Aneuploidy rate and Stemness in Low-level Mosaic Human Embryonic Stem Cells in the Presence/Absence of Bortezomib, Paclitaxel and Lapatinib.

Human embryonic stem cells (hESCs) are predisposed to aneuploidy through continual passages. Some reports indicate more sensitivity of aneuploid hESCs cells to anticancer drugs. The present study was designed to investigate the cytotoxicity of three anticancer drugs (including bortezomib, paclitaxel and lapatinib) and their effect on aneuploidy rate in hESCs. To create a low-level mosaic cell line, normal hESCs (80%) and trisomic hESCs for chromosomes 12 and 17 (20%) were mixed. The effect of the 3 mentioned anticancer drugs on the chromosomal status was assessed by metaphase spread analysis after selection of the nontoxic conditions. Expression of pluripotency genes was analyzed and an alkaline phosphatase test was performed to assess pluripotency preservation. Our data showed that treatment with bortezomib, paclitaxel and lapatinib was nontoxic at 0.01, 0.01, and 0.2µM concentrations, respectively. Alkaline phosphatase and pluripotency gene expression analyses revealed maintenance of pluripotency following treatment with above-noted nontoxic concentrations. Aneuploid cells were dominant in treated and control groups with a minimum abundance of 70%, with no significant differences between groups. Drug treatments had no negative effect on pluripotency. Insensitivity of aneuploid cells in treatment groups could be related to the specific characteristics of each cell line in response to the drug and the proliferative superiority of cells with trisomies 12 and 17.

Chromosomal instability reducing effect of paclitaxel and lapatinib in mouse embryonic stem cells with chromosomal abnormality.

Chromosomal abnormalities, as a frequent phenomenon in cultured embryonic stem cells (ESCs), is a major obstacle in the ESC application in regenerative medicine. Recent studies showed that aneuploid embryonic stem cells of humans and mice are more vulnerable to anticancer drugs, compared with normal cells. The aim of the current study was to evaluate effects of three anticancer drugs, paclitaxel, lapatinib and bortezomib, on mouse embryonic stem cells (mESCs) as a suitable and available model. To assess in vitro cell toxicity, two mESC lines were treated with the aforementioned drugs. Using G-band karyotyping and micronucleus assay, the effect of anticancer drugs in terms of reduction of chromosomal instability in the mESCs was evaluated in control and treatment groups. Also, apoptosis rate of both groups was estimated by FITC-Annexin V/Propidium Iodide (PI) double staining. In addition, the effect of these three drugs in maintaining the pluripotency was assessed through alkaline phosphatase assay and quantification of the expression of three key pluripotency genes, Nanog, Pou5f1 and Sox-2 was performed using Real Time PCR. The rate of numerical abnormalities after treatment with paclitaxel and lapatinib was lower than the control group. The expression level of pluripotency genes exhibited no significant difference between control and treatment groups. Administration of paclitaxel and lapatinib to the mESCs culture at an appropriate dose and in a timely manner could decrease chromosome stability without affecting pluripotency.

Suppression of transforming growth factor-beta signaling enhances spermatogonial proliferation and spermatogenesis recovery following chemotherapy.

STUDY QUESTION: Could small molecules (SM) which target (or modify) signaling pathways lead to increased proliferation of undifferentiated spermatogonia following chemotherapy? SUMMARY ANSWER: Inhibition of transforming growth factor-beta (TGFb) signaling by SM can enhance the proliferation of undifferentiated spermatogonia and spermatogenesis recovery following chemotherapy. WHAT IS KNOWN ALREADY: Spermatogonial stem cells (SSCs) hold great promise for fertility preservation in prepubertal boys diagnosed with cancer. However, the low number of SSCs limits their clinical applications. SM are chemically synthesized molecules that diffuse across the cell membrane to specifically target proteins involved in signaling pathways, and studies have reported their ability to increase the proliferation or differentiation of germ cells. STUDY DESIGN, SIZE, DURATION: In our experimental study, spermatogonia were collected from four brain-dead individuals and used for SM screening in vitro. For in vivo assessments, busulfan-treated mice were treated with the selected SM (or vehicle, the control) and assayed after 2 (three mice per group) and 5 weeks (two mice per group). PARTICIPANTS/MATERIALS, SETTING, METHODS: We investigated the effect of six SM on the proliferation of human undifferentiated spermatogonia in vitro using a top-bottom approach for screening. We used histological, hormonal and gene-expression analyses to assess the effect of selected SM on mouse spermatogenesis. All experiments were performed at least in triplicate and were statistically evaluated by Student's t-test and/or one-way ANOVA followed by Scheffe's or Tukey's post-hoc. MAIN RESULTS AND THE ROLE OF CHANCE: We found that administration of SB431542, as a specific inhibitor of the TGFb1 receptor (TGFbR1), leads to a two-fold increase in mouse and human undifferentiated spermatogonia proliferation. Furthermore, injection of SB to busulfan-treated mice accelerated spermatogenesis recovery as revealed by increased testicular size, weight and serum level of inhibin B. Moreover, SB administration accelerated both the onset and completion of spermatogenesis. We demonstrated that SB promotes proliferation in testicular tissue by regulating the cyclin-dependent kinase (CDK) inhibitors 4Ebp1 and P57 (proliferation inhibitor genes) and up-regulating Cdc25a and Cdk4 (cell cycle promoting genes). LIMITATIONS, REASONS FOR CAUTION: The availability of human testis was the main limitation in this study. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to report acceleration of spermatogenesis recovery following chemotherapy by administration of a single SM. Our findings suggest that SB is a promising SM and should be assessed in future clinical trials for preservation of fertility in men diagnosed with cancer or in certain infertility cases (e.g. oligospermia). STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Royan Institute and National Institute for Medical Research Development (NIMAD, grant no 963337) granted to H.B. The authors have no conflict of interest to report.

Rare case of an oligospermic male with 46,XX/46,XY tetragametic chimerism.

Chimerism, a rare human disorder, is assumed to be the result of an amalgamation of two separate zygotes in a single embryo. Studies have shown that the phenotypic spectrum of chimerism is variable and there is no definite genotype-phenotype correlation in patients with chimerism, therefore a majority of cases might remain undiagnosed. This study aims to investigate the possible mechanism of the chimerism in a 46,XX/46,XY infertile and phenotypically normal male, with 46,XX blood karyotype and normal spermatogenesis. We have used Interphase-FISH analysis to study the CEPX and CEPY regions on buccal and urine samples as well as molecular analysis of polymorphic short tandem repeats (STR) markers from 34 loci in order to discover the origin of 46,XX/46,XY. Analysis of X-linked and autosomal STR markers on blood, buccal tissue, urine, fibroblast and testis biopsy samples of the proband along with the blood sample of the patient's parents and siblings, showed divergent karyotypes in different tissues and tetragametic chimerism was diagnosed.

Enhanced generation of human embryonic stem cells from single blastomeres of fair and poor-quality cleavage embryos via inhibition of glycogen synthase kinase ß and Rho-associated kinase signaling.

STUDY QUESTION: Could selected pluripotency-enhancing small molecules (SMs) lead to efficient derivation of human embryonic stem cells (hESCs) from cleavage embryos-derived single blastomeres (SBs)? SUMMARY ANSWER: Inhibition of glycogen synthase kinase ß (GSK3ß) and Rho-associated kinase (ROCK) signaling can enhance the derivation of hESCs from cleavage embryo-derived SBs. WHAT IS KNOWN ALREADY: Parameters involved in sustaining the pluripotency of biopsied blastomeres for generating hESCs without causing injury to a viable embryo have remained obscure. This research seeks to improve the culture conditions for increasing the efficiency of deriving hESCs from SBs from cleavage-stage embryos by using SMs. STUDY DESIGN, SIZE, DURATION: In order to identify SMs which may enhance hESC generation from SBs, 11 pluripotency-enhancing SMs were screened and CHIR99021 (CH), a GSK3ß inhibitor, was selected. To optimize culture condition in hESC generation from SMs, we used ROCK inhibitor Y27632 (Y) and basic fibroblast growth factor in combination with CH or its alternative, Kenpaullone, in different time courses over 12 days. We also assessed a critical time point for CH + Y treatment of cleavage embryos from 4- to 8-cell embryo. In total, 224 embryos and 1607 SBs were used in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blastomeres of fair and poor-quality from 6- to 8-cell stage human embryos were mechanically dispersed and individually seeded into a 96-well plate that was precoated with mitotically inactivated feeder cells. Derivation of hESC line from each SB was carried out in hESC defined medium supplemented with SMs. Randomly selected hESC lines were evaluated by immunostaining for pluripotency markers, karyotype analysis and differentiation potential into the three embryonic germ layer derivatives. MAIN RESULTS AND THE ROLE OF CHANCE: We found that 3 µM CH was the only SM that was capable of directing SBs from fair and poor-quality 6-8-cell embryos into hESC lines. The application of hESC-conditioned medium had no additive effect on hESC establishment from SBs. Also, we indicated that CH combined with Y improved hESC generation efficiency by up to 31%. By using of Kenpaullone as an alternative to CH, we confirmed the involvement of GSK3 inhibition in hESC derivation from SBs. Interestingly, by treatment of 4-cell embryos, these SMs could enhance the derivation efficiency of SB-derived hESC lines up to 73% and the maximum number of hESC lines from SBs of one embryo was achieved in this state. LIMITATIONS, REASONS FOR CAUTION: The low quality of the embryos used in this study most likely had an effect on hESC generation. Furthermore, although we attempted to minimize any differences in inter-embryo quality, we cannot exclude the possibility that small differences in starting quality between embryos may have contributed to the differences observed, other than the addition of SMs. WIDER IMPLICATIONS OF THE FINDINGS: This approach would allow the establishment of autogeneic or allogeneic matched cells from embryos fertilized in vitro without destroying them. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by the National Elite Foundation and the Royan Institute for Stem Cell Biology and Technology. The authors have no conflict of interest to declare.

In vitro investigation of zinc oxide nanoparticle toxic effects in spermatogonial cells at the molecular level.

Because spermatogonia transmit genetic information across generations, their DNA must be protected from environmental damages, including exposure to zinc oxide nanoparticles (ZnO NPs), which are frequently used in modern technology. Here, we used an in vitro system enriched for spermatogonia and exposed them to 10 and 20 µg/ml ZnO NPs for one/seven days. We did not detect any significant cell death, chromosomal instability, or DNA fragmentation in the spermatogonia treated with the ZnO NPs following one-day treatment with 10 or 20 µg/ml ZnO NPs. However, ZnO NPs (both 10 and 20 µg/ml) induced chromosomal instability in the spermatogonia after seven days of treatment. Moreover, one-day exposure to these NPs induced reactive oxygen species (ROS) generation and upregulation of apoptotic pathway-related genes p53, Caspase3 and Il6, as an inflammatory factor. Taken together, our study provides preliminary evidence for possible damages induced by low concentrations of ZnO NPs in spermatogonia. We should pay increased attention when using these NPs because of the silent damages in spermatogonia that can be transmitted to the next generation and cause severe effects. However, more data and validation of these results are required to determine the extent of this concern.

Stem Cells from Human Exfoliated Deciduous Tooth Exhibit Stromal-Derived Inducing Activity and Lead to Generation of Neural Crest Cells from Human Embryonic Stem Cells.

In this article which was published in Cell J, Vol 17, No 1, Spring 2015, on pages 37-48, we found that Figure 1H, Figure 2 (OTX2, row 3), and Figure 3 (row 4) had been published incorrectly. The following figures are corrected. The authors would like to apologies for any inconvenience caused.

Establishment and characterization of Caspian horse fibroblast cell bank in Iran.

Caspian horse, a rare horse breed found in 1965 by Louise Firouz in northern Iran, is a small horse which is reported to be in danger of extinction in its original homeland. There seems to be a great need to prevent extinction of this valuable horse. In this study, 51 fibroblast cell lines from Caspian horse ear marginal tissue were successfully established by sampling 60 horses using primary explant technique. Cells were authenticated and growth curve was plotted. According to results obtained, population doubling time (PDT) was calculated 23 ± 0.5 h for all cell lines. Multiplex polymerase chain reaction (multiplex PCR) revealed that cell lines had no cross-contamination with other species. Bacteria, fungi, and mycoplasma contamination were checked using standard methods such as PCR, direct culture, and Hoechst staining. In addition to providing a valuable source for genomic, postgenomic, and somatic cloning researches, the established cell lines would preserve Caspian horse genetic resources. It will also create an accessible database for researchers.

Stem cells from human exfoliated deciduous tooth exhibit stromal-derived inducing activity and lead to generation of neural crest cells from human embryonic stem cells.

OBJECTIVE: The neural crest is a transient structure of early vertebrate embryos that generates neural crest cells (NCCs). These cells can migrate throughout the body and produce a diverse array of mature tissue types. Due to the ethical and technical problems surrounding the isolation of these early human embryo cells, researchers have focused on in vitro studies to produce NCCs and increase their knowledge of neural crest development. MATERIALS AND METHODS: In this experimental study, we cultured human embryonic stem cells (hESCs) on stromal stem cells from human exfoliated deciduous teeth (SHED) for a two-week period. We used different approaches to characterize these differentiated cells as neural precursor cells (NPCs) and NCCs. RESULTS: In the first co-culture week, hESCs appeared as crater-like structures with marginal rosettes. NPCs derived from these structures expressed the early neural crest marker p75 in addition to numerous other genes associated with neural crest induction such as SNAIL, SLUG, PTX3 and SOX9. Flow cytometry analysis showed 70% of the cells were AP2/P75 positive. Moreover, the cells were able to self-renew, sustain multipotent differentiation potential, and readily form neurospheres in suspension culture. CONCLUSION: SHED, as an adult stem cell with a neural crest origin, has stromal-derived inducing activity (SDIA) and can be used as an NCC inducer from hESCs. These cells provide an invaluable resource to study neural crest differentiation in both normal and disordered human neural crest development.