Cell Biology International (2009) 33, 1222-1229 - Koshi N. Kishimoto and others - Stimulation of the side population fraction of ATDC5 chondroprogenitors by hypoxia

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Cell Biology International (2009) 33, 1222–1229 (Printed in Great Britain)

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Stimulation of the side population fraction of ATDC5 chondroprogenitors by hypoxia

Koshi N. Kishimoto^a^*, Carol L. Oxford^b and A. Hari Reddi^a^*

^aCenter for Tissue Regeneration and Repair, Department of Orthopaedic Surgery, School of Medicine, University of California at Davis, Sacramento, CA 95817, USA
^bOptical Biology Core Facility, Department of Pathology, School of Medicine, University of California at Davis, Davis, CA 95616, USA

Abstract

The influence of oxygen tension on the side population (SP) fraction sorted from ATDC5 chondroprogenitor cells was investigated. ATDC5 cells cultured in normoxia (20%) or hypoxia (1% oxygen) were compared. The SP fraction was significantly higher in the cells cultured in hypoxia. The gene expression of 3 ABC transporters, abcb1a/b (mdr1a/b) and abcg2 (bcrp1) was quantified by RT-PCR. SP cells were characterized by the higher expression of abcb1a. The expression levels of abcb1b and abcg2 were higher than abcb1a. However, there was no significant difference between SP and non-SP fractions in the expression of abcb1b and abcg2. The telomeric repeat amplification protocol assay showed that SP cells tended to show lower telomerase activity than non-SP cells. Chondrogenic properties of ATDC5 cells derived from SP or non-SP were assessed by micromass culture. There were not significant differences between SP and non-SP derived cells in Alcian blue staining and sox9, Aggrecan, Col2a1 and SZP mRNA expression. The results demonstrate that the SP fraction was stimulated by hypoxia and chondrogenic properties of SP and non-SP fraction of ATDC5 cells were similar.

Keywords: Side population, Stem/progenitor cells, ABC transporter, Telomerase activity.

^*Corresponding authors. UC Davis Medical Center, Res Bldg I Rm, 2000 4635 Second Avenue, Sacramento, CA 95817, USA.

1 Introduction

Articular cartilage and cartilage models of developing endochondral skeleton are both avascular tissues composed of chondrocytes and surrounding extracellular matrix. Thus, chondrocytes exist in hypoxic conditions through their entire life. To develop tissue-engineering approaches for cartilage repair, the expansion of chondrocytes is a necessary and critical step. The effect of oxygen tension on the proliferation of chondrocytes is still controversial (Malda et al., 2003). The existence of stem/progenitor cells in a cell population is important for its differentiation potency. However, the potential role of hypoxia on stem cells/progenitors of chondrocytes is not clear.

The Side Population (SP) of the adult bone marrow cells was first reported as a cell population enriched in hematopoietic stem cell activity (Goodell et al., 1996). This unique staining profile was visualized by 2 wavelengths of Hoechst 33342 dye by flow cytometry. SP cells were found in various adult tissues (Asakura and Rudnicki, 2002). SP cells sorted from muscle had hematopoietic activity (Gussoni et al., 1999); and those from bone marrow had myogenic (Gussoni et al., 1999; Camargo et al., 2003) and osteogenic (Olmsted-Davis et al., 2003) activities. Therefore, SP cells might be enriched multipotent stem/progenitor cells.

The SP fraction has higher ability to efflux Hoechst 33342 dye. ABC (ATP-binding cassette) transporter subfamily B1 – also known as MDR1 or P-glycoprotein – which is encoded by abcb1a/b genes plays an important role in this phenomenon. Enforced expression of this transporter gene expanded side population cells (Bunting et al., 2000). Verapamil blocks ABCB1 transporter and SP fraction eliminates by staining with it. Another member of ABC transporter, ABCG2, also known as BCRP1 (breast cancer resistance protein1), is expressed in SP cells sorted out from a diverse source (Zhou et al., 2001).

Several reports have implied a relationship between oxygen tension and SP-related transporter genes. Hypoxia induces ABCB1 gene expression and its function (Comerford et al., 2002). ABCG2 also plays an important role in the cellular response to hypoxia. A Hypoxia Inducible Factor-1 (HIF-1) binding site in present in the murine ABCG2 promoter (Krishnamurthy et al., 2004).

Chondroprogenitor cells (ATDC5) derived from murine embryonic carcinoma provides an excellent model of chondrogenesis (Atsumi et al., 1990). In the absence of insulin (ITS+), ATDC5 cells proliferate rapidly, retaining the properties of chondroprogenitor cells. In the presence of insulin, ATDC5 cells differentiate through multistep chondrogenesis from condensation, cartilage nodule formation, hypertrophy to mineralization (Shukunami et al., 1996). We employed micromass culture system to evaluate chondrogenesis.

We have therefore examined the influence of hypoxia (1% oxygen) on SP fraction of ATDC5 cells and determine the characteristics of the SP cells.

2 Materials and methods

2.1 Cell culture

ATDC5 cells were obtained from RIKEN cell bank (Tsukuba, Japan) and expanded in the medium consisting of a 1:1 mixture of DMEM and Ham's F-12 (Invitrogen) supplemented with 5% fetal bovine serum (FBS) and antibiotics (Invitrogen: penicillin 50 U/ml, streptomycin 50mg/ml; Expansion medium). In normoxic condition cells were cultured in humidified cell incubator (Sanyo) at 37°C in 95% air (20%O2) and 5% CO2. HERAcell 150 (Heraeus) incubator filled with Nitrogen gas was used for the hypoxic condition (94% N2, 5% CO2 and 1% O2).

2.2 Hoechst 33342 staining and cell sorting

ATDC5 cells cultured in monolayer under hypoxia or normoxia were stained with Hoechst 33342, and resuspended at a working concentration of 10⁶cells/ml in Hank's balanced salt solution containing 2% FBS (HBSS+). Hoechst 33342 dye (Sigma) was added at 10 μg/ml and cells were incubated 90min at 37°C, spun down and resuspended in HBSS+ with 2μg/ml propidium iodide (PI, Sigma). Cells were analyzed and sorted with a MoFlo flow cytometer (Dako Cytomation). An argon laser tuned to 350–360nm (60mW) was used to excite the Hoechst dye. Fluorescence emission was collected with a 450/65 band pass (BP) filter for the blue and a 570/40 BP filter for red. A 555 DMLP was used to separate the emission wavelength. A second 488nm argon laser (300mW) was used to excite PI fluorescence. Fluorescence emission was measured with a 670/30 BP filter. PI-positive cells were considered dead and excluded from analysis. Sorting gate was determined with Summit v3.1 software (Dako Cytomation). Results were analyzed using FlowJo v6.3.3 (TreeStar).

2.3 Micromass culture

The micromass culture technique was modified from Ahrens et al. (1977). Ten microlitre suspension (containing 100,000 cells) was placed at the center of a well of 12-well tissue culture dish (corning). The cells were allowed to adhere for 90min before the wells were filled with 1ml of DMEM/F12 (1:1) supplemented with 1% FBS, 0.2%BSA, 50μg/ml ascorbic acid 2 phosphate, antibiotics and ITS+ (BD), i.e. differentiation medium. Experiments for chondrogenic differentiation were also conducted with differentiation medium without ITS+ and that with recombinant human BMP4 (100ng/ml R&D).

2.4 Alcian blue staining

Cultured micromass were fixed with 10% formaldehyde containing 0.1% cetylpyridinium chloride and stained with 1% Alcian blue (pH 1.0) for 30min. For quantification, alcian blue stain was solubilized in 4M guanidine HCl, 50mM Tris–HCl (pH 7.4), 0.1% CHAPS and its absorbance measured at a wavelength of 595nm.

2.5 Real-time quantitative RT-PCR

Total RNA from SP, NSP cells or micromass was extracted with RNeasy Mini or Micro kit (QIAGEN), depending on the number of cells. First strand cDNA was synthesized using High-capacity cDNA archive kit (Applied Biosystems). RT-PCR reactions were performed using ABI PRISM 7700 (Applied Biosystems) routinely in triplicate. The primer and probe sets for Sox9, Aggrecan, Col2a1, Hif1a, SZP and GAPDH were purchased from Applied Biosystems. Primer pairs for abcb1a, abcb1b and abcg2 were as follows: abcb1a forward 5′-GGATCACGTTCTCCTTCACC-3′; abcb1a reverse 5′-CAGGAGCGAATGAACTGACC-3′; abcb1b forward 5′-GGCCAAGCCTTGAAGGACA-3′; abcb1b reverse 5′-GGCCTGGGTGAAGGAGAAC-3′; abcg2 forward 5′-ATGAGCCCACGACTGGTTTG-3′; abcg2 reverse 5′-GCCAGTAAGGTGAGGCTGTCA-3′.

2.6 Telomerase activity

Telomerase activity was investigated in SP and non-SP cells using TRAPeze XL Telomerase detection Kit (Chemicon) according to the manufacturer's protocol. Sorted SP and non-SP cells were pelleted by centrifugation and resuspended in CHAPS lysis buffer at t 100,000 cells/200μl. Two microlitre (corresponding to 1000 cells) of lysate was added to the reaction mix and subjected to telomeric repeat amplification protocol (TRAP) reaction. Telomerase activity was quantified using fluorescence plate reader, Synergy HT (BioTek). The TPG (Total Product Generated) value of each sample was obtained from the standard curve of quantification controls. TRAP assay products were visualized by 10% polyacrylamide gel electrophoresis.

2.7 Statistics

Statistical significance between normoxia vs. hypoxia, or SP vs. non-SP, was assessed by paired-t test. When a p value was <0.05, the difference was considered significant.

3 Results

3.1 Hypoxia stimulates the SP fraction of chondroprogenitors

To determine the influence of oxygen tension on SP fraction, ATDC5 cells cultured under normoxia (20% oxygen) or hypoxia (1% oxygen) were subjected to Hoechst 33342 dye efflux assay. Cells were expanded from 1×10⁵ cells per 100mm plate for 4 days up to 60–70% confluency. Cells from 6 dishes were pooled as one sorting sample. We could find typical SP fractions swing out from the left upper quadrant of the main population (Fig. 1A). Hoechst staining in the presence of ABC transporter inhibitor verapamil (125μg/ml) resulted in the absence of SP subsets. The percentage of SP subset was significantly higher in hypoxia (p<0.05, n=4; Fig. 1B). Cell sizes were compared by the forward scatter value of FACS analysis. Gates for non-SP cells were set in the center of main population. The medians of forward scatter value were similar in both SP and non-SP subsets (Fig. 1C).

Fig. 1

Flow cytometry of ATDC5 cells expanded in normoxia or hypoxia (1%) and stained with Hoechst 33342. (A) Representative FACS profile of SP subset of ATDC5 cells. ATDC5 cells were grown to 60–70% confluency. (B) Comparison of percentage of SP subset between normoxia and hypoxia (n=4). (C) Comparison of forward scatter value of SP cells and non-SP cells.

3.2 Gene expression analysis of SP and non-SP cells

Total RNA was extracted from the freshly sorted cells. No significant difference was seen in Sox9 and Hypoxia inducible factor 1 alpha (Hif1a) expression between SP and non-SP cells. Further, there was no difference of Hif1a expression between normoxia and hypoxia (data not shown). SP cells had a higher expression of abcb1a than non-SP cells, the difference being significant in hypoxia (p<0.05, n=4). abcb1b and abcg2 were expressed more highly than abcb1a. However, there were no significant differences in the expression profiles of abcb1b and abcg2 between SP and non-SP cells (Fig. 2). The abcb1a gene expression profiles in SP and non-SP subsets were similar between hypoxia and normoxia.

Fig. 2

Results of quantitative RT-PCR analysis of three ABC transporter genes (abcb1a/b, abcg2). Expression of each gene was normalized to that of GAPDH. Values represents mean±standard deviation. *p<0.05 (n=3).

3.3 Telomerase activity in SP and non-SP cells

Cell lysates of SP and non-SP fractions were prepared according to the actual cell count numbers of the cell sorter. Positive fluorescence signals and DNA ladders on PAGE from PCR products were detect using samples from each fraction equivalent to 1000 cells. SP cells showed lower TPG unit value than non-SP cells in both normoxia and hypoxia. However, no statistical significance was found in SP vs. non-SP (Fig. 3, n=3). The mean TPG unit value of SP was higher in hypoxia.

Fig. 3

Telomerase activity was examined by the telomeric repeats amplifying protocol (TRAP) assay for SP and non-SP (NSP) fractions. (A) Result of TPG (Total Product Generated) units were quantified by using standard curve of quantitation controls and shown as mean±standard deviation. (n=3) (B) A representative PAGE analysis of PCR products. Each cell lysate treated with heat (+H) was used as negative control.

3.4 Cells expanded from SP and non-SP showed similar Hoechest staining profile

Sorted SP and non-SP cells from the hypoxic condition were cultured in the expansion medium in hypoxia. These cells were subjected again to Hoechst 33342 assay. Both cell types had similar staining profiles to the first assay (Fig. 4).

Fig. 4

SP cell analysis of cultured SP and non-SP cells. SP and non-SP cells were grown and expanded in DMEM/F12 supplemented with 5%FBS. Both cells showed similar pattern of SP and non-SP subset again.

3.5 Hypoxia inhibits chondrogenic matrix synthesis of ATDC5 in micromass culture

Before testing the chondrogenic property of SP and non-SP cells, we checked that of ATDC5 cells in normoxia and hypoxia. Micromass culture were maintained under normoxia or hypoxia for 7 days. In the absence of ITS+, micromass culture under both normoxia and hypoxia did not exhibit positive alcian blue staining. In the presence of ITS+, ATDC5 cells cultured in the normoxic condition stained positively for alcian blue, increased staining being observed by the supplementation of recombinant BMP4 (Fig. 5A and B, n=3). Micromass cultured in the hypoxic condition showed weak staining for alcian blue even in the presence of ITS+ and BMP4 (Fig. 5A and B, n=3). ATDC5 cells in the absence of ITS+ showed spindle to polygonal shape under the phase contrast microscope. Extracellular matrix filled the space among polygonal shaped cells in the presence of ITS+ under normoxia. Layers of round shaped cells existed in the extracellular matrix in the presence of ITS+ and BMP4 under normoxia. In contrast, ATDC5 cells cultured under hypoxia formed almost a single monolayer with less extracellular matrix even in the presence of ITS+ and BMP4 (Fig. 5C, n=3). Total RNA was extracted from cells before making micromass (Day 0), Day 3 and Day 6 of micromass culture. Sox9 expressions were upregulated in a time-dependent manner and dominant in normoxic condition. In the presence of ITS+, Aggrecan and Col2a1 expressions were upregulated under normoxia (20% oxygen). In the presence of ITS+ and BMP4, Aggrecan and Col2a1 expressions were upregulated under both normoxia and hypoxia at Day 6 (Fig. 6, n=3).

Fig. 5

Chondrogenic differentiation of ATDC5 cells cultured in micromass under normoxic or hypoxic condition. ATDC5 cells for normoxic and hypoxic experiments were expanded in the same oxygen tension. Controls were cultured without ITS. (A) Alcian blue staining of micromass. (B) Quantitative analysis of Alcian blue staining. Alcian blue staining was markedly inhibited by hypoxia. (n=3) *p<0.05, **p<0.01. (C) Phase contract microscope images of cultured micromass. Scale bar=100μm.

Fig. 6

Quantitative RT-PCR analysis of cultured micromass. Expression of each gene was normalized to that of GAPDH expression. Y axis (−δCt) represents the mean cycle number behind the GAPDH expression. Samples for Day 0 were obtained before making micromass. Values represents mean±standard deviation (n=3).

3.6 Chondrogenic differentiation of cells derived from SP of non-SP fraction

Micromass cultures with cells expanded from SP and non-SP were maintained under normoxia for 7 days. There was no significant difference between alcian blue staining of SP and non-SP derived cells (Fig. 7A and B, n=3). Gene expression assays for Sox9, Col2a1, Aggrecan and Superficial Zone Protein (SZP) showed a slightly high mean expression level in SP cells cultured with ITS+ and BMP4. However, the differences were not statistically significant (Fig. 7C n=3).

Fig. 7

Chondrogenic differentiation of ATDC5 expanded from hypoxia SP or non-SP cells. Micromass cultures were maintained under normoxia for 7 days. Controls were cultured without ITS. (A) Alcian blue staining of micromass. (B) Quantitative analysis of Alcian blue staining. (C) Quantitative RT-PCR analysis of micromass cultured with ITS and BMP4 (100ng/ml). Expression of each gene was normalized to that of GAPDH expression. Y axis (−δCt) represents the cycle number behind the GAPDH expression. Values represents mean±standard deviation (n=3).

4 Discussion

A SP subset resides in the ATDC chondroprogenitor cell line, which was enhanced by hypoxia. This novel finging shows the SP fraction of ATDC5 its enhancement by hypoxia. ATDC5 was subcloned from mouse teratocarcinoma cells (Atsumi et al., 1990). A subcloned cell line seems to consist of a uniform cell type. However, a subset with SP phenotype thought to have a different level of differentiation was found. So far, SP fractionshave been detected in several cell line, including C2C12 (Benchaouir et al., 2004), Hela, C6 glioma, MCF7, and B104 cancer cell lines (Setoguchi et al., 2004). Investigation of the SP phenomenon in cell lines may provide an excellent model for stem/progenitor cell research and insight into cell differentiation. We explored the effect of hypoxia in cells cultured below confluency, because excessive cell density may affect the oxygen supply to the cells.

Previous studies have shown that SP cells sorted from primary culture are small (Murayama et al., 2002; Umemoto et al., 2006). However, less is known about the size of SP cells from cell lines. In ATDC5 cells, similar sizes were seen in SP and non-SP. A difference in abcb1a expression between SP and non-SP was detected. Benchaouir et al. (2004) also showed higher gene expression of abcb1a in an SP subset than in non-SP of C2C12 myoblasts. Our results on ABC transporter expression profiles were consistent with that of C2C12. This suggests that abcb1a expression is a characteristic of SP cells in mouse cell lines, which is essential for the SP phenomenon. Considering the relationship between ABCB1 and hypoxia (Comerford et al., 2002), abcb1a may also playing an important role in the enhancement of SP subset by hypoxia. Immunohistochemical analysis of ABC transporters may be useful tool for the further investigation. However, a specific antibody against mouse ABCB1a is not available (Barrand and Twentyman, 1992).

mRNA of HIF-1a is constitutively expressed in most cells. Under normoxic condition, HIF-1a is degraded post-translationally at the O2-dependent degradation domain (Huang et al., 1998). ATDC5 cells cultured in hypoxia did not show a significant difference in Hif1a gene expression compared with normoxia; hence mRNA of HIF-1a is constitutively expressed in both SP and non-SP cells. Degradation of HIF-1a was instrumental in the cellular response to oxygen tension. However, it is difficult to prove the deference of nuclear translocation of HIF-1a between SP and non-SP because all the cells were exposed to normoxic condition during staining and sorting.

Telomerase activity of SP cells has been described by Umemoto et al. (2006). SP cells found in corneal epithelial cells residing in the basal layer of limbus showed quiescent cell-cycle state and lower telomerase activity than non-SP cells. Our results on telomerase activities showed no significant differences. However, SP cells tended to exhibit lower telomerase activity than non-SP cells both under normoxia and hypoxia. Hypoxia upregulates telomerase activity. HIF-1 is a mediator of telomerase gene expression (Nishi et al., 2004). Telomerase activity in SP cells under our hypoxic condition tended to show higher telomerase activity. SP cells enhanced by hypoxia might be less quiescent.

Both SP and non-SP cells of ATDC5 could be expanded in the same culture condition as their maintenance. Our result show that cells from SP lost their dye efflux capacity after cell division. Interestingly, cultured cells from non-SP gained the function of SP. This suggests that potential SP cells reside in non-SP cells of ATDC5. The SP subset was found in cultured cells arose from the non-SP subset of C2C12 cell line (Benchaouir et al., 2004), but not in those arose from the non-SP subset of C6 glioma cancer cell line (Kondo et al., 2004). Our data is similar to C2C12. These results may suggest that the potential of non-SP subset to generate SP cells differ between cancer and other cell lines.

Exogenous BMP4 stimulates the early-phase chondrogenic differentiation of ATDC5 (Shukunami et al., 2000). From our results of micromass culture, BMP4 stimulated aggrecan and collagen type 2 gene expression. However, under hypoxia, micromass showed no alcian blue staining. Based on phase contrast microscopy, the cells were rounded and did not appear layered. As a micromass itself may be a hypoxic environment, micromass culture in hypoxia might offer a harmful level of hypoxia and prevent alcian blue staining.

Cultured cells derived from SP cells of C2C12 were delayed in muscle differentiation compared with non-SP cells (Benchaouir et al., 2004). There was no significant difference in the chondrogenic differentiation between cells derived from SP and non-SP in our work. Also there was no difference in SZP (Schumacher et al., 1994), which is a known marker of superficial zone chondrocytes. C2C12 had difference in myogenic marker MyoD between SP and non-SP (Benchaouir et al., 2004). However, our results with ATDC5showed no significant difference in Sox9. This difference in marker gene expression in SP may be responsible for the difference of the stemness of SP cells.

ATDC5 differentiate into chondrocytes at a very high frequency and into unidentified pigmented cells at a very low frequency (Atsumi et al., 1990). ATDC5 is in rather advanced stage of chondrogenic differentiation. These characteristics of ATDC5 may reflect the low stemness of this cell line. However, ATDC5 exhibited a distinct SP subset. The SP phenomenon is under the influence of dye efflux capacity more directly than the stemness of the cells. Considering our results, hypoxia did not enhance stemness of ATDC5, but enhanced the dye efflux capacity of them. Nevertheless, analysis of SP subset of other chondrogenic cell lines may provide insight into the role of stem or progenitor cells in cartilage.

In conclusion, an SP cell fraction resides in ATDC5 chondroprogenitor cells, a population that is enhanced by hypoxia. This fraction was characterized by ABCB1a gene expression and lower telomerase activity. SP and non-SP fractions of ATDC5 were similar chondroprogenitor cells, showing equal chondrogenic marker gene expression profiles.

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Received 13 September 2008/18 March 2009; accepted 3 June 2009

doi:10.1016/j.cellbi.2009.06.009

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