Clonal proliferation of murine lymphohemopoietic progenitors in culture.
We have used a two-step clonal culture system to unequivocally demonstrate that individual primitive lymphohemopoietic progenitor cells have the capacity for differentiation along either the myeloid or the B-lymphoid lineage. Highly enriched murine marrow cells were plated individually in culture by micromanipulation in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium, erythropoietin, steel factor (SF), and interleukin (IL) 7. Forty-five percent of the single cells formed primary colonies expressing multiple hemopoietic lineages. When aliquots from individual colonies were replated in secondary methyl cellulose culture containing SF and IL-7, 41% of the primary colonies gave rise to lymphocyte colonies. Cells of the lymphocyte colonies were blast-like and B220+, sIg-, Mac-1-, Gr-1-, Ly-1-, L3T4-, Ly-2-, and CD3-. Thirty to 70% of the cells were Thy-1+. mu-chain mRNA was detected in most of the cells by in situ hybridization with an antisense RNA probe. When lymphocyte colonies derived from a single cell were pooled and individually injected into scid mice, donor-type IgM was measurable in the serum of mice and spleens contained donor-type B cells. We then carried out initial screening of growth factors to identify growth factors that might replace pokeweed mitogen-stimulated spleen cell conditioned medium in the primary culture. Combinations of two factors that included SF plus IL-6, IL-11, or granulocyte colony-stimulating factor were all effective in the primary culture in the maintenance of the B-lymphoid potential. Interestingly, IL-3 could neither replace nor act synergistically with SF to support the lymphoid potential of the primary cultures. Our observations demonstrate that many primitive progenitors previously believed to be myeloid-committed also possess B-lymphoid potential. This culture system should prove valuable for elucidation of the mechanisms regulating early stages of lymphohemopoiesis.
Hirayama
F
F
Department of Medicine, Medical University of South Carolina, Charleston.
Shih
J P
JP
Awgulewitsch
A
A
Warr
G W
GW
Clark
S C
SC
Ogawa
M
M
D000818:Animals; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D001402:B-Lymphocytes / Q000166:cytology*; D016207:Cytokines / Q000494:pharmacology; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D006410:Hematopoiesis / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D051379:Mice; D016513:Mice, SCID; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology
D015214:Antigens, Differentiation, Myelomonocytic; D016207:Cytokines; D012333:RNA, Messenger; D011994:Recombinant Proteins