Osteogenic Lineage Commitment of Adipose-Derived Stem Cells Is

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Osteogenic Lineage Commitment of Adipose-Derived Stem Cells is Predetermined by Three-Dimensional Cell Accumulation on Micropatterned Surface Yuichi Furuhata, Keitaro Yoshimoto, Toru Yoshitomi, Yuka Kikuchi, and Miho Sakao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15688 • Publication Date (Web): 01 Mar 2017 Downloaded from http://pubs.acs.org on March 4, 2017

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ACS Applied Materials & Interfaces

Osteogenic Lineage Commitment of AdiposeDerived Stem Cells is Predetermined by ThreeDimensional Cell Accumulation on Micropatterned Surface Yuichi Furuhataa, b, Toru Yoshitomic, Yuka Kikuchic, Miho Sakaoa and Keitaro Yoshimotoa, c, d* a

Department of Life and Cognitive Science, College of Arts and Science, The University of

Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan b

Department of Computational Biology and Medical Sciences, Graduate School of Frontier

Sciences, The University of Tokyo, Shirokanedai 4-6-1, Minato-ku, Tokyo 108-8639, Japan c

Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo,

Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan d

JST, PRESTO, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan

KEYWORDS. micropatterned surface, cell accumulation, osteogenic potential, adipose-derived stem cells, mesenchymal stem cells, cell differentiation, poly(ethylene glycol)

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ABSTRACT.

Lineage commitment of stem cells is mainly regulated by their microenvironments, which comprise soluble growth factors, extracellular matrix, mechanical forces, and cell density. Although numerous studies have investigated stem cell response to these factors in twodimensional (2D) culture, little is known about that in 3D culture. Here, we studied effects of 3D cell accumulation levels on the differentiation behavior of mesenchymal stem cells (MSCs) by using a micropatterned surface. After induction of 3D-cultured MSCs on the surface, their osteogenic differentiation was significantly promoted, while adipogenic differentiation was not. This differentiation behavior of densely packed MSCs in 3D culture is unlike that in 2D culture. Moreover, to determine the contributing factor of this commitment, the relationship between 3D cell accumulation levels and their differentiation potential was studied before differentiation induction. A series of MSCs with varied 3D accumulation levels was constructed on the micropatterned surface, where the accumulated MSCs were not in hypoxic environment. Interestingly, with increasing 3D accumulation levels, MSCs enhanced their osteogenic potential but repressed adipogenic potential in gene expression level. These results suggest that preconditioned 3D microenvironments with high cell accumulation levels promote osteogenic differentiation of MSCs and their accumulation levels help in regulating MSC differentiation.


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TEXT. Introduction Mesenchymal stem cells (MSCs) are a type of somatic stem cells and significant in regenerative medicine owing to their multilineage differentiation and self-renewal properties. MSCs can differentiate into various cells of mesodermal lineage, such as adipocytes, osteocytes, and chondrocytes1. The lineage commitment of MSCs is regulated by their microenvironment, which comprises soluble growth factors, mechanical forces, and extracellular matrix (ECM)2. Additionally, it was reported that differentiation of MSCs in monolayer culture is affected by scaffolds, cell shape, cell size, and cell density3–8. Three-dimensional (3D) cell culture systems have been proposed to create a microenvironment that mimics the native tissue and alters the cellular functions of MSCs. Compared to conventional monolayer culture, 3D culture facilitates greater cell-cell contact, cell-ECM interactions, hypoxic conditions, and a large gradient of molecular concentrations. To obtain these properties, several methods for 3D culture have been applied to MSCs, such as hydrogels, bioreactors, and micropatterned surfaces9–11. In these studies, the 3D culture methods affected therapeutic capacity and stemness properties of MSCs. Potapova et al. reported that MSC spheroid construction by hanging drop method upregulates expression levels of genes associated with hypoxia, angiogenesis, inflammation, stress response, and redox signaling12. We have also succeeded in constructing small spheroids of adipose-derived stem cells (ADSCs) using a micropatterned surface to reveal that 3D cell formation directly upregulates gene expression and protein secretion profiles of wound-healing factors13. Since 3D culture using micropatterned surfaces is advantageous in the preparation of small and uniformly sized spheroids, this approach is suitable in the evaluation of the effects of 3D culture on cellular functions.


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The differentiation behavior of MSCs is profoundly affected by 3D culture environments. For example, spheroid formation using chitosan films enhances osteogenic differentiation of ADSCs14, and that using micropatterned surfaces promotes osteogenic and adipogenic differentiation of bone marrow-derived MSCs (BMSCs)10. Although these studies showed that these 3D culture methods increase the efficiency of differentiation in the presence of induction factors, to the best of our knowledge, there have been no reports on the effect of 3D culture on cell differentiation in the absence of induction factors. Here, we focused on the effect of 3D culture on the gene expression profiles of ADSCs in the absence of induction factors. A tilted osteogenic/adipogenic balance was studied as a model of MSC differentiation, since their inverse relationship in cell differentiation is well known and has been characterized previously3,4,15. To construct cell aggregates with different accumulation levels, 3D culture was performed using a micropatterned surface as small sized cell-aggregates constructed on the surface can avoid the secondary effects of 3D culture such as hypoxia, lownutrient supply, and metabolic waste accumulation13. Initially, the effects of 3D culture on differentiation behavior of ADSCs were investigated in the presence of differentiation-inducing factors. Thereafter, we demonstrated the precision control of ADSC accumulation levels on the micropatterned surface and studied the dependency of differentiation marker gene expression on 3D accumulation levels in the absence of differentiation-inducing factors.

Materials and Methods Micropatterned surface


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96-well micropatterned culture plates (Cat. No. PP-96, Toyo Gosei Co., Ltd., Japan) were used in this study. All of the wells were coated with poly(ethylene glycol) (PEG) hydrogel and have 800 circular non-coated domains with 100 µm in diameter that are spaced, edge to edge, at 100 µm intervals on the culture surface. The seeded cells accumulate on circular non-coated domains and form spherical aggregates because the region of PEG hydrogel on the micropatterned surface inhibits cell adhesion10,16–18. Constructed cell aggregates adhere to the surface and are not easily peeled off.

Culture of ADSCs Human ADSCs (Cat. No. PT-5006, Lonza Group Ltd., Switzerland) were cultured at 37 °C under 5% CO2 in Dulbecco's modified Eagle medium (Cat. No. 044-29765, Wako Pure Chemical Industries, Ltd., Japan) supplemented with 10% (v/v) fetal bovine serum (Cat. No. FB1061/500, Biosera, France) and 1% (v/v) Penicillin-Streptomycin-Neomycin (PSN) antibiotic mixture (Cat. No. 15640055, Thermo Fisher Scientific Inc., USA). For monolayer and aggregate cultures, ADSCs (3.2 × 104 cells/well) were seeded on 24-well culture plates (Cat. No. 3820-024, AGC Techno Glass Co., Ltd., Japan) and 96-well micropatterned culture plates, respectively. For 3D cell accumulation analysis, ADSCs were seeded on 96-well micropatterned culture plates with seeding cell numbers of 0.16, 0.32, 0.64, 1.6, and 3.2 × 104 cells/well. All experiments were carried out using ADSCs with passage numbers from 3 to 5.

Analysis of mRNA expression by real-time reverse transcription PCR (Real Time RT PCR) RNA samples of ADSCs were extracted using RNeasy Micro Kit (Cat. No. 74004, Qiagen, Germany). RNA was converted into cDNA using the ReverTra Ace qPCR RT Master Mix with


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gDNA Remover (Cat. No. FSQ-301, Toyobo Co., Ltd., Japan). Real-time RT PCR was performed for actin beta (ACTB), runt related transcription factor 2 (RUNX2), alkaline phosphatase

(ALP),

peroxisome

proliferator

activated

receptor

gamma

(PPARγ),

CCAAT/enhancer binding protein alpha (C/EBPα), phosphoglycerate kinase 1 (PGK1), lactate dehydrogenase A (LDHA), and adenylate kinase 3-like 1 (AK3L1) using THUNDERBIRD SYBR qPCR Mix (Cat. No. QPS-201, Toyobo Co., Ltd., Japan) with StepOnePlusTM (Thermo Fisher Scientific Inc., USA). Thermal cycling was performed at 95 °C for 60 s, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 60 s. Data were analyzed with StepOne Software v2.2.2 (Thermo Fisher Scientific Inc., USA) and relative quantities were calculated by the ∆∆Ct method. The relative expression levels of mRNA were normalized by ACTB and then standardized to indicate samples. The analyses were repeated more than thrice, and primer sets shown in Table 1 were used.

Fluorescent staining To obtain 2D top view images of ADSCs on micro-domains, cells were rinsed with phosphate buffered saline (PBS) and incubated with 10 µg/mL of Hoechst 33258 (Cat. No. PK-CA70740044, PromoCell GmbH, Germany) and 1 µg/mL of Calcein AM (Cat. No. 341-07901, Dojindo Laboratories, Japan) at 37 °C for 30 min. The ADSCs were rinsed thrice with PBS and observed using a fluorescence microscope (AxioObserver Z1, Zeiss, Germany). To obtain 3D reconstruction images of ADSCs on micro-domains, cells were rinsed with PBS and incubated with 250 nM of MitoTracker Red CMXRos Mitochondrial Probe (Cat. No. PA-3017, Lonza Group Ltd., Switzerland) at 37 °C for 30 min. Following the incubation period, ADSCs were rinsed thrice with PBS and observed using a confocal laser scanning microscope (C2+, Nikon


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Corporation, Japan). The confocal images obtained were reconstructed and pseudocolored by zdepth using the image analysis software NIS Elements (Nikon Corporation, Japan). The maximum thickness distribution and the volume of the layers of accumulated ADSCs was determined by analyzing more than 20 and 3 domains, respectively.

Osteogenic differentiation ADSCs in monolayer and aggregate cultures were grown in DMEM supplemented with 10 nM dexamethasone (Cat. No. 047-18863, Wako Pure Chemical Industries, Ltd., Japan), 50 µM vitamin C (Cat. No. 013-19641, Wako Pure Chemical Industries, Ltd., Japan), and 10 mM βglycerophosphate (Cat. No. 046-31251, Wako Pure Chemical Industries, Ltd., Japan) for 2 weeks, with replacement of the medium every 3 days. Parallel control cultures were maintained in DMEM for 2 weeks with medium replacements every 3 days. At specific time points, alizarin red staining was performed to visualize matrix mineralization. ADSCs were rinsed with PBS, fixed by 4% paraformaldehyde for 15 min at room temperature, and rinsed thrice with water. Fixed cells were incubated with methanol for 10 min at -20 °C, rinsed with water, and incubated with 10 µg/mL alizarin red S (Cat. No. 011-01192, Wako Pure Chemical Industries, Ltd., Japan) solution at pH 4.2 for 5 min at room temperature. After the rinse with water, images were obtained using a phase contrast microscope (IX51S8F-3, Olympus Corp., Japan). Matrix mineralization was quantified by extraction of the alizarin red S stain with 5% formic acid solution and measuring the absorbance at 414 nm.

Adipogenic differentiation


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Adipogenic differentiation of ADSCs was performed using Mesenchymal Stem Cell Adipogenic Differentiation Medium (Cat. No. C-28011, PromoCell GmbH, Germany) according to the manufacturer’s instructions, with replacement of the medium every 2 days. Parallel control cultures were maintained in DMEM for 2 weeks with medium replacements every 2 days. At specified time points, oil red O staining was performed to visualize lipid droplets. Cells were rinsed with PBS, fixed with 4% paraformaldehyde for 15 min, washed twice with PBS and then with 60% v/v isopropyl alcohol, cells were stained with 0.9 mg/mL oil red O (Cat. No. 15402072, Wako Pure Chemical Industries, Ltd., Japan) solution in 60% v/v isopropyl alcohol for 15 min at room temperature, washed twice with water. Images were obtained using a phase contrast microscope (IX51S8F-3, Olympus Corp., Japan).

Statistical analysis Measurements of mRNA expression, alizarin red S quantification, and the volume of accumulated cells are presented as mean ± standard error (SE). Statistical significance was evaluated using independent-samples student’s t-test. Statistically significant values were defined as p

Osteogenic Lineage Commitment of Adipose-Derived Stem Cells Is

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