# The Molecular Signature of Human Testicular Peritubular Cells Revealed by Single-Cell Analysis
## 4 Discussion
Especially, the expression of _ACTA2_, _TAGLN_ and _SMTN_ provide support for the SMC character [36; 54; 55]. Tesis specificity is indicated, among others, by the expression of steroid receptors and steroidogenic enzymes, e.g., _AR_, _STAR_ and _CYP11A1_, which to our knowledge are not known in this combination in other human SMC types [56]. Of note, PDGFR has been reported in some types of SMCs (i.e., vascular SMCs), but it is considered also to be a myofibroblast marker [55]. It has been identified in the HTPCs together with PDGFRA, and it was also detected in situ [3; 17]. This receptor is down-regulated by the presence of FCS in the HTPCs, further indicating the dynamic and plastic nature of these cells. The changes associated with FCS in the medium indicated that the HTPCs are able to respond to stimulatory clues with an altered contractile phenotype and an altered receptor expression. Of note, other receptors, e.g., _AR_, _NR3C1_ or _EGFR_, were not or not strongly affected by FCS. PDGFRA and CD34, but of note not ACTA2, are thought to be typical for telocytes, and they have been reported in the human testis [57; 58]. The present results, thus, do not indicate that they are present in the cultured HTPCs. ECM is present between the layers of perittubular cells in the human testis, normally, and its composition and ECM amounts change in idiopathic male infertility when, for example, the DCN and BGN levels increase [10; 40]. This most likely has consequences as, e.g., high testicular BGN levels can promote a pro-inflammatory response of HTPCs [40]. The ECM of the perittubular wall is produced by the HTPCs, and our data clearly support this notion and further reveal the repertoire of ECM components, which also includes collagens and other proteoglycans (these are not shown). The changes to _BGN_ and _DCN_ upon the addition of FCS indicate that HTPCs respond to clues with altered ECM production. The expression of the MSC markers (namely, _THY1_ (_CD90_), _ENG_ (_CD105_) and _NT5E_ (_CD73_)) were also observed [45], prompting us to attempt differentiation studies. However, our attempts to differentiate the cultured HTPCs to bone or fat cells failed. _TCF21_ expression became evident. This factor, among others [46; 47; 59; 60; 61; 62; 63], was implicated with the LSCs, and this led us to attempt further differentiation studies. Following a recently published protocol [34], we performed such differentiation studies using HTPCs from two additional patients. However, this did not lead to the induction of a Leyd cell phenotype. The _STAR_ levels were increased, but the markers _INSL3_ and _LHR_ remained undetectable. Hence, the potential of the HTPCs to differentiate into mature LCs remains to be further studied. Of note, the HTPCs expressed _CYP1A1_ and _STAR_. We have previously shown [41] that forskolin, when it is added to the HTPCs, increased _STAR_ and _CYP1A1_ levels and this resulted either in the induction or in the elevation of the secretion of pregnenolone, and in some cases also of progesterone. Hence, HTPCs have the intrinsic ability to produce these steroids, but they most likely cannot synthesize androgens. It is possible that failure of HTPCs to differentiate into LCs or fat/bone cells could be due to the age of the donors and thus possible senescence-related changes of LSC and MSC [64; 65]. This has not yet been reported with regard to LSCs, to our knowledge, yet there are reports indicating that the Leydig cell population decreases in the testes of old men [66]. As the HTPCs were from human donors, studies with HTPCs from young men are not readily possible and we, therefore, are not able to further investigate these points. While HTPCs are known to be important for sperm transport [19], HTPCs also contribute to spermatogenesis. GDNF is a known growth factor, which is important for SSC renewal [43; 44; 67; 68], and its expression was confirmed in our study. It appeared not to be affected by FCS.