Human Haploid Cells Differentiated from Meiotic Competent Clonal Germ Cell Lines That Originated from Embryonic Stem Cells

Abstract

Early germ-like cells (GLCs) derived from human embryonic stem cells (hESCs) have presented new opportunities to study germ cell differentiation in vitro. However, differentiation conditions that facilitate the formation of haploid cells from the derived GLCs have eluded the field. The inability to propagate GLCs in culture is a further limitation, resulting in inconsistent rederivations of GLCs from hESCs with relatively few GLCs in these heterogeneous populations. Here we found in vitro conditions that enrich for DDX4/POU5F1+ GLCs (∼60%) and that has enabled continual propagation for >50 passages without loss of phenotype. Clonal isolation of single GLCs from these mixed cultures generated 3 GLC (>90% DDX4/POU5F1+) and 2 hESC (<0.1% DDX4+) lines that could be continually expanded without loss of phenotype. Differentiation of clonal GLC lines in serum resulted in expression of postmeiotic markers and >11% were haploid, ∼5-fold higher than previous studies. The robust clonal meiotic competent and incompetent GLC lines will be used to understand the factors controlling human germ cell meiosis and postmeiotic maturation.

Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women

“…Both proteins are specific to germ cells and are necessary for meiotic recombination to proceed 29–31 . Furthermore, chromosomal DNA content analysis of human OSC cultures 72 h after passage revealed the presence of an expected diploid (2 n ) cell population; however, we also detected peaks corresponding to 4 n and 1 n cell populations, the latter being indicative of germ cells that had reached haploid status 32 (Fig. 5i).…”

Nonviral Minicircle Generation of Induced Pluripotent Stem Cells Compatible with Production of Chimeric Chickens

“…For example, iPSCs could be generated from endangered and genetically valuable birds and stored long term as immortal stem cells. These cells could then be differentiated at any time into early germ cells (sperm and eggs) and transplanted into host domestic species for the production of endangered species gametes (Hubner et al, 2003;Nayernia et al, 2006;Toyooka et al, 2003;West et al, 2010a). These gametes could then be used in reproduction programs in zoological parks and game preserves across the world.…”

Culture of Pig Induced Pluripotent Stem Cells without Direct Feeder Contact in Serum Free Media

“…Transduction was performed using 1X TransDux (System Biosciences, CA, USA). Pig fibroblast cells were trypsinized 24 hrs after transduction and passaged onto inactivated mouse embryonic fibroblast feeder cells in embryonic stem cell expansion medium DMEM/F12 (Gibco, NY, USA) supplemented with 20% Knockout Serum Replacement (KSR; Gibco, NY, USA), 2 mM L-glutamine (Gibco, NY, USA), 0.1 mM non-essential amino acids (Gibco, NY, USA), 50 U/ml penicillin (Gibco, NY, USA), 50 μg/ml streptomycin (Gibco, NY, USA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, MO, USA) and 10 ng/ml FGF2 (Sigma-Aldrich, MO, USA and R&D Systems, MN, USA The immunostaining protocol used was previously reported [28]. Briefly, cells were washed with PBS+/+ (Thermo Scientific, UT, USA) and fixed with 4% paraformaldehyde (Sigma-Aldrich, MO, USA) at room temperature for 15 minutes.…”