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.     

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.     

Isolation and Differentiation of Mesenchymal Stem Cells From Bovine Umbilical Cord Blood

Currently, mesenchymal stem cells (MSCs) are used in veterinary clinical applications. Bone marrow and adipose tissue are the most common sources of stem cells derived from adult animals. However, cord blood which is collected non‐invasively is an alternative source of stem cells other than bone marrow and adipose tissue. Moreover, high availability and lower immunogenicity of umbilical cord blood (UCB) haematopoietic stem cells compared to other sources of stem cell therapy such as bone marrow have made them a considerable source for cell therapy, but MSCs is not highly available in cord blood and their immunogenicity is poorly understood. In this study, the cells with spindle morphology from 7 of 9 bovine UCB samples were isolated and cultured. These mesenchymal stromal cells were successfully differentiated to osteocytes, chondrocytes and adipocytes. In addition, Oct‐4 and SH3 were determined by RT‐PCR assay. It is the first report of isolation, culture, characterization and differentiation of bovine umbilical stem cells.     

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.     

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.     

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。     

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.     

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.     

崂山奶山羊脂肪间充质干细胞系的建立及鉴定

旨在建立崂山奶山羊脂肪间充质干细胞系并对其进行初步鉴定.采集崂山奶山羊脂肪组织,在Ⅰ型胶原酶的分解消化作用下,将脂肪组织中的单核细胞进行分离并扩增培养;测定其生长曲线,利用Real time RT-PCR技术对其表达的干细胞因子进行鉴定;将传至第3代的脂肪间充质干细胞进行成骨诱导分化,利用茜素红染色进行鉴定.结果显示,崂山奶山羊脂肪间充质干细胞形态为长梭形、星形等成纤维细胞样细胞形态,但比成纤维细胞饱满,第3代细胞生长至3 d左右时细胞进入指数生长期,生长至6~7 d时进入平台期;经成骨诱导分化后,经茜素红染色可见骨结节被染成深红色.综上提示,崂山奶山羊脂肪间充质干细胞具有诱导分化潜能.     

Osteogenic potential of sorted equine mesenchymal stem cell subpopulations

The objectives of this study were to use non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs), to sort equine muscle tissue-derived mesenchymal stem cells (MMSCs) and bone marrow-derived mesenchymal stem cells (BMSC) into subpopulations and to carry out assays in order to compare their osteogenic capabilities. Cells from 1 young adult horse were isolated from left semitendinosus muscle tissue and from bone marrow aspirates of the fourth and fifth sternebrae. Aliquots of 800 × 10³ MSCs from each tissue source were sorted into 5 fractions using non-equilibrium GrFFF (GrFFF proprietary system). Pooled fractions were cultured and expanded for use in osteogenic assays, including flow cytometry, histochemistry, bone nodule assays, and real-time quantitative polymerase chain reaction (qPCR) for gene expression of osteocalcin (OCN), RUNX2, and osterix. Equine MMSCs and BMSCs were consistently sorted into 5 fractions that remained viable for use in further osteogenic assays. Statistical analysis confirmed strongly significant upregulation of OCN, RUNX2, and osterix for the BMSC fraction 4 with P < 0.00001. Flow cytometry revealed different cell size and granularity for BMSC fraction 4 and MMSC fraction 2 compared to unsorted controls and other fractions. Histochemisty and bone nodule assays revealed positive staining nodules without differences in average nodule area, perimeter, or stain intensity between tissues or fractions. As there are different subpopulations of MSCs with different osteogenic capacities within equine muscle- and bone marrow-derived sources, these differences must be taken into account when using equine stem cell therapy to induce bone healing in veterinary medicine.     

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.     

Stem cell therapy in veterinary dermatology

Background –  Adult stem cells come from many sources and have the capacity to differentiate into many cell types, including those of the skin. The most commonly studied stem cells are those termed mesenchymal stem cells (MSCs), which are easily isolated from bone marrow and adipose tissue. Mesenchymal stem cells are known to produce a wide array of cytokines that modulate the regeneration process. The ease of collection, propagation and use of these MSCs in therapy of traumatic, ischaemic and immune‐mediated skin conditions is emerging. Approach and evidence –  In traumatic and ischaemic skin damage, MSCs are used in tissue‐engineered skin and by direct injection into damaged tissue. For immune‐mediated diseases, systemic administration of stem cells can modulate the immune system. The earliest clinical work has been with autologous stem cell sources, such as adipose tissue and bone marrow. In immune‐mediated diseases, the MSCs are used to downregulate production of inflammatory cytokines and to block T‐cell activation. Cells are generally given intravenously. Multiple sclerosis, rheumatoid arthritis and lupus have been successfully treated in human clinical trials. Mesenchymal stem cells can also stimulate resident local cells, such as keratinocytes and progenitor cells, to proliferate, migrate and repair skin injury and disease. Looking ahead –  The discovery of the MSC in adipose tissue has spawned a global effort to utilize these cells in therapy of a wide range of diseases of the skin. Reconstructive surgery, scar blocking and resolution and skin regeneration have all been shown to be possible in human and animal studies.     

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.     

崂山奶山羊骨髓间充质干细胞永生化细胞系的建立

为建立崂山奶山羊骨髓间充质干细胞(BMSCs)永生化细胞系,将已构建的pcDNA3.1-EGFPTERT转入崂山奶山羊BMSCs中,利用含G418的培养基筛选获得稳定转染的TERT-BMSCs;通过多次传代,测定其生长曲线;选取高代次TERT-BMSCs的分裂中期相的细胞,检测其染色体及核型的稳定性。结果表明,转入TERT基因的崂山奶山羊BMSCs能够稳定表达该基因,其生长曲线呈"S"形;在多次传代后,P40代细胞的核型正常率仍能达到88.24%。综上提示,转染得到的TERT-BMSCs具有良好的生长状态,其细胞生长稳定,符合永生化细胞特征,为间充质干细胞应用于组织修复及基因工程种子细胞的制备提供了理论基础。     

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.     

Isolation, culture and chondrogenic differentiation of canine adipose tissue- and bone marrow-derived mesenchymal stem cells–a comparative study

In the dog, mesenchymal stem cells (MSCs) have been shown to reside in the bone marrow (bone marrow-derived mesenchymal stem cells: BM-MSCs) as well as in the adipose tissue (adipose tissue-derived stem cells: ADSCs). Potential application fields for these multipotent MSCs in small animal practice are joint diseases as MSCs of both sources have shown to possess chondrogenic differentiation ability. However, it is not clear whether the chondrogenic differentiation potential of cells of these two distinct tissues is truly equal. Therefore, we compared MSCs of both origins in this study in terms of their chondrogenic differentiation ability and suitability for clinical application. BM-MSCs harvested from the femoral neck and ADSCs from intra-abdominal fat tissue were examined for their morphology, population doubling time (PDT) and CD90 surface antigen expression. RT-PCR served to assess expression of pluripotency marker Oct4 and early differentiation marker genes. Chondrogenic differentiation ability was compared and validated using histochemistry, transmission electron microscopy (TEM) and quantitative RT-PCR. Both cell populations presented a highly similar morphology and marker expression in an undifferentiated stage except that freshly isolated ADSCs demonstrated a significantly faster PDT than BM-MSCs. In contrast, BM-MSCs revealed a morphological superior cartilage formation by the production of a more abundant and structured hyaline matrix and higher expression of lineage specific genes under the applied standard differentiation protocol. However, further investigations are necessary in order to find out if chondrogenic differentiation can be improved in canine ADSCs using different protocols and/or supplements.     

Adult Multipotent Stromal Cell Technology for Bone Regeneration: A Review

Since the discovery of bone marrow derived stromal cell osteogenesis in the 1960s, tissue engineering with adult multipotent stromal cells (MSCs) has evolved as a promising approach to restore structure and function of bone compromised by injury or disease. To date, accelerated bone formation with MSCs has been demonstrated with a variety of tissue engineering strategies. Though MSC bone tissue engineering has advanced over the last few decades, limitations to clinical translation remain. A current review of this promising field is presented with a specific focus on equine investigations.     

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.