CD49f Enhances Multipotency and Maintains Stemness Through the Direct Regulation of OCT4 and SOX2§

Authors

  • Kyung-Rok Yu,

    1. Adult Stem Cell Research Center, Seoul National University, Seoul, Republic of Korea
    2. Department of Veterinary Public Health, Laboratory of Stem Cell and Tumor Biology, Seoul National University, Seoul, Republic of Korea
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  • Se-Ran Yang,

    1. Adult Stem Cell Research Center, Seoul National University, Seoul, Republic of Korea
    2. Department of Veterinary Public Health, Laboratory of Stem Cell and Tumor Biology, Seoul National University, Seoul, Republic of Korea
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  • Ji-Won Jung,

    1. Division of Intractable Diseases, Center for Biomedical Sciences, National Institute of Health, Seoul, Republic of Korea
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  • Hyongbum Kim,

    1. Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seoul, South Korea
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  • Kinarm Ko,

    1. Department of Stem Cell Biology, Konkuk University, Seoul, Republic of Korea
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  • Dong Wook Han,

    1. Department of Stem Cell Biology, Konkuk University, Seoul, Republic of Korea
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  • Sang-Bum Park,

    1. Adult Stem Cell Research Center, Seoul National University, Seoul, Republic of Korea
    2. Department of Veterinary Public Health, Laboratory of Stem Cell and Tumor Biology, Seoul National University, Seoul, Republic of Korea
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  • Soon Won Choi,

    1. Adult Stem Cell Research Center, Seoul National University, Seoul, Republic of Korea
    2. Department of Veterinary Public Health, Laboratory of Stem Cell and Tumor Biology, Seoul National University, Seoul, Republic of Korea
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  • Soo-Kyung Kang,

    1. Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany
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  • Hans Schöler,

    1. Department of Veterinary Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
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  • Kyung-Sun Kang

    Corresponding author
    1. Adult Stem Cell Research Center, Seoul National University, Seoul, Republic of Korea
    2. Department of Veterinary Public Health, Laboratory of Stem Cell and Tumor Biology, Seoul National University, Seoul, Republic of Korea
    • Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
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    • Telephone: 82-2-880-1246; Fax: 82-2-876-7610


  • Author contributions: K.-R.Y.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; S.-R.Y.: collection and/or assembly of data, data analysis and interpretation, and manuscript writing; J.-W.J.: conception and design and manuscript writing; H.K.: data analysis and interpretation and manuscript writing; K.K.: collection and/or assembly of data and data analysis and interpretation; D.W.H.: data analysis and interpretation and manuscript writing; S.-B.P. and S.W.C.: collection and/or assembly of data; S.-K.K.: data analysis and interpretation; H.S.: data analysis and interpretation and conception and design; K.-S.K.: conception and design, manuscript writing, data analysis and interpretation, and final approval of manuscript. K.R.Y. and S.R.Y. contributed equally to this article.

  • Disclosure of potential conflicts of interest is found at the end of this article.

  • §

    First published online in STEM CELLSEXPRESS February 6, 2012.

Abstract

CD49f (integrin subunit α6) regulates signaling pathways in a variety of cellular activities. However, the role of CD49f in regulating the differentiation and pluripotency of stem cells has not been fully investigated. Therefore, in this study, human mesenchymal stem cells (hMSCs) were induced to form spheres under nonadherent culture conditions, and we found that the CD49f-positive population was enriched in MSC spheres compared with MSCs in a monolayer. The expression of CD49f regulated the ability of hMSCs to form spheres and was associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. Furthermore, the forced expression of CD49f modulated the proliferation and differentiation potentials of hMSCs through prolonged activation of PI3K/AKT and suppressed the level of p53. We showed that the pluripotency factors OCT4 and SOX2 were recruited to the putative promoter region of CD49f, indicating that OCT4 and SOX2 play positive roles in the expression of CD49f. Indeed, CD49f expression was upregulated in human embryonic stem cells (hESCs) compared with hMSCs. The elevated level of CD49f expression was significantly decreased upon embryoid body formation in hESCs. In hESCs, the knockdown of CD49f downregulated PI3K/AKT signaling and upregulated the level of p53, inducing differentiation into three germ layers. Taken together, our data suggest that the cell-surface protein CD49f has novel and dynamic roles in regulating the differentiation potential of hMSCs and maintaining pluripotency. STEM CELLS 2012;30:876–887

INTRODUCTION

Human mesenchymal stem cell (hMSCs) have typically been selected and expanded in two-dimensional monolayer cultures; however, cultured hMSCs are heterogeneous, making them difficult to use for therapeutic and research applications [1, 2]. Understanding the cellular heterogeneity of adult stem cells, such as hMSCs, and isolating a cell population with higher cellular potency by identifying a surface marker have been major interests of the scientific community. It has been reported that nonadherent sphere culture methods can enrich self-renewal capability in many tissue-specific adult stem cells and primary cancers [3–5]. Sphere-forming ability is one of the properties of cancer stem cells capable of tumor regeneration. This characterization has been reported in leukemia and primary tumors of the breast, colon, and brain [6]. However, sphere-forming cells from the inner ear of mouse can differentiate into vestibular and cochlear sensory epithelia [7], and sphere-forming mesenchymal cells derived from the dermis attain hair-inducing capacity through the process of sphere formation [8]. Therefore, it is reasonable to hypothesize that a three-dimensional (3D) culture of hMSCs is required to isolate a more homogeneous population and to increase proliferation. Recently, it has been shown that growing hMSCs in 3D matrices significantly increases their ability to both expand cell populations and maintain them in an undifferentiated state [9, 10]. However, the molecular mechanisms regulating how sphere-forming cells maintain stemness are still unknown.

Given the characteristic ability of hMSCs to survive under nonadherent culture conditions, it is worth studying integrins as a plausible selection marker for this population. Integrin-mediated cell-to-cell or cell-to-extracellular matrix (ECM) interaction is an essential part of cellular behavior, adhesion, migration, proliferation, and differentiation [11, 12]. Integrin interactions cue cascades of signaling pathways involved in cell survival and proliferation, such as the focal adhesion kinase (FAK) and the phosphatidylinositol 3-kinase (PI3K)/AKT pathways [13, 14]. The PI3K signaling pathway, initiated by the activation of integrins, is important for cell migration, invasion, and proliferation [15, 16]. Integrins transduce survival signals from the ECM to cells, leading to the inhibition of a p53-regulated apoptotic pathway [17]. Furthermore, crosstalk between AKT and p53 has been found. The activation of AKT, which is driven through a kinase cascade from PI3K, can phosphorylate Mdm2, resulting in p53 inactivation and degradation [18, 19]. Additionally, p53 plays important roles in p21 induction following DNA damage and p16 induction following premature senescence [20, 21].

Integrin subunit α6 (CD49f) is required for endothelial progenitor cell migration and adhesion and the activation of postischemic vascular repair [22]. LinSca-1+CD49fHi cells derived from the prostate were capable of forming spheres in 3D cultures, eventually promoting the progression to prostate carcinoma [23]. In mice with myocardial infarcts, CD49f may be a marker of high therapeutic potency for bone marrow-derived multipotent stem cells [24]. CD49f plays a role in efficiently generating long-term multilineage grafts in hematopoietic stem cells [25]. In human embryonic stem cells (hESCs), integrin–ECM engagement is essential for cell proliferation and adhesion. Specifically, CD49f is prominently expressed in hESCs, and its ligand, laminin, can be used as a substrate for the expansion of undifferentiated hESCs [11, 26–28]. However, the role of CD49f in MSC sphere formation and the maintenance of pluripotency in hESCs through PI3K/AKT signaling remains obscure.

OCT4 and SOX2 are two core components of the pluripotency circuit for maintaining the self-renewal capacity of undifferentiated ESCs [29]. These genes, expressed in ESCs and tissue-specific adult stem cells such as MSCs, help to maintain an undifferentiated state and prevent differentiation [30, 31]. Together with other factors, transduced OCT4 and SOX2 may co-occupy each other's promoters and activate autoregulatory loops, resulting in the activation of endogenous pluripotency markers and the induction of pluripotency [32, 33]. Here, we demonstrate that CD49f can be regulated through an autoregulatory circuit through the direct binding of OCT4 and SOX2 on the CD49f promoter.

In this study, we show that CD49f plays crucial roles in maintaining the stemness of hMSCs and hESCs. Specifically, CD49f regulates proliferation and differentiation capacities through PI3K/AKT/p53 activity. Furthermore, we reveal crosstalk between the pluripotency genes OCT4 and SOX2 and CD49f in hMSCs and hESCs.

MATERIALS AND METHODS

Additional methods are described in the Supporting Information Materials and Methods section.

Cell Culture

hMSCs were isolated from human umbilical cord blood and cultured as previously described [34]. hESCs were obtained from the CHA Stem Cell Research Laboratory under a material transfer agreement. hMSC isolation methods and the culture conditions of hMSCs/hESCs are described in the Supporting Information.

MSC Sphere Formation

To generate MSC spheres, 1.5 × 105 hMSCs were plated on 100-mm culture dishes (Nunc, Rochester, NY, http://www.nuncbrand.com) coated with 1% agarose to prevent cells from attaching to the bottom of the plastic dish. A total of 15,000 cells were plated per milliliter of medium, and the MSC spheres were cultured for 7 days. After 7 days, MSC spheres were collected using a 40-μm pore cell strainer. The collected MSC spheres were washed with phosphate buffered saline (PBS) and gently centrifuged (60g/5 minutes) before further experimentation.

Immunocytochemistry

The immunocytochemical analysis of CD49f, OCT4, and SOX2 was performed as follows. Cultured cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com). The cells were incubated with 10% normal goat serum (Zymed Laboratories Inc., USA) and then labeled with primary antibodies against CD49f (1:200, ab20142; Abcam, Cambridge, U.K., http://www.abcam.com), OCT4 (1:200, ab19857; Abcam) and SOX2 (1:200, ab59776; Abcam), followed by a 1-hour incubation with Alexa 488- or Alexa 594-labeled secondary antibody (1:1,000; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). Nuclei were stained with Hoechst 33258 (1 μg/ml; 10 minutes). Images were captured using a confocal microscope (Nikon, Eclipse TE200, Japan).

Overexpression of CD49f, OCT4, SOX2, NANOG, and LIN28

To overexpress of the CD49f protein, the coding sequence of full-length human CD49f was cloned into the pCMV6 expression vector and transfected using Fugene 6 transfection reagent (Roche, Indianapolis, IN, http://www.roche-applied-science.com). To overexpress OCT4, SOX2, NANOG, and LIN28, hMSCs were infected with lentiviruses expressing these genes. The viral production and transduction process was performed as previously described [35]. Briefly, plasmids, including pSin-EF2-Oct4-Pur, pSin-EF2-Sox2-Pur, pSin-EF2-Nanog-Pur, or pSin-EF2-Lin28-Pur (Addgene, Cambridge, http://www.addgene.org/), were transfected into 293T cells together with the MISSION lentiviral packaging mix (Sigma), and viral supernatants were collected 48- and 72-hour post-transfection. The viral supernatants were used to infect human MSCs in the presence of 5 μg/ml polybrene (Sigma).

siRNA and shRNA Inhibition Study

To specifically inhibit OCT4, SOX2, CD49f, and p53, small interfering RNA knockdown studies were performed using commercial siRNA targeting OCT4, SOX2, CD49f, and p53 (Dharmacon, ON Target plus SMART pool, http://www.dharmacon.com/) along with a nontargeting siRNA (Dharmacon, ON Target plus SMART pool, Cat# D-001810-01, Lafayette, CO). The siRNA transfections were performed according to the manufacturer's instructions. Briefly, the cells were seeded at a concentration of 5 × 104 per well, and siRNA-containing media (without added antibiotics) were added when the cells reached 50% confluence. The cells were incubated with 50 nM siRNA for 48 hours to evaluate mRNA expression and for 72 hours to evaluate protein expression. After these incubations, RNA and protein extractions were performed for genetic and proteomic analyses. Control small hairpin RNA (shRNA) lentiviral particles and integrin α6 shRNA lentiviral particles were used to infect hMSCs in the presence of 5 μg/ml polybrene (Sigma-Aldrich).

Chromatin Immunoprecipitation Assays

Chromatin immunoprecipitation (ChIP) assays were performed according to the manufacturer's protocol (ChIP assay kit, Upstate Biotechnology, #17-295). Chromatin was immunoprecipitated using specific antibodies as described previously [36]. The primer sets used for the ChIP assays are listed in Supporting Information Table S1. Detailed procedures of ChIP and the in vitro differentiation assay, cumulative population doubling level (CPDL) analysis, Western blot analysis, reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR, inhibitor studies, flow cytometry analysis, sorting, and the (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) proliferation assay are described in Supporting Information.

Statistical Analysis

All experiments were conducted at least in triplicate (n = 3), and the results are expressed as the mean ± SD. The statistical analyses were conducted using an analysis of variance followed by Duncan's multiple range tests or Student's t test. A value of p < .05 was considered significant (*, p < .05; **, p < .01).

RESULTS

MSC Spheres Retain hMSC Properties with Improved Potential for Differentiation and Proliferation

hMSCs displayed a flattened and spindle-shaped morphology in monolayer culture (MSC monolayers), while under nonadherent conditions, hMSCs formed floating spheroid colonies (MSC spheres), which are characteristic of hMSCs (Fig. 1A). Under nonadherent conditions, 30–50 spheres per 1 × 104 cells were formed, and the mean diameter of the spheres was 50–150 μm. As shown in Supporting Information Figure S1A and S1B, both MSC monolayers and MSC spheres were positive for hMSC markers (CD44 and CD90) but negative for the hematopoietic stem cell markers CD34 and CD117, indicating that MSC spheres retained the properties of hMSCs.

Figure 1.

Enhancement of adipogenic and osteogenic differentiation and PI3K pathway activation in sphere-derived cells. (A): Phase-contrast images of human mesenchymal stem cells (hMSCs) cultured in a plastic culture dish or an agarose-coated dish. On average, 15,000 cells were seeded per milliliter of medium. Scale bar = 100 μm. (B–E): Phase-contrast images of monolayer cells (left panel) and sphere-derived cells (right panel) after induction into specific tissues. The lipid droplet accumulation in differentiated cells was visualized using oil Red O staining after 3 weeks of adipogenic induction (B, C). Mineral deposition was stained with Alizarin Red S after 3 weeks of osteogenic induction (D, E). (F) Oil Red O-stained dye was eluted using 100% isopropanol, and the absorbance was measured at a wavelength of 500 nm (*, p < .05). (G): A total of 100 mM cetylpyridinium chloride was used for the elution of Alizarin Red S dye, and the absorbance was measured at a wavelength of 570 nm (*, p < .05). (H): The CPDL was assessed in monolayer-derived cells and sphere-derived cells (**, p < .01, n = 3). (I): Western blot analysis was conducted after 7 days of sphere-inducing culture. MSC spheres were collected using a 40-μm strainer and washed twice. The phosphorylation levels of PI3K and its sequential downstream effectors AKT, GSK3β, p53, p21, and p16 were analyzed and compared between monolayer cells, spheres, 0.2 μM BIO-treated spheres, and 30 μM LY294002-treated spheres. (J): The number of MSC spheres cultured in growth medium containing 0 μM, 0.2 μM, or 0.5 μM BIO, a GSK3 inhibitor (**, p < .01). (K): The sphere size of 0 μM or 0.5 μM BIO-treated MSC spheres was measured. Phase-contrast images of the MSC spheres cultured with BIO at the indicated concentrations (**, p < .01). (L): The number of MSC spheres cultured in growth medium containing 0 μM or 3 μM CHIR99021, a GSK3 β inhibitor (**, p < .01). (M): The number of MSC spheres cultured in growth medium containing 0 μM, 10 μM, 30 μM, or 50 μM LY294002, a PI3K inhibitor (**, p < .01). (N): The sphere size of the 0 μM or 50 μM BIO-treated MSC spheres was measured. Phase-contrast images of the MSC spheres cultured with LY294002 at the indicated concentrations (**, p < .01). (O): The number of MSC spheres cultured in growth medium containing 0 μM or 1 μM wortmannin, a PI3K inhibitor (**, p < .01). Abbreviations: CPDL, cumulative population doubling level; GSK, glycogen synthase kinase 3; OD, optical density; PI3K, phosphatidylinositol 3-kinase.

To compare the differentiation capacity of MSC monolayers and MSC spheres, we induced the in vitro differentiation of both populations into adipogenic and osteogenic lineages. After adipogenic induction, MSC sphere-derived cells showed a higher adipogenic differentiation potential than MSC monolayer cells, as evidenced by oil Red O staining (Fig. 1B, 1C, 1F). The expression levels of adipogenic genes, such as C/EBP-β, aP2, PPAR-γ, and leptin, were significantly upregulated in sphere-derived cells compared to monolayer-cultured cells (Supporting Information Fig. S2A). Sphere-derived MSCs were also more effective at osteogenic differentiation, as shown by Alizarin Red S staining (Fig. 1D, 1E, 1G). The sphere-derived cells also showed elevated expression levels of Runx2 and osteocalcin after osteogenic induction (Supporting Information Fig. S2B). To compare the proliferative potency of hMSCs derived from monolayers and MSC spheres, the CPDL was measured. The sphere-derived cells showed an increased proliferation rate (CPDL 7.7 after 2 weeks) compared with the cells from the MSC monolayers (Fig. 1H). Taken together, these data suggest that the MSC sphere-derived cells have increased proliferation rates and a greater potential to differentiate toward both the adipogenic and osteogenic lineages.

The PI3K/AKT/GSK3β Pathway Modulates the Formation of MSC Spheres

We next treated the hMSCs with BIO and LY294002, a glycogen synthase kinase 3 (GSK3)-specific inhibitor and a PI3K inhibitor, respectively, to determine whether the PI3K/AKT/GSK3 and the p53, p21, and p16 pathways were involved in the formation of MSC spheres. As shown in Figure 1I, phospho-PI3K and phospho-AKT activities were dramatically increased in MSC spheres that were either untreated or treated with BIO compared to MSC monolayers, whereas supplementation with LY294002 led to the dephosphorylation of constitutively active PI3K (Tyr458,199), AKT (Ser473), and GSK3β (Ser9). The levels of endogenous p53, p21, and p16 were reduced in MSC spheres and BIO-treated spheres. We next assessed the efficiency of sphere formation after treatment with BIO or LY294002 to confirm whether the PI3K/AKT/GSK3β pathway was involved in MSC sphere formation. After 7 days of sphere induction under nonadherent conditions, supplementation with BIO significantly increased both the number and size of spheres formed, suggesting that GSK3β activity inhibits sphere formation (Fig. 1J, 1K). Furthermore, treatment with one of the most selective inhibitors of GSK3β, CHIR99021, increased MSC sphere-forming efficiency (Fig. 1L). In contrast, treatment with LY294002 at concentrations of 30–50 μM significantly reduced both the number and size of MSC spheres (Fig. 1M, 1N). MSC sphere formation was significantly inhibited by treatment with 1 μM wortmannin, which is a specific and direct inhibitor of PI3 kinase (Fig. 1O). Taken together, these results indicate that the PI3K/AKT/GSK3β signaling pathway is critical for MSC sphere formation.

Upregulation of CD49f Contributes to MSC Sphere Formation Via the Phosphorylation of FAK/Paxillin

Integrins are one of the key molecules that interact with the ECM. They initiate survival and activate growth-associated signal transduction pathways (e.g., PI3K/AKT) via local protein kinases, which include FAK and Paxillin. Therefore, we performed flow cytometry analysis to determine the integrin expression profiles in both the MSC monolayers and the MSC spheres. The expression levels of CD49a and CD49b showed no significant difference between the two culture conditions, while CD49e was slightly decreased in MSC spheres. However, both CD49f and CD104, which form a heterodimer, were upregulated in MSC sphere-derived cells (Fig. 2B, Supporting Information Fig. S3). In line with the flow cytometry data, immunostaining showed an increase in CD49f-positive cells in the MSC spheres. Both an elevated CD49f expression level and an increase in the number of cells positive for this marker were observed in the MSC sphere cultures compared to the MSC monolayer cultures (Fig. 2A). Using Western blot analysis, high levels of phospho-Paxillin, phospho-FAK, and CD49f were observed in MSC spheres (Fig. 2C). To confirm that integrin expression affected the efficiency of sphere formation, we purified CD49f-positive and CD49f-negative hMSCs using flow cytometry and compared the efficiency of sphere formation for these two distinct populations. The gene expression levels of Paxillin, FAK, and integrin-linked kinase (ILK), which are tyrosine kinases downstream of the integrins, were increased in the CD49f-positive population (Fig. 2D). The CD49f-positive cells were more efficient at forming spheres, and the mean diameter of the spheres was increased approximately 2.4-fold compared to the CD49f-negative cells (Fig. 2E, 2F, 2G). To determine the mechanism responsible for the increased tendency toward sphere formation in the CD49f-positive cells, we investigated the molecular signaling. Western blotting results showed that FAK, PI3K, and AKT were activated and that the protein levels of p53, p21, and p16 were lower in MSC spheres derived from CD49f-positive cells compared to hMSCs from monolayer or MSC spheres derived from CD49f-negative (Fig. 2H). Furthermore, transfection with siRNA targeting CD49f resulted in a decrease in the number and size of MSC spheres (Fig. 2I–2L). In contrast, p53 inhibition increased the number and size of MSC spheres (Supporting Information Fig. S4C, S4D). Taken together, these data suggest that the improvement in sphere formation is associated with the activation of CD49f and the PI3K/AKT/p53 signaling pathway.

Figure 2.

Upregulation of CD49f in sphere-derived cells and its regulation of sphere-forming efficiency. (A): Immunocytochemical analysis of CD49f expression in monolayer-cultured and sphere-derived human mesenchymal stem cells (hMSCs). Monolayer MSCs and dissociated MSC spheres were seeded with the same starting number of cells into four-well chamber slides. After 3 days, the cells were fixed and analyzed with the indicated antibodies. Scale bar = 100 μm. (B): The expression level of CD49f was analyzed using flow cytometry with a FITC-conjugated antibody. Measurements were performed in triplicate (**, p < .01). (C): The activities of downstream signal transducers of integrin, such as FAK and Paxillin, were estimated using Western blotting. (D): The expression levels of the integrin-related markers CD49f, Paxillin, FAK, and ILK were analyzed in both CD49f-negative and CD49f-positive populations using reverse transcription polymerase chain reaction (RT-PCR). (E): After flow cytometry sorting into CD49f-negative and CD49f-positive cell populations, the sphere-forming efficiency of 20,000 cells from each population was assessed (**, p < .01) (F): The sphere size was measured in the CD49f-negative and CD49f-positive populations. For the assessment of sphere size, 50 spheres were randomly selected and analyzed statistically (**, p < .01). (G): Phase-contrast images of a single representative sphere derived from CD49f-negative or CD49f-positive cells. (H): Western blot analysis was conducted on monolayer cells and MSC spheres from CD49f-positive and CD49f-negative populations. The phosphorylation levels of PI3K and its sequential downstream effectors AKT, GSK3β, p53, p21, and p16 were analyzed. (I): The expression level of CD49f was analyzed using RT-PCR 3 days after transfection with siCTL or siCD49f. (J): After transfection with siCTL or the siCD49f, the sphere-forming efficiency of each population was assessed (**, p < .01). (K): Sphere size was measured in the control siRNA and the siRNA targeting p53-transfected MSC spheres. For the assessment of sphere size, 50 spheres were randomly selected and analyzed statistically (**, p < .01). (L): Phase-contrast images of a single representative sphere derived from hMSCs transfected with the siCTL or the siCD49f. Scale bar = 100 μm. Abbreviations: FAK, focal adhesion kinase; GSK3, glycogen synthase kinase 3; ILK, integrin-linked kinase; PI3K, phosphatidylinositol 3-kinase; siRNA, small interfering RNA; siCTL, control siRNA; siCD49f, siRNA targeting CD49f.

Overexpression of CD49f Regulates Cellular Proliferation and Differentiation Via Regulation of the PI3K/AKT/p53 Pathway

To further assess whether CD49f gene expression was dependent upon the activation of the PI3K/AKT/GSK3β pathway, CD49f-overexpressing hMSCs were treated with LY294002 or BIO for 24 or 48 hours. In the CD49f-overexpressing hMSCs, MTT assays revealed that the growth rates of BIO-treated hMSCs were higher than those of the untreated hMSCs, whereas LY294002 inhibited cell proliferation (Fig. 3A). Because p53 is closely related to the CD49f-PI3K/AKT/GSK3β signaling pathway, the influence of p53 inhibition on the cell cycle was assessed using flow cytometry. The inhibition of p53 increased the number of cells in S-phase and decreased the number of cells in G0/G1 phase (Supporting Information Fig. S4B). These results suggest that p53 inhibition activates the cell cycle. In the sphere formation assay, BIO treatment consistently increased the number of MSC spheres, whereas LY294002 inhibited the sphere formation of the CD49f-overexpressing hMSCs (Fig. 3B). Western blot analysis showed that CD49f overexpression led to increased levels of PI3K, AKT, and GSK3β phosphorylation in the hMSCs. BIO treatment led to the induction of AKT and GSK3β phosphorylation, whereas LY294002 treatment suppressed AKT phosphorylation in the CD49f-overexpressing hMSCs (Fig. 3C). We also found that active CD49f expression was sufficient to reduce p53, p21, and p16 levels, but the decrease in p53, p21, and p16 levels was blocked by LY294002, suggesting that PI3K/AKT is required for p53, p21, and p16 regulation in hMSCs. These data suggest that the upregulation of CD49f enhances cell proliferation and sphere formation via regulation of the PI3K/AKT pathway. The CD49f-overexpressing hMSCs had a significantly enhanced osteogenic differentiation potential when compared to wild-type hMSCs. Strikingly, osteogenic differentiation was further boosted in the presence of BIO, as evidenced by an increased level of mineral deposition measured using Alizarin Red S staining. In contrast, treatment with LY294002 dramatically reduced the osteogenic differentiation potential of CD49f-overexpressing hMSCs (Fig. 3D, 3E). The gene expression levels of osteogenic markers, such as osteocalcin, Runx2, VDR, and MSX2, were consistently and significantly increased in the presence of BIO. However, LY294002 treatment inhibited osteogenic marker expression, as determined using conventional RT-PCR (Fig. 3F). Under adipogenic differentiation conditions, CD49f-overexpressing cells showed significantly increased levels of lipid accumulation with oil Red O staining compared to wild-type hMSCs, indicating that the overexpression of CD49f could also enhance the adipogenic differentiation potential of hMSCs. However, both BIO and LY294002 treatments suppressed lipid accumulation (Fig. 3G, 3H). Similarly, the gene expression levels of the adipogenic transcription factors, including C/EBP-β, aP2, PPARγ, and leptin, were significantly increased in the CD49f-overexpressing cells. However, both BIO and LY294002 treatments inhibited the transcription of adipogenic-specific markers (Fig. 3I). We further assessed the osteogenic and adipogenic differentiation capabilities of the p53-inhibited hMSCs. The inhibition of p53 led to increases in the osteogenic and adipogenic differentiation potentials (Supporting Information Fig. S4E, S4F). Therefore, these data suggest that the PI3K/AKT/p53 pathway regulates hMSC differentiation.

Figure 3.

CD49f overexpression enhances the proliferation, sphere-forming efficiency, and differentiation of human mesenchymal stem cells (hMSCs) and activates the PI3K/AKT/GSK3β pathway. (A): An MTT cell proliferation assay was performed in the presence or absence of CD49f, LY294002, or BIO. Cells were seeded in 24-well culture plates, and the OD was measured at 24 and 48 hours (*, p < .05; **, p < .01). (B): Sphere numbers were measured in the presence or absence of CD49f, LY294002, or BIO (*, p < .05; **, p < .01). (C): A Western blot analysis of PI3K/AKT/GSK3β, p53, and p16 was performed. Cells were transfected with a CD49f expression vector and treated with LY294002 and BIO. The phosphorylation levels of the protein kinases were determined using Western blot with phospho-specific antibodies against each protein. Each well was loaded with 20 μg of protein lysate. (D): hMSCs were cultured in osteogenic induction media in the presence or absence of CD49f, LY294002, or BIO. Osteogenesis was determined using Alizarin Red S staining. (E): An Alizarin Red S-stained dish was incubated with cetylpyridinium chloride. The levels of eluted Alizarin Red S solution were determined using an ELISA at 570 nm (**, p < .01). (F): The efficiency of osteogenic differentiation was evaluated by the expression of osteogenic marker genes, including osteocalcin, RUNX2, VDR, and MSX2. (G): hMSCs were cultured in adipogenic induction media in the presence or absence of CD49f, LY294002, or BIO. The accumulation of lipid droplets in the cytosol was visualized using oil Red O staining after 2 weeks of induction. (H): Oil Red O was eluted using 100% isopropanol, and the absorbance was measured via spectrometry at 500 nm (**, p < .01). (I): The efficiency of adipogenic differentiation was evaluated through the expression of adipogenic marker genes, including CEBP-β, aP2, PPAR-γ, and leptin. Abbreviations: GSK3, glycogen synthase kinase 3; MTT, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); OD, optical density; PI3K, phosphatidylinositol 3-kinase.

Overexpression of OCT4 and SOX2 Enhances CD49f Expression by Binding to the CD49f Promoter

The enhancement of hMSC multipotential by CD49f prompted us to investigate the relationship between CD49f and the pluripotency marker genes OCT4 and SOX2. First, we analyzed the expression of pluripotency markers in MSC spheres. In MSC spheres, the mRNA levels of OCT4, SOX2, NANOG, and LIN28 were increased compared with MSC monolayers (Fig. 4A). To determine whether OCT4 and SOX2 directly regulated CD49f, we introduced siOCT4 and siSOX2 into hMSCs. OCT4 and SOX2 siRNA blocked OCT4 and SOX2 expression, respectively (Fig. 4B, 4C). In addition, the siRNA targeting either OCT4 or SOX2 significantly repressed CD49f expression, resulting in decreased sphere formation (Fig. 4D, 4E). To determine how OCT4 and SOX2 regulated CD49f expression, we overexpressed either OCT4 or SOX2 in hMSCs and examined OCT4, SOX2, and CD49f expression using immunocytochemistry. The overexpression of both OCT4 and SOX2 led to increased CD49f expression (Fig. 4G). The overexpression of CD49f in hMSCs was also confirmed using flow cytometry analysis. As shown in Figure 4H, OCT4 and SOX2 overexpression led to a larger CD49f-positive subpopulation. To clarify how OCT4 and SOX2 regulated CD49f expression, we performed ChIP analysis to investigate the direct binding of both OCT4 and SOX2 to putative CD49f promoter regions. For ChIP, Tera-1 cells, hMSCs, and OCT4/SOX2/LIN28/NANOG-overexpressing hMSCs were cross-linked with formaldehyde, and the fragmented chromatin lysates were used for immunoprecipitation using the OCT4 and SOX2 antibodies. Sequentially located primers (Supporting Information Table S1) along the entire conserved promoter region of CD49f were used to analyze OCT4- and SOX2-binding DNA sites. As shown in Figure 4F, specific regions within the CD49f promoter were enriched with OCT4 and SOX2 protein. We found that the CD49f promoter region has the classic canonical octamer sequence (CD49f-1, ATTTGCAT) and the partially conserved sequence (CD49f-2, ATGTAAA) for the OCT4-binding site and the SOX2-binding site (CD49f-3, CATTATT). A negative control (IgG control) showed no significant enrichment in the surveyed region. These data suggest that OCT4 and SOX2 bind directly to the CD49f promoter and induce the expression of CD49f. We next investigated whether CD49f expression was also regulated by other reprogramming factors in hMSCs, such as LIN28 and NANOG. We introduced OCT4, SOX2, LIN28, and NANOG into hMSCs in different combinations and determined the expression level of CD49f. Again, introducing OCT4/SOX2 increased CD49f expression. Overexpressing all four factors (OCT4/SOX2/LIN28/NANOG) further enhanced the transcription level of CD49f (Fig. 4I). As observed using Western blot analysis, the protein levels of both CD49f and phosphor-FAK were increased in OCT4/SOX2- and OCT4/SOX2/LIN28/NANOG-overexpressing hMSCs (Fig. 4J). Therefore, these data suggest that OCT4, SOX2, LIN28, and NANOG form a positive feedback loop that regulates CD49f expression and integrin signaling pathway in hMSCs.

Figure 4.

OCT4 and SOX2 bind to and regulate CD49f transcription through the CD49f promoter region. (A): A quantitative estimation of mRNA expression as detected using real-time polymerase chain reaction (PCR). Relative mRNA expression levels of pluripotency markers are presented for monolayer cells and MSC spheres. All analyses were performed in triplicate and normalized to endogenous β-actin (*, p < .05; **, p < .01). (B, C): Human MSCs (hMSCs) were transfected with siOCT4 or siSOX2. Nontargeting siRNA (siCTL) was used as a control. The expression levels of OCT4 and SOX2 were analyzed using real-time PCR (**, p < .01). (D): After 48 hours of transfection, the cells were collected, and real-time PCR was performed to assess CD49f expression (**, p < .01). (E): Sphere-forming efficiency was measured in siRNA-transfected cells and control-transfected cells (**, p < .01). (F): ChIP was performed using anti-OCT4 and anti-SOX2 antibodies. Three regions on the CD49f promoter were targeted using specific primer sets, as described in Supporting Information Table S1. “NC” indicates negative control (IgG control). (G): Immunocytochemical analyses of CD49f, OCT4, and SOX2 were performed on both control virus- and OCT4 and SOX2 virus-infected cells. Nuclei were stained using Hoechst (blue). Scale bar = 50 μm. (H): The CD49f-positive population was estimated using flow cytometry for control and OCT4- and SOX2-overexpressing cells. (I): Quantitative estimates of CD49f mRNA expression were determined in control cells and cells overexpressing the indicated combinations of OCT4, SOX2, LIN28, and NANOG (**, p < .01). (J): Western blotting was performed to determine CD49f and phospho-FAK expression in the indicated combinations of virus-infected cells. Abbreviations: CTL, control; ChIP, chromatin immunoprecipitation; FAK, focal adhesion kinase; MSCs, mesenchymal stem cells; NC, negative control; siRNA, small interfering RNA; siCTL, siRNA control; siOCT4, siRNA targeting OCT4; siSOX2, siRNA targeting SOX2.

CD49f Maintains Cellular Pluripotency Through the PI3K/AKT/p53 Pathway

We next investigated the effect of CD49f on pluripotency maintenance through embryoid body formation and the depletion of CD49f in hESCs using siRNA and shRNA. Embryoid bodies were generated from hESCs for 8 days in suspension culture and were attached to gelatin-coated dishes for another 8 days to induce differentiation (Fig. 5A). The hESCs showed significantly upregulated expression of CD49f, OCT4, SOX2, and NANOG compared to hMSCs, whereas CMYC mRNA levels were comparable in both cell lines. CD49f expression and pluripotency markers decreased upon differentiation, which suggests that CD49f expression is closely related to pluripotency. In contrast, the gene expression of CMYC was not influenced by differentiation (Fig. 5B). To determine the role of CD49f in the maintenance of the pluripotency of hESCs, we treated the hESCs with siRNA against CD49f. After three rounds of consecutive transfection with siCD49f, the hESCs gradually showed typical differentiated morphologies and reduced alkaline phosphatase (AP) activity (Fig. 5C, 5D). Furthermore, a significant reduction in AP-positive colonies was observed after the two rounds of control and CD49f-targeting shRNA lentiviral particles (Fig. 5E).

Figure 5.

CD49f maintains the pluripotency of hESCs through the PI3K/AKT/GSK3β pathway. (A): Phase-contrast images of hMSCs, hESCs, and EBs. For spontaneous differentiation, the EBs were generated from hESCs after 8 days in suspension culture. To trigger further differentiation, the EBs were transferred to gelatin-coated dishes and maintained for another 8 days. (B): Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to determine the expression levels of pluripotency marker genes, CD49f, and β-actin in hMSCs, hESCs, and hESC EBs. (C): Phase-contrast images and AP staining images of hESCs transfected with the siCTL and the siCD49f. (D): A representative image and quantitative analysis of AP-positive colonies of hESCs transfected with siCTL and siCD49f. The expression levels were normalized to cells transfected with siCTL. (E): A representative image and quantitative analysis of AP-positive colonies of hESCs infected with shCTL and shCD49f viruses. (F): Immunocytochemical analyses of OCT4 and SOX2 were performed on both control siRNA- and siCD49f-transfected cells. Nuclei were stained using Hoechst (blue). (G): The expression levels of CD49f, pluripotency marker genes, and additional markers for the three germ layers in the siRNA control- and siCD49f-transfected hESCs. (H): hESCs were transfected with siCTL or siCD49f. After three rounds of consecutive transfection, cell lysates were collected and used for Western blot analysis of PI3K/AKT/GSK3β, and p53, p21, and p16. (I): hESCs were treated with the indicated concentration of LY294002, a PI3K inhibitor. NANOG and CD49f expression was measured using real-time PCR. Abbreviations: AKT, • • •; AP, alkaline phosphatase; EB, embryoid body; GSK, • • •; hESCs, human embryonic stem cells; hMSCs, human mesenchymal stem cells; PI3K, phosphatidylinositol 3-kinase; shRNA, • • •; shCTL, control shRNA; shCD49f, shRNA targeting CD49f; siRNA, • • •; siCTL, nontargeting siRNA; siCD49f, siRNA targeting CD49f.

To determine whether the inhibition of CD49f affected hESC pluripotency in addition to AP activity, we performed immunocytochemistry. The siCD49f-transfected hESCs showed significant downregulation of OCT4 and SOX2 expression (Fig. 5F). Next, we performed real-time PCR to explore pluripotency markers and lineage markers. In agreement with the immunocytochemistry results, siCD49f-transfected hESCs displayed a substantial reduction in the levels of the pluripotency marker genes. In contrast, lineage-specific marker genes were upregulated in siCD49f-transfected hESCs, demonstrating that CD49f plays a role in the maintenance of pluripotency in hESCs (Fig. 5G). In line with our observations in the MSC spheres, CD49f knockdown led to an inhibition of the PI3K/AKT/GSK3β pathway and an increase in p53, p21, and p16 expression in hESCs (Fig. 5H). To further determine whether CD49f, through the PI3K pathway, was required for maintaining the undifferentiated status of hESCs, we treated the cells with a selective PI3K/AKT inhibitor, LY294002. PI3K/AKT inhibition significantly reduced the expression of both NANOG and CD49f in a dose-dependent manner (Fig. 5I). Taken together, these findings indicate that CD49f plays a pivotal role in maintaining cellular pluripotency through the PI3K/AKT/p53 pathway.

DISCUSSION

In this study, we have isolated and characterized sphere-forming cells from hMSCs. The sphere-forming hMSCs showed higher proliferation and differentiation capabilities via activation of the PI3K/AKT/p53 axis, which is important for cell-cell and cell–ECM interactions. Integrins are a cell adhesion-related family that form heterodimeric complexes between the α and β subunits [37]. Among the integrins, we have shown that CD49f and CD104 are upregulated in sphere-derived cells. The CD49f/CD104 (integrin α6/β4) complex plays an important role in cell proliferation and PI3K/AKT activation via interactions with laminin and the anchoring filament protein kalinin [14, 37]. Downstream signal transducers of integrin, such as FAK and Paxillin, were activated in sphere cells, suggesting that integrin signaling is sufficient for triggering the PI3K/AKT/GSK3β signaling pathways. Moreover, both sphere-forming efficiency and sphere size were significantly increased in the CD49f-positive population compared to the CD49f-negative population. Interestingly, there was a difference between the MSC spheres created from the distinct populations. We found that MSC spheres generated from CD49f-positive cells showed a higher activation of the FAK, PI3K, and AKT signaling pathways compared with MSC spheres from CD49f-negative cells. Consistent with a previous report that survival signals from FAK could suppress the p53-regulated cell death pathway, we showed that p53, p21, and p16 were inhibited in MSC spheres from CD49f-positive cells with an activated FAK signaling pathway [17]. This result is compatible with previous reports that cancer cells grown under sphere-forming conditions showed preferential activation of the PI3K/AKT signaling pathway and displayed preferential sensitivity to AKT inhibition [23, 38]. Given our observation that CD49f knockdown inhibited sphere formation in hMSCs, CD49f may be a potential molecular target for interrupting cancer stem cell activity through the inhibition of PI3K/AKT signaling.

Although CD49f activates the PI3K/AKT pathway in carcinoma cells [14, 15], whether the CD49f-mediated PI3K/AKT pathway is conserved in both the proliferation and cellular potency of adult stem cells is unknown. We found that CD49f overexpression activated the PI3K/AKT pathway and led to an upregulation of sphere-forming efficiency and adipogenic and osteogenic differentiation potentials. In addition, CD49f overexpression downregulated p53 levels in hMSCs. Because the pivotal regulatory proteins PI3K/AKT can both regulate p53 and be regulated by it, we investigated p53 and its downstream effector p21, which play regulatory roles in the p53 and CDK4 in cell cycle progression [39]. Recently, studies describing the various roles of p53 in cellular processes, including self-renewal, differentiation, and reprogramming, have emerged. Cicalese et al. reported that the mammary glands of p53-knockout mice had higher number of mammosphere-forming cells that could repopulate the glands in vivo and that the p53-null mammospheres showed increased replicative potential compared to wild-type [40, 41]. Furthermore, it has been demonstrated that p53 regulates the proliferation and differentiation of mouse MSCs [42]. In agreement with those studies, our data showed that p53 levels regulated the proliferation and differentiation of hMSCs; furthermore, the downregulation of p53 through PI3K/AKT signaling was induced by CD49f.

Consistent with previous studies reporting that the PI3K/AKT network is critical for both osteogenic differentiation and bone growth [43, 44], we observed that the activation of the PI3K/AKT/GSK3β pathway mediated by CD49f appeared to promote the osteogenic differentiation of hMSCs. Furthermore, we found that hMSCs treated with BIO, an inhibitor of GSK3, showed an increased propensity for osteogenic differentiation without an effect on viability. In previous studies, it has been shown that constitutively activated PI3K/AKT induces spontaneous adipocyte differentiation [45, 46]. However, the inhibition of GSK3 blocks adipogenic differentiation by inhibiting PPARγ, the master regulator of adipogenesis [47]. In agreement with this, we observed that the treatment of BIO upregulated a set of osteogenic marker genes, while a set of adipogenic marker genes was substantially decreased, indicating that CD49f preferentially stimulates the differentiation of hMSCs into the osteogenic lineage by regulating the PI3K/AKT/p53 pathway [40, 41]. Supporting Information Figure S5 shows a schematic representation of the CD49f-PI3K/AKT/p53 pathway.

According to recent studies, OCT4 and other pluripotency marker genes can regulate the proliferative capacity, colony formation, and lineage differentiation potencies of MSCs [48–50]. In this study, we found that sphere-derived hMSCs showed an increased expression of OCT4/SOX2/LIN28/NANOG. This result suggests that the increased proliferation and differentiation potentials of sphere-forming MSCs may be mediated, at least in part, by the higher expression of these pluripotency-enhancing genes. Furthermore, we showed that the silencing of OCT4 or SOX2 led to the downregulation of CD49f activity, whereas the endogenous transcripts and proteins of CD49f were activated by the forced expression of OCT4 and SOX2. The effects of OCT4 and SOX2 siRNA and overexpression may have been indirect because changes in endogenous OCT4 and SOX2 levels may have led to different cellular states (i.e., differentiation or reprogramming). To determine whether the regulation of CD49f by OCT4 and SOX2 was due to direct binding, we performed ChIP. Using specific antibodies against OCT4 and SOX2, we showed that these two transcription factors bound to the putative promoter region of the CD49f gene (Fig. 4F). Because hMSCs display much lower expression levels of endogenous OCT4 and SOX2 than hESCs (Fig. 5B), hMSCs showed a weak enrichment of OCT4 and SOX2 in the CD49f promoter region compared to Tera-1 cells.

In this study, we elucidated the role of CD49f in the maintenance of the differentiation potential of both hMSCs and hESCs. To our knowledge, we are the first group to report that CD49f plays a significant role in the maintenance of the hMSC differentiation potential. In the case of hESCs, it has previously been reported that these cells express a broad range of integrins, including CD49f, which interacts with laminin [11, 26–28]. The laminin recognized by α6β1 integrin or α6β4 can be used as a substrate for the adhesion and expansion of undifferentiated hESCs [11, 26–28]. However, the role of CD49f in the maintenance of pluripotency and the regulation of the PI3K/AKT/GSK3β signaling pathway had yet to be elucidated in hESCs. Regarding the role of CD49f in hESCs, we showed that the depletion of CD49f led to a loss of pluripotency, as suggested by the decreased expression of OCT4 and SOX2 and the induction of differentiation in hESCs. We also showed that the CD49f-mediated maintenance of differentiation potential was linked to the PI3K/AKT/GSK3β signaling pathway in both hMSCs and hESCs. Blocking CD49f inhibited the PI3K/AKT/GSK3β pathway, which is crucial for the maintenance of pluripotency and/or viability of hESCs [51, 52]. Because active AKT can reduce the levels of p53 [19], blocking CD49f in hESCs promotes the upregulation of p53, p21, and p16. These observations suggest that CD49f helps to maintain a more stem-like state, and the loss of CD49f might trigger the progression into the differentiated state. Taken together, these findings suggest that CD49f crosstalks with OCT4 and SOX2 and may contribute to the maintenance of pluripotency through the activation of the CD49f-PI3K/AKT/p53 signaling pathway.

CONCLUSION

In summary, we conclude that CD49f, a cell-surface molecule, plays important roles in MSC sphere formation and in the determination of the differentiation potential of hMSCs. We showed that the pluripotency factors OCT4 and SOX2 bound directly to the CD49f promoter, indicating that OCT4 and SOX2 play positive roles in the expression of CD49f. In addition, the CD49f-induced activation of the PI3K/AKT/p53 pathway contributes to the maintenance of pluripotency in hESCs.

Acknowledgements

This work was supported by the Bio & Medical Technology Development Program (MEST 2010-0020265) and a Global Frontier Project grant (NRF-M1AXA002) of the National Research Foundation (NRF) funded by the Korean government.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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