Prospects of mesenchymal stem cells in veterinary regenerative medicine and drug development
How to cite this article: Chandra V, Gugjoo MB, Amarpal, Sharma GT. Prospects of mesenchymal stem cells in veterinary regenerative medicine and drug development. J Reprod Healthc Med 2021;2:2.
Stem cells are wonder cells that function silently in an individual to grow and/to regenerate. There are various stem cell types; some especially embryonic stem cells (ESCs) favor individual development while more advanced cells like adult stem cells play mostly repair and tissue matrix secretion role. Among various adult stem cell types, mesenchymal stem cells play an important role to maintain tissue homeostasis. These cells are available in almost all the tissue types and exhibit features similar to the ESCs. These cells are immunoevasive, immune modulatory, and/anti-inflammatory, and bear properties of self-renewal (although limited), multiplication, and differentiation. In addition, these cells are able to migrate and home-in to the distant tissues. All these features make these cells potential candidates for therapeutic applications and drug development. There are various studies that have favored their role in therapeutics and drug development, although more studies and further insights are desired to make stem cell therapy a definitive therapeutic option.
Mesenchymal stem cell
Stem cells are primitive cells that bear specific characteristic features such as self-renewal, multiplication, and differentiation, among others. Due to the characteristic features, these cells are considered to provide plethora of biological utilities. These cells have wide range of applications right from the basic biological features to the therapeutic applications. These cells may be utilized to understand basic mechanisms such as stem cell genome regulation and cellular behavior including proliferation, differentiation, apoptosis, immortality, senescence and/ aging, and rejuvenation process. Their applications in drug discovery, drug evaluation, genetic engineering, gene therapy, and most importantly in regenerative medicine can be a big boost to the biological sciences, in general, and medical profession/veterinary profession, in particular. Stem cell as a novel therapeutic tool has given hope to many patients suffering from incurable diseases. It has revolutionized the field of regenerative medicine and tissue engineering and is considered to provide a single stop solution to varied ailments, currently lacking any definitive treatment. Stem cell therapeutic evaluation has shown steep rise over the past two decades and has been conducted both at ex vivo and in vivo systems.[2-4]
During developmental cycle of an individual, various types of stem cells arise. At the beginning, fertilization leads to the development of the zygote with a totipotent capability giving rise to whole individual including fetal membranes. From zygote, trophoblast develops that contains external cell mass and inner cell mass. External cell mass gives rise to the fetal membranes, containing mesenchymal stem cells (MSCs) while inner cell mass harbors pluripotent embryonic stem cells that give rise to an individual except fetal membranes. MSCs are naturally present in almost all the adult tissues to take care of routine wear and tear, thus replacing the damaged cells. Furthermore, due to advanced transgenic techniques, somatic cells have been reprogrammed (dedifferentiated) to the pluripotency known as induced pluripotent stem cells (iPSCs) [Figure 1].[2,5,6] Among various stem cell types, adult stem cells especially MSCs make most of the stem cell therapeutics. It is attributed to the availability of numerous tissue sources, easy harvesting procedures, and lack of ethical and teratogenic issues. Furthermore, these cells offer properties of immunomodulation and/ anti-inflammatory, migration, and homing, in addition to those mentioned above. These cells are being evaluated in preclinical research as well as in clinical trials; although no definitive therapeutics has been developed. These cells can offer therapeutic effects either through the differentiation or through paracrine secretions, although latter one is considered as main mechanism involved in the therapeutics. This has made way for the therapeutic applications of MSCs conditioned medium (CM). MSCs CM contains high levels of angiogenic and antiapoptotic factors such as interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein -1, which inhibits the apoptosis and may promote angiogenesis. MSCs can be isolated from almost all the body tissues such as bone marrow (BM), fat, dental pulp, tendons, synovial membrane, and fetal membranes such as Wharton’s jelly, amnion, and amniotic fluid. Regardless of the source of isolation, MSCs originated from different tissues share comparable multipotency and immunomodulatory characteristics. However, there may be variations in other key features such as immune phenotype, proliferation rate, and commitment to various differentiation pathways. In addition, cultured cells may be heterogeneous group of cells. To isolate MSCs purely, the International Society for Cellular Therapy has put forth criteria of adherence to plastic, surface antigen expression (Sca-1+, CD166+, CD73+, CD90+, CD105+, CD34-, CD45-, CD11, CD14, CD19, CD79, HLA-DR, and CD31), and in vitro multipotent differentiation potential under specific differentiation conditions.[10,11]
ROLE OF MSCs IN REGENERATIVE THERAPY
MSCs have potential to repair damaged organs/tissues of the body. Transplanted MSCs can traffic and migrate to injured tissue and consequently home-in to participate in the healing of the tissue. MSCs’ regenerative potential may arise due to their differentiation and trans-differentiation into desired tissue-specific cells, being determined by the local milieu/ microenvironment. This type of repair mechanism, however, remains controversial as the implanted cells remain viable only for limited time and even the differentiated allogeneic or xenogeneic cells may elicit immune reaction. MSCs may secrete chemotactic agents to attract surrounding cells to the particular area to bring about the healing. MSCs on interaction with the resident tissue cells may secrete a wide array of biomolecules including those hampering immune and inflammatory reaction and the ones promoting healing. The MSCs’ CM too has been found to favor wound repair even in diabetic rats.[11,13] Thus, a growing common consensus among the stem cell researchers suggests that therapeutic benefit of MSCs is attributed to the release of biomolecules “paracrine,” rather than a direct cellular contribution.[13,14] Interestingly, microvesicles derived from stem cells have been found to reprogram cells that survived injury and enhance tissue regeneration. Microvesicles, membranous vesicles derived from cells, were previously thought of as artefactual resulting from cell preparatory methods or from cellular debris without any biological purpose. However, there is lot of literature demonstrating their role to transfer genetic information between cells. The microvesicles contain proteins, messenger RNAs (mRNAs), DNAs, and/or microRNAs, which are potentially involved in angiogenic pathways and microRNAs associated cell proliferation, and inhibition of apoptosis at the damages sites.
Immunomodulatory effect of transplanted MSCs
One of the interesting features of MSCs is immune evasive and immune suppression upon transplantation [Figure 1]. A foreign cell engraftment makes adhesion molecules and the major histocompatibility complex (MHC) antigens to interact with the immune cells. MSCs exert an immune tolerant phenotype by expressing low levels of MHC Class I surface antigens and the lack of expression of alloantigens and MHC Class II antigens. The adhesion molecules are constitutively expressed in MSCs, with the exception of ICAM1, being expressed upon induction. MSCs immunomodulatory functions are jointly executed by direct cell-cell contact and the secretory factors. Transplanted MSCs show immunosuppression by modulating the release of soluble anti-inflammatory molecules such as indoleamine 2,3-dioxygenase (IDO) and iNOS pathways. MSCs have been known to secrete a variety of immunosuppressive molecules in humans including programmed death ligand 1, VEGF, IL-10, IL-6, IDO, iNOS, prostaglandin-E2 (PGE 2), hepatocyte growth factor, transforming growth factor β1 (TGF-β1), CXCL-9, CXCL-10, and CXCL-11. However, most of these suppressive factors require a dynamic cross talk between MSCs and T-lymphocytes for their secretion. In addition, Nicola et al. had shown that MSCs are able to inhibit T-lymphocyte proliferation in both mixed lymphocyte culture and in the presence of polyclonal activators (IL-2 or phytohemagglutinin, PHA). CD4 and CD8 T-cells are equally inhibited in dose-dependent manner by MSCs and MSCs-T cells contact leads to T-cell arrest in the G0 phase of cell cycle. MSCs inhibit mitogen-induced T-cell proliferation, as determined by in vitro mixed lymphocyte reactions and transplantation of MSCs across MHC barriers. Other important inhibitory factors secreted by MSCs on interaction with immune effector cells, are PGE-2, IDO, and TGF-β that negatively interfere with T-cell activation and function. IDO converts tryptophan to kynurenine, which leads to T-cell inhibition and activation of immunosuppressive regulatory T-cells (Tregs). MSCs modulate immune responses by the de novo induction and expansion of CD4+ CD25+, FoxP3+, and CD8+ Tregs, which are responsible for inhibiting allogeneic lymphocyte proliferation. The MSC-mediated induction of Tregs is caused not only by direct cell contact between MSCs and CD4+ T cells but also by the secretion of PGE-2 and TGF-β1. MSCs have also potential to block the proliferation of activated B-cells in the presence of INF-ϒ and negatively interfere with antibody production, which depends on dose of MSCs and activation state of the B-cells. MSCs also inhibit chemokine secretion which is responsible for B-cell migration. MSCs inhibit IL-2-induced natural killer (NK) cell proliferation, which is mediated by soluble immunosuppressive factors TGF-β, sHLA-G and PGE-2, and by cell-cell contact. Inhibitory effect of MSCs associated with downregulated expression of the activating NK cell receptors is mediated by PGE-2 and IDO. MSCs also express the toll-like receptors 2, 3, 4, 7, and 9 at the protein level, which affect the immunomodulatory properties of these cells. It is worth mentioning here that the secretome of MSCs may vary based on the tissue source or species involved but a comparable immunomodulatory response may be observed.
MSCs’ POTENTIAL APPLICATIONS IN REGENERATIVE MEDICINE
Cutaneous wound healing is a complex process which requires coordinated cascades of cellular events including inflammation, bio-proliferation, fibroplasia, angiogenesis, and epithelialization.[31,32] In elderly patients, especially diabetics, wound healing is very slow. Delayed wound healing in diabetes is due to diminished migration and proliferation of keratinocytes and fibroblasts, increased cellular apoptosis, inflammatory macrophage phenotype (M1), activation of a pro-inflammatory mediator, reduced vascularization, and impaired recruitment of endogenous progenitor cells toward the injured area. These adverse effects might be due to high glucose microenvironment and elevated levels of inflammatory and immunomodulator cytokines. The anti-inflammatory capacity of MSCs may thus be imperative in the restoration of localized or systemic conditions that are required for normal healing. These patients are more likely to suffer from chronic wounds and thus demand therapeutic options that carries good success rate. MSCs’ application is a new hope by reducing the difficulties to heal such wounds. MSCs improve/circumvent wound healing by stimulating cellular response to injury and promote regeneration rather than scar formation. Xenogeneic canine stem cells and its CM were found to heal experimentally induced diabetic rat wounds at a significantly faster rate of wound contraction and quality of wound healing (rate of epithelization, neovascularization, and collagen deposition). In caprine and equine wound healing models, local implantation of MSCs had led to complete re-epithelialization in shorter period. The healed tissue had shown limited inflammation, thinner granulation tissue, and minimal scar tissue formation.[35-37] Even in equine clinical cases, MSCs had improved wound healing of pressure sores or decubitus ulcers.[38-40]
The current literature demonstrates that MSCs exhibit limited ability to incorporate into the particular tissue and their pro-healing effect is mainly attributed to the trophic mediators being released by them. MSCs could enhance wound healing by creating conducive microenvironment through the release of important biomolecules such as VEGF, insulin-like growth factor, epidermal growth factor, keratinocyte growth factor, stromal cell-derived factor-1, and matrix metalloproteinase-9. MSCs secrete PGE 2 which further regulates inflammation and promotes tissue healing with reduced scar formation. To enhance their delivery, several strategies such as three-dimensional allograft, hydrogel scaffold, and microsphere-based engineered skin loaded with EGF have been found useful. Apart from the stem cell scaffold, various biomolecules such as growth factors, antioxidants, and genetic engineering techniques such as plasmids and other vector-based specific gene transmission may further modulate the stem cells delivery and functionality.
MSCs’ ROLE IN NERVE INJURY/PARALYSIS
Spinal cord injury (SCI) in animals like dog is one of the commonly encountered clinical conditions with resemblance to human condition. Regarding the nature of nerve cells, it is stated that nerve cells have no new cell division after birth in mammals and little is known about its potential to contribute to endogenous repair mechanisms. However, some reports recently evidenced the presence of endogenous stem cell like cells that can differentiate into glial cells, including astrocytes or oligodendrocytes,[43,44] and by which functional neuronal recovery has been reported. Moreover, isolated perivascular MSCs from the human brain could also adopt a glial and neuronal phenotypes, at both mRNA and protein expression. MSCs may act as good in vitro model to study ovine scrapie as the cells express PrPC. MSCs from scrapie affected animal donor had normal trilineage differentiation but carried diminished neurogenic differentiation. This type of model may help to understand disease and develop therapeutics.
Canines and felines are frequently affected with SCI either accidently or mishandling or faulty injections which frequently results in permanent loss of neurological function below injured region. The partial improvement may do occur over a period of time (weeks to months) including both compensatory behavioral strategies to maximize use of spared systems and potential anatomical mechanisms of axonal sprouting[47,48] and remyelination. Injured spinal cord environment could facilitate differentiation along glial lineages. Therefore, it is possible that experimental augmentation of endogenous stem cell responses could increase recovery after injury. As these cells secrete different growth factors which ultimately enhance the recruitment of endogenous stem cells (glia cells) and better results are reported when cases are treated earlier and showed higher recovery rate than delayed treatment. Supplementation of growth factors to MSCs may be promising adjuvant to MSCs to circumvent problems of MSC proliferation and expansion, and survival in-situ.
MSCs in dog and sheep pre-clinical models involving the spinal cord or nerve injuries[51-57] and peripheral nerve injuries[58,59] have been demonstrated to improve outcome. In peripheral nerve injuries, healing although appears delayed but myelinated nerve fibers may regenerate both at distal and the proximal parts of the injury with an overall improvement in nerve action potential. Besides, MSCs and their genetic engineering, many other biological agents such as scaffolds and growth factors have been incorporated. Thus, further refinement in the experimental models is required to compare the role of MSCs and the other biological agents alone or in combination. It is worth mentioning that complete healing and recovery to normal reflexes remain a dream. Thus, further, incites are desired in the field of neurological recovery.
Bone and cartilage repair
Orthopedic problems have been found common among canines and other domestic animals species. Handling of such orthopedic cases has been the cause of concern among canine and feline practitioners worldwide. The therapeutic options, which are clinically available, are currently restricted to allografts, microvascular bone, and osteocytes, myocytes, cutaneously taken either from an autologous donor site, or following bone distraction used for reconstructive purposes. Compared to traditional medicine, stem cell regenerative therapy does not rely on a single target receptor or a single pathway for its action. MSCs improve bone healing through direct differentiation into mature osteoblasts/chondrocytes and/or paracrine effects that facilitate migration and differentiation of resident precursors. MSCs have chondrogenic potential and intraarticular injected MSCs shown chondrogenic differentiation and, in turn, actively produce extracellular matrix. MSCs secrete several biomolecules to make microenvironment favoring stimulation of locally present progenitor cells to repair the damage or by chemoattracting the circulating endogenous progenitor cells to enable repair. MSCs’ paracrine effects can be immunomodulatory, anti-scarring, and chemoattractant. The VEGF is able to activate the formation of a new network of blood capillaries, which is required during the physiological process of bone regeneration. Using biological scaffolds such as PLGA, collagen in combination with MSCs may be most effective strategy for treating bone defects.
Various experimental studies in rabbit and dog have favored the early and much improved bone healing with MSCs with or without the scaffolds and growth factors.[64-66] Even in clinical cases of non-union fracture in dog, application of MSCs, or transplantation of BMP-7 expressing MSC sheets at the fracture site had led to the healing of bone.[67,68] Comparison of MSCs derived from various tissues (adipose tissue, BM, umbilical cord blood, and Wharton’s jelly) had shown comparable improvement in bone healing. In case of cartilage repair, mostly clinical symptoms have subsided with or without actual hyaline cartilage repair.[7,69-74] In cartilage repair, more intensive studies are warranted to yield desired results. In case of bone repair, MSCs appear much promising but further studies are desired to determine the actual posology of the cell therapy.
Tendon disorders may arise from the over usage or age-related degeneration, resulting in acute or chronic tendon injuries. Research data suggest that certain tendons are more prone to such insults than others such as the rotator cuff, Achilles, tibialis posterior, and patellar tendons. Tendon and ligament injuries are extremely slow in healing and remain incomplete, posing a major challenge. Application of MSCs at the site of damage proved to be an attractive strategy to improve tendon reparative processes. The use of cultured MSCs for the treatment of tendon injuries is supported by experimental investigations in horses, dog, and laboratory animals where the MSCs were implanted locally and the clinical signs had shown significant improvement including favorable changes in tissue organization, composition, and mechanics of MSC-implanted tendons and ligaments.[1-3] In a clinical study, Renzi et al. reported that the injected BM-MSCs into horses affected by tendonitis or desmitis led to 13 out of the 18 inoculated horses returning to race competitions without any adverse effects. Clinical follow-up by Van Loon et al. reported that 77% of horses (40) returned back to work after the treatment of allogenic MSCs derived from umbilical cord blood. In dog, 9 out of 13 dogs had fully healed cranial cruciate ligament with marked neovascularization and a normal fiber pattern in the areas of injury implanted with MSCs and platelet-rich plasma. In tibial plateau leveling osteotomy repaired cases, adjunct MSCs application had failed to show improved healing as compared to those given NSAIDs. Further, research is required to investigate the exact mechanism of action of MSCs in tendon/ligament repair regarding immunomodulation and trans-differentiation potential.
MSCs IN MAMMARY HEALTH AND MILK PRODUCTION
The mammary gland is a highly specific secretory organ and growth and maintenance of the mammary epithelium depend on the function of mammary stem cells and progenitor cells. Milk production is a function of number and secretary activity of mammary epithelial cells. Transplantation studies in mice have shown that progeny of a single cell could regenerate an entire mammary gland into clear mammary fat pads. Maintaining mammary health and enhancing milk production through stem cell technology are a new and novel concept. The concept is based on the fact that multipotent mammary stem cells (MaSCs) give rise to epithelial precursor cells, the progeny of which develops into either ductal or alveolar cells. An increase in the number of functional alveolar cells will lead to increased milk production. Very less work has been conducted in this area; however, researchers could isolate and characterize MaSCs in from cattle. Appropriate regulation of milk yield, persistency, and dry period, MaSCs can potentially benefit period management and tissue repair. Suitable stimulation of mammary gland stem cells may be helpful for the enhancement of the milk production. BM-derived mesenchymal progenitor cells in conjunction with MaSC are among the most promising vascular progenitors, which can be adopted for therapy of post-mastitis to correct cytological defects. The possibility of increasing milk production by manipulating MaSC is novel. Enhancement of milk production by MaSCs transplantation is based on the hypothesis that expansion of mammary stem cell will produce cascades of proliferation involving progenitor cells followed by differentiated cells. Presence of mammary stem cells in the milk open the possibility to use a noninvasive system for the recovery of primitive cells from the mammary gland that could be a simple and rapid method which can easily provide the necessary amount of cells to monitor the functional status of the bovine mammary gland. The research on bovine stem cells in general and bovine mammary stem cell in particular is very meager and it is hoped that in near future mammary stem cells may prove beneficial in the management of bovine mastitis/ udder health. MSCs in productive mammary gland of cattle and goat may prove double rewarding: On the one hand, MSCs may secrete milk specific proteins in the presence of mammary epithelial or stem cell and, on the other hand, these cells may prevent mastitis by secreting antibacterial proteins. Even in teat fistula or fibrosis MSCs may prove promising by regenerating normal teat tissue.[84,85]
MSCs IN GENETIC ENGINEERING AND DRUG DEVELOPMENT
It has been noted that the response to drugs in animal models may not be the same as in humans. Drug evaluation using in vitro models has been a major boost not only in identifying potential therapeutic compounds but also in increasing our understanding of their absorption, distribution, metabolism, and excretion properties. The use of specialized primary culture models such as hepatocytes, human umbilical endothelial cells, and keratinocytes is limited due to their restricted expandability. Stem cells offer huge potential in the field of pharmaceutical research and regenerative therapy, using medicine. In addition to the obvious health benefits, stem cells may weed out the drugs with dangerous side effects much before they reach the market. This could save the industry millions of dollars in wasted in development costs. Furthermore, since the core competencies are largely driven by academic research, pharmaceutical companies need to gain expertise in the technology. One of the straight forward applications of stem cells lies in clarifying disease mechanisms and toxicity. Testing lead compounds for neuronal, hepatic, and cardiac toxicity would provide direct assessment of the effects and side effects of drugs. MSCs can be used as an alternative to the conventional micronucleus test for screening of genotoxic compounds thus reducing animal uses. Stem cells or iPSCs could also act as transport agents for delivery of small molecules, therapeutic agents, or unmutated normal genes. MSCs tend to grow for long although limited as compared to embryonic/iPSCs but sufficient enough to prove useful in case virus-mediated genetic engineering is a problem. Genetic engineering of MSCs with specific gene may prove useful to help evaluate therapeutic effects besides, its feasibility.
Substantial work has been conducted in the past two decades in stem cell research in animal sciences with very promising results, especially for wound and bone healing and nerve injury cases, however, their uses are still in pre-clinical experimental trials. It may be due to the costly affair of stem cell therapy to treat animals even though significant work has been done in canine and equine. Prominent mechanism by which MSCs facilitate regeneration of damaged tissue is by their secretory properties. Stem cell-derived CM has evolved as an alternative therapeutic agent and being cost effective and requiring lesser technical expertise in its application. MSCs possess very promising role in neuroregenerative therapy and the likely mechanisms suggested as the neuroprotection, angiogenesis, axon myelin remodeling, endogenous cell proliferation, and possible replacement of damaged cells. Stem cells also have lot of scope in genetic engineering and drug discovery. MSCs are better alternative for drug discovery and drug testing and this may reduce a lot of laboratory animal sacrifice. Mammary stem cell bears a great hope to enhance milk production but more efforts are needed to explore this opportunity. In animal stem cell therapeutics, though less work has been conducted but there cult to treat various types of diseases and improve productive performances in livestock has very good prospect.
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Patient’s consent not required as there are no patients in this study.
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Conflicts of interest
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