Ultrasound stimulus to enhance the bone regeneration capability of gelatin cryogels

In the present study, gelatin-based cryogels have been seeded with human SAOS-2 osteoblasts. In order to overcome the drawbacks associated with in vitro culture systems, such as limited diffusion and inhomogeneous cell-matrix distribution, this work describes the application of ultrasounds (average power, 149 mW; frequency, 1.5 MHz) to physically enhance the cell culture in vitro. The results indicate that the physical stimulation of cell-seeded gelatin-based cryogels upregulates the bone matrix production

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

New experimental model for basic research in stem cell field

The great interest in these findings is due to the promising possibility to use MSC in tissue engineering and gene therapy thanksto their plasticity and availability. Recently a subpopulation of luteinized granulosa cells (GC) derived from infertile patients during their IVF procedures showed mesenchymal stem characteristics and multipotency.I n our recent study a heterogeneous cell population were isolated from human ovarian follicular fluid (FF cells) and cultivated in minimal medium conditions without any growth factors, including leukemia-inhibiting factor. FF cells showed a different morphology, such as fibroblastic, epithelial and also neuronal shape. In particular, the cells with characteristics similar to fibroblasts expressed many specific antigens of mesenchymal stem cells (i.e. CD90, CD44, CD105, CD73) and were negative for haematopoietic and epithelial markers (CD34, CD45, cytokeratins). We confirmed the multipotency of a subset of granulosa cells by in vitro differentiation studies (e.g. osteogenic, chondrogenic and adipogenic differentiation).Therefore we propose follicular fluid cells as a cheap biological experimental model for basic research in stem cells fields thanks to their clonogenic capacity, multipotency and availability without any growth factor addiction too.FF cells could be a good model not only to study biocompatibility of engineering scaffold, but also to investigate the effect induced by mechanical conditioning

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 granu-losa and germ cells, differentiate sequentially and de novo from mesenchymal progeni-tor 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 fertili-zation 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 posi-tive 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 ob-servations 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 ex-pression in the follicular fluid derived cells at different times of minimal culture condi-tions 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 fol-low the mesenchymal stem cell population, while FSH-R was used to identify granu-losa derived cells; CNTF and beta-3-tubuline were used to discriminate between neu-ral and neuronal cells populations; epithelial and hematopoietic cells were followed us-ing 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 ob-tained immunocytochemical and morphological data to confirm the presence of these different cytotypes in the samples purified from the follicular liquid and their persis-tence, loss or amplification at different times of in vitro minimal culture conditions

Biological effects of ultrasound stimulus on cells derived from human ovarian follicular liquid

Low-Intensity Pulsed Ultrasound Stimulus (LIPUS) accelerates the bone fracture healing in animal models and in clinical studies. In this work, according to the literature, we have chosen the mesenchymal stem cells (MSCs) as precursors of bony tissue, in particular the MSCs derived from the human ovarian follicular liquid (FL), and we have investigated the effects of ultrasounds on their proliferation. We tested two different durations of ultrasound stimulus (2 and 5 min) and compared these data to the control without ultrasound treatment. To quantify the proliferation of these putative MSCs, we used the BrdU incorporation assay: in comparison with the control, the results showed that 5 min of ultrasound stimulus significantly increased the percentage number of cells in intensive proliferative activity; on the other hand, there was no significant difference using 2 min of stimulation, hypothetically because the transmitted energy was not sufficient to stimulate the cells and to consequently enhance their proliferation. In conclusion, the effects of LIPUS on putative MSCs derived from ovarian follicular liquid show potential developments in biotech or medical applications

Aquaporin-Mediated Water and Hydrogen Peroxide Transport Is Involved in Normal Human Spermatozoa Functioning.

Different aquaporins (AQPs) are expressed in human sperm cells and with a different localization. Their function has been related to cell volume control in response to the osmotic changes encountered passing from the epididymal fluid to the cervical mucus or involved in the end stage of cytoplasm removal during sperm maturation. Recently, AQPs have also shown hydrogen peroxide (H 2 O 2 ) permeability properties. Here, we investigate the expression, localization and functioning of AQPs in human sperm cells with particular attention to their role as peroxiporins in reactive oxygen species (ROS) scavenging in both normospermic and sub-fertile human subjects. Western blotting and immunocytochemistry were used to confirm and clarify the AQPs expression and localization. Water and H 2 O 2 permeability was tested by stopped flow light scattering method and by the CM-H2DCFDA (5-(and-6)-chloromethyl-2 0 ,7 0 -dichlorodihydro-fluorescein diacetate, acetyl ester) H 2 O 2 fluorescence probe, respectively. AQP3, -7, -8, and -11 proteins were found in human sperm cells and localized in the head (AQP7), in the middle piece (AQP8) and in the tail (AQP3 and -11) in both the plasma membrane and in intracellular structures. Sperm cells showed water and H 2 O 2 permeability which was reversibly inhibited by H 2 O 2 , heat stress and the AQP inhibitor HgCl 2 . Reduced functionality was observed in patients with compromised basal semen parameters. Present findings suggest that AQPs are involved in both volume regulation and ROS elimination. The relationship between sperm number and motility and AQP functioning was also demonstrate

Myeloperoxidase and lactoferrin expression in semen fluid: Novel markers of male infertility risk?

Research Question: Infections and/or inflammation processes of male genital tract are highly prevalent and often associated with risk of infertility. These conditions represent a possible cause of leukocytospermia, which is still under debate. Leukocytes are key-factors to reactive oxygen species (ROS) production and the increase of ROS in semen fluid is associated with the worsening of semen parameters. At present, there are not appropriate andrological tests to identify asymptomatic inflammatory conditions when the amount of leukocytes is in the normal range. Design: We studied the innate immunity profile of myeloperoxidase and lactoferrin (MPO/LAC) proteins expressed in the semen fluid of 39 men evaluated for couple infertility, in the absence of leukocytospermia. Results: The presence of both MPO and LAC proteins was associated with a decrease of sperm concentration and of progressive/total motility, whereas the increase of MPO-/LAC + indicated a worse sperm morphology. It is worth to report the predictive potential of MPO+/LAC + pattern (above 4.36 %) as a biological marker to distinguish normozoospermic from pathological patients. Conclusion: Our findings indicate MPO/LAC analysis as a potential diagnostic tool to identify asymptomatic conditions eventually related to male infertility, even when the number of leukocytes in semen fluid is below 1 million/mL

Cellule staminali mesenchimali da liquido follicolare ovarico umano: caratterizzazione morfologica e funzionale

La derivazione degli ovociti umani e dei follicoli primari nella donna rappresenta tuttora un argomento dibattuto. E’ provato che le componenti per i nuovi follicoli primari umani differenzino sequenzialmente e de novo da progenitori mesenchimali che risiedono nella tonaca albuginea dell’ovaio. Lo scopo del presente lavoro consiste nella caratterizzazione morfologica e funzionale di popolazioni cellulari isolate da liquido follicolare umano (altrimenti inutilizzato dopo l’isolamento degli ovociti) raccolto durante le di pick-up per la PMA. Le cellule ottenute dal liquido follicolare di pazienti in terapia con rFSH e agonisti o antagonisti del GNrh per cicli FIVET-ICSI (n=165) sono state mantenute in condizioni minime di coltura in medium privo di fattori di crescita specifici (come il LIF). In particolare si è proceduto all’analisi di una sottopopolazione di cellule, presunte staminali mesenchimali (MSC), coltivate in vitro per oltre 3 settimane, parallelamente a cellule MSC di midollo osseo umano impiegate come controllo positivo. L’analisi morfologica ha rivelato una popolazione cellulare eterogenea con fenotipi epiteliali, fibroblastici e neurali. In particolare, le cellule con aspetto fibroblastico appaiono simili alle MSC. I risultati immunocitochimici e citofluorimetrici mostrano che la maggior parte di tali cellule sono positive per marcatori tipicamente mesenchimali come vimentina, CD90, CD73, CD44 e CD105, e negative per citocheratine, CD34 e CD45. L’attività proliferativa e la capacità di formare colonie sono state valutate a differenti tempi di coltura in condizioni minime. Il saggio di incorporazione della BrdU mostra circa il 60% di cellule proliferanti nei primi 10 giorni di coltura e una diminuzione del 20% nella settimana successiva. La formazione di colonie è osservata a 13 giorni di coltura. La multipotenza delle cellule mesenchimali è stata saggiata in vitro mediante l’induzione del differenziamento in senso osteogenico. Per confermarne la caratteristica plasticità cellulare, sono inoltre stati condotti esperimenti di coltura su scaffold di criogel gelatinoso, materiale molto promettente per la sua biocompatibilità, in particolare nel campo della chirurgia ricostruttiva dell’osso. Analisi preliminari al microscopio confocale ed elettronico a scansione mostrano una capacità di crescita cellulare sia sulla superficie sia negli interstizi dello scaffold, fino alla profondità di 60μm. L’interesse di questo studio consiste nell’uso di un liquido biologico di scarto come possibile nuova fonte di MSC da impiegare per la maturazione in vitro degli ovociti o nell’ambito dell’ingegneria tissutale