Heterogeneous cell population derived from human ovarian follicular liquid: morphological studies and molecular screening

The origin of oocytes and primary follicles in ovaries of adult mammalian females is still a matter of dispute [1]. The components of new primary follicles, primitive granulosa and germ cells, differentiate sequentially and de novo from mesenchymal progenitor cells residing in the ovarian tunica albuginea (TA). It appears that mesenchymal progenitor cells contribute to the generation of epithelial cells similar to granulosa cells (GCs). The multipotency of a subset of granulosa cells was also established by in vitro differentiation into other cell types [2]. Up to now, luteinizing GCs were considered to be terminally differentiated, unavoidably becoming apoptotic a few days after ovulation. Previously, we have provided evidence for the existence of putative stem cells derived from human ovarian follicular liquid collected after routine procedures for in vitro fertilization techniques [3]. These cells grow in minimal medium condition, without any growth factor (i.e. LIF), that is considered essential according to other procedures [4]. Using immunocytochemistry and flow cytometry we showed that these cells are positive for several mesenchymal stemness markers, including CD90, CD73, CD44, CD105. However, morphological analysis revealed a heterogeneous cell population, with cells displaying a fibroblast-like, epithelial- like and neural-like shapes. These observations are also supported by the identification of cells expressing specific neural markers, such as neurofilaments and PGP9.5, in addition to vimentin and cytocheratin positive cells. All these data are suggestive of the presence of different cell populations in follicular fluids. To verify this hypothesis we select a panel of markers specific for the different cell populations previously identified and we plan a molecular screening to follow their expression in the follicular fluid derived cells at different times of minimal culture conditions in vitro. Bone marrow derived MSCs were used as a control. For each sample we performed semiquantitative RT-PCR experiments normalizing the cDNAs used as templates on the basis of the number of pseudo-mesenchymal cells morphologically identified in the sample. For this purpose OCT-4 was selected as a stem marker to follow the mesenchymal stem cell population, while FSH-R was used to identify granulosa derived cells; CNTF and beta-3-tubuline were used to discriminate between neural and neuronal cells populations; epithelial and hematopoietic cells were followed using cytokeratin (CK8 and CK10) and CD45 markers, respectively. GAPDH and β-actin specific primers were used on all samples for normalization. Here we compare the results of this molecular screening with the previously obtained immunocytochemical and morphological data to confirm the presence of these different cytotypes in the samples purified from the follicular liquid and their persistence, loss or amplification at different times of in vitro minimal culture conditions

3D culture of multipotent cells derived from waste human ovarian follicular liquid and seeded onto gelatin cryogel

Current tissue engineering uses 3D biomaterials in combination with stem cells, since mature cells are often not available in sufficient amounts or quality. Biomaterial scaffolds have been widely used in reconstructive bone surgery not only as cell carriers providing mechanical support, but also as promoters of cell attachment and proliferation (1). In particular, gelatine cryogel scaffolds are promising new biomaterials owing to their biocompatibility and to substain the differentiation of mesenchymal stromal stem cells (MSCs) (2). Human MSC proliferate onto the surfaces with fibroblastic morphology and can differentiate into osteoblasts, chondrocytes and adipocytes (3). These cells can be isolated from several sources, including bone marrow and adipose tissue (4). Our previously studies showed the possibility to obtain MSCs also from the human ovarian follicular liquid (FL) that is usually wasted during in vitro fertilization (5). In this study, we tested the ability of these FL cells to grow and differentiate on gelatine cryogel in comparison with MSCs derived from human bone marrow. Samples and controls were analyzed with confocal and scanning electron microscopes. Results demonstrated that FL cells could grow on the biomaterial not only on the top but also in the layers below till 60mm of deepness. Data suggested that the observed cells are mesenchymal since positive for vimentin and CD44 (a typical MSC marker). Preliminary results showed also the capability of induced FL cells to osteogenic differentiation to produce bone extracellular matrix, expressing some specific proteins (i.e.osteopontin). In conclusion, MSCs derived from waste human ovarian follicular liquid showed promising affinity with 3D gelatine cryogel, opening new potential developments in biotech and medical applications

Ultrastructural features of human sperm cells cryopreserved by different methods

Cryopreservation of human spermatozoa has been recognized as a key strategy for management of male fertility. Nevertheless, current protocols of sperm freezing are neither optimal nor standardized between different labs (1). In this study we compare the ultrastructural features of human normospermic sperm samples (according to WHO parameters 2010) from 5 different freezing techniques in order to identify the best methods of cryopreservation. After informed con- sent, 21 normospermic patients (from the Medically Assisted Procreation PMA Center of the Fondazione IRCCS Policlinico San Matteo in Pavia) were recruited and both traditional and improved analysis of sperm quality were applied, in order to define critical steps of cryopreservation. Cryopreservation of human spermatozoa has been related to decreased motility associated with impaired velocity and viability of sperm pre-freeze and post-thaw. For all applied methods there was a significant reduction of progressive and total motility (P) as a result of freezing. To investigate ultrastructural details, 5 additional cryopreserved samples by the best two freezing methods were analyzed with elec- tron microscopy (TEM). Preliminary data showed the minimal differences between the protocols, with a large number of queues detached and large quantities of cyto- plasmic debris after of the first protocol. Spermatozoa appear to be better preserved in the second analyzed method, despite both procedures induced deteriorations at ultrastructural level (2). Other non-routine analysis will be performed to determine whether the cooling time to +4°C may affect the procedure; Comet Assay (to assess the degree of sperm DNA fragmentation) (3) and flow cytometry (to study light scat- ters patterns and membrane integrity) (4) will be applied

Magic-factor 1, a partial agonist of Met, induces muscle hypertrophy by protecting myogenic progenitors from apoptosis.

Hepatocyte Growth Factor (HGF) is a pleiotropic cytokine of mesenchymal origin that mediates a characteristic array of biological activities including cell proliferation, survival, motility and morphogenesis. Its high affinity receptor, the tyrosine kinase Met, is expressed by a wide range of tissues and can be activated by either paracrine or autocrine stimulation. Adult myogenic precursor cells, the so called satellite cells, express both HGF and Met. Following muscle injury, autocrine HGF-Met stimulation plays a key role in promoting activation and early division of satellite cells, but is shut off in a second phase to allow myogenic differentiation. In culture, HGF stimulation promotes proliferation of muscle precursors thereby inhibiting their differentiation

HPV Infection Affects Human Sperm Functionality by Inhibition of Aquaporin-8

Human sperm cells express different aquaporins (AQPs), AQP3, 7, 8, 11, which are localized both in the plasma membrane and in intracellular structures. Besides cell volume regulation and end stage of cytoplasm removal during sperm maturation, the role of AQPs extends also to reactive oxygen species (ROS) elimination. Moreover, oxidative stress has been shown to inhibit AQP-mediated H2O2 permeability. A decrease in AQPs functionality is related to a decrease in sperm cells number and motility. Here we investigate the possible effect of human Papillomavirus (HPV) on both expression and function of AQPs in human sperm cells of patients undergoing infertility couple evaluation. Stopped-flow light-scattering experiments demonstrated that HPV infection heavily reduced water permeability of sperm cells in normospermic samples. Confocal immunofluorescence experiments showed a colocalization of HPV L1 protein with AQP8 (Pearson's correlation coefficient of 0.61), confirmed by co-immunoprecipitation experiments. No interaction of HPV with AQP3 and AQP7 was observed. A 3D model simulation of L1 protein and AQP8 interaction was also performed. Present findings may suggest that HPV infection directly inhibits AQP8 functionality and probably makes sperm cells more sensitive to oxidative stress

Multipotent mesenchymal stromal stem cells derived from waste human ovarian follicular fluid: bioengineering applications in vitro

Modern tissue engineering strategies combine living cells and scaffold materials to develop biological substitutes that can restore tissue functions. Biomaterials and bioreactor systems provide both the technological means to reveal fundamental mechanisms of cell function in a 3D environment, and the potential to improve the quality of engineered tissues. Mesenchymal stem cells (MSC) have a large capacity for self-renewal while maintaining their multipotency and represent a promising tool for regenerative medicine. Our studies showed the possibility to derive MSC from the human ovarian follicular fluid (FF cells) that picked-up during human assisted reproduction techniques and usually wasted during IVF procedures. FF cells were collected from healthy women undergone an IVF treatment during oocytes retrieval and cultured with DMEM + 10% FBS in in vitro minimal medium conditions, without any growth factor, including leukemia-inhibiting factor (LIF), previously considered essential. FF cells showed typical mesenchymal stemness markers (including CD-90, CD-44, CD-105, CD-73) and multipotent differentiation capacity in osteogenic, chondrogenic and adipogenic lineages. Now we also tested capability of FF cells to grow on gelatin cryogel scaffolds, promising new biomaterials owing to their biocompatibility, compared to MSC derived from human bone marrow. Data collected demonstrated that FF cells could grow on biomaterial not only on the top but also in the layers below till 60 μm of deepness. Results suggested that cells observed were mesenchymal ones due to their positive immunostained for vimentin and CD-44, typical markers for MSC. Finally we evaluated if ultrasound treatment on FF cells cultures have positive effects on proliferation activity. To study effects of mechanical conditioning, we tested two different times of ultrasound stimulus (LIPUS), 2 or 5 minutes, corresponding respectively to 17.88 and 44.70 joule per day on cell culture and compared data to control without any kind of mechanical stimulation. The percentage number of cells showing intensive proliferative activity was 11.26 ± 5.19% in non-stimulated control cultures, 15.60 ± 1.70% in LIPUS stimulated cultures for 2 min, and 46.43 ± 15.04% in LIPUS stimulated cultures for 5 min. Comparisons with Bonferroni test showed statistically significant differences between control and LIPUS stimulation for 5 min (p=0.003) and between the two LIPUS stimulations (p=0.021); on the other hand, the proliferation did not increase with the shorter ultrasound treatment. Our results suggest that cells provided by mesenchymal plasticity can be easily isolated by waste follicular fluid, avoiding scraping of human ovaries. Moreover, successfully growth of putative MSC derived from follicular fluid on three-dimensional cryogel scaffold and positive effect on proliferation activity induced by ultrasound stimulus, open potential developments in biotechnological or medical applications. Therefore, FF cells may be introduced as a valuable model system with which to study the mesenchymal lineages for basic research and tissue engineering

In-vitro culture system for mesenchymal progenitor cells derived from waste human ovarian follicular fluid.

To characterize different cell populations in the human ovary, morphological and functional characteristics of cell populations collected during routine IVF procedures were studied. Cells obtained from follicular fluid grew in vitro under minimal medium conditions, without growth factor, including leukaemia-inhibiting factor. Morphological analysis revealed a heterogeneous cell population, with cells displaying a fibroblast-like, epithelial-like and also neuron-like features. Morpho-functional characteristics of fibroblast-like cells were similar to mesenchymal stem cells, and, in particular, were positive for mesenchymal stemness markers, including CD90, CD44, CD105, CD73, but negative for epithelial proteins, such as cytokeratins, CD34 and CD45 antigens. Cell proliferation activity at different times and colony-forming unit capability were evaluated, and multipotency of a subset of granulosa cells was established by in-vitro differentiation studies (e.g. osteogenic, chondrogenic and adipogenic differentiation). This study suggests that cells provided by mesenchymal plasticity can be easily isolated by waste follicular fluid, avoiding scraping of human ovaries, and cultivated in minimal conditions. Successful growth of such progenitor cells on three-dimensional cryogel scaffold provides the basis for future developments in tissue engineering. This culture system may be regarded as an experimental model in which biological behaviour is not influenced by specific growth factors

Immunomagnetic separation and biological characterization of a putative mesenchymal stem cells subpopulation

Our previous data demonstrated the presence of MSCs isolated from ovarian follicular liquid. A novel immunomagnetic procedure to isolate rere cells in suspension was used, using Dynal microbeads and a dedicated multiwells magnetic device. It was possible to isolate CD44+ cells and recovere them onto coverslips for the next steps of immunostaining and/or biological characterization. Further experiments have been designed in order to verify the stemness of these cells, seeding them in culture and inducing differentiation into other cell lineages to assess in vitro their plasticity

3D culture of multipotent cells derived from waste human ovarian follicular liquid and seeded onto gelatin cryogel

Current tissue engineering uses 3D biomaterials in combination with stem cells, since mature cells are often not available in sufficient amounts or quality. Biomaterial scaffolds have been widely used in reconstructive bone surgery not only as cell carriers providing mechanical support, but also as promoters of cell attachment and proliferation (1). In particular, gelatine cryogel scaffolds are promising new biomaterials owing to their biocompatibility and to substain the differentiation of mesenchymal stromal stem cells (MSCs) (2). Human MSC proliferate onto the surfaces with fibroblastic morphology and can differentiate into osteoblasts, chondrocytes and adipocytes (3). These cells can be isolated from several sources, including bone marrow and adipose tissue (4). Our previously studies showed the possibility to obtain MSCs also from the human ovarian follicular liquid (FL) that is usually wasted during in vitro fertilization (5). In this study, we tested the ability of these FL cells to grow and differentiate on gelatine cryogel in comparison with MSCs derived from human bone marrow. Samples and controls were analyzed with confocal and scanning electron microscopes. Results demonstrated that FL cells could grow on the biomaterial not only on the top but also in the layers below till 60m of deepness. Data suggested that the observed cells are mesenchymal since positive for vimentin and CD44 (a typical MSC marker). Preliminary results showed also the capability of induced FL cells to osteogenic differentiation to produce bone extracellular matrix, expressing some specific proteins (i.e.osteopontin). In conclusion, MSCs derived from waste human ovarian follicular liquid showed promising affinity with 3D gelatine cryogel, opening new potential developments in biotech and medical applications