Isolation, characterization and propagation of mitotically active germ cells from adult mouse and human ovaries

Woods, Dori C.; Tilly, Jonathan L.

doi:10.1038/nprot.2013.047

Cited by 130 publications

(170 citation statements)

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“…To confirm whether DDX4-positive cells from post-natal ovaries can be separated independently of an antibody and used to establish a cell line, it is necessary to isolate FGSCs by using mouse strains endogenously expressing fluorescent proteins only in the germ cells. For isolating such germ cells by FACS, it is unnecessary to use an anti-DDX4 antibody for germ cell labeling, which has been a key step in previous studies (Zou et al, 2009;White et al, 2012;Woods and Tilly, 2013;Park and Tilly, 2015). In our study, without using antibody, GFP positive FGSCs have been isolated and established.…”

Section: Discussionmentioning

confidence: 99%

Production of offspring from a germline stem cell line derived from prepubertal ovaries of germline reporter mice

Zhang

2016

Mol. Hum. Reprod.

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study hypothesis: We investigated whether DEAD-box polypeptide 4 (DDX4) positive cells from post-natal ovaries of germline lineage reporter mice can be isolated based on endogenously expressed fluorescent proteins and used to establish a cell line for producing offspring. what is known already: In recent years, female germline stem cells (FGSCs) have been isolated from the ovaries of post-natal mice by magnetic-activated cell sorting or fluorescence-activated cell sorting (FACS) relying on an antibody against DDX4. However, whether DDX4-positive cells from post-natal ovaries of germline lineage reporter mice can be established without using an antibody, as well as a cell line established for producing offspring, remains unknown. study design, samples/materials, methods: To obtain the expected offspring (Ddx4-Cre;mT/mG mice), Ddx4-Cre mice were crossed with mT/mG mice. In the ovaries of Ddx4-Cre;mT/mG mice, germ cells were destined to express enhanced green fluorescent protein (EGFP) while somatic cells still express tandem dimer Tomato (tdTomato). Therefore, the germ cells could be clearly distinguished from somatic cells by fluorescent proteins. Then, we investigated the pattern of fluorescent cells in the ovaries of 21-day-old Ddx4-Cre;mT/mG mice under a fluorescent microscope. Germ cells were sorted by FACS without using antibody and used to establish a FGSC line. The FGSC line was analyzed by DDX4 immunostaining, Edu (5-ethynyl-2 ′ -deoxyuridine) labeling, and RT-PCR for germ cell markers. Finally, the physiological function of the FGSC line was examined by transplanting FGSCs into the ovaries of sterilized recipients and subsequent mating.main results and the role of chance: Firstly, we have successfully isolated FGSCs from the ovaries of 21-day-old Ddx4-Cre;mT/mG mice based on endogenously expressed fluorescent proteins. FACS was used to separate the cells and 2.3% of all viable cells was EGFP-positive germ cells. Subsequently, a FGSC line was established that was doubly positive for DDX4 immunostaining and Edu labeling. The mRNA expression of several germ cell markers in this cell line, such as Ddx4, Deleted in azoospermia-like (Dazl), B lymphocyte-induced maturation protein-1 (Blimp1), Stella and Fragilis, was detected. Lastly, the FGSC line was proven to be functional under physiological conditions, as offspring were produced after transplanting FGSCs into ovaries of sterilized recipients and a subsequent mating.limitations, reasons for caution: The molecular mechanisms of proliferation and differentiation of FGSCs in vivo and in vitro still need to be elucidated.wider implications of the findings: Our results confirm that DDX4-positive cells can be separated from post-natal mouse ovaries and used to establish cell lines that are functional in producing offspring, and provide further evidence for the existence of post-natal FGSCs in mammals. The Ddx4-Cre;mT/mG mouse strain is an ideal model for the isolation, characterization and propagation of FGSCs and is a useful tool for fully elucidating the molecu...

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Section: Discussionmentioning

confidence: 99%

Production of offspring from a germline stem cell line derived from prepubertal ovaries of germline reporter mice

Zhang

2016

Mol. Hum. Reprod.

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“…As we move toward the more investigational technologies, fertoprotection is an important emerging area of clinical intervention [using gonadotropin-releasing hormone (GnRH) analogs] and investigation (18, 19), and in vitro follicle growth and follicle transplantation are the focus of a great deal of preclinical research. Possible fertility preservation methods for the future include the development of inducible pluripotent stem cells (iPSC), production of stem cell–derived gametes, and use of germ-line stem cells isolated from the ovary (20, 21). The primary focus of this review is to describe how the application of biomaterials science to reproductive biology has led to development and advances in in vitro follicle growth and ovarian tissue transplantation, though emerging opportunities in fertility preservation are also addressed.…”

Section: Introductionmentioning

confidence: 99%

Bioengineering the Ovarian Follicle Microenvironment

Shea

Woodruff

Shikanov

2014

Annu. Rev. Biomed. Eng.

144

102

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Chemo- and radiation therapies used to treat cancer can have the unintended effect of making patients infertile. Clinically established fertility preservation methods, such as egg and embryo cryopreservation, are not applicable to all patients, which has motivated the development of strategies that involve ovarian tissue removal and cryopreservation before the first sterilizing treatment. To restore fertility at a later date, the early-stage follicles present in the tissue must be matured to produce functional oocytes, a process that is not possible using existing cell culture technologies. This review describes the application of tissue engineering principles to promote ovarian follicle maturation and produce mature oocytes through either in vitro culture or transplantation. The design principles for these engineered systems are presented, along with identification of emerging opportunities in reproductive biology.

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“…It has been suggested that oogonia-like stem cells could be harvested, cultured and expanded (Woods and Tilly, 2013) followed by in vitro culture to the mature MII stage. However, there is still controversy over the existence of such oocyte precursor cells in the female and the efficiency at which mature, developmentally competent oocytes can be derived from them.…”

Section: Gene Editing Of Male and Female Germ Cellsmentioning

confidence: 99%

Responsible innovation in human germline gene editing: Background document to the recommendations of ESHG and ESHRE

et al. 2018

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Isolation, characterization and propagation of mitotically active germ cells from adult mouse and human ovaries

Cited by 130 publications

References 50 publications

Production of offspring from a germline stem cell line derived from prepubertal ovaries of germline reporter mice

Production of offspring from a germline stem cell line derived from prepubertal ovaries of germline reporter mice

Bioengineering the Ovarian Follicle Microenvironment

Responsible innovation in human germline gene editing: Background document to the recommendations of ESHG and ESHRE

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