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.     

Cell growth characteristics and differentiation frequency of adherent equine bone marrow-derived mesenchymal stromal cells: adipogenic and osteogenic capacity

OBJECTIVES: To characterize equine bone marrow (BM)-derived mesenchymal stem cell (MSC) growth characteristics and frequency as well as their adipogenic and osteogenic differentiation potential. STUDY DESIGN: In vitro experimental study. ANIMALS: Foals (n=3, age range, 17-51 days) and young horses (n=5, age range, 9 months to 5 years). METHODS: Equine MSCs were harvested and isolated from sternal BM aspirates and grown up to passage 10 to determine cell-doubling (CD) characteristics. Limit dilution assays were performed on primary and passaged MSCs to determine the frequency of colony-forming units with a fibroblastic phenotype (CFU-F), and the frequency of MSC differentiation into adipocytes (CFU-Ad) and osteoblasts (CFU-Ob). RESULTS: Initial MSC isolates had a lag phase with a significantly longer CD time (DT=4.9+/-1.6 days) compared with the average DT (1.4+/-0.22 days) of subsequent MSC passages. Approximately 1 in 4224+/-3265 of the total nucleated BM cells displayed fibroblast colony-forming activity. Primary MSCs differentiated in response to adipogenic and osteogenic inductive conditions and maintained their differentiation potential during subsequent passages. CONCLUSIONS: The frequency, in vitro growth rate, and adipogenic and osteogenic differentiation potential of foals and young adult horses are similar to those documented for BM MSCs of other mammalian species. CLINICAL RELEVANCE: The results have direct relevance to the use of BM as a potential source of adult stem cells for tissue engineering applications in equine veterinary medicine.     

Phenotypic and functional properties of feline dedifferentiated fat cells and adipose-derived stem cells

It has been reported that mature adipocyte-derived dedifferentiated fat (DFAT) cells show multilineage differentiation potential similar to that observed in mesenchymal stem cells. Since DFAT cells can be prepared from a small quantity of adipose tissue, they could facilitate cell-based therapies in small companion animals such as cats. The present study examined whether multipotent DFAT cells can be generated from feline adipose tissue, and the properties of DFAT cells were compared with those of adipose-derived stem cells (ASCs). DFAT cells and ASCs were prepared from the floating mature adipocyte fraction and the stromal vascular fraction, respectively, of collagenase-digested feline omental adipose tissue. Both cell types were evaluated for growth kinetics, colony-forming unit fibroblast (CFU-F) frequency, immunophenotypic properties, and multilineage differentiation potential.DFAT cells and ASCs could be generated from approximately 1 g of adipose tissue and were grown and subcultured on laminin-coated dishes. The frequency of CFU-Fs in DFAT cells (35.8%) was significantly higher than that in ASCs (20.8%) at passage 1 (P1). DFAT cells and ASCs displayed similar immunophenotypes (CD44⁺, CD90⁺, CD105⁺, CD14^?, CD34^? and CD45^?). Alpha-smooth muscle actin-positive cells were readily detected in ASCs (15.2 ± 7.2%) but were rare in DFAT cells (2.2 ± 3.2%) at P1. Both cell types exhibited adipogenic, osteogenic, chondrogenic, and smooth muscle cell differentiation potential in vitro. In conclusion, feline DFAT cells exhibited similar properties to ASCs but displayed higher CFU-F frequency and greater homogeneity. DFAT cells, like ASCs, may be an attractive source for cell-based therapies in cats.     

Impact of source tissue and ex vivo expansion on the characterization of goat mesenchymal stem cells

Background: There is considerable interest in using goats as models for genetically engineering dairy animals and also for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells(MSCs) have been isolated and characterized from various species, but are poorly characterized in goats.Results: Goat MSCs isolated from bone marrow(BM-MSCs) and adipose tissue(ASCs) have the ability to undergo osteogenic, adipogenic and chondrogenic differentiation. Cytochemical staining and gene expression analysis show that ASCs have a greater capacity for adipogenic differentiation compared to BM-MSCs and fibroblasts. Different methods of inducing adipogenesis also affect the extent and profile of adipogenic differentiation in MSCs. Goat fibroblasts were not capable of osteogenesis, hence distinguishing them from the MSCs. Goat MSCs and fibroblasts express CD90, CD105, CD73 but not CD45, and exhibit cytoplasmic localization of OCT4 protein. Goat MSCs can be stably transfected by Nucleofection, but, as evidenced by colony-forming efficiency(CFE), yield significantly different levels of progenitor cells that are robust enough to proliferate into colonies of integrants following G418 selection.BM-MSCs expanded over increasing passages in vitro maintained karyotypic stability up to 20 passages in culture,exhibited an increase in adipogenic differentiation and CFE, but showed altered morphology and amenability to genetic modification by selection.Conclusions: Our findings provide characterization information on goat MSCs, and show that there can be significant differences between MSCs isolated from different tissues and from within the same tissue. Fibroblasts do not exhibit trilineage differentiation potential at the same capacity as MSCs, making it a more reliable method for distinguishing MSCs from fibroblasts, compared to cell surface marker expression.     

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.     

Characterization of Nucleated Cells From Equine Adipose Tissue and Bone Marrow Aspirate Processed for Point-of-Care Use

The objective of this study was to compare nucleated cell fractions and mesenchymal stromal cells (MSCs) from adipose tissue to bone marrow processed by a point-of-care device that are available for immediate implantation. A paired comparison using adipose and bone marrow from five horses was done. The number of nucleated cells, viability, total adherent cells on day 6 of culture and colony-forming unit fibroblasts (CFU-Fs) were determined. Gene expression for markers of stemness, adipogenic, chondrogenic, osteogenic lineage, and collagen formation was measured in total RNA isolated from adherent adipose and bone marrow cells. Day 6 adherent adipose-derived MSC was frozen briefly, whereas day 6 adherent bone marrow–derived MSC was passaged two additional times to obtain adequate cell numbers for chondrogenic, osteogenic, and adipogenic cell differentiation assays. The total cell count per gram was significantly greater for bone marrow, whereas total adherent cells per gram and the CFU-F per million nucleated cells on day 6 were significantly greater for the adipose. In undifferentiated adherent cells, relative gene expression for CD34, adipogenic, and chondrogenic markers and collagen II was significantly lower in the adipose-derived cells. Conversely, expression of collagen I was significantly higher in the undifferentiated adipose-derived cells. Cell density and total RNA were higher in differentiated adipogenic and osteogenic cultures of adipose cells and in chondrogenic cultures of bone marrow cells. This cell preparation method provides a stromal vascular fraction with a large proportion of multipotent MSCs. There are differences in the cells obtained from the two sources. This method can provide an adequate number of multipotent cells from adipose tissue for immediate implantation.     

Comparison of Chondrogenic Potential in Equine Mesenchymal Stromal Cells Derived from Adipose Tissue and Bone Marrow

Objective— To compare the chondrogenic potential of adult equine mesenchymal stem cells derived from bone marrow (MSCs) or adipose tissue (ASCs). Study Design— In vitro experimental study. Animals— Adult Thoroughbred horses (n=11). Methods— BM (5 horses; mean [±SD] age, 4±1.4 years) or adipose tissue (6 horses; mean age, 3.5±1.1 years) samples were obtained. Cryopreserved MSCs and ASCs were used for pellet cultures in stromal medium (C) or induced into chondrogenesis±transforming growth factor‐3 (TGFβ₃) and bone morphogenic factor‐6 (BMP‐6). Pellets harvested after 3, 7, 14, and 21 days were examined for cross‐sectional size and tissue composition (hematoxylin and eosin), glycosaminoglycan (GAG) staining (Alcian blue), collagen type II immunohistochemistry, and by transmission electron microscopy. Pellet GAG and total DNA content were measured using dimethylmethylene blue and Hoechst DNA assays. Results— Collagen type II synthesis was predominantly observed in MSC pellets from Day 7 onward. Unlike ASC cultures, MSC pellets had hyaline‐like matrix by Day 14. GAG deposition occurred earlier in MSC cultures compared with ASC cultures and growth factors enhanced both MSC GAG concentrations (P<.0001) and MSC pellet size (P<.004) after 2 weeks in culture. Conclusion— Equine MSCs have superior chondrogenic potential compared with ASCs and the equine ASC growth factor response suggests possible differences compared with other species. Clinical Relevance— Elucidation of equine ASC and MSC receptor profiles will enhance the use of these cells in regenerative cartilage repair.     

Impact of source tissue and ex vivo expansion on the characterization of goat mesenchymal stem cells

Background

There is considerable interest in using goats as models for genetically engineering dairy animals and also for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells (MSCs) have been isolated and characterized from various species, but are poorly characterized in goats.

Results

Goat MSCs isolated from bone marrow (BM-MSCs) and adipose tissue (ASCs) have the ability to undergo osteogenic, adipogenic and chondrogenic differentiation. Cytochemical staining and gene expression analysis show that ASCs have a greater capacity for adipogenic differentiation compared to BM-MSCs and fibroblasts. Different methods of inducing adipogenesis also affect the extent and profile of adipogenic differentiation in MSCs. Goat fibroblasts were not capable of osteogenesis, hence distinguishing them from the MSCs. Goat MSCs and fibroblasts express CD90, CD105, CD73 but not CD45, and exhibit cytoplasmic localization of OCT4 protein. Goat MSCs can be stably transfected by Nucleofection, but, as evidenced by colony-forming efficiency (CFE), yield significantly different levels of progenitor cells that are robust enough to proliferate into colonies of integrants following G418 selection. BM-MSCs expanded over increasing passages in vitro maintained karyotypic stability up to 20 passages in culture, exhibited an increase in adipogenic differentiation and CFE, but showed altered morphology and amenability to genetic modification by selection.

Conclusions

Our findings provide characterization information on goat MSCs, and show that there can be significant differences between MSCs isolated from different tissues and from within the same tissue. Fibroblasts do not exhibit trilineage differentiation potential at the same capacity as MSCs, making it a more reliable method for distinguishing MSCs from fibroblasts, compared to cell surface marker expression.

Electronic supplementary material

The online version of this article (doi:10.1186/2049-1891-6-1) contains supplementary material, which is available to authorized users.     

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.     

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。     

Isolation and characterization of bone marrow-derived equine mesenchymal stem cells

OBJECTIVE: To isolate and characterize bone marrow-derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses. SAMPLE POPULATION: Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses. PROCEDURES: Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transduction rate of equine MSCs. RESULTS: Equine MSCs can be culture-expanded. Equine MSCs undergo cryopreservation in liquid nitrogen without altering morphologic characteristics. Furthermore, equine MSCs maintain their ability to proliferate and differentiate after thawing. Immunocytochemically, the expression of the stem cell marker CD90 can be detected on equine MSCs. The multilineage differentiation potential of equine MSCs was revealed by their ability to undergo adipogenic, osteogenic, and chondrogenic differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: Our data indicate that bone marrow-derived stromal cells of horses can be characterized as MSCs. Equine MSCs have a high transduction rate and migratory potential and adapt to scaffold material in culture. As an autologous cell population, equine MSCs can be regarded as a promising cell population for tissue engineering in lesions of the musculoskeletal system in horses.     

Osteogenic proliferation and differentiation of canine bone marrow and adipose tissue derived mesenchymal stromal cells and the influence of hypoxia

The aim of this study was to compare the osteogenic and proliferative potential of canine mesenchymal stromal cells (cMSCs) derived from bone marrow (BM-cMSCs) and adipose tissue (AT-cMSCs). Proliferation potential was determined under varying oxygen tensions (1%, 5%, and 21% O(2)). Effects of reduced oxygen levels on the osteogenic differentiation of AT-cMSCs were also investigated. AT-cMSCs proliferated at a significantly faster rate than BM-cMSCs, although both cell types showed robust osteogenic differentiation. Culture in 5% and 1% O(2) impaired proliferation in cMSC from both sources and osteogenic differentiation in AT-cMSCs. Our data suggests that AT-cMSCs might be more suitable for use in a clinical situation, where large cell numbers are required for bone repair, due to their rapid proliferation combined with robust osteogenic potential. Our data also suggests that the inhibitory effects of hypoxia on both cell proliferation and differentiation should be considered when using MSCs in a potentially hypoxic environment such as a fracture site.     

CRISPR系统促进脂肪干细胞成骨分化与抑制成脂分化的研究

本研究旨在优化组织培养法分离小鼠脂肪间充质干细胞（adipose-derived stem cells,ASCs）,为研究成骨分化和成脂分化在间充质干细胞分化过程中的相互影响奠定基础。通过细胞形态学观察、细胞生长曲线和流式仪器检测所分离获得的间充质干细胞的特性,利用CRISPR-dCas9系统在快速促进间充质干细胞成骨分化的前提下观察其对成脂分化的影响,并通过生化染色、实时荧光定量PCR和免疫细胞学等手段进行分析。结果显示,接种3~5 d后可见细胞从组织块周围爬出,光镜下可见细胞形态多为成纤维细胞样的梭形细胞,且形态单一均匀,具有较高的爬出率,可以大大提高脂肪间充质干细胞的分离效率;通过CRISPR-dCas9系统激活Runx2和Osterix基因后可以促进间充质干细胞的成骨分化,实时荧光定量PCR及油红O染色结果显示,CRISPR-dCas9系统可以同时抑制间充质干细胞的成脂分化;通过CRISPR-dCas9-KRAB系统同时抑制成骨相关基因Runx2和Osterix后可以促进成脂分化。本研究利用组织贴壁法成功获得了高纯度的脂肪间充质干细胞,具有间充质干细胞的特性和分化能力;利用CRISPR系统可以同时过表达Runx2和Osterix两个基因,可以在进成骨分化的同时抑制成脂分化,表明成脂分化和成骨分化的相关性,为基因编辑在间充质干细胞诱导分化和临床应用方面提供了新的思路和方法。     

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.     

Further insights into the characterization of equine adipose tissue-derived mesenchymal stem cells

Adipose tissue-derived stem cells (ADSCs) represent a promising subpopulation of adult stem cells for tissue engineering applications in veterinary medicine. In this study we focused on the morphological and molecular biological properties of the ADSCs. The expression of stem cell markers Oct4, Nanog and the surface markers CD90 and CD105 were detected using RT-PCR. ADSCs showed a proliferative potential and were capable of adipogenic and osteogenic differentiation. Expression of Alkaline phosphatase (AP), phosphoprotein (SPP1), Runx2 and osteocalcin (OC) mRNA were positive in osteogenic lineages and peroxisome proliferator activated receptor (Pparγ2) mRNA was positive in adipogenic lineages. ADSCs show stem cell and surface marker profiles and differentiation characteristics that are similar to but distinct from other adult stem cells, such as bone marrow-derived mesenchymal stem cells (BM-MSCs). The availability of an easily accessible and reproducible cell source may greatly facilitate the development of stem cell based tissue engineering and therapies for regenerative equine medicine.     

In vitro antimicrobial activity of equine platelet lysate and mesenchymal stromal cells against common clinical pathogens

Septic arthritis is considered a medical emergency. Disease following bacterial colonization can lead to significant morbidity and mortality and requires costly treatment. Antimicrobial properties of regenerative therapies, including mesenchymal stromal cells and platelet products, have been researched extensively in human medicine. Although fewer studies have been conducted in veterinary species, they have shown promising results. The purpose of this study was to evaluate bacterial suppression by equine platelet lysate (EPL) and adipose-derived mesenchymal stromal cells (ASCs) in vitro. We hypothesized that both products would significantly inhibit the growth of Staphylococcus aureus and Escherichia coli. Pooled blood from 10 horses was used for production of EPL. Mesenchymal stromal cells were isolated from adipose tissue harvested from the gluteal region of 3 horses. The study evaluated 3 treatment groups: 10 × EPL, 1.6 million ASCs, and a control, using an incomplete unbalanced block design with repeated measurements. Optical density readings and colony-forming units/mL were calculated at 0, 3, 6, 9, 12, 18, and 24 hours. Decreased bacterial growth was seen at multiple time points for the S. aureus-ASC and S. aureus-EPL treatments, supporting our hypothesis. Increased bacterial growth was noticed in the E. coli-EPL group, with no difference in the E. coli-ASC treatment, which opposed our hypothesis. A clear conclusion of antimicrobial effects of EPL and ASCs cannot be made from this in vitro study. Although it appears that ASCs have a significant effect on decreasing the growth of S. aureus, further studies are needed to explore these effects, particularly in Gram-positive bacteria.     

Culture and characterisation of equine peripheral blood mesenchymal stromal cells

Although the use of mesenchymal stromal cells (MSCs) for the treatment of orthopaedic injuries in horses has been reported, no official guidelines exist that classify a particular cell as an equine MSC. Given the limited characterisation of peripheral blood (PB)-derived equine MSCs in particular, this study aimed to provide more detailed information in relation to this cell type. Mesenchymal stromal cells were isolated from equine PB samples and colony forming unit (CFU) assays as well as population doubling times (PDTs) (from P₀ to P₁₀) were performed.Two types of colonies, ‘fingerprint’ and dispersed, could be observed based on macroscopic and microscopic features. Moreover, after an initial lag phase (as indicated by a negative PDT at P₀ to P₁) the MSCs divided rapidly as indicated by a positive PDT at all further passages. Immunophenotyping was carried out with trypsin- as well as with accutase-detached MSC to evaluate potential trypsin-sensitive epitope destruction on particular antigens. Isolated MSC were positive for CD29, CD44, CD90 and CD105, and negative for CD45, CD79α, MHC II and a monocyte/macrophage marker, irrespective of the cell detaching agent used. Trilineage differentiation of the MSCs towards osteoblasts, chondroblasts and adipocytes was confirmed using a range of histochemical stains.     

Growth and differentiation characteristics of equine mesenchymal stromal cells derived from different sources

Multipotent mesenchymal stromal cells (MSCs) are a promising therapeutic tool for the treatment of equine tendon and other musculoskeletal injuries. While bone marrow is considered the ‘gold standard’ source of these cells, various other tissues contain MSCs with potentially useful features. The aim of this study was to compare clinically relevant characteristics of MSCs derived from bone marrow, umbilical cord blood and tissue and from adipose tissue and tendon. Cell yield, proliferation, migration, tendon marker expression and differentiation into adipocytes, chondrocytes and osteoblasts was assessed, quantified and compared.MSC numbers obtained from adipose, tendon or umbilical cord tissues were 222-fold higher than those obtained from bone marrow or cord blood. Cells derived from tendon and adipose tissues exhibited most rapid proliferation. Osteogenic differentiation was most prominent in MSCs derived from bone marrow, and was weak in MSCs derived from umbilical cord blood and tissue. In contrast, the highest levels of chondrogenic differentiation were observed in MSCs derived from these sources. Collagen 1A2 expression was highest in adipose- and tendon-derived MSCs, while scleraxis expression was highest in cord blood- and in tendon-derived MSCs. The findings indicate that MSCs from different sources display significantly diverse properties that may impact on their therapeutic application.     

Comparison of Equine Adipose Tissue-Derived Stem Cell Behavior and Differentiation Potential Under the Influence of 3% and 21% Oxygen Tension

Equine multipotent mesenchymal stem cells can be isolated from different tissues and are capable of differentiating into various organ progenitor cells. Physiological oxygen conditions in diverse tissues in vivo are hypoxic, even when standard culture conditions are normoxic. Here, equine adipose tissue-derived stem cells were used to analyze their behavior and differentiation potential into the adipogenic, osteogenic, and chondrogenic lineage under 3% and 21% oxygen tension. Hypoxia-inducible factor-1α is an indicator for hypoxic stress sensed by cells. Its expression was similar under both oxygen conditions, which could be a sign for low oxygen tension being sensed as normoxic by those stem cells. Furthermore, it was observed that hypoxia inhibits cell proliferation. Adipogenesis and chondrogenesis showed better results under 3% oxygen; for osteogenesis, an oxygen tension of 21% was more effective. This knowledge may help to improve conditions of stem cell differentiation and consequently their application in tissue engineering.