A practical miniature long-term bone marrow culture system for investigating early myelodysplasia

A method was devised to grow haemopoietic cells in long-term bone marrow culture (LTBMC) which requires only 1 x 10(6) cells/culture. Such miniature cultures were used to study growth patterns of marrow from patients with myelodysplastic syndromes (MDS). Consistent differences in LTBMC cellularity and cellular composition were noted between MDS and normal marrow. These differences were accentuated by rGM-CSF. The criteria which distinguished between and MDS marrows were: cell count at weeks 1 and 4, % neutrophils and % blasts. In 10 patients with unexplained macrocytosis or pancytopenia miniature LTBMC results clearly segregated into either 'normal' or 'MDS' growth patterns. Miniature LTBMC with rGM-CSF may therefore be a useful diagnostic test for early MDS.

CD11b+ bone marrow-derived monocytes are the major leukocyte subset responsible for retinal capillary leukostasis in experimental diabetes in mouse and express high levels of CCR5 in the circulation

We investigated the phenotype of cells involved in leukostasis in the early stages of streptozotocin-induced diabetes in mice by direct observation and by adoptive transfer of calcein-AM-labeled bone marrow-derived leukocytes from syngeneic mice. Retinal whole mounts, confocal microscopy, and flow cytometry ex vivo and scanning laser ophthalmoscopy in vivo were used. Leukostasis in vivo and ex vivo in retinal capillaries was increased after 2 weeks of diabetes (Hb A(1c), 14.2 ± 1.2) when either donor or recipient mice were diabetic. Maximum leukostasis occurred when both donor and recipient were diabetic. CD11b(+), but not Gr1(+), cells were preferentially entrapped in retinal vessels (fivefold increase compared with nondiabetic mice). In diabetic mice, circulating CD11b(+) cells expressed high levels of CCR5 (P = 0.04), whereas spleen (P = 0.0001) and retinal (P = 0.05) cells expressed increased levels of the fractalkine chemokine receptor. Rosuvastatin treatment prevented leukostasis when both recipient and donor were treated but not when donor mice only were treated. This effect was blocked by treatment with mevalonate. We conclude that leukostasis in early diabetic retinopathy involves activated CCR5(+)CD11b(+) myeloid cells (presumed monocytes). However, leukostasis also requires diabetes-induced changes in the endothelium, because statin therapy prevented leukostasis only when recipient mice were treated. The up-regulation of the HMG-CoA reductase pathway in the endothelium is the major metabolic dysregulation promoting leukostasis.

Efficient differentiation into skin cells of bone marrow cells recovered in a pellet after density gradient fractionation

We had previously demonstrated the participation of whole bone marrow cells from adult mice in the reconstitution of skin, including the epidermis and hair follicles. To get an insight into cell populations that give rise to the epithelial components of the reconstituted skin, we fractionated bone marrow cells derived from green fluorescent protein-transgenic mice by density gradient. Unexpectedly, we found that a substantial amount of mononucleated cells (approximately 30%) was recovered in the pellet fraction and that the cells in the pellet fraction preferentially differentiated into epithelial components of skin, rather than the cells in the mononuclear cell fraction. The pellet fraction contained more CD45-negative (thus uncommitted to the hematopoietic cell lineage) cells than the mononuclear cell fraction. These results indicate that density gradient fractionation results in significant loss of specific progenitor cells into the usually discarded pellet fraction.

Participation of adult mouse bone marrow cells in reconstitution of skin

Results of recent studies have indicated that bone marrow cells can differentiate into various cells of ectodermal, mesodermal, and endodermal origins when transplanted into the body. However, the problems associated with those experiments such as the long latent period, rareness of the event, and difficulty in controlling the processes have hampered detailed mechanistic studies. In the present study, we examined the potency of mouse bone marrow cells to differentiate into cells comprising skin tissues using a skin reconstitution assay. Bone marrow cells from adult green fluorescent protein (GFP)-transgenic mice were transplanted in a mixture of embryonic mouse skin cells (17.5 days post-coitus) onto skin defects made on the backs of nude mice. Within 3 weeks, fully differentiated skin with hair was reconstituted. GFP-positive cells were found in the epidermis, hair follicles, sebaceous glands, and dermis. The localization and morphology of the cells, results of immunohistochemistry, and results of specific staining confirmed that the bone marrow cells had differentiated into epidermal keratinocytes, sebaceous gland cells, follicular epithelial cells, dendritic cells, and endothelial cells under the present conditions. These results indicate that this system is suitable for molecular and cellular mechanistic studies on differentiation of stem cells to various epidermal and dermal cells.

Aging impairs peritoneal but not bone marrow-derived macrophage phagocytosis

Aging results in deterioration of the immune system, which is associated with increased susceptibility to infection and impaired wound healing in the elderly. Phagocytosis is an essential process in both wound healing and immune defence. As such, age-related impairments in phagocytosis impact on the health of the elderly population. Phagocytic efficiency in peritoneal macrophages, bone marrow-derived macrophages and bone marrow monocytes from young and old mice was investigated. Aging significantly impaired phagocytosis by peritoneal macrophages, both in vitro and in vivo. However, bone marrow-derived macrophages and bone marrow monocytes did not exhibit age-related impairments in phagocytosis, suggesting no intrinsic defect in these cells. We sought to investigate underlying mechanisms in age-related impairments in phagocytosis by peritoneal macrophages. We hypothesized that microenvironmental factors in the peritoneum of old mice impaired macrophage phagocytosis. Indeed, macrophages from young mice injected into the peritoneum of old mice exhibited impaired phagocytosis. Proportions of peritoneal immune cells were characterized, and striking increases in numbers of T cells, B1 and B2 cells were observed in the peritoneum of old mice compared with young mice. In addition, B cell-derived IL-10 was increased in resting and LPS-activated peritoneal cell cultures from old mice. These data demonstrate that aging impairs phagocytosis by tissue-resident peritoneal macrophages, but not by bone marrow-derived macrophages/monocytes, and suggest that age-related defects in macrophage phagocytosis may be due to extrinsic factors in the tissue microenvironment. As such, defects may be reversible and macrophages could be targeted therapeutically in order to boost immune function in the elderly.