The murine stromal cell line AFT024 acts specifically on human CD34+CD38- progenitors to maintain primitive function and immunophenotype in vitro.
A stromal cell line derived from murine fetal liver (AFT024) has been demonstrated to maintain long-term repopulating murine stem cells for up to 7 weeks in vitro. We evaluated the ability of AFT024 to maintain the immunophenotype and function of primitive human progenitors in vitro by comparing the cocultivation of CD34+CD38 cells on AFT024 with that on primary human stroma (HS). We have previously reported that within the CD34+CD38- population of bone marrow and cord blood, a highly primitive progenitor subpopulation can be identified functionally by its ability to generate colony forming unit-cells (CFU-Cs) in extended long-term culture (ELTC), that is, beyond 60 days of stromal cocultivation. Cocultivation of bone marrow and cord blood CD34+CD38-cells on AFT024 produced significantly greater cell expansion (p=0.0002) and CFU-C output (p=0.0007) during the ELTC period compared with culturing on HS. CFU-C production continued up to 9 weeks longer on AFT024 stroma. After 3 to 4 weeks of bulk culture on either AFT024 or HS, cells were replated in a limiting dilution to measure the number of cobblestone area-forming cells (CAFCs) maintained on each stroma. AFT024 maintained significantly more CAFCs than did HS (n=3, p=0.002). Fluorescence-activated cell sorter analysis of AFT024 and HS cocultures showed that both the frequency (p=0.018) and absolute number (p=0.027) of CD34+CD38- cells were significantly higher in cultures on AFT024 than in those on HS (n=9). The effects of AFT024 on preservation of primitive progenitors were not seen in transwell (noncontact) cultures. Thus, AFT024 acts by direct contact to maintain the phenotype and function of the most primitive and quiescent human progenitors currently identifiable by in vitro assays.
Thiemann
F T
FT
Division of Research Immunology and Bone Marrow Transplantation, Childrens Hospital Los Angeles, California 90027, USA.
Moore
K A
KA
Smogorzewska
E M
EM
Lemischka
I R
IR
Crooks
G M
GM
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD / Q000097:blood*; D018952:Antigens, CD34 / Q000097:blood*; D000943:Antigens, Differentiation / Q000097:blood*; D002478:Cells, Cultured; D006410:Hematopoiesis / Q000276:immunology*; D006801:Humans; D016130:Immunophenotyping; D008562:Membrane Glycoproteins; D051379:Mice; D009244:NAD+ Nucleosidase / Q000097:blood*; D016012:Poisson Distribution; D012016:Reference Values; D017154:Stromal Cells / Q000276:immunology
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D008562:Membrane Glycoproteins; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1