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Importance of the culture method for differential expression of osteogenic and stemness genes in the lineage commitment of bone marrow-derived mesenchymal stromal cells Directed by Professor Jin Woo Lee The Doctoral Dissertation submitted to the Department of Medical Science, the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dong Suk Yoon June 2013

This certifies that the Doctoral Dissertation of Dong Suk Yoon is approved. ------------------------------------ Thesis Supervisor: Jin Woo Lee ------------------------------------ Thesis Committee Member#1: Sahng Wook Park ------------------------------------ Thesis Committee Member#2: Yumie Rhee ------------------------------------ Thesis Committee Member#3: Jae Myun Lee ------------------------------------ Thesis Committee Member#4: Jae-woo Kim The Graduate School Yonsei University June 2013

ACKNOWLEDGEMENTS 2006년 10월이진우교수님실험실로입실해서 6년반이라는시간이흐른지금에서야이렇게감사의글을적을수있게되었습니다. 지난시간을돌아보면저에겐이논문에담긴내용들보다더많은것을얻고보다많은것을배울수있는소중한시간들이었습니다. 생물학과에편입해서 2년이라는짧은시간동안생물학을배우고이곳으로오게되었습니다. 그만큼부족한저를많은것을배우고경험할수있게이끌어주시고, 긴시간동안지켜봐주시고지도해주신이진우교수님께깊은감사를드립니다. 이곳이아니었다면배울수도경험할수도없는많은소중한것들을경험하였기에긴시간이전혀아깝지않고뜻깊었습니다. 그리고, 논문이나오기까지따뜻한관심을가져주시고세심한지도를해주신박상욱교수님, 이유미교수님, 이재면교수님, 그리고김재우교수님께깊은감사를드립니다. 교수님들께부끄럽지않은훌륭한연구자가될수있도록노력하고매진할수있도록하겠습니다. 어느날뉴스에서줄기세포관련기사를보고관심이생겨생물학과로의편입을결심하게되었습니다. 그과정에서제가올바른선택을할수있도록조언해주신김천대학교임병철교수님과, 편입생인저에게처음으로실험실이라는곳을경험할수있게해주신세종대학교소문수교수님께도깊은감사를드립니다. 끝날것같지않았던학위과정을이렇게마무리하면서함께했던많은인연들이생각납니다. 처음실험실에들어와서적응하고많은실험을가르쳐주고, 그리고무엇보다혼자서해나갈수있는방법과참된연구자로써의길을걷게해주신김윤희박사님께정말감사하다고전하고싶습니다. 지금은외국에있지만항상다정하게대해주고제가많은실험들을시도해볼수있게배려해준정호선박사님과언제나한결같던박민성박사님께도정말감사하다고전하고싶습니다. 첫번째제후배이자첫외국인친구였던백승일군과언제나밝고열심히하는모습이보기좋은황지숙양, 그리고오랜기간동안실험실막내생활을불평없이해온이슬기양에게도정말졸업축하하고앞으로도함께하고싶은인연이라고전하고싶습니다. 고된실험실생활에언제나버팀목이되어준경미누나와앞으로오랫동안많은일들을해줄현애, 곧학위를들어올실험실막내최유림학생, 앞으로저희랩에서많은일들을해주시고이끌어가실장연수박사님께도감사의

인사를전하고싶습니다. 그리고항상저희실험실에관심가져주시고많은도움주셨던한승환교수님, 처음이곳에와서지금까지도자주뵙고항상친절하게대해주신최우진교수님께도감사하고졸업축하한다고전하고싶습니다. 저희실험실중대형동물수술은다맡아서최선을다해주시는김성환교수님께도정말감사를드립니다. 공부만한다고자식노릇제대로못했지만, 항상믿고격려해주고지금까지기다려주신제아버지와어머니, 그리고장모님께도정말감사하다고전하고싶고, 항상힘이되어준든든한내동생경석이와앞으로우리가족이될미선이에게감사의인사를전하고싶습니다. 마지막으로학생남편을만나서결혼한지 4년이지났지만단한번도저한테부담주지않고항상격려해주었던제아내에게정말사랑하고고맙다고전하고싶습니다. 이렇게많은고마운분들께자랑스런과학자가될수있도록노력하겠습니다. 기회는찾아야만들어지고, 행운은노력하는자에게찾아온다고합니다. 지금도그림하나를얻기위해서그이상의노력과시간을바쳐야하는 많은후배들에게큰격려를보내고싶습니다. 윤동석올림

TABLE OF CONTENTS ABSTRACT 1 I. INTRODUCTION 4 II. MATERIALS AND METHODS 7 1. Isolation and culture of MSCs from human bone marrow aspirates 7 2. Colony-forming unit fibroblast (CFU-F) assay 7 3. Cell proliferation assay 7 4. Senescence-associated-β-galactosidase assay (SA-β-gal assay) 8 5. Alkaline phosphatase (ALP) staining 8 6. Calcium contents assay 8 7. Multi-lineage differentiation 9 A. In vitro osteogenic differentiation 9 B. In vitro adipogenic differentiation 8 C. In vitro chondrogenic differentiation 10 8. Cell cycle analysis 10 9. Size-fractionized cell sorting 10 10. Flow cytometry 11 11. Real-time quantitative polymerase chain reaction (PCR) 11 12. RNA interference 12 13. Western blot 12 14. Proteome Profiler Human Cytokine Array 13 15. Interleukin-6 enzyme-linked immunosorbent assay (ELISA) 13 16. Determination of IL-6 concentration 14 17. Determination of IL-6 receptor antibody concentration 14 18. Cignal reporter assay for Sox2-dependent GFP reporter activity 15

19. Statistical analysis 15 III. RESULTS 16 1. Confirmation of cellular senescence in BM-MSCs during prolonged passage 16 2. Decreased renewal capacity during prolonged cultivation of BM-MSCs 18 3. Decrease in multi-potency and commitment to the osteogenic lineage in late-passage MSCs 21 4. Expression of osteogenic and stemness genes during prolonged passage 24 5. Development of an alternative culture method to repopulate primitive cells in late-passage BM-MSCs 26 6. Change in proliferative capacity and cell cycle distribution of late-passage BM-MSCs under LD culture conditions 29 7. Enhanced differentiation potential in late-passage BM-MSCs cultured under LD conditions 32 8. Decrease in osteogenic gene expression and increase in stemness gene expression in late-passage BM-MSCs cultured under LD conditions 35 9. Importance of stemness genes for maintenance of renewal capacity and multi-potency in BM-MSCs 37 10. Change in the Sox2-positive cell population of heterogeneous BM-MSCs during prolonged passage 40 11. Characterization of the different subpopulations of heterogeneous BM-MSCs by cell size 42 12. Effects of LCP-secreted cytokines on cellular senescence and osteogenic lineage commitment of the SCP (primitive cell population)

46 13. Lineage commitment of late-passage BM-MSCs during cellular senescence 49 14. Effects of IL-6 on the renewal capacity and multi-potency of BM-MSCs 52 15. Effects of IL-6 on the multi-lineage differentiation potential of BM-MSCs 56 16. Effect of IL-6 on protein expression of osteogenic and stemness genes 59 17. Importance of LCP-secreted IL-6 in regulating osteogenic lineage commitment and stemness loss of the SCP 60 18. Correlation between the stemness gene Sox2 and the osteogenic transcription factor genes Runx2 and Dlx5 63 19. Effect of IL-6-induced osteogenic transcription factors, Runx2 and Dlx5, on Sox2 protein expression and transcriptional activity 65 IV. DISCUSSION 70 V. CONCLUSION 75 REFERENCES 76 ABSTRACT (IN KOREAN) 86

LIST OF FIGURES Figure 1. In vitro senescence of BM-MSCs during prolonged passage 17 Figure 2. Decrease in the number of colony-forming cells and expression of cell cycle-related genes during prolonged passage 19 Figure 3. Decrease in multi-potency and expression of alkaline phosphatase in late-passage BM-MSCs 22 Figure 4. Expression of osteogenic and stemness genes during prolonged passage of BM-MSCs 25 Figure 5. Development of an alternative culture method to repopulate the SCP in late-passage BM-MSCs 27 Figure 6. Application of LD culture conditions in late-passage BM-MSCs 28 Figure 7. Effect of LD culture conditions on the proliferation of late-passage BM-MSCs 30 Figure 8. Effects of LD culture conditions on the multi-lineage differentiation potential of late-passage BM-MSCs 33 Figure 9. Effects of LD culture conditions on the commitment to osteogenic lineage and stemness loss of late-passage BM-MSCs 36 Figure 10. Effect of Sox2 and Nanog knock-down on BM-MSC proliferation and colony-forming ability 39 Figure 11. Comparison of Sox2 expression between early- and

late-passage BM-MSCs 41 Figure 12. Scheme for the isolation of SCP and LCP from heterogeneous BM-MSCs 43 Figure 13. Characteristics of the SCP and LCP isolated from heterogeneous BM-MSCs 45 Figure 14. Effects of LCP conditioned medium on cellular senescence and osteogenic lineage commitment of the SCP 48 Figure 15. Identification of cytokines secreted from the LCP using a Human Cytokine Array 50 Figure 16. Changes in IL-6 expression in the LCP assayed by real-time PCR and ELISA 51 Figure 17. Dose- and time-dependent effects of IL-6 on STAT3 modulation in BM-MSCs 53 Figure 18. Effects of IL-6 on cellular senescence and renewal capacity of the SCP 54 Figure 19. Effects of IL-6 on multi-potency of the SCP 57 Figure 20. Changes in the protein levels of Sox2, Runx2, and Dlx5 in the presence of IL-6 59 Figure 21. Effects of tocilizumab, an IL-6 receptor antibody, on STAT3 modulation in BM-MSCs 61 Figure 22. Importance of LCP-secreted IL-6 in regulating osteogenic lineage commitment and stemness loss in BM-MSCs 62 Figure 23. Correlation between stemness and osteogenic genes 64

Figure 24. Regulation of Sox2 expression by IL-6-induced Runx2 and Dlx5 68 Figure 25. The proposed model of lineage commitment and stemness loss during senescence of heterogeneous BM-MSCs 61

ABSTRACT Importance of the culture method for differential expression of osteogenic and stemness genes in the lineage commitment of bone marrow-derived mesenchymal stromal cells Dong Suk Yoon Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Jin Woo Lee) Bone marrow-derived mesenchymal stem or stromal cells (BM-MSCs) are considered good sources for cell therapy in clinical applications. However, BM-MSCs lose their self-renewal capacity and multi-lineage differentiation potential during prolonged cell passage in vitro. Furthermore, BM-MSCs that are cultured for a long time become committed to the osteogenic lineage as they approach senescence and lose their potential to differentiate along the other lineages. The decreased stem cell properties of MSCs pose significant limitations to their application in cell-based regenerative medicine. BM-MSCs are composed of heterogeneous cell populations. However, little is known about the effects of the interaction between the different BM-MSC populations on cellular senescence. Thus, the purpose of this study was to identify mechanisms that induce cellular senescence in BM-MSCs via cell-cell interactions between the heterogeneous BM-MSC populations. In addition, - 1 -

this study proposes an alternative BM-MSCs culture method, which involves repopulating a primitive cell population from late-passage MSCs with poor multipotentiality and low cell proliferation rate by simply altering the plating density. First, we confirmed senescence in BM-MSCs following repeated serial subculture. Late-passage MSCs include a subpopulation of more committed osteogenic cells with increased expression of osteogenic transcription factors (Runx2 and Dlx5) that is further elevated during subsequent passage and diminishing stemness with decreased expression of stemness genes (Sox2 and Nanog). Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation of BM-MSCs. This result indicated that Sox2 is important for maintaining the stemness of BM-MSCs. Next, we hypothesized that cytokines secreted by the large-cell BM-MSC population would affect the cellular senescence and osteogenic lineage commitment of primitive cells (small-cell population) in heterogeneous BM-MSCs because the large-cell population has been considered the senescent population. Indeed, senescence and osteogenic lineage commitment of BM-MSCs were strongly induced in the presence of cytokines secreted by the large-cell population. Among the cytokines, the level of interleukin-6 (IL-6) secreted by large-cell population was significantly higher than that secreted by the small-cell population. Therefore, we focused our attention on the ability of IL-6 to induce cellular senescence and osteogenic lineage commitment in the small-cell population. IL-6 induced osteogenic lineage commitment and cellular senescence in the BM-MSC population by increasing Runx2 and Dlx5 protein levels, and reduced stemness by decreasing Sox2 protein expression. Furthermore, the IL-6-induced Runx2 and Dlx5 proteins decreased the transcriptional activity of Sox2. These results indicate that IL-6, one of the cytokines secreted from the large-cell population of BM-MSCs, can induce osteogenic lineage commitment by up-regulating the expression of osteogenic transcription factors (Runx2 and Dlx5) and decrease the self-renewal capacity and multi-lineage differentiation potential of primitive cells by - 2 -

down-regulating the transcriptional activity of Sox2, stemness-related gene. Further, we determined the effects of low-density culture (compared to high density culture) on late-passage BM-MSCs. We repopulated primitive cells (small-cell population) by replating late-passage BM-MSCs at low density (10-20 cells cm 2 ) regardless of donor age. The repopulated BM-MSCs derived from low-density cultures were smaller than the cells from high-density cultures had spindle-shaped morphology, and exhibited enhanced colony-forming ability, proliferation rate, and adipogenic and chondrogenic potentials. The strong expression of osteogenic genes (Runx2, Dlx5, alkaline phosphatase and type I collagen) in late-passage BM-MSCs was reduced by replating at low density, whereas expression of 3 stemness markers (Sox2, Nanog and Oct-4) reverted to levels observed in early-passage BM-MSCs. In conclusion, among the cytokines secreted by the large-cell population, IL-6 may play important roles in cellular senescence and osteogenic lineage commitment of BM-MSCs during prolonged cell passage in vitro. Therefore, molecules targeting the IL-6 signaling pathway would be useful in maintaining the primitive BM-MSC population in long-term culture. In addition, plating density should be considered a critical factor in the enrichment of primitive cells in heterogeneous BM-MSC populations. ---------------------------------------------------------------------------------------------------------- Key words: BM-MSCs, Heterogeneous population, Lineage commitment, Cellular senescence, Self-renewal, Multi-potency, Stemness, IL-6, Runx2, Dlx5, Sox2, Low density cylture - 3 -

Importance of the culture method for differential expression of osteogenic and stemness genes in the lineage commitment of bone marrow-derived mesenchymal stromal cells Dong Suk Yoon Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Jin Woo Lee) I. INTRODUCTION Mesenchymal stem cells (MSCs), also referred to as mesenchymal stromal cells, isolated from the bone marrow (BM) are believed to possess self-renewal properties and have the ability to generate multipotential progeny for musculoskeletal tissues such as bone 1-3, cartilage 4-6, and adipose tissues 7-13. However, BM-derived MSCs lose their self-renewal capacity and multi-lineage differentiation potential with increasing passage during in vitro expansion 14-19. After several passages, MSCs enter the process of senescence, characterized by enlarged and irregular cell shapes, and cessation of cell division 20. MSCs comprise a heterogeneous population of cells that that differ in size, morphology, and differentiation potential. As MSCs approach senescence in in vitro culture, they lose their multi-lineage differentiation potential. Despite the decreased multi-lineage differentiation potential, BM-MSCs have been reported to retain their ability to differentiate along the osteogenic lineage during prolonged cell passage in vitro 17,18. These results indicate that MSCs that are - 4 -

cultured for a long time become committed to the osteogenic lineage as they approach senescence, and they lose the potential to differentiate along other lineages. The loss of stem cell properties significantly limits the utility in cell-based regenerative medicine and in studies aimed at understanding their differentiation mechanisms. It has been reported that cytokines secreted by senescent cells can alter the fates of neighboring cells 21-23. Cytokines secreted by human BM cultures have been shown to exhibit certain patterns according to age or estrogen status 24, and cytokines secreted by senescent cells can stimulate tissue aging and tumor formation 25. Furthermore, it has been shown that cytokines secreted by senescent cells, such as interleukin-6 (IL-6) 26,27 and -11 (IL-11) 28, are capable of inducing cells to commit to the osteogenic lineage 24. However, less is known about the roles of the cytokines secreted by senescent cells in osteogenic lineage commitment induced prior to cellular senescence. Recently, somatic cells have been reprogrammed into induced pluripotent stem cells (ips) via forced expression of embryonic stem (ES) cell factors (Oct4, Sox2, cmyc, and Klf4), and the combination of these genes has been shown to be critical for high reprogramming efficiency. Pluripotency markers, such as Sox2, Nanog, and Oct-4, are expressed in both adult stem cells and ES cells; however, their expression in MSCs is dependent on passage number and tissue source 29. Go et al. 30 observed that overexpression of a retrovirus encoding Sox2 or Nanog in passage 5 (P5) MSCs, which displayed flattened aged morphology and reduced proliferation rates, resulted in restoration of normal morphology and proliferation levels. However, these changes were only apparent in the presence of basic fibroblast growth factor, and retroviral silencing remains an obstacle to achieving the pluripotent state and superior ips cells. To isolate BM-derived primitive cells during in vitro culture, several approaches have been reported, including assessment of surface markers such as stro-1 31, cell shape and size 32, donor age 33,34, and plating density 35-37. Some studies have demonstrated the importance of in vitro plating density in cell proliferation and differentiation potential. MSCs seeded at a cell density less - 5 -

than 5000 cells/cm 2 undergo apoptotic cell death 35 ; however, low-density (LD) cultures were shown to be sufficient for maintaining the colony-forming ability and stem cell properties ( stemness ) of early passage MSCs 36. Nevertheless, most studies have used passage-limited MSCs (less than 5 passages) 38,39 and/or have expanded MSCs at various cell densities (ranging from 10 3 to 10 5 cells/cm 2 ) 37,40-43. However, little is known about the relationship between cell culture condition and cellular senescence in BM-MSCs. Despite various trials to overcome MSC senescence and maintain their stemness, little is known about environmental factors that can negatively affect MSC quality, such as MSC senescence, commitment to the osteogenic lineage, or loss of multi-potency, in heterogeneous MSC populations. In addition, some researchers do not use late-passage MSCs cultured for a long time despite the presence of a small primitive cell population because multi-potency and renewal capacity are reduced in late-passage cell populations. Thus, the purpose of our current study was to (1) investigate whether cytokines secreted from a senescent BM-MSC population can regulate cellular senescence and stemness (or osteogenic lineage commitment) of neighboring primitive cells, and (2) repopulate a primitive cell population similar to early-passage BM-MSCs from late-passage BM-MSCs using a density-controlled culture method. - 6 -

II. MATERIALS AND METHODS 1. Isolation and culture of MSCs from human bone marrow aspirates BM aspirates were obtained from posterior iliac crests of 14 adult donors (nine males, five females) 19-69 years of age, with approval of the Institutional Review Board (IRB) of our institution. MSCs from human BM were selected based on their ability to adhere to plastic cell culture flasks. Cells were maintained in low-glucose Dulbecco s modified Eagle s medium (DMEM-LG, Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% antibiotic-antimycotic solution (Invitrogen) at 37 C in a 5% CO 2 atmosphere. Cells were grown to 80-90% confluence and then harvested by incubation with 0.25% trypsin/edta (Invitrogen) centrifuged at 1300 rpm for 3 min. Harvested cells (P1) were replated at density of 5000 cells/cm 2 and subcultured when they were 80-90% confluent up to P7. Late passage BM-MSCs (P7) were replated at high density (HD, 5000 cells/cm 2 ) or low density (LD, 17 cells/cm 2 ) and maintained for 10-12 days. 2. Colony-forming unit fibroblast (CFU-F) assay After being fixed in 1:1 acetone:methanol fixative, cultures were stained with 20% crystal violet solution (Merck, Darmstadt, Germany) for 30 min in the dark. After being washed in distilled water (DW), colony-forming ability of the stained cells was evaluated. 3. Cell proliferation assay Cell proliferation was determined using a hexosaminidase assay. Proliferative ability of density-controlled BM-MSCs was examined after 1, 4 and 7 days. Briefly, after being washed in PBS, a mixture of 0.1 M citrate buffer (Sigma, St. - 7 -

Louis, MO, USA) containing 7.5 mm p-nitrophenyl-n-acetyl-b-dglucosaminide (PNAD; ph 5.0, Sigma) and 0.5% Triton X-100 (Sigma) was added to each well and incubated at 37 _C for 3 h. After incubation, 50 mm glycine buffer (ph 10.4; Amresco, Solon, OH, USA) containing 5 mm ethylenediaminetetra acetic acid (EDTA; Sigma) was added to each well. Absorbance of released hexosaminidase was measured at 405 nm. All samples were tested in triplicate. 4. Senescence-associated-β-galactosidase assay (SA-β-gal assay) SA-β-gal assay was performed using a Cellular Senescence Assay kit (Millipore, Temecula, CA, USA) following the manufacturer s protocol. Briefly, Cells were washed with PBS, and then were fixed for 15 min at room temperature with 1X fixing solution. After washing with DW, cells were stained with freshly prepared 1X SA-β-gal detection solution [X-gal (1) : Solution A (4) : Solution B (4)] for 4h in the dark at 37 C incubator without CO 2. SA-β-gal positive cells exhibited a blue color. The number of positive cells was counted under a phase-contrast microscope. Experiments were performed in triplicate. 5. Alkaline phosphatase (ALP) staining After being fixed in 2:3 citrate buffer:acetone fixative, cultures were stained for alkaline phosphatase (ALP) using alkaline staining solution (Sigma) for 30 min in the dark. After washing in DW, cells were stained in Mayer s haematoxylin solution (Sigma) for 5 min, then rinsed in tap water. 6. Calcium contents assay To evaluate calcium content, cells were washed twice in PBS and incubated in 800 µl 0.5 N acetic acid for 24h at room temperature. After incubation, 300-8 -

µl fresh reagent (O-Cresolphthalein Complexon, ethanolamine boric acid, hydroxyquinol; Sigma) was added to 50 µl of sample supernatant, and absorbance was measured at 560 nm. Standards were prepared from a CaCl2 solution, and results were expressed as mg ml calcium equivalent per µg total protein. Experiments were performed in triplicate. 7. Multi-lineage differentiation To identify multi-lineage differentiation potential of BM-MSCs, cells were seeded at 8 10 4 cells/well in 12-well culture plates. A. In vitro osteogenic differentiation For osteogenic differentiation, cells were maintained for 14 days in osteogenic medium [DMEM-LG containing 10% FBS, 1% antibiotic-antimycotic solution, 100nM dexamethasone (Sigma), 10 mm β-glycerophosphate (Sigma), and 50 μg/ml ascorbic acid (Gibco)]. For von Kossa staining, after being fixed in 1:1 acetone:methanol, 1 ml freshly prepared 3% silver nitrate (wt/vol) (Sigma) was added, and for alizarin red S staining, 1 ml freshly prepared 3% alizarin red S solution (wt/vol) (Sigma) was added, then incubated in the dark for 30 min. B. In vitro adipogenic differentiation For adipogenic differentiation, cells were maintained for 14 days in adipogenic medium [DMEM-LG containing 10% FBS, 1% antibiotic-antimycotic solution, 1μM dexamethasone, 0.5mM isobutyltethylxanthin (Sigma), 5 μg/ml insulin (Gibco), and 200 μm indomethasin (Sigma)]. To detect lipid droplets by oil red O staining, after being fixed in 10% neutral buffered formalin, 1 ml 0.18% oil red O solution (Sigma) was added and incubated for 30 min. For quantitative analysis, absorbance was detected at 500 nm after de-staining with isopropanol for 30 min. To normalize for cell number, cells were stained with crystal violet - 9 -

(CV) for 10 min and destained with 95% ethanol; absorbance was measured at 595 nm. Each oil red O optical density (OD) value was then divided by its respective CV measurement for normalization. C. In vitro chondrogenic differentiation For chondrogenic differentiation, cells were maintained for 14 days in chondrogenic medium [DMEM-high glucose containing 1X insulin-transferrin-selenium-a (Gibco), 1% antibiotic-antimycotic solution, 50 μg/ml ascorbic acid, and 10 ng/ml TGF-β3 (R&D Systems, Minneapolis, MN, USA)]. For pellet culture, 8 10 4 cells in 15-mL tubes were harvested after centrifugation at 1200 rpm for 3 min, then chondrogenic medium with TGF-β3 was added. To detect proteoglycan synthesis 0.1% safranin O solution (Sigma) was added and incubated for 1 h. For quantitative analysis, absorbance was detected at 490 nm following de-staining with 100% ethanol for 20 min. Each safranin O value was normalized to absorbance from CV staining. 8. Cell cycle analysis Cells were harvested by incubation with 0.25% trypsin/edta and washed twice in PBS. Cells from each group (1 10 6 ) were fixed in ice-cold 70% ethanol for 1 h at -20 C, stained with 50 μg/ml propidium iodide (PI, Sigma) containing 100 μg/ml RNase A (Sigma) for 40 min at 4 C, and then analyzed using a FACS Calibur instrumentation (Becton Dickinson Instrument, San Jose, CA) to detect the cell cycle distribution. All samples were tested in triplicate (n=3). 9. Size-fractionized cell sorting BM-MSCs were harvested by incubation with 0.25% trypsin/edta and washed twice in PBS. Cells were resuspended in pre-warmed PBS, then small - 10 -

or large cell population was sorted and analysed using FACS (Beckman Coulter, Fullerton, CA, USA). 10. Flow cytometry Cultured cells were harvested with 0.02% EDTA and washed twice in PBS containing 1% FBS and 0.05% sodium azide (FACS buffer). Single cells were labeled for 20 min 4 C in FACS buffer with following conjugated antibodies: Sox2-PerCP-Cy (BD Bioscience, San Jose, CA, USA) or PerCP-Cy-mouse-IgG isotype control (BD Bioscience). After being washed in FACS buffer, cells were analysed using FACS (Beckman Coulter, Fullerton, CA, USA). 11. Real-time quantitative polymerase chain reaction (PCR) Total RNA was isolated using an RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer s instructions. One microgram of total RNA was reverse-transcribed using an Omniscript kit (Qiagen). Real-time polymerase chain reaction (PCR) was performed to determine changes in mrna expression of cell cycle-related genes, early osteogenic markers, differentiation-related genes, pluripotency-related transcription factors, and interleukin family (IL-6 and IL-11). Primer sets used were validated and purchased from Bioneer (Bioneer, Daejon, South Korea, http://sirna.bioneer.co.kr/). Primers used and product information are as follows: (GAPDH (P267613, NM_002046.3); CCNA2 (P212796, NM_001237.2); CCNB1 (P275460, NM_031966.2); CCND1 (P298560, NM_053056.2); CDK2 (P136765, NM_001798.2); CDK4 (P268249, NM_000075.2); Runx2 (P229954, NM_001015051.1); Dlx5 (P199945, NM_005221.5); PPAR-γ (P102359, NM_005037.4); Adiponectin (P160254, NM_004797.2); Sox9 (P232240, NM_000346.2); IL-6 (P211161, NM_000600.1); IL-11 (P229401, NM_000641.2); ALP (P324388, NM_000478.2); Collagen Ι (P157768, NM_000088.2); Sox2 (P200205, NM_003106.2); and Nanog (P255522, NM_024865.1). There are no validated - 11 -

primers for Oct4, typeⅡ collagen, and Osteocalcin, and thus the primer was separately designed as follows: 5 -GCAAGCCCTCATTTCACCA-3 (Oct4, sense), 5 -GCCCATCACCTCCACCAC-3 (Oct4, antisense), 5 -GAGTGGAA GAGCGGAGACTA-3 (typeⅡ collagen, sense), 5 -CTCCATGTTGCAGAAG ACTT-3 (TypeⅡ collagen, antisense), and 5 -AGAGCCCCAGTCCCCTACC C-3 (Osteocalcin, sense), 5 -AGGCCTCCTGAAAGCCGATG-3 (Osteocalcin, antisense). PCR reaction mixtures consisted of 2 SYBR Green PCR premix (Bioneer), 10 pm specific primers and 2 μl of cdna in the ABI7500 real-time machine by AppliedBiosystem (ABI, Carlsbad, CA, USA). Real-time PCR analysis underwent 40 cycles of amplification. Mean cycles threshold (CT) values from triplicate (n=3) measurements were used to calculate gene expression, with normalization to GAPDH as internal control. 12. RNA interference On-TargetPlus SmartPool sirnas for Sox2 (Cat. L-011778) and Nanog (Cat. L-014489) were purchased from Dharmacon (Boulder, CO, USA). Scramble, Runx2 (sirna No: 1132367), and Dlx5 (sirna No: 1042423) sirna were purchased from Bioneer Inc., and targeted the following sequences: scramble-sirna sense: 5`-CCUACGCCACCAAUUUCGU-3` and scramble sirna antisense: 5`-ACGAAAUUGGUGGCGUAGG-3`. Briefly, cells were plated to obtain 70-80% confluency in six-well plates and transfected with 100 nm of Sox2, Nanog, Runx2, Dlx5, or scramble (Neg) sirna using Lipofectamine TM 2000 (Invitrogen). After 6 h transfection, fresh media were added to plates, and transfection efficiency was confirmed by western blot analysis. 13. Western blot Cells were lysed in the Passive lysis buffer (Promega, Madison, WI, USA). Protein concentrations were determined by the BioRad protein assay (Bio-Rad - 12 -

Laboratories Inc., Hercules, CA, USA), and total 30 μg protein was applied and analysed by 10% SDS-PAGE (Sigma, St. Louis, MO, USA). Transferred membranes were blocked with a 5% skim milk (BD, Sparks, MD, USA) and incubated for 4 h with antibodies of Sox2 (abcam, Cambridge, UK), Nanog (abcam), Runx2 (abcam), Dlx5 (abcam), STAT3 (BD Bioscience), Tyr-p-STAT3 (Cell Signaling Technology, Inc. Boston, MA, USA) and Ser-p-STAT3 (Cell Signaling Technology, Inc.). Membranes were further probed with antibody of β-actin (Santa Cruz Biotechnology) or GAPDH (Research Diagnostics, Flanders, NJ, USA), which was provided as loading control. Sox2, Nanog, Runx2, Dlx5, STAT3, Ser-p-STAT3, and Tyr-p-STAT3 protein expression were confirmed in 3 donors, and data shown are representative. 14. Proteome Profiler Human Cytokine Array The Proteome Profiler Human Cytokine Array Panel A Array Kit (R&D Systems Inc., Cat. No; ARY005) were used to analyze cytokines secreted in small, large, and mixed cell population of BM-MSCs. 36 different anti-cytokine antibodies have been spotted in duplicate on nitrocellulose membrane provided. Membranes were incubated in blocking solution for 1h on a rocking plate at room temperature. After washing membranes with 1X wash buffer three times, each media samples were added to membrane and incubated at 4 C for overnight. After being washed with 1X wash buffer, the streptavidin-hrp was added to each membrane for 30min at room temperature. Cytokine spots bound to membranes were visualized on X-ray film using Chemi Reagent Mix provided. Signal quantification of each cytokine was measured by subtracting the background signal using the TINA 2.0 program from Fuji image scanners. 15. Interleukin-6 enzyme-linked immunosorbent assay (ELISA) - 13 -

In order to measure amount of IL-6 secreted into culture medium from small, large, or mixed-cell population, we used cell culture supernatant for experiment using IL-6 ELISA kit (KOMA Biotech Inc., Seoul, Korea) according to the manufacture s instruction. In brief, cell culture supernatants were loaded into wells coated with antibody, and wells were incubated at room temperature for 4h on a microplate shaker. After being washed with provided wash buffer, all wells were incubated with detection antibody at room temperature for 2h. Finally, plates were read by a 96-well spectrophotometric microplate reader at 450 nm wavelength. All the assays were performed in duplicate (n = 3). IL-6 concentrations were calculated from the standard curve. 16. Determination of IL-6 concentration It is known that IL-6 induces the phosphorylation of STAT3 protein. To optimize the concentration of IL-6 (KOMA Biotech Inc.) for induction of STAT3 phosphorylation in BM-MSCs in vitro, the experimental groups were organized into control group (no treatment) and 3 experimental groups (IL-6 treatment; 1, 10, 50 ng/ml). The cells were cultured in cell culture plates for 24 h and the extent of STAT3 and pstat3 expressions was measured at 1, 4, 12, and 24 h after culture by western blotting. 17. Determination of IL-6 receptor antibody concentration Tocilizumab (Actemra), humanized anti-human IL-6 receptor monoclonal antibody, was provided by Chugai Pharma Manufacturing CO., LTD via JW Pharmaceutical (Seoul, Korea). To optimize the concentration of Tocilizumab for inhibition of IL-6 signaling, the experimental groups were organized into control group (no treatment) and 2 experimental groups (Tocilizumab treatment; 25 or 50 ug/ml) under the presence of 50ng/mL of IL-6. The cells were cultured in cell culture plates for 24 h and the extent of STAT3 and pstat3 expressions - 14 -

was measured at 24 h after culture by western blotting. 18. Cignal reporter assay for Sox2-dependent GFP reporter activity The Cignal Reporter Assay for Sox2-dependent GFP reporter activity was performed following the manufacture s manuals (SABiosciences, Frederick, MD, USA). This reporter contains transcriptional regulatory elements for Sox2 (TRE: AACAAAGAGT). rhil-6 or plasmids of Runx2 and Dlx5 (100ng) plus Sox2-dependent cignal reporter (50ng) were transfected into BM-MSCs or Hela cells using Lipofectamine TM 2000 (Invitrogen). After 36h, transfected cells were analyzed using a simple dual-luciferase assay (Promega) to determine activity of Sox2 signal for IL-6 treatment or sirna effect of Runx2 and Dlx5. 19. Statistical analysis The statistical analysis for all results was performed using Student s t-test, and the data were expressed as means ± SD. Values of *, p<0.05 were considered statistically significant. - 15 -

III. RESULTS 1. Confirmation of cellular senescence in BM-MSCs during prolonged passage First, we investigated senescence of BM-MSCs during prolonged passages. It is well-known that β-galactosidase (β-gal) activity is associated with in vitro senescence of cells 44. In MSCs, β-gal activity was shown to increase during prolonged passage, but no difference was observed between BM-MSCs isolated from young and old donors 45. Furthermore, senescent BM-MSCs exhibited increased cell size 46-48 and a flattened morphology with more actin stress fibers 45. Given these results, we examined BM-MSC senescence by measuring senescence-associated (SA)-β-gal activity and observing the change in cell size during prolonged passage. SA-β-gal activity was significantly increased in late-passage BM-MSCs (P7) (Figure 1A). Cell size changes were also observed between early- and late-passage BM-MSCs. The increase in cell size was much higher in the late-passage BM-MSCs (16.2%) than in the early-passage BM-MSCs (2.75%) (Figure 1B). These results indicate that BM-MSCs senesce during prolonged serial subculture. - 16 -

Figure 1. In vitro senescence of BM-MSCs during prolonged passage. (A) SA-β-gal staining was used to examine the effects of increasing passage number on BM-MSC senescence. SA-β-gal activity is increased in late-passage (P7) BM-MSCs compared to early passage (P1) BM-MSCs (left panel). SA-β-gal positive cells were counted in triplicate by 3 independent observers (right panel). (B) FACS analysis was used to compare the distribution of cell sizes between early- and late-passage BM-MSCs. FSC indicates the size of cells analyzed by FACS, and SSC indicates the granularity of cells analyzed. These data have been confirmed in all the 3 donors tested. Representative data shown here are those of a 64-year-old female donor (A) and a 34-year-old female donor (B). - 17 -

2. Decreased renewal capacity during prolonged cultivation of BM-MSCs Next, we investigated the colony-forming ability and mrna expression of cell cycle-related genes during prolonged passage of BM-MSCs. The colony-forming abilities of later passage BM-MSCs (P3 and P7) were markedly lower than that of early-passage (P1) BM-MSCs (Figure 2A). To further assess the effects of prolonged cultivation on the expression of genes involved in cell cycle progression, real-time PCR was performed. Cell cycle progression is closely regulated by cyclins, proteins that activate cyclin-dependent kinases (CDKs). Real-time PCR analysis revealed a marked decrease in the expression of genes known to be involved in S-phase and mitosis, such as cyclin A2 (CCNA2), during the passage of cells from P1 to P7. Cyclin D1 (CCND1), a gene associated with actively proliferating cells, did not show any statistically significant changes during serial subculture (Figure 2B). In addition, we analyzed the expression of 2 genes (CDK2 and CDK4) known to be involved in G1/S phase transition. CDK2 and CDK4 expression decreased during serial subculture up to P7 (Figure 2B). Collectively, these data suggest that decrease in cell proliferation during prolonged passage is accompanied by a decrease in the expression of cell cycle-related genes, such as CCNA2, CDK2, and CDK4. - 18 -

Figure 2. Decrease in the number of colony-forming cells and expression of cell cycle-related genes during prolonged passage. (A) P1, P3, and P7-MSCs from the same donor were seeded at 1 x 10 3 cells in 100-mm culture dishes. Cells were then cultured in DMEM-LG containing 20% FBS for 12 days to examine cell colony formation (violet). *, P < 0.05 compared to P1 MSCs. (B) The - 19 -

mrna expression patterns of cell cycle-related genes in undifferentiated P1, P3, and P7-MSCs were examined using real-time PCR. After subculturing from each previous passage, P1, P3, and P7-MSCs were grown in DMEM-LG containing 10% FBS for 3 days. Cells were harvested at 80% 90% confluency for RNA extraction. Each experiment was performed in triplicate (n = 3). * P < 0.05 compared to P1-MSCs. - 20 -

3. Decrease in multi-potency and commitment to the osteogenic lineage in late-passage BM-MSCs Both early-passage (P1) and late-passage (P7) BM-MSCs readily differentiated into osteoblastic cells when cultured in osteogenic medium for 14 days, but the osteogenic potential of late-passage BM-MSCs was higher than that of early-passage BM-MSCs. The adipogenic potential of late-passage BM-MSCs was lower than that of early-passage BM-MSCs, and chondrogenic differentiation capacity was absent in late-passage BM-MSCs (Figure 3A). Furthermore, late-passage BM-MSCs expressed ALP, an early osteogenic marker, in the undifferentiated state, with decrease in colony-forming ability and increase in cell size (Figure 3B). These results indicate that BM-MSCs become committed to the osteogenic lineage as they approach senescence, and they lose their potentials to differentiate along the other lineages (adipogenic and chondrogenic lineages). - 21 -

Figure 3. Decrease in multi-potency and expression of alkaline phosphatase in late-passage BM-MSCs. (A) P1 and P7 MSCs from the same donor were seeded at 8 x 10 4 cells well in 12-well culture plates. Cells were then cultured in osteogenic, adipogenic, and chondrogenic media to determine their osteogenic, - 22 -

adipogenic and chondrogenic differentiation potential, respectively. The above data have been confirmed in all the 3 donors tested. Representative data shown here are those of a 19-year-old male donor. (B) Crystal violet (CV) staining was used to compare the colony-forming abilities of early- and late-passage BM-MSCs (upper). Alkaline phosphatase (ALP) staining was used to compare the degree of osteogenic lineage commitment in early- and late-passage BM-MSCs (lower) (n = 3, in triplicate). - 23 -

4. Expression of osteogenic and stemness genes during prolonged passage Commitment of BM-MSCs to osteogenic lineage occurs during prolonged expansion in vitro prior to cellular senescence 17 (Figure 3). Commitment to the osteogenic lineage is accompanied by increased expression of osteogenic genes, such as Runx2 and ALP, whereas expression of stemness genes involved in renewal capacity and multi-potency, such as Sox2, Oct4, and Nanog, decreased in late passage MSCs 49. Despite these reports, the correlation between osteogenic and stemness genes has not been investigated. Therefore, we examined the mrna levels of genes related to osteogenic differentiation and stemness in P1, P3, and P7 MSCs. The mrna levels of genes involved in osteogenesis, such as Runx2, Dlx5, ALP, and type Ι collagen, increased gradually from P1 to P7. Expression of Sox2 and Nanog gradually decreased as cells progressed from P1 to P7. The down-regulation of stemness genes with increase in the passage number of BM-MSCs may be related to the decrease in cell proliferation and differentiation potential, including the ability to undergo differentiation along the adipogenic or chondrogenic lineage. However, Oct4 expression levels remained constant throughout the senescence process. These data suggest that Sox2 and Nanog are involved in maintaining the renewal capacity and multi-potency of BM-MSCs. - 24 -

Figure 4. Expression of osteogenic and stemness genes during prolonged passage of BM-MSCs. The mrna expression patterns of osteogenic (A) and stemness (B) genes in undifferentiated P1, P3, and P7 MSCs were examined using real-time PCR. After subculturing cells from each previous passage, P1, P3, and P7 MSCs were grown in DMEM-LG containing 10% FBS for 3 days. Each experiment was performed in triplicate (n = 3). *, P < 0.05 compared to P1. - 25 -

5. Development of an alternative culture method to repopulate primitive cells in late-passage BM-MSCs. We examined the changes in cell morphology and proliferation of late-passage BM-MSCs after altering the cell culture method. We observed that when BM-MSCs were cultivated at 17 cells/cm 2 (LD), which is less than the widely used density of 5000 cells/cm 2 (High-density; HD), cells displayed markedly enhanced cell population doubling. Based on these results, BM-MSCs were subcultured under HD conditions (5000 cells/cm 2 ) up to P7, at which point they had dramatically lower colony-forming and multi-differentiation abilities. The P7 BM-MSCs were then replated at either HD (5000 cells/cm 2 ; P7-HD MSC) or LD (17 cells/cm 2 ; P7-LD MSC) (Figure 5). The colony-forming ability of P7 BM-MSCs was remarkably lower than that of P1 BM-MSCs; this ability was restored by switching to LD culture conditions, but not HD culture conditions (Figure 6A). We also observed that P7-HD MSCs appeared flattened and enlarged, whereas many small spindle-shaped cells reappeared in the P7-LD MSCs (Figure 6A). BM-MSCs comprise heterogeneous-sized cell populations, and the existence of small cells has been thought to indicate more efficient stem cell properties 36,50. FACS analysis of P7-HD MSCs revealed that the small-cell population (SCP) (region A, 5-10 µm) and the large-cell population (LCP) (region B, >30 µm) represented approximately 10.9% and 21.2% of the entire population, respectively. By contrast, in P7-LD MSCs, the SCP and the LCP represented approximately 30.9% and 8.7% of the entire population, respectively (Figure 6B). These results suggest that plating density is critical to the enrichment of primitive cells in heterogeneous BM-MSCs and that a much higher number of primitive cells can be repopulated, even from late-passage cells, using LD culture conditions. - 26 -

Figure 5. Development of an alternative culture method to repopulate the SCP in late-passage BM-MSCs. The overall cell culture scheme from early to late passage followed by replating under HD or LD conditions is shown. This culture scheme was maintained throughout the study. - 27 -

Figure 6. Application of LD culture conditions in late-passage BM-MSCs. (A) P1 MSCs, P7 MSCs, P7-HD, and P7-LD cells were seeded at 1 x 10 3 cells in 100-mm culture dishes. Cells were then cultured in DMEM-LG containing 20% FBS for 12 days to examine colony formation (100x). (B) P7-HD and P7-LD cells were sorted into small (<10 μm) and large (>30 um) cells using FACS, and the size distribution of the sorted cells was analyzed. Cell size was determined using an optical microscope at (100x) magnification after trypan blue staining. The above data have been confirmed in all the 10 donors tested. Representative data shown here are those of a 35-year-old male donor. - 28 -

6. Change in proliferative capacity and cell cycle distribution of late-passage BM-MSCs under LD culture conditions The proliferative ability of human BM-MSCs gradually diminishes with continuous expansion. Besides the passage number, donor age is also a critical determinant of the proliferative ability of MSCs 33, given that the colony-forming ability of MSCs noticeably decreases in donors older than 20 years 34. To determine whether the LD method was effective in restoring the proliferative ability of late-passage BM-MSCs from donors of different ages, a proliferation assay was performed in samples organized into age groups [10-20 years old (n = 3), 20-50 years old (n = 3), and 50-70 years old (n = 3)]. P1 BM-MSCs were cultured up to P7 as described in Figure 6A. Then, cells were harvested and replated at either 5000 (HD method) or 17 (LD method) cells cm 2 in 100-mm dishes. The proliferative capacities of P7-HD and P7-LD MSCs were evaluated using a hexosaminidase assay. Compared to continuous HD culture, P7-LD MSCs showed significantly higher proliferation in every age group (Figure 7A). These results suggest that the LD culture method both favors cell proliferation and increases the number of small cells in a heterogeneous population of late-passage BM-MSCs, regardless of donor age. Next, we analyzed the effect of HD and LD culture conditions on the cell cycle distribution of late-passage BM-MSCs from each age group. Cell cycle analysis revealed that on average, 87.78%, 9.53%, and 2.69% P7-HD MSCs were in G 0 /G 1, S, and G 2 /M phases, respectively, whereas, in the P7-LD MSCs, the G 0 /G 1, S, and G 2 /M phases constituted 73.53%, 20.35%, and 6.12% of the cell population respectively. The proportion of S-phase cells was higher in P7-LD MSCs than in P7-HD MSCs (Figure 7B). To further assess the effects of the LD method on the expression of genes involved in cell cycle distribution, we performed real-time PCR. The analysis revealed a marked increase in the expression of CCNA2 (cyclin A2), CCND1 (cyclin D1), CDK2, and CDK4 (Figure 7C). Thus, in P7 cells cultured under LD conditions, the expression levels of cell cycle-related genes were much higher than that in HD-cultured - 29 -

cells. Collectively, these data suggest that restoration of cell proliferation in response to altered plating density is mediated by cell cycle-related genes. Figure 7. Effect of LD culture condition on the proliferation of late-passage BM-MSCs. (A) P7 MSCs from donors aged 10 20, 20 50 and 50 70 years were replated at an appropriate density (approximately 5000 or 17 cells cm 2 ) on 100-mm dishes. The replated cells were then seeded into 24-well plates (1 x 10 4 cells well). Hexosaminidase assay was performed to determine the proliferative potential of density-controlled BM-MSCs (P7-HD and P7-LD cells). *, P < 0.05 compared to P7-HD MSCs. The representative data shown here are those of a 19-year-old male donor. (B) P7-HD and P7-LD cells from each age group were - 30 -

harvested, and 1 x 10 6 cells were used for cell cycle analysis. Each experiment was performed in triplicate. The tabulated data presented here show the mean values from 3 donors. (C) The mrna expression patterns of cell cycle-related genes in undifferentiated P7-HD and P7-LD MSCs were examined using real-time PCR. After subculturing from each previous passage, P7-HD and P7-LD cells were grown in DMEM-LG containing 10% FBS for 3 days. Cells were harvested at 80% 90% confluency for RNA extraction. Each experiment was performed in triplicate (n = 3). *, P < 0.05 compared to P7-HD MSCs. - 31 -

7. Enhanced differentiation potential in late-passage BM-MSCs cultured under LD conditions We next examined the differentiation potential of P7-HD and P7-LD MSCs. Cells were cultivated up to P7 by using the HD method, according to the general scheme shown in previous data (Figure 6A). Cells were then grown using either the HD or LD method. Each repopulated cell culture was then replated in 12-well plates at identical cell densities, was grown to confluence, and was induced to differentiate into osteoblasts and adipocytes. Both P7-HD and P7-LD MSCs expressed ALP and showed accumulation of calcium-containing mineralized nodules on performing von Kossa staining after 14 days of osteogenic induction (Figure 8A, left). In the calcium content assay, P7-HD MSCs exhibited higher calcium accumulation compared to P7-LD MSCs (Figure 8B left). Although the multi-differentiation potential of BM-MSCs decreases after expansion in culture, the potential for osteogenic differentiation is retained or increased 17,18,51. These reports indicate that MSCs differentiate into osteogenic cells with continued cell passaging. Adipogenic differentiation was examined using morphology and oil red O staining. P7-LD MSCs exhibited increased lipid droplet formation and more oil red O-stained cells compared with P7-HD MSCs (Figure 8A, middle). In addition, the absorbance of oil red O staining detected after de-staining was significantly different between the 2 cell populations (Figure 8B, middle). Chondrogenic differentiation was analyzed using safranin O staining. Similar to the results of adipogenic differentiation, P7-LD MSCs exhibited a higher chondrogenic differentiation capacity than P7-HD MSCs (Figure 8A right), and the absorbance of proteoglycans detected after de-staining was also significantly different between the 2 groups (Figure 8B, right). - 32 -

Figure 8. Effects of LD culture conditions on the multi-lineage differentiation potential of late-passage BM-MSCs. (A; left) P7-HD and P7-LD cells (8 x 10 4 cells well in 12-well plates) were incubated in osteogenic medium for 14 days. Then, ALP and von Kossa staining were performed to detect mineral deposition. (B; left) A calcium content assay was performed to confirm the results of von Kossa staining. (A; middle) P7-HD and P7-LD cells (8 x 10 4 cells well in 12-well plates) were incubated in adipogenic medium for 14 days. After incubation, oil red O staining was performed to detect lipid droplets. (B; middle) For quantitative analysis of oil red O staining, absorbance was detected at 500 nm following de-staining with isopropanol for 30 min. (A; right) P7-HD and P7-LD cells (8 x 10 4 cells well in 12-well plates) were incubated for 14 days in chondrogenic medium containing 10 ng ml TGFβ-3. After incubation, safranin O staining was performed to detect proteoglycans (100x, Con, undifferentiated control BM-MSCs; CH, chondrogenic- - 33 -

differentiated BM-MSCs). (B; right) For quantitative analysis of safranin O staining, absorbance was detected at 490 nm following de-staining with 100% ethanol for 20 min. Each experiment was performed in triplicate (n = 3). *, P < 0.05 compared to P7-HD MSCs. - 34 -

8. Decrease in osteogenic gene expression and increase in stemness gene expression in late-passage BM-MSCs cultured under LD conditions. We examined the mrna levels of genes related to osteogenic differentiation and stemness in P7-HD and P7-LD MSCs. Genes that were expressed at higher levels during HD culture, such as Runx2, Dlx5, ALP, and type Ι collagen, exhibited lower expression following LD culture (Figure 9A). These results indicate that LD culture can repress the expression of osteogenic marker genes, thereby diminishing or reversing the commitment of the osteogenic precursor cells. The lower expression of Sox2 and Nanog observed in P7 BM-MSCs was restored to normal levels following LD culture. Although the expression of Oct4 did not significantly change with increase in passage number, its expression was slightly up-regulated after LD culture (Figure 9B). The up-regulation of stemness genes during LD culture of late-passage BM-MSCs may be related to the enhanced cell proliferation and restored differentiation potential, including the ability to undergo adipogenesis and chondrogenesis. - 35 -

Figure 9. Effects of LD culture conditions on the commitment to the osteogenic lineage and stemness loss of late-passage BM-MSCs. The mrna expression patterns of (A) osteogenic and (B) stemness genes in undifferentiated P7-HD and P7-LD MSCs were examined using real-time PCR. After subculturing, P7-HD and P7-LD cells were grown in DMEM-LG containing 10% FBS for 3 days. Each experiment was performed in triplicate (n = 3). *, P < 0.05 compared to P7-HD MSCs. - 36 -

9. Importance of stemness genes for maintenance of renewal capacity and multi-potency in BM-MSCs As described above, Sox2 and Nanog but not Oct4 were significantly down-regulated from P1 to P7 (Figure 4). To investigate the roles of Sox2 and Nanog in BM-MSC proliferation and differentiation, early-passage BM-MSCs were treated with small interfering RNAs (sirnas) targeting each gene. Expression of Sox2 and Nanog was lower in Sox2 and Nanog-siRNA-transfected cells (45% and 43% reductions, respectively) than in scrambled-sirna-treated cells (Figure 10A). The proliferation rate of Sox2-siRNA- transfected cells was lower than that of scrambled-sirnatransfected cells (Figure 10B). However, Nanog knockdown had no inhibitory effect on proliferation. In a CFU assay, the number of Sox2-siRNA-transfected cell colonies was significantly lower compared to that of scrambled-sirna-transfected cells, but this was not the case for Nanog-siRNA-transfected cells (Figure 10C). Next, we examined the potential of sirna-transfected BM-MSCs to differentiate along the osteogenic, adipogenic and chondrogenic lineages. Sox2-siRNA-transfected cells had lower osteogenic, adipogenic and chondrogenic potential in all the donors tested, compared to scrambled-sirna-transfected cells, whereas the differentiation potential of Nanog-siRNA-transfected cells along the 3 lineages was not noticeably different (Figure 10D). However, results from multiple donors showed variations in the differentiation abilities (data not shown). In addition, Sox2 overexpression using a lentiviral vector enhanced colony-forming ability in late-passage (P7) BM-MSCs compared to that for mock vector-infected BM-MSCs (data not shown). These results suggest that Sox2 plays an important role in the proliferation and multi-potency of BM-MSCs, as seen by the reproducibility of results from multiple donors. - 37 -

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Figure 10. Effect of Sox2 and Nanog knock-down on BM-MSC proliferation and colony-forming ability. (A) The protein expression patterns of scrambled-, Sox2-, and Nanog-siRNA-transfected BM-MSCs were examined by western blot analysis. (B) Hexosaminidase assay was performed to determine the proliferative potential of sirna-transfected BM-MSCs. Each experiment was performed in triplicate (n = 3). *, P < 0.05 compared to scrambled-sirna-transfected BM-MSCs. (C) Scrambled-, Sox2-, and Nanog-siRNA -transfected cells were seeded at 1 x 10 3 cells in 100-mm culture dishes. Cells were then cultured in DMEM-LG containing 20% FBS for 12 days to examine colony formation (violet). Each experiment was performed in triplicate (n = 3). *, P < 0.05 compared to scrambled-sirna-transfected BM-MSCs. (D) Scrambled-, Sox2-, and Nanog-siRNA transfected cells were seeded at 8 x 10 4 cells well in 12-well culture plates. Cells were then cultured in osteogenic, adipogenic and chondrogenic media to determine their osteogenic, adipogenic, and chondrogenic differentiation potential, respectively. Each experiment was performed in triplicate (n = 3). - 39 -

10. Change in the Sox2-positive cell population of heterogeneous BM-MSCs during prolonged passage Late-passage BM-MSCs exhibited increased expression of ALP, an early osteogenic marker, in the undifferentiated state. To confirm this phenomenon, we next examined the protein expression of genes related to osteogenic differentiation and stemness in early- and late-passage BM-MSCs. Runx2 and Dlx5, which are important osteogenic transcription factors, were expressed at higher levels in late-passage BM-MSCs compared to early-passage BM-MSCs. On the other hand, expression of the Sox2 protein, one of the important factors for stemness maintenance in MSCs 30,52-55, was dramatically decreased in late-passage BM-MSCs (Figure 11A). In addition, FACS analysis of Sox2 expression revealed differences between the 2 groups: 52.84% in early-passage BM-MSCs vs. 10.72% in late-passage BM-MSCs (Figure 11B). These results suggest that late-passage BM-MSCs contain a subpopulation of more committed osteogenic cells that increases during subsequent passage, and they lose stemness with a decrease in the numbers of Sox2-positive BM-MSCs. - 40 -

Figure 11. Comparison of Sox2 expression between early and late passage BM-MSCs. (A) Protein expression levels of a stemness gene (Sox2) and osteogenic genes (Runx2 and Dlx5) were examined by western blot analysis. β-actin was used as the loading control. (B) Early- and late-passage BM-MSCs were harvested from in vitro culture, stained with an antibody against Sox2-PerCP-Cy or control IgG-PerCP-Cy, and subjected to FACS analysis. - 41 -

11. Characterization of the different subpopulations of heterogeneous BM-MSCs by cell size BM-MSCs are a heterogeneous cell population comprising cell type with different surface markers, cell size, and differentiation potential. Using these features, BM-MSCs can be divided into 2 cell types according to size and morphology 19. The SCP exhibits spindle-shaped morphology and rapid proliferation, whereas the LCP shows flattened morphology and slow proliferation. Colter et al. designated these cell populations as RS cells (rapidly self-renewing cells/small in size) and mmscs (mature MSC/large in size), respectively 56. RS cells can differentiate along the osteogenic, adipogenic, and chondorgenic lineages, whereas mmscs fail to differentiate along the adipogenic and chondrogenic lineages. These results indicate that the LCP is committed to the osteogenic lineage compared to the SCP. In a heterogeneous cell population, senescent cells can affect the fate (proliferation, senescence, and differentiation status) of neighboring cells through secretion of cytokines 21,57,58. Based on these reports, we hypothesized that the decrease in renewal capacity and multi-potency of BM-MSCs during prolonged expansion in vitro is due to interaction between the different cell populations of BM-MSCs. To determine if this was the case, we sorted BM-MSCs by cell size using the FACS cell sorter (Beckman Coulter) (Figure 12). The sorted cells exhibited small, spindle-shaped (SCP) or large, round morphology (LCP) (Figure 13A). SA-β-gal assay revealed that the LCP was a more senescent cell population compared to the SCP (Figure 13B). Furthermore, the SCP had a higher colony-forming ability than the LCP (Figure 13C). FACS analysis revealed differences in ALP levels between the 2 populations: 3.87% in the SCP and 31.4% in the LCP (Figure 13D). These results suggest that only the SCP in heterogeneous BM-MSCs can proliferate with higher efficiency compared to its larger counterparts. In addition, the LCP is thought to be senescent and committed to the osteogenic lineage. - 42 -

Figure 12. Scheme for the isolation of SCP and LCP from heterogeneous BM-MSCs. BM-MSCs were harvested by incubation with 0.25% trypsin/edta and washed twice in PBS. Cells were resuspended in pre-warmed PBS, and the SCP and LCP were sorted and analyzed using FACS. - 43 -

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Figure 13. Characteristics of SCP and LCP isolated from heterogeneous BM-MSCs. (A) The morphology of cells fractionated by size using FACS was examined under a phase-contrast microscope. Cells in the SCP are small and spindle-shaped, whereas cells in the LCP are large and exhibit a flattened morphology. (B) SA-β-gal staining was used to determine the extent of cellular senescence in the SCP and LCP. SA-β-gal activity is higher in the LCP than in the SCP. SA-β-gal-positive cells were counted in triplicate by 3 independent observers. (C) CV staining was used to evaluate the colony-forming ability of cells fractionated by size using FACS. The SCP shows better colony-forming ability than the LCP. (D) The SCP and LCP were stained with an antibody against MSCA-1-APC (PI, propidium iodide). Each experiment was performed in triplicate (n = 2). - 45 -

12. Effects of LCP-secreted cytokines on cellular senescence and osteogenic lineage commitment of the SCP (primitive cell population) We sought to determine why LD-cultured MSCs had higher stemness potential than HD-cultured MSCs. Based on previous results, we hypothesized that LCP-secreted cytokines would affect the osteogenic lineage commitment and stemness loss of the SCP in heterogeneous BM-MSCs, given that the LCP, considered the senescent cell population, was more abundant than the SCP at later passages. Therefore, we designed an appropriate experiment to investigate the effect of LCP-secreted cytokines on the cellular senescence and osteogenic lineage commitment of the SCP without direct cell-to-cell contact. Briefly, the SCP was seeded in 6-well culture plates, and cell culture inserts with 0.4-μm pore size (Falcon, Franklin, NJ, USA) were inserted into the SCP-seeded culture plates. The LCP was then seeded into the cell culture insert with 0.4-μm pore size to avoid direct contact with the SCP; the culture insert is permeable to medium, thereby exposing cells to the secreted cytokines. In this experiment, the β-gal assay showed that LCP-secreted cytokines induced cellular senescence in the SCP under conditions of confluent cell density (1 x 10 5 cells/well of a 6-well plate), but not under non-contacting conditions (1 x 10 4 cells/well) (Figure 14A and B). The LCP-secreted cytokines also induced osteogenic lineage commitment in the SCP by increasing ALP activity and upregulating Runx2 and Dlx5 mrnas, whereas Sox2 mrna expression was decreased under confluent cell density conditions (Figure 14A and C). Ho et al. reported that the cell-to-cell contact-induced senescence model is a useful system for investigating the molecular mechanisms of MSC senescence 59. They reported that cell-to-cell contact accelerates the senescence of human MSCs independently of telomere shortening and p53 activation. In the present study, we found that cell-to-cell contact alone can induce senescence and osteogenic gene expression in MSCs. However, senescence and osteogenic lineage commitment of BM-MSCs were more strongly induced by a combination of confluent cell density and LCP-secreted cytokines than by confluent density - 46 -

alone. - 47 -

Figure 14. Effects of LCP conditioned medium on cellular senescence and osteogenic lineage commitment of the SCP. (A) Cell culture inserts were used to investigate whether LCP-secreted cytokines affected cellular senescence and osteogenic lineage commitment of the SCP isolated by FACS. SCPs were seeded in 6-well culture plates at 1 x 10 4 (non-contact; NC) or 1 x 10 5 (contact; C) cells per well. The SCPs were allowed to attach to the culture plates, and cell culture inserts (0.4-μm pores) were inserted into the wells. Then, the LCPs or SCPs were seeded in the cell culture inserts at 1 x 10 5 cells per well. The cells were stained for SA-β-gal and ALP at day 7. (B) SA-β-gal-positive cells were counted in triplicate by 3 independent observers. (C) Quantitative real-time PCR was performed to investigate the expression of Runx2, Dlx5, and Sox2 mrnas. The mrna expression levels for each gene were normalized to GAPDH expression. - 48 -

13. Lineage commitment of late-passage BM-MSCs during cellular senescence We analyzed cytokines secreted from the SCP, LCP, and a mixed-cell population (MCP) using a human cytokine array. This analysis revealed the production of 5 cytokines (Groα, IL-6, IL-8, MIF, and Seprin E1) from the SCP and MCP compared to medium containing 5% serum (Figure 15A). Only 3 cytokines (IL-6, MIF, and Seprin E1) were secreted from the LCP (Figure 15A). Of these, IL-6 levels were significantly increased compared to that secreted from the SCP (6.7-fold) and MCP (3.9-fold). We focused our attention on the ability of IL-6 to induce cellular senescence and osteogenic lineage commitment in the SCP because IL-6 can induce growth arrest in lung carcinoma cells 60 and stimulate mesenchymal progenitors differentiation along the osteogenic lineage 61. We next examined the expression of IL-6 mrna in P4 MSCS, P5 SCP, P5 MCP, and P5 LCP. IL-6 mrna was highly expressed in the LCP compared to the SCP and MCP (Figure 16A). To measure the amounts of IL-6 secreted from the SCP, MCP, and LCP into the medium, the IL-6 enzyme-linked immunosorbent assay was performed using cell culture supernatants at 24 h and 48 h. IL-6 was secreted at much higher levels in the LCP (24h: 5.4-fold; 48h: 3.9-fold) than in the SCP and MCP (Figure 16B). These results suggest that the synthesis of IL-6 in the BM-MSC populations is LCP-dependent. - 49 -

Figure 15. Identification of cytokines secreted from the LCP using a Human Cytokine Array. (A) Human cytokine array panels at 48 h using conditioned media from the SCP, LCP, or MCP cultured with DMEM-LG containing 5% FBS. (B) Cytokine signals were quantified by subtracting the background signal using the TINA 2.0 program of a Fuji image scanner. - 50 -

Figure 16. Changes in IL-6 expression in the LCP assayed by real-time PCR and ELISA. (A) Real-time PCR and (B) ELISA were performed to confirm IL-6 secretion from the LCP. - 51 -

14. Effects of IL-6 on the renewal capacity and multi-potency of BM-MSCs To establish conditions for IL-6 activity in BM-MSCs, human recombinant IL-6 was used. Treatment with 50 ng/ml IL-6 significantly increased STAT3 tyr705 phosphorylation at 12 to 24 h in BM-MSCs, as determined by western blot analysis, whereas total STAT3 and STAT3 ser727 phosphorylation remained constant. By contrast, IL-6 at 1 and 10 ng/ml had no significant effect on STAT3 tyr705 phosphorylation (Figure 17). Therefore, 50 ng/ml IL-6 was determined as an appropriate dose for enhancing IL-6 activity in BM-MSCs. We next performed SA-β-gal staining after culturing BM-MSCs in the presence or absence of IL-6 for 7 days to examine the effects of IL-6 on BM-MSC senescence. The number of SA-β-gal positive cells was increased in IL-6-treated BM-MSCs (Figure 18A). Furthermore, the CFU-F assay showed that 50 ng/ml IL-6 decreased the number of colony-forming cells in BM-MSCs (Figure 18B, upper). Proliferation of BM-MSCs was measured using MTX assay. The results revealed that the proliferative capacity of BM-MSCs was somewhat inhibited by 50 ng/ml IL-6 (Figure 18B, lower). These observations indicate that IL-6, one of the candidate senescence-inducing factors in the LCP-secreted cytokines, can induce cellular senescence and decrease the self-renewal capacity of the SCP. - 52 -

Figure 17. Dose- and time-dependent effects of IL-6 on STAT3 modulation in BM-MSCs. BM-MSCs were treated with different doses of IL-6 for 12 and 24 h. Expression of p-stat3 Tyr705 is not affected by 1 and 10 ng/ml IL-6, but is enhanced at 4 to 24 hours by 50 ng/ml IL-6. However, total STAT3 and p-stat3 Ser727 are constitutively expressed, regardless of the IL-6 concentration. The data shown here are representative of that observed in all the 3 donors tested. - 53 -

Figure 18. Effects of IL-6 on cellular senescence and renewal capacity of the SCP. (A) SA-β-gal staining was performed to examine the effects of IL-6 on BM-MSC senescence. SA-β-gal activity was increased in IL-6-treated SCPs (upper). The total area of SA-β-gal-positive cells was measured using - 54 -

Metamorph Image Analyzer (Lower). (B) CV staining was performed to examine the colony-forming ability of IL-6-treated SCPs (upper). MTX assay was performed to determine the proliferative potential of IL-6-treated SCPs. Each experiment was performed in triplicate. - 55 -

15. Effects of IL-6 on the multi-lineage differentiation potential of BM-MSCs We next examined the multi-lineage differentiation potential of BM-MSCs in the presence or absence IL-6 (50 ng/ml). BM-MSCs were cultured in osteogenic medium with or without 50 ng/ml IL-6. At day 14, IL-6 enhanced the osteogenic differentiation potential by enhancing calcium deposition as seen in alizarin red S staining and by enhancing the expression of Runx2 and osteocalcin mrnas (Figure 19A). Adipogenic differentiation potential was decreased in IL-6 treated BM-MSCs compared to the cells cultured in adipogenic medium without IL-6. IL-6 inhibited the formation of lipid droplets in adipogenesis-induced BM-MSCs and suppressed the expression of PPAR-γ and adiponectin mrnas (Figure 19B). During chondrogenic differentiation of BM-MSCs in the presence of IL-6, a significant decrease in proteoglycan synthesis was observed by safranin O staining. Real-time PCR analysis of Sox9 and type ΙΙ collagen expression confirmed the chondrogenic differentiation -inhibitory effect of IL-6 (Figure 19C). These results indicate that IL-6 induces osteogenic lineage commitment of BM-MSCs by increasing the expression of Runx2 and osteocalcin mrnas, and it inhibits adipogenic and chondrogenic differentiation of BM-MSCs by suppressing the expression of PPAR-γ /adiponectin and Sox9/type ΙΙ collagen mrnas, respectively. - 56 -

Figure 19. Effects of IL-6 on multi-potency of the SCP. (A) IL-6-treated or untreated SCPs (8 x 10 4 cells per well in 12-well plates) were incubated in osteogenic medium for 14 days. After 14 days, alizarin red S staining was - 57 -

performed to detect mineral deposition (upper). For quantitative analysis of alizarin red S staining, absorbance was measured at 595nm following de-staining with 10% cetylpyridinium chloride monohydrate for 30 min (lower). Each experiment was performed in triplicate (n = 3), and representative data are shown. (B) IL-6-treated or untreated SCPs (mass culture using 8 x 10 4 cells per well in 24-well plates) were incubated in chondrogenic medium containing 10 ng/ml TGFβ-3 for 14 days. After 14 days, safranin O staining was performed to detect proteoglycans (upper). For quantitative analysis of safranin O staining, total area of safranin O-positive cells was measured using Metamorph Image Analyzer (lower). Each experiment was performed in triplicate (n = 3), and representative data are shown. (C) IL-6-treated or untreated SCPs (8 x 10 4 cells per well in 12-well plates) were incubated in adipogenic medium for 14 days. After 14 days, oil red O staining was performed to detect lipid droplets (upper). For quantitative analysis of oil red O staining, the total area of safranin O-positive cells was measured using Metamorph Image Analyzer (lower). Each experiment was performed in triplicate (n = 3), and representative data are shown. - 58 -

16. Effect of IL-6 on protein expression of osteogenic and stemness genes Next, we investigated the expression of Runx2, Dlx5, and Sox2 proteins in IL-6-treated BM-MSCs. IL-6 increased the expression of Runx2 and Dlx5, and decreased the expression of Sox2 in a dose-dependent manner (Figure 20), independent of STAT3 Tyr705 phosphorylation (data not shown). These results suggest that IL-6 induces BM-MSC commitment to the osteogenic lineage by increasing the levels of Runx2 and Dlx5, and reduces stemness by decreasing the level of Sox2 protein expression. However, these phenomena do not appear to be STAT3 pathway-dependent. Figure 20. Changes in the protein levels of Sox2, Runx2, and Dlx5 in the presence of IL-6. Expression of Sox2, Runx2, and Dlx5 in IL-6-treated SCPs were examined by western blot analysis. GAPDH was used as the loading control. - 59 -

17. Importance of LCP-secreted IL-6 in regulating osteogenic lineage commitment and stemness loss of the SCP To block the activity of LCP-secreted IL-6, a humanized IL-6 receptor antibody (tocilizumab) was used. Tocilizumab binds to the IL-6 receptor and competitively inhibits IL-6 signal transduction 62. Treatment with 25 or 50 μg/ml tocilizumab significantly decreased STAT3 tyr705 and STAT3 ser727 phosphorylation in the presence of IL-6 (50ng/mL) (Figure 21B). In particular, 50 μg/ml tocilizumab decreased total STAT3 protein levels. Therefore, we used both concentrations of tocilizumab (25 and 50 ng/ml) in this experiment. To study the role of IL-6 in the commitment to the osteogenic lineage induced by LCP-secreted cytokines, we performed the ALP assay under blockade of IL-6 signaling by tocilizumab using the cell culture insert model. As shown in Figure 22A, LCP-secreted cytokines significantly increased ALP activity in the SCP. However, tocilizumab inhibited this increase in ALP activity (Figure 22A). We also examined the protein expression levels of osteogenic (Runx2 and Dlx5) and stemness genes (Sox2) in the SCP treated with LCP-secreted cytokines in the presence of tocilizumab. LCP-secreted cytokines increased the expression of Runx2 and Dlx5, and decreased the expression of Sox2 in the SCP. The inhibitory effect of the LCP-secreted cytokines on Sox2 protein expression was overcome by the addition of 50 μg/ml tocilizumab (Figure 22B). These results suggest that IL-6 plays an important role in the induction of commitment to the osteogenic lineage and loss of stemness in the SCP of heterogeneous BM-MSCs. - 60 -

Figure 21. Effects of tocilizumab, an IL-6 receptor antibody, on STAT3 modulation in BM-MSCs. (A) Scheme for inhibition of IL-6 signaling in BM-MSCs. (B) Expression patterns of total STAT3, STAT3 tyr705, and STAT3 ser727 in the SCP treated with tocilizumab in the presence of IL-6 were examined by western blot analysis. GAPDH was used as the loading control. - 61 -

Figure 22. Importance of LCP-secreted IL-6 in regulating osteogenic lineage commitment and stemness loss in BM-MSCs. (A) ALP activity in SCPs treated with LCP-secreted cytokines in the absence or presence of tocilizumab. Each experiment was performed in triplicate, and representative data are shown. (B) Protein expression patterns of Sox2, Runx2, and Dlx5 in SCPs treated with LCP-secreted cytokines in the absence or presence of tocilizumab were examined by western blot analysis. GAPDH was used as the loading control. - 62 -

18. Correlation between the stemness gene Sox2 and the osteogenic transcription factors genes Runx2 and Dlx5 In the in vitro senescence model of BM-MSCs, we observed significant increase in the expression of osteogenic genes, Runx2 and Dlx5, and a significant decrease in the expression of the stemness gene, Sox2 with increasing passage number. To determine the correlation between osteogenic and stemness genes, we knocked down the Runx2, Dlx5, and Sox2 genes using sirnas. Knockdown of the Runx2 or Dlx5 gene in BM-MSCs increased Sox2 mrna expression (Figure 23A), whereas knockdown of the Sox2 gene had no significant effect on Runx2 or Dlx5 mrna expression (Figure 23B). These data suggest the possibility that the IL-6-induced osteogenic genes, Runx2 and Dlx5 regulate the expression of Sox2 gene via downstream signaling. - 63 -

Figure 23. Correlation between stemness and osteogenic genes. (A) Quantitative real-time PCR was performed to investigate changes in Runx2, Dlx5, and Sox2 mrna expression following Runx2 or Dlx5 sirna transfection. (B) Quantitative real-time PCR was performed to investigate changes in Runx2, Dlx5, and Sox2 mrna expression following Sox2 sirna transfection. The mrna expression levels of each gene were normalized to β-actin expression and the experiments were performed in triplicate. - 64 -

19. Effect of IL-6-induced osteogenic transcription factors, Runx2 and Dlx5 on Sox2 protein expression and transcriptional activity IL-6 induced cellular senescence in BM-MSCs by increasing the number of SA-β-gal-positive cells, and commitment to the osteogenic lineage by increasing the expression of major osteogenic transcription factors (Runx2 and Dlx5). Furthermore, IL-6 caused loss of BM-MSC self-renewal capacity and multi-lineage differentiation potential by repressing Sox2 expression. Therefore, we hypothesized that IL-6-induced Runx2 and Dlx5 regulate expression of Sox2 gene. To determine whether the repressive effects of IL-6 on Sox2 gene expression occurred via IL-6-induced Runx2 and Dlx5, we simultaneously treated BM-MSCs with IL-6 and Runx2 and Dlx5 sirnas. Introduction of Runx2 and Dlx5 sirnas into BM-MSCs without IL-6 slightly increased Sox2 protein levels. However, IL-6 successfully induced the expression of Runx2 and Dlx5 proteins, and significantly decreased Sox2 protein expression. The IL-6-induced repression of Sox2 protein was overcome by introduction of Runx2 and Dlx5 sirnas (Figure 24A). Next, we hypothesized that the transcriptional activity of Sox2 is controlled by Runx2 and Dlx5 expressed via IL-6 downstream signaling. To monitor Sox2 transcriptional activity in BM-MSCs, we performed a Cignal Sox2 Reporter Assay to determine the effects of IL-6 treatment and knockdown/ over-expression of Runx2 and Dlx5. When BM-MSCs were transfected with the Sox2 reporter vector along with negative sirna, Runx2 sirna or Dlx5 sirna, transcriptional activity of Sox2 was increased, but not significantly, in the Runx2 and Dlx5 sirna-treated groups compared to the negative sirna-treated group. IL-6 suppressed Sox2 reporter activity in the negative sirna-transfected group compared to the negative sirna-transfected group (i.e., without IL-6). Introduction of Runx2 or Dlx5 sirna restored Sox2 reporter activity in the IL-6-treated BM-MSC groups compared to the groups without IL-6 treatment (Figure 24B and C). In HeLa cells, we confirmed that introduction of Runx2 (pcmv6) or the Dlx5 (pcdna3.1) over-expression vector could significantly - 65 -

decrease the activity of the Sox2 reporter to a similar extent as in IL-6-treated HeLa cells, compared to the respective vector controls (Figure 24D). These results indicate that IL-6 can induce osteogenic lineage commitment by up-regulating the expression of osteogenic transcription factors (Runx2 and Dlx5) and decrease the self-renewal capacity and multi-lineage differentiation potential of primitive cells by down-regulating the transcriptional activity of the stemness-related gene product, Sox2. In summary, LCP-secreted cytokines (IL-6) can induce the expression of osteogenic transcription factors (Runx2 and Dlx5) in the primitive cell population of heterogeneous BM-MSCs. IL-6-induced Runx2 and Dlx5 increase the expression of an early osteogenic marker (ALP), thereby committing the primitive cell population (SCP) to the osteogenic lineage. At the same time, IL-6-induced Runx2 and Dlx5 suppress the transcriptional activity of Sox2, an important factor in maintaining the renewal capacity and multi-potency of the SCP, resulting in stemness loss in the SCP (Figure 25). - 66 -

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Figure 24. Regulation of Sox2 expression by IL-6-induced Runx2 and Dlx5. (A) Western blot analysis was performed to investigate the expression patterns of Sox2, Runx2, and Dlx5 proteins in response to IL-6 treatment, and sirunx2 and sidlx5 transfection in BM-MSCs. The above data have been confirmed in all the 3 donors tested, and representative data are shown. (B and C) BM-MSCs or HeLa cells were co-transfected with the Sox2 reporter and sirunx2, sidlx5, or negative control sirna in the absence or presence of IL-6. This reporter contains transcriptional regulatory elements for Sox2 (TRE: AACAAAGAGT) After 36 h, dual-luciferase assays were performed, and relative firefly activities were normalized to Renilla luminescence. (D) HeLa cells were co-transfected with the Cignal Sox2 reporter and PCMV6-control, PCMV6-Runx2, pcdna3.3, or pcdna3.3-dlx5. After 36 h, dual-luciferase assays were performed, and relative firefly activities were normalized to Renilla luminescence. - 68 -

Figure 25. The proposed model of lineage commitment and stemness loss during senescence of heterogeneous BM-MSCs - 69 -