EP-4739768-A1 - PROCESS FOR ESTABLISHING A HUMAN TESTICULAR TISSUE CULTURE SYSTEM

Abstract

The present disclosure provides an iterative process for identifying culture conditions that maintain identity, growth, and survival of testicular cells in vitro . Testicular cell culturing systems for supporting human spermatogenesis and culture using identified culture conditions are also provided. The methods and the culture conditions can be used to culture healthy and viable spermatozoa with lower rates of deleterious or de novo mutations or epigenetic perturbations from fertile and infertile men for future use with assisted reproductive technologies.

Inventors

• HOTALING, JIM M.
• CAIRNS, BRAD R.
• GUO, Jingtao
• NIE, Xichen

Assignees

• Paterna Biosciences Inc.

Dates

Publication Date

20260513

Application Date

20230703

Claims (20)

1. 1 . An iterative process for identifying culture conditions supportive of testicular germ cell proliferation in vitro, the process comprising: a. identifying one or more dysregulated pathways in testicular cells cultured in a first set of culture conditions by: i. 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; ii. profiling transcriptomes of single testicular cells obtained from the testicular tissue or isolated testicular germ cells; and iii. 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; b. identifying one or more proliferation factors that improve the level of proliferation of testicular germ cells by: i. 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 ii. 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; c. iteratively repeating steps (a) and (b) to identify additional factors that improve the level of proliferation of testicular germ cells; wherein the culture conditions that support testicular germ cell proliferation comprise culture media supplemented with one or more factors identified in steps (b) and (c).
2. 2. The iterative process of claim 1 , wherein profiling transcriptomes of single testicular cells comprises using single cell RNA sequencing (scRNA-seq)
3. 3. The process of claim 1 or claim 2, further comprising assigning a cell type to each single testicular cell using cell type-specific gene markers expressed in each cell.
4. 4. The iterative process of claim 3, wherein differentially expressed RNA transcripts are identified in each cell type when compared to RNA transcripts expressed in cells of corresponding cell types obtained from control tissue and 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.
5. 5. The iterative process of any one of the preceding claims, wherein control tissue is directly isolated from a subject.
6. 6. The iterative process of any one of the preceding claims, wherein control tissue is previously cultured in a discovered medium.
7. 7. The iterative process of any one of the preceding claims, wherein the culture conditions that support testicular germ cell proliferation in vitro support survival and proliferation of somatic cells that form the germ cell niche.
8. 8. The iterative process of any one of the preceding claims, wherein testicular tissue or isolated testicular germ cells cultured in vitro grown under the second set of culture conditions exhibit proper identity, growth, and survival when compared to the cells directly isolated from the testis of males.
9. 9. The iterative process of any one of the preceding claims, wherein testicular germ cell proliferation comprises proper identity, growth, development, survival, and replication of the testicular germ cells in vitro.
10. 10. The iterative process of any one of the preceding claims, wherein an improved level of proliferation of testicular germ cells comprises improved replication, survival, physiology, or development of the cultured testicular germ cells, or any combination thereof.
11. 11. The iterative process of any one of the preceding claims, wherein improving the level of proliferation comprises improved cell replication, survival, and differentiation.
12. 12. The process of any one of the preceding claims, wherein the first culture medium is base medium comprising aMEM and 10% KSR.
13. 13. The iterative process of any one of the preceding claims, wherein the first culture medium is base medium comprising aMEM and 10% KSR supplemented with factors or combinations of factors identified in a previous round of the iterative process.
14. 14. The iterative process of any one of the preceding claims, wherein cells of testicular tissue cultured in the culture media that support testicular germ cell proliferation comprise an expressed RNA transcript profile substantially similar to the expressed RNA transcript profile of cells of testicular tissue directly isolated from testis of adult males.
15. 15. The iterative process of any one of the preceding claims, wherein cells of testicular tissue cultured in culture media that support testicular germ cell proliferation comprise no dysregulated pathways.
16. 16. The iterative process of any one of the preceding claims, wherein the testicular tissue is obtained from a healthy adult subject, an infertile or sub-fertile adult subject, or a pre-pubertal subject.
17. 17. The iterative process of any one of the preceding claims, wherein the testicular tissue is directly isolated from testis of male subjects.
18. 18. The iterative process of any one of the preceding claims, wherein the testicular tissue comprises one or more seminiferous tubules comprising the testicular germ cells and testicular somatic cells, or organoids comprising the testicular germ cells and support cells.
19. 19. The iterative process of any one of the preceding claims, wherein the testicular tissue is a seminiferous tubule.
20. 20. The iterative process of claim 19, wherein the support cells of organoids comprise Sertoli cells, primary immortalized Sertoli cells, immortalized Sertoli cells, Leydig cells, myoid cells, cells identified to be useful for culturing in an organoid format, or any combination thereof.

Description

PROCESS FOR ESTABLISHING A HUMAN TESTICULAR TISSUE CULTURE SYSTEM FIELD OF THE INVENTION [0001] The present disclosure provides an iterative process for identifying culture conditions that support human testicular germ cell proliferation in vitro and testicular tissue and isolated testicular germ cell cultures supportive of germ cell proliferation while maintaining identity, growth, and survival of the testicular germ cells. BACKGROUND OF THE INVENTION [0002] 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. [0003] 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. SUMMARY OF THE INVENTION [0004] 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 SUBSTITUTE SHEET ( RULE 26) 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 RN A 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. [0005] 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. [0006] 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. [0007] 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. [0008] In some aspects, the culture medium comprises aMEM + 10% KSR, Penicillin - Streptomycin at a concentration ranging from about 0.9% to about 1.1 %, GDNF at 2 SUBSTITUTE SHEET ( RULE 26) 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