Gonzalez et al 2009

1. A putative mesenchymal stem cells population isolated from adult human testes
R. Gonzalez a
, L. Griparic a
, V. Vargas a
, K. Burgee a
, P. SantaCruz c
, R. Anderson d
, M. Schiewe d
,
F. Silva a,*, A. Patel b
a
DaVinci Biosciences LLC, 1239 Victoria Street, Costa Mesa, CA 92627, USA
b
Cardiovascular Center, University of Utah, Salt Lake City, UT, USA
c
Department of Regenerative Medicine, Omni Hospital, Guayaquil, Ecuador, USA
d
Southern California Center for Reproductive Medicine, Newport Beach, CA, USA
a r t i c l e i n f o
Article history:
Received 13 May 2009
Available online 29 May 2009
Keywords:
Adult stem cells
Human testis
Pluripotent markers
MSCs
Multilineage differentiation
Cell-based therapy
a b s t r a c t
Mesenchymal stem cells (MSCs) isolated from several adult human tissues are reported to be a promising
tool for regenerative medicine. In order to broaden the array of tools for therapeutic application, we iso-
lated a new population of cells from adult human testis termed gonadal stem cells (GSCs). GSCs express
CD105, CD166, CD73, CD90, STRO-1 and lack hematopoietic markers CD34, CD45, and HLA-DR which are
characteristic identifiers of MSCs. In addition, GSCs express pluripotent markers Oct4, Nanog, and SSEA-4.
GSCs propagated for at least 64 population doublings and exhibited clonogenic capability. GSCs have a
broad plasticity and the potential to differentiate into adipogenic, osteogenic, and chondrogenic cells.
These studies demonstrate that GSCs are easily obtainable stem cells, have growth kinetics and marker
expression similar to MSCs, and differentiate into mesodermal lineage cells. Therefore, GSCs may be a
valuable tool for therapeutic applications.
Ó 2009 Elsevier Inc. All rights reserved.
Introduction
The search for an ideal stem cell population for therapeutic pur-
poses has been a challenge for years and remains elusive. Recent
data on cell transplantation into animal models of degenerative
diseases and injuries illustrated the feasibility of the use of adult
stem cells for regenerative medicine [1–3]. Mesenchymal stem
cells (MSCs) are one of the most investigated adult stem cells. Suc-
cess in transplantation of these cells stimulated the search for
other mesenchymal cell populations from different tissues. It has
been illustrated that cells isolated from umbilical cord blood [4],
placental cord blood [5], adipose tissue [6], and dental pulp [7]
have similar properties to MSCs, yet also possess unique
characteristics.
Several groups have reported that following transplantation of
adult MSCs, patients’ symptoms improved significantly in various
disease states [8–10]. Despite the uncertainty around the mecha-
nism of adult stem cells action upon transplantation into the in-
jured site, these cells are presently the most promising tool for
cell-based therapies. Studies have demonstrated that MSCs may
be supportive to tissue recovery [11], stimulate the synthesis of
cytokines and matrix molecules [3], be angiogenic [2], have immu-
nomodulatory effects [10], and stimulate endogenous tissue pro-
genitors [3]. Nevertheless, due to the heterogeneity of disease it
is reasonable to assume that each disease condition will require
different properties from transplanted cells in order to improve
the disorder to which the cells are being applied. Thus, it is imper-
ative to investigate the use of several different cell types for ther-
apeutic applications to address the specific disease condition in the
most appropriate way.
Recently, it was demonstrated that pluripotent cells may be iso-
lated from germ-line stem cells within the human testis [12]. How-
ever, similar to embryonic stem cells, these cells generate
teratomas when transplanted into immunodeficient mice bringing
into question their potential clinical application. In order to broad-
en the array of tools for cell-based autologous therapies, we iso-
lated a novel renewable stem cell population from the adult
testes that has characteristics of MSCs, termed gonadal stem cells
(GSCs). Here we demonstrate that GSCs are easily isolated, have
similar growth kinetics, expansion rates, clonogenic capacity and
differentiation potential as MSCs.
Materials and methods
Testicular biopsies were obtained from DV Biologics LLC (Costa
Mesa, CA). Biopsies were isolated from six 22–47 years old donors
after informed consent as approved by an institutional regulatory
board (IRB) (DV Biologics).
Cell culture. Tissue was digested with 0.1% Collagenase type II
solution for 10–20 min at 37 °C. After filtration through 40 lm
0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2009.05.103
* Corresponding author. Fax: +1 949 515 2929.
E-mail address: fsilva@dvbiosciences.com (F. Silva).
Biochemical and Biophysical Research Communications 385 (2009) 570–575
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

2. strainer and centrifugation at 1500g for 10 min 4 °C, cells were
counted and plated on 10 cm dishes coated with Fibronectin
10 lg/ml (Sigma). Cells were cultured in DMEM high glucose (Gib-
co) supplemented with 10% FBS (Hyclone), FGF2 (10 ng/ml), GDNF
(10 ng/ml) (Invitrogen), and penicillin/streptomycin (Gibco). After
3 days, non adhering cells were discarded and attached cells were
cultured and expanded. At 70–80% confluency cells were detached
using Trypzen (Sigma) and re-plated on flasks without coating at
density 1000 cells/cm2
.
Colony-forming units (CFU) assay and cell cloning. For colonies,
cells were plated at a density of 150 cells/10 cm dish. After 17 days,
cells were fixed and stained in a 9% Crystal Violet Methanol solu-
tion for 1 min. Cloning efficiency was estimated as percentage of
cells which generated clones from total cell number/dish. For cell
cloning, 100 cells/10 cm dish were seeded. Selected clones were
isolated using cloning rings (Sigma Aldrich) and each clone was
re-plated in one well of a 6-well plate. After reaching 70% conflu-
ency cells were seeded in 75 cm2
flasks for further expansion.
Growth kinetics. For growth curve, cells were plated onto 24-
well plates at a density of 4000 cells/well and counted in triplicates
from day 3 to 8. Exponential intervals of the growth curve were
used to calculate doubling time as previously described [13]. For
population doublings (PD), cells were cultured on 25 cm2
flasks,
harvested, counted and re-plated when 70–80% confluency. Cell
culture was terminated when cell population failed to double after
2 weeks of culture. Population doubling was calculated using the
formula PD = [log 10(N1) À log 10(N0)/log 10(2) as previously de-
scribed [14].
Immunocytochemistry. Cells were fixed in 4% paraformaldehyde
(PFA) and stored at 4 °C. After permeabilization in 0.1% of Triton X-
100 (Promega) and blocking in 2% BSA (Sigma), primary antibody
diluted in blocking buffer was applied overnight at 4 °C. Staining
for SSEA-4 was performed without using 0.1% Triton X-100 solu-
tion. Cells were incubated with secondary antibody in blocking
buffer for 1 h at RT. Cells were counterstained with DAPI (Molecu-
lar Probes) and mounted with Fluoromount-G (Southern Biotech).
Primary antibodies used were: Oct3/4 clone H-134 (Santa Cruz Bio-
tech), Nanog (ReproCell), SSEA-4 (Millipore), vimentin (Dako), LHR
(Milliepore), and 3b HSD (Santa Cruz Biotech). Secondary antibod-
ies Alexa 488 and Alexa 594 (Molecular Probes) were used. For
negative controls incubation without primary antibody and with
corresponding specific non-immune immunoglobulins (Santa Cruz
Biotech) were used. Staining was analyzed using an Olympus IX81
inverted microscope and SlideBook software.
Flow Cytometry. Isolated cells were pelleted, resuspended in
MEM + HEPES (Gibco) with 2% BSA and counted. Directly conju-
gated antibodies were CD105, CD166, CD90, CD44, CD45, CD34,
CD11b, CD19, HLA-ABC, HLA-DP DQ DR (Serotec), CD133 (Miltenyi
Biotech) LIN, and CD73 (BD Pharmingen). For anti-SSEA-4 and anti-
STRO-1 staining (Millipore) secondary antibody goat anti-mouse
IgG + IgM-APC (Jackson Immunoresearch) was used. After staining,
cells were fixed with 4% paraformaldehyde and analyzed using
0
20,000
40,000
60,000
80,000
100,000
0 2 4 6 8 10
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12 14 16 18
days in culture passage number
cumulativepopulationdoubling
numberofcells/well
GSCs
GSC-cs
GSCs
GSC-cs
GSCs GSC-cs
Fig. 1. Characteristics of GSCs whole population and GSCs clone (GSC-cs). Phase contract images exhibit differences in morphology of cells maintained under the same culture
conditions (10Â) (A). Comparison between growth curves and cumulative population doublings for GSCs and GSC-cs (B,C). Normal karyotype of GSC-cs after seven passages
(D).
R. Gonzalez et al. / Biochemical and Biophysical Research Communications 385 (2009) 570–575 571

3. CyAn ADP Analyzer 9 color (Beckman Coulter). Histograms were
generated by using Flowjo software (Treestar Inc.).
PCR analysis. To analyze gene expression profile, cells were col-
lected in RLT buffer (Qiagen) and stored at À80 °C. NT2 cells used
as a control for pluripotency genes were purchased from ATCC
(Manassas, VA). Total RNA was isolated with the RNeasy Plus kit
(Qiagen). 200–300 ng RNA was reverse transcribed using Thermo-
Script (Invitrogen). Table 2 in Supplemental Materials shows gene
specific primers that were used in both end point and real time
PCRs. Real-time PCR was performed with a CFX96TM
Real Time Sys-
tem and iQTM
SybrGreen Supermix (Bio-Rad Laboratories) to assess
the expression of osteocalcin. GAPDH mRNA was used as a control.
Each sample was measured in triplicate. End point PCR was con-
ducted in a C1000TM
Thermal Cycler (Bio-Rad) using GoTaqÒ
Hot
Start Polymerase (Promega) and 1 ll of cDNA product for the anal-
yses of all other genes. ‘‘No RT” and ‘‘no template” controls were
included in each experiment. Student t-test was done in order to
establish statistical differences in induced samples as compared
to controls.
Cell differentiation
Adipogenic differentiation. Cells were plated in 12-well plates. At
90–100% confluency cells were switched to adipogenic induction
medium according to manufacturer’s protocol (Lonza). After
3 days, medium was changed to adipogenic maintenance medium
and kept for 1 day. Cycles of 3 days induction + 1 day maintenance
medium were repeated for 12–19 days. Control cells were kept in
regular culture medium. At 12 and 19 days cells were fixed with
4% PFA and stored at 4 °C until staining. Staining was performed
using 0.3% Oil Red O solution (Sigma–Aldrich). For quantitative as-
say, Oil Red O bound to lipid droplets was extracted with 100% Eth-
anol solution and absorbance was measured at 550 nm with
reference wavelength 650 nm. Absorbance measurements of Oil
Red O release were compared to standard titration curve of corre-
sponding dye. Obtained quantity of dye accumulation/well was
normalized to cell number determined by Hoechst 33342 (Molec-
ular Probes) staining of nuclei.
Osteogenic differentiation. Cells plated on 12-well dishes were
switched to osteogenic differentiation medium (HyClone) accord-
ing to manufacturer’s protocol. After 12 and 19 days of induction
cells were fixed in 4% PFA and stained with 2% Alizarin Red S (Sig-
ma–Aldrich). To detect calcium deposit accumulation, Ca-bound
Alizarin Red S was extracted in 10% of cetylpyridinium (Sigma) in
phosphate buffer (8 mM Na2HPO4 + 1.5 mM KH2PO4, Sigma–Al-
drich). Alizarin Red S release was measured at 550 nm with refer-
ence wavelength 650 nm. Absorbance measurements of Alizarin
Red S release were compared to standard titration curve of corre-
sponding dye. Obtained quantity of dye accumulation/well was
normalized to cell number determined by Hoechst 33342 staining
of nuclei.
Chondrogenic differentiation. Cells were placed in either control
media or chondrogenic differentiation medium according to man-
ufactures’ protocol (Lonza). Briefly, 300,000 cells/15 ml tube were
pelleted and control or chondrogenic differentiation medium was
added. After 28 days, pellets were fixed with 4% PFA. Pellets were
placed in 25% sucrose solution and frozen embedded 48 h after
in OCT compound (Sakura Finetek). Pellets were sectioned at
10 lm and stained with 1% Alcian Blue (Sigma) and counter
stained with nuclear fast red (Sigma) using standard protocols.
Karyotyping. Karyotype analysis was performed by Cell Line
Genetics using standard cytogenetic protocols. Briefly, G-banding
technique was applied to karyograms produced from at least 20
metaphases.
Fig. 2. Flow cytometry of GSCs isolated from testis at passage 3. GSCs express markers indicative of MSCs. Blue histograms—antibody staining, open histograms indicate
appropriate isotype controls. Percent of positive cells is indicated for each antigen studied. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this paper.)
572 R. Gonzalez et al. / Biochemical and Biophysical Research Communications 385 (2009) 570–575

4. Results
Expansion and growth kinetics of GSCs
In order to isolate a novel stem cell population we dissociated
cells isolated from a single small biopsy of adult human testes.
Supernatant containing spermatogonial cells and dying cells were
discarded. Attached cells were fed every 3–4 days. Under these
conditions, cells reached 70% confluency after 7–10 days and
exhibited MSC-like morphology (Fig. 1A). Clonogenic efficiency of
whole cell population was 35 + 1.8% (n = 5). In total, 7 clones
(GSC-cs) were collected and every clone was successfully ex-
panded. GSC-sc exhibited statistically significant (p < 0.001) de-
crease in clonogenic efficiency (7 + 0.6%, n = 5) in comparison
with starting population. Doubling times were similar for both
populations (33.8 ± 6.5 h GSC and 32.4 + 4.4 h GSC-cs) (Fig. 1B).
However, the proliferative capacity of GSC-cs was markedly re-
duced in comparison to that of GSCs (Fig. 1C). GSCs propagated
for 17 passages with at least 64 population doublings (Fig. 1C)
and were easily expanded to therapeutically necessary amounts
by passage 3 (>2.0 Â 108
). GSC-cs exhibit diploid cells without
chromosomal aberrations as determined by karyotype analysis
(Fig. 1D).
Characterization of GSCs
In order to characterize GSCs, we performed flow cytometry,
immunocytochemistry, and RT-PCR. GSCs exhibit characteristics
of MSCs. Flow cytometry analysis revealed that GSCs have charac-
teristics typical of MSCs isolated from bone marrow in accordance
with the International Society for Cellular Therapy minimum crite-
ria for defining MSCs [15]. GSCs were positive for CD105, CD73,
CD166 and negative for CD34, CD45, HLA-DR, CD11b and CD19
(Fig. 2). In addition, GSCs expressed high levels of CD44, CD90
and STRO-1 which are expressed on MSCs [16,17]. Interestingly,
a small percentage of GSCs express the pluripotent stem cell mar-
ker stage-specific embryonic antigen 4 (SSEA-4) (Fig. 2). Based on
morphology similar to the several distinct cell types described in
MSCs, GSCs are also a heterogeneous population. When comparing
GSCs with GSC-cs, their morphology (Fig. 1A) and antigen expres-
sion (Supplementary Table 1) were different. GSCs had a morphol-
ogy similar to MSCs while GSC-cs were much smaller and with less
processes. Specifically, GSC-cs were mostly CD90 (Thy-1) negative
and demonstrated increased expression of SSEA-4 and CD34.
Immunocytochemistry analysis demonstrates that GSCs express
the pluripotent markers Oct 4, Nanog, and SSEA-4 (Fig. 3A). RT-PCR
experiments confirms that GSCs express Oct 4 and Nanog but are
negative for Sox 2 (Fig. 3B). GSCs also express vimentin which is
a major subunit protein of the intermediate filaments of mesen-
chymal cells (Fig. 3A). There are possibilities that GSCs are derived
from other cell lineages present in testes, namely germ or Leydig.
RT-PCR for Vasa and Dazl confirms that GSCs are not of the germ
cell lineage (Fig. 3B). Additionally, the GCS are not precursors or
adult Leydig cells, based on the negative immunocytochemistry
staining for luteinizing hormone (LH) receptor and 3b-hydroxy-
steroid dehydrogenase [18] (data not shown).
Vimentin
SSEA-4
Oct4
Oct4
Merged
Nanog
Merged
MergedDAPI
Oct4
Nanog
Sox2
Vasa
Dazl
GAPDH
no RT
21 3 4 5 6
A
B
Fig. 3. Pluripotent marker expression of GSCs. Immunocytochemistry analysis revealed expression of pluripotent stem cell markers Oct4, Nanog and SSEA-4 in GSCs
population. GSCs also express the intermediate filament marker vimentin. DAPI nuclear staining is in blue (A). PCR analysis confirmed staining results. Germ cell specific
genes Vasa and Dazl were not expressed in GSCs indicating other than germ cell origin of isolated population. Lane 1, whole testes; lane 2, GSCs passage 1; lane 3, GSCs
passage 4; lane 4, GSCs passage 9; lane 5, GSC-cs passage 4; and lane 6, NT2 control cells (B). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this paper.)
R. Gonzalez et al. / Biochemical and Biophysical Research Communications 385 (2009) 570–575 573

5. GSCs differentiate into mesodermal lineage
The hallmark of MSCs is its ability to differentiate into mesoder-
mal lineage. We therefore undertook studies differentiating GCS
into adipogenic, osteogenic and chondrogenic lineage. Both GSCs
and GSC-cs were induced to the adipogenic, osteogenic, and chon-
drogenic lineages using standard MSC differentiation protocols.
Specifically, GSCs and GSC-cs induced to adipogenic lineage dis-
played lipid vacuoles (Fig. 4A and C) and increased expression of
lipoprotein lipase and PPARyIso2 (Fig. 4B) as compared to non-in-
duced controls. When subjected to osteogenic differentiation, GSCs
and GSC-cs displayed calcium deposits typical of bone (Fig. 4D and
F) and increased expression of osteocalcin and DLX5 (Fig. 4E) as
compared to non-induced controls. The chondrogenic potential of
GSCs and GSC-cs was confirmed by sulfated proteoglycans staining
(Fig. 4G) and increased expression of aggrecan and link protein
(Fig. 4H) as compared to non-induced controls after 28 days in cul-
ture. All together, these data clearly demonstrate that GSCs are eas-
ily differentiated into mesodermal lineage and have MSC
properties.
Discussion
Presently, hematopoietic stem cells (HSCs) and MSCs are the
most widely investigated stem cells for therapeutic applications
because they are easily obtained, autologous, expandable to
therapeutic amounts, and most importantly, have been shown to
improve symptoms in several disease states [19–21]. To expand
the array of cell lines for therapeutic applications, we isolated a
new population (GCS) from adult human testis. With a small
biopsy, GSCs are easily expandable to therapeutic suitable amounts
making this cell an attractive tool for regenerative medicine. GSCs
described in this study possess fundamental stem cell properties
such as clonogeneity, multipotentiality and self-renewal. Further-
more, Isolated GSCs were negative for germ cell specific markers
[12], thus representing a new cell population different from germ
cells. Interestingly, GSCs express the pluripotent markers Oct 4
and Nanog typical of embryonic stem cells (ESCs) [22,23].
GSCs are characteristically similar to MSCs isolated from bone
marrow, based on their morphology, antigen expression pattern
and differentiation potential [4,17]. GSCs exhibit substantially ex-
panded life span (>60 population doublings) when compared to
adult MSCs derived from bone marrow which normally produce
approximately 35 population doublings [4,17,24]. This may be
due to the expression of Oct-4 and Nanog in GSCs. It has been
shown that MSCs can also display marked heterogeneity in mor-
phology, growth kinetics, differentiation potential and gene
expression profiles [4,17,25]. Our flow cytometry data demon-
strates differences between GSCs population as compared to
GSC-cs in regards to surface antigen expression. GSCs were mostly
positive for CD90 and negative for CD34, while GSC-cs were mostly
negative for CD90 and had a greater percentage of SSEA-4 and
CD34 positive cells (Supplementary Table 1). Interestingly, both
SSEA-4 and CD34 are stem cell markers that are associated with
AdipogenesisChondrogenesis
0
10
20
30
40
50
60
70
80
1 2 3 4 5 6 7C CI I
GSCs GSC cs
relativeugofARS/well
GSCs GSC cs
Osteogenesis
control control
induction induction
1
10
100
1000
C CI I
Aggrecan Link
relativeexpression
0
1
2
3
4
5
6
7
8
1 2 3 4 5 6 7C CI I
GSCs GSC cs
relativeugof
ORO/well
0
1
2
3
4
5
6
7
8
9
10
day 12 day 19 day 12 day 19
lipoprotein lipase PPAR iso 2
relativeexpression
Control
Induced**
**
*
0
2
4
6
8
10
12
14
16
osteocalcin DLX5
relativeexpression
Control
Induced
***
**
day 12 day 19 day 12 day 19
Fig. 4. GSCs and GSC-c9 have multilineage differentiation potential. Staining of lipid droplets with Oil Red O—adipo differentiation for both GSCs and GSC-cs (small inserts are
controls) (A). Gene expression of lipoprotein liapase and PPAR iso 2 in cells undergoing adipogenic differentiation demonstrated upregulation in induced GSCs as compared to
controls (B). Quantification of dye accumulation/well for Oil Red O (ORO) demonstrated increased oil red O accumulation in induced cells as compared to controls for both
GSCs and GSC-cs (C). Calcium deposits with Alizarin Red S—osteo differentiation for both GSCs and GSC-cs (small inserts are controls) (D). Gene expression of osteocalcin and
DLX5 in cell subjected to osteogenic differentiation demonstrated upregulation in induced GSCs as compared to controls (E). Quantification of dye accumulation/well for
Alizarin Red S (ARS) staining demonstrated increased dye accumulation in induced cells as compared to controls for both GSCs and GSC-cs. (G) Staining with Alcian blue for
sulfated proteoglycans—representative cells undergoing chondro differentiation are shown for both GSCs and GSC-cs (F). Controls are top panel and induced are lower panels.
Gene expression of aggrecan and link in samples subjected to chondrogenic differentiation. Induced GSCs demonstrate an upregulation of studied genes as compared to
controls (H). Pictures A, D, G 40Â; inserts in G are 4Â low magnification. Data are means + SEM of triplicate samples. The ratio was calculated against the values in control that
was set to 1. *
p < 0.05; **
p < 0.01; ***
p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.)
574 R. Gonzalez et al. / Biochemical and Biophysical Research Communications 385 (2009) 570–575

6. growth; yet GSC-cs underwent replicative arrest much earlier than
GSCs (Fig. 1B). Moreover, we found that lack of CD90 expression
and increase of SSEA-4 and CD34 expression correlates with
enhancement of osteogenic differentiation potential for GSC-cs as
compared to GSCs (Fig. 4D and F). Recently, it was shown that
the expression of another marker, CD106/VCAM correlates with
preferential adipogenic versus osteogenic differentiation [26].
Thus, our study demonstrates that CD90À/SSEA-4+/CD34+ expres-
sion on cells may correlate with increased osteogenic differentia-
tion potential.
Novel cell types may be more effective in the treatment of
degenerative disorders. Although the unique properties of GSCs re-
main to be determined, we hypothesize that these cells may be
therapeutically advantageous over MSCs for a specific pathological
state, since GSCs have a greater life span and are easily differenti-
ated to mesodermal lineage cells. These cells may be used for pa-
tients with disorders like atrophic nonunion [27] for which MSCs
may not be a good candidate. Future studies involving transplant-
ing GSCs into diseased animal models will help elucidate the use of
GSCs for therapeutic application.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2009.05.103.
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