Bruna Corradetti

Comparison of equine bone marrow-, umbilical cord matrix and amniotic fluid-derived progenitor cells

The aim of the study was to compare in vitro the stemness features of horse progenitor cells derived from bone marrow (BM-MSCs), amniotic fluid (AF-MSCs) and umbilical cord matrix (EUC-MSCs). It has been suggested that there may be a stem cell population within both umbilical cord matrix and amniotic fluid. However, little knowledge exists about the characteristics of these progenitor cells within these sources in the equine species. This study wanted to investigate an alternative and non-invasive stem cell source for the equine tissue engineering and to learn more about the properties of these cells for future cell banking. Bone marrow, umbilical cord and amniotic fluid samples were harvested from different horses. Cells were analyzed for proliferation, immunocytochemical, stem cell gene expression and multilineage plasticity. BM- and AF-MSCs took similar time to reach confluence and showed comparable plating efficiency. All cell lines expressed identical stem cell markers and capability to differentiate towards osteogenic lineage. Almost all cell lines differentiated into the adipogenic lineage as demonstrated by cytochemical staining, even if no adipose gene expression was detectable for AF-MSCs. AF- and EUC-MSCs showed a limited chondrogenic differentiation compared with BM-MSCs as demonstrated by histological and biochemical analyses. These findings suggest that AF-MSCs appeared to be a readily obtainable and highly proliferative cell line from an uninvasive source that may represent a good model system for stem cell biology. More studies are needed to investigate their multilineage potential. EUC-MSCs need to be further investigated regarding their particular behavior in vitro represented by spheroid formation.

Fetal adnexa derived stem cells from domestic animal: progress and perspectives.

The fetal adnexa such as umbilical cord, amnion and amniotic fluid have been proposed as ideal sources of different stem cell lineages. Use of adnexal tissue has many potential advantages, including the noninvasive nature of the isolation procedure, the large tissue mass from which cells can be harvested with high efficiency and the potential of these cells to differentiate. Moreover, particularly in human medicine, the harvesting of these tissues is more ethically acceptable making these sources of stem cells very attractive for regenerative therapies and biotechnological applications. The adnexal tissue cells preserve some of the characteristics of the primitive embryonic layers from which they originate. Indeed, many studies indicate that these stem cells exhibit some features of embryonic stem cells as expression of embryonic markers and proliferation capability, without showing immunogenicity. However, the differentiation potential of these cells, either in vivo or in vitro, is intermediate between the pluripotent embryonic stem cells and the multipotent adult stem cells. Non-embryonic extra-fetal derived stem cells have opened new perspectives for developmental biology and for regenerative medicine, not only in humans but also in animals. In this update, we report the state of the art of fetal adnexa-derived stem cells from domestic animals and analyze their applications and potential uses in veterinary medicine.

Size-sieved subpopulations of mesenchymal stem cells from intervascular and perivascular equine umbilical cord matrix

Objectives:  Umbilical cord matrix (UCM) has been recently proposed as an alternative source of mesenchymal stem cells (MSCs). The aim of this study was to isolate and characterize presumptive stem cells from intervascular and perivascular equine UCM and to obtain homogeneous subpopulations from both sites.

Investigating the efficacy of amnion-derived compared with bone marrow–derived mesenchymal stromal cells in equine tendon and ligament injuries

BACKGROUND AIMS This is the first study to compare the treatment of horse tendon and ligament injuries with the use of mesenchymal stromal cells (MSCs) obtained from two different sources: amniotic membrane (AMSCs) and bone marrow (BM-MSCs). The objective was to prove the ability of AMSCs to exert beneficial effects in vivo. METHODS Five million allogeneic frozen-thawed AMSCs or autologous fresh BM-MSCs were injected intralesionally in horses belonging to group A (51 horses) and group B (44 horses). The interval lesion/implantation was of 6-15 days for the AMSCs and 16-35 days for the BM-MSCs. Healing was assessed clinically and ultrasonographically. Follow-up was monitored for 2 further years from return to full work. RESULTS No significant adverse effects after MSCs treatment were seen in any of the horses studied, independent of the type of stromal cell implanted. All animals belonging to group A resumed their activities between 4-5 months after treatment, whereas animals of group B resumed their activities after 4-12 months. The rate of re-injury in horses treated with AMSCs is lower (4.00%) compared with the average observed when horses were treated with BM-MSCs (23.08%). CONCLUSIONS The possibility to inject allogeneic AMSCs in real time, before any ultrasonographic change occurs within the injured tendon and ligament, together with the higher plasticity and proliferative capacity of these cells compared with BM-MSCs, represents the main features of interest for this novel approach for the treatment of equine tendon diseases. An obvious active proliferative healing in the area injected with AMSCs makes these cells more effective than BM-MSCs.

Molecular characterization and in vitro differentiation of feline progenitor-like amniotic epithelial cells

IntroductionWhile amniotic mesenchymal cells have been isolated and characterized in different species, amniotic epithelial cells (AECs) have been found only in humans and horses and are recently considered valid candidates in regenerative medicine. The aim of this work is to obtain and characterize, for the first time in the feline species, presumptive stem cells from the epithelial portion of the amnion (AECs) to be used for clinical applications.MethodsIn our study, we molecularly characterized and induced in vitro differentiation of feline AECs, obtained after enzymatic digestion of amnion.ResultsAECs displayed a polygonal morphology and the mean doubling time value was 1.94 ± 0.04 days demonstrating the high proliferating capacity of these cells. By RT-PCR, AECs expressed pluripotent (Oct4, Nanog) and some mesenchymal markers (CD166, CD44) suggesting that an epithelial-mesenchymal transition may occur in these cells that lack the hematopoietic marker CD34. Cells also showed the expression of embryonic marker SSEA-4, but not SSEA-3, as demonstrated by immunocytochemistry and flow cytometry. Moreover, the possibility to use feline AECs in cell therapies resides in their low immunogenicity, due to the absence of MHC-II antigen expression. After induction, AECs differentiated into the mesodermic and ectodermic lineages, demonstrating high plasticity.ConclusionsIn conclusion, feline AECs appear to be a readily obtainable, highly proliferative, multipotent and non-immunogenic cell line from a source that may represent a good model system for stem cell biology and be useful in allogenic cell-based therapies in order to treat tissue lesions, especially with loss of substance.

Bis-(2-ethylexhyl) phthalate impairs spermatogenesis in zebrafish (Danio rerio)

Bis-(2-ethylhexyl) phthalate (DEHP) is a widely used industrial additive for increasing plastic flexibility. Its metabolites are known to exert toxic effects on reproduction and development of mammals. The aim of this study was to evaluate the effects of environmentally relevant concentrations of DEHP (0.2 and 20 μg/L) on the reproductive biology of adult male zebrafish (Danio rerio). The effects of DEHP and 17β-ethynylestradiol (a positive control) were determined after one or three weeks of exposure by TUNEL assay, histomorphometric analysis and evaluation of reproductive performance. DEHP impaired reproduction in zebrafish by inducing a mitotic arrest during spermatogenesis, increasing DNA fragmentation in sperm cells and markedly reducing embryo production (up to 90%). In conclusion, relatively short-term exposure to environmentally relevant concentrations of DEHP is able to alter spermatogenesis and affect reproduction in zebrafish.

Tenogenic differentiation of equine mesenchymal progenitor cells under indirect co-culture.

PURPOSE Adult bone marrow mesenchymal stem cells (BM-MSCs) are a potential cell source for tendon repair in direct cell therapy and tissue engineering investigations. The purpose of this study was to evaluate the tenogenic induction of undifferentiated BM-MSCs under indirect co-culture technique with trimmed native tendon tissue. Since the horse represents a preferred species to study tendon regenerative strategies, this work was conducted on equine BM-MSCs. METHODS Equine BM-MSCs were co-cultured in a transwell system with tendon tissue fragments. The BM-MSC tenogenic differentiation was evaluated by cytochemical staining and real time PCR for gene expression. Cell viability in tendon fragments and cultured cells was analyzed. RESULTS Our results indicate that under indirect co-culture with native and healthy tendon tissue the BM-MSCs expressed tendon-specific markers such as decorin, tenomodulin, tenascin-C, and collagen type I. They also retained a tenocyte-like phenotype during monolayer culture. CONCLUSIONS Data are very encouraging for future in vitro investigations into committing cells to the tenogenic lineage without adding growth factors or serum to the culture medium for both cell therapy and tissue engineering.

Amniotic membrane-derived mesenchymal cells and their conditioned media: potential candidates for uterine regenerative therapy in the horse.

Amniotic membrane-derived mesenchymal cells (AMCs) are considered suitable candidates for a variety of cell-based applications. In view of cell therapy application in uterine pathologies, we studied AMCs in comparison to cells isolated from the endometrium of mares at diestrus (EDCs) being the endometrium during diestrus and early pregnancy similar from a hormonal standpoint. In particular, we demonstrated that amnion tissue fragments (AM) shares the same transcriptional profile with endometrial tissue fragments (ED), expressing genes involved in early pregnancy (AbdB-like Hoxa genes), pre-implantantion conceptus development (Erα, PR, PGRMC1 and mPR) and their regulators (Wnt7a, Wnt4a). Soon after the isolation, only AMCs express Wnt4a and Wnt7a. Interestingly, the expression levels of prostaglandin-endoperoxide synthase 2 (PTGS2) were found greater in AM and AMCs than their endometrial counterparts thus confirming the role of AMCs as mediators of inflammation. The expression of nuclear progesterone receptor (PR), membrane-bound intracellular progesterone receptor component 1 (PGRMC1) and membrane-bound intracellular progesterone receptor (mPR), known to lead to improved endometrial receptivity, was maintained in AMCs over 5 passages in vitro when the media was supplemented with progesterone. To further explore the potential of AMCs in endometrial regeneration, their capacity to support resident cell proliferation was assessed by co-culturing them with EDCs in a transwell system or culturing in the presence of AMC-conditioned medium (AMC-CM). A significant increase in EDC proliferation rate exhibited the crucial role of soluble factors as mediators of stem cells action. The present investigation revealed that AMCs, as well as their derived conditioned media, have the potential to improve endometrial cell replenishment when low proliferation is associated to pregnancy failure. These findings make AMCs suitable candidates for the treatment of endometrosis in mares.

A1-3 chromosomal translocations in Italian populations of the peach potato aphid Myzus persicae (Sulzer) not linked to esterase-based insecticide resistance

Esterase-based resistance in the peach-potato aphid, Myzus persicae (Sulzer), is generally due to one of two alternative amplified carboxylesterase genes, E4 or FE4 (fast E4). The E4 amplified form is distributed worldwide and it is correlated with a particular translocation between autosomes 1 and 3, whereas the FE4 form, which has hitherto not been found to be associated with chromosomal rearrangements, is typical of the Mediterranean regions. In this study, we present for the first time cytogenetic and molecular data on some M. persicae parthenogenetic lineages, which clearly show a chromosomal A1-3 translocation associated with esterase FE4 genes and unrelated to high levels of esterase-based resistance.

Leptin and leptin receptor are detectable in equine spermatozoa but are not involved in in vitro fertilisation

In human and swine, leptin (OB) has been identified in seminal plasma and leptin receptors (OB-R) on the cell surface of spermatozoa, indicating that spermatozoa are a target for OB. This hormone has also been detected in follicular fluid (FF) in women and mares, although its role requires further study. The aims of this study were to investigate the immunolocalisation and the expression of OB and OB-R in equine spermatozoa and to evaluate the involvement of OB in equine in vitro fertilisation (IVF). Since progesterone (P) and OB are both found in FF, the individual and combined effects of these two hormones were studied in equine IVF and compared with the results obtained from the use of FF for in vitro sperm preparation. For the first time, we were able to identify OB and OB-R mRNA and their corresponding proteins in equine spermatozoa. When spermatozoa were treated with OB, there was a decrease in the three motility parameters VSL, STR and LIN, commonly associated with hyperactivation, whilst the acrosome reaction rate increased (P<0.05). The fertilisation rate was 51% with FF, 46.15% with P, 43.64% with P+OB and 0% with OB alone. The percentage of eight-cell stage embryos was 18.7% with FF, 17.1% with P and 16.7% with OB+P. OB alone did not permit oocyte fertilisation, indicating that, in the horse, OB is involved in capacitation and hyperactivation but not in sperm penetration.