Low oxygen tension potentiates proliferation and stemness but not multilineage differentiation of caprine male germline stem cells

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O2) environment, whereas, their in-vitro expansion is typically performed under normoxia (20–21% O2). The comparative information about the effects of low and normal O2 levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O2) and normoxia (21% O2). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p < 0.05) improved, thus, yielding a significantly (p < 0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O2 conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.


Introduction
The spermatogonial stem cells (SSCs), a group of most primitive spermatogonia in testis, are male germline stem cells (mGSCs) with self-renewal and differentiation ability, and thus considered as the foundation of spermatogenesis through their pluripotent characteristics [1]. These cells are present on the basement membrane of seminiferous tubules (ST) and are almost completely enclosed by the Sertoli cells [2].
Existing reports have demonstrated that both intrinsic and extrinsic variables regulate the growth and functional characteristics of mGSCs [3]. Among these, oxygen (O 2 ) tension is an important variable, which is closely associated with the maintenance of stemness and proliferation of mGSCs [4]. However, the effect of lower O 2 tension on mGSC proliferation, while maintaining stemness characteristics, under an in-vitro culture system has rarely been reported [5]; hence, the appropriate mechanism of expansion and differentiation of these cells has remained an enigma.

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The testis is considered as a naturally O 2 -deprived organ [6] as the ST are poorly vascularized and operate normally at lower O 2 levels i.e. approximately 20% of the testicular artery blood O 2 pressure (12-15 mm Hg) [7]. Therefore, the in-vivo niche of mGSCs is characterized by low physiological O 2 tension [7,8]. Since low O 2 levels induce various responses in cultured cells [9], the derivation of cell populations with stemness characteristics and their differentiation should therefore be affected by the hypoxic culture [9], although it is unknown whether caprine mGSCs (cmGSCs) can do so. Moreover, culturing cmGSCs under low O 2 tension can mimic their natural intra-testicular niche and allow investigations of self-renewal and other biological characteristics of these cells [10,11]. However, the effect of hypoxia on culture characteristics of cmGSCs and maintenance of stemness through the expression of pluripotency and adhesion markers, and the extent of differentiation have yet to be investigated.
Therefore, in this study, we hypothesized that culturing cmGSCs under hypoxic conditions would have an impact on their culture and differentiation characteristics. For this, we compared the variation in functional properties of enriched cmGSCs such as the cell proliferation rate, viability, growth characteristic, senescence, immunophenotypic properties, stemness, multilineage differentiation (adipogenic, chondrogenic, and neurogenic); and expression of stemness, adhesion, and differentiation-specific key genes when grown under culture conditions with low (5%) and normal (21%) O 2 tension.

Collection of tissue and cell harvest
Testes from four healthy prepubertal bucks (aged ~ 3 months) were collected and transported to the laboratory in a normal saline solution containing streptomycin (500 μg/mL; Sigma-Aldrich, Cat#S9137) and penicillin (400 IU/mL; Sigma-Aldrich, Cat#P3032), within 30 min of the sacrifice of the animals. All the experimental procedures were carried out following good veterinary practices and approved by the Animal Ethics Committee of the Institute (ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, India; Ref. No. 5/2018-BT/PR27544/AAQ/1/715/2018).
After 3 to 4 washings with Dulbecco's phosphatebuffered saline (DPBS; Sigma-Aldrich, Cat#D5773) and removal of connective tissue, testes were processed for collection and enrichment of cmGSCs. Isolation of cmGSCs was performed by two-step enzymatic digestion method, as described earlier [12], with minor modifications. Briefly, for the first digestion, minced tissue was suspended in DMEM (fivefold v/w) containing 1 mg/mL collagenase IV from Clostridium histolyticum (Sigma-Aldrich, Cat#C5138), 1 mg/mL hyaluronidase type II from sheep testes (Sigma-Aldrich, Cat#H2126), 5 μg/mL DNase type I (Sigma-Aldrich, Cat#DN25), 1 mg/mL trypsin (Sigma-Aldrich, Cat#T7409) and antibiotics (1% w/v), and incubated at 37 °C in an orbital shaker for 45 min. The supernatant was discarded after washing at 1000 rpm for 5 min with DMEM media containing antibiotics for the removal of interstitial cells. Subsequently, the second digestion was then given for 30 min, as described for the first digestion. After second digestion, twice washing of cells was done with DMEM with 10% (v/v) fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA, Cat#10082-147) to stop the enzymatic digestion. The supernatant after centrifugation was filtered through nylon mesh filters of pore size 80 μm (Merck Millipore, Cat#NY8002500) and 60 μm (Merck Millipore, NY6002500) to enrich the cmGSC population.

Enrichment and expansion of cmGSCs
The enrichment of putative cmGSCs was performed by differential plating and Percoll density gradient centrifugation methods as described previously [12]. The enriched cmG-SCs thus obtained were grown under either normoxic [5% CO 2 , 95% air (20% O 2 ), 37 °C; control] or hypoxic (5% O 2 , 5% CO 2 , and remaining N 2 , 37 °C; treatment) culture conditions. For all assays, the cultured cells of both the groups were used and all experiments were performed in triplicates.

Survival rate and growth kinetics
The cells were evaluated for 7 days to analyze the survival rate and growth characteristics in a culture system with low and normal O 2 tension. The number of total and live cells were counted during different days in culture (day 0, 3, 5, and 7) using an automatic cell counter (Countess™ II FL, Invitrogen Inc.), and the survival rate was calculated according to the formula i.e., Survival rate(%) = (Live cell count∕Total cell count) × 100 For the initial 3 days and then for subsequent days, the number of cell divisions was measured with the formula i.e., Number of cell divisions = Log 2 N∕N 0 , where N 0 and N are the initial and final number of cells after either 3 or 7 days of culture, respectively [13]. For this, each observation the cells were counted by trypan blue method after trypsinization with 0.25% Trypsin-EDTA (Sigma-Aldrich, Cat#T4049), using an automatic cell counter (Countess™ II FL, Invitrogen Inc.).
To examine PDT, cells were seeded at a density of 2 × 10 3 per cm 2 and PDT calculated for two different time intervals of the culture of cmGSCs using an algorithm available online (http:// doubl ing-time. com) [14] Morphometry and alkaline phosphatase (AP) staining After 3rd day of culture, cell growth and the cell colonies in each 24-well plate in normoxia or hypoxia were observed by inverted microscopy, and the areas under the colonies were measured using ZEISS Zen 3.1 software (Carl Zeiss Microscopy, GmbH Carl-Zeiss-Promenade 10 07745 Jena, Germany). The mean of the five largest colonies of three wells of the culture plates was used for comparison in each independent experiment that was performed in triplicate. AP staining was performed according to the manufacturer's instructions (Sigma-Aldrich, Cat#86R) and protocol described earlier [15].

Colony-forming assay
To examine cellular viability of cultured cells through assessment of colony-forming units under normoxic and hypoxic conditions, the cell colonies were washed with PBS and fixed with 4% paraformaldehyde solution for 15 min at RT before staining with 1% aqueous solution crystal violet (Sigma-Aldrich, Cat#V5265) for 30 min at RT. After thorough washing with distilled water, colonies were observed under an optical microscope, and then the stain was solubilized with 1% SDS solution for 10 min at RT. The absorbance was measured as 590 nm using a microtiter plate reader (Sun Rise, Tecan, Männedorf, Switzerland). Each test was conducted in triplicate.

Metabolic viability and cellular senescence assay
A tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric kit (Merck Millipore, Cat#CT02) was used for the determination of metabolic viability of cmGSCs in different culture conditions. Four thousand cells per well were seeded into a 96-well flat-bottom microplate with 0.1 mL culture media. After 24 h of culture at 37 °C in a CO 2 incubator, the cells were treated with 10 µL of an MTT solution per well and then incubated at 37 °C for an additional 4 h. After the addition of 0.1 mL of isopropanol with 0.04 N HCl to each well and mixed thoroughly, absorbance was measured at 570 nm Population doubling time = Duration × log (2) log (final cell count) − log (initial cell count) using a microtiter plate reader and data were analyzed with Magellan 4.0 Software (Männedorf, Switzerland).
A senescence-associated β-galactosidase (SA-β-gal) activity was evaluated to assess the senescence in cmGSCs grown under different culture conditions following the manufacturer's protocol (Cellular senescence assay kit; Merck Millipore, Cat#KAA002).

BrdU incorporation assay
The proliferation of cmGSCs was determined using immunocytochemical staining based on 5-bromo-2-deoxyuridine (BrdU; Sigma-Aldrich, Cat#B5002) incorporation by DNA synthesis. For this, cmGSCs were seeded in a 24-well culture plate in triplicates and incubated under either normoxic or hypoxic culture conditions. After 3 days of incubation, culture media was replaced with the media containing 30 μg/ mL BrdU. The cells were incubated at 37 °C for 2 h in a CO 2 incubator before they were washed and fixed with cold 70% ethanol. Then, the cells were treated with 1.5 M HCl for 30 min at RT. After twice washing with 1 × PBS, blocking, a process of permeabilization (2% BSA in 1 × PBS and 0.3% Triton X-100) was done for 60 min at RT. Further, the cells were incubated with a detection antibody (anti-BrdU-Alexa 488 antibody; Merck Millipore, Cat#FCMAB101A4; 1:200) and DAPI (Sigma-Aldrich; Cat#D8417, 300 ng/mL in blocking buffer) for staining of cells and nuclei, respectively. To estimate proliferation rate, cells were counted on three wells and four images/well. The ratio of BrdU and DAPI positive (proliferative), and BrdU negative and DAPI positive (non-proliferative) cells was calculated out of the total number of cells.
Immunocytochemistry cmGSCs were seeded into 24-well plates and cultured in normoxic or hypoxic conditions to obtain about 80% cell confluence. After 7 days of culture, the cells were fixed with 4% paraformaldehyde for 15-20 min. and permeabilized by incubating them with Triton X-100. Thereafter, blocking was performed with 4% bovine serum albumin for 60 min at RT. The fixed and permeabilized cmGSCs were incubated overnight at 4 °C with affinity-purified rabbit Ab specific for a pluripotent marker [rabbit anti-OCT 4 (Sigma-Aldrich, Cat#AB3209; 1:200 dilution), rabbit anti-THY-1 (Invitrogen, Cat#PA5-11917; 1:500 dilution) and rabbit anti-PGP9.5 or UCHL-1 (Invitrogen, Cat#PA1-10024; 1:500 dilution)]. After rinsing in PBS (three-times, 5 min each), cells were exposed to Donkey anti-rabbit IgG (Alexa Fluor 488, Invitrogen, Cat#R37118; 1:1000 dilution) as a secondary antibody for 60 min in the dark at RT. Additionally, cell nuclei were counterstained with DAPI with antifade mountant (ProLong™ Gold Antifade Mountant with DAPI, Invitrogen, Cat#P36931). All the steps for negative control were processed in a similar to the samples except for the use of primary Ab and the imaging of the labeled cells was performed by fluorescence microscope (Zeiss Axiovert A1, Germany).

Neurogenic, adipogenic, and chondrogenic differentiation of cmGSCs
The cmGSCs from the passage (P) three were selected for differentiation that were grown in 24 well plates. Once the cultured cells reached 60-70% confluency in the basal medium, the cells were grown in the specific induction media that permit the differentiation of cmGSCs into different lineages such as neuron-like cells, chondrocytes, or adipocytes. The differentiating cells were observed and images were obtained with an inverted microscope (Nikon ECLIPSE TE2000-U).

Adipogenic differentiation of cmGSCs and confirmation
For adipogenic differentiation, cmGSCs were cultured in two laboratory-made media formulations [16]. The induction media were referred as medium A (90% DMEM, 10% FBS, 5.5 × 10 −5 M β-ME, 1 µM Decaderol, 0.1 mg/L insulin and 5 µM IBMX) and medium B (90% DMEM, 10% FBS, 5.5 × 10 −5 M β-ME, and 0.1 mg/L insulin). Induction medium A was added for three days followed by induction medium B for a day. The procedure of using media A and B was repeated three times. Then the cells were maintained in medium B until day 15 of the experiment. During the entire experiment, culture plates were kept at the normoxic or hypoxic conditions in the incubators.
After 15 days, differentiating cmGSCs were assessed by cell morphology and staining of lipid droplets with Oil Red O. To quantify differentiation into adipocytes, the cells were fixed with 4% paraformaldehyde for 30 min at RT and stained with 0.5% Oil Red O solution in isopropanol (Sigma-Aldrich, Cat#O1391). The adipogenic differentiation-specific genes PPARγ and C/EBPα were detected by RT-PCR [16].

Chondrogenic differentiation and confirmation
The cmGSCs were harvested with trypsinization and centrifuged into a pellet. The cells were seeded with commercially available StemPro® chondrogenic supplement (Gibco, Thermo Fisher Scientific, Cat#A10064-01) mix with Stem-Pro® basal differentiation medium (Gibco, Thermo Fisher Scientific, Cat#A10069-01). The medium was changed every third day of culture until d 15. Chondrogenic differentiation was evaluated first by microscopic observations. After differentiation steps were complete, gently wash the cells with DPBS (Sigma-Aldrich, Cat#D8537), and colonies were fixed in 4% paraformaldehyde for 30 min at RT. Then, after twice washing with DPBS, the cells were stained with 1% Alcian blue (Sigma-Aldrich, Cat#A3157) solution prepared in 0.1 N HCl for 30 min. After three times rinsing with 0.1 N HCl, distilled water was added to neutralize the acidity, and visualized under a light microscope. Moreover, the expression profile of the COL2A1 gene was evaluated to confirm chondrogenic differentiation [17].

cDNA synthesis and quantitative real time-PCR (qRT-PCR)
The total RNA was isolated by using a total RNA extraction reagent (RNAiso Plus) as per the manufacture's protocol (DSS Takara, Japan, Cat#9108). After quantification of RNA using Quantus™ Fluorometer (Promega, USA) and synthesis of respective cDNA using Primescript™ 1st strand cDNA synthesis kit (DSS Takara, Japan, Cat#6110A) using a thermocycler (C1000™ Thermal Cycler, Bio-Rad), qRT-PCR reactions were performed using SYBR green chemistry with respective primers (Supplementary Table 1) in a StepO-nePlus® Real-time PCR system (Applied Biosystem, USA). No template control has been kept as a reaction negative control along with the GAPDH gene as an endogenous control in all the cmGSCs samples. ΔΔCT method as described by [18] was used in determining the relative expression (fold change) of target genes.

Statistical analysis
Statistical analysis was done on the final relative foldchange (RQ) i.e. 2 −ΔΔCt . The n-fold expressions of different groups of samples were compared with the normal samples. For two-way comparisons, e.g., normoxia vs. hypoxia, an unpaired t-test was performed (version 20.0 of the SPSS software package). For multiple comparisons, data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Data are presented as mean ± SEM and differences of p < 0.05 were considered significant.

Morphological characteristics of cmGSCs and alkaline phosphates (AP) staining
In both the culture conditions, cmGSCs began to adhere after 4-6 h of seeding. Gradually the cells grown and formed paired and cluster colonies in 3-4 days in culture. Cells had grown to 80-85% confluency after 7-8 days and 9-10 days under hypoxic and normoxic conditions, respectively [Supplementary Fig. 1a]. The proliferation activity reduced with the advancement of days in culture in both normoxia and hypoxia. The results of morphometry of cmGSCs grown under either normoxic or hypoxic culture conditions are presented in Supplementary Table 2.
The AP staining of cmGSCs was performed to identify the pluripotent characteristics of cmGSCs cultured under normoxia or hypoxia. The cells grown in different culture conditions were positive for AP staining, though the intensity of staining varied among the groups. A significantly (p < 0.05) higher intensity was observed in hypoxia compared to normoxia on days 7 and 10 of culture (Supplementary Fig. 1b).

Survival rate and growth characteristics
The comparison of total cell count and count of live cells after different days in culture under normoxic or hypoxic conditions is presented in Fig. 2. The results suggest that the number of total and live cells were similar in normoxia or hypoxia up to day 3 in culture. Thereafter, on days 5 and 7 significantly (p < 0.05) higher survival and the number of total and live cells were recorded in the hypoxia group compared to the normoxia ( Supplementary Fig. 2).
The PDT of cmGSCs expanded under hypoxic conditions was 1.8 fold (on day 5) and 1.3 fold (on day 7) lower than cmGSCs cultured under normoxic conditions (Fig. 1a). The overall PDT (from day 0 to day 7) was 1.4 fold lower under hypoxic culture conditions (2.86 days) compared to the normoxic condition (3.99 days). Similarly, the number of cell division were higher in hypoxic conditions (2.46) compared to the normoxia (1.77). The shorter PDT and more cell cycles indicate a higher growth rate of cmGSCs under hypoxic conditions compared to normoxic conditions (Fig. 1a). Thus, low O 2 tension in the in-vitro culture system prevents the increase in PDT and decrease in the number of cell divisions of cmGSCs over time.

Cellular viability, MTT, SA-β-GAL senescence, and BrdU proliferation assays
The colony-forming unit assay by crystal violet staining was performed to assess the cellular viability and proliferative potential of cmGSCs grown under normal or lowered O 2 levels. The representative images of crystal violet staining and results of absorbance are presented in Fig. 1 c and d. A significantly (p < 0.05) higher number of colonies and optical density in hypoxia compared with normoxia, indicate the favorable effect of hypoxic conditions on the proliferation of cultured cmGSCs. cmGSCs obtained from three different donors were cultured for 7 days under hypoxia (5% O 2 ) or normoxia (20% O 2 ), and the effect of O 2 level on viability and proliferation were studied during different days (day 3 and day 7) in culture. For viability assay, MTT assay was performed on day 3 and day 7. On day 7, a significantly higher optical density (0.88 ± 0.05 vs 1.061 ± 0.10; p < 0.05) indicates a higher number of viable cells under hypoxic conditions compared to the normoxic conditions (Fig. 2a) was performed on days 3 and 7 of cmGSCs cultured under normoxic or hypoxic conditions (Fig. 2 c-d). The number of senescent cells was similar on day 3, whereas a significantly (p < 0.01) higher number of SA-β-GAL-positive cmGSCs were observed under normoxic conditions than in hypoxia. The proportion of SA-β-gal-positive cells were 52.33 ± 2.33% and 28.00 ± 1.15% of total cmGSCs counted under normoxia or hypoxia, respectively (Fig. 2b). Representative images of BrdU proliferation assay of cmGSCs cultured in normoxic or hypoxic conditions at days 3 and 7 of culture are presented in Fig. 3. Over time, higher expression of BrdU in the cmGSCs cultured in hypoxic conditions, demonstrate that the proliferation was distinctly higher for cmGSCs expanded under hypoxic conditions than for those under normoxic conditions (Fig. 3). Overall, hypoxic culture condition improves viability and increased proliferation whereas decrease senescence in cmGSCs compared to the normoxic culture conditions.

Immunophenotypic characterization
The immunophenotype of cmGSCs expanded under normoxia or hypoxia for 5-8 days were characterized by staining for GC markers (octamer-binding transcription factor-4, OCT-4; T-cell antigen, THY-1, and ubiquitin carboxyl-terminal esterase L-1, UCHL-1) and the representative images of immunofluorescence staining of cultured cmGSCs are presented as Fig. 4. As confirmed by immunocytochemical analysis, the expression of the pluripotency specific cell surface (THY-1) and intracellular (OCT-4 and UCHL-1) markers were higher in cmGSCs cultured under hypoxia compared to the cells in normoxia (Fig. 4), implying that hypoxia improves the expression of pluripotent stem cell characteristic of cmGSCs over time in culture.

Neurogenic differentiation
Neurogenic differentiation of cmGSCs was confirmed by toluidine blue staining (Fig. 5a). After feeding cmGSCs with neurogenic-inducing media, blue color neuron-like cells were seen with toluidine blue staining. The neuronlike structures were relatively larger and more confluent in cmGSCs culture under normoxia compared to hypoxic conditions (Fig. 5a). The expression of nestin, β-tubulin, and doublecortin mRNA was detected ( Supplementary Fig. 3), indicating the differentiation of cmGSCs into neuronal cells.

Adipogenic differentiation
Adipogenic differentiation of cmGSCs was confirmed by Oil Red-O staining. After culturing of cmGSCs with adipogenic-inducing media for 15 days, oil droplets were observed in the cytoplasm (Fig. 5b). A greater number of cells with larger oil droplets were appeared by bright field microscopy and specific staining of differentiated cmGSCs under normoxia compared to the hypoxic condition. The results indicated that cmGSCs had differentiated into fat cells following adipogenic induction in both the culture conditions, though the higher number were present in the normoxia compared to the hypoxia group.

Chondrogenic differentiation
After 15 days of induction in culture with differentiation media, chondrogenic differentiation of cmGSCs was confirmed by Alcian blue staining (Fig. 5c). The cmGSCs cultured with induction media under normoxia or hypoxia showed the formation of the distinct lacuna structure of cartilage (Fig. 5c). Comparing the effect of culture conditions on chondrogenic differentiation of cmGSCs by bright field and specific staining method, cartilage lacunae, and representative morphological structures were more distinctly found under hypoxic culturing compared to the normoxia. The results indicated that cmGSCs had differentiated into chondrocytes following chondrogenic induction with normoxia as a more favorable condition for such differentiation.

Real-time PCR for quantification of pluripotency and adhesion-related genes
The expression of pluripotency (OCT-4, THY-1, and UCHL-1) and adhesion (β-integrin and E-cadherin) related functional genes were investigated using cmGSCs cultured under either normoxic or hypoxic condition for 1 week. Their expressions in different culture systems were studied by comparing their expression levels relative to the housekeeping gene (GAPDH). The qRT-PCR based gene expression analysis showed that the expression of pluripotency  Fig. 3a). The qRT-PCR results represent that the cultured cmG-SCs showed the amplification of differentiation markers such as adipogenesis (PPARγ and C/EBPα), neurogenesis (nestin, β-tubulin, and doublecortin), and chondrogenesis (COL2A1). The expressions level of these genes in different culture systems were studied by comparing their levels relative to the housekeeping gene. Under hypoxic conditions, the expression of differentiation markers was significantly (p < 0.05) down-regulated in hypoxic conditions compared to normoxia. The expression level of C/EBPα, nestin, and COL2A1 were downregulated by 3.76 ± 2.68, 4.50 ± 1.67, and 6.34 ± 1.17 folds compared to normoxia, respectively. The results indicate a lower extent of multilineage differentiation of cmGSCs under hypoxic conditions compared to normoxia.

Discussion
The level of O 2 is an important variable of the stem cell milieu and plays an important role in the regulation of the biological characteristics of stem cells. Therefore, O 2 tension in the culture system is believed to play a vital role in cell proliferation, development, and homeostasis [4]. Usually, in-vitro expansion of stem cells is performed under ambient O 2 level (20-21% O 2 ); which is nearly 4-10-folds higher than the O 2 level in the natural milieu of mGSCs [7]. Thus, the testis has been considered as a naturally O 2 -deprived organ [6] and the mGSC population preferentially resides in a low O 2 (avascular) microenvironment along the basement membrane of the testis. Therefore, as an initial step to better understand the responsiveness and functional characteristic of cmGSCs to the O 2 tension, in this study we investigated the effect of low O 2 concentration on expansion and multiple differentiation of cmGSCs through cell viability, proliferation rate, senescence, immunophenotype, and gene expression of specific markers, and compared with the normoxic condition.
In the study, we demonstrated that cultured cmGSCs under hypoxia had significantly (p < 0.05) higher proliferation rates than cmGSCs cultured under normoxia. We observed a faster expansion and earlier attainment of confluency while culturing cmGSCs under lower O 2 conditions. The higher growth rate and viability coupled with lower senescence of cmGSCs under hypoxic conditions resulted in shorter PDT of the cells. These findings are in agreement with earlier reports that demonstrated higher proliferation of other stem cells such as human BM-MSCs cultured under hypoxia [19,20]. The proliferative effect of cmGSCs under low O 2 tension suggests that the relative physiological environment may provide a suitable condition for the viability and proliferation of these cells. Overall, we found that the culturing cmGSCs under hypoxic conditions augment their proliferation, viability, and other biological characteristics during ex-vivo expansion. Overall, these results could be useful to develop suitable culture conditions for cmGSCs and methodologies for improving the output of cmGSCsbased cell therapies and other downstream applications to upgrade animal production.
Senescence adversely affects cell proliferation as the senescent cells lose their pluripotent characteristics, and undergo cell cycle arrest during aging [21]. The delay in the development of senescence in cmGSCs under hypoxic conditions may be due to the maintenance of their higher rate of proliferation under hypoxia than normoxic culturing. This may be applicable to obtain a larger population of cmGSCs with desired characteristics during long-term culture. The higher survival and proliferation of cmGSCs under hypoxic conditions may be because such an environment represents the physiological growing conditions of these cells [7,8]. It is also suggested that mGSCs preferentially reside in hypoxic regions of the ST, which facilitates metabolic pathways that are integral for the ongoing regenerative capacity of the cells [22]. Though beneficial effects of hypoxic culture on proliferation and differentiation potentials of specific cell lines have been suggested by several groups [19,20,23], other studies showed contrasting results with negative or no effects of hypoxia on stem cell proliferation and differentiation [24,25]. These discrepancies in the results may be due to the variation in the O 2 level, the duration of the trial, range of biological characteristics compared with type and number of the stem cell population, and the variation in the sensitivity of the system that was used to regulate the O 2 level. Moreover, the duration and degree of hypoxia used in previous studies vary greatly and may account, in part, for the inconsistent effects of hypoxia on the functional characteristics of cmGSCs [26,27].
In the present study, we demonstrated that cmGSCs cultured under hypoxia for 7 days had higher rates of cell proliferation and expression of stem cell markers (OCT-4, THY-1, UCHL-1) and adherence specific marker genes (β-integrin and E-cadherin) than cells cultured under normoxia. The upregulation of stemness marker genes (OCT-4, THY-1, and UCHL-1) was observed with concurrent down-regulation of the differentiation-specific genes such as C/EBPα, nestin, β-tubulin, and COL2A1. This inverse association among the expression profile of stemness and differentiation-specific marker genes suggest that cmGSCs loose stemness characteristic during the process of differentiation towards certain lineages, as described earlier for the other proliferating cells [28].
The higher expression of adhesion molecules (both β-integrin and E-cadherin) and subsequently higher adhesion rate of cultured cells onto the surface may be important for faster expansion of the cells under hypoxic conditions [29]. As observed in the present study, the higher survival and proliferation of cmGSCs under hypoxic condition may be because such an environment represents the physiological growing conditions of these cells [8], that facilitates metabolic pathways that are integral for the ongoing regenerative capacity of the cells [22]. A recent study on mice suggests that hypoxia promotes in-vitro proliferation of testicular germ cells through upregulation of anti-apoptotic gene expression and suppression of the proapoptotic gene pathway [11]. Thus, the beneficial effects of hypoxia on cmGSCs proliferation and senescence may be mediated through its effect on differential expression of genes involved in cell adhesion, metabolic and apoptotic pathways. Overall, the O 2 has been shown to act as a communication signal that can affect characteristics of stem cells such as self-renewal, stemness, and differentiation properties [30,31].
In this study, we also observed that the exposure of cmG-SCs to a hypoxic environment reduced their differentiation into the neurogenic, adipogenic, and chondrogenic lineages. The absence of significant difference in the doublecortin (marker of immature neurons) gene among the groups suggests a similar extent of cmGSCs differentiation up to the state of immature neurons in both the culture conditions. A similar effect of hypoxic condition was reported earlier for chondrogenic or osteogenic differentiation of adipose tissuederived stem cells [27]. Further studies into the possible effects of long-and transient/short-term hypoxic culturing with different O 2 levels on lineage-committed differentiation of cmGSCs are required.
A large number of viable cells may be required for downstream applications of mGSCs. The clinical applications of mGSCs may include the preservation of the breeding potential of elite or genetically valuable animals [32], donorderived spermatogenesis [33], and transplantation of genetically altered male germ cells to maintain male fertility and produce transgenic farm animals with desired characteristics [34]. The other important potential clinical application of mGCSc includes restoration of fertility by transplantation of cells from rare or valuable prepubertal male animals that die before sexual maturity or from animals that undergo castration but exhibit superior phenotypic traits later in life [35] or treatment of animals suffering from congenital reproductive disorders [36].
In the present study, we have shown that the hypoxic condition (5% O 2 ) had reassuring effects on viability, proliferation, and stemness, and delayed the senescence of cmGSCs.
Our results indicate that under hypoxic conditions, cmGSCs overexpress key makers of stemness and adhesion, thereby positively influencing their self-renewal and proliferation. Nonetheless, hypoxic culturing inhibits the differentiation of cmGSCs into different cell types. Thus, faster expansion of cultured cmGSCs coupled with the differential expression pattern of stemness, adhesion, and differentiation-specific key markers in cmGSCs provides correlative evidence that a low O 2 microenvironment (5% O 2 ) provides a better culture condition for the self-renewal and proliferation of cmGSCs without adversely affecting their stemness properties. Overall, a culture microenvironment with low O 2 tension is supportive for maintaining proliferation and stemness but not for multilineage differentiation (plasticity) of this stem cell population. These observations add to the understanding of cmGSCs responses to defined culture conditions, which is the most critical issue for their maintenance and downstream clinical applications.