In Vitro Differentiation of Leydig Cells From hiPSCs: A First Step Towards a Cellular Therapy for Hypogonadism?

The hypothalamic–pituitary–gonadal (HPG) axis has a central role in fetal development, sexual maturity, and male aging. Disruption of hormones produced by the HPG axis, caused by either genetic mutation or environmental insult, results in impaired reproductive function, delayed puberty, and hypogonadism at adulthood. In adulthood, the most notable clinical symptom of hypogonadism is the inadequate functional activity of the testes, with deficient testosterone production leading to reduced circulating serum testosterone levels. Additionally, poor spermatogenesis is usually a sign of hypogonadism (1). Low testosterone levels resulting from hypothalamic or pituitary failure (eg, due to genetic defects, neoplasm, or trauma) lead to hypogonadotropic hypogonadism (also called central or secondary hypogonadism). When reduced testosterone levels result from a testicular malfunction of steroidogenesis due to genetic causes, inflammation, or infection, it can cause hypergonadotropic hypogonadism (also known as primary hypogonadism) (1). Adult-onset hypogonadism in men due to aging, obesity, or poor health is generally known as late-onset hypogonadism (LOH). LOH is a relatively pandemic disorder in which the incidence of biochemical hypogonadism in aging males (>40 years of age) varies from 2% to 15% within the general population, as evaluated by the European Male Aging Study (EMAS). Over time, men with hypogonadism will develop erectile dysfunction, infertility, myophagism, osteoporosis, and depression, which have severely deleterious impacts on human reproductive health. Depending on its etiology, LOH may be permanent or potentially reversible (1).

For decades, testosterone replacement therapy (TRT) has been used to treat LOH and help reverse the signs and symptoms of low testosterone levels. TRT treatments are currently available as injection of testosterone cyprionate, testosterone enanthate, or testosterone undeconoate; application of testosterone-containing gels or patches to the skin; implantable pellets containing testosterone; and a nasal spray. Recently, gonadotropin treatment in boys has also been used to induce testicular and penile growth, spermatogenesis, and testosterone production. However, TRT has a number of adverse potential side effects, such as heart attack, stroke, and prostate cancer. Furthermore, TRT is not suitable for long-term therapy due to inhibition of the feedback loop within the HPG axis regulating intratesticular testosterone production (2). Therefore, developing a physiological alternative therapy that can be used for long-term testosterone supplementation is of great need.

Leydig cells (LCs) located in the testicular interstitial compartment produce testosterone in a luteinizing hormone (LH)–responsive manner, which maintains the male phenotype and regulates male reproductive functions (3). In aging males, the progressive decline of serum testosterone levels and LC number may be primary causes of LOH, although it is also associated with some comorbidities, such as obesity, hyperglycemia, and hypertension (4). Therefore, LC transplantation has been considered as an ideal alternative approach to enhance testicular testosterone production for the treatment of male hypogonadism. Given the limited proliferative capacity of adult LCs, direct transplantation of LCs may provide only a short-term treatment for LOH. A previous study reported that transplanted human stem Leydig cells (SLCs) could replace disrupted LCs and restore testosterone production (5). However, if applied to clinical use, this method would have some limitations due to a lack of specific markers for isolation and the small proportion of SLCs in patients with hypogonadism. Therefore, a long-term treatment method for primary male hypogonadism by transplanting Leydig-like cells could be useful in future clinical applications. A recent study developed a well-defined approach to differentiate human induced pluripotent stem cells (hiPSCs), first into early mesenchymal progenitors and then to induce Leydig-like cells through ectopic persistent expression of steroidogenic factor 1 (SF1; official name NR5A1), a key gene driving differentiation of steroidogenic cells (6). Although this approach provided a promising long-term therapy for deriving functional LCs, exogenous genomic insertion may be unsafe for future clinical applications. A small-molecule–based strategy induced Leydig-like cells from hiPSCs without genetic modifications, but only produced low levels of testosterone; furthermore, this method required a long incubation period (approximately 1 month) and complicated recipes (2 different media with complex compositions) to induce Leydig-like cells (7). Therefore, there is a need to develop a safer, feasible, and rapid strategy for testosterone secretion by artificially induced human LCs to treat LOH syndrome.

Ishida et al. (8) have reported a fast and robust protocol to directly induce the differentiation of hiPSCs into functional testosterone-producing Leydig-like cells. In their strategy, the overexpression of SF1 only during the process of in vitro differentiation in a doxycycline-dependent manner reduces potential risks from ectopic gene overexpression in a clinical application. In addition, the protocol is simpler and time-saving, consisting of only 2 steps with basic medium and using 2 molecular compounds with medium replacement required just once a week.

The authors developed a strategy in which hiPSCs can be differentiated into human Leydig-like or adrenal-like cells by doxycycline-inducible overexpression of SF1 and confirmed that the concentration of testosterone and aldosterone/cortisol in the culture supernatant was high. The patterns of gene expression in the 2 steroidogenic cell types have a significant difference: Leydig-like cells strongly expressed not only the steroidogenic genes STAR, CYP11A1, CYP17A1, and HSD3B2, but also the specific marker genes of adult LCs HSD17B3, INSL3, and LHCGR, whereas adrenal-like cells expressed the adrenal-cortex–specific steroid hormone metabolizing enzymes CYP21A2, CYP11B1, and CYP11B2. The authors demonstrated that testosterone secreted by the hiPSC-derived Leydig-like cells could stimulate the proliferation of LNCaP cells, which is a testosterone-sensitive prostate cancer cell line, demonstrating its functionality. Furthermore, the authors also found that only functional adrenal-like cells, rather than Leydig-like cells, were induced from female-derived hiPSCs (8), suggesting that the induction of tissue-specific cell types from hiPSCs may be associated with gender.

Although this study demonstrates a big improvement in technology, some issues remain to be resolved. The proportion of Leydig-like and adrenal-like cells from a population of hiPSCs was not addressed, nor if adrenal-like cells could lead to immune rejection, and even tumorigenicity, in future cell transplantation therapy. Importantly, if these induced Leydig-like cells can maintain long-term steroidogenesis to increase the levels of serum circulating testosterone levels and whether they are LH responsive are issues that need to be explored further. Overall, autologous Leydig-like cells produced from hiPSCs are an intriguing prospect for the treatment of LOH syndrome by cell transplantation therapy.