Expression of gonadotropins and testicular markers in multipotent stroma cells and amnion derived induced pluripotent stem cells

Multipotent stromal cells (MSCs) are the most reliable candidates for stem cell therapies because of their plasticity, immunoregulatory features and availability. Unlike embryonic stem cells (ESCs), MSCs do not face ethical, legislative and clinical safety issues. Although there is much controversy about MSC definition, they are conventionally defined as a class of cells with potential to self-renewal and certain 'stemness' abilities to differentiate into multiple cell lineages within the same germ layer, displaying a spindle-shaped morphology, adherence to plastic and expression of certain surface markers, such as CD105+, CD90+ and CD73 while being negative for hematopoietic markers, such as CD34- and CD45-. Placental MSCs particularly attract attention in the field of research and clinical application, due to the virtual absence of ethical concerns and ease of obtaining, but reports about possible culture duration of MSCs, expression of immune-relevant molecules, such as MHC class I, and pluripotency potential represented by markers like Oct4, Sox2, Nanog, Klf4, c-Myc, Lin28 remain controversial. To address these topics, we performed a comprehensive long-term characterization of MSCs derived from amnion and bone marrow from the human and our preclinical non-human primate model, the common marmoset monkey (Callithrix jacchus). While ESC and iPS lines from Callithrix jacchus are well established and characterized, there is little known on marmoset MSCs, especially in comparison with the human.

In terms of immunogenicity, expression of MHC class I complex was significantly reduced in amnion MSCs in early vs. late passages (P3 – P12), whereas in bone marrow MSCs samples MHC class I presence was close to 90% from the beginning. Surprisingly, pluripotency genes Oct4 and Nanog were significantly expressed in early MSC passages, accompanied by partial demethylation status of their Oct4 promoter. Sox2 was significantly higher expressed in the human than in the marmoset, while the other way round for Lin28 expression, whereas c-Myc and Klf4 were barely detectable in all samples. From our findings we speculated about the existence of a small ''true'' pluripotent population of cells at least in the amnion with unknown determination of developmental origin. Therefore, we screened reprogrammed and extracted amnion cells and iPS for early developmental and germ cell markers like VASA, SALL4, CRiPTO, PLZF, GPR125, STELLA and PGP95 as well as reproductive gonadotropins and their respective receptors (LH/CG(R), FSH(R)) by qPCR. In amnion derived MSCs and iPS, significantly high expression of STELLA and SALL4 was observed, whereas only ESCs and amnion iPS expressed detectable levels of CRIPTO. Furthermore, only amnion derived MSCs expressed GPR125 and PGP95 levels. FSH production could be found in amnion tissue but not in ESCs, its production was maintained further to our surprise in amnion iPS cells. CG/LH and its corresponding receptor was found in even higher levels after reprogramming. Due to our findings of germ cell markers in combination with gonadotropin expression, the postulated cell population might be migrated from early epiblast, but not from trophoectoderm or primitive endoderm. They might be the ideal candidates for further differentiation into germ cells or co-cultures or might simply serve as “natural drug stores” for the immunological neutral production of gonadotropins.