A. Lange Consiglio

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.

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

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.

Effects of lipid peroxidation on chromatin in rabbit and mouse spermatozoa: A cytochemical approach

Abstract To extend present knowledge on the possible interactions between lipid peroxidation and chromatin, we studied rabbit and mouse spermatozoa during spontaneous lipid peroxidation. Epididymal spermatozoa were aerobically incubated in high sodium or high potassium media to obtain spontaneous lipid peroxidation in the absence of added promoters. Quantitative cytochemical assays were performed in situ, in individual spermatozoa processed by Feulgen reaction and Gallocyanin-chrome alum (GCA) staining. Our findings show that in both species examined, the spermatozoa chromatin undergoes destabilization associated with marked alterations of the physico-chemical state of the DNA-protein complex. The decrease of Feulgen and GCA-positive material may be explained by DNA damage and implies the release from the nucleus of DNA fragments. Moreover, our data support the idea that during spontaneous lipid peroxidation, the spermatozoa chromatin undergoes destabilization sometimes correlated with increased acid lability of the DNA-protein complex. There are differences in the two species, probably correlated with differences in major protective enzymatic systems against cell damage by autoxidation and, especially, against different kinds of oxygen radicals responsible for initiation of lipid peroxidation.

Time course of in vitro maturation of compact cumulus horse oocytes after roscovitine-induced meiotic inhibition: effects on the coordination between nuclear and cytoplasmic maturation.

This study was carried out to evaluate the usefulness of a pre-maturation step in improving the coordination between cytoplasmic and nuclear maturation of horse compact cumulus oocytes by the addition of roscovitine (ROSC). Oocytes were collected by scraping and pre-cultured for 18 h in a maturation medium TCM199 supplemented with pyruvate, LH, FSH, insulin growth factor (IGF), epidermal growth factor (EGF), insulin, transferrin and selenium (IVM-ROSC) or in a simple medium (M199-ROSC). After pre-maturation, oocytes from both the groups were in part denuded and fixed-stained and in part in vitro matured to assess the kinetic of in vitro maturation (IVM). The nuclear progression and the cytoskeletal organization of microfilaments and cortical granules (CG) of treated and untreated oocytes were assessed by fluorescent probes. Oocytes immediately fixed after recovery and oocytes pre-cultured in M199-ROSC for 18 h did not show metaphase II (MII) plates, whereas in IVM-ROSC group, 6/69 oocytes (8.7%) showed MII plates. After inhibition, during maturation kinetics at 11, 18 and 29 h, maturation rate of M199-ROSC group progressively increased and at 29 h of IVM, reached the maturation rate of control group (13/66, 19.7% vs 31/125, 24.8%). No statistically significant differences in cytoplasmic maturation were found. The number of MII plates after 29 h of IVM, was significantly higher (p < 0.05) in IVM-ROSC group (34/90) compared with M199-ROSC (13/66) and control groups (31/125) as well as the number of oocytes with microfilaments and CG distributed in cortical region (25/34 vs 3/13 and 7/31 respectively). Our results showed that pre-culturing in the presence of Roscovitine in a fully supplemented maturation medium containing gonadotropins and growth factors partially suppressed the meiotic maturation, but established a more suitable environment for improving cytoplasmic maturation of horse compact cumulus oocytes as defined by microfilaments and CG configuration.

Cytochrome oxidase and lactate dehydrogenase activities in peroxidized rabbit epididymal spermatozoa.

Summary: We report a quantitative cytochemical study on cytochrome oxidase and lactate dehydrogenase activities on rabbit epididymal spermatozoa during spontaneous lipid peroxidation.

Cytochemical study on human spermatozoa metabolism during in vitro capacitation.

Summary: In eutherian mammalian spermatozoa the capacitation is coupled to a specific type of metabolism, that is glycolysis or oxidative respiration. A cytochemical study was carried out on cytochrome oxidase and lactate dehydrogenase in human spermatozoa collected at different times during in vitro capacitation.

Reconstruction of calf oocytes by germinal vesicle transfer in mature bovine oocytes: preliminary results

Reconstruction of calf oocytes by germinal vesicle transfer in mature bovine oocytes: preliminary results A. Lange Consiglio & A. Bignotti & A. M. Pecile & F. Cremonesi Published online: 14 July 2009 # Springer Science + Business Media B.V. 2009

Effects of deep freezing on the energy metabolism of bovine spermatozoa during in vitro capacitation: A cytochemical approach.

In order to study the effects of deep freezing on the energy metabolism of bovine spermatozoa, a cytochemical quantitative study was carried out by a microdensitometric method on cytochrome oxidase and lactate dehydrogenase (LDH) activities. These were evaluated in situ on individual frozen-thawed bull spermatozoa collected at different times during in vitro capacitation. The results showed that in bull spermatozoa both the initiation of motility and capacity to fertilize eggs were associated with the anaerobic rather than aerobic glycolysis. The freezing-thawing processes and storage in liquid nitrogen induced a general enhancement of both the enzymatic activities examined. The high ionic strength treatment gave rise to a significant but reversible decrease in both the cytochrome oxidase and LDH activities in the fresh as well as in the frozen-stored sperm. The findings, based on cytochemical observations of energy metabolism of spermatozoa and evaluated during in vitro capacitation, suggest that the respiration and the anaerobic glycolysis of spermatozoa seem to be slightly impaired by the freezing-thawing and storage processes.

Secretome derived from different cell lines in bovine embryo production in vitro

The present study investigated the effects of conditioned medium (CM), composed of microvesicles (MVs) and soluble factors present in the supernatant (SN), of bovine endometrial and amniotic cells on embryo quality and rate of blastocyst production. Presumptive zygotes were randomly assigned on Days 1, 3 and 5 after fertilisation to synthetic oviducal fluid with amino acids (SOFaa; control) or to SOFaa supplemented with either 20% endometrial or amniotic CM, 20% SN or 100×106MVsmL-1. Embryos were evaluated on Day 7. For groups supplemented with MVs derived from either endometrial or amniotic cells on Day 1 of culture, blastocysts had developed, but at a lower rate than in the control group. Blastocysts had developed in all groups in which endometrial or amniotic cell-derived CM or MVs were added on Day 3 of culture, but the rate of blastocyst development was significantly lower in both CM groups than in the MVs groups. The addition of all secretome fractions (CM, MVs and SN) derived from either bovine endometrial or amniotic cells on Day 5 of culture resulted in blastocyst production, but only amniotic MVs resulted in a blastocyst production rate comparable to that in the control group. Supplementation of SOFaa on Day 5 resulted in a qualitatively higher number of inner cell mass cells compared with the control group only for the amniotic CM and MVs groups. At day 7, these data were confirmed by RT-qPCR evaluation of genes (Bcl-2-associated X protein (BAX) and glutathione peroxidase 1 (GPX1) involved in apoptosis and protection against reactive oxygen species. In conclusion, of the different secretome fractions tested, only amniotic MVs added to SOFaa resulted in better outcomes than in the control group.