Effect of matrigel on the osteogenic potential of canine adipose tissue-derived mesenchymal stem cells

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) are a promising source of cells for bone tissue engineering. Matrigel is a basement membrane extract containing multiple extracellular components. This mixture may promote the osteogenic differentiation of MSCs and provide a more appropriate microenvironment for transplanted cells. Here, we investigated the effect of Matrigel on the osteogenic potential of Ad-MSCs. Canine Ad-MSCs were cultured in 2D and 3D matrices and implanted into subcutaneous pouches of dogs either with or without Matrigel. Culture mineralization, cell adhesion efficiency, cell proliferation, osteoid matrix production and alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase activities were quantified and compared. Ad-MSCs grown in 2D cultures with Matrigel showed higher levels of calcium deposition and ALP activity than those grown in the absence of Matrigel under osteogenic conditions. In 3D cultures, the cells cultivated with Matrigel showed greater attachment, proliferation and osteogenic differentiation than those grown without Matrigel. In vivo, Ad-MSCs implanted with Matrigel showed higher osteogenic potential than those without Matrigel. In conclusion, these data suggest that the use of Matrigel can increase the osteogenic potential of canine Ad-MSCs.     

Expression of neural markers on bone marrow-derived canine mesenchymal stem cells

OBJECTIVE: To evaluate cell surface markers of bone marrow-derived canine mesenchymal stem cells (MSCs) by use of flow cytometric analysis and determine whether canine MSCs express proteins specific to neuronal and glial cells. SAMPLE POPULATION: Bone marrow aspirates collected from iliac crests of 5 cadavers of young adult dogs. PROCEDURES: Flow cytometric analysis was performed to evaluate cell surface markers and homogeneity of third-passage MSCs. Neural differentiation of canine MSCs was induced by use of dibutyryl cAMP and methyl-isobutylxanthine. Expressions of neuronal (beta III-tubulin) and glial (glial fibrillary acidic protein [GFAP] and myelin basic protein) proteins were evaluated by use of immunocytochemical and western blot analyses before and after neural differentiation. RESULTS: Third-passage canine MSCs appeared morphologically homogeneous and shared phenotypic characteristics with human and rodent MSCs. Immunocytochemical and western blot analyses revealed that canine MSCs constitutively expressed beta III-tubulin and GFAP. After induction of neural differentiation, increased expression of GFAP was found in all samples, whereas such change was inconsistent in beta III-tubulin expression. Myelin basic protein remained undetectable on canine MSCs for these culture conditions. CONCLUSIONS AND CLINICAL RELEVANCE: Canine bone marrow-derived mononuclear cells yielded an apparently homogeneous population of MSCs after expansion in culture. Expanded canine MSCs constitutively expressed neuron or astrocyte specific proteins. Furthermore, increases of intracellular cAMP concentrations induced increased expression of GFAP on canine MSCs, which suggests that these cells may have the capacity to respond to external signals. Canine MSCs may hold therapeutic potential for treatment of dogs with neurologic disorders.     

Stem cell factor supports migration in canine mesenchymal stem cells

Adult Mesenchymal Stem Cells (MSC) are cells that can be defined as multipotent cells able to differentiate into diverse lineages, under appropriate conditions. These cells have been widely used in regenerative medicine, both in preclinical and clinical settings. Initially discovered in bone marrow, MSC can now be isolated from a wide spectrum of adult and foetal tissues. Studies to evaluate the therapeutic potential of these cells are based on their ability to arrive to damaged tissues. In this paper we have done a comparative study analyzing proliferation, surface markers and OCT4, SOX9, RUNX2, PPARG genes expression in MSC cells from Bone marrow (BMMSC) and Adipose tissue (ASC). We also analyzed the role of Stem Cell Factor (SCF) on MSC proliferation and on ASCs metalloproteinases MMP-2, MMP-9 secretion. Healthy dogs were used as BMMSC donors, and ASC were collected from omentum during elective ovariohysterectomy surgery. Both cell types were cultured in IMDM medium with or without SCF, 10% Dog Serum (DS), and incubated at 38 °C with 5% CO2. Growth of BMMSCs and ASCs was exponential until 25–30 days. Flow citometry of MSCs revealed positive results for CD90 and negative for CD34, CD45 and MCH-II. Genes were evaluated by RT-PCR and metalloproteinases by zymografy. Our findings indicate morphological and immunological similarities as well as expression of genes from both origins on analyzed cells. Furthermore, SCF did not affect proliferation of MSCs, however it up-regulated MMP-2 and MMP-9 secretion in ASCs. These results suggest that metalloproteinases are possibly essential molecules pivoting migration.     

Contrasting effect of perlecan on adipogenic and osteogenic differentiation of mesenchymal stem cells in vitro

Perlecan, a basement membrane component, shows diverse functions in different organs and tissues. However, the role of perlecan in differentiation of mesenchymal stem cells (MSCs) has been barely investigated. In this study, we examined the effect of perlecan on adipogenic and osteogenic differentiation of MSCs in vitro by adding extrinsic perlecan to culture media or blocking the function of intrinsic perlecan expressed into culture media by differentiating MSCs. Extrinsic perlecan suppressed adipogenic differentiation; however, it promoted osteogenic differentiation. These functions were further confirmed by a study of blocking intrinsic perlecan. Perlecan treated with heparitinase‐I also showed the suppressive effect on adipogenic differentiation. In contrast, the promotive effect on osteogenic differentiation was found to be heparan sulfate‐dependent. Intrinsic perlecan was suggested to be effective at the late stage of adipogenic differentiation by a study of perlecan‐blocking performed at distinct periods, but was suggested to be effective at the early stage of osteogenic differentiation. Our results showed perlecan has contrasting effect on adipogenic and osteogenic differentiation of MSCs due to its diverse actions. Based on these outcomes, we recognized that employing extrinsic perlecan or blocking intrinsic perlecan is effective for regulating adipogenic and osteogenic differentiation of MSCs by restricting its direction.     

Isolation and characterization of canine umbilical cord blood-derived mesenchymal stem cells

Human umbilical cord blood-derived mesenchymal stem cells (MSCs) are known to possess the potential for multiple differentiations abilities in vitro and in vivo. In canine system, studying stem cell therapy is important, but so far, stem cells from canine were not identified and characterized. In this study, we successfully isolated and characterized MSCs from the canine umbilical cord and its fetal blood. Canine MSCs (cMSCs) were grown in medium containing low glucose DMEM with 20% FBS. The cMSCs have stem cells expression patterns which are concerned with MSCs surface markers by fluorescence-activated cell sorter analysis. The cMSCs had multipotent abilities. In the neuronal differentiation study, the cMSCs expressed the neuronal markers glial fibrillary acidic protein (GFAP), neuronal class III β tubulin (Tuj-1), neurofilament M (NF160) in the basal culture media. After neuronal differentiation, the cMSCs expressed the neuronal markers Nestin, GFAP, Tuj-1, microtubule-associated protein 2, NF160. In the osteogenic & chondrogenic differentiation studies, cMSCs were stained with alizarin red and toluidine blue staining, respectively. With osteogenic differentiation, the cMSCs presented osteoblastic differentiation genes by RT-PCR. This finding also suggests that cMSCs might have the ability to differentiate multipotentially. It was concluded that isolated MSCs from canine cord blood have multipotential differentiation abilities. Therefore, it is suggested that cMSCs may represent a be a good model system for stem cell biology and could be useful as a therapeutic modality for canine incurable or intractable diseases, including spinal cord injuries in future regenerative medicine studies.     

The canine epiphyseal-derived mesenchymal stem cells are comparable to bone marrow derived-mesenchymal stem cells

Mesenchymal stem cells (MSCs) hold great potential in cell therapy and have attractedincreasing interests in a wide range of biomedical sciences. However, the scarcity of MSCsand the prolonged isolation procedure limited the clinical application. To address these 2issues, we developed a method to isolate MSCs from bone biopsy tissues of euthanizedcanine body donors. Compared to the traditional method to isolate MSCs from aspirated bonemarrow (BMSCs), the isolation procedure for MSCs from harvested epiphyseal cancellous bone(EMSCs) was less time-consuming. The isolated EMSCs had similar plastic-adherence,tri-lineage differentiation and consistent surface marker profiles compared to BMSCs. Weharvested BMSCs and EMSCs from 24 euthanized cases from clinics and 42 euthanized donorsfrom a local shelter. The successful rate for EMSC isolation is significantly highercompared to BMSC isolation, while the other properties of the isolated MSCs including theclonogenicity, proliferative potentials and molecular phenotypes were not discerniblydifferent between the MSCs established by the two methods. In conclusion, we demonstrateda new procedure to harvest MSCs by bone biopsy at the epiphyseal region. This method isless time consuming and more reliable, and the resulting MSCs are comparable to thoseharvested by bone marrow aspiration. The combination of the two methods can greatlyimprove the efficiency to harvest MSCs.     

Sequential sub-passage decreases the differentiation potential of canine adipose-derived mesenchymal stem cells

Adipose-derived mesenchymal stem cells (AD-MSCs) are abundant in adipose tissue from animals of all ages, are easily isolated, can differentiate into multi-lineage cells, and have a clinical application. This promising potential may only be achieved if the cells are expanding in a large number while maintaining their stemness in sequential passages. In this study, canine AD-MSCs (cAD-MSCs) were individually isolated from five dogs and subjected to proliferative culture with seven sub-passages. The cells at each sub-passage were characterized for properties associated with multipotent MSCs such as proliferation kinetics, expression of MSCs-specific surface markers, expression of molecules associated with self-renewal and differentiation capabilities into mesodermal lineage cells. Proliferation of the cells plateaued at passage 5 by cumulative population doubling level, while cell doubling time gradually increased with passage. MSCs surface markers (CD44, CD90, and CD105) and molecules (Oct 3/4, Sox-2, Nanog and HMGA2) associated with self-renewal were all expressed in the cells between passages 1 to 6 by RT-PCR. In addition, the cells at passage 1, 3 or 6 underwent adipogenic and chondrogenic differentiation under specific induction conditions. However, the level of adipogenic and chondrogenic differentiation was negatively correlated with the number of sub-passage. The present study suggests that sequential sub-passages affect multipotent properties of cAD-MSCs, which should be considered in their therapeutic application in regenerative medicine.     

In vitro neuronal and osteogenic differentiation of mesenchymal stem cells from human umbilical cord blood

Mesenchymal stem cells (MSCs) have the capabilities for self-renewal and differentiation into cells with the phenotypes of bone, cartilage, neurons and fat cells. These features of MSCs have attracted the attention of investigators for using MSCs for cell-based therapies to treat several human diseases. Because bone marrow-derived cells, which are a main source of MSCs, are not always acceptable due to a significant drop in their cell number and proliferative/differentiation capacity with age, human umbilical cord blood (UCB) cells are good substitutes for BMCs due to the immaturity of newborn cells. Although the isolation of hematopoietic stem cells from UCB has been well established, the isolation and characterization of MSCs from UCB still need to be established and evaluated. In this study, we isolated and characterized MSCs. UCB-derived mononuclear cells, which gave rise to adherent cells, exhibited either an osteoclast or a mesenchymal-like phenotype. The attached cells with mesenchymal phenotypes displayed fibroblast-like morphologies, and they expressed mesenchym-related antigens (SH2 and vimentin) and periodic acid Schiff activity. Also, UCB-derived MSCs were able to transdifferentiate into bone and 2 types of neuronal cells, in vitro. Therefore, it is suggested that the MSCs from UCB might be a good alternative to bone marrow cells for transplantation or cell therapy.     

Characterization of adipose-derived equine and canine mesenchymal stem cells after incubation in agarose-hydrogel

Adult stem cells are of particular interest for the therapeutic approach in the field of regenerative medicine. Due to their ease of harvest, adipose-derived mesenchymal stem cells (ASCs) are an attractive stem cell source that has become increasingly popular. Critical aspects of applied cell therapies are the circumstances of transport from the laboratory towards the site of operation and cell delivery into the desired area. With regard to these issues, agarose-hydrogel was analyzed as a cell carrier matrix of equine and canine ASCs in vitro, which can be used for minimally invasive application. Isolated ASCs were expanded and 2.5 × 10⁶ cells were combined with agarose-hydrogel to build a 0.4% hydrogel-cell solution which was stored at two temperatures (room temperature (RT) vs. 37°C). Cell viability was investigated (live-dead assay) at different time points (0, 1, 6 and 24 h) in order to determine i) the effect of different temperatures on the cell survival as well as ii) the maximum possible time span before implantation. CFU-assay and WST-1 assay were performed after 24 h incubation in agarose-hydrogel and the cells were induced into adipogenic and osteogenic differentiation to analyze the effects of the incubation on the cell behaviour. No negative effect of the agarose-hydrogel incubation was determined on the different species’ cell behaviour at either RT or 37°C with any of the assays used. We can recommend agarose-hydrogel as a cell carrier for cell implantation with a storage period of up to 24 h at room temperature or at 37°C prior to implantation.     

Effect of serum-derived albumin scaffold and canine adipose tissue-derived mesenchymal stem cells on osteogenesis in canine segmental bone defect model

Composite biological and synthetic grafts with progenitor cells offer an alternative approach to auto- or allografts for fracture repair. This study was conducted to evaluate osteogenesis of autologous serum-derived albumin (ASA) scaffolds seeded with canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) in a canine segmental bone defect model. ASA scaffold was prepared with canine serum using cross-linking and freeze-drying procedures. Beta-tricalcium phosphate (β-TCP) was mixed at the cross-linking stage. Ad-MSCs were seeded into the scaffold and incubated for one day before implantation. After 16 weeks, the grafts were harvested for histological analysis. The dogs were divided into five groups: control, ASA scaffolds with and without Ad-MSCs, and ASA scaffolds including β-TCP with and without Ad-MSCs. ASA scaffolds with Ad-MSCs had a significantly larger area of increased opacity at the proximal and distal host cortex-implant interfaces in radiographs 16 weeks after implantation compared to the groups with β-TCP (p < 0.05). Histomorphometric analysis showed that ASA scaffolds with Ad-MSCs had significantly greater new bone formation than other groups (p < 0.05). These results suggest that Ad-MSCs seeded into ASA scaffolds enhanced osteogenesis in the bone defect model, but that β-TCP in the ASA scaffold might prevent penetration of the cells required for bone healing.     

A double-blinded placebo-controlled evaluation of adipose-derived mesenchymal stem cells in treatment of canine atopic dermatitis

Mesenchymal stem cells (MSCs) have emerged as a new therapy for various immune-mediated inflammatory diseases. In this study we perform the first double-blinded, placebo-controlled evaluation of the efficacy of adipose-derived allogenic canine MSCs for the treatment of canine atopic dermatitis (cAD). Enrolled canine patients were randomly divided into placebo (PBS saline), low-dose (5?×?10⁵ cells/kg), and high-dose (5?×?10⁶ cells/kg) treatment groups. Each patient received three subcutaneous MSCs treatments or PBS saline at four-week intervals with injections at five sites. Patients were monitored by physical exams, pruritus visual analog scales (PVAS) signed by the primary caretaker, canine atopic dermatitis extent and severity index-4 (CADESI-4) scores by two veterinarians, and complete blood count and serum chemistry analysis along with laboratory analysis for potential biomarkers. Patients were kept off any immune-modulating drugs during the study period, and oral antibiotics and topicals were used for managing pruritus and secondary infections. The PVAS scores and the serum miR-483 levels were significantly lower in the high dose group compared to the placebo group at day90 post first-treatment. The CADESI-4 scores of the high dose group also showed downward trends. No severe adverse effects were observed in any patient in this study. The high dose MSC treatment is efficacious in alleviating the clinical signs of cAD until 30 days after the last subcutaneous administration of MSCs, and miRNA-483 may be a reliable prognostic biomarker for cAD. The MSCs efficacy and potential biomarkers should be further explored by a larger scale clinical trial.

     

Comparing the osteogenic potential of canine mesenchymal stem cells derived from adipose tissues, bone marrow, umbilical cord blood, and Wharton's jelly for treating bone defects

Alternative sources of mesenchymal stem cells (MSCs) for replacing bone marrow (BM) have been extensively investigated in the field of bone tissue engineering. The purpose of this study was to compare the osteogenic potential of canine MSCs derived from adipose tissue (AT), BM, umbilical cord blood (UCB), and Wharton''s jelly (WJ) using in vitro culture techniques and in vivo orthotopic implantation assays. After canine MSCs were isolated from various tissues, the proliferation and osteogenic potential along with vascular endothelial growth factor (VEGF) production were measured and compared in vitro. For the in vivo assay, MSCs derived from each type of tissue were mixed with β-tricalcium phosphate and implanted into segmental bone defects in dogs. Among the different types of MSCs, AT-MSCs had a higher proliferation potential and BM-MSCs produced the most VEGF. AT-MSCs and UCB-MSCs showed greater in vitro osteogenic potential compared to the other cells. Radiographic and histological analyses showed that all tested MSCs had similar osteogenic capacities, and the level of new bone formation was much higher with implants containing MSCs than cell-free implants. These results indicate that AT-MSCs, UCB-MSCs, and WJ-MSCs can potentially be used in place of BM-MSCs for clinical bone engineering procedures.     

不同代次大鼠骨髓间充质干细胞和脂肪间充质干细胞成骨分化比较

本研究旨在观察不同代次骨髓间充质干细胞(BMSCs)和脂肪间充质干细胞(ADSCs)体外培养的生长特点和体外诱导成骨能力。通过密度梯度离心和贴壁培养法分离培养大鼠骨髓间充质干细胞和脂肪间充质干细胞,用含地塞米松、抗坏血酸、β-甘油磷酸钠的培养液定向诱导传代细胞向成骨细胞分化,并利用茜素红染色、碱性磷酸酶染色及PCR方法检测成骨细胞。结果表明骨髓及脂肪间充质干细胞呈成纤维细胞样生长,增殖能力强,生长迅速。第5、10、15、20代BMSCs及ADSCs经诱导培养后茜素红染色呈阳性并且出现"矿化"、碱性磷酸酶活性强,随着细胞代次的递增,诱导后细胞碱性磷酸酶活性呈递减趋势;诱导后的两类细胞传代后细胞仍能继续分化,并形成正常的"矿化"结节,且碱性磷酸酶染色均弱于初次诱导。结果提示,BMSCs及ADSCs易于分离培养及体外扩增,诱导条件下成骨能力强且成骨细胞传代培养仍具有成骨能力,适合作为再生医学骨组织工程的种子细胞。     

Impact of the source and serial passaging of goat mesenchymal stem cells on osteogenic differentiation potential:implications for bone tissue engineering

Background: Adult mesenchymal stem cells(MSCs) can be conveniently sampled from bone marrow, peripheral blood, muscle, adipose and connective tissue, harvested from various species, including, rodents, dogs, cats, horses,sheep, goats and human beings. The MSCs isolated from adult tissues vary in their morphological and functional properties. These variations are further complicated when cells are expanded by passaging in culture. These differences and changes in MSCs must be considered prior to their application in the clinic or in a basic research study. Goats are commonly used as animal models for bone tissue engineering to test the potential of stem cells for bone regeneration. As a result, goat MSCs isolated from bone marrow or adipose tissue should be evaluated using in vitro assays, prior to their application in a tissue engineering project.Results: In this study, we compared the stem cell properties of MSCs isolated from goat bone marrow and adipose tissue. We used quantitative and qualitative assays with a focus on osteogenesis, including, colony forming unit, rate of cell proliferation, tri-lineage differentiation and expression profiling of key signal transduction proteins to compare MSCs from low and high passages. Primary cultures generated from each source displayed the stem cell characteristics,with variations in their osteogenic potentials. Most importantly, low passaged bone marrow MSCs displayed a significantly higher and superior osteogenic potential, and hence, will be the preferred choice for bone tissue engineering in future in vivo experiments. In the bone marrow MSCs, this process is potentially mediated by the p38 MAPK pathway. On the other hand, osteogenic differentiation in the adipose tissue MSCs may involve the p44/42 MAPK pathway.Conclusions: Based on these data, we can conclude that bone marrow and fat-derived MSCs undergo osteogenesis via two distinct signaling pathways. Even though the bone marrow MSCs are the preferred source for bone tissue engineering, the adipose tissue MSCs are an attractive alternative source and undergo osteo-differentiation differently from the bone marrow MSCs and hence, might require a cell-based enhancer/inducer to improve their osteogenic regenerative capacity.     

小鼠脂肪间充质干细胞向胰岛素分泌细胞的诱导分化研究

为了通过特定转录因子将小鼠脂肪间充质干细胞(mADSCs)定向诱导分化为胰岛素分泌细胞(IPCs)。本研究分别构建Pdx1(胰十二指肠同源盒基因1)、MafA(V-maf肌肉腱膜纤维肉瘤癌基因同源物A)、NeuroD1(神经分化因子1)3种基因的慢病毒过表达载体,使用293T细胞对3种因子进行慢病毒包装,将3种慢病毒过表达载体以单因子侵染、双因子侵染、三因子联合侵染的方式对mADSCs进行定向分化诱导,于诱导分化第15天对不同方式诱导的IPCs进行检测鉴定,并对不同方式诱导组的IPCs进行高糖刺激,刺激后30~120min检测培养基中含糖量的变化。结果显示,构建的慢病毒过表达载体pHBLV-CMV-IRES-ZsGreen-Pdx1、pHBLV-CMV-PGK-RFPMafA、pHBLV-CMV-PGK-RFP-NeuroD1所含目的片段基因序列与小鼠全基因编码序列完全一致,三种基因慢病毒过表达载体构建成功;诱导分化第15天,三因子联合诱导组所形成的IPCs克隆双硫腙(DTZ)染色呈阳性,并可表达胰岛素生物合成及分泌相关基因;在高糖刺激条件下,三因子联合诱导组糖分解速度、分解量远优于单因子或双因子诱导组。结果表明,Pdx1、MafA、NeuroD1 3种因子联合作用,可以将小鼠脂肪间充质干细胞定向诱导分化为胰岛素分泌细胞,并可在高糖刺激下,有效发挥降糖作用。