Long-term culture: evidence of the capacity of stroma to sustain hematopoiesis of a second inoculum

• Published in: Sangre (Zaragoza) Vol. 41; no. 5; p. 345
• Main Authors: López Holgado, N, Consuelo del Cañizo, M, Tabernero, M D, Hernández, M D, Vallejo, C, San Miguel, J F
• Format: Journal Article
• Language: Spanish
• Published: Spain 01.10.1996
• Subjects: Adipose Tissue - cytology; Adipose Tissue - radiation effects; Blood Cells - physiology; Bone Marrow - radiation effects; Bone Marrow Cells; Cell Count; Coculture Techniques; Colony-Forming Units Assay; Connective Tissue - radiation effects; Connective Tissue Cells; Female; Hematopoiesis - radiation effects; Hematopoietic Stem Cells - physiology; Hematopoietic Stem Cells - radiation effects; Humans; In Situ Hybridization; Male
• ISSN: 0036-4355

To analyze, in two-stages long term bone marrow cultures (LTBMC), the efficiency of the method used for irradiation of the stroma and to check if after that, it's capable to support the hematopoiesis. We have used for the first inoculum, bone marrow cells from eight controls. They were cultured at 2 x 10(6) cells/ml concentration until obtaining a fully confluent stroma which was irradiated with 15 Gy. After that, over this layer, we put a second inoculum with bone marrow cells in four cases, and with peripheral blood cells in four others, at a concentration of 1 x 10(5) cells/mL. In four cases the first inoculum was from a man and the second from a woman. The culture's haematopoietic activity was determined by the number of CFU-GM, total cell counts and the differential counts during four weeks. At the end of the study we recovered the cells from the supernatant of the culture and they were analyzed by in situ hybridization with an alpha centromeric probe specific to the X chromosome. In all cases we obtained a confluent stroma with production of haematopoiesis (cobblestone areas). The cells recovered at the end of the culture were, in all cases, from the second inoculum because they had a double signal with the X probe. The number of CFU-GM obtained was higher in bone marrow than in peripheral blood (34.5 and 9.2 respectively) however, the total cells were similar in both cases (2.3 x 10(5) and 2.9 x 10(5)) although the cellular subtypes varied depending on the second inoculum (B.M. or P.B.). Our data confirm that the dose of 15 Gy eradicate the haemopoiesis of first inoculum, which allowed to analyze stroma and progenitor cells, separately. In addition, the stroma is capable to support the hematopoiesis generated by progenitor cells from peripheral and bone marrow.