Publications
Here is a selection of publications where different laminin isoforms were used to create more authentic cell culture systems.
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
This publication by the groups of Drs. Hovatta, Lanner, and Kvanta describe the production of hESC-RPE cells in a xeno-free and defined manner. In the paper, they describe an effective differentiation methodology using a 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.
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 the transplantation of OpRegen into the subretinal space of RCS rats protected the 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. The 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 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 ongoing, NCT02286089.
N-Terminomics identifies HtrA1 cleavage of thrombospondin-1 with generation of a proangiogenic fragment in the polarized retinal pigment epithelial cell model of age-related macular degeneration
Chen C-Y., Melo E., Jakob P., Friedlein A., Elsässer B., Goettig P., Kueppers V., Delobel F., Stucki C., Dunkley T., Fauser S., Schilling O., Iacone R. Matrix Biology, 2018
Here, the authors investigate the impact of elevated HtrA1 levels on the retinal pigment epithelial (RPE) secretome using a polarized culture system. The authors suggest a mechanism by which increased levels of HtrA1 may contribute to AMD pathogenesis. Human primary Retinal Pigmented Epithelium (RPE) cells (Sciencell, 6540) were seeded in transwells coated with Laminin 521. A model that recapitulates the structural, molecular and apical/basolateral signatures of adult RPE cells was achieved. The upregulation of HtrA1 alters the abundance of key proteins involved in angiogenesis and extracellular matrix remodeling. Thrombospondin-1, an angiogenesis modulator, was identified as a substrate for HtrA1 using terminal amine isotope labeling of substrates in conjunction with HtrA1 specificity profiling. HtrA1 cleavage of thrombospondin-1 was further corroborated by in vitro cleavage assays and targeted proteomics together with small-molecule inhibition of HtrA1. While thrombospondin-1 is anti-angiogenic, the proteolytically released N-terminal fragment promotes the formation of a tube-like structure by endothelial cells.
Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method
Hongisto H., Ilmarinen T., Vattulainen M., Mikhailova A., Skottman H. Stem cell research and therapy, 2017
Here the authors describe a robust xeno- and feeder cell-free culture system for undifferentiated hPSCs along with efficient and scalable methods to derive high-quality retinal pigment epithelial (RPE) cells and corneal limbal epithelial stem cells (LESCs). Multiple genetically distinct hPSC lines were adapted to a robust, defined, xeno-, and feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial cells were established utilizing xeno-free media and a combination of extracellular matrix proteins laminin-521 and Collagen IV. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to cell populations expressing LESC markers such as p40/p63α by day 24. Finally, the authors established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE cells, and hPSC-LESCs, and demonstrated successful recovery after thawing on laminin-521 and Collagen IV. The simple xeno-free methods described here could be upgraded to GMP-quality for future preclinical testing and safety and functional efficacy testing of the hPSC-RPE and hPSC LESCs produced with these protocols are currently ongoing in non-human primates and rabbit models of LSCD, respectively.
Generation of Retinal Pigment Epithelial Cells Derived from Human Embryonic Stem Cells Lacking Human Leukocyte Antigen Class I and II
Petrus-Reurer S. Winblad N., Kumar P., Gorchs L., Chrobok M., Kathleen Wagner A.K., Bartuma H., Lardner E., Aronsson M., Plaza Reye A., Andre H., Alici E., Kaipe H., Kvanta A., Lanner F.Stem Cell Reports, 2020
In this Stem Cell Reports article, the authors developed a strategy to evade the immune recognition triggered during transplants therapies with human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells. They generated and characterized hESCs and hESC-RPEs lacking surface presentation of HLA-I and -II through CRISPR/Cas9 targeting of B2M and CIITA. They established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. The activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. The results show that hESC-RPEs lacking HLA-I and -II evade T-cell response. Moreover, hESC-RPEs lacking HLA-I and -II do not increase NK cell cytotoxic activity. After transplantation of these immune-modified hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model. The results support the strategy to overcome host-donor mismatch in non-autologous cell-based treatment of AMD and other hESC-derived cell replacement therapies.
Identification of cell surface markers and establishment of monolayer differentiation to retinal pigment epithelial cells
Plaza Reyes A., Petrus-Reurer S. Padrell Sánchez S., Kumar P., Douagi I, Bartuma H., Aronsson M., Westman S., Lardner E., André H Falk A., Nandrot E.F., Kvanta A., Lanner F.Nature Communications, 2020
Here, the authors have performed a comprehensive antibody screening and identify cell surface markers for RPE cells. They identified CD140b, CD56, GD2, and CD184 as central cell surface markers to evaluate hPSC-RPE differentiation efficiency, as well as a potential tool for the enrichment of hPSC-RPE during and after differentiation. They show that these markers can be used to isolate RPE cells during in vitro differentiation and to track, quantify and improve differentiation efficiency. Using these markers together with single-cell RNA-sequencing to evaluate the differentiation process, they have established an efficient, robust, direct and scalable xeno-free and defined monolayer differentiation protocol, where culture on supportive human recombinant Biolaminin 111 and 521 eliminates the need for manual selection, allowing large-scale production of pure hPSC-RPE.
Blood vessels of human islets of Langerhans are surrounded by a double basement membrane
Virtanen I., Banerjee M., Palgi J., Korsgren O., Lukinius A., Thornell L.E., Kikkawa Y., Sekiguchi K., Hukkanen M., Konttinen Y.T., Otonkoski T. Diabetologia, 2008
Immunohistochemistry revealed a unique organization for human laminin-511/521 as a peri-islet BM, which co-invaginated into islets with vessels, forming an outer endocrine BM of the intra-islet vascular channels, and was distinct from the vascular BM that additionally contained laminin-411/421. These findings were verified by electron microscopy. Lutheran glycoprotein, a receptor for the laminin alpha5 chain, was found prominently on endocrine cells, as identified by immunohistochemistry and RT-PCR, whereas alpha(3) and beta(1) integrins were more diffusely distributed. High Lutheran content was also found on endocrine cell membranes in a short-term culture of human islets. The adhesion of dispersed beta cells to laminin-511 was inhibited equally effectively by antibodies to integrin and alpha(3) and beta(1) subunits and by the soluble Lutheran peptide.
Unique basement membrane structure of human pancreatic islets: implications for b-cell growth and differentiation
Otonkoski T., Banerjee M., Korsgren O., Thornell L.-E., Virtanen I.Diabetes, Obesity and Metabolism, 2008
The authors showed that in the human islets, a double BM structure surrounding each blood vessel within the islet. In addition, a continuous peri-islet BM was surrounding the entire islet, invaginating into the islet tissue together with the arterioles. The capillaries are surrounded by a double BM both in fetal and adult tissues. The B-cells are facing a BM that is separate from the endothelia, unlike the situation in mouse where the B-cells interact directly with BMs of capillary endothelial cells. Here they show that (i) a1 is not expressed in the adult human pancreas; (ii) a2 is only expressed in the exocrine pancreas; (iii) a4 is expressed in the blood vessel BMs; (iv) a5 and b1 are expressed similarly, both in the endocrine and endothelial BMs in the islets. Taken together, this suggested that there is a double-layered BM organization around the vascular channels of human islets: the inner vascular leaflet of the duplex contains Lms-411/421 and -511/521 whereas the outer leaflet facing the parenchymal endocrine cells contains only Lm-511. In contrast to the adult pancreas, a laminin a1 chain could be found in the acinar BMs of the fetal pancreas and faintly also in the developing islets. Similarly, as in the adult pancreas, immunoreactivity for laminin a5 chain was distinctly surrounding the developing islets and also in BMs of intra-islet vessels. Strong expression of laminin B1 and g1 in the fetal pancreas. a3 and b1 integrin subunits were found both on the vascular channels and the endocrine cells. However, the integrin a6 subunit was clearly absent from the endocrine cells and only expressed in the endothelial cells. The islet cells facing this BM have a strong and polarized expression of Lutheran glycoprotein, which is a well-known receptor for the laminin a5 chain. Dispersed human islet cells adhere to purified human laminin-511 and the binding is equally effectively blocked by a soluble form of Lutheran as by antibody against integrin B1. The results reveal that the BM structure of human islets, different from rodents.
Importance of Cell-Matrix Interactions in Rat Islet Bets-Cell Secretion In Vitro: Role of a6B1 Integrin
Bosco D., Meda P., Halban P.A, Rouiller D.G. Diabetes, 2000
The present work addresses the influence of short-term cell-matrix interactions on islet beta-cell function and provides the first insight into the molecular basis of these interactions. When primary rat beta-cells were allowed to attach to a laminin-332 rich matrix (804G matrix), there was an increased insulin secretory response to secretagogues. This change was the result of an increase in the proportion of actively secreting beta-cells and in the amount of insulin secreted per active cell. In turn, the spreading or flattening of beta-cells on this matrix was enhanced by secretagogues, and flattened cells secreted more insulin than rounded cells. a6B1 integrins are present heterogeneous at the surface of islet cells in situ and is upregulated by insulin secretagogues. a6B1 expression is higher in spreading cells and anti-a6B1–specific antibodies decrease spreading. These observations demonstrate that islet cell-matrix interactions can improve the sensitivity of insulin cells to glucose and are mediated, at least in part, by a6B1 integrins, suggesting that outside-in signaling through a6B1 integrin plays a major role in the regulation of B-cell function.
Extracellular Matrix Protects Pancreatic Beta-Cells Against Apoptosis: Role of Short- and Long-Term Signaling Pathways
Hammar E., Parnaud G., Bosco D., Perriraz N., Maedler K., Donath M., Rouiller D.G., Halban P. A.Diabetes, 2004
Beta-cells cultured on laminin-332 rich surfaces improve their functions. This study provides evidence for the activation of signaling pathways and gene expression by laminin-332 leading to improved beta-cell survival. Laminin-332 protect sorted rat beta-cells against apoptosis under standard conditions (11.2 mmol/l glucose, 10% serum), after serum deprivation (1% serum), and in response to interleukin-1 (IL-1; 2 ng/ml), compared with the poly-L-lysine control. Caspase-8 activity was reduced in cells cultured on laminin-332, whereas FAK, Akt and ERK phosphorylation was augmented. Treatment with an anti-1 integrin antibody, with the ERK pathway inhibitor PD98059, and/or with the phosphatidylinositol 3-kinase inhibitor LY294002 augmented cell death on the laminin-332 rich matrix but not on pLL.