How to maintain and transport equine adipose tissue for isolating mesenchymal stem cells?

BACKGROUND: Adipose tissue (AT) is one of the most important mesenchymal stem cell (MSC) sources because of its high quantities, availability and ease of collection. After being collected samples, they should be transported to a laboratory for stem cell (SC) isolation, culture and expansion for future clinical application. Usually, laboratories are distant from animal husbandry centers; therefore, it is necessary to provide suitable conditions for adipose tissue transportation, such that adipose-derived MSCs are minimally affected. In the current study, the impact of tissue maintenance under different conditions on MSCs derived from these tissues was evaluated. We aimed at finding suitable and practical transportation methods in which ASCs go through the slightest changes. RESULTS: In the current study, after being collected, equine AT was randomized into eight groups: four samples were maintained in stem cell culture media at 25 οC and 4 οC for 6 and 12 hrs. as transportation via SC media groups. Three samples were frozen at three different temperatures (- 20, - 75 and - 196 οC) as cryopreserved groups; these samples were defrosted 1 week after cryopreservation. Fresh and unfrozen AT was evaluated as a control group. The tissue samples were then initiated into enzymatic digestion, isolation and the culturing of SCs. Cells at passage three were used to evaluate the ability to form colonies, proliferation rate, plotting of the cell growth curve, and viability rate. All experiments were performed in triplicate. Stem cell isolation was successful in all groups, although purification of SCs from the first series of cryopreservation at - 196 οC and two series of - 20 οC was unsuccessful. There was no significant difference between the surface area of colonies in all groups except for - 20 οC. The growth rate of transportation via stem cell media at 25 οC for 6 hrs. was similar to that of the control group. MTT analysis revealed a significant difference between 25 οC 12 hrs. Group and other experimental groups except for control, 4 οC 12 hrs. and - 196 οC group. CONCLUSION: Data have shown freezing at - 75 οC, transportation via stem cell media at 4 οC for 12 hrs. and 25 οC for 6 hrs. are acceptable tissue preservation and transportation methods due to minor effects on MSCs features.

Differentiation of human adipose-derived mesenchymal stem cells toward tenocyte by platelet-derived growth factor-BB and growth differentiation factor-6.

Mesenchymal Stem Cells (MSCs) are important in regenerative medicine and tissue engineering and will be a very sensible choice for repair and regeneration of tendon. New biological practices, such as cellular therapy using stem cells, are promising for facilitating or expediting tendon therapy. Before using these cells clinically, it is best to check and confirm the optimal conditions for differentiation of these cells in the laboratory. Hence, in the present study, the impacts of PDGF-BB and GDF-6 supplementation on adipose-derived MSCs (ASCs) culture were studied. The frozen ASC were recovered and expanded in basic culture medium (DMEM with 10%FBS). The cells after passage five (P5) were treated with basic medium containing L-Prolin, Ascorbic Acid and only PDGF-BB or GDF-6 (20 ng/ml) or both of them (mix) as 3 groups for 14 days to investigate efficiency of ASCs differentiation towards tenocytes. The cells culturing in basic medium were used as control group. To validate tenogenic differentiation, H&E and Sirius Red staining were used to assess cell morphology and collagen production, respectively. In addition, mRNA levels of collagen I and III, Scleraxis and Tenomodulin as tenogenic markers were analyzed using qPCR. In all test groups, cells appeared slenderer, elongated cytoplasmic attributes compared to the control cells. The intensity of Sirius Red staining was significantly higher in GDF-6, PDGF-BB alone, than in group without supplements. The optical density was higher in the GDF-6 than PDGF-BB and mix-group. QPCR results showed that Col I and III gene expression was increased in all groups compared to the control. SCX expression was significantly increased only in the PDGF-BB group. TNMD mRNA expression was not significant among groups. In this study, we have corroborated that human ASCs are reactionary to tenogenic induction by GDF-6 and PDGF-BB alone or in combination. These outcomes will help greater insight into GDF-6 and PDGF-BB driven tenogenesis of ASCs and new directions of discovery in the design of ASC-based treatments for tendon healing.

The effect of equine bone marrow-derived mesenchymal stem cells on the expression of apoptotic genes in neutrophils.

BACKGROUND: Bone marrow mesenchymal stem cells (BM-MSCs), as multipotent cells with self-renewal and plastic-adherent properties, have immunomodulatory effects on immune cells, including neutrophils. These cells are in close proximity in bone marrow (BM) sinusoids with non-multiplicative immature neutrophils. BM-MSCs exert their immunomodulatory effects on adjacent cells both directly (cell-to-cell contact) and indirectly (secretion of soluble factors). OBJECTIVES: The aim of this study was to evaluate the effect of equine bone marrow mesenchymal stem cells (BM-MSCs) on the expression of some pro- and anti-apoptotic genes (p53, survivin and Bcl2 ) in neutrophils co-cultured with BM-MSCs. METHODS: For this purpose, peripheral blood neutrophils were isolated and separately co-cultured for 12 hr with both BM-MSCs and the BM-MSCs΄ supernatant. Four groups were included: neutrophils with only culture media (as control), neutrophils co-cultured with BM-MScs, neutrophils cultured with BM-MSCs' supernatant and neutrophils cultured with lipopolysaccharide (LPS, as positive control). Then, the expression of mentioned genes (p53, survivin and Bcl2 ) was evaluated by quantitative polymerase chain reaction (qPCR). RESULTS: Compared with control neutrophils, in neutrophils co-cultured with both BM-MSCs and BM-MSCs' supernatant, the mRNA expression levels of p53, as pro-apoptotic gene, and survivin and Bcl2 , as anti-apoptotic genes, were remarkably increased and decreased (p < .05), respectively. CONCLUSIONS: These data revealed the notion that the direct contact of BM-MSCs is not obligatory for their effects on the apoptotic status of neutrophils and they affect neutrophils via soluble secreted factors, which is promising for clinical implications in equine medicine.

Prognostic role of follicular fluid tumor necrosis factor alpha in the risk of early ovarian hyperstimulation syndrome.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic condition characterized by capillary hyperpermeability which can be predicted by preovulatory ovarian responses such as number of follicles. A variety of cytokines are thought to be involved in pathophysiology of this syndrome. METHODS: A prospective cohort study invloving sixty intracytoplasmic sperm injection (ICSI) patients. On the day of hCG injection, we explored the threshold of larger follicles ≥11 mm diameter with a count of ≥18 follicles for the high-risk moderate-to-severe OHSS and 13-18 follicles for the low-risk moderate-to-severe OHSS. Whereas larger follicles count of less than 13 were classified as normoresponders. Pooled follicular fluid (FF) samples of each patient were collected on the day of oocyte retrieval. Magnetic multiplex immunoassay was explored to measure the concentrations of some intrafollicular cytokines including: GM-CSF, INF-γ, TNF-α, IL-10, CXCL8/IL-8, IL-6, IL-5, IL-4, IL-2, and IL-1ß. All sixty patients underwent controlled ovarian hyperstimulation (COH) with either GnRH agonist or antagonist protocols. RESULTS: Intrafollicular TNF-α concentration was significantly different (p < 0.05) in the high-risk moderate-to-severe OHSS patients compared to low-risk moderate-to-severe OHSS patients and normoresponders. TNF-α in FF had a negative correlation with the chance of high-risk moderate-to-severe OHSS. The differences in the risk of OHSS between patients who received GnRH agonist or antagonist were not significant (p > 0.05). CONCLUSIONS: In accordance to the negative correlation of TNF-α and high risk of early OHSS, we did not expect TNF-α to play a role in increasing vascular permeability in ovarian tissues. In addition, the risk of early moderate-to-severe OHSS was not affected by different GnRH superovulation protocols.

Induction of tenogenic differentiation of equine adipose-derived mesenchymal stem cells by platelet-derived growth factor-BB and growth differentiation factor-6.

Managing tendon healing process is complicated mainly due to the limited regeneration capacity of tendon tissue. Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and have been considered for tendon repair and regeneration. This study aimed to evaluate the capacity of equine adipose tissue-derived cells (eASCs) to differentiate into tenocytes in response to platelet-derived growth factor-BB (PDGF-BB) and growth differentiation factor-6 (GDF-6) in vitro. Frozen characterized eASCS of 3 mares were thawed and the cells were expanded in basic culture medium (DMEM supplemented with 10% FBS). The cells at passage 5 were treated for 14 days in different conditions including: (1) control group in basic culture medium (CM), (2) induction medium as IM (CM containing L-prolin, and ascorbic acid (AA)) supplemented with PDGF-BB (20 ng/ml), (3) IM supplemented with GDF-6 (20 ng/ml), and (4) IM supplemented with PDGF-BB and GDF-6. At the end of culture period (14th day), tenogenic differentiation was evaluated. Sirius Red staining was used to assess collagen production, and H&E was used for assessing cell morphology. mRNA levels of collagen type 1 (colI), scleraxis (SCX), and Mohawk (MKX), as tenogenic markers, were analyzed using real-time reverse-transcription polymerase chain reaction (qPCR). H&E staining showed a stretching and spindle shape (tenocyte-like) cells in all treated groups compared to unchanged from of cells in control groups. Also, Sirius red staining data showed a significant increase in collagen production in all treated groups compared with the control group. MKX expression was significantly increased in PDGF-BB and mixed groups and COLI expression was significantly increased only in PDGF-BB group. In conclusion, our results showed that PDGF-BB and GDF-6 combination could induce tenogenic differentiation in eASCs. These in vitro findings could be useful for cell therapy in equine regenerative medicine.

Equine adipose mesenchymal stem cells (eq-ASCs) appear to have higher potential for migration and musculoskeletal differentiation.

Equine adipose-derived mesenchymal stem cells (eq-ASCs) possess excellent regeneration potential especially for treatment of musculoskeletal disorders. Besides their common characteristics, MSCs harvested from different species reveal some species-specific and donor-dependent behaviors. Hence, the molecular analysis of MSCs may shed more light on their future clinical application of these cells. This study aimed to investigate some behavioral aspects of eq-ASCs in vitro which may influence the efficacy of stem cell therapy. For this purpose, MSCs of a donor horse were isolated, characterized and expanded under normal culture conditions. During continuous culture condition, eq-ASCs were started to formed aggregated structures that was accompanied with the up-regulation of migratory related genes including transforming growth factor beta 1 (TGFB1) and its receptor 3 (TGFBR3), and snail family transcriptional repressor 1 (SNAI1), E-cadherin (CDH1) and ß-catenin (CTNNB1). Moreover, the expression of a musculoskeletal progenitor marker, scleraxis bHLH transcription factor (SCX), was also increased after 3 days. In order to clarify the impact of TGFB signaling pathway on cultured cells, gain- and loss-of-function treatment by TGFB3 and SB431542 (TGFB inhibitor) were performed, respectively. We found that TGFB3 treatment exaggerated the aggregate formation effects, in some extend via induction of cytoskeletal actin rearrangement, while inhibition of TGFB signaling pathway by SB431542 reversed this phenomenon. Overall, our findings support the fact that eq-ASCs have an inherent capacity for migration, which was enhanced by TGFB3 treatment and, this ability may play crucial role in cell motility and wound healing of transplanted cells.

IL-5 in follicular fluid as a negative predictor of the intracytoplasmic sperm injection outcome.

PURPOSE: To determine the prognostic value of intrafollicular concentrations of some cytokines from women undergoing ovarian stimulation in the outcome of intracytoplasmic sperm injection/embryo transfer (ICSI/ET) cycles. METHODS: A total of 80 patients were included in this study following ovarian stimulation and ICSI. Follicular fluids (FF) were collected at the day of oocyte retrieval. Ten cytokines including: tumor necrosis factor- alpha (TNF-α), interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, CXCL8/IL-8, IL-10, granulocyte-macrophage colony stimulating factor (GM-CSF), and interferon gamma (IFN-γ) were measured using magnetic multiplex immunoassays. RESULTS: Only the concentration of IL-5, IL-4, and GM-CSF in FF were significantly different (p < 0.05) between ICSI cycles that resulted in pregnancy and those that failed. Elevated FF IL-5 levels were associated with poor oocyte quality, which decreases the chance of both biochemical and clinical pregnancy. Higher FF GM-CSF associated with decrease of mature oocytes, while higher FF IL-4 concentrations were linked to good ICSI outcome through increased fertilization rate. CONCLUSIONS: The elevated intrafollicular concentrations of IL-5 seem to be a negative predictor to the pregnancy outcome in ICSI cycles.

Equine bone marrow-derived mesenchymal stem cells: optimization of cell density in primary culture.

BACKGROUND: The primary cell seeding density of bone marrow-derived mononuclear cells (BM-MNCs) affects several cellular behaviors, including attachment to the culture dish, proliferation, and differentiation. METHODS: The aim of this study was to determine the best density of equine BM-MNCs in primary culture (P0) for obtaining the maximum bone marrow-derived mesenchymal stem cell (BM-MSC) yields at the end of P0. Bone marrow samples of two healthy mares were aspirated. The MNCs were isolated and cultured at different densities (1×105, 2×105, 4×105, 8×105, and 1×106 cells/cm2). Within the 7th and 14th days after seeding, the colonies containing more than 15 cells were counted and the percentage of confluency and the number of cells were calculated on day 21. RESULTS: The lowest density of MNCs was associated with the least number of colonies, number of adherent cells, and confluency percentage, whereas the highest density was associated with the maximum number of colonies and confluency percentage (P<0.05). However, the maximum number of cells at the end of P0 was associated with the intermediate (4×105 cells/cm2) and the highest concentration (P<0.05). CONCLUSIONS: The maximum number of MSCs at the end of P0 was obtained at the densities of 1×106 and, especially, at 4×105 cells/cm2.

Immunomodulatory effects of mesenchymal stem cells on leukocytes with emphasis on neutrophils.

Mesenchymal stem cells (MSCs) are a population of multipotent cells with the ability of expansion and plastic-adherence in vitro. MSCs can differentiate into chondrocytes, osteocytes and adipocytes; they lack co-stimulatory molecules and have small amount of MHC-I that makes no immunogenicity. These characteristics are empowering MSCs' huge in vivo applications. In addition, MSCs possess the ability of regulating the immune responses in many diseases. Many studies have shown that MSCs have immunosuppressive as well as immunoenhancing properties such as inhibition of T-lymphocytes proliferation and cytokines production which lead to the balance of Th1 and Th2. Some other immunomodulatory features of MSCs are increasing suppressive capacity of Treg, reducing activity of B-lymphocytes and immunoglobulins secretion, inhibition of dendritic cells maturation and antigen presenting capacity, and inhibition of NK-cells activity. MSCs also exert inhibitory effects on neutrophil apoptosis and reduce reactive oxygen species production. The purpose of this paper is to focus on the MSCs' effects on immune cells, especially neutrophils.

Equine adipose-derived mesenchymal stem cells: phenotype and growth characteristics, gene expression profile and differentiation potentials.

OBJECTIVE: Because of the therapeutic application of stem cells (SCs), isolation and characterization of different types of SCs, especially mesenchymal stem cells (MSCs), have gained considerable attention in recent studies. Adipose tissue is an abundant and accessible source of MSCs which can be used for tissue engineering and in particular for treatment of musculoskeletal disorders. This study was aimed to isolate and culture equine adipose-derived MSCs (AT-MSCs) from little amounts of fat tissue samples and determine some of their biological characteristics. MATERIALS AND METHODS: In this descriptive study, only 3-5 grams of fat tissue were collected from three crossbred mares. Immediately, cells were isolated by mechanical means and enzymatic digestion and were cultured in optimized conditions until passage 3 (P3). The cells at P3 were evaluated for proliferative capacities, expression of specific markers, and osteogenic, chondrogenic and adipogenic differentiation potentials. RESULTS: Results showed that the isolated cells were plastic adherent with a fibroblast-like phenotype. AT-MSCs exhibited expression of mesenchymal cluster of differentiation (CD) markers (CD29, CD44 and CD90) and not major histocompatibility complex II (MHC-II) and CD34 (hematopoietic marker). Cellular differentiation assays demonstrated the chondrogenic, adipogenic and osteogenic potential of the isolated cells. CONCLUSION: Taken together, our findings reveal that equine MSCs can be obtained easily from little amounts of fat tissue which can be used in the future for regenerative purposes in veterinary medicine.