Long-Term Efficacy of GMP Grade Xeno-Free hESC-Derived RPE Cells Following Transplantation

McGill T.J., Bohana-Kashtan O., Stoddard J.W., Andrews M.D., Pandit N., Rosenberg-Belmaker L.R., Wiser O., Matzrafi L., Banin E., Reubinoff B., Netzer N., Irving C.
Trans Vis Sci Tech., 2017

Publication from researchers at Cell Cure Neurosciences where they display the efficacy of RPE cells derived under xeno-free conditions from clinical and xeno-free grade human embryonic stem cells following transplantation into the subretinal space of Royal College of Surgeons (RCS) rats. BioLamina’s laminin cell culture substrate is being used in the differentiation protocol. The results of this study demonstrate that transplantation of OpRegen into the subretinal space of RCS rats protected retinal structure, rescued visual function, preserved rod and cone photoreceptors long-term (up to 180 days). Transplanted RPE cells were identified in both the subretinal space and integrated into the host RPE monolayer in animals of all age groups, and often contained internalized photoreceptor outer segments. Optomotor tracking was rescued in a dose-dependent manner. Outer nuclear layer was significantly thicker in cell-treated eyes than controls up to P150. No pathology was observed. This data combined with data collected in a definitive safety studies (tumorigenicity and spiking and safety/biodistribution) has resulted in an FDA approved IND and a Phase 1/2a clinical trial for AMD patients is currently ongoing, NCT02286089.


Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model

Plaza Reyes A., Petrus-Reurer S., Antonsson L., Stenfelt S., Bartuma H., Panula S., Mader T., Douagi I., Andre H., Hovatta O., Lanner F., Kvanta A.
Stem Cell Reports, 2015

A publication by the groups of Drs. Hovatta, Lanner and Kvanta describe production of hESC-RPE cells in a xeno-free and defined manner. In the paper they describe an effective differentiation methodology using human recombinant laminin-521 matrix with a xeno-free and defined medium. The differentiated RPE cells exhibit native characteristics including morphology, pigmentation, marker expression, monolayer integrity, polarization and phagocytic activity. The authors also established a large-eyed geographic atrophy model that allowed in vivo imaging of the hESC-RPE and host retina. Cells were transplanted in suspension and showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity.


Retinal Pigment Epithelial Cells Synthesize Laminins, Including Laminin 5, and Adhere to Them through a3- and a6-Containing Integrins

Aisenbrey S., Zhang M., Bacher D., Yee J., Brunken W.J., Hunter D.D.
Invest Ophthalmol Vis Sci., 2006

The multilayered extracellular matrix underlying the retina is Bruch’s membrane (BM). Here they show that BM contains laminin chains that could form laminin-111, -332, -511, and -521. RPE cells synthesized these laminin chains in vitro, hence, RPE cells may synthesize BM laminins. The RPE cells adhered to the BM component collagen IV, but preferentially adhered to laminins. Of the laminins tested, the RPE cells adhered preferentially to laminin 332. The RPE cells interacted with these laminins via a3 and a6 containing integrins.


Laminin Expression in Adult and Developing Retinae: Evidence of Two Novel CNS Laminins

Libby R.T., Champliaud M-F, Claudepierre T., Xu Y., Gibbons E.P., Koch M., Burgeson R.E., Hunter D.D, Brunken W.J.
The Journal of Neuroscience, 2000

Here, they examine the expression of all known laminin chains within the retina. The interphotoreceptor matrix (and, during early development, the subretinal space) contains the laminin a3, a4, a5, b2, b3, g2, and g3 chains. This suggests the presence of three laminins: laminin-332, laminin-423 and laminin-523. These laminin isoforms could exert important effects on photoreceptor development and may play a role in photoreceptor production, stability and synaptic organization.


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

Stenzel D., Franco C.A., Estrach S., Mettouchi A., Sauvaget D., Rosewell I., Schertel A., Armer H., Domogatskaya A., Rodin S., Tryggvason K., Collinson L., Sorokin L., Gerhardt H.
EMBO reports, 2011

Here the authors show that laminin α4 regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. α4 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. The authors 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.


North Carolina Macular Dystrophy Is Caused by Dysregulation of the Retinal Transcription Factor PRDM13

Small K.W., DeLuca A.P, Whitmore S.S, Rosenberg T., Silva-Garcia R., Udar N., Puech b., Garcia C.A., Rice T.A., Fishman G.A, Héon E., Folk J.C, Streb L.M., Haas C.M., Wiley L.A., Scheetz T.E., Fingert J.H., Mullins R.F., Tucker B.A., Stone E.M.
American Academy of Ophtalmology, 2015

iPSCs were maintained in Essential 8 media on 521-To-Go plates and then differentiated by a 3D differentiation protocol to retinal tissues. Genome sequencing of patient DNA to identified rare mutations involved in macular dystrophy. Found 5 rare mutations involved in macular development. Strongest indication for the PRDM13 gene.


Molecular interactions in the retinal basement membrane system: A proteomic approach

Balasubramani M., Schreiber E.M., Candiello J., Balasubramani G.K., Kurtz J., Halfter W.
Matrix biology, 2010

A direct analysis of retinal BM isolated from embryonic chick eyes using a mass spectrometry (MS) based proteomics approach. A semi-quantitative measure of protein abundance distinguished, nidogens-1 and -2, laminin subunits α1, α5, β2, and γ1, agrin, collagen XVIII, perlecan, FRAS1 and FREM2 as the most abundant BM protein components. Laminin subunits α3, β1, γ2, γ3 minor constituents. Conclusion is that laminin-521 is found in the native retinal inner limiting membrane. 

Laminin-511 and -521 Enable Efficient In Vitro Expansion of Human Corneal Endothelial Cells (HCEC)

Okumura N., Kakutani K., Numata R., Nakahara M., Schlötzer-Schrehardt U., Kruse F., Kinoshita S., Koizumi N.
IVOS Cornea, 2015

The purpose of this study was to investigate the usefulness of laminin isoforms as substrates for culturing human corneal endothelial cells (HCECs) for clinical applications. Laminin-511 and -521 were expressed in Descemet’s membrane and corneal endothelium. These laminin isoforms significantly enhanced the in vitro adhesion and proliferation, and differentiation of HCECs compared to uncoated control, fibronectin and collagen I. iMatrix also supported HCEC cultivation with a similar efficacy to that obtained with full-length laminin. Functional blocking of a3b1 and a6b1 integrins suppressed the adhesion of HCECs even in the presence of laminin-511.


Identification and Potential Application of Human Corneal Endothelial Progenitor Cells

Hara S., Hayashi R., Soma T., Kageyama T., Duncan T., Tsujikawa M., Nishida K.
Stem Cells Dev. 2014

This article demonstrates for the first time that Laminin-511 is an optimal, human matrix for the isolation and expansion of corneal endothelial progenitors. The authors show that the proliferative capacity of these endothelial progenitors is very high on Laminin-511 compared to conventional methods. Laminin-511 can be used to rapidly isolate and expand a homogenous population of a endothelial progenitor cells that can be differentiated to endothelial cells in a biorelevant environment. The authors demonstrate that the proliferative capacity of these endothelial progenitors is very high on Laminin-511 compared to conventional methods. Laminin-511 can thus be used to rapidly isolate and expand a homogenous population of endothelial progenitors that can be differentiated to endothelial cells in a biorelevant environment. Main points of the article are: 1) High proliferative capacity in serum-free media compared to standard methods, 2) Large numbers of cells generated, 3) Facilitates rapid isolation of a homogenous population of endothelial progenitors, 4) Enables differentiation to endothelial cells in a biorelevant environment, 5) Cells can be subcultured for at least 5 passages.


Laminin synthesis and the adhesion characteristics of immortalized human corneal epithelial cells to laminin isoforms

Filenius S., Hormia M., Rissanen J., Burgeson R.E., Yamada Y., Araki-Sasaki K., Nakamura M., Virtanen I., Tervo T.
Exp. Eye Res., 2001

We have studied the synthesis of laminins and determined the specific integrins mediating the adhesion of immortalized human corneal epithelial cells to mouse laminin-111, and human laminin-332 and laminin-511.The cells produced a3, b3 and g2 chains of laminin-332, but not laminin-111 and laminin-511. Integrin a3B1 complex mediated the adhesion of corneal epithelial cells to human laminin-332 and laminin-511. Integrin complex a3B, as well as laminin a3 chain, was also shown to mediate cell adhesion to newly produced endogenous laminin-332. The results also show that among corneal basement membrane laminins, laminin-332 is synthetized by epithelial cells while laminin-511 may be a product of keratocytes.


The Different Binding Properties of Cultured Human Corneal Endothelial Cell Subpopulations to Descemet’s Membrane Components

Toda M., Ueno M., Yamada J., Hiraga A., Tanaka H., Schlötzer-Schrehardt U., Sotozono C., Kinoshita S., Hamuro J.
Invest Ophthalmol Vis Sci. 2016

In culture, human corneal endothelial cell (cHCEC) tend to enter into cell-state transition (CST), such as epithelial-to-mesenchymal transition (EMT) or fibrosis, thus resulting in the production of different subpopulations. In this study, the authors examined the binding ability of cHCECs subpopulations to major Descemet’s membrane components that distribute to the endothelial face; that is, laminin-511, -411, Type-IV collagen, and proteoglycans. Each subpopulation was prepared by controlling the culture conditions or by using magnetic cell separation, and then confirmed by staining with several cell-surface markers. Binding abilities of HCEC subpopulations were examined by adding the cells to culture plates immobilized with collagens, laminins, or proteoglycans, and then centrifuging the plates. The cHCECs showed best attachment to laminin laminin-521 and -511. The cells showed a weaker binding to laminin-411, laminin-332, Type-IV collagen. The minimum concentrations necessary for the observed cell binding in this study were as follows: laminin-521 and -511, 3 ng/mL; laminin-411, 2.85 ug/mL; Type-IV collagen, 250 ng/mL. Cells suspended in Opti-MEM-I or Opeguard-MA were bound to laminin, yet no binding was observed in cells suspended in BSS-Plus. Both the fully differentiated, mature cHCEC subpopulations and the epithelial-to-mesenchymal– transitioned (EMT)-phenotype subpopulation were found to attach to laminin- or collagen-coated plates. Interestingly, the binding properties to laminins differed among those subpopulations. Although the level of cells adhered to the laminin-411–coated plate was the same among the cHCEC subpopulations, the fully differentiated, mature cHCEC subpopulations was significantly more tightly bound to laminin-511 than was the EMT-phenotype subpopulations. These findings suggest that the binding ability of cHCECs to major Descemet’s membrane components is distinct among cHCEC subpopulations, and that Opti-MEM-I and Opeguard-MA are useful cell-suspension vehicles for cell-injection therapy. This research group focused on developing a novel medical approach, termed cell-injection therapy, for the treatment of patients with endothelial dysfunction.


Adhesion, Migration, and Proliferation of Cultured Human Corneal Endothelial Cells by Laminin-5

Yamaguchi M., Ebihara N., Shima N., Kimoto M., Funaki T., Yokoo S., Murakami A., Yamagami S.
Investigative Ophthalmology & Visual Science, 2011

Here, the authors investigate the expression of laminin-332 and its receptors by human corneal endothelial cells (HCECs) and effect on adhesion, proliferation, and migration of cultured HCECs. Adult HCECs expressed the laminin-332 receptor a3B1 integrin, but not laminin-332 itself. Laminin-332  is expressed in the basement membrane of the corneal epithelium, but not in the corneal endothelium. Significantly more HCEC cells became adherent to recombinant laminin-332-coated dishes than to uncoated dishes in the cell adhesion assay. The proliferation of cultured HCECs was moderately promoted by laminin-332. A significantly higher percentage of wound closure was obtained with medium containing soluble laminin-332 than with control medium in the wound-healing assay. To conclude, recombinant laminin-332 promotes adhesion, migration, and moderate proliferation of cultured HCECs. The results suggest that immature or undifferentiated HCECs express laminin-332, whereas it is suppressed during development or differentiation.


Changes in Corneal Basal Epithelial Phenotypes in an Altered Basement Membrane

Wang I-J., Jui-Fang Tsai R., Yeh L-K., Yao-Nien Tsai R., Hu F-R., Kao W.W.Y.
PLOS ONE, 2011

Analysis of human healthy donors show that the BM of the limbal epithelium differs from that of the central cornea. Aside from laminin-111 and laminin-332, the limbal BM also contains laminin α2β2 chains, while the corneal BM does not.


Compositional Differences between Infant and Adult Human Corneal Basement Membranes

Kabosova A., Azar D.T., Bannikov G.A., Campbell K.P, Durbeej M., Ghohestani R.F,. Jones J.C.R, Kenney M.C, Koch M., Ninomiya Y., Patton B.L., Paulsson M., Sado Y., Sage E.H., Sasaki T., Sorokin L.M., Steiner-Champliaud M-F, Sun T-T, SundarRaj N., Timpl R., Virtanen I., Ljubimov A.V.
Invest Ophthalmol Vis Sci. 2007

The purpose of the study was to identify changes of human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) during postnatal corneal development. Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from α1-α2 to α3-α4 chains after 3 years of life; in the adult, α1-α2 chains were retained only in the limbal BM. Laminin α2 and β2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, β2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin γ3 chain. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult.


Corneal integrins and their functions

Stepp M.A.
Experimental Eye Research, 2005

There are a minimum of 12 different integrin heterodimers reported to be expressed by the major resident cells of the cornea: the corneal and limbal epithelial cells, keratocytes/fibroblasts, and corneal endothelial cells. These different integrin heterodimers play important and varied roles in maintaining the cornea and organizing how its cells interact with their surrounding extracellular matrix to maintain corneal clarity. In this review, an overview of the discovery and functions of integrins is provided along with a description of the current state of our knowledge of this large family of important proteins.


Human laminin β2 deficiency causes congenital nephrosis with mesangial sclerosis and distinct eye abnormalities

Zenker M., Aigner T, Wendler O, Tralau T, Müntefering H, Fenski R, Pitz S, Schumacher V, Royer-Pokora B, Wühl E, Cochat P, Bouvier R, Kraus C, Mark K, Madlon H, Dötsch J, Rascher W, Maruniak-Chudek I, Lennert T, Neumann LM, Reis A.
Human Molecular Genetics, 2004

Here, the authors demonstrate that LAMB2 mutations can be consistently found in patients with Pierson syndrome, a newly delineated entity characterized by CNS and distinct ocular anomalies. They point out the importance of laminin β2 for the proper development of structures of the anterior eye segment.


The functions of exogenous and endogenous laminin-5 on corneal epithelial cells

Ebihara N, Mizushima H, Miyazaki K, Watanabe Y, Ikawa S, Nakayasu K, Kanai A.
Exp. Eye Res., 2000

In this study, the authors investigated the functions of laminin-332 on SV-40 transfected human corneal epithelial cells (HCE cells). We also revealed different functions between exogenous and endogenous laminin-332 on HCE cells. HCE cells themselves secreted laminin-5 endogenously. Exogenously added laminin-5 strongly promoted cell adhesion via a3b1 integrin, cell spreading, assembly of hemidesmosomes and mildly inhibited cell migration. Using an anti laminin-5 monoclonal antibody (mAb) or anti integrin a3b1 mAbs, the blocking of the interaction between endogenously secreted laminin-5 and HCE cells caused strong inhibition of cell migration. Integrin a3b1 and a6b4 were expressed in HCE cells. These results indicated that endogenous (unprocessed) laminin-5 has a crucial role in cell migration on HCE cells via a3b1 integrin. In conclusion, structural differences between exogenous and endogenous laminin-5 regulated their functions on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion, cell spreading and assembly of hemidesmosomes. Endogenously secreted laminin-5 had a crucial role in cell migration.


Collagens and proteoglycans of the cornea: importance in transparency and visual disorders

Massoudi D., Malecaze F., Galiacy S.D.
Cell Tissue Res., 2016

The cornea represents the external part of the eye and consists of an epithelium, a stroma and an endothelium. The organization of the extracellular matrix contained in the corneal stroma allows a constant refractive power. The stroma contains mesenchymal cells called keratocytes that secrete an extracellular matrix (ECM) composed mainly of collagen fibrils consisting mostly of type I collagen. Corneal epithelial basal cells secrete Corneal epithelial basement membrane (CEBM) components. These components include type IV collagen, type VII collagen, laminin 332, nidogens and heparan sulfate proteoglycans. The Descemet’s membrane (DM) is the BM of the endothelium. This structure is composed of type IV collagen, type VIII collagen, laminin 332, laminin 411, laminin 511 as well as perlecan and nidogens. The aim of this review is to describe the different corneal collagens and proteoglycans by highlighting their importance in corneal transparency as well as their implication in corneal visual disorders.