Abstract
Mesenchymal stem cells from a variety of sites are a natural resource that using appropriate skills can be cultured in the laboratory, in scaffolds, to provide differentiated-cell replacement tissues, for clinical application. To perform such work with human cells, strict ethical integrity must be observed at all stages. Adipocytes, osteocytes and chrondrocytes are amongst the most desirable end-point cells. Hydrolytic degradable scaffolds allow implanted cells to synthesise their own extracellular matrix in situ after implantation, degeneration of the foreign scaffold to temporally match creation of the new innate one. For preliminary in vitro stem cell differentiation protocols, initial investigation is commonly performed with stem cells in commercially available porous collagen sponges or cell-free small intestinal submucosa. Differentiation of stem cells to a specific phenotype is achieved by culturing them in apposite culture media under precise conditions. Once the cells have differentiated, they are checked and characterised in a wide variety of systems. This chapter describes differentiation media for adipocytes, osteocytes, chondrocytes, myocytes and neural precursors and methods of observing their characteristics by microscopy using phase contrast microscopy, standard light microscopy and electron microscopy with tinctorial, immunocytochemical and electron dense stains, respectively. Cell sorting techniques are not dealt with here. Immunocytochemistry/microscopy staining for specific differentiated-cell antigens is an invaluable procedure, and the range of commercially available antibodies is wide. Precautions need to be considered for using actively proliferating cells in vivo, so that implanted cells remain controlled by the body’s molecular signals and avoid development of malignancy.
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References
Sarraf, C.E., Harris, A.B., McCulloch, A.D. and Eastwood, M. (2002) Tissue engineering of biological cardiovascular system surrogates. Heart Lung Circ. 11, 142–150.
Du, M., Yin, J. and, Zhu, M.J. (2010) Cellular signaling pathways regulating the initial stage of adipogenesis and marbling of skeletal muscle. Meat Sci. 86, 103–109.
Owen, M.E., Cavé, J. and Joiner, C.J. (1987) Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F. J Cell Sci. 87, 731–738.
Gazit, D., Karmish, M., Holzman, L. and Bab, I. (1990) Regenerating marrow induces systemic increase in osteo- and chondrogenesis. Endocrinology 126, 2607–2613.
Hoerstrup, S.P., Sodian, R., Daebritz, S., Wang, J., Bacha, E.A., Martin, D.P. et al. (2000) Functional living tri-leaflet heart valves grown in vitro. Circulation 102 (3), 11144–11149.
Schmidt, D., Dijkman, P.E., Driessen-Mol, A., Stenger, R., Mariani, C., Puolakka, A. et al (2010) Minimally-invasive implantation of living tissue engineered heart valves: a comprehensive approach from autologous vascular cells to stem cells. J. Am. Coll. Cardiol. 56, 510–520.
Sahoo, S., Lok Toh, S. and Hong Goh, J.C. (2010) PLGA nanofiber-coated silk microfibrous scaffold for connective tissue engineering. J. Biomed. Mater. Res. B Appl. Biomater. 95(1), 19–28.
Babaeijandaghi, F., Shabani, I., Seyedjafari, E., Safaei Naraghi, Z., Vasei, M. and Haddadi-Asi, V. et al. (2010) Accelerated epidermial regerneration and improved dermal reconstruction achieved by polyethersulfone nanofibres. Tissue Engineering Part A. 16(11), 3527–3536.
Matheny, R.G., Hutchison, M.L., Dryden, P.E., Hiles, M.D., Shaar, C.J. (2000) Porcine small intestine submucosa as a pulmonary valve leaflet substitute. J. Heart Valve Dis. 9, 769–774.
Bader, A. and Macchiarini, P. (2010) Moving towards in situ tracheal regeneration: the bionic tissue engineered transplantation approach. J. Cell Mol. Med. 14(7), 1877–1889.
Lin, N., Lin, J., Liu, B., Weidong, P., Chen, S., Xu, R. (2010) Differentiation of bone marrow-derived mesenchymal stem cells into hepatocye-like cells in an alginate scaffold. Cell Proliferation 43(5), 427–434.
Liu, T., Teng, W.K., Chan, B.P., Chew, S.Y. (2010)Photochemical crosslinked electrospun collagen nanofibers: synthesis, characterisation and neural stem cell interactions. J. Biomed. Mater. Res A. 95(1), 276–282.
Bianco, A., Del Gaudio, C., Baiguera, S., Armentano, I, Bertarelli C., and Dottori, M. (2010) Microstructure and cytocompatability of electrospun nanocomposites based on poly (epsilon-caprolactone) and carbon nanostructures. Int. J. Artif. Organs 33, 271–282.
Sarraf, C.E., Harris, A.B., McCulloch, A.D. and Eastwood M (2002) Tissue engineering of cardiovascular system surrogates. Heart Lung Circ. 11, 142–150.
Sun, L-Y., Hsieh, D-K., Syu, W-S., Li, Y-S., Chiu, H-T., Chiou, T-W. (2010) Cell proliferation of human bone marrow mesenchymal stem cells on biodegradable microcarriers enhances in vitro differentiation potential. Cell Proliferation 43(5), 445–456.
Smith, A.G., Heath, J.K., Donaldson, D.D., Wong, G.G. Moreau, J. et al. (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 15, 688–690.
Dick, E., Rajamohan, D., Ronksley, J. and Denning, C. (2010) Evaluating the utility of cardiomyocytes from human pluripotent stem cells for drug screening. Biochem. Soc. Trans. 38, 1037–1045.
Carrasco de Paula, I. (2008) General principles regarding the use of adult stem cells. Cell Prolif. 41(1), 78–84.
Gluckman, E. (2000) Ethical and legal aspects of placental/cord blood banking and transplant. Eurocord Netcord Organisation. Hematol. J. 1, 67–69.
Friedenstein, A. J., Gorskaja, J.F. and Kulagina, N.N. (1976) Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp. Hematol. 4, 267–274.
von Drygalski, A. and Adamson, J.W. (2000) Placental/umbilical cord blood (PCB) stem cells for transplantation: early clinical outcomes and the status of ex vivo expansion strategies. Keio J. Med. 49, 141–151.
Sasayama, N., Kashiwakura, I., Tokushima, Y., Wada, S., Murakami, M. Hayase, Y. et al. (2001) Expansion of megakaryocyte progenitors from cryopreserved leukocyte concentrates of human placental and umbilical cord blood in short-term liquid culture. Cytotherapy 3, 117–126.
Mizuno, H. and Hyakusoku, H. (2001). Fat grafting to the breast and adipose-derived stem cells: recent scientific consensus and controversy. Aesthet. Surg. J. 30, 387–389.
Thalmeier, K., Meissner, P., Moosmann, S., Sagebiel, S., Wiest, I., Huss, R. (2001) Mesenchymal differentiation and organ distribution of established human stromal cell lines in NOD/SCID mice. Acta Haematol.105(3), 159–165.
Quirici, N., Scavullo, C., de Girolamo, L., Lopa, S., Arrigoni, E., Deliliers, G.L., Brini, A.T. (2010) Anti-L-NGFR and -CD34 monoclonal antibodies identify multipotent mesenchymal stem cells in human adipose tissue. Stem Cells Dev. 19, 915–925.
Williams, R.L., Hilton, D.J., Pease, S., Willson, T.A., Stewart, C.L., Gearing, D.P., Wagner, E.F., Metcalf, D., Nicola, N.A., Gough, N.M. (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature. 336(6200), 684–687.
Lennon, D.P., Haynesworth, S.E., Young, R.G., Dennis, J.E., Caplan, A.I. (1995) A chemically defined medium supports in vitro proliferation and maintains the osteochondral potential of rat marrow-derived mesenchymal stem cells. Exp. Cell Res. 219, 211–222.
Otto, W.R., Rao, J. (2004)Tomorrow’s skeleton staff: mesenchymal stem cells and the repair of bone and cartilage. Cell Prolif. 37, 97–110.
Jung, S., Sen, A., Rosenberg, L., Behiel, L.A. (2010) Identification of growth and attachment factors for the serum-free isolation and expansion of human mesenchymal stromal cells. Cytotherapy 12, 637–657.
Lindner, U., Kramer, J., Behrends, J., Driller, B., Wendler, N.-O., Boehrnsen, F., Rohwedel, J., Schlenke, P. (2010) Improved proliferation and differentiation capacity of human mesenchymal stromal cells cultured with basement-membrane extracellular matrix proteins. Cytotherapy 12(8), 992–1005.
Xu, W., Zhang, X., Qian, H., Zhu, W., Sun, X., Hu, J. et al. (2004) Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro. Exp. Biol. Med. 229, 623–631.
Takahashi, T., Lord, B., Schulze, P.C., Fryer, R.M., Sarang, S.S., Gullans, S.R., Lee, R.T. (2003) Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes. Circulation 107, 1912–1916.
Sittichokechaiwut, A., Edwards, J.H., Scutt, A.M., Reilly, G.C. (2010) Short bouts of mechanical loading are as effective as dexamethasone at inducing matrix production by human bone marrow mesenchymal stem cell. Eur. Cell Mater. 20, 45–57.
Anand, U., Otto, W.R., Casula, M.A., Day, N.C., Davis, J.B., Bountra, C., Birch, R., Anand, P. (2006) The effect of neurotrophic factors on morphology, TRPV1 expression and capsaicin responses of cultured human DRG sensory neurons. Neurosci. Lett. 399, 51–56.
Lunn, G., Lawler, G. (2003) Unit 2A: Laboratory Safety, in Current Protocols in Protein Science, John Wiley & Sons, Chichester, UK. DOI: 10.1002/0471140864.psa02as28.
Additional Reading
IHCWorld: http://www.ihcworld.com/_protocols/em/em_routine_tissue.htm Routine transmission electron microscopy staining protocol for tissues: Reynold’s Lead Citrate Solution. Visited 20.9.10.
Page KM (1982) Bone and the preparation of bone sections, in Bancroft, JD and Stevens A (Eds) Theory and practice of histological techniques. Churchill Livingstone, Edinburgh, pp 297–331.
Acknowledgements
The authors would like to thank George Elia (CRUK, London) for exemplary histochemical expertise, Dr Uma Anand (Imperial College, London) for advice on neuronal cell culture, Professor Ketan Patel and Dr Anthony Otto (Reading University) for muscle cell growth conditions.
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Otto, W.R., Sarraf, C.E. (2012). Culturing and Differentiating Human Mesenchymal Stem Cells for Biocompatible Scaffolds in Regenerative Medicine. In: Mitry, R., Hughes, R. (eds) Human Cell Culture Protocols. Methods in Molecular Biology, vol 806. Humana Press. https://doi.org/10.1007/978-1-61779-367-7_27
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DOI: https://doi.org/10.1007/978-1-61779-367-7_27
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