# The Molecular Signature of Human Testicular Peritubular Cells Revealed by Single-Cell Analysis
## 3 Results
### Human Testicular Peritubular Cells Are a Testicular Subtype of Smooth Muscle Cells  
The contractile abilities, i.e. a key feature of SMCs, are retained by the isolated HTPCs [19; 35], and HTPCs were described to structurally resemble SMCs [9]. A number of distinct SMCs exist, which share typical marker combinations, as it has been recently reviewed [36]. The robust presence of the prototype SMC markers _ACTA2_, _VIM_, _SM22A_ (_TAGLN_), calponin (_CNNI_), caldesmon (_CALD1_) and smoothelin (_SMTN_) were not or only marginally affected by the absence/presence of FCS, and this reveals the SMC character of the HTPCs (Figure 3). Additional SMC markers [36], namely, _SMMHC_ (_MYH11_), desmin (_DES_), myocardi (_MYOCD_), and serum response factor (_SRF_) or (not shown) _CSPG4_, _RGSS_, _EZH2_ and _LGR5_, were expressed only by a few subtypes of HTPCs, and they did not notably change upon FCS addition to the medium. _PDGFRB_, which is found in vascular SMCs, but not in airway or intestinal SMCs [36], was present in the HTPCs. Of note, this receptor is also considered to be a marker for myofibals [37], and it was increased in the absence of FCS. The corresponding proteins were represented in the peritubular cells in situ as indicated by images provided by the Human Protein Atlas. Table 1 depicts a comparison of our data (HTPC-1/HTPC-2; no FCS) with the ex vivo data of [PERSON] et al. [3] and [PERSON] et al. [17]. Human Testicular Peritubular Cells Are Producers of ECM, Which Is a Constituent of the Peritubular Wall  
Known ECM components of the human peritubular wall compartment include DCN, BGN and COL1A1, among others [10; 35; 38]. Figure 4 shows that _BGN_ and _DCN_ expression levels are increased in the absence of FCS in the culture medium. The corresponding proteins are represented in the peritubular cells in situ (Human Protein Atlas). These ECM components were also previously readily found, specifically, in the secretome ofHTTPCs [39]. Hence, they are produced by HTTPCs, and their production is regulated. 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 2: (**A**): UMAP plot of samples derived from HTTC-1 with and without FCS. (**B**): Venn diagram indicates overlap of both of the samples. The exposure of HTTCs to FCS results in the expression of 1505 additional genes. (**C**): Cell cycle stages [31] display one distinct proliferating cluster in the FCS sample. (**D–F**): Four distinct states of cellular differentiation were observed, indicating plasticity and proliferation. (**D**) Dot plot demonstrates expression of most abundant genes in each cluster. (**E**) Velocity analysis was embedded into the PAGA graph, which indicates trajectory of the proliferating cluster into three functional subgroups. (**F**) Representative GO terms after string analysis with upregulated genes. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Gene Name** & **Description** & **HTPC-1** & **HTPC-2** & **TPCs of [PERSON] et al. [17]** & **TPCs of [PERSON] et al. [3]** \\ \hline _ACTA2_ & Smooth-muscle actin & \(\surd\) & \(\surd\) & \(\surd\) & \(\surd\) \\ _MYH11_ & Myosin heavy chain 11 & \(\surd\) & \(\surd\) & \(\surd\) & \(\surd\) \\ _TAGLN_ & Transgenic & \(\surd\) & \(\surd\) & \(\surd\) & \(\surd\) \\ _CNN1_ & Calponin 1 & \(\surd\) & \(\surd\) & \(\timesd\) & \(\surd\) \\ _CALD1_ & Caldesmon 1 & \(\surd\) & \(\surd\) & \(\surd\) & \(\surd\) \\ _SRF_ & Serum response factor & \(\surd\) & \(\surd\) & \(\surd\) & \(\surd\) \\ _MYOCD_ & Myocardi & (\(\surd\)) & \(\timesd\) & \(\surd\) & \(\surd\) \\ _VIM_ & Vimentin & \(\surd\) & \(\surd\) & \(\surd\) \\ _DES_ & Desmin & \(\surd\) & \(\surd\) & \(\surd\) & \(\surd\) \\ _SMTN_ & Smoothelin & \(\surd\) & \(\surd\) & \(\surd\) & \(\surd\) \\ _PDGFRB_ & Platelet derived growth factor & \(\surd\) & \(\surd\) & \(\surd\) & \(\surd\) \\  & receptor B & & & & \\ \hline \hline \end{tabular}
\end{table}