Application of a novel sorting system for equine mesenchymal stem cells (MSCs)

The objective of this study was to validate non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs) into subpopulations, for use with MSCs derived from equine muscle tissue, periosteal tissue, bone marrow, and adipose tissue. Cells were collected from 6 young, adult horses, postmortem. Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and left supragluteal subcutaneous adipose tissue. Aliquots of 800 × 10³ MSCs from each tissue source were separated and injected into a ribbon-like capillary device by continuous flow (GrFFF proprietary system). Cells were sorted into 6 fractions and absorbencies [optical density (OD)] were read. Six fractions from each of the 6 aliquots were then combined to provide pooled fractions that had adequate cell numbers to seed at equal concentrations into assays. Equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells were consistently sorted into 6 fractions that remained viable for use in further assays. Fraction 1 had more cuboidal morphology in culture when compared to the other fractions. Statistical analysis of the fraction absorbencies (OD) revealed a P-value of < 0.05 when fractions 2 and 3 were compared to fractions 1, 4, 5, and 6. It was concluded that non-equilibrium GrFFF is a valid method for sorting equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells into subpopulations that remain viable, thus securing its potential for use in equine stem cell applications and veterinary medicine.     

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.     

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.     

蒙古羊骨髓间充质干细胞的分离培养及多向诱导分化

本研究旨在建立蒙古羊骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)体外分离、纯化、增殖方法,诱导其向脂肪细胞、成骨细胞及软骨细胞分化。穿刺法抽取蒙古羊骨髓组织,采用密度梯度离心法结合贴壁培养法分离纯化BMSC,增殖,并测定其生长曲线及细胞倍增时间。对第3代蒙古羊BMSC进行成脂、成骨及成软骨诱导,观察诱导后的细胞形态。采用油红O、茜素红、HE等染色法在组织学水平对BMSC进行成脂、成骨和成软骨分化鉴定。结果显示,蒙古羊BMSC呈现均一梭形成纤维细胞样生长,生长曲线呈S型,生长增殖能力良好。在不同分化诱导之后,细胞呈现脂肪细胞、成骨细胞及软骨细胞的表型特征。表明获得的蒙古羊BMSC具有多向分化潜能,所分离细胞确为骨髓间充质干细胞。     

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.     

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.     

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.     

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.     

Osteoinductivity of gelatin/β-tricalcium phosphate sponges loaded with different concentrations of mesenchymal stem cells and bone morphogenetic protein-2 in an equine bone defect model

Fracture is one of the most life-threatening injuries in horses. Fracture repair is often associated with unsatisfactory outcomes and is associated with a high incidence of complications. This study aimed to evaluate the osteogenic effects of gelatin/β-tricalcium phosphate (GT) sponges loaded with different concentrations/ratios of mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) in an equine bone defect model. Seven thoroughbred horses were used in this study. Eight bone defects were created in the third metatarsal bones of each horse. Then, eight treatments, namely control, GT, GT/M-5, GT/M-6, GT/M-5/B-1, GT/M-5/B-3, GT/M-6/B-1, and GT/M-6/B-3 were applied to the eight different sites in a randomized manner (M-5: 2?×?10⁵ MSCs; M-6: 2?×?10⁶ MSCs; B-1: 1 μg of BMP-2; B-3: 3 μg of BMP-2). Repair of bone defects was assessed by radiography, quantitative computed tomography (QCT), and histopathological evaluation. Radiographic scores and CT values were significantly lower in the control group than in the other groups, while they were significantly higher in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The amount of mature compact bone filling the defects was greater in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The present study demonstrated that the GT sponge loaded with MSCs and BMP-2 promoted bone regeneration in an equine bone defect model. The GT/MSC/BMP-2 described here may be useful for treating horses with bone injuries.     

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)和茜素红染色鉴定成骨分化,RT-PCR检测成骨相关基因(COL1、OSX、RUNX2、ALP、OCN、OPG、OPN、RANKL)表达变化。结果显示,1~10μmol/L的镉显著促进BMSCs增殖,20μmol/L的镉显著抑制其增殖(P<0.05);5μmol/L以上的镉可显著抑制ALP活性,并以浓度依赖方式极显著抑制成骨相关基因(COL1、OSX、RUNX2、ALP、OCN、OPG、OPN)mRNA表达,上调RANKL mRNA表达(P<0.01)。表明,一定浓度镉可抑制鸡BMSCs体外增殖及其向成骨细胞(OB)的分化。     

骨髓间充质干细胞的研究进展

骨髓间充质干细胞是存在于骨髓中的除造血干细胞以外的另一类具有多向分化潜能的干细胞。在一定的诱导条件下 ,这类细胞可定向分化为多种造血以外组织 ,特别是中胚层和神经外胚层来源的组织细胞。例如成骨细胞、成软骨细胞、脂肪细胞、腱细胞、肌肉细胞、神经细胞等。骨髓间充质干细胞具有贴壁生长的特性 ,在体外易分离和扩增 ,还易于外源基因的转入和表达 ,在人类医学上被认为是一种理想的治疗性细胞和基因治疗中的靶细胞。本文针对骨髓间充质干细胞的研究进展和在临床医学上的应用进行综述     

Proliferation of equine bone marrow-derived mesenchymal stem cells in gelatin/β-tricalcium phosphate sponges

A three dimensional scaffold is essential in mesenchymal stem cells (MSCs) delivery in cell-based therapy for facilitating cell adherence, migration, proliferation, and differentiation. The objectives of this study were to evaluate the possibility of β-tricalcium phosphate incorporated gelatin sponges (Gelatin/β-TCP sponge) as scaffolds for equine MSCs and to examine the effects of seeding density and seeding method on the proliferation of equine MSCs in the Gelatin/β-TCP sponges. Mononuclear cells and MSCs isolated from bone marrow were seeded into Gelatin/β-TCP sponges at different densities by different seeding methods-static or agitated methods. Proliferation of the MSCs in Gelatin/β-TCP was assessed using the Cell Counting Kit-8 assay and histological examination. Distribution and proliferation of MSCs in the Gelatin/β-TCP sponge were observed, and the Gelatin/β-TCP sponge supported limited growth when seeded at high density. We also found that the agitated seeding method enhanced the proliferation of MSCs. This study demonstrated the suitability of Gelatin/β-TCP sponges for the proliferation and maintenance of equine MSCs. These results contribute to the application of MSC-seeded Gelatin/β-TCP sponges in equine 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.     

The comparison of equine articular cartilage progenitor cells and bone marrow-derived stromal cells as potential cell sources for cartilage repair in the horse

A chondrocyte progenitor population isolated from the surface zone of articular cartilage presents a promising cell source for cell-based cartilage repair. In this study, equine articular cartilage progenitor cells (ACPCs) and equine bone marrow-derived stromal cells (BMSCs) were compared as potential cell sources for repair. Clonally derived BMSCs and ACPCs demonstrated expression of the cell fate selector gene, Notch-1, and the putative stem cell markers STRO-1, CD90 and CD166. Chondrogenic induction revealed positive labelling for collagen type II and aggrecan. Collagen type X was not detected in ACPC pellets but was observed in all BMSC pellets. In addition, it was observed that BMSCs labelled for Runx2 and matrilin-1 antibodies, whereas ACPC labelling was significantly less or absent. For both cell types, osteogenic induction revealed positive von Kossa staining in addition to positive labelling for osteocalcin. Adipogenic induction revealed a positive result via oil red O staining in both cell types. ACPCs and BMSCs have demonstrated functional equivalence in their multipotent differentiation capacity. Chondrogenic induction of BMSCs resulted in a hypertrophic cartilage (endochondral) phenotype, which can limit cartilage repair as the tissue can undergo mineralisation. ACPCs may therefore be considered superior to BMSCs in producing cartilage capable of functional repair.     

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.     

Equine adipose-tissue derived mesenchymal stem cells and platelet concentrates: their association in vitro and in vivo

Equine mesenchymal stem cells (MSC) are of particular interest both for basic research and for the therapeutic approach to musculoskeletal diseases in the horse. Their multilineage differentiation potential gives them the capability to contribute to the repair of tendon, ligament and bone damage. MSCs are also considered a promising therapeutic aid in allogeneic cell transplantation, since they show low immunogenicity and immunomodulating functions.Adipose tissue-derived adult equine stem cells (AdMSC) can be isolated, expanded in vitro and then inoculated into the damaged tissue, eventually in the presence of a biological scaffold. Here we report our preliminary experience with adipose-derived mesenchymal stem cells in allogeneic cell-therapy of tendonitis in the horse. MSCs, derived from visceral adipose tissue, were grown in the presence of autologous platelet lysate and characterized for their differentiation and growth potential. Expanded AdMSC were inoculated into the damaged tendon after their dispersion in activated platelet-rich plasma (PRP), a biological scaffold that plays an important role in maintaining cells in defect sites and contributes to tissue healing. Fourteen out of sixteen treated horses showed a functional recovery and were able to return to their normal activity.     

Isolation of equine bone marrow‐derived mesenchymal stem cells: a comparison between three protocols

Reason for performing the study: There is a need to assess and standardise equine bone marrow (BM) mesenchymal stem cell (MSC) isolation protocols in order to permit valid comparisons between therapeutic trials at different sites. Objective: To compare 3 protocols of equine BM MSC isolation: adherence to a plastic culture dish (Classic) and 2 gradient density separation protocols (Percoll and Ficoll). Materials and methods: BM aspirates were harvested from the sternum of 6 mares and MSCs isolated by all 3 protocols. The cell viability after isolation, MSC yield, number of MSCs attained after 14 days of culture and the functional characteristics (self‐renewal (CFU) and multilineage differentiation capacity) were determined for all 3 protocols. Results: The mean ± s.d. MSC yield from the Percoll protocol was significantly higher (6.8 ± 3.8%) than the Classic protocol (1.3 ± 0.7%). The numbers of MSCs recovered after 14 days culture per 10 ml BM sample were 24.0 ± 12.1, 14.6 ± 9.5 and 4.1 ± 2.5 × 10 ⁶ for the Percoll, Ficoll and Classic protocols, respectively, significantly higher for the Percoll compared with the Classic protocol. Importantly, no significant difference in cell viability or in osteogenic or chondrogenic differentiation was identified between the protocols. At Passage 0, cells retrieved with the Ficoll protocol had lower self‐renewal capacity when compared with the Classic protocol but there was no significant difference between protocols at Passage 1. There were no significant differences between the 3 protocols for the global frequencies of CFUs at Passage 0 or 1. Conclusions and clinical relevance: These data suggest that the Percoll gradient density separation protocol was the best in terms of MSC yield and self‐renewal potential of the MSCs retrieved and that MSCs retrieved with the Ficoll protocol had the lowest self‐renewal but only at passage 0. Then, the 3 protocols were equivalent. However, the Percoll protocol should be considered for equine MSC isolation to minimise culture time.