The present invention relates to female germline stem cells and their progenitors, methods of isolation thereof, and methods of use thereof.

This invention provides a method of producing an epiblast-like cell (EpiLC) from a pluripotent stem cell, which comprises culturing the pluripotent stem cell in the presence of activin A; a method of producing a primordial germ cell-like (PGC-like) cell a pluripotent stem cell, which comprises culturing the EpiLC obtained by the method above in the presence of BMP4 and LIF. Also provided are a cell population containing PGC-like cells as obtained by the method, and reagent kits for the EpiLC- and PGC-like cell-induction from a pluripotent stem cell.

Human pluripotent embryonic stem cells produced by somatic cell nuclear transfer as well as methods of making and using said human pluripotent embryonic stem cells are disclosed.

A sustained culture of isolated avian gonocytes is provided, as well as a method of making and using the same. A chimeric avian containing an isolated gonocyte and a transgenic avian produced using the chimeric avian are also provided. The cell and method may be employed to make, among other things, transgenic avian that produce a heterologous protein, e.g., a therapeutic protein.

The present invention is long-term cultures of avian PGCs and techniques to produce germline chimeric and transgenic birds derived from prolonged PGC cultures. In some embodiments, these PGCs can be transfected with genetic constructs to modify the DNA of the PGC, specifically to introduce a transgene encoding an exogenous protein. When combined with a host avian embryo by known procedures, those modified PGCs are transmitted through the germline to yield transgenic offspring. These germline chimeric birds do not have substantial contributions of PGC-derived phenotypes in somatic cells or tissues. This invention includes compositions comprising long-term cultures of PGCs that can be genetically modified by gene targeting, that can accept large amounts of foreign DNA and that contribute to the germline of recipient embryos.

The present teachings provide for a method of breeding livestock in vitro. Provided are steps to create embryonic stem cells from a plurality of blastocysts, genotype the embryonic stem cells to select the best embryos for mating to create offspring, and induce the embryonic stem cells into primordial germ cell-like cells (PGCLCs). Male PGCLCs are further induced into spermatogonial stem cell-like cells, and then spermatid-like cells. Female PGCLCs are induced into oocytes, which are then matured. The resulting gametes can then be mixed with each other or with opposite sex gametes from animals with desirable genetics to create the next generation of embryos, which can then be run through the process again. This method of in vitro breeding can be used to increase the speed of genetic progress in livestock.

Human pluripotent embryonic stem cells produced by somatic cell nuclear transfer as well as methods of making and using said human pluripotent embryonic stem cells are disclosed.

Methods and compositions are provided for producing a primordial germ cell like cell (PGCLC) from a pluripotent stem cell, e.g., an induced pluripotent stem cell (iPSC), via GATA5 overexpression. GATA5 expression can be induced using any convenient method, e.g., by introducing an exogenous nucleic acid encoding GATA5 or by using a tool such as CRISPRa to stimulate expression from the GATA5 endogenous locus.

The present invention relates to a method for differentiating hair follicle cells into germline stem cells, germline stem cells differentiated by the method, and use of the same germline stem cells. A method of differentiating hair follicle cells into germline stem cells according to the present invention can differentiate hair follicle cells into germline stem cells using culture conditions only, without genetic modification. Capable of inducing differentiation of cells of specific individual types, such as hair follicle cells, into cells of different types such as germline cells, the present invention is therefore expected to be usefully used for the understanding of reproductive biology and the clinical application thereof.

Reserves of immortalized genetic material are stored in a bank for providing a resource (e.g., artificial gametes) for couples (e.g., same sex couples) to produce biologically-related children. The reserves provide the ability to derive sperm from induced pluripotent stem cells (iPSC's) of one partner and/or eggs from iPSC's of the other partner. For example, a biological sample is stored as iPSC's that can be used to generate an unlimited supply of genetic material (e.g., artificial gametes for conception) when needed by a user (e.g., a year or more after the sample is provided, e.g., 5 years or more after the sample is provided). Such a bank is helpful for an individual in a same sex (or infertile) couple who desires to have biological children at some point during his/her lifetime, but does not plan to have children in the immediate timeframe, for example.