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

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.


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.