# The Molecular Signature of Human Testicular Peritubular Cells Revealed by Single-Cell Analysis
## 3 Results
### Human Testicular Peritubular Cells Are a Source of Factors Involved in the Regulation of Spermat  
The factors that are produced by murine testicular peritubular cells and HTPCs can influence the SSC (e.g., GDNF) and spermatogenesis [43; 44]. In the HTPCs, heterogeneity and weak regulatory influences of FCS were found for _GDNF_, _CXCL12_, _LIF_ and _NGF_, as well as for _ALDHIA1_ and _ALDHIA3_ (Figure 6). GDNF, CXCL2 and NGF production were previously verified [40; 44], and they showed variability with regard to their production by different patient-derived HTPCs. At this point in the study, we addressed the possibility that retinoic acid may be derived from HTPCs. The biosynthetic enzymes (ALDH1A1 and ALDH1A3) are also represented by the HTPCs in situ (Human Protein Atlas). Retinoic acid production by the HTPCs in vitro was confirmed (\(n=2\)). Higher levels were found in the supernatant of the cells without FCS in the culture medium, i.e., a condition, in which also the levels of _ALDHIA1_ were elevated. Table 1 (Appendix B) shows the comparison of our data (HTPC-1/HTPC-2; no FCS) with the data of [PERSON] et al. [3] and [PERSON] et al. [17]. Figure 4: ECM components. UMAP plots of ECM components produced by HTPCs and typically found in the peritubular wall, namely, _BGN_, _DCN_ and _COLLA1_ (**left side**). Violin plots comparing expression with and without FCS of HTPC-1 (**middle**) and corresponding protein detection with HPA images (**right side**; scale bars = 50 \(\upmu\)m). Please, note that _BGN_ and _DCN_ levels are increased in the absence of FCS in the culture medium. A comparison between HTPCs and ex vivo data is provided in Table 1.  
Human Testicular Peritubular Cells Express Mesenchymal Stromal Cell Markers and Leydig Stem Cell Markers  
Based on the minimal criteria for defining multipotent MSCs by [PERSON] et al. [45], MSCs must be plastic adherent when they are maintained in standard culture conditions, must express CD105 (ENG), CD73 (NT5E) and CD90 (THY1), and they must lack the expression of CD45 (PTPRC), CD34, CD14 or CD11B (ITGAM), CD79A or CD19 and HLA-DR (HLA-DRA/HLA-DRB1) surface molecules. These criteria are met by HTTCs, in general, although some cells were found to express _CD34_, _CD14_, _CD79A_ or _CD19_. Of note, the expression of _ENG_, _NT5E_ and _THY1_ was increased when FCS was added to the culture medium (Figure 7). The available images of the immunostined peritubular cells that are provided in the Human Protein Atlas support this pattern of expression. Table A3 (Appendix B) shows the comparison of our data (HTPC-1/HTPC-2; no FCS) with the data of [PERSON] et al. [3] and [PERSON] et al. [17]. Figure 5: Receptors and ion channels. UMAP plots of previously described receptors and ion channels of HTTCs (**left side**). Violin plots comparing expression with and without FCS of HTTC-1 (**middle**) and corresponding protein detection with HPA images (**right side**; scale bars = 50 \(\upmu\)m). No distinct differences in the expression levels are apparent when FCS was absent in the culture medium. A comparison between HTTCs and ex vivo data is provided in Table A3 (Appendix B). A third criterium, namely, that the MSCs must differentiate into osteoblasts, adipocytes and chondroblasts in vitro was not confirmed as our attempts to differentiate HTPCs to adipocytes and bone cells were not successful (Table A4, Appendix B). The human peritubular wall is being considered to be a niche for Leydig stem cells (LSC). A number of markers for LSCs had been previously suggested in human and nonhuman species. They include THY1 (CD90), CD51 (ITGAV), COUP-TFII (NR2F2), PDGFRA, TCF21 and Endoglein (ENG) [46; 47]. These were readily detected, yet Nestin (_NES_) and _ARX_ were not readily found. The available images of the immunostagined peritubular cells that are provided in the Human Protein Atlas support this expression (Figure 7). Figure 6: Summary of factors relevant for spermatogenesis.