Isolation and Propagation of Human Corneal Stromal Keratocytes for Tissue Engineering and Cell Therapy

Yusoff, Nur Zahirah Binte M; Riau, Andri K.; Yam, Gary Hin‐Fai; Halim, Nuur Shahinda Humaira binte

doi:10.3390/cells11010178

Cited by 22 publications

(10 citation statements)

References 39 publications

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“…Furthermore, good viability on day 7 indicates that the corneal stroma-specific bioink provides a suitable environment for the bioprinted hASC–CSKs. After 10 days, bioprinted hASC–CSKs demonstrated native hCSK-like morphology which is reported to be stellate and show more round cell bodies . Compared to our previous study with bioprinting hASC–CSKs in a bioink without decellularized ECM, here the cell morphology was more stellate with longer extensions even after shorter culture period postprinting.…”

Section: Discussioncontrasting

confidence: 56%

Cornea-Specific Human Adipose Stem Cell-Derived Extracellular Matrix for Corneal Stroma Tissue Engineering

Puistola,

Kethiri,

Nurminen

et al. 2024

ACS Appl. Mater. Interfaces

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Utilizing tissue-specific extracellular matrices (ECMs) is vital for replicating the composition of native tissues and developing biologically relevant biomaterials. Human-or animal-derived donor tissues and organs are the current gold standard for the source of these ECMs. To overcome the several limitations related to these ECM sources, including the highly limited availability of donor tissues, cell-derived ECM offers an alternative approach for engineering tissue-specific biomaterials, such as bioinks for three-dimensional (3D) bioprinting. 3D bioprinting is a state-of-the-art biofabrication technology that addresses the global need for donor tissues and organs. In fact, there is a vast global demand for human donor corneas that are used for treating corneal blindness, often resulting from damage in the corneal stromal microstructure. Human adipose tissue is one of the most abundant tissues and easy to access, and adipose tissue-derived stem cells (hASCs) are a highly advantageous cell type for tissue engineering. Furthermore, hASCs have already been studied in clinical trials for treating corneal stromal pathologies. In this study, a corneal stroma-specific ECM was engineered without the need for donor corneas by differentiating hASCs toward corneal stromal keratocytes (hASC− CSKs). Furthermore, this ECM was utilized as a component for corneal stroma-specific bioink where hASC−CSKs were printed to produce corneal stroma structures. This cost-effective approach combined with a clinically relevant cell type provides valuable information on developing more sustainable tissue-specific solutions and advances the field of corneal tissue engineering.

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Section: Discussioncontrasting

confidence: 56%

Cornea-Specific Human Adipose Stem Cell-Derived Extracellular Matrix for Corneal Stroma Tissue Engineering

Puistola,

Kethiri,

Nurminen

et al. 2024

ACS Appl. Mater. Interfaces

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“…To date, CSKs have been mainly preferred as a cell source for 3D bioprinting of cornea stroma [4,67]. However, expanding CSKs in vitro is challenging and there is a huge shortage of cornea donor tissue limiting their use in corneal TE and cell therapy [68,69]. Human mesenchymal stem cells such as hASCs have proven to be capable of producing new ECM proteins within the host cornea stroma, modulate pre-existing scars and enhance transparency by corneal stroma remodeling [70].…”

Section: Discussionmentioning

confidence: 99%

Hyaluronic acid based next generation bioink for 3D bioprinting of human stem cell derived corneal stromal model with innervation

Mörö

Samanta²,

Honkamäki

et al. 2022

Biofabrication

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Corneal transplantation remains gold standard for the treatment of severe cornea diseases, however, scarcity of donor cornea is a serious bottleneck. 3D bioprinting holds tremendous potential for cornea tissue engineering (TE). One of the key technological challenges is to design bioink compositions with ideal printability and cytocompatibility. Photocrosslinking and ionic crosslinking are often used for the stabilization of 3D bioprinted structures, which can possess limitations on biological functionality of the printed cells. Here, we developed a hyaluronic acid (HA)-based dopamine containing bioink using hydrazone crosslinking chemistry for the 3D bioprinting of corneal equivalents. First, the shear thinning property, viscosity, and mechanical stability of the bioink were optimized before extrusion-based 3D bioprinting for the shape fidelity and self-healing property characterizations. Subsequently, human adipose stem cells (hASCs) and hASC -derived corneal stromal keratocytes (hASC-CSKs) were used for bioprinting corneal stroma structures and their cell viability, proliferation, microstructure and expression of key proteins (lumican, vimentin, connexin 43, α-SMA) were evaluated. Moreover, 3D bioprinted stromal structures were implanted into ex vivo porcine cornea to explore tissue integration. Finally, human pluripotent stem cell derived neurons (hPSC-neurons), were 3D bioprinted to the periphery of the corneal structures to analyze innervation. The bioink showed excellent shear thinning property, viscosity, printability, shape fidelity and self-healing properties with high cytocompatibility. Cells in the printed structures showed good tissue formation and 3D bioprinted cornea structures demonstrated excellent ex vivo integration to host tissue as well as in vitro innervation. The developed bioink and the printed cornea stromal equivalents hold great potential for cornea TE applications.

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“…Proliferative markers such as marker of proliferation Ki-67 (MKI67) 32,33 and proliferating cell nuclear antigen (PCNA) 34,35 were highly expressed in the presence of FBS compared to SF and TGF-β1 (Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

Corneal keratocytes, fibroblasts, and myofibroblasts exhibit distinct transcriptional profilesin vitro

Poole,

Iyer,

Schmidtke

et al. 2024

Preprint

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After a stromal injury in the cornea, the release of growth factors and pro-inflammatory cytokines typically results in the activation of quiescent keratocytes toward migratory fibroblast and/or fibrotic myofibroblast phenotypes. The persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. The primary goal of this study was to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which changes in phenotype may occur. Here, we cultured primary rabbit corneal keratocytes on collagen-coated glass coverslips in serum free media (SF), serum containing media (FBS), or in the presence of TGF-β1 to induce keratocyte, fibroblast, and myofibroblast phenotypes, respectively. Total RNA was collected and sent to Novogene for bulk RNA sequencing. Subsequent bioinformatic analysis included gene expression quantification, differential expression, and functional analysis. When comparing FBS and TGF-β1 conditions to SF, genes characteristic of a quiescent keratocyte phenotype were downregulated (e.g. KERA, LUM, ALDH1A1), while genes commonly associated with fibroblasts or myofibroblasts were upregulated (e.g. VIM, TNC, FN1, ITGA5, ACTA2). Functional analysis of genes differentially expressed between fibroblasts and keratocytes highlighted pathways related to proliferation (e.g. DNA replication, PI3K-Akt signaling) and cell migration (e.g. Rap1 signaling, ECM-receptor interactions). Enriched pathways for the comparison of myofibroblasts to keratocytes included focal adhesion, regulation of actin cytoskeleton, hippo signaling, and ECM-receptor interaction pathways. Together, these pathways support changes in cytoskeletal organization, cell contractility, mechanotransduction, and cell-ECM interactions in myofibroblasts compared to keratocytes. Overall, these data demonstrate that there are distinct transcriptional differences between cultured corneal keratocytes, fibroblasts, and myofibroblasts. In our initial analysis, we have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to proliferation, cell mechanical activity, and ECM interactions. Furthermore, our findings reveal novel markers for each cell type as well as possible targets for modulating cell behavior and differentiation to promote physiological corneal wound healing.

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Isolation and Propagation of Human Corneal Stromal Keratocytes for Tissue Engineering and Cell Therapy

Cited by 22 publications

References 39 publications

Cornea-Specific Human Adipose Stem Cell-Derived Extracellular Matrix for Corneal Stroma Tissue Engineering

Cornea-Specific Human Adipose Stem Cell-Derived Extracellular Matrix for Corneal Stroma Tissue Engineering

Hyaluronic acid based next generation bioink for 3D bioprinting of human stem cell derived corneal stromal model with innervation

Corneal keratocytes, fibroblasts, and myofibroblasts exhibit distinct transcriptional profilesin vitro

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