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

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

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

Origin and development Origin and development of the NCof the NC

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

Migration of NCMigration of NC cellscells in vivoin vivo and and in vitroin vitro

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::

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

Methods of labeling of the NC cellsMethods of labeling of the NC cells

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

QQ

CC

QQCC

CC

QQ

GrimGrim M, M, HalataHalata Z. Z. AnatAnat EmbryolEmbryol 202: 401, 2000202: 401, 2000

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)

BgalBgalXgal

ED 9.5

IdentificationIdentification ofof neuralneural crestcrest cellscells

in in Wnt1Wnt1--lacZlacZ/ / ++mousemouse

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

Cell types differentiated from the NCCell types differentiated from the NC

ED13, B gal

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

Migration of cranial neural crest cells

Wnt1-cre/R26R mouse

B.M. Carlson (1999)

ED 9.5Xgal

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:

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

JiangJiang et et alal., ., DevDev BiolBiol 241:106, 2002241:106, 2002

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

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

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

Developmental disorders of the NCDevelopmental disorders of the NC

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

SpSp1H1H/+/+ SpSp1H1H/ Sp/ Sp1H1H

ED 13.5

Pax3Pax3 mutationmutation in in mousemouse ((splotchsplotch mutationmutation))

PiebaldismPiebaldism ((pigmentationpigmentation defectsdefects, sterility, , sterility, anemiaanemia) ) mutationmutation ofof KITKIT gene in man gene in man andand KitKit gene in gene in mousemouse

NNeuraleural crestcrest ccells in ells in epidermis epidermis –– in in hair folliclehair follicle

Merkel cellMerkel cells, melanocytes, stem s, melanocytes, stem cellscells

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

F. S. F. S. MerkelMerkel (1875) : (1875) : „„TastzellenTastzellen““ ofof thethe skin skin ofof birdsbirds andand mammalsmammals

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

Merkel cells in whisker hair follicle

K8

ED 10

ED 13

ED 13

BgalBgal/H /H Wnt1Wnt1--crecre/R26R/R26R

NeuralNeural crestcrest cellscells in epidermis in epidermis –– in in hairhair folliclefollicle

E.M.J.Peters (2002)

c-kit-positive melanoblasts

c-kit / hem

NeuralNeural crestcrest stem stem cellscells in in thethe hairhair folliclefollicle

Growth cycle of human scalp hair

Anagen2-6 years

Catagen1-2 weeks

Telogen5-6 weeks

Math1 TRITC Ki67 FITC

Dissection of the bulge from adult whisker follicle

Xgal + NCCs emigrated 4 days after explantation

Bulge explant-derived NCCsexpress nestin and Sox10

(day 5 in culture)

Anti-nestin Ab + DAPI Anti-Sox10 Ab + DAPI

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

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

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

Coexpressionof Sox10 and Nestin, Expression of Nanog

Sox10 + Nestin Nanog + DAPI

hEPI-NCSCs in tissue culture after emigration from back skin follicles

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

•• 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.

Folia Biologica (Praha) 56, 149-157 (2010)

The use of NCSCThe use of NCSCss in regenerative in regenerative medicinemedicine

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

NestinNestin GAD67GAD67 RIPRIP

Morphology of EPIMorphology of EPI--NCSC implants in the NCSC implants in the lesionedlesioned spinal cordspinal cord

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.

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]

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

Thanks for your attention

Institute of Anatomy, First Faculty of Medicine, Charles University Prague, Czech Republic