Laminin α5 substrates promote survival, network formation and functional development of human pluripotent stem cell-derived neurons in vitro

Abstract

Laminins are one of the major protein groups in the extracellular matrix (ECM) and specific laminin isoforms are crucial for neuronal functions in the central nervous system in vivo. In the present study, we compared recombinant human laminin isoforms (LN211, LN332, LN411, LN511, and LN521) and laminin isoform fragment (LN511-E8) in in vitro cultures of human pluripotent stem cell (hPSC)-derived neurons. We showed that laminin substrates containing the α5-chain are important for neuronal attachment, viability and network formation, as detected by phase contrast imaging, viability staining, and immunocytochemistry. Gene expression analysis showed that the molecular mechanisms involved in the preference of hPSC-derived neurons for specific laminin isoforms could be related to ECM remodeling and cell adhesion. Importantly, the microelectrode array analysis revealed the widest distribution of electrophysiologically active neurons on laminin α5 substrates, indicating most efficient development of neuronal network functionality. This study shows that specific laminin α5 substrates provide a controlled in vitro culture environment for hPSC-derived neurons. These substrates can be utilized not only to enhance the production of functional hPSC-derived neurons for in vitro applications like disease modeling, toxicological studies, and drug discovery, but also for the production of clinical grade hPSC-derived cells for regenerative medicine applications.

Screening of Hydrogels for Human Pluripotent Stem Cell–Derived Neural Cells: Hyaluronan‐Polyvinyl Alcohol‐Collagen‐Based Interpenetrating Polymer Network Provides an Improved Hydrogel Scaffold

“…Vinculin is a link protein between cell adhesion receptors and the actin cytoskeleton, and its expression is increased on focal adhesion points . As mentioned earlier, integrin α6β4 is related to neural cell laminin adhesion …”

“…The 08023‐derived neurons had the best neurite and neuronal network forming capacity, while hNP1 and 10212 neurons were less efficient (Figure B). Previously, we showed that similarly differentiated hESC and hiPSC neurons do not express ECM‐ and adhesion‐related molecules at the same levels, nor do they form neurites or neuronal networks similarly in different laminin formats . Even though hPSC cell lines have differential initial differentiation capacity despite their origin, the differentiation method used does not necessarily make them more similar in their cell type–specific behavior.…”

“…The controls were as follows: positive control PM and negative control nonfunctionalized GG . In addition, in‐house 2D laboratory standard coating control mouse laminin (Sigma‐Aldrich) was used . For PM, cells were mixed with 0.25% PM diluted in 10% sucrose in phosphate‐buffered saline (PBS).…”

“…Integrins are cell membrane–bound proteins mediating these cell–ECM interactions and thus play an important role in cell attachment and behavior . Some integrins are especially associated with neurons such as integrin α6β4, which is found to act as a laminin receptor . Moreover, neuronal cells have various specific ECM receptors, such as 40S ribosomal protein SA (RPSA), that have important roles in growth cones .…”

Distribution of Collagen I, III, and IV and Laminin in the Human Liver during Prenatal Development

“…Neurite formation and collagenase IV production are important biological activities of laminin. Laminin, but also collagens I, III, and IV, promote the mobility of various cells (during embryonic development, including neural crest cell migration, and in wound healing) [Yu et al, 2008;Hyysalo et al, 2017]. Moreover, Roskams [2006] and Brizzi et al [2012] reported that the cells in the liver are part of a single and specific biological compartment (stem cell niche) in which all of the cells exercise their functions only when coexisting together inside the ECM.…”