Laminin-guided highly efficient endothelial commitment from human pluripotent stem cells

Abstract

Obtaining highly purified differentiated cells via directed differentiation from human pluripotent stem cells (hPSCs) is an essential step for their clinical application. Among the various conditions that should be optimized, the precise role and contribution of the extracellular matrix (ECM) during differentiation are relatively unclear. Here, using a short fragment of laminin 411 (LM411-E8), an ECM predominantly expressed in the vascular endothelial basement membrane, we demonstrate that the directed switching of defined ECMs robustly yields highly-purified (>95%) endothelial progenitor cells (PSC-EPCs) without cell sorting from hPSCs in an integrin-laminin axis-dependent manner. Single-cell RNA-seq analysis revealed that LM411-E8 resolved intercellular transcriptional heterogeneity and escorted the progenitor cells to the appropriate differentiation pathway. The PSC-EPCs gave rise to functional endothelial cells both in vivo and in vitro. We therefore propose that sequential switching of defined matrices is an important concept for guiding cells towards desired fate.

Selective Laminin-Directed Differentiation of Human Induced Pluripotent Stem Cells into Distinct Ocular Lineages

“…Recombinant E8 fragments of laminin-111 (Taniguchi et al, 2009) N/A Recombinant E8 fragments of laminin-211 (Taniguchi et al, 2009) N/A Recombinant E8 fragments of laminin-332 (Miyazaki et al, 2012) N/A Recombinant E8 fragments of laminin-411 (Ohta et al, 2016) N/A Recombinant E8 fragments of laminin-511 (Ido et al, 2004(Ido et al, , 2007Taniguchi et al, 2009) N/A…”

“…The expression of laminin is spatiotemporally regulated, especially during development (Miner et al, 1997;Nguyen and Senior, 2006), with several laminin isoforms able to support hPSC proliferation in undifferentiated states (Miyazaki et al, 2008;Rodin et al, 2010Rodin et al, , 2014Xu et al, 2001). Truncated versions of the protein-the laminin E8 fragments (LNE8)-permit robust proliferation of hPSCs (Miyazaki et al, 2012), while other isoforms are known to be effective in the long-term expansion or generation of specific somatic cell types from hPSCs (Fusaoka-Nishioka et al, 2011;Kanninen et al, 2016;Ohta et al, 2016;Takayama et al, 2013Takayama et al, , 2016.…”

Preparation of iPS cell-derived CD31+ endothelial cells using three-dimensional suspension culture

“…Although the yield of EC remains insufficient in our system, adaptation of chemically defined medium and novel marker for cardiovascular progenitor cells [19] may help to improve the efficiency of our differentiation system. Moreover, endothelial lineage was highly induced from hiPS cells by sequential switching of defined ECMs [25] , therefore, the yield of EC may be improved by supplement with optimized ECM-derived fragment in bioreactor system. In an attempt to scale up the culture volume for KDR + cell induction, the cell density on day 7 was at the same level in 30- and 100-mL cultures (data not shown).…”

Cardiac Extracellular Matrix Modification as a Therapeutic Approach

“…A polyurethane elastomer functionalized with RGD, another cell binding domain YIGSR, and heparin has been shown to reduce platelet adhesion and enhance attachment of endothelial cells to the surface [43]. An E8 fragment of laminin 411 which represents the C-terminal region of the protein has also been shown to increase differentiation of human iPSCs toward endothelial cells and could be combined with a blank slate to study endothelial differentiation [44]. The fragment improves endothelial differentiation over the entire laminin 411 protein, and endothelial cells created this way showed higher expression of genes associated with vascular development and angiogenesis.…”