In Vitro and In Vivo Differentiation of Progenitor Stem Cells Obtained After Mechanical Digestion of Human Dental Pulp.

Human population is facing a revolutionary change in the demographic structure with an increasing number of elderly people requiring an unmet need to ensure a smooth aging process and dental care is certainly an important aspect that has to be considered. To date, dentistry has been conservative and the need of transferring the scientific models of regenerative dentistry into clinical practice is becoming a necessity. The aim of this study was to characterize the differentiation commitment (in vitro) and the clinical grafting ability (in vivo) of a population of progenitor stem cells obtained after mechanical digestion of dental pulp with an innovative system recently developed. This approach was successfully used in previous studies to obtain a clinical-grade ready to use dental pulp fragments that could be grafted in autologous tissues to obtain bone. We are thus showing that micro grafts resulting from mechanical digestion contain stem cells with a mesenchymal phenotype, able to differentiate toward different cell types and to generate new bone in patients. We are providing data for the establishment of standardized and routinely oral surgery approaches, having outlined the cellular properties of human stem cells obtained from the dental pulp. This method can represent a valid tool for both regenerative medicine and tissue engineering purposes not only applicable to the cranio-maxillofacial region but, likely, to different bone pathologies for a fastening and healing recovering of patients. J. Cell. Physiol. 232: 548-555, 2017. © 2016 Wiley Periodicals, Inc.

Achilles' heel of pluripotent stem cells: genetic, genomic and epigenetic variations during prolonged culture.

Pluripotent stem cells differentiate into almost any specialized adult cell type of an organism. PSCs can be derived either from the inner cell mass of a blastocyst-giving rise to embryonic stem cells-or after reprogramming of somatic terminally differentiated cells to obtain ES-like cells, named induced pluripotent stem cells. The potential use of these cells in the clinic, for investigating in vitro early embryonic development or for screening the effects of new drugs or xenobiotics, depends on capability to maintain their genome integrity during prolonged culture and differentiation. Both human and mouse PSCs are prone to genomic and (epi)genetic instability during in vitro culture, a feature that seriously limits their real potential use. Culture-induced variations of specific chromosomes or genes, are almost all unpredictable and, as a whole, differ among independent cell lines. They may arise at different culture passages, suggesting the absence of a safe passage number maintaining genome integrity and rendering the control of genomic stability mandatory since the very early culture passages. The present review highlights the urgency for further studies on the mechanisms involved in determining (epi)genetic and chromosome instability, exploiting the knowledge acquired earlier on other cell types.

A New Medical Device Rigeneracons Allows to Obtain Viable Micro-Grafts From Mechanical Disaggregation of Human Tissues.

Autologous graft is considered the gold standard of graft materials; however, this approach is still limited due to both small amount of tissue that can be collected and to reduced cell viability of cells that can be obtained. The aim of this preliminary study was to demonstrate the efficacy of an innovative medical device called Rigeneracons® (CE certified Class I) to provide autologous micro-grafts immediately available to be used in the clinical practice. Moreover, Rigeneracons® is an instrument able to create micro-grafts enriched of progenitors cells which maintain their regenerative and differentiation potential. We reported preliminary data about viability cell of samples derived from different kind of human tissues, such as periosteum, cardiac atrial appendage biopsy, and lateral rectus muscle of eyeball and disaggregated by Rigeneracons®. In all cases we observed that micro-grafts obtained by Rigeneracons® displayed high cell viability. Furthermore, by cell characterization of periosteum samples, we also evidenced an high positivity to mesenchymal cell markers, suggesting an optimal regenerative potential.

FSH and LH receptors are differentially expressed in cumulus cells surrounding developmentally competent and incompetent mouse fully grown antral oocytes.

Cumulus cells (CCs) maintain strict functional relationships with the enclosed antral oocyte and are thought to reflect its developmental competence. Several studies have described a correlation between CC gene expression and oocyte quality. Herein, we tested whether CC-specific FSH and LH receptors (FSHR and LHR, respectively) are differentially expressed in CCs enclosing developmentally competent or incompetent oocytes. To this end, mouse fully grown cumulus-oocyte complexes were isolated and their CCs and oocytes analysed separately. Based on their chromatin organisation, oocytes were classified as those with a surrounded nucleolus (SN) or a non-surrounded nucleolus (NSN), the former being developmentally competent, whereas the latter arrest at the 2-cell stage. The CCs were then analysed to compare the pattern of expression of the Fshr and Lhr genes and their proteins. Quantitative reverse transcription-polymerase chain reaction analysis revealed that only Lhr is significantly differentially expressed. Immunofluorescence analysis revealed that both FSHR and LHR proteins are significantly upregulated in CCs surrounding oocytes arrested at the 2-cell stage, reflecting their developmental incompetence.

Transcriptome based identification of mouse cumulus cell markers that predict the developmental competence of their enclosed antral oocytes.

BACKGROUND: The cumulus cells (CCs) enveloping antral and ovulated oocytes have been regarded as putative source of non-invasive markers of the oocyte developmental competence. A number of studies have indeed observed a correlation between CCs gene expression, embryo quality, and final pregnancy outcome. Here, we isolated CCs from antral mouse oocytes of known developmental incompetence (NSN-CCs) or competence (SN-CCs) and compared their transcriptomes with the aim of identifying distinct marker transcripts. RESULTS: Global gene expression analysis highlighted that both types of CCs share similar transcriptomes, with the exception of 422 genes, 97.6% of which were down-regulated in NSN-CCs vs. SN-CCs. This transcriptional down-regulation in NSN-CCs was confirmed by qRT-PCR analysis of CC-related genes (Has2, Ptx3, Tnfaip6 and Ptgs2). Only ten of the 422 genes were up-regulated with Amh being the most up-regulated in NSN-CCs, with an average 4-fold higher expression when analysed by qRT-PCR. CONCLUSIONS: The developmental incompetence (NSN) or competence (SN) of antral oocytes can be predicted using transcript markers expressed by their surrounding CCs (i.e., Has2, Ptx3, Tnfaip6, Ptgs2 and Amh). Overall, the regulated nature of the group of genes brought out by whole transcriptome analysis constitutes the molecular signature of CCs associated either with developmentally incompetent or competent oocytes and may represent a valuable resource for developing new molecular tools for the assessment of oocyte quality and to further investigate the complex bi-directional interaction occurring between CCs and oocyte.

Developmental arrest and mouse antral not-surrounded nucleolus oocytes.

The antral compartment in the ovary consists of two populations of oocytes that differ by their ability to resume meiosis and to develop to the blastocyst stage. For reasons still not entirely clear, antral oocytes termed surrounded nucleolus (SN; 70% of the population of antral oocytes) develop to the blastocyst stage, whereas those called not-surrounded nucleolus (NSN) arrest at two cells. We profiled transcriptomic, proteomic, and morphological characteristics of antral oocytes and observed that NSN oocyte arrest is associated with lack of cytoplasmic lattices coincident with reduced expression of MATER and ribosomal proteins. Cytoplasmic lattices have been shown to store maternally derived mRNA and ribosomes in mammalian oocytes and embryos, and MATER has been shown to be required for cytoplasmic lattice formation. Thus, we isolated antral oocytes from a Mater(tm/tm) mouse and we observed that 84% of oocytes are of the NSN type. Our results provide the first molecular evidence to account for inability of NSN-derived embryos to progress beyond the two-cell stage; these results may be relevant to naturally occurring preimplantation embryo demise in mammals.

The frequency of heterologous synapsis increases with aging in Robertsonian heterozygous male mice.

The house mouse is characterised by highly variable chromosome number due to the presence of Robertsonian (Rb) chromosomes. During meiosis in Rb heterozygotes, intricated chromosomal figures are produced, and many unsynapsed regions are present during the first prophase, triggering a meiotic silencing of unsynapsed chromatin (MSUC) in a similar mode to the sex chromosome inactivation. The presence of unsynapsed chromosome regions is associated with impaired spermatogenesis. Interestingly, in male mice carrying multiple Rb trivalents, the frequency of germ cell death, defective tubules, and altered sperm morphology decreases during aging. Here, we studied whether synapsis of trivalent chromosomes and MSUC are involved in this improvement. By immunocytochemistry, we analysed the frequency of unsynapsed chromosomes and of those positive to γH2AX (a marker of MSUC) labelling in spermatocytes of 3-, 5- and 7-month-old Rb heterozygotes. With aging, we observed a decrease of the frequency of unsynapsed chromosomes, of spermatocytes bearing them and of trivalents carrying γH2AX-negative unsynapsed regions. Our quantitative results show that both synapsis and MSUC processes are better accomplished during male aging, partially accounting for the improvement of spermatogenesis.

Embryonic stem cells, derived either after in vitro fertilization or nuclear transfer, prolong survival of semiallogeneic heart transplants.

Tolerance induction toward allogeneic organ grafts represents one of the major aims of transplantation medicine. Stem cells are promising candidates for promoting donor-specific tolerance. In this study, we investigated the immunomodulatory properties of murine embryonic stem cells (ESCs), obtained either by in vitro fertilization (IVF-ESCs) or by nuclear transfer (NT-ESCs), in heart transplant mouse models. IVF-ESCs did not prolong the survival of fully allogeneic cardiac transplants but significantly prolonged the survival of semiallogeneic hearts from the same ESC donor strain for >100 d in 44% of the animals. However, 28% of transplanted animals infused with IVF-ESCs experienced development of a teratoma. NT-ESCs similarly prolonged semiallogeneic heart graft survival (>100 d in 40% of the animals) but were less teratogenic. By in vitro studies, IVF-ESC and NT-ESC immunoregulation was mediated both by cell contact-dependent mechanisms and by the release of soluble factors. By adding specific inhibitors, we identified PGE(2) as a soluble mediator of ESC immunoregulation. Expansion of regulatory T cells was found in lymphoid organs and in the grafts of IVF-ESC- and NT-ESC-tolerized mice. Our study demonstrates that both IVF-ESCs and NT-ESCs modulate recipient immune response toward tolerance to solid organ transplantation, and that NT-ESCs exhibit a lower tendency for teratoma formation. Because NT-ESCs are obtained by NT of a somatic cell from living individuals into an enucleated oocyte, they could represent a source of donor-derived stem cells to induce tolerance to solid organ allograft.

Mouse embryonic stem cells irradiated with γ-rays differentiate into cardiomyocytes but with altered contractile properties.

Embryonic stem cells (ESCs) for their derivation from the inner cell mass of a blastocyst represent a valuable in vitro model to investigate the effects of ionizing radiation on early embryonic cellular response. Following irradiation, both human and mouse ESCs (mESCs) maintain their pluripotent status and the capacity to differentiate into embryoid bodies and to form teratomas. Although informative of the maintenance of a pluripotent status, these studies never investigated the capability of irradiated ESCs to form specific differentiated phenotypes. Here, for the first time, 5Gy-irradiated mESCs were differentiated into cardiomyocytes, thus allowing the analysis of the long-term effects of ionizing radiations on the differentiation potential of a pluripotent stem cell population. On treated mESCs, 96h after irradiation, a genome-wide expression analysis was first performed in order to determine whether the treatment influenced gene expression of the surviving mESCs. Microarrays analysis showed that only 186 genes were differentially expressed in treated mESCs compared to control cells; a quarter of these genes were involved in cellular differentiation, with three main gene networks emerging, including cardiogenesis. Based on these results, we differentiated irradiated mESCs into cardiomyocytes. On day 5, 8 and 12 of differentiation, treated cells showed a significant alteration (qRT-PCR) of the expression of marker genes (Gata-4, Nkx-2.5, Tnnc1 and Alpk3) when compared to control cells. At day 15 of differentiation, although the organization of sarcomeric α-actinin and troponin T proteins appeared similar in cardiomyocytes differentiated from either mock or treated cells, the video evaluation of the kinematics and dynamics of the beating cardiac syncytium evidenced altered contractile properties of cardiomyocytes derived from irradiated mESCs. This alteration correlated with significant reduction of Connexin 43 foci. Our results indicate that mESCs populations that survive an ionizing irradiation treatment are capable to differentiate into cardiomyocytes, but they have altered contractile properties.

FTIR spectral signatures of mouse antral oocytes: molecular markers of oocyte maturation and developmental competence.

Mammalian antral oocytes with a Hoescht-positive DNA ring around the nucleolus (SN) are able to resume meiosis and to fully support the embryonic development, while oocytes with a non-surrounded nucleolus (NSN) cannot. Here, we applied FTIR microspectroscopy to characterize single SN and NSN mouse oocytes in order to try to elucidate some aspects of the mechanisms behind the different chromatin organization that impairs the full development of NSN oocyte-derived embryos. To this aim, oocytes were measured at three different stages of their maturation: just after isolation and classification as SN and NSN oocytes (time 0); after 10h of in vitro maturation, i.e. at the completion of the metaphase I (time 1); and after 20h of in vitro maturation, i.e. at the completion of the metaphase II (time 2). Significant spectral differences in the lipid (3050-2800cm(-1)) and protein (1700-1600cm(-1)) absorption regions were found between the two types of oocytes and among the different stages of maturation within the same oocyte type. Moreover, dramatic changes in nucleic acid content, concerning mainly the extent of transcription and polyadenylation, were detected in particular between 1000 and 800cm(-1). The use of the multivariate principal component-linear discriminant analysis (PCA-LDA) enabled us to identify the maturation stage in which the separation between the two types of oocytes took place, finding as the most discriminating wavenumbers those associated to transcriptional activity and polyadenylation, in agreement with the visual analysis of the spectral data.

Mouse embryonic stem cells that survive γ-rays exposure maintain pluripotent differentiation potential and genome stability.

This study aimed to investigate the cell cycle, apoptosis, cytogenetics and differentiation capacity of mouse embryonic stem cells (mESCs) that survived a single dose of 2 or 5 Gy γ-rays during a period of up to 96 h of culture. After 2 Gy irradiation and 24 h culture, compared to control, a significant majority of cells was blocked at the G2/M phase and a massive apoptosis was recorded. Between 48 and 72 h post-irradiation, the parameters used to describe the cell cycle and apoptosis returned similar to those of control samples. When mESCs were irradiated with 5 Gy, a small fraction of cells, even after 96 h of culture, still presented clear evidences of a G2/M block and apoptosis. The cytogenetic analysis performed at 96 h showed that the structural stability of the aberrations did not change significantly when comparing control and 2 or 5 Gy-treated populations. However, the chromosomal damage observed in the progeny of the survived cells after 5 Gy exposure is significantly higher than that observed in control samples, although it is mostly of the stable and transmissible type. Ninety-six hours after irradiation, the survived mESCs maintained their undifferentiated status and capability to differentiate into the three germ layers. Overall, these results indicate a commitment of mESCs to maintain pluripotency and genome stability.

Stem cells: sources and therapies.

The historical, lexical and conceptual issues embedded in stem cell biology are reviewed from technical, ethical, philosophical, judicial, clinical, economic and biopolitical perspectives. The mechanisms assigning the simultaneous capacity to self-renew and to differentiate to stem cells (immortal template DNA and asymmetric division) are evaluated in the light of the niche hypothesis for the stemness state. The induction of cell pluripotency and the different stem cells sources are presented (embryonic, adult and cord blood). We highlight the embryonic and adult stem cell properties and possible therapies while we emphasize the particular scientific and social values of cord blood donation to set up cord blood banks. The current scientific and legal frameworks of cord blood banks are reviewed at an international level as well as allogenic, dedicated and autologous donations. The expectations and the challenges in relation to present-day targeted diseases like diabetes mellitus type I, Parkinson's disease and myocardial infarction are evaluated in the light of the cellular therapies for regenerative medicine.

Fully-mature antral mouse oocytes are transcriptionally silent but their heterochromatin maintains a transcriptional permissive histone acetylation profile.

PURPOSE: The final stages of antral mouse oocytes maturation are characterised by the transition from transcriptionally active NSN to inactive SN oocytes. Here, we studied the profile of histone acetylation changes occurring during the NSN-to-SN transition. METHODS: During the NSN-to-SN transition, oocytes were classified based on their chromatin organisation and the immunocytochemical profile of histones H2B, H3 and H4 acetylation was analysed. RESULTS: We described four patterns of immunostaining common to the three acetylated histones, corresponding to stages of progressive localisation: From a diffused distribution in NSN oocytes to the association with constitutive heterochromatin and then to the nucleolar surface region in SN oocytes. CONCLUSIONS: The maintenance of a favourable transcriptional epigenetic context, in the heterochromatin of transcriptionally silent SN oocytes, might be related to the early stages of development when transcripts from these heterochromatic regions are functional to preimplantation progression.

Quantitative variation of LINE-1 sequences in five species and three subspecies of the subgenus Mus and in five Robertsonian races of Mus musculus domesticus.

The quantitative variation of a conserved region of the LINE-1 ORF2 sequence was determined in eight species and subspecies of the subgenus Mus (M. m. domesticus, M. m. musculus, M. m. castaneus, M. spicilegus, M. spretus, M. cervicolor, M. cookii, M. caroli) and five Robertsonian races of M. m. domesticus. No differences in LINE-1 ORF2 content were found between all acrocentric or Robertsonian chromosome races, whereas the quantitative variation of the LINE-1 ORF2 sequences detected among the eight taxa partly matches with the clades into which the subgenus is divided. An accumulation of LINE-1 ORF2 elements likely occurred during the evolution of the subgenus. Within the Asiatic clade, M. cervicolor, cookii, and caroli show a low quantity of LINE-1 sequences, also detected within the Palearctic clade in M. m. castaneus and M. spretus, representing perhaps the ancestral condition within the subgenus. On the other hand, M. m. domesticus, M. m. musculus and M. spicilegus showed a high content of LINE-1 ORF2 sequences. Comparison between the chromosomal hybridization pattern of M. m. domesticus, which possesses the highest content, and M. spicilegus did not show any difference in the LINE-1 ORF2 distribution, suggesting that the quantitative variation of this sequence family did not involve chromosome restructuring or a preferential chromosome accumulation, during the evolution of M. m. domesticus.

Expression of phase I and phase II genes in mouse embryonic stem cells cultured in the presence of 2,3,7,8-tetrachlorodibenzo-para-dioxin.

Embryonic stem (ES) cells have features that resemble the pluripotent cells of peri-implantation embryos and have been used as an in vitro model to assess the effects of test substances on these stages of development. Here, for the first time, we report on the effects of the xenobiotic 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) on mouse ES cells cultured with TCDD at concentrations ranging from 0.0001 to 100 nM for 15 min to 48 h. TCDD effects were determined by analysing the induction of Cyp1A1, Cyp1A2, Cyp1B1 (phase I) and Nqo1, Gsta1, Ugt1a6 (phase II) genes. Cyp1A1 was the phase I gene most rapidly induced (4 h at 1 nM); Cyp1B1 was induced at 48 h (1 nM), whereas Cyp1A2 expression was not affected. TCDD did not alter phase II gene expression, which remained at basal levels throughout the 48 h of culture. We studied more accurately the expression of Cyp1A1, the earliest gene to respond to the presence of TCDD. We found that: 1) Cyp1A1 gene induction is dependent on the duration of exposure (precisely it is first induced after 3 h of culture at 1 nM, the minimum effective-dose); 2) Cyp1A1 induction requires the continuous presence of TCDD, being interrupted 4 h after removal of the xenobiotic; and 3) induced expression of CYP1A1 protein is dependent on TCDD concentration, the higher the concentration the earlier the production of the enzyme. Furthermore, after 48 h of treatment, TCDD did not promote either apoptosis or changes to the differentiation status of the ES cells. These results are the first important step to investigate the effects of dioxin on the very early stages of mammalian development.

Embryonic stem cell differentiation studied by FT-IR spectroscopy.

We propose, here, an FT-IR method to monitor the spontaneous differentiation of murine embryonic stem (ES) cells in their early development. Principal component analysis and subsequent linear discriminant analysis enabled us to segregate stem cell spectra into separate clusters - corresponding to different differentiation times - and to identify the most significant spectral changes during differentiation. Between days 4 to 7 of differentiation, these spectral changes in the protein amide I band (1700-1600 cm(-1)) and in the nucleic acid absorption region (1050-850 cm(-1)) indicated that mRNA translation was taking place and that specific proteins were produced, reflecting the appearance of a new phenotype. The DNA/RNA hybrid bands (954 cm(-1) and 899 cm(-1)) were also observed, suggesting that the transcriptional switch of the genome started at this stage of differentiation. As confirmed by cytochemical assays, the FT-IR approach presented here allows to detect at molecular level the biological events of ES cell differentiation as they take place and to monitor in a rapid way the temporal evolution of the ES cell culture.

Oct-4 regulates the expression of Stella and Foxj2 at the Nanog locus: implications for the developmental competence of mouse oocytes.

BACKGROUND: Our knowledge of what determines the mammalian oocyte developmental competence is meagre. By comparing the transcriptional profiles of developmentally competent surrounded nucleolus (SN) and incompetent not surrounded nucleolus (NSN) mouse MII oocytes, we recently demonstrated that Oct-4 and Stella are key factors in the establishment of the oocytes' developmental competence. METHODS: Using RT-PCR, microarray and immunocytochemistry assays, we analysed expression of genes and proteins in oocytes isolated throughout folliculogenesis and classified based on their SN- or NSN-type of chromatin organization. RESULTS: We show that: (1) Oct-4 and Stella are expressed concurrently at the beginning of oocytes' growth and only in SN oocytes; (2) Germ Cell Nuclear Factor is a putative regulator of Oct-4 expression in MII oocytes; (3) the function of Oct-4 is directed at the Nanog locus, regulating the expression of Stella and Foxj2. CONCLUSIONS: (1) A number of factors that act upstream and downstream of Oct-4 emerge as candidate players in the acquisition of the oocyte's developmental competence; (2) we define molecular markers that identify a specific group of ovarian oocytes (SN) that have a potential to acquire developmental competence; (3) the presence of SN and NSN oocytes in human ovaries extends the interest of these results to the field of human reproduction.

The position of the germinal vesicle and the chromatin organization together provide a marker of the developmental competence of mouse antral oocytes.

Based on their chromatin organization, antral oocytes can be classified into two classes, namely surrounded nucleolus (SN, chromatin forms a ring around the nucleolus), and not surrounded nucleolus (NSN, chromatin has a diffuse pattern). Oocytes of both classes are capable of meiotic resumption, but while SN oocytes, following fertilization, develop to term, NSN oocytes never develop beyond the two-cell stage. A recent study has shown that the position of the germinal vesicle (GV) can be used as a morphological marker predictive of oocyte meiotic competence, i.e. oocytes with a central GV have a higher meiotic competence than oocytes with an eccentric GV. In the present study, we have associated both markers with the aim of identifying, with more accuracy, the oocytes' developmental competence. Following their isolation, antral oocytes were classified on the basis of both SN and NSN chromatin configuration and their GV position, matured to metaphase II and fertilized in vitro. We demonstrated that the position of the GV is a good marker to predict the oocytes' developmental competence, but only when associated with the observation of the chromatin organization.

Identification, isolation, and RT-PCR analysis of single stage-specific spermatogenetic cells obtained from portions of seminiferous tubules classified by transillumination microscopy.

The protocol here described allows the analysis of gene expression in single specific mouse spermatogenetic cells. Germ cells were singularly isolated by microdissection of portions of seminiferous tubules classified, based on their transillumination pattern, into four distinct zones along their length. Single portions of a seminiferous tubule, corresponding to specific zones, were mechanically disaggregated into single cells that were (1) identified as spermatogonia, spermatocytes, round or elongated spermatids, (2) isolated using a micromanipulator, and (3) singularly transferred into a test tube for retro-transcription PCR analysis. On each single isolated cell, we have determined the quantitative profile of expression of Gapdh, an endogenous housekeeping gene known to be expressed throughout spermatogenesis. The protocol described allows an accurate analysis of the temporal and quantitative profile of gene expression throughout the whole male gamete differentiation process which so far has mainly been performed on enriched population of cells.

Role of Oct-4 during acquisition of developmental competence in mouse oocyte.

Knowledge of what determines the developmental competence of oocytes during folliculogenesis is poor. This review, through the analysis of the expression profile of developmentally competent or incompetent mouse oocytes, summarizes the results of recent studies showing that the Oct-4 transcription factor regulating the expression of Stella and Foxj2 at the Nanog locus could play a pivotal role in the establishment of the oocyte's developmental competence.