Tissue and Cell 75 (2022) 101742

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Tissue and Cell

journal homepage: www.elsevier.com/locate/tice

Immunophenotyping of progenitor cells from articular cartilage of New

Zealand Rabbits (Oryctolagus cuniculus)
Rafael Gonçalves Hayashi a, *, Jéssica Borghesi a, Lara Carolina Mario a,
Ana Carolina Silveira Rabelo a, Adriana Raquel de Almeida da Anunciação a,
Mariana Ferreira Lima a, Maria Angélica Miglino a, Phelipe de Oliveira Favaron a,
Ana Claudia Oliveira Carreira a, b, **
a
Departament of Surgery, School of Veterinary Medicine and Animal Science, University of São Paulo (FMVZ-USP), São Paulo, Brazil
b
Cell and Molecular Terapy Center (NUCEL) and School of Medicine-Chemistry Institute, Biochemistry Departament, Universtity of São Paulo, São Paulo, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Mesenchymal stem cell (MSC) have immunomodulatory and anti-inflammatory effects, allowing its application
Stem cells in the therapy of different diseases, including articular cartilage injuries, which induce the establishment of a
Mesenchymal pro-regenerative microenvironment in the injured tissue. Therefore, our objective was to isolate, characterize
Chondrocytes
and differentiate cartilage cells from different joints of New Zealand rabbit (Oryctolagus cuniculus), in order to
Lagomorph
Culture
verify their potential as MSC for future clinical use. For this, cartilage fragments were isolated from the humerus-
radio-ulnar joints, humeral scapula, femoro-tibio-patellar, and lame femoris from rabbits. The results showed
that the cells were rounded in the center of the plate and fibroblastoids in the periphery. After thawing, the cells
did not change their growth time in culture, nor their morphology. The cells showed labeling for mesenchymal
stem cell, cytoskeleton, pluripotency and cell proliferation, but not for hematopoiesis markers (CD105+ and
CD34-). We also observed that, when induced, they were able to differentiate into osteogenic, adipogenic, and
chondrogenic cells. After application of these cells in nude mice, no tumor growth was observed in spleen,
kidney, liver, lung and heart. Therefore, we conclude that cells isolated from the articular cartilage of rabbits
present characteristics of MSC with potential for future clinical applications.

1. Introduction
Hyaline cartilage present in articular cartilage, is a specialized con­
nective tissue and it is present in synovial joints, also called diarthrosis,
which comprise the movable joints of the shoulder, elbow, knee and hip.
Its function is to make the joint movement support, absorb and
distribute the weight of impacts, thus reducing friction (Taffetani et al.,
2015; Camarero-Espinosa et al., 2016). The main constituents of artic­
ular cartilage are specialized cells called chondrocytes, which are
embedded in a highly hydrated and organized extracellular matrix,
consisting predominantly of collagen fibers and proteoglycans (Hoffman
et al., 2010). This organized structure provides the necessary visco­
elasticity for the mechanical properties of the hyaline cartilage that
covers the articular surfaces of long bones (Piermattei et al., 2009).
In articular cartilage trauma, inflammatory processes, periarticular
bone growth and changes in the subchondral bone are commonly found,
which promote a disruption of the balance between the anabolic and
catabolic pathways, with consequent induction and/or increased activ­
ity of pro-inflammatory molecules and degradation cartilage (Hoffman
et al., 2010; Mainil-Varlet et al., 2010). Damage to articular cartilage has
important clinical implications, since cartilage has a limited potential
for intrinsic healing and tends to incomplete regeneration by local
chondrocytes, accompanied by formation of inferior fibrocartilage
(Cucchiarini et al., 2014; Naranda et al., 2017).
In the last decade, several viable cartilage regeneration options have
been introduced in clinical practice, including mesenchymal stem cell

* Corresponding author at: Avenida Professor Doutor Orlando Marques de Paiva, 87, Cidade Universitária, Faculdade de Medicina Veterinária e Zootecnia, 05508-
270, São Paulo, SP, Brazil.
** Corresponding author at: Grupo NUCEL. Faculdade de Medicina - Universidade de São Paulo. Avenida Doutor Arnaldo, 455 (Cerqueira Cesar) - São Paulo - CEP:
01246-903 - SP.
E-mail addresses: rafael.hayashi@alumni.usp.br (R.G. Hayashi), ancoc@iq.usp.br (A.C.O. Carreira).

https://doi.org/10.1016/j.tice.2022.101742
Received 23 August 2021; Received in revised form 14 January 2022; Accepted 17 January 2022
Available online 19 January 2022
0040-8166/© 2022 Elsevier Ltd. All rights reserved.
R.G. Hayashi et al.
(MSC) therapy (Camarero-Espinosa et al., 2016; Naranda et al., 2017).
MSCs have gained significant interest, triggering the design of several
trials that prove safety and at the same time provide promising pre­
liminary results of effectiveness (Colombini et al., 2019). MSCs have
immunomodulatory and anti-inflammatory effects, which induce the
establishment of a pro-regenerative microenvironment in the injured
tissue (Parolini et al., 2008; Colombini et al., 2019). These cells have
been used through local implantation, systemic application, combined
with gene therapy; and through tissue engineering (Bakhtina et al.,
2014; Park et al., 2017). However, studies have shown that the use of
MSCs for the treatment of articular cartilage damage may encounter
some problems, especially related to the heterogeneity of MSCs, which is
a reflection of the diversity of MSCs in the range of embryonic origins,
anatomical locations, properties biological functions and functions.
Since heterogeneity represents a significant obstacle in the research and
application of MSCs, it is of great importance that specific MSCs are
selected to enhance the repair of cartilage damage. Although MSCs from
the same tissue may present heterogeneity, at least they share the same
embryonic origins and anatomical locations, which may reduce some
obstacles in this regard (Zha et al., 2021).
The rabbit is an animal commonly used in preclinical orthopedic
treatments, more specifically for bones, articular cartilage, ligament
reconstruction, spinal lesion and cardiovascular regenerative medicine
strategies (Mapara et al., 2012; Bakhtina et al., 2014). Although rabbit
MSCs are investigated for these purposes, they have not been fully
characterized in terms of immunophenotype and differentiation poten­
tial. Thus, the immunophenotypic characteristics of progenitor cells
from articular cartilage in rabbits were determined through qualitative
and quantitative assays; and the potential for multipotent differentiation
of MSCs was also characterized; in addition to the evaluation of its
tumorigenic potential.
2. Materials and methods
2.1. Animals
All procedures were approved by the Ethics Committee on the Use of
Animals from Faculty of Veterinary Medicine and Zootechnics of the
University of São Paulo under protocol 1215180216. New Zealand
rabbits (Oryctolagus cuniculus) (n = 2), with approximately one year old,
and no sex predilection, were used to obtain progenitor cells from
articular cartilage. For this purpose, cartilage fragments of approxi­
mately 6 mm2 were isolated from the humerus-radio-ulnar joints, hu­
meral scapula, femoro-tibio-patellar, and lame femoris.
2.2. Joint cartilage cells culture
After collection, the cartilage fragments were washed three times in
PBS solution with 5% penicillin/streptomycin (Invitrogen, Cat. 15140-
122, Carlsbad, CA, USA) and subjected to enzymatic digestion with
collagenase (1:10) for six hours. After this period, the cells were
centrifuged at 1500 rpm for 3 min, and the supernatant was removed.
The pellet generated was resuspended in 3 different culture mediums:
DMEM High Glucose (LGC Biotecnologia, Cat. BR30003-05, Cotia, SP),
ALPHA MEM (LGC Biotecnologia, Cat. BR3007-05, Cotia, SP); and
HAM’s F-12 (LGC Biotechnology, Cat. BR30004-05, Cotia, SP), supple­
mented with 15 % fetal bovine serum (SFB) (LGC, Biotechnology, Cat.
BR330110 01, Cotia, SP), 1% amino acids essentials (cat. BR30238-01;
LGC Biotechnology), and 1% penicillin/streptomycin. The plates were
kept in a humidified oven at 37 ◦ C with 5% CO2.
2.3. Morphological analysis
Cells were evaluated periodically and photographed on average
every three days through an inverted microscope (NIKON ECLIPSE TS-
100) to observe the cellular morphology and cell growth.
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Tissue and Cell 75 (2022) 101742
2.4. Cryopreservation test
The cells were submitted to enzymatic digestion with 0.25 % trypsin
(LGC Biotecnologia, Cat. BR30042-01, Cotia, SP) for 5 min. Subse­
quently, 1 × 106 cells were resuspended in 1 mL of freezing medium (90
% SFB and 10 % dimethylsulfoxide (DMSO) and distributed in cryotubes
(Corning Cat.430055, NY, USA). After this, the cryotubes were stored in
the Mister Froozen apparatus and kept in a freezer − 20 ◦ C for 2 h. Then,
the tubes were transferred to liquid nitrogen, where they remained
stored. When necessary, the cells were subjected to rapid thawing in the
water bath at 37 ◦ C, and then placed again in culture in 100 mm plates.
2.5. Scanning electron microscopy
For the scanning electron microscopy assay a 24-well plate was
prepared with coverslips. Soon after, 1 × 104 cells were plated in each
well for adhesion. After approximately 24 h, the cells were fixed in 2.5 %
glutaraldehyde, washed in 0.1 M phosphate buffer pH 7.4, and powders
fixed in 1% osmium tetroxide, followed by continuous dehydration in
alcohol. Finally, they were dry dehydrated at a critical point (Balzers
CPD 020). Subsequently, the samples were placed in a metallic support
for gold plating (“sputtering” Emitech K550). To analyze the results, the
ME Leo 435 V P electron microscope was used.
2.6. Cell metabolism
Cell metabolism was determined using colorimetric MTT (3-[4,5-
dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay as
described previously by Borghesi et al. (2017). Briefly, 1 × 103 cells
were cultured in a 96-well plate with three different culture media
(DMEM High Glucose, ALPHA MEM, and HAM’S F12). After 24, 48 and
72 h, 10 μl of MTT (5 mg/mL) (cat. V-13154; Life) was added and the
plate was incubated for 1 h at 37 ◦ C. After that, the MTT solution was
removed and 100 μl of DMSO was added. The reading was performed on
a Thermo Scientific Multiskan FC spectrophotometer (Thermo Scienti­
fic, Wilmington, DE, USA) at a wavelength of 540 nm. The results ob­
tained were plotted on a graph using the program GraphPad version 5
(GraphPad Software, CA, USA).
2.7. Immunofluorescence
For the immunofluorescence assay, a 24-well plate was prepared
with coverslips. A total of 1 × 104 cells were plated per well and incu­
bated for 48 h. Then, the culture medium was removed, and the cells
fixed in 4% paraformaldehyde. The protocol was the same used by
Rabelo et al. (2021) and, for this, the primary antibodies were used:
Oct-4 (sc-4420, Santa Cruz Biotechnology, Inc, Europe), Nanog (n-17,
sc30331, Santa Cruz Biotechnology, Inc, Europe), Vimentin (sc-73259,
Santa Cruz Biotechnology, Inc, Europe), ß-tubulin (sc47751, Santa Cruz
Biotechnology, Inc, Europe), PCNA-3 (sc-46, Santa Cruz Biotechnology,
Inc, Europe), CD105 (Abcam, ab53321, Cambrige, UK), CD34 (555824,
BD Pharmygen, São Paulo, Brazil), CD90 (Abcam, ab225, Cambrige, UK)
diluted in PBS at 1:50 concentration. The LSM 510 fluorescence mi­
croscope (Carl Zeiss Microscopy, Jena, Germany) was used to visualize
the reactions in the 20x and 40x objectives.
2.8. Flow cytometry
Immunophenotypic analysis was performed using flow cytometry
and for this, 1 × 105 cells were fixed in 4% paraformaldehyde and
incubated with primary antibodies (dilution 1: 100) for 30 min at 4 ◦ C.
After this, the cells were incubated with the secondary antibody (dilu­
tion 1: 500) for 30 min at 4 ◦ C. Finally, the cells were washed with PBS
and analyzed by a BD FACSariaIIu flow cytometer (Becton Dickinson,
San Jose, CA, USA). Controls were performed using unlabeled cells
exposed only to the non-specific secondary antibody. For each sample,
R.G. Hayashi et al.
100,000 events were counted. The primary antibodies used were as
follows: Oct ¾ (sc-4420), Nanog (N17, SC-30331), Vimentin (SC-
73259), ß-tubulin (SC-47751), Cytokeratin 18 (SC-323291), CD34
(555824, BD Pharmygen, São Paulo, Brazil), CD73 (SC-14684), CD105
(AB53321), CD117 (A4502, DAKO), and PCNA (SC-46).
2.9. Adipogenic and osteogenic differentiation
Approximately 1 × 104 cells (test) and 5 × 103 (control) were
cultured in 24-well plates until 80 % confluence. On this occasion, the
entire culture medium was removed and 1 mL of induction medium for
adipogenic differentiation (A1007001, Thermo Fisher Scientific, Mas­
sachusetts, USA) and osteogenic differentiation (A1007201, Thermo
Fisher Scientific, Massachusetts, USA) were added to the test cells and
DMEM High Glucose medium plus 15 % SFB for control cells. After 14
days, the cells differentiated into adipocytes, osteocytes and their con­
trols had their medium removed and were fixed in 4% para­
formaldehyde for 15 min at room temperature.
The cells submitted to adipogenic differentiation and their controls
were stained with the Oil Red solution for 5 min, washed in 70 % alcohol
and in running water and counterstained with hematoxylin for 2 min.
The cells differentiated into osteocytes and their controls were washed
in distilled water and stained with a 5% silver nitrate solution in dark
room for 30 min. Then they were washed in distilled water, exposed to a
100 W lamp for 60 min for Von Kossa staining and then washed quickly
with 5% sodium thiosulfate and then counterstained with hematoxylin.
2.10. Chondrogenic differentiation
The cells were washed with PBS, trypsinized and approximately 2 ×
106 cells resuspended in 2 mL of the chondrogenic differentiation me­
dium (A1007101, Thermo Fisher Scientific, Massachusetts, USA). The
cells were centrifuged, and the cell button was placed in the oven. Half
of the medium was changed twice a week for 21 days.
After the differentiation period, the cells were washed with PBS and
fixed in 4% paraformol for 2 h. Then, they were cleared in successive
baths of ethyl alcohol in increasing concentrations (50, 70 and 90 %),
followed by 3 baths in 100 % alcohol lasting 10 min each. At the end, the
sample was submitted to two xylol baths lasting 5 min, then placed in a
liquid paraplast bath at 60 ◦ C in a dry oven for 30 min and included in
paraplast. After inclusion, 5 μm sections were performed in an automatic
microtome and placed on slides and, at the end, the samples were
stained with Masson’s trichrome.
2.11. Tumorigenic potential analysis
To verify the safety in the use of cells derived from articular cartilage,
approximately 1 × 106 cells were inoculated intramuscularly into the
right pelvic member of two nude mice for eight weeks. The animals were
evaluated weekly to observe the possible tumor formation. At the end of
this period the animals were euthanized and samples of heart, lung,
liver, kidney and spleen were collected. These samples were fixed in
paraformaldehyde 4% and after 48 h, the tissues were processed and
included in paraffin. 5 μm sections were adhered to histological slides
and stained in hematoxylin and eosin (H.E.) and then analyzed in the
Olympus light microscope (CX 31 RBSFA).
3. Results
3.1. Isolation and morphological analysis of joint cartilage progenitor
cells
During the primary culture, after 8 h, even with the enzymatic
digestion of the cartilage, it was not possible for all material to be
digested, leaving explants with cells still being released and, therefore,
were maintained in the primary culture. After 24 h, it was possible to
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Tissue and Cell 75 (2022) 101742
visualize the first cells adhering to the culture plate and, after 3 days, the
colonies were visualized, with a high density of progenitor cells. The
cells in the center of the plate had a rounded shape, while those in the
periphery had a fibroblastoid shape. (Fig. 1A). In some of the
fibroblastoid-shaped cells, a process of cell division was observed, evi­
denced by the presence of two nuclei in their cytoplasm (Fig. 1B). After 5
days the plate reached 80 % confluence, and the cells were frozen for 7
days and then thawed to check their viability. After thawing, the cells
did not change their growth time in culture (Fig. 1C), besides main­
taining the fibroblast morphological aspects with elongated cytoplasm
and centralized nucleus (Fig. 1D). The cultivation was expanded until
passage 7, with no change in its morphology.
3.2. Articular cartilage progenitor cells grow better in DMEM High
Glucose medium
Through the MTT assay it was possible to choose the most efficient
culture medium in terms of cell growth of the articular cartilage pro­
genitor cells. Among the evaluated media, it was observed that DMEM
High Glucose medium was the most efficient of all, as it was the one that
had a more continuous growth line with greater cell growth during the
72 h analyzed. The ALPHA MEM promotes evident cell growth up to 48
h, however, afterwards it promotes a delay in cell growth. DMEM F-12
was less efficient, as it had a lower cell growth rate than the other media
(Fig. 1E).
3.3. Articular cartilage progenitor cells show labeling for mesenchymal
stem cell, cytoskeleton, pluripotency and cell proliferation, but not for
hematopoiesis markers
The immunofluorescence analysis was performed in order to verify
the expression of the mesenchymal stem cell markers (CD90 and
CD105); cytoskeleton (β-tubulin; vimentin); pluripotency (Nanog and
Oct-4); cell proliferation (PCNA); and hematopoiesis (CD34). A positive
and strong marking was observed for β-tubulin and vimentin, confirm­
ing the presence of cytoskeleton markers. Strong labeling for CD105 was
observed, and although weak labeling for CD90 was observed, there is
signaling from mesenchymal stem cell markers. Pluripotency was also
observed, as there was positive marking for Nanog and Oct-4. Prolifer­
ation was observed in the cells through the positive labeling of PCNA.
However, there was no expression for the CD34, showing that there is no
hematopoiesis marking (Fig. 2).
In order to confirm the antibody markings found in the immuno­
fluorescence, the cells were analyzed by flow cytometry, allowing
quantitative data to be obtained. In relation to cytoskeletal markers,
vimentin expression was 52.56 %; β-tubulin 28.49 %; and CK-18 was
21.49 % (Fig. 3A). High expression of the mesenchymal marker CD105
(71.15 %) was observed, and median for CD73 (10 %), however for
CD90 the expression was only 1.39 % (Fig. 3B). When pluripotency was
analyzed, it was possible to observe a low expression of Nanog (6.56 %)
and Oct-4 (4.2 %) (Fig. 3C). For the hematopoietic precursor cell
marker, CD117, an expression of 44.92 % was found; while for he­
matopoietic cells, CD34, there was a low expression (3.59 %) (Fig. 3D).
In contrast, there was a high expression of PCNA (68.5 %) (Fig. 3E).
3.4. Articular cartilage progenitor cells show chondrogenic, osteogenic
and adipogenic differentiation
The progenitor cells were maintained in osteogenic and adipogenic
induction medium for 14 days. In chondrogenic differentiation stained
with Masson’s trichrome, the elongated cells lost their original archi­
tecture to a shape like chondrocytes, with collagen in their extracellular
matrix (Fig. 4A). For osteogenic differentiation, Von Kossa staining was
used, and it was observed that the cells lost their original shape, pre­
senting an extracellular matrix with calcification points (Fig. 4B). And
the adipogenic differentiation stained with Oil Red showed cells that lost
R.G. Hayashi et al. Tissue and Cell 75 (2022) 101742

Fig. 1. Cell culture visualized by light microscopy; scanning electron microscopy; and colorimetric cell viability assay (MTT). [A] 4x increase. observe colony and
population of progenitor cells; [B] 20x increase. Progenitor cell with characteristic elongated cytoplasm (CYT) and its nucleus (NUC) in the process of replication; [C]
4x increase. Cultivation plate with 90 % confluence, liable to expansion; [D] note, in the scanning electron microscopy, progenitor cells with homogeneous
morphology, presenting a flattened, elongated aspect and fibroblast with nucleus (NUC) and cytoplasm (CYT); [E] graphs representing the cell viability assessment
method (MTT) using the three culture media.

their fibroblastoid shape, with a more rounded cell membrane and a less
centralized nucleus and with vacuoles in their cytoplasm (Fig. 4C).
3.5. Articular cartilage progenitor cells do not have tumorigenic potential
Two nude mice received application of cartilage progenitor cells in
order to verify their tumorigenic potential. The cells remained inocu­
lated for 60 days in the animals, which during this period did not show
any macroscopic morphological changes. After euthanasia, a new
macroscopic evaluation was performed, being possible to observe that
there was no tumor nodule at the application site or in the rest of the
body (Fig. 4D). In addition, there was no microscopic change in the liver,
lung, kidney, heart and spleen (Fig. 4E–I).
4. Discussion
Mesenchymal stem cells (MSC) have become an important tool in the
treatment of lesions of the cartilaginous tissue, due to their potential for
self-renewal and differentiation in cells that form the mesodermal tissue
(Abdallah and Kassem, 2008). As the rabbit is widely used in preclinical
models to evaluate regenerative medicine approaches, studies are
needed to assess the potential of MSCs of this species to be a viable
translation model (Bakhtina et al., 2014). Therefore, we investigated the
ability of rabbit cartilage progenitor cells to serve as a source of
mesenchymal stem cells, with a focus on establishing a protocol for
culture and characterization of these cells, in addition to assessing their
potential for differentiation by immunophenotyping.
According to the International Society for Cellular Therapy (ISCT),
one of the established criteria for a cell to be classified as MSC is that it
can be isolated from a population of mononuclear cells based on their

4
R.G. Hayashi et al. Tissue and Cell 75 (2022) 101742

Fig. 2. Immunophenotyping of progenitor cells from articular cartilage through immunofluorescence. Labeling for antibodies vimentin, β-tubulin, CD105, Oct-4,
Nanog and PCNA. Weak expression of CD90 and absence of CD34 marker. DAPI (blue) and FITC (green).

selective adhesion, in culture, to the plastic surface (Bydlowski et al.,
2009; Galipeau et al., 2016). In our study, the articular cartilage pro­
genitor cells were isolated and demonstrated adhesion to plastic, even
after successive passages and thawing. Furthermore, the fibroblastoid
shape of these cells remained constant after all the procedures, going
according to the findings by Fei et al. (2013) and Borghesi et al. (2017).
When the cells were exposed to three different culture media (DMEM
HIGH, ALPHA MEM and HAM’s F12) and evaluated by the colorimetric
assay with MTT, it was possible to observe a greater cellular metabolism
in the presence of DMEM HIGH medium. These data show that the cells
needed a higher concentration of glucose to develop their metabolic
activities, a fact that has already been shown to be a crucial step in the
adaptation of mesenchymal stem cells after implantation. This is due to
the inability of MSCs to rapidly use intracellular reserves, requiring free

5
R.G. Hayashi et al.
Fig. 3. Histogram of partial immunophenotyping of progenitor cells from New Zealand rabbits in P4 by flow cytometry. [A] Cytoskeletal markers: Vimentin (52.56
%); B-tubulin (28.49 %); and CK18 (21.49 %). [B] Mesenchymal markers: CD105 (71.15 %); CD90 (1.39 %); and CD73 (10 %). [C] Pluripotency markers: Nanog
(6.56 %) and Oct-4 (4.2 %). [D] Hematopoietic precursor cell markers: CD34 (3.59 %) and CD117 (44.92 %). [E] Proliferation marker: PCNA (68.5 %).
glucose as the primary source of energy (Moya et al., 2018).
It is well established that for a cell to be classified as MSC it is
necessary to have the expression of mesenchymal markers and nega­
tivity for hematopoietic markers (Wilson et al., 2019). Therefore, we
analyzed the immunophenotypes of cells during passage four, both by
flow cytometry and by immunofluorescence. Our results showed that
there was no expression of the hematopoietic marker CD34, going ac­
cording to that recommended by ISCT and other studies that also found
no expression for this marker in mesenchymal cells isolated from rabbits
(Borghesi et al., 2017; Zomer et al., 2018).
Cytoskeletal and mesenchymal stem cells markers, such as vimentin
and CK-18, which are intermediate filaments, and β-tubulin, a micro­
tubule membrane protein, were also evaluated (Coulombe and Omary,
2002; Chang and Goldman, 2004). A high expression of these markers
was found in progenitor cells isolated from rabbit cartilage, suggesting
that our culture was heterogeneous in composition, with mesenchymal
and epithelial cells. According to Borghesi et al. (2017) cells isolated
from the amniotic membrane of rabbits also showed this heterogeneity.
However, it can be assumed that there was a prevalence of mesenchymal
cells, since the method used for cell isolation was collagenase and the
fibroblastoid format was observed throughout the culture along cell
passages (Parolini et al., 2008).
When more specific mesenchymal markers were evaluated in our
study, a high expression of CD105 and CD73 was noted, but low for
CD90. These contradictory results have also been described by other
authors (Martínez-Lorenzo et al., 2009; Bakhtina et al., 2014). The study
conducted by Bakhtina et al. (2014) found an absence of CD90 expres­
sion in rabbit bone marrow mesenchymal cells. In addition, Martíne­
z-Lorenzo et al. (2009) found 40.5 % of CD90 expression and negative
expression of CD73 and CD105 in MSCs of rabbit adipose tissue. Ac­
cording to Zomer et al. (2018) the disparities in the results may be due to
non-specific antibodies, since anti-rabbit antibodies are not widely
available. In addition, differences in the passage of cells used in each
6
Tissue and Cell 75 (2022) 101742
study and variations in isolation methodologies can affect the results
(Zomer et al., 2018).
It is known that the Nanog and Oct-4 transcription factors are
involved in the regulation of pluripotency, self-renewal and prolifera­
tion of embryonic and adult stem cells (Musiał-Wysocka et al., 2019).
When these markers were analyzed in our study, we found low expres­
sion of both in the progenitor cells isolated from the cartilage. However,
these transcription factors can be expressed at low levels in pre-transit
MSCs and can gradually decrease over time (Han et al., 2014). In
addition, there was high expression of the PCNA marker, both by flow
cytometry and by immunofluorescence, showing that the capacity of cell
proliferation was not affected. These characteristics make us infer that
the progenitor cells isolated from the rabbit’s cartilage have the po­
tential to respond to injury and migrate to defective areas of the carti­
lage (Xue et al., 2019).
Despite presenting a distinct immunophenotype, progenitor cells
from articular cartilage were able to differentiate in vitro into three
different cells with characteristics of adipocytes, osteocytes and chon­
drocytes. A great potential for differentiating rabbit MSCs has also been
demonstrated by other authors (Bakhtina et al., 2014; Liu et al., 2018).
After isolation, characterization and differentiation of progenitor
cells isolated from rabbit articular cartilage, which are prerequisites for
defining a stem cell, the tumorigenic potential test was carried out to
assess the safety of its potential use in regenerative medicine and after
60 days of application of the cells in Balb-c NUDE mice, it was found that
there was no formation of a thrombus, tumor, or any macro and
microscopic changes in the skin, spleen, liver, kidneys, heart and lung.
This result confirms previous descriptions about the safety of mesen­
chymal cells from rabbits (Borghesi et al., 2017).
5. Conclusion
Based on the analyses, it appears that cells isolated from articular
R.G. Hayashi et al. Tissue and Cell 75 (2022) 101742

Fig. 4. Phenotyping of the articular cartilage progenitor cells through their differentiation and tumorigenic test in an immunodepressed mouse. [A] chondrogenic
differentiation stained with Masson’s trichrome; [B] osteogenic differentiation stained by the Von Kossa method; [C] adipogenic differentiation stained through Oil
Red staining; [D] Immune-depressed mouse after euthanasia and it’s viscera with hematoxylin eosin staining: [E] of the spleen; [F] kidney with the presence of
glomeruli (arrow); [G] liver showing a hepatocyte (arrow); [H] lung; with alveolus (arrow) [I] heart with myocardial cells (arrow).

cartilage of New Zealand Rabbits (Oryctolagus cuniculus) have common Fundings

characteristics of MSC and may have a potential for future therapeutic
application of cartilage lesions.
Author statement
Rafael Gonçalves Hayashi: Investigation; Writing - Original Draft;
Writing - Review & Editing.
Jéssica Borghesi: Investigation; Writing - Original Draft; Writing -
Review & Editing.
Lara Carolina Mario: Investigation; Writing - Original Draft.
Ana Carolina Silveira Rabelo: Investigation; Formal analysis; Writing
- Original Draft; Writing - Review & Editing.
Adriana Raquel de Almeida da Anunciação: Investigation.
Mariana Ferreira Lima: Investigation.
Maria Angélica Miglino: Resources.
Phelipe de Oliveira Favaron: Methodology.
Ana Claudia Oliveira Carreira: Validation; Supervision.
Contributors
All authors have approved the final article.
This article was carried out with the support of the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) –
Financing Code 001. And with the support of the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) – Código de Finan­
ciamento169317/2017-0.
Data availability
Data will be made available on request.
Declaration of Competing Interest
The authors report no declarations of interest.
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