Step-by-step protocols for non-viral derivation of transgene-free induced pluripotent stem cells from somatic fibroblasts of multiple mammalian species.

Potentials of immortal proliferation and unlimited differentiation into all the three germ layers and germ cells in induced pluripotent stem cells (iPSCs) render them important bioresources for in vitro reconstitution and modeling of intravital tissues and organs in various animal models, thus contributing to the elucidation of pathomechanisms, drug discovery and stem cell-based regenerative medicine. We previously reported promising approaches for deriving transgene-free iPSCs from somatic fibroblasts of multiple mammalian species by episomal vector or RNA transfection, although the respective step-by-step protocols and the combinatorial usage of these methods, which achieved high induction efficiency, have not been described in the literature so far. Here, we provide a detailed step-by-step description of these methods with critical tips and slight modifications (improvements) to previously reported methods. We also report a novel method for the establishment of iPSCs from the Syrian hamster (also known as golden hamster; Mesocricetus auratus), a unique animal model of hibernation. We anticipate this methodology will contribute to stem cell biology and regenerative medicine research.

Photoperiod-independent testicular development in the model newt Pleurodeles waltl.

Urodele amphibian newts have unique biological properties in male gametogenesis, in addition to their extreme regenerative capacity. Male newts are able to regenerate new testes even after reaching sexual maturity and can possess multiple testes. Notably, these animals maintain primordial germ cell-like cells in a tissue adjacent to the testis. Spermatogenesis proceeds while synchronizing in a region-specific manner in the testis. However, the newt species that have been used most commonly require 2-3 years to achieve sexual maturity, and spermatogenesis in these species shows seasonality. These traits have restricted the use of newts for studies on testicular development and spermatogenesis, and testis development in newts remains poorly characterized. Recently, the Iberian ribbed newt Pleurodeles waltl has been established as an emerging model organism. P. waltl reaches sexual maturity more quick after birth than do other newts and is capable of breeding year-round. Thus, P. waltl is expected to serve as an appealing experimental model for studying the mechanisms of male gametogenesis in the urodeles. In the present study, we use P. waltl to describe the entire developmental process of the newt testis from primordial gonad to maturity. Notably, the mature testes show synchronized progression of spermatogenesis along the anteroposterior axis. Additionally, we demonstrate that the process of spermatogenesis in P. waltl proceeds irrespective of day length. Our results show that P. waltl newts are a suitable model for investigating the process of testicular development. We also expect that these results will be useful for the maintenance of P. waltl bioresources.

Cell cycle gene-specific control of transcription has a critical role in proliferation of primordial germ cells.

Transcription elongation is stimulated by positive transcription elongation factor b (P-TEFb), for which activity is repressed in the 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. We show here a critical role of 7SK snRNP in growth control of primordial germ cells (PGCs). The expression of p15(INK4b), a cyclin-dependent kinase inhibitor (CDKI) gene, in PGCs is selectively activated by P-TEFb and its recruiting molecule, Brd4, when the amount of active P-TEFb is increased due to reduction of the 7SK snRNP, and PGCs consequently undergo growth arrest. These results indicate that CDKI gene-specific control of transcription by 7SK snRNP plays a pivotal role in the maintenance of PGC proliferation.

MVH in piRNA processing and gene silencing of retrotransposons.

VASA is an evolutionarily conserved RNA helicase essential for germ cell development. The mouse PIWI family proteins MILI and MIWI2 are involved in production of Piwi-interacting RNAs (piRNAs) in fetal male germ cells through a ping-pong amplification cycle. Expression of retrotransposons is elevated in MILI- and MIWI2-deficient male germ cells due to defective de novo DNA methylation, which is presumably caused by impaired piRNA expression. Here, we report that essentially the same abnormalities are observed in MVH (mouse VASA homolog)-deficient mice. Comprehensive analysis of piRNAs in MVH-deficient fetal male germ cells showed that MVH plays crucial roles in the early phase of the ping-pong amplification cycle.

A heterozygous mutation of GALNTL5 affects male infertility with impairment of sperm motility.

For normal fertilization in mammals, it is important that functionally mature sperm are motile and have a fully formed acrosome. The glycosyltransferase-like gene, human polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5), belongs to the polypeptide N-acetylgalactosamine-transferase (pp-GalNAc-T) gene family because of its conserved glycosyltransferase domains, but it uniquely truncates the C-terminal domain and is expressed exclusively in human testis. However, glycosyltransferase activity of the human GALNTL5 protein has not been identified by in vitro assay thus far. Using mouse Galntl5 ortholog, we have examined whether GALNTL5 is a functional molecule in spermatogenesis. It was observed that mouse GALNTL5 localizes in the cytoplasm of round spermatids in the region around the acrosome of elongating spermatids, and finally in the neck region of spermatozoa. We attempted to establish Galntl5-deficient mutant mice to investigate the role of Galntl5 in spermiogenesis and found that the heterozygous mutation affected male fertility due to immotile sperm, which is diagnosed as asthenozoospermia, an infertility syndrome in humans. Furthermore, the heterozygous mutation of Galntl5 attenuated glycolytic enzymes required for motility, disrupted protein loading into acrosomes, and caused aberrant localization of the ubiquitin-proteasome system. By comparing the protein compositions of sperm from infertile males, we found a deletion mutation of the exon of human GALNTL5 gene in a patient with asthenozoospermia. This strongly suggests that the genetic mutation of human GALNTL5 results in male infertility with the reduction of sperm motility and that GALNTL5 is a functional molecule essential for mammalian sperm formation.

Meiotic wave adds extra asymmetry to the development of female chicken gonads.

Development of female gonads in the chicken is asymmetric. This asymmetry affects gene expression, morphology, and germ cell development; consequently only the left ovary develops into a functional organ, whereas the right ovary remains vestigial. In males, on the other hand, both gonads develop into functional testes. Here, we revisited the development of asymmetric traits in female (and male) chicken gonads between Hamburger Hamilton stage 16 (HH16) and hatching. At HH16, primordial germ cells migrated preferentially to the left gonad, accumulating in the left coelomic hinge between the gut mesentery and developing gonad in both males and females. Using the meiotic markers SYCP3 and phosphorylated H2AFX, we identified a previously undescribed, pronounced asymmetryc meiotic progression in the germ cells located in the central, lateral, and extreme cortical regions of the left female gonad from HH38 until hatching. Moreover, we observed that--in contrast to the current view--medullary germ cells are not apoptotic, but remain arrested in pre-leptotene until hatching. In addition to the systematic analysis of the asymmetric distribution of germ cells in female chicken gonads, we propose an updated model suggesting that the localization of germ cells--in the left or right gonad; in the cortex or medulla of the left gonad; and in the central part or the extremities of the left cortex--has direct consequences for their development and participation in adult reproduction.

Oligo-astheno-teratozoospermia in mice lacking Cnot7, a regulator of retinoid X receptor beta.

Spermatogenesis is a complex process that involves cooperation of germ cells and testicular somatic cells. Various genetic disorders lead to impaired spermatogenesis, defective sperm function and male infertility. Here we show that Cnot7(-/-) males are sterile owing to oligo-astheno-teratozoospermia, suggesting that Cnot7, a CCR4-associated transcriptional cofactor, is essential for spermatogenesis. Maturation of spermatids is unsynchronized and impaired in seminiferous tubules of Cnot7(-/-) mice. Transplantation of spermatogonial stem cells from male Cnot7(-/-) mice to seminiferous tubules of Kit mutant mice (Kit(W/W-v)) restores spermatogenesis, suggesting that the function of testicular somatic cells is damaged in the Cnot7(-/-) condition. The testicular phenotypes of Cnot7(-/-) mice are similar to those of mice deficient in retinoid X receptor beta (Rxrb). We further show that Cnot7 binds the AF-1 domain of Rxrb and that Rxrb malfunctions in the absence of Cnot7. Therefore, Cnot7 seems to function as a coregulator of Rxrb in testicular somatic cells and is thus involved in spermatogenesis.

Generation of a tyrosine hydroxylase-2A-Cre knockin non-human primate model by homology-directed-repair-biased CRISPR genome editing.

Non-human primates (NHPs) are the closest animal model to humans; thus, gene engineering technology in these species holds great promise for the elucidation of higher brain functions and human disease models. Knockin (KI) gene targeting is a versatile approach to modify gene(s) of interest; however, it generally suffers from the low efficiency of homology-directed repair (HDR) in mammalian cells, especially in non-expressed gene loci. In the current study, we generated a tyrosine hydroxylase (TH)-2A-Cre KI model of the common marmoset monkey (marmoset; Callithrix jacchus) using an HDR-biased CRISPR-Cas9 genome editing approach using Cas9-DN1S and RAD51. This model should enable labeling and modification of a specific neuronal lineage using the Cre-loxP system. Collectively, the current study paves the way for versatile gene engineering in NHPs, which may be a significant step toward further biomedical and preclinical applications.

A New Horizon in Reproductive Research with Pluripotent Stem Cells: Successful In Vitro Gametogenesis in Rodents, Its Application to Large Animals, and Future In Vitro Reconstitution of Reproductive Organs Such as "Uteroid" and "Oviductoid".

Recent success in derivation of functional gametes (oocytes and spermatozoa) from pluripotent stem cells (PSCs) of rodents has made it feasible for future application to large animals including endangered species and to ultimately humans. Here, we summarize backgrounds and recent studies on in vitro gametogenesis from rodent PSCs, and similar approaches using PSCs from large animals, including livestock, nonhuman primates (NHPs), and humans. We also describe additional developing approaches for in vitro reconstitution of reproductive organs, such as the ovary (ovarioid), testis (testisoid), and future challenges in the uterus (uteroid) and oviduct (oviductoid), all of which may be derived from PSCs. Once established, these in vitro systems may serve as a robust platform for elucidating the pathology of infertility-related disorders and ectopic pregnancy, principle of reproduction, and artificial biogenesis. Therefore, these possibilities, especially when using human cells, require consideration of ethical issues, and international agreements and guidelines need to be raised before opening "Pandora's Box".

Generation of a common marmoset embryonic stem cell line CMES40-OC harboring a POU5F1 (OCT4)-2A-mCerulean3 knock-in reporter allele.

POU class 5 homeobox 1 (POU5F1, also known as OCT4) is critical for maintenance of pluripotency, germ cell fate, reprogramming into a pluripotent state, and early embryogenesis. We generated an embryonic stem cell (ESC) line of the common marmoset (Callithrix jacchus) harboring a heterozygous knock-in allele of OCT4-P2A-mCerulean-T2A-pac. The ESC line (CMES40-OC) will be valuable for investigation of primed/naïve pluripotency and germ cell fate. Homozygous OCT4 knock-in clones were generated but could not be sustained in an undifferentiated state in long-term culture. The OCT4 knock-in system facilitated simultaneous knock-in of a reporter construct at another locus, DDX4 (VASA).

Non-viral Induction of Transgene-free iPSCs from Somatic Fibroblasts of Multiple Mammalian Species.

Induced pluripotent stem cells (iPSCs) are capable of providing an unlimited source of cells from all three germ layers and germ cells. The derivation and usage of iPSCs from various animal models may facilitate stem cell-based therapy, gene-modified animal production, and evolutionary studies assessing interspecies differences. However, there is a lack of species-wide methods for deriving iPSCs, in particular by means of non-viral and non-transgene-integrating (NTI) approaches. Here, we demonstrate the iPSC derivation from somatic fibroblasts of multiple mammalian species from three different taxonomic orders, including the common marmoset (Callithrix jacchus) in Primates, the dog (Canis lupus familiaris) in Carnivora, and the pig (Sus scrofa) in Cetartiodactyla, by combinatorial usage of chemical compounds and NTI episomal vectors. Interestingly, the fibroblasts temporarily acquired a neural stem cell-like state during the reprogramming. Collectively, our method, robustly applicable to various species, holds a great potential for facilitating stem cell-based research using various animals in Mammalia.

Identification of a novel human UDP-GalNAc transferase with unique catalytic activity and expression profile.

A novel member of the human ppGalNAc-T family, ppGalNAc-T20, was identified and characterized. Amino acid alignment revealed a high sequence identity between ppGalNAc-T20 and -T10. In the GalNAc transfer assay towards mucin-derived peptide substrates, the recombinant ppGalNAc-T20 demonstrated to be a typical glycopeptide GalNAc-transferase that exhibits activity towards mono-GalNAc-glycosylated peptide EA2 derived from rat submandibular gland mucin but no activity towards non-modified EA2. The in vitro catalytic property of ppGalNAc-T20 was compared with that of ppGalNAc-T10 to show different acceptor substrate specificities and kinetic constants. The ppGalNAc-T20 transcript was found exclusively in testis and brain. In situ hybridization further reveals that ppGalNAc-T20 was specifically localized in primary and secondary spermatocytes of the two meiotic periods, suggesting that it may involve in O-glycosylation during mouse spermatogenesis.

Induction of primordial germ cells from mouse induced pluripotent stem cells derived from adult hepatocytes.

Pluripotent stem cells can be established by various methods, but they share several cytological properties, including germ cell differentiation in vitro, independently of their origin. Although mouse induced pluripotent stem (iPS) cells can produce functional gametes in vivo, it is still unclear whether or not they have the ability to produce presumptive germ cells in vitro. Here, we show that mouse iPS cells derived from adult hepatocytes were able to differentiate into presumptive germ cells marked by mouse vasa homolog (Mvh) expression in feeder-free or suspension cultures. Embryoid body (EB) formation from iPS cells also induced the formation of round-shaped cells resembling immature oocytes. Mvh(+) cells formed clumps by co-aggregation with differentiation-supporting cells, and increased expression of germ cell markers was detected in these cell aggregates. Differentiation culture of presumptive germ cells from iPS cells could provide a conventional system for facilitating our understanding of the mechanisms underlying direct reprogramming and germline competency.

Generation of a male common marmoset embryonic stem cell line DSY127-BV8VT1 carrying double reporters specific for the germ cell linage using the CRISPR-Cas9 and PiggyBac transposase systems.

BLIMP1 (PRDM1) and VASA (DDX4) play pivotal roles in the development of the germ cell linage. Importantly, these genes are specifically expressed in germ cells; BLIMP1 in primordial germ cells (PGCs) to early-stage gonocytes, and VASA in migration-stage PGCs to mature gametes. The high reproductive efficiency of common marmosets (marmosets; Callithrix jacchus) makes them advantageous for use in germ cell research. We herein report the generation of a male marmoset embryonic stem cell (ESC) line harboring BLIMP1 and DDX4 double reporters. This ESC line will be a useful tool for investigating male gametogenesis in non-human primates.

Differences and similarities in the developmental status of embryo-derived stem cells and primordial germ cells revealed by global expression profiling.

Embryonic germ-line cells are unipotent cells that give rise to either sperm or oocytes. However, pluripotent stem cells can be derived from primordial germ cells (PGCs) or spermatogonia, suggesting that germ-line cells retain a capacity for pluripotency. Here, we made genome-wide comparisons of the gene expression profiles of freshly isolated PGCs, in vitro-formed PGCs (iPGCs), and other stem cell lines, including embryonic stem cells (ESCs), embryonic germ cells (EGCs) and germ-line stem (GS) cells. Comparing PGC with ESC, 382 genes/transcripts were significantly up-regulated in ESC, while 188 were elevated in PGC. This suggests that PGCs possess transcription program distinct from that of ESC, although both share expression of many pluripotency-associated genes. Our micro-array analysis showed that the analyzed samples could be classified into two groups: one consisting of all the ESCs and most of EGCs, and the other containing PGC samples, iPGC, one type of female EGC and GS cells. We then identified "signature" genes for the two groups, and used them to characterize GS cells, EGC, and iPGCs, and revealed developmental status of each cell type. The relationships between PGCs and stem cells derived from embryos or germ cells are discussed in light of these findings.

Identification of transcripts commonly expressed in both hematopoietic and germ-line stem cells.

Germ-line stem cells (GSCs) constitute a stem cell population with remarkable stability and proliferative potential in vitro and are a useful model for studying the mechanism of self-renewal and "stemness" function of committed tissue stem cells. To identify GSC-specific genes, we performed subtractive hybridization using cDNA from GSCs, testis, and embryonic stem (ES) cells, and successfully identified 11 genes highly expressed in GSCs. Histological analysis confirmed expression of Cry alpha b, Mcpt8, Cxcl5, Fth1, Ctla2 alpha, and Spp1 in undifferentiated spermatogonia on the basement membrane area of the seminiferous epithelium of the testis, where the GSC niche is thought to be located. Among GSC-specific genes, quantitative PCR analysis showed seven genes-Fth1, Cry alpha b, Spp1, Bcap31, Arhgap1, Ctla2 alpha, and Serpina3g-to be common transcripts highly expressed in hematopoietic stem cells (HSCs). Histological analysis confirmed that Ctla2 alpha-, Serpina3g-, and Spp1-expressing cells were observed in the trabecular bone region of the bone marrow, where the HSC niche is located. Furthermore, histological analysis revealed that only Spp1 was expressed in the hair follicle bulge in the area of the hair follicle stem cell niche. Thus, identifying stemness genes by comparative analysis to GSCs is a powerful tool with which to explore the fundamental commonalities of HSCs and other stem cell types.

Production of functional spermatids from mouse germline stem cells in ectopically reconstituted seminiferous tubules.

Testicular germ cell transplantation into the seminiferous tubules is at present the only way to induce spermatogenesis from a given source of spermatogonial stem cells. Here we show an alternative method that harnesses the self-organizing ability of testicular somatic cells. The testicular cells of embryonic or neonatal mice or rats and of newborn pigs were dissociated into single cells. Each of them reorganized into a tubular structure following implantation into the subcutis of immunodeficient mice. When mouse germline stem (GS) cells derived from spermatogonial stem cells and expanded in culture were intermingled with testicular cells of rodents, they were integrated in the reconstituted tubules and differentiated beyond meiosis into spermatids. Normal offspring were produced by the microinjection of those spermatids into oocytes. This method could be applicable to various mammalian species and useful for producing functional gametes from GS cells in a xenoectopic environment.

A noncoding RNA containing a SINE-B1 motif associates with meiotic metaphase chromatin and has an indispensable function during spermatogenesis.

A search for early response genes that are activated following germ cell induction from mouse embryonic stem cells in vitro led us to the isolation of a long noncoding RNA that contains a SINE (short interspersed element)-B1F motif that was named R53. In situ hybridization and northern blot analyses revealed that the R53 subfragment RNA bears a B1F motif, is processed from the primary transcript, is expressed in adult testis and is predominantly localized in meiotic metaphase chromatin during spermatogenesis. Recent studies of chromosome-associated RNAs have explored novel functions of noncoding RNAs. Specifically, chromosome-bound noncoding RNAs function not only as structural components of chromosome but also as scaffolds that recruit epigenetic modulators for transcriptional regulation, and they are dynamically rearranged during the cell cycle. However, few studies have explored meiotic chromatin; thus, R53 RNA appears to be the first long noncoding RNA to be tightly associated with the metaphase chromatin during spermatogenesis. Furthermore, R53 knockdown using a lentivirus-mediated RNAi injected into mouse testis and organ culture of the fragments revealed a remarkable reduction in postmeiotic cells and irregular up-regulation of several postmeiotic genes, which suggests the possibility that the SINE-B1-derived noncoding RNA R53 plays an indispensable role in the transcriptional regulation of key spermatogenesis genes.

JMJD1C Exhibits Multiple Functions in Epigenetic Regulation during Spermatogenesis.

Jmjd1C is one of the Jmjd1 family genes that encode putative demethylases against histone H3K9 and non-histone proteins and has been proven to play an indispensable role in mouse spermatogenesis. Here, we analyzed a newly-bred transgenic mouse strain carrying a Jmjd1C loss-of-function allele in which a ß-geo cassette was integrated into the intron of the Jmjd1C locus. Jmjd1C gene-trap homozygous testes exhibited malformations in postmeiotic processes and a deficiency in the long-term maintenance of undifferentiated spermatogonia. Some groups of spermatids in the homozygous testis showed abnormal organization and incomplete elongation from the first wave of spermatogenesis onwards. Moreover, histone H4K16 acetylation, which is required for the onset of chromatin remodeling, appeared to be remarkably decreased. These effects may not have been a result of the drastic decrease in gene expression related to the events but instead may have been due to the lack of interaction between JMJD1C and its partner proteins, such as MDC1 and HSP90. Additionally, significant decreases in Oct4 expression and NANOG- and OCT4-expressing spermatogonia were found in the Jmjd1C homozygous mature testis, suggesting that JMJD1C may participate in the maintenance of spermatogonial stem cell self-renewal by up-regulating Oct4 expression. These results indicate that JMJD1C has multiple functions during spermatogenesis through interactions with different partners during the spermatogenic stages.

Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment.

Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs.