Isolation and characterization of equine amniotic membrane-derived mesenchymal stem cells

Recent studies have shown that mesenchymal stem cells (MSCs) are able to differentiate into multi-lineage cells such as adipocytes, chondroblasts, and osteoblasts. Amniotic membrane from whole placenta is a good source of stem cells in humans. This membrane can potentially be used for wound healing and corneal surface reconstruction. Moreover, it can be easily obtained after delivery and is usually discarded as classified waste. In the present study, we successfully isolated and characterized equine amniotic membrane-derived mesenchymal stem cells (eAM-MSCs) that were cultured and maintained in low glucose Dulbecco''s modified Eagle''s medium. The proliferation of eAM-MSCs was measured based on the cumulative population doubling level (CPDL). Immunophenotyping of eAM-MSCs by flow cytometry showed that the major population was of mesenchymal origin. To confirm differentiation potential, a multi-lineage differentiation assay was conducted. We found that under appropriate conditions, eAM-MSCs are capable of multi-lineage differentiation. Our results indicated that eAM-MSCs may be a good source of stem cells, making them potentially useful for veterinary regenerative medicine and cell-based therapy.     

Expansion under hypoxic conditions enhances the chondrogenic potential of equine bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are widely used in regenerative medicine in horses. Most of the molecular characterisations of BM-MSCs have been made at 20% O₂, a higher oxygen level than the one surrounding the cells inside the bone marrow. The present work compares the lifespan and the tri-lineage potential of equine BM-MSCs expanded in normoxia (20% O₂) and hypoxia (5% O₂). No significant differences were found in long-term cultures for osteogenesis and adipogenesis between normoxic and hypoxic expanded BM-MSCs. An up-regulation of the chondrogenesis-related genes (COL2A1, ACAN, LUM, BGL, and COMP) and an increase of the extracellular sulphated glycosaminoglycan content were found in cells that were expanded under hypoxia. These results suggest that the expansion of BM-MSCs in hypoxic conditions enhances chondrogenesis in equine BM-MSCs.     

Comparative characteristic study from bone marrow-derived mesenchymal stem cells

Tissue engineering has been extensively investigated and proffered to be a potential platform for novel tissue regeneration. The utilization of mesenchymal stem cells (MSCs) from various sources has been widely explored and compared. In this regard, MSCs derived from bone marrow have been proposed and described as a promising cell resource due to their high yield of isolated cells with colony-forming potential, self-renewal capacity, MSC surface marker expression, and multi-lineage differentiation capacities in vitro. However, there is evidence for bone marrow MSCs (BM-MSCs) both in vitro and in vivo from different species presenting identical and distinct potential stemness characteristics. In this review, the fundamental knowledge of the growth kinetics and stemness properties of BM-MSCs in different animal species and humans are compared and summarized. Finally, to provide a full perspective, this review will procure results of current information studies focusing on the use of BM-MSCs in clinical practice.     

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.     

In vitro comparison of feline bone marrow-derived and adipose tissue-derived mesenchymal stem cells

Mesenchymal stem cells (MSC) are increasingly being proposed as a therapeutic option for a variety of different diseases in human and veterinary medicine. At present, MSC are most often collected from bone marrow (BM) or adipose tissue (AT) and enriched and expanded in vitro before being transferred into recipients. However, little is known regarding the culture characteristics of feline BM-derived (BM-MSC) versus AT-derived MSC (AT-MSC). We compared BM-MSC and AT-MSC from healthy cats with respect to in vitro growth and cell surface phenotype. Mesenchymal stem cells isolated from AT proliferated significantly faster than BM-MSC. Phenotypic differences between BM-MSC and AT-MSC were not present in the surface markers assessed. We conclude that BM-MSC and AT-MSC are similar phenotypically but that cultures of AT-MSC are easier to generate because of their higher intrinsic proliferative rate. Thus, AT-MSC may be the preferred MSC for clinical applications where rapid and efficient generation of MSC is important.     

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.     

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.     

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.     

Characterization of equine adipose tissue-derived stromal cells: adipogenic and osteogenic capacity and comparison with bone marrow-derived mesenchymal stromal cells

Objective— To characterize equine adipose tissue-derived stromal cell (ASC) frequency and growth characteristics and assess of their adipogenic and osteogenic differentiation potential.
Study Design— In vitro experimental study.
Animals— Horses (n=5; aged, 9 months to 5 years).
Methods— Cell doubling characteristics of ASCs harvested from supragluteal subcutaneous adipose tissue were evaluated over 10 passages. Primary, second (P2), and fourth (P4) passage ASCs were induced under appropriate conditions to undergo adipogenesis and osteogenesis. Limit dilution assays were performed on each passage to determine the frequency of colony-forming units with a fibroblastic (CFU-F) phenotype and the frequency of ASC differentiation into the adipocyte (CFU-Ad) and osteoblast (CFU-Ob) phenotype.
Results— ASC isolates exhibited an average cell-doubling time of 2.1±0.9 days during the first 10 cell doublings. Approximately 1 in 2.3±0.4 of the total stromal vascular fraction nucleated cells were ASCs, based on the CFU-F assays, and 1 in 3.6±1.3 expressed alkaline phosphatase, an osteogenic marker. Primary ASCs differentiated in response to adipogenic (1 in 4.9±5.4, CFU-Ad) and osteogenic (1 in <2.44, CFU-Ob) inductive conditions and maintained their differentiation potential during subsequent passages (P2 and P4).
Conclusion— The frequency, in vitro growth rate, and adipogenic and osteogenic differentiation potential of equine ASCs show some differences to those documented for ASCs in other mammalian species.
Clinical Relevance— Adipose tissue is a potential source of adult stem cells for tissue engineering applications in equine veterinary medicine.     

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.     

野猪骨髓间充质干细胞的分离培养与生物学特性

采用全骨髓培养法对野猪股骨中骨髓间充质干细胞（Bone mesenchymal stem cells,BMSC）进行分离、培养并传代,建立野猪骨髓间充质干细胞体外培养方法及对其生物学特性进行观察研究。结果显示,细胞形态呈梭形,漩涡状生长,生长曲线为典型S型,在诱导液作用下,可分化为成脂肪样细胞。结果表明,通过本方法能够分离到较纯的BMSC,为野猪资源保存和体细胞核移植提供技术支持。     

猪骨髓间充质干细胞分离培养和诱导分化脂肪细胞

采用全骨髓培养法分离猪骨髓间充质干细胞(Mesenchymal stem cells,MSCs)并传代培养;取第4代纯化的MSCs在成脂诱导培养基中诱导分化;分化的成脂细胞用形态学和油红O染色法进行鉴定;用实时荧光定量PCR(Real-time PCR)检测成脂分化标志基因PPARγ2和LPL mRNA的表达情况。结果显示,分离培养的猪MSCs细胞经连续传代形态上无明显改变;MSCs在成脂分化培养液中诱导分化2d开始有少量脂滴出现,油红O染色成阳性,诱导18d成脂转化率可达59.8%;在诱导分化第5、10、15天时,PPARγ2mRNA相对表达量分别是(5.065±0.159)、(6.268±0.340)、(9.277±0.261),LPL mRNA的相对表达量分别是(10.995±1.473)、(13.130±0.712)、(15.762±0.934)。结果表明,用本诱导条件诱导猪MSCs向脂肪细胞分化,经形态学和油红O染色鉴定,成脂细胞分化率可达60%,且随分化时间的延长,脂肪细胞标志基因表达增加。     

猪骨髓间充质干细胞分离培养及生物学特性

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