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2 State of the Science
Pages 7-30

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From page 7...
... from pluripotent stem cells (PSCs) and creation of model systems for early human ­embryo organization and development, can inform research into in vitro gametogenesis (IVG)
From page 8...
... Early Germ Cell Development in Humans Surani outlined the initial stages of human embryonic germ cell development (see Figure 2-1)
From page 9...
... Experiments using PGCLCs identified the three key transcriptional regulators controlling the human germ cell fate: SOX17, TFAP2C, and PRDM1 (Tang et al., 2016)
From page 10...
... Animal Models of Germ Cell Development Many of the foundational IVG experiments were performed using rodent model systems. Despite several key conserved regulatory features (i.e., the role of proteins such as TFAP2C and PRDM1)
From page 11...
... As Surani explained, PGCs rely on signals from gonadal somatic cells to differentiate into sperm in a testicular environment or eggs in an ovarian environment. Therefore, fully reconstituting gametogenesis in vitro requires coculturing PGCLCs and gonadal somatic cell–like cells under optimal conditions (Hayashi et al., 2017; Hikabe et al., 2016; Yoshino et al., 2021)
From page 12...
... THE "UNFINISHED AGENDA"3 IN HUMANS AND NONHUMAN PRIMATES Scientists at the forefront of IVG research, Mitinori Saitou of Kyoto University and Kotaro Sasaki of the University of Pennsylvania, addressed efforts to reconstitute gametes from PSCs in nonhuman primates and humans. Neither oogenesis nor spermatogenesis has been reconstructed in full.
From page 13...
... 4 Xenogeneic culture systems involve cells belonging to different species: in this case, PGCLCs derived from humans and fetal ovarian somatic cells derived from mice. 5 Organoids are three-dimensional, organ-like structures made by growing stem cells under specialized culture conditions.
From page 14...
... . Human and cynomolgus monkey fetal ovaries, in which oogonia are the predominant germ cell population, were dissociated into single cells and reaggregated to form reconstituted ovaries that were cultured under floating conditions.
From page 15...
... NOTE: E = embryonic day; P = postnatal day; PGC = primordial germ cell; wpf = weeks postfertilization; yo = years old. SOURCES: Presented by Sasaki, April 19, 2023; Sasaki and Sangrithi, 2023.
From page 16...
... ASSAYING THE FUNCTIONAL POTENTIAL OF IVG-DERIVED GAMETES If gametogenesis is successfully reconstituted, then these in vitro– derived cells need to be able to perform all the functions of a healthy gamete. As an expert in spermatogonial stem cell (SSC)
From page 17...
... This system could be used to validate the function of in vitro–derived gametes from nonhuman primates. Xenotransplant Assays Considering that SSC transplantation cannot be used as a routine bioassay for every species, other functional assays are needed to evaluate in vitro–derived gametes from nonhuman primates and humans, Orwig said.
From page 18...
... ROADBLOCKS FOR ACHIEVING IVG Germ cells are distinct from somatic cells in many ways, and these differences pose some of the greatest barriers to IVG. Three experts in 7 These cells were transplanted to a region of the body outside of the testis.
From page 19...
... Ultimately, Paula Cohen emphasized that "all of these features must be recapitulated in an in vitro or ex vivo system" to yield functional gametes. Sex Differences in Meiosis Male and female germ cells progress through meiotic prophase differently.
From page 20...
... The Importance of the Epigenetic Program Epigenetic resetting and genomic imprinting are processes unique to germ cells that are "fundamental for normal embryonic development and healthy physiological postnatal development"; Hajkova discussed these critical processes and the problems they pose to authentically reconstituting gametogenesis in vitro. Epigenetic Programming After their specification early in development, PGCs undergo global DNA demethylation and erasure of genomic imprints via "epigenetic resetting" (Figure 2-6)
From page 21...
... Epigenetic resetting also impacts chromatin and nuclear structure; whereas somatic cells at this stage of development have clusters of highly compacted chromatin, PGCs have a euchromatic and open chromatin structure. After epigenetic resetting, "the developmental and epigenetic routes diverge" for male and female germ cells, Hajkova said.
From page 22...
... Goriely discussed research on DNA mutation rates and clonality in germ cells and how these factors could be considered for gametogenesis in vitro. Germline Mutation Rates The human genomic mutation rate in germ cells is much lower and more tightly regulated than that of somatic cells, Goriely said, resulting in only ~70 de novo point mutations (DNMs)
From page 23...
... The "Selfishness" of the Male Germline Within the testis, a class of pathogenic DNMs that are up to 1,000 times more frequent than other DNMs are clonally selected because they provide an advantage to mutant SSCs. Goriely said, these mutations are "good for the testis" -- increasing sperm production by colonizing seminiferous tubules at the expense of other spermatogonia -- "but nasty for the next generation." These DNMs cause gain-of-function mutations that lead to debilitating disorders, including Apert Syndrome, achondroplasia, and Noonan syndrome.
From page 24...
... , Goriely said, but only mutation rates observed in somatic cells have been used for comparison, and these are several orders of magnitude higher than germline mutation rates. Moreover, both mutation rates and clonality must be considered in reconstituting gametogenesis.
From page 25...
... Paula Cohen added that timing in culture systems needs to account for how germ cells behave differently ex vivo or in vitro; for instance, human fetal ovaries grafted into mice performed oogenesis but at an accelerated time line. Surani noted how helpful it would be to compare these cells to in vivo–derived gametes to determine if they are authentically recapitulating gametogenesis.
From page 26...
... Clark echoed Hajkova and Sasaki, saying that "sequencing is a really good first step," particularly considering that the cell culture passages and tools needed to ensure iPSCs are competent for IVG can introduce anomalies. Generating Offspring Clark asked Hayashi about the limited efficiency of two-cell stage embryos derived from an in vitro egg to produce mouse pups.
From page 27...
... Clark asked Hayashi and Paula Cohen to discuss when the ­aneuploidy is likely arising, during meiosis prophase I or meiosis II (MII)
From page 28...
... . Paula Cohen mused that it could be possible to make an XX "universal gamete," but introducing the Y chromosome would likely cause several problems.
From page 29...
... 13The ISSCR Guidelines for Stem Cell Research and Clinical Translation indicate that with the proper oversight, in vitro culture of human embryos for research can proceed until the formation of the primitive streak or 14 days from fertilization, whichever occurs first.


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