Sertoli-cell-conditioned medium increases the expression of the germ-cell specific VASA gene in differentiating human embryonic stem cells

Introduction: Research on embryonic stem cell (ESC) derived gametes has gained more and more attention in the last years. Several studies in mice reported that mESC can differentiate into gametes that can even generate offspring. Recently, human ESC also showed differentiation potential towards the germ cell lineage. After spontaneous differentiation of hESC to embryoid bodies (EBs), germ cell markers could be detected in a small number of differentiated cells. The addition of exogenous factors (BMP4, BMP7 and BMP8) to the differentiation medium increased the number of these germ-cell-like cells.
In our study, we aimed to investigate the effect of Sertoli-cell-conditioned medium (ScCM) on the potential of hESC to differentiate in vitro towards the germ cell lineage compared to spontaneous differentiation, by evaluating the mRNA expression of VASA. In human, VASA expression is restricted to the germ cell lineage, starting from the migratory primordial germ cells up to the spermatid stage. In contrast to earlier germ cell markers such as DAZL or POU5F1, VASA is not expressed in hESC and is therefore a reliable marker of later stage germ cell lineage differentiation.

Materials and methods: Four hESC lines (VUB01, VUB02, VUB07 and VUB09_FSHD) and one immortalised mouse Sertoli cell line (TM4, ATCC) were used in this study. Basic differentiation medium consisted of Knock-out DMEM containing 20% foetal calf serum, 2 mM L-glutamine, 1% NEAA and 0.1 mM ?-mercaptoethanol. ScCM was produced by incubating 30ml of basic differentiation medium in a 175cm flask on a confluent layer of Sertoli cells for 24h. Sertoli cells were also used as feeder layers for culture of hESC in direct contact or in indirect contact, via well inserts. In some experiments, BMP4 (100ng/ml), BMP7 (50ng/ml) and BMP8 (50ng/ml) were added to the basic medium. VASA transcripts were measured by relative quantification real-time RT-PCR.

Results:
When hESC were left to differentiate as aggregates in ScCM, VASA expression was higher than compared to spontaneous differentiation to EBs.
VASA expression was comparable in cells that were growing as aggregates in ScCM or in inserts with indirect contact with a Sertoli cell feeder layer. Both conditions gave better results than when the hESC were plated directly on the Sertoli cell feeder layer.
Both the addition of BMPs or ScCM could increase VASA expression when compared to spontaneous differentiated hESC. However, a combination of these two components did not have an additional effect on the germ cell differentiation.

Discussion: As reported previously, we show that hESC can differentiate towards the germ cell lineage. Mouse ScCM can increase the expression of the germ cell marker VASA, comparable to the effect of BMP addition. However, the proportion of cells undergoing this differentiation remains low and therefore enrichment of these cells would be necessary if used for further experiments. A thorough analysis of the ScCM to identify the factor(s) responsible for the increase in VASA expression could give new insight in the molecular mechanisms controlling germ cell differentiation in vivo. Also, further exploration of the characteristics of the obtained germ-like cells is required. For our study, immunostainings for POU5F1, C-Kit or SCP-3 in combination with VASA are ongoing to determine the exact time point of germ cell differentiation of the VASA-expressing cells within the EBs.