Equine umbilical cord blood was collected without complications to the mare or foal at the time of foaling using a standard commercially available blood transfusion bag system. Mesenchymal stem cells with in vitro differentiation potency towards the osteogenic, chondrogenic and adipogenic cell fates were successfully isolated from the cord blood. The undifferentiated MSCs have been cryopreserved, thawed and expanded post-thawing without obvious loss of morphology, proliferative potential and differentiation capacity.
The volume of blood collected was markedly higher (mean 168 ml) than what has been reported in human studies (mean 42.8 ml, range 13–80 ml [12
]; mean 60.9 ml, range 17–141,5 ml [33
]). The MSCs isolation frequency of 57% is very similar to reported isolation frequencies in human studies of up to 63% [12
]. However, these high isolation frequencies are often only obtained in a subset of human cord blood samples after applying various critical parameters [12
]. Correlating the number of primary colonies to the volume of blood processed might help determine whether this apparently high success rate is due to the higher volume of equine blood collected or due to higher inherent precursor frequency of these cells in equine cord blood compared to human cord blood. Here, the precursor frequency was very low with primary colonies varying between 1 and 5 per sample, indicating that this stem cell population is a very rare cell type, which is in correlation with human studies [12
]. The different morphologies of undifferentiated cells are intriguing since they might reflect inherently different stem cell phenotypes. Morphological and differentiation potency differences between USSCs and "true" MSCs have been proposed [32
]. Mesenchymal stem cells apparently form Alizarin Red positive nodules after six to eight passages and lack endodermic potential; whereas USSCs retain spindle-shaped fibroblastoid cell morphology in monolayer and have endodermic potential [13
]. Whether the different morphologies noted in this study reflect USSCs and MSCs is undetermined, but warrants further investigation. So-far reduced growth rates have not been observed during sub-culturing. However, the number of samples is insufficient to draw any conclusion regarding the proliferative potential of these cells and how their proliferative potential might compare to equine bone marrow, and possibly equine adipose-derived MSCs.
Although further molecular characterization is required, the osteogenic and chondrogenic potency of these cells has been convincingly demonstrated in this study. No monoclonal antibodies are presently available for immunophenotyping equine mesenchymal stem cells and a specific panel of MSC markers has not been reported in the scientific literature. If and when such an equine MSC marker panel is available, screening and cell separation of the undifferentiated umbilical cord blood derived MSCs would be very interesting in order to compare the immunophenotye profile of these cells with that of human cord blood derived MSCs and that of equine bone marrow and possibly adipose tissue derived MSCs. Nevertheless, osteogenesis has been demonstrated by both morphological and functional criteria as established in previous studies of MSCs [12
]. Chondrogenic assessment was used using established methods and our histological findings of typical hyaline cartilage morphology with glycosaminoglycan containing matrix is in accordance with other similar studies of MSCs [13
]. Koerner and colleagues (2006) reported that adult equine peripheral blood progenitor cells did not show any capacity to produce cartilage at the histological level [25
]. This apparent difference in chondrogenic differentiation potential between equine cord blood and adult peripheral blood is undetermined but warrants further investigation. Adipogenesis of peripheral blood derived MSCs from adult horses was previously shown successfully, although reduced adipogenic potential was noted compared to human MSCs [25
]. Here, rabbit serum was added to the commercial adipogenic induction media based on the work by Janderova and colleagues (2003) in which superior adipogenesis was demonstrated [35
]. Adipogenic differentiation of the equine cord blood derived MSCs were only successful with the addition of rabbit serum to the media. Conclusions to the adipogenic potential of adult equine peripheral blood MSCs versus equine cord blood MSCs cannot be made at this point in time, since the adipogenic potential observed for both cell types was demonstrated under different culture conditions. Whether there is an inherent difference between these blood-derived blood MSCs responsible for our failure to induce adipogenesis without the addition of rabbit serum is undetermined. Also, the effect of rabbit serum on the adipogenic differentiation potential of adult equine blood MSCs is unknown. Adipogenesis was unsuccessful for human cord blood derived MSCs cultured in rabbit serum deficient media [16
]. Whether this is a methodological difference or a cellular difference between equine and human cord blood MSCs warrants further studies.
Equine cord blood MSCs might have superior cellular characteristic to other equine stem cells with regard to immune tolerance, proliferative potential and differentiation potency, which are desirable traits in selection of cells for tissue engineering. Comparative studies of human bone marrow, adipose tissue and cord blood derived MSCs revealed lower isolation frequency from cord blood compared to bone marrow and adipose tissue, but cord blood derived MSCs had higher proliferative potential than both bone marrow and adipose tissue derived MSCs [16
]. Longer telomeres, higher telomerase activity and superior in vitro
proliferation capacity and differentiation potential of human cord blood MSCs compared to human bone marrow and adipose tissue derived MSCs have also been reported [12
]. Human leukemia patients transfused with HLA mismatched cord blood compared to transfusions of HLA matched bone marrow showed no difference in treatment outcome [24
] indicating that cord blood derived stem cells might be immunologically privileged compared to bone marrow derived stem cells. The exact immunogenic potential of umbilical cord blood derived MSCs remain undetermined. Banking of ELA matched cord blood derived stem cells would therefore be the safest approach for allogenic-based tissue engineering or stem cell-based therapies at the moment. Isolation of MSCs from cryopreserved cord blood is very difficult and MSCs isolation and expansion should be carried out from fresh cord blood prior to long-term cryopreservation [31
The impact of orthopaedic injuries to the health and revenue of the individual racehorse can be dramatic as was recently illustrated by the catastrophic and life-threatening injury that the 2006 Kentucky Derby champion "Barbaro" sustained during the 2006 Preakness Stakes. The financial magnitude of Thoroughbred racing in general is illustrated by the fact that in 2004 a total of 171 thousand Thoroughbred races were completed worldwide competing for a total purse value of 4 billion US dollars [1
]. Bearing this in mind there is naturally a great interest in new possible treatment modalities for equine orthopaedic injuries which is also illustrated by the experimental application of autologous bone marrow MSCs and adipose tissue derived cell products to race and non-race horses with especially tendon problems [28
]. The horse is a recognized animal model of OA induced injuries at various orthopaedic research facilities around the world for several reasons; the pathophysiology of OA appears similar between horses and humans, there are significant similarities in joint cartilage composition between horse and man and the shear size of horse joints and structures makes surgical manipulation easy compared to other domestic animal models [36
]. The horse also provides a unique opportunity of studying naturally occurring injuries, which in many cases have considerable resemblance to injuries in human athletes. Deliveries of racehorses are generally observed making large-scale cord blood collection very feasible. For all of the above-mentioned similarities between horse and man, the horse now appears as a very favourable pre-clinical animal model for cord blood MSC research.