Process for establishing a human testicular tissue culture system
AssigneePaterna Biosciences Inc.
InventorsJim M. Hotaling, Brad R. Cairns, Jingtao Guo, Xichen Nie
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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.
Claims19
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 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 using single cell RNA sequencing (scRNA-seq);
- iii. assigning a cell type to each single testicular cell using cell type-specific gene markers expressed in each cell;
- iv. 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;
- b. identifying one or more factors that improve the level of proliferation of testicular germ cells by:
- i. 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 a biological pathway identified in (a);
- 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 or to identify combinations of factors identified in step (b) that improve the level of proliferation of testicular germ cells in vitro;
- wherein culture conditions supportive of testicular germ cell proliferation in vitro comprise one or more factors identified in steps (b) and (c).
Dependencies are inferred from the claim text.