3D migration of human amniotic fluid stem cells involves mesenchymal and amoeboid modes and is regulated by mTORC1.

3D cell migration is an integral part of many physiologic processes. Although being well studied in the context of adult tissue homeostasis and cancer development, remarkably little is known about the invasive behavior of human stem cells. Using two different kinds of invasion assays, this study aimed at investigating and characterizing the 3D migratory capacity of human amniotic fluid stem cells (hAFSCs), a well-established fetal stem cell type. Eight hAFSC lines were found to harbour pronounced potential to penetrate basement membrane (BM)-like matrices. Morphological examination and inhibitor approaches revealed that 3D migration of hAFSCs involves both, the matrix metalloprotease (MMP)-dependent mesenchymal, elongated mode and the Rho-associated protein kinase (ROCK)-dependent amoeboid, round mode. Moreover, hAFSCs could be shown to harbour transendothelial migration capacity and to exhibit a motility-associated marker expression pattern. Finally, the potential to cross extracellular matrix (ECM) was found to be induced by mTORC1-activating growth factors and reduced by blocking mTORC1 activity. Taken together, this report provides the first demonstration that human stem cells exhibit mTORC1-dependent invasive capacity and can concurrently make use of mesenchymal and amoeboid 3D cell migration modes what represents an important step towards the full biological characterization of fetal human stem cells with relevance to both, developmental research and stem cell-based therapy. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: The array of established human stem cells contains cells of various sources, developmental stages and differentiation capacities. Still, their effective use in stem cell-based therapies is limited. Although being first described in 2003 and meanwhile well-studied, the in vivo biological function and the therapeutic application of hAFSCs are still elusive. This study reports on the identification and characterization of the 3D migratory capacity of hAFSCs, a so far undescribed hAFSC trait which ultimately governs cell function. These data represent an important step towards the full biological characterization of fetal human stem cells with implications for research and stem cell-based therapy.