ECMatrix™ Laminin Substrates are Superior Xeno-free Extracellular Matrix Coatings for Feeder-free Human Pluripotent Stem Cell Cultures

The stem-cell niche refers to the immediate microenvironment where stem cells physically interact with diverse ECM (extracellular matrix) proteins and signaling molecules to regulate cell fate. To create better in vitro models of the cellular environment, researchers have historically coated plasticware with distinct ECM proteins and adhesion molecules to facilitate binding by cellular integrin proteins. The basement membrane, which separates the epithelium from underlying tissue, is composed of about fifty kinds of ECM proteins including laminins, collagens, and assorted  other glycoproteins. Laminins are heterotrimeric proteins composed of an α, β, and γ chain, and are among the largest basement membrane proteins, having a typical molecular weight of ~800 kDa. Laminin isoforms are referred to with three-digit numbers that represent their chain composition. For example, Laminin-511 refers to the isoform consisting of α5, β1, and γ1 chains.

Figure 1. Structure of full length laminin protein, laminin-511 and laminin-511 E8 isoforms.

Table 1. ECMatrix™-E8 Laminin Substrate Product Available

Laminin Isoform	111	211	121	221*	332	311	321	411*	421	511*	521	213
α	α1	α2	α1	α2	α3	α3	α3	α4	α4	α5	α5	α2
β	β1	β1	β2	β2	β3	β1	β2	β1	β2	β1	β2	β1
γ	γ1	γ1	γ1	γ1	γ2	γ1	γ1	γ1	γ1	γ1	γ1	γ3

Human pluripotent stem cells (ES and iPS cells) express the major integrin species α6β1, and therefore can be maintained stably and expanded efficiently in feeder-free conditions on culture vessels coated with  laminin-511, the binding partner of integrin α6β1 . However, laminin-511 is not suitable for large-scale production because of its large molecular weight and heterotrimeric nature. Professor Kiyotoshi Sekiguchi’s group (Matrixome, Inc.) have solved this problem by producing a recombinant E8 fragment of laminin-511 at large-scale that retains full integrin-binding activity. ECMatrix™-511 E8 Laminin Substrates can be used to culture pluripotent stem cells under feeder-free conditions with numerous advantages over traditional coating methods including:

• Animal-free, xeno-free formulation: Consistent from lot-to-lot, with no prescreening required
• No plate precoating required: Save time by simply adding to media while passaging cells
• Supports single-cell passaging without ROCKi: Ideal for CRISPR editing or clonal isolation
• Higher adhesion and growth rates: Achieve densities needed for experiments sooner
• Easy to handle: No chilling of cell culture consumables required

Additional laminin E8 fragments can also be used as substrates to differentiate human pluripotent stem cells into different lineages including endothelial progenitors (Laminin-411) and cardiomyocytes (Laminin-221).

Table 2. Substrate Overview Table

	Stem Cell Qualified ECM Gel Matrix (BME)	Vitronectin	ECMatrix™-511 E8
Part Number	CC131	CC130	CC160, CC161
Derivation	EHS Sarcoma (Undefined)	Recombinant (Defined)	Recombinant (Defined)
Species	Mouse	Human	Human
Consistency	+	++	+++
Lot Specific Testing	Required	Not Required	Not Required
Pre-coating Step	Required	Required	Not Required
Time for Coating	4-24 Hours	4-24 Hours	1 Hour
Single Cell Passaging	+	+	+++
Clump Passaging	+++	++	+
Cell Growth	+	++	+++
Cell Adhesion	++	+	+++
Prechilled Equipment	Required	Not Required	Not Required
Coating Concentration	Varies by lot	0.5 µg/cm2	0.25 µg/cm2

Results

Figure 2. Growth of human induced pluripotent stem cells. Human iPSCs grown on ECMatrix™-511 E8 Laminin Substrates demonstrate higher cell proliferation rates compared to Matrigel^® basement membrane extract when passaged as single cells using Accutase^®.

Figure 3. Human iPSC pluripotency markers. Human iPSCs grown on ECMatrix™-511 E8 Laminin Substrates over 5 passages maintain pluripotency markers SSEA-4 (red, A), Oct-4 (green, B) and TRA-1-60 (red, C).

Figure 4. Adhesion and growth of human iPSCs on ECMatrix™-511 E8 Laminin Substrates vs. Matrigel^® Adhesion and growth of human iPSCs on ECMatrix™-511 E8 Laminin Substrates vs. Matrigel^®. A) Human iPS cells adhere more strongly to Laminin-511 E8 fragment than to the intact Laminin-511 or Matrigel^®. OD570 absorbance represents the relative number of attached cells normalized against the values at the maximum effect on Matrigel^®. Matrigel^® control was arbitrarily set=1. B) Human iPS cells cultured using the Laminin-511 E8 Laminin Substrate resulted in 200-fold more cells when compared with Matrigel^® coating methods.

Figure 5. Cell adhesion of human iPSCs cultured on ECMatrix™-511 E8 Laminin Substrates vs. laminin-521 and vitronectin. ECMatrix™-511 E8 laminin supports adhesion and growth of human iPSCs using both precoated and non-precoated culture methods at low protein concentrations (<0.5 µg/cm2). Unlike other ECM substrates, such as full-length laminin-521 and vitronectin, ECMatrix™-511 E8 is simply added to cell suspensions that have been detached from cultureware during iPS cell passaging.

Ordering Info

Description	Recommended Cell Type	Part Number
ECMatrix™-511 E8 Laminin Substrate	Human ES/iPS Cells	CC160-350UG
	Human ES/iPS Cells	CC160-1050UG
	Human ES/iPS Cells	CC160-100ML
ECMatrix™-511 Silk E8 Laminin Substrate	Human ES/iPS Cells	CC161-1050UG
	Human ES/iPS Cells	CC161-100ML
ECMatrix™-411 E8 Laminin Substrate	Endothelial Progenitors	CC162-350UG
	Endothelial Progenitors	CC162-1050UG
ECMatrix™-221 E8 Laminin Substrate	Cardiomyocytes	CC163-350UG
	Cardiomyocytes	CC163-1050UG
Stem Cell Qualified ECM Gel Matrix	Human ES/iPS Cells	CC131-5ML
	Human ES/iPS Cells	CC131-10X5ML
PluriSTEM-XF™ Recombinant Vitronectin	Human ES/iPS Cells	CC130
ECMatrix™-111 E8 Laminin Substrate	Hepatocytes and Neurons	CC164-350UG
	Hepatocytes and Neurons	CC164-1050UG
ECMatrix™-332 E8 Laminin Substrate	Skin, Hair and RPE Cells	CC165-350UG
	Skin, Hair and RPE Cells	CC165-1050UG

References

1. Miyazaki T, Futaki S, Suemori H, Taniguchi Y, Yamada M, Kawasaki M, Hayashi M, Kumagai H, Nakatsuji N, Sekiguchi K, et al. 2012. Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nat Commun. 3(1): https://doi.org/10.1038/ncomms2231

2. Nakagawa M, Taniguchi Y, Senda S, Takizawa N, Ichisaka T, Asano K, Morizane A, Doi D, Takahashi J, Nishizawa M, et al. 2015. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep. 4(1): https://doi.org/10.1038/srep03594

3. Miyazaki T, Isobe T, Nakatsuji N, Suemori H. 2017. Efficient Adhesion Culture of Human Pluripotent Stem Cells Using Laminin Fragments in an Uncoated Manner. Sci Rep. 7(1): https://doi.org/10.1038/srep41165

4. 2019. CiRA: Center for iPS Cell Research and Application. [Internet]. Kyoto: Kyoto University: Available from: https://www.cira.kyoto-u.ac.jp/e/research/protocol.