Reprogramming of chimpanzee fibroblasts into a multipotent cancerous but not fully pluripotent state by transducing iPSC factors in 2i/LIF culture.

To induce and maintain naïve pluripotency in mouse embryonic and induced pluripotent stem cells (ESCs/iPSCs), chemically defined N2B27 medium with PD0325901, CHIR99021, and leukemia inhibitory factor (2i/LIF) is a classic and simple condition. However, this method cannot be simply extrapolated to human ESCs/iPSCs that are principally stabilized in primed pluripotency and become primitive neuroepithelium-like cells in N2B27+2i/LIF culture. Here, we assessed iPSC reprogramming of fibroblasts from chimpanzee, our closest living relative, in N2B27+2i/LIF culture. Under this condition, chimpanzee cells formed alkaline phosphatase-positive dome-shaped colonies. The colony-forming cells could be stably expanded by serial passaging without a ROCK inhibitor. However, their gene expression was distinct from iPSCs and neuroepithelium. They expressed the OCT3/4 transgene and a subset of transcripts associated with pluripotency, mesenchymal-epithelial transition, and neural crest formation. These cells exhibited a differentiation potential into the three germ layers in vivo and in vitro. The current study demonstrated that iPSC reprogramming in N2B27+2i/LIF culture converted chimpanzee fibroblasts into a multipotent cancerous state with unique gene expression, but not fully pluripotent stem cells.

Sphere-formation culture of testicular germ cells in the common marmoset, a small New World monkey.

Spermatogonia are specialized cells responsible for continuous spermatogenesis and the production of offspring. Because of this biological property, in vitro culture of spermatogonia provides a powerful methodology to advance reproductive biology and engineering. However, methods for culturing primate spermatogonia are poorly established. We have designed a novel method for culturing spermatogonia in the common marmoset (Callithrix jacchus), a small primate. By using our method with a suite of growth factors, adult marmoset testis-derived germ cells could be cultured in the form of a floating sphere for several weeks. Notably, this method could be applied not only to freshly isolated cells but also to cryopreserved cell stocks. The spheres enriched spermatogonia and early spermatocytes, and could be assembled from a C-KIT(+) spermatogonial population. Techniques for culturing spermatogonia could facilitate increased understanding of primate reproduction as well as the preservation of valuable biomaterials from nonhuman primates.

Gene expression ontogeny of spermatogenesis in the marmoset uncovers primate characteristics during testicular development.

Mammalian spermatogenesis has been investigated extensively in rodents and a strictly controlled developmental process has been defined at cellular and molecular levels. In comparison, primate spermatogenesis has been far less well characterized. However, important differences between primate and rodent spermatogenesis are emerging so it is not always accurate to extrapolate findings in rodents to primate systems. Here, we performed an extensive immunofluorescence study of spermatogenesis in neonatal, juvenile, and adult testes in the common marmoset (Callithrix jacchus) to determine primate-specific patterns of gene expression that underpin primate germ cell development. Initially we characterized adult spermatogonia into two main classes; mitotically active C-KIT(+)Ki67(+) cells and mitotically quiescent SALL4(+)PLZF(+)LIN28(+)DPPA4(+) cells. We then explored the expression of a set of markers, including PIWIL1/MARWI, VASA, DAZL, CLGN, RanBPM, SYCP1 and HAPRIN, during germ cell differentiation from early spermatocytes through round and elongating spermatids, and a clear program of gene expression changes was determined as development proceeded. We then examined the juvenile marmoset testis. Markers of gonocytes demonstrated two populations; one that migrates to the basal membrane where they form the SALL4(+) or C-KIT(+) spermatogonia, and another that remains in the lumen of the seminiferous tubule. This later population, historically identified as pre-spermatogonia, expressed meiotic and apoptotic markers and were eliminated because they appear to have failed to correctly migrate. Our findings provide the first platform of gene expression dynamics in adult and developing germ cells of the common marmoset. Although we have characterized a limited number of genes, these results will facilitate primate spermatogenesis research and understanding of human reproduction.

Small RNA profiling and characterization of piRNA clusters in the adult testes of the common marmoset, a model primate.

Small RNAs mediate gene silencing by binding Argonaute/Piwi proteins to regulate target RNAs. Here, we describe small RNA profiling of the adult testes of Callithrix jacchus, the common marmoset. The most abundant class of small RNAs in the adult testis was piRNAs, although 353 novel miRNAs but few endo-siRNAs were also identified. MARWI, a marmoset homolog of mouse MIWI and a very abundant PIWI in adult testes, associates with piRNAs that show characteristics of mouse pachytene piRNAs. As in other mammals, most marmoset piRNAs are derived from conserved clustered regions in the genome, which are annotated as intergenic regions. However, unlike in mice, marmoset piRNA clusters are also found on the X chromosome, suggesting escape from meiotic sex chromosome inactivation by the X-linked clusters. Some of the piRNA clusters identified contain antisense-orientated pseudogenes, suggesting the possibility that pseudogene-derived piRNAs may regulate parental functional protein-coding genes. More piRNAs map to transposable element (TE) subfamilies when they have copies in piRNA clusters. In addition, the strand bias observed for piRNAs mapped to each TE subfamily correlates with the polarity of copies inserted in clusters. These findings suggest that pachytene piRNA clusters determine the abundance and strand-bias of TE-derived piRNAs, may regulate protein-coding genes via pseudogene-derived piRNAs, and may even play roles in meiosis in the adult marmoset testis.

Generation of germ cells in vitro in the era of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are stem cells that can be artificially generated via "cellular reprogramming" using gene transduction in somatic cells. iPSCs have enormous potential in stem-cell biology as they can give rise to numerous cell lineages, including the three germ layers. An evaluation of germ-line competency by blastocyst injection or tetraploid complementation, however, is critical for determining the developmental potential of mouse iPSCs towards germ cells. Recent studies have demonstrated that primordial germ cells obtained by the in vitro differentiation of iPSCs produce functional gametes as well as healthy offspring. These findings illustrate not only that iPSCs are developmentally similar to embryonic stem cells (ESCs), but also that somatic cells from adult tissues can produce gametes in vitro, that is, if they are reprogrammed into iPSCs. In this review, we discuss past and recent advances in the in vitro differentiation of germ cells using pluripotent stem cells, with an emphasis on ESCs and iPSCs. While this field of research is still at a stage of infancy, it holds great promises for investigating the mechanisms of germ-cell development, especially in humans, and for advancing reproductive and developmental engineering technologies in the future.

Aberrant gene expression and sexually incompatible genomic imprinting in oocytes derived from XY mouse embryonic stem cells in vitro.

Mouse embryonic stem cells (ESCs) have the potential to differentiate into germ cells (GCs) in vivo and in vitro. Interestingly, XY ESCs can give rise to both male and female GCs in culture, irrespective of the genetic sex. Recent studies showed that ESC-derived primordial GCs contributed to functional gametogenesis in vivo; however, in vitro differentiation techniques have never succeeded in generating mature oocytes from ESCs due to cryptogenic growth arrest during the preantral follicle stages of development. To address this issue, a mouse ESC line, capable of producing follicle-like structures (FLSs) efficiently, was established to investigate their properties using conventional molecular biological methods. The results revealed that the ESC-derived FLSs were morphologically similar to ovarian primary-to-secondary follicles but never formed an antrum; instead, the FLSs eventually underwent abnormal development or cell death in culture, or formed teratomas when transplanted under the kidney capsule in mice. Gene expression analyses demonstrated that the FLSs lacked transcripts for genes essential to late folliculogenesis, including gonadotropin receptors and steroidogenic enzymes, whereas some other genes were overexpressed in FLSs compared to the adult ovary. The E-Cadherin protein, which is involved in cell-to-cell interactions, was also expressed ectopically. Remarkably, it was seen that oocyte-like cells in the FLSs exhibited androgenetic genomic imprinting, which is ordinarily indicative of male GCs. Although the FLSs did not express male GC marker genes, the DNA methyltransferase, Dnmt3L, was expressed at an abnormally high level. Furthermore, the expression of sex determination factors was ambiguous in FLSs as both male and female determinants were expressed weakly. These data suggest that the developmental dysfunction of the ESC-derived FLSs may be attributable to aberrant gene expression and genomic imprinting, possibly associated with uncertain sex determination in culture.

Cell-intrinsic reprogramming capability: gain or loss of pluripotency in germ cells.

In multicellular organisms, germ cells are an extremely specialized cell type with the vital function of transmitting genetic information across generations. In this respect, they are responsible for the perpetuity of species, and are separated from somatic lineages at each generation. Interestingly, in the past two decades research has shown that germ cells have the potential to proceed along two distinct pathways: gametogenesis or pluripotency. Unequivocally, the primary role of germ cells is to produce gametes, the sperm or oocyte, to produce offspring. However, under specific conditions germ cells can become pluripotent, as shown by teratoma formation in vivo or cell culture-induced reprogramming in vitro. This phenomenon seems to be a general propensity of germ cells, irrespective of developmental phase. Recent attempts at cellular reprogramming have resulted in the generation of induced pluripotent stem cells (iPSCs). In iPSCs, the intracellular molecular networks instructing pluripotency have been activated and override the exclusively somatic cell programs that existed. Because the generation of iPSCs is highly artificial and depends on gene transduction, whether the resulting machinery reflects any physiological cell-intrinsic programs is open to question. In contrast, germ cells can spontaneously shift their fate to pluripotency during in-vitro culture. Here, we review the two fates of germ cells, i.e., differentiation and reprogramming. Understanding the molecular mechanisms regulating differentiation versus reprogramming would provide invaluable insight into understanding the mechanisms of cellular reprogramming that generate iPSCs.

Derivation of induced pluripotent stem cells by retroviral gene transduction in mammalian species.

Pluripotent stem cells can provide us with an enormous cell source for in vitro model systems for development. In 2006, new methodology was designed to generate pluripotent stem cells directly from somatic cells, and these cells were named induced pluripotent stem cells (iPSCs). This method consists of technically simple procedures: donor cell preparation, gene transduction, and isolation of embryonic stem cell-like colonies. The iPSC technology enables cell biologists not only to obtain pluripotent stem cells easily but also to study the reprogramming events themselves. Here, we describe the protocols to generate iPSCs from somatic origins by using conventional viral vectors. Specifically, we state the usage of three mammalian species: mouse, common marmoset, and human. As mouse iPSC donors, fibroblasts are easily prepared, while mesenchymal stem cells are expected to give rise to highly reprogrammed iPSCs efficiently. Common marmoset (Callithrix jacchus), a nonhuman primate, represents an alternative model to the usual laboratory animals. Finally, patient-specific human iPSCs give us an opportunity to examine the pathology and mechanisms of dysregulated genomic imprinting. The iPSC technology will serve as a valuable method for studying genomic imprinting, and conversely, the insights from these studies will offer valuable criteria to assess the potential of iPSCs.

Molecular signatures to define spermatogenic cells in common marmoset (Callithrix jacchus).

Germ cell development is a fundamental process required to produce offspring. The developmental program of spermatogenesis has been assumed to be similar among mammals. However, recent studies have revealed differences in the molecular properties of primate germ cells compared with the well-characterized mouse germ cells. This may prevent simple application of rodent insights into higher primates. Therefore, thorough investigation of primate germ cells is necessary, as this may lead to the development of more appropriate animal models. The aim of this study is to define molecular signatures of spermatogenic cells in the common marmoset, Callithrix jacchus. Interestingly, NANOG, PRDM1, DPPA3 (STELLA), IFITM3, and ZP1 transcripts, but no POU5F1 (OCT4), were detected in adult marmoset testis. Conversely, mouse testis expressed Pou5f1 but not Nanog, Prdm1, Dppa3, Ifitm3, and Zp1. Other previously described mouse germ cell markers were conserved in marmoset and mouse testes. Intriguingly, marmoset spermatogenic cells underwent dynamic protein expression in a developmental stage-specific manner; DDX4 (VASA) protein was present in gonocytes, diminished in spermatogonial cells, and reexpressed in spermatocytes. To investigate epigenetic differences between adult marmoset and mice, DNA methylation analyses identified unique epigenetic profiles to marmoset and mice. Marmoset NANOG and POU5F1 promoters in spermatogenic cells exhibited a methylation status opposite to that in mice, while the DDX4 and LEFTY1 loci, as well as imprinted genes, displayed an evolutionarily conserved methylation pattern. Marmosets have great advantages as models for human reproductive biology and are also valuable as experimental nonhuman primates; thus, the current study provides an important platform for primate reproductive biology, including possible applications to humans.