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Colonization of collagen scaffolds by adipocytes derived from mesenchymal stem cells of the common marmoset monkey
Biochemical and Biophysical Research Communications 411 (2011) 317–322
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Colonization of collagen scaffolds by adipocytes derived from mesenchymal stem cells of the common marmoset monkey Inga Bernemann a,⇑, Thomas Mueller b, Rainer Blasczyk b, Birgit Glasmacher a, Nicola Hofmann a a b
Institute for Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany
a r t i c l e
i n f o
Article history: Received 3 June 2011 Available online 25 June 2011 Keywords: Common marmoset monkey MSCs Collagen scaffolds Non-human primate Adipogenic differentiation
a b s t r a c t In regenerative medicine, human cell replacement therapy offers great potential, especially by cell types differentiated from immunologically and ethically unproblematic mesenchymal stem cells (MSCs). In terms of an appropriate carrier material, collagen scaffolds with homogeneous pore size of 65 lm were optimal for cell seeding and cultivating. However, before clinical application and transplantation of MSCderived cells in scaffolds, the safety and efficiency, but also possible interference in differentiation due to the material must be preclinically tested. The common marmoset monkey (Callithrix jacchus) is a preferable non-human primate animal model for this aim due to its genetic and physiological similarities to the human. Marmoset bone marrow-derived MSCs were successfully isolated, cultured and differentiated in suspension into adipogenic, osteogenic and chondrogenic lineages by defined factors. The differentiation capability could be determined by FACS. Specific marker genes for all three cell types could be detected by RT-PCR. Furthermore, MSCs seeded on collagen I scaffolds differentiated in adipogenic lineage showed after 28 days of differentiation high cell viability and homogenous distribution on the material which was validated by calcein AM and EthD staining. As proof of adipogenic cells, the intracellular lipid vesicles in the cells were stained with Oil Red O. The generation of fat vacuoles was visibly extensive distinguishable and furthermore determined on the molecular level by expression of specific marker genes. The results of the study proved both the differential potential of marmoset MSCs in adipogenic, osteogenic and chondrogenic lineages and the suitability of collagen scaffolds as carrier material undisturbing differentiation of primate mesenchymal stem cells. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Regenerative medicine aims in the replacement of tissue due to loss or organ failure by implantation of an engineered biological substitute. This implant is either directly functional at the time of implantation or matures to the desired functional tissue type during or after the integration. One particular challenge in tissue engineering poses the combination of appropriate cells on a suitable biomaterial as a carrying structure. Besides the usage of stem cells or reprogrammed cells, the utilization of autologous cells, obtained relatively uninvasive from e.g. skin, bone marrow, blood or hair, is a feasible approach, due to the absence of allogenicity and immune rejection. Although autologous somatic cells can be isolated, cultured, and expanded in vitro in large numbers and seeded on diverse matrices [1] the outcome and survival of these cells re⇑ Corresponding author. Address: Institute for Multiphase Processes, Leibniz Universität Hannover, Callinstr. 36, D-30167 Hannover, Germany. Fax: +49 511 762 19389. E-mail address: [email protected] (I. Bernemann). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.06.134
mains critical [2]. Furthermore, the transplantation into a human patient remains impossible due to ethical reasons. Before direct clinical application of mesenchymal stem cells (MSCs) and cell seeded substitutes in cell and tissue replacement therapy, the safety and feasibility must be preclinically tested. This should be preferably carried out in a non-human primate animal model, since mouse stem cells have proven to display great differences from the human [2]. The common marmoset (Callithrix jacchus) is such a model readily used in biomedical research due to its genetic and physiological similarities to the human [3,4]. Over the last years there has been an increasing usage of marmoset as an alternative non-rodent species in preclinical safety evaluations on new pharmaceuticals [4–7] stem cell research [7–9] in, not only investigation of multiple sclerosis research [10,11] in infectious and autoimmune diseases [12,13] but also in reproductive medicine, transplantation research [14] and immunology [15–17]. C. jacchus (cj) and homo sapiens are thought to have diverged from a common ancestor, a primitive anthroid, which existed approximately 30 million years ago [18]. Despite many years of evolution separation, the human DNA
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sequences are still similar enough to anneal to marmoset metaphases, which simplifies a lot of different standard molecular biological applications. Therefore, and due to the small size and easy handling of the animals, the usage of marmoset cells in particular marmoset MSCs in reconstructive medicine applications become more and more attractive. The scaffolding material in cell-based tissue engineering decides the characteristics of the subsequent constructs. In this application, collagen is the most abundant protein in the human and animal body, and thus an excellent and potential biomaterial for scaffolds for various applications in regenerative medicine. In biological systems, it allows cell attachment, differentiation, organogenesis, tissue regeneration and repair. Collagen scaffolds are mechanically stable with a high tensile strength and can be altered into different sizes and shapes with various physical and chemical modifications [1,19,20]. Collagen scaffolds are successful matrices for adipose tissue engineering, because they are biocompatible, support tissue formation and demonstrate considerable stability to allow successful integration between the newly formed tissue and the surrounding host tissue [21]. The homogeneous pore structure of the used scaffolds [1,19,20] may be decisively responsible for the attachment, the distribution and the differentiation of the cells in three dimensions. Collagen type I scaffolds are also already well-accepted for human implantation [1]. With a porosity of 98% and excellent degradability and biocompatibility properties, this material is predestinated for functional human tissue engineering. Beside the matrix itself, the choice of an appropriate cell type for engineered biological substitute is quite important. Bone marrow-derived MSCs are the progenitors of multiple lineages, including bone, cartilage, muscle, and fat. They have manifold application and a high therapeutic potential. This combination of marmoset MSCs and collagen I scaffolds is the first step in clinical practice. The adipogenic differential potential of the cells in collagen matrix, especially behind the background of adipose tissue engineering, offers valuable clues to the applicability in human research. In plastic and reconstructive surgery, autologous fat grafts are utilized as filling material for the reconstruction of soft tissue defects [22,23]. The current standard of care for soft tissue reconstruction and augmentation includes the utilization of synthetic and tissue transfer [24–27]. The need for secondary surgical procedures for autologous tissue harvest and the average of 40–60% reduction loss in graft volume over time are considered drawbacks of current autologous fat transplantation procedures [24,28,29]. Hence, for adipose tissue engineering, the use of adult MSCs may be advantageous over the use of differentiated adipocytes
[18,30,31]. MSCs offer a potentially unlimited source of cells for tissue engineering application and the use of stem cells to produce adipose tissue has become more and more popular [32]. And, if the patient’s own cells are used, biocompatibility complications can be eliminated [33]. Regarding adipose tissue engineering, we characterized the differentiation capability of cjMSCs into adipogenic lineage engrafted into collagen scaffolds. To proof the functionality of cjMSC before seeding, we established multilineage differentiation of cjMSCs in suspension. We used isolated bone marrow-derived MSCs from the common marmoset monkey for the production of engineered adipose tissue replacement which will allow implantations in a non-human primate animal model in vivo at a later point in time. 2. Materials and methods All chemicals and reagents were obtained from Sigma–Aldrich GmbH (Munich, Germany) unless otherwise specifically indicated. 2.1. Collagen scaffolds Collagen scaffolds (Fig. 1A) were produced as described before [1,19,20,34] by directional solidification and subsequent freezedrying by power-down technology. This process generated collagen scaffolds with a homogeneous pore structure and defined pore sizes (Fig. 1B and C) [35]. Due to their biodegradable and biocompatible behaviour collagen scaffolds are suitable 3D matrices for a multitude of cell types in tissue engineering [1,34]. Collagen type I is also well-accepted for human implantation [1]. In this study we used collagen scaffolds with a height of 3 mm, a diameter of 15 mm, an average pore size of 65 lm and a porosity of 98%. 2.2. Animal experiments All experiments were in accordance the district government of Lower Saxonia, Hannover, and North-Rhine Westfaelia, Germany. Euthanasia of the animals and retrieval of the bone marrow was approved by the Institutional Animal Care. It should be highlighted that all animals were initially killed for other studies or as veterinary measure, but not primarily for our experiments. 2.3. Isolation of marmoset bone marrow stem cells The bone marrow of marmoset was isolated by rupture of the tibia and femur of each animal immediately after death was confirmed by a veterinarian. The cavity was flushed with a hypodermic needle attached to a syringe. For preventing coagulation and
Fig. 1. (A) Collagen scaffolds (bar = 3 mm). (B) The homogeneous and parallel pore structure of the collagen scaffolds allows optimized cell distribution, adhesion and proliferation in the 3D matrix, SEM (bar = 100 lm). (C) Close-up visualizing of the pore structure (bar = 50 lm).
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for cell singularization, a heparin PBS mix was utilized (5 IU/ml) and the bone marrow was separated by pipetting thoroughly for 3 min. After singularization, the cell suspension was transferred into red cell lysis buffer (NH4Cl 0.15 M, KHCO3 10 mM, EDTA 0.1 mM) for 5 min and centrifuged at 200g for 10 min. The cell pellet was resuspended in the MSC culture medium consisting of Dulbecco’s Modified Eagle Medium (DMEM) (Biochrom AG, Berlin, Germany), 15% FCS (Biochrom AG, Berlin, Germany), 1% penicillin/streptomycin (Invitrogen GmbH, Karlsruhe, Germany), 10 lmol nystatin, 50 lM L-ascorbin acid-2-phosphate and plated on a cell culture dish. 2.4. Differentiation potential of marmoset MSCs To demonstrate the general differentiation capability of marmoset MSCs (cjMSCs) into adipogenic, osteogenic and chondrogenic lineage different supplements have to add to the culture medium and the cells have been cultivated for several weeks. All cells were visualized using Axiovert 200 microscope. About 7.5 103 cells per cm2 were cultured for 3 weeks. Then adipogenic differentiation was achieved by the addition of 1 lM dexamethasone, 0.2 mM indomethacin, 0.5 mM IBMX and 10 lg/ ml insulin in DMEM with 10% FBS. After 3 days the medium was replaced by culture medium (DMEM, 10% FBS, insulin (20 lg/ml)) for 2 days. This cultivation circle was repeated for four times. Oil Red O dye was used to detect the generation of fat vacuoles. In order to do this, cells were washed with PBS and fixed for 20 min in 4% paraformaldehyde, then washed in H2O twice, followed by a short rinse in 50% ethanol. After incubation for 10 min in Oil Red O in acetone and 50% ethanol, removal of the staining dye and differentiation in 50% ethanol. The osteogenic differentiation was induced by the addition of osteogenic differentiation medium supplemented with 0.1 lM dexamethasone, 10 mM ß-glycerophosphate, 0.05 mM L-ascorbin acid-2-phosphate and 1% ITS (BD Biosciences, Heidelberg, Germany) in DMEM with 15% FBS. Von Kossa staining characterized the biological mineralization. The cells were washed in PBS and fixed with 10% phosphate-buffered paraformaldehyde, washed in PBS and distilled water. 1% silver nitrate solution was added. Then the dish was exposed to sunlight for 30 min, after which the plate was rinsed with water. Five percentage sodium thiosulfate was added for 5 min, and then the plates were rinsed in water. For the chondrogenic differentiation, 2 105 cjMSCs were pelleted by centrifugation at 500g for 5 min. The chondrogenic differentiation of the pellet micromass was induced by incubation for 3 weeks in chondrogenic differentiation medium with 0.1 lM dexamethasone, 1 mM sodium pyruvate, 0.17 mM L-ascorbin acid-2-phosphate, 0.35 mM L-proline, 10 ng/ml transforming growth factor-beta 3 (RELIATech, Braunschweig, Germany) and 1% ITS + Premix in DMEM with 15% FBS. Medium was changed every 2–3days. After 3 weeks, the pellet was fixed with 10% paraformaldehyde, sectioned at 7 lm and stained with alcian blue (Merck, Darmstadt, Germany) as an indicator of sulfated glycosaminoglycan [sGAG]-rich extracellular matrix and Hoechst 33342 (Invitrogen, Karlsruhe, Germany), according to standard protocols using cryosections. The detection of extracellular matrix production was done by using an aggrecan marker (Acris Antibodies, Herford, Germany). 2.5. Flow cytometric analysis The quantification of differentiation of cjMSCs was analysed in a FACSCalibur flow cytometer (BD Bio-Sciences, Heidelberg, Germany). Therefore, differentiated cells were suspended and singularized. After 14 days of adipogenic differentiation, the cells
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were fixated in 4% paraformaldehyde for 10 min. Then cells were resuspended in 150 ll Nile Red (1 lg/ml) and incubated for 60 min at RT. Partly the cells could not be pelletized. These cells were stained separately according to the protocol and then analysed. For analysis the osteogenic differentiated cells were stained on day 14 with calcein in (12.5 lg/ml) over night at 4 °C. Then the cells were trypsinized and analyzed. The quantification of chondrogensis was done after 3 weeks. Therefore, the cells of the pellet culture were singularized and suspended. The primary antibody Aggrecan (1:200) incubated for 2 h at 37 °C and Alexa Fluor 488 rabbit anti mouse IgG secondary antibody (1:400) at 37 °C for 1 h. After washing with PBS the cells were transferred into a FACS tube and measured. Undifferentiated MSCs were used in all experiments as control. 2.6. Adipogenic differentiation in collagen scaffolds The collagen scaffolds were seeded with 1 106 cells resolved in 400 ll culture medium. On day 1 after seeding, the culture medium (DMEM with 10% FBS) was complemented with adipogenic supplements. The control scaffolds were cultivated for 4 weeks in culture medium without differentiation supplements. The cell viability was determined using LIVE/DEAD Viabilty/Cytotoxicity Kit (Invitrogen, Karlsruhe, Germany). After 4 weeks of adipogenic differentiation, intracellular lipid vesicles were detected by Oil Red O staining. 2.7. Differentiation analysis by RT-PCR The analysis of the general differentiation capability of MSCs and their adipogenic differentiation in collagen scaffolds was also determined on a molecular level by RT-PCR. To analyze the expression of typical marker genes for the respective differentiated cell types, we selected for adipocytes PPAR-g, aP2 and CEBPa. For chondrocyte development ACAN, ColA1 and CYR6.1 expression was investigated. Osteoblast formation was displayed by TIMP2, MMP2 and RUNX2 expression. For quantification and control we utilized ß-actin and the ribosomal gene RPS29. For the RT-PCR we select the following primer: PPAR-g: 50 -CTGTGAAGTTCAATGCACTG-30 ; 50 -TGTTCCGTGACAA TCTGTCTG-30 aP2: 50 -GATGGCAATTGGAAGGTAGA-30 ; 50 -AGAACTGAATACTGT CAGAG-30 CEBPa: 50 -GTGGACAAGAACAGCAACGA-30 ; 50 -GTCATTGTCAC TGGTCAGCT-30 TIMP2: 50 -CAAGATGCACATCACCCTCT-30 ; 50 -TTCTTCTCTGTGAC CCAGTC-30 MMP2: 50 -GACACGCTGAAGAAGATGCA -30 ; 50 -CTGCCTCTCCATCAT GGATTC-30 RUNX2: 50 -AACTTCCTCTGCTCCGTGCT-30 ; 50 -TGCGGTAGCATT TCTCAGCT-30 ColA1: 50 -GAGACTACTGGATTGACCCC-30 ; 50 -CCAGGTTGTCATCTC CATAG-30 CYR6.1: 50 -CTGTGGAACTGGTATCTCCA-30 ; 50 -CTTCACACTCGAACATCCAG-30 ACAN: 50 -GTGTTCCATTACAGAGCCATTT-30 ; 50 -TCTCATTGGTGT CTCGGATG-30 ß-Actin: 50 -CATTGTCACCAACTGGGAC-30 ; 50 -CAGCCATGTATGTG GCCATC-30 RPS29: 50 -GCAAGATGGGTCACCAGCAG-30 ; 50 -CCAGTGTTTCCGTCAGTACG-30 RNA of 5 105 cells respectively was isolated with the RNeasy kit (Quiagen, Hilden, Germany) according to the manufacturer’s instructions and normalized with a Nanodrop ND-1000 (PeqLab, Erlangen, Germany). 0.5 lg RNA was transcribed with a RT-kit (Applied Biosystems, Forster City, USA). The PCR-reaction mix
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contained 30 ll volume with 1 ll cDNA, 0.3 lM of each primer (TIB Biomol, Berlin, Germany), 1U Taq DNA polymerase (NatuTec, Frankfurt, Germany) and 333 lM DNTP mix under standard PCR conditions (initial denaturation 5 min 94 °C; 30 cycles 1 min at 94 °C, annealing 1 min 62 °C, elongation 1 min 72 °C, final extension 15 min 72 °C). PCR products were separated by electrophoresis (1.5% agarose gel), and pictures were taken (Gel Doc Universal Hood II, BioRad, Munich, Germany).
3. Results 3.1. Analysis of the differentiation capability of cjMSCs The adipogenic medium-treated cjMSCs showed positive staining after 3 weeks of in vitro incubation (Fig. 2B). Flow cytometric analysis after 2 weeks showed from the content of fat vesicles, only 10.0 ± 6.3% of the pelletized cells positively
Fig. 2. Marmoset MSCs differentiated into adipogenic lineage (B) (Oil Red O; bar = 100 lm) and osteogenic lineage (A) (von Kossa, bar = 100 lm). Qualitative chondrogenic analysis in a high density MSC pellet culture by aggrecan, Hoechst 33342 staining (C) (bar = 200 lm), and alcian blue staining (D) (bar = 200 lm).
Fig. 3. Fluorescence live/dead images of adipogenic differentiated marmoset MSC seeded collagen scaffolds demonstrated cell viability and cell proliferation after 4 weeks. Calcein AM, EthD ((C) bar = 1 mm, (D) bar = 100 lm). Fat vacuoles are identifiable inside the green stained cytoplasm of the differentiated living cells and by Oil Red O staining ((A) bar = 5 mm, (B) bar = 100 lm).
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specific marker genes was measured (Fig.4A). The increase after 4 weeks is shown in Fig.4A. The results of the RT-PCR confirmed adipogenic differentiation capacity of cjMSCs in collagen scaffolds and verified the results from the Oil Red O staining (Fig.3A and B).
4. Discussion
Fig. 4. RT-PCR analyse: detection of typical marker gene for adipogenic differentiation of cells in suspension on d15 and into scaffolds on d32 (A) and also for osteogenic (B) and chondrogenic differentiation (C) confirm the differentiation potential of cjMSCs.
identified as adipocytes, whereas 85.5% were positive in the supernatant. Overall, 22.6 ± 4.7% of the cells could be successfully differentiated into adipocytes after 14 days. In general, the longer the period of differentiation, the greater the quantity of lipid a fat cell in culture can accumulate [36]. After 3 weeks of osteogenic differentiation, the generation of calcium deposits could distinct qualitative be proved by von Kossa staining (Fig. 2A). Flow cytometric analysis after 14 days showed that 11.34 ± 2.61% of the investigated cells positively identified as osteoblasts. The chondrogenesis was qualitative and quantitative proven by histological investigations (Fig. 2C and D) and flow cytometric analysis. The increase of aggregan expression after 21 days indicates the chondrogenic differentiation (Fig. 2C). Additionally images of alcian blue-stained cryosections verified qualitative chondrogenic differentiation potential of the cells (Fig. 2D) and proved the presence of sulfated glycosaminoglycans (sGAG). Flow cytometric analysis after 3 weeks showed that 9.46 ± 1.6% of the cells in the pellet culture positively identified as chondrocytes. Specific marker genes for all three cell types (adipocytes, osteocytes and chondrocytes) could be detected by RT-PCR (Fig.4). These experiments showed that isolated cjMSC can differentiate in a controlled manner to multiple lineages. 3.2. Analysis of the adipogenic differentiation potential of cjMSCs in collagen scaffolds The viability after 4 weeks adipogenic differentiation was evaluated and we observed a high cell proliferation and a high cell amount of viable cells and only a small quantity of dead cells (Fig. 3C and D). The cells adhered and proliferated three dimensionally into the scaffold. After 4 weeks there is a high density of evenly distributed cells in and on the collagen scaffold. Fig. 3C and D illustrates that the cells were grown multidimensional in the scaffold. After 4 weeks the generation of fat vacuoles in collagen scaffolds was detected (Fig. 3A and B). The histological investigation yielded that the cell distribution in collagen scaffold was dense and evenly distributed (Fig. 3A–D). Fat cells in all sections of the scaffold generate comprehensive intracellular lipid vesicles. The macroscopic and microscopic results proved the distinct adipogenic differentiation capability of cjMSCs in collagen scaffolds. Gene expression of adipogenic marker genes in differentiated MSCs in scaffolds was demonstrated by RT-PCR. On day 0 and after 4 weeks of adipogenic differentiation, the expression of this
In the last years there has been tremendous progress in the field of regenerative medicine and stem cell research [37]. Not even nearly all cell types could be as proof of principle generated, but also somatic cells reprogrammed into pluripotent lineages to overcome immunogenicity of allogenic embryonic stem cells [38]. However, tissue functions are dependent on the arrangement, connection, and interaction of their composed cells and matrix elements in 3D indicating that sole generation of certain cell types in vitro display limited information about cell efficiency and function in a transplant in vivo. To obtain more information about generated biological tissue substitutes in terms of cell behaviour, differentiation potential and survival in human bodies we utilized a non-human primate animal model to mimic their integration in human environment. Our animal model of the common marmoset fulfils a multitude of required conditions, like its direct translation to embryonic, autologous, and allogenic somatic stem cells and potentially also to induced pluripotent cells (iPS). In the past it has been demonstrated that collagen type I scaffolds prepared from porcine collagen suspension is a suitable carrier for stem cells and a compatible biodegradable material in the field of tissue engineering [1,19,20,34,35,39]. It has been demonstrated that various cell types can attach, differentiate and proliferate in collagen scaffolds to form a specific tissue or organ, however this was never performed in a non-human primate model with human or primate MSCs. Isolated marrow-derived mesenchymal stem cells of the common marmoset monkey displayed a surprisingly excellent differentiation capability in adipogenic, osteogenic and chondrogenic lineage using defined protocols. In this study we established the differentiation of the cjMSCs and confirmed the applicability of diverse functional and morphological assays for the marmoset species. Furthermore, cjMSCs integrated well in collagen type I scaffolds where they differentiated excellently into adipogenic cells. Hence, for adipose tissue engineering research this combination is particularly suitable. The existence of a large number of lipid vesicles and gene expression of several adipogenic markers could be demonstrated even after 4 weeks of cultivation, which is proof of function and cell survival. The study also demonstrated that cjMSCs could be seeded and evenly distributed in collagen scaffolds. Even after adipogenic differentiation, the cells in the scaffold showed high cell density and cell viability. In conclusion, in this study we have successfully established the cultivation and differentiation of MSCs of a non-human primate model into different somatic lineages, namely adipogenic, osteogenic and chondrogenic lineages. The generation of an adipose substitute by interaction of cjMSCs and collagen scaffolds could also be observed and characterized. The non-human primate MSCs attached and penetrated the collagen scaffolds surprisingly well and displayed an excellent adipogenic differentiation capability and survival. This study strongly suggests that the common marmoset monkey is a suitable model for soft tissue engineering in human regenerative medicine. In further studies our substitutes will be utilized in transplantation studies as a preclinical approach. Thereby, the perspective of clinical application of adipose tissue engineering developments into human regenerative medicine increases and opens manifold medical application possibilities.
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Acknowledgments We thank ResorbaÒ, Germany, for the provision of the collagen and the Centre for Reproductive Medicine and Andrology Muenster (CeRA) for donation of the marmoset bone marrow. Thanks to A. Deiwick and C. Marx from IMP, Leibniz Universität Hannover, K. Egler from the MH Hannover for their outstanding technical support and W. Hake for his excellent photographic work (Fig. 1A). This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German research Foundation) for the Cluster of Excellence REBIRTH (from Regenerative Biology to Reconstructive Therapy) (EXC 62/1). References [1] M. Kuberka, I. Heschel, B. Glasmacher, G. Rau, Preparation of collagen scaffolds and their applications in tissue engineering, Biomed. Tech. 47 (2002) 485–487. [2] T. Cantz, G. Key, M. Bleidißel, A. Brenne, H.R. Schöler, Absence of Oct4 expression in somatic tumor cell lines, Stem Cells 26 (2008) 692–697. [3] Nature Publishing Group, The common marmoset: Biomedical supermodel, Nature 459 (2009) 7246. [4] K. Mansfield, Marmoset models commonly used in biomedical research, Comp. Med. 53 (2003) 383–392. [5] D. Smith, P. Trennery, D. Farningham, J. Klapwijk, The selection of marmoset monkeys (Callithrix jacchus) in pharmaceutical toxicology, Lab. Anim. 35 (2001) 117–130. [6] U. Zühlke, G. Weinbauer, The common marmoset (Callithrix jacchus) as a model in toxicology, Toxicol. Pathol. 31 (2003) 23–127. [7] T. Mueller, G. Fleischmann, K. Eildermann, K. Maetz-Resing, P.A. Horn, E. Sasaki, R. Behr, A novel embryonic stem cell line derived from common marmoset monkey (Callithrix jacchus) exhibiting germ cell-like characteristics, Hum. Reprod. 24 (2009) 1359–1372. [8] T. Mueller, T. Hupfeld, J. Roessler, M. Simoni, J. Gromoll, R. Behr, Molecular cloning and functional characterization of endogenous recombinant common marmoset monkey (Callithrix jacchus) follicle stimulating hormone, J. Med. Primatol. (2010), doi:10.1111/j.1600-0684.2010.00453.x [Epub ahead of print]. [9] E. Sasaki, K. Hanazawa, R. Kurita, A. Akatsuka, T. Yoshizaki, H. Ishii, Y. Tanioka, Y. Ohnishi, H. Suemizu, A. Sugawara, N. Tamaoki, K. Izawa, Y. Nakazaki, H. Hamada, H. Suemori, S. Asano, N. Nakatsuji, H. Okano, K. Tani, Establishment of novel embryonic stem cell lines derived from the common marmoset (Callithrix jacchus), Stem Cells 23 (2005) 1304–1313. [10] B.A. t ‘Hart, L. Massacesi, Clinical, pathological, and immunologic aspects of the multiple sclerosis model in common marmosets (Callithrix jacchus), J. Neuropathol. Exp. Neurol. 68 (2009) 341–355. [11] B.A. t’Hart, M. van Meurs, H.P. Brok, L. Massacesi, J. Bauer, L. Boon, R.E. Bontrop, J.D. Laman, A new primate model for multiple sclerosis in the common marmoset, Immunol. Today 21 (2000) 290–297. [12] D.H. Crawford, G. Janossy, C.M. Hetherington, G.E. Francis, A.J. Edwards, A.V. Hoffbrand, H.G. Prentice, Immunological characterization of hemopoietic cells in the common marmoset, rhesus monkey, and man. In search of a model for human marrow transplantation, Transplantation 31 (1981) 245–250. [13] S. Potkay, Diseases of the Callitrichidae: a review, J. Med. Primatol. 21 (1992) 189–236. [14] C.M. Luetjens, J.B. Stukenborg, E. Nieschlag, M. Simoni, J. Wistuba, Complete spermatogenesis in orthotopic but not in ectopic transplants of autologously grafted marmoset testicular tissue, Endocrinology 149 (2008) 1736–1747. [15] A. Averdam, H. Kuhl, M. Sontag, T. Becker, A.L. Hughes, R. Reinhardt, L. Walter, Genomics and diversity of the common marmoset monkey NK complex, J. Immunol. 178 (2007) 7151–7161.
[16] M. Barros, V. Boere, J.P. Husto, C. Tomaz, Measuring fear and anxiety in the marmoset (Callithrix penicillata) with novel predator confrontation model: effects of diazepam, Behav. Brain Res. 108 (2000) 205–211. [17] L. Boon, H.P. Brok, J. Bauer, A. Ortiz-Buijsse, M.M. Schellekens, S. RamdienMurli, E. Blezer, M. van Meurs, J. Ceuppens, M. de Boer, B.A. t’Hart, J.D. Laman, Prevention of experimental autoimmune encephalomyelitis in the common marmoset (Callithrix jacchus) using a chimeric antagonist monoclonal antibody against human CD40 is associated with altered B-cell responses, J. Immunol. 167 (2001) 2942–2949. [18] J.K. Sherlock, D.K. Griffin, J.D. Delhanty, J.M. Parrington, Homologies between human and marmoset (Callithrix jacchus) chromosomes revealed by comparative chromosome painting, Physiol. Genomics 33 (2) (1996) 214–219. [19] H. Schoof, J. Apel, I. Heschel, G. Rau, Control of pore structure and size in freeze-dried collagen scaffolds, J. Biomed. Mater. Res. 58 (2002) 352–357. [20] H. Schoof, L. Bruns, A. Fischer, I. Heschel, G. Rau, Dendritic ice morphology in unidirectionally solidified collagen scaffolds, J. Cryst. Growth 209 (2000) 122– 129. [21] M.N. Rahaman, J.J. Mao, Stem cell based composite tissue constructs for regenerative medicine, Biotechnol. Bioeng. 91 (3) (2005) 261–284. [22] M.W. Strickberger, Evolution, third ed., Jones and Barclett Publishers, London, UK, 2000. [23] A.J. Katz, R. Llull, M.H. Hedrick, J.W. Futrell, Emerging approaches to the tissue engineering of fat, Clin. Plast. Surg. 26 (1999) 587–603. [24] A. Alhadlaq, M. Tang, J.J. Mao, Engineered adipose tissue from human mesenchymal stem cells maintains predefined shape and dimension: implications in soft tissue augmentation and reconstruction, Tissue Eng. 11 (3/4) (2005) 556–566. [25] A. Atala, Future perspectives in reconstructive surgery using tissue engineering, Urol. Clin. North Am. 26 (1999) 157–165. [26] K.J. Walgenbach, M. Voigt, A.W. Riabikhin, C. Andree, D.J. Schaefer, T.J. Galla, G. Bjorn, Tissue engineering in plastic reconstructive surgery, Anat. Rec. 263 (2001) 372. [27] E.S. Garfein, D.P. Orgill, J.J. Pribaz, Clinical applications of tissue engineered constructs, Clin. Plast. Surg. 30 (2003) 485. [28] C.W.J. Patrick, Adipose tissue engineering: the future of breast and soft tissue reconstruction following tumor resection, Semin. Surg. Oncol. 19 (2000) 302– 311. [29] S.R. Coleman, Long-term survival of fat transplants, controlled demonstrations, Aesth. Plast. Surg. 19 (1995) 421. [30] C.W. Patrick, Tissue engineering strategies for adipose tissue repair, Anat. Rec. 263 (2001) 361–366. [31] J. Smahel, Adipose tissue in plastic surgery, Ann. Plast. Surg. 16 (1986) 444– 453. [32] C.T. Gomillion, K.J.L. Burg, Stem cells and adipose tissue engineering, Biomaterials 27 (2006) 6052–6063. [33] F.M. Gregoire, C.M. Smas, H. S Sul, Understanding adipocyte differentiation, Physiol. Rev. 78 (3) (1998) 783–809. [34] D. von Heimburg, M. Kuberka, R. Rendchen, K. Hemmrich, G. Rau, N. Pallua, Preadipocyte-loaded collagen scaffolds with enlarged pore size for improved soft tissue engineering, Int. J. Artif. Organs 26 (2003) 1064–1076. [35] M. Kuberka, D. von Heimburg, H. Schoof, I. Heschel, G. Rau, Magnification of the pore size in biodegradable collagen sponges, Int. J. Artif. Organs 25 (2002) 67–73. [36] Y.-H. Lee, S.-Y. Chen, R.J. Wiesner, Y.-F. Huang, Simple flow cytometric method used to assess lipid accumulation in fat cells, J. Lipid Res. 45 (2004) 1162– 1167. [37] T. Vazin, W.J. Freed, Human embryonic stem cells: derivation, culture, and differentiation: a review, Restor. Neurol. Neurosci. 28 (2010) 589–603. [38] H. Zaehres, J.B. Kim, H.R. Schöler, Induced pluripotent stem cells, Methods Enzymol. 476 (2010) 309–325. [39] M. Kuberka, I. Bernemann, Y. Marquardt, G. Rau, B. Glasmacher, Influence of seeding methods on cellular growth in 3D collagen scaffolds for tissue engineering, Biomed. Tech. 49 (2) (2004) 634–635.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Colonization of collagen scaffolds by adipocytes derived from mesenchymal stem cells of the common marmoset monkey Inga Bernemann a,⇑, Thomas Mueller b, Rainer Blasczyk b, Birgit Glasmacher a, Nicola Hofmann a a b
Institute for Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany
a r t i c l e
i n f o
Article history: Received 3 June 2011 Available online 25 June 2011 Keywords: Common marmoset monkey MSCs Collagen scaffolds Non-human primate Adipogenic differentiation
a b s t r a c t In regenerative medicine, human cell replacement therapy offers great potential, especially by cell types differentiated from immunologically and ethically unproblematic mesenchymal stem cells (MSCs). In terms of an appropriate carrier material, collagen scaffolds with homogeneous pore size of 65 lm were optimal for cell seeding and cultivating. However, before clinical application and transplantation of MSCderived cells in scaffolds, the safety and efficiency, but also possible interference in differentiation due to the material must be preclinically tested. The common marmoset monkey (Callithrix jacchus) is a preferable non-human primate animal model for this aim due to its genetic and physiological similarities to the human. Marmoset bone marrow-derived MSCs were successfully isolated, cultured and differentiated in suspension into adipogenic, osteogenic and chondrogenic lineages by defined factors. The differentiation capability could be determined by FACS. Specific marker genes for all three cell types could be detected by RT-PCR. Furthermore, MSCs seeded on collagen I scaffolds differentiated in adipogenic lineage showed after 28 days of differentiation high cell viability and homogenous distribution on the material which was validated by calcein AM and EthD staining. As proof of adipogenic cells, the intracellular lipid vesicles in the cells were stained with Oil Red O. The generation of fat vacuoles was visibly extensive distinguishable and furthermore determined on the molecular level by expression of specific marker genes. The results of the study proved both the differential potential of marmoset MSCs in adipogenic, osteogenic and chondrogenic lineages and the suitability of collagen scaffolds as carrier material undisturbing differentiation of primate mesenchymal stem cells. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Regenerative medicine aims in the replacement of tissue due to loss or organ failure by implantation of an engineered biological substitute. This implant is either directly functional at the time of implantation or matures to the desired functional tissue type during or after the integration. One particular challenge in tissue engineering poses the combination of appropriate cells on a suitable biomaterial as a carrying structure. Besides the usage of stem cells or reprogrammed cells, the utilization of autologous cells, obtained relatively uninvasive from e.g. skin, bone marrow, blood or hair, is a feasible approach, due to the absence of allogenicity and immune rejection. Although autologous somatic cells can be isolated, cultured, and expanded in vitro in large numbers and seeded on diverse matrices [1] the outcome and survival of these cells re⇑ Corresponding author. Address: Institute for Multiphase Processes, Leibniz Universität Hannover, Callinstr. 36, D-30167 Hannover, Germany. Fax: +49 511 762 19389. E-mail address: [email protected] (I. Bernemann). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.06.134
mains critical [2]. Furthermore, the transplantation into a human patient remains impossible due to ethical reasons. Before direct clinical application of mesenchymal stem cells (MSCs) and cell seeded substitutes in cell and tissue replacement therapy, the safety and feasibility must be preclinically tested. This should be preferably carried out in a non-human primate animal model, since mouse stem cells have proven to display great differences from the human [2]. The common marmoset (Callithrix jacchus) is such a model readily used in biomedical research due to its genetic and physiological similarities to the human [3,4]. Over the last years there has been an increasing usage of marmoset as an alternative non-rodent species in preclinical safety evaluations on new pharmaceuticals [4–7] stem cell research [7–9] in, not only investigation of multiple sclerosis research [10,11] in infectious and autoimmune diseases [12,13] but also in reproductive medicine, transplantation research [14] and immunology [15–17]. C. jacchus (cj) and homo sapiens are thought to have diverged from a common ancestor, a primitive anthroid, which existed approximately 30 million years ago [18]. Despite many years of evolution separation, the human DNA
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sequences are still similar enough to anneal to marmoset metaphases, which simplifies a lot of different standard molecular biological applications. Therefore, and due to the small size and easy handling of the animals, the usage of marmoset cells in particular marmoset MSCs in reconstructive medicine applications become more and more attractive. The scaffolding material in cell-based tissue engineering decides the characteristics of the subsequent constructs. In this application, collagen is the most abundant protein in the human and animal body, and thus an excellent and potential biomaterial for scaffolds for various applications in regenerative medicine. In biological systems, it allows cell attachment, differentiation, organogenesis, tissue regeneration and repair. Collagen scaffolds are mechanically stable with a high tensile strength and can be altered into different sizes and shapes with various physical and chemical modifications [1,19,20]. Collagen scaffolds are successful matrices for adipose tissue engineering, because they are biocompatible, support tissue formation and demonstrate considerable stability to allow successful integration between the newly formed tissue and the surrounding host tissue [21]. The homogeneous pore structure of the used scaffolds [1,19,20] may be decisively responsible for the attachment, the distribution and the differentiation of the cells in three dimensions. Collagen type I scaffolds are also already well-accepted for human implantation [1]. With a porosity of 98% and excellent degradability and biocompatibility properties, this material is predestinated for functional human tissue engineering. Beside the matrix itself, the choice of an appropriate cell type for engineered biological substitute is quite important. Bone marrow-derived MSCs are the progenitors of multiple lineages, including bone, cartilage, muscle, and fat. They have manifold application and a high therapeutic potential. This combination of marmoset MSCs and collagen I scaffolds is the first step in clinical practice. The adipogenic differential potential of the cells in collagen matrix, especially behind the background of adipose tissue engineering, offers valuable clues to the applicability in human research. In plastic and reconstructive surgery, autologous fat grafts are utilized as filling material for the reconstruction of soft tissue defects [22,23]. The current standard of care for soft tissue reconstruction and augmentation includes the utilization of synthetic and tissue transfer [24–27]. The need for secondary surgical procedures for autologous tissue harvest and the average of 40–60% reduction loss in graft volume over time are considered drawbacks of current autologous fat transplantation procedures [24,28,29]. Hence, for adipose tissue engineering, the use of adult MSCs may be advantageous over the use of differentiated adipocytes
[18,30,31]. MSCs offer a potentially unlimited source of cells for tissue engineering application and the use of stem cells to produce adipose tissue has become more and more popular [32]. And, if the patient’s own cells are used, biocompatibility complications can be eliminated [33]. Regarding adipose tissue engineering, we characterized the differentiation capability of cjMSCs into adipogenic lineage engrafted into collagen scaffolds. To proof the functionality of cjMSC before seeding, we established multilineage differentiation of cjMSCs in suspension. We used isolated bone marrow-derived MSCs from the common marmoset monkey for the production of engineered adipose tissue replacement which will allow implantations in a non-human primate animal model in vivo at a later point in time. 2. Materials and methods All chemicals and reagents were obtained from Sigma–Aldrich GmbH (Munich, Germany) unless otherwise specifically indicated. 2.1. Collagen scaffolds Collagen scaffolds (Fig. 1A) were produced as described before [1,19,20,34] by directional solidification and subsequent freezedrying by power-down technology. This process generated collagen scaffolds with a homogeneous pore structure and defined pore sizes (Fig. 1B and C) [35]. Due to their biodegradable and biocompatible behaviour collagen scaffolds are suitable 3D matrices for a multitude of cell types in tissue engineering [1,34]. Collagen type I is also well-accepted for human implantation [1]. In this study we used collagen scaffolds with a height of 3 mm, a diameter of 15 mm, an average pore size of 65 lm and a porosity of 98%. 2.2. Animal experiments All experiments were in accordance the district government of Lower Saxonia, Hannover, and North-Rhine Westfaelia, Germany. Euthanasia of the animals and retrieval of the bone marrow was approved by the Institutional Animal Care. It should be highlighted that all animals were initially killed for other studies or as veterinary measure, but not primarily for our experiments. 2.3. Isolation of marmoset bone marrow stem cells The bone marrow of marmoset was isolated by rupture of the tibia and femur of each animal immediately after death was confirmed by a veterinarian. The cavity was flushed with a hypodermic needle attached to a syringe. For preventing coagulation and
Fig. 1. (A) Collagen scaffolds (bar = 3 mm). (B) The homogeneous and parallel pore structure of the collagen scaffolds allows optimized cell distribution, adhesion and proliferation in the 3D matrix, SEM (bar = 100 lm). (C) Close-up visualizing of the pore structure (bar = 50 lm).
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for cell singularization, a heparin PBS mix was utilized (5 IU/ml) and the bone marrow was separated by pipetting thoroughly for 3 min. After singularization, the cell suspension was transferred into red cell lysis buffer (NH4Cl 0.15 M, KHCO3 10 mM, EDTA 0.1 mM) for 5 min and centrifuged at 200g for 10 min. The cell pellet was resuspended in the MSC culture medium consisting of Dulbecco’s Modified Eagle Medium (DMEM) (Biochrom AG, Berlin, Germany), 15% FCS (Biochrom AG, Berlin, Germany), 1% penicillin/streptomycin (Invitrogen GmbH, Karlsruhe, Germany), 10 lmol nystatin, 50 lM L-ascorbin acid-2-phosphate and plated on a cell culture dish. 2.4. Differentiation potential of marmoset MSCs To demonstrate the general differentiation capability of marmoset MSCs (cjMSCs) into adipogenic, osteogenic and chondrogenic lineage different supplements have to add to the culture medium and the cells have been cultivated for several weeks. All cells were visualized using Axiovert 200 microscope. About 7.5 103 cells per cm2 were cultured for 3 weeks. Then adipogenic differentiation was achieved by the addition of 1 lM dexamethasone, 0.2 mM indomethacin, 0.5 mM IBMX and 10 lg/ ml insulin in DMEM with 10% FBS. After 3 days the medium was replaced by culture medium (DMEM, 10% FBS, insulin (20 lg/ml)) for 2 days. This cultivation circle was repeated for four times. Oil Red O dye was used to detect the generation of fat vacuoles. In order to do this, cells were washed with PBS and fixed for 20 min in 4% paraformaldehyde, then washed in H2O twice, followed by a short rinse in 50% ethanol. After incubation for 10 min in Oil Red O in acetone and 50% ethanol, removal of the staining dye and differentiation in 50% ethanol. The osteogenic differentiation was induced by the addition of osteogenic differentiation medium supplemented with 0.1 lM dexamethasone, 10 mM ß-glycerophosphate, 0.05 mM L-ascorbin acid-2-phosphate and 1% ITS (BD Biosciences, Heidelberg, Germany) in DMEM with 15% FBS. Von Kossa staining characterized the biological mineralization. The cells were washed in PBS and fixed with 10% phosphate-buffered paraformaldehyde, washed in PBS and distilled water. 1% silver nitrate solution was added. Then the dish was exposed to sunlight for 30 min, after which the plate was rinsed with water. Five percentage sodium thiosulfate was added for 5 min, and then the plates were rinsed in water. For the chondrogenic differentiation, 2 105 cjMSCs were pelleted by centrifugation at 500g for 5 min. The chondrogenic differentiation of the pellet micromass was induced by incubation for 3 weeks in chondrogenic differentiation medium with 0.1 lM dexamethasone, 1 mM sodium pyruvate, 0.17 mM L-ascorbin acid-2-phosphate, 0.35 mM L-proline, 10 ng/ml transforming growth factor-beta 3 (RELIATech, Braunschweig, Germany) and 1% ITS + Premix in DMEM with 15% FBS. Medium was changed every 2–3days. After 3 weeks, the pellet was fixed with 10% paraformaldehyde, sectioned at 7 lm and stained with alcian blue (Merck, Darmstadt, Germany) as an indicator of sulfated glycosaminoglycan [sGAG]-rich extracellular matrix and Hoechst 33342 (Invitrogen, Karlsruhe, Germany), according to standard protocols using cryosections. The detection of extracellular matrix production was done by using an aggrecan marker (Acris Antibodies, Herford, Germany). 2.5. Flow cytometric analysis The quantification of differentiation of cjMSCs was analysed in a FACSCalibur flow cytometer (BD Bio-Sciences, Heidelberg, Germany). Therefore, differentiated cells were suspended and singularized. After 14 days of adipogenic differentiation, the cells
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were fixated in 4% paraformaldehyde for 10 min. Then cells were resuspended in 150 ll Nile Red (1 lg/ml) and incubated for 60 min at RT. Partly the cells could not be pelletized. These cells were stained separately according to the protocol and then analysed. For analysis the osteogenic differentiated cells were stained on day 14 with calcein in (12.5 lg/ml) over night at 4 °C. Then the cells were trypsinized and analyzed. The quantification of chondrogensis was done after 3 weeks. Therefore, the cells of the pellet culture were singularized and suspended. The primary antibody Aggrecan (1:200) incubated for 2 h at 37 °C and Alexa Fluor 488 rabbit anti mouse IgG secondary antibody (1:400) at 37 °C for 1 h. After washing with PBS the cells were transferred into a FACS tube and measured. Undifferentiated MSCs were used in all experiments as control. 2.6. Adipogenic differentiation in collagen scaffolds The collagen scaffolds were seeded with 1 106 cells resolved in 400 ll culture medium. On day 1 after seeding, the culture medium (DMEM with 10% FBS) was complemented with adipogenic supplements. The control scaffolds were cultivated for 4 weeks in culture medium without differentiation supplements. The cell viability was determined using LIVE/DEAD Viabilty/Cytotoxicity Kit (Invitrogen, Karlsruhe, Germany). After 4 weeks of adipogenic differentiation, intracellular lipid vesicles were detected by Oil Red O staining. 2.7. Differentiation analysis by RT-PCR The analysis of the general differentiation capability of MSCs and their adipogenic differentiation in collagen scaffolds was also determined on a molecular level by RT-PCR. To analyze the expression of typical marker genes for the respective differentiated cell types, we selected for adipocytes PPAR-g, aP2 and CEBPa. For chondrocyte development ACAN, ColA1 and CYR6.1 expression was investigated. Osteoblast formation was displayed by TIMP2, MMP2 and RUNX2 expression. For quantification and control we utilized ß-actin and the ribosomal gene RPS29. For the RT-PCR we select the following primer: PPAR-g: 50 -CTGTGAAGTTCAATGCACTG-30 ; 50 -TGTTCCGTGACAA TCTGTCTG-30 aP2: 50 -GATGGCAATTGGAAGGTAGA-30 ; 50 -AGAACTGAATACTGT CAGAG-30 CEBPa: 50 -GTGGACAAGAACAGCAACGA-30 ; 50 -GTCATTGTCAC TGGTCAGCT-30 TIMP2: 50 -CAAGATGCACATCACCCTCT-30 ; 50 -TTCTTCTCTGTGAC CCAGTC-30 MMP2: 50 -GACACGCTGAAGAAGATGCA -30 ; 50 -CTGCCTCTCCATCAT GGATTC-30 RUNX2: 50 -AACTTCCTCTGCTCCGTGCT-30 ; 50 -TGCGGTAGCATT TCTCAGCT-30 ColA1: 50 -GAGACTACTGGATTGACCCC-30 ; 50 -CCAGGTTGTCATCTC CATAG-30 CYR6.1: 50 -CTGTGGAACTGGTATCTCCA-30 ; 50 -CTTCACACTCGAACATCCAG-30 ACAN: 50 -GTGTTCCATTACAGAGCCATTT-30 ; 50 -TCTCATTGGTGT CTCGGATG-30 ß-Actin: 50 -CATTGTCACCAACTGGGAC-30 ; 50 -CAGCCATGTATGTG GCCATC-30 RPS29: 50 -GCAAGATGGGTCACCAGCAG-30 ; 50 -CCAGTGTTTCCGTCAGTACG-30 RNA of 5 105 cells respectively was isolated with the RNeasy kit (Quiagen, Hilden, Germany) according to the manufacturer’s instructions and normalized with a Nanodrop ND-1000 (PeqLab, Erlangen, Germany). 0.5 lg RNA was transcribed with a RT-kit (Applied Biosystems, Forster City, USA). The PCR-reaction mix
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contained 30 ll volume with 1 ll cDNA, 0.3 lM of each primer (TIB Biomol, Berlin, Germany), 1U Taq DNA polymerase (NatuTec, Frankfurt, Germany) and 333 lM DNTP mix under standard PCR conditions (initial denaturation 5 min 94 °C; 30 cycles 1 min at 94 °C, annealing 1 min 62 °C, elongation 1 min 72 °C, final extension 15 min 72 °C). PCR products were separated by electrophoresis (1.5% agarose gel), and pictures were taken (Gel Doc Universal Hood II, BioRad, Munich, Germany).
3. Results 3.1. Analysis of the differentiation capability of cjMSCs The adipogenic medium-treated cjMSCs showed positive staining after 3 weeks of in vitro incubation (Fig. 2B). Flow cytometric analysis after 2 weeks showed from the content of fat vesicles, only 10.0 ± 6.3% of the pelletized cells positively
Fig. 2. Marmoset MSCs differentiated into adipogenic lineage (B) (Oil Red O; bar = 100 lm) and osteogenic lineage (A) (von Kossa, bar = 100 lm). Qualitative chondrogenic analysis in a high density MSC pellet culture by aggrecan, Hoechst 33342 staining (C) (bar = 200 lm), and alcian blue staining (D) (bar = 200 lm).
Fig. 3. Fluorescence live/dead images of adipogenic differentiated marmoset MSC seeded collagen scaffolds demonstrated cell viability and cell proliferation after 4 weeks. Calcein AM, EthD ((C) bar = 1 mm, (D) bar = 100 lm). Fat vacuoles are identifiable inside the green stained cytoplasm of the differentiated living cells and by Oil Red O staining ((A) bar = 5 mm, (B) bar = 100 lm).
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specific marker genes was measured (Fig.4A). The increase after 4 weeks is shown in Fig.4A. The results of the RT-PCR confirmed adipogenic differentiation capacity of cjMSCs in collagen scaffolds and verified the results from the Oil Red O staining (Fig.3A and B).
4. Discussion
Fig. 4. RT-PCR analyse: detection of typical marker gene for adipogenic differentiation of cells in suspension on d15 and into scaffolds on d32 (A) and also for osteogenic (B) and chondrogenic differentiation (C) confirm the differentiation potential of cjMSCs.
identified as adipocytes, whereas 85.5% were positive in the supernatant. Overall, 22.6 ± 4.7% of the cells could be successfully differentiated into adipocytes after 14 days. In general, the longer the period of differentiation, the greater the quantity of lipid a fat cell in culture can accumulate [36]. After 3 weeks of osteogenic differentiation, the generation of calcium deposits could distinct qualitative be proved by von Kossa staining (Fig. 2A). Flow cytometric analysis after 14 days showed that 11.34 ± 2.61% of the investigated cells positively identified as osteoblasts. The chondrogenesis was qualitative and quantitative proven by histological investigations (Fig. 2C and D) and flow cytometric analysis. The increase of aggregan expression after 21 days indicates the chondrogenic differentiation (Fig. 2C). Additionally images of alcian blue-stained cryosections verified qualitative chondrogenic differentiation potential of the cells (Fig. 2D) and proved the presence of sulfated glycosaminoglycans (sGAG). Flow cytometric analysis after 3 weeks showed that 9.46 ± 1.6% of the cells in the pellet culture positively identified as chondrocytes. Specific marker genes for all three cell types (adipocytes, osteocytes and chondrocytes) could be detected by RT-PCR (Fig.4). These experiments showed that isolated cjMSC can differentiate in a controlled manner to multiple lineages. 3.2. Analysis of the adipogenic differentiation potential of cjMSCs in collagen scaffolds The viability after 4 weeks adipogenic differentiation was evaluated and we observed a high cell proliferation and a high cell amount of viable cells and only a small quantity of dead cells (Fig. 3C and D). The cells adhered and proliferated three dimensionally into the scaffold. After 4 weeks there is a high density of evenly distributed cells in and on the collagen scaffold. Fig. 3C and D illustrates that the cells were grown multidimensional in the scaffold. After 4 weeks the generation of fat vacuoles in collagen scaffolds was detected (Fig. 3A and B). The histological investigation yielded that the cell distribution in collagen scaffold was dense and evenly distributed (Fig. 3A–D). Fat cells in all sections of the scaffold generate comprehensive intracellular lipid vesicles. The macroscopic and microscopic results proved the distinct adipogenic differentiation capability of cjMSCs in collagen scaffolds. Gene expression of adipogenic marker genes in differentiated MSCs in scaffolds was demonstrated by RT-PCR. On day 0 and after 4 weeks of adipogenic differentiation, the expression of this
In the last years there has been tremendous progress in the field of regenerative medicine and stem cell research [37]. Not even nearly all cell types could be as proof of principle generated, but also somatic cells reprogrammed into pluripotent lineages to overcome immunogenicity of allogenic embryonic stem cells [38]. However, tissue functions are dependent on the arrangement, connection, and interaction of their composed cells and matrix elements in 3D indicating that sole generation of certain cell types in vitro display limited information about cell efficiency and function in a transplant in vivo. To obtain more information about generated biological tissue substitutes in terms of cell behaviour, differentiation potential and survival in human bodies we utilized a non-human primate animal model to mimic their integration in human environment. Our animal model of the common marmoset fulfils a multitude of required conditions, like its direct translation to embryonic, autologous, and allogenic somatic stem cells and potentially also to induced pluripotent cells (iPS). In the past it has been demonstrated that collagen type I scaffolds prepared from porcine collagen suspension is a suitable carrier for stem cells and a compatible biodegradable material in the field of tissue engineering [1,19,20,34,35,39]. It has been demonstrated that various cell types can attach, differentiate and proliferate in collagen scaffolds to form a specific tissue or organ, however this was never performed in a non-human primate model with human or primate MSCs. Isolated marrow-derived mesenchymal stem cells of the common marmoset monkey displayed a surprisingly excellent differentiation capability in adipogenic, osteogenic and chondrogenic lineage using defined protocols. In this study we established the differentiation of the cjMSCs and confirmed the applicability of diverse functional and morphological assays for the marmoset species. Furthermore, cjMSCs integrated well in collagen type I scaffolds where they differentiated excellently into adipogenic cells. Hence, for adipose tissue engineering research this combination is particularly suitable. The existence of a large number of lipid vesicles and gene expression of several adipogenic markers could be demonstrated even after 4 weeks of cultivation, which is proof of function and cell survival. The study also demonstrated that cjMSCs could be seeded and evenly distributed in collagen scaffolds. Even after adipogenic differentiation, the cells in the scaffold showed high cell density and cell viability. In conclusion, in this study we have successfully established the cultivation and differentiation of MSCs of a non-human primate model into different somatic lineages, namely adipogenic, osteogenic and chondrogenic lineages. The generation of an adipose substitute by interaction of cjMSCs and collagen scaffolds could also be observed and characterized. The non-human primate MSCs attached and penetrated the collagen scaffolds surprisingly well and displayed an excellent adipogenic differentiation capability and survival. This study strongly suggests that the common marmoset monkey is a suitable model for soft tissue engineering in human regenerative medicine. In further studies our substitutes will be utilized in transplantation studies as a preclinical approach. Thereby, the perspective of clinical application of adipose tissue engineering developments into human regenerative medicine increases and opens manifold medical application possibilities.
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