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PubMed1985

Thymus regeneration by bone marrow cell suspensions differing in the potential to form early and late spleen colonies.

We have determined the thymus-repopulating capacity of purified hemopoietic stem cells, bone marrow cells from mice injected four days previously with 5-fluorouracil (5-FUBM), and bone marrow cells cultured in the presence of stem-cell-activating factor (SAF; SAFBM). SAF is identical to interleukin 3 (IL-3). Purified stem cells are more enriched in day-12 CFU-S than in day-8 CFU-S. 5-FUBM consists of CFU-S that give rise to late (day-12) spleen colonies. SAFBM contains predominantly CFU-S that give rise to early spleen colonies (days 6-8). There is also a net increase in the number of spleen colony-forming units (CFU-S) in these cultures. Thymus regeneration after transplantation with either purified stem cells or 5-FUBM was delayed approximately three days as compared with that after transplantation with normal bone marrow cells. This delay can be ascribed to the absence of prothymocytes in these preparations. Thymus regeneration by SAFBM was delayed approximately ten days as compared with that after transplantation with normal bone marrow cells. The most likely explanation of these results is as follows. The prothymocytes in normal bone marrow produce a relatively limited offspring in the thymus soon after transplantation. This is rapidly replaced by the offspring of newly formed prothymocytes, the results of differentiation of the pluripotent stem cells. These stem cells also give rise to late spleen colonies. Stem cells that give rise to early spleen colonies appear to have lost the capacity for differentiation into the T-cell lineage.

Animals↗
PubMed2002

Frequent detection of the replicative form of TT virus DNA in peripheral blood mononuclear cells and bone marrow cells in cancer patients.

The TT virus (TTV), a member of a family of human viruses related to the circoviridae viruses, was associated initially with acute and chronic liver diseases. TTV consists of a single-stranded, circular DNA genome of 3.8 kilobases (kb) and at least three open reading frames (ORFs). The objective of the present study was to determine whether or not TTV replicated in peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs). DNA was extracted from the PBMCs or BMCs of 153 cancer patients and from the PBMCs of 50 healthy blood donors (the controls). By using a single round of polymerase chain reaction (PCR), TTV was detected in 98.6% (141 of 143) of the PBMCs and in 90% (9 of 10) of the BMCs from cancer patients. TTV DNA was detected in significantly fewer control subjects at 86% (43 of 50; P < 0.05). Strand-specific PCR (SSPCR) targeting the ORF2 of the common genotypes of TTV was developed specifically to detect TTV positive or negative strand DNA and to examine TTV replication. TTV positive strand DNA, which may be an intermediate of viral replication, was detected in 55.3% (78 of 141) of the TTV-infected PBMCs of the cancer patients and in 7% (3 of 43) of the controls (P < 0.001). The replicative form of TTV was also detectable in 55.6% (5 of 9) of the TTV-infected BMCs. The existence of double-strand (positive and negative strands) TTV DNA in PBMCs and BMCs of the cancer patients was also supported by the finding that TTV DNA extracted from these cells was resistant to S1 nuclease. Using in situ hybridization, TTV DNA was also demonstrated to be present in the nucleus of PBMCs. It is concluded that replicative intermediate forms of TTV DNA are present in both PBMCs and BMCs, indicating that blood cells may be a site of TTV replication.

Bone Marrow Cells↗
PubMed2000

[beta1 Integrin Dysfunction in Adult Chronic Myeloid Leukemia Bone Marrow Cells]

According to our previous experiments, Ph(+) chronic myeloid leukemia (CML) cell line K562 cells have defects in beta 1 integrin activation. In order to search the same regularity in Ph(+) CML bone marrow cells, bone marrow mononuclear cells (BMMNC) from 12 cases of Ph(+) CML and 10 cases of normal individuals were studied. Their expression rate of 9EG7 epitope on beta1 integrin post treatment by 8A2 or GM- or G-CSF and cell adhesion ability with soluble fibronectin (FN) were evaluated by flow cytometry; in addition, the effects of CGP57148B, a highly specific ABL tyrosine kinase inhibitor, were observed. Our results showed that 9EG7 expression rate and FN binding rate were very low in all the inactivated cells. The parameter increased markedly post 8A2 activation in both NBMMNCs and CMLBMMNCs, but the degree of increase in CMLBMMNCs was significantly lower than that in NBMMNCs; GM-CSF or G-CSF could significantly increase the parameters in NBMMNCs while had no effects on that in CMLBMMNCs. CGP57148B could increase the beta1 integrin activation potential of CMLBMMNCs but had no effects on that of NBMMNCs. The results indicate that decreased activation potential of beta1 integrin in CMLBMMNCs is the major cause of adhesion defects of Ph(+) CML cells; beta1 integrin functional insufficiency in CMLBMMNCs could not be directly reversed by ABL tyrosine kinase inhibitor CGP57148B.

Journal Article↗
PubMed2004

Skeletal muscle differentiation potential of human adult bone marrow cells.

Murine bone marrow (BM) cells have been shown to undergo myogenic differentiation and participate in muscle repair in different muscle regeneration models. In the present paper, we report on a subset of cells (CD45+/desmin+) with myogenic potential being present at very low frequencies in human adult BM. By a simple culture method, we were able to obtain in vitro multinucleated myotubes in up to 20% of the cultures. Myotubes were generated using both BM flushed from rib fragments obtained during thoracotomy and BM derived from iliac crest aspirates. Cells of the different adherent and non-adherent fractions expressed numerous muscle specific markers by immunocytochemistry, real-time RT-PCR, flow cytometry, and Western blot analyses. Moreover, direct injection of whole BM into the right tibialis anterior muscle of immunodeficient mice (NOD/RAG) that had previously been treated with cardiotoxin to induce muscle degeneration, showed a variable but significant level of human cell engraftment (from 0.06 to 0.26% Dys+/FISH+ fibers). These data suggest that cells with skeletal muscle differentiation potential are present in adult human BM can differentiate in vitro and give rise to myogenic cells in vivo in immunodeficient mice after muscle damage. Further improvements might allow new approaches to cell-mediated therapies for muscular diseases.

Alkaline Phosphatase↗
PubMed1979

Quantitative development of adherent cell colonies in bone marrow cell culture in vitro.

Quantification of the formation of adherent cell colonies in bone marrow cell culture was attempted. By secondary transfer of the bone marrow cells as a single cell suspension after 4 days' culture of fine marrow fragments, a linear relationship was obtained between the number of adherent cell colonies developing and the number of cells secondarily inoculated into the culture bottle. This suggests that 4 days' culture of the bone marrow cells with close intercellular interactions is sufficient for the 'conditioning' of the cells to develop adherent cell colonies. Activity of such colonies to support haemopoietic stem cell proliferation was also shown.

Animals↗
PubMed1982

Bone marrow cell count per cubic millimeter bone marrow: a new parameter for quantitating therapy-induced cytoreduction in acute leukemia.

A new technique is introduced for determining the number of bone marrow cells per cubic millimeter marrow, providing an accurate and objective means for quantitating therapy-induced cytoreduction. The method requires a correction for admixed peripheral blood in bone marrow aspirates to measure the fraction of remaining pure marrow. While cell kinetic differences between blood, aspirates, and biopsies identify the proportion of contaminating blood cells, the ratio of red cell hematocrits in blood and aspirate gives the volume of trapped blood. By combining both procedures, bone marrow cell counts per unit volume pure marrow result (BMC/cu mm BM), which were found highly reproducible. Blast cell counts (BMBC/cu mm BM) were obtained by additional morphological differentiation. BMC and BMBC/cu mm BM were monitored in 16 patients with acute nonlymphoblastic leukemia treated with daunorubicin, cytosine arabinoside, and 6-thioguanine in combination and in 4 patients with end-stage acute leukemias and non-Hodgkin's lymphomas during high-dose thymidine therapy. Total and daily therapy-induced cytoreduction rates were significantly greater (P less than 0.01) in responders than nonresponders to either regimen. Changes in BMC/cu mm BM were also found representative for changes in BMBC/cu mm BM, since the majority of bone marrow cells were blasts. In acute leukemia. BMC/cu mm BM thus provides accurate and objective measurements of treatment efficacy in vivo and after short periods of drug exposure. Differences in cytoreduction rates within the group of responders also suggest possible prognostic implications.

Acute Disease↗
PubMed1990

Argyrophilic nucleolar organiser region (AgNOR) staining in normal bone marrow cells.

Fifteen normal bone marrow aspirates were stained with the agyrophilic nucleolar organiser region (AgNOR) method. The results of the specific staining AgNORs as well as nuclear and cytoplasmic staining were analysed. A system was devised to characterise precisely the AgNORs present in the nuclei of bone marrow cells. Particular types of bone marrow cells had a characteristic AgNOR and non-AgNOR staining pattern. The bone marrow cells were identified easily and reliably with AgNOR staining and the method was especially useful for lymphocytes, plasma cells, erythroid cells, basophils/mast cells, monocytes and cells containing haemosiderin. The immature haemopoietic cells exhibited more and larger AgNORs than the more mature cells. It is concluded that AgNOR staining can be used to study bone marrow cells by providing additional information when used in conjunction with conventional stains.

Bone Marrow↗
PubMed2004

Non-adherent bone marrow cells are a rich source of cells forming bone in vivo.

Syngeneic, allogeneic and xenogeneic (rat) freshly isolated bone marow cells + stromal cell cultures maintained in vitro for 10-30 days, as well as non-adherent cells removed from these cultures on 3rd-4th day were injected into the kidney parenchyma of mice, immunosuppressed with hydrocortisone. In syngeneic grafts the immunosuppression was omitted. In all transplant systems bone tissue was formed inside the kidney with 20% to 32% variation. Bone produced by allogeneic and xenogeneic cells is subject to rejection when immunosuppression ceases, as the bone formed is of donor origin. The "floating" cells, regardless of the transplant system, normally discarded during media replacement, turned out to be efficient bone producers. This notion is of practical implication when bone marrow cells are used for bone healing.

Animals↗
PubMedDate not supplied

Behaviour of heat-treated bone marrow cells in long-term bone marrow cultures.

The effect of prior heat treatment on the ability of bone marrow cells to form long-term bone marrow culture has been studied. Bone marrow cells were heated for various times in the temperature range of 39-43 degrees C and then cultured in the modified Dexter type suspension culture. At weekly intervals, the behaviour of the cultures in terms of stroma formation and confluency, cellular viability, and myelopoiesis were evaluated. The results show that there was a dose-dependent decrease in the number of viable cells in the non-adherent fraction of the cultures. Cytological analysis of these cells showed a strong shift towards macrophage population in the successive weeks of the cultures and also as a function of heat dose delivered to these cultures. The stroma formation was delayed or inhibited as a function of the heat dose. The number of granulocyte-macrophage colony forming cells (CFU-GM) in both adherent and non-adherent fraction of the cultures were decreased substantially after hyperthermia treatment. At 41 degrees C and higher temperatures, the CFU-GM were severely diminished in both fractions. The dose response experiments showed that the decrease in the number of CFU-GM was dependent on the heat dose. The results suggest that CFU-GM is an extremely sensitive target in the hyperthermia treatment of bone marrow cells and heat-treated bone marrow cells lose their ability to maintain long-term cultures.

Animals↗
PubMed1980

[Transfer of the hematopoietic microenvironment during heterotopic transplantation of a mixture of isolated bone marrow cells].

New heterotopic bone marrow was formed after syngeneic transplantation of bone marrow cell suspension placed on millipore filters and grafted under kidney capsule. Bone marrow cells produced a bone marrow cavity which later became populated by hemopoietic cells. Bone marrow cells responsible for hemopoietic territory formation could be withdrawn from cell suspension after treatment with carbonyl iron powder.

Animals↗
PubMed1993

Studies on the bone marrow cells from an excised pipe bone.

Samples of rib bone were collected and preserved under various conditions. The effect of preserving a piece of bone on the yield of hematopoietic stem cells was analyzed. Bone marrow cells were collected by means of flushing the bone marrow cavity with medium. The initial number of nucleated bone marrow cells collected from the donor's excised rib ranged from 0.8 x 10(8) to 2.3 x 10(8) per 1 cm of rib with an average of 1.21 x 10(8) (n = 7). In addition, the initial number of mononuclear bone marrow cells ranged from 1.1 x 10(7) to 4.8 x 10(7) per 1 cm of rib with an average of 2.47 x 10(7) (n = 7). The percentage loss of mononuclear bone marrow cells from rib preserved in medium for 48 hours at 4 degrees C, was less than 30%. A colony-forming assay by the methylcellulose method, complemented with r-G-CSF, r-GM-CSF, r-IL3, r-IL6, and r-erythropoietin, was done serially using bone marrow buffy-coat cells. These cells were obtained from the pieces of rib that were stored under various conditions. The number of CFU-GM and BFU-E that proliferated in this culture was 41.1 +/- 5.5 and 15.2 +/- 2.7 per dish. If the bone was preserved at 4 degrees C for 48 hours following excision, the recovery of CFU-GM and BFU-E from the bone marrow cells was greater than 50%. The effect of cryopreserving bone marrow mononuclear cells on the yield of hematopoietic cells was also analyzed. The percentage loss of proliferated hematopoietic cells by cryopreservation was less than 30% for bone marrow cells. Bone marrow cells were collected from pieces of rib obtained from cadaveric donors by means of the same method. The average number of nucleated bone marrow cells and MNCs was 1.26 x 10(7) and 2.30 x 10(7) per 1 cm of rib, respectively. The average number of CFU-GM and BFU-E that was detected by the colony-forming assay was 33.4 +/- 5.35 and 11.7 +/- 1.75 per dish, respectively. The percentage loss of proliferated hematopoietic cells by cryopreservation was less than 40% for bone marrow cells from cadaveric donors. These data, which reveal that hematopoietic bone marrow stem cells could be collected from a piece of pipe bone that was without circulation, suggest that it is possible to use pipe bone from cadaveric donors as a source of hematopoietic stem cells for bone marrow transplants.(ABSTRACT TRUNCATED AT 400 WORDS)

Aged↗
PubMed2005

Development of cell therapy using autologous bone marrow cells for liver cirrhosis.

The plasticity of bone marrow has been confirmed by the autopsy of a female recipient of bone marrow cell transplantation from a male donor. To establish new clinical cell therapies using autologous bone marrow cells for patients with liver failure, we developed a new in vivo model named the green fluorescent protein (GFP)/carbon tetrachloride (CCl4) model. Using the GFP/CCl4 model, we found that transplanted Liv8-negative cells efficiently repopulated into cirrhotic liver tissue and differentiated into albumin-producing hepatocytes under persistent liver damage induced by carbon tetrachloride. Moreover, bone marrow cell transplantation into mice with liver cirrhosis improved liver function and liver fibrosis with the strong expression of matrix metalloproteinases (MMPs), especially MMP-9 activity, resulting in an improved survival rate. Results from the GFP/CCl4 model showed that cell therapy using autologous bone marrow cells has the potential to become an effective treatment for patients with liver failure. A summary of findings from the GFP/CCl4 model is described.

Animals↗
PubMed2003

Enhancement of spine fusion using combined gene therapy and tissue engineering BMP-7-expressing bone marrow cells and allograft bone.

STUDY DESIGN: Prospective study to assess the enhancement of spine fusion using a tissue engineering construct consisting of bone marrow cells genetically modified by adenovirus (Ad) vector-encoding bone morphogenetic protein-7 (BMP-7) seeded onto an allograft scaffold in a rat model. OBJECTIVES: To evaluate Ad transgene expression at the fusion site and the effect of AdBMP-7-treatment on fusion rates, mechanical stability, microscopic anatomy, and bone formation rates. SUMMARY OF BACKGROUND DATA: Nonunion is a major complication of spine fusion. Gene transfer may be an effective method for locally overexpressing BMP-7, a gene important for bone formation and regeneration to enhance allograft spine fusion. MATERIALS AND METHODS: Bone marrow cells were treated with AdBMP-7 or Adbetagal (encoding the marker gene beta-galactosidase), AdNull (with no gene), or no vector and implanted with allograft in a site of posterior spine fusion. Marker gene expression was assessed up to 14 days after administration. Fusions were evaluated at 8 weeks. RESULTS: Ad gene expression was maximal on day 3, waning to background levels by 14 days. With AdBMP-7 treatment, radiographic fusion rate was 70% and mechanical fusion rate was 80% versus 0% by either parameter in control groups. Fused AdBMP-7-treated spines had a 2.5-fold to 3.0-fold lower range of motion and 1.7-fold to 1.9-fold lower hysteresis than controls. Fusion masses of AdBMP-7-treated spines had the microscopic appearance of normal trabecular bone and showed a 23-fold higher uptake of fluorochrome indicating increased bone formation. CONCLUSIONS: Addition of AdBMP-7-modified marrow cells can enhance allograft spine fusion.

Adenoviridae↗
PubMed1984

Strong natural killer (NK) cell activity in bone marrow of myeloma patients: accelerated maturation of bone marrow NK cells and their interaction with other bone marrow cells.

Bone marrow natural killer (NK) cell activity was studied in patients with multiple myeloma. Bone marrow mononuclear cells from myeloma patients expressed considerable levels of cytotoxicity against K562 in a 4 h 51Cr-release assay, which was comparable to that of blood lymphocytes. In contrast, NK-cell activity was markedly low or absent in bone marrow of normal donors and control patients. Fractionation of bone marrow cells from myeloma patients on linear bovine serum albumin gradients enriched NK effector cells in the upper, lymphocyte-enriched fraction, with no reactivity in the middle fraction containing mainly myeloma cells. Treatment with either Leu-7 or OKMI monoclonal antibody plus complement reduced or abrogated bone marrow NK-cell activity of myeloma patients as well as blood NK-cell activity. However, OKMI plus complement failed to reduce control bone marrow NK-cell activity, although the activity was reduced by Leu-7 plus complement. Overnight exposure to interferon (IFN) of bone marrow cells resulted in an augmentation of NK-cell activity in myeloma patients, but not in controls. Furthermore, adherent bone marrow cells from controls suppressed IFN-induced enhancement of NK-cell activity of blood lymphocytes, whereas bone marrow of myeloma patients did not contain such suppressor cells. No cytotoxicity was induced in control bone marrow cells by cocultivation with bone marrow myeloma cells. Bone marrow NK cells from myeloma patients were able to lyse control bone marrow cells in a 18 h assay and their lytic activity was augmented by IFN treatment. However, neither bone marrow cells nor blood lymphocytes were cytotoxic to autologous and allogeneic fresh myeloma cells even after activation with IFN. These results suggest that NK precursor cells differentiate into HNK-I- and OKMI-positive mature NK cells in bone marrow of myeloma patients, but not in control bone marrow, and that these functional bone marrow NK cells may interact with other bone marrow elements.

Bone Marrow↗
PubMed2002

Treatment of streptozotocin-induced diabetes mellitus by transplantation of islet cells plus bone marrow cells via portal vein in rats.

BACKGROUND: We have established a new method for the transplantation of allogeneic pancreatic islets (PIs) using sublethal irradiation (9 Gy) plus simultaneous transplantation of PIs and bone marrow cells (BMCs) via the portal vein (PV) followed by intravenous (i.v.) injection of donor BMCs (9 Gy + PV + i.v.). METHODS: Approximately 600 PIs of Brown Norway (BN: RT1An, RT1Bn) rats were transplanted into the liver of streptozotocin-induced diabetic Fischer 344 (F344: RT1Al, RT1Bl) rats via the PV. BMCs (3x108) of BN rats were injected via the PV or i.v. into the recipients simultaneously. In some groups, additional i.v. injections of BMCs from BN rats were given 5 days after the PI transplantation. RESULTS: All the recipients (10 of 10) in the 9 Gy + PV + i.v. group showed normoglycemia for more than 1 year, whereas PIs were rejected within 30 days after transplantation in the group of 9 Gy + i.v. + i.v. CONCLUSIONS: These results suggest that simultaneous transplantation of PIs and BMCs via the PV is effective in inducing persistent tolerance.

Animals↗