Growth of bone marrow and skeletal muscle side population stem cells in suspension culture.

The ability to efficiently isolate and expand various stem cell populations in vitro is crucial for successful translation of cell-based therapies to the clinical setting. One such heterogeneous population that possesses a remarkable potential for the development of cell-based treatments for a variety of degenerative diseases and disorders is called the Side Population (SP). For many years, investigators have isolated these primitive cells based upon their ability to efflux the fluorophore Hoechst 33342. This attribute enabled separation of SP cells derived from multiple tissue sources from other endogenous cell populations using fluorescence-activated cell sorting (FACS). While all tissue-specific SP fractions appear to contain cells with multi-potent stem cell activity, the therapeutic utility of these cells has yet to be fully realized because of the scarcity of this fraction in vivo. In view of that, we developed a method to expand adult murine bone marrow and skeletal muscle-derived SP cells in vitro. Here, we describe a spinner-flask culture system that supports the growth of SP cells in suspension when they are combined with feeder cells cultured on spherical microcarriers. In this way, their distinguishing biological characteristics can be maintained, attachment-stimulated differentiation is avoided, and therapeutically relevant quantities of SP cells are generated. Modification of the described procedure may permit expansion of the SP from other relevant tissue sources and our method is amenable to establishing compliance with current good manufacturing practices.

[1]  V. Edgerton,et al.  Identification of myogenic-endothelial progenitor cells in the interstitial spaces of skeletal muscle , 2002, The Journal of cell biology.

[2]  Douglas B. Cowan,et al.  Microcarrier-Based Expansion of Adult Murine Side Population Stem Cells , 2013, PloS one.

[3]  M. Goodell,et al.  Hematopoietic stem cell characterization and isolation. , 2011, Methods in molecular biology.

[4]  S. McKinney-Freeman,et al.  Muscle-derived hematopoietic stem cells are hematopoietic in origin , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[5]  M. Sampaolesi,et al.  New therapies for muscular dystrophy: cautious optimism. , 2004, Trends in molecular medicine.

[6]  L. Kunkel,et al.  Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation parameters. , 2004, Experimental cell research.

[7]  R. Liao,et al.  Methods to study the proliferation and differentiation of cardiac side population (CSP) cells. , 2013, Methods in molecular biology.

[8]  M. Rudnicki,et al.  The post‐natal heart contains a myocardial stem cell population , 2002, FEBS letters.

[9]  G. Cossu.  Fusion of bone marrow-derived stem cells with striated muscle may not be sufficient to activate muscle genes. , 2004, The Journal of clinical investigation.

[10]  A. L. Wezel.  Growth of Cell-strains and Primary Cells on Micro-carriers in Homogeneous Culture , 1967, Nature.

[11]  S. McKinney-Freeman,et al.  Isolation and characterization of side population cells. , 2005, Methods in molecular biology.

[12]  H. Okano,et al.  Growth and differentiation potential of main- and side-population cells derived from murine skeletal muscle. , 2003, Experimental cell research.

[13]  M. Rudnicki,et al.  Myogenic specification of side population cells in skeletal muscle , 2002, The Journal of cell biology.

[14]  L. Kunkel,et al.  Systemic delivery of human microdystrophin to regenerating mouse dystrophic muscle by muscle progenitor cells. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[15]  S. Itescu,et al.  New directions in strategies using cell therapy for heart disease , 2003, Journal of Molecular Medicine.

[16]  S. McKinney-Freeman,et al.  Altered phenotype and reduced function of muscle-derived hematopoietic stem cells. , 2003, Experimental hematology.

[17]  M. Goodell,et al.  Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration. , 2003, The Journal of clinical investigation.

[18]  J. DiMaio,et al.  Cellular and Molecular Regulation of Skeletal Muscle Side Population Cells , 2004, Stem cells.

[19]  M. Goodell,et al.  Phenotype and hematopoietic potential of side population cells throughout embryonic development. , 2003, Blood.