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Bone and Cartilage Cells

The role of laminins in cartilaginous tissues: from development to regeneration

Sun Y., Wang T.L., Toh W.S., Pei M.
European Cells and Materials, 2017

In this review, the authors facilitate an understanding of the spatial and temporal interactions between cartilage-forming cells and laminin microenvironment to eventually advance cell-based cartilage engineering and regeneration. The expression of laminins in various developmental stages of cartilage and cartilage-like tissues, including developing, adult and pathological cartilage is being discussed. They delineated the expression of laminins in hyaline cartilage, fibrocartilage and cartilage-like tissue (nucleus pulposus) throughout several developmental stages. They also examined the effect of laminins on the biological activities of chondrocytes, including adhesion, migration and survival. Furthermore, we scrutinized the potential influence of various laminin isoforms on cartilage-forming cells’ proliferation and chondrogenic differentiation.

 

Osteoblast-derived Laminin-332 is a novel negative regulator of osteoclastogenesis in bone microenvironments

Uehara N., Kukita A., Kyumoto-Nakamura Y., Yamaza T., Yasuda H., Kukita T.
Laboratory Investigation, 2017

In this article, the authors show that laminin 332 is expressed in primary osteoblasts, and is implicated in the regulation of osteoclast differentiation. Immunofluorescence analysis and RT-PCR analysis indicated specific expression of laminin 332 in osteoblast-like cells localized on bone surface. Laminin 332 markedly inhibited osteoclastogenesis induced by receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) when bone marrow-derived macrophages (BMMs) were cultured on laminin 332-coated plates. Laminin 332 also blocked RANKL-induced activation of mitogen-activated protein kinases (MAPKs) (ERK, JNK, and p38) and expression of NFATc1, c-Fos, and c-Jun. Laminin 332 suppressed osteoclast differentiation while retaining macrophage phenotypes, including nonspecific esterase activity and gene expression of lysozyme and EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1). Furthermore, the treatment of primary osteoblasts with osteoclastogenic factors dramatically suppressed expression of laminin 332. These findings suggest that laminin 332 produced by osteoblasts in bone tissues has a pivotal role in controlling normal bone remodeling through suppressing osteoclastogenesis. 

 

Laminin-521 Promotes Rat Bone Marrow Mesenchymal Stem Cell Sheet Formation on Light-induced Cell Sheet Technology

Jiang Z., Xi Y., Lai K., Wang Y., Wang H., Yang G.
BioMed Research International, 2016

The cell sheet technology is an area of research that is of great interest for tissue engineering and regenerative medicine. Here, the authors investigated the effects of an ECM coating on rat bone marrow mesenchymal stem cells (rBMSC) cultured on light-induced TiO2 nanodot films. Cell sheets can be detached on a TiO2 nanodot-coated quartz substrate by using UV365 illumination. The effects of rat fibronectin, human recombinant laminin-521, -511, -421, and -111 on the formation of cell sheets were investigated and also compared to uncoated films. The result showed the highest success for laminin-521. rBMSCs rapidly attached and spread on films coated with laminin-521 (1.2 ug/ml) and formed intact cell sheets after 5 days of culture. Laminin-521 promote the formation of rBMSC sheets with good viability under hyperconfluent conditions (4 to 8 layers of cells). The cells also maintained multilineage potential, including osteogenic, adipogenic, and chondrogenic differentiation. The cell sheets formed had rich ECM (including collagen I) and cells were connected with each other in a dense network-like tissue. The cell sheets can survive along with the insertion of BMSC sheet−implant complexes and enhance osteogenesis and integration in vivo. Compared with mechanical scraping, this method provided a more robust strategy to fabricate BMSC sheet−implant complexes with enhanced osteogenesis.

 

Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells

Mittag F., Falkenberg E-M, Janczyk A., Götze M., Felka T., Aicher W.K. Kluba T.
Orthopedic Reviews, 2012

In this article, the authors show that laminin 332 and type I collagen promote attachment, and that laminin 332 promote osteogenic differentiation of MSC. Expansion of MSC in animal serum-free, GMP-conforming media yielded vital cells meeting all minimal criteria for MSC. Attachment assay revealed a favourable binding of MSC to laminin 332 and type I collagen. Compared to plastic, osteogenic differentiation was significantly increased by laminin 332 after 28 days of culture, with no significant differences in gene expression patterns observed. Their data also confirm that laminin 332 serve better in bone repair, as this material promotes both firm attachment and osteogenic differentiation of MSC.

 

Tissue distribution of the laminin β1 and β2 chain during embryonic and fetal human development

Roediger M., Miosge N., Gersdorff N.
J Mol Hist., 2010

Here, the authors investigated the tissue distribution of the laminin b1 and b2 chains on the protein level in various developing embryonic and fetal human organs between gestational weeks 8 and 12. The laminin b1 chain was ubiquitously expressed in the basement membrane zones of the brain, ganglia, blood vessels, liver, kidney, skin, pancreas, intestine, heart and skeletal system. Furthermore, the laminin b2 chain was present in the basement membrane zones of the brain, ganglia, skin, heart and skeletal system. The findings of this study support and expand upon the theory that these two laminin chains are important during human development. In cartilage, the laminin b1 chain was expressed from gw 10 onwards but not during gw 8 and 9, whereas the detection of the laminin b2 chain was limited to gw 8 and 9. This indicates a developmental switch in the laminin b chain and suggests that the laminin b1 chain does not play a role in human cartilage development until the fetal stage. In human fetal cartilage (gw 17 and 24), a strong pericellular immunohistochemical reaction for laminin 111 was shown. In embryo chick sternum and mouse limb bud, laminin b1 and b2 chains are present in the cytoplasm of chondrocytes.

 

Regulation of Proliferation and Chondrogenic Differentiation of Human Mesenchymal Stem Cells by Laminin-5 (Laminin-332)

Hashimoto J., Hashimoto U., Kariya Y., Miyazaki K.
STEM CELLS, 2006

In this study, the authors examined a possible role of laminin 332 in the proliferation and differentiation of human MSCs. When MSCs were incubated in the presence of a coated or soluble form of laminin 332 in a growth medium, they proliferated more rapidly than nontreated cells, keeping their differentiation potential. On the other hand, laminin 332 potently suppressed the chondrogenic differentiation of MSCs. These activities were mediated mainly by integrin a3B1. However, laminin-5 had no effect on the osteogenic differentiation of MSCs. These results suggest that laminin-5 may contribute to the development of bone tissues by promoting the proliferation and by suppressing the chondrogenic differentiation of MSCs.

 

Laminin-5 suppresses chondrogenic differentiation of murine teratocarcinoma cell line ATDC5

Hashimoto J., Ogawaa T., Tsubota Y., Miyazaki K.
Experimental Cell Research, 2005

Here, the authors examined the possible role of laminin 332 in chondrogenesis. The murine teratocarcinoma cell line ATDC5 transiently and weakly expressing laminin 332 were stimulated for differentiation. Exogenous laminin 332 in either insoluble or soluble form strongly inhibited the differentiation phenotypes, i.e. formation of cartilaginous cell aggregates and production of chondrogenic marker proteins through its integrin-binding domain LG3 in the a3 chain. Laminin 332 had no effect on cell growth. In addition, they found that the laminin 332 with the 105-kDa, processed g2 chain suppressed differentiation more strongly than one with the 150-kDa g2 chain. This indicated that the proteolytic processing of g2 chain regulated the activity of laminin 332. However, a g2 chain short arm fragment had no effect on the chondrogenesis, and it rather suppressed the differentiation at excessive concentrations. These results suggest that laminin 332 and its processing modulate chondrogenic differentiation during development.

 

Characterization and functional analysis of laminin isoforms in human bone marrow

Siler U., Seiffert M., Puch S., Richards A., Torok-Storb B., Müller C.A, Sorokin L., Klein G.
Blood, 2000

Based on gene expression, laminin-411/421 and laminin-511/521 are the most abundant laminin isoforms synthesized by human bone marrow stromal cells. Laminin-511/521 preparations showed strong adhesive interactions with human CD34+ cell lines. Antibodies against the B1 integrin subunit inhibited these interactions. In addition to its adhesion-mediating properties, laminin-511/521 preparations also showed a mitogenic activity for human hematopoietic progenitor cells. Other laminin isoforms tested, especially laminin-111 and laminin-211 and 221, showed only weak or no adhesive interactions with the hematopoietic cell lines tested and are suggested to play a minor role in the hematopoietic microenvironment. In the bone marrow, LN‐511 and 521 are the major laminins and show strongly adhesive and mitogenic activities toward early developing HSCs.

 

Bone Marrow Mesenchymal Stem Cells Adhesion Assay

Yang Z. and Xiao R.
Bio-protocol, 2016

Here, the authors present a protocol for culture of bone marrow MSC (BM-MSCs) on laminin-521 or laminin-511. The protocol is based on the method by Siler et al., 2000, and can easily be translated to MSCs from other origin. Both laminin isoforms show a significantly better efficient attachment compared to uncoated wells and support seeding of a lower cell number compared to uncoated plats. The BM-MSCs adhere to the laminin-coated wells within 10 min, while for non-coated wells, it may take longer time. In this protocol, a final coating concentration of 2 μg/cm2 is used but can effectively be lowered 4-10 times without loss of function.

 

Immunohistochemical Distribution of Laminin-5 γ 2 Chain and its Developmental Change in Human Embryonic and Foetal Tissues

Lu W., Miyazaki K., Mizushima H., Nemoto N.
The Histochemical Journal, 2001

Here, immunohistochemical distribution of laminin γ2 chain, a subunit of the basement membrane protein laminin 332, was examined in 19 cases of human embryos and foetuses ranging from 4 to 25 weeks of gestation. Laminin γ2 was first detected in the basement membranes underlying ectodermal epithelial tissues, such as the skin and tooth, as early as 5–6 weeks of gestation. Between 6–7 and 12–13 weeks, laminin γ2 was detected in the basement membranes of various endodermal epithelial tissues, such as the bronchus, oesophagus, stomach, intestines, urinary bladder, gallbladder and hepatopancreatic duct. The deposition of laminin γ2 in basement membrane was associated with the process of morphogenesis. In the small intestine, laminin γ2 first appeared in the basement membrane of the primitive short villi, and its level gradually increased in the villus region but decreased in the cryptic region during the maturation of the organ. In addition, non-basement membrane immunoreactivity for laminin γ2 was detected in some mesoderm-derived tissues, such as the cartilage and skeletal and smooth muscle fibres. These results suggest a common role of laminin 332 and some specific roles of its γ2 chain in the morphogenesis of human tissues.

The role of laminins in cartilaginous tissues: from development to regeneration

Sun Y., Wang T.L., Toh W.S., Pei M.
European Cells and Materials, 2017

In this review, the authors facilitate an understanding of the spatial and temporal interactions between cartilage-forming cells and laminin microenvironment to eventually advance cell-based cartilage engineering and regeneration. The expression of laminins in various developmental stages of cartilage and cartilage-like tissues, including developing, adult and pathological cartilage is being discussed. They delineated the expression of laminins in hyaline cartilage, fibrocartilage and cartilage-like tissue (nucleus pulposus) throughout several developmental stages. They also examined the effect of laminins on the biological activities of chondrocytes, including adhesion, migration and survival. Furthermore, we scrutinized the potential influence of various laminin isoforms on cartilage-forming cells’ proliferation and chondrogenic differentiation.

 

Collagen Type IV and Laminin Expressions during Cartilage Repair and in Late Clinically Failed Repair Tissues from Human Subjects

Foldager C.B., Toh W.S., Christensen B.B., Lind M., Gomoll A.H., Spector M.
Cartilage, 2016

Here, the authors evaluate the expressions of Col4 and laminin in the pericellular matrix (PCM) in damaged cartilage and during cartilage repair. Laminin isoform111 has been identified in adult articular cartilage and laminin 332 in embryonic cartilage. While the interterritorial ECM is composed mainly of collagen type II and proteoglycans, the PCM consists mainly of collagen type IV and laminin. Collagen type IV and laminin were not found in the interterritorial ECM of normal cartilage and traumatically damaged cartilage. Depleted laminin expression levels in osteoarthritic articular cartilage.

 

Basement membrane components are key players in specialized extracellular matrices

Kruegel J. and Miosge N.
Cell. Mol. Life Sci., 2010

Basement membranes (BMs) are specialized extracellular matrices (sECMs) with unique components that support important functions including differentiation, proliferation, migration, and chemotaxis of cells during development. The composition of these sECM is as unique as the tissues to which they are localized, and changes in BM composition play significant roles in facilitating the development of various diseases. Furthermore, tissues have to provide sECM for their stem cells during development and for their adult life. Here, the authors briefly review the latest research on these unique sECM and their components with a special emphasis on embryonic and adult stem cells and their niches.

 

Laminins and Nidogens in the Pericellular Matrix of Chondrocytes - Their Role in Osteoarthritis and Chondrogenic Differentiation

Schminke B., Frese J., Bode C., Goldring M.B., Miosge N.
American Society for Investigative Pathology, 2015

This is an investigation of cartilage tissue and isolated chondrocytes in three-dimensional culture obtained from patients with late-stage knee OA and nidogen-2 knockout mice. Chondrogenic progenitor cells (CPCs) produced high levels of laminin-a1, laminin-a5, and nidogen-2 in their pericellular matrix, and laminin-a1 enhanced collagen type II and reduced collagen type I expression by cultured CPCs. Nidogen-2 increased SOX9 gene expression. This study reveals that the influence of the pericellular matrix on CPCs is important for the expression of the major regulator transcription factors, SOX9 and RUNX2. Our novel findings that laminins and nidogen-2 drive CPCs toward chondrogenesis may help in the elucidation of new treatment strategies for cartilage tissue regeneration. In summary, the expression of nidogen-2 and laminin is increased in human OA cartilage, and they act as chondrogenic regulators, especially for CPCs. Laminin also promotes chondrogenesis, enhancing collagen type II, COMP, and aggrecan expression, and down-regulating collagen type I. Therefore, these findings on laminin and nidogen-2 may aid in the elucidation of new treatment options, especially for tissue regeneration.

 

Tissue distribution of the laminin β1 and β2 chain during embryonic and fetal human development

Roediger M., Miosge N., Gersdorff N.
J Mol Hist, 2010

Here, the authors investigated the tissue distribution of the laminin b1 and b2 chains on the protein level in various developing embryonic and fetal human organs between gestational weeks 8 and 12. The laminin b1 chain was ubiquitously expressed in the basement membrane zones of the brain, ganglia, blood vessels, liver, kidney, skin, pancreas, intestine, heart and skeletal system. Furthermore, the laminin b2 chain was present in the basement membrane zones of the brain, ganglia, skin, heart and skeletal system. The findings of this study support and expand upon the theory that these two laminin chains are important during human development. The laminin b1 chain is present in developing bone.

 

Expression of laminin isoforms, receptors, and binding proteins unique to nucleus pulposus cells of immature intervertebral disc

Chen J., Jing L., Gilchrist C.L., Richardson W.J., Fitch R.D., Setton L.A.
Connect Tissue Res.,  2009

Intervertebral disc (IVD) disorders are believed to be related to aging-related cell loss and phenotypic changes, as well as biochemical and structural changes in the extracellular matrix of the nucleus pulposus (NP) region. In this study, The authors evaluate the zonal-specific expression of the laminin chains, receptors (i.e., integrins), and other binding proteins in immature tissue and isolated cells of rat, porcine and human intervertebral disc. Our goal was to reveal features of cellular environment and cell-matrix interactions in the immature NP. Laminin gamma1 chain is more highly expressed in immature NP porcine tissues, in parallel with the expression pattern for a laminin receptor, integrin alpha6 subunit, as compared to adjacent anulus fibrosus region. This result suggests that cell-matrix interactions may be unique to the immature NP. Results from both immunohistochemical staining and flow cytometry analysis found that NP cells expressed higher levels of the laminin alpha5 chain, laminin receptors (integrin alpha3, alpha6, beta4 subunit, and CD239), and related binding proteins (CD151), as compared to cells from adjacent anulus fibrosus. These differences suggest that laminin interactions with NP cells are distinct from that of the anulus fibrosus and that laminins may be important contributors to region-specific IVD biology.

 

Regulated expression of fibronectin, laminin and related integrin receptors during the early chondrocyte differentiation

Tavella S, Bellese G, Castagnola P, Martin I, Piccini D, Doliana R, Colombatti A, Cancedda R, Tacchetti C.
Journal of Cell Science, 1997

Here, the authors investigate the expression and localization of fibronectin, laminin, and their receptors, and we used an in vitro chick chondrocyte differentiation model to define a time hierarchy for their appearance in early chondrogenesis and to determine their role in the cell condensation process. They show that fibronectin contributes to the initial cell-cell interactions that occur during condensation. In later stages, a downregulation of both fibronectin and of its a5b1 integrin receptor occur, as demonstrated by mRNA and protein kinetics. Immunolocalisation studies suggest that the reduction of fibronectin in discrete areas is involved in local activation of the cell differentiation program. Furthermore, we show that laminin is expressed during the in vitro cell condensation process in areas that are negative for fibronectin staining. The types of laminin as well as the timing of expression have been determined by northern blot and RT-PCR analyses. The highest levels of expression are coincident with maximal cell aggregation. Our data show that the a1, b1, and g1 mRNAs are maximally expressed at the higher level of cell aggregation, and progressively decrease during the differentiation to stage I chondrocytes; stage II hypertrophic chondrocytes express only low levels of g1. We do not exclude the possibility that other forms of LN, for which chick specific probes are not yet available, may also be expressed. The increase in a1, b1, and g1, LN mRNAs is paralleled by a switch in the isotype of a6 integrin subunits synthesized, from B to A. Immunolocalisationstudies suggest that laminin is involved in the definition of differentiating areas as opposed to non-differentiating areas of the condensed region, i.e. the periphery, which eventually gives rise to the perichondrium.

 

Osteoblast-derived Laminin-332 is a novel negative regulator of osteoclastogenesis in bone microenvironments

Uehara N., Kukita A., Kyumoto-Nakamura Y., Yamaza T., Yasuda H., Kukita T.
Laboratory Investigation, 2017

In this article, the authors show that laminin 332 is expressed in primary osteoblasts, and is implicated in the regulation of osteoclast differentiation. Immunofluorescence analysis and RT-PCR analysis indicated specific expression of laminin 332 in osteoblast-like cells localized on bone surface. Laminin 332 markedly inhibited osteoclastogenesis induced by receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) when bone marrow-derived macrophages (BMMs) were cultured on laminin 332-coated plates. Laminin 332 also blocked RANKL-induced activation of mitogen-activated protein kinases (MAPKs) (ERK, JNK, and p38) and expression of NFATc1, c-Fos, and c-Jun. Laminin 332 suppressed osteoclast differentiation while retaining macrophage phenotypes, including nonspecific esterase activity and gene expression of lysozyme and EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1). Furthermore, the treatment of primary osteoblasts with osteoclastogenic factors dramatically suppressed expression of laminin 332. These findings suggest that laminin 332 produced by osteoblasts in bone tissues has a pivotal role in controlling normal bone remodeling through suppressing osteoclastogenesis. 

 

Laminin-521 Promotes Rat Bone Marrow Mesenchymal Stem Cell Sheet Formation on Light-induced Cell Sheet Technology

Jiang Z., Xi Y., Lai K., Wang Y., Wang H., Yang G.
BioMed Research International, 2016

The cell sheet technology is an area of research that is of great interest for tissue engineering and regenerative medicine. Here, the authors investigated the effects of an ECM coating on rat bone marrow mesenchymal stem cells (rBMSC) cultured on light-induced TiO2 nanodot films. Cell sheets can be detached on a TiO2 nanodot-coated quartz substrate by using UV365 illumination. The effects of rat fibronectin, human recombinant laminin-521, -511, -421, and -111 on the formation of cell sheets were investigated and also compared to uncoated films. The result showed the highest success for laminin-521. rBMSCs rapidly attached and spread on films coated with laminin-521 (1.2 ug/ml) and formed intact cell sheets after 5 days of culture. Laminin-521 promote the formation of rBMSC sheets with good viability under hyperconfluent conditions (4 to 8 layers of cells). The cells also maintained multilineage potential, including osteogenic, adipogenic, and chondrogenic differentiation. The cell sheets formed had rich ECM (including collagen I) and cells were connected with each other in a dense network-like tissue. The cell sheets can survive along with the insertion of BMSC sheet−implant complexes and enhance osteogenesis and integration in vivo. Compared with mechanical scraping, this method provided a more robust strategy to fabricate BMSC sheet−implant complexes with enhanced osteogenesis.

 

Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells

Mittag F., Falkenberg E-M, Janczyk A., Götze M., Felka T., Aicher W.K. Kluba T.
Orthopedic Reviews, 2012

In this article, the authors show that laminin 332 and type I collagen promote attachment, and that laminin 332 promote osteogenic differentiation of MSC. Expansion of MSC in animal serum-free, GMP-conforming media yielded vital cells meeting all minimal criteria for MSC. Attachment assay revealed a favourable binding of MSC to laminin 332 and type I collagen. Compared to plastic, osteogenic differentiation was significantly increased by laminin 332 after 28 days of culture, with no significant differences in gene expression patterns observed. Their data also confirm that laminin 332 serve better in bone repair, as this material promotes both firm attachment and osteogenic differentiation of MSC.

 

Tissue distribution of the laminin β1 and β2 chain during embryonic and fetal human development

Roediger M., Miosge N., Gersdorff N.
J Mol Hist., 2010

Here, the authors investigated the tissue distribution of the laminin b1 and b2 chains on the protein level in various developing embryonic and fetal human organs between gestational weeks 8 and 12. The laminin b1 chain was ubiquitously expressed in the basement membrane zones of the brain, ganglia, blood vessels, liver, kidney, skin, pancreas, intestine, heart and skeletal system. Furthermore, the laminin b2 chain was present in the basement membrane zones of the brain, ganglia, skin, heart and skeletal system. The findings of this study support and expand upon the theory that these two laminin chains are important during human development. In cartilage, the laminin b1 chain was expressed from gw 10 onwards but not during gw 8 and 9, whereas the detection of the laminin b2 chain was limited to gw 8 and 9. This indicates a developmental switch in the laminin b chain and suggests that the laminin b1 chain does not play a role in human cartilage development until the fetal stage. In human fetal cartilage (gw 17 and 24), a strong pericellular immunohistochemical reaction for laminin 111 was shown. In embryo chick sternum and mouse limb bud, laminin b1 and b2 chains are present in the cytoplasm of chondrocytes.

 

Regulation of Proliferation and Chondrogenic Differentiation of Human Mesenchymal Stem Cells by Laminin-5 (Laminin-332)

Hashimoto J., Hashimoto U., Kariya Y., Miyazaki K.
STEM CELLS, 2006

In this study, the authors examined a possible role of laminin 332 in the proliferation and differentiation of human MSCs. When MSCs were incubated in the presence of a coated or soluble form of laminin 332 in a growth medium, they proliferated more rapidly than nontreated cells, keeping their differentiation potential. On the other hand, laminin 332 potently suppressed the chondrogenic differentiation of MSCs. These activities were mediated mainly by integrin a3B1. However, laminin-5 had no effect on the osteogenic differentiation of MSCs. These results suggest that laminin-5 may contribute to the development of bone tissues by promoting the proliferation and by suppressing the chondrogenic differentiation of MSCs.

 

Laminin-5 suppresses chondrogenic differentiation of murine teratocarcinoma cell line ATDC5

Hashimoto J., Ogawaa T., Tsubota Y., Miyazaki K.
Experimental Cell Research, 2005

Here, the authors examined the possible role of laminin 332 in chondrogenesis. The murine teratocarcinoma cell line ATDC5 transiently and weakly expressing laminin 332 were stimulated for differentiation. Exogenous laminin 332 in either insoluble or soluble form strongly inhibited the differentiation phenotypes, i.e. formation of cartilaginous cell aggregates and production of chondrogenic marker proteins through its integrin-binding domain LG3 in the a3 chain. Laminin 332 had no effect on cell growth. In addition, they found that the laminin 332 with the 105-kDa, processed g2 chain suppressed differentiation more strongly than one with the 150-kDa g2 chain. This indicated that the proteolytic processing of g2 chain regulated the activity of laminin 332. However, a g2 chain short arm fragment had no effect on the chondrogenesis, and it rather suppressed the differentiation at excessive concentrations. These results suggest that laminin 332 and its processing modulate chondrogenic differentiation during development.

 

Characterization and functional analysis of laminin isoforms in human bone marrow

Siler U., Seiffert M., Puch S., Richards A., Torok-Storb B., Müller C.A, Sorokin L., Klein G.
Blood, 2000

Based on gene expression, laminin-411/421 and laminin-511/521 are the most abundant laminin isoforms synthesized by human bone marrow stromal cells. Laminin-511/521 preparations showed strong adhesive interactions with human CD34+ cell lines. Antibodies against the B1 integrin subunit inhibited these interactions. In addition to its adhesion-mediating properties, laminin-511/521 preparations also showed a mitogenic activity for human hematopoietic progenitor cells. Other laminin isoforms tested, especially laminin-111 and laminin-211 and 221, showed only weak or no adhesive interactions with the hematopoietic cell lines tested and are suggested to play a minor role in the hematopoietic microenvironment. In the bone marrow, LN‐511 and 521 are the major laminins and show strongly adhesive and mitogenic activities toward early developing HSCs.

 

Bone Marrow Mesenchymal Stem Cells Adhesion Assay

Yang Z. and Xiao R.
Bio-protocol, 2016

Here, the authors present a protocol for culture of bone marrow MSC (BM-MSCs) on laminin-521 or laminin-511. The protocol is based on the method by Siler et al., 2000, and can easily be translated to MSCs from other origin. Both laminin isoforms show a significantly better efficient attachment compared to uncoated wells and support seeding of a lower cell number compared to uncoated plats. The BM-MSCs adhere to the laminin-coated wells within 10 min, while for non-coated wells, it may take longer time. In this protocol, a final coating concentration of 2 μg/cm2 is used but can effectively be lowered 4-10 times without loss of function.

 

Immunohistochemical Distribution of Laminin-5 γ 2 Chain and its Developmental Change in Human Embryonic and Foetal Tissues

Lu W., Miyazaki K., Mizushima H., Nemoto N.
The Histochemical Journal, 2001

Here, immunohistochemical distribution of laminin γ2 chain, a subunit of the basement membrane protein laminin 332, was examined in 19 cases of human embryos and foetuses ranging from 4 to 25 weeks of gestation. Laminin γ2 was first detected in the basement membranes underlying ectodermal epithelial tissues, such as the skin and tooth, as early as 5–6 weeks of gestation. Between 6–7 and 12–13 weeks, laminin γ2 was detected in the basement membranes of various endodermal epithelial tissues, such as the bronchus, oesophagus, stomach, intestines, urinary bladder, gallbladder and hepatopancreatic duct. The deposition of laminin γ2 in basement membrane was associated with the process of morphogenesis. In the small intestine, laminin γ2 first appeared in the basement membrane of the primitive short villi, and its level gradually increased in the villus region but decreased in the cryptic region during the maturation of the organ. In addition, non-basement membrane immunoreactivity for laminin γ2 was detected in some mesoderm-derived tissues, such as the cartilage and skeletal and smooth muscle fibres. These results suggest a common role of laminin 332 and some specific roles of its γ2 chain in the morphogenesis of human tissues.

 

The major basement membrane components localize to the chondrocyte pericellular matrix--a cartilage basement membrane equivalent?

Kvist AJ, Nyström A, Hultenby K, Sasaki T, Talts JF, Aspberg A.
Matrix Biology, 2008

In this study, the authors studied basement membrane components in cartilage and conclude that articular cartilage chondrocytes are surrounded by the defining basement membrane proteins laminin, collagen type IV, nidogen and perlecan, deposited in a thin pericellular structure. This structure is presumably involved in maintaining chondrocyte phenotype and viability and may well allow a new understanding of cartilage development and provide clues to the progression of degenerative joint disorders. Imunoelectron microscopy confirmed high laminin concentration in the pericellular matrix. Interestingly, individual laminin subunits (α1, α2, α4, α5, β1 and γ1) were located in different zones of the cartilage. Of the laminin subunits surveyed, α1, α5, β1 and γ1 showed the highest staining intensity (mouse femoral head cartilage). Interestingly, there was distinct cell-to-cell variability in the levels of laminin surrounding the cells. In cartilage from newborn mice, basement membrane components are widespread in the territorial and interterritorial matrix, while in mature cartilage of adult mice the basement membrane components are localized mainly to a narrow pericellular zone. With progression into old age, this layer becomes less distinct, especially in areas of obvious mechanical attrition. Laminin α1 showing preferential localization around a select population of superficial layer chondrocytes. In general, a gradual shift from diffuse generalized staining of basement membrane components in the new born towards a pericellular localization in the mature cartilage was observed. In new born mouse knee cartilage, laminin 111 showed staining in the extracellular matrix, while laminin α5 (511 and 521) stained weakly at this point only to increase with age.