Expression data from E12.5 male fatal PGCs, PGCLC in vitro, round spermatids and spermatids like cells produced in vitro
ABSTRACT: Generation of haploid gametes in vitro can potentially address gamete failure-based infertility.This study reports complete in vitro meiosis from murine ESC-derived PGCLCs resulting in the formation of male spermatid-like cells (SLCs) capable of producing viable fertile offspring via intracytoplasmic sperm injection (ICSI).Our findings provide the basis for generation of haploid spermatids in vitro in human, the generation of transgenic animals, and the use of this system to investigate mechanisms of meiosis. We used microarrays to compare gene expression profiles of in vivo and in vitro derived PGC cells and round spermatids. We collected E12.5 male fatal PGCs, PGCLC in vitro, round spermatids and spermatids like cells produced in vitro, each sample has 3 replications.
Project description:We reasoned that the pi-RISC, by virtue of the enormous sequence portfolio of piRNAs, might mediate elimination of a large variety of mRNAs in late stages of spermiogenesis. Both Miwi-null and Caf1-null mice showed early spermiogenic arrest, preventing us from determining the role of MIWI and CAF1 in elongating spermatids using the existing genetic models. We therefore used microarrays to detail the global effect of MIWI or CAF1 on mRNA levels in mouse elongating spermatids. Using GFP+ elongating spermatids sorted from mouse testes transduced with shMiwi:GFP, shCaf1:GFP, or control pSilencer:GFP, we performed transcriptome profiling on Affymetrix mouse arrays.
Project description:Generation of functional spermatids from human spermatogonial stem cells (SSCs) in vitro is of utmost importance for uncovering mechanisms underlying human germ cell development and treating infertility. Here we report a three-dimensional-induced (3D-I) system by which human SSCs were efficiently differentiated into functional haploid spermatids. Human SSCs were isolated and identified phenotypically. Meiotic chromatin spreads and DNA content assays revealed that spermatocytes and haploid cells were effectively generated from human SSCs by 3D-I system. Haploid cells derived from human SSCs harbored normal chromosomes and excluded Y chromosome microdeletions. RNA sequencing and bisulfite sequencing analyses reflected similarities in global gene profiles and DNA methylation in human SSCs-derived spermatids and normal round spermatids. Significantly, haploid spermatids generated from human SSCs via 3D-I system were capable of fertilizing mouse oocytes, which subsequently enabled the development of hybrid embryos. This study thus provides invaluable human male gametes for treating male infertility.
Project description:Haploid cells are amenable for genetic analysis because they contain only one set of chromosomes. Here,we report the derivation of haESCs from monkey parthenogenic blastocysts. These cells, which we designated PG-haESCs (parthenogenic haploid embryonic stem cells), express classical ESC markers, are pluripotent, and can differentiate to different cell lines from all three embryonic germ layers in vivo and in vitro. We used microarrays to compare the gene expression levels among PG-haESC, ICSI-derived ESCs and female monkey somatic fibroblasts. We used ICSI-derived ESCs and somatic fibroblasts isloated from female individuals as control. Gene expression profiles of all the cell lines were analysed on an Affymetrix Rhesus Macaque array.
Project description:Generation of functional spermatids from azoospermia patients is of unusual significance in the treatment of male infertility. Here, we report an efficient approach to obtain human functional spermatids from cryptorchid patients. Spermatogonia remained whereas meiotic germ cells were rare in cryptorchid patients. Expression of numerous markers for meiotic and postmeiotic male germ cells was enhanced in human spermatogonial stem cells (SSCs) of cryptorchidism patients by retinoic acid (RA) and stem cell factor (SCF) treatment. Meiotic spreads and DNA content assays revealed that RA and SCF induced a remarkable increase of SCP3-, MLH1-, and CREST-positive cells and haploid cells. Single-cell RNA sequencing analysis reflected distinct global gene profiles in embryos derived from round spermatids and nuclei of somatic cells. Significantly, haploid spermatids generated from human SSCs of cryptorchid patients possessed fertilization and development capacity. This study thus provides an invaluable source of autologous male gametes for treating male infertility in azoospermia patients.
Project description:Haploid stem cells offer an easy-to-manipulate genetic system and therefore have great values for studies of recessive phenotypes. Here, we show that mouse androgenetic haploid ES (ahES) cell lines can be established by transferring sperm into enucleated oocyte. The ahES cells maintain haploidy and stable growth over 30 passages, express pluripotent markers, possess the ability to differentiate into all three germ-layers in vitro and in vivo, and contribute to germline of chimeras when injected into blastocysts. Although epigenetically distinct from sperm cells, the ahES cells can produce viable and fertile progenies after intracytoplasmic injection into mature oocytes. The oocyte injection procedure can also produce viable transgenic mice from genetically engineered ahES cells. We used microarrays to compare the global programme of gene expression among ahES cells, normal diploid ES cells, MEF cells and round sperm cells and found that gene expression pattern of ahES cells was highly similar with ES cells but was distinct from MEF cells and round sperms. Androgenetic haploid ES cells were FACS sorted to harvest the G0/G1 phase haploid cells. Total RNA were extracted from three ahES cell lines (AH129-5, AH129-N1, AH129-NC1, all 129Sv genetic background), two ES cell lines ( CS1-1, R1, 129Sv background), MEF cells and round sperm and hybridized with Affymetrix GeneChip 430 2.0 array. Data were collected and analyzed to compare their gene expression pattern.
Project description:This SuperSeries is composed of the following subset Series: GSE35785: mRNA expression data from AG-haESC, E14 and MEF GSE35786: CGH analysis of AG-haESCs (androgenetic haploid embryonic stem cells) Refer to individual Series
Project description:Stable cytoplasmic bridges (or ring canals) connecting the clone of spermatids are assumed to facilitate the sharing of haploid gene products and synchronous development of the cells. We have visualized these cytoplasmic bridges under phase-contrast optics and recorded the sharing of cytoplasmic material between the spermatids by a digital time-lapse imaging system ex vivo. A multitude of small (ca. 0.5 microm) granules were seen to move continuously over the bridges, but only 28% of those entering the bridge were actually transported into other cell. The average speed of the granules decreased significantly during the passage. Immunocytochemistry revealed that some of the shared granules contained haploid cell-specific gene product TRA54. We also demonstrate the novel function for the Golgi complex in acrosome system formation by showing that TRA54 is processed in Golgi complex and is transported into acrosome system of neighboring spermatid. In addition, we propose an intercellular transport function for the male germ cell-specific organelle chromatoid body. This mRNA containing organelle, ca. 1.8 microm in diameter, was demonstrated to go over the cytoplasmic bridge from one spermatid to another. Microtubule inhibitors prevented all organelle movements through the bridges and caused a disintegration of the chromatoid body. This is the first direct demonstration of an organelle traffic through cytoplasmic bridges in mammalian spermatogenesis. Golgi-derived haploid gene products are shared between spermatids, and an active involvement of the chromatoid body in intercellular material transport between round spermatids is proposed.
Project description:Haploid cells are amenable for genetic analysis because they contain only one set of chromosomes.Here,we report the derivation of haESCs from androgenetic blastocysts. These cells, which we designated AG-haESCs, express classical ESC markers, are pluripotent, and contribute to various tissues including the germline upon injection into diploid blastocysts. We used microarrays to compare the gene expression levels among androgenetic haploid embryonic stem cell lines(AG-haESC) E14 and male mouse embryonic fibroblasts (MEFs) and identified that most paternally imprinted genes were down-regulated and the maternally imprinted genes were up-regulated. To avoid the influence of diploidized cells on the expression profile, we collected samples from FACS of cells at G1/G0 stage by staining Hochest 33342. We used E14,which was a male embryonic stem cell lines, and MEFs isloated from male individuals as control. Gene expression profiles of all the cell lines were analysed on an Affymetrix GeneChip 430 2.0 array.
Project description:When PDMSCs were induced to heptocytes in vitro, cells mophology, stem cell markers, mitochondrial metabolism will change according to the differentiated status.But dedifferentiation reverses differentiated cells to a more primitive phenotype and PDMSCs will retain the multilineage potency. Furthermore, it will leads to the alteration of gene expression pattern. We used microarrays to detail the global programme of gene expression underlying dedifferentiation and hepatogenic differentiation prcocesses, we intend to identify distinct classes of differentiated genes during these processes. Human PDMSCs at passage 5 were induced to hepatocytes for 11 days, then the inductive medium was replaced by general culture medium for 1 day. Then human PDMSCs, hepatogenic PDMSCs at 11 days, dedifferentiated PDMSCs were selected for RNA extraction and hybridization on Affymetrix microarrays. To that end, we hand-selected cells at three time-points: before hepatogenic induction (P), hepatogenic PDMSCs at 11 days (H) and dedifferentiated PDMSCs for 1 day (DH) .
Project description:The direct conversion, or trans-differentiation, of non-cardiac cells into cardiomyocytes by forced expression of transcription factors and microRNAs provide promising ways of cardiac regeneration. However, genetic manipulations are still not desirable in real clinical applications. we report the generation of automatically beating cardiomyocyte-like cells from mouse fibroblasts with only chemical cocktails. These chemical-induced cardiomyocyte-like cells (CiCMs) express cardiomyocyte-specific markers, exhibit sarcomeric organization, and possess typical cardiac calcium flux and electrophysiological features. Microarray-bassed gene expression patterns of Mouse embryonic fibroblasts (MEFs), CiCMs, and cardiomyocytes(CMs) indicated a clear transition from dividing MEFs to differentiated cardiomyocyte-like state in CiCM samples. Mouse embryonic fibroblasts were treated with a small-molecule combination CRFVPT (10 μM CHIR99021 (C); 10 μM RepSox (R); 50 μM Forskolin (F); 0.5 mM VPA (V); 5 μM Parnate, (P); 1 μM TTNPB (T)) to induce transdifferentiation to chemical-induced cardiomyocyte-like cells. CiCMs beating clusters were picked at day 24 for analysis. MEFs were isolated from mouse embryos, and CMs were isolated from mouse hearts. Total RNA of MEFs, CiCMs and CMs were extracted and hybridization on Affymetrix microarrays.