Stem cells (embryonic stem cells, somatic stem cells such as neural stem cells, and cardiac stem cells) and cancer cells are known to aggregate and form spheroid structures. This behavior is common in undifferentiated cells and may be necessary for adapting to certain conditions such as low-oxygen levels or to maintain undifferentiated status in microenvironments including stem cell niches. In order to decipher the meaning of this spheroid structure, we established a cardiosphere clone (CSC-21E) derived from the rat heart which can switch its morphology between spheroid and nonspheroid. Two forms, floating cardiospheres and dish-attached flat cells, could be switched reversibly by changing the cell culture condition. We performed differential proteome analysis studies and obtained protein profiles distinct between spherical forms and flat cells. From protein profiling analysis, we found upregulation of glycolytic enzymes in spheroids with some stress proteins switched in expression levels between these two forms. Evidence has been accumulating that certain chaperone/stress proteins are upregulated in concert with cellular changes including proliferation and differentiation. We would like to discuss the possible mechanism of how these aggregates affect cell differentiation and/or other cellular functions. 1. Introduction Two epoch accomplishments in the first decade of 21st century are changing the scope of biomedical research. The first was the completion of the human genome project , which enabled the onset of “Omics” or the integrative approach (System Biology) . The second was the discovery of adult stem cells in human  followed by induction of pluripotency by Yamanaka factors (Oct3/4, Sox, Klf4, and c-Myc) in both mouse and human somatic cells [4, 5]. Adult stem cells are undifferentiated cells found throughout the body after development. They have the ability to self-renew indefinitely and have the developmental potential to generate many other cell types due to cell fate switching induced by extracellular environmental signals . Plasticity of stem cells as well as the induction and reprogramming of somatic cells ignited the hope of discovering cellular therapy for the regeneration of damaged body parts. The revelation of the involvement of extracellular factors in switching cell types resulted in paradigm shift from “genetic determinism”, the paradigm that all biological processes follow the one-way instruction stored in genomes to an “environment-genome interaction” understanding. Studies on the regulatory molecular mechanisms
R. R. Smith, L. Barile, H. C. Cho et al., “Regenerative potential of cardiosphere-derived cells expanded from percutaneous endomyocardial biopsy specimens,” Circulation, vol. 115, no. 7, pp. 896–908, 2007.
S. Miyamoto, N. Kawaguchi, G. M. Ellison, R. Matsuoka, T. Shin'Oka, and H. Kurosawa, “Characterization of long-term cultured c-kit+ cardiac stem cells derived from adult rat hearts,” Stem Cells and Development, vol. 19, no. 1, pp. 105–116, 2010.
N. Kawaguchi, A. J. Smith, C. D. Waring et al., “ -4 high rat cardiac stem cells foster adult cardiomyocyte survival through IGF-1 paracrine signalling,” PLoS ONE, vol. 5, no. 12, Article ID e14297, 2010.
N. Kawaguchi, R. Nakao, M. Yamaguchi, D. Ogawa, and R. Matsuoka, “TGF-β superfamily regulates a switch that mediates differentiation either into adipocytes or myocytes in left atrium derived pluripotent cells (LA-PCS),” Biochemical and Biophysical Research Communications, vol. 396, no. 3, pp. 619–625, 2010.
M. K. Hasan, Y. Komoike, S.-I. Tsunesumi et al., “Myogenic differentiation in atrium-derived adult cardiac pluripotent cells and the transcriptional regulation of GATA4 and myogenin on ANP promoter,” Genes to Cells, vol. 15, no. 5, pp. 439–454, 2010.
H. Hosseinkhani, M. Hosseinkhani, S. Hattori, R. Matsuoka, and N. Kawaguchi, “Micro and nano-scale in vitro 3D culture system for cardiac stem cells,” Journal of Biomedical Materials Research A, vol. 94, no. 1, pp. 1–8, 2010.
M. Machida, Y. Takagaki, R. Matsuoka, and N. Kawaguchi, “Proteomic comparison of spherical aggregates and adherent cells of cardiac stem cells,” International Journal of Cardiology, vol. 153, no. 3, pp. 296–305, 2011.
H. Oh, S. B. Bradfute, T. D. Gallardo et al., “Cardiac progenitor cells from adult myocardium: Homing, differentiation, and fusion after infarction,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 21, pp. 12313–12318, 2003.
A. Linke, P. Müller, D. Nurzynska et al., “Stem cells in the dog heart are self-renewing, clonogenic, and multipotent and regenerate infarcted myocardium, improving cardiac function,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 25, pp. 8966–8971, 2005.
D. C. Andersen, P. Andersen, M. Schneider, H. B. Jensen, and S. P. Sheikh, “Murine “cardiospheres” are not a source of stem cells with cardiomyogenic potential,” Stem Cells, vol. 27, no. 7, pp. 1571–1581, 2009.
D. R. Davis, E. Kizana, J. Terrovitis et al., “Isolation and expansion of functionally-competent cardiac progenitor cells directly from heart biopsies,” Journal of Molecular and Cellular Cardiology, vol. 49, no. 2, pp. 312–321, 2010.
P. V. Johnston, T. Sasano, K. Mills et al., “Engraftment, differentiation, and functional benefits of autologous cardiosphere-derived cells in porcine ischemic cardiomyopathy,” Circulation, vol. 120, no. 12, pp. 1075–1083, 2009.
I. Chimenti, R. R. Smith, T.-S. Li et al., “Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice,” Circulation Research, vol. 106, no. 5, pp. 971–980, 2010.
K. Cheng, T.-S. Li, K. Malliaras, D. R. Davis, Y. Zhang, and E. Marbán, “Magnetic targeting enhances engraftment and functional benefit of iron-labeled cardiosphere-derived cells in myocardial infarction,” Circulation Research, vol. 106, no. 10, pp. 1570–1581, 2010.
K. Malliaras, T.-S. Li, D. Luthringer et al., “Safety and efficacy of allogeneic cell therapy in infarcted rats transplanted with mismatched cardiosphere-derived cells,” Circulation, vol. 125, no. 1, pp. 100–112, 2012.
S.-T. Lee, A. J. White, S. Matsushita et al., “Intramyocardial injection of autologous cardiospheres or cardiosphere-derived cells preserves function and minimizes adverse ventricular remodeling in pigs with heart failure post-myocardial infarction,” Journal of the American College of Cardiology, vol. 57, no. 4, pp. 455–465, 2011.
R. Lautam？ki, J. Terrovitis, M. Bonios et al., “Perfusion defect size predicts engraftment but not early retention of intra-myocardially injected cardiosphere-derived cells after acute myocardial infarction,” Basic Research in Cardiology, vol. 106, no. 6, pp. 1379–1386, 2011.
C. A. Carr, D. J. Stuckey, J. J. Tan et al., “Cardiosphere-derived cells improve function in the infarcted rat heart for at least 16 weeks—an mri study,” PLoS ONE, vol. 6, no. 10, Article ID e25669, 2011.
J. Ye, A. Boyle, H. Shih et al., “Sca-1+ cardiosphere-derived cells are enriched for isl1-expressing cardiac precursors and improve cardiac function after myocardial injury,” PLoS ONE, vol. 7, no. 1, Article ID e30329, 2012.
R. R. Makkar, R. R. Smith, K. Cheng et al., “Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial,” The Lancet, vol. 379, no. 9819, pp. 895–904, 2012.
D. Torella, C. Indolfi, D. F. Goldspink, and G. M. Ellison, “Cardiac stem cell-based myocardial regeneration: towards a translational approach,” Cardiovascular and Hematological Agents in Medicinal Chemistry, vol. 6, no. 1, pp. 53–59, 2008.
G. R. Martin and M. J. Evans, “Differentiation of clonal teratocarcinoma cells: formation of embryoid bodies in vitro,” Proceedings of the National Academy of Sciences of the United States of America, vol. 72, no. 4, pp. 1441–1445, 1975.
O. N. Suslov, V. G. Kukekov, T. N. Ignatova, and D. A. Steindler, “Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 22, pp. 14506–14511, 2002.
D. Khaitan, S. Chandna, M. B. Arya, and B. S. Dwarakanath, “Establishment and characterization of multicellular spheroids from a human glioma cell line; implications for tumor therapy,” Journal of Translational Medicine, vol. 4, article 12, 2006.
G. Francia, S. Man, B. Teicher, L. Grasso, and R. S. Kerbel, “Gene expression analysis of tumor spheroids reveals a role for suppressed DNA mismatch repair in multicellular resistance to alkylating agents,” Molecular and Cellular Biology, vol. 24, no. 15, pp. 6837–6849, 2004.
Y. L. Tang, W. Zhu, M. Cheng et al., “Hypoxic preconditioning enhances the benefit of cardiac progenitor cell therapy for treatment of myocardial infarction by inducing CXCR4 expression,” Circulation Research, vol. 104, no. 10, pp. 1209–1216, 2009.
D. Torella, G. M. Ellison, S. Méndez-Ferrer, B. Ibanez, and B. Nadal-Ginard, “Resident human cardiac stem cells: role in cardiac cellular homeostasis and potential for myocardial regeneration,” Nature Clinical Practice Cardiovascular Medicine, vol. 3, supplement 1, pp. S8–S13, 2006.
D. Torella, G. M. Ellison, I. Karakikes, and B. Nadal-Ginard, “Growth-factor-mediated cardiac stem cell activation in myocardial regeneration,” Nature Clinical Practice Cardiovascular Medicine, vol. 4, supplement 1, pp. S46–S51, 2007.
G. M. Ellison, D. Torella, I. Karakikes, and B. Nadal-Ginard, “Myocyte death and renewal: modern concepts of cardiac cellular homeostasis,” Nature Clinical Practice Cardiovascular Medicine, vol. 4, supplement 1, pp. S52–S59, 2007.
G. M. Ellison, D. Torella, I. Karakikes et al., “Acute β-adrenergic overload produces myocyte damage through calcium leakage from the ryanodine receptor 2 but spares cardiac stem cells,” Journal of Biological Chemistry, vol. 282, no. 15, pp. 11397–11409, 2007.
D. Torella, G. M. Ellison, I. Karakikes, and B. Nadal-Ginard, “Cardiovascular development: towards biomedical applicability—resident cardiac stem cells,” Cellular and Molecular Life Sciences, vol. 64, no. 6, pp. 661–673, 2007.
K. G. A. Rani, K. Jayakumar, G. Srinivas, R. R. Nair, and C. C. Kartha, “Isolation of ckit-positive cardiosphere-forming cells from human atrial biopsy,” Asian Cardiovascular and Thoracic Annals, vol. 16, no. 1, pp. 50–56, 2008.
Y. N. Tallini, S. G. Kai, M. Craven et al., “c-kit expression identifies cardiovascular precursors in the neonatal heart,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 6, pp. 1808–1813, 2009.
E. Prinsloo, M. M. Setati, V. M. Longshaw, and G. L. Blatch, “Chaperoning stem cells: a role for heat shock proteins in the modulation of stem cell self-renewal and differentiation?” BioEssays, vol. 31, no. 4, pp. 370–377, 2009.
G. Saretzki, L. Armstrong, A. Leake, M. Lako, and T. Von Zglinicki, “Stress defense in murine embryonic stem cells is superior to that of various differentiated murine cells,” Stem Cells, vol. 22, no. 6, pp. 962–971, 2004.
F. Q. Schafer and G. R. Buettner, “Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple,” Free Radical Biology and Medicine, vol. 30, no. 11, pp. 1191–1212, 2001.
J. D. Shao, H. Li, Y. Bian, and Y. Zhong, “Heat-shock protein 90α1 is required for organized myofibril assembly in skeletal muscles of zebrafish embryos,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 2, pp. 554–559, 2008.
F. Guilak, D. M. Cohen, B. T. Estes, J. M. Gimble, W. Liedtke, and C. S. Chen, “Control of Stem Cell Fate by Physical Interactions with the Extracellular Matrix,” Cell Stem Cell, vol. 5, no. 1, pp. 17–26, 2009.
B. A. Bryan, D. C. Mitchell, L. Zhao et al., “Modulation of muscle regeneration, myogenesis, and adipogenesis by the Rho family guanine nucleotide exchange factor GEFT,” Molecular and Cellular Biology, vol. 25, no. 24, pp. 11089–11101, 2005.
H. Kondoh, M. E. Lleonart, Y. Nakashima et al., “A high glycolytic flux supports the proliferative potential of murine embryonic stem cells,” Antioxidants and Redox Signaling, vol. 9, no. 3, pp. 293–299, 2007.
O. Toussaint, G. Weemaels, F. Debacq-Chainiaux, K. Scharffetter-Kochanek, and M. Wlaschek, “Artefactual effects of oxygen on cell culture models of cellular senescence and stem cell biology,” Journal of Cellular Physiology, vol. 226, no. 2, pp. 315–321, 2011.
T. Ezashi, P. Das, and R. M. Roberts, “Low O2 tensions and the prevention of differentiation of hES cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 13, pp. 4783–4788, 2005.
R. H. Wenger, “Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression,” FASEB Journal, vol. 16, no. 10, pp. 1151–1162, 2002.
H. C. Beck, J. Petersen, O. Felthaus, G. Schmalz, and C. Morsczeck, “Comparison of neurosphere-like cell clusters derived from dental follicle precursor cells and retinal Müller cells,” Neurochemical Research, vol. 36, no. 11, pp. 2002–2007, 2011.
H. R. Kumar, X. Zhong, D. J. Hoelz et al., “Three-dimensional neuroblastoma cell culture: proteomic analysis between monolayer and multicellular tumor spheroids,” Pediatric Surgery International, vol. 24, no. 11, pp. 1229–1234, 2008.
L. Gaedtke, L. Thoenes, C. Culmsee, B. Mayer, and E. Wagner, “Proteomic analysis reveals differences in protein expression in spheroid versus monolayer cultures of low-passage colon carcinoma cells,” Journal of Proteome Research, vol. 6, no. 11, pp. 4111–4118, 2007.
A. Fathi, M. Pakzad, A. Taei et al., “Comparative proteome and transcriptome analyses of embryonic stem cells during embryoid body-based differentiation,” Proteomics, vol. 9, no. 21, pp. 4859–4870, 2009.