Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions

ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE 1 2 3 4 5 6 7 8 9 10 11 12 a,b 13 Charlotte Rombaut, M.Sc.,c Carlos Simon, M.D.,b Antonio Pellicer, M.D.,a Q4 Jose V. Medrano, Ph.D., 14 and Ellen Goossens, Ph.D.c 15 a
n Sanitaria La Fe (IIS La Fe), Valencia, Spain; b Fundacio
n Instituto Reproductive Medicine Unit, Instituto de Investigacio 16 Valenciano de Infertilidad (FIVI), INCLIVA, Department of Pediatrics, Obstetrics and Gynecology, Valencia University, 17 Valencia, Spain; and c Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Brussels, Belgium 18 19 20 21 22 Objective: To study the ability of human spermatogonial stem cells (hSSCs) to proliferate in vitro under mouse spermatogonial stem 23 cells (mSSCs) culture conditions. Design: Experimental basic science study. 24 Setting: Reproductive biology laboratory. 25 Patient(s): Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a 26 prostate cancer treatment. 27 Intervention(s): Testicular cells used to create in vitro cell cultures corresponding to the following groups: [1] unsorted human testic28 ular cells, [2] differentially plated human testicular cells, and [3] cells enriched with major histocompatibility complex class 1 (HLA
)/ 29 epithelial cell surface antigen (EPCAMþ) in coculture with inactivated testicular feeders from the same patient. Main Outcome Measure(s): Analyses and characterization including immunocytochemistry and quantitative reverse-transcription 30 polymerase chain reaction for somatic and germ cell markers, testosterone and inhibin B quantification, and TUNEL assay. 31 Result(s): Putative hSSCs appeared in singlets, doublets, or small groups of up to four cells in vitro only when testicular cells were 32 cultured in StemPro-34 medium supplemented with glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor 33 (LIF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Fluorescence-activated cell sorting with HLA
/ 34 EPCAMþ resulted in an enrichment of 27% VASAþ/UTF1þ hSSCs, compared to 13% in unsorted controls. Coculture of sorted cells 35 with inactivated testicular feeders gave rise to an average density of 112 hSSCs/cm2 after 2 weeks in vitro compared with unsorted 36 cells (61 hSSCs/cm2) and differentially plated cells (49 hSSCS/cm2). However, putative hSSCs rarely stained positive for the proliferation marker Ki67, and their presence was reduced to the point of almost disappearing after 4 weeks in vitro. 37 Conclusion(s): We found that hSSCs show limited proliferation in vitro under mSSC culture conditions. Coculture of HLA
/EPCAMþ 38 sorted cells with testicular feeders improved the germ cell/somatic cell ratio. (Fertil SterilÒ 2016;-:-–-. Ó2016 by American Society 39 for Reproductive Medicine.) 40 Key Words: Human spermatogonial stem cells, in vitro propagation, male fertility preservation 41 Discuss: You can discuss this article with its authors and with other ASRM members at 42 43 44 45 46 perm banking is the gold standue to a disorder or gonadotoxic medisperm cells before puberty, this strategy 47 dard for fertility preservation in cal therapies employed against cancer cannot be applied to prepubertal cancer 48 men at risk of germ cell damage (1). However, due to the lack of mature patients undergoing gonadotoxic treat49 ments. On the other hand, because 50 prepubertal testes already contain sperReceived May 11, 2016; revised June 17, 2016; accepted July 11, 2016. 51 J.V.M. has nothing to disclose. C.R. has nothing to disclose. C.S. has nothing to disclose. A.P. has matogonial stem cells (SSCs) with the nothing to disclose. E.G. has nothing to disclose. 52 potential to initiate complete spermatoSupported by a Sara Borrell grant conceded to J.V.M. (CD12/00568) by the Instituto de Salud Carlos III 53 (ISCIII) and a grant from VUB/UZ Brussel (Scientific Fund Willy Gepts) (to E.G.). genesis (2–6), the cryopreservation Reprint requests: Ellen Goossens, Ph.D., Biology of the testis (BITE), Faculty of Medicine & Pharmacy, 54 of testicular tissue has been applied Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels (Jette), Belgium (E-mail: ellen. 55 [email protected]). in several health centers around 56 the world as a fertility preservation Fertility and Sterility® Vol. -, No. -, - 2016 0015-0282/$36.00 57 strategy for prepubertal boys at risk of Copyright ©2016 Published by Elsevier Inc. on behalf of the American Society for Reproductive 58 Medicine germ cell depletion (7–9). http://dx.doi.org/10.1016/j.fertnstert.2016.07.1065 59

Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions

S

VOL. - NO. - / - 2016

1

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:57 pm
ce E

60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118

ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177

Although not yet applied as clinical procedure in humans, resident SSCs within prepubertal tissue have shown their ability to completely regenerate spermatogenesis after germ cell transplantation in several animal models in vivo, including primates (6, 10–14). Mature sperm cells obtained either by germ cell transplantation or alternative in vitro maturation strategies can be employed for fertilizing oocytes by assisted reproduction technologies and achieve pregnancies (7–9, 15). However, due to the limited efficiency of these techniques, their success in generating mature sperm depends on the quality and number of SSCs employed (16). Moreover, due to the small size of the testes in prepubertal patients, the amount of prepubertal testicular tissue and thus the number of SSCs within is limited. Taking all this together, in vitro propagation of SSCs is presented as an essential initial step in fertility restoration strategies (9). Long-term in vitro propagation of mouse SSCs (mSSCs) was first reported in 2003 (17), and it has been replicated by several groups as a model for human SSCs (hSSCs) (18, 19). For their in vitro survival and proliferation, mSSCs were found to be dependent of glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), secreted by somatic cells in the SSC niche within the testis in vivo (17). More recently, a model for longterm in vitro propagation of hSSCs was reported based on mSSC requirements (20, 21). In the proposed model, a differential plating of testicular cells based on different abilities of the germ/somatic cell fractions to attach to plastic allowed the enrichment of germ cells that were cultured for up to 4 months and gave rise to two different types of stem cells in vitro: cell clusters of hSSCs and human embryonic stem cell-like (hESC-like) colonies. However, long-term in vitro propagation of hSSCs has been shown difficult to replicate, and the germ cell identity of the hESC-like phenotype has been questioned in other reports (22–24). Indeed, in a recent report by our group replicating the same experimental conditions and using a combined approach of two markers to unequivocally identify germ cells in vitro, we observed a limited survival of germ cells along time, accompanied with an overgrowth of the somatic fraction (25). Here, we characterize the phenotype of the cell populations that appear in vitro after culturing adult human testicular samples to study whether mSSC culture conditions are sufficient to promote the survival and proliferation of hSSCs in vitro for the long term. For this, we compared the phenotype of whole human testicular cell suspensions and differentially plated testicular cells cultured in standard and in mSSC culture conditions along time. Also, we prevented the in vitro somatic fraction overgrowth by coculturing enriched spermatogonial cells based on their major histocompatibility complex class 1 (HLA
)/epithelial cell surface antigen (EPCAMþ) phenotype (26, 27) onto g-irradiated testicular somatic cells employed as mitotically inactivated testicular feeders. Our results may shed light on the controversy of in vitro amplification of hSSCs and help future studies establish a robust model for hSSC in vitro culture.

MATERIALS AND METHODS Human Testis Material Testicular tissue from three donors undergoing bilateral orchiectomy as part of a prostate cancer treatment was split in small fragments of 3–6 mm3 and subjected to uncontrolled slow freezing in Dulbecco's minimum essential medium/ Ham's F-12 medium (GIBCO/Life Technologies) with 10% fetal bovine serum (GIBCO/Life Technologies), 0.15 M sucrose (VWR), and 1.5 M dimethyl sulfoxide (Sigma-Aldrich) as previously described elsewhere (28). All testicular samples used in this study showed normal spermatogenesis as proven by histology before and after cryopreservation (data not shown). This study was approved by the ethics committee of UZ Brussel (approval no. 2013/185).

Testicular Cell Isolation and Culture Cryopreserved samples were quickly thawed at 37 C and washed in Dulbecco's minimum essential medium/Ham's F-12 medium before enzymatic tissue digestion for single-cell isolation. Briefly, testicular tissue pieces were mechanically dissociated into 0.5–1.0 mm3 pieces and subjected to a twostep enzymatic digestion with collagenase/hyaluronidase/ trypsin (Sigma-Aldrich) as previously described elsewhere (29). The resulting single-cell suspensions were filtered through nylon membranes with a pore diameter of 30 mm (Durviz), counted in a hemocytometer, and split into three different experimental set-ups (Fig. 1A). [1] In the unsorted whole testicular culture setup (whole culture), cell suspensions were directly seeded with a density of 10,000 cells/cm2 onto culture plates and cultured at 37 C, 5% CO2 in either standard medium (SM) or mouse spermatogonial stem cells medium (mSSCM) until confluence for 14 days. [2] In the differential plating group, single cells were seeded with a density of 10,000 cells/cm2 onto culture plates overnight at 37 C, 5% CO2 in SM for differential attachment of somatic cells. The nonattached germ cell enriched fraction was subsequently seeded in new culture plates. After differential plating separation of cells, both attached (attached cells) and nonattached (differentially plated cells) cell fractions were cultured at 37 C, 5% CO2 in either SM or mSSCM until confluence for 14 days. Additionally, some confluent plates from the attached fraction cultured in mSSCM were gamma-irradiated at 50 Gy in a Vero SBRT irradiator machine for their mitotic inactivation and used as testicular feeders for the third experimental setup. [3] In a third experimental setup, single-cell suspensions were employed for spermatogonial enrichment by fluorescence-activated cell sorting (FACS) as described later. After FACS, the sorted cells were seeded onto inactivated testicular feeders and cultured at 37 C, 5% CO2 in mSSC medium for up to 28 days. All media were replaced by fresh media every 2–3 days. Composition of SM and mSSCM can be found in Supplemental Tables 1 and 2 (available online).

Gene Expression Analysis Total RNA from the cell cultures was extracted with the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Additionally, total RNA from intact frozen/thawed VOL. - NO. - / - 2016

2

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:57 pm
ce E

178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236

Fertility and Sterility®

FIGURE 1

print & web 4C=FPO

237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295

Experimental design and characterization of the phenotype of human testicular cells in vitro. (A) Experimental design of the study with the three main experimental conditions tested: [1] differential plating, [2] whole-cell culture, and [3] fluorescence-activated cell sorting (FACS) enrichment of human spermatogonial stem cells (hSSCs) and coculture with inactivated testicular feeders. (B) Illustrative pictures of the phenotype of human testicular cells in vitro over time. (C) Time-course analysis at day 7 and 14 of culture in both standard medium (SM) and mouse spermatogonial stem cells (mSSC) medium for the mRNA expression of testicular somatic cell markers. Relative expression found in frozen/thawed testicular samples is also shown as a reference of physiologic expression. Data are presented as mean  standard error. Statistically significant differences: *P<.05 and **P<.01. (D) Illustrative pictures of the stainings for aSMA, STAR, and SOX9 on human testicular cells in vitro at day 14. Medrano. Limited proliferation of hSSCs in vitro. Fertil Steril 2016.

tissue was extracted with TRIzol reagent (Life Technologies) and used as positive control for quantitative reversetranscription polymerase chain reaction (qRT-PCR). After RNA quantification by Nanodrop (ThermoFisher), 1 mg of total RNA was used for reverse transcription into cDNA using the first-strand cDNA synthesis kit (GE Healthcare) with NotId(T)18 primers according to the manufacturer's instructions. Gene expression was assessed using the ViiA 7 Real-Time PCR System (Life Technologies) and normalized to the average of the housekeeping gene glucuronidase beta (GUSB) with the 2
DCt method. The PCR program consisted of 2 minutes at 50 C, followed by 10 minutes at 95 C for the activation of Taq polymerase, and 40 PCR cycles comprising 15 seconds at 95 C for cDNA denaturalization

and 1 minute at 60 C for elongation. The list of Taqman Gene Expression assays (Life Technologies) and probes/ primers employed (IDT) are shown in Supplemental Table 3 (available online).

Immunostainings Cytospins from FACS and cultured cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature. In the case of intracellular antigens, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich). Samples were blocked with 4% of serum (of the animal in which the secondary antibody was raised) in phosphate-buffered saline (PBS) with 1% bovine serum albumin and 0.05% Tween-20 (PBS-

VOL. - NO. - / - 2016

3

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:57 pm
ce E

296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354

ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413

T) for 1 hour at room temperature, followed by incubation of primary antibodies in PBS-T (Supplemental Table 4, available online). A dilution of 1:1,000 of secondary Alexa fluor antibodies (Life Technologies) in PBS was added to the slides, which were then incubated for 1 hour in darkness at room temperature before mounting with ProLong Gold antifade reagent with 40 ,6-diamidino-2-phenylindole (Life Technologies). Negative controls were performed by omission of the first antibody. Mounted samples were examined using an Olympus IX81 inverted microscope with cell^F software, version 2.8 (Olympus). For VASA/UTF1 cytospin cell counts, the phenotype of all cells found in six 10 fields was considered. For VASA/UTF1 cultured cell counts, seven P24 stained wells/sample were considered.

DNAse I, and 25 mM HEPES (Sigma-Aldrich), pH 7.0. To isolate putative spermatogonial populations (26), the cells were stained with phycoerythrin-conjugated anti-human EPCAM and allophycocyanin-conjugated anti-human HLAABC antibodies following the manufacturer's instructions (Biolegend) for 30 minutes on ice protected from light. Stained cells from each patient were sorted into four different tubes using a FACSARIA III FACS machine (BD Biosciences) through a 85-mm nozzle. Sorted cells were directly seeded onto inactivated confluent testicular feeders from the same donor at a density of 5,000–10,000 cells/cm2 and cultured in mSSC medium at 37 C, 5% CO2 for up to 28 days. A small fraction of 10,000–20,000 cells from each sorted tube was employed for cytospin preparations as previously described elsewhere (31).

Proliferation and Apoptosis Assay To analyze the proliferation of cells in vitro, culture wells were stained for Ki67 antigen (30) as described in the previous section and in Supplemental Table 4. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Roche) was applied following the manufacturer's instructions for the detection of apoptosis in cultured cells. Wells from each culture condition were incubated with DNAse I as positive control for the technique.

Hormone Measurements Free testosterone and inhibin B concentrations were quantified in culture medium conditioned for 48 hours with the cells in culture. Testosterone was measured with the Elecsys Testosterone II competitive immunoassay in a Cobas 6000 instrument (Roche Diagnostics). We incubated 20 mL of sample with a biotinylated monoclonal testosterone-specific antibody. After 9 minutes, streptavidin-coated microparticles and a testosterone derivative labeled with a ruthenium complex were added so that the formed complex would bind to the solid phase via interaction of biotin and streptavidin. The reaction mixture was aspirated into the measuring wells, where the microparticles were magnetically captured onto the surface of the electrode. Unbound substances were then removed with ProCell/ProCell M. Application of a voltage to the electrode induced chemiluminescent emission, which was measured by a photomultiplier with a functional sensitivity of the assay of 0.12 mg/L. Serum inhibin B was measured using a solid-phase sandwich enzyme-linked immunoassay (ELISA) (Inhibin B Gen II Elisa; Beckman Coulter). The samples were incubated in microtiter plates coated with a monoclonal antibody against anti-activin-B. After incubation and washing, the wells were incubated with the substrate tetramethylbenzidine. The absorbance was measured at 450 nm with a limit of quantification of 4.8 ng/L.

Fluorescence Activated Cell Sorting (FACS) Single cells were suspended in a concentration of 1,000,000 cells every 100 mL of FACS staining buffer consisting of Hank's balanced salt solution with 1% fetal bovine serum (GIBCO/Life Technologies), 1 mM EDTA, 100 mg/mL

Statistics Statistical analysis of qRT-PCR results and cell counts was performed with one-way analysis of variance and Student's t test pairwise comparisons by SPSS software (SPSS Inc.). P< .05 was considered statistically significant.

RESULTS Characterization of Cultured Human Testicular Somatic Cells Reveals Phenotypic Differences Due to the Culture Conditions In a first set of experiments, we tested the phenotype of human testicular cells cultured under mSSCs propagation conditions with mSSC medium (mSSCM) compared with a standard culture medium with serum (SM). For that, testicular cell suspensions were seeded onto plastic culture plates and maintained at 37 C, 5% CO2 until first confluence at day 14 in both media compositions. In parallel, we performed differential plating of testicular cells as previously described elsewhere (20) and cultured both cell fractions in the same way described above (see Fig. 1A). As a result of differential plating, we observed an attachment rate of 64.33%  8.76%. In agreement with previous reports, we also observed the formation of a few cell clusters and flattened cell aggregations resembling hESC colonies during the first 10 days of culture both in SM and mSSCM (20–23). However, the number of both cell clusters and hESC-like colonies decreased over time and almost disappeared at day 14, at the same time that fibroblast-like cells became confluent. The TUNEL assay demonstrated very few apoptotic cells either at day 3, 7, or 14, indicating that the disappearance of cell clusters and hESC-like colonies was due to cell dilution and not a cell death phenomenon (Supplemental Fig. 1, available online). Compared with SM, testicular cells cultured in mSSCM acquired a more spindleshaped morphology and became confluent earlier, forming a homogeneous monolayer of fibroblast-like cells with some single round cells on top (see Fig. 1B). Time-course qRT-PCR analysis at 7 and 14 days of culture indicated a significant up-regulation of the peritubular cell markers a2 actin (ACTA2) (32) and Desmin (DES) (33) in SM-cultures compared with mSSCM cultures, and this VOL. - NO. - / - 2016

4

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:57 pm
ce E

414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472

Fertility and Sterility® 473 up-regulation increased over time. The Leydig cell marker ste474 roidogenic acute regulatory protein (STAR) (34) was signifi475 cantly up-regulated in all mSSCM cultures at day 14, 476 whereas other Leydig cell markers such as 3b-hydroxysteroid 477 dehydrogenase-isomerase (HSD3B1) (35) and insulin-like 478 factor 3 (INSL3) (36) showed an initial up-regulation only in 479 whole culture-mSSCM at day 7. The Sertoli cell marker SRY 480 box 9 (SOX9) (37) was up-regulated in all mSSCM conditions 481 without evident changes over time, whereas GATA binding 482 protein 4 GATA4 (38) expression increased over time in SM, 483 and Wilm's tumor antigen 1 (WT1) (39) expression was 484 down-regulated over time in all culture conditions (see 485 Fig. 1C). 486 Further immunocytologic staining analysis for the peri487 tubular cell marker ACTA2, the Leydig cell marker STAR 488 and the Sertoli cell marker SOX9 supported the presence of 489 the three somatic populations in all culture conditions tested 490 (see Fig. 1D). Also, vimentin staining (33) supported the so491 matic identity of most of the cells in all culture conditions 492 (Fig. 2B). 493 Taking all these observations together, confluent cultures 494 at day 14 showed very similar phenotype among samples, 495 despite some notable differences due to differential plating 496 in the case of INSL3 and HSD3B1, indicating that the effect 497 of differential plating disappeared as somatic cells became 498 confluent. However, results indicate a different somatic cell 499 composition due to the culture medium employed that may 500 influence their ability to support the growth of germ cells. 501 502 Human Germ Cells Are Able to Survive In Vitro 503 Only under mSSC Culture Conditions 504 505 We next analyzed the germ cell presence in testicular cell cul506 tures. Expression analysis by qRT-PCR indicated a statisti507 Q1 cally significant up-regulation of the germ cell markers 508 deleted in azoospermia-like (DAZL) (40) and DDX4 (VASA) 509 only in mSSC conditions, especially in the whole culture at 510 D7 and in the differentially plated cells at D14 (see Fig. 2A). 511 Additionally, spermatogonial markers promyelocytic leuke512 mia zinc finger protein (PLZF, also known as ZBTB16) (41), 513 undifferentiated embryonic cell transcription factor 1 514 (UTF1) (42), and fibroblast growth factor receptor 3 (FGFR3) 515 Q2 (43) showed a statistically significant enrichment in whole 516 culture mSSCM at D7, indicating the presence of spermatogo517 nial cells at the earlier stages of culture in mSSCM. However, 518 the expression levels of putative spermatogonial markers 519 decreased at D14, with the exception of FGFR3 showing com520 parable levels between D7 and D14 in SM. In the case of ubiq521 uitin carboxyl-terminal esterase l 1 (UCHL1) (44), all 522 conditions shared a similar profile (see Fig. 2A). 523 We identified the presence of putative hSSCs by costain524 ing cultured cells with the germ cell marker VASA (45, 46) in 525 combination with the putative SSC markers UTF1 and UCHL1. 526 Double positive cells (VASAþ/UTF1þ and VASAþ/UCHL1þ) 527 presented a small round morphology on top of somatic cells 528 only in mSSCM cultures, suggesting that SM was not an 529 appropriate environment to support hSSCs in vitro. These 530 putative hSSCs were negative for vimentin, supporting their 531 germ cell origin, and appeared in singlets, doublets, and

even groups of four cells (see Fig. 2B). No evidence of VASA positive staining was presented in cell clusters and hESC-like colonies (data not shown). It is interesting that whereas all the observed VASA positive cells coexpressed UTF1 and UCHL1, we found that UCHL1 was also expressed by most of the somatic testicular cells in vitro, supporting our previous results and highlighting that UCHL1 may not be a good marker for in vitro cultured hSSCs (25). Thus, we employed the VASAþ/UTF1þ phenotype to identify and quantify the number of hSSCs in vitro, observing that both whole culture (62 cells/cm2) and differentially plated cells (50 cells/cm2) conditions had higher amounts of VASAþ/UTF1þ cells/cm2 than the attached cells condition (8 cells/cm2) at day 14 of culture. However, the number of VASAþ/UTF1þ cells in whole culture and differentially plated cells conditions did not statistically significantly differ (Fig. 3D). Moreover, the number of VASAþ/UTF1þ increased from an average of 2 putative hSSCs/cm2 at day 7 to an average of 62 hSSCs/cm2 at day 14, whereas this number only reached an average of 8 hSSCs/cm2 in the attached cell fraction after differential plating (see Fig. 3B). However, the colocalization rate of VASA and UTF1 with the proliferation marker Ki67 (47) was very low, indicating the quiescent behavior of hSSCs after 14 days in vitro (see Fig. 3A).

FACS of Human Testicular Cells Based on HLAL/ EPCAMD Phenotype Results in Spermatogonial Enrichment Although somatic cells are necessary for supporting SSC survival (24, 25, 48), some reports have highlighted that their overgrowth may be deleterious for hSSC in vitro proliferation (25, 48, 49). To avoid this somatic overgrowth, in a following set of experiments we enriched the population of hSSCs within testicular cell suspensions and coculture them onto mitotically inactivated testicular feeder cells from the same patient (see Fig. 1A). We selected a combination of antibodies for the major histocompatibility complex class 1 (HLA-ABC) and for the epithelial cell surface antigen (EPCAM) to stain testicular cells and sort out enriched hSSC populations by flow cytometry (26) (Fig. 4A). Further colocalization staining of VASA and UTF1 allowed us to quantify the percentage of somatic cells (VASA
/UTF1þ and VASA
/UTF1
cells), hSSCs (VASAþ/ UTF1þ cells), and the rest of germ cells (VASAþ/UTF1
cells) within sorted populations (see Fig. 4B and C; Supplemental Fig. 2, available online). Our results indicated an enrichment of VASAþ/UTF1þ cells within the HLA
/EPCAMþ sorted cells compared with unsorted testicular cells (27% versus 13%). Moreover, the total fraction of HLAþ sorted cells showed a very low number of VASAþ cells (3%) compared with unsorted (28%) and HLA
sorted cells (43% in HLA
/EPCAMþ sorted cells and 20% in HLA
/EPCAM
sorted cells). However, VASA
cells were detected in all sorted populations ranging from 57% in HLA
/EPCAMþ sorted cells to 97% in the total fraction of HLA
sorted populations, indicating that this strategy may

VOL. - NO. - / - 2016

5

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:57 pm
ce E

532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590

ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE

FIGURE 2

print & web 4C=FPO

591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649

Identification and characterization of human spermatogonial stem cells (hSSCs) within human testicular cells in vitro. (A) Time-course analysis at day 7 and 14 of culture in both standard medium (SM) and mouse spermatogonial stem cells (mSSC) medium for the mRNA expression of germ cell markers. Relative expression found in frozen/thawed testicular samples is also shown as a reference of physiologic expression. Data are presented as mean  standard error. Statistically significant differences: *P<.05 and **P<.01. (B) Illustrative pictures of the staining colocalizations for VIM, VASA, UCHL1, and UTF1 on human testicular cells in vitro at day 14. Medrano. Limited proliferation of hSSCs in vitro. Fertil Steril 2016.

be useful to enrich but not to purify germ cells from testicular cell suspensions (see Fig. 4C). Cell suspensions from FACS were cocultured with mitotically inactivated testicular feeders from the same donor

in mSSCM for a period of up to 4 weeks. Additionally, sorted populations were seeded directly on plastic, resulting in very poor cell attachment in the case of HLA
/EPCAMþ and HLA
/EPCAM
sorted populations (data not shown). Due to VOL. - NO. - / - 2016

6

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:57 pm
ce E

650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708

Fertility and Sterility® FIGURE 3

print & web 4C=FPO

709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767

Proliferation analysis and quantification of hSSCs in vitro. (A) Illustrative staining colocalizations for Ki67, VASA, and UTF1 at day 14. (B) Time-course quantification at days 7 and 14 of the number of VASAþ/UTF1þ cells/cm2 in whole culture and attached cells conditions cultured in mouse spermatogonial stem cells (mSSC) medium. Due to low number of cells at day 7 in differentially plated cells condition, counts at day 7 are missed. (C) Time-course quantification at days 14 and 28 of the number of VASAþ/UTF1þ cells/cm2 in HLA
/EPCAMþ and HLA
/EPCAM
sorted cells cocultured in mSSC medium with inactivated testicular feeders. (D) Comparison of the number of VASAþ/UTF1þ cells/cm2 among culture conditions at day 14. Data are presented as mean  standard error. Statistically significant differences: *P<.05 and **P<.01. Medrano. Limited proliferation of hSSCs in vitro. Fertil Steril 2016.

VOL. - NO. - / - 2016

7

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:57 pm
ce E

768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826

ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE

FIGURE 4

print & web 4C=FPO

827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885

Results of HLA/EPCAM fluorescence-activated cell sorting (FACS) and coculture with inactivated testicular feeders in mouse spermatogonial stem cells (mSSC) medium. (A) Illustrative graph plots and descriptive statistics for the FACS of human testicular cell suspensions based on HLA/EPCAM double staining. (B) Illustrative picture of the VASA (red)/UTF1 (green) colocalization staining shown in cytospins from HLA
/EPCAMþ sorted cells. (C) Circular graphs showing the percentage of somatic cells (VASA
/UTF1þ and VASA
/UTF1
cells), hSSCs (VASAþ/UTF1þ cells), and the rest of germ cells (VASAþ/UTF1
cells) within HLA/EPCAM sorted populations. (D) Time-course analysis at day 14 and 28 for the mRNA expression of germ cell markers in coculture conditions. Relative expression found in frozen/thawed testicular samples is also shown as a reference of physiologic expression. Data are presented as mean  standard error. Statistically significant differences: *P<.05 and **P<.01. Medrano. Limited proliferation of hSSCs in vitro. Fertil Steril 2016.

the limited number of cells obtained from the HLAþ/EPCAMþ sorted population, we decided to merge them with the HLAþ/ EPCAM
and name this culture condition as HLAþ. The phenotype of cocultures was very similar to the one previously described for cultures in mSSCM.

On the other hand, molecular expression analysis revealed few differences in the somatic markers among experimental conditions, with a significant down-regulation over time for HSD3B1 and GATA4 in inactivated feeder controls (Supplemental Fig. 3, available online). However, we found VOL. - NO. - / - 2016

8

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:58 pm
ce E

886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944

Fertility and Sterility® 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003

a statistically significant up-regulation of germ cell markers DAZL and VASA in the coculture of HLA
/EPCAMþ cells at D14 that decreased at D28 until levels were comparable to the feeder control, suggesting a low germ cell presence in the late stages of coculture (see Fig. 4D). It is interesting that this up-regulation was not accompanied by the upregulation of any of the spermatogonial markers analyzed. Supporting our previous findings, we found that HLA
/ EPCAMþ sorting and coculture with inactivated feeders resulted in a statistically significant increase in the number of putative hSSCs after 14 days of coculture compared with previous experiments, with an average of 112 putative hSSCs/cm2 (see Fig. 3A). However, quantification of putative hSSCs at D28 resulted in a statistically significant decrease of their number until they had almost disappeared, supporting our qRT-PCR observations (see Fig. 3C). Further trypsinization of cultures followed by differential plating before reaching this loss of hSSCs was attempted, but any hSSC was found after passaging (data not shown).

Discussion Our results support previous findings that identified the presence of derivatives from the three main testicular somatic cell types (peritubular, Leydig, and Sertoli cells) in vitro (see Fig. 1C and D) (25). We observed a lower representation of peritubular cells in vitro when human testicular cells were cultured in mSSCM, indicating their preference to grow in a serum-enriched medium like SM. On the other hand, although the presence of Leydig cell derivatives was confirmed by STAR staining, it was surprising to see that this marker was up-regulated over time in mSSCM, whereas other Leydig cell markers such as INSL3 and HSD3B1 were downregulated and almost disappeared at day 14. Because the expression of STAR is necessary for the earliest stages of steroidogenesis in Leydig cells (34), the down-regulation of later steroidogenic actors such as HSD3B1 (35) and the functional marker of Leydig cell secretion INLS3 (36) may be indicative of an early blockade of Leydig cell functionality in vitro. Testosterone quantifications of media employed for testicular cell cultures indicated insignificant changes in the levels of testosterone compared with fresh medium controls, supporting this hypothesis (Supplemental Fig. 4, available online). However, it would be interesting to analyze if the in vitro stimulation of Leydig cells by the addition of LH/hCG to the culture medium may improve steroidogenic production. Finally, our data also support the presence of Sertoli cell derivatives in vitro during at least the first 14 days of culture. However, the cytoplasmic localization of SOX9 observed in vitro may suggest dedifferentiation of Sertoli cells because this pattern is typical of immature fetal Sertoli cells (50). Insignificant changes of inhibin B among conditioned culture media and fresh controls (see Supplemental Fig. 4) and the absence of a clear correlation among the expression levels of GATA4, WT1, and SOX9 may also be a direct consequence of the dysregulation of Sertoli cell derivatives due to dedifferentiation. Future studies to detect the presence of other immature Sertoli cell markers such as antim€ ullerian hormone

(AMH) and cytokeratin 18 (CK18) may shed light on the possible dedifferentiation of Sertoli cells in response to in vitro culture conditions (51). In agreement with other reports (17, 20, 21, 25, 52), we clearly showed that only mSSCM conditions supported the survival of germ cells in vitro (see Fig. 2). Expression analysis indicated that whole testicular cell culture in mSSCM resulted in a statistically significant up-regulation of the spermatogonial markers at day 7, followed by their down-regulation, whereas DAZL and VASA statistically significantly up-regulated at day 14 in differentially plated cells. An exception for this behavior occurred with UCHL1, the expression of which cannot be considered exclusive for spermatogonia in vitro because it was shown to stain positive in most of somatic cells in vitro, as previously described elsewhere (25). When we compared our data with previous reports, although differential plating demonstrated its efficiency to eliminate most germ cells from culture in the attached cell fraction, we did not observe a statistically significant difference after 14 days in vitro compared with the whole culture of testicular cells (see Fig. 3D). More importantly, although the formation of germ cell clusters has been widely reported in mSSC culture in vitro (17), in our study the hSSCs identified by their VASAþ/UTF1þ phenotype never appeared in cell clusters (20, 21). In contrast, hSSCs appeared in singlets, doublets, or groups of four cells (see Fig. 2B) in the same line that previous reports had described (24, 25, 49), highlighting the need to use more than just one marker to ensure the SSC identity of cultured cells (25). The low colocalization of hSSCs with the proliferation marker Ki67 (see Fig. 3A) prompted us to sort out an enriched hSSC population for coculture on mitotically inactivated testicular feeder cells to study whether prevention of somatic cell overgrowth may improve the long-term culture of hSSCs (see Fig. 4A). For this, we selected HLA-ABC as a generic marker for somatic cells (53) to separate them from the germ cell fraction within testicular cell suspensions, and EPCAM as an epithelial marker previously reported to be expressed in spermatogonia (54, 55) to separate them from the rest of germ cells. Compared with previous reports that used the same combination of markers to sort out putative hSSCs (26), we were able to sort out up to four cell subpopulations, but we did not observe the appearance of EPCAMþdim and EPCAMþbright subpopulations within the EPCAMþ population. However, further VASA/UTF1 colocalization staining supported the efficiency of HLA
/EPCAMþ FACS as a method to sort out an enriched population of putative hSSCs, whereas HLAþ sorted populations were composed mainly of somatic cells (see Fig. 4B and C). Among the sorted cells, only the HLA
/EPCAMþ cocultures with inactivated testicular feeders showed the appearance of VASAþ/UTF1þ putative hSSCs. It is interesting that HLA
sorted cells were unable to properly attach to plastic and thus we discarded them, highlighting the need of a somatic feeder for the proper attachment of germ cells (data not shown). Further quantification of VASAþ/UTF1þ cells indicated a statistically significant enrichment of putative hSSCs in cocultures following HLA
/EPCAMþ FACS

VOL. - NO. - / - 2016

9

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:58 pm
ce E

1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062

ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121

compared with previous conditions (see Fig. 3D), supporting the efficiency of the design to enrich hSSCs for in vitro culture. However, putative hSSCs showed very poor proliferation and almost disappeared after 28 days of culture (see Fig. 3C). Taken together, our results indicate that sorting of HLA
/ EPCAMþ cells and coculturing them with inactivated testicular feeders improved the germ cell/somatic cell ratio in vitro. However, although the mouse-based culture conditions supported the survival of hSSCs for up to 14 days in vitro, our results indicate that these conditions are not enough to promote their long-term proliferation. It is interesting that this phenomenon correlates with the behavior of hSSCs after transplantation into the mouse seminiferous epithelium in vivo, where hSSCs colonize but are unable to divide or differentiate into spermatocytes, whereas transplanted mSSCs are able to colonize, divide, and fully regenerate spermatogenesis (4–6, 56). Based on this, we suggest that in vitro conditions based on the mSSC niche may keep hSSCs in a quiescent state, recapitulating the same behavior that is shown in vivo after transplantation into the mouse seminiferous epithelium. The reasons for this are intriguing and may be due to the different nature of mouse and human SSC niches (3). In the mouse model, single spermatogonia A (As) undergo several cycles of division and form chains of 2 (Apaired), 4, 8, 16, and even 32 spermatogonia A (Aaligned) interconnected before differentiating. However, although only As and Apaired are considered SSCs, it is thought that longer chains of Aaligned spermatogonia can be broken down into As and Apaired, inducing them to recover their mSSC phenotype. On the other hand, in humans and other primates, two kinds of spermatogonia exist depending on their nuclei condensation named as A dark (Ad) and A pale (Ap). In the primate model, Ad spermatogonia remain quiescent most of the time, representing the SSC pool, and rarely undergo asymmetric divisions to produce Ap spermatogonia which eventually differentiate into B spermatogonia and enter spermatogenesis. Taking these observations together, it is obvious that mSSCs represent a much more plastic phenotype than hSSCs and this may influence the needs of each cell type for their propagation in vitro. Although our study has important limitations due to the use of cryopreserved samples from elderly donors that may have biased our results compared with those previously reported using prepubertal tissue (21), we conclude that the in vitro culture conditions we employed for mSSCs are insufficient for their human counterparts because they induce a quiescent state instead of promoting their amplification. In this sense, further research specifically focused on hSSC is mandatory to create reliable in vitro models for their longterm in vitro propagation. As an alternative to the twodimensional in vitro culture system that we employed in this study, recent reports have explored three-dimensional in vitro systems that mimic the natural niche of SSCs in the seminiferous tubules (57–60). In vitro systems based on the creation of a three-dimensional niche may represent an optimal model for the study of hSSC biology, allowing proliferation of SSCs and even their differentiation into haploid cells (61) that eventually could be employed in reproductive

medicine, paving the way for in vitro spermatogenesis as an alternative option to restore fertility of infertile patients. Acknowledgments: The authors thank all members of the Biology of the Testis (BITE) laboratory for their technical assistance and support, especially Yoni Baert for helpful discussions. We also thank Thierry Gevaert for his advice with the irradiator, Jean Marc Lazou for his technical assistance with FACS, and Johan Schiettecatte for hormone measurements.

REFERENCES 1. 2. 3. 4. 5.

6. 7. 8.

9.

10.

11. 12. 13.

14.

15.

16.

17.

18.

19.

20.

Sharma V. Sperm storage for cancer patients in the UK: a review of current practice. Hum Reprod 2011;26:2935–43. de Rooij DG. The spermatogonial stem cell niche. Microsc Res Tech 2009;72: 580–5. Phillips BT, Gassei K, Orwig KE. Spermatogonial stem cell regulation and spermatogenesis. Philos Trans R Soc Lond B Biol Sci 2010;365:1663–78. Avarbock MR, Brinster CJ, Brinster RL. Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nat Med 1996;2:693–6. Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci U S A 1994; 91:11303–7. Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 1994;91:11298–302. Gassei K, Orwig KE. Experimental methods to preserve male fertility and treat male factor infertility. Fertil Steril 2016;105:256–66. Picton HM, Wyns C, Anderson RA, Goossens E, Jahnukainen K, Kliesch S, et al. A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys. Hum Reprod 2015;30: 2463–75. Goossens E, Van Saen D, Tournaye H. Spermatogonial stem cell preservation and transplantation: from research to clinic. Hum Reprod 2013;28: 897–907. Hermann BP, Sukhwani M, Winkler F, Pascarella JN, Peters KA, Sheng Y, et al. Spermatogonial stem cell transplantation into rhesus testes regenerates spermatogenesis producing functional sperm. Cell Stem Cell 2012;11:715–26. Schlatt S, Foppiani L, Rolf C, Weinbauer GF, Nieschlag E. Germ cell transplantation into X-irradiated monkey testes. Hum Reprod 2002;17:55–62. Honaramooz A, Megee SO, Dobrinski I. Germ cell transplantation in pigs. Biol Reprod 2002;66:21–8. Kim Y, Selvaraj V, Dobrinski I, Lee H, McEntee MC, Travis AJ. Recipient preparation and mixed germ cell isolation for spermatogonial stem cell transplantation in domestic cats. J Androl 2006;27:248–56. Kim Y, Turner D, Nelson J, Dobrinski I, McEntee M, Travis AJ. Production of donor-derived sperm after spermatogonial stem cell transplantation in the dog. Reproduction 2008;136:823–31. Sadri-Ardekani H, Atala A. Testicular tissue cryopreservation and spermatogonial stem cell transplantation to restore fertility: from bench to bedside. Stem Cell Res Ther 2014;5:68. Ishii K, Kanatsu-Shinohara M, Shinohara T. Cell-cycle-dependent colonization of mouse spermatogonial stem cells after transplantation into seminiferous tubules. J Reprod Dev 2014;60:37–46. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, et al. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 2003;69:612–6. Martin LA, Seandel M. Serial enrichment of spermatogonial stem and progenitor cells (SSCs) in culture for derivation of long-term adult mouse SSC lines. J Vis Exp 2013:e50017. Martin LA, Assif N, Gilbert M, Wijewarnasuriya D, Seandel M. Enhanced fitness of adult spermatogonial stem cells bearing a paternal ageassociated FGFR2 mutation. Stem Cell Rep 2014;3:219–26. Sadri-Ardekani H, Mizrak SC, van Daalen SK, Korver CM, RoepersGajadien HL, Koruji M, et al. Propagation of human spermatogonial stem cells in vitro. JAMA 2009;302:2127–34. VOL. - NO. - / - 2016

10

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:58 pm
ce E

1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180

Fertility and Sterility® 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36. 37.

38.

39. 40.

41.

Sadri-Ardekani H, Akhondi MA, van der Veen F, Repping S, van Pelt AM. In vitro propagation of human prepubertal spermatogonial stem cells. JAMA 2011;305:2416–8. Chikhovskaya JV, Jonker MJ, Meissner A, Breit TM, Repping S, van Pelt AM. Human testis-derived embryonic stem cell-like cells are not pluripotent, but possess potential of mesenchymal progenitors. Hum Reprod 2012;27: 210–21. Chikhovskaya JV, van Daalen SK, Korver CM, Repping S, van Pelt AM. Mesenchymal origin of multipotent human testis-derived stem cells in human testicular cell cultures. Mol Hum Reprod 2014;20:155–67. Eildermann K, Gromoll J, Behr R. Misleading and reliable markers to differentiate between primate testis-derived multipotent stromal cells and spermatogonia in culture. Hum Reprod 2012;27:1754–67. Baert Y, Braye A, Struijk RB, van Pelt AM, Goossens E. Cryopreservation of testicular tissue before long-term testicular cell culture does not alter in vitro cell dynamics. Fertil Steril 2015;104:1244–52.e1–4. Dovey SL, Valli H, Hermann BP, Sukhwani M, Donohue J, Castro CA, et al. Eliminating malignant contamination from therapeutic human spermatogonial stem cells. J Clin Invest 2013;123:1833–43. Valli H, Sukhwani M, Dovey SL, Peters KA, Donohue J, Castro CA, et al. Fluorescence- and magnetic-activated cell sorting strategies to isolate and enrich human spermatogonial stem cells. Fertil Steril 2014;102:566–80.e7. Baert Y, Van Saen D, Haentjens P, In't Veld P, Tournaye H, Goossens E. What is the best cryopreservation protocol for human testicular tissue banking? Hum Reprod 2013;28:1816–26. van Pelt AM, Morena AR, van Dissel-Emiliani FM, Boitani C, Gaemers IC, de Rooij DG, et al. Isolation of the synchronized A spermatogonia from adult vitamin A-deficient rat testes. Biol Reprod 1996;55:439–44. Ohta Y, Ichimura K. Proliferation markers, proliferating cell nuclear antigen, Ki67, 5-bromo-20 -deoxyuridine, and cyclin D1 in mouse olfactory epithelium. Ann Otol Rhinol Laryngol 2000;109:1046–8. Panula S, Medrano JV, Kee K, Bergstrom R, Nguyen HN, Byers B, et al. Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells. Hum Mol Genet 2011;20:752–62. Cowan G, Childs AJ, Anderson RA, Saunders PT. Establishment of long-term monolayer cultures of somatic cells from human fetal testes and expansion of peritubular myoid cells in the presence of androgen. Reproduction 2010; 139:749–57. Arenas MI, Bethencourt FR, De Miguel MP, Fraile B, Romo E, Paniagua R. Immunocytochemical and quantitative study of actin, desmin and vimentin in the peritubular cells of the testes from elderly men. J Reprod Fertil 1997; 110:183–93. Dong L, Wang H, Su Z, Niu S, Wang R, Wu L, et al. Steroidogenic acute regulatory protein is a useful marker for Leydig cells and sex-cord stromal tumors. Appl Immunohistochem Mol Morphol 2011;19:226–32. Raucci F, D'Aniello A, Di Fiore MM. Stimulation of androgen production by D-aspartate through the enhancement of StAR, P450scc and 3b-HSD mRNA levels in vivo rat testis and in culture of immature rat Leydig cells. Steroids 2014;84:103–10. Ivell R, Wade JD, Anand-Ivell R. INSL3 as a biomarker of Leydig cell functionality. Biol Reprod 2013;88:147. Hemendinger RA, Gores P, Blacksten L, Harley V, Halberstadt C. Identification of a specific Sertoli cell marker, Sox9, for use in transplantation. Cell Transplant 2002;11:499–505. Kyronlahti A, Euler R, Bielinska M, Schoeller EL, Moley KH, Toppari J, et al. GATA4 regulates Sertoli cell function and fertility in adult male mice. Mol Cell Endocrinol 2011;333:85–95. Zhao C, Bratthauer GL, Barner R, Vang R. Diagnostic utility of WT1 immunostaining in ovarian sertoli cell tumor. Am J Surg Pathol 2007;31:1378–86. Anderson RA, Fulton N, Cowan G, Coutts S, Saunders PT. Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis. BMC Dev Biol 2007;7:136. Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, Sukhwani M, et al. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet 2004;36:653–9.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56. 57.

58.

59.

60.

61.

van Bragt MP, Roepers-Gajadien HL, Korver CM, Bogerd J, Okuda A, Eggen BJ, et al. Expression of the pluripotency marker UTF1 is restricted to a subpopulation of early A spermatogonia in rat testis. Reproduction 2008;136:33–40. von Kopylow K, Staege H, Schulze W, Will H, Kirchhoff C. Fibroblast growth factor receptor 3 is highly expressed in rarely dividing human type A spermatogonia. Histochem Cell Biol 2012;138:759–72. Luo J, Megee S, Dobrinski I. Asymmetric distribution of UCH-L1 in spermatogonia is associated with maintenance and differentiation of spermatogonial stem cells. J Cell Physiol 2009;220:460–8. Castrillon DH, Quade BJ, Wang TY, Quigley C, Crum CP. The human VASA gene is specifically expressed in the germ cell lineage. Proc Natl Acad Sci U S A 2000;97:9585–90. Medrano JV, Marques-Mari AI, Aguilar CE, Riboldi M, Garrido N, MartinezRomero A, et al. Comparative analysis of the germ cell markers c-KIT, SSEA-1 and VASA in testicular biopsies from secretory and obstructive azoospermias. Mol Hum Reprod 2010;16:811–7. Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984;133:1710–5. Kubota H, Brinster RL. Culture of rodent spermatogonial stem cells, male germline stem cells of the postnatal animal. Methods Cell Biol 2008;86: 59–84. Zheng Y, Thomas A, Schmidt CM, Dann CT. Quantitative detection of human spermatogonia for optimization of spermatogonial stem cell culture. Hum Reprod 2014;29:2497–511. de Santa Barbara P, Moniot B, Poulat F, Berta P. Expression and subcellular localization of SF-1, SOX9, WT1, and AMH proteins during early human testicular development. Dev Dyn 2000;217:293–8. Steger K, Rey R, Louis F, Kliesch S, Behre HM, Nieschlag E, et al. Reversion of the differentiated phenotype and maturation block in Sertoli cells in pathological human testis. Hum Reprod 1999;14:136–43. Koruji M, Shahverdi A, Janan A, Piryaei A, Lakpour MR, Gilani Sedighi MA. Proliferation of small number of human spermatogonial stem cells obtained from azoospermic patients. J Assist Reprod Genet 2012;29:957–67. Fujita K, Tsujimura A, Miyagawa Y, Kiuchi H, Matsuoka Y, Takao T, et al. Isolation of germ cells from leukemia and lymphoma cells in a human in vitro model: potential clinical application for restoring human fertility after anticancer therapy. Cancer Res 2006;66:11166–71. Anderson R, Schaible K, Heasman J, Wylie C. Expression of the homophilic adhesion molecule, Ep-CAM, in the mammalian germ line. J Reprod Fertil 1999;116:379–84. Ryu BY, Orwig KE, Kubota H, Avarbock MR, Brinster RL. Phenotypic and functional characteristics of spermatogonial stem cells in rats. Dev Biol 2004;274:158–70. Nagano M, Patrizio P, Brinster RL. Long-term survival of human spermatogonial stem cells in mouse testes. Fertil Steril 2002;78:1225–33. Huleihel M, Nourashrafeddin S, Plant TM. Application of threedimensional culture systems to study mammalian spermatogenesis, with an emphasis on the rhesus monkey (Macaca mulatta). Asian J Androl 2015;17:972–80. Stukenborg JB, Wistuba J, Luetjens CM, Elhija MA, Huleihel M, Lunenfeld E, et al. Coculture of spermatogonia with somatic cells in a novel threedimensional soft-agar-culture-system. J Androl 2008;29:312–29. Stukenborg JB, Schlatt S, Simoni M, Yeung CH, Elhija MA, Luetjens CM, et al. New horizons for in vitro spermatogenesis? An update on novel three-dimensional culture systems as tools for meiotic and post-meiotic differentiation of testicular germ cells. Mol Hum Reprod 2009;15:521–9. Reda A, Hou M, Landreh L, Kjartansdottir KR, Svechnikov K, Soder O, et al. In vitro spermatogenesis—optimal culture conditions for testicular cell survival, germ cell differentiation, and steroidogenesis in rats. Front Endocrinol (Lausanne) 2014;5:21. Sato T, Katagiri K, Gohbara A, Inoue K, Ogonuki N, Ogura A, et al. In vitro production of functional sperm in cultured neonatal mouse testes. Nature 2011;471:504–7.

VOL. - NO. - / - 2016

11

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:58 pm
ce E

1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298

ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE

SUPPLEMENTAL FIGURE 1

web 4C=FPO

1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 1354 1355 1356 1357

Results from TUNEL assay on human testicular cells in vitro. Illustrative pictures correspond to day 14 of culture except for spermatogonial stem cell (SSC)-like clusters that correspond to day 3 due to their later disappearance in vitro. Medrano. Limited proliferation of hSSCs in vitro. Fertil Steril 2016.

VOL. - NO. - / - 2016

11.e1

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:58 pm
ce E

1358 1359 1360 1361 1362 1363 1364 1365 1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 1403 1404 1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416

Fertility and Sterility® 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 1511 1512 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534

SUPPLEMENTAL FIGURE 2

web 4C=FPO

1417 1418 1419 1420 1421 1422 1423 1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473 1474 1475

Illustrative pictures of the VASA (red)/UTF1 (green) colocalization staining in cytospins from unsorted and HLA/EPCAM sorted cells. Medrano. Limited proliferation of hSSCs in vitro. Fertil Steril 2016.

VOL. - NO. - / - 2016

11.e2

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:58 pm
ce E

ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 1564 1565 1566 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591 1592 1593

SUPPLEMENTAL FIGURE 3

Time-course analysis at days 14 and 28 for the mRNA expression of germ testicular somatic markers in coculture conditions. Relative expression found in frozen/thawed testicular samples is also shown as a reference of physiologic expression. Data are presented as mean  standard error. Statistically significant differences: *P<.05 and **P<.01. Medrano. Limited proliferation of hSSCs in vitro. Fertil Steril 2016.

VOL. - NO. - / - 2016

11.e3

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:58 pm
ce E

1594 1595 1596 1597 1598 1599 1600 1601 1602 1603 1604 1605 1606 1607 1608 1609 1610 1611 1612 1613 1614 1615 1616 1617 1618 1619 1620 1621 1622 1623 1624 1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 1637 1638 1639 1640 1641 1642 1643 1644 1645 1646 1647 1648 1649 1650 1651 1652

Fertility and Sterility® 1653 1654 1655 1656 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667 1668 1669 1670 1671 1672 1673 1674 1675 1676 1677 1678 1679 1680 1681 1682 1683 1684 1685 1686 1687 1688 1689 1690 1691 1692 1693 1694 1695 1696 1697 1698 1699 1700 1701 1702 1703 1704 1705 1706 1707 1708 1709 1710 1711

1712 1713 1714 1715 1716 1717 1718 1719 1720 1721 1722 1723 1724 1725 1726 1727 1728 1729 1730 1731 1732 1733 1734 1735 1736 1737 1738 1739 1740 1741 1742 1743 1744 1745 1746 1747 1748 1749 1750 1751 1752 1753 1754 1755 1756 1757 1758 1759 1760 1761 1762 1763 1764 1765 1766 1767 1768 1769 1770

SUPPLEMENTAL FIGURE 4

Hormone quantifications in fresh control media and media used in culture conditions for 48 hours. Data are presented as mean  standard error. Statistically significant difference: *P<.05. Medrano. Limited proliferation of hSSCs in vitro. Fertil Steril 2016.

VOL. - NO. - / - 2016

11.e4

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:58 pm
ce E

ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE 1771 1772 1773 1774 1775 1776 1777 1778 1779 1780 1781 1782 1783 1784 1785 1786 1787 1788 1789 1790 1791 1792 1793 1794 1795 1796 1797 1798 1799 1800 1801 1802 1803 1804 1805 1806 1807 1808 1809 1810 1811 1812 1813 1814 1815 1816 1817 1818 1819 1820 1821 1822 1823 1824 1825 1826 1827 1828 1829

1830 1831 1832 1833 1834 1835 1836 1837 1838 1839 1840 1841 1842 1843 1844 1845 1846 1847 1848 1849 1850 1851 1852 1853 1854 1855 1856 1857 1858 1859 1860 1861 1862 1863 1864 1865 1866 1867 1868 1869 1870 1871 1872 1873 1874 1875 1876 1877 1878 1879 1880 1881 1882 1883 1884 1885 1886 1887 1888

SUPPLEMENTAL TABLE 1 Composition of standard medium. Component DMEM/Ham's F-12 with Glutamax Fetal bovine serum Penicillin/streptomycin

Reference

Concentration

31331028, Gibco/Thermo Fisher 10106-169, Gibco/Thermo Fisher 15140122, Gibco/Thermo Fisher

89% 10% 1%

Note: DMEM ¼ Dulbecco's minimum essential medium. Medrano. Limited proliferation of hSSCs in vitro. Fertil Steril 2016.

VOL. - NO. - / - 2016

11.e5

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:58 pm
ce E

Fertility and Sterility® 1889 1890 1891 1892 1893 1894 1895 1896 1897 1898 1899 1900 1901 1902 1903 1904 1905 1906 1907 1908 1909 1910 1911 1912 1913 1914 1915 1916 1917 1918 1919 1920 1921 1922 1923 1924 1925 1926 1927 1928 1929 1930 1931 1932 1933 1934 1935 1936 1937 1938 1939 1940 1941 1942 1943 1944 1945 1946 1947

1948 1949 1950 1951 1952 1953 1954 1955 1956 1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006

SUPPLEMENTAL TABLE 2 Composition of mouse spermatogonial stem cell medium (mSSCM). Component StemPro-34 SFM StemPro nutrient supplement Bovine albumine D(þ) Glucose Ascorbic acid Transferrin Piruvic acid d-Biotin 2b-Mercaptoethanol DL-Lactic acid MEM nonessential aminoacids Human insulin Sodium selenite Putrescine L-Glutamine MEM vitamine solution b-Estradiol Progesterone Human EGF Human bFGF Human LIF Human GDNF Fetal bovine serum Penicillin/streptomycin

Reference

Concentration

10639-011, Gibco/Thermo Fisher 10639-011, Gibco/Thermo Fisher 10735094001, Roche G7021, Sigma-Aldrich A4544, Sigma-Aldrich T1147, Sigma-Aldrich P2256, Sigma-Aldrich B4501, Sigma-Aldrich M7522, Sigma-Aldrich L4263, Sigma-Aldrich 11140-035, Gibco/Thermo Fisher I9278, Sigma-Aldrich S2651, Sigma-Aldrich P7505, Sigma-Aldrich 25030–024, Gibco/Thermo Fisher 11120–037, Gibco/Thermo Fisher E2758, Sigma-Aldrich P8783, Sigma-Aldrich AF-100, Peprotech 100–18B, Peprotech 300–05, Peprotech 450–10, Peprotech 10106–169, Gibco/Thermo Fisher 15140122, Gibco/Thermo Fisher

90% 26 mL/mL 5 mg/mL 6 mg/mL 0.1 mM 100 mg/mL 30 mg/mL 10 mg/mL 0.05 mM 1 mL/mL 10 mL/mL 25 mg/mL 30 nM 60 mM 2 mM 10 mL/mL 30 ng/mL 60 ng/mL 20 ng/mL 10 ng/mL 10 ng/mL 10 ng/mL 1% 1%

Note: EGF ¼ epidermal growth factor; bFGF ¼ basic fibroblast growth factor; GDNF ¼ glial cell-derived neurotrophic factor; LIF ¼ leukemia inhibitory factor; MEM ¼ minimum essential medium; SFM ¼ serum-free medium. Medrano. Limited proliferation of hSSCs in vitro. Fertil Steril 2016.

VOL. - NO. - / - 2016

11.e6

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:58 pm
ce E

ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025 2026 2027 2028 2029 2030 2031 2032 2033 2034 2035 2036 2037 2038 2039 2040 2041 2042 2043 2044 2045 2046 2047 2048 2049 2050 2051 2052 2053 2054 2055 2056 2057 2058 2059 2060 2061 2062 2063 2064 2065

2066 2067 2068 2069 2070 2071 2072 2073 2074 2075 2076 2077 2078 2079 2080 2081 2082 2083 2084 2085 2086 2087 2088 2089 2090 2091 2092 2093 2094 2095 2096 2097 2098 2099 2100 2101 2102 2103 2104 2105 2106 2107 2108 2109 2110 2111 2112 2113 2114 2115 2116 2117 2118 2119 2120 2121 2122 2123 2124

SUPPLEMENTAL TABLE 3 Taqman Gene Expression assays and probes/primers used for quantitative reverse-transcription polymerase chain reaction expression analyses. Target mRNA ACTA2 DES STAR INSL3 HSD3B1 SOX9

WT1 GATA4 DDX4 DAZL PLZF UTF1 UCHL1 FGFR3 GUSB

Assays/primers and probes sequences Hs00426835_g1 Hs00157258_m1 Hs00264912_m1 Hs01895076_s1 Hs04194787_g1 Probe: TCTGAACGA GAGCGAGAAGC Forward: AGTACCCG CACTTGCACAAC Reverse: GTAATCCG GGTGGTCCTTCT Hs01103751_m1 Hs01034629_m1 Hs00987133_m1 Hs00154706_m1 Hs00957433_m1 Hs00864535_s1 Hs00985157_m1 Hs00179829_m1 Hs99999908_m1

Company Life Technologies Life Technologies Life Technologies Life Technologies Life Technologies IDT IDT IDT Life Technologies Life Technologies Life Technologies Life Technologies Life Technologies Life Technologies Life Technologies Life Technologies Life Technologies

Medrano. Limited proliferation of hSSCs in vitro. Fertil Steril 2016.

VOL. - NO. - / - 2016

11.e7

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:58 pm
ce E

Fertility and Sterility® 2125 2126 2127 2128 2129 2130 2131 2132 2133 2134 2135 2136 2137 2138 2139 2140 2141 2142 2143 2144 2145 2146 2147 2148 2149 2150 2151 2152 2153 2154 2155 2156 2157 2158 2159 2160 2161 2162 2163 2164 2165 2166 2167 2168 2169 2170 2171 2172 2173 2174 2175 2176 2177 2178 2179 2180 2181 2182 2183

2184 2185 2186 2187 2188 2189 2190 2191 2192 2193 2194 2195 2196 2197 2198 2199 2200 2201 2202 2203 2204 2205 2206 2207 2208 2209 2210 2211 2212 2213 2214 2215 2216 2217 2218 2219 2220 2221 2222 2223 2224 2225 2226 2227 2228 2229 2230 2231 2232 2233 2234 2235 2236 2237 2238 2239 2240 2241 2242

SUPPLEMENTAL TABLE 4 Antibodies and experimental conditions for immunostaining. Antibody Mouse antiVMENTIN Mouse anti-aSMA Rabbit anti-STAR Rabbit anti-SOX9 Goat anti-hVASA Rabbit anti-UCHL1 Mouse anti-UTF1 Rabbit anti-Ki67

Reference

Dilution

Incubation

M072529, Dako

1/100

4 C, ON

A2547, Sigma-Aldrich SC-25806, Santa Cruz Biotechnologies AB5535, Millipore AF2030, R&D Systems 7863–0504, AbD Serotec MAB4337, Millipore AB16667, Abcam

1/900 1/50

4 C, ON 4 C, ON

1/200 1/200 1/200

4 C, ON 4 C, ON 4 C, 2 h

1/50 1/200

4 C, ON RT, 1 h

Note: ON ¼ overnight; RT ¼ room temperature. Medrano. Limited proliferation of hSSCs in vitro. Fertil Steril 2016.

VOL. - NO. - / - 2016

11.e8

FLA 5.4.0 DTD
FNS30405_proof
1 August 2016
1:58 pm
ce E