Primary human testicular PDGFRα+ cells are multipotent and can be differentiated into cells with Leydig cell characteristics in vitro
Authors: Eliveld J , van den Berg E A , Chikhovskaya J V , van Daalen S K M , de Winter-Korver C M , van der Veen F , Repping S , Teerds K , van Pelt A M M 
Published: June 2019

Abstract

STUDY QUESTION
Is it possible to differentiate primary human testicular platelet-derived growth factor receptor alpha positive (PDGFRα+) cells into functional Leydig cells?

SUMMARY ANSWER
Although human testicular PDGFRα+ cells are multipotent and are capable of differentiating into steroidogenic cells with Leydig cell characteristics, they are not able to produce testosterone after differentiation.

WHAT IS KNOWN ALREADY
In rodents, stem Leydig cells (SLCs) that have been identified and isolated using the marker PDGFRα can give rise to adult testosterone-producing Leydig cells after appropriate differentiation in vitro. Although PDGFRα+ cells have also been identified in human testicular tissue, so far there is no evidence that these cells are true human SLCs that can differentiate into functional Leydig cells in vitro or in vivo.

STUDY DESIGN, SIZE, DURATION
We isolated testicular cells enriched for interstitial cells from frozen–thawed fragments of testicular tissue from four human donors. Depending on the obtained cell number, PDGFRα+-sorted cells of three to four donors were exposed to differentiation conditions in vitro to stimulate development into adipocytes, osteocytes, chondrocytes or into Leydig cells. We compared their cell characteristics with cells directly after sorting and cells in propagation conditions. To investigate their differentiation potential in vivo, PDGFRα+-sorted cells were transplanted in the testis of 12 luteinizing hormone receptor-knockout (LuRKO) mice of which 6 mice received immunosuppression treatment. An additional six mice did not receive cell transplantation and were used as a control.

PARTICIPANTS/MATERIALS, SETTING, METHODS
Human testicular interstitial cells were cultured to Passage 3 and FACS sorted for HLA-A,B,C+/CD34−/PDGFRα+. We examined their mesenchymal stromal cell (MSC) membrane protein expression by FACS analyses. Furthermore, we investigated lineage-specific staining and gene expression after MSC trilineage differentiation. For the differentiation into Leydig cells, PDGFRα+-sorted cells were cultured in either proliferation or differentiation medium for 28 days, after which they were stimulated either with or without hCG, forskolin or dbcAMP for 24 h to examine the increase in gene expression of steroidogenic enzymes using qPCR. In addition, testosterone, androstenedione and progesterone levels were measured in the culture medium. We also transplanted human PDGFRα+-sorted testicular interstitial cells into the testis of LuRKO mice. Serum was collected at several time points after transplantation, and testosterone was measured. Twenty weeks after transplantation testes were collected for histological examination.

MAIN RESULTS AND THE ROLE OF CHANCE
From primary cultured human testicular interstitial cells at Passage 3, we could obtain a population of HLA-A,B,C+/CD34−/PDGFRα+ cells by FACS. The sorted cells showed characteristics of MSC and were able to differentiate into adipocytes, chondrocytes and osteocytes. Upon directed differentiation into Leydig cells in vitro, we observed a significant increase in the expression of HSD3B2 and INSL3. After 24 h stimulation with forskolin or dbcAMP, a significantly increased expression of STAR and CYP11A1 was observed. The cells already expressed HSD17B3 and CYP17A1 before differentiation but the expression of these genes were not significantly increased after differentiation and stimulation. Testosterone levels could not be detected in the medium in any of the stimulation conditions, but after stimulation with forskolin or dbcAMP, androstenedione and progesterone were detected in culture medium. After transplantation of the human cells into the testes of LuRKO mice, no significant increase in serum testosterone levels was found compared to the controls. Also, no human cells were identified in the interstitium of mice testes 20 weeks after transplantation.

LARGE SCALE DATA
N/A

LIMITATIONS, REASONS FOR CAUTION
This study was performed using tissue from only four donors because of limitations in donor material. Because of the need of sufficient cell numbers, we first propagated cells to passage 3 before FACS of the desired cell population was performed. We cannot rule out this propagation of the cells resulted in loss of stem cell properties.

WIDER IMPLICATIONS OF THE FINDINGS
A lot of information on Leydig cell development is obtained from rodent studies, while the knowledge on human Leydig cell development is very limited. Our study shows that human testicular interstitial PDGFRα+ cells have different characteristics compared to rodent testicular PDGFRα+ cells in gene expression levels of steroidogenic enzymes and potential to differentiate in adult Leydig cells under comparable culture conditions. This emphasizes the need for confirming results from rodent studies in the human situation to be able to translate this knowledge to the human conditions, to eventually contribute to improvements of testosterone replacement therapies or establishing alternative cell therapies in the future, potentially based on SLCs.

STUDY FUNDING/COMPETING INTEREST(S)
This study was funded by Amsterdam UMC, location AMC, Amsterdam, the Netherlands. All authors declare no competing interests.