{"paper_id":"dc346686-9ea0-47e2-aa97-37becd502df0","body_text":"Stem cells have a capacity for differentiation and self-renewal. The great interest in science by these cells occur due to its immunomodulatory factors and tissue repair that make possible the use in therapies and innovative treatments ( Faita  et al ., 2016 ).\nSeveral adult tissue are source of stem cells with therapeutic potential, such a adipose tissue ( Zuk  et al ., 2002 ;  Rada  et al ., 2011 ), bone marrow ( Kastrinaki  et al ., 2008 ) and peripheral blood ( Villaron  et al ., 2004 ). There is possible to use the fetal membranes as source too, such as amniotic membrane (AMSC) ( Park  et al ., 2012 ), amniotic fluid ( Perin  et al ., 2007 ; Steigma and Fauza, 2007;  You  et al ., 2008 ) and placenta ( Steigman an Fauza, 2007 ;  Barlow  et al ., 2008 ).\nThe fetal membranes are essential for embryonic development, as they provide maternofetal exchange. It consists of four different membranes: amnion, chorion, the vitelline sac and the allantoic sac ( Mossman, 1987 ). The amnion is the innermost layer of fetal membranes, and contains a thick basement membrane and an avascular stroma ( Malak and Bell, 1994 ) it acts as an additional maternofetal barrier ( Leiser and Kaufmann, 1994 ). Other potential studies involved embryo development and stem cell niches is also highlighted in our data in canines ( Martins  et al ., 2011 ;  Pieri  et al ., 2015 ; De Souza  et al . 2018).\nAmniotic membrane stem cells (AMSC) have previously been described for their immunoregulatory properties ( Bossolasco  et al ., 2006 ), their differentiate and self-renew potential ( Kita  et al ., 2010 ) and vigorous cell proliferation. The AMSC are isolated from different species as canine, feline, equine, suine, murine and humans. The application of AMSC occur especially in dogs ( Park  et al ., 2012 ;  Cardoso  et al ., 2016 ) and the cats ( Vidane  et al ., 2017 ), although the murine and suine are good specie models for humans studies.\nThus, this study aimed to carry out amniotic stem cells review about their applicability in the treatment in veterinary medicine.\n\nDiscovered by  Becker  et al . (1963 ), the stem cells are undifferentiated cells able to perform self-renewal and differentiate into the most diverse functional cell types ( Park  et al ., 2015 ;  Dai  et al ., 2016 ;  Sobhani  et al ., 2017 ), thus being considered promising sources for the use in tissue engineering and organ regeneration.\nStem cells can be classified according to their potential for differentiation. Totipotent cells are those able to differentiate in all cell lines, including extra-embryonic tissues, examples of totipotent cells include the zygote and its early cleavage products. The pluripotent cells are those capable of differentiating only in cells belonging to the three germ layers ( Morgani  et al ., 2013 ) as embryonic stem cells (ESCs) ( Slack, 2018 ) and induced pluripotent stem cells (iPSCs), somatic cells reprogramed do pluripotency ( Takahashi and Yamanaka 2006 ). The iPSCs were initially produced of cells of mice and humans, however, there is currently reprogramming cells several animal species (Takahashi and Yamanaka, 2006; Takahsshi  et al ., 2007;  Zhang  et al ., 2015 ;  Gonçalves  et al ., 2017 ). Thus, besides the great importance for studies with a therapeutic objective, the IPSCs are also important in veterinary medicine for the maintenance of genetic material of animals with great economic importance ( Kumar  et al ., 2015 ).\nAnother type of classification are the multipotent cells, which have ability to differentiate into all cell types within a given lineage ( Ratajczak  et al ., 2014 ;  Khanlarkhani  et al , 2016 ). Although multipotent cells have a lower potential for differentiation, they are considered an important source for cellular therapy ( Mirzaei  et al ., 2018 ), since they have genetic stability, low immunological profile and slower metabolism when compared to pluripotent stem cells, characteristics that decrease the risk of rejection ( Liao and Tse, 2013 ). A further advantage of multipotent stem cells is the accessibility, and the fact that they can be isolated from the most diverse cell types (Khanlarkhani  et al ., 2016; Mirzaei  et al ., 2018).\nThe mesenchymal stem cell (MSC) is a type of multipotent stem cell present in most adult tissues, a heterogeneous cell that plays a key role in the development and renew of organs ( Klein, 2016 ). Several reports have shown that MSCs are commonly isolated and characterized from adipose tissue, bone marrow, umbilical cord, liver, as well a fetal attachments such as yolk sac and amnion ( Seo  et al ., 2009 ;  Uranio  et al ., 2011 ;  Wenceslau  et al ., 2011 ;  Reich  et al ., 2012 ;  Mançanares  et al ., 2015 ). This adaptability and easy access make this stem cell a great candidate for use in transplants and therapies.\n\nThe MSCs were described by  Friedenstein  et al . (1970 ) in bone marrow as a component of the marrow stromal cell population that collectively supports hematopoietic stem cell renew and differentiation ( Largeault, 2004 ; Martins  et al ., 2007). There are MSC niches in many adult tissues and organs. These cells play an active role in the homeostasis of these sites and can be isolated from the umbilical cord blood and matrix, adipose tissue, synovial membranes and embryonic and extraembryonic tissues.\nAccording to the International Society of Cell Therapy the criteria for the characterization of human MSC are adherence to the plastic, positive expression for the surface markers CD105, CD73 and CD90, negative expression for the markers CD45, CD34, CD14 or CD11b, CD79a or CD19 and the HLA-DR surface molecules, capability to differentiate (osteoblasts, adipocytes and chondrocytes)  in vitro  ( Dominici  et al ., 2006 ). According to  Casteilla  et al . (2011 ),  Tharasanit  et al . 2011 ),  Mançanares  et al . (2015 ), MSCs exhibit fibroblastoid morphology in culture.\nMSC are easy to isolate, cultivate, and manipulate. These cells have immunoregulation and immunosuppressive characteristics, great plasticity and the potential for therapeutic applications for a variety of clinical conditions ( Nardi and Meirelles, 2006 ;  Oliveira  et al ., 2014 ;  Samsoraj  et al ., 2017 ).\n\nThe amnion is the most internal extra-embrionary membrane, composed of a thin, elastic, translucent and semi-permeable membrane, derived from the ectoderm that covers the fetus and is closely connected to the chorionic membrane ( Fernandes  et al ., 2012 ;  Favaron  et al ., 2015 ).\nAmniotic membrane (AM) is avascular tissue ( Miglino  et al ., 2006 ;  Dua  et al ., 2004 ). It has the function of involving the embryo and delimits the amniotic cavity, which is filled by amniotic liquid to avoid mechanic shocks ( Moore and Persaud, 2008 ;  Mamede  et al ., 2012 ;  Koob  et al ., 2014 ).\nBasically, the amniotic membrane is composed of two cell types. The epithelial layer composed by the epithelial amniotic cells which present a cuboidal/columnar morphology derived from ectoderm. And the second cell type is composed of mesenchymal stromal cells that are derived from mesoderm. Both populations have similar markers and have the potential to differentiate  in vitro  in the main mesodermal lineages (Chang  et al ., 2010;  Díaz-Prado  et al ., 2011 ).\nThe AM has particular characteristics such as anti-inflammatory, anti-bacterial, anti-viral and immunological action. Also contains numerous growth factors, cytokines, and signaling molecules that play important roles in fetal development and gestation ( Kogan  et al ., 2018 ).\nDue to the multipotent properties, cells from the amniotic membrane have been investigated for being an attractive source for tissue transplantation ( Mamede  et al ., 2012 ).\n\nThe AMSC are derived from extraembryonic mesoderm and are randomly distributed in the extracellular matrix of amnio which are rich in collagen and laminin fibers. They are multipotent cells with characteristics that favor their use, such as low immunogenicity and carcinogenicity, production of regulatory molecules of the immune system, high plasticity and propagation in culture and presence of high concentration of lysosomes (Chang  et al ., 2010;  Cremonesi  et al ., 2011 ;  Vidane  et al ., 2014 ;  Cardoso  et al ., 2016 ;  Faita  et al ., 2016 ;  Magatti  et al ., 2016 ;  Miki  et al ., 2016 ).\nThe AMSC demonstrates absent immunogenic properties and production of anti-inflammatory and bactericidal substances that provide their use in the treatment of skin wounds, cutaneous ulcers, and ophthalmic disorders such as corneal ulcers ( Díaz-Prado  et al ., 2011 ;  Parolini  et al ., 2008 ).\nFurthermore, the AMSC are considered excellent sources of stem cells for regenerative therapies of the nervous system due to neural differentiation ability ( Uranio  et al ., 2011 ;  Park  et al ., 2012 ).\nThese cells have advantages over adult stem cells because they preserve embryonic, immunosuppressive and pluripotent characteristics, which are confirmed by the expression of embryonic markers such as OCT-4 and NANOG ( Cremonesi  et al ., 2011 ;  Vita  et al ., 2012 ;  Saulnier  et al ., 2016 ). Other advantage of AMSC is because they are easy to acquire, offer no damage to donors, and have a lower immune response, making them important in research for regenerative medicine, since inflammation and immunogenicity are crucial factors for successful transplantation ( Kim  et al . 2014 ).\nThe cells derived from the amniotic membrane have the same characteristics of mesenchymal stem cells, according to the International Society of Cell Therapy criteria ( Cardoso  et al ., 2016 ), and their morphology shows fibroblast characteristics ( Fig. 1 ).\nFigure 1 A and B: Canine fetus developing, with possible visualization of placental (P) structures, marginal hematoma (H), chorioallantoic membrane (CA), yolk sac (YS) and amniotic membrane (*). C and D: photomicrographs of AMCs in primary culture on different days of culture. C, with 4 days and D with 10 days.\n\nThere are many types of isolation protocols for AMSC in each species, although they are quite similar. In this review, we will describe the protocols most use with different species in our lab.\nFor species which the site of collection is more contaminated, as swine and horses, we strongly suggest the refrigeration of samples (1h - 2h) before the beginning of the mechanic process, in order to reduce the bacterial activity. So, the amniotic membrane must be mechanically dissect and isolated from embryonic attachments. Under sterile conditions, the tissue must be washed with PBS supplemented with 1% penicillin/streptomycin (P/S, Sigma, USA). After this step, the sample will be transfer to a clean dish and mince with scalpels to become homogeneous and pasty.\nAfter the above-described process, the sample is submitted to chemical digestion using collagenase type I (Sigma, USA) which the concentration and time are described below to each species ( Tab. 1 ).\nAfter digestion the collagenase enzyme has to be neutralizing using the same volume of expansion media to the cell suspension. So, the digested sample is centrifuged at 1600 rpm/5 min, and then cell pellet is obtained to be culture with the expansion media and keep at 37°C and 5% CO2.\nTable 1 Culture protocols of AMSC from different species. Animal % collagenase Time Expanded Medium Reference Canine 1-2 mg/mL 3 - 4 h high-glucose Dulbecco’s modified Eagle’s medium  \n(HG- DMEM) + 10%  \nSFB + 1% P/S + 2 mM L-glutamine \n Park  et al ., 2012 \n Feline 1 mg/mL 3 h HG-DMEM + 10% SFB + 1%  \nP/S + 2 mM L-glutamine \n Vidane  et al ., 2017 \n Equine 0,93 mg/mL  \n+ 20 mg/mL DNAse 3 h HG-DMEM + 10% SFB + 10 ng/ml epidermal  \ngrowth factor (EGF; Sigma) + 1%  \nP/S + 0.25 µg/ml amphotericin B + 2 mM L-glutamine \n Lange-Consiglio,  et al ., 2013a \n Swine 0,93 mg/mL  \n+ 20 mg/mL DNAse 3 h HG-DMEM +10% SFB + 10 ng/mL EGF + 1%  \nP/S + 0.25 µg/ml amphotericin B + 2 mM L-glutamine Lange-Consiglio  \n et al ., 2015 Human 25 mg/mL 1h HG-DMEM + 10% SFB + 1% P/S Dizaji  et al ., 2017\n\nThe AMSC is considered a source of election to treat spinal cord injury.  Sankar  et al . (2003 ), showed good interaction of human amniotic membrane stem cells when applied in  iatrogenic spinal cord injury  in monkeys, and they observed a significant remyelination, as well as their ability to modulate the glial scar. And a similar study was performed by  Zhi-Yuan  et al . (2006 ) who observed a returning of motor function in the hind limbs of treated animals. Furthermore,  Meng  et al . (2008 ) studied the co-transplantation of AMSC with stem cells of neuronal origin in rats with  chronic spinal cord injury . And again, the results demonstrated a significant locomotor improvement in addition to neuronal survival and differentiation.\nKakishita  et al . (2003 ) verified that human amniotic epithelial cells secrete biologically active neurotrophins and can enhance the survival of dopamine neurons, being a promising therapeutic tool in Parkinson's disease. As well as  Yang  et al . (2009 ) who also observed a increasing of dopamine and its metabolic products in the striatum in rats with Parkinson’s disease.\nKim and Choi (2011 ) observed neovascularization in mouses suffering of limb ischemia. They administered intramuscularly hAMSCs on the leg whose femoral vessels were ligated. The blood flow recovery was significantly higher in the transplanted group, when compared with the control group, being an attractive source for the treatment of ischemic diseases.\nAnd  Tao  et al . (2012 ) transplanted human amniotic membrane stem cells transfected with the brain derived neurotrophic factor (BDNF) into the brains of rats with induced stroke. This technique ameliorated the behavioral dysfunction and reduced the infarct volume, improving functional recovery.\nThe stem cell therapy is a routine treatment in horses, and the amnion plays an important role in this scenario. In mares with endometriosis, the AMSC cells were used because of their potential to improve cell replenishment based on gene expression profiling when low proliferation of uterine cells is associated to pregnancy failure in an  in vivo  study, showing lower rate of reinjury and faster resume to their activities ( Corradetti  et al ., 2014 ). In tendon repair, the re-injury was lower in treatment with AMSC than compared with BM-MSC ascertained by ultrasonography ( Lange-Consiglio  et al ., 2013b ). Still, biomaterials composed for amniotic membrane has attracted attention. They have being associated to better mechanical properties in tendons injuries, plus this membrane has been used to accelerate healing wounds ( Violini  et al ., 2012 ;  Hortensius  et al ., 2016 ).\nA promising use of large animal models must be done before clinical trial and confirm efficacy of therapy and controversial results must be followed ( Gonçalves  et al , 2014 ).  Kamiya  et al . (2005 ) showed efficacy in suppressing corneal inflammatory reactions, when topically applied human amniotic membrane culture supernatant in mices with induced corneal neovascularization.\nVidane  et al . (2017 ) evaluated the effects of intravenous administration of allogeneic feline amniotic membrane stem cells in cats with naturally occurring chronic kidney disease. Despite the kidney architecture and morphology did not change during the treatment, the tranplantation showed renoprotective effect, improved renal function, delaying the progression of the disease and stabilizing the clinical condition of the animals.\nAnd finally, the AMSC are described into application in humans to aid in treatment of cutaneous wounds, burns, and superficial ocular reconstruction ( Ward and Bennett, 1984 ;  Avila  et al ., 2001 ;  Gomes  et al ., 2005 ).","source_license":"CC-BY-4.0","license_restricted":false}