The type of stromal feeder used in limiting dilution assays influences frequency and maintenance assessment of human long-term culture initiating cells.
The goal of this study was to evaluate if differences in culture conditions used in long-term culture assays affect enumeration of LTC-IC in freshly sorted or ex vivo expanded CD34+/HLA-DRdim/CD2-/CD7- (34+/Lin-) cells. The variables examined included different stromal feeders (murine bone marrow fibroblast cell line, M2-10B4 and murine fetal liver cell line, AFT024) and presence or absence of cytokines (MIP-1alpha + IL-3). The absolute LTC-IC frequency in 34+/Lin- cells measured in limiting dilution assays (LDA) on AFT024 (4.45 +/- 0.69%) was significantly higher than in M2-10B4 (1.45 +/- 0.20%) LDA. Addition of MIP-1alpha and IL-3 to AFT024 LDA increased the measured LTC-IC frequency to 6.8 +/- 0.9%. We also determined the fraction of LTC-IC that persisted after 34+/Lin cells were cultured for 5 weeks by replating progeny in the three LDA readout systems. The measured LTC-IC maintenance was significantly lower when M2-10B4 LDA (13.1 +/- 3.5%, P < 0.05) were used compared with AFT024 LDA (36.6 +/- 5.5%) or AFT024 LDA supplemented with MIP-1alpha and IL-3 (29.1 +/- 6.3%). Thus, the number of LTC-IC measured in freshly sorted 34+ cells depends on the stromal feeder used in LDA assays. Furthermore, and most important, assessment of LTC-IC expansion or maintenance may vary significantly depending on the type of stromal feeder used to enumerate LTC-IC.
Department of Medicine, and Stem Cell Biology Program, University of Minnesota, Minneapolis 55455, USA.
M
M
Punzel
K A
KA
Moore
I R
IR
Lemischka
C M
CM
Verfaillie
D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D018920:Coculture Techniques; D006684:HLA-DR Antigens / Q000032:analysis; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D051379:Mice; D017154:Stromal Cells / Q000166:cytology; D013997:Time Factors
D015703:Antigens, CD; D006684:HLA-DR Antigens