A selective culture system for generating terminal deoxynucleotidyl transferase-positive lymphoid cells in vitro. III. Structure of the bone marrow microenvironment for early lymphopoiesis.

Autor:	Medlock ES; Department of Pathology, School of Medicine, University of Connecticut Health Center, Farmington., McKenna SD, Goldschneider I
Jazyk:	angličtina
Zdroj:	Laboratory investigation; a journal of technical methods and pathology [Lab Invest] 1993 Nov; Vol. 69 (5), pp. 616-28.
Abstrakt:	Background: We have previously demonstrated the feasibility of generating terminal deoxynucleotidyl transferase-positive (TdT+) lymphoid precursor cells in vitro in the nonadherent compartment of a long-term xenogeneic culture system in which rat bone marrow (BM) cells are seeded onto established mouse BM adherent cell layers. We have also noted that the appearance of TdT+ cells in these cultures is preceded by the formation of clusters of lymphoblasts in close association with the mouse BM adherent cell layer. Inasmuch as the selective generation of such primitive lymphoid cells is not ordinarily observed in homogeneic (i.e., mouse: mouse, rat:rat) BM cultures, the nature of the microenvironment for the generation of committed lymphoid stem/progenitor cells has not yet been detailed. Consequently, the aim of this study was to define the cellular components in the adherent compartment of our xenogeneic culture system that are associated with the earliest stages of lymphopoiesis in vitro. Experimental Design: The nature of the interactions between rat BM lymphoid precursor cells and mouse BM adherent microenvironmental cells was investigated by a combination of immunophenotyping and scanning and transmission electron microscopy of primary cultures. The kinetics of formation and composition of lymphoid clusters were also determined morphologically and phenotypically. Results were compared with those of other investigators who have studied lymphopoiesis in intact BM or in homogeneic cultures of pre-B cells. Results: Two distinct microenvironmental regions are represented within the mouse BM adherent cell layer: (a) paucilayer (PL) regions, composed of two or three horizontally oriented layers of alkaline phosphatase-positive mouse stromal cells; and (b) multilayer (ML) regions, containing 4 to 8 layers of such stromal cells. In both regions, proliferating rat lymphoid cells, expressing the HIS24 (B220) and/or HIS50 (heat stable antigen) early B-lineage antigens, are "sandwiched" between adjacent layers of stromal cells and enveloped by cytoplasmic processes from interdigitating mouse macrophages (pseudoemperipolesis). More than 95% of the lymphoid cells are of rat origin, whereas more than 95% of the nonlymphoid cells are of mouse origin. Large clusters, containing up to 1,000 lymphoid cells, preferentially develop in the ML regions and are comprised primarily of TdT+ cells. Small clusters containing 5 to 50 lymphoid cells, preferentially develop in the PL regions and are comprised primarily of TdT- cells, that can generate TdT+ cells upon transfer onto fresh adherent cells layers. Formation of individual small clusters, which outnumber large clusters by approximately 10-fold, is initiated by as few as 25 unfractionated rat BM cells. This process is not preceded by a lag period, and is linear with respect to time and cell dose. Formation of large clusters requires approximately 30 times more input cells, and is linear with respect to time after a lag of 5 days. Conclusions: The number of small lymphoid clusters formed in vitro closely approximates the frequency of lymphoid stem/progenitor cells in the BM inoculum (3 to 5%). This suggests that, under ideal conditions, individual clusters are clonally derived and the seeding efficiency of the culture system approaches 100%. The results further suggest that large clusters are formed by the coalescence of numerous small clusters within ML regions of the adherent cell layer; and that the formation of ML regions may be supported by an underlying monolayer of macrophages. A novel aspect of this system appears to be the frequency of pseudoemperipolesis, rather than phagocytosis, of primitive lymphoid cells by macrophages, that has also been noted in vivo. Moreover, the ML regions themselves bear a close resemblance to the recently described pro-B cell-enriched, multicellular aggregate fraction of freshly harvested mouse BM. Hence, this system appears to structurally recreate in vitro the
Databáze:	MEDLINE
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