Amniotic fluid stem cell-based models to study the effects of gene mutations and toxicants on male germ cell formation

Abstract

Male infertility is a major public health issue predominantly caused by defects in germ cell development. In the past, studies on the genetic regulation of spermatogenesis as well as on negative environmental impacts have been hampered by the fact that human germ cell development is intractable to direct analysis in vivo. Compared with model organisms including mice, there are fundamental differences in the molecular processes of human germ cell development. Therefore, an in vitro model mimicking human sperm formation would be an extremely valuable research tool. In the recent past, both human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been reported to harbour the potential to differentiate into primordial germ cells and gametes. We here discuss the possibility to use human amniotic fluid stem (AFS) cells as a biological model. Since their discovery in 2003, AFS cells have been characterized to differentiate into cells of all three germ layers, to be genomically stable, to have a high proliferative potential and to be non-tumourigenic. In addition, AFS cells are not subject of ethical concerns. In contrast to iPS cells, AFSs cells do not need ectopic induction of pluripotency, which is often associated with only imperfectly cleared epigenetic memory of the source cells. Since AFS cells can be derived from amniocentesis with disease-causing mutations and can be transfected with high efficiency, they could be used in probing gene functions for spermatogenesis and in screening for male reproductive toxicity.

Introduction

Spermatogenesis starts shortly after birth with gonocytes and so-called spermatogonial stem cells as founder populations. At puberty, mitotically dividing spermatogonial cells maintain the ability of self-renewal and occupy niches in the seminiferous tubule in the testes. Early in spermatogenesis, these spermatogonial cells face the choice of self-renewal or differentiation. The end-product of spermatogenesis is the male gamete, called sperm. Germ cell development (male and female) is unique in generating haploid cells, which are responsible for the maintenance of the species. Errors in spermatogenesis can cause infertility, which is an increasing public health issue. On the one hand, it is well known that mutations and environmental toxicants can mediate severe negative effects on male germ cell development. On the other hand, for different reasons discussed below, the underlying molecular mechanisms still remain elusive.^{1, 2, 3}

The relevance of animal models to study human germ cell development

Our knowledge on the molecular processes controlling human germ cell formation is limited, mainly due to the inaccessibility of early stages of human development to experimentation. Exploration of the unknown causes of human infertility is hampered by many ethical and practical difficulties associated with the collection of relevant human tissue samples. Accordingly, most of our understanding on the regulation of infertility is based on epidemiological studies.⁴ Another reason for our limited knowledge on germ cell development in humans is that extrapolation of results obtained from animal models, such as mice, is not really possible (see the discussion in Ref. 5). In no other species, infertility caused by errors in germ cell formation and meiotic missegregation of chromosomes are nearly as common as in humans.^{6, 7}

Whereas women require two X chromosomes for oocyte development, mice are fertile with just a single X chromosome.^{8, 9} The homology between human and mice of well known reproductive genes, such as STELLAR, GDF3 or NANOG, is very low with only 30% identity at the amino acid level in some cases.^{10, 11} Regarding the topic of this review, it is of importance to consider that several Y chromosome genes, including the azoospermia-associated deleted in azoospermia (DAZ) genes, are absent in mice.^{12, 13} Taken together, these fundamental differences in the molecular regulation of human germ cell development compared with model organisms underline the demand to establish stem cell-based approaches for the in vitro investigation of germ cell formation.

Embryonic stem (ES) and induced pluripotent stem (iPS) cell-based models to study male germ cell development in vitro

Using in vitro differentiation approaches, mouse ES cells^{14, 15, 16} and human ES cells^{17, 18, 19, 20, 21} have been demonstrated to harbour the potential to form primordial germ cells and gametes. Recently, iPS cells derived from reprogramming of somatic cells also have been shown to differentiate into primordial germ cells and even to generate haploid germ cells.^{5, 22, 23, 24} These pioneering studies allowed new important insights into the regulation of germ cell formation in vitro.

A variety of protocols have been established to promote germ cell formation from pluripotent stem cells. One potent approach is to isolate SSEA1-positive cells from embryoid bodies by immunomagnetic beads. Retinoic acid treatment for several days triggers rapid differentiation of ES cells, while stimulating proliferation of primordial germ cells. Retinoic acid treatment can therefore be used to distinguish between these two cell populations.²⁵ Although meiosis is inefficient in embryoid bodies, it is possible to flow cytometrically isolate a small population of haploid cells (representing round spermatids) expressing FE-J1, which is specific for male meiotic germ cells¹⁶ (see also Figure 1). Spontaneous differentiation of pluripotent stem cells into primordial germ cells can also be induced by several days of cultivation in a medium containing bone morphogenetic proteins (BMP-4, BMP-7 and BMP-8b). The RNA binding protein VASA is specifically expressed in the germ cell lineage and is essential for germ cell formation.²⁶ An elegant approach allowing flow cytometric isolation of germ cells after bone morphogenetic protein-induced differentiation is prior transfection of the pluripotent stem cells with a reporter system carrying GFP under the VASA promoter. Since spontaneous induction of meiosis is low, overexpression of genes of the DAZ gene family can be performed to induce meiosis for haploid spermatids^{5, 27} (Figure 1). Finally, in mice it is possible to test the biological function of so-obtained haploid male gametes either by transplantation into busulfan-mediated sperm-depleted testes to check in vivo differentiation, or by intracytoplasmic injection into recipient oocytes to test whether this supports cleavage^{14, 15, 16} (Figure 1).

Figure 1.

AFS cells as a model to study gene functions for spermatogenesis and to screen for male reproductive toxicity. Monoclonal AFS cell lines can be established from human and mouse amniotic fluid. These clonal stem cell lines can be tested for their potential to form primordial germ cells and male gametes. The effects of gene modulations and of a variety of different toxicants on spermatogenesis can be tested. For details, see the text. AFS, amniotic fluid stem; FACS, flow-activated cell sorting; GFP, green fluorescent protein; MACS, magnetic cell sorting; VASA, vasa.

The approaches described above are readily available in vitro models mimicking human gamete development and are therefore extremely valuable research tools. Stem cell-based models have already been shown to be useful to investigate the role of specific genes for germ cell formation.^{27, 28} It is well known that spermatogenesis is a complex biological process, which is particularly sensitive to environmental insults, such as toxicants,²⁹ and stem cell-based in vitro models have already been used for reproductive toxicity screening.^{30, 31}

Amniotic fluid stem (AFS) cells could be a useful tool to study the molecular regulation of spermatogenesis and the effects of toxicants

It is very likely that undifferentiated and differentiated cells of varying lineages and origins are present in amniotic fluid. In 2003, first evidence for the existence of human AFS cells was published describing a highly proliferative stem cell type in human amniotic fluid expressing the pluripotent marker Oct4.³² Meanwhile, many independent research groups have confirmed the existence of this Oct4⁺/c-Kit⁺ AFS cell type and have shown that these stem cells harbour the potential to differentiate, e.g. upon haematopoietic, neurogenic, osteogenic, chondrogenic, adipogenic, renal and hepatic lineages. Still, the precise origin of this stem cell type remains elusive. From their biological properties and their marker expression pattern, AFS cells appear to be closer to ES cells than e.g. to trophoblast cells. Most of the studies have been performed with AFS cells derived via amniocenteses, but there are also some data available with AFS cells isolated out of amniotic fluid at birth.^{33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48} It is now known that descending from one single AFS cell of this type, it is possible to induce both differentiation into cells of all three embryonic tissue types and embryoid body formation. These findings on the pluripotency of AFS cells were obtained using monoclonal cell lines generated via magnetic cell sorting (e.g., for c-Kit) and minimal dilution approaches from human amniocentesis samples. Monoclonal AFS cell lines can be cultivated in an undifferentiated status with high proliferation rate and without the need of feeder cells.^{38, 41, 49} In this article, we suggest to test the usability of human (and mouse) AFS cells as a model to study sperm cell development in vitro using the differentiation protocols described above for ES and iPS cells (Figure 1). We suggest to use the well-known and widely used approach to isolate monoclonal Oct4⁺/c-Kit⁺ AFS cell lines. These AFS cell lines can be generated by minimal dilution starting expansion from one single stem cell. Several human cell lines of this kind exist and they exhibit identical behaviour. In a recent paper,⁴⁹ we have compared three such monoclonal AFS cell lines proving that they are not heterogenous.

The generation of ES cell lines via destroying a human embryo raises a variety of ethical issues and furthermore ES cells are tumourigenic per se.^{50, 51} In the case of iPS cells, the process of ectopic induction of pluripotency might somehow interfere with its usefulness as a biological model for germ cell formation.^{52, 53, 54} The same is true for the facts, that the epigenetic memory of the original differentiated state is not perfectly erased during reprogramming^{55, 56} and that iPS cells have been reported to accumulate karyotypic abnormalities and gene mutations during propagation in culture.^{57, 58, 59, 60} AFS very likely do not harbour accumulated somatic mutations, because they are primary cells of a very early stage of human development. Furthermore, monoclonal human AFS cell lines have been reported to maintain genome stability during expansion and do not induce tumour formation in severe combined immunodeficient mice.^{41, 44, 46, 47, 49, 61, 62}

To analyse the genetic mechanisms of sperm cell development, it is of the highest relevance to use stem cell-based models, which allow the investigation of the consequences of a specific genetic aberration.^{63, 64} One approach would be to use stem cells with natural occurring genetic aberrations, which are of relevance for human pathological phenotypes associated with infertility. ES cell lines with such disease-causing mutations can be generated using embryos, which in the course of in vitro fertilisation have been excluded from transfer into the uterus via preimplantation genetic diagnosis of single-gene disorders or structural and numerical chromosome aberrations.^{65, 66} In the recent past, also a variety of iPS lines from single-gene disorders, chromosome syndromes and complex diseases have been generated and it would be of great interest to use iPS lines from infertile men as in vitro models for germ cell formation.^{67, 68} However, the spectrum of usable ES cells with an inherited defect is limited, because in vitro fertilisation with preimplantation genetic diagnosis is only rarely applied. And whereas the usage of iPS cells derived from adults has the advantage that the detailed clinical history of the patient is known, one must take into account that such cells might have already accumulated other mutations, which might then interfere with the in vitro studies on spermatogenesis.⁶⁴ Accordingly, here we want to suggest to generate AFS cell lines with prenatally diagnosed single-gene disorders, unbalanced segregated parental chromosome translocations, aneuploidies or de novo structural chromosomal aberrations.⁶⁹ Such stem cell lines could then be useful for the exploration of the unknown genetic causes of male human infertility (Figure 1).

Another approach to obtain insights into the genetic regulation of male germ cell development is the modulation of gene expression in the here discussed stem cell-based models. As already described above, ES cell-based models have been successfully used to study the role of specific genes, such as DAZL, DAZ, BOULE or NANOS3, for germ cell formation.^{27, 28} In this context, it is interesting to note that DAZL gene expression has been found in human amniotic fluid cell samples.⁷⁰ However, it still must be clarified which cells within the amniotic fluid express this germ cell marker and whether this is of any physiological relevance. We here also want to suggest the usage of small interfering RNA-mediated knockdown of endogenous genes in human monoclonal AFS cells to investigate their roles during gamete formation. We have recently established a protocol for efficient small interfering RNA-mediated prolonged gene silencing in AFS cells allowing a 96%–98% downregulation of endogenous expressions over a time period of about 14 days.⁷¹ Spermatogenesis is a strictly regulated biological process, which is very sensitive to environmental toxicants, such as mercury and lead. Human are exposed to these ubiquitous environmental pollutants predominantly by ingestion of contaminated food and beverages and by inhalation of contaminated air. Both metals are known to accumulate in the human body and to target a variety of developmental processes. There is evidence that both metals can reduce male fertility by interfering with male germ cell development resulting in altered sperm density, motility and viability.^{29, 72, 73, 74} ES cell-based approaches to study germ cell development have been shown to be useful tools for the direct examination of the effects of environmental toxicants on the molecular regulation of human germ cell formation.^{30, 31} Very recently, we found that mercury and lead can affect the biological properties of human AFS cells.⁷⁵ The differentiation of AFS cells into cell of the germ cell lineage would allow to investigate the role of these two toxicants on human male germ cell development, but also to screen additional substances for their toxic potential for spermatogenesis. AFS cell-based germ cell models may even be more useful to study the effects of toxicants than that of mutations, since in vitro manipulations might induce other interfering mutations that may complicate the investigation of the original question. The efficiency of differentiation from ES or iPS cells into haploid male germ cells is generally reported to be low.^{14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24} Although there is no evidence for the speculation that this efficiency might be higher using AFS cells, to our opinion, the here reviewed knowledge warrants testing this new stem cell type for their usability for the in vitro investigation of sperm production (Figure 1).

The authors declare no competing financial interest.