the neural crest: its derivatives and stem cellsanat.lf1.cuni.cz/english/vyuka/pvp/grimnc.pdf ·...
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W. HisW. His ((18681868))
The Neural Crest: The Neural Crest: its Derivatives and Stem Cells its Derivatives and Stem Cells
MiloMilošš GrimGrimInstitute of Anatomy, First Faculty of Medicine,
Center for Cell Therapy and Tissue RepairCharles University Prague
April 13, 2011
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The structure of the lecture:The structure of the lecture:1)1) Why to study the neural crest (NC) Why to study the neural crest (NC) 2)2) Origin and development of the NCOrigin and development of the NC3)3) Methods of labeling of the NC cellsMethods of labeling of the NC cells4)4) Cell types differentiated from the NC Cell types differentiated from the NC 5)5) Developmental disorders of the NCDevelopmental disorders of the NC6)6) NC cells in epidermis NC cells in epidermis –– in hair follicle in hair follicle 77) Neural crest stem cells (NCSCs) in the hair follicle) Neural crest stem cells (NCSCs) in the hair follicle88) The use of NCSCs in regenerative medicine) The use of NCSCs in regenerative medicine
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WWhyhy to study to study tthe neural crest (NC) cells?he neural crest (NC) cells?
•• Cells of NC participate in development of almost all organs originating from ectoderm, mesoderm and endoderm
•• NC is the 4th embryonic layer of craniates•• Model system for studies of embryonic induction, cell determination, differentiation and epithelial-mesenchymal transition
•• Model system for the study of cell invasivity –development of carcinoma metastasis
•• Defective development of the NC leads to developmental malformations
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Origin and development Origin and development of the NCof the NC
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Induction and epitheliInduction and epithelialal--mesenchymal transition mesenchymal transition of NC cells of NC cells
B.M. Carlson (1999)
HH 10ISH ISH ofof mRNA for mRNA for SlugSlug
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Migration of NCMigration of NC cellscells in vivoin vivo and and in vitroin vitro
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HNK-1tenascin
fibronectin
Molecular mechanisms of NC cells migrationMolecular mechanisms of NC cells migration• Permisive contactPermisive contact--guidance + chemorepelent moleculesguidance + chemorepelent molecules
• ScatterScatter factorfactor /c/c--met receptor, Pax3 met receptor, Pax3 •• SteelSteel factorfactor (stem cell (stem cell factorfactor) /c) /c--kitkit receptorreceptor•• ChemokinChemokin SDFSDF--1 / CXCR4 receptor1 / CXCR4 receptor
• ParacrineParacrine signalingsignalingsystemssystems::
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Local signaling during migration of NC cells induces Local signaling during migration of NC cells induces differentiated gene expression. Pluripotent NC cells differentiated gene expression. Pluripotent NC cells successively differentiate in bipotent and unipotent successively differentiate in bipotent and unipotent cellcell types types
Regional identity and differentiation of NC cellsRegional identity and differentiation of NC cells
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Methods of labeling of the NC cellsMethods of labeling of the NC cells
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ED 17
ED 3 LabelingLabeling systemsystem exploringexploring differentdifferentorganisationorganisation ofof perinucleolarperinucleolarchromatine in chromatine in chickchick (C) (C) andand japanesejapanesequailquail (Q) in (Q) in theirtheir embryonicembryonic chimeraschimeras
CC
QQCC
CC
GrimGrim M, M, HalataHalata Z. Z. AnatAnat EmbryolEmbryol 202: 401, 2000202: 401, 2000
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LabelingLabeling of NC cells in mammalsof NC cells in mammals
•• using reporter using reporter gene gene lacZ lacZ ((E. coliE. coli)) inintransgentransgenicic mouse mouse Wnt1/lacZWnt1/lacZ
•• permanent expression of permanent expression of lacZ genelacZ geneinin ccrere--lox system lox system inintransgentransgenicic mouse mouse Wnt1Wnt1--cre/cre/R26RR26R
•• detection of detection of laclacZ gene expression Z gene expression indigogenindigogenicic method (method (XXggalal) at pH 7.5 ) at pH 7.5 imimmmunohistochemicalyunohistochemicaly ((BgalBgal) () (rabbit rabbit antianti--E. coliE. coli --galaktosidase; Chemicon)galaktosidase; Chemicon)
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BgalBgalXgal
ED 9.5
IdentificationIdentification ofof neuralneural crestcrest cellscells
in in Wnt1Wnt1--lacZlacZ/ / ++mousemouse
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The Wnt1The Wnt1--cre / R26R twocre / R26R two--componentcomponentsystem to label neural crest cellssystem to label neural crest cells
WntWnt--1 promoter1 promoter(neural crest specific)(neural crest specific)
Cre-recombinase
Wnt1Wnt1--cre / + mousecre / + mouse
CreCre--recombinaserecombinase recognizesrecognizesloxPloxP sites and cutssites and cuts
--galgal
--galactosidasegalactosidaseis expressed is expressed permanentlypermanently
R26R / + mouseR26R / + mouse
loxP loxP
STOP --galgalR26RR26Rpromoter (ubiquitous)promoter (ubiquitous)
Wnt1Wnt1--cre / R26R mousecre / R26R mouseloxP loxP
STOP -galR26RR26RPromoterPromoter
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Cell types differentiated from the NCCell types differentiated from the NC
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ED13, B gal
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4a) Derivatives of NC of the trunk4a) Derivatives of NC of the trunk
HNK-1
Neurons of spinal ganglia, of autonomic ganglia, enteric neuronsNeurons of spinal ganglia, of autonomic ganglia, enteric neurons, , Schwann cells, pigment cells, cells of adrenal medullaSchwann cells, pigment cells, cells of adrenal medulla
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Migration of cranial neural crest cells
Wnt1-cre/R26R mouse
B.M. Carlson (1999)
ED 9.5Xgal
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Migration of NC cells of the headMigration of NC cells of the head
osteoblastsosteoblasts, fibroblasts,, fibroblasts,chondroblastschondroblasts, smooth , smooth muscle cells, muscle cells, odontoblastsodontoblastsCardiac NC (R4Cardiac NC (R4--R8)R8)::for cfor cardiacardiac ooutflowutflow tract tract N. LeDouarin (1999)ED4 chick
Ectomesenchyme:
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Derivatives of the NC in the headDerivatives of the NC in the head a part of sensory ganglia of V., VII., IX., X. a part of sensory ganglia of V., VII., IX., X. parasymp. ganglia and their satellite cells parasymp. ganglia and their satellite cells Schwann cells of glomus caroticum Schwann cells of glomus caroticum parafolicullar C parafolicullar C –– cells (calcitonin),cells (calcitonin), melanocyte, melanocyte, EEcctomesenchymetomesenchyme –– osteoblasts, fibroblasts, osteoblasts, fibroblasts, chondroblasts, smooth muscle cells in anterior part chondroblasts, smooth muscle cells in anterior part of the head, odontoblasts, pia mater, arachnoidea, of the head, odontoblasts, pia mater, arachnoidea, stromal cells of cornea, smooth muscle cells of iris stromal cells of cornea, smooth muscle cells of iris stromal cells of thymus, thyroid and parathyroid stromal cells of thymus, thyroid and parathyroid gland, salivary glands, lacrimal gland gland, salivary glands, lacrimal gland
Cardiac neural crest Cardiac neural crest –– outflow tract, wall of large outflow tract, wall of large branches of aortic arch branches of aortic arch
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JiangJiang et et alal., ., DevDev BiolBiol 241:106, 2002241:106, 2002
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Fate Maps of Neural Crest and Mesoderm in the Mammalian EyeP. J. Gage, W. Rhoades, S. K. Prucka and T. Hjalt, Invest Ophthalm & Vis Sci. 46:4200 – 8, 2005
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ToothTooth developmentdevelopment((Wnt1Wnt1--crecre/R26R) (/R26R) (ChaiChai et et alal. . DevelopmentDevelopment 127:1671, 2000)127:1671, 2000)FromFrom thethe NC NC originateoriginateodontoblastsodontoblasts, , cementoblastscementoblasts, , periodontiumperiodontium
Xgal
Bgal
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CardiacCardiac neuralneural crestcrestR 4. R 4. –– R 8. R 8.
Wnt1Wnt1--crecre/R26R /R26R transgenictransgenic mousemouse, , Xgal , Xgal , 7. 7. –– 9. 9. weekweekJiangJiang et et alal.: .: DevelopmentDevelopment 127:1607, 2000127:1607, 2000
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Developmental disorders of the NCDevelopmental disorders of the NC
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DevelopmentalDevelopmental defectsdefects ofof thethe NCNC CHARGE syndromCHARGE syndrom ((CColobomaoloboma iridisiridis, , HHearteart
defectsdefects, , AAtresiatresia choanaechoanae, , RRetardationetardation ofofdevelopmentdevelopment, , GGenitalenital hypoplasiahypoplasia in in malesmales, , EEarar anomaliesanomalies))
DiGeorgeDiGeorge syndromsyndrom ((hypofunctionhypofunction ofofparathyroidparathyroid andand thyroidthyroid glandgland, , thymusthymushypoplasiahypoplasia, , defectsdefects ofof septationseptation ofof aorta aorta andandpulmonarypulmonary trunktrunk
anomaliesanomalies ofof teethteeth albinismalbinism WaardenburgWaardenburg syndromsyndrom ((Pax3Pax3 mutationmutation ––
pigmentationpigmentation defectsdefects, , defectsdefects ofof limb limb musclesmuscles, , cleftcleft palatepalate, , cardiovascularcardiovascular defectsdefects, , hypertelorismhypertelorism))
HirschsprungHirschsprung diseasedisease PiebaldismPiebaldism
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SpSp1H1H/+/+ SpSp1H1H/ Sp/ Sp1H1H
ED 13.5
Pax3Pax3 mutationmutation in in mousemouse ((splotchsplotch mutationmutation))
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PiebaldismPiebaldism ((pigmentationpigmentation defectsdefects, sterility, , sterility, anemiaanemia) ) mutationmutation ofof KITKIT gene in man gene in man andand KitKit gene in gene in mousemouse
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NNeuraleural crestcrest ccells in ells in epidermis epidermis –– in in hair folliclehair follicle
Merkel cellMerkel cells, melanocytes, stem s, melanocytes, stem cellscells
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Merkel cells Merkel cells -- large light cells in the basal layer of large light cells in the basal layer of epidermis and epidermis and in in mucous membranes of ectodermal origin mucous membranes of ectodermal origin in vertebratesin vertebrates
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F. S. F. S. MerkelMerkel (1875) : (1875) : „„TastzellenTastzellen““ ofof thethe skin skin ofof birdsbirds andand mammalsmammals
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Merkel cells are transducers of tactile stimuli in slowly adapting mechanoreceptors of the skin
Human MCs represent 3.6 - 5.7% of basal epidermal cellsfrom glabrous and hairy skin (Fradette et al., 2003)
Halata Z, Grim M, Bauman K: Anat Rec 271A: 225, 2003
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Merkel cells in whisker hair follicle
K8
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ED 10
ED 13
ED 13
BgalBgal/H /H Wnt1Wnt1--crecre/R26R/R26R
NeuralNeural crestcrest cellscells in epidermis in epidermis –– in in hairhair folliclefollicle
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E.M.J.Peters (2002)
c-kit-positive melanoblasts
c-kit / hem
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NeuralNeural crestcrest stem stem cellscells in in thethe hairhair folliclefollicle
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Growth cycle of human scalp hair
Anagen2-6 years
Catagen1-2 weeks
Telogen5-6 weeks
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Math1 TRITC Ki67 FITC
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Dissection of the bulge from adult whisker follicle
Xgal + NCCs emigrated 4 days after explantation
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Bulge explant-derived NCCsexpress nestin and Sox10
(day 5 in culture)
Anti-nestin Ab + DAPI Anti-Sox10 Ab + DAPI
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Bulge explant-derived NCCs are pluripotentSchwann cells
MelEM
anti-SMA anti-ß-III tubulin DAPI
Xgal
Smooth muscle cells Neurons
Xgal
anti-S-100
SCIP Ab
Melanocytes Chondrocytes
Anti-collagen II Ab
Dev Dyn 231:258-269, 2004; Embryo Today 72:162-172, 2004Supp. by LN 00A065 and VZ 111100003-3
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Bulge-derived NCCs undergo self-renewal(determined by serial cloning)
6 h 18 h 72 h48 h
Primary clone
5-day-old secondary clone
cells from 2-weekssecondary clones
ß-III tubulin
anti-SMA
Xgal
Xgal
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Tissue source: skin biopsy from different body locations
Isolation of humanhuman epidermal neural creststem cells (hEPI- NCSCs) from hair follicles
Epidermis with folliclesafter dispase treatment
Primary culture cellsafter emigration fromisolated follicle
Sphere after readhesion
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Coexpressionof Sox10 and Nestin, Expression of Nanog
Sox10 + Nestin Nanog + DAPI
hEPI-NCSCs in tissue culture after emigration from back skin follicles
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Differentiation of human epidermal neural crest stem cells fom hairfollicles into neural crest progeny Folia Biol. (Praha) 56, 149-157, 2010
Smooth muscle actin
Beta III tubulin
S100
GFAP
Smooth muscle cells
NeuronsSchwann cells
Schwann cells
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•• UnitedUnited StatesStates Patent ApplicationPatent Application2006028117720060281177•• KindKind CodeCode A1 A1 SieberSieber--Blum; Blum; MayaMaya et et alal. . DecemberDecember 14, 2006 14, 2006 •• MethodMethod ofof isolatingisolating epidermalepidermal neuralneural crestcrest stem stem cellscells
•• InventorsInventors::•• SieberSieber--Blum Blum MayaMaya ((BrookfieldBrookfield, WI), WI)•• GrimGrim MilosMilos ((PraguePrague, CZ), CZ)
•• CorrespondenceCorrespondence NameName andand AddressAddress: QUARLES & BRADY LLP FIRSTAR PLAZA, : QUARLES & BRADY LLP FIRSTAR PLAZA, ONE SOUTH PINCKNEY STREET P.O BOX 2113 SUITE 600 MADISON ONE SOUTH PINCKNEY STREET P.O BOX 2113 SUITE 600 MADISON WI 53701WI 53701--2113 2113 USSerialUSSerial No.: 376498Series No.: 376498Series CodeCode: 11 : 11 FiledFiled: : MarchMarch 15, 200615, 2006
•• U.S. U.S. CurrentCurrent ClassClass:435/368; 435/371 U.S. :435/368; 435/371 U.S. ClassClass atat PublicationPublication:435/368; 435/371 :435/368; 435/371 InternIntern'l 'l ClassClass: C12N 5/08 20060101 C12N005/08: C12N 5/08 20060101 C12N005/08
•• AbstractAbstract•• TheThe presentpresent inventioninvention describesdescribes novel novel methodsmethods for for isolatingisolating a a
substantiallysubstantially purepure cell cell populationpopulation ofof nonnon--embryonicembryonic epidermalepidermalneuralneural crestcrest stem stem cellscells fromfrom thethe bulgebulge--region region ofof mammalianmammalian hairhairfolliclesfollicles. . AlsoAlso discloseddisclosed isis thethe substantiallysubstantially purepure cell cell populationpopulation ofoffollicularfollicular bulgebulge--derivedderived neuralneural crestcrest stem stem cellscells for for medicalmedical researchresearchandand therapeutictherapeutic use. use.
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Folia Biologica (Praha) 56, 149-157 (2010)
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The use of NCSCThe use of NCSCss in regenerative in regenerative medicinemedicine
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M. Sieber-Blum, L. Schnell, M.Grim,R.Schneider, ME Schwab: Characterization of epidermal neural creststem cell (EPI-NCSC) grafts in the lesioned spinal cord. Mol Cell Neurosci 32: 67 - 81, 2006
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NestinNestin GAD67GAD67 RIPRIP
Morphology of EPIMorphology of EPI--NCSC implants in the NCSC implants in the lesionedlesioned spinal cordspinal cord
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Epidermal neural crest stem cells (EPI-NCSC) are remnants of the embryonic neural crest that reside in an adult location, the bulge of hair follicles, and which can differentiate into all major neural crest derivatives.
Human EPI-NCSC can potentialy be used to treatfollowing conditions and diseses: Spinal cord injury, tissue engineering of heart valves, cardiac birthdefects, Hirschprung disease, craniofacialmalformations/injuries, bone fractures, peripheral neuropathies, pigmentation defects.
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Hu YF, Gourab K, Wells C, Clewes O, Schmit BD, Sieber-Blum M.Epidermal neural crest stem cell (EPI-NCSC)--mediated recovery of sensory functionin a mouse model of spinal cord injury. Stem Cell Rev. 2010 Jun;6(2):186-98.
Clewes O, Narytnyk A, Gillinder KR, Loughney AD, Murdoch AP, Sieber-Blum M. Human EpidermalNeural Crest Stem Cells (hEPI-NCSC)-Characterization and Directed Differentiation intoOsteocytes and Melanocytes. Stem Cell Rev. 2011 Apr 1. [Epub ahead of print]
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Halata Z, Grim M, Christ B: Origin of spinal cord meninges, sheaths of peripheral nerves, andcutaneous receptors including Merkel cells. An experimental and ultrastructural study with avianchimeras. Anat Embryol 182: 529 – 537, 1990
Grim M, Halata Z, Franz T: Schwann cells are not required for guidance of motor nerves in thehindlimb in Splotch mutant mouse embryos. Anat Embryol 186: 311 – 318, 1992
Grim M, Christ B: Neural crest cell migration into the limb bud of avian embryos. In: Limb development and regeneration. JF Fallon, PF Goetinck, RO Kelley, DL Stocum (eds). J. Wiley-Liss, Inc. 1993, pp. 391 - 402
Grim M, Halata Z: Developmental origin of Merkel cells in birds. In: Merkel Cells, Merkel Cell Carcinoma and Neurobiology of the Skin. eds.: H Suzuki, T Ono, pp. 23 - 32 Excerpta MedicaInternat. Congress Series 1187, Elsevier, 2000
Grim M, Halata Z: Developmental origin of avian Merkel cells Anat Embryol 202: 401 - 410, 2000 Grim M, Riedlbauchová L, Valášek P: Interaction of head mesoderm and cells of neural crest in the
chick. In: Origin and Fate of Somites. Eds.:E.J.Sanders, J.W.Lash, C.P. Ordahl, IOS Press, 2001,NATO Sci. Ser. I. Vol. 329, pp. 48 - 55
Szeder V, Grim M, Halata Z, Sieber-Blum M: Neural Crest Origin of Mammalian Merkel Cells. Dev Biol 253: 258–263, 2003
Halata Z, Grim M, Baumann KI: Merkelova buňka: morfologie, vývojový původ, funkce. Čas Lék čes 142: 4–10, 2003
Halata Z, Grim M, Baumann KI: Friedrich Sigmund Merkel and his “Merkel Cell”, morphology, development and physiology review and new results. Anat Rec 271A: 225-239, 2003
Grim M, Halata Z, Szeder V and M Sieber-Blum: Merkel Cells are Postmitotic Cells of Neural CrestOrigin. In: The Merkel Cell, eds. Baumann KI, Halata Z, Moll I, Springer-Verlag Berlin Heidelberg, 2003 pp. 89 – 96
Sieber-Blum M and Grim M: The adult hair follicle – cradle for pluripotent neural creststem cells. Embryo Today 72: 162 - 172, 2004
Sieber-Blum M, Grim M, Hu YF, Szeder V: Pluripotent Neural Crest Stem Cells in the Adult HairFollicle. Dev Dyn 231: 258 – 269, 2004
Sieber-Blum M, Schnell L, Grim M, Yao Fei Hu, Schneider R, Schwab ME: Characterization ofEpidermal Neural Crest Stem Cell (EPI-NCSC) Grafts in the Lesioned Spinal Cord. Molecular andCellular Neuroscience April 2006
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Thanks for your attention
Institute of Anatomy, First Faculty of Medicine, Charles University Prague, Czech Republic