Reprogramming Human Female Adipose Mesenchymal Stem Cells into Primordial Germ Cell-Like Cells

In the last two decades, considerable progress has been made in the derivation of mammalian germ cells from pluripotent stem cells such as Embryonic Stem Cells (ESCs) and induced Pluripotent Stem Cells (iPSCs). The pluripotent stem cells are generally first induced into pre-gastrulating endoderm/mesoderm-like status and then specified into putative primordial germ cells (PGCs) termed PGC-like cells (PGCLCs) which possess the potential to generate oocytes and sperms. Adipose-derived mesenchymal stromal cells (ASCs) are multipotent cells, having the capacity to differentiate into cell types such as adipocytes, osteocytes and chondrocytes. Since no information is available about the capability of female human ASCs (hASCs) to generate PGCLCs, we compared protocols to produce such cells from hASCs themselves or from hASC-derived iPSCs. The results showed that, providing pre-induction into a peri-gastrulating endoderm/mesoderm-like status, hASCs can generate PGCLCs. This process, however, shows a lower efficiency than when hASC-derived iPSCs are used as starting cells. Although hASCs possess multipotency and express mesodermal genes, direct induction into PGCLCs resulted less efficient

FGF9 – Nodal signaling negatively control meiotic entry of postnatal male germ cells

Fibroblast growth factor 9 (FGF9) produced by the somatic cells of the testis acts directly on germ cells to inhibit meiosis by upregulating levels of the RNA binding protein Nanos2, both in fetal and postnatal gonad [1]. Several studies have recently demonstrated that the anti-meiotic effect of FGF9 in the fetal testis is mediated by Nodal, a member of the TGF-Beta morphogen family. In fetal male germ cells FGF9 upregulates Cripto, which is a Nodal co-receptor, making these cells unable to enter meiosis [2]. However it is currently unknown how FGF9 acts postnatally to inhibit entry into meiosis of male mitotic germ cells. We first investigated which was the signal transduction pathway activated by FGF9 on postnatal mouse spermatogonia. We found that FGF9 signaling is dependent on ERK but not on AKT activation, which is instead triggered by Kit ligand (an inducer of meiotic entry that cooperates with retinoic acid). By qRT-PCR we found that Nodal and Cripto are expressed in postnatal spermatogonia and that FGF9 (but not Kit ligand) treatment promotes their upregulation. By western blot we found that phospho-Smad2, an indicator of active Nodal signaling, is increased in spermatogonia upon FGF9 treatment, suggesting that Nodal mediates FGF9 action also in postnatal male germ cells

Microgravity Promotes Differentiation and Meiotic Entry of Postnatal Mouse Male Germ Cells

A critical step of spermatogenesis is the entry of mitotic spermatogonia into meiosis. Progresses on these topics are hampered by the lack of an in vitro culture system allowing mouse spermatogonia differentiation and entry into meiosis. Previous studies have shown that mouse pachytene spermatocytes cultured in simulated microgravity (SM) undergo a spontaneous meiotic progression. Here we report that mouse mitotic spermatogonia cultured under SM with a rotary cell culture system (RCCS) enter into meiosis in the absence of any added exogenous factor or contact with somatic cells. We found that isolated Kit-positive spermatogonia under the RCCS condition enter into the prophase of the first meiotic division (leptotene stage), as monitored by chromosomal organization of the synaptonemal complex 3 protein (Scp3) and up-regulation of several pro-meiotic genes. SM was found to activate the phosphatidyl inositol 3 kinase (PI3K) pathway and to induce in Kit-positive spermatogonia the last round of DNA replication, typical of the preleptotene stage. A PI3K inhibitor abolished Scp3 induction and meiotic entry stimulated by RCCS conditions. A positive effect of SM on germ cell differentiation was also observed in undifferentiated (Kit-negative) spermatogonia, in which RCCS conditions stimulate the expression of Kit and Stra8. In conclusion, SM is an artificial environmental condition which promotes postnatal male germ cell differentiation and might provide a tool to study the molecular mechanisms underlying the switch from mitosis to meiosis in mammals

Role of Pten deletion and BRafV600E mutation in the generation of testicular germ cell tumors

Testicular germ cell tumors (TGCT) represent the most common solid malignancy affecting males between the ages of 15 and 35, while ovarian germ cell tumours (OGCT) are a type of ovarian neoplasm principally affecting young women. Germ cell tumors (GCTs) account for about 95 % of testicular cancer cases and for only 2-3% of ovarian cancer cases (Siegel et al., 2011). Most TGCT are potentially curable, however approximately 5% of patients with TGCT develop chemoresistance and die from the disease. PTEN deletion and mutational activation of BRAF are frequent genetic alterations found in human TGCTs, suggesting that they might be directly involved in germ cell tumorigenesis. Furthermore, BRAF mutation positively correlates with the acquisition of resistence to cisplatin, the most commonly chemotherapic agent employed for the treatment of human TGCTs. We obtained heterozygous floxed Ptenloxp/+ BRafCA Spo11Cre mice showing ovarian teratomas and testicular tumors with an incidence of about 30% at 20 days post partum (dpp) . Since Spo- 11Cre is active at around 13.5 days post coitum (dpc) in female germ cells and at around 7 dpp in male germ cells (18), these results suggest that ovarian teratomas origin from early meiotic germ cells in the fetal period whereas GCT formation in males can be a postnatal event. By histological inspection, we found that cancer cells in testes showed features reminiscent of seminoma such as a diffuse, confluent multinodular pattern. However, by immunohistochemical staining, we observed that the cells within the tumor showed heterogeneous positivity for the pluripotency markers Oct4, Sox2, Nanog, Kit and Prdm14, suggesting that they can represent a mixed form of seminoma and embryonal carcinoma cells. Our results indicate that deregulated MAP and PI3 Kinase activation can lead to postnatal male germ cells transformation

Characterization of c-Kit receptor function in cardiac regeneration by using transgenic mouse models

Background. Cardiac stem cells expressing the tyrosine kinase receptor c-kit have been recently used in in vivo and in vitro cardiac regenerative studies. However, it remains to be clarified whether the c-kit receptor itself plays a critical role in the process of cardiac regeneration. In order to clarify this point, we will explore whether c-Kit receptor affects cardiac stem cells proliferation, survival, migration and differentiation after heart injury. Methods and Results. We have generated transgenic mice in which an activatory point mutation (c-KitD814Y mice) has been introduced in the kinase domain of the c-kit gene. Initially, we have analyzed c-kit expression in tissues and organs at different stages of embryonal and post-natal development through immunohystochemical and biochemical analyses. We have found that in two transgenic lines the receptor is highly expressed and activated in heart, testis and cerebellum, compared to wild type mice. In order to follow the fate of the c-Kit transgenic stem cells we crossed c-KitD814Y mice with mice expressing GFP under c-Kit regulative sequences control. By cytofluorimetric and fluorescence microscopy analyses, we observed a 2 fold of increase in the number of c-kit positive cells on heart samples from double transgenic mice at different ages. To verify the c-kit role in cardiac regeneration we performed a necrotic heart damage in vivo and monitored cardiac repair in transgenic mice versus wild-type mice. After 9 days the wounded hearts of transgenic mice presented a larger connectival tissue area compared to wild-type mice. On the contrary, after 45 days a consistent reduction of fibrotic area was observed in transgenic mice. These preliminary results suggest a faster repair of damaged heart area that contain stem cells with an activated c-kit receptor. Further in vitro and in vivo experiments will be performed to assess whether transgenic c-kit cells directly transdifferentiate into cardiomyocytes or whether they act in a paracrine manner. In summary, the generation of transgenic mice carrying a constitutively activated c-kit in cardiac stem cells, will allow to investigate the role of the receptor and to highlight the molecular mechanism underlying heart regeneration

Ontogenetic Profile of the Expression of Thyroid Hormone Receptors in Rat and Human Corpora Cavernosa of the Penis

Introduction. In the last few years, various studies have underlined a correlation between thyroid function and male sexual function, hypothesizing a direct action of thyroid hormones on the penis. Aim. To study the spatiotemporal distribution of mRNA for the thyroid hormone nuclear receptors (TR) alpha 1, alpha 2 and beta in the penis and smooth muscle cells (SMCs) of the corpora cavernosa of rats and humans during development. Methods. We used several molecular biology techniques to study the TR expression in whole tissues or primary cultures from human and rodent penile tissues of different ages. Main Outcome Measure. We measured our data by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) amplification, Northern blot and immunohistochemistry. Results. We found that TR alpha 1 and TR alpha 2 are both expressed in the penis and in SMCs during ontogenesis without development-dependent changes. However, in the rodent model, TR beta shows an increase from 3 to 6 days post natum (dpn) to 20 dpn, remaining high in adulthood. The same expression profile was observed in humans. While the expression of TR beta is strictly regulated by development, TR alpha 1 is the principal isoform present in corpora cavernosa, suggesting its importance in SMC function. These results have been confirmed by immunohistochemistry localization in SMCs and endothelial cells of the corpora cavernosa. Conclusions. The presence of TRs in the penis provides the biological basis for the direct action of thyroid hormones on this organ. Given this evidence, physicians would be advised to investigate sexual function in men with thyroid disorders. Carosa E, Di Sante S, Rossi S, Castri A, D'Adamo F, Gravina GL, Ronchi P, Kostrouch Z, Dolci S, Lenzi A, and Jannini EA. Ontogenetic profile of the expression of thyroid hormone receptors in rat and human corpora cavernosa of the penis. J Sex Med 2010;7:1381-1390

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Sox2 induces Mesenchymal to Epithelial transition in somatic cells

During late embryonic development mesodermal cells generate epithelial organs, such as kidney and ovary, via a process named Mesenchymal to Epithelial Transition (MET) (1). This process can also occur reversely giving rise to Epithelial to Mesenchimal transition (EMT) (2), which represents the mechanism by which metastasis originate from primary tumors. Many lines of evidence underlie a continuous interaction between these two programs. Indeed MET is believed to participate in the establishment and stabilization of distant metastases by allowing cancerous cells to regain epithelial properties and integrate into distant organs. It has been shown that fibroblasts undergo MET at some point during reprogramming to mimic undifferentiated ESCs (2).Since SOX2 is an essential factor to reprogram somatic cells to stem cell fate, we investigated if it might play a role in inducing MET in mouse embryonic fibroblasts. Using a lentiviral vector we found that Sox2 transduced cells changed morphology as early as two days after infection compared to their mock infected controls. Although cells showed an epithelial-like morphology, they were E-cadherin negative. Furthermore, Sox2 negatively regulated the expression of some of the mTOR pathway such as 4EBP1, EIF4E and S6. FACS analysis in Sox2-transduced somatic cells also revealed a cell cycle arrest in G1-phase. Together our data suggest a role of Sox2 in cell fate and commitment