Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential

Barberini et al. Stem Cell Research & Therapy 2014, 5:25 http://stemcellres.com/content/5/1/25 RESEARCH Open Access Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential Danielle Jaqueta Barberini1, Natália Pereira Paiva Freitas1, Mariana Sartori Magnoni2, Leandro Maia1, Amanda Jerônimo Listoni3, Marta Cristina Heckler1, Mateus Jose Sudano4, Marjorie Assis Golim2, Fernanda da Cruz Landim-Alvarenga5 and Rogério Martins Amorim1* Abstract Introduction: Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank. Methods: The BM-MSCs, AT-MSCs and UC-MSCs were cultured and evaluated in vitro for their osteogenic, adipogenic and chondrogenic differentiation potential. Additionally, MSCs were assessed for CD105, CD44, CD34, CD90 and MHC-II markers by flow cytometry, and MHC-II was also assessed by immunocytochemistry. To interpret the flow cytometry results, statistical analysis was performed using ANOVA. Results: The harvesting and culturing procedures of BM-MSCs, AT-MSCs and UC-MSCs were feasible, with an average cell growth until the third passage of 25 days for BM-MSCs, 15 days for AT-MSCs and 26 days for UC-MSCs. MSCs from all sources were able to differentiate into osteogenic (after 10 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs), adipogenic (after 8 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs) and chondrogenic (after 21 days for BM-MSCs, AT-MSCs and UC-MSCs) lineages. MSCs showed high expression of CD105, CD44 and CD90 and low or negative expression of CD34 and MHC-II. The MHC-II was not detected by immunocytochemistry techniques in any of the MSCs studied. Conclusions: The BM, AT and UC are feasible sources for harvesting equine MSCs, and their immunophenotypic and multipotency characteristics attained minimal criteria for defining MSCs. Due to the low expression of MHC-II by MSCs, all of the sources could be used in clinical trials involving allogeneic therapy in horses. However, the BM-MSCs and AT-MSCs showed fastest ‘‘in vitro’’ differentiation and AT-MSCs showed highest cell growth until third passage. These findings suggest that BM and AT may be preferable for cell banking purposes. * Correspondence: 1 Departament of Veterinary Clinics, College of Veterinary Medicine and Animal Science, São Paulo State University, UNESP, Botucatu, SP, Brazil Full list of author information is available at the end of the article © 2014 Barberini et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Barberini et al. Stem Cell Research & Therapy 2014, 5:25 http://stemcellres.com/content/5/1/25 Introduction Mesenchymal stem cells (MSCs) are non-hematopoietic, multipotent progenitor cells that are easily isolated from various adult tissues. MSCs are characterized by extensive proliferative ability, as well as the ability to differentiate in vitro into various mesenchymal lineages in response to an appropriate stimulus. These lineages include osteoblasts, adipocytes, chondrocytes, tenocytes and myocytes [1,2]. The use of MSCs has been demonstrated in the cartilage, bone and tendon of horses [3-5]. Although controversial, MSCs can also differentiate in response to specific stimuli in germ cells of other lineages, such as neurons, glial cells and hepatocytes [6-8]. In equine species, bone marrow (BM) is one of the most studied and used sources for obtaining adult stem cells [9,10]. However, adipose tissue (AT) is also an abundant and accessible source of MSCs that can provide a large number of cells required for use in cell therapy [11,12]. Additionally, cells from the amniotic membrane [13] and umbilical cord (UC) are a promising source of MSCs because they are less immunogenic, their collection is non-invasive, and they have the potential to differentiate into neural and endothelial cells [14,15]. Equine MSCs are mainly identified by their adherence to plastic and their ability to differentiate into multiple lineages [16] because immunophenotyping in horses is hindered by the lack of specific markers, limited availability of monoclonal anti-horse antibodies [17-19] and evidence that certain markers of other species do not cross-react with equine species [11]. Therefore, several markers have been tested and used, such as the positive markers CD44, CD90 CD29 [11,15,17,20], CD105 [21-23], MHC-I [5,15,20] and the negative markers CD14 [17], CD34 [21,23], MHC-II [5,17,20,23,24], CD45 [21,24], based on minimal criteria established by the International Society for Cellular Therapy (ISCT) to define human MSCs [25] and adipose-tissue derived stromal/stem cells [26]. Evidence suggests that these cells improve regeneration and tissue function by their ability to self-renew [3], their ability to differentiate into mesodermal, neuroectodermal and endodermal lineages [6], their synthesis of growth factors and their release of anti-inflammatory and immunomodulatory cytokines [2,18,20,27]. Autologous therapy with MSCs is widely used because it does not result in any significant deleterious effects at the time of implantation or later [28], and shows antiinflammatory and immunosuppressive effects [29]. However, treatment with autologous MSCs has limitations, such as in acute injuries, because expansion of MSCs by culturing takes 10 to 21 days [5], or in elderly patients because there is a decrease in the quantity, proliferation and differentiation potential of MSCs [30]. Nevertheless, adipose-derived nucleated cells have a short interval for Page 2 of 11 isolation of an injectable uncultured cell pool (24 to 48 hours), providing distinct advantages with regard to timeliness compared with an injection of cultured MSCs from other sources [29,31]. Allogeneic treatment in horses offers advantages in acute injuries because MSCs can be injected quickly. Allogeneic treatment then eliminates the time needed for the isolation and expansion of autologous MSCs. This treatment also allows the use of a more homogeneous cell population with a proven capacity for differentiation into various lineages [5,18,31], by taking MSCs from a cell bank of horse donors [27,32]. However, a heterogeneous cell population can be more effective de (...truncated)