Analysis of the Pro- and Anti-Inflammatory Cytokines Secreted by Adult Stem Cells during Differentiation

Adipose-derived stromal/stem cells (ASCs) are adult stem cells that have the potential to differentiate into mesenchymal lineage cells. The abundance of ASCs in adipose tissue and easy accessibility with relatively little donor site morbidity make them attractive candidate cells for tissue engineering and regenerative medicine. However, the underlying inflammatory process that occurs during ASC differentiation into adipocytes and osteoblast has not been extensively investigated. ASCs cultured in osteogenic and adipogenic differentiation medium were characterized by oil red o staining and alizarin red staining, respectively. ASCs undergoing osteogenic and adipogenic differentiation were isolated on days 7, 14, and 21 and assessed by qRT-PCR for the expression of pro- and anti-inflammatory cytokines. ASCs undergoing osteogenic differentiation expressed a distinct panel of cytokines that differed from the cytokine profile of ASCs undergoing adipogenic differentiation at each of the time points analyzed. Mapping the cytokine expression profile during ASC differentiation will provide insight into the role of inflammation in this process and identify potential targets that may aid in enhancing osteogenic or adipogenic differentiation for the purposes of tissue engineering and regenerative medicine.


Introduction
Adipose-derived stromal/stem cells (ASCs) are adult stem cells with multipotential differentiation capacity. The ability for ASCs to differentiate along osteogenic and adipogenic lineage cells makes them ideal candidates for regenerative medicine [1,2]. Furthermore, the abundance and easy accessibility in harvesting large volumes of adipose tissues allow for large-scale expansion of ASCs for therapeutic purposes [3,4].
The osteogenic and adipogenic differentiation of ASCs has been shown to require the activation of key transcriptional factors that govern cell fate. RUNX2 has previously been shown to be a master regulator of osteoblast differentiation, as RUNX2 activates and regulates many osteogenic signaling pathways, including but not limited to transforming growth factor beta (TGF-), bone morphogenetic protein (BMP), Wingless type Wnt, and Hedgehog [5,6]. ASCs cultured in osteogenic differentiation medium have also been shown to upregulate a key osteogenic factor dickkopf Wnt signaling pathway inhibitor 1 (DKK-1), as early as one day. Additional osteogenic transcriptional factors (connective tissue growth factor (CTGF), platelet-derived growth factor receptor beta (PDGFR-), TGF-, insulin-like growth factor binding protein 3 (IGFBP3), and tenascin C (TNC)) were induced after 7 days in osteogenic differentiation medium [7]. In contrast, peroxisome proliferator-activated receptor gamma (PPAR ) is principally regarded as the master regulator of adipogenesis, since no factor can rescue adipocyte formation when PPAR is knocked out [8]. Induction of CCAAT-enhancer-binding proteins (C/EBP , C/EBP ) and peroxisome proliferator-activated receptor delta (PPAR ) expression occurs during early adipogenic differentiation, while fatty acid binding protein 4 (FABP4), C/EBP , lipoprotein lipase (LPL), leptin, and glucose transporter 4 (GLUT4) expression is upregulated during late adipogenic differentiation [9,10].
While many studies have explored the mechanism(s) governing ASC differentiation, few studies have investigated the associated expression of inflammatory gene expression that occurs during the differentiation of these cells. The expression profile of mRNA encoding these inflammatory cytokines may provide information regarding the mechanism governing ASC differentiation. Herein, ASCs were induced to differentiate into osteogenic and adipogenic lineage cells and assessed by qRT-PCR for the expression of pro-and antiinflammatory cytokines. These studies demonstrated a systemic and robust upregulation of pro-and anti-inflammatory cytokines that was time-dependent. These studies demonstrated the plasticity of ASCs and identified inflammatory cytokines secreted at different stages of differentiation that may govern the ultimate cell fate of ASCs.

Human Subjects.
Primary human ASCs were obtained from subcutaneous abdominal adipose tissue of 3 Caucasian females (mean age 34.6 ± 8.4 and mean body mass index 22.2 ± 1.1) undergoing elective liposuction. Tissues were obtained with written informed consent under a protocol reviewed and approved by the Pennington Biomedical (Baton Rouge, LA) Institutional Review Board. Lipoaspirates were processed by incubating tissue in 0.1% type I collagenase and 1% BSA dissolved in 100 mL of phosphate buffered saline (PBS) supplemented with 2 mM calcium chloride. The mixture was placed in a 37 ∘ C shaking water bath at 75 rpm for 60 min and then centrifuged to remove oil, fat, primary adipocytes, and collagenase solution, leaving behind a pellet of cells. Cells were resuspended in medium, which consisted of Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12; Life Technologies, Grand Island, NY) and 10% fetal bovine serum (FBS; HyClone; Logan, UT), plated on 150 cm 2 culture dishes (NUNC, Rochester, NY) and maintained in a humidified 5% CO 2 incubator. Fresh medium was added every 2-3 days until cells achieved 80-90% confluence and were harvested with 0.25% trypsin/1 mM EDTA (Life Technologies) and cryopreserved prior to experimental use.

Flow Cytometry.
ASCs were harvested with 0.25% trypsin/1 mM EDTA for 3-4 minutes at 37 ∘ C. A total of 3 × 10 5 cells were suspended in 50 L PBS and incubated with fluorescence-labeled antibodies. The samples were incubated for 30 minutes at room temperature and washed with PBS. The samples were then analyzed with Gallios Flow Cytometer (Beckman Coulter, Brea, CA) running Kaluza software (Beckman Coulter). To assay cells by forward and side scatter, FACScan was standardized with microbeads (Dynosphere uniform microspheres; Bangs Laboratories Inc.; Thermo Scientific; Waltham, MA). At least 10,000 events were analyzed and compared with isotype controls.

Colony Forming Unit Assay.
ASCs were plated at a density of 100 cells on a 10 cm 2 plate (NUNC) in CCM and incubated for 14 days. Plates were then rinsed with PBS and stained with 3% crystal violet (Sigma) for 30 minutes at room temperature. Plates were washed with PBS and once with tap water. Colonies that were larger than 2 mm in diameter were counted.

Differentiation Protocols
Osteogenic Differentiation. ASCs were cultured in six-well plates (NUNC) in CCM until 70% confluence. Medium was replaced with fresh osteogenic differentiation medium (ODM) consisting of 50 M ascorbate 2-phosphate, 10 mM -glycerol phosphate, and 10 nM dexamethasone. After 14 days, cells were fixed in 10% formalin for 1 hour, washed with distilled water, and stained with 1% alizarin red (pH 4.1) to visualize calcium deposition in the extracellular matrix. Images were acquired at 4x magnification on an Eclipse TE200 (Nikon, Melville, NY) with Digital Camera DXM1200F (Nikon) using ACT-1 software (Nikon). For quantification, alizarin red was extracted from each well with 10% CPC and read at 584 nm (FLUOstar optima). Protein extraction with RIPA buffer (Pierce; Thermo Scientific; Waltham, MA) and protein quantification with the BCA assay (Thermo Scientific) were performed according to manufacturer's instructions. Samples were normalized to the amount of protein in each sample.
Adipogenic Differentiation. ASCs were cultured in six-well plates in CCM until cells achieved 70% confluence. Medium was replaced with fresh adipogenic differentiation medium (ADM) consisting of CCM supplemented with 0.5 M dexamethasone, 0.5 mM isobuytlmethylxanthine, and 50 M

Discussion
The interest in ASCs for tissue engineering purposes and regenerative medicine has grown significantly due to their accessibility, abundance, and capacity to differentiate into mesenchymal lineage cells. While studies have begun to investigate the mechanism by which ASCs differentiate into adipogenic or osteogenic lineage cells, the precise role of inflammatory cytokines has not been explored extensively. The mRNA levels of many pro-and anti-inflammatory cytokines expressed by ASCs during osteogenic and adipogenic differentiations were assessed. ASCs undergoing osteogenic differentiation expressed a distinct panel of cytokines that differed from the cytokine profile of ASCs undergoing adipogenic differentiation at each of the time intervals analyzed.
A quantitative comparison of the proinflammatory cytokines and anti-inflammatory cytokines expressed during osteogenic differentiation of ASCs demonstrates three distinct groups of cytokines. These groups of cytokines are categorized based on their induction at early, mid, and late stages of osteogenic differentiation ( Figure 6). While the current study investigated the inflammatory cytokines secreted during the osteogenic differentiation of these cells in a cocktail of growth factors, others have taken the approach of treating progenitor cells with a similar cocktail of growth factors and supplemented the medium with additional inflammatory cytokines, such as TNF-and IL-1 [11][12][13][14]. Most of these studies were conducted in bone marrow-derived mesenchymal stem cells (BMSCs), which are derived from the mesodermal lineage and have a similar differentiation potential as ASCs. Human BMSCs treated with TNFresulted in the activation of NF-B, leading to increased mineralization and enhanced expression of osteogenic proteins, such as BMP2 and alkaline phosphatase, and transcription factors such as RUNX2 and Osterix [11,12]. Human BMSCs treated with IL-1, likewise, enhanced differentiation into osteoblasts through the Wnt-5a/receptor tyrosine kinase-like orphan receptor 2 pathway [13]. Ferreira et al. found that IL-1 also enhanced mineralization through both nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) and mitogen-activated protein kinase (MAPK) pathways [14]. The results presented in the current study are consistent with previously published reports showing an upregulation of TNF-and IL-1 during differentiation. The present study supplements the current body of literature and highlights specific inflammatory factors that should be investigated further based on their induction during osteogenic differentiation. Additional studies, however, are necessary to elucidate the precise mechanism by which these cytokines effect osteogenic differentiation. Furthermore, it should be noted that the effects of proand anti-inflammatory cytokines on osteogenesis might be determined by the physical location. For instance, cytokines have been shown to contribute to a decrease in bone mineral density by inhibiting osteoblast proliferation and differentiation and enhancing the rate of osteoclast differentiation in patients with severe inflammatory disease [15,16]. Thus, patients diagnosed with such diseases as rheumatoid arthritis and osteoarthritis have a higher incidence of osteoporosis [15,16]. In contrast, inflammatory cytokines are strongly suspected to induce ectopic bone formation, for instance, in arteries during atherosclerosis or in postburn heterotopic ossification [17,18]. Consistent with these observations, antiinflammatory drugs have been shown to reduce the incidence and severity of ectopic bone formation [19].
With respect to adipogenesis, the current study demonstrated a time-dependent expression of cytokines in ASCs during adipogenic differentiation ( Figure 6). While limited studies have been conducted on the cytokine profile of ASCs undergoing adipogenic differentiation, it has been previously shown that cytokines such as IL-1, IL-6, and TNFhave the ability to inhibit adipogenic differentiation of BMSCs. PPAR is suppressed by IL-1 and TNF-, and this suppression is mediated through NF-B [20]. In the context of obesity, increased proinflammatory cytokines are secreted by the adipose tissue due to tissue hypoxia that results from hypertrophy and hyperplasia of adipocytes [21]. Based on previous reports, this increase in proinflammatory cytokines, such as IL-1 and TNF-, should result in decreased adipogenesis. However, due to the obesity-associated dysregulation of adipocytes and ASCs, these cells no longer respond to these cytokines properly [22]. Thus, the increase in IL-1 and TNFdoes not inhibit adipogenesis. Additional studies focusing on the impact of other pro-and anti-inflammatory cytokines on adipogenesis will shed light on ASC differentiation for the purposes of soft tissue reconstruction. Furthermore, they may provide insight into what other factors mediate adipogenesis in the context of obesity.

Conclusion
The osteogenic and adipogenic differentiation of ASCs alter the expression of an array of cytokines. The levels of induction of these pro-and anti-inflammatory cytokines are dependent on the stage (early, middle, or late) and type (osteogenic or adipogenic) of differentiation. The data presented here provides a framework for understanding the role that cytokine expression may play in tissue engineering projects using ASCs. By understanding which cytokines are upregulated during osteogenic or adipogenic differentiation, it will be possible to specifically target these molecules to enhance osteogenic or adipogenic differentiation for soft tissue defects or bone defects. Furthermore, paracrine expression of these cytokines by differentiating ASCs may change the local environment. By understanding the local environment produced by differentiation ASCs, we will also be better equipped to assess the engraftment and survival of tissue engineered constructs prepared with ASCs.