F. Cremonesi

Characterization and potential applications of progenitor-like cells isolated from horse amniotic membrane.

The aim of this work was to isolate, for the first time, progenitor‐like cells from the epithelial (AECs) and mesenchymal (AMCs) portions of the horse amniotic membrane, and to define the biological properties of these cells. AECs displayed polygonal epithelial morphology, while AMCs were fibroblast‐like. Usually, six to eight passages were reached before proliferation decreased, with 13.08 and 26.5 cell population doublings attained after 31 days for AECs and AMCs, respectively. Immunocytochemical studies performed at passage 3 (P3) showed that both cell populations were positive for the expression of specific embryonic markers (TRA‐1‐60, SSEA‐3, SSEA‐4 and Oct‐4). Meanwhile, RT–PCR performed at P1 and P5 showed expression of mesenchymal stem/stromal cell markers (CD29, CD105, CD44 and CD166) with negativity for CD34 at P1, although this marker began to be expressed by P5. The cells also expressed MHC‐I at both P1 and P5, but lacked MHC‐II expression at P1. Both AECs and AMCs demonstrated high plasticity, differentiating in vitro toward the osteogenic, adipogenic, chondrogenic and neurogenic lineages. Equine amnion‐\derived cells could also be frozen and recovered without loss of their functional integrity in terms of morphology, presence of specific stemness markers and differentiation ability, although the renewal capacity was lower than that observed for freshly isolated cells. To investigate potential therapeutic effects and cell tolerance in vivo, horse amnion‐derived cells were allogeneically injected into three horses with tendon injuries, resulting in a quick reduction in tendon size and ultrasonographic cross‐sectional area measurements. These results suggest that horse amnion‐derived cells may be useful for cell therapy applications. Copyright

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.

Isolation, in vitro culture and characterization of foal umbilical cord stem cells at birth

ABSTRACT Isolation, in vitro culture and characterizationof foal umbilical cord stem cells at birth F. Cremonesi & S. Violini & A. Lange Consiglio & P. Ramelli & G. Ranzenigo & P. Mariani Published online: 8 August 2008 # Springer Science + Business Media B.V. 2008 Keywords Foal.Stemcells.UmbilicalcordAbbreviationsEGF epidermal growth factorFCS foetal calf serumGADPH glyceraldehyde-3-phosphate dehydrogenaseMSCs mesenchymal stem cellsOct-4 octamer binding protein 3/4SOX-2 SRY-box containing gene 2IntroductionOver the past few years, mesenchymal stem cells (MSC) have become a promising tool fortherapeutic applications in regenerative medicine both for humans and animals. Likehaematopoietic cells, mesenchymal cells have been shown to proliferate and formfibroblast-like colonies in vitro. MSCs can be isolated directly from bone marrow, adiposetissue, umbilical cord and other foetal tissues. In specific culture conditions in vitro, theyare able to differentiate into several cell types such as osteoblasts, condroblasts, tenocytes,myocytes and adipocytes. Bone marrow represents the most common source for MSCs,however, cell number and proliferation/differentiation capabilities decrease with donor ageand culture passages in vitro (D’Ippolito et al. 1999). Therefore, umbilical cord couldrepresent a good source of stem cells other than bone marrow, considering the pluripotencycharacteristics of these cells and the fact that it is easy to sample from the umbilical cord.Protocols for tissue sampling and the isolation of MSCs from bone marrow are welldefined in horses, however, to date no investigations of the use and characterization of cells

Effects of a trichothecene, T-2 toxin, on proliferation and steroid production by porcine granulosa cells

Fusarium mycotoxins, such as trichothecenes and zearalenone, are produced by molds and contaminate a large variety of grains and feedstuffs worldwide. Mycotoxins of Fusarium fungi include the trichothecenes, deoxynivalenol and T-2 toxin (T2), and zearalenone, and have been implicated in poor reproductive performance in pigs. However, direct ovarian effects of T2 toxin have not been reported. Therefore, porcine granulosa cells (GC) from small follicles (1-5mm) were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing various doses of FSH, insulin-like growth factor-I and T2 (at various doses/combinations) to evaluate the influence of T2 on steroid production and cell proliferation. T2 at 1, 3, 30 and 300 ng/mL completely inhibited FSH plus IGF-I-induced estradiol production, whereas 0.3 ng/mL of T2 inhibited estradiol production by 40%. Progesterone production was less sensitive to the inhibitory effects of T2 with 0.3 ng/mL having no effect and 1 ng/mL inhibiting progesterone production by only 30%. At 30 and 300 ng/mL, T2 completely inhibited FSH plus IGF-I-induced progesterone production. The impact of T2 on the dose-response to IGF-I (0, 3, 10 and 30 ng/mL) was also evaluated; T2 blunted the stimulatory effect of 3-30 ng/mL of IGF-I on steroid production and cell proliferation. Serum-induced granulosa cell proliferation was decreased (P<0.05) by 40% after 1 day and by 56% after 2 days of T2 treatment. The present studies indicate for the first time that T2 may be able to alter in growth of the granulosa layer within ovarian follicles in addition to their effect on steroidogenesis. In conclusion, T2 has potent direct dose-dependent effects on granulosa cell proliferation and steroidogenesis. These direct ovarian effects could be one mechanism whereby contaminating Fusarium mycotoxins in feedstuffs could impact reproductive performance in swine.

Conditioned Medium from Horse Amniotic Membrane-Derived Multipotent Progenitor Cells: Immunomodulatory Activity In Vitro and First Clinical Application in Tendon and Ligament Injuries In Vivo

We have recently demonstrated that heterologous transplantation of horse amniotic membrane-derived mesenchymal cells (AMCs) can be useful for cell therapy applications in tendon diseases, and hypothesized that these cells may promote tendon repair via paracrine-acting molecules targeting inflammatory processes. To test this hypothesis, here we examined the immunomodulatory characteristics of AMCs and of their conditioned medium (AMC-CM) in vitro, and studied the potential therapeutic effect of AMC-CM in thirteen different spontaneous horse tendon and ligament injuries in vivo. Our results demonstrate that AMCs are capable of inhibiting peripheral blood mononuclear cell (PBMC) proliferation after allogenic stimulation either when cocultured in cell-to-cell contact, or when the two cell types are physically separated by a transwell membrane, suggesting that soluble factors are implicated in this phenomenon. Our hypothesis is further supported by the demonstration that PBMC proliferation is inhibited by AMC-CM. In our in vivo studies, no significant adverse effects were observed in treated tendons, and clinical and ultrasonographical evaluation did not reveal evidence of inappropriate tissue or tumor formation. Clinical outcomes were favorable and the significantly lower rate (15.38%) of reinjuries observed compared to untreated animals, suggests that treatment with AMC-CM is very efficacious. In conclusion, this study identifies AMC-CM as a novel therapeutic biological cell-free product for treating horse tendon and ligament diseases.

Mesenchymal stem cells from amnion and amniotic fluid in the bovine

Amnion and amniotic fluid (AF) are noncontroversial and inexhaustible sources of mesenchymal stem cells (MSCs) that can be harvested noninvasively at low cost. As in humans, also in veterinary field, presumptive stem cells derived from these tissues reveal as promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. The aim of this work is to obtain and characterize, for the first time in bovine species, presumptive MSCs from the epithelial portion of the amnion (AECs) and from the AF (AF-MSCs) to be used for clinical applications. AECs display a polygonal morphology, whereas AF-MSCs exhibit a fibroblastic-like morphology only starting from the second passage, being heterogeneous during the primary culture. For both lines, the proliferative ability has been found constant over the ten passages studied and AECs show a statistically lower (P<0.05) doubling time with respect to AF-MSCs. AECs express MSC-specific markers (ITGB1 (CD29), CD44, ALCAM (CD166), ENG (CD105), and NT5E (CD73)) from P1 to P3; in AF-MSCs, only ITGB1, CD44, and ALCAM mRNAs are detected; NT5E is expressed from P2 and ENG has not been found at any passage. AF-MSCs and AECs are positive for the pluripotent markers (POU5F1 (OCT4) and MYC (c-Myc)) and lack of the hematopoietic markers. When appropriately induced, both cell lines are capable of differentiating into ectodermal and mesodermal lineages. This study contributes to reinforce the emerging importance of these cells as ideal tools in veterinary medicine. A deeper evaluation of the immunological properties needs to be performed in order to better understand their role in cellular therapy.

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.

Seroprevalence of feline immunodeficiency virus, feline leukaemia virus and Toxoplasma gondii in stray cat colonies in northern Italy and correlation with clinical and laboratory data

Stray cat colonies in urban and rural areas of Lombardy, northern Italy, were surveyed for seroprevalence of feline immunodeficiency virus (FIV) antibodies, feline leukaemia virus (FeLV) antigen and Toxoplasma gondii IgG. Of 316 cats tested, 6.6% were positive for FIV and 3.8% were positive for FeLV infection; 203 cats were tested for T gondii IgG antibodies and a prevalence of 30.5% was detected. Statistical analysis tested the influence of provenience, age, gender, health status and laboratory results on seroprevalence and found male gender and adult age were risk factors for FIV infection. FIV-infected cats were more likely to have a decreased red blood cell count than FIV seronegative cats. No predictors were significantly associated with FeLV and T gondii seropositivity. Colony cats in this study posed a limited risk for retrovirus infection to pet cats allowed outdoors, whereas toxoplasmosis exposure was comparable with the worldwide data.