Anna Domogatskaya

Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511

We describe a system for culturing human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells on a recombinant form of human laminin-511, a component of the natural hES cell niche. The system is devoid of animal products and feeder cells and contains only one undefined component, human albumin. The hES cells self-renewed with normal karyotype for at least 4 months (20 passages), after which the cells could produce teratomas containing cell lineages of all three germ layers. When plated on laminin-511 in small clumps, hES cells spread out in a monolayer, maintaining cellular homogeneity with approximately 97% OCT4-positive cells. Adhesion of hES cells was dependent on α6β1 integrin. The use of homogeneous monolayer hES or iPS cell cultures provides more controllable conditions for the design of differentiation methods. This xeno-free and feeder-free system may be useful for the development of cell lineages for therapeutic purposes.

Functional Diversity of Laminins

Laminins are a large family of conserved, multidomain trimeric basement membrane proteins that contribute to the structure of extracellular matrix and influence the behavior of associated cells, such as adhesion, differentiation, migration, phenotype stability, and resistance to anoikis. In lower organisms such as Hydra there is only one isoform of laminin, but higher organisms have at least 16 trimeric isoforms with varying degrees of cell/tissue specificity. In vitro protein and cell culture studies, gene manipulation in animals, and laminin gene mutations in human diseases have provided insight into the specific functions of some laminins, but the biological roles of many isoforms are still largely unexplored, mainly owing to difficulties in isolating them in pure form from tissues or cells. In this review, we elucidate the evolution of laminins, describe their molecular complexity, and explore the current knowledge of their diversity and functional aspects, including laminin-mediated signaling via membrane receptors, in vitro cell biology, and involvement in various tissues gained from animal model and human disease studies. The potential use of laminins in cell biology research and biotechnology is discussed.

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

Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

Laminin‐511 but Not ‐332, ‐111, or ‐411 Enables Mouse Embryonic Stem Cell Self‐Renewal In Vitro

We tested specific laminin (LN) isoforms for their ability to serve as substrata for maintaining mouse embryonic stem (ES) cells pluripotent in vitro in the absence of leukemia inhibitory factor or any other differentiation inhibitors or feeder cells. Recombinant human LN‐511 alone was sufficient to enable self‐renewal of mouse ES cells for up to 169 days (31 passages). Cells cultured on LN‐511 maintained expression of pluripotency markers, such as Oct4, Sox2, Tert, UTF1, and Nanog, during the entire period, and cells cultured for 95 days (17 passages) were used to generate chimeric mice. LN‐332 enabled ES cells proliferation but not pluripotency. In contrast, under the same conditions LN‐111, Matrigel, and gelatin caused rapid differentiation, whereas LN‐411 and poly‐d‐lysine did not support survival. ES cells formed a thin monolayer on LN‐511 that differed strikingly from typical dense cluster ES cell morphology. However, expression of pluripotency markers was not affected by morphological changes. The effect was achieved at low ES cell density (<200 cell/mm2). The ability of LN‐511 and LN‐332 to support ES cell proliferation correlated with increased cell contact area with those adhesive substrata. ES cells interacted with LN‐511 via β1‐integrins, mostly α6β1 and αVβ1. This is the first demonstration that certain extracellular matrix molecules can support ES cell self‐renewal in the absence of differentiation inhibitors and at low cell density. The results suggest that recombinant laminin isoforms can provide a basis for defined surface coating systems for feeder‐free maintenance of undifferentiated mammalian ES cells in vitro.

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4‐mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin‐induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.

Impeded Interaction between Schwann Cells and Axons in the Absence of Laminin α4

The Schwann cell basal lamina (BL) is required for normal myelination. Loss or mutations of BL constituents, such as laminin-2 (α2β1γ1), lead to severe neuropathic diseases affecting peripheral nerves. The function of the second known laminin present in Schwann cell BL, laminin-8 (α4β1γ1), is so far unknown. Here we show that absence of the laminin α4 chain, which distinguishes laminin-8 from laminin-2, leads to a disturbance in radial sorting, impaired myelination, and signs of ataxia and proprioceptive disturbances, whereas the axonal regenerative capacity is not influenced. In vitro studies show poor axon growth of spinal motoneurons on laminin-8, whereas it is extensive on laminin-2. Schwann cells, however, extend longer processes on laminin-8 than on laminin-2, and, in contrast to the interaction with laminin-2, solely use the integrin receptor α6β1 in their interaction with laminin-8. Thus, laminin-2 and laminin-8 have different critical functions in peripheral nerves, mediated by different integrin receptors.

Integrin-laminin interactions controlling neurite outgrowth from adult DRG neurons in vitro

A prerequisite for axon regeneration is the interaction between the growth cone and the extracellular matrix (ECM). Laminins are prominent constituents of ECM throughout the body, known to support axon growth in vitro and in vivo. The regenerative capacity of adult neurons is greatly diminished compared to embryonic or early postnatal neurons. Since most lesions in the nervous system occur in the adult, we have examined neurite outgrowth from adult mouse DRG neurons on four laminin isoforms (laminin-1/LM-111, laminin-2/LM-211, laminin-8/LM-411 and laminin-10/LM-511) in vitro. The growth on laminin-1 and -10 was trophic factor-independent and superior to the one on laminin-2 and -8, where growth was very poor in the absence of neurotrophins. Among other ECM proteins, laminins were by far the most active molecules. Using function-blocking antibodies to laminin-binding integrins, we identified non-overlapping functions of integrins alpha3beta1, alpha7beta1 and alpha6beta1 on different laminin isoforms, in that alpha3beta1 and alpha7beta1 integrins appeared to be specific receptors for both laminin-1 and-2, whereas integrin alpha6beta1 was a receptor for laminin-8 and-10. Lastly, by use of immunohistochemistry, expression of subunits of laminin-1, -2, -8 and -10 in sensory organs in the human epidermis could be demonstrated, supporting an important role for these laminins in relation to primary sensory axons.

Laminin deposition is dispensable for vasculogenesis but regulates blood vessel diameter independent of flow

Basement membranes (BMs) consisting of laminins, collagens, and heparan sulfate proteogly cans (HSPGs) are vital for proper endothelial cell function, but many aspects of their role in vascular development remain unknown. Here, we demonstrate that vascular structures within differentiating embryoid bodies are wrapped in a BM composed of α4‐ and α5‐chain laminins, fibronectin, collagen IV, and HSPGs. In sprouting angiogenesis, laminins were pro duced by stalk cells, as well as the leading tip cell, and deposited along the sprout length, including tip cell filopodia. In embryonic stem cells deficient in laminins, due to lamc1 (laminin γ1) deletion, vascular develop ment and organization were largely unaffected. How ever, the frequency of vessels with wide lumens was increased 4‐fold. Laminin‐deficient vessels were more over characterized by increased fibronectin levels and enhanced endothelial cell proliferation. We conclude that laminins are dispensable for vascular development but that they regulate lumen formation in the absence of flow and vascular tone.—Jakobsson, L., Domogatskaya, A., Tryggvason, K., Edgar, D., Claesson‐Welsh, L. Laminin deposition is dispensable for vascu‐ logenesis but regulates blood vessel diameter independent of flow. FASEBJ. 22, 1530–1539 (2008)

The safety of human pluripotent stem cells in clinical treatment.

Abstract Human pluripotent stem cells (hPSCs) have practically unlimited proliferation potential and a capability to differentiate into any cell type in the human body. Since the first derivation in 1998, they have been an attractive source of cells for regenerative medicine. Numerous ethical, technological, and regulatory complications have been hampering hPSC use in clinical applications. Human embryonic stem cells (ESCs), parthenogenetic human ESCs, human nuclear transfer ESCs, and induced pluripotent stem cells are four types of hPSCs that are different in many clinically relevant features such as propensity to epigenetic abnormalities, generation methods, and ability for development of autologous cell lines. Propensity to genetic mutations and tumorigenicity are common features of all pluripotent cells that complicate hPSC-based therapies. Several recent advances in methods of derivation, culturing, and monitoring of hPSCs have addressed many ethical concerns and technological challenges in development of clinical-grade hPSC lines. Generation of banks of such lines may be useful to minimize immune rejection of hPSC-derived allografts. In this review, we discuss different sources of hPSCs available at the moment, various safety risks associated with them, and possible solutions for successful use of hPSCs in the clinic. We also discuss ongoing clinical trials of hPSC-based treatments.

Melanoma cells produce multiple laminin isoforms and strongly migrate on α5 laminin(s) via several integrin receptors.

Melanoma cells express and interact with laminins (LMs) and other basement membrane components during invasion and metastasis. In the present study we have investigated the production and migration-promoting activity of laminin isoforms in melanoma. Immunohistochemistry of melanoma specimens and immunoprecipitation/western blotting of melanoma cell lines indicated expression of laminin-111/121, laminin-211, laminin-411/421, and laminin-511/521. Laminin-332 was not detected. In functional assays, laminin-111, laminin-332, and laminin-511, but not laminin-211 and laminin-411, strongly promoted haptotactic cell migration either constitutively or following stimulation with insulin-like growth factors. Both placenta and recombinant laminin-511 preparations were highly active, and the isolated recombinant IVa domain of LMα5 also promoted cell migration. Function-blocking antibodies in cell migration assays revealed α6β1 integrin as the major receptor for laminin-111, and both α3β1 and α6β1 integrins for laminin-332 and laminin-511. In contrast, isolated LMα5 IVa domain-promoted melanoma cell migration was largely mediated via αVβ3 integrin and inhibited by RGD peptides. Given the ubiquitous expression of α5 laminins in melanoma cells and in melanoma-target tissues/anatomical structures, as well as the strong migration-promoting activity of these laminin isoforms, the α5 laminins emerge as putative primary extracellular matrix mediators of melanoma invasion and metastasis via α3β1 and other integrin receptors.