Culture surface protein coatings affect the barrier properties and calcium signalling of hESC-RPE

Viheriälä, Taina; Sorvari, Juhana; Ihalainen, Teemu O.; Mörö, Anni; Grönroos, Pyry; Schlie-Wolter, Sabrina; Chichkov, Boris N.; Skottman, Heli; Nymark, Soile; Ilmarinen, Tanja

doi:10.1038/s41598-020-79638-8

Cited by 14 publications

(16 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…5 c in the Results-section) and their average intensity values were extracted as a function of time using the ImageJ Multi measure tool. Raw intensity data were analyzed and normalized curve plots representing single cell responses were extracted using a custom self-developed MATLAB script package (MATLAB R2017b, The MathWorks Inc.), as reported previously [ 43 , 44 ]. For quantifying cells that expressed Ca 2+ oscillations, kymographs were extracted from 5 randomly selected segments from each parallel sample video using the command Slice [/] in ImageJ (see Fig.…”

Section: Methodsmentioning

confidence: 99%

Corneal epithelial differentiation of human pluripotent stem cells generates ABCB5+ and ∆Np63α+ cells with limbal cell characteristics and high wound healing capacity

et al. 2021

Self Cite

View full text Add to dashboard Cite

Background Differentiation of functional limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) is an important objective which can provide novel treatment solutions for patients suffering from limbal stem cell deficiency (LSCD). Yet, further characterization is needed to better evaluate their immunogenicity and regenerative potential before clinical applications. Methods Human PSCs were differentiated towards corneal fate and cryopreserved using a clinically applicable protocol. Resulting hPSC-LSC populations were examined at days 10–11 and 24–25 during differentiation as well as at passage 1 post-thaw. Expression of cornea-associated markers including PAX6, ABCG2, ∆Np63α, CK15, CK14, CK12 and ABCB5 as well as human leukocyte antigens (HLAs) was analyzed using immunofluorescence and flow cytometry. Wound healing properties of the post-thaw hPSC-LSCs were assessed via calcium imaging and scratch assay. Human and porcine tissue-derived cultured LSCs were used as controls for marker expression analysis and scratch assays at passage 1. Results The day 24–25 and post-thaw hPSC-LSCs displayed a similar marker profile with the tissue-derived LSCs, showing abundant expression of PAX6, ∆Np63α, CK15, CK14 and ABCB5 and low expression of ABCG2. In contrast, day 10–11 hPSC-LSCs had lower expression of ABCB5 and ∆Np63α, but high expression of ABCG2. A small portion of the day 10–11 cells coexpressed ABCG2 and ABCB5. The expression of class I HLAs increased during hPSC-LSCs differentiation and was uniform in post-thaw hPSC-LSCs, however the intensity was lower in comparison to tissue-derived LSCs. The calcium imaging revealed that the post-thaw hPSC-LSCs generated a robust response towards epithelial wound healing signaling mediator ATP. Further, scratch assay revealed that post-thaw hPSC-LSCs had higher wound healing capacity in comparison to tissue-derived LSCs. Conclusions Clinically relevant LSC-like cells can be efficiently differentiated from hPSCs. The post-thaw hPSC-LSCs possess functional potency in calcium responses towards injury associated signals and in wound closure. The developmental trajectory observed during hPSC-LSC differentiation, giving rise to ABCG2+ population and further to ABCB5+ and ∆Np63α+ cells with limbal characteristics, indicates hPSC-derived cells can be utilized as a valuable cell source for the treatment of patients afflicted corneal blindness due to LSCD. Graphical Abstract

show abstract

Section: Methodsmentioning

confidence: 99%

Corneal epithelial differentiation of human pluripotent stem cells generates ABCB5+ and ∆Np63α+ cells with limbal cell characteristics and high wound healing capacity

et al. 2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…For Ca 2+ imaging, hESC-RPE cells were cultured on hanging inserts. Ca 2+ imaging of the hESC-RPE monolayers was performed as previously described [ 12 , 22 ] at time points of 3 weeks and 12 weeks. Briefly, the cells were loaded with Ca 2+ sensitive dye fluo-4-acetoxymethyl ester (1 mM, fluo-4 AM; Molecular Probes, Thermo Fischer Scientific) for 45 min.…”

Section: Methodsmentioning

confidence: 99%

“…Although early polarized RPE cultures may be more plastic to environmental changes than cells cultured for longer time periods, they could also be functionally more immature and not possess intact signaling pathways for critical cellular functions. Among essential RPE functions are its barrier properties that ensure the proper movement of components between the blood supply and retina [ 12 ]. With phagocytosis, RPE disposes photoreceptor outer segments (POS) that photoreceptors renew daily [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

“…Importantly, Ca 2+ signaling is linked to purinergic signaling in RPE: extracellular adenosine triphosphate (ATP) induces elevations in intracellular Ca 2+ concentration regulating the chemical composition and the amount of water in the subretinal space and ensuring proper communication between RPE and the retina [ 16 , 17 ]. We have previously shown that culture time of hESC-RPE improves the intercellular homogeneity of Ca 2+ response properties in cell population [ 12 ]. In the current study, we evaluated the impact of culture time on the ability of the hESC-RPE to endure treatments with chemical stressors.…”

Section: Introductionmentioning

confidence: 99%

“…The emphasis was placed on using physiologically relevant functional assays including phagocytosis and calcium imaging to assess the properties of intact RPE monolayers maturated on porous carrier substrate for 3 or 12 weeks. Time points were chosen to represent early and late maturation status based on our previous experience and earlier study following the development of barrier function of in vitro cultured hESC-RPE over time [ 12 ]. Our results indicate that the late maturity status of hESC-RPE cells can tolerate cellular stress more effectively than the population with early maturation status, with potential implications to hPSC-RPE cell therapy.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Cell maturation influences the ability of hESC-RPE to tolerate cellular stress

et al. 2022

Self Cite

View full text Add to dashboard Cite

Background Transplantation of human pluripotent stem cell-derived retinal pigment epithelium (RPE) is an urgently needed treatment for the cure of degenerative diseases of the retina. The transplanted cells must tolerate cellular stress caused by various sources such as retinal inflammation and regain their functions rapidly after the transplantation. We have previously shown the maturation level of the cultured human embryonic stem cell-derived RPE (hESC-RPE) cells to influence for example their calcium (Ca2+) signaling properties. Yet, no comparison of the ability of hESC-RPE at different maturity levels to tolerate cellular stress has been reported. Methods Here, we analyzed the ability of the hESC-RPE populations with early (3 weeks) and late (12 weeks) maturation status to tolerate cellular stress caused by chemical cell stressors protease inhibitor (MG132) or hydrogen peroxide (H2O2). After the treatments, the functionality of the RPE cells was studied by transepithelial resistance, immunostainings of key RPE proteins, phagocytosis, mitochondrial membrane potential, Ca2+ signaling, and cytokine secretion. Results The hESC-RPE population with late maturation status consistently showed improved tolerance to cellular stress in comparison to the population with early maturity. After the treatments, the early maturation status of hESC-RPE monolayer showed impaired barrier properties. The hESC-RPE with early maturity status also exhibited reduced phagocytic and Ca2+ signaling properties, especially after MG132 treatment. Conclusions Our results suggest that due to better tolerance to cellular stress, the late maturation status of hESC-RPE population is superior compared to monolayers with early maturation status in the transplantation therapy settings.

show abstract

Cytocompatibility of electrospun poly‐L‐lactic acid membranes for Bruch's membrane regeneration using human embryonic stem cell‐derived retinal pigment epithelial cells

Abbasi,

O'Neill

2024

J Biomedical Materials Res

View full text Add to dashboard Cite

Cell replacement therapy is under development for dry age‐related macular degeneration (AMD). A thin membrane resembling the Bruch's membrane is required to form a cell‐on‐membrane construct with retinal pigment epithelial (RPE) cells. These cells have been differentiated from human embryonic stem cells (hESCs) in vitro. A carrier membrane is required for cell implantation, which is biocompatible for cell growth and has dimensions and physical properties resembling the Bruch's membrane. Here a nanofiber electrospun poly‐L‐lactic acid (PLLA) membrane is tested for capacity to support cell growth and maturation. The requirements for laminin coating of the membrane are identified here. A porous electrospun nanofibrous PLLA membrane of ∼50 nm fiber diameter was developed as a prototype support for functional RPE cells grown as a monolayer. The need for laminin coating applied to the membrane following treatment with poly‐L‐ornithine (PLO), was identified in terms of cell growth and survival. Test membranes were compared in terms of hydrophilicity after laminin coating, mechanical properties of surface roughness and Young's modulus, porosity and ability to promote the attachment and proliferation of hESC‐RPE cells in culture for up to 8 weeks. Over this time, RPE cell proliferation, morphology, and marker and gene expression, were monitored. The functional capacity of cell monolayers was identified in terms of transepithelial electrical resistance (TEER), phagocytosis of cells, as well as expression of the cytokines, vascular endothelial growth factor (VEGF) and pigment epithelium‐derived factor (PEDF). PLLA polymer fibers are naturally hydrophobic, so their hydrophilicity was improved by pretreatment with PLO for subsequent coating with the bioactive protein laminin. They were then assessed for amount of laminin adsorbed, contact angle and uniformity of coating using scanning electron microscopy (SEM). Pretreatment with 100% PLO gave the best result over 10% PLO treatment or no treatment prior to laminin adsorption with significantly greater surface stiffness and modulus. By 6 weeks after cell plating, the coated membranes could support a mature RPE monolayer showing a dense apical microvillus structure and pigmented 3D polygonal cell morphology. After 8 weeks, PLO (100%)‐Lam coated membranes exhibited the highest cell number, cell proliferation, and RPE barrier function measured as TEER. RPE cells showed the higher levels of specific surface marker and gene expression. Microphthalmia‐associated transcription factor expression was highly upregulated indicating maturation of cells. Functionality of cells was indicated by expression of VEGF and PEDF genes as well as phagocytic capacity. In conclusion, electrospun PLLA membranes coated with PLO‐Lam have the physical and biological properties to support the distribution and migration of hESC‐RPE cells throughout the whole structure. They represent a good membrane candidate for preparation of hESC‐RPE cells as a monolayer for implantation into the subretinal space of AMD patients.

show abstract

Culture surface protein coatings affect the barrier properties and calcium signalling of hESC-RPE

Cited by 14 publications

References 58 publications

Corneal epithelial differentiation of human pluripotent stem cells generates ABCB5+ and ∆Np63α+ cells with limbal cell characteristics and high wound healing capacity

Corneal epithelial differentiation of human pluripotent stem cells generates ABCB5+ and ∆Np63α+ cells with limbal cell characteristics and high wound healing capacity

Cell maturation influences the ability of hESC-RPE to tolerate cellular stress

Cytocompatibility of electrospun poly‐L‐lactic acid membranes for Bruch's membrane regeneration using human embryonic stem cell‐derived retinal pigment epithelial cells

Contact Info

Product

Resources

About