A Human Homozygous HELQ Missense Variant Does Not Cause Premature Ovarian Insufficiency in a Mouse Model.

Disruption of meiosis and DNA repair genes is associated with female fertility disorders like premature ovarian insufficiency (POI). In this study, we identified a homozygous missense variant in the HELQ gene (c.596 A>C; p.Gln199Pro) through whole exome sequencing in a POI patient, a condition associated with disrupted ovarian function and female infertility. HELQ, an enzyme involved in DNA repair, plays a crucial role in repairing DNA cross-links and has been linked to germ cell maintenance, fertility, and tumour suppression in mice. To explore the potential association of the HELQ variant with POI, we used CRISPR/Cas9 to create a knock-in mouse model harbouring the equivalent of the human HELQ variant identified in the POI patient. Surprisingly, Helq knock-in mice showed no discernible phenotype, with fertility levels, histological features, and follicle development similar to wild-type mice. Despite the lack of observable effects in mice, the potential role of HELQ in human fertility, especially in the context of POI, should not be dismissed. Larger studies encompassing diverse ethnic populations and alternative functional approaches will be necessary to further examine the role of HELQ in POI. Our results underscore the potential uncertainties associated with genomic variants and the limitations of in vivo animal modelling.

Diverse genetic causes of amenorrhea in an ethnically homogeneous cohort and an evolving approach to diagnosis.

RESEARCH QUESTION: Premature ovarian insufficiency (POI) is characterised by amenorrhea associated with elevated follicle stimulating hormone (FSH) under the age of 40 years and affects 1-3.7% women. Genetic factors explain 20-30% of POI cases, but most causes remain unknown despite genomic advancements. DESIGN: We used whole exome sequencing (WES) in four Iranian families, validated variants via Sanger sequencing, and conducted the Acyl-cLIP assay to measure HHAT enzyme activity. RESULTS: Despite ethnic homogeneity, WES revealed diverse genetic causes, including a novel homozygous nonsense variant in SYCP2L, impacting synaptonemal complex (SC) assembly, in the first family. Interestingly, the second family had two independent causes for amenorrhea - the mother had POI due to a novel homozygous loss-of-function variant in FANCM (required for chromosomal stability) and her daughter had primary amenorrhea due to a novel homozygous GNRHR (required for gonadotropic signalling) frameshift variant. WES analysis also provided cytogenetic insights. WES revealed one individual was in fact 46, XY and had a novel homozygous missense variant of uncertain significance in HHAT, potentially responsible for complete sex reversal although functional assays did not support impaired HHAT activity. In the remaining individual, WES indicated likely mosaic Turners with the majority of X chromosome variants having an allelic balance of ∼85% or ∼15%. Microarray validated the individual had 90% 45,XO. CONCLUSIONS: This study demonstrates the diverse causes of amenorrhea in a small, isolated ethnic cohort highlighting how a genetic cause in one individual may not clarify familial cases. We propose that, in time, genomic sequencing may become a single universal test required for the diagnosis of infertility conditions such as POI.

Enhancing differentiation of menstrual blood-derived stem cells into female germ cells using a bilayer amniotic membrane and nano-fibrous fibroin scaffold.

Three-dimensional nanofiber scaffolds offer a promising method for simulating in vivo conditions within the laboratory. This study aims to investigate the influence of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold on the differentiation of human menstrual blood mesenchymal stromal/stem cells (MenSCs) into female germ cells. MenSCs were isolated and assigned to four culture groups: (i) MenSCs co-cultured with granulosa cells (GCs) using the scaffold (3D-T group), (ii) MenSCs using the scaffold alone (3D-C group), (iii) MenSCs co-cultured only with GCs (2D-T group), and (iv) MenSCs without co-culture or scaffold (2D-C group). Both MenSCs and GCs were independently cultured for two weeks before co-culturing was initiated. Flow cytometry was employed to characterize MenSCs based on positive markers (CD73, CD90, and CD105) and negative markers (CD45 and CD133). Additionally, flow cytometry and immunocytochemistry were used to characterize the GCs. Differentiated MenSCs were analyzed using real-time PCR and immunostaining. The real-time PCR results demonstrated significantly higher levels of VASA expression in the 3D-T group compared to the 3D-C, 2D-T, and 2D-C groups. Similarly, the SCP3 mRNA level in the 3D-T group was notably elevated compared to the 3D-C, 2D-T, and 2D-C groups. Moreover, the expression of GDF9 was significantly higher in the 3D-T group when compared to the 3D-C, 2D-T, and 2D-C groups. Immunostaining results revealed a lack of signal for VASA, SCP3, or GDF9 markers in the 2D-T group, while some cells in the 3D-T group exhibited positive staining for all these proteins. These findings suggest that the combination of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold with co-culturing GCs facilitates the differentiation of MenSCs into female germ cells.

Deficiency of the mitochondrial ribosomal subunit, MRPL50, causes autosomal recessive syndromic premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a common cause of infertility in women, characterised by amenorrhea and elevated FSH under the age of 40 years. In some cases, POI is syndromic in association with other features such as sensorineural hearing loss in Perrault syndrome. POI is a heterogeneous disease with over 80 causative genes known so far; however, these explain only a minority of cases. Using whole-exome sequencing (WES), we identified a MRPL50 homozygous missense variant (c.335T > A; p.Val112Asp) shared by twin sisters presenting with POI, bilateral high-frequency sensorineural hearing loss, kidney and heart dysfunction. MRPL50 encodes a component of the large subunit of the mitochondrial ribosome. Using quantitative proteomics and western blot analysis on patient fibroblasts, we demonstrated a loss of MRPL50 protein and an associated destabilisation of the large subunit of the mitochondrial ribosome whilst the small subunit was preserved. The mitochondrial ribosome is responsible for the translation of subunits of the mitochondrial oxidative phosphorylation machinery, and we found patient fibroblasts have a mild but significant decrease in the abundance of mitochondrial complex I. These data support a biochemical phenotype associated with MRPL50 variants. We validated the association of MRPL50 with the clinical phenotype by knockdown/knockout of mRpL50 in Drosophila, which resulted abnormal ovarian development. In conclusion, we have shown that a MRPL50 missense variant destabilises the mitochondrial ribosome, leading to oxidative phosphorylation deficiency and syndromic POI, highlighting the importance of mitochondrial support in ovarian development and function.

A Bibliometric and In Silico-Based Analysis of Anti-Lung Cancer Compounds from Sea Cucumber.

Lung cancer is one of the most lethal malignancies in the world. However, current curative approaches for treating this type of cancer have some weaknesses. Therefore, scientists are attempting to discover new anti-lung cancer agents. Sea cucumber is a marine-derived source for discovering biologically active compounds with anti-lung cancer properties. To explore the anti-lung cancer properties of sea cucumber, we analyzed surveys using VOSviewer software and identified the most frequently used keywords. We then searched the Google Scholar database for compounds with anti-lung cancer properties within that keyword family. Finally, we used AutoDock 4 to identify the compounds with the highest affinity for apoptotic receptors in lung cancer cells. The results showed that triterpene glucosides were the most frequently identified compounds in studies examining the anti-cancer properties of sea cucumbers. Intercedenside C, Scabraside A, and Scabraside B were the three triterpene glycosides with the highest affinity for apoptotic receptors in lung cancer cells. To the best of our knowledge, this is the first time that anti-lung cancer properties of sea cucumber-derived compounds have been examined in in silico conditions. Ultimately, these three components displayed anti-lung cancer properties in in silico conditions and may be used for the manufacture of anti-lung cancer agents in the near future.

Epigenetic Modification Factors and microRNAs Network Associated with Differentiation of Embryonic Stem Cells and Induced Pluripotent Stem Cells toward Cardiomyocytes: A Review.

More research is being conducted on myocardial cell treatments utilizing stem cell lines that can develop into cardiomyocytes. All of the forms of cardiac illnesses have shown to be quite amenable to treatments using embryonic (ESCs) and induced pluripotent stem cells (iPSCs). In the present study, we reviewed the differentiation of these cell types into cardiomyocytes from an epigenetic standpoint. We also provided a miRNA network that is devoted to the epigenetic commitment of stem cells toward cardiomyocyte cells and related diseases, such as congenital heart defects, comprehensively. Histone acetylation, methylation, DNA alterations, N6-methyladenosine (m6a) RNA methylation, and cardiac mitochondrial mutations are explored as potential tools for precise stem cell differentiation.

Integral Role of the Mitochondrial Ribosome in Supporting Ovarian Function: MRPS7 Variants in Syndromic Premature Ovarian Insufficiency.

The mitochondrial ribosome is critical to mitochondrial protein synthesis. Defects in both the large and small subunits of the mitochondrial ribosome can cause human disease, including, but not limited to, cardiomyopathy, hypoglycaemia, neurological dysfunction, sensorineural hearing loss and premature ovarian insufficiency (POI). POI is a common cause of infertility, characterised by elevated follicle-stimulating hormone and amenorrhea in women under the age of 40. Here we describe a patient with POI, sensorineural hearing loss and Hashimoto's disease. The co-occurrence of POI with sensorineural hearing loss indicates Perrault syndrome. Whole exome sequencing identified two compound heterozygous variants in mitochondrial ribosomal protein 7 (MRPS7), c.373A>T/p.(Lys125*) and c.536G>A/p.(Arg179His). Both novel variants are predicted to be pathogenic via in-silico algorithms. Variants in MRPS7 have been described only once in the literature and were identified in sisters, one of whom presented with congenital sensorineural hearing loss and POI, consistent with our patient phenotype. The other affected sister had a more severe disease course and died in early adolescence due to liver and renal failure before the reproductive phenotype was known. This second independent report validates that variants in MRPS7 are a cause of syndromic POI/Perrault syndrome. We present this case and review the current evidence supporting the integral role of the mitochondrial ribosome in supporting ovarian function.

Premature Ovarian Insufficiency in CLPB Deficiency: Transcriptomic, Proteomic and Phenotypic Insights.

CONTEXT: Premature ovarian insufficiency (POI) is a common form of female infertility that usually presents as an isolated condition but can be part of various genetic syndromes. Early diagnosis and treatment of POI can minimize comorbidity and improve health outcomes. OBJECTIVE: We aimed to determine the genetic cause of syndromic POI, intellectual disability, neutropenia, and cataracts. METHODS: We performed whole-exome sequencing (WES) followed by functional validation via RT-PCR, RNAseq, and quantitative proteomics, as well as clinical update of previously reported patients with variants in the caseinolytic peptidase B (CLPB) gene. RESULTS: We identified causative variants in CLPB, encoding a mitochondrial disaggregase. Variants in this gene are known to cause an autosomal recessive syndrome involving 3-methylglutaconic aciduria, neurological dysfunction, cataracts, and neutropenia that is often fatal in childhood; however, there is likely a reporting bias toward severe cases. Using RNAseq and quantitative proteomics we validated causation and gained insight into genotype:phenotype correlation. Clinical follow-up of patients with CLPB deficiency who survived to adulthood identified POI and infertility as a common postpubertal ailment. CONCLUSION: A novel splicing variant is associated with CLPB deficiency in an individual who survived to adulthood. POI is a common feature of postpubertal female individuals with CLPB deficiency. Patients with CLPB deficiency should be referred to pediatric gynecologists/endocrinologists for prompt POI diagnosis and hormone replacement therapy to minimize associated comorbidities.

Meiotic genes in premature ovarian insufficiency: variants in HROB and REC8 as likely genetic causes.

Premature ovarian insufficiency (POI), affecting 1 in 100 women, is characterised by loss of ovarian function associated with elevated gonadotropin, before the age of 40. In addition to infertility, patients face increased risk of comorbidities such as heart disease, osteoporosis, cancer and/or early mortality. We used whole exome sequencing to identify the genetic cause of POI in seven women. Each had biallelic candidate variants in genes with a primary role in DNA damage repair and/or meiosis. This includes two genes, REC8 and HROB, not previously associated with autosomal recessive POI. REC8 encodes a component of the cohesin complex and HROB encodes a factor that recruits MCM8/9 for DNA damage repair. In silico analyses, combined with concordant mouse model phenotypes support these as new genetic causes of POI. We also identified novel variants in MCM8, NUP107, STAG3 and HFM1 and a known variant in POF1B. Our study highlights the pivotal role of meiosis in ovarian function. We identify novel variants, consolidate the pathogenicity of variants previously considered of unknown significance, and propose HROB and REC8 variants as new genetic causes while exploring their link to pathogenesis.

The Clinical Trials of Mesenchymal Stromal Cells Therapy.

Mesenchymal stromal cells (MSCs) are a heterogeneous population of adult stem cells, which are multipotent and possess the ability to differentiate/transdifferentiate into mesodermal and nonmesodermal cell lineages. MSCs display broad immunomodulatory properties since they are capable of secreting growth factors and chemotactic cytokines. Safety, accessibility, and isolation from patients without ethical concern make MSCs valuable sources for cell therapy approaches in autoimmune, inflammatory, and degenerative diseases. Many studies have been conducted on the application of MSCs as a new therapy, but it seems that a low percentage of them is related to clinical trials, especially completed clinical trials. Considering the importance of clinical trials to develop this type of therapy as a new treatment, the current paper is aimed at describing characteristics of MSCs and reviewing relevant clinical studies registered on the NIH database during 2016-2020 to discuss recent advances on MSC-based therapeutic approaches being used in different diseases.

A recessive variant in TFAM causes mtDNA depletion associated with primary ovarian insufficiency, seizures, intellectual disability and hearing loss.

Mitochondrial disorders are collectively common, genetically heterogeneous disorders in both pediatric and adult populations. They are caused by molecular defects in oxidative phosphorylation, failure of essential bioenergetic supply to mitochondria, and apoptosis. Here, we present three affected individuals from a consanguineous family of Pakistani origin with variable seizures and intellectual disability. Both females display primary ovarian insufficiency (POI), while the male shows abnormal sex hormone levels. We performed whole exome sequencing and identified a recessive missense variant c.694C > T, p.Arg232Cys in TFAM that segregates with disease. TFAM (mitochondrial transcription factor A) is a component of the mitochondrial replisome machinery that maintains mtDNA transcription and replication. In primary dermal fibroblasts, we show depletion of mtDNA and significantly altered mitochondrial function and morphology. Moreover, we observed reduced nucleoid numbers with significant changes in nucleoid size or shape in fibroblasts from an affected individual compared to controls. We also investigated the effect of tfam impairment in zebrafish; homozygous tfam mutants carrying an in-frame c.141_149 deletion recapitulate the mtDNA depletion and ovarian dysgenesis phenotypes observed in affected humans. Together, our genetic and functional data confirm that TFAM plays a pivotal role in gonad development and expands the repertoire of mitochondrial disease phenotypes.

Vitamin D3 affects mitochondrial biogenesis through mitogen-activated protein kinase in polycystic ovary syndrome mouse model.

Polycystic ovarian syndrome (PCOS) is a disorder characterized by oligomenorrhea, anovulation, and hyperandrogenism. Altered mitochondrial biogenesis can result in hyperandrogenism. The goal of this study was to examine the effect of vitamin D3 on mitochondrial biogenesis of the granulosa cells in the PCOS-induced mouse model. Vitamin D3 applies its effect via the mitogen-activated pathway kinase-extracellular signal-regulated kinases (MAPK-ERK1/2) pathway. The PCOS mouse model was induced by the injection of dehydroepiandrosterone (DHEA). Isolated granulosa cells were subsequently treated with vitamin D3, MAPK activator, and MAPK inhibitor. Gene expression levels were measured using real-time polymerase chain reaction. MAPK proteins were investigated by western blot analysis. We also determined reactive oxygen species (ROS) levels with 2', 7'-dichlorofluorescein diacetate. Mitochondrial membrane potential (mtMP) was also measured by TMJC1. Mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1-α and nuclear respiratory factor), antioxidant (superoxide dismutase, glutathione peroxidase, and catalase), and antiapoptotic (B-cell lymphoma-2) genes were upregulated in the PCOS mice that treated with vitamin D3 compared with the PCOS mice without any treatment. Vitamin D3 and MAPK activator-treated groups also reduced ROS levels compared with the nontreated PCOS group. In summary, vitamin D3 and MAPK activator increased the levels of mitochondrial biogenesis, MAPK pathway, and mtMP markers, while concomitantly decreased ROS levels in granulosa cells of the PCOS-induced mice. This study suggests that vitamin D3 may improve mitochondrial biogenesis through stimulation of the MAPK pathway in cultured granulosa cells of DHEA-induced PCOS mice which yet to be investigated.

Assessment of PGC1α-FNDC5 Axis in Granulosa Cells of PCOS Mouse Model.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is a metabolic and endocrine disorder which is characterized by hyperandrogenism, anovulation or oligomenorrhea and polycystic ovarian morphology. It is believed that modulation in metabolism of granulosa cells of PCOS patients may lead to infertility. One of the metabolic modulators is FNDC5 and its cleaved form, irisin. The axis of PGC1α-FNDC5 pathway is one of the main factors affecting cellular energy balance the purpose of this study was to evaluate this pathway in granulosa cells derived from PCOS mice model in comparison with control group. METHODS: In the present study, PCOS mouse model was developed by injection of dehydroepiandrosterone (DHEA) hormone in 20 mice for a period of 20 days. Also, 20 uninjected mice were used as the control. Meanwhile, a vehicle group consisted of mice which received daily subcutaneous sesame oil injection (n=20). Relative expressions of PGC1α and FNDC5 in granulosa cells were evaluated by RT-qPCR. Analysis of gene expressions was calculated by the ΔΔCT method and the relative levels of mRNA were normalized to GAPDH transcript levels. Differences in genes expression among three groups were assessed using one-way ANOVA, Tukey's Post Hoc test. RESULTS: Our results showed that expression of FNDC5 was significantly reduced in granulosa cells of DHEA-induced PCOS mice compared with control and vehicle groups (p<0.05), while there was no significant differences in PGC1α expression among different groups. CONCLUSION: Down regulation of FNDC5 transcript level may contribute in metabolic disturbance of granulosa cells derived from PCOS ovary apart from PGC1α levels which remained unchanged.

Vitamin D3 regulates steroidogenesis in granulosa cells through AMP-activated protein kinase (AMPK) activation in a mouse model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disorder in reproductive-aged women. Hormonal abnormality caused by steroidogenesis disturbances appears to be the main culprit of the clinical picture in PCOS. Vitamin D3 could regulate steroidogenesis in granulosa cells, but the mechanism of action of vitamin D3 on steroidogenesis remains unknown. AMP-activated protein kinase (AMPK) has a modulating role in steroid hormone production. We investigated the effect of vitamin D3 on steroidogenesis in cultured granulosa cells of dehydroepiandrosterone-induced PCOS mice and studied the involvement of AMPK signalling pathway in the current process. Immunoblotting assay showed that vitamin D3 could increase phosphorylation of AMPK alpha and acetyl-CoA carboxylase, main substrate of AMPK. Vitamin D3 and 5-aminoimidazole-4-carboxamide-1-ß-D-riboside or Aicar (AMPK activator) not only reduced gene expression of steroidogenic enzymes (P450scc or Cyp11a1, StAR, Cyp19a1 and 3B-HSD), but also reduced production of progesterone and 17B-estradiol assessed by radioimmunoassay. Pretreatment with compound C (AMPK inhibitor) decreased APMK phosphorylation and eliminated the effects of vitamin D3 and Aicar on steroidogenic enzymes expression and estradiol and progesterone production. This study showed that vitamin D3 has the main role in regulating of steroidogenesis in granulosa cells of mouse polycystic ovary through activation of the AMPK signalling pathway. SIGNIFICANCE OF THE STUDY: Polycystic ovarian syndrome (PCOS) is an endocrine disorder of women in reproductive age. This disorder is partly related to disruption in steroidogenesis pathway and dysregulation of estradiol and progesterone production in granulosa cells of polycystic ovaries. Previously, we have shown that vitamin D3 could modulate steroidogenesis pathway in PCOS granulosa cells. In this study, we investigate the molecular mechanism of vitamin D3 in regulation of steroidogenesis pathway. We have shown that vitamin D3 has a modulating role in steroidogenesis pathway of granulosa cells by regulation of AMP-activated protein kinase (AMPK) as an underlying molecular mechanism in mouse polycystic ovary.

Cryoprotective effect of resveratrol on DNA damage and crucial human sperm messenger RNAs, possibly through 5' AMP-activated protein kinase activation.

This work aimed at investigating the effect of resveratrol on (1) DNA integrity and (2) fertilizing capacity of sperm by quantifying the presence of key paternal transcripts considered as markers for male fertility (protamine 1 [PRM1] and protamine 2 [PRM2]) and pregnancy success (adducin 1 alpha [ADD1]) in cryopreserved human spermatozoa through modulation of AMP-activated protein kinase (AMPK). The study populations was drawn from 22 normozoospermic healthy volunteers which were incubated with or without AMPK activator (resveratrol [RSV], 15 µM) or inhibitor (Compound C [CC], 30 µM) for 1 h and were then cryopreserved. Untreated frozen-thawed spermatozoa served as controls. The RSV-induced AMPK activation decreased the level of DNA fragmentation in comparison with the control (21.18 ± 0.92 vs. 22.50 ± 0.40; p < 0.01). The relative mRNA expression levels of protamines (1 and 2) and ADD1 in RSV pretreated frozen-thawed human spermatozoa were also improved significantly compared to the control (p < 0.05). Conversely, the inhibitory effect of CC on AMPK activity deteriorated the deleterious effects of cryopreservation on these parameters (p < 0.01). In conclusion, these results demonstrated the cryoprotective effect of the RSV-induced increase in AMPK activity on DNA integrity and key paternal transcripts of cryopreserved human spermatozoa. These findings are of great importance for improving the available cryopreservation protocols in terms of the number of lesions that produced over key genes and the dramatic effects on sperm DNA fragmentation.

Modulation of steroidogenesis by vitamin D3 in granulosa cells of the mouse model of polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder of women of reproductive age characterized by polycystic ovarian morphology, anovulation or oligomenorrhea, and hyperandrogenism. It is shown that disruption in the steroidogenesis pathway caused by excess androgen in PCOS is a critical element of abnormal folliculogenesis and failure in dominant follicle selection. Vitamin D plays an important role in the regulation of ovulatory dysfunction and can influence genes involved in steroidogenesis in granulosa cells. In the present study, we investigated the effects of vitamin D3 on steroidogenic enzyme expression and activities in granulosa cell using a PCOS mouse model. In our study, the PCOS mouse model was developed by the injection of dehydroepiandrosterone (DHEA) for 20 days. The mRNA and protein expression levels of genes involved in steroidogenesis in granulosa cells were compared between polycystic and normal ovaries using real-time PCR and Western blotting assays. Granulosa cells of DHEA-induced PCOS mice were then cultured with and without vitamin D3 and mRNA and protein expression levels of steroidogenic enzymes and serum 17beta-estradiol and progesterone levels were investigated using qRT-PCR, western blot, and radioimmunoassay, respectively. Steroidogenic enzymes including Cyp11a1, StAR, Cyp19a1, and 3ß-HSD were upregulated in granulosa cells of PCOS mice when compared to normal mice. Treatment with vitamin D3 decreased mRNA and protein expression levels of steroidogenic enzymes in cultured granulosa cells. Vitamin D3 also decreased aromatase and 3ß-HSD activity that leads to decreased 17beta-estradiol and progesterone release. This study suggests that vitamin D3 could modulate the steroidogenesis pathway in granulosa cells of PCOS mice that may lead to improving follicular development and maturation. This is a step towards a possible conceivable treatment for PCOS. ABBREVIATIONS: AMHR-II: anti-müllerian hormone receptor-II; 3ß-HSD: 3ß-hydroxysteroid dehydrogenase; Cyp11a1: Cytochrome P450 Family 11 Subfamily A Member 1; Cyp19a1: cytochrome P450 aromatase; DHEA: dehydroepiandrosterone; FSH: follicle stimulating hormone; FSHR: follicle stimulating hormone receptor; IVF: in vitro fertilization; 25OHD: 25-hydroxy vitamin D; OHSS: ovarian hyperstimulation syndrome; PCOS: polycystic ovarian syndrome; P450scc: P450 side-chain cleavage enzyme; StAR: steroidogenic acute regulatory protein; VDRs: vitamin D receptors.

Production of stable GFP-expressing neural cells from P19 embryonal carcinoma stem cells.

Murine P19 embryonal carcinoma (EC) cells are convenient to differentiate into all germ layer derivatives. One of the advantages of P19 cells is that the exogenous DNA can be easily inserted into them. Here, at the first part of this study, we generated stable GFP-expressing P19 cells (P19-GFP+). FACS and western-blot analysis confirmed stable expression of GFP in the cells. We previously demonstrated the efficient induction of neuronal differentiation from mouse ES and EC cells by application of a neuroprotective drug, selegiline In the second part of this study selegiline was used to induce differentiation of P19-GFP+ into stable GFP-expressing neuron-like cells. Cresyl violet staining confirmed neuronal morphology of the differentiated cells. Furthermore, real-time PCR and immunoflourescence approved the expression of neuron specific markers. P19-GFP+ cells were able to survive, migrate and integrated into host tissues when transplanted to developing chick embryo CNS. The obtained live GFP-expressing cells can be used as an abundant source of developmentally pluripotent material for transplantation studies, investigating the cellular and molecular aspects of early differentiation.

The Potential of Menstrual Blood-Derived Stem Cells in Differentiation to Epidermal Lineage: A Preliminary Report.

BACKGROUND: Menstrual blood-derived stem cells (MenSCs) are a novel source of stem cells that can be easily isolated non-invasively from female volunteered donor without ethical consideration. These mesenchymal-like stem cells have high rate of proliferation and possess multi lineage differentiation potency. This study was undertaken to isolate the MenSCs and assess their potential in differentiation into epidermal lineage. METHODS: About 5-10 ml of menstrual blood (MB) was collected using sterile Diva cups inserted into vagina during menstruation from volunteered healthy fertile women aged between 22-30 years. MB was transferred into Falcon tubes containing phosphate buffered saline (PBS) without Ca2(+) or Mg2(+) supplemented with 2.5 µg/ml fungizone, 100 µg/mL streptomycin, 100 U/mL penicillin and 0.5 mM EDTA. Mononuclear cells were separated using Ficoll-Hypaque density gradient centrifugation and washed out in PBS. The cell pellet was suspended in DMEM-F12 medium supplemented with 10% FBS and cultured in tissue culture plates. The isolated cells were co-cultured with keratinocytes derived from the foreskin of healthy newborn male aged 2-10 months who was a candidate for circumcision for differentiation into epidermal lineage. RESULTS: The isolated MenSCs were adhered to the plate and exhibited spindle-shaped morphology. Flow cytometric analysis revealed the expression of mesenchymal markers of CD10, CD29, CD73, and CD105 and lack of hematopoietic stem cells markers. An early success in derivation of epidermal lineage from MenSCs was visible. CONCLUSION: The MenSCs are a real source to design differentiation to epidermal cells that can be used non-invasively in various dermatological lesions and diseases.

Effects of selegiline, a monoamine oxidase B inhibitor, on differentiation of P19 embryonal carcinoma stem cells, into neuron-like cells.

Selegiline, the irreversible inhibitor of monoamine oxidase B (MAO-B), is currently used to treat Parkinson's disease. However, the mechanism of action of selegiline is complex and cannot be explained solely by its MAO-B inhibitory action. It stimulates gene expression, as well as expression of a number of mRNAs or proteins in nerve and glial cells. Direct neuroprotective and anti-apoptotic actions of selegiline have previously been observed in vitro. Previous studies showed that selegiline can induce neuronal phenotype in cultured bone marrow stem cells and embryonic stem cells. Embryonal carcinoma (EC) cells are developmentally pluripotene cells which can be differentiated into all cell types under the appropriate conditions. The present study was carried out to examine the effects of selegiline on undifferentiated P19 EC cells. The results showed that selegiline treatment had a dramatic effect on neuronal morphology. It induced the differentiation of EC cells into neuron-like cells in a concentration-dependent manner. The peak response was in a dose of selegiline significantly lower than required for MAO-B inhibition. The differentiated cells were immunoreactive for neuron-specific proteins, synaptophysin, and ß-III tubulin. Stem cell therapy has been considered as an ideal option for the treatment of neurodegenerative diseases. Generation of neurons from stem cells could serve as a source for potential cell therapy. This study suggests the potential use of combined selegiline and stem cell therapy to improve deficits in neurodegenerative diseases.