ROLE OF MESENCHYMAL STEM CELLS (MSCS ) IN VETERINARY REGENERATIVE THERAPY & DRUG DEVELOPMENT
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. 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. 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 . 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
Wound healing
Cutaneous wound healing is a complex process which requires coordinated cascades of cellular events including inflammation, bio-proliferation, fibroplasia, angiogenesis, and epithelialization. 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. Even in equine clinical cases, MSCs had improved wound healing of pressure sores or decubitus ulcers.
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,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 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 and peripheral nerve injuries 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.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. 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. 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/ligament repair
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.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.
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.
The nature of mesenchymal stem cells
The term “stem cell” emerged in the nineteenth century, describing mitotically quiescent primordial germ cells capable of regeneration of a variety of tissues . Stem cells are defined by their ability to self-renew and by their potential to differentiate into functional cells under appropriate conditions . In animals, two classes of stem cells have been identified: embryonic stem cells (ESC) and adult (somatic) stem cells (ASC) , which include mesenchymal stem cells, hematopoietic stem cells, and tissue-specific stem/progenitor cells .
MSCs are responsible for tissue turnover; therefore, when tissue repair is necessary, these cells can be stimulated to proliferate and differentiate, resulting in their presence in many , if not all , tissues. In addition, MSCs display important features that render them valuable for cell therapy and tissue engineering such as their low immunogenicity, high anti-inflammatory potential , ability to modulate innate immune responses , bioactive mediation and adhesion capacity to inhibit scar formation and apoptosis, increased angiogenesis, and stimulation of intrinsic progenitor cells to regenerate their functionality . Due to their clinically relevant characteristics, MSCs have received more attention than the other ASC types.
During early embryogenesis, the trophectoderm differentiates into extraembryonic tissues, while the inner cell mass of the embryo, populated by embryonic stem cells, gives rise to the embryo itself, thus being able to differentiate into all cell types that form the body . In contrast, it was a generally held belief that MSCs have restricted differentiation ability, being able to differentiate into mesenchymal lineages only. In the early 2000s, some discussion took place regarding the veracity of the definition of mesenchymal stem cells, concerning their potential to differentiate into non-mesenchymal lineages and whether the differences that seemed to exist between ESC and MSC had narrowed to a point that it was questionable whether they existed at all . In 2002, it was shown that bone marrow-derived cells expressed some pluripotent markers, such as Oct-4, Rex-1, and SSEA; were able to differentiate into three germ layers in vitro; and when injected into an early blastocyst, were able to contribute to all organs . The number of studies investigating the pluripotent ability of MSC has grown recently, and many researchers have reported cells derived from bone marrow , adipose tissue , ovarian tissue , placenta , and uterus that express pluripotent markers. MSCs derived from several different species, including bovine, have been shown to differentiate into mesodermal, endodermal, and ectodermal lineages . A relevant clinical difference between ESC and MSC is that MSCs do not form teratomas when injected in vivo , which is favorable for their clinical use.
Rigorous evaluation of the differentiation capacity of MSC is a critical step in the solidification of support for their redefinition as pluripotent. In order to study the functionality of MSC, experiments were performed to evaluate the transdifferentiation of MSC in vivo. Studies have shown the ability of MSC to transdifferentiate into various types of skin cells, islet-like cell clusters, and renal epithelium cells . These three studies are just a few examples of the considerable amount of data that has been collected over the past decade supporting the transdifferentiation potential of MSC when transplanted in vivo. Considering these results together, MSCs have been proven to functionally differentiate into three germ layers. If MSCs express pluripotent markers and have the ability to differentiate in vitro into three germ layers and transdifferentiate in vivo into three germ layers, perhaps there is a lack of precision concerning terminology in some papers when they are called multipotent.
Mesenchymal stem cell culture
Cell culture begins after mechanical or enzymatic disaggregation of the original tissue and can be performed under various conditions such as in an adhesive layer, a solid substrate, or in a suspension culture. It is well established that MSCs adhere to plastic substrate culture plates , a characteristic condition of MSC that arises after tissue disaggregation. Disaggregation is achieved by proteolytic enzyme digestion that is very effective at isolating cells from a tissue; however, it also has the potential to damage them. According to Gazit [, MSC derived from adipose tissue can be easily isolated after enzymatic treatment with collagenase. This enzyme is the most frequently used for isolation of MSC due to its ability to cleave collagen connections . The optimum concentration of the enzyme, the incubation time, and the temperature must be carefully monitored during isolation .
Different protocols have been used to isolate, expand, and characterize MSC. One common protocol, based on cell adherence to the plastic during the first 48–72 h of culture, is effective, though typically results in a heterogeneous population of cells . To select a homogenous or a desirable population of MSC, more stringent isolation protocols have been proposed. These include the use of different cell culture media , cell sorting , and cell adherence to the plastic during the first 3 h of culture .
MSCs have the capacity to expand several times in culture, maintaining their growth potential and plasticity, with a doubling time which is variable according to the tissue and initial plating density . Each time that the cells fill the flask culture area, they need to be enzymatically removed from the flask for further re-cultivation, a process defined as cell passage .
In order for the cells to become able to survive, proliferate, and differentiate in vitro, the culture system must emulate the in vivo conditions of the cells’ original tissue . The cells must be maintained in an incubator with 5% CO2, which facilitates pH maintenance in the culture medium , at the physiological temperature optimal for the donor species.
Supplementation of the medium should be performed to mimic in vivo conditions in order to sustain cell growth. Fetal bovine serum (FBS) is used in the cell culture medium as a source of growth factors and a vital nutrient, which supports expansion and attachment of MSC to the culture plate . The use of antibiotics is important to prevent contamination, and it is necessary to evaluate the type of contamination that cells may be exposed to and potential toxicity of the dose when choosing which antibiotic to use. The most commonly used antibiotics are penicillin and streptomycin, making an effective and relatively non-toxic combination at the concentrations of 100 U/mL and 100 mg/mL, respectively
Sources of bovine mesenchymal cells
Bone marrow
Bone marrow was the first tissue described as a source of plastic-adherent, fibroblast-like cells that develop fibroblastic colony-forming units (CFU-F) when seeded on tissue culture plates. MSCs derived from bone marrow were first isolated and identified in mice and were described as non-hematopoietic cells with the potential to differentiate into mesodermal tissues, such as adipocytes, osteoblasts, chondrocytes, and skeletal muscle cells .
In cattle, bone marrow has been the source for MSC in several studies. In this procedure, marrow cells are aspirated from calves and isolated for further analysis. Many reports with bovine BM-MSC focused on chondrogenic differentiation . Spontaneous chondrogenesis of bovine MSC in pellet culture occurred without the addition of any external bioactive stimulators, i.e., factors from the transforming growth factor (TGF)-β family, previously considered necessary . The same group isolated bovine MSC from eight calves and induced them to undergo osteogenic, chondrogenic, and adipogenic differentiation . One year later, the same group analyzed the MSC chondrogenic response during culture on different types of extracellular matrices (ECM). Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum-free medium and medium supplemented with TGF-β1. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. TGF-β1 treatment in the presence of collagen type II provided more favorable conditions for the expression of the chondrogenic phenotype. It was concluded that collagen type II has the potential to induce and maintain MSC chondrogenesis, but in the presence of TGF-β1, the cells expressed higher transcript levels of genes associated with differentiation, suggesting a higher fidelity differentiation . The presence of BM-derived MSC with a pluripotent profile was demonstrated in later experiments. The cells were adherent to plastic surfaces and exhibited fibroblast-like morphology. In addition, the cells expressed pluripotent markers, such as OCT4, SOX2, and NANOG, as well as typical MSC markers, including CD29, CD90, and CD105. When the cells were isolated from fetal BM, they exhibited fibroblast-like morphology and were able to differentiate into hepatogenic and neurogenic lineages. The cells were not only positive for MSC markers CD29 and CD73 but also for the pluripotency markers, whereas they were negative for hematopoietic markers CD34 and CD45 .
Adipose tissue
Currently, bone marrow and adipose tissue are the main sources of MSC in veterinary medicine . However, AT-MSCs have some advantages over BM-MSC, including faster development in vitro , easier isolation, and higher density stromal cells . To date, there are only two studies in bovine species with MSC isolated from adipose tissue . In both studies, cells exhibited fibroblast-like morphology and were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages; they expressed different MSC markers in each of the studies. In one study, cells were positive for CD105, CD73, CD29, CD90, and H2A markers and negative for CD45, CD34, and CD44 markers , while in the other study, cells were positive for CD90, CD105, and CD79 and the negative for CD45, CD34, and CD73 . MSCs are known to demonstrate considerable variability between populations in their proliferation, differentiation, and molecular phenotype .
Umbilical cord
The umbilical cord has two sources of MSC. One is the cord blood, from which the cells are isolated by density gradient, and the other is the cord tissue, from which the cells can be disaggregated by enzymatic action. The cord blood is collected non-invasively and represents an alternative source of stem cells when compared to adipose tissue and bone marrow. In addition, the high availability and lower immunogenicity of umbilical cord blood cells compared to other sources of stem cells such as bone marrow have made them a viable and valuable source for cell therapy .
It was reported that cells isolated from the umbilical cord blood of humans have more MSC volume and greater plasticity, are genetically more flexible than bone marrow MSC, and also, as noted above, produce a less prominent immune response . While MSCs derived from umbilical cords in human, murine, and avian species have been the subject of many investigations, little is known about these cells in livestock species . The first study that isolated bovine MSC from the umbilical cord blood observed that the cells grew into monolayer cell sheets and could be expanded into high passages. In addition, the cells expressed OCT4 and CD73 and were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages . In another study, isolated cells were sub-cultured to passage 32 and expressed CD29, CD44, CD73, CD90, and CD166 . Moreover, those cells were able to differentiate into osteoblasts, lipoblasts, hepatocytes, islet cells, and neurocytes, indicating their potential use for experimental and clinical applications for bovine, and very importantly showing evidence that MSCs have the potential to differentiate into non-mesodermal lineages .
Placenta and fetal fluids
The placenta performs a number of very important roles during pregnancy, including being responsible for the supply of nutrients, production of hormones, elimination of waste, and facilitation of gas exchange . The placenta can be isolated easily by non-invasive harvest after delivery without any ethical or moral concern . Only one study with bovine placenta-derived mesenchymal stem cells has been published, in which the authors successfully differentiated islet-like cells from the placental stem cells. The isolated cells expressed typical mesenchymal stem cell markers, including CD73 and CD166, and a pluripotent marker, OCT4, but not hematopoietic markers, such as CD45 .
Regarding fetal fluids, it has been reported that the amnion and amniotic fluid are abundant sources of mesenchymal stem cells that can be harvested at low cost and without ethical conflict . The authors isolated MSC from amniotic fluid, and the cells exhibited fibroblast-like morphology only starting from the fourth passage, being heterogeneous during the primary culture. Immunofluorescence results showed that amniotic fluid MSCs were positive for CD44, CD73, and CD166 but negative for CD34 and CD45. In addition, the cells expressed OCT4 and, when appropriately induced, were able to differentiate into ectodermal and mesodermal lineages .
Uterus
The endometrial stromal cells are dynamic, growing, and differentiating throughout the estrous cycle and pregnancy . In addition, these cells are known to modulate the immune system and could have clinical applications for human and animal health . Some studies have isolated and characterized bovine mesenchymal stem cells in the endometrium . The cells had fibroblast-like morphology, and when cultured in a specific osteogenic medium, they rapidly developed the characteristics of mineralized bone . The endometrium-derived cells were found to express MSC markers such as CD29 and CD44 and pluripotent markers such as OCT4, SOX2, and c-KIT . Moreover, the cells demonstrated excellent clonicity, differentiation potential in mesodermal lineages, and excellent maintenance of quality after the cryopreservation process . A recent report showed the ability of endometrial cells to adhere to the plastic culture dishes, displaying fibroblast-like morphology, high proliferative capacity, and the ability to differentiate into chondrogenic, osteogenic, and adipogenic lineages.
Therapeutic delivery of mesenchymal stem cells
To achieve the best response after cell therapy, the general health of the patient, time of cell application, cell type, delivery route, and number of applications must be considered . Following stem cell derivation, cell expansion is needed for subsequent transplantation into the patient . In addition, cryopreservation of these cells can provide a ready source of abundant autologous stem cells . Cryopreservation of bovine MSC may be achieved successfully with no change in the characteristics between fresh and thawed cells . The delivery of the cell preparation should take place rapidly in order to avoid changes in cell viability and to prevent biological contamination of the cells . Moreover, it has been suggested that early administration of stem cells is presumed to be more advantageous than attempting treatment when fibrous scar tissue has already been formed .
The most effective delivery method depends on the condition that is being treated. Intravenous administration is possible due to the ability of MSC to migrate across the endothelium and home to injured tissues . However, cells can become trapped in the lungs . Thus, direct injection to the injured tissue provides a more convenient method , aiming a high concentration of MSC at the injury site without the risk of cell migration to other sites in the body . In cases in which relevant structural defects are present, such as segmental bone, articular cartilage, and soft tissue defects, the cells need to be delivered by a carrier in order to have a substrate to control cell adhesion as well as the location of the cells in vivo, and to form a template for the formation of new tissue . Recently, decellularized tissue has proven to be a promising option for scaffold construction . The bovine model in particular has an advantage when compared to smaller animal models such as mice, due to the larger quantity of tissue to be decellularized, providing a much closer analogy to human conditions for eventual translational applications in organ construction and tissue engineering .
Bovine mesenchymal stem cell therapy
Mastitis
The dairy industry is a multi-billion dollar industry, with 811 million tons of milk produced in 2017 . Clinical mastitis significantly reduces milk production and animal value. It has a severe impact on udder tissue and is also an animal welfare issue. Very importantly, the damage caused by mastitis cannot be mediated or reversed with current therapeutic strategies. Bovine mammary stem cell therapy offers significant potential for the regeneration of the udder tissues such that they could be replaced/repaired with minimal side effects . Furthermore, the anti-inflammatory properties of the MSC could potentially reduce the severity of the disease.
Stem cells modified with therapeutic agents may also be employed to combat mastitis. It has been reported that cloning the bovine lactoferricin (LFcinB) gene into the PiggyBac transposon vector is a feasible means of creating MSCs with heterologous expression of the hybrid antibacterial peptide LfcinB . These cells would then confer their high antibacterial properties against bovine mastitis origin Staphylococcus aureus and Escherichia coli directly into the mammary gland, providing strong innate udder immunity to fight against intramammary infections . This study represents a template for cost-effective expression of other antimicrobial peptides in genetic engineering. In addition to the therapeutic advantage of this approach, because of the high milk production ability, bovine mammary glands can be used as bioreactors for the production of proteins on a large scale for the pharmaceutical industry .
Biotechnology applied in animal reproduction
Nuclear transfer was successfully performed in amphibians in the 1950s and in mammals some 30 years later. Dolly the sheep was the first mammal to be cloned by somatic nuclear transfer . The goal of nuclear transfer research was to introduce precise genetic modifications in livestock species by making the desirable modifications in cells used as nuclear donors . MSC could be used to produce transgenic animals for the improvement of the animal’s health as well as for biomedical interest, for example, to produce cows resistant to mastitis and to recover proteins, such as human α-lactalbumin, from milk .
Another interesting possibility that arose from the development of nuclear transfer was that of cloned human embryos produced with the purpose of further establishment of patient-specific ES cells for regenerative medicine . However, bioethical issues and related regulations hampered the attempts at production of human embryonic stem cells. To overcome that issue, in 2006 , somatic cells were reprogrammed to a pluripotent state by introducing transcription factors (OCT3/4, SOX2, KLF4, and C-MYC) into their genome. These cells were called induced pluripotent stem cells (iPS) and had similar characteristics to ESC, including the ability to originate tissues from the three germ layers both in vitro and in vivo . Despite the advantages of iPS, there are still several ethical issues related to their application, such as genetic instability, tumorigenicity, and differentiation. Also, efficient methods for cell transplantation need to be investigated further . The low tumorigenicity and high differentiation potential have made MSC a very promising source of cells for the treatment of degenerative and inherited diseases .
Nuclear transfer technique is based on the transfer of the nucleus from a donor cell into an oocyte or early embryo from which the chromosomes have been removed . The most important drawback of this technique is the inability of the ooplasm to eliminate epigenetic markers and restore the genetic material of the donor nucleus to the embryonic totipotent state . Many studies have focused on resolving this inability, due to the importance of chromatin structure in the cell reprogramming process . One of the areas that have been explored by these studies is the use of mesenchymal stem cells for somatic nuclear transfer, which has been suggested in bovine species. For example, it was shown that the epigenetic status of bovine adipose-derived MSC was variable during culture. Of the cell passages examined in this study, passage 5 seemed to be the most efficient in the performance of nuclear transfer due to its high level of stemness, multipotency, and the low level of chromatin compaction . The embryo production rate was also shown to improve when embryos were co-cultured with MSC , representing in yet another way the importance of MSC in addressing commercial goals.
Bone injuries
Although some bone fractures and small defects can regenerate, there are conditions in which tissue loss is too extensive, as well as cases of non-union fractures and other critical-size defects where osteogenesis does not physiologically occur . This represents another opportunity in which the application of MSC could upregulate the body’s regenerative process to improve patient recuperation.
The events associated with bone healing have been chronicled reviewed . When a bone fracture occurs, the inflammatory response increases the blood supply to the region. Cellular recruitment initially leads to the replacement of the fracture hematoma with fibrous tissues and, progressively, cartilaginous matrix, which is subsequently replaced by bone through endochondral ossification in both the periosteal and endosteal callus. MSCs reside in the bone marrow in low densities, and the recruitment of MSC to the fracture site is critical. This recruitment occurs by way of a chemotactic stimulus and results in the homing of circulating stem cells to the site of injury. Once these cells arrive, they begin participating in repair mechanisms .
The reconstruction of large bone segments is a relevant clinical problem. Preclinical and clinical data are accumulating to support the use of MSC to enhance bone repair and regeneration . There are no clinical data on the use of mesenchymal stem cells for bone repair in cattle, although the ability of MSC to differentiate into the osteogenic lineage has been shown .
Attitudes in the livestock industry have shifted towards the preservation of the commercial viability of individual animals with high genetic value, leading in turn to an increase in medical expenditure to keep those animals healthy. Owners are frequently willing to elect expensive treatments, even when the prognosis is poor, when cattle have high economic or genetic potential . This notwithstanding, a number of criteria should be carefully analyzed when deciding the best treatment for a bone fracture, such as cost and success rates of the treatment, the value of the animal, and the location and type of fracture. Unlike horses, only rodeo livestock cattle need to perform athletically; thus, musculoskeletal integrity is less of an issue. However, fractures can result in a loss of meat and milk production and interfere with reproductive efficiency, including nefarious effects on natural breeding and impairment of embryo and semen production as well . Thus, MSC could represent an important auxiliary source in the treatment of bone fracture for cattle for multiple reasons, including their anti-inflammatory potential , their ability to increase angiogenesis, and their ability to stimulate intrinsic progenitor cells to regenerate tissue functionality . MSC treatment has the potential to reduce animal recovery time and reduce economic loss associated with bone injury, reducing the time for repair that can negatively influence milk and meat production and interfere with natural breeding, as mentioned above. In addition, the reduction of the recovery period can improve the outcomes for cattle with aggressive behavior, in which conventional treatment would be impractical due to the necessary motion constraints and temperament issues.
Joint injuries
In cattle, chronic osteoarthritis (OA) has been reported to be a significant cause of infertility in bulls , leading to economic loss and decrease in animal value. OA is a degenerative disease of the articular cartilage, which causes the release of pro-inflammatory cytokines . The molecules involved in the OA process include growth factors, transforming growth factor β (TGF-β1), and cytokines and chemokines such as IL-8 . These molecules influence a wide range of biological processes that include cell proliferation, differentiation, migration, and apoptosis . In horses, the efficacy of stem cells for the treatment of OA has been evaluated in the form of experimental and clinical studies, with more favorable results for bone marrow-derived cells than adipose-derived cells. The fact that MSCs secrete paracrine signaling molecules and trophic factors that influence cell response to injury and modulate the innate immune response demonstrates the potential use of those cells for OA treatment in cows. In this species, there are no current clinical data, although some studies have demonstrated the isolation of MSC and their potential to differentiate into the chondrogenic lineage . Methods are evolving to achieve this goal. To induce MSC to undergo chondrogenic differentiation, factors that support strong cell-cell interaction, growth factors, and an environment which maintains spherical morphology such as polymer gels have been shown to be required . It has further been reported that the age of the cell donor and the biochemical microenvironment are the major determinants of both bovine chondrocyte and MSC functional capacity .
Diabetes mellitus
Currently, experimental and clinical data have provided support for the use of MSC for the treatment of diabetes mellitus . Diabetes mellitus occurs in cattle and is similar to juvenile onset diabetes mellitus in humans, in that it is often immunomediated . In cattle, no genetic background for diabetes has yet been confirmed . Other etiologic factors have been implicated. Cases of diabetes have been reported in cattle infected with bovine diarrhea virus , and with foot and mouth disease . Two mechanisms have been proposed to explain how the virus causes diabetes mellitus: (1) the beta cells in the pancreas are directly destroyed by the virus or (2) the immune response against the virus infection could induce an autoimmune response in the host . The lack of insulin in animals with diabetes mellitus results in elevated glucose levels in the blood and urine. In addition, fatty acid synthesis in the liver is impaired in the diabetic animal and this leads to acid-base balance impairment, ketoacidosis, and dehydration, resulting in collapse, coma, and death . MSCs were shown to transdifferentiate into islet-like clusters expressing insulin and glucagon . At present, there are no clinical data available to validate MSC treatment for diabetes in bovine species. However, recent and promising evidence demonstrates that bovine MSCs have been successfully differentiated into islet-cells . More studies need to be done in order to prove the functionality of those cells for eventual use in preclinical trials and pharmaceutical studies.
Potential of the bovine model for improvements to human health
The use of domestic animals as models has an essential role in narrowing the gap between translational research and clinical practice . In regenerative medicine, the greatest advantage of using these models is to answer questions regarding the benefits and potential risks of stem cell treatments . Each treatment needs to be tested in animal models, outlining human phenotypes, such as the size of the organs and more similar physiology . Once the safety and efficacy of the treatment are proven, it can be applied in human therapy . The traditional model used for stem cell biology is the mouse, mainly because of its low cost, rapid reproduction, and ease of genetic modification . Despite these advantages, the mouse model fails to precisely reproduce certain human diseases . Additionally, mice have a short lifespan, small body size, and different physiology when compared to humans . Moreover, it is difficult to mimic the complexity of genetically heterogeneous human populations when studies are done with small groups of inbred mice . To effectively study regenerative medicine and make the jump from the laboratory to human health applications, different animal models need to be used, allowing for better and more complete evaluations of cell-based therapies. In order to achieve this goal, it is important to select the most appropriate animal model, considering both size and experimental tractability, for example, ease of surgical manipulation, abundance of blood and tissues, efficiency of cloning, and feasibility of xenotransplantation . Generally, larger animals are a better choice of model than mice for this purpose, specifically because they have a longer life span, which enables longitudinal studies, and because their physiological parameters are closer to those of humans . Moreover, large animal species are more appropriate for mimicking human clinical settings due to their anatomy and physiology .
The increase of genetic information can lead to new and more effective methodologies for the elimination or treatment of factors that negatively impact human health, such as cancer, cardiovascular disease, low birth weight, and infertility . An important advantage of using cattle as a model is the possibility to study genetic and environmental influences on animal production and human disease . The cattle genome contains a minimum of 22,000 genes, of which approximately 80% are shared with humans . Due to these advantages over the mouse model, it is clear that more widespread adoption of the bovine model would have positive consequences for human health. In the field of tissue engineering, large animal models represent a promising tool that allows for the translation of novel experimental scaffolds into clinical practice .
An important advantage of large animals in tissue engineering is the fact that they provide large amounts of tissue that, after decellularization, can be used as scaffolds with similar organ size to that of humans as proven, for example, with the bovine placenta . In order to elucidate physiological processes important to human health, the bovine model can be used for the study of reproduction regarding aging, physiology, gametogenesis, and infertility, as well as for bone structure formation, fat deposition, altitude and heat tolerance, hematopoiesis, leukemia, tuberculosis, xenotransplantation, gene therapy, and stem cells .
Although the use of a large animal model confers considerable advantages for translational applications, there are also some drawbacks that are important to consider when making a choice of model for an experiment. The major disadvantages of bovine models include the expenses of animal care, facility maintenance, necessity of veterinary support, and lesser availability of antibodies, probes, and reagents. However, due to the fact that they are more appropriate to mimic human scenarios than rodent models, these studies are essential to justify the risks and costs of clinical trials . Research done in less translatable models such as mice necessitates repetition in more applicable organisms, leading to additional costs and delays developing critically needed therapies.
Stem cells are the undifferentiated and uncommitted cells that give rise to deferent cell types or lineage on dividing. These stem cells are used as regenerative medicine for the treatment of various diseases in human and animals.
In the present study mesenchymals Stem cells (MSC) used for the treatment of mastitis animal and it found that diseased animal treated with MSC has been cured which suggest that stem cell therapy use as regenerative medicine providing a promising area for the treatment of various diseases in animals.
Stem cells “the hope cells”:
A stem cell is a specialized cell which has the unique characteristic to develop into specialized cell types in the body and these stem cells may be used to replace cells and tissues that have been damaged due to disease.
Due to their ability to repair, regenerate, and develop into specialized cell types, Stem cells hold considerable promise as a source of cells for therapeutic applications in various conditions including metabolic, degenerative and inflammatory diseases for the repair and regeneration of damaged or lost tissues.
Stem cell therapy for animals has seen breakthroughs over past few years. The past decade has witnessed an exponential growth in treatment of diseases. In a few instances, stem-cell-based therapies produced remarkable clinical results and had a striking impact on incurable diseases.
A Sub set of adult stem cells called mesenchymal stem cells, first described by friedenstein , are multipotent cells that can be differentiated into many lineages.
They can exhibit anti inflammatory antimicrobial , immunomodulatory, anti apoptotic ,low immunogenicity, tissue regeneration capacity which fascinates researchers to explore its applications in different fields of biology.
In both human and veterinary research stem cells derived from adult tissue promises in treatment of many chronic diseases.
Application in livestock industry:
Although from past few years stem cells used as a therapeutic tool for treatment in humans but it is still in an infant stage in animal husbandry. Among the entire domesticated species cow play a important role in economy of live stock industry with the production of 811 million tons of milk.
There are certain conditions like lameness, mastitis, metritis etc which negatively reflects on the milk production as well as the reproductive efficiency of the cattle. This causes loss of economically and genetically sound animals. Our attempt to explore the use of mesenchymal stem cells for treating mastitis gives promising results in treating these diseases in effective way.
Mesenchymal Stem Cells (MSC) in bovine Mastitis:
Bovine Mastitis is an inflammation of mammary gland causing heavy economic losses worldwide. There is a 200 billion dollar loss through mastitis only in US. Bovine mastitis causes physical, chemical, usually bacteriological changes in milk and pathological changes in glandular tissues of the udder which affect the quality and quantity of milk.
Many reports suggested that MSCs have antibacterial properties against mastitis-causing pathogen S. aureus. At the infection site, exposure of MSC increases production of several paracrine factors including VEGF, SDF-1, and IL-6, that are involved in the activation of inflammatory cells to infected area and reduce the bacterial infection on the target site and help in the prevention cure of mastitis.
MSC in wound healing:
Normal wound healing is a vigorous and complex process which involves a series of events, including bleeding, coagulation, acute inflammation, cell migration, proliferation, differentiation etc. however this process required long time to heal.
Apart from this treatment with MSC require comparable less time to other chemical or drug treatments. Region behind is that after administration of MSCs to wounds area improves wound healing by speed up epithelialization, granulation tissue formation and increasing angiogenesis.
Mesenchymal Stem Cells therapy is promising area particularly critical wounds which are difficult to heal heal.
Conclusion:
Using of MSC as regenerative medicine has the potential to revolutionize the treatment of many diseases and injuries. Form the above results it can be safely stated that MSCs can be used as an alternative regenerative medicine and it shows more healing when applied locally in udder or on wounds.
DR A KUMAR, BIOTECHNOLOGY ,IVRI
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