title: Enrichment and characterization of thymus-repopulating cells in stroma-dependent cultures of rat bone marrow.
abstract: The bone marrow precursor cells seeding the thymus have been difficult to investigate using fresh bone marrow and in vivo thymus reconstitution assays. We have therefore designed a short-term bone marrow culture system allowing the study of thymus-repopulating cells in the marrow microenvironment. Low-density rat bone marrow cells were grown on pre-established mouse bone marrow stromal cell layers. Cocultured cells were maintained either under steroid-free conditions (Whitlock/Witte-type culture) or in the presence of 10(-7) M hydrocortisone (Dexter-type culture). After 3 days in vitro, the unanchored cell fractions were tested for their ability to colonize and repopulate fetal mouse thymic lobes in vitro. Both fresh low-density cells and Whitlock/Witte-type cultures, but not Dexter-type cultures, gave rise intrathymically to significant numbers of rat donor-type Thy-1.1high CD2+ CD5low CD43+ cells accounting for 50% to 90% of the organ-cultured cells at day 14. Repopulation of fetal mouse thymic lobes by rat Thy-1.1high cells could be used as a readout assay for initiation of thymopoiesis from bone marrow precursor cells, since 90% of the cells were CD3-/low and TCR alpha beta-/low and 15% of the cells co-expressed CD4 and CD8. Dose-response analysis showed that thymus repopulating cells were at least maintained, if not amplified during the 3-day culture period, leading to at least a 10-fold enrichment as compared to unfractionated bone marrow. Unlike fresh low-density cells before culture, short-term Whitlock/Witte-type cultures were depleted in myeloid-restricted precursor cells. In culture, the thymus-repopulating activity was predominantly associated with a 10% lymphoid cell subset which did not express the B-lineage-associated antigens revealed by HIS24 (the rat B220 equivalent) and HIS50 mAbs. We propose that unanchored thymus-repopulating cells enriched in Whitlock/Witte-type cultures may represent lymphoid-restricted, T-cell precursors of the bone marrow capable of emigrating and colonizing the thymus.
authors.affiliation: Laboratoire de Physiopathologie du Développement, CNRS URA 1337, Paris, France.
authors.forename: Z
authors.initials: Z
authors.lastname: Prakapas
authors.forename: M
authors.initials: M
authors.lastname: Denoyelle
authors.forename: C
authors.initials: C
authors.lastname: Dargemont
authors.forename: F G
authors.initials: FG
authors.lastname: Kroese
authors.forename: J P
authors.initials: JP
authors.lastname: Thiery
authors.forename: M A
authors.initials: MA
authors.lastname: Deugnier
mesh_terms: D000818:Animals; D016175:B-Lymphocyte Subsets / Q000166:cytology; D001853:Bone Marrow / Q000196:embryology; D001854:Bone Marrow Cells; D002478:Cells, Cultured; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D017208:Rats, Wistar; D017154:Stromal Cells / Q000502:physiology; D013601:T-Lymphocytes / Q000166:cytology; D013950:Thymus Gland / Q000166:cytology* / Q000196:embryology; D013997:Time Factors