Markus Neubauer,1 Leoni Kunz-Schughart,2 Marit Hoffmann,2 Achim Goepferich,1 Torsten Blunk1
1 Department of Pharmaceutical Technology, University of Regensburg, Universitätsstrasse 31, 93040 Regensburg, Germany
2 Department of Pathology, University of Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany
Chapter 5 bFGF-influenced adipogenesis of MSCs under clonal conditions
-98-
Abstract
Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating at least towards the osteogenic, chondrogenic, and adipogenic lineage. Modulators of the differentiation are a wide variety of growth factors such as transforming growth factor-β, bone morphogenetic proteins, and basic fibroblast growth factor (bFGF). Recently, we demonstrated the enhancing effect of bFGF on the adipogenic conversion of MSCs in 2-D and 3-D cell culture (see chapters 4 and 7). However, mechanisms by which bFGF exerts its effects on MSCs are poorly investigated.
The presence of multiple cell populations in the MSC culture renders the determination of the underlying mechanism more difficult and requires a system with which single cells can be investigated under clonal conditions. The first goals of this study was to evaluate the potential of different media to stimulate the growth of MSCs under clonal conditions. A medium consisting of α-MEM, fetal bovine serum, ascorbic acid, and the B27 supplement, denoted as the cloning medium, was found to be suitable for the expansion of MSCs under cloning condition. This medium ensured the maintenance of the differentiation potential and the responsiveness to bFGF as enhancer of the adipogenesis of MSCs. Administration of the cloning medium allowed for the investigation of the mechanism of action of bFGF on the adipogenesis of MSCs under clonal conditions. In conclusion, differentiation experiments under clonal conditions in which bFGF was supplemented either only in the single cell culture or in the entire culture suggests bFGF to act mainly via the preferential proliferation of a subset of the MSCs capable of undergoing adipogenesis.
Chapter 5 bFGF-influenced adipogenesis of MSCs under clonal conditions
Introduction
Mesenchymal stem cells (MSCs) represent intensively investigated stem cells which have the capacity of multipotential differentiation into at least chondrocytes, osteoblasts, and adipocytes [1]. The fate of MSCs depends on the microenvironmental conditions such as absence or presence of inducing stimuli and differentiation and growth factors such as transforming growth factors [2], bone morphogenic proteins [2-4], and basic fibroblast growth factor (bFGF) [5-7].
Recently, we could demonstrate the enhancing effect of bFGF on the adipogenesis of MSCs in 2-D (Chapter 4) and 3-D cell culture (Chapter 7). Basic FGF increases the number of differentiated adipocytes and their maturation and yields elevated expression levels of adipogenic markers such as glycerol-3-phosphate dehydrogenase (GPDH), glucose transporter 4 (GLUT4), and peroxisome proliferator-activated receptor γ (PPARγ) at the molecular level following the administration of hormonal induction regimen (Chapter 4). In 2-D cell culture, supplementation of bFGF leads to an increased expression of PPARγ even prior to adipogenic induction accompanied by a very high responsiveness of bFGF-treated MSCs to a PPARγ ligand, the thiazolidinedione troglitazone.
Principally, two possible mechanisms of the action of bFGF are discussed [5,6]: (1) Basic FGF leads to preferential proliferation of a subset of the MSC population which is responsible for the increased PPARγ message and (2) supplementation of bFGF exerts direct effects on the commitment level of MSCs, that is, bFGF induces the expression of PPARγ in certain cells.
Bone marrow is composed of at least three cellular systems: haematopoietic, endothelial and stromal. In adult bone marrow, macrophages, adipocytes, osteogenic cells, haematopoietic cells, cells originating from blood vessels and “reticular” cells coexist and partially cooperate [8]. When MSCs are isolated by adherence to cell culture plastics, as was the case in the studies mentioned above, the adherent cells represent a mixture of different cell types. The presence of multiple cell populations renders the determination of the underlying mechanism more difficult and requires a system with which single cells can be investigated under clonal conditions.
In this study, single cells were sorted with means of FACS analysis. As reasonable clonal growth of certain cell types often is hard to achieve, the first goal of this study was to evaluate an appropriate growth medium for the clonal expansion of MSCs by testing different
Chapter 5 bFGF-influenced adipogenesis of MSCs under clonal conditions
-100- Eagle’s medium (α-modification) (α-MEM), and RPMI supplemented with additives such as the B27 supplement and conditioned media. The optimum medium revealed to be α-MEM, 10% FBS, 1% antibiotics, 50 µg/ml ascorbic acid, and the B27 supplement, in the following referred to as cloning medium. The cloning medium was used in the further experiment in order to study the mechanisms of the effect of bFGF on the adipogenesis of MSCs. Basic FGF was administered either (1) in the complete culture, that is, during the proliferation phase and in the subsequent single cell culture, or (2) only in the single cell culture, or (3) not at all as a control group. Supplementation of bFGF only in the single cell culture might be effective if bFGF is capable to exert direct effects on the commitment level, whereas bFGF also in the proliferation phase might support the preferential proliferation of a distinct cell subset which are prone to undergo adipogenic conversion.
Materials and Methods
Materials
If not otherwise stated, chemicals were obtained from Sigma (Steinheim, Germany). The B27 Supplement (in the following abbreviated as B27) was obtained as a 50-fold concentrate from Invitrogen (Karlsruhe, Germany). Basic FGF was obtained from PeproTec (Rocky Hill, NJ, USA). Insulin was kindly provided by Hoechst Marion Roussel (Frankfurt am Main, Germany). Cell culture plastics were purchased from Corning Costar (Bodenheim, Germany).
Evaluation of the cloning medium: Cell isolation and expansion in the proliferation phase Marrow stromal cells were obtained from six-week old male Sprague Dawley rats (weight:
170 - 180 g, Charles River, Sulzfeld, Germany). MSCs were flushed from the tibiae and femora according to the protocol of Ishaug [9]. Cells were centrifuged at 1,200 rpm for 5 min.
The resulting cell pellet was resuspended in basal medium consisting of DMEM (Biochrom, Berlin, Germany), 10 % fetal bovine serum (FBS, Gemini Bio-Products Inc., Calabasas, CA, USA), 1% penicillin/streptomycin (Invitrogen, Karlsruhe, Germany), and 50 µg/ml ascorbic acid (Fig. 1c). Cells were seeded in T75 flasks and cultured at 37°C and 5% CO2. Cells were allowed to adhere to the substratum for three days. The flasks were rinsed twice with phosphate-buffered saline (PBS, Invitrogen, Karlsruhe, Germany) in order to remove non-adherent cells. In the following experiments, cells were expanded (proliferation phase) with media based on either α-MEM, DMEM (Biochrom, Berlin, Germany), or RPMI 1640 (BioWhittaker Europe, Verviers, Belgium) respectively, supplemented with 10 % fetal bovine serum (Gemini Bio-Products Inc., Calabasas, CA, USA), 1% penicillin/streptomycin
Chapter 5 bFGF-influenced adipogenesis of MSCs under clonal conditions (Invitrogen, Karlsruhe, Germany), and 50 µg/ml ascorbic acid (Table 1). In some cases, B27 (50-fold concentrate) was additionally supplemented (Table 1). RPMI 1640 was generally supplemented with L-glutamine, MEM sodium pyruvate (C.C. Pro, Neustadt, Germany), MEM non-essential amino acids, and MEM vitamin solution according to the manufacturers’
instructions. After reaching confluence, cells were passaged with 0.25% trypsin and EDTA (Invitrogen, Karlsruhe, Germany) and resuspended in DMEM without any other additives at a density of 1.5 to 2 million cells/ml for cell sorting. Cell suspensions were stored on ice until the cell sorting was performed. In the following, the culture phase from day 3 after cell isolation to the time point of the passage is designated as proliferation phase (PP) (Fig. 1a).
Fig. 1a: Time scheme of the cell culture. For the evaluation of the cloning medium, cells were propagated in the proliferation phase, sorted following the passage, and expanded under clonal conditions using various media (Table 1). Subsequently, the number of grown clones was determined. The most favorable cloning medium (Fig. 1c) was used in all further experiments: For the mechanistic investigation of the effect of bFGF on the adipogenesis of MSCs, cells were propagated using the cloning medium during the proliferation phase, the attachment phase, and the cloning expansion. Adipogenic differentiation was induced with the induction medium and MSCs were differentiated five more days with the maintenance medium(Fig. 1c). After 39 days, the number of grown and differentiated clones was determined. (* The proliferation phase lasted seven days in the presence of the cloning medium, but required up to twelve days with other media depending on their composition.)
d0 d3 d10 d31 d39
Chapter 5 bFGF-influenced adipogenesis of MSCs under clonal conditions
-102- Fig.1b Basic FGF supplementation in different periods of the cell culture. MSCs were exposed to bFGF only in the single cell culture (C-F) or throughout the complete culture period, that is, in the proliferation phase and in the single cell culture (F-F). Cells grown in the absence of bFGF (C-C) served as a control. Grey boxes represent cloning medium without bFGF (B), black boxes represent cloning medium supplemented with bFGF (F).
Media Description Basal medium DMEM, 10% FBS, 1% PS, 50 µg/ml ascorbic acid
Cloning medium α-MEM, 10% FBS, 1% PS, 50 µg/ml ascorbic acid, B27
Induction medium
clonal conditions: cloning medium supplemented with the hormonal cocktail (10 nM dexamethasone, 0.5 mM IBMX, 60 µM indomethacin, 10 µg/ml insulin)
non-clonal conditions: basal medium supplemented with the hormonal cocktail
Maintenance medium
clonal conditions: cloning medium supplemented with 10 µg/ml insulin
non-clonal conditions: basal medium supplemented with 10 µg/ml insulin
Fig. 1c: Definition of important and frequently used terms concerning the composition of various media.
Evaluation of the cloning medium: Cell sorting and expansion under clonal conditions
Propidium iodide (PI, 1 µg/ml) was added to the cell suspensions in order to exclude dead cells from the sorting procedure. Cells were monitored in the FACS analysis (FACStar, Beckton Dickinson, Heidelberg, Germany) in two-parameter dot plots, forward scatter (FSC) against sideward scatter (SSC) in order to observe the cell population(s), and FSC against the fluorescence channel 3 (FL3) in order to exclude dead cells which were stained with PI. PI fluorescence was measured on the FL3 emission channel through a 670 nm longpass filter, following excitation with an argon ion laser source at 488 nm. The cells to be sorted were gated in the region 2 (R2). Single cells were sorted in one well each of a 96-well plate. 94