Characterization and retroviral transduction of an early human lymphomyeloid precursor assayed in nonswitched long-term culture on murine stroma.
In the hierarchy of human hematopoietic progenitors, long-term culture-initiating cells (LTC-IC) and extended LTC-IC belong to the earliest cell populations that can be assayed in vitro. We report the identification of a multipotential lymphomyeloid progenitor detected in a nonswitch culture system. We observed the emergence of CD33+ myeloid and CD19+ B-lymphoid cells following plating of lineage-depleted (Lin-) CD34 -enriched or purified CD34+ CD38- cord blood cells on MS-5 stroma in the absence of exogenous cytokines. Both CD19+ CD20- pro-B and CD19+ CD20+ pre-B lymphocytes coexist with myeloid cells in long-term culture. A limiting dilution approach was used to show that a single CD34+ CD38- cell can generate lymphomyeloid progeny in conventional (5-week) and extended (10-week) cultures. Most of the clones in long-term culture or extended long-term culture contained not only lymphoid and myeloid cells, but also myeloid clonogenic progenitors. A high proportion of CD34+ CD38- cells gave rise to lymphomyeloid clones after 5 and 10 weeks of culturing (up to 48% and 16%, respectively), which distinguishes the assay reported here from those using switch culture conditions. We performed retroviral gene transfer experiments involving 1-3 days of exposure of Lin CD34+ -enriched cells to virus encoding enhanced green fluorescent protein. Monitoring of gene transfer efficiency into LTC-IC by enhanced green fluorescent protein fluorescence showed that it is possible to achieve marking of lymphomyeloid LTC-IC, albeit to a lesser extent than myeloid-restricted LTC-IC.
Gan
O I
OI
Department of Cancer and Blood Research, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Dorrell
C
C
Pereira
D S
DS
Ito
C Y
CY
Wang
J C
JC
Dick
J E
JE
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD; D018941:Antigens, CD19 / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D000943:Antigens, Differentiation / Q000032:analysis; D001402:B-Lymphocytes / Q000166:cytology; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D005312:Fetal Blood / Q000166:cytology; D018014:Gene Transfer Techniques; D006098:Granulocytes / Q000166:cytology*; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008214:Lymphocytes / Q000166:cytology*; D008562:Membrane Glycoproteins; D051379:Mice; D009244:NAD+ Nucleosidase / Q000032:analysis; D012190:Retroviridae / Q000235:genetics; D017154:Stromal Cells; D013997:Time Factors
D015703:Antigens, CD; D018941:Antigens, CD19; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D049452:Green Fluorescent Proteins; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1