Patent Application
20230399608
The present disclosure provides an iterative process for identifying culture conditions that support human testicular germ cell proliferation in vitro and testicular tissue and germ cell cultures supportive of germ cell proliferation while maintaining identity, growth, and survival of the testicular germ cells.
A key need is the ability to culture human germ cells long term, at a scale needed for analysis at a transcriptome-scale manner, and in a manner that fully preserves their identity and functionality for spermatogenesis. However, the field of human male fertility is impeded by the lack of tools for studying spermatogenesis. There are no current successful ways of accomplishing these needs.
Through a wide range of approaches, considerable progress in understanding gametogenesis and germline-niche communication has been achieved in mice. In addition, spermatogonia have been successfully cultured, and methods developed to produce functional sperm from cultured spermatogonia that are capable of successful in vitro fertilization and generation of viable and fertile mice. In contrast, in humans, although adult testis physiology is well described, less is known about spermatogonial stem cells (SSCs), proliferative spermatogonia and their regulation, and long-term culturing of human spermatogonia coupled to genomics approaches to ensure their identity has not been achieved. Accordingly, methods and systems for in vitro culture of testicular germline cells (spermatogonia) are needed, which are the predecessors of spermatogenesis. To accomplish, a new approach is needed.
One aspect of the instant disclosure encompasses an iterative process for identifying culture conditions supportive of testicular germ cell proliferation in vitro. The process comprises identifying one or more dysregulated pathways in testicular cells cultured in a first set of culture conditions by (1) culturing testicular tissue or isolated testicular germ cells in vitro in a first culture medium, wherein the testicular tissue comprises seminiferous tubules and testicular germ cells, and wherein the first culture medium supports a first level of proliferation of germ cells; (2) profiling transcriptomes of single testicular cells obtained from the testicular tissue or isolated testicular germ cells; and (3) identifying RNA transcripts differentially expressed in cultured tissue or germ cells when compared to RNA transcripts expressed in cultured tissue or germ cells of corresponding cell types obtained from control testicular tissue or isolated testicular germ cells, wherein differentially expressed RNA transcripts identify one or more dysregulated biological pathways in cells of the testicular tissue or isolated testicular germ cells cultured in the first set of culture conditions.
The process then comprises identifying one or more proliferation factors that improve the level of proliferation of testicular germ cells by: (1) culturing testicular tissue or isolated testicular germ cells in vitro in one or more second culture media, wherein the one or more second culture media comprise the first culture medium supplemented with one or more factors that regulate a biological pathway identified in (a); and (2) identifying one or more second culture media that support improved levels of germ cell proliferation when compared to the first level of germ cell proliferation in the first culture medium, thereby identifying the one or more factors that improve the level of proliferation of testicular germ cells.
The process can be repeated iteratively to identify additional factors that improve the level of proliferation of testicular germ cells. The culture conditions that support testicular germ cell proliferation comprise culture media supplemented with one or more factors identified in the process or the iterated processes.
Another aspect of the instant disclosure encompasses a culture medium supportive of testicular germ cell proliferation in vitro, the culture medium comprising a base medium supplemented with proliferation factors that improve the level of proliferation of testicular germ cells in vitro. The culture medium can be a medium identified using a process described above.
In some aspects, the culture medium comprises αMEM+10% KSR, Penicillin-Streptomycin at a concentration ranging from about 0.9% to about 1.1%, GDNF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, FGF2 at a concentration ranging from about 19 ng/ml to about 21 ng/ml, Insulin at a concentration ranging from about 9 ug/ml to about 11 ug/ml, EGF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, Testosterone at a concentration ranging from about 9 uM to about 11 uM, and Echinomycin at a concentration ranging from about 4 ng/ml to about 6 ng/ml. (Condition 2) In other aspects, the culture medium comprises αMEM+10% KSR, Penicillin-Streptomycin at a concentration ranging from about 0.9% to about 1.1%, GDNF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, FGF2 at a concentration ranging from about 19 ng/ml to about 21 ng/ml, Insulin at a concentration ranging from about 9 ug/ml to about 11 ug/ml, EGF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, and Testosterone at a concentration ranging from about 9 uM to about 11 uM. (C2) In yet other aspects, the culture medium comprises αMEM+10% KSR, Penicillin-Streptomycin at a concentration ranging from about 0.9% to about 1.1%, GDNF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, FGF2 at a concentration ranging from about 19 ng/ml to about 21 ng/ml, Insulin at a concentration ranging from about 9 ug/ml to about 11 ug/ml, EGF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, and Testosterone at a concentration ranging from about 78 uM to about 82 uM. (C2 with eight times testosterone)
An additional aspect of the instant disclosure encompasses a testicular cell culture for culturing testicular germ cells in vitro. The cell culture comprises testicular tissue comprising germ cells or testicular germ cells; and a culture medium comprising base media, and one or more factors that improve the level of proliferation of testicular germ cells in vitro.
In some aspects, the testicular tissue comprises seminiferous tubules comprising the testicular germ cells and the culture medium comprises αMEM+10% KSR, Penicillin-Streptomycin at a concentration ranging from about 0.9% to about 1.1%, GDNF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, FGF2 at a concentration ranging from about 19 ng/ml to about 21 ng/ml, Insulin at a concentration ranging from about 9 ug/ml to about 11 ug/ml, EGF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, Testosterone at a concentration ranging from about 9 uM to about 11 uM, and Echinomycin at a concentration ranging from about 4 ng/ml to about 6 ng/ml.
In some aspects, the testicular tissue comprises seminiferous tubules comprising the testicular germ cells and the culture medium comprises αMEM+10% KSR, Penicillin-Streptomycin at a concentration ranging from about 0.9% to about 1.1%, GDNF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, FGF2 at a concentration ranging from about 19 ng/ml to about 21 ng/ml, Insulin at a concentration ranging from about 9 ug/ml to about 11 ug/ml, EGF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, Testosterone at a concentration ranging from about 9 uM to about 11 uM, and Echinomycin at a concentration ranging from about 4 ng/ml to about 6 ng/ml.
In yet other aspects, the testicular germ cells comprise SSC, developing spermatogonia, or a combination thereof and wherein the culture medium comprises αMEM+10% KSR, Penicillin-Streptomycin at a concentration ranging from about 0.9% to about 1.1%, GDNF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, FGF2 at a concentration ranging from about 19 ng/ml to about 21 ng/ml, Insulin at a concentration ranging from about 9 ug/ml to about 11 ug/ml, EGF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, and Testosterone at a concentration ranging from about 78 uM to about 82 uM. (C2 with eight times testosterone)
The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure. Certain embodiments can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
The present disclosure encompasses processes for identifying culture conditions that support growth and development of testicular germ cells in vitro, both germline and somatic. The processes make use of genomic approaches to identify testicular germ cell proliferation factors by identifying dysregulated biological pathways in cultured cells and receptor ligands that can be used to identify the in vitro culture conditions. Surprisingly, it was discovered that culture conditions identified using the process of the instant disclosure faithfully recreate conditions needed for stages of spermatogenesis and maintaining the identity, growth, and survival of the testicular germ cells in vitro. Accordingly, media and cell cultures comprising the identified culture conditions that can be used to support testicular germ cell growth in vitro are also disclosed. The processes and compositions can comprise spermatogonial stem cells and spermatogonia grown in vitro that can be used for infertility treatment.
One aspect of the present disclosure encompasses processes for identifying culture conditions supportive of testicular germ cell proliferation in vitro. The processes comprise identifying factors (proliferation factors) that, when added to culture media, can improve the level of proliferation of testicular germ cells cultured in the medium. Optionally, the processes can be iteratively repeated to identify additional proliferation factors, or to identify combinations and concentrations of factors optimized for germ cell proliferation.
The process can identify culture conditions for germ cell growth and development all while maintaining the identity and survival of the germ cells. Culture conditions identified using processes of the instant disclosure can be utilized to obtain sperm with low rates of deleterious or de novo mutations or epigenetic perturbations. Further, the process can be used to help treat male infertility by manipulating germline, help restore fertility for childhood cancer survivors, and provide a useful platform to study human germline.
Mammalian spermatogenesis involves the differentiation of adult spermatogonial stem cells (SSCs) into mature spermatozoa through a complex developmental process (
The overall efficiency and success of spermatogenesis relies on the presence of the stem cell niche.
A process of the instant disclosure comprises identifying culture conditions supportive of testicular germ cell proliferation in vitro. As used herein, the term “testicular germ cell” refers to testicular germ cells at any stage of development from SSCs to mature spermatozoa. Accordingly, a process of the instant disclosure can identify culture conditions supportive of SSC self-renewal and differentiation as well as transitions between multiple cell states and cellular processes during spermatogenesis.
The stages of development can be identified and verified by the distinct transcriptional/developmental states of germ cells, or by identification of markers specific for each cell type. The identity of germ cells at each stage of development can be identified using methods known in the art and can be as described in Guo et al., Cell Stem Cell, 2017; Guo et al., Cell Research, 2018; and Guo et al., Cell Stem Cell, 2020, the disclosures of all of which are incorporated herein in their entirety.
The process comprises culturing testicular tissue comprising germ cells in vitro in a culture medium. Testicular tissue can be tissue comprising germ cells isolated from a subject, or can be testicular tissue produced in vitro, such as testicular organoids comprising germ cells. If the testicular tissue is an organoid, the organoid can assume the function of the testis niche in guiding testicular germ cells during spermatogenesis. The process can also comprise culturing testicular germ cells in vitro in a culture medium. Accordingly, testicular germ cells can comprise isolated germ cells, germ cells associated with testicular tissue, or germ cells associated with organoids. In some aspects, the testicular tissue is tissue dissected from a subject and comprising seminiferous tubules and testicular germ cells. In some aspects, the testicular tissue is seminiferous tubules comprising germ cells. In some aspects, the process comprises culturing isolated SSCs and/or spermatogonia.
A process of the instant disclosure can be used to identify culture conditions that can support division, growth, and development (proliferation) of testicular germ cells in vitro at any stage in the developmental process. As used herein, the term “proliferation” when referring to testicular germ cells refers to the ability of germ cells to grow, divide, survive (e.g., dish life of stem cells), and develop, as well as maintain the identity and survival of the germ cells. Accordingly, the process can identify culture conditions that support self-renewal and transition of SSCs to proliferative spermatogonia, from proliferative spermatogonia to entering meiosis, from differentiating spermatogonia to primary and secondary spermatocytes, spermatids, through sperm maturation, or any combination thereof. In some aspects, the process is used to identify culture conditions supportive of SSC and spermatogonial proliferation.
The process can be used to identify culture conditions that can support proliferation of testicular germ cells of any mammalian animal in vitro. “Mammalian,” as used herein refers to both human subjects (and cells sources) and non-human subjects (and cell sources or types), such as dog, cat, mouse, monkey, etc. (e.g., for veterinary purposes). In some aspects, a process of the instant disclosure is used to identify culture conditions that can support proliferation of human testicular germ cells in vitro. This is important because there are no current successful ways of successfully culturing human testicular germ cells.
The testicular tissue or germ cells can be from prepubertal or adult fertile or infertile males. The testicular tissue or germ cells can be from a live subject or a cadaveric subject. The testicular tissue or germ cells can also be freshly harvested or can be cryopreserved cells. For instance, the cryopreserved testicular tissue or germ cells can be from a subject expected to have germ-damaging treatment (e.g., chemotherapy) for future use with assisted reproductive technologies. In some aspects, testicular germ cells can be from a pre-pubertal subject.
One aspect of a process of the instant disclosure encompasses identifying one or more factors supportive of testicular germ cell proliferation in vitro by identifying one or more dysregulated pathways in cells of cultured testicular tissue, identifying factors that can regulate the dysregulated pathways (referred to hereinafter as pathway factors), and identifying among the pathway factors, factors that improve the level of proliferation of testicular germ cells by screening the identified pathway factors for factors that can regulate the identified dysregulated pathways to identify the factors among them that can improve germ cell proliferation. The inventors discovered that testicular cells cultured in media that cannot support sufficient germ cell proliferation comprise dysregulated biological pathways. The inventors also discovered that some factors that can regulated identified dysregulated pathways can improve proliferation of germ cells when the factors are used to supplement culture media.
A process of identifying dysregulated pathways comprises the use of a genomic approach to identify dysregulated pathways in the cultured testicular tissue or testicular germ cells. A process of the instant disclosure further comprises screening factors that can regulate the identified dysregulated pathways to identify the factors among them that can improve germ cell proliferation. Factors that can regulate the biological pathways and methods of identifying factors that can regulate pathways are known in the art or can be identified by individuals of skill in the art using known methods. The factors can be ligands, inhibitors, small molecule factors, as well as peptides among others. Screening the identified factors can be as described in Section I(d) herein below. In some aspects, identified pathway factors for screening are as listed in Table 7 herein below.
In some aspects, identifying the one or more dysregulated pathways in testicular cells cultured in a first set of culture conditions comprises (1) culturing testicular germ cells or testicular tissue comprising germ cells in vitro in a first culture medium, wherein the first culture medium supports a first level of proliferation of the germ cells; (2) profiling transcriptomes of testicular cells obtained from the testicular tissue; and (3) identifying RNA transcripts differentially expressed in cells grown in the first culture medium when compared to RNA transcripts expressed in cells obtained from control tissue. The differentially expressed RNA transcripts identify one or more dysregulated biological pathways in the testicular cells cultured in the first set of culture conditions when compared to the biological pathways of the control tissue or cells. Testicular tissues and cells can be as described in Section I(a) herein above.
In some aspects, the process further comprises assigning a cell type to each single testicular cell using cell type-specific gene markers expressed in each cell. When the process comprises assigning a cell type to single testicular cells, the process can further comprise identifying RNA transcripts differentially expressed in each cell type when compared to RNA transcripts expressed in cells of corresponding cell types obtained from control tissue.
As explained above, testicular germ cells can comprise isolated germ cells independent of other testicular tissue, germ cells associated with testicular tissue cultured in vitro, or germ cells associated with organoids in vitro. Accordingly, if the process comprises identifying factors by identifying dysregulated pathways in cells of cultured testicular tissue or in cells of cultured organoids, the process can comprise identifying one or more dysregulated pathways in cells of cultured testicular tissue or cells of cultured organoids and identifying dysregulated pathways of germ cells associated with testicular tissue or organoids. In some aspects, a process of the instant disclosure encompasses identifying dysregulated pathways in cells that form a niche for germ cell proliferation. For instance, the process can comprise identifying dysregulated pathways in somatic cells, including Sertoli cells, Leydig cells, endothelial cells, myoid cells, or any combination thereof. In some aspects, a process of the instant disclosure encompasses identifying dysregulated pathways in cells of seminiferous tubules, and germ cells in the seminiferous tubules. In some aspects, the process comprises identifying one or more factors that improve the level of proliferation of testicular germ cells cultured in vitro independently from other testicular tissue by identifying one or more dysregulated pathways in the cultured germ cells.
The dysregulated pathways are identified by first culturing testicular tissue or germ cells in vitro in a first culture medium that can support a first level of proliferation of testicular germ cells. In some aspects, the first culture medium is a base medium. The base medium can be as described in Section II herein below. In some aspects, the first culture medium comprises base medium supplemented with factors identified in a previous round of pathway factor-identification using the process of the instant disclosure and various combinations and concentrations of the previously identified pathway factors (See, e.g., Section I(d) herein below).
After culturing the testicular tissue or germ cells in the first culture medium, a genomics approach is used to identify the dysregulated pathways. In essence, identifying dysregulated pathways comprises identifying differentially expressed RNA transcripts in single testicular cells grown in vitro in the first culture medium when compared to RNA expressed in control tissue or germ cells. In some aspects, the genomes of cultured testicular tissue or cells are profiled to obtain the transcriptional profile of each isolated cell. When the process further comprises assigning a cell type to each single testicular cell, the transcriptional profile can be the transcriptional profile of each cell type. In some aspects, control testicular tissue is testicular tissue obtained directly from a subject. In some aspects, control testicular tissue is tissue directly obtained from a subject and the level of RNA transcripts in each cell type in the tissue obtained directly from a subject can be as described in Guo et al. 2018, the disclosure of all of which is incorporated herein in its entirety.
Any method of transcriptional profiling can be used in a process of the instant disclosure. Methods of obtaining transcriptional profiles of single cells are known in the art and include, without limitation, single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, Single-Cell Combinatorial Indexing RNA Sequencing (sci-RNA-seq), and Single-nucleus RNA sequencing (snRNA-seq). scRNA-seq involves the isolation of individual cells, followed by reverse transcription of RNA to create a cDNA library which is then sequenced. In spatial transcriptomics, RNA transcripts are sequenced without first isolating single cells, allowing for the preservation of the spatial context of the transcriptome in tissue sections. The cells' transcriptomes can be related to their spatial position within the tissue. Single-Cell Combinatorial Indexing RNA Sequencing (sci-RNA-seq) allows for high-throughput single-cell transcriptome profiling. It works by using combinatorial labeling of cells and pooling steps to greatly increase the throughput. Single-nucleus RNA sequencing (snRNA-seq) is used when the cells are difficult to dissociate or are sensitive to the process (e.g. Sertoli cells can be too large), or when the analysis of archived frozen material is required. It profiles the transcriptome of single nuclei rather than whole cells. In some aspects, the cells are dissociated to obtain single cells of all types of testicular tissue cell types of the tissue and the genomes of isolated cells are profiled to obtain the transcriptional profile of each isolated cell.
In some aspects, the profiling step of the process comprises using scRNA-seq approaches to identify the differentially expressed RNA transcripts in each cell type when compared to the level of RNA transcripts in the corresponding cell type in control tissue or germ cells, such as, from tissue obtained directly from a donor. For instance, identification of differentially expressed RNA transcripts using scRNA-seq can be as described in Guo et al., Cell Stem Cell, 2017; Guo et al., Cell Research, 2018; and Guo et al., Cell Stem Cell, 2020, the disclosures of all of which are incorporated herein in their entirety.
The differentially expressed RNA transcripts identify one or more dysregulated biological pathways in the in vitro cultured cells. In some aspects, Gene Ontology (GO) terms of genes expressing the RNAs in the cells or cell types is used to group differentially expressed genes into pathways. In some aspects, dysregulated pathways in cultured testicular tissue can be identified using a process detailed in the diagram shown in
A dysregulated biological pathway can be any biological pathway essential for testicular germ cell proliferation. For instance, the identified dysregulated pathways can be metabolic pathways, transcription pathways, signaling pathways, survival pathways, cell cycle pathways, physiological pathways, and developmental pathways among others. In some aspects, dysregulated pathways are identified in cultured testicular tissue, and the identified dysregulated pathways are pathways associated with extracellular exosome, negative regulation of apoptotic process, cytokine, response to hypoxia, actin cytoskeleton, extracellular matrix, and muscle contraction.
In some aspects, the one or more dysregulated pathways comprise one or more pathways of hypoxia-inducible factor (HIF;
Another aspect of the instant disclosure encompasses identifying one or more factors supportive of testicular germ cell proliferation in vitro by identifying ligands of receptors that can improve germ cell proliferation in testicular tissue or germ cells cultured in vitro. In essence, the process comprises identifying one or more receptors expressed in testicular tissue or germ cells, identifying ligands of the identified receptors, and screening the identified ligands in testicular tissue or germ cells cultured in vitro to identify receptor ligands that can improve germ cell proliferation in in vitro cultures of testicular tissue or germ cells.
Cell surface receptors play crucial roles in the proliferation and development of testicular germ cells, the precursors to sperm cells. Receptors mediate the signaling pathways that regulate germ cell growth, differentiation, and function. The binding of specific ligands, such as hormones or growth factors, to these cell surface receptors triggers intracellular signaling cascades, influencing gene expression and subsequent cellular behavior. For instance, follicle-stimulating hormone (FSH) receptors and luteinizing hormone (LH) receptors, both present on the cell surface, are critically involved in the endocrine regulation of spermatogenesis. FSH stimulates Sertoli cells, which support and nourish developing germ cells, while LH acts on Leydig cells to stimulate the production of testosterone, a hormone essential for spermatogenesis. Additionally, growth factors like glial cell line-derived neurotrophic factor (GDNF), acting through its receptor GFRα1, play vital roles in the self-renewal of spermatogonial stem cells.
A process of identifying receptors expressed in testicular tissue and germ cells comprises the use of a genomic approach. Testicular tissues and cells can be as described in Section I(a) herein above. In some aspects, databases of receptor expression patterns can be mined to identify receptors expressed in testicular tissue or germ cells. For instance, receptors expressed in testicular tissue and cells can be identified by identified receptor genes comprising a level of expression indicative of substantial expression in the cells and/or an expression pattern indicative of testicular tissue-specific or testicular cell-type specific expression. Any database of receptor expression and ligands can be used in a process of the instant disclosure. Databases of receptors and ligands are known in the art. Further, it will be recognized that databases continue to be updated and new databases continue to be compiled. Accordingly, a database of the instant disclosure can be an existing database of receptors and ligands as well as databases compiled or updated in the future. Non-limiting examples of databases of receptors and ligands suitable for use in a process of the instant disclosure include CellphoneDB, CellChat, huARdb (human Antigen Receptor database; Wu et al., Nucleic Acids Research, Volume 50, Issue D1, 7 Jan. 2022, Pages D1244-D1254), Cellinker (Zhang et al., Bioinformatics. 2021 Jan. 20), among others. It will be noted that, as these databases may not necessarily encompass all expressed receptors at all stages of development of all tissues and cells, a process of the instant disclosure, could identify different receptors and ligands depending on the database used in the process and depending on the testicular tissue or germ cells and developmental stages of testicular tissue and germ cells being investigated. As shown in Example 3 herein below, extensive experimentation showed that other methods of identifying receptors and corresponding ligands such as using CellphoneDB and CellChat to analyze cell-cell interaction to find potential ligands generated different results depending on the receptor ligand database used.
In some aspects, the process can comprise identifying receptors in cells of testicular tissue or cells of cultured organoids. In some aspects, a process of the instant disclosure encompasses identifying receptors in cells that form a niche for germ cell proliferation. For instance, the process can comprise identifying receptors expressed in somatic cells, including Sertoli cells, Leydig cells, endothelial cells, myoid cells, or any combination thereof. In some aspects, a process of the instant disclosure encompasses identifying receptors in cells of seminiferous tubules. In some aspects, the process comprises identifying receptors of testicular germ cells. In some aspects, the receptors are identified in SSCs, differentiating spermatogonia, or both. In some aspects, the receptors are identified in State 1 SSCs (active stem cells) and State 2/3 differentiating spermatogonia (differentiated germ cells with strong proliferation ability).
In some aspects, the receptors are identified by determining the level of expression of RNA transcripts of receptor genes expressed in the various cells and cell types of interest. In some aspects, the level of expression of RNA transcripts in testicular cells can be as described in Guo et al. 2018, the disclosure of all of which is incorporated herein in its entirety. In some aspects, identifying ligands can be as depicted in
A process of the instant disclosure further comprises identifying among the pathway factors that can regulate the dysregulated pathways in Section I(b) or the receptor ligands identified in Section I(c) the one or more factors that can improve the level of proliferation of testicular germ cells in vitro. As used herein, the term “improved proliferation” when referring to testicular germ cells refers to improved growth, division, survival, physiology, or development of the germ cells and can be measured by an increase in the number of germ cells over time during culture, the duration of life of the cells in the culture medium, proper physiology of the cells, or any combination thereof.
Identifying one or more proliferation factors comprises culturing testicular tissue in vitro in culture media supplemented with one or more factors that regulate that can regulate the dysregulated pathways in Section I(b), supplemented with the receptor ligands identified in Section I(c), or any combination of the factors and ligands. The second cultures that support improved levels of germ cell proliferation comprise one or more proliferation factors, thereby identifying the one or more factors that improve the level of proliferation of testicular germ cells. In some aspects, improved proliferation of testicular germ cells in the new culture conditions can be confirmed by testing for improved growth, division, survival, physiology, or development of the germ cells and can be measured by an increase in the number of germ cells over time during culture, the duration of life of the cells in the culture medium, proper physiology of the cells, or any combination thereof. Germ cells grown in the identified culture conditions can maintain their identity at all stages of development. When the testicular cells comprise cells other than germ cells such as cells other than germ cells in tissue obtained from subjects or cells other than germ cells in organoids, cells grown in the identified culture conditions can maintain their identity at all stages of development. In some aspects, improved proliferation of testicular germ cells in the new culture conditions comprise maintained testicular size and germ cell division and survival. Culture media can be as described in Section II herein below and culturing testicular tissue comprising testicular germ cells or isolated testicular germ cells can be as described in Section III herein below.
In some aspects, identifying culture conditions can be informed by a previously performed round of the process. More specifically, identifying culture conditions comprises can be iteratively repeating to identify additional proliferation factors, various combinations of identified factors, various levels of the factors, and any combination thereof. When replication factors are identified by identifying dysregulated pathways, the process of identification of dysregulated pathways and pathway factors, screening the identified pathway factors, or any combination thereof can be iteratively repeated to identify proliferation factors to identify additional factors, various combinations of identified factors, various levels of the factors, and any combination thereof that improve the level of proliferation of testicular germ cells can be iteratively repeated to identify additional factors that improve the level of proliferation of testicular germ cells.
Importantly, the inventors discovered that a process of identifying proliferation factors using testicular tissue can be used as a learning platform for identifying proliferation factors that can improve proliferation of germ cells grown in vitro in an alternative growth format. More specifically, proliferation factors identified using a process that uses testicular tissue to identify proliferation factors can in turn be used in a process of identifying proliferation factors using isolated testicular germ cells, thereby greatly increasing the efficiency of identifying the germ cell proliferation factors.
In some aspects, proliferation factors identified using a process of the instant disclosure can be as described in Section II herein below. In some aspects, ligands of the identified receptors can be as described in Section II herein below.
One aspect of the instant disclosure encompasses an iterative process for identifying culture conditions supportive of testicular germ cell proliferation in vitro. The process first comprises identifying one or more dysregulated pathways in testicular cells cultured in a first set of culture conditions. In some aspects, identifying one or more dysregulated pathways comprises culturing testicular tissue in vitro in a first culture medium, profiling transcriptomes of single testicular cells obtained from the testicular tissue, and identifying RNA transcripts differentially expressed in each cell type when compared to RNA transcripts expressed in cells of corresponding cell types obtained from control tissue. Culturing testicular tissue comprising testicular germ cells or isolated testicular germ cells can be as described in Section III herein below.
After identifying pathways dysregulated in testicular cells cultured in the first set of culture conditions, the process then comprises identifying one or more proliferation factors that improve the level of proliferation of testicular germ cells. Identifying one or more proliferation factors that improve the level of proliferation comprises culturing testicular tissue in vitro in one or more second culture media, wherein the one or more second culture media comprise the first culture medium supplemented with one or more factors that regulate an identified biological pathway. The tissue and cells are then cultured and one or more second culture media that support improved levels of germ cell proliferation when compared to the first level of germ cell proliferation in the first culture medium are identified. The second cultures that support improved levels of germ cell proliferation comprise one or more proliferation factors, thereby identifying the one or more factors that improve the level of proliferation of testicular germ cells.
In some aspects, the process of identification of dysregulated pathways and pathway factors, screening the identified pathway factors, or any combination thereof can be iteratively repeated to identify proliferation factors to identify additional factors, various combinations of identified factors, various levels of the factors, and any combination thereof that improve the level of proliferation of testicular germ cells can be iteratively repeated to identify additional factors that improve the level of proliferation of testicular germ cells.
In some aspects, the process of identification of receptors and receptor ligands, screening the ligands, or any combination thereof can be iteratively repeated to identify proliferation factors to identify additional factors, various combinations of identified factors, various levels of the factors, and any combination thereof that improve the level of proliferation of testicular germ cells.
In some aspects, the dysregulated pathway is selected from the HIF pathway, pathways downstream of the HIF pathway, and any combination thereof. In some aspects, the dysregulated pathway comprises the HIF pathway, inflammatory pathways, fibrosis pathways, angiogenesis/VEGFA-VEGFR1/2 signaling pathways, and any combination thereof. In some aspects, the one or more dysregulated pathways comprise one or more pathways of hypoxia-inducible factor (HIF;
Another aspect of the instant disclosure encompasses an iterative process for identifying culture conditions supportive of testicular germ cell proliferation in vitro. The process comprises identifying one or more dysregulated pathways in testicular cells cultured in a first set of culture conditions by: culturing testicular tissue in vitro in a first culture medium, wherein the testicular tissue comprises seminiferous tubules and testicular germ cells, and wherein the first culture medium supports a first level of proliferation of germ cells; profiling transcriptomes of single testicular cells obtained from the testicular tissue using single cell RNA sequencing (scRNA-seq); assigning a cell type to each single testicular cell using cell type-specific gene markers expressed in each cell; and identifying RNA transcripts differentially expressed in each cell type when compared to RNA transcripts expressed in cells of corresponding cell types obtained from control tissue, wherein differentially expressed RNA transcripts identify one or more dysregulated biological pathways in the testicular cell types cultured in the first set of culture conditions.
The process also comprises identifying one or more proliferation factors that improve the level of proliferation of testicular germ cells in vitro by: culturing testicular tissue in vitro in one or more second culture media, wherein the one or more second culture media comprise the first culture medium supplemented with one or more identified factors that regulate one or more of the identified biological pathways; identifying one or more second culture media that support improved levels of germ cell proliferation when compared to the first level of germ cell proliferation in the first culture medium, thereby identifying the one or more factors that improve the level of proliferation of testicular germ cells. The culture conditions that support testicular germ cell proliferation comprise culture media supplemented with one or more of the identified factors. Culture media can be as described in Section II herein below and culturing testicular tissue comprising testicular germ cells or isolated testicular germ cells can be as described in Section II herein below.
The process also comprises iteratively repeating the steps of pathway identification and screening factors that can regulate the identified pathways to identify additional factors that improve the level of proliferation of testicular germ cells.
An additional aspect of the instant disclosure encompasses an iterative process for identifying culture conditions supportive of testicular germ cell proliferation in vitro. The process comprises identifying one or more receptors expressed in testicular tissue or germ cells and identifying ligands of the identified receptors. The process also comprises identifying one or more proliferation factors that improve the level of proliferation of testicular germ cells. Identifying one or more proliferation factors comprises culturing testicular tissue in vitro in one or more second culture media, wherein the one or more second culture media comprise the first culture medium supplemented with one or more factors that regulate an identified biological pathway. The second cultures that support improved levels of germ cell proliferation comprise one or more proliferation factors, thereby identifying the one or more factors that improve the level of proliferation of testicular germ cells. Culture media can be as described in Section II herein below and culturing testicular tissue comprising testicular germ cells or isolated testicular germ cells can be as described in Section III herein below.
In some aspects, the process of identifying receptors and receptor ligands, screening the ligands, or any combination thereof can be iteratively repeated to identify proliferation factors to identify additional factors, various combinations of identified factors, various levels of the factors, and any combination thereof that improve the level of proliferation of testicular germ cells.
Yet another aspect of the instant disclosure encompasses an iterative process for identifying culture conditions supportive of testicular germ cell proliferation in vitro. The process comprises (1) identifying the receptors from a database of receptors, receptors specifically expressed in testicular identified using scRNA-seq transcriptional profiles of testicular tissue or testicular germ cells, receptors specifically expressed in testicular identified using scRNA-seq transcriptional profiles of testicular tissue or testicular germ cells; (2) identifying ligands of the identified receptors; (3) culturing testicular germ cells or testicular tissue in vitro in a culture medium supplemented with one or more of the identified ligands; (4) identifying culture media that support improved levels of germ cell proliferation when compared to the level of germ cell proliferation in un-supplemented culture medium, thereby identifying the one or more factors that improve the level of proliferation of testicular germ cells. The process further comprises iteratively repeating the process of identifying receptors and receptor ligands, screening the ligands, or any combination thereof can be iteratively repeated to identify proliferation factors to identify additional factors, various combinations of identified factors, various levels of the factors, and any combination thereof that improve the level of proliferation of testicular germ cells. The culture media that support testicular germ cell proliferation comprise one or more of the identified ligands.
Identifying the one or more receptors expressed in testicular tissue or germ cells comprises selecting about 150 receptors comprising the highest level of expression in the testicular cells, ranking the receptors by expression level in decreasing order, and excluding receptors that have ubiquitous expression in testicular tissue, i.e., expressed in testicular cells outside of SSCs, developing spermatogonia, or both. Identifying ligands of the identified receptors comprises identifying from a database of ligands, ligands expressed in human testes. The database of receptors and receptor ligands is CellTalkDB.
The process also comprises identifying one or more proliferation factors that improve the level of proliferation of testicular germ cells. Identifying one or more proliferation factors comprises culturing testicular tissue in vitro in one or more second culture media, wherein the one or more second culture media comprise the first culture medium supplemented with one or more factors that regulate an identified biological pathway. The second cultures that support improved levels of germ cell proliferation comprise one or more proliferation factors, thereby identifying the one or more factors that improve the level of proliferation of testicular germ cells. Culture media can be as described in Section II herein below and culturing testicular tissue comprising testicular germ cells or isolated testicular germ cells can be as described in Section III herein below.
Using the process of the instant disclosure, the inventors were able to identify receptors, receptor ligands, and the ligands among them that can improve proliferation of SSCs and differentiating spermatogonia. In some aspects, ligands of the identified receptors can be as described in Section II herein below. In some aspects, the identified receptors specifically expressed in SSCs and differentiating spermatogonia, the ligands of the receptors, and ligands that can improve proliferation of germ cells can be as listed in Table 5.
Another aspect of the instant disclosure encompasses a culture medium supportive of testicular germ cell proliferation in vitro. The terms “medium”, “media”, “culture medium”, or “culture media,” as used herein, refers to an aqueous based solution that is provided for the growth, viability, or storage of cells used in carrying out the present invention. A “base media,” as used herein, refers to a basal salt nutrient or an aqueous solution of salts and other elements that provide cells with water and certain bulk inorganic ions essential for normal cell metabolism and maintains intracellular and/or extra-cellular osmotic balance. Base media can comprise energy sources such as glucose or galactose, amino acids, vitamins, salts, a buffering system to maintain the medium within the physiological pH range, or any combination thereof. Base media for culturing mammalian cells are known in the art and can be available commercially. Non-limiting examples of base media include phosphate buffered saline (PBS), Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), Roswell Park Memorial Institute Medium (RPMI) 1640, MCDB 131, Click's medium, McCoy's 5A Medium, Medium 199, William's Medium E, insect media such as Grace's medium, Ham's Nutrient mixture F-10 (Ham's F-10), Ham's F-12, α-Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM) and Iscove's Modified Dulbecco's Medium.
In the context of testicular cells, certain specialized media have been developed. For instance, F12/DMEM, often supplemented with fetal bovine serum (FBS), is commonly used. Other media, like M199, can also be utilized. In addition, specific supplements may be included depending on the cell type. For instance, for the culture of Sertoli cells (a type of testicular cell), supplements can include insulin, transferrin, and biotin, among others. Furthermore, in some instances, growth factors such as glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor (FGF) might be used to support spermatogonial stem cell cultures. However, the specific formulation can and will vary widely depending on the specific requirements of the testicular cells in question. In some aspects, the culture medium further comprises components that support the proliferation of stem cells in culture. Such a medium can be obtained commercially, e.g., STEMPRO-34, Pluripotent Stem Cell SFM XF/FF, or derived from a stem cell culture medium known in the art. In addition to a basal medium, the culture medium can include additional components or supplements to support the proliferation of testicular cells Such supplements include, but are not limited to, fetal bovine serum, Knockout Serum Replacement (KSR), B27 supplement, glial cell-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), stem cell factor (SCF) retinoic acid, follicle-stimulating hormone (FSH), an antibiotic, an antifungal, and the like.
In some aspects, the culture medium comprises a base medium supplemented with proliferation factors that improve the level of proliferation of testicular germ cells in vitro. In some aspects, a base culture medium is αMEM supplemented with KSR. In some aspects, a base culture medium is αMEM+10% KSR.
In some aspects, the culture medium comprises base medium supplemented with proliferation factors previously identified using a process of the instant disclosure. A process of the instant disclosure can be as described in Section I herein above. Proliferation factors previously identified using a process of the instant disclosure can be as described herein.
In some aspects, a medium of the instant disclosure comprises a culture medium supplemented with proliferation factors identified by identifying one or more dysregulated pathways in cells of cultured testicular tissue, identifying factors that can regulate the dysregulated pathways (referred to hereinafter as pathway factors), and identifying among the pathway factors, factors that improve the level of proliferation of testicular germ cells by screening the identified pathway factors for factors that can regulate the identified dysregulated pathways to identify the factors among them that can improve germ cell proliferation. In some aspects, the pathway factors can be identified as described in Section I(b), and the proliferation factors can be as identified in Section I(d).
In some aspects, a medium of the instant disclosure is supplemented with one or more of an inhibitor of hypoxia-inducible factor (HIF), an anti-apoptosis factor, an anti-inflammation factor, a ROS inhibitor, a gonadocorticoid, a gonadotropin, a member of the GDNF family of ligands (GFL), an activin, a fibroblast growth factor receptor (FGFR) protein ligand, an interleukin 6 cytokine, a chemokine, a retinoic acid receptor ligand, a ligand of receptor of Table 5, or any combination thereof. In some aspects, HIF can be HIF-1a, VHL E3 ubiquitin ligase (VHL), or a combination thereof. In some aspects, HIF-1a inhibitor can be a polyamide (disrupts the HIF-1-DNA interface), acriflavine (inhibits dimerization of HIF-1), chetomin (disruptes the HIF-1-p300 interaction), YC1 (inactivates the transcriptional activity of HIF-1a), amphotericin B (inactivates the transcriptional activity of HIF-1a), AJM290 (inactivates the transcriptional activity of HIF-1α), AW464 (inactivates the transcriptional activity of HIF-1α), PX-12 (inhibits HIF-1α protein levels), PX-478 (inhibits HIF-1α protein levels), aminoflavone (inhibits HIF-1α protein levels), EZN-2968 (an RNA antagonist of HIF1α), echinomycin (disrupts the HIF-1-DNA interface), or any combination thereof.
In some aspects, proliferation factors are selected from Testosterone, Activin A, FSH, GDNF, FGF2, LIF, RA, CXCL12, WNT-3A, Neurturin (NRTN), Netrin-1 (NTN1), BMP2, rh beta-NGF, rh Midkine Protein, rh HB-EGF, rh Holo-Transferrin, rh MIF, rh CXCL4, rh LIF, insulin, proliferation factors listed in Table 12, and any combination thereof.
In some aspects, a proliferation factor of the instant disclosure is a HIF-1α inhibitor. In some aspects, the HIF-1α inhibitor is echinomycin, PX-12, vitexin, or any combination thereof. In one aspect, the HIF-1α inhibitor is echinomycin, and the concentration of echinomycin in the culture media can range from about 0.1 nM to about 100 nM, about 1 nM to about 50 nM, or about 2 nM to about 7 nM.
In some aspects, a proliferation factor of the instant disclosure is a gonadocorticoid. In some aspects, the gonadocorticoid can be an androgen. The androgen can be testosterone, FSH, hCG, LH, GDNF, or a combination thereof. In some aspects, the androgen is testosterone, and the concentration of testosterone in the culture media ranges from about 10−5M to about 10−9M, from about from about 10−6M to about 10−8M, or from about 1.5×10−6M to about 0.5×10−8M. In some aspects, the androgen is testosterone, and the concentration of testosterone in the culture media ranges from about 8 uM to about 150 uM, from about 18 uM to about 140 uM, from about 28 uM to about 130 uM, from about 38 uM to about 120 uM, from about 48 uM to about 110 uM, from about 58 uM to about 110 uM, from about 68 uM to about 110 uM, or from about 78 uM to about 110 uM. In some aspects, the androgen is testosterone, and the concentration of testosterone in the culture media ranges from about 78 uM to about 82 uM.
In some aspects, a proliferation factor of the instant disclosure is a member of GFL. In some aspects, the member of GFL can be GDNF. In some aspects, the concentration of GDNF in the culture media ranges from about 0.1 ng/mL to about 100 ng/mL, about 1 ng/mL to about 50 ng/mL, or about 7 ng/mL to about 12 ng/mL.
In some aspects, a proliferation factor of the instant disclosure is a fibroblast growth factor receptor (FGFR) protein ligand. In some aspects, the FGFR protein ligand can be bFGF (FGF2). In some aspects, the concentration of bFGF in the culture media ranges from about 0.1 ng/mL to about 100 ng/mL, about 1 ng/mL to about 50 ng/mL, or about 7 ng/mL to about 12 ng/mL.
In some aspects, a proliferation factor of the instant disclosure is a gonadotropin. The gonadotropin can be human chorionic gonadotropin (hCG), leutenizing hormone (LH), or both. The activin can be activin A. The concentration of activin A in the culture media can range from about 0.1 ng/mL to about 200 ng/mL, about 1 ng/mL to about 150 ng/mL, or about 25 ng/mL to about 75 ng/mL.
In some aspects, a proliferation factor of the instant disclosure is an interleukin 6 cytokine. The interleukin 6 cytokine can be leukemia inhibitory factor (LIF). In some aspects, the concentration of LIF in the culture media ranges from about 1 ng/mL to about 500 ng/mL, about 10 ng/mL to about 200 ng/mL, or about 75 ng/mL to about 125 ng/mL.
In some aspects, a proliferation factor of the instant disclosure is a chemokine. The chemokine can be CXCL12. In some aspects, the concentration of CXCL12 in the culture media ranges from about 1 ng/mL to about 500 ng/mL, about 10 ng/mL to about 200 ng/mL, or about 75 ng/mL to about 125 ng/mL.
In some aspects, a proliferation factor of the instant disclosure is a retinoic acid. The retinoic acid receptor ligand can be retinoic acid. In some aspects, the concentration of retinoic acid in the culture media ranges from about 10−5M to about 10−9M, from about from about 10−6M to about 10−8M, or from about 2.5×10−7M to about 3.5×10−7M.
In some aspects, a proliferation factor of the instant disclosure is insulin. In some aspects, the concentration of insulin in the culture media ranges from about 0.1 ng/mL to about 100 ng/mL, about 1 ng/mL to about 50 ng/mL, about 7 ng/mL to about 12 ng/mL, or about 9 ng/mL to about 11 ng/mL.
In some aspects, a medium of the instant disclosure comprises αMEM with KSR base medium supplemented with one or more factors selected from echinomycin, testosterone, RA, and FSH and any combination thereof. In other aspects, a medium of the instant disclosure comprises αMEM with KSR base medium supplemented with one or more factors selected from echinomycin, testosterone, and GDNF. In yet other aspects, a medium of the instant disclosure comprises αMEM with KSR base medium supplemented with one or more factors selected from echinomycin, testosterone, GDNF, HCG, and FSH. In some aspects, a medium of the instant disclosure comprises αMEM with KSR base medium supplemented with one or more factors selected from Testosterone, Activin A, FSH, GDNF, FGF2, LIF, RA, CXCL12, and any combination thereof.
In some aspects, a culture medium of the instant disclosure comprises, Testosterone, GDNF, and FGF2. In some aspects, a culture medium of the instant disclosure comprises αMEM+10% KSR, echinomycin at a concentration ranging from about 4 nM to about 6 nM, Testosterone at a concentration ranging from about 10−6 M to about 10−8 M, GDNF at a concentration ranging from about 9 ng/ml to about 11 ng/mL, and FGF2 at a concentration ranging from about 9 ng/ml to about 11 ng/mL.
In some aspects, a culture medium of the instant disclosure comprises αMEM+10% KSR, Penicillin-Streptomycin, GDNF, FGF2, Insulin, EGF, Testosterone, and Echinomycin. In some aspects, a culture medium of the instant disclosure comprises αMEM+10% KSR, Penicillin-Streptomycin at a concentration ranging from about 0.9% to about 1.1%, GDNF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, FGF2 at a concentration ranging from about 19 ng/ml to about 21 ng/ml, Insulin at a concentration ranging from about 9 ug/ml to about 11 ug/ml, EGF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, Testosterone at a concentration ranging from about 9 uM to about 11 uM, and Echinomycin at a concentration ranging from about 4 ng/ml to about 6 ng/ml. (Condition 2)
In some aspects, a culture medium of the instant disclosure comprises αMEM+10% KSR, Penicillin-Streptomycin, GDNF, FGF2, Insulin, EGF, Testosterone, and Echinomycin. In some aspects, a culture medium of the instant disclosure comprises αMEM+10% KSR, Penicillin-Streptomycin at a concentration ranging from about 0.9% to about 1.1%, GDNF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, FGF2 at a concentration ranging from about 19 ng/ml to about 21 ng/ml, Insulin at a concentration ranging from about 9 ug/ml to about 11 ug/ml, EGF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, and Testosterone at a concentration ranging from about 9 uM to about 11 uM. (C2) In some aspects, a culture medium of the instant disclosure comprises αMEM+10% KSR, Penicillin-Streptomycin at a concentration ranging from about 0.9% to about 1.1%, GDNF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, FGF2 at a concentration ranging from about 19 ng/ml to about 21 ng/ml, Insulin at a concentration ranging from about 9 ug/ml to about 11 ug/ml, EGF at a concentration ranging from about 19 ng/ml to about 21 ng/ml, and Testosterone at a concentration ranging from about 78 uM to about 82 uM. (C2 with eight times testosterone)
In some aspects, media comprising proliferation factors can be further supplemented with antioxidant molecules. In some aspects, the antioxidant molecules comprise GSH, VC, VA, or any combination thereof. In some aspects, the antioxidant molecules comprise GSH, VC, and VA.
An additional aspect of the instant disclosure encompasses a testicular cell culture for culturing testicular germ cells in vitro. The cell culture comprises testicular tissue comprising germ cells or testicular germ cells in a culture medium comprising proliferation factors. The testicular tissue comprising germ cells or testicular germ cells can be as described in Section I(a) herein above. The culture medium and proliferation factors can be as described in Section II herein above. In some aspects, the factors that improve the level of proliferation of testicular germ cells in vitro can be identified using any of the processes described in Section I herein above.
In some aspects, the testicular cell culture comprises testicular tissue comprising testicular germ cells. In some aspects, the testicular tissue comprises seminiferous tubules. In some aspects, the testicular cell culture comprises isolated testicular germ cells. In some aspects, the testicular cell culture comprises isolated spermatogonia. In some aspects, the tissue culture comprises C2 media. In some aspects, the tissue culture comprises Control 2 media.
Importantly, the inventors surprisingly discovered that to successfully culture germ cells, methods of preparing testicular tissue differ from methods of preparing testicular germ cells for culture. For instance, the inventors developed a method of preparing testicular tissue for successfully culturing testicular germ cells. In some aspects, testicular tissue comprising testicular germ cells is prepared as described in Example 2 herein below. The inventors also developed a method of preparing testicular germ cells for successful proliferation. In some aspects, the spermatogonia are isolated as described in Example 6 herein below.
Equally importantly, the inventors also discovered that suitable media for successful culturing germ cells differ from methods of preparing testicular germ cells for culture. For instance, control 2 media was found to be suitable for culturing testicular tissue, whereas C2 media was better at culturing isolated testicular germ cells. Accordingly, in some aspects, a testicular cell culture of the instant disclosure can comprise control 2 media and testicular tissue comprising testicular germ cells. In some aspects, a testicular cell culture of the instant disclosure can comprise control 2 media comprising eight times testosterone and testicular tissue comprising testicular germ cells. This notable because to date, such an elevated concentration of testosterone has not been described to be effective or useful for culturing testicular tissue, testicular germ cells, or any other tissue. In other aspects, a testicular cell culture of the instant disclosure comprises C2 media and isolated testicular germ cells. Accordingly, in some aspects, a testicular cell culture of the instant disclosure can comprise control 2 media and testicular tissue comprising testicular germ cells, wherein the tissue is isolated using a method described in Example 2. Accordingly, in some aspects, a testicular cell culture of the instant disclosure can comprise control 2 media comprising eight times testosterone and testicular tissue comprising testicular germ cells, wherein the tissue is isolated using a method described in Example 2. In other aspects, a testicular cell culture of the instant disclosure comprises C2 media and isolated testicular germ cells, wherein the germ cells are isolated using a method described in Example 6.
Another aspect of the instant disclosure encompasses a method of culturing testicular germ cells in vitro. The method comprises culturing testicular tissue comprising germ cells or isolated germ cells in culture media comprising proliferation factors. In some aspects, the method of culturing can further comprise preparing testicular tissue for culture or isolating testicular germ cells for culture. Proliferation factors can be factors identified using a process described in Section I herein above. In some aspects, proliferation factors and proliferation media can be as described in Section II herein above. The testicular tissue and testicular germ cells can be as described in Section I(a) herein above. Preparing testicular tissue and isolating testicular germ cells can be as described in Section III.
Importantly, despite the essential role of the testis niche in spermatogenesis, a process of the instant disclosure was able to identify culturing isolated testicular germ cells independent of other testicular tissue to identify culture conditions supportive of testicular germ cell proliferation in vitro. This is in part due to the discovery of germ cell proliferation factors identified using a process of the instant disclosure that permit proliferation of the isolated germ cells in vitro independent of the testis niche. The inventors surprisingly discovered that the process of the instant disclosure can identify culture conditions that allows germ cell culture where the germ cells maintain their identity, growth, survival, and replication of the germ cells for extended periods of time. For instance, germ cells can be cultured in the identified culture conditions for 1 week or longer, 2 weeks or longer, 3 weeks or longer, 1 month or longer, 2 months or longer, 1 year or longer, or indefinitely. In some aspects, germ cells can be cultured in the identified culture conditions for 14 days or longer. In some aspects, germ cells can be cultured in the identified culture conditions for 21 days or longer. In some aspects, germ cells can be cultured in the identified culture conditions for 28 days or longer.
In some aspects, a culture method of the instant disclosure comprises culturing testicular germ cells in a culture comprising testicular tissue for a period of time, followed by isolating the testicular germ cells in the cultured tissue and culturing the isolated testicular germ cells independently of the tissue. In some aspects, such a hybrid method of culturing testicular germ cells can be used to culture the germ cells for 28 days and longer. In some aspects, such a hybrid method of culturing testicular germ cells can be as described in Example 6.
As explained herein above, the inventors surprisingly discovered that for successfully culturing germ cells, methods of preparing testicular tissue differ from methods of preparing testicular germ cells for culture. For instance, the inventors developed a method of preparing testicular tissue for successfully culturing testicular germ cells. In some aspects, testicular tissue comprising testicular germ cells is prepared as described in Example 2 herein below. Accordingly, when a method of culturing testicular germ cells of the instant disclosure comprises culturing testicular tissue, the method can comprise preparing the testicular tissue using the developed method of preparing testicular tissue. In some aspects, when a method of culturing testicular germ cells of the instant disclosure comprises culturing testicular tissue, the method comprises preparing the testicular tissue using a method of preparing testicular tissue described in Example 2 herein below.
The inventors also developed a method of preparing testicular germ cells for successful proliferation. In some aspects, the spermatogonia are isolated as described in Example 6 herein below. Accordingly, when a method of culturing testicular germ cells of the instant disclosure comprises culturing isolated testicular germ cells, the method can comprise preparing the germ cells using the developed method of preparing testicular tissue. In some aspects, when a method of culturing testicular germ cells of the instant disclosure comprises culturing isolated testicular germ cells, the method comprises preparing the testicular tissue using a method of preparing isolated testicular germ cells described in Example 6 herein below.
Yet another aspect of the present disclosure encompasses a method of obtaining spermatozoa from fertile and infertile men through culturing. The method comprises culturing more than one spermatogonial stem cell using culture conditions identified using the process described in Section I herein above. Each SSC is separately cultured. The method further comprises identifying a spermatogonial stem cell culture comprising sperm produced by the cultured SSC. The sperm do not contain deleterious heritable mutations and/or contain lower rates of de novo mutations, and comprise an expressed RNA transcript profile substantially similar to the expressed RNA transcript profile of the SSC in the SSC culture. Additionally, the method comprises harvesting spermatozoa from the identified culture conditions. The method can further comprise the step of freezing the spermatozoa for future use. In some aspects, the method further comprises the step of using the spermatozoa with assisted reproductive technologies such as intrauterine insemination or in vitro fertilization.
One aspect of the present disclosure encompasses a method of producing viable spermatozoa. The method comprises obtaining or having obtained testicular tissue from a subject; and culturing the testicular tissue in culture conditions identified using the process described in Section I herein above, the testicular cell culturing system described herein above, or both.
A further aspect of the present disclosure encompasses a kit for culturing testicular germ cells in vitro under conditions identified using the process described in Section I herein above, the testicular cell culturing system described herein above, or both.
Kits according to the present disclosure can include one or more additional reagents useful for culturing testicular tissue and germ cells according to the present disclosure. A kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as admixture where the compatibility of the reagents will allow. The test kit can also include other material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or any other material useful in processing or conducting any other step of the tagging method.
Kits according to the present disclosure preferably include instructions for culturing testicular tissue and germ cells. Instructions included in kits of the present disclosure can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site that provides the instructions.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.
“Mammalian,” as used herein refers to both human subjects (and cells sources) and non-human subjects (and cell sources or types), such as dog, cat, mouse, monkey, etc. (e.g., for veterinary purposes).
As various changes could be made in the above-described cells and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.
All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the present disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
The publications discussed throughout are provided solely for their disclosure before the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
The following examples are included to demonstrate the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the disclosure. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes could be made in the disclosure and still obtain a like or similar result without departing from the spirit and scope of the disclosure, therefore all matter set forth is to be interpreted as illustrative and not in a limiting sense.
Human testis development in prenatal life involves complex changes in germline and somatic cell identity. To better understand, 32,500 single-cell transcriptomes of testicular cells from embryonic, fetal, and infant stages were profiled and analyzed. The results show that at 6-7 weeks postfertilization, as the testicular cords are established, the Sertoli and interstitial cells originate from a common heterogeneous progenitor pool, which then resolves into fetal Sertoli cells (expressing tube-forming genes) or interstitial cells (including Leydig-lineage cells expressing steroidogenesis genes). Almost 10 weeks later, beginning at 14-16 weeks post-fertilization, the male primordial germ cells exit mitosis, downregulate pluripotent transcription factors, and transition into cells that strongly resemble the state 0 spermatogonia originally defined in the infant and adult testes. Therefore, these fetal spermatogonia were called “state f0.” Overall, multiple insights into the coordinated and temporal development of the embryonic, fetal, and postnatal male germline together with the somatic niche were reveal.
Within the developing fetal testicular niche, recent genomics profiling and immunofluorescence (IF) imaging approaches have revealed that male germline cells undergo major developmental changes (Gkountela et al., 2013, 2015; Guo et al., 2015; Li et al., 2017; Tang et al., 2015). Notably, the germline transitions from pluripotent-like PGCs migrating to and into the developing gonad to pluripotent-like and mitotically active PGCs in the gonad (called fetal germ cells [FGCs] or gonocytes), followed by the transition to “mitotically arrested” germ cells that repress the pluripotency-like program at/after weeks 14-18 (Li et al., 2017). Here, a key unanswered question during this stage of germline development involving the relationship between the mitotically arrested germ cells that arise during weeks 14-18 and the postnatal SSCs is as follows: are prenatal germ cells nearly identical to postnatal SSCs or are there major additional developmental stages that occur during prenatal stages? Notably, prior work by the inventors on the adult testis identified five distinct spermatogonial states (called states 0-4) accompanying human spermatogonial differentiation, with state 0 identified as the most naive and undifferentiated state (Guo et al., 2017, 2018, 2020), a result supported by single-cell RNA sequencing (scRNA-seq) profiling from other groups (Hermann et al., 2018; Li et al., 2017; Shami et al., 2020; Sohni et al., 2019; Wang et al., 2018). Consistent with this notion, state 0 is the predominant SSC state present in the infant testis, and state 0 SSCs express hundreds of state-specific markers, including PIWIL4, TSPAN33, MSL3, and EGR4 (Guo et al., 2018). The key markers identified in state 0 SSCs are also expressed in the undifferentiated spermatogonial states identified by others in recent studies, such as the SSC1-B (Sohni et al., 2019) or SPG-1 adult spermatogonia population (Shami et al., 2020), as well as in spermatogonia profiled from human neonates (Sohni et al., 2019) and in undifferentiated spermatogonia from macaques (Shami et al., 2020). Here, it is explored whether the previously identified mitotically arrested prenatal germ cells transcriptionally resemble state 0 postnatal spermatogonia, or instead represent a unique precursor that undergoes additional prenatal changes before birth.
The testis niche plays an important role in guiding the survival and differentiation of the male germline. In the adult testis, somatic niche cells, including Sertoli, Leydig, and myoid cells, provide physical and hormonal support for the successful execution of spermatogenesis from SSCs (Guo et al., 2018). The development of the functional adult testis and its organized tubule-like structure is completed at puberty, during which the final specification and maturation of all somatic niche cells takes place. Prior work by the inventors, which used scRNA-seq to study human testis development during puberty, revealed a common progenitor for Ley-dig and myoid cells that exists before puberty in humans, which is analogous to the somatic progenitor observed in fetal mice (Guo et al., 2020). However, during prenatal life, several key issues remain elusive, such as how the human testicular niche cell lineages are initially specified, whether they have a common progenitor, how the nascent gonad initially forms cords, and how niche cells differentiate further during subsequent fetal developmental stages to arrive at their postnatal states.
To address these questions, a total of 32,500 unsorted single testicular cells from embryonic, fetal, and postnatal samples were profiled through the 10× Genomics Chromium platform. This unbiased profiling allowed us to examine the specification process in the somatic cell niche and the development of both the germline and niche cells; this enabled a detailed comparison of the cell types and developmental processes in infant, pubertal, and adult testis.
Human testis tissues were obtained from 3 embryonic stages (6, 7, and 8 weeks postfertilization), 3 fetal stages (12, 15, and 16 weeks postfertilization), and 1 young infant stage (5 months postbirth) for comparisons to prior datasets from older infants, juveniles, and adults. To systematically investigate both germ cell and somatic cell development across embryonic and fetal stages, single-cell suspensions were prepared from these testicular tissues and performed scRNA-seq using the 10× Genomics platform. For embryonic and fetal samples, 5,000 single cells per sample were profiled; for the young infant sample, 2 replicates were performed, and profiled 2,500 single cells. From a total of 32,500 cells, 30,045 passed standard quality control dataset filters and were retained for downstream analysis (see Method details). 80,000-120,000 reads/cell were obtained, which enabled the analysis of 1,800-2,500 genes/cell.
To analyze the dataset, UMAP (uniform manifold approximation and projection dimension reduction analysis) was first performed on the combined datasets using the Seurat package (
Development of the male germline was examined by parsing out and analyzing the germ cells separately from the somatic cells of the prenatal and postnatal (5 months) testes (cluster 12 from
To validate the scRNA-seq profiles at the protein level, IF staining for key markers was performed. The proportion of NANOG+ (PGC marker) and MKI67+ (proliferation marker) decreased from 5 to 19 weeks (
To define candidate key genes and networks linked to germline developmental stages and transitions, network analysis was conducted. Using weighted correlation network analysis (WGCNA) (Langfelder and Horvath, 2008), gene-gene interactions that display dynamic expression patterns during PGC differentiation to state f0 spermatogonia were identified. Here, for the PGC up-regulated network (“PGC network;”
The cell type analyses revealed that the human embryonic and fetal testis stages consist primarily of somatic niche cells, including Sertoli cells and interstitial cells (including Leydig cells) (
To further define the gene expression programs that accompany male sex determination, gene expression clustering analysis (k-means) was performed to identify the gene groups that display dynamic expression patterns along the pseudotime developmental trajectories (
This population of cells then bifurcates into distinct transcriptional programs consistent with embryonic Leydig or Sertoli cell progenitors. Along the Sertoli lineage, expressed genes are associated with chromatin assembly, extracellular region, and filament formation. Along the Leydig lineage, cells first express genes related to DNA replication, proliferation, and cell cycle, indicating a phase of Leydig lineage amplification, consistent with a much higher number of cells present on the Leydig lineage trajectory at and after 8 weeks compared to the Sertoli lineage (
The datasets provided an opportunity to compare and contrast fetal versus postnatal human Leydig and Sertoli cells. 396 or 703 genes were found to be differentially expressed (upregulated or down-regulated, respectively) when comparing fetal to infant Leydig cells, respectively (bimodal test; adjusted p<0.01; |log FC|>0.25) (
Prior work by the inventors based on juvenile human testes showed that Leydig and myoid cells share a common progenitor at prepubertal stages (Guo et al., 2020). To gain a deeper understanding of the relationship between the fetal interstitial progenitors and prepubertal Leydig/myoid progenitors, as well as insight into how the common progenitor for the Leydig and myoid lineage is specified from fetal and postnatal precursor cells, additional analysis was performed. Here, in silico scRNA-seq datasets from fetal interstitial cells (clusters C, D, and E from
Key Factors Correlated with Embryonic Specification of Interstitial and Sertoli Lineages
Whereas testicular niche cells from 8 to 16 weeks expressed transcription factors characteristic of advanced interstitial or Sertoli cell lineages, the cells from the 6-week gonads lack these late markers, which initially emerge at week 7 (
Next, it was sought to identify candidate key transcription factors that may participate in initial somatic lineage specification (
Finally, extensive IF was performed to validate the genomics findings. GATA3 was observed throughout the genital ridge epithelium at week 5, which became restricted to a subpopulation of interstitial cells at weeks 6-7, and by week 8, GATA3 protein becomes undetectable (
PGCs are specified in the early embryo, followed by migration to the genital ridge (Chen et al., 2019; Tang et al., 2016; Witchi, 1948). The genital ridge then undergoes exquisite developmental programming to form the somatic cells of the testicular niche that support the survival and differentiation of the male germline during fetal life. Although prior studies from mice provide rich knowledge of the formation and lineage specification in the embryonic testis (reviewed in Svingen and Koopman, 2013), understanding of human embryonic and fetal testis development has been much less studied, particularly in regard to the specification of the somatic lineages. Here, through the application of single-cell sequencing of unselected testicular cells, together with IF staining, a detailed molecular overview of human fetal testis development is provided, to help delineate the temporal molecular changes involved in human embryonic and fetal testis development and further differentiation.
One critical question it was aimed to address is the transition of PGCs into spermatogonia, specifically the transcriptional relationship of differentiating male human PGCs during fetal life to postnatal state 0 SSCs, which have been identified as the most undifferentiated male germline stem cells in human infants and adults (Guo et al., 2018; Sohni et al., 2019), as well as primates (Shami et al., 2020). Combined with prior work (Guo et al., 2017, 2018, 2020; Sohni et al., 2019), the current work provides an evidence-based and detailed model for human germline development that spans embryonic, fetal, infant, pubertal, and adult stages (
Prior work in mouse models has revealed several factors and pathways that play important roles in lineage specification and progression of testicular somatic cells in mice (Liu et al., 2016; Svingen and Koopman, 2013; Yao et al., 2002). Recently, scRNA-seq has proven to be a powerful tool to study embryonic and neonatal mouse testis development (Stévant et al., 2019; Tan et al., 2020). Here, the work demonstrates that several key factors in early somatic lineages (e.g., WT1, NR2F1, SOX9, SRY, DMRT1) are shared between humans and mice. Furthermore, through the systematic examination of prenatal human testes via single-cell profiling and IF staining, many additional candidate factors are provide for future characterization, and reveal multiple human-mouse differences. For example, through IF staining of the genital ridge epithelium, no evidence of Sertoli cell or Leydig cell identity was found before 6 weeks postfertilization. Then, starting at week 6, the unbiased/unselected single cell transcriptome profiling identified rare fetal Leydig- and Sertoli-like cells. A large, closely related population of cells that is heterogeneously positive for developmental transcription factors, notably NR2F1, GATA3, and GATA4 RNA was also identified in pseudotime a. GATA3 protein analysis demonstrated that GATA3 is uniformly expressed by the genital ridge epithelium at week 5 postfertilization before specification of Sertoli and Ley-dig cells. Notably, at week 6, when cord formation initiates, GATA3 expression is restricted to a subpopulation of cells in the interstitium. In counterdistinction, GATA4 expression is evident and broad at 6-7 weeks postfertilization, and remains detectable at 17 weeks postfertilization. In the mouse embryo, GATA4 is known to be critical for genital ridge formation, and in the absence of GATA4, the bipotential gonads do not form (Hu et al., 2013). Given that GATA3 is expressed in the genital ridge epithelium before GATA4, it is speculated that GATA3 may have a role in specifying the genital ridge in humans, whereas GATA4 instead may be involved in maintaining the somatic cell lineages after 6 weeks postfertilization, when GATA3 expression is reduced. In the mouse, NR5A1 (also called SF1) is another major transcription factor required for specifying the genital ridge epithelium (Hatano et al., 1996; Luo et al., 1994). However, clear expression of NR5A1 in the GATA3+ human progenitors was not observed, providing a second example in which formation of the genital ridge epithelium in human embryos appears different from the mouse (
In addition to being specified at an equivalent developmental stage, it was also discovered that the 6- and 7-week somatic niche progenitors expressed markers consistent with their ability to differentiate into interstitial/Leydig and Sertoli lineages by transiently expressing (in a small subset of cells) key transcription factors, including ARX, NR0B1, or SRY. This identity is further reinforced at 8 weeks, when all cells are distinguishable as interstitial/Leydig or Sertoli lineage cells. Notably, the establishment of the male somatic cell lineages in the embryonic testis occurs almost 2 months before the PGCs begin differentiating into state f0 (at 14-18 weeks). In contrast, in mice, there is only a 2-day delay in the timing of the male niche cell differentiation (at day 12) to the initiation of mouse PGC differentiation into prospermatogonia (at embryonic day 14) (Saitou and Yamaji, 2012; Svingen and Koopman, 2013; Western et al., 2008). The purpose of this 2-month delay in which human PGCs are shielded from initiating differentiation into state f0 spermatogonia in the seminiferous cord niche may be related to the need to increase the number of male germ cells through proliferation, given that these cells are MKI67+, before initiation of state f0 differentiation and male-specific epigenetic reprogramming (
The testis produces gametes in adult males through continuous niche-guided differentiation of SSCs, and a deep understanding of this biology is needed to improve male reproductive health. Here, the work provides major insights into defining the timing and strategy of human testis formation and its development before and after birth. Notably, the state f0 germ cells that emerge at 15 weeks during fetal life display remarkable similarities to the infant and adult state 0 cells, and thus allow us to link and depict the complete developmental progression of PGCs to adult state 0 cells. Furthermore, detailed molecular characterization of a common somatic progenitor pool and its amplification and transition to testicular niche cells, as well as initial insights into testicular cord formation and possible roles in guiding germ cell development are provided. These results should provide a foundation for future hypothesis-driven research, and could also help guide the reconstruction and study of the human early testis in vitro.
Prenatal male gonads from 6 to 16 weeks post-fertilization were obtained from three collaborating laboratories at University of Washington Birth Defects Research Laboratory (BDRL), University of Tubingen and Karolinska Institutet. At BRDL, the prenatal gonads were obtained with regulatory oversight from the University of Washington IRB approved Human Subjects protocol, combined with a Certificate of Confidentiality from the Federal Government. The research project was also approved by the research ethics committee of the University of Tubingen. All consented material was donated anonymously and carried no personal identifiers. Human first trimester tissue was collected after elective surgical terminations with maternal written informed consent. The Regional Human Ethics Committee, Stockholm, Sweden, approved the collection (Dnr 2007/1477-31 with complementary permissions 2011/1101-32 and 2013/564-32. The ethical approval to perform the gonadal studies: Dnr 2013/457-31/4). Developmental age was documented by BDRL and University of Tu€bingen as days post fertilization using a combination of prenatal intakes and Carnegie staging. Developmental age was documented by Karolinska Institutet as days post fertilization by the examination of anatomical landmarks such as nervous system, limb, eye and gonadal development according to the atlas of England. Formalin fixed and paraffin embedded adult testis from biobank samples without underlaying testicular pathologies was obtained at the Department of Pathology at the Karolinska Institutet, and Karolinska University Hospital (ethical approval: Dnr 2014/267-31/4).
Postnatal human testicular sample (5 months old) was obtained through the University of Utah Andrology laboratory and Donor-Connect. This sample was removed from deceased individuals who consented to organ donation for transplantation and research.
The prenatal samples collected at BDRL used for single cell transcriptome profiling were shipped overnight in HBSS with an ice pack for immediate processing in Los Angeles. From University of Tu€bingen samples were delivered to UCLA within 24-48 hours after the procedure.
The postnatal whole testis was transported to the research laboratory on ice in saline and processed within 1 hour of removal by surgery. Around 90% of each testis was divided into smaller portions (·500 mg—1 g each) using scissors and directly transferred into cryovials (Corning cat #403659) in DMEM medium (Life Technologies cat #11995073) containing 10% DMSO (Sigma-Aldrich cat #D8779), 15% fetal bovine serum (FBS) (GIBCO cat #10082147) and cryopreserved in Mr. Frosty container (Thermo Fisher Scientific cat #5100-0001) at a controlled slow rate, and stored at −80° C. for overnight. Cryovials were transferred to liquid nitrogen for longterm storage.
Prenatal tissues were processed within 24-48 hours after termination. Upon arrival to UCLA tissues were gently washed with PBS and placed in dissociation buffer containing collagenase IV 10 mg/ml (Life Technologies #17104-019), Dispase II 250 ug/ml (Life Technologies #17105041), DNase I 1:1000 (Sigma 4716728001), 10% FBS (Life Technologies 10099141) in 1×PBS. After every 5 minutes tissues were gently pipetted with P1000 pipette against the bottom of Eppendorf tube. This process was repeated 3 times for a total of 15 minutes. Afterward, cells were centrifuged for 5 minutes at 500 g and pellet was resuspended in 1×PBS with 0.04% BSA and strained through 40 mm strainer and counted using automated cell counter (Thermo Fisher, Countess II). The cell concentration was adjusted to 800-1200 cells per microliter and immediately used for scRNA-seq. For postnatal tissues, 1 cryovial of tissue was thawed quickly, which was then washed twice with PBS, and subject to digestion as described previously (Guo et al., 2018). Tissues were washed twice in 1×PBS and minced into small pieces for better digestion outcome. Tissues were then treated with trypsin/ethyl-enediaminetetraacetic acid (EDTA; Invitrogen cat #25300054) for 20-25 min and collagenase type IV (Sigma Aldrich cat #C5138-500MG) at 37° C. Single testicular cells were obtained by filtering through 70 mm (Fisher Scientific cat #08-771-2) and 40 mm (Fisher Scientific cat #08-771-1) strainers. The cells were pelleted by centrifugation at 600 g for 15 min and washed with PBS twice. Cell number was counted using a hemocytometer, and the cells were then resuspended in PBS+0.4% BSA (Thermo Fisher Scientific cat #AM2616) at a concentration of 1,000 cells/uL ready for single-cell sequencing.
It was targeted to capture 6,000-7,000 cells. The prenatal sequencing was conducted in UCLA, and the postnatal sequencing was conducted at University of Utah. Briefly, cells were diluted following manufacturer's instructions, and 33.8 mL of total mixed buffer together with cells were loaded into 10× Chromium Controller using the Chromium Single Cell 3′ v3 reagents. The sequencing libraries were prepared following the manufacturer's instructions, using 13 cycles of cDNA amplification, followed by an input of 100 ng of cDNA for library amplification using 12 cycles. The resulting libraries were then sequenced on a 2×150 cycle paired-end run on an Illumina Novaseq 6000 instruments.
Raw data were demultiplexed using mkfastq application (Cell Ranger v2.2.0) to make Fastq files. Fastq files were then run with count application (Cell Ranger v2.2.0) using default settings, which performs alignment (using STAR aligner), filtering and UMI counting. The UMI count tables were used for further analysis.
Intact testes were fixed in 4% PFA at room temperature for 2 hours on a platform rocker. Tissues were washed 3 times with PBS for 10 minutes each wash then placed into paraffin blocks (Histogel, Thermo Scientific HG4000012) for sectioning onto slides. Sections were deparaffinized and rehydrated in a Xylene then ethanol series (100%, 95%, 70%, 50%, water) respectively. Antigen retrieval was performed in either Tris-EDTA solution (pH 9.0) or Sodium Citrate Solution (pH 6.0) in a hot water bath (95° C.) for 40 minutes. Sections were washed in PBS, 0.2% Tween-20 (PBS-T) 3 times, 5 minutes each then permeabilized in PBS, 0.05% Trition X-100 for 20 minutes. Sections were blocked with blocking solution (10% Normal Donkey Serum (NDS), PBS-T) for 30 minutes at room temperature in a humid chamber. Primary Antibodies were diluted in 2.5% NDS, PBS-T at the appropriate dilutions (see Key resources table) and incubated overnight at 4° C. in a humid chamber. After 3 washes in PBS-T (5 minutes each) secondary antibodies were added and allowed to incubate at room temperature for 1 hour in a humid chamber. After 3 washes in PBS-T, DAPI was added to the sections for approximately 5 minutes, then washed 3 times 5 minutes each in PBS-T. Prolong Gold antifade mountant (Invitrogen P10144) was added to the sections. Coverslips were placed onto slides then sealed with nail polish. Slides were allowed to cure overnight, in the dark, at room temperature then subsequently stored at 4° C. until ready to image. For sections stained with PIWIL4 antibody, the blocking buffer used was Superblock blocking buffer (Thermo Scientific 37580). In addition, the SignalBoost Immunoreaction Enhancer Kit (Millipore 407207) was used to dilute primary and secondary antibodies for experiments involving PIWIL4 antibody.
A Zeiss LSM 880 with Airyscan controlled by the Zen Black software, equipped with the Plan-Apochromat 203/0.8 NA and the Plan-Apochromat 633/1.4 NA M27 oil immersion objective, was used to acquire confocal images. Saved CZI files were converted to Imaris format files (.ims) using the Imaris File converter (Bitplane), then processed using the image analysis software IMARIS 9.3 (Bit-plane). An Olympus BX-61 light microscope was used to examine Hematoxylin and Eosin (H&E) stained slides. The ImageJ stitch function uses similar features/structures from a collection of images to make a fused image, therefore each image has some overlap with the previous image taken. Briefly, H&E images were taken with the 20× objective. In ImageJ under the Plugins dropdown box the Stitching plugin was chosen and then selected the Grid/Collection Stitching function. In the “Type” box “unknown position” was selected and “all files in directory” was chosen for the “Order”. Linear Blending was chosen for the Fusion Method used. The Regression threshold was set at 0.30. The Max/avg displacement threshold was set at 2.50 and the Absolute displacement threshold was set to 3.50. Stitched images were built using the ImageJ2(NIH) Grid/Collection Stitching plugin.
The Seurat program was used as a first analytical package. To start with, UMI count tables from both replicates from all four juvenile donors were loaded into R using Read10× function, and Seurat objects were built from each experiment. Each experiment was filtered and normalized with default settings. Specifically, cells were retained only if they contained >500 expressed genes and had <25% reads mapped to mitochondrial genome. t-SNE and clustering analysis were first run on each replicate, which resulted in similar t-SNE map. Data matrices from different donors and replicates were then combined with the previously published infant and adult data (Guo et al., 2018). Next, cells were normalized to the total UMI read counts, as instructed in the tutorial. t-SNE and clustering analyses were performed on the combined data using the top 6,000 highly variable genes and 1-30 PCs, which showed the most significant p values.
Detailed pseudotime for different cell types were performed using the Monocle package (v2.10.1) following the default settings. After pseudotime coordinates/order were determined, gene clustering analysis was performed to establish the accuracy of pseudo-time ordering. Here, cells (in columns) were ordered by their pseudotime, and genes (in rows) were clustered by k-means clustering using Cluster 3.0. Different k-mean numbers were performed to reach the optimal clustering number. Cell cycle analysis was performed using scran program R Package; v1.6.5).
Hub genes in PGC, spermatogonia and State 0 were found by WGCNA. When finding hub genes in PGC and spermatogonia, gene expression data of 40 cells from PGC and State 0 respectively were randomly extracted from the UMI count tables of scRNA-seq data. Genes were filtered by selecting those genes expressed in more than 20 cells since scRNA-seq data had a high drop-out rate and low expression genes may represent noise. Then the counts were normalized by total reads (x*100000/total reads) and then log-transformed (log 2(x+1)). Afterward, one-step network construction and module detection were performed. In this step, parameters including signed hybrid network type, Pearson correlation method and the default soft-threshold power b were chosen to reach the scale-free network topology. To identify the modules that were significantly correlated with PGC or spermatogonia, bi-weight mid-correlation (robustY=FALSE) was used. The quality of the modules was checked by the strong correlation between module eigengenes and traits of interest as well as the strong correlation between gene module membership and gene-trait correlation. Finally, hub genes inside those modules were selected from the top 40 genes with the highest intramodular connectivity (sum of in-module edge weights). Specifically, in order to find hub genes in State 0 rather than spermatogonia, gene expression data of 40 cells from State1 was added to rule out the genes expressing broadly in States 0-4 and performed the same analysis to determine the modules that were significantly correlated with State 0. Ten hub genes were selected by the same standard. Finally, the networks were visualized by Cytoscape Software 3.7.2.
To identify culture conditions that support growth and development of testicular germ cells in vitro, both germline and somatic, a genomic approach was used to identify dysregulated biological pathways in cultured testicular tissue comprising tubules and germ cells that can be used to identify the in vitro culture conditions. Using tissue analysis approach to analyze in vitro cultured tissue, it was discovered that spermatogonia were able to proliferate/replicate in vitro and differentiating spermatogonia were able to proliferate/replicate and enter meiosis (
To identify dysregulated pathways in cultured tissue, a process using a genomic approach using scRNA-seq was used to further reveal the molecular changes of cultured testicular tissue using a process detailed in the diagram shown in
Using the genomic approach described above, it was discovered that expression of the genes associated with the following were altered in cultured Leydig and myoid cells: extracellular exosome, negative regulation of apoptotic process, cytokine, response to hypoxia, actin cytoskeleton, extracellular matrix, and muscle contraction (
Among the dysregulated pathways, it was discovered that the HIF pathway was activated in somatic cells of cultured testicular tissue. Dysregulation of the HIF pathway and genes having altered expression in the somatic cells of cultured tissue are shown in
Considering the above, small molecule inhibitors (Table 1) were used against some pathways in the HIF pathways to determine if and which inhibitor can reverse the effects of culturing and maintain testicular tissue structure. To accomplish this, the iterative method of identifying factors that could improve culture conditions was used to test the effect of the small molecule inhibitors described in Table 1 with parameters described further below. It was discovered that the HIF-1 inhibitor echinomycin helps maintain testicular tissue structure (
Culture media was also supplemented with ligands (Table 2) informed by the genomic work here and previously discovered by the inventors (Guo et al., Cell Stem Cell, 2017; Guo et al., Cell Research, 2018; and Guo et al., Cell Stem Cell, 2020).
It was discovered that the ligands were effective but not sufficient for restoring tissue structure when echinomycin was not also added. Conversely, when echinomycin, testosterone, FSH, and RA are added to the medium, resulted in better germ cell proliferation when compared to echinomycin alone or ligands alone (
Tissue preparation: Whole testes are removed from cadaveric organ donors by DonorConnect staff, which are picked up by Utah team and transported to research lab on ice. Testicular tissues are cut by 3-5 mm by dimeter in size using surgical scissors and tweezers.
Culture media preparation: the base media is αMEM+10% KSR. We first tested various small molecular inhibitors/ligands with different concentrations in the base media to culture testicular tissues. We then chose to use the inhibitor/ligand combinations with better outcomes based on morphology change and germ cell proliferation status (i.e. maintained testicular size with germ cell proliferation; see below for more details). One of the most effective combination of proliferation factors we current have to date is echinomycin (HIF inhibitor; concentration: 5 nM)+Testosterone (concentration: 10-7M)+GDNF (concentration: 10 ng/mL)+bFGF (concentration: 10 ng/mL).
Tissue culture: Place three pieces of testicular tissue into one well of a 6-well plate with 2 ml of media in each well. The tissue should be fully immersed in media. Place the plate at 34° C. in 5% CO2 in an incubator with culture media changed every other day.
Morphology examination: Measure the sizes of the cultured testicular tissues at different time points, including Day 1, Day 7, Day 14, and Day 21. And then fix the sample for H&E staining. The detailed method has been described previously (Guo et al., 2018, 2020).
Germ cell proliferation examination: Add Edu (2 ul into each well) at different time points at Day 0, Day 6, Day 13, Day 20. Harvest the tissue 24 hours later for the germ cell proliferation test. Samples are washed three times by PBS and digested to isolated tubules by collagenase type IV at 37° C. Then wash 3 times by PBS to terminate the digestion and perform whole-mount staining of the tubules with Edu and DDX4/UTF1/SYCP3. The detailed method has been described previously (Gassei et al., 2014).
Single cell-RNA seq profiling of cultured tissues: Single cell transcriptome of the cultured testicular tissues in the most effective combination is obtained. The detailed method for tissue dissociation and sequencing execution has been described previously (Guo et al., 2018, 2020). We make comparisons of the cultured profile with non-cultured healthy testicular profiles, which allows us to refine our culture media by testing more small molecular inhibitors/ligands.
Extract Testicular Somatic Cells for tSNE/UMAP Analysis
A process for identification and selection of receptor ligands capable of improving testicular germ cell proliferation was developed. The process uses expression data obtained by mining scRNA-seq data obtained from testicular tissue (
More specifically, the approach was based on the single-cell RNA sequencing transcriptome profile of the adult human testis atlas (Guo, J., Grow, E. J., Mlcochova, H., Maher, G. J., Lindskog, C., Nie, X., Guo, Y., Takei, Y., Yun, J., Cai, L., et al. (2018). The adult human testis transcriptional cell atlas. Cell Research 28, 1141. 10.1038/s41422-018-0099-2; the disclosure of all of which is incorporated herein in its entirety), which provides a comprehensive characterization of cell types in the human adult testis. Pseudo-bulk RNA-seq analysis was used, which involves aggregating individual cells into groups to enhance the receptor gene expression signal on SSCs and differentiating spermatogonia. Each individual cell is assigned to its corresponding cell type cluster based and summing the expression values of all cells within each cluster to create pseudo-bulk samples.
The CellTalkDB database was used to identify receptors expressed in SSCs/differentiating spermatogonia. Specifically, the receptors whose expression in SSCs/differentiating spermatogonia ranks within the top 150 were selected, as expression levels below this point are generally low in these cell populations. Next, the gene expression patterns of the top 150 receptors were examined in the single-cell RNA-seq data, excluding receptors that exhibit ubiquitous expression in the testis. This filtering step helps to focus on receptors that are more specific to the target cell populations. Using the CellTalkDB database, the corresponding ligands for the selected receptors were then identified. The ligand candidates were prioritized based on their expression levels in the testes, and their known functionality.
The identified ligands were then used in the iterative process of the instant disclosure for identifying culture conditions that support testicular germ cell proliferation in vitro to refine the culture conditions. Ligands identified using this process and the concentrations of ligands that were or can be used in the iterative process are listed in Table 5. Ligands that were found to be effective to date are also noted.
Importantly, after extensive experimentation, it was surprisingly discovered that other methods of identifying receptors and corresponding ligands such as using CellphoneDB and CellChat to analyze cell-cell interaction to find potential ligands was not successful. As shown in
The iterative process of the instant disclosure and the process for identifying SSC ligands were used to identify a combination of factors that can extend germ cell proliferation (e.g., dish life of stem cells) to at least 21 days in cultured tubules (
Inhibitors of dysregulated pathways identified in Example 2 were screened using the iterative process of the instant disclosure comparing cultured to uncultured tissue to identify culture conditions that can improve the level of proliferation of germ cells.
The inhibitors used for screening were selected based on scRNA-seq results comparing cultured and uncultured somatic cells by scRNA-seq to identify dysregulated pathways (
A protocol for culturing isolated spermatogonia, i.e., spermatogonia without niche cells, and independently from tubules was developed. In some aspects, tubule culture systems such as those described in Examples 2-5 can be used a learning platform to inform SPG culture.
According to the developed protocol, spermatogonia are first isolated and the isolated cells are cultured.
Materials and Reagents.
Method. Spermatogonia are isolated from testicular tissue as follows. 2 g of tissue are scraped with a razor on a petri dish with razor blades to spread the tissue. The scraped tissue is added to 50 ml Eppendorf Conical Tubes with digestion I solution and shaken vigorously (a rotor at 60 rpm was used) for 5 mins. At the end of digestion, separated tubules can be detected. The tissue is then filtered with a 40 um strainer, washed with 5 mL MACS buffer, and the flow through is discarded. The tissue is then added into a new tube with 5 ml digestion II solution, and shaken vigorously (a rotor at 60 rpm was used) for 30 mins. The tissue is again filtered with a 40 um strainer, washed with 15 mL MACS buffer, and the cell suspension is transferred into a new 50 ml tube and placed on ice. 600 ul of FBS is added into the new tube to terminate the reaction and the tube is centrifuged at 300 g for 5 mins at 4° C. The supernatant is aspirated without disturbing the cell pellet, and the cells are resuspended in 1 ml MACS buffer. The cells are pelleted again and the supernatant is aspirated without disturbing the cell pellet. The cells in the cell pellet is then resuspend in 1 ml MACS buffer and filtered with a 40 um strainer. The cells in the filtrate is now ready for concentration calculation and the subsequent single-cell sequencing.
Culture media. Spermatogonia cell culture media used is as shown in Table 13 and is referred to herein throughout as C2 media. The composition of the C2 media was informed by the iterative process of the instant disclosure and/or results of the iterative process used with cultured testicular tissue.
Cells are plated at a concentration of ˜90,000 cells/cm2 in petri plates and incubated for 24 hours at 35° C. After incubation, the supernatant is removed and laminin is added at a concentration of 0.25 mg/cm2. The supernatant is also replated to a new plate. The media is changed every other day, at which time the top 50% of the media is removed and replaced with a similar volume of media.
As it can be seen in
Through the iterative process of the instant disclosure, the concentration of testosterone in C2 media (Table 13) described in Example 6 was adjusted to include eight times the levels of testosterone in C2 media improves SPG proliferation when compared to culture in C2 media as measured by the number of proliferating SPG in media after 14 days of culture.
As it can be seen in
After extensive experimentation, it was discovered that using a hybrid system comprising a combination of the testicular tissue culture system of Example 2 using the control 2 culture media SPG culture system of using the C2 medium, was able to significantly increase the length time germ cells can proliferate in culture. More specifically, it was discovered that by culturing testicular tissue using the tissue culture system of Example 2 in the control 2 culture media followed by culturing SPG using the SPG culture system of Example 7, the germ cells were able to continue proliferating in culture for at least 21 days (
As shown in
Samples were received from Donor Connect. The samples were processed and fertile samples were chosen to set up the experiment. The tissue was cut into small pieces of an approximate size of 0.5 cm×0.5 cm. The pieces were cultured in two 6 well cell culture plates with three pieces in a well. Media used for culture was condition 2 media with. The tissue pieces were incubated at 35° C. with 5% CO2 for 7 days with media change done every alternative day. On day 7 of the culture, all the tissue pieces were pooled and cells were isolated from them. Two different SPG cell isolation methods were followed.
Two g of tissue are added to a petri dish and scraped with razor blades to spread the tissue. The scraped tissue is transferred to 50 ml Eppendorf Conical Tubes with digestion I solution, incubated at 37° C., and shaken vigorously (a rotor at 60 rpm was used) for 3 mins. At the end of digestion, separated tubules can be detected. The tissue is then filtered with a 40 μm strainer and the flow through is discarded. The tissue is then added into a new 50 ml tube with digestion solution II, incubated at 37° C., and shaken vigorously about every 5 min (a rotor at 60 rpm can be used) for 30 mins. At the end of digestion, the wall of tubules is blurred. The tissue is again filtered with a 40 μm strainer and the wash and the cell suspension is transferred into a new 50 ml tube and placed on ice. 600 ul of FBS is added into the new tube to terminate the reaction and the tube is centrifuged at 300 g for 5 mins at 4° C. The supernatant is aspirated without disturbing the cell pellet, and the cells are resuspended in 1 ml MACS buffer. The cells are pelleted again in a 1.5 ml tube for 5 min at 300 g and the supernatant is aspirated without disturbing the cell pellet. The cells in the cell pellet is then resuspend in C2 media. The cells are counted and plated per the required cell density per well.
Two g of tissue are added to a petri dish and scraped with razor blades to spread the tissue. The scraped tissue is transferred to 50 ml Eppendorf Conical Tubes with digestion I solution, incubated at 37° C., and shaken vigorously (a rotor at 60 rpm was used) every 1 min for 3-5 mins. At the end of digestion, separated tubules can be detected. The tubules are filtered with a 40 μm strainer, rinsed with ˜5 ml cold MACS buffer, and tissue on top of the strainer is collected into a tube with 5 ml Digestion II buffer. Discard the cells passed through. The tissue is digested in Digestion II at 37° C. in a water bath for 30 min, with vigorous shaking every 5 mins. At the end of digestion, the wall of tubules should be blurred. The tissue from the previous step is filtered through a 40 μm strainer. The cell suspension is added into a new 50 ml tube (on ice) and 600 ul FBS as well as 100 ul DNase I are added into the new tube to terminate the reaction, add 15 ml cold MACS buffer and centrifuged at 300 g for 5 mins at 4° C. The supernatant is aspirated without disturbing the cell pellet and the cells are resuspended in 1 ml MACS buffer, transferred to 1.5 ml tube, spun at 300 g for 5 min and resuspended in C2 media. The cells are counted.
The isolated cells were cultured in 96 well cell culture plates with a seeding density of 30,000 live cells per well in 200 ul media. The media tested were as in Table 15. MatriClone at 0.16 ul was added to each well. The cell culture plates were incubated at 35° C. with 5% CO2. Media changes were done every other day by aspirating 100 ul of old media and supplementing the same amount of fresh media. Four different plates were set-up to stain and observe the cells at day 2, day 7, day 14 and day 21. The cells were stained with EdU and DDX4. EdU stains the cells that are in the process of replication and DDX4 stains germ cells.
Method for Staining
The plates were observed under the microscope with three different channels of light.
The present application is a continuation-in-part of International Application number PCT/US2022/011179, filed on Jan. 4, 2022, which claims the benefit of U.S. Provisional Application No. 63/133,633, filed Jan. 4, 2021, the entire contents of each of the aforementioned applications is hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63133633 | Jan 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US22/11179 | Jan 2022 | US |
| Child | 18346691 | US |
