Department of Transplantation Immunology

Research of this Department is focused on the isolation, characterization and cultivation of stem cells and their use for treatment of severe injuries or so far uncurable diseases. Stem cells are propagated in tissue cultures and using various nanofiber scaffolds transferred onto mechanically or chemically damaged ocular surface. The ability of transferred cells to inhibit a harmful inflammatory immune reaction ocurring in the site of injury and to support healing process is evaluated. The ultimate goal of the research is to get insights into the mechanisms of specific immune response after transplantation of stem cells with the aim to increase their antiinflammatory and therapeutic potential. The experience with the study of transplantation immunity and the combination of nanotechnologies with stem cell research enable to propose and test novel therapeutic approaches. The recent study is extended on the development of stem cell-based therapy for currently uncurable serious sight-threatening retinal diseases.

1. Comparative study on the therapeutic potential of mesenchymal stem cells and tissue-specific limbal stem cells
The ability of mesenchymal stem cels (MSC) and limbal stem cells (LSC) to produce various immunoregulatory molecules and to modulate immune response was compared. The therapeutic potential of these stem cells was evaluated in the model of treatment of chemically damaged ocular surface in the rabbit. It was found that stem cells produce a number of immunoregulatory molecules and that MSC have comparable therapeutic properties as do tissue-specific LSC. These results bring the evidence that MSC can replace tissue-specific LSC in the cases when LSC are absent or difficult to obtain.

Characterization of MSC. The ability of MSC to express genes for immunomodulatory molecules was determined by real-time PCR. The cells were cultured untreated or were stimulated with LPS, IFN-γ with LPS plus IFN-γ

Therapeutic potential of MSC and LSC. Stem cells were transferred using nanofiber scaffold onto damaged ocular surface and their therapeutic potential was evaluated. The figure shows healthy rabbit eye, the eye shortly after chemical damage, damaged eye covered with nanofiber scaffold, untreated damaged eye two weeks after the injury and damaged eye treated with MSC or LSC

Publications:

Holáň V., Trošan P., Čejka Č., Javorková E., Zajícová A., Heřmánková B., Chudíčková M, Čejková J.: A comparative study of the therapeutic potential of mesenchymal stem cells and limbal epithelial stem cells for ocular surface reconstruction. Stem Cells Translat. Med. 4, 1052-1063, 2015. IF 5.709.
Heřmanková B., Zajícová A., Javorková E., Chudíčková M., Trošan P., Hájková M., Krulová M., Holáň V.: Suppression of IL-10 production by activated B cells via a cell contact-dependent cyclooxygenase-2 pathway upregulated in IFN-γ-treated mesenchymal stem cells. Immunobiology 221, 129-136, 2016. IF 3.044. 

1. The effects of cytokines on migration and therapeutic potential of mesenchymal stem cells in a model of local inflammatory reaction in damaged eye 

Using a well established model of chemically damaged ocular surface in mice we showed that systemically administered mesenchymal stem cells (MSCs) selectively migrate into the site of injury and inhibit infiltration with cells of of the immune system. The ability of MSCs to inhibit local inflammatory reaction can be modulated by their preincubation with cytokines which determine the development of T and B lymphocytes and regulate the secretory capacity of MSCs. The results thus show the possibilities to use cytokine-pretreated and systemically administered MSCs for the treatment of local inflammatory reaction.

Phenotype characterization of MSCs (A), labeling of MSCs with vital dye PKH26 (B, C) and detection of labeled MSCs in the injured eye (D).

Publications:

Javorková E, Trošan P, Zajícová A, Krulová M, Hajková M, Holáň V, (2014): Modulation of the early infl ammatory microenvironment in alkaliburned eye by systemically administered interferon treated mesenchymal stem cells. Stem Cells Dev. 23, 2490-2500. IF 4.202

Holáň V, Zajícová A, Javorková E, Trošan P, Chudičková M, Pavlíková M, Krulová M, (2014): Distinct cytokines balance the development of regulatory T cells and IL-10-producing regulatory B cells. Immunology 141, 577-586. IF 3.3735

1. Regeneration of severely damaged ocular surface by stem cells

Limbal and mesenchymal stem cells were cultured in vitro on nanofiber scaffolds and transferred on the damaged ocular surface in experimental animal models. The therapeutic effects of stem cells were evaluated by histology and according to the ability to inhibit expresssion of genes for proinflammatory molecules IL-2, IFN-gama, IL-17 and iNOS. The results thus demonstrate the ability of stem cells and nanofiber scaffolds to regenerate ocular surface after the injury. 

Collaboration: Evropská oční klinika Lexum, Praha. 

A scheme of immunoregulatory and therapeutic effects of mesenchymal stem cells. There are number of diferent mechanisms by which MSC contribute to the healing process. Differentiation into epithelial-like cells, immunomodulation and the production of growth and trophic factors are the most important mechanisms.

A structure of nanofiber scaffold (A) and the growth of mesenchymal stem cells on nanofiber scaffold (B, C). (A) Scanning electron microscopy image of nanofibers from copolymer polyamid 6/12, the avarage nanofiber diameter is 380 nm, (B) Mouse MSC growing on nanofibers (red filaments – F-actin stained with phalloidin, blue – DAPI staining of the nuclei), (C) Scanning electron microscopy of mouse MSC growing on nanofibers.

Chemically damaged eye of the rabbit covered by a nanofiber scaffold with stem cells. The application of a nanofiber scaffold seeded with MSC onto the damaged ocular surface of a rabbit.

Publications:

Holáň, V., Javorková, E.: (2013) Mesenchymal stem cells, nanofiber scaffolds and ocular surface reconstruction. Stem Cells Rev. Rep. 9(5), 609-619. IF 4.523

Čejková J., Tošan P., Čejka Č., Lenčová A., Zajícová A., Javorková E., Kubinová Š., Syková E., Holáň V.: (2013) Suppression of alkali-induced oxidative injury to the cornea by mesenchymal stem cells growing on nanofiber scaffolds and transferred onto the damaged corneal surface. Exp. Eye Res. 116, 312-323. IF 3.026

Holáň, V., Javorková, E., Trošan, P.: The growth and delivery of mesenchymal and limbal stem cells using copolymer polyamide 6/12 nanofiber scaffolds. In: Wright, B. and Connon, C. J. (eds), Corneal Regenerative Medicine, Methods Mol. Biol., Humana Press – Springer, New York, London 2013, S. 187-199. ISBN 978-1-62703-431-9.

2. Distinct roles of cytokines in the development of regulatory T cells and regulatory B cells

The role of cytokines in development of regulatory T (Treg) and B (Breg) cells was described. TGF-beta was shown as the main cytokine determining the development of Tregs, but this cytokine inhbited activation of IL-10-producing Breg. On the contrary, another two cytokines, IL-12 and IFN-gama, enhanced the development of Breg. These results show distinct roles of cytokines in development of Treg and Breg, and suggest new approaches for targeted regulation of the immune system.

Collaboration: Přírodovědecká fakulta UK, Praha

(A) Characterization of B cell populations by flow cytometry. (B) Detection of cells producing IL-10 using technology ELISPOT.

Publications:

Holáň, V., Zajícová, A., Javorková, E., Trošan, P., Chudičková, M., Pavlíková, M., Krulová, M.: (2013) Distinct cytokines balance the development of regulatory T cells and IL-10-producing regulatory B cells. Immunology. In press. IF 3.705

Kubera M., Curzytek K., Duda W., Leskiewicz M., Basta-Kaim A., Budziszewska B., Roman A., Zajícová A., Holáň V., Szczesny E., Lason W., Maes M.:. (2013) A new animal model of (chronic) depression induced by repeated and intermittent lipopolysaccharide administration for 4 months. Brain Behav. Immun. 31, 96-104. IF 5.612

Holáň, V., Krulová, M.: (2013) Common and small molecules as the ultimate regulatory and effector mediators of antigen-specific transplantation reactions. World J. Transplant. 3(4), 54-61. (nový časopis, zatím bez IF)

The Laboratory of Eye Histochemistry and Pharmacology examines the causes of the bad healing or non-healing lesions of the anterior eye segment after various injuries or ocular diseases and conditions necessary for regeneration of tissues of the anterior eye segment, particularly of the cornea, with the aim to restore visual functions. Great attention is devoted to the role of oxidative stress in the initiation or development of the intraocular inflammation. In regenerative processes of ocular tissues the antiinflammatory and antioxidative effects of limbal epithelial stem cells and mesenchymal stem cells are investigated. For this reason the immunohistochemical, biochemical and biophysical methods are employed.

Assoc. Prof. Jitka Čejková, MD, DSc | Head of Laboratory

E-mail: cejkova@biomed.cas.cz
Phone: +420 241 062 208

Research Scientists:

Assoc. Prof. Jitka Čejková, MD, DSc
Čestmír Čejka, MSc, PhD

Laboratory technician:

Jana Herlova

Graduated students:

Tomanová Aneta Bc.

Vašková Věrča Bc.

Švandová Ivana Bc.

Bayerová Martina Bc.

Important results in 2015

Limbal epithelial stem cells (LSCs) and mesenchymal stem cells (MSCs) cultured in vitro on nanofiber scaffolds and transferred on the damaged rabbit cornea (with 0.25N NaOH) highly suppressed corneal neovascularization appearing in untreated alkali injured eyes. Corneal neovascularization is dangerous particularly from the decrease of visual acuity and changes in corneal optical properties. MSCs prepared from bone marrow (BM-MSCs) revealed better results than MSCs from adipose tissue (Ad-MSCs). In suppression of corneal neovascularization and in the corneal healing without untransparent scar formation the therapeutic effect of BM-MSCs was comparable with the effect of LSCs.

Immunohistochemical detection of VEGF expressions and corneal neovascularization. On day 12 after corneal alkali injury and tretment with BM-MSCs or LSCs incubated and treansferred on nanofiber scaffolds onto the damaged corneal surface, the corneal neovascularization was highly suppressed.

A - control (healthy cornea), B - untreated alkali injured cornea, C - alkali injured cornea treated with stem cell free nanofibers, D - alkali injured cornea treated with BM-MSCs, E - alkali injured cornea treated with LSCs, F - alkali injured cornea treated with Ad - MSCs, G - negative control, H - the number of vessels per sector of the corneal surface. Scale bars : 50 µm.

Publications:

Holáň V., Trošan P., Čejka Č., Javorková E., Zajícová A., Heřmánková B., Chudíčková M, Čejková J.: A comparative study of the therapeutic potential of mesenchymal stem cells and limbal epithelial stem cells for ocular surface reconstruction. Stem Cells Translat. Med. 4, 1052-1063, 2015. IF 5.709.

Čejka Č., Čejková J. : Oxidative stress to the cornea, changes in corneal optical properties, and advances in treatment of corneal oxidative injuries. Oxid Med Cell Longev. 2015;2015:591530. doi: 10.1155/2015/591530. Epub 2015 Mar 11. IF 3.516

1. The importance of oxidative stress in the cornea after its injury

Oxidative stress in the cornea has an important role in the initiation as well as propagation of corneal disturbances after its injury, e.g. after irradiation with UV rays, chemical injury, mainly after alkali burns. In the injured cornea an imbalance between oxidants and antioxidants appears, naturally occurring inhibitors of matrix metalooproteinases are inhibited. The intracorneal inflammation develops, the corneal transparency is lost and corneal neovascularization appears.

Oxidative stress plays and important role in ocular diseases and injuries. Early diagnosis of oxidative stress plays the key role for choice of the mode of treatment. We found that impression cytologies of corneal epithelial cells with immunoghistochemical markers enable rapid and easy diagnostics of oxidative injuries, usable in clinical medicine. Thus the method of corneal impression cytology is not only a method suitable for the examination of severity of corneal damage, e.g. in dry eye disease as was described previously by us, however, also for sensitive detection of early corneal disturbances evoked by oxidative injury. Nitrotyrosine expression, a marker of oxidative stress, a – in corneal impression cytology after corneal injury, b- in cryostat corneal section after corneal injury, c – negative expression of nitrotyrosine incorneal imporession cytology, healthy eye, d - negative expression of nitrotyrosine in cryostat section of the cornea, healthy eye. Similarly e – h is expression of malondialdehyde, a marker of lipid peroxidation in corneal impression cytology and corneal cryostat section of injured and healthy corneas.

The role of oxidative stress after various injuries was compared in the cornea, e.g. after UVB radiation or alkali burns. The antioxidant/prooxidant imbalance appears in the cornea (oxidative stress) leading to the inhibition of naturally existing inhibitors of metalloproteinases and serine proteases. Destructive proteases, pro-inflammatory cytokines and nitric oxide synthases are generated and activated. Nitrotyrosine and malondialdehyde (markers of oxidative stress and lipid peroxidation) occur in the cornea. The intraocular inflammation develops and cornea is vascularized.

Publication: Čejková J, Čejka C, (2015): The role of oxidative stress in corneal diseases and injuries. Histol Histopathol. 11611– Epub. [ahead of print] IF 2.236

1. New regenerating agent (RGTA, CACICOL 20) for the healing of chronic corneal injuries and diseases

We proved that new regenerating agent (RGTA, CACICOL20) is effective for the healing of corneal injuries, particularly of poorly healing chronic ulcers. Following RGTA topical application the expression of proteolytic enzymes (mainly metalloproteinases) and enzymes that generate nitric oxide gradually decreased, accompanied by the positive corneal healing without scarring. Corneal neovascularization and inflammation were reduced. Corneal transparency recovered totally or at least partially.

Representative photographs of rabbit eyes with ulcers developed after the injury of the cornea with 1.0 N NaOH and subsequently treated with RGTA or placebo. a - The control eye; b - The cornea of the first eye one day after the injury with 1.0 N NaOH. Corneal transparency is lost; c - The cornea of the first eye after one month following the injury (the cornea without any treatment). Corneal ulcer develops (black arrow); d - The cornea of the first eye after subsequent treatment with RGTA for one month. The ulcer is healed (black arrow); e – The cornea of the second eye one day after the injury with 1.0 N NaOH. The corneal transparency is lost. f – The cornea of the second eye after one month – for this time interval the cornea was without any treatment. The central corneal ulcer develops (arrow). g – The cornea of the second eye after four weeks of subsequent placebo treatment. The ulcer is developed (black arrow) and highly persisted (h). The cornea is vascularized.

Expression of genes for proinflammatory cytokines in alkali-injured corneas from placebo- and RTGA-treated eyes. Corneas from the eyes injured by alkali and treated with placebo or RGTA were harvest on day 7 (after 0.15 N NaOH) (A) or day 21 (after 1.0 N NaOH) (B) and the expression of genes for IL-1beta (a), IFN- (b) and iNOS (c) were determined by real-time PCR. Each bar represents the mean +-S.D. from at least 3 determinations. Values with asterisks are significantly (*P<0.05, ***P<0.01) different from placebo treated corneas.

Collaboration: Thea Laboratoires, Clermont Ferrand, France (Dr.Celine Olmiere), Department of Transplantation Immunology, (prof. RNDr Vladimír Holáň, DrSc.)

Publication: Cejkova J, Olmiere C, Cejka C, Trosan P, Holan V (2013). The healing of alkali-injured cornea is stimulated by a novel matrix regenerating agent (RGTA, CACICOL20): a biopolymer mimicking heparan sulfates reducing proteolytic, oxidative and nitrosative damage. Histol Histopathol 2013 Oct 9. (29,457-78,2014).IF 2,281.

2. Rabbit mesenchymal stem cells growing on nanofiber scaffolds heal oxidative injury of the cornea evoked by alkali burns

Rabbit mesenchymal stem cells growing on nanofiber scaffolds and transferred onto the damaged corneal surface healed oxidative injury of the cornea evoked by alkali. Alkali injury evoked the imbalance between corneal oxidants and antioxidants leading to the increased expression of pro-inflammatory cytokines, and to the formation of nitrotyrosine and malondialdehyde. Mesenchymal stem cells significantly reduced corneal oxidative stress and healed the cornea with the restoration of transparency.

Representative photograps of alkali-injured corneas (with 0.15 N NaOH) (A-G), corneal neovascularization (H) and vascular endothelial growth factor (VEGF) (I) on day 15 after the injury. A - healthy eye; B - immediately after the injury the cornea turs grayish; C - injured cornea on which nanofiber scaffolds with mesenchymal stem cells (MSCs) were transferred and sutured with conjunctiva; D, E - In alkali-injured cornea untreated with MSCs high neovascularization (grade 4) (D) or middle neovascularization (grade 3) (E) appeared. E, F - In contrast, in alkali injured cornea treated with MSCs only low neovascularization developed (grade 2) (F) or only solely vessels could be found (grade 1) (G). H - Quantification of corneal neovascularization by counting vessels. I – Quantification of corneal neovascularization using real-time PCR (the expression of genes for VEGF).

Haematoxylin-eosin staining (HE) (a,b), expression of Vascular endothelial factor (VFGF) (C-F) and expression of macrophages (g-j) on day 15 after the injury of the cornea with 0.15 N NaOH. a – In the injured untreated cornea numerous inflammatory cells are present in the vascularized stroma (arrows); b – In contrast, the injured cornea treated with MSCs is without vessels and the number of inflammatory cells is highly reduced; c, e – In untreated injured cornea VEGF expression is highly pronounced, whereas in injured cornea treated with MSCs VEGF expression is low, nearly as in the control cornea (f). In untreated injured cornea high number of macrophages is seen (g, i). In contrast, in injured cornea treated with MSCs only solely macrophages can be found (h); j – Control cornea, macrophages are not present.

Collaboration: Department of Neuroscience (prof. MUDr Eva Syková, DrSc.), Department of Transplantation Immunology (prof. RNDr Vladimír Holáň, DrSc.)

Publication: Cejkova J, Trosan P, Cejka C, Lencova A, Zajicova A, Javorkova E, Kubinova S, Sykova E, Holan V (2013). Suppression of alkali-induced oxidative injury in the cornea by mesenchymal stem cells growing on nanofiber scaffolds and transferred onto the damaged corneal surface. Exp Eye Res 116:312-323.IF 3,026.