Recent Developments in In Vitro Spermatogenesis and Future Directions

: Recent developments in stem cell technologies have made signiﬁcant advancements in the ﬁeld of in vitro gametogenesis. In vitro gametogenesis (IVG) is a promising technology where functional gametes (sperm or egg cells) can be generated from stem cells. Scientists have made continuous advancements in the ﬁeld and successfully derived fully functional sperm from stem cells in mice. Two recent papers generated excitement in IVG by generating bi-maternal and bi-paternal mice from embryonic stem cells (ESCs) and pluripotent stem cells (PSCs). IVG is a promising technology with potential applications that include infertility treatment, fertility preservation, same-sex reproduction, bypassing oocyte depletion in women with advanced age, conservation biology, genetic disorder prevention, and research into human germ cell development. In vitro spermatogenesis (IVS) is the attempt to recreate the process of spermatogenesis in a culture system. Spermatogenesis is essential for male fertility and reproductive health, but it can be impaired by various factors such as genetic defects, environmental toxicants, infections, aging, or medical therapies. Spermatogenesis is a complex and highly regulated process involving multiple cell proliferation, differentiation, and maturation stages. The main challenges of IVS are to provide a suitable microenvironment that mimics the testis in vivo, to support the survival and development of all the cell types involved in spermatogenesis, and to achieve complete and functional spermatogenesis. Therefore, there is a great interest in developing methods to study spermatogenesis in vitro, both for basic research and clinical applications. This review covers recent developments in in vitro spermatogenesis in the past two years. Advances in tissue engineering and regenerative medicine have introduced techniques like ex vivo tissue culture and technologies such as bioreactors, microﬂuidic systems, and organoids. Bioreactors and microﬂuidic systems replicate physiological conditions for tissue and cell cultivation, while organoids model organ functionality. Meanwhile, scaffolds, made from various materials, provide essential structural support, guiding the growth and organization of cells into functional tissues.


Introduction
Recent developments in stem cell technologies, especially induced pluripotent stem cells (iPSCs) derived from patient-specific cell sources, have brought many promises and potential applications in many research fields.There are many applications of iPSCs, such as disease modeling, drug discovery and development, regenerative medicine, personalized medicine, gene therapy, and developmental biology, to name a few.Although iPSCs hold immense promise, some obstacles and concerns must be addressed, such as improving reprogramming efficiencies, epigenetic carryovers from the cell source, and ethical and regulatory matters.However, the field of stem cell research keeps progressing.One of the most promising fields is regenerative medicine, where cells, tissues, or organs can be generated by reprogramming cells into iPSCs from patient-specific (i.e., allogeneic) sources.
In vitro gametogenesis (IVG) is a promising technology where functional gametes (sperm or egg cells) can be generated from pluripotent stem cells (PSCs).Although it is still in development, potential applications include infertility treatment, fertility preservation, same-sex reproduction, bypassing oocyte depletion in women with advanced age, genetic disorder prevention, and research into human germ cell development.Efforts to derive functional gametes in vitro have been made since the 1920s [1], with early efforts mainly focused on ex vivo organ cultures to produce mature gametes.Success in deriving fully functional sperm from stem cells in mice [2] and haploid spermatid-like cells from human PSCs [3] and primates have been reported [4].The successful derivation of testicular sperm was reported in rodents, whereby rhesus spermatid-like haploid cells derived from rhesus PSCs successfully fertilized oocytes and triggered blastocyst development [4].The protocol has been independently replicated in other studies [5][6][7][8][9], demonstrating the potency and efficacy of in vitro spermatogenesis in more relevant systems to humans.Two recent papers on mice generated excitement in the field of IVG by generating bi-maternal and bi-paternal mice from ESCs and PSCs [10,11].
In vitro spermatogenesis (IVS) is the attempt to recreate the process of spermatogenesis in a culture system.The main challenges of IVS are to provide a suitable microenvironment that mimics the testis in vivo, to support the survival and development of all the cell types involved in spermatogenesis (germ cells, Sertoli cells, Leydig cells, and peritubular myoid cells), and to achieve complete and functional spermatogenesis.Spermatogenesis produces male gametes (spermatozoa) from spermatogonial stem cells (SSCs) in the testis of mammals.It is a complex and highly regulated process involving multiple cell division, differentiation, and maturation stages.Spermatogenesis is essential for male fertility and reproductive health, but it can be impaired by various factors such as genetic defects, environmental toxicants, infections, aging, or medical therapies.Therefore, there is a great interest in developing methods to study spermatogenesis in vitro, both for basic research and clinical applications.Different methods have been utilized for IVS, such as cell cultures, ex vivo organ cultures, bioreactors, microfluidics, organoids, and scaffold methods.In vitro spermatogenesis is a crucial research area due to its potential in treating male infertility by artificially inducing sperm maturation outside the body.It can aid young cancer patients who risk sterility from treatments by using previously harvested spermatogonial stem cells to produce sperm later in life.The study of this process also offers valuable insights into developmental biology and genetic disorders.Additionally, it provides a platform for drug and toxicology testing without live subjects.Lastly, there is potential for treating genetic diseases by modifying genes during the process before conception.
Two recent publications have covered extensively the topic of in vitro spermatogenesis (IVS) in mice and human [12,13], and two publications on IVG [14,15].Therefore, this review will only cover the recent developments in in vitro spermatogenesis in the past two years and focus on nonmurine studies.
Rodents have the most transit-amplifying division, with 12-13 divisions between the spermatogonial stem cell and the mature sperm, while nonhuman primates have 8-9 divisions, and humans have only 5-6 divisions [51].The transitional 2 (T 2 ) prospermatogonia state, an intermediate precursor state to SSC, is unique to rodent species [57].Another difference between rodent and primate spermatogenesis is the timing of meiosis [45,[50][51][52].In rodents, meiosis occurs continuously throughout adulthood, whereas in primates, meiosis is restricted to discrete periods during early postnatal development and adulthood [51][52][53].In humans, for example, meiosis starts around puberty and continues throughout life, with the production of sperm cells taking about 64-72 days [52,54,55].Also, the duration of spermatogenesis differs among rodents, primates, and humans, with 35 days, 45 days, and 74 days, respectively [51].The hormonal regulation of spermatogenesis also differs in rodents, nonhuman primates, and humans.Rodents rely more on testosterone to regulate spermatogenesis [52,53], while nonhuman primates and humans rely on testosterone and FSH to regulate spermatogenesis [58].
The process of spermiation, which is the release of mature sperm cells from the seminiferous tubules, also differs between rodents and primates.In rodents, spermiation is continuous, whereas in primates, it is episodic and coordinated with hormonal changes [56].Additionally, the morphology of sperm cells can vary between species.For example, in rodents, geometric morphometrics of the head shape are slanted and sickle-shaped with long flagella, whereas, in primates, the heads are typically oval-shaped with a shorter flagellum [59].
Finally, there are some differences in the molecular regulation of spermatogenesis between rodents and primates.For example, some genes involved in spermatogenesis are expressed differently between the two groups, and specific signaling pathways that are critical for germ cell development may also differ [45,56].GFRA1, ITGA6, ZBTB16, GPR125, SALL4, and THY1 are expressed in undifferentiated spermatogonia in mice, monkeys, and humans, but, at odds with the mouse, UTF1, MAGEA4, and FGFR3 are only expressed in type A spermatogonia in humans [60][61][62].Recent single-cell studies have identified many conserved and divergent gene expressions among different cell types within testis [43,45,63].For example, when gene expression profiles from human, macaque, and mouse testes were analyzed together, six spermatogonia (SPG) states, four meiosis states, ten round spermatids states, and five to six elongated spermatids states were identified [45].SPG6 state cells represented type B of preleptotene spermatocytes in all three species and expressed FMR1NB, ZCWPW1, DPEP3, IQBP1, and CALR, but BEND2, ZRANB2, PAGE4, ZNFX1, HLTF, and ZBED5 were only expressed in primates [45].Moreover, their study showed that Sertoli and Leydig cells have the least interaction with SPG cell populations in mice [45], which highlights conserved pathways, but the timing of activation and the origin or target of the signal might have diverged during the evolutionary process.Some epigenetic regulations also differ among rodents, nonhuman primates, and humans.The percentage of histone switching for protamines during spermatogenesis varies among rodents, nonhuman primates, and humans.In rodents, about 98-99% of the histones are replaced by protamines, while in nonhuman primates and humans, about 80-96% are replaced by protamines [64].Overall, methylation profiles are also different between humans and mice.About 80% of rodent and 75% of human genes are methylated, and the number of imprinted genes also considerably differs in rodents and humans (~125-252 and ~50-90) [65].
Overall, while the general process of spermatogenesis is conserved between rodents and primates, there are several differences in the timing, morphology, and molecular regulation between these groups.Understanding these differences is essential for developing spermatogenesis models and advancing research in male infertility and contraception.

Stem Cells and Regenerative Medicine
In vitro PSC differentiation into spermatogenic cells has become a promising approach for studying spermatogenesis and treating male infertility.At the same time, ex vivo organ cultures and tissue culture methods are still the most common approaches to derive/expand SSCs and sperm/spermatids.Multiple in vitro differentiation methods have been developed, each with advantages and disadvantages.This review will summarize the recently published protocols on the differentiation methods of in vitro stem cells (spermatogonial stem cells and pluripotent stem cells) into spermatogenic cells in the past two years, mainly focused on nonrodent studies.

Ex Vivo Organ/Tissue Culture
Ex vivo culture involves maintaining and studying cells or tissues outside their original organism in a controlled environment.Ex vivo tissue culture has been used extensively with various successes since the beginning of IVS.Researchers used frozen goat testis tissue to compare the efficacy of the hanging drop culture method and organ culture methods, where they were able to observe the development of elongated spermatozoa in ex vivo organ culture on an agarose gel block with high-glucose Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (FBS), AlbuXL, L-glutamine, nonessential amino acids (NEAA), and penicillin-streptomycin in 5% CO 2 in air [66].The same group used a scaffold (agarose matrix) to culture prepubertal farming goat testis in α-MEM supplemented with nucleosides, 10% FBS, AlbuXL, and antibiotics [67].After 60 days, researchers observed sperm-like cells with tails in the lumen of the tissue [67].

Bioreactor
A bioreactor is a device that supports the growth of cells and tissues in a biologically active environment by maintaining optimal temperature, pH, oxygen, and nutrient conditional for the cell growth while removing waste.The topic of utilizing microfluidic systems, scaffolds, and bioreactors used in in vitro spermatogenesis has been reviewed elsewhere [75].In 2022, a group generated human 3D organoids in mini-spin bioreactors from hPSCs [76].The human embryonic stem cell (hESC) line H1 was differentiated in large clumps in a maintenance medium supplemented with 8 µM CHIR 99,021 and 5 µM ROCK inhibitor Y-27632 and transferred to low-adhesion cell culture dishes for embryo body (EB) formation [76].The medium was changed to a chemically defined medium (CDM) composed of DMEM/F12, 64 mg/L L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA2P), 1 X insulin-transferrin-selenium (ITS), and 100 U/100 µg pen/strep [76].The medium was supplemented with 8 µM CHIR on the first day, and 10 ng/mL of FGF9 was supplemented on day 3 [76].On day 6, EBs were transferred into a mini-spin bioreactor, and 50 ng/mL of BMP4 and 50 ng/mL of SHH were added on day 10-16 [76].On days 20-24 and 30-38, 10 IU/mL of hCG and 25 mIU/mL PMSG were added [76].They ob-served the development of mesonephros-like organoids in D10-16, nontubular progenitors in D16-22, regression of mesonephric tubules and ingression of progenitor cells in D22-30, and cord-like structures and distinct cell clusters in D30-40 [76].The researchers reported the expression of PAX2 and WT1 in D16 EBs and DMRT1 in D26 and D35 EBs [76].The gonadal ridge markers, GATA4 and WT1, were expressed in D16-20 EBs, and SOX9 was expressed in D20 [76].They also observed AMH and Inhibin B expression in D26-40 EBs and the development of steroidogenic cells (CYP11A1 + , StaR + , and 3β-HSD + ) [76].Their model showed vasculature development and coelomic vessel formation in EBs (VE-Cad + and CD31 + ) [76].However, the expression of early-stage primitive gametogenic marker (OCT4) and post-migratory germ cells (DAZL) were not observed in the EBs [76].The authors of the study speculated that EBs generated in bioreactors might be able to provide microenvironments necessary for further development of in vitro-generated PGCLCs [76].

Microfluidic System
A microfluidic system is a system where multiple channels with a micrometer diameter control a culture condition by manipulating fluids at a microscale.The microfluidic system can closely mimic the in vivo condition by balancing hormones, growth factors, temperature, pH, pressure, and other requirements necessary for spermatogenesis.The most significant advantage is the ability to control the fluid flow rate within the system, which can help stimulate fluid movement, provide nutrients, and remove waste products.A recent study compared the efficacy of immature porcine testicular tissue culture via four culture methods: a static system with a polytetrafluoroethylene membrane, agarose gel, agarose gel with a polydimethylsiloxane chamber, and a microfluidic system [77].The study showed that agarose gel with a polydimethylsiloxane chamber moderately improved the number of meiotic and post-meiotic germ cells [77].The culture medium was based on a previous publication [78], and is only composed of DMEM/F-12, 10% Knockout Serum Replacement (KSR), FSH, and antibiotics [77].

Organoid
An organoid is a miniaturized and simplified version of an organ with micro-anatomy produced in vitro in a three-dimensional culture.In in vitro differentiation studies, organoids serve as a bridge between traditional two-dimensional cell cultures and whole-organ experiments.They can be derived from stem cells or dissociated tissues and can self-organize into structures that closely resemble the functionality and architecture of organs.In this context, organoids are utilized to study organ development, disease modeling, drug testing, and potentially regenerative medicine applications, offering a more accurate representation of in vivo conditions than traditional cell culture methods.
Spermatogonial stem cell isolation and later differentiation into advanced spermatogenic cells have significantly progressed.Isolated cell cultures involve the enzymatic dissociation of testis tissue into single cells or cell clusters cultured in various media and conditions.This method allows the effects of specific factors (such as hormones, growth factors, or feeder cells) on the proliferation and differentiation of germ cells or other testicular cells to be investigated.However, isolated cell cultures have several limitations, such as the loss of cell-cell interactions, tissue architecture, and physiological signals essential for spermatogenesis.Moreover, in most species, isolated cell cultures have yet to achieve complete spermatogenesis beyond certain stages (such as meiosis or spermiogenesis).
Fragment cultures involve the culture of small pieces of testis tissue that retain some of their original structure and cell composition.This method preserves some critical cell-cell interactions and tissue organization for spermatogenesis.However, fragment cultures also have drawbacks, such as difficulty controlling the oxygen and nutrient supply, accumulating waste products, and the heterogeneity of tissue fragments.Fragment cultures have supported partial or complete spermatogenesis in some species (such as mice or rats) but not in others (such as humans or monkeys).
These involve culturing stem cells or dissociated testis tissue in a nonadherent environment that induces them to form 3D aggregates known as embryoid bodies.Three-dimensional cultures involve generating organ-like structures from testicular cells that self-organize into a seminiferous epithelium and an interstitial compartment separated by a basement membrane.These structures are called testicular organoids (TOs), aiming to mimic the testicular microenvironment in vitro.TOs can be formed via multiple methods, such as microwell aggregation, bioprinting, or microfluidics.TOs have several advantages over other IVS methods, such as maintaining the cell-cell interactions, tissue architecture, and physiological signals essential for spermatogenesis.Under specific culture conditions, TOs differentiate into germ cells, including spermatogenic cells.The efficiency of germ cell differentiation can be low, and the resulting cells often need proper maturation.TOs have also shown promising results in supporting partial or complete spermatogenesis in some species (such as pigs or mice).Still, more work is needed to optimize their generation and function.
While the generation of fully mature sperm cells from pluripotent stem cells and testicular organoids in humans is still a relatively new field of research, these advancements hold significant promise for developing new treatments for male infertility and other reproductive disorders.More research is needed to optimize the culture conditions and improve the efficiency and reproducibility of these techniques.Additionally, ethical considerations regarding using human fetal tissue and genetic manipulation must be carefully addressed.A recent review paper extensively covered the topic of testicular organoids [85,86], so this review will only focus on the recent developments in testicular organoids in the past two years.

Scaffolds
Scaffolds used in in vitro studies offer structural supports that mimic the in vivo environment, aiding cell attachment, proliferation, and differentiation.These scaffolds support three-dimensional cell interactions, guide cell growth, allow efficient nutrient and waste diffusion, and can be modified to release specific growth factors or drugs.In in vitro spermatogenesis research, the extracellular matrix (ECM) maintains the necessary complex interactions for germ cell maturation.Commonly utilized ECM components include collagen, which offers a natural environment for cell adhesion; laminin, which supports cell differentiation and migration; fibronectin, promoting cell attachment and growth; gelatin, often used as a hydrogel for cell encapsulation; and Matrigel, a protein mixture resembling the intricate extracellular environment in tissues.Thus, selecting suitable scaffold and ECM components is vital for replicating in vivo conditions and ensuring accurate differentiation studies.
Spermatogenesis requires spatiotemporal regulation of SSCs, which interact with the extracellular matrix (ECM), paracrine factors, hormones, and testicular somatic cells.To mimic such highly orchestrated interactions, various scaffolds have been developed to further enhance the in vitro maturation of SSCs.

Future Direction
Despite progress in in vitro spermatogenesis and the development of testicular organoids, several challenges still need to be addressed.Different approaches and methods covered in this review are summarized in Table 1 and Figure 3.These include the need for more efficient culture methods, the ability to generate functional sperm cells, and the development of reproducible and scalable protocols.In addition, ethical and regulatory considerations are associated with using human testicular tissue for research.
Recent single-cell studies have discovered complex crosstalk among cells in seminiferous tubules and the importance of different factors during development and spermatogen-esis.Therefore, it will be necessary to investigate further the coculture methods involving culturing stem cells with testicular somatic cells, such as Sertoli cells, which provide a supportive microenvironment for spermatogenesis.However, the use of testicular somatic cells can introduce variability in the resulting cells, and the regulatory requirements associated with using human testicular tissue can limit the applicability of this method.Still, various workarounds exist to purify advanced spermatogenic cells, such as round spermatids and spermatozoa.
Also, the step-wise differentiation method can provide accurate temporal regulation to enhance proper transcriptional and epigenetic regulations.The step-wise differentiation method involves the induction of pluripotent stem cells or somatic cells into primordial germ cell (PGC)-like cells, which can further differentiate into male germ cells.PGCs are the precursors of male and female germ cells and are responsible for forming gametes.The step-wise differentiation method uses several growth factors and culture conditions to mimic the in vivo developmental process.For example, adding BMP4 and bFGF to the culture medium can induce the differentiation of pluripotent stem cells into PGC-like cells, which can be further differentiated into spermatogonia by adding RA and Kit ligands.The spermatogonia can then differentiate into spermatocytes via the addition of RA and testosterone, which induce the initiation of meiosis.Finally, spermatids can be generated by adding FSH and hCG, which activate signaling pathways that promote the formation of mature sperm.For example, researchers in a recent rodent study developed functional spermatozoa from mouse embryonic stem cells using the step-wise differentiation method [80,94].The generated spermatozoa could fertilize eggs and produce healthy offspring [80,94].Similarly, another study demonstrated the generation of haploid germ cells from human pluripotent stem cells [3], which has been successfully adopted in primates for successful fertilization and blastocyst development [4].Despite the progress made in the step-wise differentiation method, several challenges still need to be addressed in generating functional spermatozoa.These challenges include optimizing culture conditions and identifying specific signaling pathways in germ cell differentiation.In past years, studies in rodent models have significantly improved the development of spermatogenesis into functional spermatid/sperm in vitro, especially in coculture and organoid models [81][82][83][84].Groundbreaking studies in germ cell developments led to elucidation of the intricate processes involved in specific developmental pathways to generate mature gametes, such as the role of various genes and proteins, the interaction of germ cells with surrounding somatic cells, the role of the spermatogonial niche, and the epigenetic modifications required for germ cell development [31][32][33][43][44][45]48,51,95,96].The success in spermatogonial stem cells in rodents and monkeys demonstrated the efficacy of regenerative medicine in restoring male fertility [97][98][99][100][101].The ethical concerns associated with using human pluripotent stem cells must be addressed before this technology can be translated into clinical applications.
Future studies will need to investigate the combinations of methods, which will provide more accurate spatiotemporal signals to bring us closer to successfully generating functional sperm in vitro.

Conclusions
The research on in vitro spermatogenesis holds transformative potential across various domains of medicine, social sciences, and ethics.Its most immediate application is in reproductive medicine, which could provide new avenues for treating male infertility.By creating mature sperm cells outside the body, men with low sperm counts, poor sperm quality, or even those rendered infertile due to medical treatments could have an alternative means to father biological children.Additionally, it could be invaluable for preserving the fertility of young men undergoing treatments like chemotherapy, which may compromise their reproductive capabilities.This could alleviate not just the physical, but also the emotional and psychological challenges of infertility, offering hope to millions of families.The research also has significant implications for drug discovery and toxicology testing.Generating sperm in vitro would allow for high-throughput screening of substances that could affect male fertility, speeding up the safety profiling of new drugs and identifying potential reproductive hazards among existing ones.The overall significance of in vitro spermatogenesis research is immense.It stands to revolutionize reproductive medicine, contribute significantly to genetic research, and offer new pathways in drug discovery.
In vitro spermatogenesis and testicular organoids are promising technologies that have the potential to revolutionize the field of male reproductive biology.While many challenges still need to be addressed, the progress in these areas provides hope for developing new treatments for infertility and other male reproductive disorders.So far, ex vivo organ/tissue culture has achieved complete spermatogenesis in vitro in nonrodent species (Figure 3).However, even with an ex vivo organ/tissue culture system, it is not certain that continuous spermatogenesis can be achieved in vitro.Since the ex vivo organ/tissue culture system is the oldest approach to in vitro spermatogenesis, the newest approaches, such as organoids, bioprinting, and microfluidics, will improve differentiation efficiency.
In conclusion, IVS is a challenging but promising research field with potential applications for fertility preservation, drug discoveries, infertility treatment, and a basic understanding of spermatogenesis.Among the different methods of IVS, TOs represent a novel and advanced model that can better simulate the testicular microenvironment in vitro.However, TOs are still in their early stages of development and require further refinement and validation before they can be widely used for research or clinical purposes.

Figure 1 .
Figure 1.Conserved expression markers during male germ cell development from zygote to mature spermatozoa in vivo.After fertilization, cells undergo a differentiation process where primordial germ cells (PGCs) develop in the embryo and migrate toward the genital ridge, where early PGCs arrest and differentiate into gonocytes.Gonocytes mature during development and eventually develop into spermatogonial stem cells.Spermatogenesis resumes at puberty.Complex orchestrated stage-specific gene expressions regulate the development of gonocytes, and markers and relative expression levels are depicted [43-49].

Figure 3 .
Figure 3. Summary of different approaches used to achieve in vitro spermatogenesis covered in this review.

Table 1 .
Summary of different in vitro spermatogenesis methods used in recent publications.