A method for clonal analysis of epidermal growth factor-responsive neural progenitors.
Epidermal growth factor (EGF) responsive neural progenitors are defined by clonal growth from single cells. In previous studies we were unable to obtain clones at single cell densities using trypsinized cells and trituration alone always gave cellular aggregates. Here we report on single cell derived clones using a technique involving trituration of EGF responsive neurospheres, cell filtration, and single cell sorting using a MoFlo high speed fluorescence activated cell sorter. Single cell deposition was confirmed by labeling cells with Hoechst 33342 and Flow-check Fluorospheres, and visualization by fluorescence microscopy. The cells were deposited into liquid medium and grown from single cells in 10-20 ng/ml EGF for 12-14 days. This gave a cloning efficiency of 2.12%+/-0.37. New colonies occurred as late as day 18 post-sort. Tritiated thymidine suicide indicates that a percentage of these cells are cycling. Immunohistochemical analysis for oligodendrocytes, astroglia, and neuronal lineages performed on colonies at 10-14 and 21-28 days gave 39% uni-lineage, 36% bi-lineage, and 25% tri-lineage colonies. A total of five different types of progenitor cells were observed. In individual colonies, oligodendrons predominated with a lesser presence of astroglial or neuronal cell types. This approach establishes a reliable and reproducible method for single cell cloning of neurosphere cells.
Engstrom
Caron M
CM
Department of Neurology, Cancer Center, University of Massachusetts Medical Center, Worcester, MA, USA.
Demers
Delia
D
Dooner
Mark
M
McAuliffe
Christina
C
Benoit
Brian O
BO
Stencel
Kimberly
K
Joly
Marguerite
M
Hulspas
Ruud
R
Reilly
Judith L
JL
Savarese
Todd
T
Recht
Lawrence D
LD
Ross
Alonzo H
AH
Quesenberry
Peter J
PJ
D000818:Animals; D000963:Antimetabolites; D015415:Biomarkers; D001973:Bromodeoxyuridine; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D019070:Cell Lineage; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D002999:Clone Cells; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects
D000963:Antimetabolites; D015415:Biomarkers; D004815:Epidermal Growth Factor; D001973:Bromodeoxyuridine