Optimization of adipogenic differentiation conditions for canine adipose-derived stem cells

BackgroundCanine adipose-derived stem cells (cADSCs) exhibit various differentiation properties and are isolated from the canine subcutaneous fat. Although cADSCs are valuable as tools for research on adipogenic differentiation, studies focusing on adipogenic differentiation methods and the underlying mechanisms are still lacking.ObjectivesIn this study, we aimed to establish an optimal method for adipogenic differentiation conditions of cADSCs and evaluate the role of peroxisome proliferator-activated receptor gamma (PPARγ) and estrogen receptor (ER) signaling in the adipogenic differentiation.MethodsTo induce adipogenic differentiation of cADSCs, 3 different adipogenic medium conditions, MDI, DRI, and MDRI, using 3-isobutyl-1-methylxanthine (M), dexamethasone (D), insulin (I), and rosiglitazone (R) were tested.ResultsMDRI, addition of PPARγ agonist rosiglitazone to MDI, was the most significantly facilitated cADSC into adipocyte. GW9662, an antagonist of PPARγ, significantly reduced adipogenic differentiation induced by rosiglitazone. Adipogenic differentiation was also stimulated when 17β-estradiol was added to MDI and DRI, and this stimulation was inhibited by the ER antagonist ICI182,780.ConclusionsTaken together, our results suggest that PPARγ and ER signaling are related to the adipogenic differentiation of cADSCs. This study could provide basic information for future research on obesity or anti-obesity mechanisms in dogs.     

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 expansion and differentiation of fresh and revitalized adult canine bone marrow-derived and adipose tissue-derived stromal cells

The objective of this study was to determine the tissue density, in vitro expansion and differentiation of canine adipose tissue-derived (ASC) and bone marrow-derived (BMSC) stromal cells. Primary (P0) and cell passages 1-6 (P1-6) cell doubling numbers (CD) and doubling times (DT) were determined in fresh cells. The P0, P3, and P6 adipogenic (CFU-Ad), osteogenic (CFU-Ob), and fibroblastic (CFU-F) colony forming unit frequencies, lineage specific mRNA levels in differentiated P3 cells and composition of P3 and P6 chondrogenic pellets were assessed in cryogenically preserved cells. Cell yields from bone marrow were significantly higher than adipose tissue. Overall ASC and BMSC CDs and DTs and P3 and P6 CFU-F, CFU-Ad, and CFU-Ob were comparable. The P0 BMSC CFU-Ob was significantly higher than ASC. Lineage specific mRNA levels were higher in differentiated versus control cells, but similar between cell types. Protein was significantly greater in P3 versus P6 ASC chondrogenic pellets. Based on these findings, fresh and revitalized canine ASCs are viable alternatives to BMSCs for stromal cell applications.     

Effects of different media on proliferation and differentiation capacity of canine, equine and porcine adipose derived stem cells

Adult stem cells are of particular interest for therapeutic use in the field of regenerative medicine. Adipose-derived mesenchymal stem cells (ASCs) are an attractive stem cell source for all fields of regenerative medicine because adipose tissue - and therewith cells - can easily be harvested from each donor. However, common expansion using fetal bovine serum (FBS) can not be used for clinical applications as xenogenic proteins must be avoided. Adipose tissue from equine, canine and porcine donors was digested with collagenase to isolate ASCs. ASCs were either expanded in a cell culture medium supplemented with FBS or in a serum-free medium (UltraCulture; UC) supplemented with a serum substitute (UltroserG). From all three animal species, the adipogenic and osteogenic differentiation potential of ASCs cultured with different media was analyzed in vitro. Cell proliferation analysis showed a population doubling time of 48-68 h for canine cells, 54-65 h for porcine cells and 54-70 h for equine cells, expanded in different media. Except for porcine ASCs, cells cultured in media supplemented with FBS grew faster than cells expanded in UC medium with UltroserG. Yet, all cells maintained their potential to differentiate into adipocytes and osteoblasts. UltraCulture medium containing UltroserG can for all examined species be recommended if FBS needs to be avoided in the expansion of donor-derived (stem) cells.     

Isolation and characterisation of cancer stem cells from canine osteosarcoma

There is increasing evidence that cancer is a stem cell disease. This study sought to isolate and characterise cancer stem cells from canine osteosarcoma. One human and three canine cell lines were cultured in non-adherent culture conditions using serum-starved, semi-solid media. Primitive sarcosphere colonies from all cell lines were identified under these conditions and were characterised using molecular and cytochemical techniques for embryonic stem cell markers. Expression of the embryonic stem cell-associated genes Nanog, Oct4 and STAT3 indicated a primitive phenotype. Sarcospheres could be reproduced consistently when passaged multiple times and produced adherent cell cultures when returned to normal growth conditions. Similarities between human and canine osteosarcoma cell lines add credence to the potential of the dog as a model for human disease.     

Isolation,proliferation and characterization of endometrial canine stem cells

In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT‐PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue‐derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue‐derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the “Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy” for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue‐derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow‐derived MSC in dog.     

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.     

胚胎干细胞定向分化研究进展

胚胎干细胞是一类从早期胚胎内细胞团或原始生殖细胞分离和克隆出的 ,具有自我更新和发育全能性并产生分化后代能力的早期细胞 ,可分化形成各种组织类型。在合适的条件下 ,胚胎干细胞可发育成造血细胞、神经细胞、肌细胞等各种细胞。探索胚胎干细胞分化机理及其体外定向诱导分化为其他组织细胞的条件 ,就可以通过改变体外培养条件来探索胚胎干细胞向不同组织细胞分化规律 ,对揭开动物个体发育之迷 ,具有极其重要的理论意义 ;也可为细胞替代治疗、器官移植、建立药物筛选模型以及进行发育及细胞生物学的研究奠定基础。本文仅就胚胎干细胞定向分化的理论基础 ,定向分化研究现状作一综述     

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.     

In vitro differentiation of canine celiac adipose tissue-derived stromal cells into neuronal cells

To investigate in vitro differentiation of canine adipose tissue-derived stromal cells (ATSCs) into neuronal cells, ATSCs from celiac adipose tissue in clinically healthy beagle dogs were treated with 100 muM dibutyryl cyclic adenosine monophosphate (dbcAMP) and 125 muM isobuthylmethylxanthine (IBMX). ATSCs were morphologically changed into differentiated ATSCs from spindle-shaped cells to neuron-like cells with numerous processes after the treatment. Expression of neuron-specific enolase (NSE) as an early neuron specific marker protein was detected in both ATSCs and differentiated ATSCs, however diachronic increase of NSE expression was observed in differentiated ATSCs after the treatment with dbcAMP/IBMX. In addition, neurofilament-68 (NF-68) as an early to mature neuron specific marker protein was weakly expressed in differentiated ATSCs. Neuron specific glutamate and glucose transporter (EAAC1 and GLUT-3, respectively) mRNAs were strongly expressed in differentiated ATSCs compared with those in ATSCs, although glia specific glutamate transporter mRNA (GLT-1) was also detected in differentiated ATSCs. ATSCs can differentiate into early to mature neuronal cells and are candidate cells for autologous nerve regeneration therapy, although additional research is needed to examine functional characteristics of differentiated ATSCs.     

In vitro retinoid-induced growth inhibition and morphologic differentiation of canine osteosarcoma cells

OBJECTIVE: To determine differentiation and growth inhibition effects of retinoids on canine osteosarcoma cells. SAMPLE POPULATION: 3 osteosarcoma cell lines established from osteosarcomas in dogs. PROCEDURE: Osteosarcoma cells were incubated with various concentrations of all-trans-retinoic acid and 9-cis-retinoic acid or control medium, counted daily for 10 days, and evaluated for morphologic changes. Synthesis of DNA was measured by use of a cell proliferation ELISA. To analyze effect of retinoids on colony formation on plastic dishes, cells were cultured for 14 days, fixed, and stained; number of colonies was counted. RESULTS: In a dose-dependent manner, both retinoids induced morphologic differentiation and growth inhibition in the 3 osteosarcoma cell lines and inhibited each cell's ability to form anchorage-dependent colonies. CONCLUSION AND CLINICAL RELEVANCE: Retinoids induced differentiation of osteosarcoma cells of dogs, resulting in altered expression of their malignant phenotype. Induction of differentiation by retinoids may have potential as an adjunctive treatment for osteosarcoma in dogs.     

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.     

Characterisation of the third component of canine and feline complement

Canine and feline C3 have been found by immune precipitation to have a molecular weight of 198K and 197K respectively. Each comprises two polypeptide chains α and β (canine α - 126K β - 72K; feline α - 125K β - 72K). The α chain is subsequently cleaved by C3b ina to produce two fragments α^edc (canine 65K; feline 64K) and α^fb (canine 40K; feline 46K). The findings are compared to the documented molecular structure of human C3.     

胚胎干细胞标记基因在大鼠成体干细胞中的表达

为了给组织工程筛选合适的种子细胞,分别对大鼠脂肪间充质干细胞(rat adipose-derived stem cells,rADSCs)、骨髓间充质干细胞(rat bone marrow stromal stem cells,rBMSCs)和骨骼肌卫星细胞(rat skeletal muscle-derived stem cells,rMDSCs)进行胚胎干细胞标记基因Oct-4、Sox-2、Nanog、IL-6和Tert分析,并确定这些基因在这3种细胞中的表达差异性。首先分别应用酶消化法和机械分离法体外分离培养rADSCs、rMDSCs和rBMSCs 3种细胞。其次应用免疫荧光染色检测Oct-4、Sox-2、Nanog、IL-6和Tert在这3种细胞中的表达,然后采用实时定量PCR和Western blot分别在RNA和蛋白质水平定量检测、分析这些基因在不同细胞中的表达量及其差异。免疫荧光染色结果显示这3种细胞中均表达Oct-4、Sox-2、Nanog、IL-6和Tert,但在RNA水平和蛋白质水平均未检测到Nanog的表达,而IL-6和Tert的相对表达量在这3种细胞中均高于Oct-4和Sox-2。研究结果表明,rADSCs、rBMSCs和rMDSCs虽然具有自我更新和多向分化潜能,但是不具有与胚胎干细胞完全相同的特性。     

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.     

Isolation, expansion, and differentiation of goat adipose-derived stem cells

A goat adipose-derived stem cell (ADSC) line was established and compared to a rat line. Goat ADSC cells had normal diploidy after subculture. Proliferation of goat ADSCs was faster than rat cells in the same conditions. Both rat and goat ADSCs stained positively for vimentin, CD49d, CD44 and CD13, but stained negatively for CD34 and CD106. Bone nodules were apparent, and alizarin staining was positive after osteogenic induction. Cells expressing osteocalcin were positive by alkaline phosphatase (ALP) staining. After osteogenic induction, ossification nodules of goat ADSCs were larger than in rats, with dense ALP staining. Adipogenic induction resulting in lipid droplets and peroxisome proliferator-activated receptor (PPARγ2) expression were observed. Cartilage lacunae were formed and COL2A1 was expressed. More cartilage lacunae with better morphology were seen following differentiation of goat ADSC's using the hang-drop method. For goat ADSCs, results with both adherent-induced and hanging-drop induced cultures were better than for three-dimensional cultures.