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.

Chimaerism following allogeneic bone marrow transplantation:detection of residual host cells using the polymerase chain reaction

Chimaerism was assessed in five recipients following sex mismatched allogeneic bone marrow transplantation. Techniques included karyotyping of bone marrow cells, dot blot DNA analysis of blood and bone marrow suspensions, and in vitro amplification of DNA by the polymerase chain reaction (PCR) using blood and bone marrow suspensions and stored bone marrow slides. Results of karyotypic analysis suggested complete chimaerism in four patients, while in one patient mixed chimaerism was detected. Mixed chimaerism was also detected, however, in a second patient using PCR and confirmed by dot blot analysis on all tissues examined. PCR is a sensitive tool for investigation of chimaerism following bone marrow transplantation. Since this technique does not require radioactivity, it is an attractive method for use in a clinical laboratory. This technique represents a further development in the use of DNA methodologies in the assessment of haematological disease.

Long-term bone marrow culture profiles in patients with myelodysplastic syndromes are not explicable by defective apoptosis

It has been suggested that increased intramedullary apoptosis may explain the paradox between peripheral blood cytopenias and the hyper- or normo-cellular bone marrow observed in the myelodysplastic syndromes (MDS). We wished to see if culture performance could be related to the presence of apoptotic cells in a group of patients with MDS (12 patients) and other patients with peripheral blood cytopenias (six patients) which caused diagnostic difficulty. There was no correlation between LTBMC or adherent cell growth and the presence of apoptotic cells in the original marrow sample. A variable degree of apoptosis was observed in both groups of patients. LTBMC profiles correlated well with diagnosis but were unrelated to the extent of intramedullary apoptosis. This suggests that apoptosis is a much more ubiquitous process in disease than previously thought. (C) 1998 Elsevier Science Ltd. All rights reserved.

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.

Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, approximately 24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1 alpha, and HNF4alpha. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases.

Characterization of NKR+ T-cell subsets in human bone marrow:implications for immunosurveillance of neoplasia

In addition to hematopoietic progenitors, human bone marrow contains mature T/NK lymphocytes. Valpha24Vbeta11 NKT-cells, a subset of NK receptor+ (NKR+) T-cells in humans, are rare in bone marrow, suggesting the presence of other NKR+ T-cells which may contribute to tumor surveillance. NKR+/- T-cells were examined in blood (PB), and bone marrow from donors (DM) and patients with active hematopoietic malignancy (PM), or in remission (PR). T-cells in PR & PM were enriched for CD56+ and CD57+ subsets, compared to DM. All marrow NKR+/- T-cell subsets were more activated than PB. PM and, surprisingly, PR marrow contained more activated cells than DM. CD8+ cells were significantly increased in all patient marrows and there was evidence of the formation of an effector/memory pool in malignant marrow. These data suggest that NKR+ T-cell enrichment in human bone marrow that has been exposed to neoplastic transformation is compatible with a role in localized tumor surveillance/eradication.

Influence of ultraviolet-B irradiation on engraftment, graft-versus-host disease and graft-versus-leukemia effect in a rat model for allogeneic bone marrow transplantation

Ultraviolet-B (UVB) irradiation is known to inhibit lymphocyte activity and consequently to reduce the incidence of graft-versus-host disease (GVHD) in experimental models for allogeneic bone marrow transplantation (BMT). GVHD is frequently associated with morbidity and mortality, but also with the beneficial graft-versus-leukemia (GVL) effect, demonstrated by a reduction in the incidence of leukemia relapse. In this study, we investigated whether UVB treatment of allogeneic T cells could prevent GVHD while sparing the beneficial GVL effect following allogeneic BMT in the Brown Norway myelocytic leukemia (BNML) rat model analogous to human acute myelocytic leukemia (AML). The dose of UVB required to abolish lethal GVHD in the rat allogeneic BMT model (WAG/Rij donors into BN recipients) was 4000 J/m2. However, this UVB dose simultaneously abrogated all GVL activity mediated by the T cells in the graft, while the radio-protective capacity of rat BM cells was strongly reduced. The number of allogeneic BM cells required to protect lethally irradiated BN rats was increased 50 to 100-fold. It is concluded that UVB acts as a non-selective form of T cell inactivation, and that UVB pretreatment of an allogeneic marrow graft is unlikely to be useful clinically as a preventive measure for GVHD, since other means of reduction of the number of functional T cells are less damaging to bone marrow stem cells.

A rapid dot-blot assay to assess chimerism following sex-mismatched bone marrow transplantation

Mixed chimerism may occur more frequently than previously thought following allogeneic bone marrow transplantation and may have implications in terms of relapse, graft-versus-host disease and immune reconstitution. DNA analysis using single or multilocus polymorphic probes cannot reliably discriminate between donor and recipient cells below a level of 10%. We used probe pHY2.1, a cloned segment of tandemly repeated DNA (2000 copies) on the long arm of chromosome Y. A dot blot procedure allowed us to immobilize DNA directly from 50 microliter of peripheral blood or bone marrow. Cross-reactivity was eliminated by hybridization at conditions of extreme stringency (65 degrees C, 50% formamide). Mixing experiments detected male DNA at a level of 0.1% after 10 h exposure. Five patients were studied serially post-bone marrow transplantation. One patient showed mixed chimerism for 12 months, one had complete autologous recovery and the remaining three showed complete engraftment. All results were verified by standard karyotyping on bone marrow cells. This technique is a simple, rapid and sensitive assay for chimerism following sex mismatched bone marrow transplantation.

Bioreactor Expansion of Human Adult Bone Marrow-Derived Mesenchymal Stem Cells

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell transplantation (HSCT) alleviates complications such as graft-versus-host disease, leading to a speedy recovery of hematopoiesis. To meet such clinical demand, a fast MSCs expansion method is required. In the present study, we examined the feasibility of expanding MSCs from the isolated bone marrow mononuclear cells using a rotary bioreactor system. The cells were cultured in a rotary bioreactor with Myelocult� medium containing a combination of supplementary factors, including stem cell factor (SCF), interleukin 3 and 6 (IL-3, IL-6). After 8 days of culture, total cell numbers, Stro-1+CD44+CD34- MSCs and CD34+CD44+Stro-1- HSCs were increased 9, 29, and 8 folds respectively. Colony forming efficiency-fibroblast per day (CFE-F/day) of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSCs markers endoglin (SH2) and vimentin, whereas markers associated with lineage differentiation including osteocalcin (osteogenesis), Type II collagen (chondrogenesis) and C/EBPα (adipogenesis) were not detected. Upon induction, the bioreactor-expanded MSCs were able to differentiate into osteoblasts, chondrocytes and adipocytes. Taken together, we conclude that the rotary bioreactor with the modified Myelocult� medium reported in this study may be used to rapidly expand MSCs.

Chondrogenic Differentiation Alters the Immunosuppressive Property of Bone Marrow-Derived Mesenchymal Stem Cells, and the Effect is Partially due to the Upregulated Expression of B7 Molecules

To investigate the immunosuppressive properties of mesenchymal stem cells (MSCs), in the present study we examined the immunogenicity of undifferentiated and tri-lineage (chondrocytes, osteoblasts and adipocytes) differentiated rat bone marrow-derived MSCs under xenogeneic conditions. After chondrogenic-differentiation, rat bone marrow-derived MSCs stimulated human peripheral blood monocyte-derived DCs (hDCs), leading to 8- and 4-fold higher lymphocyte proliferation and cytotoxicity than that of undifferentiated MSCs. The Chondrogenic-differentiated MSCs were chemotactic to hDCs in Dunn chamber chemotaxis system and were rosetted by hDCs inrosette assays. Flow cytometry analysis revealed that chondrogenic-differentiated MSCs had promoted hDCs maturation causing higher CD83 expression in hDCs, whereas undifferentiated MSCs, osteogenic-and adipogenic-differentiated MSCs showed inhibitory effect on hDCs maturation. The co-stimulatory molecules B7 were up-regulated only in the chondrogenic-differentiated MSCs. After blocking B7 molecules with specific monoclonal antibodies in the chondrogenic-differentiated MSCs, CD83 expression of co-cultured hDCs was greatly reduced. In conclusion, chondrogenic differentiation may increase the immunogenicity of MSCs, leading to stimulation of DCs. The up-regulated expression of B7 molecules on the chondrogenic differentiated MSCs may be partially responsible for this event.

Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells

Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFB mechanism, since pharmacological inhibition of IKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).