Jan Friberg M.D., Ph.D
TESTICULAR REJUVINATION – CREATING SPERM WITH STEM CELLS
In rodents, mesenchymal stem cells (MSC) and small vesicles created by MSC called exosomes, can protect spermatogenetic tissue from destruction after exposure to gonadotoxic treatment. When placed in the right environment – the testicular “niche” the MSC also develop into mature sperm that have full fertilizing capacity and create embryos. These embryos can the develop to normal fertile pups.
Direct development of sperm from MSC in humans has not yet been demonstrated. Progress in the field continues and some isolated reports indicate that stem cell and stem cell products can induce sperm production in patients with azoospermia.
Non-obstructive azoospermia (NOA) or severe oligozoospermia (cryptozoospermia) is observed in approximately 3-5 % of infertile couples. The absence or extremely low sperm production needs verification in multiple sperm samples. It can be caused by genetic, hormonal, social and environmental factors. During surgical exploration of testicular tissue from patients with NOA functional spermatozoa may be recovered and used for IVF with ICSI treatment, but sperm are obtained in less than 50% of these patients. If no sperm are detected the use of donor sperm remains the only alternative treatment. Sine most couples prefer to have their own genetic children, science has continued to explore new techniques to induce sperm production.
In the testicular “niche” spermatogonia self-renew and also undergo mitotic and meiotic division to generate mature sperm. In this “niche” supporting cells and interstitial tissue produce hormones, cytokines and growth factors that are transported to the sperm producing cells. The development of spermatozoa is a complex process highly dependent on intra and intercellular communication between germ cells, Sertoli cells, Leydig cells, and the surrounding matrix cells. (Saitou & Miyauch; 2016, Nikolic et al 2016). This process is genetically very complicated and over 2000 genes and a large number of growth factors are activated.
The earliest precursors of spermatozoa, the spermatogonia develop in close proximity to the Sertoli cells, on the basal membrane (a modified extra cellular matrix) of the seminiferous tubules. However, hormone-sensitive Leidig cells outside of the basal membrane as well as Sertoli cells and spermatogonia on the inside, are growing in the extra cellular matrix (ECM) which is crossed by vessels. On these vessels there are large amounts of stem cells positioned as pericytes that constantly receive messages from circulating exosomes, the conductor of all cell function in the body and these perivascular stem cells can multiply the exosome message into the EMC which surrounds all cells. The ECM anchors and supports all cells and also regulate cell survival, proliferation, polarity, adhesions and migration. The ECM is also involved in growth mechanisms, cell regulation and healing processes and as such it is the primary factor involved in regeneration and formation of new tissue. Growth factors in the EMC also greatly influence the early development of sperm cell precursors. The EMC is there fore the key to the function of all stage of spermatogenesis and spermiogenesis.
The development of a sperm cell from a spermatogonia to a mature spermatozoa in the testis takes 72 days in the human and the transport through the extra-testicular sperm ducts takes another 10-21 days before the spermatozoa shows up in the ejaculate.
MESENCHYMAL STEM CELLS (MSC) FOR TESTICULAR REJUVINATION
MSC were identified in the 1970’s (Friedenstein et al 1970) and over the past 50 years they have been a focus for an intense scientific interest. Because of their regenerative and proliferative properties MSC have been used extensively in various medical disciplines. MSC have been shown not to have any toxic or carcinogenic effects and found to be extremely safe to use. They are present in all body tissues but because it is relatively easy to obtain them from adipose tissue harvesting them through liposuction and special treatment has become one of the preferred techniques. MSC’s are a heterogenous cell population and contain multilineage pluripotent stem cells which function in the same way as embryonic stem cells (ESC). They have the capacity to differentiate into all cells from three germ layers spontaneously.
The detection of spermatogonial stem cells initiated an intensive research in male infertility. Animal experiments were performed were sperm cell destruction was induced with various gonadotoxic regiments and azoospermic animals were created. It was then shown that MSC from bone marrow, human amnion, human placenta, adipose tissue, umbilical cord blood cells and urine derived stem cells could partially or completely restore spermatogenesis (reviewed by Fazeli et al 2018, Gauthier-Fisher et al 2019). Offspring was created from MSC with special markers and the created pups had the same markers as the initially used MSC (Cakici et al 2013).
How the MSC helps with the sperm cell recovery is poorly understood. A possibility is that MSC directly develop into germ cells that then differentiate. Undifferentiated human MSC injected/transplanted into the testicular tissue colonizes the seminiferous tubules and differentiate into germ cells both in vivo and in vitro. They develop to sperm-like cells with genes that are typically present to develop meiotic cells (Chen et al 2015, Allah et al 2016). However, the most favored theory is that injected undifferentiated MSC through paracrine mechanisms in the functional testicular “niche” layer self-renew and differentiate. A possibility also exists that special stem cells (VSEL – very small embryonic stem cell – like cells), present in the testicular tissue get activated when all germ and “niche” cells get destroyed by chemotherapy or radiation. Injection of MSC could help recruitment and differentiation of the small primitive progenitor cells into spermatogonial stem cells (Bhartiya et al 2016)
Medium from the cultures of MSC is also capable of inducing spermatogenesis when injected into the testicular tissue of animals that had been made azoospermic (Cai et al 2019, Cai et al 2021). The culture medium contains extra cellular communication particles called exosomes. Those exosomes carry the full cascade of stimulatory and regulatory factors such as growth factors, enzyme receptors, transcription factors, and maturation proteins that governs cell structure and function through autocrine, paracrine, and endocrine signaling. They also contain messenger RNA (mRNA) and micro RNA (miRNA) but no DNA and consequently have no tumorigenic effect and do not cause a graft verses host reaction.
INDUCTION OF SPERMATOZOA FROM SOMATIC CELLS
The use of specific transcription factors can lead to reprogramming of somatic cells into induced pluripotent stem cells (iPSC) in a mouse model (Takahashi & Yamanaka 2006). This is done with the animals own somatic cell types such as fibroblasts, keratinocytes or blood cells so no immunorejection occur. When these iPSC are introduced into the “niche” of spermatogenesis and supporting cells in the testicular tissue, the cells can develop into primordial germ cells and further differentiate into spermatozoa (reviewed by Fang et al 2018). With the use of intracellular sperm injection (ICSI) and in vitro fertilization (IVF) techniques embryos have been created that developed to healthy fertile pups (Hayashi et al 2011, Hayashi et al 2012, Zhou et al 2016). Using human iPSC testicular Leydig cells with normal testosterone production and Sertoli cells have also been produced. (Rodriguez-Gutierrez et al 2018, Chen et al 2019) but production of mature human spermatozoa has not yet been achieved.
The idea of making functional spermatozoa from patients own somatic cells is very attractive. However, limited knowledge of the technique in primates and a potential risk for genetic and epigenetic mutations to be induced during the reprogramming to iPSC is an important concern. Therefore, reprogramming of human stroma cells to spermatozoa is still in the preclinical stages and not ready for human application (Fang et al 2018), but the techniques is in use on humans to create new heart cells after myocardial ischemia (Wang et al 2015).
STIMULATION OF SPERM PRODUCTION IN MEN
Progress in research on stem cells and stem cell products have been introduced into human infertility research. Clinical trials are usually reported to the U.S. government and a review of their web site (www.clincaltrials.gov) reveals that at least six human trials are on the way worldwide to stimulate sperm production. Most studies are aiming to help patients with NOA but one is directed to “male infertility” and one is directed to the azoospermia seen in Klinefelter’s syndrome. Autologous MSC from bone marrow are mostly preferred to be injected into the testicular tissue but one study applies bone marrow stem cells directly into the testicular artery. Adipose tissue derived stem cells are also used. The studies are all ongoing and no results have been published yet.
Scattered reports in the literature have indicated that the injection of stem cell products both intravenously and into the testicular tissue appears to be a possible way to induce spermatogenesis. Cassim and Mohamed (2019) followed a man with NOA who failed gonadotropin therapy and was given 3 courses of intravenous infusion and one intratesticular injection of adipose tissue derived MSC and he produced enough sperm to be a candidate for IVF-ICSI. In another report. Spiel (personal communication) had one man with a long standing azoospermia who developed normozoospermia and fertility after intravenous exosome treatment.
Clinic’s have also been set up for treatment of male infertility using stem cell derived techniques. The main approach has been to inject cytokines and growth factors obtained from platelet-rich plasma (PRP) combined with stem cells obtained (from adipose tissue at liposuction) directly into the testicular tissue. Few results are available but a success rate of 56% has been mentioned in obtaining spermatozoa in the ejaculate from previously azoospermic men (Ozyigit O; personal communication).
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