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Epithelial Cells

Decrease of laminin‑511 in the basement membrane due to photoaging reduces epidermal stem/progenitor cells

Iriyama S., Yasuda M., Nishikawa S., Takai E., Hosoi J., Amano S.
Scientific reports, 2020

Here, the authors examine how photoaging affects the function of Inter-follicular epidermal stem cells (IFE-SCs) which regulate epidermal proliferation and differentiation. It is known that daily sunlight disrupts epidermal homeostasis and the authors found that sun-exposed skin showed a decrease of MCSP-positive and β1-integrin-positive cells concomitantly with a decrease of laminin-511 at the dermal-epidermal junction (DEJ), as compared with sun-protected skin. Higher levels of laminin-511 were associated with increased colony formation efficiency, higher expression levels of MCSP as well as other stem cell markers in human skin. UVB exposure to cultured human skin impaired laminin-511 integrity at the dermal-epidermal junction and reduced MCSP-positive basal epidermal cells as well as K15-positive cells. Combined treatment with matrix metalloproteinase and heparanase inhibitors protected the integrity of laminin-511 and inhibited the reduction of MCSP-positive cells and K15-positive cells. These results suggest that photoaging may reduce the levels of MCSP-positive and K15-positive epidermal stem/ progenitor cells in the epidermis via loss of laminin-511 at the dermal-epidermal junction.


Chemically defined and xenogeneic-free culture method for human epidermal keratinocytes on laminin-based matrices

Tjin M.S., Chua A.W.C, Tryggvason K. 
Nature Protocols, 2020

In this protocol, the authors describe how to implement a defined, xeno-free culture system that supports long-term ex vivo expansion of functional human epidermal keratinocytes. Laminins, skin-specific basement membrane proteins play important roles in the maintenance of phenotypic integrity and in supporting the survival of keratinocytes that are adhered to them. The fully human keratinocyte culture system is presented in this article is ‘regulatory friendly’ and increases the potential of epithelial cellular therapy, which can be expanded to treat less severe burns and other skin defects, such as chronic diabetic wounds. It takes between 7 and 14 d to obtain an initial culture and a secondary culture from the primary culture can be expanded up to 20-fold within 4–5 d once cells reach confluency.


Biologically relevant laminin as a chemically defined and fully human platform for human epidermal keratinocyte culture

Tjin M.S., Chua A.W.C, Moreno-Moral A., Chong L.Y. , Tang P.Y., Harmston N.P., Cai Z., Petretto E., Tan B.K., Tryggvason K. 
Nature Communications, 2018

The current expansion of autologous human keratinocytes to resurface severe wound defects still relies on murine feeder layer and calf serum in the cell culture system. Here, the authors report a completely xeno-free and defined culture system utilizing skin-specific laminin cell culture substrates which enables robust expansion of adult human skin keratinocytes. The authors characterized the human skin basement membrane and murine feeder layer 3T3- J2 and have identified two biologically relevant recombinant laminins, laminin 511 and 421, as potential candidates to replace the murine feeder. The authors report a completely xeno-free and defined culture system utilizing these laminins which enables robust expansion of adult human skin keratinocytes, comparable to the 3T3-J2 co-culture system in terms of basal markers’ profile, colony-forming efficiency and the ability to form a normal stratified epidermal structure in both in vitro and in vivo models. Human keratinocytes cultured either on laminin 511 or 421 were able to generate a fully stratified epidermal layer in vivo similar to that on the 3T3 co-culture system. The results show that the proposed system may not only provide safer keratinocyte use in the clinics but also facilitate the broader use of other cultured human epithelial cells in regenerative medicine.


Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

Ishihara J., Ishihara A., Fukunaga K., Sasaki K., White M.J.V., Briquez P.S., Hubbell J.A.
Nature Communications, 2018

In this article, the authors show that multiple laminin isoforms promiscuously bind to growth factors with high affinity, through their heparin-binding domains located in the α chain laminin-type G (LG) domains. These domains also bind to syndecan cell-surface receptors, promoting attachment of fibroblasts and endothelial cells. The authors explore the application of these multifunctional laminin HBDs in wound healing in the type-2 diabetic mouse and demonstrate that covalent incorporation of laminin HBDs into fibrin matrices improve retention of GFs and significantly enhances the efficacy of vascular endothelial cell growth factor (VEGF-A165) and platelet-derived growth factor (PDGF-BB) in promoting wound healing in vivo, under conditions where the GFs alone in fibrin are inefficacious.


Polymerized Laminin-332 Matrix Supports Rapid and Tight Adhesion of Keratinocytes, Suppressing Cell Migration

Kariya Y., Sato H., Katou N., Kariya Y., Miyazaki K.
PLOS ONE, 2012

Laminin-332 is known to supports the stable anchoring of basal keratinocytes to the epidermal basement membrane but is also a motility factor for wound healing and cancer invasion. Here they investigated Laminin-332 matrices deposited by normal human keratinocytes and many cancer cell lines. All types of cells efficiently deposited Laminin-332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ß1 and/or c1 chains (such as Lm511 and Lm311) were not deposited on the culture plates even if secreted into the culture medium. The deposited Laminin-332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. Laminin-332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332 (not a BioLamina product), which highly promoted cell migration. The Lm332 matrix support adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin a3ß1 bound to the Lm332 matrix at a three times higher level than purified Lm332. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion whereas unassembled soluble Lm332 supports cell migration. The question is though how the purified Ln-332 looked like. Difficult to purify and might be fractionated.


Development of a biomaterial associated with mesenchymal stem cells and keratinocytes for use as a skin substitute

Steffens D., Mathor M.B., Santi B.T.S., Luco D.P., Pranke P.
Regen. Med., 2015

Here they developed s scaffolds of poly-DL-lactic acid with and without the linkage of laminin-332, bringing together MSCs and keratinocytes aimed for treatment as a new skin substitute. Three groups of scaffolds were studied: 1) poly-DL-lactic acid (PDLLA), 2) hydrolyzed PDLLA (PDLLA/NaOH) and 3) PDLLA/Lam which is a PDLLA/NaOH scaffold linked to laminin-332. The results corroborate the hypothesis that laminin influenced the adhesion of the MSCs. Laminin significantly promoted the adhesion and spreading of proliferating oral and epidermal keratinocytes compared with collagen nanofibers only. The use of biocompatible and biodegradable polymers associated with the properties of laminin leads to an improvement in the adherence and viability of the cells, showing LN-332 is beneficial for the growth of MSCs and keratinocytes.


Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

Pouliot N., Saunders N.A., Kaur P.
Exp Dermatol., 2002

The authors investigated the expression and function of laminin-511 and -521 in neonatal and adult human skin. They found that the laminin-a5 chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well-studied laminin-5 isoform did not change significantly with age, laminin-511 and -521 appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-511 and -521 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin.


FK506 positively regulates the migratory potential of melanocyte-derived cells by enhancing syndecan-2 expression

Hyejung Jung and Eok-Soo Oh
Pigment Cell & Melanoma Research, 2016

Here they investigate the mechanism through which FK506 regulates the migration of melanocytes. B16F10 melanoma cells were cultured on laminin-332, an optimal substrate for regulating the adhesion and migration of melanocytes and melanoma cells. FK506 treatment enhanced cell spreading on laminin-332 and increased migration in both melanocytes and melanoma cells.

Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment. Champliaud et al. J Cell Biol., 1996

PLCgamma1 is essential for early events in integrin signaling required for cell motility. Jones et al. J Cell Sci., 2005

Molecular organization of the cutaneous basement membrane zone. Ghohestani et al. Clin Dermatol., 2001

Current insights into the formation and breakdown of hemidesmosomes. Litjens et al. Trends Cell Biol., 2006

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1, and alpha6beta4 integrins. Nishiuchi et al. Matrix Biol., 2006 

Laminin 5 binds the NC-1 domain of type VII collagen. Rousselle et al. J Cell Biol., 1997

Mutation analysis and molecular genetics of epidermolysis bullosa. Pulkkinen & Uitto. Matrix Biol., 1999

Cloning of the laminin alpha 3 chain gene (LAMA3) and identification of a homozygous deletion in a patient with Herlitz junctional epidermolysis bullosa. Vidal et al. Genomics, 1995

Keratinocyte-derived laminin-332 promotes adhesion and migration in melanocytes and melanoma. Chung et al. J Biol Chem., 2011

FK506 positively regulates the migratory potential of melanocyte-derived cells by enhancing syndecan-2 expression. Jung & Oh, Pigment Cell & Melanoma Research, 2016

An unusual N-terminal deletion of the laminin alpha3a isoform leads to the chronic granulation tissue disorderlaryngo-onycho-cutaneous syndrome. McLean et al. Hum Mol Genet., 2003 

Laminin 5 deposition regulates keratinocyte polarization and persistent migration. Frank & Carter. J Cell Sci., 2004


Functional diversity of laminins. Domogatskaya et al. Annu Rev Cell Dev Biol., 2012

Laminin-10 is crucial for hair morphogenesis

Li J., Tzu J., Chen Y., Zhang Y.P., Nguyen N.T., Gao J., Bradley M., Keene D.R., Oro A.E., Miner J.H., Marinkovich M.P.
EMBO J., 2003

Here, the authors describe the essential role of laminin-511 in hair follicle development. Treatment of human scalp xenografts with antibodies to laminin-10, or its receptor β1 integrin, produced alopecia. Skin from lama5-null mice fails to support hair development. Absence of laminin-511 in transgenic mice led to a number of developmental abnormalities, including arrest of hair development and deficient Shh expression in hair follicles. Transplantation of skin grafts from these Lama5-null mouse embryos onto healthy mice failed to support hair growth. Intriguingly, purified laminin-511 (isolated from A549 lung carcinoma cell conditioned medium) was able to restore hair development in the Lama5-null skin. The authors conclude that laminin-511 is required for hair follicle development and report the first use of exogenous protein to correct a cutaneous developmental defect.


Spatial and temporal control of laminin-332 (5) and -511 (10) expression during induction of anagen hair growth

Sugawara K., Tsuruta D., Kobayashi H., Ikeda K., Hopkinson S.B., Jones J.C., Ishii M.
J Histochem Cytochem., 2007

Here they characterized changes in laminin isoform expression during hair cycling. At the mRNA level, laminin-511 expression undergo a steady increase during anagen stages. In contrast, laminin-332 expression is initially upregulated in outer root sheath (ORS) keratinocytes at anagen II and then transiently downregulated. Immunohistochemistry demonstrated that laminin-332 and α6β4 integrin were well colocalized, but their signals were remarkably decreased in the lower half of follicles after anagen VI. In hair follicle culture, laminin-511/521-rich human placental laminin enhanced hair growth, whereas recombinant laminin-332 antagonized hair growth induced by laminin-511. Our results indicate a positive role for laminin-511 and a negative role for laminin-332 on hair growth.


Laminin-511 is an epithelial message promoting dermal papilla development and function during early hair morphogenesis

Gao J., DeRouen M.C., Chen C.H., Nguyen M., Nguyen N.T., Ido H., Harada K., Sekiguchi K., Morgan B.A., Miner J.H., Oro A.E., Marinkovich M.P.
Genes Dev., 2008.

Here the authors demonstrate the mechanism of how laminin-511 controls hair morphogenesis. Dermal papilla (DP) from laminin-511 mutants showed developmental defects by E16.5 in mice, including a failure to maintain expression of the key morphogen noggin. Laminin-511 mutant DP showed decreased length and structure of primary cilia in vitro and in vivo. Laminin-511, but not laminin-111, restored primary cilia formation in lama5−/− mesenchyme and triggered noggin expression in an Shh- and PDGF-dependent manner. Hair development required the B1 integrin binding but not the heparin binding domain of laminin-511. Inhibition of laminin-511 receptor B1 integrin disrupted DP primary cilia formation as well as hair development. These studies show that epithelial-derived laminin-511 is a critical early signal that directs ciliary function and DP maintenance as a requirement for hair follicle downgrowth.


Laminin-511 and integrin beta-1 in hair follicle development and basal cell carcinoma formation

DeRouen M.C., Zhen H., Tan S.H., Williams S., Marinkovich M.P., Oro A.E.
BMC Dev Biol., 2010

With the use of a basal cell carcinoma (BCC) model system and mouse mutants the authors re-evaluate the role of laminin-511 in epithelial invagination in the skin. Blocking laminin 511 and 332 in BCCs maintains primary cilia and Shh signalling, but prevents invagination. Similarly, in laminin-511 and dermal beta-1 integrin mutants, dermal papilla development and primary cilia formation are normal. Dermal beta-1 integrin mutants have normal hair follicle development. They conclude that laminin-511 has a primary role of promoting hair follicle epithelial downgrowth without affecting dermal primary cilia and Shh target gene induction.


FOXC1 maintains the hair follicle stem cell niche and governs stem cell quiescence to preserve long-term tissue-regenerating potential

Lay K., Kumeb T., Fuchsa E.
PNAS, 2016

Here, the authors suggest that hair follicle stem cells (HFSCs) have restricted potential in vivo, which they conserve by coupling quiescence to adhesion-mediated niche maintenance, thereby achieving long-term tissue homeostasis. They examine whether parsimonious stem cells use is essential to conserve long-term tissue-regenerating potential during normal homeostasis by conditionally ablating a key transcription factor Forkhead box C1 (FOXC1).  FOXC1-deficient HFSCs spend less time in quiescence, leading to markedly shortened resting periods between hair cycles. The enhanced hair cycling accelerates HFSC expenditure, and impacts hair regeneration in aging mice. Interestingly, although FOXC1-deficient HFs can still form a new bulge that houses HFSCs for the next hair cycle, the older bulge is left unanchored. Hair follicle stem cells lacking the protein FOXC1 can only retain one old bulge in their hair follicles, while normal stem cells can keep up to four. In vitro cell adhesion assay they show that FACS-purified WT and Foxc1-KO HFSCs attach very well to LN-511 with about 6 times higher well area coverage compared to collagen I, fibronectin or Matrigel-coated plates.


Laminin 10/11- an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

Pouliot N., Saunders N.A., Kaur P.
Experimental Dermatology, 2002

They investigated the expression and function of the laminin-511 and -521 in neonatal and adult human skin. These isoforms are expressed abundantly in the basement membrane underlying the inter-follicular epidermis and the blood vessels in the dermis. The expression levels did not change significantly with age, but appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-511 and -521 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. An in vitro cell adhesion assays demonstrated that laminin-511 and -521 is potent adhesive substrates for both neonatal and adult keratinocytes and that this adhesion is mediated by the a3b1 and a6b4 integrins. Further, laminin-511 and -521 provide a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type-18 transformed tumorigenic keratinocyte cell line in vitro. Laminin-511 and -521 also stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin-10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.


Integrin-linked kinase regulates the niche of quiescent epidermal stem cells

Morgner J., Ghatak S., Jakobi T., Dieterich C., Aumailley M., Wickström S.A.
Nat Commun., 2015

In the present study the authors conclude that the precise ratio between LN-332 and LN-511 adjusts activities of key signalling pathways that determine SC activation within the hair follicle bulge niche. They suggest that integrin-linked kinase (ILK) is required for the maintenance of quiescent bulge SCs through remodelling of the ECM niche, thereby governing the activation and maintenance of hair follicle stem cells (HFSCs). ILK mediates deposition of inverse laminin-332 and -511 gradients within the basement membrane (BM) wrapping the hair follicles. The precise ratio of LN-511 and LN-332 regulates core SC fate-determining signalling pathways and that this ratio is disturbed in the absence of ILK, leading to aberrant SC activation and failure to re-establish quiescence. The BM composition tunes activities of Wnt and transforming growth factor-β pathways and subsequently regulates HFSC activation. LN-511, present at low levels around the bulge and at higher levels around the hair germ/TACs, promotes Tgf-β signalling, whereas LN-332, highly expressed along the IFE and to a lesser extent along the upper regions of HFs, suppresses Wnt/β-catenin signalling. Notably, reconstituting an optimal laminin microenvironment restores the altered signalling in ILK-deficient cells.


Functional diversity of laminins. Domogatskaya et al. Annu Rev Cell Dev Biol., 2012

Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment. Champliaud et al. J Cell Biol., 1996

PLCgamma1 is essential for early events in integrin signalling required for cell motility. Jones et al. J Cell Sci., 2005

Abnormal Wnt and PI3Kinase Signaling in the Malformed Intestine of lama5 Deficient Mice

Ritié L., Spenlé C., Lacroute J.I., Bolcato-Bellemin A-L., Lefebvre O., Bole-Feysot C., Jost B., Klein A., Arnold C., Kedinger M., Bagnard D., Orend G., Simon-Assmann P.
PLOS ONE, 2012

Laminin-511 is highly expressed in the intestine. To understand the mechanistic role of laminin-511 in tissue homeostasis, the researchers used RNA profiling of embryonic intestinal tissue of lama5 knockout mice and identified a lama5 specific gene expression signature. They show that laminin a5 plays a crucial role in both epithelial and mesenchymal (smooth muscle) cell behavior by inhibiting Wnt and activating PI3K signaling. We conclude that conflicting signals are elicited in the absence of lama5, which alter cell adhesion, migration as well as epithelial and muscle differentiation. The LMa5 deficient intestine also displays a smooth muscle defect and myogenic differentiation markers are affected. Laminin-511 supports adhesion of epithelial cells and Akt phosphorylation. Laminin-511 stimulates the spreading of epithelial and muscle cells (compared to laminin-111). Inhibition of Akt with wortmannin abolished spreading of epithelial cells on laminin-511 as evidenced by cell laminin-511 specifically activates Akt through the PI3K pathway in intestinal epithelial but not in mesenchymal cells. Cell migration was also higher on Laminin-511. Laminin-511 also protects cells against H2O2-induced apoptosis.


Laminins in the Developing and Adult Human Small Intestine: Relation With the Functional Absorptive Unit

Teller I.C., Auclair J., Herring E., Gauthier R., Ménard D., Beaulieu J-F.
Developmental dynamics, 2007

Here, the expression of the five laminin a-chains was analyzed in the developing and mature human small intestine at the protein and transcript levels in order to further delineate specific involvement of individual laminins in relation to the epithelial cell state as defined along the functional crypt-villus axis. The results show that all of the a-laminins are expressed in significant amounts in the small intestine relative to a panel of other tissues and organs. Distinct epithelial and mesenchymal origins, as well as a differential occurrence in intestinal basement membranes according to developmental stage, along the crypt-villus axis and in compartment-related experimental intestinal cell models. Taken together, the data point out the prime importance of a2-, a3-, and a5-containing laminins for the development and maintenance of the functional human intestinal epithelium.


Laminin α5 influences the architecture of the mouse small intestinal mucosa

Mahoney Z.X., Stappenbeck T.S., Miner J.H
J Cell Sci. 2008

The villus basement membrane is rich in laminin α5. Here the authors show that diminution of laminin α5 in a mouse model led to a compensatory deposition of colonic laminins that resulted in a transformation from a small intestinal to a colonic mucosal architecture. The alteration in mucosal architecture was associated with reduced levels of nuclear p27Kip1, a cell cycle regulator, and altered intestinal epithelial cell proliferation, migration, and differentiation. The results suggest that laminin α5 plays a crucial role in establishing and maintaining the specific mucosal pattern of the mouse small intestine.


Laminin a5 chain is required for intestinal smooth muscle development

Bolcato-Bellemin A-L., Lefebvre O., Arnold C., Sorokin L., Miner J. H., Kedinger M., Simon-Assmann P.
Developmental Biology, 2003

Here, the function of the laminin a5 chain in the developing intestine was defined by analyzing laminin a5 -/- mutants and by grafting experiments. The authors show that laminin a5 plays a major role in smooth muscle organization and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin a5 -/- mice. Loss of a5 expression was paralleled by ectopic or accelerated deposition of laminin a2 and a4 chains; this may explain why no obvious defects were observed in the villous form and enterocytic differentiation. Lack of the laminin a5 chain was accompanied by a decrease in epithelial a3B1 integrin receptor expression adjacent to the epithelial basement membrane and of Lutheran blood group glycoprotein in the smooth muscle cells, indicating that these receptors are likely mediating the a5 interactions. Taken together, the laminin a5 chain is essential for the normal development of the intestinal smooth muscle.


Developmental Expression and Cellular Origin of the Laminin a2, a4, and a5 Chains in the Intestine

Lefebvre O., Sorokin L., Kedinger M., Simon-Assmann P.
Developmental Biology, 1999

In this study, the authors examine the expression patterns and the cellular origins of the laminin a2, a4, and a5 chains in the developing mouse intestine and in in vitro mouse/chick or chick/mouse interspecies hybrid intestines. All three laminin chains are highest in the fetal intestine undergoing intense morphogenetic movements. Laminin a4 is associated with mesenchyme-derived cell populations such as endothelium and smooth muscle. In contrast, laminin a2 and a5 chains participate in the structural organization of the subepithelial basement membrane and, in the mature intestine, show a complementary pattern of expression. All three laminin chains occur in the smooth muscle basement membrane. Laminin a2 was found to be deposited into the basement membrane exclusively by mesenchymal cells, and the laminin a5 chain was deposited by both epithelial and mesenchymal cells in an apparently developmentally regulated pattern.