. In the current study,
we asked whether Wharton’s jelly-derived MSCs (WJ-MSCs) which express
stage-specific embryonic antigen-4 (SSEA-4) can be considered as a
pluripotent-like stem cell population.
Methods: SSEA-4 expression in different culture conditions was compared and
the efficiency of two cell separation methods were assessed: Magnetic Activated
Cell Sorting (MACS) and Fluorescence Activated Cell Sorting (FACS). After
isolation, SSEA-4+ cells were analyzed for the following parameters: the
maintenance of the SSEA-4 antigen expression after cell sorting, stem cell-
related gene expression, proliferation potential, clonogenicity, secretome
profiling, and the ability to form spheres under 3D culture conditions.
Results: FACS allowed for the enrichment of SSEA-4+ cell content in the
population that lasted for six passages after sorting. Despite the elevated
expression of stemness-related genes, SSEA-4+ cells neither differed in their
proliferation and clonogenicity potential from initial and negative populations nor
exhibited pluripotent differentiation repertoire. SSEA-4+ cells were observed to
form smaller spheroids and exhibited increased survival under 3D conditions.
OPEN ACCESS
EDITED BY
Selim Kuci,
University Hospital Frankfurt, Germany
REVIEWED BY
Tokiko Nagamura-Inoue,
The University of Tokyo, Japan
Ajoy Aloysius,
University of Kentucky, United States
*CORRESPONDENCE
Anna Sarnowska,
asarnowska@imdik.pan.pl,
RECEIVED 23 May 2023
ACCEPTED 17 January 2024
PUBLISHED 22 February 2024
CITATION
Smolinska A, Chodkowska M, Kominek A,
Janiec J, Piwocka K, Sulejczak D and
Sarnowska A (2024), Stemness properties of
SSEA-4+ subpopulation isolated from
heterogenous Wharton’s jelly mesenchymal
stem/stromal cells.
Front. Cell Dev. Biol. 12:1227034.
doi: 10.3389/fcell.2024.1227034
COPYRIGHT
© 2024 Smolinska, Chodkowska, Kominek,
Janiec, Piwocka, Sulejczak and Sarnowska. This
is an open-access article distributed under the
terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that the
original publication in this journal is cited, in
accordance with accepted academic practice.
No use, distribution or reproduction is
permitted which does not comply with these
terms.
Abbreviations: AD-MSCs, adipose-derived mesenchymal stem/stromal cells; BDNF, brain-derived
neurotrophic factor; bFGF, basic fibroblast growth factor; Cal-AM, Calcein AM; CCL2, chemokine
ligand 2; CFU, colony forming unit; EGF, epithelial growth factor; ESC, embryonic stem cells; EthD-
1, Ethidium homodimer-1; FACS, Fluorescence Activated Cell Sorting (FACS); GDNF, glial cell line-
derived neurotrophic factor; ICAM-1, intercellular adhesion molecule 1; iPSCs, induced pluripotent stem
cells; LIF, leukemia inhibitory factor; MACS, Magnetic Activated Cell Sorting (MACS); MSCs, mesenchymal
stem/stromal cells; PDT, population duplication time; RT-qPCR, Real Time-Quantitative Polymerase
Chain Reaction; SSEA-4, Specific stage embryonic antigen-4; UC-MSCs, umbilical cord-derived
mesenchymal stem/stromal cells; VEGF-c, vascular endothelial growth factor-c; WJ-MSCs, Wharton
jelly mesenchymal stem/stromal cells; WJ-MSC-SSEA-4 +, SSEA-4-positive WJ-MSC population; WJ-
MSC-SSEA-4-, SSEA-4-negative WJ-MSC population.
Frontiers in Cell and Developmental Biology frontiersin.org01
TYPE Original Research
PUBLISHED 22 February 2024
DOI 10.3389/fcell.2024.1227034
Conclusion: Despite the transient expression of stemness-related genes, our
findings could not fully confirm the undifferentiated pluripotent-like nature of
the SSEA-4+ WJ-MSC population cultured in vitro.
KEYWORDS
mesenchymal stem/stromal cells, SSEA-4, stemness, pluripotent, FACS, MACs,
heterogeneity
1 Introduction
According to the guidelines published in 2006 by the
International Society for Cell & Gene Therapy, mesenchymal
stem/stromal cells (MSCs) are multipotent adult cells that
differentiate toward mesodermal lineage tissues: osteocytes,
chondrocytes, and adipocytes (Dominici et al., 2006). However,
many research groups suggested a wider MSC differentiation
potential by providing protocols to obtain other cells such as
neurons or hepatocytes (Zhao et al., 2016). Some researchers
went even further and claimed that MSCs might manifest the
properties of pluripotent-like cells by the expression of stemness-
related transcription factors (such as Sox2, Nanog, and Oct4) and
differentiation toward cells from all three germ layers. Even though
the reported observations are controversial and disputable, the
observed discrepancies between research groups can be explained
by MSC heterogeneity.
Heterogeneity poses a serious issue for further research as only a
small fraction of cells in MSC populations appear to fulfill functional
criteria for stem cells (Ivanovic, 2023). The existence of surface
antigens associated with other cell types is one of the observed
aspects of MSC heterogeneity. Researchers propose numerous
candidates for a genuine stem population to improve the
efficiency of MSC therapies (Lv et al., 2014) based on induced
pluripotent stem (iPS) and embryonic stem cell studies. Cells
positive for SSEA-3, an early embryonic antigen, were confirmed
to differentiate toward cells from all three germ layers (Kuroda et al.,
2010) although they were present in the initial population in a
negligible percentage, which does not correspond to the plasticity of
MSCs. MSCs positive for CD271, an antigen typical of neural crest-
derived cells, proliferated more rapidly and contained more cells
capable of forming colonies (Mikami et al., 2011;Barilani et al.,
2018). MSCs expressing CD146, an antigen associated with
endothelial cells, exhibited a greater ability to migrate to
damaged tissue (Wangler et al., 2019). CD133, a surface antigen
associated with glioblastoma cells, was also suggested as a potential
marker of stemness population in umbilical cord-derived MSCs
(UC-MSCs) and MSCs derived from adipose tissue (AD-MSCs)
(Doshmanziari et al., 2021). Another iPS- and embryonic stem cell
(ESC)-expressed marker found in a much higher proportion of
MSCs is stage-specific embryonic antigen-4 (SSEA-4).
SSEA-4 appears during early embryonic development
(Henderson et al., 2002) but is also found on undifferentiated
cells such as embryonic stem cells (ESCs) (Draper et al., 2002;
Kallas et al., 2011), induced pluripotent stem cells (iPSCs) (Ojima
et al., 2015), and various types of tumor cells (Sivasubramaniyan
et al., 2015;Nakamura et al., 2019;Lee et al., 2021). Many
publications reported the expression of SSEA-4 within MSC
populations within the range of 30%–90% (Drela et al., 2016;
Musiał-Wysocka et al., 2019). Despite the abundance of evidence
on pluripotent-like properties of SSEA-3, researchers debate
whether SSEA-4 may also be a prognostic marker for genuine
stem cell populations (Gang et al., 2007;Kawanabe et al., 2012).
Targeting SSEA-4 is a strategy for stem population selection in
undifferentiated ESCs from differentiated derivatives (Fong et al.,
2009) and neural stem/progenitor cells from the human embryonic
forebrain (Barraud et al., 2007). SSEA-4 was also suggested as an
identifier of tumor-initiating subpopulations and proposed as a
target for the therapies (He and Garcia, 2004;Sivasubramaniyan
et al., 2015;Soliman et al., 2020). In our previous paper, long-term
3D culture was observed to increase the content of SSEA-4+ cells,
thereby suggesting that SSEA-4 could help toward survival under
harsh 3D conditions (Kaminska et al., 2021).
This study was carried out to determine the therapeutic benefits
of the SSEA-4-positive cells from Wharton’s jelly MSCs (WJ-MSCs)
as a potential pluripotent-like stem cell population responsible for
so-called “MSCs plasticity”with restorative (replacing injured cells)
properties. To identify the MSC-SSEA-4 + subpopulation’s unique
properties, it was compared both to the negative population, without
SSEA-4, (WJ-MSC-SSEA-4-) and to the heterogenous MSC
populations (unsorted WJ-MSC) (experimental steps explained in
Supplementary Figure S1). Our experiments allowed us to establish
the most favorable conditions for SSEA-4 expression and separation
while the positive subpopulation analyses provided full
characteristics of stemness-related properties.
2 Materials and methods
2.1 WJ-MSCs isolation and primary culture
Human umbilical cords were acquired from full-term deliveries
with the written consent of the mother according to the Warsaw
Medical University Ethics Committee Guidelines (KB/213/2016).
The cords (15–20 cm long) were first transported in phosphate-
buffer saline solution (PBS; Sigma-Aldrich, Saint Louis, MO,
United States) with a mixture of penicillin-streptomycin-
amphotericin B (1:100, Gibco, Thermo Fisher Scientific,
Walthman, United States) and then cut into slices with a lancet
(slice thickness: 2–3 mm). Wharton’s jelly cylindrical fragments of
2–3 mm in diameter were obtained from the umbilical cord using
the diameter biopsy punch (Miltex, GmbH, Viernheim, Germany).
The explants were transferred to 6-well culture plates and cultured
in the following medium standard for WJ-MSC culture: DMEM
(Gibco), 5% human platelet cell lysate (Mill Creek Life Sciences,
Rochester, MN, United States), and penicillin-streptomycin-
amphotericin B (1:100; Gibco). The following cell culture
conditions were applied: adherent surface, 37
O
C temperature,
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95% humidity, 5% CO
2
concentration, and 5% O
2
concentration.
The culture medium was replaced every 2 days for 14 days in vitro.
The cells were cultured until they migrated out of the explant and the
culture reached semiconfluence; then the cells were detached with
Accutase Cell Detachment Solution (Beckton Dickinson, Franklin
Lakes, NJ, United States) and counted.
We compared SSEA-4 expression in populations cultured with
different human platelet cell lysates such as PLTGold Clinical Grade
(Mill Creek Life Sciences), MultiPL’30 (Macopharma), and
MultiPL’100 (Macopharma, Tourcoing, France). In further
experiments, PLT Gold Clinical Grade was used as a lysate. The
WJ-MSCs were cultured under the conditions described above for
three passages. The cells were collected and cell sorting
was performed.
2.2 Flow cytometry
The cells were detached with Accutase Cell Detachment Solution
(BD), washed in PBS, and resuspended in Stain Buffer (BD). Flow
cytometry analyses were performed with antibodies listed in Table 1.
The cells were incubated in diluted antibodies in the dark for 30 min.
After incubation, the cells were washed twice with Stain Buffer (BD)
and resuspended in Stain Buffer. The resuspended cells were
analyzed using FACS Canto II (BD) with FACSDiva software
(BD) and FlowJo 10 (BD). The following laser configurations
were applied: violet - 407 nm (detectors: 510/50, 450/50), blue -
488 nm (detectors: 488,10, 530/30, 585/42, 670LP, 780/60), and red -
633 nm (detectors: 660/20, 780/60). The gating strategy was
presented in online resources (Supplementary Figure S2).
2.3 AD-MSCs culture
AD-MSCs isolation and culture were accepted by the Bioethical
Committee at the Centre of Postgraduate Medical Education (No.
63/PB/2013) on 25 September 2013, according to the guidelines of
the Declaration of Helsinki. Adipose tissue was collected during
liposuction in the Plastic Surgery Department at Orlowski’s Clinical
Hospital in Warsaw. The AD-MSCs were isolated according to the
previously described protocol (Figiel-Dabrowska et al., 2021;
Rybkowska et al., 2023). The isolated AD-MSCs were in MEM α
(Gibco), 5% human platelet lysate (Mill Creek Life Sciences), and 1%
penicillin-streptomycin-amphotericin B (1:100; Gibco). The
following cell culture conditions were applied: adherent surface,
37
O
C temperature, 95% humidity, 5% CO
2
concentration, and 5%
O
2
concentration. The culture medium was changed every 2–3 days
and AD-MSCs were passaged when the culture reached
semiconfluence. AD-MSCs from the third passage were detached
with Accutase Cell Detachment Solution and washed with PBS
twice. AD-MSCs were used for flow cytometry and prepared in
the manner described above.
2.4 Magnetic activated cell sorting (MACS)
isolation of SSEA-4+ cells
The WJ-MSCs from the third passage were used for MACS
separation of WJ-MSC-SSEA-4+ cells. WJ-MSCs were detached and
counted. The collected cells (~2-10 × 10
6
) were incubated with
magnetic beads using an anti-SSEA-4 MicroBead kit for 20 min in
the dark. Then, the cells were washed with PBS. The cells were
resuspended in PBS with 1% BSA and loaded into the autoMACS
Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany). The
cells were run through the magnetic field with the Possel S. For
further analysis, we used a positive cell population retained within
the column and eluted as the second fraction was collected. After
MACS sorting, the cells were counted with Trypan Blue on a
hemocytometer to calculate the total cell number and cell
viability. Then, the cells were subcultured for further experiments
as described above.
2.5 Fluorescence-activated cell sorting
(FACS) isolation of SSEA-4+ cells
The WJ-MSCs from the third passage were used for FACS
separation of WJ-MSC-SSEA-4+ cells. The cells were stained as
was the case in flow cytometry staining described in Section 2.2.
After incubation and washing, the cells were sorted using FACS Aria
IIu (BD) in the Laboratory of Cytometry, Nencki Institute of
Experimental Biology, Warsaw. The following laser
TABLE 1 List of antibodies used in flow cytometry analysis.
Specificity Fluorochrome Isotype Company Catalog number
SSEA-4 PerCP-Cy5.5 Mouse IgG3 κBD 561565
Isotype control PerCP-Cy5.5 Mouse IgG3 κBD 561572
CD271 PE Mouse IgG1 κBD 560927
CD146 PE Mouse IgG1 κBD 550315
Isotype control PE Mouse IgG1 κBD 555749
CD133 Brilliant Violet 421 Mouse IgG2B κBD 566598
Isotype control Brilliant Violet 421 Mouse IgG2B κBD 562748
CD49F PE Rat IgG2A κThermoFischer Scientific 12-0495-81
Isotype control PE Rat IgG2A κThermoFischer Scientific 12-4321-80
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configurations were applied: violet - 407 nm (detectors: 450/40, 530/
30), blue - 488 nm (detectors: 488/10, 530/30, 585/42, 616/23, 695/
40, 780/60), and red - 633 nm (detectors: 660/20, 780/60). The gating
strategy is presented in online resources (Supplementary Figure S3).
The cells were collected from both populations, SSEA-4- and SSEA-
4+, and resuspended in the cell culture medium. Directly after FACS
sorting, the obtained population was analyzed with FACS Aria again
to confirm the purity of sorting. Then, the cells were transported to
our laboratory where we counted the total cell number and viability
with Trypan Blue on a hemocytometer. Then, the cells were seeded
in a culture dish and subcultured for further experiments as
described above.
2.6 Parameters of cell sorting
The following parameters of MACS and FACS sorting, which
are recovery, survival, and yield, were compared in order to
determine a more efficient method of SSEA-4+ cell separation.
Recovery was expressed as the ratio of the number of cells
obtained in the positive fraction to the number of cells used in
the sorting. To calculate recovery, we counted cells prior to sorting
and in post-sorting fractions. To describe survival, we investigated
the mortality of cells in samples received after cell separation with
Trypan Blue staining. The yield was expressed as the ratio of positive
cell content before and after cell separation. Purity was described as
the percentage of SSEA-4+ cells received in a positive population
sample. To estimate yield, the samples were analyzed with
flow cytometry.
2.7 Immunocytochemistry
Immunocytochemistry was performed to detect SSEA-4 and
CD90, one of the surface antigen characteristics of MSCs, for the
following populations: unsorted WJ-MSC, WJ-MSC-SSEA-4-, and
WJ-MSC-SSEA-4+. WJ-MSCs were washed with PBS and fixed in 4%
PFA for 15 min. The samples were incubated with a blocking mixture
consisting of 10% Goat Serum (Sigma Aldrich) and 1% bovine serum
albumin (Sigma Aldrich) for 1 h at room temperature (RT). In the
next step, primary antibodies were applied for 24 h at 4°C(Table 2).
The next day, the cells were washed with PBS and then incubated with
secondary antibodies conjugated with fluorochrome for 1 h (Table 2).
Finally, the samples were mounted with Fluoromont-G with DAPI
(Gibco) that stained cell nuclei. The analysis was performed using a
confocal microscope (Zeiss, Oberkochen, Germany).
2.8 Real time-quantitative polymerase chain
reaction (RT-qPCR)
Total RNA was isolated from the following groups: unsorted
WJ-MSCs, negative and positive populations after FACS sorting
(WJ-MSC-SSEA-4- p0 and WJ-MSC-SSEA4+ p0, respectively), and
negative and positive fractions cultured for 1 passage in vitro (WJ-
MSC-SSEA-4- p1 and WJ-MSC-SSEA4+ p1, respectively). RNA
isolation was performed using the following kits depending on
the cell number: Total RNA Mini Plus kit (A&A Biotechnology,
Gdynia, Poland) and Total RNA Mini Plus Concentrator (A&A
Biotechnology) according to the manufacturer’s protocols. After
isolation, the RNA was eluted with 20 µL of RNase-free H
2
O (Sigma
Aldrich). The quantity and quality of RNA were assessed using a
NanoDrop 2000 spectrophotometer (Thermo Scientific). Genomic
DNA (gDNA) contamination was eliminated in all RNA samples
using a Clean-up RNA Concentrator (A&A Biotechnology, Thermo
Fisher Scientific, Walthman, United States).
The reverse transcription process was generated using a High-
Capacity RNA-to-cDNA™Kit (Applied Biosystems) according to
the manufacturer’s instructions. After receiving complementary
strand DNA (cDNA), the samples were diluted in RNase-free
water. Quantitative polymerase chain reactions were performed
using SYBR green Master Mix (Applied Biosystems) and specific
primers (Supplementary Table S1) with the 7,500 Real-Time PCR
System (Applied Biosystems). The relative amount of RNA was
calculated using the comparative delta-delta Ct method (2
−ΔΔCT
) and
gene expression was normalized using β-actin (ACTB), while the
unsorted population was used as a reference group. Gene expression
was compared with the mean level of corresponding gene expression
in cells of the unsorted population and expressed as an n-fold ratio.
Gene expression was compared with the mean level of
corresponding gene expression in cells of the unsorted
population and expressed as an n-fold ratio.
2.9 Three germ layer differentiation
potential evaluation
Three germ layer differentiation potential was determined for
unsorted, positive, and negative populations. After FACS separation,
the cells were seeded on a 6-well plate and cultured in a standard
culture medium with the addition of basic fibroblast growth factor
(bFGF) (Gibco) until they reached 70%–80% confluency.
Differentiation assay was performed with the Human Pluripotent
Stem Cell Functional Identification Kit (Biotechne, R&D Systems,
TABLE 2 List of antibodies used for immunocytochemistry.
Antigen Isotype Dilution Company Applied secondary antibody Secondary antibody fluorochrome
SSEA-4 Mouse IgG3 1:200 Merck Goat anti-IgG3 Alexa Fluor 488
CD90 Mouse IgG1 1:200 Santa Cruz Goat anti-IgG1 Alexa Fluor 546
SOX17 Goat IgG H + L 1:100 R&D Donkey anti-IgG Alexa Fluor 488
Otx2 Goat IgG H + L 1:100 R&D Donkey anti-IgG Alexa Fluor 488
Brachyury Goat IgG H + L 1:100 R&D Donkey anti-IgG Alexa Fluor 488
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Minneapolis, MN, United States), which is dedicated to the
differentiation of iPSCs. The cells were cultured according to the
manufacturer’s protocol, with a culture medium based on DMEM.
After 4 days of in vitro culture, the cells were collected for RNA
isolation and evaluation for gene expression of OTX2, Brachyury,
and SOX17 (scheme of the experiment presented in Supplementary
Figure S4; primers sequences can be found in
Supplementary Table S1).
2.10 Colony forming unit (CFU) assay
To perform the CFU assay, unsorted WJ-MSCs, WJ-MSC-
SSEA-4-, and WJ-MSC-SSEA-4 + were seeded on a 6-well plate,
10 cells per well. The cells were cultured for 10 days in vitro under
standard conditions. The cells were washed with PBS, fixed with 4%
PFA for 15 min, and washed with PBS again. The fixed cells were
stained with 0.5% toluidine blue for 20 min and washed with
distilled water after staining. The number of colonies containing
50 cells or more was counted and fibroblast colony-forming units
(CFU-F) were calculated as a percentage of seeded cells.
2.11 Proliferation analysis
Cell proliferation was estimated as population duplication time
(PDT) for four passages after cell sorting for the following
populations: unsorted WJ-MSC, WJ-MSC-SSEA-4-, and WJ-
MSC-SSEA-4+. The cells from each group were counted in
Trypan Blue, seeded at a density of 2000 cells/cm2, and cultured
under standard conditions. After 5 days of in vitro culture, the cells
were collected, counted, and reseeded again at initial density. The
PDT value was calculated according to the following equation:
PDT (t−t0)×log 2
logN−log N0, where N is the number of cells obtained at the
end of the passage, N
0
is the initial number of the seeded cells and
t-t
0
is the duration of the passage (counted in days).
2.12 Soluble secretome analysis
Soluble secretome was analyzed with human Magnetic Luminex
Assay (R&D Systems, Minneapolis, MN, United States) for
unsorted, positive, and negative populations. For this purpose,
the cells after FACS sorting were seeded at a density of
2,000 cells per cm
2
. The medium from the cell culture was
collected at two time points: 3 days and 5 days in vitro after
FACS sorting. The standard culture medium was used as a
negative control. The levels of the following molecules were
measured: epithelial growth factor (EGF), bFGF, glial cell line-
derived neurotrophic factor (GDNF), brain-derived neurotrophic
factor (BDNF), chemokine ligand 2 (CCL2), leukemia inhibitory
factor (LIF), angiogenin, vascular endothelial growth factor-c
(VEGF-c), and intercellular adhesion molecule 1 (ICAM-1). The
actual levels of secreted factors were determined by subtraction of
the negative control values from the obtained results. Luminex assay
was performed according to the manufacturer’s protocol and
measured in Bio-Plex 200 System (Bio-Rad Bio-Rad, Hercules,
CA, United States).
2.13 3D culture of WJ-MSCs
Unsorted WJ-MSCs from the third passage and WJ-MSCs
directly after cell sorting (WJ-MSC-SSEA-4- and WJ-MSC-SSEA-
4+) were collected, counted, and seeded in antiadhesive 6-well plates
(Nunclon Sphera, Thermo Fischer Scientific) at a density of 10
5
cells
per 1 mL. The cells were cultured as spheroids in culture medium
and the conditions described above for 72 h in vitro. The diameters
and numbers were measured after 24, 48, and 72 h of 3D culture.
After 72 h of 3D culture, spheroids were collected and dissociated
with Accutase Cell Detachment Solution (BD) for further
viability analysis.
2.14 Viability test after 3D culture
To estimate the number of alive and dead cells after 3D culture, a
viability test was performed using a mix of ethidium homodimer-1
(8 μM, EthD-1, Invitrogen) Calcein AM (Cal-AM) (0,1 μM,
Invitrogen) and Hoechst 33,342 dye (1 μg/mL; Sigma Aldrich).
Spheroids or single cells derived from dissociated spheroids were
incubated with a staining mixture for 45 min at room temperature in
darkness. The stained cells were observed in the Axio
Vert.A1 fluorescence microscope (Zeiss). Dead and alive cells
were calculated automatically with the ZEISS ZEN 2.0 Blue
Edition software.
2.15 Statistics
The experiments were performed on the cells obtained from at
least three WJ donors (n ≥3). Normality was examined with the
Shapiro-Wilk normality test. The unpaired t-student test was used
for the data from two groups with normal distribution. The data
from multiple groups with normal distribution were analyzed by
using a one-way analysis of variance (ANOVA), followed by
Tuckey’s multiple comparison test. For the non-moral
distribution, the data was analyzed using the Kruskal–Wallis test,
followed by Dunn’s multiple comparison test. The results are
presented as mean ± standard deviation (SD) for parametric tests
or as median ±95% confidence interval (95% CI) for non-parametric
tests. The results were considered statistically significant when the
p-value was higher than 0.05. Statistical analysis was conducted with
the GraphPad Prism 7 software.
3 Results
3.1 Expression of SSEA-4 in the
heterogenous WJ-MSCs population
WJ-MSCs used for the experiments exhibited surface antigens
recommended by The International Society for Cell & Gene Therapy
for MSC characteristics (Supplementary Figure S5;Supplementary
Table S2) and differentiated toward mesodermal lineage cells:
osteocytes, adipocytes, and chondrocytes (Supplementary Figure
S6). All MSC-SSEA-4+ cells expressed the CD90, which is one of
the recommended MSC antigens (Figure 1A; negative controls are
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presented in online resources: Supplementary Figure S7). To
establish the most favorable conditions, SSEA-4+ cells present
in heterogenous MSCs were estimated with regard to i) the source
of tissue, ii) the passage number, and iii) the culture medium.
SSEA-4+ cell content was compared in two MSC populations
derived from different tissues: Wharton’s jelly and adipose
tissue (Figures 1B,C). Nearly 3.5 times more SSEA-4+ cells were
detected in WJ-MSCs than in AD-MSCs (70% ± 8.3% and 20.3% ±
11.7, respectively). No significant changes were observed in SSEA-
4 expression in WJ-MSCs obtained from the first, third, and fifth
passage of in vitro culture (Figure 1D). To choose the most optimal
culture medium composition, three commercially available platelet
lysates were compared as a source of proteins and tropic factors:
MultiLP’30, MultiLP’100, and PLTGold Clinical Grade (Figures
1E,F). MultiLP’100 and PLT Gold lysates were more abundant in
growthfactorsthanMultiLP’30. The proportion of positive cells
was the lowest under cell culture with MultiLP’30 lysate (35.7% ±
11.1), while it was found to increase in cultures using
MultiLP’100 and PLTGold lysates (74% ± 9.7% and 70% ± 8.3,
respectively) (Figure 1F). The application of a higher
concentration of platelet lysate was also observed to increase
the content of double-positive SSEA-4+ CD271+ cells in WJ-
MSC populations (Supplementary Figure S8). Although almost
all WJ-MSC-SSEA-4+ cultured with lysates at a higher
concentration expressed CD271, a similar analysis in AD-MSC
populations revealed that it was CD271+ cells that were a separate
subpopulation of SSEA-4+ cells (Supplementary Figure S6D).
Consequently, WJ-MSCs from the third passage, cultured with
PLTGold human platelet lysate, were selected for further
experiments and analysis.
FIGURE 1
SSEA-4+ cells content in the heterogenous population of WJ-MSCs. (A) SSEA-4 surface antigen together with CD90 in the heterogenous WJ-MSC
populations; immunocytochemical staining. The white scale bar indicates 100 µm . (B) Representative histograms from flow cytometry analysis for SSEA-
4+ population in WJ-MSCs and AD-MSCs. (C) Comparison of SSEA-4+ cells in MSCs isolated from different sources: Wharton’s Jelly (WJ-MSCs) and
adipose tissue (AD-MSCs). Passage number of analyzed cells: third passage; applied lysate platelet: PLTGold. (D) SSEA-4+ cell content in the WJ-
MSCs from first, third, and fifth passage of cell culture; flow cytometry. (E) SSEA-4+ cell content in the WJ-MSC populations cultured with different
platelet lysates: MultiLP’30 (Macopharma), MultiLP’100 (Macopharma), and PLTGold (Mill Creek Life Sciences); flow cytometry analysis. (F) Representative
histograms from flow cytometry analysis for the SSEA-4+ population from WJ-MSCs cultured in different platelet lysates: MultiLP’30, MultiLP’100, and
PLTGold. For (B),(D),and(E) results are presented as mean values of three experiments ±SD. p-value for ** <0.01.
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3.2 Comparison of sorting methods for WJ-
MSC-SSEA-4+ cell separation
To choose a more optimal WJ-MSC-SSEA-4+ cell separation
method, MACS and FACS were considered. The following
parameters were compared: recovery, which was expressed as the
ratio of the positive cell numbers obtained to the cell number used in
sorting; survival, which was expressed as cell mortality and yield and
as the ratio of positive cells before and after sorting. FACS allowed us
to obtain more than 13 times as many cells as with MACS(MACS:
1.6% ± 0.9 vs. FACS: 21.4% ± 7.4) (Figure 2A). We observed a
decrease in the cell viability after MACS sorting, but the differences
were not statistically significant due to substantial discrepancies in
the values obtained in MACS sorting (MACS: 62.75% ± 24.3 vs.
FACS: 89% ± 2) (Figure 2B). Our calculations performed with
Trypan Blue staining were also supported by EthD-1 staining
used in the pilot experiment (Supplementary Figure S9). A
slightly higher yield was recorded for the FACS method, with the
differences being statistically insignificant (MACS: 99.9% ± 11.9 vs.
FACS: 148.9% ± 44.4) (Figure 2C). SSEA-4+ cell content recorded
before and after sorting was compared to calculate yield value
(Figure 2D). Overall, FACS sorting was selected for our further
experiments as the method showed superior cell recovery and
tendencies toward decreased mortality and increased yield.
Figure 2D shows a significant difference in SSEA4 expression in
the initial population, which resulted from the difference in the
cytometer assigned to the method. MACS sorting was analyzed
using FACS Canto. For FACS sorting, we used the integrated FACS
Aria IIu (BD), which differed in the detector settings (as described in
the materials and method section). The results obtained may explain
the discrepancies in the SSEA4 expression of the different study
groups. Nevertheless, the same cytometer was always used in our
further experiments, such as the persistence of SSEA-4+ cells after
sorting or co-expression of other surface antigens.
3.3 SSEA-4+ cells after FACS separation
As a result of FACS, two groups of cells were received, which are the
negative subpopulation (WJ-MSC-SSEA-4-) and the positive
FIGURE 2
Parameter comparison of sorting methods: Magnetic Activated Cell Sorting (MACS) and Fluorescence Activated Cell Sorting (FACS). (A) Percent of
recovered SSEA-4+ cells after cell sorting; p-value for **<0.01. (B) Viability of cells in population after cell sorting. (C) So rting yield expressed as a change
in SSEA-4+ cell content calculated before and after sorting. (D) Representative histograms from flow cytometry analyses of SSEA-4 cell content before
and after sorting for MACS and FACS were used to calculate yield. For (B) and (D) results are presented as mean values of at least four
experiments ±SD. p-value for ** <0.01.
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subpopulation (WJ-MSC-SSEA-4+) (Figure 3). The content in the
unsorted population before FACS and in populations received after
FACS was measured to examine purity and yield (Figure 3A). The
FACS method resulted in 87.4% ± 4.3 of SSEA-4+ cells in the positive
population, while the negative population contained 1.2% ± 1.6 of
SSEA-4+ cells (Figure 3B). SSEA-4+ cell content in positive and
negative populations was monitored for the next six passages
(Figures 3B,C). For the first four passages, both populations differed
significantly in SSEA-4+ percentage. SSEA-4+ cell content was observed
to increase in the negative population. In the sixth passage after FACS,
no differences between the analyzed populations were recorded.
Immunocytochemical staining revealed notably more SSEA-4+ cells
in the positive population after FACS separation (Figure 3D).
3.4 Pluripotent-like properties of WJ-MSC-
SSEA-4+ subpopulation
The expression of genes associated with pluripotent stem cells
(Nanog, Oct4, and Sox2) was compared to confirm the
undifferentiated state of the SSEA-4+ population (Figure 4A).
RNA was collected directly after cell sorting (p0) and after one
passage of in vitro cell culture (p1) for the populations received with
FACS. WJ-MSC-SSEA-4- + population exhibited increased
expression of Nanog and Oct4 directly after cell sorting, which
decreased with cell culture. The expression of early neural genes
connected with the ectoderm germ layer was also analyzed
(Figure 4B). An increased expression of H3TUBULIN, NESTIN,
FIGURE 3
Persistence of SSEA-4+ cells after FACS in WJ-MSC populations: unsorted WJ-MSCs, WJ-MSC-SSEA-4+, and WJ-MSC-SSEA-4-. (A) Purity and
yield of FACS—the comparison of unsorted WJ-MSCs before FACS and in positive and negative populations after FACS; representative histograms. (B)
SSEA-4+ cells content dynamics during cell culture after FACS. The results are presented as mean values of three experiments ±SD, p-value for
** <0.01 and *** <0.001. (C) SSEA-4+ cell content changes after 1, 2, 4, and 6 passages after FACS; representative histograms. (D) SSEA-4 co-
expression with CD90 for unsorted populations and positive and negative populations at the end of the first passage after FACS. White scale bars
indicate 100 µm.
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FIGURE 4
Stemness properties of WJ-MSC populations. (A, B) Relative gene expression level (fold change, mean ± SD) of pluripotency (A) and neural (B) associated
genes. Quantitation was determined relative to ACTB by quantitative real-time PCR. Changes in gene expression are shown relative to the unsorted WJ-MSC
populations (value = 1). The expression of the following groups was analyzed: unsorted WJ-MSCs, negative fraction collected directly after sorting (WJ-MSC-SSEA-
4- p0), positive fraction collected directly after sorting (WJ-MSC-SSEA-4+ p0), negative fraction cultured for one passage in vitro (WJ-MSC-SSEA-4- p1), and
positive fraction cultured for one passage in vitro (WJ-MSC-SSEA-4+ p1). The results shown are the mean of three independent RNA isolations, with the p-value of
*<0.05, ** <0.01, ***<0.001, and ****<0.0001. (C) Immunocytochemical analysis of Brachyury, Otx2, and Sox17 (green) expression. Nuclei were counterstained
with DAPI (blue). Scale bar = 100 μm. (D) The relative gene expression level (fold change, mean ± SD) of genes associated with specific germ layers, OTX2
(ectoderm), BRACHYURY (mesoderm), and SOX17 (endoderm), after differentiation. Quantitation was determined relative to ACTB by quantitative real-time PCR.
Changes in gene expression are shown relative to the unsorted WJ-MSC populations (value = 1). The results shown are the mean of three independent RNA
isolations. (E) Colony forming unit (CFU) frequency assay. (F) Population Doubling Time (PDT). The results are presented as mean values of three experiments ±SD.
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and GFAP was observed in the positive population directly after
FACS sorting; the expression of H3TUBULIN was still elevated after
one passage of cell culture.
Subsequently, the potential to differentiate toward cells derived
from three germ layers was compared between unsorted and sorted
populations (Figures 4C,D;Supplementary Figure S4). Final effects
were evaluated with the measurement of specific gene
expression–OTX2 for ectodermal differentiation, BRACHYURY
for mesodermal differentiation, and SOX17 for endodermal
differentiation–and visualized with immunocytochemical staining.
Both populations received in FACS sorting, WJ-MSC-SSEA-4+, and
WJ-MSC-SSEA-4- exhibited increased expression of BRACHYURY,
compared to the unsorted population, but the differences were not
statistically significant. Unchanged OTX2 and SOX17 expression
indicated that the WJ-MSC-SSEA-4+ population lacked pluripotent
potential (Figure 4D). The immunocytochemical analysis confirmed
the abovementioned results: high expression for the mesodermal
marker (Brachyury) and no clear expression for ectodermal and
endodermal lineage. No difference between all three populations was
observed (Figure 4C). Additionally, the observed mesodermal
differentiation capacity toward osteocytes, adipocytes, and
chondrocytes was similar for both analyzed populations
(Supplementary Figure S10).
The physiological properties of the analyzed
subpopulations–clonogenicity and proliferation–between passages
were also investigated. Clonogenicity was examined with the CFU
assay (Figure 4E). Proliferation was described as the PDT for five
passages after FACS (Figure 4F). No significant differences were
recorded between unsorted, negative, and positive subpopulations in
the CFU assay and PDT measurements. With regard to proliferation
and clonogenicity, the SSEA-4+ population propagated in the
in vitro culture as was the case in the negative and initial
populations.
3.5 Expression of other surface antigens
within WJ-MSC populations after SSEA-4+
enrichment
We examined whether the SSEA-4+ cell enrichment influenced
the expression of other surface antigens associated with other stem
cells still found within MSC populations, which are CD49F, CD133,
CD146, and CD271 (Figure 5). We did not observe significant
changes in the percentage of the surface antigens before and after
cell sorting. WJ-MSC subpopulations contained 84%–94% of
CD49F + cells (Figures 5A,B), 1.4%–3.5% of CD133+ cells
(Figures 5C,D), 74%–79% of CD146+ cells (Figures 5E,F), and
2.4%-4% of CD271+ cells (Figures 5G,H). For each antigen, the
fold in expression was calculated in relation to the unsorted
population (Figure 5I). The most significant changes were
observed for antigens that were sparse in population, such as
CD133 and CD271. However, we did not record any statistically
significant differences again, potentially due to considerable
discrepancies between samples received from different donors.
Additionally, the same analysis was performed for AD-MSCs
isolated from adult donors and a significantly lower number of
CD49F cells (34.2% ± 14.5) was found, while the CD271+
subpopulation was more numerous than in the WJ-MSCs
(32.4% ± 7.8) (Supplementary Figure S11). Our surface antigen
analysis revealed that the SSEA-4+ population could hardly be
connected with other unique subpopulations.
3.6 Secretory profiles of subpopulations
In the next step, we compared the secretory profiles of unsorted
WJ-MSCs, WJ-MSC-SSEA-4-, and WJ-MSC-SSEA-4+ subpopulations
on days 3 and 5 in vitro after FACS sorting (Figure 6). We measured the
levels of selected trophic factors (EGF, bFGF, GDNF, and BDNF),
cytokines and chemokines (CCL2 and LIF), and vascular factors
(angiogenin, VEGF-c, and ICAM-1). The unsorted population
exhibited an increased secretion of BDNF, HGF, and GDNF on the
third day of our observation. The WJ-MSC-SSEA-4- population
secreted less VEGF-c on the third day after FACS sorting. The
secretion profile slightly differed on the fifth day after FACS sorting.
TheWJ-MSC-SSEA-4-populationsecretedhigherlevelsofbFGFand
LIF, compared to other variants. The WJ-MSC-SSEA-4+ population
secreted a higher level of CCL2 and a lower level of VEGF-c. Except for
soluble molecules described above, no significant differences in
secretion of other factors were recorded between compared groups
while the levels of EGF were found to be lower than the levels observed
in the culture media of negative controls (Supplementary Figure S12).
3.7 Sphere formation ability in the WJ-MSC-
SSEA-4+ population
Finally, we analyzed the influence of WJ-MSC-SSEA-4+
enrichment on the sphere formation ability in the 3D culture. The
3D culture was carried out with anti-adherent culture plates for 72 h
in vitro. All the analyzed populations were observed to form spheroids
with a small diameter (15–50 µm) (Figure 7A). For each day, we
counted the cell number per 10,000 seeded cells (Figure 7B)and
measured their diameters (Figures 7C–E); spheroids were grouped
according to their size: small spheres (smaller than 20 µm), medium
spheres (20–50 µm), and large spheres (larger than 50 µm) (Figures
7F–H). A decrease in spheroid number was observed in 3D culture
(Figure 7B). After the first 24 h, WJ-MSC-SSEA-4- cells formed
significantly smaller spheroids than the unsorted subpopulation
(95% CI, 25.3–27.6 vs. 29.15–32.4, respectively), while WJ-MSC-
SSEA-4+ cells were found to form the smallest spheroids (95% CI
range: 23.4–25.6 µm) (Figure 7C). After 48 h of 3D culture, unsorted
WJ-MSCs formed significantly bigger spheres than negative and
positive populations (95% CI ranges, 35.89–39.47 vs.
31.41–34.82 vs. 31.65–34.03, respectively) (Figure 7D). After 72 h,
no significant differences were detected in the diameter of the
spheroid between the analyzed variants (Figure 7E). The medium-
sized spheroid predominated in all the compared groups in 3D culture
(Figures 7F,G). The percentage of spheres of different sizes varied over
time. A significantly larger number of small spheroids was identified
in negative and positive subpopulations during the first 24 h than in
subsequent days of 3D culture. Similarly, we observed fewer medium-
size spheres in the positive subpopulations and larger spheres in
unsorted and negative subpopulations. WJ-MSC-SSEA-4+ cells
formed smaller spheres during the first 24 h of 3D culture;
however, the differences diminished with the duration of 3D culture.
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The viability of cells after 3D culture was evaluated with live-
dead staining using Cal AM, EthD-1, and Hoechst after 72 h of 3D
culture (Figure 8A). Cal AM-stained live cells in green, while EthD-1
bonded with nucleic acid, indicating dead cells. Dead cells were
mostly observed in the spheroid core. WJ-MSC-SSEA-4+
subpopulation contained significantly fewer dead cells than
unsorted and negative subpopulations (6.6 ± 1.7 for WJ-MSC-
SSEA-4+ vs. 8.4 ± 2.7 for unsorted WJ-MSCs vs. 11.4 ± 2.8 for
WJ-MSC-SSEA-4-) (Figure 8B). Our experiments revealed that
SSEA-4+ cells formed smaller spheres during the first 24 h of 3D
culture; WJ-MSC-SSEA-4+ cells were characterized with better
survival during 3D culture.
4 Discussion
The heterogeneity of the MSCs arises from multiple factors
ranging from differences between donors, isolation sites, and
FIGURE 5
Unique surface antigens before and after FACS for the following populations: unsorted WJ-MSCs, WJ-MSC-SSEA-4- and WJ-MSC-SSEA-4+.
(A–H). Flow cytometry analyses –representative histograms and gathered results for CD49F (A, B), CD133 (C, D), CD146 (E, F), CD271 (G, H). Grey
histogram –isotype control. (I). Change in the content of the analyzed antigens after cell sorting compared to the content observed in the unsorted
population. For (B, D, F, H and I) the results are presented as mean values of 3 experiments ± SD.
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methods to a variety of proposed culture conditions (Lech et al.,
2016;Costa et al., 2021;Wedzinska et al., 2021). Moreover, the
heterogeneity issue is even more multifaceted as the cells within an
established in vitro culture differ in morphology, size, phenotype,
and differentiation potency (Sun et al., 2020;Wang et al., 2021;
Zhang et al., 2022). Confirmed differentiation of multipotent MSCs
toward neuron-like cells could result from intrinsic cell plasticity or
contamination by cells of different origins (Somoza et al., 2008). The
heterogeneity of MSCs causes a serious limitation in the translation
of MSC studies for further clinical research. The application of a
homogeneous subpopulation of morphologically similar cells would
not only overcome this problem but would also result in better
therapeutic outcomes as separated cells could exhibit outstanding
properties such as faster proliferation (Kawamura et al., 2018)or
unique differentiation directions (Khaki et al., 2018). Separation by
surface antigen is one of the ideas in search of such a promising
subpopulation, especially because MSCs present a variety of markers
associated with other cell types. This study investigated whether
SSEA-4+ could distinguish genuine (pluripotent-like) stem cells
within MSC populations, especially in light of our previous
studies, in which this subpopulation survived significantly longer
in 3D culture (in spheres) (Kaminska et al., 2021).
Heterogenous MSC populations can be divided at least into two
distinct classes that differ significantly in terms of ontogenetic origin
and relatedly basic biological characteristics. MSCs isolated from
perinatal tissues, i.e., umbilical cord, umbilical cord blood, placenta,
FIGURE 6
Secretory profiles 3 days in vitro (A) and 5 days in vitro (B) after FACS. The following groups were analyzed: unsorted WJ-MSCs, negative population
(WJ-MSC-SSEA-4-), and positive population (WJ-MSC-SSEA-4+). The results are presented as mean values ±SD of three experiments. p-value for
*<0.05, ** <0.01, *** <0.001, and **** <0.0001.
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or amniotic fluid, are related to the early stages of fetal development
and their spectrum of differentiation seems to be broader. The
second class represents MSCs isolated from the adult tissue, e.g.,
bone marrow or adipose tissue, with lower clonogenic and
proliferative potential (Drela et al., 2016). MSCs from neonatal
and mid-gestational fetal tissues exhibit extremely low
immunogenicity; they are more plastic and grow faster. WJ-
MSCs also possess the spontaneous potential to express neural
markers, which have almost been undetected in BMSC. The
ontogenetic origin of “primitive”MSCs was described by
Takashima et al. (Takashima et al., 2007), who used Cre
recombinase-mediated lineage tracing analysis which revealed
that a primitive class of immature somatic progenitors with
pluripotent potential and a preference for neuronal
differentiation may originate from the embryonic neural crest
neuroepithelium. After undergoing the first developmental
epithelial-mesenchymal transition (EMT), these cells could form
a cohort of primitive mesenchymal cells (pre-MSCs) that transiently
populate all fetal tissue niches and then are gradually replaced by the
mesoderm-recruited, post-gastrulation, adult MSCs.
Based on the above data, in order to find the marker of
“primitive MSCs”, we selected an ontogenetically younger
mesenchymal stem/stromal cell source, i.e., umbilical cord stroma
(Wharton’s jelly). WJ-MSCs used in our experiments expressed
surface antigen characteristics of MSCs and possessed multipotent
capacities to differentiate toward mesodermal cells (Supplementary
Materials). On the basis of previous studies (Musiał-Wysocka et al.,
2019), despite reports of slightly lower SSEA4 expression in 5%
oxygen concentration, we decided to apply hypoxic/physioxic
conditions that are considered closer to physiological conditions
within the cell niche than atmospheric 21% concentration (Ivanovic,
2009). Following the flow cytometry analysis performed for MSCs
derived from different tissues and passages, and cultured in different
media, we decided to apply WJ-MSCs from the third passage
cultured in platelet lysate with a higher concentration. The
estimations of SSEA-4 expression in MSC populations vary
FIGURE 7
3D spheroid culture for unsorted WJ-MSCs, negative population (WJ-MSC-SSEA-4-), and positive population (WJ-MSC-SSEA-4+): time
characteristics. (A) Morphology during the first 72 h of spheroid culture. Black scale bars represent 100 µm. (B) The number of spheroids formed from
10,000 cells during the first 72 h of spheroid culture. (C–E): spheroid diameter during 24, 48, and 72 h of 3D culture, respectively. (F–H): percentage of
spheroids depending on the diameter during 24, 48, and 72 h of 3D culture, respectively. The results are presented as mean values ±SD (B, F, G, and
H) or median values ±95% Confidence Interval (C–E) of three experiments. p-value for * <0.05, ** <0.01, *** <0.001, and **** <0.0001; significant
differences for (F–H): & - 24 h vs. 48 h, for $ - 24 h vs. 72 h, single symbol –p-value <0.05, double symbol –p-value <0.01.
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across scientific literature and depend on several factors. Petrenko
et al. reported differences between SSEA-4-cell percentage values
observed in different MSC sources: 10% for AD-MSCs, 55% for bone
marrow-derived MSCs (BM-MSCs), and 60% for WJ-MSCs
(Petrenko et al., 2020). BM-MSCs from younger donors
contained more SSEA-4+ cells (5.2% vs. 4% for elderly donors)
(Kawamura et al., 2018) while MSCs isolated from female donors
expressed fewer SSEA-4+ cells (72% vs. 79.8% observed in male
donors) (Selle et al., 2022).
The available literature and our observations strongly suggest
that the initial optimization should be vital for SSEA-4+ expression
determination in MSC populations. Culture media components
were also reported to affect SSEA-4 expression (He et al., 2014). He
et al. confirmed that a higher concentration of fetal bovine serum
increased SSEA-4+ cell content for WJ-MSC and BM-MSC (He
et al., 2014), which is in agreement with our observation. It is
especially important to emphasize this observation because sera
and platelet lysates do differ between manufacturers and even
batches. For the described experiments, we used PLT Gold for cell
culture even though the analysis of its influence on MSC
characteristics is lacking in scientific literature. However, studies
performed for the older generations of this platelet lysate showed
that it did not alter MSC characterization such as cell morphology,
expression of MSC markers (CD73, CD90, and CD105),
multipotent differentiation capacity, and proliferation ratio
when compared with other human platelet lysates (Juhl et al.,
2016;Lensch et al., 2018;Bhat et al., 2021). Low oxygen
concentration is another factor that could reduce SSEA-4
expression (Musiał-Wysocka et al., 2019).In addition to
environmental factors and the cell source, the technique of
isolation may also be crucial. When isolating MSCs from WJ,
the non-enzymatic method was reported to be the optimal one to
obtain cells with higher clonogenic and proliferative potential
expressing spontaneous neural markers (Lech et al., 2016).
Unfortunately, this method applied in our experiments did not
allow for an assessment of SSEA4 expression in freshly isolated,
uncultured cells.
With our non-enzymatic method, it was not possible to analyze
WJ-MSC without culture. To obtain the cells straight from the
tissue, it would have been necessary to use the enzymatic method
which does not seem to be optimal for UC-MSCs (Lech et al., 2016).
In our so-called “0 passage”culture, the number of SSEA4+ cells
varied and ranged from 30% to 90%. Other researchers reported that
they isolated WJ-MSC from three patients and SSEA4+ cells
accounted for 51%, 67%, and 70%, respectively (Musiał-Wysocka
et al., 2019).
Furthermore, it is still debated whether MACS or FACS is a
better sorting option to favorably affect the process efficiency and
the cell quality. Some researchers reported that MACS allowed for
the isolation of positive populations with reduced cell stress and
increased yield (Bowles et al., 2019), while others found FACS-based
selection less variable (Muratore et al., 2014;Cheng et al., 2017).
FACS was shown to produce better outcomes in SSEA-4+ cell
isolation from ESC populations (Fong et al., 2009). Sutermaster
and Darling observed inefficient cell sorting and high false-negative
rates when MACS was used according to the manufacturer’s
protocols (antibody and microbead concentration). After
optimization, however, comparable MACS and FACS outcomes
were obtained (Sutermaster and Darling, 2019). A dual MACS-
FACS sorting procedure was recorded as the most effective (Kerényi
et al., 2016). Our study demonstrated that FACS resulted in a more
satisfactory recovery. In our study, reduced mortality of cells and
better yield after FACS sorting were recorded but the observed
differences were not statistically significant. Ultimately, FACS
processing was selected to separate SSEA-4+ cells as the method
proved to result in better recovery of positive cells.
In this study, two populations were received with FACS
separation: negative (WJ-MSC-SSEA-4-) and positive (WJ-MSC-
FIGURE 8
Viability of populations: unsorted WJ-MSCs, WJ-MSC-SSEA-4-, and WJ-MSC-SSEA-4+ after 72 h of 3D spheroid culture. (A) Calcein AM (Cal AM),
ethidium homodimer-1 (EthD-1), and Hoechst staining. Green signals indicate alive cells (Cal AM), red signals indicate dead cells (EthD-1), and blue signals
indicate nuclei (Hoechst). White scale bars represent 100 µm. (B) Analysis of dead and alive cell percentage in populations from CalAM-EthD-1 staining.
The results are presented as mean values of three experiments ±SD. p-value for * <0.05, ** <0.01, and **** <0.0001.
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SSEA-4+), and both were compared to unsorted WJ-MSCs. Post-
sorting cytometric analysis to assess the purity was performed
immediately after isolation. Increased values of SSEA-4+
percentage were recorded in the positive population and almost
no SSEA-4+ cells were detected in the negative population directly
after FACS. The purity of the following subpopulations was
examined until the sixth passage of cell culture when we did not
observe differences in the content of SSEA-4 + cells between the
groups. Other research groups also reported the presence of SSEA-
4+ cells in the negative population after cell sorting (Rosu-Myles
et al., 2013;He et al., 2014). Although He et al. confirmed the purity
of the negative population directly after cell sorting, they observed
the SSEA-4 expression on similar levels both in positive and negative
populations (He et al., 2014). Induction of SSEA-4 in a negative
population could be associated with serum/platelet lysate
concentration; this probably could provide an SSEA-4 substrate
for cells. Rosu-Myles et al. reported a decrease in SSEA-4+ number
during 28 days (approximately four passages) of culture in unsorted
WJ-MSCs and both positive and negative subpopulations (Rosu-
Myles et al., 2013). Glycosphingolipids, as well as other lipids, are
not encoded by genes, while lipid cell composition is usually defined
by enzymes involved in metabolic pathways (Dowhan, 2009). To
detect SSEA-4 expression by qPCR, other studies used gene
encoding sialyltransferase ST3GAL3 (ST3 beta-galactoside alpha-
2,3-sialyltransferase 3) that is necessary for SSEA-4 synthesis
(Hatzfeld et al., 2007). However, the main disadvantage of this
approach is that it does not allow for direct measurement of SSEA-4
levels in cells.
Our quantitative PCR analysis revealed an increased expression of
such stemness-related transcription factors as Oct4 and Nanog, which
are associated with pluripotency. Small CD105+ SSEA-4+ cells
isolated from bovine embryonic fibroblasts expressed pluripotent
markers and differentiated toward cells from all three germ layers
(Pan et al., 2015). He et al. did not observe an increased expression of
pluripotency genes in the sorted SSEA-4+ subpopulation (He et al.,
2014). In our earlier studies, we observed a spontaneous neural
differentiation of MSCs manifested in the presence of genes and
proteins associated with early neurons and glial cells (Figiel-
Dabrowska et al., 2021;Tomecka et al., 2021). The SSEA-4+
population also exhibited increased expression of H-III-Tubulin,
Nestin, and GFAP, thereby suggesting a better potential for
ectoneural differentiation through their undifferentiated state.
However, WJ-MSC-SSEA-4+ cells were incapable of efficient
differentiation toward cells from all three germ layers, which
definitely contradicts their pluripotent potential. Multipotential
differentiation toward mesodermal lineage (osteocytes, adipocytes,
and chondrocytes) was also performed by other researchers, but no
notably significant differences were recorded in differentiation
between cells from unsorted, positive, and negative populations
(Rosu-Myles et al., 2013;He et al., 2014). He et al. did not only
observe differences in proliferation between SSEA-4+ and SSEA-4-
populations but also reported that SSEA-4+ expression was not
correlated with cell proliferation (He et al., 2014). Furthermore, in
our study, neither better proliferation nor colony-forming capacities
by SSEA-4+ cells were detected, which is also consistent with other
reports (He et al., 2014;Matsuoka et al., 2015). In contrast, Rosu-
Myles et al. reported increased proliferation and clonogenicity
potential for SSEA-4+ cells (Rosu-Myles et al., 2013). Interestingly,
SSEA-4- subpopulation derived from limbal epithelial cells exhibited
better clonogenicity than SSEA-4+ cells (Truong et al., 2011).
As MSCs are a highly heterogenous cell group, we investigated
the impact of SSEA-4+ sorting on the expression of other surface
antigens which have not been typically referred to by other
researchers so far. We also assessed the expression of the
markers in cells from two different sources: Wharton’s jelly and
adipose tissue. Following the latest literature, the following markers
were selected to be analyzed: CD49F, CD133, CD146, and CD271
(Bakondi et al., 2009;Krebsbach and Villa-Diaz, 2017;Wangler
et al., 2019). The distribution of CD49F (integrin α6) in various stem
cell populations suggests its involvement in the stemness
(pluripotent) maintenance; it was identified on the surface of, i.a.,
embryonic stem cells, hair follicle stem cells, hematopoietic stem
cells, neural stem cells, and some cancer stem cells (Krebsbach and
Villa-Diaz, 2017). AD-MSC-CD49F + exhibited a greater
proliferation and mesenchymal differentiation potential. In one
of the available studies, mouse and rat AD-MSCs were found to
contain a maximum of 30% CD49F + cells, depending on the culture
of the passage (Zha et al., 2021). Contrastingly, in our study, almost
90% of the cells in WJ-MSC populations were CD49F while AD-
MSCs contained only approximately 17% of CD49F + cells. To our
knowledge, this is the first paper to report CD49F+ in the human
MSC populations derived from neonatal sources. CD133 (prominin
1) is another surface antigen not only associated with cancer stem
cells but also found on the surface of hematopoietic stem cells and
neural stem cells (Glumac and LeBeau, 2018). CD133+ cells isolated
from MSCs derived from peripheral blood and adipose tissue-
derived MSCs were already reported to express pluripotent
markers at a higher level than unsorted MSCs (González-Garza
et al., 2018). In our study, CD133+ appeared sparse for both WJ-
MSCs and AD-MSCs. The expression of CD146 (melanoma cell
adhesion molecule - MCAM) is associated with vascular smooth
muscle cell lineage commitment (Espagnolle et al., 2014). We
observed a higher percentage of CD146+ cells in WJ-MSC
populations than in AD-MSCs. Finally, we investigated the
expression of CD271 (low affinity nerve growth factor
receptor—LANGFR/p75), which indicates cells of
neuroectodermal, neural crest origin (Sowa et al., 2013;Coste
et al., 2017). CD271+ cells were self-renewed and differentiated
into neurons and glial cells after transplantation in vivo (Morrison
et al., 1999). MSC-CD271+ cells were found to exhibit faster
proliferation and better clonogenicity and expression of
pluripotent and neural genes (Mikami et al., 2011;González-
Garza et al., 2018;Kawamura et al., 2018). Originally, we also
intended to separate CD271 + subpopulation. However, the
number of CD271+ within the WJ-MSC populations was not
sufficiently high for all the planned analyses to be made. We
found that SSEA-4+ enrichment enhanced the CD271+ cell
population, but the differences between the groups were not
statistically significant. SSEA-4+ sorting was not found to
significantly affect the expression of the surface antigens, which
could be explained by extensive deviations between cells isolated
from different donors.
Secretory properties of MSCs have been widely investigated in
the context of therapeutic application but a limited number of
reports focused on the secretion abilities of specific MSC
subpopulations. We tested the hypothesis that surface markers
Frontiers in Cell and Developmental Biology frontiersin.org15
Smolinska et al. 10.3389/fcell.2024.1227034
are strictly connected with stromal cell function by tuning the
cytokines released (Islam et al., 2019) and modulating the tissue
microenvironment. Here, we compared the correlation between the
presence/lack of the SSEA4 marker and the levels of secreted,
different regeneration-related molecules such as trophic factors,
cytokines, chemokines, and factors associated with
vasculogenesis. The secretion profiles were found to differ
between the third and fifth day of the experiment. Reduced levels
of some molecules (HGF, BDNF, and GDNF) observed in both
sorted populations on the third day suggested the impact of FACS
on the cells’condition. On the fifth day after FACS, for some trophic
factors, the highest levels of secretion were observed in the negative
population and the lowest levels were recorded in the positive
population, which suggests that the SSEA-4-deficient cells may be
the population that is more specialized in the secretion of trophic
factors. However, the large standard deviations imply that the
factors secretion could be more of an individual matter, as
reported in our other paper (Sypecka et al., 2022).
Our study also examined whether the WJ-MSC-SSEA-4+
population would exhibit better survival in 3D conditions.
According to the latest literature, 3D conditions could resemble the
native niche of MSCs more accuratelythanstandardlyused2Dculture
systems and could be more effective in stemness maintenance (Jauković
et al., 2020;Rybkowska et al., 2023).Ourpreviouspaperconfirmed that
long-term spheroid culture affected WJ-MSCs’survival, proliferation,
and senescence, as well as increased SSEA-4+ expression (Kaminska
et al., 2021). In this study, we cultured WJ-MSCs from the analyzed
groups for 3 days in vitro as spheroids and compared the number,
diameter, and cell viability of the spheroids. Changes in diameter were
recorded between variants for the first 48 h of 3D culture. The spheres
formed with WJ-MSC-SSEA-4+ cells were the smallest in the first 24 h.
At the endpoint, the differences between the groups ceased to be
noticeable. SSEA-4+ cells derived from different tissues were also
reported to form spheres (Barraud et al., 2007;Lopez-Lozano et al.,
2022). SSEA-4+ isolated from the bovine embryonic fibroblast
population formed larger spheres after the seventh day of 3D
culture than SSEA-4 cells (Pan et al., 2015). The viability assay
revealed that our WJ-MSC-SSEA-4+ subpopulation exhibited the
lowest number of dead cells after 3D culture.
It would be an interesting aspect of the project if the differentiation
potential of all analyzed populations cultured in 3D conditions could be
assessed. Unfortunately, long-term 3D spheroids culture resulted in
increased senescence and led to sphere disintegration of heterogenous
WJ-MSCs (Kaminska et al., 2021).
Finally, the association of SSEA-4 with pluripotency was the last
aspect we addressed in this study. Derivation of iPSCs from SSEA-3/
4 knockout mice raised the question of whether SSEA-4 was essential
(Hamamura et al., 2020). Moreover, the increased transient expression
of pluripotent genes by the WJ-MSC-SSEA-4+ subpopulation did not
affect the proliferation and colony-forming capacity. In fact, two states
of pluripotency can be distinguished: naïve (observed for embryonic
cells before implantation into the uterus) and primed (observed for cells
after implantation) (Weinberger et al., 2016;Nishihara, 2017). SSEA-3
and SSEA-4 were associated only with primed pluripotency while
SSEA-1 was observed in the naïve state (Nishihara, 2017). In both
states, the cells were observed to express Nanog, Sox2, and Oct4 genes,
and they did differentiate toward cells from all three germ layers and
form teratomas in vivo (Nishihara, 2017). Knockout of B3GALT5, an
enzyme involved in SSEA-3/4 synthesis, was reported to facilitate the
transition of human ESCs from primed to naïve state (Lin et al., 2020).
Contrastingly, SSEA-3+ cells isolated from the amniotic membrane
appeared to represent a naïve state of pluripotency, which was
confirmed by the presence of SSEA-1 and expression of KLF4—a
gene characteristic only of this state (Ogawa et al., 2022). However,
those observations were not confirmed in the SSEA-3+ population
derived from other tissue sources. It remains debatable whether the
presence of two states explains the observed results in the WJ-MSC-
SSEA-4+ population.
Some limitations of this study should be acknowledged. A
decrease in the cell viability observed in both methods of cell
sorting is a downside of the methodology used. Slightly higher
viability of cells from unsorted populations could influence the
outcomes received in PDT and CFU assays. To minimize this
effect, unsorted cells were transported to the sorting facility in
similar conditions applied for both positive and negative
subpopulations. Furthermore, if indeed the sorting procedure had
such a profound effect on the condition of the cells, differences
between passages in cell culture would have been noticed. The next
limitation concerns the similarity of SSEA-4+ cell content between
unsorted and positive populations. Most researchers report the
results observed in positive and negative populations. We decided
to analyze the outcomes from the initial population to confirm
whether SSEA-4 enrichment indeed impacted WJ-MSC
populations. Nevertheless, if SSEA-4 surface antigen had such a
huge impact, we would have observed significantly different results
at least in the negative population. In fact, some of the analyzed
aspects such as proliferation, CFU, and expression of other surface
antigens were almost similar in all study groups.
5 Conclusion
This study described the characteristics of SSEA-4+ cells separated
from the heterogenous WJ-MSC populations. WJ-MSCs contained
approximately 35%–70% SSEA-4+ cells, depending on the applied
culture condition. The environment richer in proteins and trophic
factors appeared to be more favorable for SSEA-4 cell enrichment
probably due to providing the essential substrate for synthesis. FACS
allowed for the selection of positive SSEA-4+ cells and its number
increased during the further in vitro culture. Elevated relative
expression of the investigated stemness-related genes suggested an
undifferentiated state of the WJ-MSC-SSEA-4 + subpopulation, which
could also affect the differentiation potential toward ectoneural cells.
However, this effect was transient and diminished with further cell
culture, which could account for the unchanged pluripotent
differentiation potential, proliferation ratio, and colony-forming
capacities observed in the positive population. The SSEA-4+
population was not found to be associated with other potential
stemness surface antigens. SSEA-4 enrichment influenced such
aspects of 3D culture as diameter during the first 24 h and viability of
cells inside the spheres. Our hypothesis that WJ-MSC-SSEA-4+ cells
couldbeamorefavorablesubpopulationduetouniquepluripotent-like
features and restorative/replacing properties could not be confirmed as
no unequivocal results were obtained. However, the search for such a
marker is an important direction for further research on mesenchymal
stem cells.
Frontiers in Cell and Developmental Biology frontiersin.org16
Smolinska et al. 10.3389/fcell.2024.1227034
Data availability statement
The datasets presented in this article are not readily available
because of privacy issues to make sure that confidentiality of tissue’s
donor is preserved. Requests to access the datasets should be
directed to Anna Sarnowska (contact: asarnowska@imdik.pan.pl)
Ethics statement
The studies involving human participants were reviewed and
approved by Ethics Committee of Warsaw Medical University (date:
11 October 2016, no. KB/213/2016) and Bioethical Committee at the
Centre of Postgraduate Medical Education (date: 8 October 2013,
No. 63/PB/2013). The patients provided their written informed
consent to participate in this study.
Author contributions
ASm was responsible for designing the study, performing
experiments, analyzing data and writing the manuscript; AK, JJ
and KP performed FACS sorting and analyzed data; MC performed
experiments with AD-MSCs and analyzed data; DS performed
Luminex assay and analyzed data; ASa designed the studies,
supervised experiments, analyzed data and reviewed the
manuscript. All authors contributed to the article and approved
the submitted version.
Funding
This study was funded by National Science Centre grant NCN
2018/31/B/NZ4/03172, statutory funds to Mossakowski Medical
Research Institute and ESF, POWR.03.02.00-00-I028/17-00.
Acknowledgments
We would like to thank Natalia Krześniak from the Prof. W.
Orlowski Memorial Hospital in Warsaw for providing the
adipose tissue for ADSCs isolation, the Laboratory of
Advanced Microscopy Techniques for assistance with
Confocal Microscopy imaging, and the Laboratory of
Cytometry from Nencki Institute of Experimental Biology for
their advice and help regarding flow cytometry. Finally, we are
thankful to our colleagues from Translational Platform for
Regenerative Medicine.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fcell.2024.1227034/
full#supplementary-material
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