Proliferation capacity, neuronal differentiation potency and microstructures after the differentiation of canine bone marrow stromal cells into neurons

We examined the proliferation capacity and neuronal differentiation potency of canine bone marrow stromal cells (BMSCs). In addition, the microstructures of neuron-like cells after neuronal differentiation were observed under a scanning electron microscope. Canine BMSCs grew to confluency at 10.0 ± 2.5 days, and 3.8 ± 2.1 × 10(6) BMSCs were collected in one passage. Approximately 65% of canine BMSCs changed to neuron-like morphology after neuronal differentiation, and nearly all neuron-like cells stained positive against neuron-specific enolase. In addition, microstructures such as the cellular organelles, filaments and growth cones of these cells bore a close resemblance to those of the original mature neurons. These results suggested that canine BMSCs might be capable of differentiating into neurons.     

Characterization of neuron-like cells derived from canine bone marrow stromal cells

Regenerative therapy using bone marrow stromal cells (BMSCs) has begun to be clinically applied in humans and dogs for neurological disorders such as spinal cord injury. Under appropriate conditions in vitro, BMSCs differentiate into neuronal cells, which may improve the effects of regenerative therapy. In this study, we evaluated canine neuron-like cells (NLCs) derived from BMSCs. We speculated on their suitability for neuro-transplantation from the point of view of their morphological features, long-term viability, abundant availability, and ability to be subcultured. Canine NLCs were differentiated as follows: third-passage BMSCs were maintained in pre-induction medium containing 2-mercaptoethanol and dimethylsulfoxide for 5 h, and then cells were transferred to neuronal induction medium containing fetal bovine serum, basic fibroblast growth factor, epidermal growth factor, dibutyryl cyclic AMP, and isobutylmethylxanthine for 7 or 14 days. Canine NLCs fulfilled the transplantation criteria and expressed markers of both immature neurons (nestin, 84.7 %) and mature neuronal cells (microtubule-associated protein-2, 95.7 %; βIII-tubulin protein, 12.9 %; glial fibrillary acidic protein, 9.2 %). These results suggest that canine BMSCs can be induced to differentiate into neuronal cells and may be suitable for neuro-transplantation. This study may provide information for improving cellular therapy for neurological diseases.     

Use of autologous bone marrow mononuclear cells and cultured bone marrow stromal cells in dogs with orthopaedic lesions

The aim of the study is to evaluate the clinical application in veterinary orthopedics of bone marrow mononuclear cells (BMMNCs) and cultured bone marrow stromal cells (cBMSCs) for the treatment of some orthopaedic lesions in the dog. The authors carried out a clinical study on 14 dogs of different breed, age and size with the following lesions: 1 bone cyst of the glenoid rime; 2 nonunion of the tibia; 3 nonunion of the femur; 2 lengthening of the radius; 1 large bone defect of the distal radius;1 nonunion with carpus valgus; 4 Legg-Calvé-Perthés disease. In 9 cases the BMMCNs were used in combination with a three dimensional resorbable osteogenic scaffold the chemical composition and size of which facilitates the ingrowth of bone. In these cases the BMMNCs were suspended in an adequate amount of fibrin glue and then distribuited uniformly on a Tricalcium-Phosphate (TCP) scaffold onto which were also added some drops of thrombin. In 1 case of nonunion of the tibia and in 3 cases of Legg-Calvè-Perthés (LCP) disease the cultured BMSCs were used instead because of the small size of the dogs and of the little amount of aspirated bone marrow. X-ray examinations were performed immediately after the surgery. Clinical, ultrasounds and X-ray examinations were performed after 20 days and then every month. Until now the treated dogs have shown very good clinical and X-ray results. One of the objectives of the study was to use the BMMNCs in clinical application in orthopaedic lesions in the dog. The advantages of using the cells immediately after the bone marrow is collected, are that the surgery can be performed the same day, the cells do not need to be expanded in vitro, they preserve their osteogenic potential to form bone and promote the proper integration of the implant with the bone and lastly, the technique is easier and the costs are lower.     

Use of autologous bone marrow mononuclear cells and cultured bone marrow stromal cells in dogs with orthopaedic lesions

The aim of the study is to evaluate the clinical application in veterinary orthopedics of bone marrow mononuclear cells (BMMNCs) and cultured bone marrow stromal cells (cBMSCs) for the treatment of some orthopaedic lesions in the dog. The authors carried out a clinical study on 14 dogs of different breed, age and size with the following lesions: 1 bone cyst of the glenoid rime; 2 nonunion of the tibia; 3 nonunion of the femur; 2 lengthening of the radius; 1 large bone defect of the distal radius;1 nonunion with carpus valgus; 4 Legg-Calvé-Perthés disease. In 9 cases the BMMCNs were used in combination with a three dimensional resorbable osteogenic scaffold the chemical composition and size of which facilitates the ingrowth of bone. In these cases the BMMNCs were suspended in an adequate amount of fibrin glue and then distribuited uniformly on a Tricalcium-Phosphate (TCP) scaffold onto which were also added some drops of thrombin. In 1 case of nonunion of the tibia and in 3 cases of Legg-Calvè-Perthés (LCP) disease the cultured BMSCs were used instead because of the small size of the dogs and of the little amount of aspirated bone marrow. X-ray examinations were performed immediately after the surgery. Clinical, ultrasounds and X-ray examinations were performed after 20 days and then every month. Until now the treated dogs have shown very good clinical and X-ray results. One of the objectives of the study was to use the BMMNCs in clinical application in orthopaedic lesions in the dog. The advantages of using the cells immediately after the bone marrow is collected, are that the surgery can be performed the same day, the cells do not need to be expanded in vitro, they preserve their osteogenic potential to form bone and promote the proper integration of the implant with the bone and lastly, the technique is easier and the costs are lower.     

Separation of bovine bone marrow into maturation-related myeloid cell fractions

A prerequisite for studies on bovine myeloid cells in relation to maturity is a reliable separation method, in order to obtain enriched and partially purified cell fractions of different maturation stages. Since current techniques for bovine bone marrow cell isolation fall short of this requirement, a technique for fractionating bovine bone marrow using a three-layer discontinuous Percoll gradient was developed. Three maturation-dependent myeloid cell fractions were obtained at specific densities, as maturation of cells is accompanied with a progressive density increase. Early immature myeloid cells, i.e. myeloblasts and promyelocytes, were found at a density of 1.060g/ml. Late immature myeloid cells, i.e. myelocytes and metamyelocytes, were retrieved at 1.080g/ml. Bands and segmented cells, representing the mature fraction, accumulated in the high-density pellet (>1.080g/ml). Myeloid cell populations were identified in each fraction by flow cytometry based on their forward and side scatter pattern. Confirmation was provided by light microscopy of flow cytometrically sorted myeloid populations, using morphological characteristics. The developed method provides a unique tool for studying maturation-dependent functions in bovine bone marrow.     

Culture of bovine bone marrow progenitor cells in vitro.

In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400 g). The use of 3% bovine leucocyte-conditioned medium, produced by stimulation of blood lymphocytes with 4 microg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte-conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose-based medium requires different culture conditions, which are different from the culture conditions for human cells.     

Differentiation of canine bone marrow stromal cells into voltage- and glutamate-responsive neuron-like cells by basic fibroblast growth factor

We investigated the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into voltage- and glutamate-responsive neuron-like cells. BMSCs were obtained from the bone marrow of healthy beagle dogs. Canine BMSCs were incubated with the basal medium for neurons containing recombinant human basic fibroblast growth factor (bFGF; 100 ng/ml). The viability of the bFGF-treated cells was assessed by a trypan blue exclusion assay, and the morphology was monitored. Real-time RT-PCR was performed to evaluate mRNA expression of neuronal, neural stem cell and glial markers. Western blotting and immunocytochemical analysis for the neuronal markers were performed to evaluate the protein expression and localization. The Ca²⁺ mobilization of the cells was evaluated using the Ca²⁺ indicator Fluo3 to monitor Ca²⁺ influx. To investigate the mechanism of bFGF-induced neuronal differentiation, the fibroblast growth factor receptor inhibitor, the phosphoinositide 3-kinase inhibitor or the Akt inhibitor was tested. The bFGF treatment resulted in the maintenance of the viability of canine BMSCs for 10 days, in the expression of neuronal marker mRNAs and proteins and in the manifestation of neuron-like morphology. Furthermore, in the bFGF-treated BMSCs, a high concentration of KCl and L-glutamate induced an increase in intracellular Ca²⁺ levels. Each inhibitor significantly attenuated the bFGF-induced increase in neuronal marker mRNA expression. These results suggest that bFGF contributes to the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells and may lead to the development of new cell-based treatments for neuronal diseases.     

Comparative study of equine bone marrow and adipose tissue-derived mesenchymal stromal cells

Reasons for performing study: Mesenchymal stromal cells (MSCs) represent an attractive source for regenerative medicine. However, prior to their application, fundamental questions regarding molecular characterisation, growth and differentiation of MSCs must be resolved. Objectives: To compare and better understand the behaviour of equine MSCs obtained from bone marrow (BM) and adipose tissue (AT) in culture. Methods: Five horses were included in this study. Proliferation rate was measured using MTT assay and cell viability; apoptosis, necrosis and late apoptosis and necrosis were evaluated by flow cytometry. The mRNA expression levels of 7 surface marker genes were quantified using RT‐qPCR and CD90 was also analysed by flow cytometry. Differentiation was evaluated using specific staining, measurement of alkaline phosphatase activity and analysis of the mRNA expression. Results: High interindividual differences were observed in proliferation in both cell types, particularly during the final days. Statistically significant differences in viability and early apoptosis of cultured AT‐ and BM‐MSCs were found. The highest values of early apoptosis were observed during the first days of culture, while the highest percentage of necrosis and late apoptosis and lowest viability was observed in the last days. Surface marker expression pattern observed is in accordance to other studies in horse and other species. Osteogenic differentiation was evident after 7 days, with an increasing of ALP activity and mRNA expression of osteogenic markers. Adipogenic differentiation was achieved in BM‐MSCs from 2 donors with one of the 16 media tested. Chondrogenic differentiation was also observed. Conclusions: Proliferation ability is different in AT‐MSCs and BM‐MSCs. Differences in viability and early apoptosis were observed between both sources and CD34 was only found in AT‐MSCs. Differences in their osteogenic and adipogenic potential were detected by staining and quantification of specific tissue markers. Potential relevance: To provide data to better understand AT‐MSCs and BM‐MSCs behaviour in vitro.     

Epidemiology: Culture of bovine bone marrow progenitor cells in vitro

Summary

In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400g). The use of 3% bovine leucocyte‐conditioned medium, produced by stimulation of blood lymphocytes with 4 pg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte‐conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose‐based medium requires different culture conditions, which are different from the culture conditions for human cells.     

Influence of an autologous serum-supplemented medium on the proliferation and differentiation into neurons of canine bone marrow stromal cells

We investigated the influence of autologous serum (AS)-supplemented medium on the proliferation and differentiation into neurons of canine bone marrow stromal cells (BMSCs). Canine BMSCs were cultured using α-MEM only, α-MEM with 10% fetal bovine serum (FBS), and 5, 10 and 20% AS-supplemented α-MEM. Growth of canine BMSCs was observed in all AS groups. The proliferation capacity of canine BMSCs in the AS groups was similar to that in the FBS group. No significant differences between the FBS and AS groups were observed in the percentage of the cells that changed to the neuron-like morphology and neuron-specific enolase-positive ratio after neuronal differentiation. Canine BMSCs cultured using AS-supplemented medium were able to proliferate and showed neuronal differentiation potency.     

Expression and distribution of the Kit receptor in bovine bone marrow cells

OBJECTIVE: To characterize the expression and distribution of the Kit receptor in bovine bone marrow cells (BMC) and to define the function of its ligand, stem cell factor (SCF). ANIMALS: Six 7- to 70-day-old healthy male Holstein-Friesian calves. PROCEDURES: Expression and distribution of the Kit receptor were assessed by use of flow cytometry with monoclonal antibodies (mAb) against the bovine Kit protein. Using Giemsa-stained centrifuged preparations, the histologic appearance of Kit receptor positive (Kit+) BMC were evaluated. Semisolid cultures supplemented with granulocyte colony-stimulating factor (G-CSF) and SCF were used to measure the colony formation capacity of Kit+ BMC. RESULTS: The Kit receptor was expressed on approximately 18% of total BMC. Most of Kit+ BMC did not coexpress lineage markers, but a small subset of this population did coexpress CD3. The Kit+CD3- BMC were a heterogeneous cell population comprising blast-like cells such as myeloblasts, promyelocytes, rubriblasts, and prorubricytes. Conversely, Kit+CD3+ BMC had a lymphocyte-like appearance. Kit+ BMC formed colonies in semisolid culture with G-CSF, whereas Kit- BMC failed to grow. Addition of SCF to G-CSF resulted in superadditive enhancement in colony numbers and size. CONCLUSIONS: The Kit receptor is expressed primarily on immature blood cells in bovine bone marrow, and Kit+ BMC contain hematopoietic progenitor cells that are reactive to G-CSF. In addition, SCF synergizes with G-CSF to stimulate colony formation by these cells. Our results suggest that the Kit receptor and its ligand, SCF, are involved in early stages of granulopoiesis in calves.     

Culture of macrophages from bovine bone marrow

Macrophages perform important immunoregulatory and host defense functions. Examination of this cell type in the bovine has been restricted because of lack of a means to obtain pure bovine macrophage populations reproducibly. We have developed a system for production of large numbers of macrophages from this species with greater than 99% purity. Stem cells were obtained from the bone marrow of neonatal calves and cultured in vitro in the presence of macrophage-colony-stimulating factor. Bovine bone marrow culture-derived macrophages were esterase-positive, expressed Fc receptors for aggregated IgG, and bovine macrophage differentiation markers. In addition, they displayed class I and class II major histocompatibility (MHC) antigens. The level of MHC antigen expressed could be further enhanced by treatment with recombinant bovine interferons. The macrophages exhibited expected functions, for example, Fc-mediated ingestion of opsonized sheep red blood cells. Augmentation of phagocytic capacity by either alpha or gamma interferon could also be demonstrated. The data reported here confirm that bone marrow culture is a convenient, reliable source of macrophages for investigations of this bovine cell type.     

Clinical safety of intratesticular transplantation of allogeneic bone marrow multipotent stromal cells in stallions

Although stem cell therapy is a promising alternative for treatment of degenerative diseases, there are just few reports on the use of stem cells therapy in horse's reproductive system. This study aims to evaluate the effect of intratesticular injection of bone marrow mesenchymal stromal/stem cells (MSCs) in healthy stallions, and its outcome on seminal parameters and fertility. In Experiment 1, 24 stallions were divided into treatment group (TG) and control group (CG). In the TG, an intratesticular application of MSC was performed, and in the CG, only PBS was used. Measurements of testicular volume, surface temperature and Doppler ultrasonography were performed 24 and 48 hr after treatments. Fifteen days after application, the testicles were removed and submitted to histological analysis. In Experiment 2, 3 fertile stallions received similarly treatment with MSCs. Physical examination and sperm analysis were performed weekly during 60 days after treatment, and at the end, semen from one of them was used for artificial inseminations of 6 healthy mares. In Experiment 1, clinical examinations showed no signals of acute inflammation on both groups according to the analysed variables (p > .05). Also, no signal of chronic inflammation was observed on histological evaluation. In Experiment 2, stallions presented no physical alterations or changes in sperm parameters, and a satisfactory fertility rate (83%; 5/6) was observed after AI. The results support the hypothesis that intratesticular application of bone marrow MSCs is a safe procedure, and this could be a promising alternative to treat testicular degenerative conditions.     

镉对体外培养鸡骨髓基质细胞成骨分化的毒性作用

骨是镉毒性作用的主要靶器官之一,但其对鸡骨髓基质细胞(bone marrow stromal cells,BMSCs)增殖和成骨分化的毒性作用仍不清楚.本研究利用差速贴壁纯化法获得鸡BMSCs,加入不同浓度镉处理不同时间,采用CCK-8法检测细胞增殖,碱性磷酸酶(alkaline phosphatase,ALP)和茜素...     

Interactions of porcine alveolar macrophages and bone marrow cells with African swine fever virus and virus-infected cells

Virus yields from porcine alveolar macrophages (AM) infected with African swine fever virus (ASFV) were greater and were achieved more rapidly, when inoculated at a high multiplicity of infection (MOI) than at low MOI. The difference was related to a lower percentage of cells becoming infected after low MOI inoculation. The reduced yields after low MOI were not caused by prolongation of the culture time, by bacterial endotoxins or by production of inhibitory substances by infected AM. Virus-infected AM were not susceptible to lysis in antibody-dependent cell mediated cytotoxicity (ADCC) assays and this was apparently due to a paucity of viral antigen expressed on the cell surface. Uninfected AM did not act as effectors in ADCC.Porcine bone marrow (PBM) cells were effective in mediation of ADCC and their activity was reduced after ASFV infection. Cells separated into adherent and non-adherent populations, depleted by carbonyl iron treatment or separated by Ficoll-Hypaque centrifugation, all showed effector activity in ADCC. The effector cells were not mature neutrophils or lymphocytes and were probably granulocytic precursors.     

体外诱导牛BMSC向上皮样细胞分化的研究

为培育转基因肉牛提供种子细胞以及进一步丰富牛骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)的多向分化潜能,利用细胞免疫荧光染色和分子生物学方法,初步探讨表皮生长因子和胰岛素体外诱导牛BMSC向上皮样细胞分化的可能性。利用含细胞因子的诱导液对纯化稳定的P4代牛BMSC进行体外诱导,并对诱导后的细胞进行细胞角蛋白18的细胞免疫荧光观察和细胞角蛋白19的RT-PCR鉴定。结果表明,诱导后细胞经细胞角蛋白18免疫荧光染色后出现明显的荧光。RT-PCR结果显示诱导分化后细胞角蛋白19基因在细胞中表达。因此,在体外,表皮生长因子和胰岛素可诱导牛BMSC初步分化为上皮样细胞。     

Flow cytometric analysis of the DNA content of bovine and human bone marrow cells

The defence against infection in high-yielding dairy cows is correlated with the number and function of circulating neutrophils and depends on their production in bone marrow. Therefore, the DNA content of isolated bone marrow cell suspensions from 7 calves, 7 cows and 14 humans was assayed by flow cytometry. Bovine sternal bone marrow samples were collected within 30 min of death, and human marrow samples were collected by sternal puncture and aspiration. Mononucleated cells were isolated by gradient centrifugation. In the bone marrow samples from calves and cows, 35 +/- 2.6% and 31.8 +/- 1.5% of the isolated bone marrow cells respectively were in the S/G2/M-phase. The difference between calves and cows was not significant. In the human samples, only 12 +/- 0.8% of the cells were in the S/G2/M-phase. A significant (P < 0.001) difference was observed between the two species. These results indicated that the proliferative, in activity of haematopoietic cells is significantly higher in cattle than in humans.     

猪骨髓间充质干细胞分离培养及生物学特性

采集健康猪股骨骨髓,利用percoll梯度离心法分离单个核细胞,进行体外原代和传代培养,并用不同代数细胞进行了细胞生长能力、膜表面抗原(CD105、CD90、CD45)及诱导分化能力的检测.结果显示分离培养的单个核细胞贴壁生长,形态呈成纤维细胞样及涡旋状克隆团;细胞传代至第17代生长状况仍然良好;用F3代细胞进行膜表面抗原标记显示,CD90和CD105阳性表达率分别为(97.4±1.8)％和(99.6±0.9)％,而CD45阳性表达率仅为(1.8±0.55)％,证实这些细胞具有猪骨髓间充质干细胞(Mesenchymal stem Cells,MSCs)表面抗原;经地塞米松、维生素C和β-磷酸甘油等诱导F3代细胞,21d后出现钙物质沉积细胞群,用茜素红和Von kossa染色呈阳性,证实这些细胞已分化形成成骨细胞;经地塞米松、IBMX、胰岛素及吲哚美辛等诱导F3代细胞,21d后细胞质出现脂滴样结构,用油红O染色呈阳性,证实已分化形成成脂细胞;冷冻-解冻细胞的膜表面抗原及多能分化能力与未冻存细胞的检测结果基本一致.结果证实,从猪骨髓中分离培养及经传代扩增获得的贴壁细胞是纯化的MSCs.     

Incorporation of dietary polyunsaturated fatty acids into pork fatty tissues.

The incorporation and elimination rate of dietary PUFA in pork fat was investigated in this study. Experiment 1 examined the incorporation of dietary PUFA into backfat (BF) and into the triglyceride (TG) and phospholipid (PL) fractions of the i.m. fat of the loin. Experiment 2 examined the elimination of PUFA from BF due to withdrawal of PUFA from the diet. In Exp. 1, Piétrain x Seghers Hybrid pigs (70 barrows and 70 gilts averaging 11 wk of age and 30 kg initially) were assigned to five dietary treatments in a 2 x 5 factorial arrangement during a 16-wk feeding period. Pigs received a diet containing about 2.5% tallow (T) for 8, 10, 12, 14, or 16 wk, followed by a diet containing about 15% full-fat soybeans (FFS) for 8, 6, 4, 2, or 0 wk, respectively. Additionally, BF biopsies were taken every 2 wk from eight pigs on the 8-wk FFS dietary treatment, starting from the time pigs were switched to the FFS diet. Linoleic acid [18:2(n-6)], linolenic acid [18:3(n-3)], eicosadienoic acid [20:2(n-6)], arachidonic acid [20:4(n-6)], and PUFA contents in BF increased (P < .01) with time on the FFS diet, but contents of these fatty acids were similar for pigs fed FFS for 6 or 8 wk. The PUFA content of the biopsies rose throughout the FFS treatment; the greatest increase in PUFA occurred during the first 2 wk of feeding FFS. The PUFA content of the TG and PL fractions in i.m. fat of the longissimus muscle tended to increase with time on the FFS diet. The increase was significant (P < .01) in the TG fraction for 18:3(n-3) and in the PL fraction for 20:4(n-6) and 22:6(n-3). In Exp. 2, 11-wk-old pigs (10 barrows and 10 gilts) were fed a FFS-based diet from 11 to 19 wk of age, followed by the T-diet for an additional 8 wk. During the latter period, biopsies were taken every 2 wk. The elimination of PUFA from BF was greatest during the first 2 wk after the dietary change. The PUFA reached the lowest level at the age of 25 wk. These experiments show that the PUFA: saturated fatty acid ratio of BF can be increased from .34, corresponding with a T-based diet, to .55, by feeding a FFS diet for 6 wk before slaughter.