Intra-renal arterial injection of autologous bone marrow mesenchymal stromal cells ameliorates cisplatin-induced acute kidney injury in a rhesus Macaque mulatta monkey model

BackgroundClinically, acute kidney injury (AKI) is a potentially devastating condition for which no specific therapy improves efficacy of the repair process. Bone marrow mesenchymal stromal cells (BM-MSCs) are proven to be beneficial for the renal repair process after AKI in different experimental rodent models, but their efficacy in large animals and humans remains unknown. This study aims to assess the effect of autologous rhesus Macaque mulatta monkey BM-MSC transplantation in cisplatin-induced AKI.MethodsWe chose a model of AKI induced by intravenous administration of 5 mg/kg cisplatin. BM-MSCs were transplanted through intra-arterial injection. The animals were followed for survival, biochemistry analysis and pathology.ResultsTransplantation of 5 × 10⁶ cells/kg ameliorated renal function during the first week, as shown by significantly lower serum creatinine and urea values and higher urine creatinine and urea clearance without hyponatremia, hyperkalemia, proteinuria and polyuria up to 84 d compared with the vehicle and control groups. The superparamagnetic iron oxide nanoparticle-labeled cells were found in both the glomeruli and tubules. BM-MSCs markedly accelerated Foxp3+ T-regulatory cells in response to cisplatin-induced damage, as revealed by higher numbers of Foxp3+ cells within the tubuli of these monkeys compared with cisplatin-treated monkeys in the control and vehicle groups.ConclusionsThese data demonstrate that BM-MSCs in this unique large-animal model of cisplatin-induced AKI exhibited recovery and protective properties.     

Association of a synthetic bone graft and bone marrow cells as a composite biomaterial

Porous calcium phosphates have osteoconductive properties. The aim of this study was to obtain synthetic calcium phosphate bone graft substitute. X-ray diffraction was employed to investigate the formation of the beta-tricalcium phosphate (β-TCP) phase. We evaluated the effects of bone marrow on the osteoconductivity and mechanical properties of synthetic bone graft (SG). SG cylinders loaded with bone marrow (SGBM) and SG alone were implanted into rabbits femoral condyle bone defects. Histological examinations revealed the resorption of the SG, trabecular bone with osteoblasts and osteoid substance around the implants, and colonization inside the porous β-TCP by newly formed bone. Histomorphometry conducted after three months revealed the osteoid surface to be higher in SGBM than SG (p < 0.05). The compressive strengths of SG and SGBM were significantly higher than the anatomic control at all time periods. The elastic modulus of SBG and SGBM became weaker after implantation. The present results indicate that gB-TCP is a good matrix for bone marrow, which contributes osteoinductive properties in an orthotopic. The composite biomaterial may be useful in reconstructive bone surgery.     

Role of bone marrow imprints in haematological diagnosis: a detailed study of 3781 cases

X. Gong, X. Lu, X. Wu, R. Xu, Q. Tang, G. Xu, L. Wang, X. Zhang and X. Zhao Role of bone marrow imprints in haematological diagnosis: a detailed study of 3781 cases Objectives: To explore the role of imprints in routine bone marrow (BM) diagnosis. Methods: The cellularity and diagnostic accuracy of BM imprints, aspirate smears and trephine biopsy sections from 3781 patients were assessed using routine cytochemical staining. Seventy‐nine cases of lymphoma and 114 cases of plasma cell myeloma (PCM) were selected for correlation analysis of tumour cell infiltration patterns. Another 21 cases of lymphoma were selected to detect t(14;18)(q32;q21) and t(11;14)(q13;q32) by fluorescent in situ hybridization (FISH) on BM imprints, and the G‐banding technique was performed for comparison. Results: BM imprints were better than smears for evaluating cellularity. In the BM imprint group, diagnostic accuracy for metastatic carcinoma, myeloproliferative neoplasm, myelodysplastic/myeloproliferative neoplasm and PCM was better than in the smear group, while accuracy for megaloblastic anaemia, acute myeloid leukaemia, refractory cytopenia with unilineage or multilineage dysplasia, refractory anaemia with excess blasts and lymphoplasmacytic lymphoma was higher than in the section group, but not statistically different from the smear group. Good correlation of infiltration patterns of lymphoma and myeloma cells was found between BM imprints and sections (r = 0.90 and 0.78, respectively). Detection of t(11;14)(q13;q32) by FISH on imprints was higher than G‐banding analysis. Conclusions: BM imprints show features of both smears and trephine sections. Imprints are superior to smears for evaluation of cellularity, and are also better than sections for analysis of cytological changes. In addition, FISH on BM imprints markedly improves the identification of chromosomal abnormalities.     

RANKL from bone marrow adipose lineage cells promotes osteoclast formation and bone loss

Receptor activator of NF‐κB ligand (RANKL) is essential for osteoclast formation and bone remodeling. Nevertheless, the cellular source of RANKL for osteoclastogenesis has not been fully uncovered. Different from peripheral adipose tissue, bone marrow (BM) adipose lineage cells originate from bone marrow mesenchymal stromal cells (BMSCs). Here, we demonstrate that adiponectin promoter‐driven Cre expression (Adipoq^Cre ) can target bone marrow adipose lineage cells. We cross the Adipoq^Cre mice with rankl^fl/fl mice to conditionally delete RANKL from BM adipose lineage cells. Conditional deletion of RANKL increases cancellous bone mass of long bones in mice by reducing the formation of trabecular osteoclasts and inhibiting bone resorption but does not affect cortical bone thickness or resorption of calcified cartilage. Adipoq^Cre; rankl^fl/fl mice exhibit resistance to estrogen deficiency and rosiglitazone (ROS)‐induced trabecular bone loss but show bone loss induced by unloading. BM adipose lineage cells therefore represent an essential source of RANKL for the formation of trabecula osteoclasts and resorption of cancellous bone during remodeling under physiological and pathological conditions. Targeting bone marrow adiposity is a promising way of preventing pathological bone loss.     

Peritoneal repairing cells: a type of bone marrow derived progenitor cells involved in mesothelial regeneration

The peritoneal mesothelium exhibits a high regenerative ability. Peritoneal regeneration is concomitant with the appearance, in the coelomic cavity, of a free‐floating population of cells whose origin and functions are still under discussion. We have isolated and characterized this cell population and we have studied the process of mesothelial regeneration through flow cytometry and confocal microscopy in a murine model lethally irradiated and reconstituted with GFP‐expressing bone marrow cells. In unoperated control mice, most free cells positive for mesothelin, a mesothelial marker, are green fluorescent protein (GFP). However, 24 hrs after peritoneal damage, free mesothelin⁺/ GFP⁺ cells appear in peritoneal lavages. Cultured lavage peritoneal cells show colocalization of GFP with mesothelial (mesothelin, cytokeratin) and fibroblastic markers. Immunohistochemical staining of the peritoneal wall also revealed colocalization of GFP with mesothelial markers and with procollagen‐1 and smooth muscle α‐actin. This was observed in the injured area as well as in the surrounding not‐injured peritoneal surfaces. These cells, which we herein call peritoneal repairing cells (PRC), are very abundant 1 week after surgery covering both the damaged peritoneal wall and the surrounding uninjured area. However, they become very scarce 1 month later, when the mesothelium has completely healed. We suggest that PRC constitute a type of monocyte‐derived cells, closely related with the tissue‐repairing cells known as ‘fibrocytes’ and specifically involved in peritoneal reparation. Thus, our results constitute a synthesis of the different scenarios hitherto proposed about peritoneal regeneration, particularly recruitment of circulating progenitor cells and adhesion of free‐floating coelomic cells.     

孕期应激对子代大鼠骨髓淋巴干细胞发育的影响

孕期应激对子代产生的影响是多方面的,这种影响是复杂的。研究表明,出生前的应激经历可导致出生后子代长期的免疫功能改变。这些改变追其根源与骨髓淋巴干细胞的改变有关。本文综述了大鼠孕期经历应激的子代骨髓淋巴干细胞所受的影响及免疫系统的相关改变,并根据现有的研究提出假说,为进一步研究孕期应激导致子代免疫系统改变的机理研究提供新的思路。     

Adenovirus-mediated BMP2 expression in human bone marrow stromal cells

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.     

Differentiation of embryonic stem cells in adult bone marrow

Embryonic stem cells (ESCs) are a potential source of generating transplantable hematopoietic stem and progenitor cells, which in turn can serve as "seed" cells for hematopoietic regeneration. In this study, we aimed to gauge the ability of mouse ESCs directly differentiating into hematopoietic cells in adult bone marrow (BM). To this end, we first derived a new mouse ESC line that constitutively expressed the green fluorescent protein (GFP) and then injected the ESCs into syngeneic BM via intra-tibia. The progeny of the transplanted ESCs were then analyzed at different time points after transplantation. Notably, however, most injected ESCs differentiated into non-hematopoietic cells in the BM whereas only a minority of the cells acquired hematopoietic cell surface markers. This study provides a strategy for evaluating the differentiation potential of ESCs in the BM micro-environment, thereby having important implications for the physiological maintenance and potential therapeutic applications of ESCs.     

NK cells in allogeneic bone marrow transplantation

NK cells, until recently an ignored subset of lymphocytes, have begun to emerge as important cytotoxic effectors. It is now accepted that NK cells together with T cells constitute major actors in graft-versus-leukemia reaction after allogeneic bone marrow transplantation (BMT). Over the last several years the mechanisms regulating the activation of NK cells have been the subject of intense investigations encouraged by the clinical implications that these studies will have. This article provides a general overview of NK-cells biology and regulation pertinent to their function in allogeneic BMT, followed by a review of the in vivo preclinical and clinical evidence for the beneficial effect of NK cells in the adoptive immunotherapy of leukemia.     

The frequency of bone marrow cells that bind erythropoietin

The frequencies of rat and mouse bone marrow cells capable of binding erythropoietin were studied by both direct fluorescence and indirect immunofluorescence. We found that between 1-2% of the cells bound erythropoietin, that the binding was specific, and that the number of cells that bound erythropoietin was, in part, a function of the erythropoietic state of the donor animal. A statistical method for evaluating the data obtained is included.     

孕期应激对子代大鼠骨髓淋巴干细胞发育的影响

孕期应激对子代产生的影响是多方面的,这种影响是复杂的。研究表明,出生前的应激经历可导致出生后子代长期的免疫功能改变。这些改变追其根源与骨髓淋巴干细胞的改变有关。本文综述了大鼠孕期经历应激的子代骨髓淋巴干细胞所受的影响及免疫系统的相关改变,并根据现有的研究提出假说,为进一步研究孕期应激导致子代免疫系统改变的机理研究提供新的思路。     

X-ray microanalytical studies on cryofixed blood cells of the ascidian Phallusia mammillata

Summary Cryofixed blood morula cells of Phallusia mammillata (Cuvier), which are considered to be vanadium-accumulating cells, were examined by X-ray microanalysis using STEM (scanning transmission electron microscopy) and SEM (scanning electron microscopy). It is thought that cryopreparation preserves the native distribution of diffusible elements such as sodium, chlorine, and potassium, and prevents the displacement of vanadium, all of which may occur during conventional preparation. The results show that morula cell globules contain a large amount of sulphur and chlorine, and some sodium, magnesium, bromium and potassium, but very little or no vanadium.     

Modulation of one of three murine bone marrow stromal cell lines to adipose cells by serum and insulin

Adipose cells have been recognized as an integral component of the bone marrow hematopoietic microenvironment in vivo and as an essential cell type required for in vitro maintenance of stem cells. Four stromal cell lines obtained from the adherent cell population of murine bone marrow cultures have been enriched and purified by multiple trypsinizations. We noted that these cell lines exhibited an accumulation of vacuoles of lipid, the extent of which varied be-tween cell lines in response to a change from medium containing 10% fetal calf serum to medium containing 20% horse serum. The lipid was lost when the cell lines were transferred back into the medium supplemented with fetal calf serum. In light of the reported lipogenic and antilipolytic effects of insulin on fibroblasts and adipocytes, we investigated the ability of insulin to induce adipocyte transformation of these bone marrow stromal cell populations. Three cell lines were exposed to bovine insulin at concentrations ranging from 10^?9 to 10^?6 M. All three cell lines responded to the insulin by accumulating lipid, but the extent of accumulation and the insulin concentration at which maximum lipid content was attained were population specific. One cell line (MC₁) responded fully at physiological levels of insulin (10^?9 M), whereas the other two showed lipid accumulation only at pharmacological concentrations. The initial growth of MC₁ was inhibited in the presence of 10^?9 M insulin which is compatible with the observed differentiation to adipocytes. The growth of MC₃ was unaltered in the presence of physiological concentrations of insulin, whereas that of MC₄ was accelerated. Grafts of organ cultures of the cell lines under the kidney capsule of syngeneic mice developed specific characteristics rep-resentative of the different cell lines. In particular, the majority of the grafts of MC₁ consisted primarily of fat cells which were not observed in the grafts of MC₃ and MC₄. These data strongly suggest that these cell lines comprise cells with different potentialities and that the MC₁ line represents a preadipocyte stromal cell of bone marrow.     

Cyclic nucleotides and haemoglobin concentration in rabbit bone marrow cell suspensions stimulated by purified erythropoietin

This study was conducted to determine the possible correlations between cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP), and haemoglobin (Hb) concentration in nucleated cell suspensions of rabbit bone marrow incubated with erythropoietin (Ep). The levels of cAMP and cGMP were measured following the addition of different Ep concentrations to the suspensions. The Hb concentration was also measured in suspensions treated with Ep, dibutyryl cAMP (db-cAMP) or dibutyryl cGMP (db-cGMP), respectively. The following results were obtained: (1) upon the addition of 1 IU ml-1 Ep, an increase of cAMP levels was related to an increase in Hb concentration; while a decrease of Hb concentration was related to an increase of cGMP levels obtained when 0.1 IU ml-1 Ep was present in the incubation mixture. (2) A mimetic effect on Hb concentration was obtained upon the addition of db-cAMP or db-cGMP to the suspensions. (3) A quantitative correlation was found between the cAMP/cGMP ratio and Hb levels in cellular suspensions. This rapport was reviewed with respect to the controls as a decrease in Hb concentration when the ratio is less than one and an increase in Hb concentration when the ratio is greater than one.     

Characterization and hepatogenic differentiation of mesenchymal stem cells from human amniotic fluid and human bone marrow: a comparative study

Since stem cells can differentiate into hepatocyte, stem cell-based therapy becomes a potential alternative treatment for terminal liver diseases. However, an appropriate source of human mesenchymal stem cells (hMSCs) for hepatocytes has not yet been clearly elucidated. The aim of the present study was to investigate the in vitro biological characterization and hepatic differentiation potential of human amniotic fluid-derived mesenchymal stem cells (AF-hMSCs) and human bone marrow-derived mesenchymal stem cells (BM-hMSCs). Our results show that AF-hMSCs possess higher proliferation and self-renewal capacity than BM-hMSCs. Cytogenetic studies indicate that AF-hMSCs are as genetically stabile as BM-hMSCs. Following incubation with specific hepatogenic agents, AF-hMSCs showed a higher hepatic differentiation potential than BM-hMSCs. Expression of several liver-specific markers was significantly greater in AF-hMSCs than in BM-hMSCs, as shown by real time RT-PCR and immunofluorescence (IF). In conclusion, AF-hMSCs possess superior potential for hepatic differentiation, making them more suitable for diverse terminal liver diseases.     

骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用

本文通过制备小鼠骨髓内皮细胞无血清条件培养液(serum-free murine bone marrow endothelial cell conditioned medium, mBMEC-CM),经超滤分为分子量>10 kDa组分和<10 kDa组分,分别观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞集落生成的影响。用Wright’S Giemsa染色计数内皮细胞集落及检测骨髓内皮细胞的vWF,通过[3H]- TdR掺入量,观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞增殖的影响,并用分子杂交方法检测内皮细胞表达的细胞因子,从几个方面来研究mBMEC-CM对骨髓内皮细胞增殖的作用。结果显示,骨髓内皮细胞vWF 检测阳性。mBMEC-CM原液及其分子量>10 kDa组分能刺激骨髓内皮细胞集落增殖,且能明显增加骨髓内皮细胞[3H]-TdR 掺入量;分子量<10 kDa组分对骨髓内皮细胞集落增殖无明显刺激作用,也不能增加骨髓内皮细胞[3H]-TdR掺入量。外源加入IL-6、IL-11、SCF、GM-CSF、VEGF、bFGF 6种细胞因子能明显刺激骨髓内皮细胞集落增殖,SCF、VEGF、bFGF能明显增加骨髓内皮细胞[3H]-TdR掺入量。Atlas array膜杂交实验显示骨髓内皮细胞内源性表达GM-CSF、SCF、MSP-1、endothelin-2、thymosin β10、connective tissue GF、PDGF-A chain、MIP-2α、PlGF、neutrophil activating protein ENA-78、INF-γ、IL-1、IL-6、IL-13、IL-11、inhibin-α等细胞因子的mRNA。上述结果提示,骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖具有促进作用。     

Retracted: Icariin attenuates methotrexate chemotherapy-induced bone marrow microvascular damage and bone loss in rats

Methotrexate (MTX), a widely used antimetabolite in paediatric cancer to treatment, has been widely reported to cause bone loss and bone marrow (BM) microvascular (particularly sinusoids) damage. Investigations must now investigate how MTX-induced bone loss and microvasculature damage can be attenuated/prevented. In the present study, we examined the potency of icariin, an herbal flavonoid, in reducing bone loss and the dilation/damage of BM sinusoids in rats caused by MTX treatment. Groups of young rats were treated with five daily MTX injections (0.75 mg/kg) with and without icariin oral supplementation until Day 9 after the first MTX injection. Histological analyses showed a significant reduction in the bone volume/tissue volume (BV/TV) fraction (%) and trabecular number in the metaphysis trabecular bone of MTX-treated rats, but no significant changes in trabecular thickness and trabecular spacing. However, the BV/TV (%) and trabecular number were found to be significantly higher in MTX + icariin-treated rats than those of MTX alone-treated rats. Gene expression analyses showed that icariin treatment maintained expression of osteogenesis-related genes but suppressed the induction of adipogenesis-related genes in bones of MTX-treated rats. In addition, icariin treatment attenuated MTX-induced dilation of BM sinusoids and upregulated expression of endothelial cell marker CD31 in the metaphysis bone of icariin + MTX-treated rats. Furthermore, in vitro studies suggest that icariin treatment can potentially enhance the survival of cultured rat sinusoidal endothelial cells against cytotoxic effect of MTX and promote their migration and tube formation abilities, which is associated with enhanced production of nitric oxide.     

运动诱导的低铁状态大鼠骨髓细胞铁摄入的变化

本文观察了运动性低铁状态大鼠骨髓细胞转铁蛋白 (Tf)结合铁和非Tf结合铁摄入的变化。大鼠随机分为 6个月的运动组 (EG)和对照组 (SG)。SG平均每个幼红细胞Tf受体数为 890 15 0± 16 4849个 ,而在EG为 2 17536 0± 46 2 737个 (P <0 0 5 ) ,但受体的解离常数不受运动影响。EG中Tf的内吞平台和胞内铁聚积速度显著高于SG ,胞浆和胞内膜性成分中Tf结合铁和Fe(Ⅱ )摄入增加。EG的胞浆内Fe(Ⅱ )摄入的米氏常数值降低 ;细胞膜性成分中Fe(Ⅱ )摄入的最大速度增加。上述结果表明 ,运动不仅通过增加Tf受体的表达促进Tf结合铁的内吞 ,而且增强非Tf结合铁的内吞途径。尽管这些变化的机制尚不清楚 ,但它们有利于运动时血红素的合成     

Human bone marrow stromal cells express an osteoblastic phenotype in culture

Summary This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two “clonings” and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [³H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D₃ by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of β-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by “budding” structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with⁴⁵Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.     

Standardization of the immunocytochemical detection of neuroblastoma cells in bone marrow.

Standard cytomorphological examination of bone marrow (BM) aspirates does not appear to be sensitive enough to detect single neuroblastoma cells. The SIOPEN Neuroblastoma Bone Marrow Committee developed a sensitive and reproducible anti-GD2 immunocytochemical assay and introduced morphological and immunocytological criteria for the interpretation of results. Fixed cytospins were incubated with a commercially available anti-GD2 monoclonal antibody and an APAAP kit. Cells fulfilling all morphological and immunocytological criteria were called criteria-positive cells (CPCs). Not convincingly interpretable cells fulfilled some, but not all, criteria, and negative cells displayed only exclusion criteria. The genetic profile of doubtful cells was checked by fluorescence in situ hybridization. Ideally, 3 x 10(6) cells were analyzed to reach a 95% probability of detecting one tumor cell in 1 x 10(6) mononuclear cells. Four quality control rounds were organized to validate the method. A total of 111 quality control samples were analyzed. Two main improvements were achieved: in discordant cases, the range between the lowest and highest reported result was reduced by half, and discordant results were only found in samples with less than 10 CPCs per 1 x 10(6). This article describes the first internationally standardized protocol to detect and quantify rare neuroblastoma cells by immunocytochemistry. This method is an indispensable tool for multicenter studies evaluating the clinical significance of minimal residual disease in neuroblastoma.