Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells

Abstract

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the potential to provide an infinite source of tissues for regenerative medicine. Although defined xeno-free media have been developed, culture conditions for reliable propagation of hESCs still require considerable improvement. Here we show that recombinant E8 fragments of laminin isoforms (LM-E8s), which are the minimum fragments conferring integrin-binding activity, promote greater adhesion of hESCs and hiPSCs than do Matrigel and intact laminin isoforms. Furthermore, LM-E8s sustain long-term self-renewal of hESCs and hiPSCs in defined xeno-free media with dissociated cell passaging. We successfully maintained three hESC and two hiPSC lines on LM-E8s in three defined media for 10 passages. hESCs maintained high level expression of pluripotency markers, had a normal karyotype after 30 passages and could differentiate into all three germ layers. This culture system allows robust proliferation of hESCs and hiPSCs for therapeutic applications.

Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment

“…To date, at least 16 laminin isoforms are known to exist in mammalian tissues, and most of them have been produced in our laboratory as recombinant proteins 10,11 . Other investigators have reported that recombinant vitronectin 12 , a hydrogel 13 , and a LN-511 fragment 14 can serve as alternatives to Matrigel or feeder cells for culturing of hES cells. However, none of the cell culture substrata allows clonal survival of hES cells without the use of apoptosis inhibitors [15][16][17] or chemically undefined substances 18,19 , and the cells are often passaged in small clumps that can contain a significant proportion of differentiated cells.…”

Integrating Mechanical and Biological Control of Cell Proliferation Through Bioinspired Multieffector Materials

“…Additionally, Yang and co-workers observed a decrease in the levels of soluble aβ-40 aβ-42 and amyloid-β plaques probably derived from the IKVAV functionality (143) . (102) Silk fibroin Electrospun matrices Enhances adhesion of different cell types in vitro (101) Fibronectin Coating surfaces Enhances adhesion and retains regenerative capacity of human hematopoietic Stem Cells ex vivo (144) Laminin E8 Coating surfaces Support efficient (145) fragment adhesion and expansion of dissociated human pluripotent stem cells Vitronectin Coating surfaces Promote adhesion and osteogenic diferenciation of human mesenchymal stem cells (146) GFOGER Coating synthetic polycaprolactone (PCL) scaffolfs…”

Feeder Cell Sources and Feeder-Free Methods for Human iPS Cell Culture

“…The MEF conditioned medium can also be replaced, however, as the combination of a human laminin coating with defined medium supplements, such as recombinant bFGF and the additional growth factors flt3-L, SCF, and LIF, was shown to support the growth and maintenance of undifferentiated human ESCs [67]. It has also been shown that recombinant laminin E8 fragments (LM-E8s), which are truncated peptides composed of the C-terminal regions of the α, β, and γ chains of laminin, enable robust propagation of human ESCs and iPSCs in an undifferentiated state in cultures with defined and xeno-free media [68]. We have confirmed that human gingiva-derived iPSCs [30] can be maintained in an undifferentiated state on LM-E8-coated plates after dissociation and passaging ( Fig.…”