International Journal of

Germline stem cells
     Spermatogenesis depends on stem cells, which have the ability to self-renew and generate a large number of differentiated germ cells in most species. In mammals, millions of spermatozoa are produced every day from spermatogonial stem cells (SSCs), the germ-line stem cells in the testis. Although SSCs are infrequent in the testis, presumably ~1 in 3,000 – 4,000 cells in adult mouse testis, SSCs can be identified unequivocally by a functional transplantation assay. To maintain spermatogenesis, the processes of self-renewal and differentiation of SSCs must be precisely regulated by intrinsic gene expression in the stem cells and extrinsic signals, including soluble factors or adhesion molecules from the surrounding microenvironment, the stem cell niche. We identified and characterized several genes playing essential role during germ cell development (1-5).
     Spermatogonial stem cells are descendants of the primordial germ cells (PGCs), which migrate from extra-embryonic sites to colonize the gonadal ridge early during embryonic life. In the females, the primordial germ cells proliferate extensively and enter meiotic prophase at around the time of birth. In males, cessation of germ cell proliferation and blockade of meiotic entry are important steps in the morphogenetic cascade initiated by the expression of the SRY gene. The male germ cells associate with somatic cells of the presumptive gonad and form testicular cords, which mark the differentiation of PGCs into gonocytes.
     We established different in vitro culture systems for study the process of germ cell differentiation and development (6-9). On molecular level, we are studying molecular mechanism involving germline stem cell proliferation and differentiation with focusing on Piwil2 gene which plays very important role in this process and interestingly in tumorigenesis (10-15)
 
 From stem cells to germ cells
     Today, there is much evidence suggesting that organ-specific stem cells need not rely completely on their own sources for maintenance and regeneration of an organism. In certain circumstances, mostly related to tissue damage, stem cell populations residing past the affected organ can contribute to its recovery--that means from different cell lines and also in tissues from another germ layer. The key factor in formation of self-renewing cellular clones is the presence of stem cells either from the tissue of origin or stem cells migrating from other areas and their successful settlement in an empty niche of the damaged tissue. Stem cell plasticity is the ability of adult tissue-specific stem cells to switch to new identities. The term plasticity also means stem cell phenotypic potential, which is broader than phenotypes of differentiated cells in their original tissues.
     One study published in recent month has dramatically changed the outlook on infertility and germ cell development, as the message it delivered is that male and female gametes can be produced in vitro from embryonic or teratocarcinoma stem cells (16). Recently, we showed that ES cells can differentiate to spermatogonial stem cells using a promoter based selection strategy (Figure 1).  These cells are able to undergo meiosis, generate haploid male gametes in vitro during 72 h and are functional, as shown by fertilization after intra-cytoplasmic injection into mouse oocytes. Resulting two cell embryos were transferred into oviduct and live mice were born. Seven of eight animals developed to normal adult mice. This is a clear indication that male gametes derived from embryonic stem cells by this strategy are able to induce normal fertilization and development. Although we succeeded in obtaining progeny from ES-cell derived male gametes, technical improvements are necessary. Improvement of oocyte microinjection is important to obtain more 2 cell embryos. The fertilization rate might have been affected by damage during oocyte injection or by heterogeneity of microinjected male gametes. This research is particularly important in helping us to understand more about spermatogenesis, the biological process in which sperm is produced. We must know this if we are to get to the root of infertility (9).
     In another approach, we demonstrated the potential of bone marrow stem cell to differentiate to male germ cells. Bone marrow-derived stem cells can be trans-differentiated into multilineage cells, such as muscle of mesoderm, lung and liver of endoderm, and brain and skin of ectoderm. We showed that bone marrow stem cells are able to Trans-differentiate into male germ cells. For derivation of male germ cells from adult bone marrow stem (BMS) cells we used a genetic selection strategy using a transgenic mouse line expressing EGFP specifically in male germ cells. BMS cell-derived germ cells expressed the known molecular markers of primordial germ cells like fragilis, stella, Rnf17, Mvh and Oct4, as well as molecular markers of spermatogonial stem cells and spermatogonia like Rbm, c-Kit, Tex18, Stra8, Piwil2, Dazl, Hsp90a, beta1- and alpha6-integrins. However, the BMS-derived germ cells were not able to enter meiosis.  Our finding addresses one of the critically important questions in the field of stem cell plasticity. For the first time, we demonstrated that a somatic adult stem cell can differentiate to germ cells.
 

Figure 1- Schematic presentation of promoter based strategy used for generation of ES cell derived male gametes.

From germ cells to stem cells      Our approach provides an accessible in vitro model system for studies of mammalian gametogenesis, as well as for developing new strategies for reproductive engineering and infertility treatment (Figure 3).