Laminins in stemness and germ cell development in human

Sammanfattning: Stem cell niches regulate the fine balance between self-renewal and differentiation of various stem cells. Although crucial hallmarks of stem cell niches, such as the mechanical signalling, neural inputs communicating distant cues, the proximity to blood vessels and the structural support by the surrounding basement membrane (BM), have been evaluated in different niches, little much attention has been directed to the specific composition of the BM. As a crucial component of the BM, laminins have shown major importance for physiological development, with lack of laminin α (LAMA) 1 and 5 resulting in major developmental disruptions. Although, the importance of laminin 521 (LN521) in the embryonic inner cell mass (ICM) from which pluripotent stem cells (PSCs) are derived and in tissues such as the pancreas, nervous and muscular system and vasculature has been established, recent mass spectrometry studies of decellularized adult testicular tissue have suggested the presence of LN521 as well as LN121 in the testis. Hence, we aimed to investigate the role of LN521 in stemness behaviour of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) as well as the potential role of LN521 and 121 in the testicular stem cell niche. Utilizing five human foreskin fibroblast cultured hESC lines, pluripotency after short-term culture on LN521, LN121 and Matrigel was evaluated. We found that LN521, not only supports cell adhesion and stem cell maintenance but further homogenises the pluripotency expression pattern between the cell lines. Further, by applying LN521 as a culture substrate for derivation of Klinefelter syndrome (KS) hiPSCs, we demonstrated the beneficial effect of LN521 on fibroblast reprogramming efficiency. By utilizing LN521 as culture substrate of KS derived hiPSCs we additionally revealed a similar X chromosome inactivation (XCI) behaviour to female cultured PSCs, observed by either the maintenance of XCI with one inactive X chromosome or erosion of XCI. Finally, testicular laminin composition was evaluated during gonadal development and its importance for spermatogonial stem cell maintenance evaluated. We found LAMA 5 to be restricted to the vasculature while LAMA 1 was the sole α laminin chain of prenatal and preand peripubertal seminiferous cords and tubules. LAMA 1 was further restricted to the innermost basal lamina of the adult seminiferous tubules. Interestingly, LAMA 1 loss, as a result of inadequate culture conditions, correlated with a loss of germ cells. In conclusion we demonstrated the importance of LN521 in stem cell maintenance of hESCs and hiPSCs and revealed a correlation between germ cell maintenance and presence of LN121.