Labeled Bone Marrow Cells Research Articles

The effect of transforming growth factor β(1) in the transition of bone marrow-derived macrophages into myofibroblasts during renal fibrosis

To study which subgroup of bone marrow derived cells formed myofibroblasts and the mechanism that transforming growth factor β(1)(TGFβ(1)) regulates the formation of bone marrow derived macrophages into myofibroblasts during renal fibrosis. Chimeric mice were generated by lethally irradiation of C57 mice followed by transfusion of green fluorescent protein (GFP) labeled bone marrow cells. Complete marrow reconstitution was developed until 12 weeks after transplantation. The mice were randomly divided into Sham operation group, unilateral ureteral obstruction (UUO) 3 days group, UUO5 days group, UUO7 days group and UUO7 with TGFβ(1) treatment group. Each group had four mice. Flow cytometry was used to evaluate cell components. Compared with Sham operation group the proportions of GFP(+) CD(14)(+)α-smooth muscle actin(α-SMA)(+) cells, GFP+ CD(44)(+)CD(105)(+)α-SMA(+) cells and GFP(+) F4/80(+) α-SMA(+) cells in each UUO group were progressively increased and the transformation rate in UUO7 day group was the highest. The GFP(+) F4/80(+) α-SMA(+) cells accounted for the largest population. TGFβ(1) promoted the transformation of bone marrow derived macrophages into myofibroblasts. Compared with Sham operation group or UUO7 day group, the proportion of GFP(+) F4/80(+) α-SMA(+) cells increased in UUO7 day TGFβ(1) treatment group. Compared with Sham operation group (or UUO7 days group) the protein expressions of F4/80, α-SMA, Collagen Ⅰ increased in UUO7 with TGFβ(1) group. Bone marrow derived macrophages are considered as the main type of myofibroblast precursors during the development of renal fibrosis. TGFβ(1) regulates the transformation of bone marrow-derived macrophages into myofibroblasts. This process contributes to progressive renal fibrosis and deterioration of renal function.

Bone Marrow Cell Trafficking Analyzed by 89Zr-oxine Positron Emission Tomography in a Murine Transplantation Model.

Purpose: The success of hematopoietic stem cell transplantation (HSCT) depends on donor cell homing to the bone marrow. However, there is no reliable method of noninvasively monitoring the kinetics and distribution of transferred cells. Using zirconium-89 (89Zr)-oxine cell labeling combined with PET imaging, we sought to visualize and quantify donor cell homing in a mouse bone marrow transplantation model.Experimental Design: The effect of 89Zr-oxine labeling on bone marrow cell viability and differentiation was evaluated in vitro89Zr-labeled bone marrow cells (2 × 107 cells, 16.6 kBq/106 cells) were transferred intravenously, and serial microPET images were obtained (n = 5). The effect of a CXCR4 inhibitor, plerixafor (5 mg/kg) and G-CSF (2.5 μg) on bone marrow homing and mobilization were examined (n = 4). Engraftment of the transferred 89Zr-labeled cells was evaluated (n = 3).Results:89Zr-oxine-labeled bone marrow cells showed delayed proliferation, but differentiated normally. Transferred bone marrow cells rapidly migrated to the bone marrow, spleen, and liver (n = 5). Approximately 36% of donor cells homed to the bone marrow within 4 hours, irrespective of prior bone marrow ablation. Inhibition of CXCR4 by plerixafor alone or with G-CSF significantly blocked the bone marrow homing (P < 0.0001, vs. nontreated, at 2 hours), confirming a crucial role of the CXCR4-CXCL12 system. Mobilization of approximately 0.64% of pretransplanted bone marrow cells induced a 3.8-fold increase of circulating bone marrow cells. 89Zr-labeled donor cells engrafted as well as nonlabeled cells.Conclusions:89Zr-oxine PET imaging reveals rapid bone marrow homing of transferred bone marrow cells without impairment of their stem cell functions, and thus, could provide useful information for optimizing HSCT. Clin Cancer Res; 23(11); 2759-68. ©2016 AACR.

Submyeloablative conditioning with busulfan permits bone marrow-derived cell accumulation in a murine model of Alzheimer’s disease

Previous work has suggested that bone marrow (BM)-derived cells (BMDCs) accumulate within the CNS and could potentially associate with β-amyloid plaques in Alzheimer’s disease (AD). To explore the accumulation of BMDCs in murine AD, we transplanted green fluorescent protein (GFP)-labeled BM cells into triple transgenic (3×Tg) and wild-type (wt) mice using non-irradiative myelosuppresive conditioning with busulfan (BU). We find that BU (80mg/kg) is sufficient to obtain adequate chimerism (>85%) in wt mice. In order to obtain appreciable non-irradiative chimerism in the 3×Tg mice (>80%), anti-asialo ganglio-N-tetraosylceramide (α-ASGM-1) antibody was also used to reduce natural killer cell function and thereby abrogate the hybrid resistance of the 3×Tg mouse strain. Using BU conditioning and α-ASGM-1 together, we observed sustained BM chimerism and BMDC accumulation within the CNS of the 3×Tg and wt mice. In cortex and hippocampus, BMDC accumulation was perivascular in distribution and similar between 3×Tg and wt mice, with no clear association between BMDCs and AD plaques. We conclude that non-irradiative BM chimerism can be achieved with BU in 3×Tg mice, but requires α-ASGM-1 (or similar appropriate NK-cell depletion). Use of this chimerism protocol permits BMDCs accumulation in the CNS of mixed strain recipient mice although BMDCs appear to be largely perivascular within cortex and hippocampus.

Resident microglia, and not peripheral macrophages, are the main source of brain tumor mononuclear cells.

Gliomas consist of multiple cell types, including an abundant number of microglia and macrophages, whereby their impact on tumor progression is controversially discussed. To understand their unique functions and consequently manipulate either microglia or macrophages in therapeutic approaches, it is essential to discriminate between both cell populations. Because of the lack of specific markers, generally total body irradiated chimeras with labeled bone marrow cells were used to identify infiltrated cells within the brain. However, total body irradiation (TBI) affects the blood-brain barrier integrity, which in turn potentially facilitates immune cell infiltration. In this study, changes on the blood-brain barrier were avoided using head-protected irradiation (HPI). Head protection and total body irradiated chimeras exhibited similar reconstitution levels of the myeloid cell lineage in the blood, enabling the comparable analyses of brain infiltrates. We demonstrate that the HPI model impeded a massive unspecific influx of donor-derived myeloid cells into naive as well as tumor-bearing brains. Moreover, experimental artifacts such as an enlarged distribution of infiltrated cells and fourfold increased tumor volumes are prevented in head-protected chimeras. In addition, our data evidenced for the first time that microglia are able to up-regulate CD45 and represent an inherent part of the CD45(high) population in the tumor context. All in all, HPI allowed for the unequivocal distinction between microglia and macrophages without alterations of tumor biology and consequently permits a detailed and realistic description of the myeloid cell composition in gliomas.

Percutaneous Bone Marrow Transplantation Using Fractional Ablative Erbium:YAG Laser

Topical application of therapeutic agents has been a mainstay in Dermatology for the treatment of skin disorders but is not commonly used for systemic delivery. For a topically applied agent to reach distant body sites it must first overcome the barrier function of the skin and then penetrate into deeper structures before reaching the systemic circulation. This has limited the use of topically applied agents to those having specific charge, solubility and size restrictions. Pretreatment of the skin with ablative fractional laser appears to enhance the uptake of some topically applied drugs but the ability to effectively deliver agents to distant sites is largely unproven. In this report we used a fractional ablative Erb:YAG (Erbium/Yttrium Aluminum Garnet) laser to facilitate the transfer of bone marrow stem cells through the skin in a murine bone marrow transplant model. Chimerism could be detected in the peripheral blood of recipient C57BL/6 mice that were pretreated with ablative fractional laser and had topically applied enhanced green fluorescent protein (GFP) labeled bone marrow cells from syngeneic donor transgenic mice. This study indicates that fractional laser can be used to deliver stem cells through the skin and remain functionally intact.

The ETS Transcription Factor ETV7 Exhausts Hematopoietic Stem Cells By Enhancing The Cell Cycle Entry and Cell Proliferation

Although ETS-transcription factors play a role in normal and malignant hematopoiesis, their function in hematopoietic stem cells (HSCs) and leukemia initiating cells (LICs) is largely unknown. We originally identified the novel ETS transcription factor ETV7, which is highly homologous to ETV6/TEL, a frequent target of chromosomal translocation in human leukemia. Previously we have shown that ETV7 is a hematopoietic oncoprotein that requires cooperating mutations to induce leukemic transformation. Microarray analysis revealed that ETV7 expression is upregulated in 70% of pediatric ALL and AML samples. This indicates a possible oncogenic function of ETV7 in a variety of leukemias, although the molecular mechanism of ETV7-mediated leukemogenesis remains to be elucidated. ETV7 is widely but not abundantly expressed in various human tissues. Recently we found that overexpression of ETV7 in human cord blood-derived CD34+ cells depletes the number of CD34+CD38- HSCs. In addition, ETV7-transduced cells slightly accerelated cell proliferation. These results suggest that overexpression of ETV7 activates cell proliferation in primary human CD34+cells and depletes the number of HSCs. Here, by using a mouse model, we show that ectopic expression of ETV7 in quiescent HSCs accelerates their cell cycle entry and proliferation, leading to the exhaust of HSCs in mice.The ETV7 gene locus is deleted in part of the rodents including the mouse despite its high level of conservation among vertebrates. To circumvent this limitation, we have generated an ETV7 BAC transgenic mouse that carries a single copy of a human BAC DNA containing the ETV7 gene locus. In flow cytometry (FCM) analysis of wild type (WT) and ETV7 bone marrow (BM)-derived Lin-Sca1+cKit+(LSK) cells, the size and frequency of LT(long term)-HSCs (CD48-CD150+LSK) in ETV7 LSK was 2-fold lower than that in WT LSK, while the frequency of LSK and hematopoietic common progenitor cells in WT and ETV7 BM are similar. As compared with WT-LSK, ETV7-LSK showed a significantly decreased number of myeloid progenitor colonies in both the initial plating (MC1) and replating of MC1 colonies (MC2) in methylcellulose colony formation assay in vitro. To assess the ETV7 HSC function contributing to blood cell generation in vivo, we performed competitive repopulation assays. In agreement with the in vitro results, the repopulation ability of HSC is significantly compromised in ETV7 mice as measured 7 weeks post transplantation. This defect was even more pronounced 16 weeks post transplantation. Since enhanced cell cycle entry is known to cause loss of hematopoietic stem/progenitor cells (HSPCs) through the activation of a tumor suppressor response, we quantified p19ARF, p16INK4a, and p21CDKN1A gene expression in LSK cells by qRT-PCR. At day 6 and day 9 of in vitro culture, ETV7 LSK cells activated the p19ARF, p16INK4a, and p21CDKN1A genes about 2-fold greater than WT LSK cells. To measure the de novo DNA replication of HSPCs in vivo, BrdU-pulse labeled BM cells were harvested and BrdU incorporation was quantified by FCM analysis. ETV7 LSK cells showed elevated BrdU incorporation compared with that of WT. In addition, Hoechst33342/Pyronin Y staining revealed that ETV7 LSK enhanced transition from G0 to the G1 phase of the cell cycle, suggesting that ETV7 forced cell cycle entry of quiescent HSCs. Finally to clarify the involvement of the CDKN2A tumor suppressor in ETV7-associated HSC exhaustion, we examined the frequency of HSPCs in CDKN2A-/- and ETV7+/-CDKN2A-/- LSK cells in vivo by FCM analysis. Loss of CDKN2A but not ARF restored the depletion of ETV7 LT-HSCs. Moreover, loss of CDKN2A rescued the defect of repopulation ability in vivo and self-renewal activity in vitro of ETV7 HSPCs. These results indicate that exhaustion of HSC in ETV7 BM occurred through ETV7-induced activation of cell proliferation and the CDKN2A tumor suppressor pathway in mice. Disclosures:No relevant conflicts of interest to declare.

Suppression of Choroidal Neovascularization and Quantitative and Qualitative Inhibition of VEGF and CCL2 by Heparin

To study the effect of heparin on the development of laser-induced choroidal neovascularization (CNV) and to assess the underlying molecular mechanisms. Bone marrow transplantation (BMT) was conducted by intravenous injection of green fluorescence protein (GFP)-labeled bone marrow cells (1 × 10(7) cells) into irradiated (9 Gy) C57BL/6J mice. Laser photocoagulation was applied to induce CNV; subsequently, unfractionated heparin or phosphate-buffered saline was injected into mice that did or did not undergo BMT. The area of CNV, distribution of injected heparin, and quantities of infiltrating cells positive for Griffonia simplicifolia (GS) and GFP inside and outside the CNV were evaluated. Effects of heparin on the secretion of VEGF, CCL2, and TNF-α by ARPE19 cells and on the binding of VEGF, CCL2, TNF-α, and their receptors were analyzed in vitro. Intravitreal injection of heparin at higher doses reduced the size of the CNV. Heparin localized at the vascular structures and photoreceptor layers adjacent to the laser scar. Only GS-positive cells infiltrating outside the CNV were reduced significantly, but not those inside the CNV or those expressing GFP. Relative decreases in VEGF and CCL2 levels were observed in media of ARPE19 cells at higher heparin concentrations. In vitro binding assays revealed that heparin and porcine ocular fluid, respectively, suppressed the binding of VEGF to VEGFR2 and CCL2 to CCR2. Intravitreal heparin injection inhibited CNV development. Reduced VEGF and CCL2 secretion by RPE cells and suppression of VEGF-VEGFR2 and CCL2-CCR2 interactions at the laser site mediated by heparin may contribute to the pharmacologic effect.

Good Vibrations?

The beneficial effects of exercise on obesity--and associated conditions such as diabetes--are widely thought to depend on increased metabolic expenditure and are thus expected to correlate with how strenuous and long-lasting a particular physical activity is. Noting that daily exposure to brief periods of high-frequency, low-intensity mechanical signals is anabolic for bone and muscle and that adipocytes, osteoblasts, and myocytes all derive from a common progenitor (the mesenchymal stem cell), Rubin et al . investigated the effects of low-magnitude mechanical signals (LMMS) on adipogenesis. After several months’ exposure to a high-frequency mechanical stimulus of lower magnitude than would be incurred by walking (15 minutes 5 days a week of whole-body vibration on an oscillating platform), the fat volume of the torsos of LMMS mice was reduced about 27% relative to that of control mice. This difference, which could not be attributed to changes in food intake, was accompanied by a decrease in liver nonesterified free fatty acid and triglyceride content (both risk factors for type II diabetes). Flow cytometry analysis of mice that had received transplants of green fluorescent protein (GFP)-labeled bone marrow cells indicated that a smaller fraction of marrow-derived mesenchymal stem cells had differentiated into adipocytes in mice subjected for 6 weeks to LMMS. Thus, the authors conclude that brief exposure to high-frequency LMMS can decrease adiposity, at least in part by influencing the generation of adipocytes from mesenchymal stem cells, and speculate that these findings may lead to nonpharmacologic interventions against obesity that do not require prolonged and strenuous physical activity. C. T. Rubin, E. Capilla, Y. K. Luu, B. Busa, H. Crawford, D. J. Nolan, V. Mittal, C. J. Rosen, J. E. Pessin, S. Judex, Adipogenesis is inhibited by brief, daily exposure to high-frequency, extremely low-magnitude mechanical signals. Proc. Natl. Acad. Sci. U.S.A. 104 , 17879-17884 (2007). [Abstract] [Full Text]

In vivo MR imaging of bone marrow cells trafficking to atherosclerotic plaques

To develop a magnetic resonance imaging (MRI)-based method to monitor in vivo trafficking of bone marrow (BM) cells to atherosclerotic lesions. BM cells from LacZ-transgenic mice were labeled with a superparamagnetic iron oxide (Feridex) and then transplanted into ApoE(-/-) recipient mice that were fed an atherogenic diet. Twenty-four ApoE(-/-) mice were divided into three study groups: 1) group I with Feridex-labeled BM transplantation (BMT) cells (N = 9), 2) group II with unlabeled BMT cells (N = 10), and 3) group III with no BMT cells (N = 5). Migrated Feridex/LacZ-BM cells to atherosclerotic aortic walls were monitored in vivo using a 4.7T MR scanner and correlated with histopathological findings. In group I with Feridex-BMT cells, histology examination displayed plaques in five of nine animals. In four of these five animals, in vivo MRI showed large MR signal voids of the aorta walls (due to the "blooming" effect of migrated Feridex-BM cells in plaques), which were correlated with Feridex- and/or LacZ-positive cells detected in the atherosclerotic lesions. No signal voids could be visualized in the two control animal groups (groups II and III). This study demonstrates the potential use of in vivo MRI to monitor the trafficking of magnetically labeled BM cells to atherosclerotic lesions.

Bone Marrow Cells Transdifferentiate to Cardiomyocytes When Introduced into the Embryonic Heart

Since rates of cardiomyocyte generation in the embryo are much higher than within the adult, we explored whether the embryonic heart would serve as useful experimental system for examining the myocardial potential of adult stem cells. Previously, we reported that the long-term culturing of adult mouse bone marrow produced a cell population that was both highly enriched for macrophages and cardiac competent. In this study, the myocardial potential of this cell population was analyzed in greater detail using the embryonic chick heart as recipient tissue. Experiments involving the co-incubation of labeled bone marrow cells with embryonic heart tissue showed that bone marrow (BM) cells incorporated into the myocardium and immunostained for myocyte proteins. Reverse transcription-polymerase chain reaction analysis demonstrated that the heart tissue induced bone marrow cells to express the differentiated cardiomyocyte marker alpha-cardiac myosin heavy chain. The cardiomyocyte conversion of the bone marrow cells was verified by harvesting donor cells from mice that were genetically labeled with a myocardial-specific beta-galactosidase reporter. Embryonic hearts exposed to the transgenic bone marrow in culture exhibited significant numbers of beta-galactosidase-positive cells, indicating the presence of bone marrow-derived cells that had converted to a myocardial phenotype. Furthermore, when transgenic mouse BM cells were injected into living chick embryos, donor cells incorporated into the developing heart and exhibited a myocardial phenotype. Immunofluorescence analysis demonstrated that donor BM cells exhibiting myocyte markers contained only nuclei from mouse cells, indicating that differentiation and not cell fusion was the predominant mechanism for the acquisition of a myocyte phenotype. These data confirm that adult mouse bone marrow contain cells with the ability to form cardiomyocytes. In addition, the predominance of the macrophage phenotype within the donor bone marrow cell population suggests that transdifferentiation of immune response cells may play a role in cellular regeneration in the adult.

Molecular persistence of chronic myeloid leukemia caused by donor T cells specific for lineage-restricted maturation antigens not recognizing immature progenitor-cells

Although donor lymphocyte infusion (DLI) induces complete remissions in 70% of patients with relapsed chronic myeloid leukemia (CML) after allogeneic stem-cell transplantation (SCT), some patients are refractory to DLI by showing disease persistence. In a patient who received DLI for relapsed CML, we observed persisting molecular disease despite a hematological and cytogenetic remission in the absence of graft-versus-host disease (GVHD). To determine the nature of this immune response, we isolated leukemia-reactive donor T-cell clones from the bone marrow (BM) of the patient at the time of clinical response. Four different types of CD8+ HLA class I restricted T-cell clones were obtained that were cytotoxic against Ebstein-Barr virus-transformed B-cell lines (EBV-LCL) of the patient, but not the donor, indicating recognition of minor histocompatibility antigens (mHags). By using survival studies with CFSE labelled BM cells populations, a hematopoietic progenitor cell inhibition assay and direct morphological examination we showed that the T-cell clones recognized mature monocytic and myeloid cells, whereas immature BM progenitor cells were insufficiently lysed. This patient's refractoriness for DLI appears to be caused by inadequate lysis of progenitor cells by these cytotoxic T cells. These findings support the hypothesis that for eradication of CML a cytotoxic T-cell response against leukemic progenitor cells is essential.

Origin and distribution of bone marrow‐derived cells in the central nervous system in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is associated with increased numbers of microglia within the central nervous system (CNS). However, it is unknown whether the microgliosis results from proliferation of CNS resident microglia, or recruitment of bone marrow (BM)-derived microglial precursors. Here we assess the distribution and number of BM-derived cells in spinal cord using transplantation of green fluorescent protein (GFP)-labeled BM cells into myelo-ablated mice over-expressing human mutant superoxide dismutase 1 (mSOD), a murine model of ALS. Transplantation of GFP+ BM did not affect the rate of disease progression in mSOD mice. Mean numbers of microglia and GFP+ cells in spinal cords of control mice were not significantly different from those in asymptomatic mSOD mice and showed no change with animal age. The number of GFP+ cells and microglia (F4/80+ and CD11b+ cells) within the spinal cord of mSOD mice increased compared to age-matched controls at a time when mSOD mice exhibited disease symptoms, continuing up to disease end-stage. Although we observed an increase in the number of GFP+ cells in spinal cords of mSOD mice with disease symptoms, mean numbers of GFP+ F4/80+ cells comprised less than 20% of all F4/80+ cells and did not increase with disease progression. Furthermore, the relative rates of proliferation in CD45+GFP- and CD45+GFP+ cells were comparable. Thus, we demonstrate that the microgliosis present in spinal cord tissue of mSOD mice is primarily due to an expansion of resident microglia and not to the recruitment of microglial precursors from the circulation.

The Tale of Early Hematopoietic Cell Seeding in the Bone Marrow Niche

Since introduction of the notion of a "niche" that hosts engraftment and activity of hematopoietic cells, there is a massive effort to discover its structure and decipher its function. Our understanding of the niche is continuously changing with reinterpretation of traditional concepts and apprehension of new insights into the biology of hematopoietic cell homing, seeding, and engraftment. Here we discuss some of the early events in hematopoietic stem cell seeding and engraftment and propose a perspective based on visualization of labeled bone marrow cells in real time in vivo. Primary seeding of hematopoietic cells in the bone marrow niches evolves as a complex and dynamic process; however, it follows discrete topological and chronological patterns. Initial seeding occurs on the endosteal surface of the marrow, which includes heterogeneous niches for primary seeding. Several days after transplantation the endosteal niches become more restrictive, hosting primarily mitotically quiescent cells, and gradual centripetal migration is accompanied by engagement in proliferation and differentiation. The hematopoietic niches evolve as heterogeneous three-dimensional microenvironments that are continuously changing over time.

A strategy of retrograde injection of bone marrow mononuclear cells into the myocardium for the treatment of ischemic heart disease

Objective. – Bone marrow cells implantation (BMI) has been reported to efficiently improve ischemic heart disease. However, BMI strategies are generally invasive. To establish a BMI strategy for ischemic heart disease, we performed implantation of autologous cryopreserved mononuclear cells (MNCs) from bone marrow (BM) retrogradely into the myocardium via the coronary vein in pigs with acute myocardial infarction (AMI) and old myocardial infarction (OMI). Methods. – BM cells were harvested from the pigs' fumurs. MNCs were collected by centrifugation and were cryopreserved. Anterior myocardial infarction was induced by occlusion of the midportion of the left anterior descending coronary artery without surgical intervention. Frozen BM cells were quickly thawed and injected retrogradely via the coronary vein into the myocardium through a single balloon infusion catheter 6 h and 2 weeks after the induction of infarction. Four weeks after implantation, coronary arteriograms were obtained, cardiac function was analyzed with the use of a conductance catheter, and histopathologic anlysis was performed with a confocal laser microscope. Plasma levels of natriuretic peptides and angiogenic growth factors were measured after BMI. Results. – Flow cytometric analysis revealed that 90% of cryopreserved BM cells were viable in vitro. Labeled BM cells were entirely distributed around in the infarcted area of maycardium in pigs. BMI increased collateral neovascuralization in infarcted hearts. BMI significantly improved cardiac function in AMI with BMI and OMI with BMI groups. BMI also increased the formation of microcapillary arteries in infarcted hearts. Levels of natriuretic peptides were significantly decreased, and levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor ( FGF2) were significantly increased after BMI. Confocal laser microscopy revealed the presence of proliferative and activated myocardial cells in infarcted hearts after BMI. Conclusion. – The retrograde infusion of cryopreserved BM cells into myocardium efficiently induced angiogenesis and improved cardiac function in pigs with AMI or OMI. These results suggest that the present strategy of BMI will be safe and feasible as an angiogenic cell therapy for ischemic heart disease.

Transfer of Endothelial Progenitor and Bone Marrow Cells Influences Atherosclerotic Plaque Size and Composition in Apolipoprotein E Knockout Mice

Recent clinical trials use cell therapy with bone marrow (BM) cells or endothelial progenitor cells (EPCs) for ischemic syndromes. We explored the effect of BM cell- or spleen cell-derived EPC transfer on plaque size and stability markers in the apolipoprotein E knockout (apoE KO) mouse model. ApoE KO mice aged 10 weeks served as recipients. Labeled BM cells and spleen cell-derived EPCs from age-matched apoE KO mice were injected intravenously to 2 groups of recipient mice each. Additional mice served as controls receiving saline. Both protocols were repeated 3 times at 2 weekly intervals. On killing, plaque size and character were studied, lipid profile analyzed, and serum and aortic cytokines assayed. Spleen cell-derived cells contained a significantly larger number of endothelial cell precursors. Labeled EPCs and BM cells were found abundantly in the spleens, yet also in the lesions of the recipient mice. Aortic sinus lesion size was significantly increased in mice receiving BM cells (n=10) in the EPC-treated group (n=10) compared with controls (n=10; a 54% and a 34% increase in aortic sinus plaque area, respectively). Mice receiving EPCs exhibited plaques with larger lipid cores and thinner fibrous caps and a higher number of infiltrating CD3 cells. RT-PCR analysis of aortas revealed reduced expression of mRNA for interleukin-10 (IL-10) in both cell transfer groups. Higher serum concentrations of IL-6 and monocyte chemoattractant protein-1 were found in sera from BM recipients, whereas lower IL-10 levels were found in mice transfused with spleen-derived EPCs. Transfer of BM cells and EPCs may result in an increase in atherosclerotic lesion size, whereas EPC transfer could also potentially influence plaque stability.

Systemic recruitment of osteoblastic cells in fracture healing

We hypothesise that following a bone fracture there is systemic recruitment of bone forming cells to a fracture site. A rabbit ulnar osteotomy model was adapted to trace the movement of osteogenic cells. Bone marrow mesenchymal stem cells from 41 NZW rabbits were isolated, culture-expanded and fluorescently labelled. The labelled cells were either re-implanted into the fracture gap (Group A); into a vein (Group B); or into a remote tibial bone marrow cavity 48 h after the osteotomy (Group C) or 4 weeks before the osteotomy was established (Group D), and a control group (Group E) had no labelled cells given. To quantify passive leakage of cells to an injury site, inert beads were also co-delivered in Group B. Samples of the fracture callus tissue and various organs were harvested at discrete sacrifice time-points to trace and quantify the labelled cells. At 3 weeks following osteotomy, the number of labelled cells identified in the callus of Group C, was significantly greater than following IV delivery, Group B, and there was no difference in the number of labelled cells in the callus tissues, between Groups C and A, indicating the labelled bone marrow cells were capable of migrating to the fracture sites from the remote bone marrow cavity. Significantly fewer inert beads than labelled cells were identified in Group B callus, suggesting some of the bone-forming cells were actively recruited and selectively chosen to the fracture site, rather than passively leaked into the circulation and to bone injury site. This investigation supports the hypothesis that some osteoblasts involved in fracture healing were systemically mobilised and recruited to the fracture from remote bone marrow sites.

Biological aspects of limb transplantation: I. Migration of transplanted bone marrow cells into recipient.

The transplanted limb contains bone marrow tissue. The hematopoietic cells contained in the bone of the graft normally differentiate after transplantation and can be released to the recipient. The cells migrate to the recipient bone marrow cavities and lymphoid organs. This causes the immune reaction between the donor and the recipient, which develops not only in the graft itself but also in the recipient immune organs where donor bone marrow cells home. The purpose of this study was to investigate the process of migration of the hematopoietic cells from the donor limb to the recipient bone marrow cavities and lymphoid tissues. The questions the authors asked were: what is the rate of release of bone marrow cells from the transplanted bone, where do the released bone marrow cells home in the recipient, how fast are donor bone marrow cells rejected by the recipient, and can some bone marrow cells homing in the recipient tissues survive and create a state of microchimerism. Experiments were performed on Brown Norway and Lewis inbred rat strains (n = 30). Limb donors received intravenous chromium-51-labeled bone marrow cells. Twenty-four hours later, the limb with homing labeled bone marrow cells was transplanted to an allogeneic or syngeneic recipient. The rate of radioactivity of bone marrow cells released from the graft and homing in recipient tissues was measured after another 24 hours. To eliminate factors adversely affecting homing such as the "crowding effect" and allogeneic elimination of bone marrow cells by natural killer cells, total body irradiation and antiasialo-GM1 antiserum were applied to recipients before limb transplantation. In rats surviving with the limb grafts for 7 and 30 days, homing of donor bone marrow cells was studied by specific labeling of donor cells and flow cytometry as well as by detecting donor male Y chromosome. The authors found that transplantation of the limb with bone marrow in its natural spatial relationship with stromal cells and blood perfusion brings about immediate but low-rate release of bone marrow cells and their migration to recipient bone marrow and lymphoid tissues. Large portions of allogeneic bone marrow cells are rapidly destroyed in the mechanism of allogeneic elimination by radioresistant but antiasialo-GM1-sensitive natural killer cells. Some transplanted bone marrow cells remain in the recipient's tissues and create a state of cellular and DNA microchimerism. A low number of physiologically released donor bone marrow cells do not seem to adversely affect the clinical outcome of limb grafting. Quite the opposite, a slight prolongation of the graft survival time was observed.

Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axons.

The potential of bone marrow cells to differentiate into myelin-forming cells and to repair the demyelinated rat spinal cord in vivo was studied using cell transplantation techniques. The dorsal funiculus of the spinal cord was demyelinated by x-irradiation treatment, followed by microinjection of ethidium bromide. Suspensions of a bone marrow cell fraction acutely isolated from femoral bones in LacZ transgenic mice were prepared by centrifugation on a density gradient (Ficoll-Paque) to remove erythrocytes, platelets, and debris. The isolated cell fraction contained hematopoietic and nonhematopoietic stem and precursor cells and lymphocytes. The cells were transplanted into the demyelinated dorsal column lesions of immunosuppressed rats. An intense blue beta-galactosidase reaction was observed in the transplantation zone. The genetically labeled bone marrow cells remyelinated the spinal cord with predominately a peripheral pattern of myelination reminiscent of Schwann cell myelination. Transplantation of CD34(+) hematopoietic stem cells survived in the lesion, but did not form myelin. These results indicate that bone marrow cells can differentiate in vivo into myelin-forming cells and repair demyelinated CNS.

Prevention of GVHD by modulation of rat bone marrow with UV-B irradiation: II. Kinetics of migration of UV-B-irradiated bone marrow cells in naive and lethally irradiated rats

UV-B irradiation (700 J/m 2) of bone marrow (BM) cells prior to transplantation into lethally γ-irradiated (1050 rad) allogeneic rats prevents the development of GVHD and results in a stable mixed lymphohematopoietic chimerism. To better understand the underlying mechanisms of the development of stable radiation chimeras in this model, this study was designed to examine whether the dose (700 J/m 2) of UV-B irradiation used for the modulation of the BM inoculum would affect the homing pattern of radiolabeled BM cells compared to that of thoracic duct lymphocytes (TDL) in the naive and lethally irradiated recipients. The results showed that intravenously administered, 111Indium-oxine-labeled, unmodified TDL home specifically to the spleen, lymph nodes, and BM compartments with a subsequent recirculation of a large number of cells from the spleen to the lymph nodes. In contrast, radiolabeled, unmodified BM cells migrate specifically to the spleen, liver, and BM with the lymph nodes, thymus, and nonlymphoid organs containing very little amounts of radioactivity. The stable concentrations of radioactivity in the lymphoid and nonlymphoid compartments between 3 and 72 hr after injection suggest that BM cells, unlike TDL, do not recirculate. The migration pattern of BM cells in the naive recipient was not significantly different from that seen in lethally irradiated animals except for the higher concentration of radioactivity in the spleen and BM of irradiated animals compared to that seen in naive recipients. The similarity of tissue localization of BM cells in naive or in irradiated syngeneic recipients to that of allogeneic recipients suggests that the homing of BM cells is not MHC restricted. Our findings of similarity between tissue localization of UV-B-irradiated labeled BM cells and unmodified BM cells in naive and lethally irradiated recipients suggest that a dose of 700 J/m 2 of UV-B irradiation is not capable of impairing BM cell migration although it is sufficient to abolish the homing of TDL to the HEV-bearing organs. Thus, our results show that BM cells are less susceptible to cell damage by UV-B irradiation than lymphocytes thereby providing the rationale for ex vivo modulation (rather than elimination) of mature T-lymphocytes in the donor BM inoculum with UV-B irradiation. This relatively simple and effective approach to modulation of T-cells in donor BM inoculum may be potentially useful in preventing GVHD without endangering successful engraftment in larger animals and in man.

Cell proliferation and thymocyte subset reconstitution in sublethally irradiated mice: compared kinetics of endogenous and intrathymically transferred progenitors.

After sublethal (6 Gy) whole-body irradiation, the C57BL/Ba (Thy-1.1) murine thymus regenerated in two waves, on days 3-10 and 25-32, separated by a severe relapse. The second phase of depletion-reconstitution reproduced the first one, in a less synchronous manner. The depletion affected all cell subsets, but CD4+ CD8- cells decreased later than immature cells. Cell proliferation, measured by BrdUrd incorporation, started on day 3 after irradiation and concerned CD4- CD8-, CD4- CD8+, and CD4+ CD8+ cells, sequentially. CD4+ CD8- cells never represented a significant percentage of cycling cells. When irradiation was immediately followed by an intrathymic injection of 10(5) C57BL/Ka (Thy-1.2) bone marrow cells, the relapse in thymus reconstitution was no longer observed. Detected with anti-Thy-1.2 antibodies, donor cells started cycling on day 14 and showed only one wave of proliferation. In these chimeras, recipient thymocytes behave exactly like thymocytes of solely irradiated mice. Intrathymically transferred CD4- CD8- thymocytes (10(5] showed the same proliferation kinetics as endogenous cells, with a peak in number on day 10 but completely disappeared from the thymus on days 14-21. These data reflect maturational differences between intrathymic and bone marrow precursor cells and suggest different radiosensitivities not linked to proliferative status. The resting state of the thymus immigrants was shown by the absence of Thy-1 acquisition by bone marrow cells continuously labeled for 10 days with BrdUrd in vivo before intrathymic transfer. When such labeled bone marrow cells were injected in the thymus, only the minor BrdUrd- subset gave rise to Thy-1+ cells.