|
|
Blood, Vol. 108, Issue 2, 737-744, July 15, 2006
Enhanced purification of fetal liver hematopoietic stem cells using SLAM family receptors
Blood Kim et al.
108: 737
Supplemental materials for: Kim et al
SUPPLEMENTARY METHODS Analysis of central and peripheral nervous system cells
For flow-cytometric analysis of SLAM receptor expression on fetal tissue, telencephalon and whole gut from wild type E14.5 timed-pregnant mouse embryos were dissected, dissociated, stained for SLAM family markers, and analyzed by flow-cytometry. For analysis of adult nervous system tissue, lateral ventricle subventricular zone (SVZ) and outer gut wall from adult wild type mice were dissected, enzymatically dissociated, stained for SLAM family markers, and analyzed by flow-cytometry. Cells were dissected and dissociated as previously described30-32 with the exception of adult gut cells, which were first treated with 1mg/ml Collagenase type 4 (CLS4, Worthington, NJ) for 20min at 37°C, followed by 10min dissociation in 10U/ml papain (PAP2, Worthington, NJ) at 37°C. Dissociated cells were resuspended in staining medium (90% Leibovitz L-15 medium (Gibco), 10% ddH2O (BioWhittaker), 1% Penn/Strep (Gibco), 10mM HEPES, 1mg/ml BSA. Antibody staining was performed at 1:100 dilution for CD150-PE (clone: TC15-12F12.2, 115904, Biolegend), CD244-PE (clone: 2B4, 553306, Pharmingen) and CD48-PE (clone: HM48-1, 557485, Pharmingen); and 1:500 dilution for CD45-APC (clone 30-F11, 17-0451-83, eBioscience) in staining medium for 20min on ice. Stained cells were resuspended in 0.1ug/ml propidium iodide to exclude dead cells during flow-cytometry. For fetal gut or telencephalon cells, 5000 unfractionated cells or the equivalent number of fractionated cells were sorted into each well of 6-well ultra-low binding plates (Corning) for neurosphere formation as previously described30-32. For adult SVZ cells, 1000 unfractionated cells or the equivalent number of fractionated cells were sorted into each well. Cultures were incubated at 37°C with 6% CO2. Neurospheres were counted at day 10, then transferred to adherent cultures for four days before being assessed for multipotency as previously described30-32.
Files in this Data Supplement:
- Figure S1. CD150, CD244, and CD48 are not expressed by neural progenitors from the developing central or peripheral nervous systems (JPG, 87.7 KB)
-
Representative flow-cytometry plots show typical staining profiles of CD150 (A), CD244 (B), and CD48 (C) on freshly dissociated fetal (E14.5) telencephalon (first column) or gut (containing gut epithelium as well as enteric nervous system; second column) cells. Statistics in each quadrant of the plots represent mean ± SD for four or five independent experiments. SLAM family receptors were sometimes expressed by CD45+ hematopoietic cells from each tissue but were rarely expressed by CD45— from these tissues. The positive and negative cell fractions for each SLAM marker were sorted into culture to determine the percentage of cells in each fraction that were capable of forming multilineage neural stem cell colonies in culture (D). Based on the size of each population, and the percentage of cells within each population that formed stem cell colonies in culture, we also calculated the percentage of all neural stem cell activity from the tissue that was contained in each fraction of cells (E). While neural stem cell activity from the CNS and PNS was consistently detected in the negative cell fractions, cells that stained positively for SLAM family markers rarely formed neural stem cell colonies in culture (all data represent mean ± SD for 3 to 4 independent experiments). Note that when multilineage colonies arose in culture from SLAM receptor positive fractions, never more than one or two such colonies were observed. Given that unstained cells contaminate positively stained cell fractions isolated by flow-cytometry, it is likely that rare neural stem cell colonies that arose from SLAM receptor positive fractions arose from contaminating unstained cells.
- Figure S2. CD150, CD244, and CD48 are not expressed by neural progenitors from the adult central or peripheral nervous systems (JPG, 69.7 KB)
-
Representative flow-cytometry plots show typical staining profiles of CD150 (A), CD244 (B), and CD48 (C) on freshly dissociated adult lateral ventricle subventricular zone (SVZ; first column) or outer gut wall (containing muscle layers as well as enteric nervous system; second column). Statistics in each quadrant of the plots represent mean ± SD for four independent experiments. SLAM family receptors were sometimes expressed by CD45+ hematopoietic cells from each tissue but were rarely expressed by CD45— cells from these tissues. The positive and negative CNS SVZ cell fractions for each SLAM marker were sorted into culture to determine the percentage of cells in each fraction that were capable of forming multilineage neural stem cell colonies in culture (D). Based on the size of each population, and the percentage of cells within each population that formed stem cell colonies in culture, we also calculated the percentage of all neural stem cell activity from the SVZ that was contained in each fraction of cells (E). While CNS stem cell activity was consistently detected in the negative cell fractions, cells that stained positively for SLAM family markers never formed neural stem cell colonies in culture (all data represent mean ± SD for 3 independent experiments).
|
|
