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Updated on 07/05/2021

Use of mesenchymal stromal cells in horse medicine

Authors : S. Boullier, J-L Cadoré and P. Carnat-Gautier

Mesenchymal stromal cells as advanced therapies in veterinary medicines may have a therapeutic use in the treatment of osteo-articular degenerative affection in horses. Other therapeutic interests have yet to be discovered.
A centralized marketing authorization was delivered for two veterinary medical product based on allogenic mesenchymal stromal cells, veterinarians also perform autologous ones.
However, products based on autologous mesenchymal stromal cell, have to be produced by the veterinarian on expert platform, with all the necessary safety and quality controls because of the lack of standardized procedure.
This article is available in the version of the authors above or in the technical supplement of  the journal La Dépêche Vétérinaire n° 180 of October 2020.

Mesenchymal stromal cells (MSC) are part of advanced therapies including (according to the definition of article 4(43) of Regulation (EU) n°2019/6 of the European Parliament and of the Council on veterinary medicinal products):
•    a Veterinary product specifically engineered for gene therapy, regenerative medicine, tissue engineering, therapy by blood products or by phages,
•    a medicine product based on nanotechnology or
•    whatever other therapy considered as emergent field in Veterinary medicine.
They are currently fast growing in Veterinary medicine. The recent obtainment of a marketing authorization (MA) for two products using mesenchymal stromal cells in horses is an opportunity to share the current knowledge on these therapies. 

Definition
According to the International Society of Cell Therapy (ISCT), mesenchymal stromal cells are defined as adherent plastic cells, expressing a specific set of surface antigens and that have the potential to differentiate into osteogenic, chondrogenic or adipogenic cells (Dominici and al, 2006). This definition, somewhat vague, leads to heterogeneity of the mesenchymal stromal cells reported in the literature (Wilson and al, 2019): 
1- Initially, mesenchymal stromal cells were obtained from hematopoietic bone marrow then they have been isolated from various tissues including peripheral blood, cord blood, placenta, adipose tissue, or dental pulp leading a degree of heterogeneity of their biological activity. 
2- As for other primary cells, specific features of the donor such as the genetic/epigenetic properties, physiological and health conditions or age impact the derived mesenchymal stromal cells.
3- The Culture medium, the growth factors as well as the maintenance in cell culture may impact the nature of the derived cells and induce changes of their phenotype. However, there is no standard protocol for the derivation of MSC and their amplification in vitro is a crucial step for the production of therapeutic products. During these in vitro amplifications, other cell types may emerge and negatively impact the final product.

Biological properties and mode of action
The variability is a limit for the clear definition of the mesenchymal stromal cells biological activities. However, most studies point to the fact that unlike the first assumptions, injected mesenchymal stromal cells do not differentiate themselves locally into cells able to replace injured tissues (Von Bahr and al. 2012). Injected MSC only persist few weeks in the body and with their mode of action through a paracrine activity, via the secretion of trophic factors. Consequently, there has been a change in nomenclature of these cells from mesenchymal “stem” cells to mesenchymal “stromal” cells. Overall, mesenchymal stromal cells are established to proceed thanks to immunomodulatory angiogenic and hematopoietic properties (Noronha NC and al. 2019).

Conditions of use
Other factors may drive therapeutic activities of mesenchymal stromal cells (Caplan and al, 2019). Most of initial studies performed in horses looked at osteo-articular affections. Only limited data are available for other indications (Maddens and al, 2018, Cousty 2013). Number of cells as well as injection mode may impact the response. While loco-regional route seems to optimize the quantity of effector cells in the injured zone, very little cells find their way to target tissue after intravenous injection .The quantity of injected MSC may be impact by other cells that may contaminate the MSC stock. This point is crucial to assure a biological effect. For the treatment of degenerative joint diseases, between 2 and 20 millions of cells per injection are currently used in human and horses trials. Storage and preparation must ensure the better cell viability possible

Autologous or allogeneic use
MSC may be used to treat the animal they derived from (autologous) or to treat a receiver (allogeneic). In 2019, a centralized marketing authorization was delivered for two veterinary medical product based on allogenic MSC for their application in osteo-articular degenerative (Horstem®) or inflammatory (Arti-Cell Forte®) diseases in horses. Under the terms laid down in Article L. 5143-4 of the French Public Health Code named “Veterinary therapeutic cascade”, only these two products can be administered for allogeneic treatment for the given indications. This rule applies to all European member states because it comes from the transposition of Articles 10 and 11 of the Directive (EU) 2001/82. 
There is currently no other authorized MSC-based products for other species or indications. The use of autologous products without marketing authorization is possible; the MSC stock must be prepared in lab facilities with suitable means and appropriate technicity to ensure its innocuity. These products cannot be considered as extemporaneous preparation since they have to go through an industrial process.  Both MSC-based autologous and allogenic products are subjected to the same technical constraints in terms of sampling, selection, characterization, amplification, preparation conditions and quality control. The only difference between an autologous product and an allogeneic product is linked to the amplification scale.

Health risks associated with the use of MSC-based products
Like any other drugs, the use of MSC can induce short or long-term side effects (Caplan and al, 2019) among them, an inflammatory response in the receiver due to the production of trophic factors by the inoculated MSC including angiogenic factors, but also to presence of foreign cells from target tissue. It has been hypothesized that MSC, thanks to their particular properties, were invisible to the immune system and then, they did not induce a specific response from their host. It is now well established that MSC are recognized and eliminated by the immune system and this occurs more quickly for the allogenic cells participating to the induction of an inflammatory response. Horstem® and Arti-Cell Forte®, the two authorized MSC-based products frequently induce local inflammatory reactions after an intra-articular administration.
Neoplastic side effects have been reported but the risk for the injected cells to become tumor cells is now considered to be very low, since the injected cells are rapidly eliminated. Injected cells may encourage the development of preexisting pre-tumor cells by modifying their  immunological environment (immune-modulation) associated with the production of angiogenic factors. Even if this risk stays theoretical it should be consider. The formation of ectopic tissue has been reported in rare cases in humans (Bauer G 2018). The level of differentiation of the injected MSC may contribute to an unwanted and deleterious immune response. This remains to be explored in experimental trials. Lastly, an infectious risk due to the presence of unwanted infectious agents in the cellular preparation must be avoided. The MSC donors must be tested for the absence of major pathogens. The contamination can also occur during the in vitro production of the cellular stock. This can be avoid by good laboratory practices and adequate control of the production before injection. To note that these controls are required for authorized products. Before any treatment with MSC-based drugs, the Veterinary will have to be vigilant to the origin of the cells as well as to their production and storage conditions. Traceability is a key element before considering using these products for treatment.  The practitioner’s liability maybe sought. In any case, monitoring of side effects and pharmacovigilance are essential when using MSC-based products with or without marketing authorization.

Differences with other products derived from blood 
Other advanced therapy products for indications similar to those of MSC-based products are already available in horses, mainly blood derivatives with IRAP (IL-1 Receptor Antagonist Protein) and PRP (Platelet Rich Plasma). IRAP is produced from serum, which would be enriched with anti-inflammatory factors; its composition and mode of action are not yet completely elucidated. PRP is a platelet-enriched plasma; the platelets would have healing and regenerative properties on the tissues. Like for MSC, the production of IRAP and PRP is not fully standardized and attention must to be paid to ensure the quality of the injected product, especially in terms of absence of infectious agents. Up to day, nor IRAP or PRP received a marketing authorization; autologous or allogeneic administration are proposed (Cousty M, 2017).

Based on our knowledge on MSC-based products, several critical points should be established before considering their use in horse for therapeutic purposes. Among them, we would like to point out to:
•    Conditions of MSC derivations and production, absence of infectious agents for any products designed for animal use with or without MA, 
•    Mechanisms of action,
•    Autologous or allogeneic origin,
•    Cellular viability, cell number, route(s) of administration and doses,
•    Indication, and target species,
•    Reported or potential side effects,
•    Practicability of supply, storage and preservation, packaging, availability of the product,
•    Cost,
•    Effectiveness reported in scientific publications and/or MA files.

In summary, MSC as advanced therapies may have a therapeutic use for example in the treatment of osteo-articular degenerative affection. A lot needs are to be done to understand their mechanisms of action and to extend their use in horses.  Preparations by the veterinarian have to be only autologous ones because there are some medicines with MA for allogeneic preparations. A standardized procedure, from the cell derivation to the delivery of injectable doses, needs to be established to secure the use of MSC in equine medicine. Side effects such as inflammatory, auto-immune and potentially neoplastic reactions as well as infectious risks must be carefully monitored and reported. 
To this day, the use of MSC-based products without MA in other species than horse has become common. We wish to underline the lack of efficacy and safety data when using these products in other species than the ones targeted by the MA.

In conclusion, if MSC-based products are considered to treat a horse, we suggest to use allogeneic MSC products that already have a MA. Autologous MSC products without MA are a possible option only if they are produced on expert platform, with all the necessary safety and quality controls. In case of side effects, the practitioner’s liability maybe sought. The vigilance and the knowledge of all the actors engaged is a prerequisite.


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