Secreted factors from adipose tissue increase adipogenic differentiation of mesenchymal stem cells

Abstract

Objectives: Adipose tissue engineering is one of the hottest topics in the field of regenerative medicine. Fat tissue has been considered as an abundant and accessible source of adult stem cells by tissue engineers, since it gives rise to adipose stem cells. However, recent reports have pointed out that adipose tissue, as a secretory and endocrine organ, might secrete cytokines that regulate body functions such as metabolism, infammation and more. In this study, we aim to investigate the adipogenic-inducing factors secreted by fat tissue.

Materials and methods: Conditioned medium were collected by culturing fat tissue fragments in plastic flasks. Mesenchymal stem cells (MSCs) cultured in conditioned medium (CM) to test the adipogenic-inducing factors. Oil red O staining, reverse transcription/polymerase chain reaction and immunocytofluorescent staining were performed to examine the differentiation of MSCs in CM.

Results: MSCs cultured in CM of adipose tissue spontaneously differentiated into adipocytes. Furthermore, supplementation of insulin or dexamethasone to CM accelerated the process of lipid accumulation of differentiated MSCs.

Discussion: Results from this study demonstrated that fat tissues secrete small molecules, which induce adipogenic differentiation of MSCs.

Conclusions: Our study provides clues for improving adipose tissue engineering by using fragmented adipose tissue as sources of fat-inducing factors.

Figure 1

Spontaneous differentiation of adipose‐derived stromal cells. (a) Fat tissue isolated from the groin fat pad, cut into small pieces and cultured in flasks, for up to 2 weeks. Adipose stromal cells migrated out of fat tissue fragments and gradually turned into fully mature adipocytes. Bar = 50 μm (b) Conditioned medium was formed by culturing fat tissue with DMEM containing 10% FBS for 1 week. Bone marrow MSCs cultured in conditioned medium for 1 week differentiation into adipocytes. Upper panel – pictures with no staining, while lower panel shows images of oil‐red O staining. Bar = 50 μm

Figure 2

Serum‐free conditioned medium from adipose tissue, induces adipogenic differentiation of bone marrow MSC s. (a) CM with FBS was obtained by culturing fat tissue with DMEM containing 10% FBS for 1 week. CM without FBS was obtained by culturing fat tissue with serum‐free medium for 1 week and then supplemented with 10% FBS. Both CM with FBS and CM without FBS induced adipogenic differentiation of MSCs after 1 week's culture. Bar = 50 μm. (b) RT‐PCR analysis of adipogenic genes on MSCs, cultured in different media. (c) Imunofluorescence staining showed that PPAR‐γ was expressed by MSCs cultured either in CM with FBS or in CM without FBS. Nuclei counterstained with DAPI. Bar = 10 μm.

Figure 3

Conditioned medium supplemented with dexamethasone or insulin induced stronger adipogenic differentiation of MSC s. (a) Control medium (DMEM+10%FBS) and CM without FBS (collected as described before) were supplemented either with dexamethasone (Dex) or insulin, and these media were used to culture MSCs for 1 week. CM without FBS supplemented with insulin induced strongest lipid accumulation. Bar = 100 μm. (b) MSCs were then fixed and stained with oil‐red O. Pixels of positive area were calculated using imagej, to show amounts of lipid formed in MSCs. **P < 0.01. (c) Expression of PPAR‐γ was detected by RT‐PCR. GAPDH was used as internal control. (d) Signal intensities of PPAR‐γ and GAPDH on agarose gel were quantified to estimate relative levels of PPAR‐γ. **P < 0.01. (e) Expression of PPAR‐γ was visualized by secondary antibodies conjugated to rhodamine. Nuclei were counterstained with DAPI. Bar = 10 μm.

Figure 4

Active components of conditioned medium, between 3 and 5 kDa. (a) Different components of CM without FBS were separated by ultra centrifugation. All three components, as well as controls, were supplemented with 10% FBS and insulin. MSCs cultured in these media for 1 week showed different levels of adipogenic differentiation. Bar = 50 μm (b) MSCs were then fixed and stained in oil‐red O. Pixels of positive areas were calculated using imagej, to show amounts of lipid formed in MSCs. **P < 0.01. (c) Expression of PPAR‐γ was detected by RT‐PCR. GAPDH was used as internal control. (d) Signal intensities of PPAR‐γ and GAPDH on agarose gel were quantified to estimate relative levels of PPAR‐γ. **P < 0.01. (e) Expression of PPAR‐γ was visualized using secondary antibodies conjugated to rhodamine. Nuclei were counterstained with DAPI. Bar = 10 μm.