The present invention is of methods of establishing and propagating human embryonic stem cell lines using feeder cells-free, xeno-free culture systems and stem cells which are capable of being maintained in an undifferentiated, pluripotent and proliferative state in culture which is free of xeno contaminants and feeder cells.

The present invention provides a method for maintaining and expanding a human primordial germ cell-like cell (hPGCLC) derived from a human primordial germ cell (hPGC) or a human pluripotent stem cell, including culturing hPGCLC in the presence of (i) a forskolin or phosphodiesterase 4 (PDE4) inhibitor, and (ii) one or more cytokines selected from the group consisting of a basic fibroblast growth factor (bFGF), a leukemia inhibitory factor (LIF), and an epidermal growth factor (EGF).

A method of differentiating a primordial germ cell into a primordial follicle in vitro includes culturing a primordial germ cell and a supporting cell adjacent to the primordial germ cell under a pressurized condition or a low oxygen concentration condition.

The invention relates to in vitro generation of organized 3D cell structures recapitulating various degrees of early organogenesis, including head-trunk embryo-like structures, using epigenetic remodeling factors. The invention relates in particular to methods of obtaining such organized 3D cell structures from mammalian cells, and to devices, in particular microfluidic platform, to perform such methods. The invention also concerns the use of the thus obtained 3D cell structures in applications of molecule screening, developmental testing, production of physiologically active substances and models for therapeutic investigation or use.

A problem of this invention it to provide a method for differentiate a primordial germ cell into a functional GV stage oocyte by in vitro culture.

This invention relates to a method for differentiating a primordial germ cell into a functional GV stage oocyte by in vitro culture, comprising: (a) a step of producing a secondary follicle by culturing the primordial germ cell and supporting cells adjacent to the primordial germ cells under conditions that eliminate the effects of estrogen or a factor having a similar function to estrogen; (b) a step of partially dissociating cells between a granulosa cell layer and a thecal cell layer, wherein an oocyte, the granulosa cell layer, and the thecal cell layer constitute the produced secondary follicle; and (c) a step of differentiating the oocyte into a functional GV stage oocyte by culturing the oocyte, the granulosa cell layer, and the thecal cell layer that constitute the secondary follicle in a medium containing a high-molecular-weight compound.

The present invention is of methods of establishing and propagating human embryonic stem cell lines using feeder cells-free, xeno-free culture systems and stem cells which are capable of being maintained in an undifferentiated, pluripotent and proliferative state in culture which is free of xeno contaminants and feeder cells.

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 (iPSCs) of one partner and/or eggs from iPSCs of the other partner. For example, a biological sample is stored as iPSCs 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.

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 a primordial germ cell-like cell (PGCLC) from an epiblast isolated from an embryo or an epiblast-like cell (EpiLC) induced from a pluripotent stem cell (PSC), which comprises allowing the epiblast or EpiLC to express exogenous transcription factor(s) selected from the group consisting of: (i) Blimp1, Prdm14 and Tfap2c; ii) Blimp1 and Prdm14; (iii) Blimp1 and Tfap2c; (iv) Prdm14 and Tfap2c; and (v) Prdm14; thereby inducing the epiblast or EpiLC into a PGC state without acquiring transient mesodermal program.

The invention provides a method for inducing human primordial germ cell-like (PGC-like) cells from human pluripotent stem cells, with high efficiency and high reproducibility, and a cell surface marker for identifying human PGC-like cells. In particular, the invention provides a method for producing a human PGC-like cell from a human pluripotent stem cell, includes a step of producing a mesoderm-like cell by culturing a human pluripotent stem cell in a culture medium comprising activin A and a GSK3 inhibitor, and a step of culturing the mesoderm-like cell in a culture medium containing BMP. The invention also provides a method for producing an isolated human PGC-like cell, which includes the aforementioned two steps and the additional step of selecting a cell positive to at least one cell surface marker selected from the group consisting of PECAM (CD31), INTEGRIN6 (CD49f), INTEGRIN3 (CD61), KIT (CD117), EpCAM, PODOPLANIN and TRA1-81.