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Histology of healed corneas. Sections from corneas 14 days after wounding were stained with H&E (A, B) or immunostained with antibody to collagen type III as described in "Methods" section. (A, C) Show corneas treated with CSSC in CCG whereas (B, D) show corneas treated with CCG only. Scale bars 5 50 mm. Abbreviations: CCG, compressed collagen gel; CSSC, corneal stromal stem cells.
Source publication
Stem cells from human corneal stroma (CSSC) suppress corneal stromal scarring in a mouse wound‐healing model and promote regeneration of native transparent tissue (PMID:25504883). This study investigated efficacy of compressed collagen gel (CCG) as a vehicle to deliver CSSC for corneal therapy. CSSC isolated from limbal stroma of human donor cornea...
Contexts in source publication
Context 1
... assess the stromal regeneration of wounded corneas after CCG treatment with or without CSSC, histologic sections using H&E staining were analyzed 14 days after wounding. Figure 5B shows that healing with CCG only led to tissue edema, hypercellularity and uneven epithelial coverage in the healed wound area. Corneas exposed to CSSC-loaded CCG, however, showed (Fig. 5A) histology indistinguishable from that of normal cornea, demonstrating regen- eration of native corneal tissue in the region removed by Alger- Brush debridement. Figure 5D shows that the edematous region of cornea healed without CSSC cells contained collagen III, a well- known marker of corneal scarring, whereas the tissue treated with stem cell-containing CCG did not stain for collagen III (Fig. ...
Context 2
... assess the stromal regeneration of wounded corneas after CCG treatment with or without CSSC, histologic sections using H&E staining were analyzed 14 days after wounding. Figure 5B shows that healing with CCG only led to tissue edema, hypercellularity and uneven epithelial coverage in the healed wound area. Corneas exposed to CSSC-loaded CCG, however, showed (Fig. 5A) histology indistinguishable from that of normal cornea, demonstrating regen- eration of native corneal tissue in the region removed by Alger- Brush debridement. Figure 5D shows that the edematous region of cornea healed without CSSC cells contained collagen III, a well- known marker of corneal scarring, whereas the tissue treated with stem cell-containing CCG did not stain for collagen III (Fig. ...
Context 3
... assess the stromal regeneration of wounded corneas after CCG treatment with or without CSSC, histologic sections using H&E staining were analyzed 14 days after wounding. Figure 5B shows that healing with CCG only led to tissue edema, hypercellularity and uneven epithelial coverage in the healed wound area. Corneas exposed to CSSC-loaded CCG, however, showed (Fig. 5A) histology indistinguishable from that of normal cornea, demonstrating regen- eration of native corneal tissue in the region removed by Alger- Brush debridement. Figure 5D shows that the edematous region of cornea healed without CSSC cells contained collagen III, a well- known marker of corneal scarring, whereas the tissue treated with stem cell-containing CCG did not stain for collagen III (Fig. ...
Context 4
... assess the stromal regeneration of wounded corneas after CCG treatment with or without CSSC, histologic sections using H&E staining were analyzed 14 days after wounding. Figure 5B shows that healing with CCG only led to tissue edema, hypercellularity and uneven epithelial coverage in the healed wound area. Corneas exposed to CSSC-loaded CCG, however, showed (Fig. 5A) histology indistinguishable from that of normal cornea, demonstrating regen- eration of native corneal tissue in the region removed by Alger- Brush debridement. Figure 5D shows that the edematous region of cornea healed without CSSC cells contained collagen III, a well- known marker of corneal scarring, whereas the tissue treated with stem cell-containing CCG did not stain for collagen III (Fig. ...
Citations
... The excimer laser-induced haze model is a very 'clean' model of haze induction, as opposed to the others mentioned, such as chemical injury and microbial keratitis models, which often are dictated by the extent of vascularization 31,32 . In addition, more patients may present with such conditions in the clinic. ...
Intrastromal cell therapy utilizing quiescent corneal stromal keratocytes (qCSKs) from human donor corneas emerges as a promising treatment for corneal opacities, aiming to overcome limitations of traditional surgeries by reducing procedural complexity and donor dependency. This investigation demonstrates the therapeutic efficacy of qCSKs in a male rat model of corneal stromal opacity, underscoring the significance of cell-delivery quality and keratocyte differentiation in mediating corneal opacity resolution and visual function recovery. Quiescent CSKs-treated rats display improvements in escape latency and efficiency compared to wounded, non-treated rats in a Morris water maze, demonstrating improved visual acuity, while stromal fibroblasts-treated rats do not. Advanced imaging, including multiphoton microscopy, small-angle X-ray scattering, and transmission electron microscopy, revealed that qCSK therapy replicates the native cornea’s collagen fibril morphometry, matrix order, and ultrastructural architecture. These findings, supported by the expression of keratan sulfate proteoglycans, validate qCSKs as a potential therapeutic solution for corneal opacities.
... Biomaterials serve dual purposes in both clinical and experimental contexts (19)(20)(21)(22). ...
Background/Aim: Biomaterials are essential in modern medicine, both for patients and research. Their ability to acquire and maintain functional vascularization is currently debated. The aim of this study was to evaluate the vascularization induced by two collagen-based scaffolds (with 2D and 3D structures) and one non-collagen scaffold implanted on the chick embryo chorioallantoic membrane (CAM). Materials and Methods: Classical stereomicroscopic image vascular assessment was enhanced with the IKOSA software by using two applications: the CAM assay and the Network Formation Assay, evaluating the vessel branching potential, vascular area, as well as tube length and thickness. Results: Both collagen-based scaffolds induced non-inflammatory angiogenesis, but the non-collagen scaffold induced a massive inflammation followed by inflammatory-related angiogenesis. Vessels branching points/Region of Interest (Px^2) and Vessel branching points/Vessel total area (Px^2), increased exponentially until day 5 of the experiment certifying a sustained and continuous angiogenic process induced by 3D collagen scaffolds. Conclusion: Collagen-based scaffolds may be more suitable for neovascularization compared to non-collagen scaffolds. The present study demonstrates the potential of the CAM model in combination with AI-based software for the evaluation of vascularization in biomaterials. This approach could help to reduce and replace animal experimentation in the pre-screening of biomaterials. Biomaterials assume a pivotal role in the design and development of an extensive array of biomedical products and devices, encompassing, among others, prosthetic heart valves, hip joint replacements, dental implants, and ocular contact lenses. Of particular note is a substantial subset of 620 in vivo 38: 620-629 (2024)
... As keratocyte progenitors, CSSCs differentiate and express keratocyte-specific collagens and proteoglycans (keratocan, lumican, and decorin) that comprise the stromal matrix [6,10]. They also produce matrix metalloproteinases (MMP), which break down the excess collagen linking in scar tissues [11]. Moreover, the treatment upregulated tumor necrosis factor α stimulated gene 6 (TSG-6) inhibiting CD11b + /Ly6G + neutrophil infiltration, hence suppressing stromal fibrosis [12]. ...
Background
Mesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities.
Methods
A total of 24 donor corneal rims with informed consent were used—18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC’s conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring.
Results
By comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters—stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)—were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring.
Conclusions
We established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC–in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments.
... Studies in recent years showed the differentiation ability of CSSCs to keratocytes and functional restoration of scarred corneas [9,10]. ...
Although extensive studies have evaluated the regulation effect of microenvironment on cell phenotype and cell differentiation, further investigations in the field of the cornea are needed to gain sufficient knowledge for possible clinical translation. This study aims to evaluate the regulation effects of substrate stiffness and inflammation on keratocyte phenotype of corneal fibroblasts, as well as the differentiation from stem cells towards keratocytes. Soft and stiff substrates were prepared based on polydimethylsiloxane (PDMS). HTK and stem cells were cultured on these substrates to evaluate the effects of stiffness. The possible synergistic effects between substrate stiffness and inflammatory factor IL-1β were examined by qPCR and immunofluorescence staining. In addition, macrophages were cultured on soft and stiff substrates to evaluate the effect of substrate stiffness on the synthesis of inflammatory factors. The conditioned medium of macrophages (Soft-CM and Stiff-CM) was collected to examine the effects on HTK and stem cells. It was found that inflammatory factor IL-1β promoted keratocyte phenotype and differentiation when cells were cultured on soft substrate (~130 kPa), which were different from cells cultured on stiff substrate (~2×103 kPa) and TCP (∼106 kPa). Besides, macrophages cultured on stiff substrates had significantly higher expression of IL-1β and Tnf-α as compared to the cells cultured on soft substrates. And Stiff-CM decreased the expression of keratocyte phenotype markers as compared to Soft-CM. The results of our study indicate a stiffness-dependent dynamic effect of inflammation on keratocyte phenotype and differentiation, which is of significance not only in gaining a deeper knowledge of corneal pathology and repair, but also in being instructive for scaffold design in corneal tissue engineering and ultimate regeneration.
... In order to minimize this condition, the authors performed the compression of the collagen polymer followed by cell association in its polymeric scaffold. The complex formed between the collagen and the cells presented a high tensile strength with sufficient malleability to shape the injured corneal surface, in addition to increasing cell viability in this microenvironment and post-freezing regeneration of the injured tissue [99]. In this context, another study employed type I collagen associated with agarose to microencapsulate mesenchymal cells. ...
One of the limitations in organ, tissue or cellular transplantations is graft rejection. To minimize or prevent this, recipients must make use of immunosuppressive drugs (IS) throughout their entire lives. However, its continuous use generally causes several side effects. Although some IS dose reductions and withdrawal strategies have been employed, many patients do not adapt to these protocols and must return to conventional IS use. Therefore, many studies have been carried out to offer treatments that may avoid IS administration in the long term. A promising strategy is cellular microencapsulation. The possibility of microencapsulating cells originates from the opportunity to use biomaterials that mimic the extracellular matrix. This matrix acts as a support for cell adhesion and the syntheses of new extracellular matrix self-components followed by cell growth and survival. Furthermore, by involving the cells in a polymeric matrix, the matrix acts as an immunoprotective barrier, protecting cells against the recipient's immune system while still allowing essential cell survival molecules to diffuse bilaterally through the polymer matrix pores. In addition, this matrix can be associated with IS, thus diminishing systemic side effects. In this context, this review will address the natural biomaterials currently in use and their importance in cell therapy.
... These cells express stem cell markers CD90, CD73, CD105 and possess ability to differentiate into cells of various lineages 12,13 . CSSC can suppress corneal scar formation in lumican knockout 10 and Col3a1-EGFP 14 genetic mouse corneal scar models and prevent corneal scar formation in an acute corneal wound model 11,15 . This finding opens the door for stem cell-based therapy for the treatment of corneal scars without corneal transplantation. ...
... Next, it was examined whether the harvested secretome can induce regeneration in wounded mouse corneas. A corneal wound removing the corneal epithelium, Bowman's member, and superficial stroma was induced using an Algerbrush II as previously described 11,15,19 . Immediately after wound, 0.5µl of 100U/ml thrombin was applied to the wound site, and1µl of 1:1 mixture of 10mg/ml fibrinogen and 25X concentrated secretome (either from CSSC or fibroblasts) or medium as sham control was added to the thrombin on top of the corneal wound to form fibrin gel attaching to the wounded corneal surface. ...
... ; https://doi.org/10.1101/2022.05.07.490347 doi: bioRxiv preprint Similar to our previously reports 1,18 , the CSSC used in this study expressed the stem cell markers CD90, CD73, CD105, OCT4, ABCG2 etc., indicating their stemness. Our group and others have reported the potential of CSSC to promote corneal wound healing in a variety of corneal scarring models 10,11,15,34 . Previous reports have explored the role of stem cell-derived paracrine factors and extracellular vesicles (EVs) for corneal wound healing by upregulation of Akt signaling pathway 35 or by delivery of miRNAs 36 . ...
Corneal scarring is a leading cause of blindness in the world. In this study, we explored the therapeutic potential of corneal stromal stem cell (CSSC)-derived secretome in a mechanical debridement mouse model of corneal scarring. CSSC secretome was able to promote scarless corneal wound healing. The mechanisms include 1) dampening inflammation with reduced CD45+, CD11b+/GR1+, and CD11b+/F4/80+ inflammatory cells in the wounded corneas; 2) reducing fibrotic extracellular matrix deposition such as collagen IV, collagen 3A1, SPARC, and α-SMA; 3) rescuing sensory nerves. The proteomic analysis shows upregulated proteins related to wound healing and cell adhesion which boost scarless wound healing. It also shows upregulated neuroprotective proteins in CSSC secretome related to axon guidance, neurogenesis, neuron projection development, and neuron differentiation. Four unique complement inhibitory proteins CD59, vitronectin, SERPING1, and C1QBP found in CSSC secretome contribute to reducing a complement cascade mediating cell death and membrane attacking complex autoantibodies after corneal injury. This study provides novel insights into mechanisms of stem cell secretome induced scarless corneal wound healing and neuroprotection and identifies regenerative proteins in the CSSC secretome.
Significance Statement
In this study, we report the therapeutic role of corneal stromal stem cell (CSSC) secretome for scarless corneal wound healing and corneal sensory nerve rescuing. We uncovered that CSSC secretome dampens inflammation, reduces fibrosis, induces sensory nerve regeneration, and rescues corneal cells by inhibiting the complement system in the wounded mouse corneas. This study provides pre-clinical evidence for the use of CSSC secretome as a biologic treatment for corneal scarring to prevent corneal blindness. We delineated a plethora of proteins in the CSSC secretome, which individually or in combination have the potential as future therapies for scarless corneal wound healing.
... These beneficial features lead to regenerating clear corneas in pre-clinical models of corneal wounding. Both topical application and stromal injection of CSSCs remodelled the defective stromal structure, replaced the fibrotic ECM proteins (like Col3A1, hyaluronan and FN, produced by the activated stromal fibroblasts), and restored the native stromal ECM, resulting in better light passage with reduced deviation and scattering [7,10,11,40]. Moreover, the antiscarring effect of CSSCs was shown to be associated with significant reduction of corneal inflammation (inhibiting neutrophil infiltration) [9]. ...
Introduction: Corneal blindness due to scarring is treated with corneal transplantation. However, a global problem is the donor material shortage. Preclinical and clinical studies have shown that cell-based therapy using corneal stromal stem cells (CSSCs) suppresses corneal scarring, potentially mediated by specific microRNAs transported in extracellular vesicles (EVs). However, not every CSSC batch from donors achieves similar anti-scarring effects. Objectives: To examine miRNA profiles in EVs from human CSSCs showing “healing” versus “non-healing” effects on corneal scarring and to design a tool to select CSSCs with strong healing potency for clinical applications. Methods: Small RNAs from CSSC-EVs were extracted for Nanostring nCounter Human miRNA v3 assay. MicroRNAs expressed > 20 folds in “healing” EVs (P
... Unlike keratocytes, CSSCs are able to undergo extensive expansion in vitro without losing the ability to differentiate into keratocyte phenotype [29]. CSSCs embedded in compressed collagen gel has been injected into the injured cornea of mouse which resulted in successful regeneration without scar formation after 2 weeks [39]. Compared with the CSSCs, MSCs are easier to obtain from either bone marrow (BMSC) or adipose tissues (ASC), and their self-renewal ability has been proven. ...
Corneal injury is a commonly encountered clinical problem which led to vision loss and impairment that affects millions of people worldwide. Currently, the available treatment in clinical practice is corneal transplantation, which is limited by the accessibility of donors. Corneal tissue engineering appears to be a promising alternative for corneal repair. However, current experimental strategies of corneal tissue engineering are insufficient due to inadequate differentiation of stem cell into keratocytes and thus cannot be applied in clinical practice. In this review, we aim to clarify the role and effectiveness of both biochemical factors, physical regulation, and the combination of both to induce stem cells to differentiate into keratocytes. We will also propose novel perspectives of differentiation strategy that may help to improve the efficiency of corneal tissue engineering.
... Besides, MSCs can differentiate and transdifferentiate into dermal or epidermal cells [86]. Human corneal stem cells suppress corneal scarring in the mouse model even after general cryopreservation [87]. ...
Over the last decade, stem cell-associated therapies are widely used because of their potential in self-renewable and multipotent differentiation ability. Stem cells have become more attractive for aesthetic uses and plastic surgery, including scar reduction, breast augmentation, facial contouring, hand rejuvenation, and anti-aging. The current preclinical and clinical studies of stem cells on aesthetic uses also showed promising outcomes. Adipose-derived stem cells are commonly used for fat grafting that demonstrated scar improvement, anti-aging, skin rejuvenation properties, etc. While stem cell-based products have yet to receive approval from the FDA for aesthetic medicine and plastic surgery. Moving forward, the review on the efficacy and potential of stem cell-based therapy for aesthetic and plastic surgery is limited. In the present review, we discuss the current status and recent advances of using stem cells for aesthetic and plastic surgery. The potential of cell-free therapy and tissue engineering in this field is also highlighted. The clinical applications, advantages, and limitations are also discussed. This review also provides further works that need to be investigated to widely apply stem cells in the clinic, especially in aesthetic and plastic contexts.
... As the progress in tissue engineering field, development of new techniques will provide an innovation to solve the existing problems of stem cell transplantation. It was reported that improvement the therapeutic effect of stem cells has been achieved by tissue engineering modified methods in the treatment of a variety of diseases [12,13]. In TBI models, s series of hydrogels were developed as neural scaffolds by encapsulating stem cells and bioactive factors to repair cerebral function due to its three-dimensional network structure which is similar to neural tissue [14][15][16]. ...
Injectable hydrogel has the advantage to fill the defective area and thereby shows promise as therapeutic implant or cell/drug delivery vehicle for tissue repair. In this study, an injectable hyaluronic acid hydrogel in situ dual-enzymatically cross-linked by galactose oxidase (GalOx) and horseradish peroxidase (HRP) was synthesized and optimized, and the therapeutic effect of this hydrogel encapsulated with bone mesenchymal stem cells (BMSC) and nerve growth factors (NGF) for traumatic brain injury (TBI) mice was investigated. Results from in vitro experiments showed that either tyramine-modified hyaluronic acid hydrogels (HT) or NGF loaded HT hydrogels (HT/NGF) possessed good biocompatibility. More importantly, the HT hydrogels loaded with BMSC and NGF could facilitate the survival and proliferation of endogenous neural cells probably by neurotrophic factors release and neuroinflammation regulation, and consequently improved the neurological function recovery and accelerated the repair process in a C57BL/6 TBI mice model. All these findings highlight that this injectable, BMSC and NGF-laden HT hydrogel has enormous potential for TBI and other tissue repair therapy.
