EARLY ONTOGENY OF THE HUMAN MARROW FROM LONG BONES - AN IMMUNOHISTOCHEMICAL STUDY OF HEMATOPOIESIS AND ITS MICROENVIRONMENT

We examined long bones from 42 human embryos and fetuses whose gestati onal ages ranged from 6 to 28 weeks. Bone rudiment sections were stain ed using a panel of monoclonal antibodies directed at antigens express ed by hematopoietic cells, endothelial cells, smooth muscle cells, fib roblasts, and stromal cells, to describe the events preceding and acco mpanying the onset of hematopoiesis in the diaphyseal region. Five dis tinct stages were identified. Stage I (6.6 to 8.5 gestational weeks [g w]) was that of entirely cartilaginous rudiments: chondrocytes were di lated and capillaries with CD34(+) endothelial cells were observed in the perichondral limb mesenchyme. At stage II (8.5-9 gw) chondrolysis was actively proceeding; numerous CD68(+) cells were observed, intersp ersed within the marrow cavity. Stage III (9 to 10.5 gw) was character ized by the development of the vascular bed in the absence of detectab le hematopoiesis. At mid-diaphysis, specific structures that we named primary logettes were discernible; they consisted of small chambers of connective tissue, framed by a loose network of CD45-negative cells o rganized around an arteriole and limited from the surrounding sinus by a clearcut lining of CD34(+) endothelial cells flanked on their ablum inal side by alpha SM actin(+) myoid cells. Stage IV (10.5-15 gw) was characterized by the onset of hematopoiesis. Hematopoietic cells were found exclusively in the primary logettes that had considerably increa sed in size. Logettes filled with hematopoietic cells were immersed wi thin large and almost empty vascular sinuses. Logettes were attached b y a short pedicle to connective tissue adjacent to bone/cartilage rema ining formations; this tissue contained very rare hematopoietic cells. Logettes were few, usually less than 10 per long bone, and found sole ly in the diaphyseal area. Most hematopoietic cells found inside loget tes were CD15(+) myelocytes; rarely seen were glycophorin A(+) immatur e erythroblasts and CD34(+) nonendothelial cells. Hematopoietic cells within the logettes were in contact with alpha SM actin(+) myoid cells and flattened endothelial-like (although consistently CD34-negative), aligned cells limiting small capillary lumina. Stage V (16 gw onward) was that of final organization of the long hones with areas of fully calcified bone and areas of dense hematopoiesis where logettes were no longer visible. This study shows three major features of incipient lo ng bone hematopoiesis: 1) absence of CD34(+) hematopoietic precursors before the onset of hematopoiesis and extreme rarity of those in the e merging blood-forming marrow, 2) predominance of granulopoiesis. and 3 ) exclusive development in specific structures organized by vascular c ells. This study also suggests that CD68(+) cells are instrumental in the chondrolysis process while vascular cells (endothelial and myoid c ells) may be the critical microenvironment at the onset of hematopoies is. (C) 1996 by The American Society of Hematology.