Table 1 Current understanding and comparison of PGCs with VSELs isolated from mouse bone marrow and adult 720 mouse and human ovary and testis

1

PGCs versus VSELs isolated from mouse bone marrow [[40],[50],[59]]

VSELs have been studied in details and compared to PGCs based on available literature but a direct comparison of the two has yet not been made

• PGCs are pluripotent cells derived from epiblast stage embryo whereas VSELs are pluripotent stem cells detected in mouse bone marrow (and other adult body organs). Both have a distinct spherical shape, large nucleus, a thin rim of cytoplasm and long telomeres · VSELs are more differentiated than inner cell mass-derived ESC and share a lot of markers with EpiSC (Gbx2, Fgf5 and Nodal).

• VSELs exhibit similarity in gene expression and epigenetic signatures to epiblast-derived migratory PGCs (but not post-migratory PGCs). However some differences do exist in the gene expression which could be explained by the effect of the niche where they reside (genital ridge for PGCs and bone marrow for VSELs).

• VSELs and the PGCs exhibit similar mechanism of imprinting erasure. Global erasure of parental imprints occurs in PGCs whereas in VSELs erasure mainly on paternally imprinted DMRs (H19-Igf2, RasGRF1) has been reported. DMRs for selected maternally methylated genes (Kcnq1, Igf2R) in PGCs – are hypermethylated in VSELs. In contrast to PGCs, VSELs exhibit an erasure of imprint especially for paternally imprinted DMRs

• Stella promoter in VSELs like in PGCs is partially demethylated and shows transcriptionally active histone modifications (H3Ac and H3K4me3)

• Bone marrow VSELs express at both mRNA and protein levels, genes specific to epiblast (e.g. Stella, Fragilis and Blimp 1) and also those specific to migratory PGCs specification e.g. Dppa2, Dppa4 and Mvh. Both express pluripotent markers including nuclear Oct-4, Nanog, Rex-1, SSEA-1. Both express CXCR4 which is responsible for their migration/mobilization.

• VSELs express several pituitary and gonadal hormone receptors and Sall4 (an early marker shared by germ and hematopoietic cells).

• VSELs similarly like PGCs could be specified to hematopoietic lineage.

2

Adult mouse and human testis & ovary VSELs compared to bone marrow VSELs [[45],[46],[63],[74]]

• Mouse bone marrow VSELs are LIN-/CD45-/SCA-1 +. Similarly VSELs isolated from the mouse ovary and testis are LIN-/CD45-/SCA-1 + .

• Both are very small in size, spherical in shape and have high nucleo- cytoplasmic ratio

• Both express pluripotent markers at mRNA and protein level including nuclear OCT-4, Nanog, Rex-1, SSEA-1 (SSEA-4 in humans)

• Gonadotropin receptors (FSHR) have been reported on both gonadal and bone marrow VSELs

• Human ovarian and testicular VSELs were FACS sorted using SSEA-4 as a marker. Both PGCs and human ovarian and testicular VSELs stain positive for alkaline phosphatase. VSELs express several genes related to pluripotency and self-renewal (POU5F1 (OCT4), SALL4, CDH1, LIN28B, NANOG, SOX2, SOX11, DPPA3 (STELLA), LEFTY1, ZIC3, ZIC5, PRDM14, GAL, PPP1R9A, RNF2, LASS1 (CERS1), SMO, MMP25, GULP1, MLLT4, BMP7, MYBL2, DNMT3B, ZFP42, HESRG, ZSCAN10, TRO, GLI2, FBN3 and DDX11) and PGC related genes (VASA, PRDM1, DPPA3)

3

VSELs compared to embryonic stem (ES) cells [[47],[59],[70],[74]]

• In contrast to ES cells isolated from inner cell mass and iPS cells – VSELs do not form teratomas in SCID mice nor complement blastocyst development. This basic difference between these two populations of pluripotent stem cells is due to novel epigenetic mechanism of imprint erasure on paternally imprinted DMRs (H19-Igf2, RasGRF1) exhibited by VSELs

• VSELs express pluripotent transcripts like ES cells but several folds low expression is reported

• 341 genes were down regulated and 435 genes were up regulated when hES cells were compared to VSELs isolated from human ovary. Interestingly genes like H19 (maternally imprinted gene), VASA (germ cell marker) and PLD6 (required for gametogenesis and meiosis) were up regulated in VSELs compared to hES cells [70].

• ES cells undergo symmetric cell divisions, are immortal in vitro and give rise to cells of all three lineages. VSELs remain quiescent and do not readily divide in culture. They self-renew under special conditions and are capable of giving rise to cells of all three lineages e.g. VSELs in ovary surface epithelium respond to FSH, undergo self-renewal and clonal expansion (evidenced by germ cell nest formation) followed by differentiation into oocytes both in vitro[70] and in vivo[47].

• Distinct expression profile of VSELs shows that they are more related to PGCs than ES cells and thus have the potential to spontaneously differentiate into gametes.