Hypoxia Preconditioning Enhances Self-Renewal and Multi-Differentiation Potential of Tendon-Derived Stem Cells

Research Article

Austin Sports Med. 2016; 1(1): 1004.

Hypoxia Preconditioning Enhances Self-Renewal and Multi-Differentiation Potential of Tendon-Derived Stem Cells

Huang H1,3 and Zhang J2*

1Department of Orthopaedic Surgery, Nanjing Medical University, China

2Department of Orthopaedic Surgery, University of Pittsburgh, USA

3Department of Orthopaedic Surgery, China Orthopaedic Regenerative Medicine Group (CORMed), China

*Corresponding author: Jianying Zhang, MechanoBiology Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, USA

Received: March 03, 2016; Accepted: March 22, 2016; Published: March 25, 2016


Recently, Tendon-Derived Mesenchymal Stem Cells (TMSCs) have been successfully isolated and shown that they possess self-renewal potential and multilineage differentiation capacity which serves them as a promising cell source for tendon tissue engineering.

However, long-term culture in vitro may alter the biology of adult MSCs and result in significant changes on their primitive characteristics. Given that oxygen concentrations in vivo are significantly less at tissue level, it is conceivable that many cells would function more normally in vitro at oxygen concentrations lower than 20%.

To determine the hypoxic effects on rabbit Tendon-Derived Mesenchymal Stem Cells (TMSCs), they were cultured in normoxia (21% 2) vs. hypoxia (5% O2) for up to passage 5 (P5), and their differentiation potential, stem cell marker expression and proliferation rate were compared at early and late passages. We found that TMSCs at 5% O2 significantly increased in proliferation compared to those at 20% O2. Moreover, the expression of two stem cell marker genes, Nanog and Oct-4, was upregulated in the cells cultured at 5% O2. Similarly, more TMSCs expressed three stem cell markers including SSEA-4, nucleostemin and Nanog. The total collagen production released by these stem cells was highly expressed in normoxia group compared to hypoxia group at each passage. In addition to these, higher expression of markers for adipogenesis, osteogenesis and chondrogenesis were observed by qRT-PCR and western blotting.

We conclude that hypoxia conditions have a beneficial effect on TMSCs for their maintenance of stem cell properties and improvement of multidifferentiation potential. Oxygen tension does play a critical role in the niche of TMSCs in vitro.

Keywords: Tendon-derived stem cells; Hypoxia conditions; Normoxia conditions; Self-renewal; Multi-differentiation potential


TMSCs: Tendon-Derived Mesenchymal Stem Cells; BMSCs: Bone Marrow Stem Cells; MSCs: Mesenchymal Stem Cells; ESCs: Embryonic Stem Cells; IACUC: Institutional Animal use and Care committee; PBS: Phosphate Buffered Saline; DMEM: Dulbecco’s Modified Eagle’s Medium; FBS: Fetal Bovine Serum; PDT: Population Doubling Time; SSEA-4: Stage-Specific Embryonic Antigen-4; GAG: Glycosaminoglycans; FACS: Flow Cytometeryl; FITC: Fluorescein Isothiocyanate; FITC: Fluorescein Isothiocyanate; PE: Phycoerythrin; PPARγ: peroxisome proliferators-activated receptor γ; GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase; SD: Standard Deviation; ANOVA: Analysis Of Variance; PLSD: Predicted Least- Square Difference


Injured or degenerative tendon demonstrates limited capacity for spontaneous repair. The absence of vasculature prevents reparative cells from penetrating the tissue and maintaining its integrity.

Thus the restoration of damaged tendon still remains an ongoing challenge. In recent years, tendon regenerations using stem cell-based therapies and tissue engineering techniques have been attempted, which provide a promising alternative for repair of tendon rupture and tendinopathy. Of particular interest is the use of Bone Marrow- Derived Mesenchymal Stem Cells (BMSCs) to regenerate functional tendons, however, unfortunately, several reports have shown that ectopic bone formation is observed after transplantation [1-3]. Furthermore, it has also been verified that tumor can be induced by undifferentiated BMSCs in some specific circumstances [4]. Recently, a rare cell population from tendons called Tendon-Derived Mesenchymal Stem Cells (TMSCs) has been successfully isolated and confirmed that they possess several universal criteria of stem cell, including clonogenicity, self-renewal and multipotent differentiation capacity, indicating that TMSCs may be an appealing cell source for tendon tissue engineering [5].

Although Mesenchymal Stem Cells (MSCs) show high cell renewal potential, they are also vulnerable to replicative senescence.

The bottleneck of any sources of MSCs for cell therapy is the low survival rate after transplantation of cells. In addition, long-term culture in vitro may alter the biology of adult MSCs and result in significant changes in cell cycle kinetics [6]. There is an increasing body of evidence that MSCs may become senescent during protracted culture as indicated by their decreased differentiation potential, morphologic alterations and reduced telomerase activity [7,8]. More work in these areas needs to be pursued before the clinical application of MSCs.

It is well known that self-renewal and multilineage differentiation are the marked abilities for all kinds of stem cells [9]. These are maintained within a niche composed of various factors, including cytokines, growth factors, adhesion molecules, and extracellular matrix [10]. Apart from these, a hypoxic environment is also denoted as an important regulator which plays a role in the maintenance of multipotency and extension of survival [11,12].

It has been accepted that the oxygen tension in most in vitro settings is considerably higher than that found in most mammalian tissues. These tensions correspond to an oxygen concentration of approximately 4-7% [13,14]. Thus, it is conceivable that many cells would function more normally in vitro at oxygen concentrations lower than 20%. Low O2, therefore, appears to be a niche component for tendon-derived mesenchymal stem cells. As we all know, Oxygen Can Generate Reactive Oxygen Species (ROS), and exposure to aberrant levels of ROS may induce senescence dysfunction in stem cells [15]. Thus, we have sufficient reason to speculate that it is advantageous for TMSCs to localize within a hypoxic niche where the ROS source O2 is limited.

However, the degree and duration of hypoxia described in the literature vary greatly and may result in opposite effects on the proliferation and differentiation capacities of MSCs [16-18]. So far only one study described the effect of low O2 tension (2%) on the in vitro expansion and maintenance of undifferentiated stem cell characteristics of human TMSCs and showed higher clonogenicity, cell proliferation but lower differentiation potential [19]. In the present study we investigated the effect of reduced oxygen (5%) in vitro on rabbit patella Tendon-Derived Mesenchymal Stem Cells (TMSCs), this oxygen tension approximates that of tendon in vivo, as described above. Rabbit TMSCs were cultured in normoxia (21% O2) versus hypoxia (5% O2) for up to passage 5 (P5) and their differentiation potential, stem cell marker expression and proliferation rate were compared at early and late passages. We observed that hypoxia was conducive to maintenance of the stem-cell characteristics and enhancement of the multi-differentiation capabilities during their expansion in vitro.

Materials and Methods

Control of hypoxic and normoxic culture conditions

We used a tri-gas incubator to achieve hypoxic culture conditions (Thermo Scientific Heracell 150i, Thermo Scientific, Pittsburgh, PA). In the tri-gas incubator, the concentration of oxygen was precisely controlled by two gas controllers and one oxygen sensor. The supply of nitrogen and carbon dioxide gases was achieved by using a nitrogen gas controller and a carbon dioxide gas controller which were connected to two nitrogen tanks and two carbon dioxide tanks, respectively. The gas tank could be automatically switched to another when the gas in one tank ran out. To avoid extra air was brought into the incubator by opening the door, the incubator was separated into three isolation chambers and each chamber was sealed by double doors. The oxygen in the incubator was further controlled by an oxygen sensor. With these control devices in place, the oxygen concentration in the incubator was kept at the constant level of 5% during our cell culture experiments.

For normoxic culture conditions, a regular tissue culture incubator (Thermo Scientific) was used, where 95% air and 5% carbon dioxide were fed into the incubator and as a result, a 20% O2 concentration inside the incubator was achieved.

Isolation of rabbit TMSCs

Five female New Zealand white rabbits (8-10 week-old, 3.0-4.0 kg) were used in all experiments. The protocol for use of the rabbits was approved by the IACUC of University of Pittsburgh. TMSCs were isolated from rabbit patellar tendons. The procedures for isolation of TMSCs were similar to our previously published protocol [20].

Cell culture

Rabbit TMSCs were seeded in 6-well plate at a density of 1.5×104/ well and cultured with 3 ml of 20% FBS-DMEM/well in the tri-gas incubator as described above to achieve a 5% 2 culture condition, or in the regular incubator to realize a 20% 2 culture condition. When changed medium for the cells cultured in the tri-gas incubator, we placed the replacement medium inside the tri-gas incubator for 30 min before being used. The medium was changed every 3 days under both hypoxic and normaxic conditions. The cell proliferation was determined by cell counting at each time point according to the method published previously [21].

Cellular production of total collagen

Rabbit TMSCs were seeded in 6-well plates at a density of 4.5×104 per well and grown in growth medium in the tri-gas or regular incubator for 5 days. The assay of collagen production was performed when the cell culture became confluent at 80%. After the cell-conditioned medium was collected, cells were detached by trypsinization. Cell numbers were then counted using auto cellometer (Nexcelom Bioscience LLC). To measure total soluble collagen in cell-conditioned media, we used a Sircol collagen assay (Biodye Science, Biocolor Ltd, Carrickfergus, Northern Ireland and UK). Briefly, the cell-conditioned medium was mixed with Sircol dye reagent on an orbital shaker for 30 minutes. This solution was then centrifuged to obtain a collagen-dye complex pellet, which was solubilized with an alkali reagent. A microplate reader (Spectra MAX 190, Molecular Devices, Sunnyvale, California) was used to measure absorbance of the samples at a wavelength of 540 nm. A standard curve for calculating collagen concentration was obtained using a manufacturer-supplied acid soluble type I collagen calibration standard solution. Finally, to compare the hypoxia group with the normoxia group at different passages, we normalized the amounts of collagen with the total cell number produced by each group.

Expression of stem cell markers

Immunocytochemical assay was used for the expression of the following stem cell markers: nucleostemin, Nanog, stagespecific embryonic antigen-4 (SSEA-4) on TMSCs. To perform immunostaining, the cells were seeded in two 12-well plates at a density of 1.5×104/well with 1.5 ml medium and cultured in either 5% O2 or 20% O2 conditions for 3 days. All of them were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature and treated with 0.1% Triton X-100 for 30 min for Nanog and nucleostemin staining. After washing the cells with PBS, either mouse anti-Nanog (1:350, Santa Cruz Biotechnology, Inc., cat. # sc-33759, Santa Cruz, CA) or goat anti-nucleostemin (1:400, Neuromics, Cat. # GT15050, Edina, MN) was applied for 2 h at room temperature. The cells were washed with PBS for three times, and either Cy-3-conjugated goat anti-mouse IgG antibodies (1:500 for Nanog, Millipore, Cat. # AP124C, Billerica MA) or Cy3-conjugated donkey anti-goat IgG antibodies (1:500 for nucleostemin, Millipore, Cat. # AP18°C, Billerica, MA) was applied for 1 h at room temperature. In order to stain for SSEA-4, fixed cells were incubated with mouse anti-human SSEA-4 antibodies (1:500, Invitrogen, Cat. # 414000, Frederick, MD) for 2 hours at room temperature. After washing the cells with PBS, cells were treated with Cy3-conjugated goat anti-mouse IgG antibodies (1:500, Millipore, Cat. # AP124C, Billerica MA) for 1h at room temperature. To quantify the expression of stem cell markers, the stained samples were examined using an inverted fluorescence microscope and images were taken with a 20 × objective using a CCD camera.

Multilineage differentiation potential

Multilineage differentiation potential was tested in vitro for adipogenesis, chondrogenesis, and osteogenesis, respectively. The cells at passage 2 were seeded in 6-well plates at a density of 2.4 × 105 cells/well in basic growth medium (DMEM plus 10% FBS) and cultured either under 5% O2 or 20% O2 tension. To test adipogenic potential, cells were cultured in adipogenic induction medium (Millipore, Billerica, MA) consisting of basic growth medium added with dexamethasone (1 m?), insulin (10 mg/ml), indomethacin (100 m?), and isobutylmethylxanthine (0.5 m?). As a test of chondrogenic potential, they were cultured in basic growth medium supplemented with proline (40 mg/ml), dexamethasone (39 ng/ml), TGF-β3 (10 ng/ml), ascorbate 2-phosphate (50 mg/ml), sodium pyruvate (100 mg/ml), and insulintransferrin-selenious acid mix (50 mg/ml) (BD Bioscience, Bedford, MA). Finally, the osteogenic potential was tested by culturing cells in osteogenic induction medium (Millipore, Billerica, MA) consisting of basic growth medium supplemented with dexamethasone (0.1 mM), ascorbic 2-phosphate (0.2 mM), and glycerol 2-phosphate (10 mM). After culturing for 21 days, Oil red O assay, Safranin O assay, and Alizarin red S assay, as descried previously [21], were used to assess adipogenesis, chondrogenesis, and osteogenesis of TMSCs when grown in 5% O2 and 20% O2 culture conditions.

Semi-quantification of the extent of cell differentiation

The stained samples were examined using an inverted microscope and images were taken with a 20 × objective using a CCD camera. A total number of eight views from each well were randomly chosen. The areas of positive staining were identified manually and computed by a SPOT imaging software (Diagnostic Instruments, Inc., Sterling Heights, MI). The ratio of positive staining was calculated by dividing the stained area by the view area. The values of all views from three duplicate wells (24 views in total) were averaged to obtain the percentage of positive staining, which represented the extent of cell differentiation in the respective induction medium.

Quantitative real-time PCR (qRT-PCR)

To measure the stemness and multipotential of TMSCs under hypoxic and normaxic culture conditions, we performed qRT-PCR. Total RNA was extracted using an RNeasy Mini-Kit with an oncolumn DNase I digest (Qiagen). First-strand cDNA was synthesized in a 20 μl reaction of 1μg total RNA through reverse transcription with Super-Script II (Invitrogen). The conditions for the cDNA synthesis were: 65°C for 5 min and cooling for 1 min at 4°C, then 42°C for 50 min, and finally 72°C for 15 min. The qRTPCR was carried out using QIAGEN QuantiTect SYBR Green PCR Kit (Qiagen) [22]. In a 50 μl PCR reaction mixture, 2 μl cDNA (total 100 ng RNA) were amplified in a Chromo 4 Detector (MJ Research). Rabbit-specific primers were used for stem cell genes expression, including Oct- 4 and Nanog. For differentiated TMSCs, rabbit-specific primers were used for collagen type II, peroxisome proliferators-activated receptor γ (PPAR γ), Sox9, osteocalcin, and Runx2. Glyceraldehyde- 3-Phosphate Dehydrogenase (GAPDH) was used as an internal control. The forward and reverse primer sequences and the resultant products were designed according to published methods, and are listed in (Table 1) [23-26]. All primers were synthesized by Invitrogen (Carlsbad, CA). The relative gene expression levels were calculated from 2-?CT, where ?CT was determined by the formula: ?CT = (Cttarget-CTGAPDH) differentiation-(Cttarget- CTGAPDH) control. In the formula, CTtarget and CTGAPDH are the cycle thresholds of target gene and GAPDH gene, respectively, for each RNA sample. The Standard Deviation (SD) of the ?CT was determined from at least three parallel tests.