Science Room

Show Menu

PRODUCT RECOMMENDATION

BIOLAMININ 521 

 
                                             Go to the BioLamina shop

Do you want us to contact you? Please click here


PROTOCOL

Effective production of hESC-RPE cells in an animal origin-free and defined manner


Production and transplantation of GMP grade animal origin-free hESC-derived RPE cells

Animal origin-free and feeder-free differentiaƒtion of human pluripotent stem cells to RPE cells

 


VIEW ON DEMAND

Defined and animal origin-free differentiation of clinically compliant hESC-RPE cells

GMP-compliant stem cell differentiation to dopaminergic neurons on laminin


APPLICATION NOTE

Get the RPE culture application note here


ARTICLES

HOW TO CULTURE RETINAL CELLS ON BIOLAMININ SUBSTRATES

 

Effective monolayer differentiation protocol for the generation of hESC-RPE cells under defined and animal origin-free conditions

In a publication by the groups of Drs. Hovatta, Lanner and Kvanta at Karolinska Institutet, Sweden, the authors describe a protocol for effective differentiation of hESC-RPE cells on Biolaminin 521 in a defined and animal origin-free manner (Plaza Reyes, 2015). 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 allows imaging of the hESC-RPE and host retina. Cells are transplanted in suspension and show long-term integration and form polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity.

In 2020, the same authors published an article in Nature Communications (Plaza Reyes, 2020) where they 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.

The same research group also published an article in Stem Cell Reports (Petrus-Reurer, 2020) where the present a strategy to evade the immune recognition triggered during transplants therapies with human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells. 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.

These results are in accordance with data published by Finnish scientists (Hongisto, 2017) where the authors describe a robust animal origin- 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, animal origin-, and feeder-free culture system of Essential 8™ medium and Biolaminin 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. In addition, the authors established xeno-free cryo-banking protocols for pluripotent hPSCs, hPSC-RPE cells, and hPSC-LESCs, and demonstrated successful recovery after thawing on Biolaminin 521 and Collagen IV (Hongisto, 2017). 

Biolaminin 521 also supports the culture of human primary RPE cells. Chen et al. show that culturing of human fetal RPE cells on laminin 521 PET transwell inserts creates a model that recapitulates the structural, molecular and apical/basolateral signatures of adult RPE cells (Chen, 2018).

 

Protocols for ocular cell replacement therapy

The simple animal origin-free methods described by Hongisto et al. 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 (Hongisto, 2017).


In a recent publication, researchers at Cell Cure Neurosciences Ltd. display the efficacy of RPE cells derived under defined conditions from clinical and animal origin-free grade human embryonic stem cells following transplantation into the subretinal space of Royal College of Surgeons (RCS) rats (McGill, 2017). Again, the laminin cell culture substrate is being used in the differentiation protocol. The results demonstrate that transplantation of hESC-RPE cells into the subretinal space of RCS rats protected the retinal structure, rescued visual function, preserved rod and cone photoreceptors long-term. OKT was rescued in a dose-dependent manner and the outer nuclear layer (ONL) was significantly thicker in cell-treated eyes than controls. This data combined with data collected in definitive safety studies (tumorigenicity and spiking and safety/biodistribution) has resulted in an FDA approved IND (investigational new drug) and a Phase 1/2a clinical trial for AMD patients is currently ongoing, NCT02286089.


Laminin expression in Bruch’s membrane

It has been shown that the beta-2 chain of the laminin molecule is important for the RPE cells (Libby, 1996). It surrounds RPE cells at the onset of rod photoreceptor birth and is present on the apical surface of the retinal neuroepithelium. At birth, laminin that contains the beta-2 chain fills the matrix between the juxtaposed surfaces of the RPE and neuroepithelium. It is present throughout postnatal development and is also is found in association with blood vessels in the neural retina. A publication by Aisenbrey and colleagues show that the multi-layered extracellular matrix underlying the retina, Bruch’s membrane, contains several laminins that retinal pigment epithelial cells grow on. The main isoforms expressed are laminin 521, 511, 332 and 111. RPE cells adhere robustly to all laminins of Bruch´s membrane and actively synthesize these laminins (Aisenbrey, 2006).

BIOLAMININ KEY ADVANTAGES

  • Defined and animal origin-free monolayer differentiation method for clinically compliant hESC-RPE

  • Biolaminin 521 supports high seeding efficiency and cell migration

  • Biolaminin 521 cultured hESC-RPE cells exhibit native characteristics including morphology, marker expression, monolayer integrity, pigmentation, polarization, and phagocytic activity

  • Cells transplanted in suspension in a large-eyed animal model incorporate as a nicely polarized monolayer

  • Transplanted cells show long-term, in vivo functionality, including phagocytic activity and photoreceptor rescue

  • CD140b, CD56, GD2, and CD184 identified 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

  • Biolaminin 111 and 521 eliminates the need for manual selection, allowing large-scale production of pure hPSC-RPE

  • Laminin culture substrates is a part of FDA approved IND, and a Phase 1/2a clinical trial for AMD patients is currently ongoing

  • Main isoforms expressed in Bruch’s membrane are laminin 521, 511, 332 and 111

  • A biologically relevant culture environment for human ES and iPS cells

  • No lot-to-lot variability for standardized experiments with less variation 

  • x
  • Expansion of human PSC

  • Mesenchymal stem cells

  • Clonal cell culture applications

  • Eye cells

  • Cardiac cells

  • Neural cells

  • Skeletal muscle cells

  • Kidney cells

  • Hepatic cells

  • Cancer cells

  • Lung cells

  • Animal stem cells

  • Endothelial cells

  • Pancreatic cells

  • Intestinal cells

  • Normal and cancerous mammary cells

  • Epithelial cells

Biolaminin 521 CTG

Biolaminin 521 CTG cell therapy grade cell culture matrix makes pluripotent stem cell culture easy. A defined, animal origin-free and biologically relevant cell culture system for better cell models.

  • x
  • Expansion of human PSC

  • Clonal cell culture applications

  • Eye cells

  • Cardiac cells

  • Neural cells

  • Skeletal muscle cells

  • Kidney cells

  • Hepatic cells

  • Cancer cells

  • Lung cells

  • Animal stem cells

  • Mesenchymal stem cells

  • Endothelial cells

  • Pancreatic cells

  • Intestinal cells

  • Normal and cancerous mammary cells

  • Epithelial cells

Biolaminin 521 MX

Biolaminin 521 MX research-grade cell culture matrix makes pluripotent stem cell culture easy. A defined, animal origin-free and biologically relevant cell culture system for better cell models.

  • x
  • Expansion of human PSC

  • Mesenchymal stem cells

  • Clonal cell culture applications

  • Eye cells

  • Cardiac cells

  • Neural cells

  • Skeletal muscle cells

  • Kidney cells

  • Hepatic cells

  • Cancer cells

  • Lung cells

  • Animal stem cells

  • Endothelial cells

  • Pancreatic cells

  • Intestinal cells

  • Normal and cancerous mammary cells

  • Epithelial cells

Biolaminin 521 LN

Biolaminin 521 LN research-grade cell culture matrix makes pluripotent stem cell culture easy. A defined, animal origin-free and biologically relevant cell culture system for better cell models.