Objective: Regenerative medicine aims at repairing damaged tissues using new cells. Different cell types have been proposed to this purpose with different advantages and limitations. Adult stem cells are well characterized but show a limited proliferation ability, differentiation potency, and are difficult to access. An alternative is represented by embryonic stem cells that display unlimited self-renewal and the ability to differentiate into all cell types of the body. However these cells are difficult to control and bring with them an increased risk of neoplastic transformation. More recently induced pluripotent stem cells have been proposed as a new promising technology. These cells, on the other hand, require the permanent integration of viral vectors into the host genome. This limits their possible use in regenerative medicine. A similar limitation accompanies the use of transdifferentiated cells, where reprogramming of a mature cell into another is obtained through viral transfections. We recently proposed a new strategy based on the direct conversion of an adult mature cell into another, avoiding the stable pluripotent state and the use of any gene transfection. This approach, designated “epigenetic conversion” is achieved through the use of an epigenetic modifier that drives the cells to a less committed and “high permissive” state and allows them to be re-addressed to a different lineage. We propose epigenetic conversion as a simple, direct and safe way to obtain cells to be used in cell therapy. EpigEnEtic modificAtion And gEnE ExprEssion All cells in a multicellular organism contain the same genome. Nevertheless, they can adopt a specific fate and specialize in the several tissues that constitute the body. This is possible because each cell express different sets of genes which are responsible for a distinct phenotype. Cell commitment and differentiation occur without alteration in the sequence of DNA, but rather through modifications “on top of it”. These are defined as epigenetic modifications and are responsible of heritable changes that stably maintain the genomic region activity state. Two major mechanisms are involved in these regulatory processes: DNA methylation and histone modifications (Figure 1). The first consists in the addition of a methyl group to the cytosine or adenine DNA nucleotides. The other is characterized by the attachment of different molecules on the histone tail, allowing or preventing transcription factors and other proteins to access the DNA. These differences in gene expression drive development and differentiation. In particular, mammalian development is characterized by a bimodal DNA methylation reprogramming that takes place during germ cell development, resetting parent-of-origin based genomic imprints and restoring totipotency to gametes, and then during the embryonic pre-implantation phase1. This second phase of methylation reprogramming occurs between fertilization and the formation of the blastocyst, in the absence of transcription or DNA replication and it is known as “active de-methylation”. At the time of fertilization a rapid paternal-specific asymmetric loss of methylation is clearly visible, while the initiation of the de novo methylation starts at the time of the first differentiative event, namely at the time 2 G. Pennarossa, S. Maffei, F. Gandolfi, T.A.L. Brevini isms through a gradual loss of differentiative potency5 that leads to a progressive restriction in their options6. This state, which is achieved and maintained through the epigenetic regulation of gene expression7, is physiologically very stable and its complete reversal requires an extensive reprogramming process that makes it inefficient and prone to errors8. However, several recent studies have demonstrated that differentiation is a bi-directional process, since terminally differentiated cells can be forced back to an increased potency state. Using the Waddington’s landscape metaphor, the ball can be pushed from the bottom of the valley up to the top of the hill in counter-current direction. A clear application of this concept is represented by the induced pluripotent stem (iPS) cell technology (Figure 2). However, this process requires levels of gene expression higher than those required once that state is reached, equivalent to an “activation energy”9. This reflects the necessity to initiate epigenetic reprogramming events and is normally obtained through the use of retroviruses, integrated in the host genome to force over-expression of the four transcription factors needed to reach the pluripotent state, namely OCT4, KLF4, SOX2 and MYC1013. This results in the reactivation of endogenous genes, regaining a developmental potency akin to that of embryonic stem cells (ESCs). It is fundamental to highlight that this approach suffers from a number of severe limitations that, in our opinion, prevent its possible use in regenof the inner cell mass (ICM) and trophectoderm (TE) formation. The ICM, which gives rise to all the tissues of the adult, becomes hyper-methylated, while the TE, that forms most of the structure of the placenta, remains under-methylated2,3. During the following developmental phases embryo/fetus cells undergo further specification process characterized by differential gene expression and epigenetic restrictions that gradually limit cell potency to a more limited phenotype-related expression pattern, producing highly specialized committed populations. A nice description of this concept was proposed by Conrad Hal Waddington4 in his famous “epigenetic landscape”, a metaphor for biological development. In his landscape the embryonic cell is represented by a ball that rolls from a non-committed status (pluripotent status) down a hill marked by slopes and valleys that symbolize the many different and complex mechanisms involved in cell differentiation process. Rolling down, the ball is addressed, by slopes and valleys, towards a progressively more restricted potency pathway, down to a tissue specific differentiated state (unipotent status). rEvErsion of cEllulAr fAtE The acquisition of epigenetic marks culminates with the fixation of a specific lineage fate by differential DNA methylation, which has been considered stable and potentially irreversible for many years. In particular, as we have discussed, mature cells acquire the differentiated state in an adult organFigure 1. Epigenetic mechanisms establishing cell phenotype related expression pattern. Gentle Makeover: Epigenetic Conversion of One Cell into Another 3 All these problems have stimulated the development of virus-free protocols for iPS derivation25,26 but, at present, these are generally more technical demanding, less efficient27 and do not solve the problems arising from the use of transgenes.

Recently carbon nanostructured materials are more attractive for the supercapacitor applications. Pure carbon nanotubes (CNTs) and double walled nitrogen doped carbon nanotubes (N-DWCNTs) have been synthesized for electrode material for the Supercapacitor applications. The N-DWCNTs were synthesized by Thermal Chemical Vapor Depositions (T-CVD) and they are coil shaped. The synthesized N-DWCNTs were characterized by powder X-ray diffraction (XRD) technique. The morphology of coil shaped N-DWCNTs were revealed by the Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The Electrochemical performance of pure CNTs and N-DWCNTs was analyzed using Cyclic Voltammetry (CV) with 0.5 M H2SO4 electrolyte. It showed the good pseudocapacitance behavior for N-DWCNTs. Electrochemical impedance spectroscopy (EIS) was measured for N-DWCNTs and showed the smaller charge transfer resistance (Rct – 7.731 Ω) which is much smaller than that of CNTs (23.69 Ω). The specific capacitance was measured for pure CNTs (23.40 F/g) and N-DWCNTs (51.29 F/g) with current density 0.125 A/g.

Purpose In this study we hypothesized that the mRNA vector Staufen mediates RNA relocalization during meiotic maturation, and by virtue of its interactions with endoplasmic reticulum, provides a possible mechanism by which protein synthesis is regulated. Methods We assessed the expression of staufen ( STAU ) and calreticulin ( CALR ), the latter adopted as a marker of the endoplasmic reticulum, in human oocytes at different stages of maturation: GV, metaphase MI and MII. Oocytes were subjected to polymerase chain reaction in order to investigate the expression of STAU and CALR . The corresponding protein products were identified by immunofluorescence and confocal laser scanning microscopy. Results STAU and CALR were constantly expressed and selectively localized during oocyte maturation. At the GV stage the both proteins displayed a dispersed distribution localization throughout the cytoplasm. Progressing to the MII stage, STAU tended to compartmentalize towards the cortical area of the oocyte clustering in granules of larger sizes. At the MII stage, CALR assumed a pattern reminiscent and possibly coincident with the position of the meiotic spindle. Conclusions The changing pattern of STAU distribution during meiotic maturation of human oocytes implicates a novel mechanism for the regulation of protein synthesis based on mRNA localization. Moreover, the unique disposition of CALR at the MII spindle uncovers a physical interaction with endoplasmic reticulum that may mediate cytoskeletal remodelling during oocyte maturation.

Different cell types have been suggested as candidates for use in regenerative medicine. Embryonic pluripotent stem cells can give rise to all cells of the body and possess unlimited self-renewal potential. However, they are unstable, difficult to control and have a risk of neoplastic transformation. Adult stem cells are safe but have limited proliferation and differentiation abilities and are usually not within easy access. In recent years, induced pluripotent stem (iPS) cells have become a new promising tool in regenerative medicine. However, the use of transgene vectors, commonly required for the induction of iPS cells, seriously limits their use in therapy. The same problem arising from the use of retroviruses is associated with the use of cells obtained through transdifferentiation. Developing knowledge of the mechanisms controlling epigenetic regulation of cell fate has boosted the use of epigenetic modifiers that drive cells into a 'highly permissive' state. We recently set up a new strategy for the conversion of an adult mature cell into another cell type. We increased cell plasticity using 5-aza-cytidine and took advantage of a brief window of epigenetic instability to redirect cells to a different lineage. This approach is termed 'epigenetic conversion'. It is a simple, direct and safe way to obtain both cells for therapy avoiding gene transfection and a stable pluripotent state.

Cryopreservation and retransplantation of ovarian tissue is a real option to preserve fertility in young cancer patients. However, a high risk of retransmission of malignancy exists in several tumours. In these patients, cryopreserved whole ovaries could provide an appealing source of oocytes to be grown and matured in vitro. The aim of this study was to develop a perfusion system for ex vivo culture of fresh and cryopreserved whole ovaries. Upon arrival to the laboratory, all ovaries were perfused via the ovarian artery with Ringer's solution and 10 UI L–1 of heparin for 10 min. Ovaries to be frozen were subsequently perfused with cryoprotectant solution [L-15 medium, 10% FBS, and 1.5 M dimethyl sulfoxide (DMSO)] and then frozen using Multi Thermal Gradient freezing technology (Core Dynamics Ltd., Ness Ziona, Israel), pushing the samples along the thermal gradient (4 to –70°C) at 0.01 mm s–1, resulting in a cooling rate of 0.3°C min–1. Samples were thawed at 37°C and immediately perfused with L-15 medium supplemented with decreasing sucrose concentrations (0.25, 0.125, and 0 M). In a closed-circuit perfusion system, 100 mL of recirculating medium (M199, 25 mM HEPES, 1% BSA, 2 mM glutamine, and antibiotic/antimycotic) was pumped into the ovarian artery using a peristaltic pump. The flow rate through the ovary was maintained between 1 and 1.5 mL min–1. Whole sheep ovaries were cultured at 38.5°C for 1 or 3 days. After culture, ovaries were fixed with 10% formaldehyde. Statistical analysis was performed using Student's t-test (SPSS 20, IBM Corp., Armonk, NY, USA). Morphological analysis showed that the rate of intact follicles was inversely related to the days of culture but was not affected by cryopreservation. In fact, the percentage of morphologically normal follicles in fresh and frozen ovaries cultured for 1 day (87 ± 3.4 and 83 ± 3.2%, respectively; P = 0.058) was higher (P = 0.048) than in ovaries cultured for 3 days (75 ± 2.9 and 71 ± 2.8%, respectively; P = 0.053). Cell proliferation, measured as Ki67-positive stromal cells, decreased during culture (P = 0.028) and was affected by cryopreservation both on Day 1 (13 ± 7 v. 15 ± 4%; P = 0.047) and Day 3 (10 ± 4 v. 12 ± 6%; P = 0.039). Similar results were observed for the apoptotic index that increased during culture both in fresh and cryopreserved ovaries (P = 0.028). The number of apoptotic cells per millimeter squared was lower (P = 0.031) in fresh (23 ± 10%) than in frozen ovaries (27 ± 15%) both on Day 1 and on Day 3 (30 ± 14 v. 33 ± 20%, respectively; P = 0.03). Cell viability and active endocrine function during culture is confirmed by steroid secretion, which is conserved in both fresh and cryopreserved ovaries for up to 3 days. Our results show that it is possible to culture both fresh and cryopreserved whole ovaries for up to 3 days. Although fresh ovaries, on average, did better than cryopreserved ones, we observed large individual variations, with positive and negative results overlapping between fresh and frozen samples. Further studies are in progress to explain the reason of such variations. Supported by AIRC IG 10376, Carraresi Foundation, and by Legge 7 (R.A.S).

The different structures of a mammalian ovary require complex 3-dimensional interactions to function properly. It is difficult to access the ovary in vivo and to study its physiology in vitro, it is necessary to dissect its different parts and culture them individually. Although informative, this approach prevents the understanding of the role played by their interactions. Perfusion systems are available for ovaries of laboratory animals while organs of larger species have been maintained in culture only for a few hours. This has prompted us to develop a system that can preserve the function of a whole sheep ovary for a few days ex vivo so that it is available for analysis in controlled conditions. Twenty-four sheep ovaries were collected at the local abattoir; 18 were assigned randomly to 3 experimental groups (media A, B, and C) and 6 were immediately fixed in 10% formaldehyde and used as fresh controls. Whole ovaries were cultured for up to 4 days using a semi-open perfusion system. Organs were perfused through the ovarian artery, at a flow rate of 1.5 mL min–1 with basal medium (M199, 25 mM HEPES, 2 mM l-glutamine and 100 µg mL–1 antibiotic-antimycotic solution) supplemented with 0.4% fatty acid free BSA (medium A); or 0.4% BSA heat shock fraction (medium B); or 10% FBS, 50 ng mL–1 IGF-1, and 50 mg bovine insulin (medium C). Ovaries were stimulated with FSH (Folltropin®-V, Bioniche Animal Health Inc., Belleville, Ontario, Canada) changing medium in a pulsatile manner (1 mg mL–1 for 2 h; 0.5 mg mL–1 for 2 h; 0 mg mL–1 for 20 h), with the same cycle repeated each day of culture. At every change, aliquots were collected for oestradiol (E2) and progesterone (P4) quantification. After culture, ovaries were examined for follicular morphology, cell proliferation, and apoptotic rate. Statistical analysis was performed using one-way ANOVA (SPSS 20, IBM, Armonk, NY, USA). In media A and B, all morphological parameters showed a small but significant decrease compared to fresh control, only after 3 days of culture. The different BSA in medium B did not affect follicle morphology but significantly increased cell proliferation (medium A, 28.59 ± 3.26%; medium B, 32.04 ± 2.67%) and decreased apoptosis (medium A, 32.51 ± 5.92%; medium B, 24.55 ± 2.55%). In both media, steroid concentration increased after FSH pulses (E2 range 1.95–10.50 pg mL–1; P4 range 0.34–3.08 ng mL–1), reaching levels similar to those measurable in peripheral plasma. The presence of FBS, IGF-1, and insulin in medium C allowed extension of the culture period to 4 days with a percentage of intact follicles comparable to that observed after 3 days in media A and B. Moreover, proliferation rates were comparable to fresh controls. Steroid pattern changed with P4 values dropping close to zero (range 0.03–1.18 ng mL–1) and E2 level (range 23.59–94.98 pg mL–1) increasing 10-fold, achieving a concentration similar to that measured in the ovarian vein around oestrous. Our data indicate that it is possible to support viability of large animal whole ovaries for up to 4 days, providing a physiologically relevant model for studying ovarian functions in vitro. Research was supported by AIRC IG 10376 and by the Carraresi Foundation.

Formation of lipid oxidation products was evaluated in dietary vegetable oils by using a novel analytical approach that consisted of derivatization of TAG into FAME and HPLC analysis with two detectors in series, UV and evaporative light scattering detector (ELSD). Three sunflower oils with different contents of oleic and linoleic acids, i.e., high-linoleic (HLSO), high-oleic (HOSO) and high-stearic high-oleic (HSHOSO), and two oils containing linolenic acid, soybean (SbO) and rapeseed (RO) oils, were heated at 40 °C and analyzed up to the total exhaustion of tocopherols. Results showed that oxidation products of linoleate were predominant in all cases, whereas no significant formation of oleate oxidation products was observed in the five oils in the presence of substantial contents of the tocopherols naturally occurring. Formation of oleate hydroperoxides and monoepoxystearates derived from oleic acid was only detected when tocopherols were exhausted in the monounsaturated oils, i.e., HOSO, HSHOSO and RO. The analysis of the main oxidation products of linoleate by the method applied proved to be a good analytical approach to evaluate the global oxidation extent of oils containing oleic and linoleic acids as the only oxidizable substrates. The method used enabled the quantitative determination of the simple hydroperoxydienes of linoleate and linolenate as a whole. Results suggested occurrence of hydroperoxy compounds other than those determined by the method in the oils containing linolenic acid, showing the low stability of simple hydroperoxydienes and their participation in further reactions.

Mammalian differentiation is obtained through epigenetic regulations that shape the genome, which is identical in all cells, to distinct phenotypes and tissue specific identities. The differentiated state of mature cells in an adult organism is therefore acquired through epigenetic restrictions that lead to a gradual loss of differentiative potency. In agreement with this, recent experiments demonstrate that terminally differentiated cells can be induced to de-differentiate in vitro and increase their plasticity in response to epigenetic modifiers that are capable of reverting cells from their lineage commitment to a more plastic state. Here we describe experiments where we prepared porcine skin fibroblasts and granulosa primary cultures and exposed them to an inhibitor of DNA methylation, the 5-aza-cytidine (5-aza-CR), to increase cell plasticity. Taking advantage of the obtained increased permissivity window, we investigated the ability of 5-aza-CR treated cells to respond to specific differentiation conditions and be re-addressed to a different cell lineage either within the same germ layer or to a different germ layer. Cells were evaluated for their morphological changes and assessed using RT-PCR and immunocytochemical studies during the treatment. Following the exposure to 5-aza-CR the phenotype of both cell types changed. Treated cells displayed an oval or round shape, and appeared smaller with larger nuclei and granular and vacuolated cytoplasm. This was accompanied by an active expression of the main pluripotency-related genes OCT4, NANOG, SOX2, and REX1, originally undetectable in untreated fibroblasts and granulosa cells. 5-aza-CR treated granulosa cells cultured with recombinant human vascular endothelial growth factor to induce myogenic specification (different lineage within the same germ layer) suppressed the expression of granulosa specific marker (Cytokeratin) as well as of the pluripotency genes, and expressed MYOD, MYF5, and MYOG (earliest myogenic markers that are involved in the coordination of skeletal muscle development or myogenesis). In order to trans-differente 5-aza-CR treated fibroblasts to cells of a different germ layer, they were exposed to activin A to promote endoderm commitment. Cells down-regulated Vimentin (fibroblast marker) as well as pluripotent gene expression and transcribed Nestin (transiently involved in multi-lineage progenitor cell differentiation), SOX17, FOXA2 (induction of definitive endoderm), and HNF4A, HNF1 (primitive gut tube specific genes). Altogether these results suggest that it is possible to obtain a direct inter-lineage conversion by removing epigenetic restriction, using demethylating agents such as 5-aza-CR, and avoiding a stable pluripotent state. This novel approach may represent a promising tool for regenerative medicine because it does not involve the use of any transgenic modifications, retroviral transfection, or both. Supported by Network Lombardo iPS (NetLiPS) Project ID 30190629.

The morphology of the testis may be altered in various developmental, physiological and pathological conditions and these changes are reflected by the alterations in the reproductive capacity. Studying testicular morphology under these conditions or following therapeutic interventions relies on quantitative data. Design-based stereology provides quantitative morphological data on the most important characteristics of the testis. The total volume of the testis, seminiferous tubules, interstitial tissues and germinal epithelium, length, diameter and cross sectional area of the tubules as well as the number of Sertoli, Leydig, myoid cells, spermatogonia, spermatocytes and spermatids can be estimated. The present paper explains and demonstrates accurate and efficient stereological methods of sampling and analysis of testicular specimens according to the basics made by the pioneer stereologists. Second-order stereology can provide additional information on the spatial arrangement of the content of the tissues, cells or organelles in testis. These methods are valuable enough to be included in the toolbox of testicular research and are essential whenever quantitative data on morphological characteristics of the testis are required including testicular biopsy of human or in experimental studies.

Large animal models provide useful data for pre-clinical research including regenerative medicine. However whereas the derivation of tissue specific stem cells has been successful. pluripotent stem cells so far have been difficult to obtain in these species. A possible alternative could be direct reprogramming but this has only been described in mouse and human. We have recently described an alternative method for reprogramming human somatic cells based on a brief demethylation step immediately followed by an induction protocol. Aim of the present paper was to determine whether this method is applicable to pig in the attempt to achieve cell reprogramming in a large animal model for the first time. Pig dermal fibroblasts were exposed to DNA methyltransferase inhibitor 5-aza-cytidine (5-aza-CR) for 18 h. After a brief recovery period, fibroblast were subjected to a three-step protocol for the induction of endocrine pancreatic differentiation that was completed after 42 days. During the process pig fibroblast rapidly lost their typical elongated form and gradually became organized in a reticular pattern that evolved into distinct cell aggregates. After a brief expression of some pluripotency genes, cells expression pattern mimicked the transition from primitive endoderm to endocrine pancreas. Not only converted cells expressed insulin but were able to release it in response to a physiological glucose challenge in vitro. Finally they were able to protect recipient mice against streptozotocin-induced diabetes. This work shows, that the conversion of a somatic cell into another, even if belonging to a different germ layer, is possible also in pig.

Cartilage repair strategies increasingly focus on the in vitro development of cartilaginous tissues that mimic the biological and mechanical properties of native articular cartilage. However, current approaches still face problems in the reproducible and standardized generation of cartilaginous tissues that are both biomechanically adequate for joint integration and biochemically rich in extracellular matrix constituents. In this regard, the present study investigated whether long-term continuous compressive loading would enhance the mechanical and biological properties of such tissues. Human chondrocytes were harvested from 8 knee joints (n = 8) of patients having undergone total knee replacement and seeded into a collagen type I hydrogel at low density of 2 × 105 cells/ml gel. Cell-seeded hydrogels were cut to disks and subjected to mechanical stimulation for 28 days with 10% continuous cyclic compressive loading at a frequency of 0.3 Hz. Histological and histomorphometric evaluation revealed long-term mechanical stimulation to significantly increase collagen type II and proteoglycan staining homogenously throughout the samples as compared to unstimulated controls. Gene expression analyses revealed a significant increase in collagen type II, collagen type I and MMP-13 gene expression under stimulation conditions, while aggrecan gene expression was decreased and no significant changes were observed in the collagen type II/collagen type I mRNA ratio. Mechanical propertywise, the average value of elastic stiffness increased in the stimulated samples.In conclusion, long-term mechanical preconditioning of human chondrocytes seeded in collagen type I hydrogels considerably improves biological and biomechanical properties of the constructs, corroborating the clinical potential of mechanical stimulation in matrix-associated autologous chondrocyte transplantation (MACT) procedures.

Recent studies have shown that the use of specific inhibitors for signalling pathways known to drive murine embryonic stem cell (ESC) differentiation may represent a tool to derive and maintain pluripotent cell lines. The application of this novel approach could provide a new strategy to overcome the limitations still existing for the derivation of ESC in large animal species. These molecules, also known as 3i factors, include CHIR99021 (GSK3 inhibitor), PD173074 (FGF inhibitor) and PD0325901 (MAPK/ERK kinase or MEK inhibitor). However only scattered information are available on the involvement of these pathways in the maintenance of pluripotency in domestic animals. The aim of this study was to investigate the presence of receptors for these inhibitors in porcine inner cell mass (ICM) and to isolate and culture porcine pluripotent lines in a serum-free medium supplemented with the 3i factors, without any additional growth factor. Ovaries were collected at the local abattoir and cumulus–oocyte complexes (COC) were aspirated from antral follicles. In vitro maturation was then performed for 46 h. Frozen–thawed spermatozoa were purified and live spermatozoa were co-cultured with denuded oocytes for 24 h. Putative embryos were cultured in NCSU-23 medium until the blastocyst stage and then subjected to immuno-surgery. Isolated ICM were analysed by RT-PCR. Poly(A)+RNA was extracted using Dynabeads® mRNA DIRECT Micro-kit (Invitrogen, Carlsbad, CA, USA) and immediately reverse-transcribed with Superscript™ II Reverse Transcriptase (Invitrogen). Specific primers were designed for FGF4, FGFR-1, FGFR-2, FGFR-4, GSK3, and MEK genes. The PCR amplified products were sequenced and aligned using ClustalW. The RT-PCR results showed that porcine ICM actively transcribe for GSK3, MEK, FGFR-2, FGFR-4, and FGF4 genes, whereas no signal was detectable for FGFR-1. Based on these observations, IVF-derived ICM were plated onto inactivated STO feeder cells and cultured in N2B27 medium supplemented with 3 µM CHIR99021, 100 nM PD173074, and 0.4 µM PD0325901. Outgrowth formation was monitored and cells were passaged to a new STO monolayer every 7 days, as previously described (Brevini et al. 2010 Stem Cell Rev.). Assessment of pluripotency markers was carried out both by RT-PCR and immunocytochemical analysis at every passage for up to 15 passages. The results obtained indicate that porcine cells cultured in 3i medium, without additional growth factors, expressed pluripotency markers; namely OCT4, NANOG, SOX2, and REX1, preserving their pluripotent state over time. Our data indicate that porcine ICM express 3i factor target molecules. In agreement with this, the use of 3i medium allows the establishment and proliferation of pluripotent cell lines. Together, these findings suggest the involvement of the GSK3, FGF, and MEK pathways in porcine pluripotency network and advocate the use of the 3i medium as an efficient tool for ESC derivation in porcine. Supported by NetLiPS Project ID: 30190629.

STUDY QUESTION Does directional freezing improve the structural and functional integrity of ovarian fragments compared with conventional slow freezing and to whole ovary cryopreservation? SUMMARY ANSWER Compared with slow freezing, the use of directional freezing significantly improves all structural and functional parameters of ovarian fragments assessed in vitro and, overall, whole ovaries were better preserved than ovarian fragments. WHAT IS KNOWN ALREADY Directional freezing has been developed to provide an alternative way to cryopreserve large biological samples and it is known to improve the structural and functional integrity of whole ovaries. Conventional slow freezing of ovarian fragments is the procedure more widely used in clinical settings but it causes substantial structural damage that limits the functional period after transfer back into the patient. STUDY DESIGN, SIZE, DURATION We performed a 2 × 2 factorial design experiment on a total of 40 sheep ovaries, divided into four groups (n = 10 ovaries per group): (i) directional freezing of whole ovary (DFwo); (ii) directional freezing of ovarian fragments (DFof); (iii) conventional freezing of whole ovary (CFwo); (iv) conventional freezing of ovarian fragments (CFof). An additional eight ovaries were used as fresh controls. PARTICIPANTS/MATERIALS, SETTING, METHODS Ewe ovaries were randomly assigned to one of the experimental groups and frozen accordingly. Upon thawing, ovarian tissue was examined morphologically and cultured in vitro for 7 days. Samples were analyzed for cell proliferation and apoptosis, for DNA damage and repair activity, and for the presence of a panel of heat shock proteins (HSPs) by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE Most studied parameters were significantly improved (P < 0.05) in all samples cryopreserved with directional compared with slow freezing. The proportion of primordial follicles, which developed to the primary stage in whole ovaries (53 ± 1.7%) and in ovarian fragments (44 ± 1.8%) cryopreserved with directional freezing, was greater than with slow frozen whole ovaries (6 ± 0.5%, P = 0.001) or fragments (32 ± 1.5%, P = 0.004). After 7 days of culture, cell proliferation in DFwo (28 ± 0.73%) was the highest of all groups (P < 0.05) followed by DFof (23 ± 0.81%), CFof (20 ± 0.79%) and CFwo (9 ± 0.85%). Directional freezing also resulted in a better preservation of the cell capacity to repair DNA damage compared with slow freezing both in whole ovaries and ovarian fragments. Apoptosis and HSP protein levels were significantly increased only in the CFwo group. Direct comparison demonstrated that, overall, DFwo had better parameters than DFof and was no different from the fresh controls. LIMITATIONS, REASONS FOR CAUTION The study is limited to an in vitro evaluation and uses sheep ovaries, which are smaller than human ovaries and therefore may withstand the procedures better. WIDER IMPLICATIONS OF THE FINDINGS Improved integrity of ovarian morphology may translate to improved outcomes after transplantation. Alternatively, the particularly good preservation of whole ovaries suggests they could provide a source of ovarian follicles for in vitro culture in those cases when the presence of malignant cells poses a substantial risk for the patient. STUDY FUNDING/COMPETING INTEREST(S) Supported by: Associazione Italiana per la Ricerca sul Cancro (AIRC) IG 10376, Carraresi Foundation and by Legge 7 Regione Autonoma Sardegna (R.A.S). There are no conflicts of interest.

Ovarian tissue cryobanking is proposed as an effective option for preserving female fertility in cancer patients. At present 2 options are available: cryopreservation of ovarian cortical fragments or of the whole ovary. The use of whole ovary reduces ischemic insult. However, the larger the sample volume, the more difficult it is to introduce the cryoprotective agents and to ensure an adequate cooling rate that minimizes tissue damage. For this reason, we used the multi-thermal gradient method, based on running the sample through a temperature gradient. This allows a homogeneous cooling rate through the whole sample independently from its volume. The aim of the study was to determine whether multi-thermal gradient freezing allows a substantial reduction of the damages induced by cryopreservation of large samples by comparing the viability of cortical fragments versus whole ovaries after thawing and grafting in nude mice. Sheep ovaries were collected at the local abattoir and randomly divided into 3 groups: A) ovaries frozen as cortical fragments, B) ovaries frozen as whole organs, and C) fresh ovaries immediately processed for further analysis (control). Ovarian fragments (10 × 5 × 1 mm) were sliced from the cortical region and immersed into cryoprotectant solution (Leibovitz L-15 medium, 10% FCS, and 1.5 M dimethyl sulfoxide), while whole ovaries were perfused with the same solution. Samples were placed into glass freezing tubes 16 mm in diameter filled with cryoprotectant solution. Samples were frozen with the multi-thermal gradient freezing apparatus (Core Dynamics, Ness Ziona, Israel) progressing along the thermal gradient at a rate of 0.01 mm s–1, resulting in a cooling rate of 0.3°C min–1. Two weeks later, samples were thawed by plunging the tubes into a 37°C water bath with gentle shaking. Whole ovaries were perfused with 10 mL of HEPES-Talp medium, 0.5 M sucrose, and 10 IU mL–1 of heparin and their cortical region was cut into fragments. These fragments and those derived from group A were rehydrated in L-15 medium with decreasing sucrose concentrations. Fragments (2 × 2 × 1 mm) were xenografted in the dorsal region of 6 nude mice for each group. Mice were killed after 8 weeks and grafts were collected for analysis. Cryopreserved samples were compared with each other and fresh controls (group C). Morphologically normal follicles at primordial, primary, and secondary stages were visible in all samples. Cell proliferation was assessed measuring Ki-67 mRNA and counting immunohistochemically positive cells. The FSH receptor and GDF9 gene expression were used to evaluate tissue viability. No significant differences for any of these parameters were measured amongst the groups. We conclude that directional freezing is an effective method for ovarian tissue cryopreservation independently from the sample volume, thus overriding the limitations usually associated with whole-organ banking. Supported by AIRC IG 10376 and by Carraresi Foundation.

Pluripotent stem cells are the focus of an extremely active field of investigation that is bringing new light on our understanding of the mechanisms that control pluripotency and differentiation. Rodent and primates are the only species where true, or bona fide, pluripotent stem cells have been derived. The attempts to derive pluripotent stem cells from domestic ungulates have been going on for more than 20 years with little progress. Cell lines from these species present a series of limitations that have precluded their use for both basic and clinically oriented studies. However, in the last 3 years, some substantial progress have been made making the currently available ungulate pluripotent stem cells closest than ever before to their human and mouse counterpart. This result has been achieved through both conceptual and technical progress that will be illustrated and discussed in this review.

Whole-ovary cryopreservation followed by retransplantation with vascular anastomosis is a promising method to restore cancer-patient fertility. However, protocols need to be improved to minimize the damages related to an inadequate cooling rate caused by the large volume of whole organs. The aim of this study was to compare conventional slow-freezing (SF) versus directional-freezing (DF) techniques. Of 30 sheep ovaries collected at the local abattoir, 6 were used as fresh controls and 24 were perfused for 5 min with cryoprotectant solution made of Leibovitz L-15 medium, 10% FCS, and 1.5-M dimethyl sulfoxide. Samples were inserted in glass freezing tubes filled with the same solution. Twelve ovaries were frozen with a Kryo 560M apparatus (Planer, UK) using a cooling rate of 0.5°C min–1 from 4 to –40°C and 5°C min–1 from –40 to –100°C followed by direct plunging in liquid nitrogen. The other 12 ovaries were frozen using an MTG 1315 apparatus (Core Dynamics, Ness Ziona, Israel). Tubes were pushed along the thermal gradient (4 to –70°C) at 0.01 mm s–1 resulting in a cooling rate of 0.3°C min–1 and then plunged in liquid nitrogen. After 2 weeks, samples were thawed at 37°C for 2 min with gentle shaking. Morphological analysis showed that the rate of intact follicles was 94% in control samples, 87% in DF samples, and 58.3% in SF samples (P < 0.05). No significant differences were found in stromal cell density between fresh and DF ovaries (17 833 and 17 041 nuclei mm–2, respectively), whereas SF ovaries showed a lower density (10 875 nuclei mm–2; P < 0.001). To assess vessel integrity, thawed ovaries were perfused with 25% Indian ink/167 UI of heparin mL–1 saline solution and embedded in paraffin. Fresh and DF ovaries displayed a similar rate of perfused vessels (82.7 and 80.2%, respectively), whereas SF ovaries significantly lower number of perfused vessels (52.3%), indicating the occurrence of vascular injury. Follicle viability after freezing was evaluated by culturing individual cortical fragments (2 × 2 × 1 mm) for 7 days. A high rate of morphologically normal follicles was found in both fresh (85%) and DF ovaries (78%). The number of intermediate and primary follicles was significantly increased in both groups when compared to Day 0 (P < 0.001). By contrast, cortical slices obtained from SF ovaries showed a higher rate of degenerated follicles, oocytes with pyknotic nuclei, and a significant decrease of follicle number. Furthermore, qPCR and immunohistochemical assay for Ki-67 showed a similar cell proliferation in fresh and DF ovary but a significantly lower one in SF samples. We conclude that directional freezing allows a better preservation of both cortical and stromal tissue, as well as the vasculature, thus overcoming 2 of the main restrictions associated with whole-organ freezing. Supported by AIRC IG 10376 and by Carraresi Foundation.