Monitoring the fate of autologous and allogeneic mesenchymal progenitor cells injected into the superficial digital flexor tendon of horses: preliminary study

Autologous mesenchymal progenitor cells (MPCs) purified from bone marrow aspirates are being used in the treatment of superficial digital flexor tendon (SDFT) injuries in the horse with promising results. In this study the fate of autologous and allogeneic MPCs following injection into the SDFT was monitored by stable transfection of MPCs with green fluorescent protein (GFP). Small lesions were created manually in one forelimb SDFT of 2 horses and injected with autologous MPCs, allogeneic MPCs or bone marrow supernatant alone. Post mortem examinations performed after 10 or 34 days revealed GFP labelled cells located mainly within injected lesions, but with a small proportion integrated into the crimp pattern of adjacent healthy areas of tendon. Furthermore, there was no visible cell mediated immune response to allogeneic MPCs in either of the host horses.     

Implantation of bone marrow-derived mesenchymal stem cells demonstrates improved outcome in horses with overstrain injury of the superficial digital flexor tendon

Reasons for performing study: Mesenchymal stem (progenitor; stromal) cell (MSC) therapy has gained popularity for the treatment of equine tendon injuries but without reports of long‐term follow‐up. Objectives: To evaluate the safety and reinjury rate of racehorses after intralesional MSC injection in a large study of naturally occurring superficial digital flexor tendinopathy and to compare these data with those published for other treatments. Methods: Safety was assessed clinically, ultrasonographically, scintigraphically and histologically in a cohort of treated cases: 141 client‐owned treated racehorses followed‐up for a minimum of 2 years after return to full work. Reinjury percentages were compared to 2 published studies of other treatments with similar selection criteria and follow‐up. The number of race starts, discipline, age, number of MSCs injected and interval between injury and treatment were analysed. Results: There were no adverse effects of the treatment with no aberrant tissue on histological examination. The reinjury percentage of all racehorses with follow‐up (n = 113) undergoing MSC treatment was 27.4%, with the rate for flat (n = 8) and National Hunt (n = 105) racehorses being 50 and 25.7%, respectively. This was significantly less than published for National Hunt racehorses treated in other ways. No relationship between outcome and age, discipline, number of MSCs injected or injury to implantation interval was found. Conclusions: Whilst recognising the limitations of historical controls, this study has shown that MPC implantation is safe and appears to reduce the reinjury rate after superficial digital flexor tendinopathy, especially in National Hunt racehorses. Potential relevance: This study has provided evidence for the long‐term efficacy of MSC treatment for tendinopathy in racehorses and provides support for translation to human tendon injuries.     

The effect of intralesional injection of bone marrow derived mesenchymal stem cells and bone marrow supernatant on collagen fibril size in a surgical model of equine superficial digital flexor tendonitis

Reasons for performing study: Collagen fibril size is decreased in repair tissue following tendon injury compared to normal tendon matrix in horses. Mesenchymal stem cells have been suggested to promote regeneration of tendon matrix rather than fibrotic repair following injury, although this concept remains unproven. Objectives: To explore the hypothesis that implantation of autologous mesenchymal stem cells derived from bone marrow into a surgically created central core defect in the superficial digital flexor tendon (SDFT) of horses would induce the formation of a matrix with greater ultrastructural similarities to tendon matrix than the fibrotic scar tissue formed in control defects. Methods: Tissue was collected 16 weeks after induction of injury and 12 weeks after treatment from normal and injured regions of control and treated limbs of 6 horses and examined using transmission electron microscopy. Collagen fibril diameters were measured manually with image analysis software and surface areas calculated. Three parameters assessed for normal and injured tissue were mass average diameter (MAD), collagen fibril index (CFI) and the area dependent diameter (ADD). Results: Normal regions from both treated and control limbs displayed higher MAD and CFI values, as well as a characteristic bimodal distribution in fibril size. Injured regions from both treated and control limbs displayed significantly lower MAD and CFI values, as well as a unimodal distribution in fibril size. There were no significant differences between treated and control limbs for any of the parameters assessed. Conclusions: Intralesional injection of autologous bone marrow derived mesenchymal stem cells had no measurable effect on the fibril diameter of collagen in healing tissue in the SDFT of this experimental model 16 weeks after injury. Potential relevance: Favouring matrix regeneration over fibrotic repair may not be the mechanism by which autologous mesenchymal stem cells assist healing of tendon injury.     

Ultrasound‐guided lipoaspiration for mesenchymal stromal cell harvest in the horse

Mesenchymal stromal cells (MSC) are being used to treat a variety of conditions in the horse. Traditional lipectomy and bone marrow aspiration for MSC harvest have disadvantages including cosmetic issues with lipectomy and extensive time for cell expansion after bone marrow harvests. This article describes a new technique of adipose harvest in the horse, utilising ultrasound and liposuction that can be safely and effectively performed in an ambulatory environment. Ultrasound‐guided lipoaspiration offers a minimally invasive technique using small portals and a diffuse area of collection significantly improving aesthetic outcome and increasing the likelihood of treating within a matter of days vs. a matter of weeks.     

Differentiation and characterization of rat adipose tissue mesenchymal stem cells into endothelial‐like cells

In this study, mesenchymal stem cells were isolated from rat adipose tissue (AD‐MSCs) to characterize and differentiate them into endothelial‐like cells. AD‐MSCs were isolated by mechanical and enzymatic treatments, and their identity was verified by colony‐forming units (CFU) test and by differentiation into cells of mesodermal lineages. The endothelial differentiation was induced by plating another aliquot of cells in EGM‐2 medium, enriched with specific endothelial growth factors. Five subcultures were performed. The expression of stemness genes (OCT4, SOX2 and NANOG) was investigated. The presence of CD90 and the absence of the CD45 were evaluated by flow cytometry. The endothelial‐like cells were characterized by the evaluation of morphological changes and gene expression analysis for endothelial markers (CD31, CD144, CD146). Characterization of AD‐MSCs showed their ability to form clones, to differentiate in vitro and the OCT‐4, SOX‐2, NANOG genes expression. Immunophenotypic characterization showed the CD90 presence and the CD45 absence. The endothelial‐like cells showed morphological changes, the expression of CD31, CD144, CD146 genes and the presence of CD31 membrane receptor. Matrigel assay showed their ability to form network and vessels‐like structures. This study lays the foundations for future evaluation of the potential AD‐MSCs pro‐angiogenic and therapeutic role.     

Autologous point‐of‐care cellular therapies variably induce equine mesenchymal stem cell migration,proliferation and cytokine expression

Reasons for performing study: Autologous cellular therapy products including adipose‐derived stromal vascular fraction (SVF), bone marrow mononuclear cells (BMMNs), cord blood mononuclear cells (CBMNs) and platelet rich plasma are options for treatment of acute orthopaedic lesions while mesenchymal stem cells (MSCs) are culture expanded. These products may contribute to healing by secreting matrix proteins or growth factors, but they may also act on endogenous MSCs to facilitate healing. Objectives: To determine the effects of cell therapy products on MSCs function in vitro. The hypothesis was that cell therapy products promote MSCs functions including proliferation, migration and mediator release. Methods: Fat, bone marrow (BM), cord blood and platelets were obtained from 6 Quarter Horses. The BM‐MSCs and their autologous cell therapy products were co‐incubated in transwells. Mesenchymal stem cells proliferation, migration, gene expression and cytokine concentrations were determined. Results: All cell therapy products increased MSCs proliferation, but SVF induced significantly more proliferation than any other product. Also SVF elicited more MSCs chemotaxis and, along with BMMNs, significantly more MSCs chemoinvasion. Cord blood mononuclear cells stimulated MSCs to produce high concentrations of interleukin‐6 (IL‐6), transforming growth factor‐β1 (TGF‐β1), and prostaglandin E₂ (PGE₂). Stromal vascular fraction and platelet lysate did not stimulate MSCs but SVF and platelet lysate themselves contained high concentrations of PGE₂ and IL‐6 (SVF) and TGF‐β1 (platelet lysate). Conclusions: Autologous cell products variably stimulate MSCs functions with 2 primary patterns apparent. Products either contained preformed mediators that may have intrinsic healing function, or products stimulated MSCs to secrete mediators. Potential relevance: The specific clinical indications for these products may differ to include administration as a sole treatment modality prior to MSCs injection for intrinsic cell and cytokine activity (i.e. SVF) or administration concurrently with MSCs to activate MSCs for treatment of chronic lesions (i.e. CBMNs).     

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.     

Isolation and biological characteristics of multipotent mesenchymal stromal cells derived from chick embryo intestine

ABSTRACT

1. Over the past decade, rapid advancement in isolation methods for identifying markers of the once elusive intestinal stem cell (ISC) populations has laid the foundation for unravelling their complex interrelationships during homeostasis. Study on ISC in avian intestinal tissue might play a pivotal foundation for further studies on the epithelial-to-mesenchymal transition (EMT) in gastrointestinal disease and cell-based therapy as well as intestinal tissue engineering.

2. The following experiment isolated a population of fibroblast-like, plastic adhering cells derived from chick embryo intestine, showing a strong self-renewing and proliferative ability, which was maintained in vitro up to passage 25. The findings included growth characteristics, detected expression of cell surface markers and characterised the capability of these cells to differentiate towards the osteogenic, adipogenic, and chondrogenic cell lineages.

3. RT-PCR analysis showed that these cells from chick embryos expressed mesenchymal stromal cell markers CD44, CD90 and VIMENTIN as well as ISC-specific genes LGR5, MI1, SMOC2, BMI1, and HOPX. Immunofluorescence and flow cytometry confirmed this biology characterisation further.

4. In conclusion, cells were isolated from the intestine of 18-day-old chicken embryos that exhibited the biological characteristics of mesenchymal stromal cells as well as markers of intestinal stem cells. Our findings may provide a novel insight for in vitro cell culture and characteristics of ISCs in avian species, which may also indicate a benefit for obtaining cell source for intestinal tissue engineering as well as cell-based investigation for gastrointestinal disease and treatment.     

Relationship between the ultrasonographic findings of suspected superficial digital flexor tendon injury and the prevalence of subsequent severe superficial digital flexor tendon injuries in Thoroughbred horses: a retrospective study

The onset of severe injury to the superficial digital flexor tendon (SDFT) is extremely difficult to predict from slight changes in ultrasonographic findings in cases with no apparent clinical signs. This study investigated the relationship between an increased cross-sectional area (CSA) or edema in the subcutaneous tissue around the tendon and the subsequent onset of severe SDFT injury in Thoroughbred racehorses. Horses were classified into three groups based on ultrasound diagnosis (USD) findings: Group A included cases with enlarged tendons; Group B included cases with tendons of normal size but with prominent edema in the peritendinous tissue; and Group C (control group) included cases with no abnormal USD findings. The incidence of subsequent severe tendon injury was significantly higher in the horses in Groups A (25.7%, 28/101) and B (28.3%, 65/212) than in those in Group C (4.9%, 2/41). There were no significant differences in the median period and the median number of races from the first examination to the subsequent tendon injury between Groups A (140 days, 1 race) and B (120 days, 1 race). The results of this study revealed that horses with increased CSA and peritendinous edema are likely to suffer a subsequent severe tendon injury. Also, these two USD findings, i.e., increased CSA and peritendinous edema, indicate the risk of onset of severe SDFT injury.     

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.     

Generation of embryonic stem‐like cells from in vivo‐derived porcine blastocysts at a low concentration of basic fibroblast growth factor

Although basic fibroblast growth factor (bFGF) is an essential factor supporting the maintenance of porcine embryonic stem (ES) cell self‐renewal and pluripotency, its high cost has limited previous studies, and the development of a low‐cost culture system is required. For these systems, in vivo blastocysts were progressively cultured under various conditions consisting of different culture mediums and/or different feeder cell numbers at a low concentration of bFGF. As the results, the sequential culture of in vivo‐derived porcine blastocysts on 5.0 × 10⁵ mouse embryonic fibroblast (MEF) feeder cells in alpha minimum essential medium‐based medium for primary culture, on 2.5 × 10⁵ MEF feeder cells in Mixture medium for the 1st subpassage, and on 2.5 × 10⁵ MEF feeder cells in DMEM/Ham's F10‐based medium for the post‐2nd subpassage could support the establishment and maintenance of porcine ES‐like cells at the low concentration of bFGF. The established porcine ES‐like cells showed ES cell‐specific characteristics such as self‐renewal and pluripotency. We confirmed that porcine ES‐like cells could be generated from in vivo‐derived porcine blastocysts at a low concentration of bFGF.     

The combination of BMP12 and KY02111 enhances tendon differentiation in bone marrow-derived equine mesenchymal stromal cells (BM-eMSCs)

The Wingless and Int-1 (WNT) and bone morphogenic protein/growth differentiation factor (BMP/GDF) signalling pathways contribute significantly to the development of the musculoskeletal system. The mechanism by which they contribute is as follows: BMP/GDF signalling usually promotes tendon differentiation, whereas WNT signalling inhibits it. We hypothesised that inhibiting WNT and subsequently stimulating BMP signalling may enhance the tenogenic differentiation of stem cells. The objective of this study was to determine whether a combination of WNT inhibitor (KY02111) and BMP12/GDF7 protein could enhance the differentiation of bone marrow-derived equine mesenchymal stromal cells (BM-eMSCs) into tenocytes. Cells were cultured in five treatments: control, BMP12, and three different combinations of BMP12 and KY02111. The results indicated that a 1-day treatment with KY02111 followed by a 13-day treatment with BMP12 resulted in the highest tenogenic differentiation score in this experiment. The effect of KY02111 is dependent on the incubation time, with 1 day being better than 3 or 5 days. This combination increased tenogenic gene marker expression, including SCX, TNMD, DCN, and TNC, as well as COL1 protein expression. In conclusion, we propose that a combination of BMP12 and KY02111 can enhance the in vitro tenogenic differentiation of BM-eMSCs more than BMP12 alone. The findings of this study might be useful for improving tendon differentiation protocols for stem cell transplantation and application to tendon regeneration.     

Horses with equine recurrent uveitis have an activated CD4+ T‐cell phenotype that can be modulated by mesenchymal stem cells in vitro

Equine recurrent uveitis (ERU) is an immune‐mediated disease causing repeated or persistent inflammatory episodes which can lead to blindness. Currently, there is no cure for horses with this disease. Mesenchymal stem cells (MSCs) are effective at reducing immune cell activation in vitro in many species, making them a potential therapeutic option for ERU. The objectives of this study were to define the lymphocyte phenotype of horses with ERU and to determine how MSCs alter T‐cell phenotype in vitro. Whole blood was taken from 7 horses with ERU and 10 healthy horses and peripheral blood mononuclear cells were isolated. The markers CD21, CD3, CD4, and CD8 were used to identify lymphocyte subsets while CD25, CD62L, Foxp3, IFNγ, and IL10 were used to identify T‐cell phenotype. Adipose‐derived MSCs were expanded, irradiated (to control proliferation), and incubated with CD4⁺ T‐cells from healthy horses, after which lymphocytes were collected and analyzed via flow cytometry. The percentages of T‐cells and B‐cells in horses with ERU were similar to normal horses. However, CD4⁺ T‐cells from horses with ERU expressed higher amounts of IFNγ indicating a pro‐inflammatory Th1 phenotype. When co‐incubated with MSCs, activated CD4⁺ T‐cells reduced expression of CD25, CD62L, Foxp3, and IFNγ. MSCs had a lesser ability to decrease activation when cell‐cell contact or prostaglandin signaling was blocked. MSCs continue to show promise as a treatment for ERU as they decreased the CD4⁺ T‐cell activation phenotype through a combination of cell‐cell contact and prostaglandin signaling.