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Review
. 2018 Jul 5;373(1750):20170230.
doi: 10.1098/rstb.2017.0230.

Developing defined substrates for stem cell culture and differentiation

Louise Hagbard  1 , Katherine Cameron  2 , Paul August  3 , Christopher Penton  3 , Malin Parmar  4 , David C Hay  2 , Therése Kallur  5
Affiliations
Review

Developing defined substrates for stem cell culture and differentiation

Louise Hagbard et al. Philos Trans R Soc Lond B Biol Sci. .

Abstract

Over the past few decades, a variety of different reagents for stem cell maintenance and differentiation have been commercialized. These reagents share a common goal in facilitating the manufacture of products suitable for cell therapy while reducing the amount of non-defined components. Lessons from developmental biology have identified signalling molecules that can guide the differentiation process in vitro, but less attention has been paid to the extracellular matrix used. With the introduction of more biologically relevant and defined matrices, that better mimic specific cell niches, researchers now have powerful resources to fine-tune their in vitro differentiation systems, which may allow the manufacture of therapeutically relevant cell types. In this review article, we revisit the basics of the extracellular matrix, and explore the important role of the cell-matrix interaction. We focus on laminin proteins because they help to maintain pluripotency and drive cell fate specification.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Keywords: cell culture; cell therapy; defined substrates; human pluripotent stem cells; human recombinant laminins.

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Conflict of interest statement

L.H. and T.K. are both employed by BioLamina AB and P.A. and C.P. by Icagen Inc. D.C.H. is a founder, director and shareholder in Stemnovate Limited. D.C.H. is also a director and shareholder in HigherSteaks Limited.

Figures

Figure 1.
Figure 1.
(a–c) Laminins are abundant components of the BMs and have essential roles in structural organization. Via binding to cell-surface receptors, such as integrins, laminins are also key regulators of cell behaviour (a). The molecular structure of laminin isoforms is cross-shaped. The Greek letters indicate the specific chains. The α chains have five globular domains (LG1–5) at the C terminus. The short arms are composed of a globular laminin N-terminal domain (LN), globular laminin IV domain (L4), or laminin four domain (LF). Each of these domains is separated by rod-like regions formed by multiple laminin epidermal-growth-factor-like (LE) domains (b). The α5-chain laminins are key cell adhesion proteins of the natural stem cell niche. It is naturally expressed and secreted by cells in the inner cell mass ICM of the embryo whereas the α1-chain laminins are mostly expressed in the Reichert's membrane which supports the outer extra-embryonic layer of trophoblasts (c).
Figure 2.
Figure 2.
(a–d) hPSCs grow as homogeneous monolayers on LN-521, exhibiting a high nuclear-to-cytoplasm ratio and prominent nucleoli. Owing to the biologically relevant support from the matrix, hESCs and hiPSCs can be seeded as single cells on LN-521 without the need to use apoptosis inhibitors (e.g. ROCKi) for survival. Within 1 h after seeding, the cells have attached and are evenly distributed across the surface (a, day 0). The cells show high motility on LN-521, migrate and form small colonies and start dividing (a, day 1). The colonies merge (a, day 3) into a homogeneous monolayer without signs of spontaneous differentiation (a, day 4). LN-521 supports higher post-seeding survival (24 h) of single-cell dissociated hPSCs, compared with Matrigel and non-relevant laminin matrices (b). In addition, hPSCs proliferate faster on LN-521 compared with other feeder-free matrices (EHS extract; Engelbreth–Holm–Swarm mouse sarcoma extract), with about 10-fold increase every 4 days (c). LN-521 even enables large-scale automated processes for robust hPSC expansion in bioreactors. The cells easily attach, spread evenly on the surface of the microcarriers and give a 20-fold expansion within 4 days (d).
Figure 3.
Figure 3.
(a–f) Differentiation of hESCs on LN-111 towards DA cells results in more than a 40-fold increase of transplantable DA progenitors at day 16 (a) and the human progenitors innervate the striatum, become tyrosine hydroxylase (TH+) neurons and contribute to complete behavioural recovery after transplantation into animals modelling Parkinson's disease (b; scale bar represents 1.5 mm). LN-521 improves muscle cell proliferation and differentiation, giving larger myotubes, higher amounts of nuclei per myotube (express myosin heavy chain, MHC) and more consistent differentiation in long-term cultures (c; MHC in green) compared with cells on Matrigel (d; DAPI in blue). Human ESCs differentiated on LN-521 and LN-111 demonstrate efficient hepatocyte maturation with significant improvements in cell function [43]. The hepatocyte-like cells are highly organized on LN-521, express transporter protein MRP1 (e; green) and HNF4a (red), and a majority of the cells are albumin+ (f; green) Cell nuclei are visualized with DAPI (blue).
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