title: Microcarrier-based expansion of adult murine side population stem cells.
abstract: The lack of reliable methods to efficiently isolate and propagate stem cell populations is a significant obstacle to the advancement of cell-based therapies for human diseases. One isolation technique is based on efflux of the fluorophore Hoechst 33342. Using fluorescence-activated cell sorting (FACS), a sub-population containing adult stem cells has been identified in a multitude of tissues in every mammalian species examined. These rare cells are referred to as the 'side population' or SP due to a distinctive FACS profile that results from weak staining by Hoechst dye. Although the SP contains multi-potent cells capable of differentiating toward hematopoietic and mesenchymal lineages; there is currently no method to efficiently expand them. Here, we describe a spinner-flask culture system containing C2C12 myoblasts attached to spherical microcarriers that act to support the growth of non-adherent, post-natal murine skeletal muscle and bone marrow SP cells. Using FACS and hemocytometry, we show expansion of unfractionated EGFP⁺ SP cells over 6 wks. A significant number of these cells retain characteristics of freshly-isolated, unfractionated SP cells with respect to protein expression and dye efflux capacity. Expansion of the SP will permit further study of these heterogeneous cells and determine their therapeutic potential for regenerative and reparative therapies.
authors: Department of Anesthesiology, Perioperative and Pain Medicine, Boston Children's Hospital, Boston, Massachusetts, United States of America.
authors: Christina A
authors: CA
authors: Pacak
authors: Mau-Thek
authors: MT
authors: Eddy
authors: Lindsey
authors: L
authors: Woodhull
authors: Kai-Roy
authors: KR
authors: Wang
authors: Ivan
authors: I
authors: Alpatov
authors: Shelby
authors: S
authors: Fullen
authors: Rory P
authors: RP
authors: Dowd
authors: Yeong-Hoon
authors: YH
authors: Choi
authors: Douglas B
authors: DB
authors: Cowan
mesh_terms: D000818:Animals; D001562:Benzimidazoles; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D032446:Myoblasts / Q000502:physiology; D058985:Side-Population Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D001562:Benzimidazoles; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C017807:bisbenzimide ethoxide trihydrochloride
references: Circ Res. 2012 May 11;110(10):1355-63
references: 22581921
references: Nat Med. 2001 Sep;7(9):1028-34
references: 11533706
references: J Cell Biol. 2011 Oct 3;195(1):147-63
references: 21949413
references: Nat Med. 1997 Dec;3(12):1337-45
references: 9396603
references: Nature. 1981 Jul 9;292(5819):154-6
references: 7242681
references: Methods Mol Biol. 2005;290:343-52
references: 15361673
references: J Cell Biol. 2002 Oct 14;159(1):123-34
references: 12379804
references: Proc Natl Acad Sci U S A. 2004 Sep 28;101(39):14228-33
references: 15381773
references: Exp Cell Res. 2005 Feb 15;303(2):360-74
references: 15652349
references: Nature. 1967 Oct 7;216(5110):64-5
references: 4292963
references: J Cell Sci. 2008 May 1;121(Pt 9):1426-34
references: 18397996
references: Cell Stem Cell. 2009 Mar 6;4(3):217-25
references: 19265661
references: Exp Hematol. 2002 Nov;30(11):1339-45
references: 12423688
references: J Clin Invest. 2004 Dec;114(11):1577-85
references: 15578090
references: J Histochem Cytochem. 2012 Jan;60(1):31-44
references: 22205679
references: Stem Cells. 2004;22(7):1305-20
references: 15579648
references: Biochem Biophys Res Commun. 1998 Jul 9;248(1):134-9
references: 9675099
references: J Exp Med. 1996 Apr 1;183(4):1797-806
references: 8666936
references: Exp Cell Res. 2004 Aug 1;298(1):144-54
references: 15242769
references: Nature. 1999 Sep 23;401(6751):390-4
references: 10517639