Characterization of stem cells associated with seminiferous tubule of adult rat testis for their potential to form Leydig cells

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Characterization of stem cells associated with seminiferous tubule of adult rat testis for their potential to form Leydig cells Xiaoju Guan , Xingxing Zhao , Xinrui Hao , Fenfen Chen , Panpan Chen , Minpeng Ji , Xin Wen , Han Lin , Leping Ye Ph.D. , Haolin Chen Ph.D. PII: DOI: Reference:

S1873-5061(19)30223-5 https://doi.org/10.1016/j.scr.2019.101593 SCR 101593

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Stem Cell Research

Received date: Revised date: Accepted date:

27 April 2019 12 August 2019 17 September 2019

Please cite this article as: Xiaoju Guan , Xingxing Zhao , Xinrui Hao , Fenfen Chen , Panpan Chen , Minpeng Ji , Xin Wen , Han Lin , Leping Ye Ph.D. , Haolin Chen Ph.D. , Characterization of stem cells associated with seminiferous tubule of adult rat testis for their potential to form Leydig cells, Stem Cell Research (2019), doi: https://doi.org/10.1016/j.scr.2019.101593

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Characterization of stem cells associated with seminiferous tubule of adult rat testis for their potential to form Leydig cells Xiaoju Guana,b,*, Xingxing Zhaoa, *, Xinrui Haoa, Fenfen Chenb, Panpan Chenb, Minpeng Jia, Xin Wenb, Han Lina,c, Leping Yed,#, Haolin Chena,b,c,# a

Department of Anesthesiology, Perioperative Medicine, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027, China b

Department of Gynecology and Obstetrics, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027, China c

Zhejiang Province Key Lab of Anesthesiology, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027, China d

Department of Pediatrics, Peking University First Hospital, No.1 Xi'an Men Street, West District, Beijing, 100034, China

Running title: Characterization of CD90+ Stem Leydig Cells *These authors contributed equally to the work. #

To whom correspondence should be addressed:

Haolin Chen, Ph.D. The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Building 11-723, 109 Western Xueyuan Road, Wenzhou, Zhejiang, 325027, China Email: [email protected] Phone: 86-13566251050 Leping Ye, Ph.D. Department of Pediatrics, Peking University First Hospital, No.1 Xi'an Men Street, West District, Beijing, 100034, China 1

Email: [email protected] Phone: 86-13957708960 Disclosure Statement: The authors have nothing to disclose.

Classification: Biological Sciences: Cell Biology

Author contributions: HL, LY and HC designed the research; XG, XZ, XH, FC, PC, MJ and XW conducted the experiments; XG, XZ and HC analyzed the data; and XG, LY and HC wrote the paper.

Highlights 

CD51+ cells of adult mice testis contain two sub-populations.



The weakly-positive CD51 cells in adult mice testis are stem Leydig cells.

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The strongly-positive CD51 cells in adult mice testis are macrophages.

Abstract Adult testicular Leydig cells arise from stem cells in the neonatal and adult testis. The nature of these stem Leydig cells (SLCs) have not been well characterized. We have found previously that a group cells expressing CD90, a cell surface glycoprotein that may play roles in cell-cell and cell-matrix interactions and associated with the seminiferous tubule surface, have the ability to form Leydig cells. As yet, the relationship between this CD90+ cell population and SLCs reported previously by other groups is still unknown. In the present study, we systematically characterized these CD90+ cells by their ability to express multiple potential 2

SLC markers and to proliferate and differentiate into Leydig cells in vitro. First, we have found by qPCR and immunohistochemical staining that the CD90+ cells do not express any of the markers of the common seminiferous tubular cells, including myoid, Sertoli, germ and Leydig cells, as well as macrophages. Moreover, when the CD90+ cells were isolated by fluorescent-sorting, the cells expressed high levels of all the potential SLC marker genes, including Nestin, Cd51, Coup-tf2, Arx, Pdgfra and Tcf21. Also, CD90-positive, but not -negative, cells were able to form Leydig cells in vitro with the proper inducing medium. Overall, the results indicated that the tubule-associated CD90+ cells represent a population of SLC in adult testis.

Keywords: Stem Leydig cells, CD90, Testosterone, Seminiferous tubules, Testis

Highlights 

CD90+ cells of seminiferous tubules do not express common testicular cell markers.



CD90+ cells of seminiferous tubules do express common stem Leydig cell markers.



CD90+ cells of seminiferous tubules can form Leydig cells in vitro.

Abbreviations SLC: Stem Leydig cells CD90: Cluster of differentiation 90 (Thy-1) GFRA1: GDNF family receptor alpha-1: 3

ACTA2: Alpha-actin-2 (alpha smooth muscle actin) COUP-TFII: COUP transcription factor 2 (NR2F2) ARX: Aristaless related homeobox gene CD51: Cluster of differentiation 51 (Integrin alpha V) p75NTR: p75 Neurotropin receptor PDGF: Platelet-derived growth factor PDGFRα: Platelet-derived growth factor receptor alpha TCF21: Transcription factor 21 (POD-1) F4/80: An antigen marker for macrophage (EMR1, ADGRE1) SAG: Smoothened agonist EGF: Epidermal growth factor LIF: Leukemia inhibitory factor IGF-1: Insulin-like growth factor FBS: Fetal bovine serum CYP11A1: Cytochrome P450, family 11, subfamily A, polypeptide 1 (P450scc) EDS: Ethane dimethane sulfonate ITS: Insulin/transferrin/selenite tissue culture supplement HBSS: Hank's balanced salt solution BMP-4: Bone morphogenetic protein 4 LH: Luteinizing hormone 4

DHH: Desert hedgehog SOX9: SRY (sex determining region Y)-box 9 DDX4: DEAD-box helicase 4 (VASA) RPS16: Ribosomal protein S16

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1. Introduction Testicular Leydig cells are the primary source of testosterone in males. Testosterone is essential for the development of the male reproductive system and for the maintenance of male reproductive functions (Nef and Parada, 2000; Smith and Walker, 2014). Testosterone deficiency in the adult may also contribute to a broad range of symptoms, including changes in body composition, decrease in muscle mass, increased fatigue, depressed mood, decreased cognitive function (Wu et al., 2010; Huhtaniemi, 2014; McHenry, 2012), and reduced immune response (Malkin et al., 2004; Bobjer et al., 2013). The maintenance of a functional Leydig cell population, and therefore of normal levels of serum testosterone throughout adult life, is of fundamental importance. In rodents as well as humans, testosterone production gradually increases from the peripubertal period through the adult, coincident with the development of adult Leydig cells (Teerds and Huhtaniemi, 2015). In previous studies, we and others have found that adult Leydig cells developed from a group of stem Leydig cells (SLC) during puberty (Ge et al., 2006). There was strong evidence to suggest that similar SLCs were also present in the adult testis (Stanley et al., 2012). However, these cells have not been well characterized. Cluster Differentiation 90 (CD90) is a cell adhesion molecule belonging to the immunoglobulin superfamily. Since the protein was initially identified in thymocytes, it is also referred to as thymocyte differentiation antigen-1 (Thy-1). CD90 is a cell surface glycoprotein that may play roles in cell–matrix and cell–cell adhesion. Since expression of the protein is often associated with stem cells of various tissues, such as pancreas (Stevenson et al., 2009), endometrium (Cheng et al., 2017), dental pulp (Ngoc Tran et al., 2017), bone marrow (Calloni et al., 2013; Logan et al., 2012), mesenchyme (Sousa et al., 2014) and cancers (Shaikh et al., 2016), it has been frequently used as a marker to identify stem cells. In a previous study, we have found that a group of CD90 expressing cells associated with the seminiferous tubule surface have the ability to give rise to Leydig cells (Li et al., 2016). 6

However, there have also been reports that spermatogonia stem cells may express CD90 (Hou et al., 2011). Also, macrophages that were found associated with seminiferous tubule surface (DeFalco et al., 2015) have similar morphology to that of the CD90+ cells identified by us. The relationships among the CD90+ cells with these other seminiferous tubule-associated somatic- or germcells are still unclear. In addition to CD90, several protein markers have been reported for SLCs in neonatal or adult testes. These include Nestin (Jiang et al., 2014; Davidoff et al., 2004), COUP-TFII (Kilcoyne et al., 2014; Qin et al., 2008), ARX (Miyabayashi et al., 2013), CD51 (Zang et al., 2017), p75NTR (Zhang et al., 2017), PDGFRα (Ge et al., 2006; Landreh et al., 2013) and TCF21 (Barsoumand Yao, 2010). However, since these markers were identified by cells across different species and/or different ages, it is still unclear whether these markers are expressed universally by a single group of SLCs or by different cell populations, since recent studies suggest that multiple origins for Leydig cells may exist (Rotgers et al., 2018; Kumar and DeFalco, 2018; Shima et al., 2018). In the present study, we have systematically characterized the CD90+ cells we have identified by examined their expressions of all these markers reported previously. Our results indicated that the tubule surface associated CD90+ cells do express high levels of all the potential SLC marker genes, but do not express any of the known markers for the seminiferous tubule-associated differentiated cells. These results suggest strongly that the CD90+ cells associated with seminiferous tubule surface represent a homogenous SLC population that expresses all the major potential SLC markers reported so far.

2. Materials and methods 2.1. Chemicals

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Culture media (M-199, DMEM/F12), Hank's Balanced Salt Solution (HBSS) and PBS were purchased from Invitrogen (Carlsbad, CA). Smoothened Agonist (SAG) was from EMD Bioscience (Milwaukee, WI). ITS (insulin/transferrin/selenite) were from Sigma (St. Louis, MO). BSA was from MP Biochemicals (Solon, OH). Chicken embryo extract was from US Biologicals (Salem, MA). B-27and N-2 Supplements, Non-Essential amino acids and Type IV collagenase were from Thermo Fisher (Carlsbad, CA). Rat PDGFAA was obtained from R&D Systems (Minneapolis, MN). Murine FGF2, EGF, PDGFBB and Oncostatin-M were from Peprotech (Rocky Hill, NJ). [1,2,6,7,16,17-3H(N)]-Testosterone (115.3 Ci/mmol) was from Perkin Elmer Life Sciences, Inc (Boston, MA). Testosterone antibody was from ICN (Costa Mesa, CA). Human LH was from MyBiosource (San Diego, CA). Ethane dimethane sulfonate (EDS) was synthesized according to the method described by Jackson and Jackson (1984). The manufacture of the antibodies used are listed in Supplemental File Table S1. 2.2. Animals and treatments Adult male Sprague Dawley rats of 90 days of age were purchased from Shanghai Animal Centre (Shanghai, China). Rats were housed in the animal facilities of the Second Affiliated Hospital of Wenzhou Medical University at 22°C, 12-hour light, 12hour dark with free access to water and rat chow. All animal procedures were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University and were performed in accordance with the Guide for the Care and Use of Laboratory Animals of NIH (NIH publication #85-23, revised in 1985). To eliminate Leydig cells from the testes, rats were injected with a single dose of EDS (i.p., 80 mg/kg of BW) dissolved in a mixture of DMSO:PBS (1:3). Testes were collected 4 days after EDS treatment, by which time all adult Leydig cells had been eliminated (Jackson and Jackson, 1984). 2.3. Isolation and culture of seminiferous tubules

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Seminiferous tubules were dissected with fine forceps from the testes of normal untreated (control) or 4-day post EDS treated rats under a transillumination dissection microscope (Kotaja et al. 2004).Tubules were stained with CD90-FITC or CD90-PE antibodies immediately after their isolation or after culture with PDGFAA (10ng/ml) or FGF2 (10ng/ml) for 7 days in vitro. To assay the ability of CD90+ cells to form Leydig cells, equal lengths (5 cm) of isolated tubules with or without one-week PDGFAA or FGF2 treatment were further differentiated in vitro for 2 two weeks at 34C and 5% CO2 with a Leydig cell inducing medium. The medium contained DMEM/F-12 supplemented with 0.1% BSA, 15 mM HEPES, 2.2 mg/ml sodium bicarbonate, penicillin/streptomycin (100 U/ml/100 μg/ml), 1 X ITS, 5mM lithium chloride and 10 ng/ml LH. At the end of experiments, media were collected and frozen at -20°C for testosterone measurement. Testosterone levels were determined by radioimmunoassay (RIA).The sensitivity and intra-assay and inter-assay coefficients of variation of the RIA were 13 pg/tube and 8.9 and 13.6%, respectively. Duplicate wells were used for each treatment, and each experiment was repeated at least three times. 2.4. Purification and culture of CD90+ cells by flow cytometry To isolate CD90+ cells, the isolated tubules were digested with 1 mg/ml collagenase-IV in DMEM/F12 medium at 34C for 30 min with slow shaking (90 cycles/min). After allowing seminiferous tubules to settle, the dispersed cells were filtered through a 50µm pore nylon mesh and stained by CD90-PE antibody (1:100 in Ca2+/Mg2+-free HBSS, 0.5% BSA, 5mM EDTA) for 45 min, and then sorted by flow cytometry (MoFlo Sorter, Beckman-Coulter, Brea, CA). Two groups of cells (CD90- and CD90+) were collected and expanded with a modified protocolas published previously (Jiang et al., 2014; Li et al., 2016). Cells were cultured in DMEM/F12 medium containing 0.1% BSA, 0.5 nM dexamethasone, 1X ITS, 0.5 ng/ml LIF, 2.5% chicken embryo extract, 50 μM β-mercaptoethanol, 0.5 % nonessential amino acids, 0.5% N2 and 1% B27 supplements, 10 ng/ml FGF2, 10 ng/ml EGF, 10 ng/ml PDGFBB, 10 ng/ml oncostatin-M and 2.5% FBS. The cells were cultured 9

at 34C with 5% CO2. The medium was changed every 3 days. When cells reached about 90% confluence, they were switched to Leydig cell differentiation inducing medium containing LH (10ng/ml) and DHH agonist SAG (0.5μM). After 3 weeks, the medium was collected for the testosterone assay. Some of the freshly digested cells were stained with PDGFRα primary antibody (1:200) followed by Alexa Fluor 488-secondary antibody (1:1000). After 3 washings, the cells were suspended in Ca2+/Mg2+-free HBSS (0.5% BSA and 5 mM EDTA) for flow cytometric sorting (Li et al., 2016). The CD90+ and PDGFRα+ cells were then cultured for 48 hours and co-stained each for PDGFRα and CD90-PE. Double positive cells were then counted under a microscope with an eyepiece grid under 20X objective for each isolation. The single-positive and double-positive cells were expressed as percentages of the total cells counted. Experiments were repeated 3 times. A total of 4 fields were counted for each individual experiment. 2.5. Immunofluorescence and HSD3β activity staining. To stain tubule surface CD90 cells, fresh or cultured tubules were washed with Ca2+and Mg2+ free PBS (0.5% BSA) and then incubated with CD90-PE or CD90-FITC antibody (1:100) for 45 min. In some experiments, positive cells were counted along the surface of the tubules and expressed as the number per unit. The unit was defined as a square area with the four sides of the square equal to the diameter of a given tubule. For each treatment, at least 60 square areas were counted from three different experiments. For 2 color co-staining, seminiferous tubules or cultured cells were washed with Ca2+and Mg2+ free PBS (0.5% BSA) and then incubated with CD90-PE or CD90-FITC antibody (1:100) for 45 min. After washing 3 times with PBS, the tubules or cells were then fixed with formalin for 30 mins and incubated with primary antibodies for PDGFRα, ACTA2, F4/80 or GFRA1 for 60 min followed by incubation with Alexa Fluor-conjugated second antibodies (1:1000) for 45 min. After three washes, the tissues were examined under a Nikon Eclipse 800 microscope and photos were taken with a Princeton Instruments 5-Mhz cooled CCD camera, 10

custom CRI color filter, and IP-Lab digital image analysis software (Scanalytics). To get clear photos of spermatogonia cells, a small piece of tubule was immerged into 50ul BSA solution after staining. A piece of coverslip was then carefully applied on top. The siphon effect from the coverslip squeezed germ cells out of both ends of the tubule. The cells were then examined under microscope. For enzymatic staining of HSD3β, cultured cells were dried at room temperature for 30 minutes. The samples were then stained for 45 min with a solution containing 0.4 mM 5β-androstan-3β-ol-17-one steroid substrate, 1 mg/ml NAD, and 0.2 mg/ml tetranitro blue tetrazolium, as described previously (Stanley et al., 2012). 2.6. RNA extraction and qPCR Total RNA was extracted from whole tubules or cells right after isolation by cell sorting, using an RNeasy minikit (Qiagen) according to the manufacturer’s protocol. The concentrations of RNAs were measured by reading OD values at 260 nm by a NanoDrop 2000 (Thermo Scientific). Total RNA was used as the template for cDNA synthesis primed with random hexamers (Bio-Rad, 170-8890). The reaction mixture was incubated at 42C for 30 min followed by 5 min at 85C. SYBR Green qPCR Kit (Takara, Otsu, Japan) was used to analyze the mRNA levels of testicular cell marker genes, including Cd51, Pdgfra, Coup-tf2, Arx, Nestin, Tcf21,Cd90, Sox9, Ddx4, F4/80, Cyp11a1 and Acta2. The PCR reaction mixture contained 7.5 μl SYBR Green mix, 1.5 μl forward and reverse primer mix, 0.02 μg diluted cDNA, and 4 μl RNase-free H2O. The procedure of qPCR was set as the following: 95C for 5 min, followed by 40 cycles of 95C for 10 s, and 60C for 30s. The house-keeping gene, ribosomal protein S16 (Rps16), was used as the internal control. The mRNA level of each gene was read as the Ct value and calculated using a double delta Ct method with Rps16 as an internal reference. The primers were synthesized by Generay Biotechnology Inc (Shanghai, China). The sequences are listed in Supplemental Table S2. 11

2.7. Statistical analyses Data are expressed as the mean ± standard error of the mean (SEM). For the comparisons of two groups, a student t test was used. For the comparisons of multiple groups one-way ANOVA was applied. If group differences were revealed by ANOVA (P<0.05), differences between individual groups were determined with the Student-Neuman-Kuels test, using Sigma Stat software (Systat Software Inc., Richmond, CA). Values were considered significant at P<0.05. 3. Results 3.1. Proliferation and differentiation of seminiferous tubule-associated CD90+ in vitro. When the seminiferous tubules of adult rat testis were dissected and stained with CD90-PE antibody, positive cells with square to polygon-shapes were clearly visible (Fig 1A). If the focus of the microscope was adjusted to the sides of the tubule, the CD90+ cells on the edges showed clearly with a very thin cytoplasm lining along the tubule surface (Fig 1B). Since EDS treatment of the rats completely eliminates adult Leydig cells in vivo, the treatment can trigger a wave of SLC proliferation, which significantly increases SLC numbers (Chen et al., 2015). Indeed, pretreatment of animals with EDS 4 days in advance significantly increased the number of CD90+ cells (Fig 1C vs 1B). When the tubules of EDS-treated rats were cultured in basal, serum-free medium (DEME/F12 with 1X ITS) for a week in vitro, the number of CD90+ cells doubled compared to freshly isolated tubules (Fig 1D vs 1C). However, adding the known SLC mitogens PDGFAA (Fig 1E) or FGF2 (Fig 1F) to the culture medium increased the numbers of the CD90+ cells even more dramatically. Interestingly, unlike PDGFAA which does not affect cellular shape notably, FGF2 affected cell morphology significantly (Fig 1F). Cells adopted elongated “fibroblast-like” shapes after one week of FGF2 treatment. Also, the cells accumulated in multiple-layers along the tubular surface probably due to the extensive proliferation. These results indicated that the tubule surface-associated CD90+ cells responded to the two most important SLC mitogens 12

PDGFAA and FGF2. The number of CD90+ cells were also quantified from 3 repeated experiments (Fig 1G). To further examine whether these CD90+ cells can be induced to form Leydig cells, the tubules with different in vivo or in vitro treatments were cultured for 2 more weeks in the presence of Leydig cell differentiation-inducing medium (LH + lithium). As expected, the testosterone concentrations accumulated in the medium by the end of differentiation were in proportional to the number of CD90+ cells formed by various treatments (Fig 1H vs 1G). These results further support the conclusion that the CD90+ cells associated with tubule surface belong to a stem Leydig cell population.

3.2. Do tubule-associated CD90+ cells express typical SLC lineage markers? In order to further characterize the tubule-associated CD90+ cells, freshly isolated tubules from EDS-treated rats were digested with collagenase to release peritubular cells. The cells were then stained with CD90-PE antibody (Fig 2C) and further isolated with fluorescence-activated cell sorting (Fig 2A, 2B). Since testicular cells have high auto-fluorescence, it is necessary to correct signals for background readings to avoid false positive cells. We used FITC channel (X-axis) readings to correct the possible interference of background signals in PE channel (Y-axis). For the unstained cell preparations, all cells located along the diagonal line, suggesting that the auto-fluorescence distributes evenly across the two channels (Figs. 2A), which makes it possible to use FITC channel readings to correct PE channel signals. For the cells stained with CD90-PE, one group of the cells appeared on the top of the diagonal line, suggesting their specific staining for the CD90-PE antibody (Figs.2B, 2.3±0.4%). The cells below the diagonal line were collected as CD90-negative cells. The isolated cells (CD90+) were checked again under fluorescent microscope and consistently found to be more than 95% pure (Fig 2D). We then examined the CD90- and CD90+ cells for their expression of the typical SLC marker genes, including Cd51, Pdgfra, Coup-tf2, Arx, Nestin and Tcf21. RNAs from the raw tubules were included as a control. As shown in Figure 3, all the 6 potential 13

SLC marker genes were highly expressed by CD90+ cells while they were either undetectable or expressed at significantly reduced levels by RNAs from the CD90- cells or the tubules. To further confirm these gene expression results, we reciprocally costained with the marker proteins of PDGFRα and CD90 with the cells sorted by CD90-PE antibody or PDGFRα antibody (Fig 4). For the cells sorted with CD90 antibody (Fig 4A1-A3), 95.2% were PDGFRα positive (Fig 4A3, 4C). In contrast, only 74.7% of the cells sorted with PDGFRα antibody (Fig 4B) were CD90+ (Fig 4B, 4C), suggesting that CD90-cells were included in the PDGFRα+ population. Overall, these results strongly support the conclusion that the CD90+ cells present as typical SLCs using an array of known markers, and CD90 itself is an excellent marker for these tubule-associated SLCs. 3.3. Do CD90+ cells express markers of other tubular cells? Seminiferous tubule contains multiple lineages of somatic cells and various developing stages of germ cells, and it has been suggested that the CD90+ cells could represent cells other than pure SLCs. We first checked whether CD90+ cells express any marker genes for these cells (Fig 5). First, as expected, the CD90+ and CD90- cells indeed expressed differentiated levels of the Cd90 gene itself, which confirmed the purity of the sorted cells and validated the qPCR and cell-sorting methods (Fig 5A). We then looked at 5 genes known to be expressed by other testicular cells but not SLC, such as Ddx4 (germ cells), Sox9 (Sertoli cells), F4/80 (macrophages), Cyp11a1 (Leydig cells) or Acta2 (myoid cells), and found that none of them was expressed at any significant level by CD90+ cells. Instead, they were expressed at high levels in either un-isolated tubular cells or CD90- cells. These results support the conclusion that CD90+ cells do not belong to any of these differentiated cell types. To further confirm some of these gene expression results, we have co-stained with the marker proteins of myoid cells (ACTA2), spermatogonia stem cells (GFRA1) or macrophages (F4/80) with the CD90-PE antibody (Fig 6, Fig S1). Staining of the freshly isolated tubules with ACTA2 antibody resulted in labeling a layer of large and round cells that cover the whole seminiferous tubule surface, a typical characteristic of peritubular myoid cells (Fig 6A1). CD90+ cells, however, were scattered 14

across the tubule surface individually or in small clusters (Fig 6A2). Clearly, there was no overlap between the two (Fig 6A3). Similarly, no co-staining was found between the macrophage marker F4/80 and CD90 (Fig S1). GFRA1 specifically detected the single, pair and aligned spermatogonia stem cells up to 8-cells chains (Fig 6B1) while CD90+ cells were much larger with mostly square to hexagon shapes (Fig 6B2). Again, there was no co-localization between the 2 markers (Fig 6B3). Early reports suggested that spermatogonia cells may also express CD90 (Hou et al., 2011). However, we did not find any significant staining of germ cells in the whole mount tubules. In order to approach this in more details, we carefully squeezed the germ cells out of tubule ends by gently applying a cover-slip on top of the isolated tubules. The germ cells outside of the tubule wall were indeed stained positively for CD90 (Fig 6C2), though very pale compared to the positive CD90+ cells stained on the tubular surface (Fig 6B2). Interestingly, some of these lightly positive cells also co-localized with GFRA1 staining (Fig 6C3), suggesting that the lightly stained CD90+ germ cells included early spermatogonia stem cells, as reported previously (Hou et al., 2011). Due to the significant differences in the intensity in CD90 staining, the lightly stained germ cells did not interfere with the strong staining of the tubule surface CD90+ cells during the cell sorting process. Overall, these results confirmed the qPCR results and indicated that the strong CD90+ cells associated with tubule surface is a specific population different from that of peritubular myoid cells, germ cells or tubule-associated macrophages. 3.4. Can tubule-associated CD90+ cells form Leydig cells in vitro To compare their ability to form Leydig cells, CD90- and CD90+ sorted cells were expanded in vitro for a week (Fig 7A, 7B). The CD90- cells spread very thin with large round shapes, a typical morphology of myoid cells or perivascular smooth muscle cells (Fig 7A). The cells divided little during the week of culture. The CD90+ cells, however, attached to the plate and spread-out quickly (Fig 7B). They appeared to proliferate actively. The morphology of CD90+ cells were similar to that of typical mesenchymal stem cells before differentiation (Fig 7B). However, after 3 weeks in culture with Leydig cell differentiation15

inducing medium (LH+SAG), the CD90+ cells rounded up (Fig 7C). Enzymatic- and immunohistochemical-staining indicated that these cells begin to express high levels of HSD3B (Fig 7D) and CYP11A1 (Fig 7E), the markers of Leydig cells. Also, the CD90+ cells, but not CD90- cells, began to produce testosterone in the culture medium after 3 weeks of differentiation, though both cell type produced no testosterone before the treatment (Fig 7F). Overall, these results support the conclusion that the CD90+ cells associated with seminiferous tubules are SLCs.

4. Discussion Adult stem cells have the capacity to repair damaged tissues and maintain tissue homeostasis. The observation that a new generation of Leydig cells forms after an experimental elimination of pre-existing Leydig cells from the adult rat testis with EDS (Jackson and Jackson, 1984; Chen et al., 2015) suggested that there must be stem cells in the adult testis that are capable of giving rise to new cells (Davidoff et al., 2004; Stanley et al., 2012). Indeed, putative SLCs have been identified and cultured from different adult animals, including rat (Ge, et al., 2006; Stanley et al., 2012; Landreh et al., 2013; Kilcoyne et al., 2014; Li et al., 2016), mice (Jiang et al., 2014; Zang et al., 2017) and human (Zhang et al., 2017; Landreh et al., 2014). These cells were identified based on different surface markers, appeared capable of self-renewing in vitro for prolonged periods of time, and also able to differentiate into testosterone-producing cells in vitro or in vivo (Zang et al., 2017; Zhang et al., 2017; Arora et al., 2019). However, it was unknown how consistent these markers were and whether the markers identified the same cells or different groups of cells. In the present study, we focused on CD90 positive cells associated with the seminiferous tubule surface in adult rat testis and further characterized the cells by their abilities to express all the potential SLC markers. Our results indicated that the peritubular CD90+ cells expressed high levels of all these reported SLC markers, including Nestin (Davidoff et al., 2004; Jiang et al., 2014), COUP-TFII (Kilcoyne et al., 2014; Qin et al., 2008), ARX (Miyabayashi et al., 2013), CD51 (Jiang et al., 2014; Zang et 16

al., 2017), PDGFRα (Ge, et al., 2006; Stanley et al., 2012; Landreh et al., 2013) and TCF21 (Barsoum et al., 2010). Also, the cells proliferated extensively in response to PDGFAA and FGF2, the two well-known SLC mitogens, and differentiated into testosterone-producing cells in the presence of LH and DHH agonist SAG. All the evidence supports the conclusion that the CD90+ cells associated with seminiferous tubules are SLCs. When the seminiferous tubules were isolated from normal adult rat testis, CD90+ cells were visible on the tubule surface, but the number was very limited. However, if the rats were given a dose of EDS, which eliminates all adult Leydig cells from the testis, the number of CD90+ cells increase significantly, suggesting that the cells began to divide in response to the loss of the Leydig cell population. This is consistent with the behavior of adult stem cells that are typically quiescent, but only begin to proliferate in response to tissue-damage signals. When the tubules were cultured in vitro, the cells kept dividing and numbers increased dramatically within a week, especially in the presence of PDGFAA or FGF2, two well-known SLC mitogens. These cells, although there were morphological changes in culture, particularly with FGF2, still maintained their potential to form testosterone producing cells when they were switched into Leydig cell differentiation inducing medium, and the testosterone production was proportional to the number of Leydig cell marker positive cells. These results support the conclusion that the CD90+ cells associated with tubule surface are SLCs. To further characterize the cells, we have sorted the cells and have the ability of the cells to express other SLC marker genes compared between the positive- and negative-populations. Interestingly, the positive cells expressed significantly higher levels of all available marker genes very consistently, and the negative cells did not. This was a little surprising to us since the changes were so consistent. It indicates that all these markers indeed identified the same group of cells, which is different from some of the previous evidence suggesting that there may be more than one group of Leydig producing SLCs in the testis. To further study the relationship between CD90+ cells and PDGFRα+ cells, we have sorted the two populations individually and reciprocally co17

stained the cells by the antibodies to the two proteins. The results indicated that almost all (95%) of CD90+ cells were also PDGFRα+ positive, but the reverse was not true. For the PDGFRα+ population isolated, only 75% were CD90+. This indicates that the PDGFRα+ population is larger than CD90+ population and the former contains the latter, which implies that CD90 could be a better marker than PDGFRα in identifying and isolating tubule-associated SLCs. The conclusion that CD90 is a good SLC marker is also supported by the observation that CD90+ cells did not express any of the marker genes for the known seminiferous tubular cells, including germ (Ddx4), Sertoli (Sox9), myoid (Acta2), macrophage (F4/80) or Leydig (Cyp11a1) cells, suggesting that CD90+ cells are a different population from any of these well-known differentiated cell types. Co-staining with the antibodies also confirmed some of the qPCR results. There was no co-staining found with ACTA2 (myoid cell), GFRA1 (spermatogonia stem cell) or F4/80 (macrophage) antibodies. However, we detected, though very faint, CD90 staining in the early stages of germ cells as had been shown previously (Hou et al., 2011). Due to the extremely low intensity, we did not detect any interference from the germ cells in the sorting process for the CD90+ SLCs, however. The conclusion that the tubule-associated CD90+ cells are SLC was further reinforced by the observation that the sorted CD90+ cell population was capable of forming testosterone producing cells in vitro. In the presence of a relatively simple medium (LH plus DHH signaling agonist SAG for 3 weeks), CD90+ cells, but not CD90- cells, began to express Leydig cell marker proteins HSD3B and CYP11A1 and to produce testosterone. Cell surface marker proteins play critical roles in the identification and isolation of stem cells from the complex cell mixtures of various organs. CD90 (Thy-1) is a 25–37 kDa, heavily N-glycosylated, glycophosphatidylinositol anchored cell surface protein with a single V-like immunoglobulin domain. CD90 has been used frequently to identify and/or purify stem/progenitor cells from various tissues, such as pancreas (Stevenson et al., 2009), endometrium (Cheng et al., 2017), dental pulp (Ngoc Tran et al., 2017), bone marrow (Calloni et al., 2013; Logan et al., 2012), mesenchyme (Sousa et al., 2014) and cancers (Shaikh et al., 2016).The 18

function of the protein has not yet been fully elucidated. It has been speculated that the protein plays multiple roles in cell-cell and cell-matrix interactions, with implications in neurite outgrowth, nerve regeneration, apoptosis, metastasis, inflammation, and fibrosis. In stem cells, CD90 may play a role in maintaining the “stemness” of the cells, since reduction in CD90 expression was associated with differentiation of the cells (Sibov et al., 2012; Moraes et al., 2016). The full role played by CD90 in SLCs, however, is still unclear and is currently under study in our laboratory. In human testis, a group of CD90+ cells were identified with the ability to establish colonies and expand in vitro (Smith et al., 2014). Further characterization of the cells revealed distinct mesenchymal characteristics with the ability to differentiate into adipocytes, chondrocytes and osteocytes. The cells were also capable of functioning as feeder cells in supporting the survival and differentiation of spermatogonia stem cells in vitro (Smith et al., 2014). However, since the study did not carefully characterize the cells’ potential using appropriate markers, it is still unclear whether these cells were capable of giving rise to adult Leydig cells and whether these cells are the human version of our CD90+ cells described here. The relationship between the two cells deserves further study. 5. Conclusions In summary, we have identified a group of CD90+ cells associated with the surface of seminiferous tubules that showed the characteristics of SLC with the ability to undergo extensive proliferation in vitro and to differentiate to give arise to testosterone producing cells. The cells expressed high levels of most of known SLC marker genes, but not any of the markers for the known seminiferous tubular cell types. The evidences suggest that these CD90+ cells represent a population of SLC. Funding: This work is supported by grants from Chinese central or local governments, including National Natural Science Foundation 81471448 (HL), 81771635 (LY), Research Fund for Lin He’s Academician Workstation of New Medicine and 19

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Li, X., Wang, Z., Jiang, Z., Guo, J., Zhang, Y., Li, C., Chung, J., Folmer, J., Liu, J., Lian, Q., Ge, R., Zirkin, B.R., Chen, H., 2016. Regulation of seminiferous tubule-associated stem Leydig cells in adult rat testes. Proc. Natl. Acad. Sci. USA. 113, 2666–2671. Logan, A.C., Weissman, I.L.,Shizuru, J.A., 2012. The road to purified hematopoietic stem cell transplants is paved with antibodies. Curr. Opin. Immunol. 24, 640-648. Malkin, C.J., Pugh, P.J., Jones, R.D., Kapoor, D., Channer, K.S., Jones, T.H., 2004. The effect of testosterone replacement on endogenous inflammatory cytokines and lipid profiles in hypogonadal men. J. Clin. Endocrinol. Metab. 89, 3313–3318. McHenry, M.C., 2012. Testosterone deficiency in older men: a problem worth treating. Consult. Pharm. 27, 152-163. Miyabayashi, K., Katoh-Fukui, Y., Ogawa, H., Baba, T.,Shima, Y., Sugiyama, N., Kitamura, K., Morohashi, K.,2013. Aristaless related homeobox gene, Arx, is implicated in mouse fetal Leydig cell differentiation possibly through expressing in the progenitor cells. PLoS One 8, e68050. Moraes, D.A., Sibov, T.T., Pavon, L.F., Alvim, P.Q., Bonadio, R.S., D.a., Silva, J.R., Pic-Taylor, A., Toledo, O.A., Marti, L.C., Azevedo, R.B., Oliveira, D.M., 2016. A reduction in CD90 (THY-1) expression results in increased differentiation of mesenchymal stromal cells. Stem Cell Res. Ther. 7, 97. Nef, S., Parada, L.F., 2000. Hormones in male sexual development. Genes Dev. 14, 3075-3086. Ngoc, Tran, T.D., Stovall, K.E., Suantawee, T., Hu, Y., Yao, S., Yang L.J., Adisakwattana, S., Cheng, H., 2017. Transient receptor potential melastatin 4 channel is required for rat dental pulp stem cell proliferation and survival. Cell Prolif. 50, e12360. Qin, J., Tsai, M.J., Tsai, S.Y., 2008. Essential roles of COUP-TFII in Leydig cell differentiation and male fertility. PLoS One 3, e3285. Rotgers, E., Jørgensen, A., Yao, H.H., 2018. At the crossroads of fate-somatic cell lineage specification in the fetal gonad. Endocr. Rev. 39, 739-759.

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Figure Legends

Figure 1. CD90 expressing cells associated with seminiferous tubules and their ability to form testosterone producing cells. CD90+ cells on the tubule-surface of control (A, B) or EDS-treated (C-F) rat testis. The freshly isolated tubules (C) were cultured in vitro for one week with basal (D) or PDGFAA (E) or FGF2 (F) stimulation. The CD90+ cells were quantified (G) and their ability to produce testosterone (H) was compared in vitro by culture of the tubules with Leydig cell differentiation medium (LH plus lithium) for 3 weeks. The data are expressed as mean±SEM from cells of 4 individual experiments. ND, not detectable. *Significantly different from testis controls at P<0.05. Bar, 50μm. Figure 2. Flow cytometry sorting of CD90 positive cells. (A) Collagenase digested testicular cells without CD90-PE antibody staining were analyzed by PE and FITC channels. (B) CD90 stained cells were separated into two groups: negative (-) cells and positive (+) cells. (C) CD90-PE stained cells before sorting. (D) CD90+ cells after sorting. 24

Figure 3. Expression of the reported SLC marker genes of the sorted cells. RNAs from whole seminiferous tubules were used as controls. The positive (CD90+) cells consistently expressed significantly high levels of all the reported SLC marker genes, including Cd51, Pdgfra, Coup-tf2, Arx, Nestin and Tcf21. The tubules and CD90- cells expressed low or undetectable levels of the marker genes. The data are expressed as mean±SEM from cells of 4 individual experiments. ND, not detectable. *Significantly different from tubule controls at P<0.05. Figure 4. Co-staining of CD90 and PDGFRα of cells sorted by CD90-PE (A1-A3) or PDGFR-Alexa Fluor 488 (B) antibodies. The CD90-sorted cells were co-stained with CD90-PE and PDGFR-Alexa Fluor 488 antibodies and were examined with PE (A1) and Alexa Fluor 488 (A2) filters. The photos taken with the two filters was also merged (A3, red for CD90 and green for PDGFR). The PDGFR-sorted cells were co-stained with CD90-PE and PDGFR-Alexa Fluor 488 antibodies and the photos taken with the two filters was merged (B, red for CD90 and green for PDGFR). Double-positive cells were quantified from the two sorted populations and were expressed as the percentages of the sorted markers. The data are expressed as mean±SEM from cells of 3 individual experiments. Bar, 50μm. Figure 5. Expressions of marker genes of seminiferous tubular cells by the CD90-PE sorted cells. RNAs from the whole tubules were used as controls. (A) As expected, CD90+ cells expressed high Cd90 gene and the CD90- cells expressed undetectable level of the gene. In contrast, the CD90+ cells were either negative or low in the expressions of marker genes of seminiferous tubular cells, including Sox9 (Sertoli), Ddx4 (germ), F4/80 (macrophage), Cyp11a1 (Leydig) and Acta2 (myoid). These genes were highly expressed by either tubular cells or negative cells. The data are expressed as mean±SEM from cells of 4 individual experiments. ND, not detectable. *Significantly different from testis controls at P<0.05.

25

Figure 6. Co-staining of ACTA2 (myoid cell) or GFRA1 (spermatogonia stem cells) with CD90 (SLC) on whole mounted seminiferous tubules of EDS-treated rats. The ACTA2-Alexa Fluor/CD90-PE co-stained tubules were examined with Alexa Fluor-488(A1) or PE (A2) filters. The photos taken with the two filters was merged (A3, green for ACTA2 and red for CD90). GFRA1-Alexa Fluor/CD90-PE co-stained tubules were examined with Alexa Fluor-488 (B1, C1) or PE (B2, C2) filters. The photos taken with the two filters was also merged (B3, C3; green for GFRA1 and red for CD90). C1-C3 were germ cells “squeezed” out of tubules. Note that the spermatogonia stem cell bridges were broken (B1, B3 vs C1, C3). Bars, 50μm. Figure 7. Culture and differentiation of CD90- and + cells in vitro. The sorted cells were expanded in vitro for a week (A, CD90- ; B, CD90+). The CD90+ cells were differentiated with Leydig cell differentiation-inducing medium for 3 weeks (C). Enzymatic(HSD3B, D) and immunohistochemical-(CYP11A1, E) staining of the differentiated CD90+ cells. The CD90+ cells, but not CD90- cells, began to produce testosterone in the cultured medium after 3 weeks differentiation, whereas both cells produced no testosterone before differentiation (F). The data are expressed as mean±SEM from cells of 3 individual experiments. ND, not detectable. *Significantly different from CD90- cells at P<0.05. Bars, 50μm.

26

A

B

C

D

E

F

G

H

*

*

* *

*

* * 27

Fig 1

A CD90PE Log

B

FL1 Log

FL1 Log

C

Fig 2

D

28

*

0.15 0.10

0.05

0.06 0.05 0.04 0.03 0.02

ND

0.00

Tubule

CD90-

CD90+

7.0

Arx

6.0

*

5.0 4.0 3.0 2.0 1.0 0.0 Tubule

CD90-

CD90+

E mRNA (Ratio to RSP16X103)

mRNA (Ratio to RSP16X103)

D

0.07

0.01

0.00

*

Tubule

mRNA (Ratio to RSP16)

0.20

Pdgfra

0.08

CD90-

CD90+

Nestin

*

3.0 2.0

*

1.0

0.05

Coup-tf2

*

CD90-

CD90+

0.04 0.03 0.02 0.01

Tubule

F

5.0 4.0

0.06

0.00

0.25

mRNA (Ratio to RSP16)

Cd51

C

B 0.09

0.25

mRNA (Ratio to RSP16)

mRNA (Ratio to RSP16)

A

Tcf21 0.20

*

0.15 0.10

0.05

ND 0.00

0.0 Tubule

CD90-

Fig 3 29

CD90+

Tubule

CD90-

CD90+

A1

B

A2

A3

C

PDGFRα+ among CD90+ fraction

95.2±3.6%

30

Fig 4

CD90+ among PDGFRα+ fraction

74.7±5.1%

*

0.04 0.03 0.02 0.01

0.01

0.01 0.01 0.00 0.00

*

*

0.00

0.00 Tubule

CD90-

Tubule

CD90+

E

F4/80 (Macrophage) 0.20

0.15 0.10

0.05

* 0.00

mRNA (Ratio to RSP16)

mRNA (Ratio to RSP16)

Sox9 (Sertoli)

ND

D 0.25

mRNA (Ratio to RSP16)

0.05

C

CD90-

0.12

Cyp11a1 (Leydig)

0.10

0.08 0.06 0.04 0.02

*

ND

CD90-

CD90+

0.00 Tubule

CD90-

CD90+

CD90+

Testis

Fig 5 31

0.45

Ddx4 (Germ)

0.40 0.35 0.30

*

0.25 0.20 0.15 0.10

*

0.05 0.00 Tubule

F mRNA (Ratio to RSP16)

mRNA (Ratio to RSP16)

Cd90

mRNA (Ratio to RSP16)

B0.01

A 0.06

CD90-

CD90+

0.16

Acta2 (myoid)

0.14 0.12 0.10

0.08 0.06 0.04 0.02 0.00 Tubule

CD90-

CD90+

A1

A1

A2

A3

B1

B2

B3

C1 B

C2

C3

Fig 6

32

A

D

C

B

E

F

*

ND

Fig 7 33