Stem/Cell/Systematics/And/Potentials/For/Modeling/And/Therapy

Stem/cell/systematics/ and/potentials/for/modeling/and/therapy/

Poul Hyttel University/of/Copenhagen The$Anatomy$Building University$of$Copenhagen,$Frederiksberg Contents

• Stem*cell*definitions • Cell*differentiation*potency • The*stem*cell*hierarchy:*Pluripotent,*multipotent*and* unipotent*stem*cells • Induced*pluripotent*stem*cells*for*disease*modeling • Stem*cell*therapy • Conclusions Stem'cell'definitions • Self:renew'(indefinite'multiplication) • Differentiate'into'specialized'cell'types' (differentiation'potency)

Self:renewal

Stem'cell Limited expansion Intermediate'progenitors

Conrad' Waddington' (1957) Terminally'differentiated Contents

• Stem*cell*definitions • Cell*differentiation*potency • The*stem*cell*hierarchy:*Pluripotent,*multipotent*and* unipotent*stem*cells • Induced*pluripotent*stem*cells*for*disease*modeling • Stem*cell*therapy • Conclusions Totipotency Cell$differentiation$potency hED1 hED1.5 hED2 • Totipotency:$All$ embryomic and$ extraCembryonic$ cell$types • Pluripotency:$All$ embryonic$cell$ types • Multipotency:$ Several$cell$types • Unipotency:$A$ Pluripotency Multipotency single$cell$type hED3 hED5 hED7pED11 Contents

• Stem*cell*definitions • Cell*differentiation*potency • The*stem*cell*hierarchy:*Pluripotent,*multipotent*and* unipotent*stem*cells • Induced*pluripotent*stem*cells*for*disease*modeling • Stem*cell*therapy • Conclusions Most%common%stem%cell%types • Pluripotent%stem%cells%(PSCs) – Embryonic%stem%cells%(ESCs) – Induced%pluripotent%stem%cells%(iPSCs) • Multipotent%stem%cells – Multipotent%stem%cells%are%often%referred%to%as%adult%stem%cells% (even%though%they%are%also%found%in%embryos) – Hematopoietic%stem%cells%(HSCs) – Mesenchymal%stem%cells%(MSCs) – Neural%stem%cells%(NSCs) • Unipotent%stem%cells – Epidermal%stem%cells%(EpiSCs) Most%common%stem%cell%types • Pluripotent%stem%cells%(PSCs) – Embryonic%stem%cells%(ESCs) – Induced%pluripotent%stem%cells%(iPSCs) • Multipotent%stem%cells – Multipotent%stem%cells%are%often%referred%to%as%adult%stem%cells% (even%though%they%are%also%found%in%embryos) – Hematopoietic%stem%cells%(HSCs) – Mesenchymal%stem%cells%(MSCs) – Neural%stem%cells%(NSCs) • Unipotent%stem%cells – Epidermal%stem%cells%(EpiSCs) Embryonic*stem*cells*(ESCs)

Ectoderm Mesoderm Endoderm Human&embryonic&stem&cells&(hESCs)&7 1998

Thompson&et&al.&(1998)

Most%common%stem%cell%types • Pluripotent%stem%cells%(PSCs) – Embryonic%stem%cells%(ESCs) – Induced%pluripotent%stem%cells%(iPSCs) • Multipotent%stem%cells – Multipotent%stem%cells%are%often%referred%to%as%adult%stem%cells%(even% though%they%are%also%found%in%embryos) – Hematopoietic%stem%cells%(HSCs) – Mesenchymal%stem%cells%(MSCs) – Neural%stem%cells%(NSCs) • Unipotent%stem%cells – Epidermal%stem%cells%(EpiSCs) Induced pluripotent stem&cells (iPSCs)

Mouse& fibroblasts

Oct4 Sox2 Klf4 C-Myc Shinya Yamanaka

Induced pluripotent& stem&cells (iPSC) Cell$differentiation$potency

Conrad$ Waddington (1957)

somatic$cells + Reprogramming$factors Nobelprize 2012-in-medicine

John-B-Gurdon Shinya Yamanaka Most%common%stem%cell%types • Pluripotent%stem%cells%(PSCs) – Embryonic%stem%cells%(ESCs) – Induced%pluripotent%stem%cells%(iPSCs) • Multipotent%stem%cells – Multipotent%stem%cells%are%often%referred%to%as%adult%stem%cells% (even%though%they%are%also%found%in%embryos) – Hematopoietic%stem%cells%(HSCs) – Mesenchymal%stem%cells%(MSCs) – Neural%stem%cells%(NSCs) • Unipotent%stem%cells – Epidermal%stem%cells%(EpiSCs) Most%common%stem%cell%types • Pluripotent%stem%cells%(PSCs) – Embryonic%stem%cells%(ESCs) – Induced%pluripotent%stem%cells%(iPSCs) • Multipotent%stem%cells – Multipotent%stem%cells%are%often%referred%to%as%adult%stem%cells% (even%though%they%are%also%found%in%embryos) – Hematopoietic%stem%cells%(HSCs) – Mesenchymal%stem%cells%(MSCs) – Neural%stem%cells%(NSCs) • Unipotent%stem%cells – Epidermal%stem%cells%(EpiSCs) Hematopoietic*stem*cells*(HSCs)

Reside*in*bone*marrow

Most*well*defined*stem*cell*hierarchy,* but*still*developing

Transplantation*of*bone*marrow@ derived*hematopoietic*stem*cells* commonly*used

Eaves*(2015) Most%common%stem%cell%types • Pluripotent%stem%cells%(PSCs) – Embryonic%stem%cells%(ESCs) – Induced%pluripotent%stem%cells%(iPSCs) • Multipotent%stem%cells – Multipotent%stem%cells%are%often%referred%to%as%adult%stem%cells% (even%though%they%are%also%found%in%embryos) – Hematopoietic%stem%cells%(HSCs) – Mesenchymal%stem%cells%(MSCs) – Neural%stem%cells%(NSCs) • Unipotent%stem%cells – Epidermal%stem%cells%(EpiSCs) Mesenchymal+stem+cells+(MSCs)

BMIMSCs+and+ADIMSCs+ fundamentally+different+cells+ Reside+in+different+organs types+albeit+with+same+ differentiation+potentials Definition • Adhere+to+plastic • Positive+for+CD73,+CD90+and+CD105 • Negative+for+CD14,+CD34,+CD45+and+ HLAIDR • In+vitro+differentiation+into • Chondroblasts • Osteoblasts • Adipocytes

• Further+differentiation+potentials • Myocytes • Neurons • Oligodendrocytes • Hepatocytes • Pancreocytes Oryan et+al.+(2017)Q+Ullah+et+al.+(2015)Q+Noël+et+al.+(2008) Most%common%stem%cell%types • Pluripotent%stem%cells%(PSCs) – Embryonic%stem%cells%(ESCs) – Induced%pluripotent%stem%cells%(iPSCs) • Multipotent%stem%cells – Multipotent%stem%cells%are%often%referred%to%as%adult%stem%cells% (even%though%they%are%also%found%in%embryos) – Hematopoietic%stem%cells%(HSCs) – Mesenchymal%stem%cells%(MSCs) – Neural%stem%cells%(NSCs) • Unipotent%stem%cells – Epidermal%stem%cells%(EpiSCs) Neural'stem'cells'(NSCs)

Reside'in'CNS

Differentiate'into • Neuronal'subtypes • Astrocytes • Oligodendrocytes Most%common%stem%cell%types • Pluripotent%stem%cells%(PSCs) – Embryonic%stem%cells%(ESCs) – Induced%pluripotent%stem%cells%(iPSCs) • Multipotent%stem%cells – Multipotent%stem%cells%are%often%referred%to%as%adult%stem%cells% (even%though%they%are%also%found%in%embryos) – Hematopoietic%stem%cells%(HSCs) – Mesenchymal%stem%cells%(MSCs) – Neural%stem%cells%(NSCs) • Unipotent%stem%cells – Epidermal%stem%cells%(EpiSCs) Epidermal*stem*cells*(EpiSC)

Reside*in*skin

Differentiate*into • Keratinocytes Contents

Access%to%the% human%target: iPSC4derived% patient4specific% neurons Frontotemporal+dementia+ localized+to+chromosome+3+(FTD3) Mutation+in CHMP2B'on+ chromosome+3+ resulting+in+ dementia+with+ early+onset

FReJa Consortium

CHMP2B mutation iPSC4based6modeling6of6FTD3 CRISPR/Cas9 Plasmid%iPSC% reprogramming

FTD3% Gene% iPSC corrected% iPSC Neural% differentiation

Skin% fibroblasts Gene% FTD3% corrected% neurons neurons

Rescue%from% Identification6of6disease% ROS FTD36phenotype6phenotype ROS and6 Endosomestherapeutic6targets Endosomes

Mitochondria Mitochondria FTD3%patient iPSC4based6modeling6of6FTD3 CRISPR/Cas9 Plasmid%iPSC% reprogramming

FTD3% Gene% iPSC corrected% iPSC Neural% differentiation

Skin% fibroblasts Gene% FTD3% corrected% neurons neurons

Rescue%from% Identification6of6disease% ROS FTD36phenotype6phenotype ROS and6 Endosomestherapeutic6targets Endosomes

Mitochondria Mitochondria FTD3%patient Generation)of)integration,free patient,specific)iPSCs Fibroblasts from)skin)biopsy iPSC

L1MYC Day)26 LIN28 OCT3/4 Shp53+ SOX2 KLF4

Okita et)al.)2011 Rasmussen)et)al.)2011 Mikkel Rasmussen,)Kristine)Freude and)Yu)Zhang iPSC4based6modeling6of6FTD3 CRISPR/Cas9 Plasmid%iPSC% reprogramming

FTD3% Gene% iPSC corrected% iPSC Neural% differentiation

Skin% fibroblasts Gene% FTD3% corrected% neurons neurons

Rescue%from% Identification6of6disease% ROS FTD36phenotype6phenotype ROS and6 Endosomestherapeutic6targets Endosomes

Mitochondria Mitochondria FTD3%patient Importance+for+gene+editing+in+iPSC disease+modeling

Age+and+gender+ matched+control

Isogenic/gene+edited+

control

Courtesy+of+Kristine+Freude Gene'editing'of'iPSCs

Courtesy'of' Trinity'College,'Dublin Jacob'Corn' Gene$editing$of$iPSCs

FTD3$iPSC Gene3corrected iPSC

ssODN with$correct$template$sequence

C/T GuideRNA

Cas9

Anna$Poon,$Benjamin$Schmid and$Kristine$Freude iPSC4based6modeling6of6FTD3 CRISPR/Cas9 Plasmid%iPSC% reprogramming

FTD3% Gene% iPSC corrected% iPSC Neural% differentiation

Skin% fibroblasts Gene% FTD3% corrected% neurons neurons

Rescue%from% Identification6of6disease% ROS FTD36phenotype6phenotype ROS and6 Endosomestherapeutic6targets Endosomes

Mitochondria Mitochondria FTD3%patient

Neural'differentiation'of'iPSCs Neural+differentiation+of+iPSCs

iPSC

Neural+progenitor+cells Neurons+and+glia+cells iPSC4based6modeling6of6FTD3 CRISPR/Cas9 Plasmid%iPSC% reprogramming

FTD3% Gene% iPSC corrected% iPSC Neural% differentiation

Skin% fibroblasts Gene% FTD3% corrected% neurons neurons

Rescue%from% Identification6of6disease% ROS FTD36phenotype6phenotype ROS and6 Endosomestherapeutic6targets Endosomes

Mitochondria Mitochondria FTD3%patient Electron'microscopy'of' iPSC:derived'neurons Isogenic) Patient control

Indføj'”Sted'og'dato”'i'feltet'for'

Nanett K'Nicolaisen RNAseq analyses%of% iPSC=derived%neurons

FTD3% isogenic% controls

Healthy% person

FTD3% patients

Yu%Zhang,%Alun and%Kristine%Freude RNAseq analyses%of% iPSCHderived%neurons

FTD3% isogenic% controls

Healthy% person

FTD3% patients

Major%FTD3%misregulated genes:%Endosomes,%mitochondria%and%iron%homeostasis Yu%Zhang,%Alun and%Kristine%Freude Mitochondrial'defects

RNAseq Mitochondrial'genes FTD3'vs isogenic'control

Nanett K'Nicolaisen and'Blanca'Garcia Astrocyte)reactivity)and)impaired)autophagy

Katarina)Stoklund)Dittlau Contents

• Stem)cell)definitions • Cell)differentiation)potency • The)stem)cell)hierarchy:)Pluripotent,)multipotent)and) unipotent)stem)cells • Induced)pluripotent)stem)cells)for)disease)modeling • Stem)cell)therapy • Conclusions “Stem&cell&tourism”&– false& hope&for&real&money “one$of$the$most$dangerous$elements$of$ our$culture:$that$we$have$forgotten$how$to$ die.”&(Jill&Lepore) Important)issues)in)stem)cell)therapy • Cell)type – Multipotent)(e.g.)MSCs,)NSCs) – Pluripotent)(e.g.)ESCs,)iPSCs) – Autologous)or)allogenic)cells • The)“super)donor”)– homozygous)for)human)leucocyte)antigen)(HLA))– will) match)heterozygous)patients)with)just)one)match – ”Super)donor”)iPSC)banks)established)in)Japan,)China)and)US • Large)scale)manufacturing)of)cells • Principle)for)administration – Scaffolds – Homing – Engraftment)vs.)stimulatory)effects)of)secretome • Legal)regulation – Special)“fast)track”)approval)processes)in)US,)EU)and)Japan A"brief"overview"of" clinical"trials"in"stem"cell"therapy categorized"according"to"stem"cell"types

(Mid"July)"""ClinicalTrials.gov@"U.S."National"Library"of"Medicine Hematopoietic*stem*cells*– the*forerunner Indication #studies Results Reference Bone*marrow* 3,309 Reestablishment*of*hematopoietic* (Gatti et*al.,*1968) transplantation function First*allogenic*transplantation*1968 Dr. E*Donnall Thomas*Nobel*Prize*1990 Cancer 324 Chimeric*antigen*receptor*(CAR)*CAR*T* (Androulla and* Cell*therapy Papadopoulou.*2018) Several*FDA*approved*treatments

ClinicalTrials.gov8*U.S.*National*Library*of*Medicine Epidermal*stem*cells – a*superficial*speciality

Indication #studies Results Reference Burns,*chronic*wounds,* 20 Restoration*of*normal*skin*function (Jackson*et*al.,*2017) autoimmunity*and* urethral*reconstruction

ClinicalTrials.gov9*U.S.*National*Library*of*Medicine Mesenchymal+stem+cells+– a+wide+field Indication #studies Results Reference Ischemic+heart+disease+ 244 Improved+cardiac+function,+effect+ (Jeong et+al.,+2018) potentially+due+to+secreted+chemokines Bone+and+cartilage+ 194 Accelerated+healing,+mechanisms+still+ (Oryan et+al.,+2017) defects+ controversial Degenerative+disc+ 25 Significant+pain+relief (Orozco+et+al.,+2011) disease+ Immunomodulation 58 Clear+immunosuppressive properties,+ (Wang+et+al.,+2018) mechanisms+still+controversial Diabetes 56 Positive+effects,+mechanisms+still+ (Wehbe and+Hawat,+ controversial 2017) Spinal+cord+injury+ 46 Functional+repair,+mechanisms+still+ (Qu++and+Zhang,+2017) controversial Parkinson’s+disease+ 9 Lack+of+convincing+results (Palmer+et+al.,+2016) Retinopathy+incl.+ 6 Some+visual+improvement+but+also+side+ (Öner,+2018) macular+degeneration effects ClinicalTrials.gov9+U.S.+National+Library+of+Medicine Neural'stem'cells'– a'niche Indication #studies Results Reference Parkinson’s'disease' 7 Some'clinical'improvement'but' (Palmer'et'al.,'2016) inconsistent Spinal'cord'injury' 4 Functional'repair'but'inconsistent (Wright'et'al.,'2018)

Retinopathy'incl.' 1 Lack'of'updated'results (Tsukamoto'et'al.,'2013) macular'degeneration'

ClinicalTrials.gov6'U.S.'National'Library'of'Medicine Embryonic*stem*cell*– a*potent*challenge Indication #studies Results Reference Retinopathy*incl.* 19 ESCDderived*retinal*pigmented* (Schwartz*et*al.,*20158* macular*degeneration* epithelium*visual*improvement*but*not* Öner,*2018) consistent Diabetes 3 Lack*of*updated*results (Cheng*et*al.,*2016)

Spinal*cord*injury* 1 Significant*clinical*improvements (Schroff,*2016)

Parkinson’s*disease* 1 Lack*of*updated*results (Palmer*et*al.,*2016)

Ischemic*heart*disease* 1 Improved*cardiac*function (Menasché et*al.,*2015)

ClinicalTrials.gov8*U.S.*National*Library*of*Medicine Induced'pluripotent'stem'cells' – a'new'era Indication #studies Results Reference Retinopathy'incl.' 2 No'results'posted,'Japanese'study' (Öner,'2018) macular'degeneration' discontinued'due'to'safety'reasons Parkinson’s'disease' 1 Lack'of'updated'results (Cheng'et'al.,'2016)

Spinal'cord'injury' 1 Significant'clinical'improvements (Schroff,'2016)

Parkinson’s'disease' 0 Projected (Palmer'et'al.,'2016)

ClinicalTrials.gov8'U.S.'National'Library'of'Medicine Parkinson’s*disease*and*iPSC Parkinson’s*disease

Dopamine* release

Dopaminergic* neurons Parkinson’s1disease Transplantation

Science11990

6481week fetal Suspension1of Uni4 or1bilateral1stereotaxic injections to1 mesencephalon dopaminergic immunosuppressed1patients progenitor cells Agnete Kirkeby How/to/produce/dopaminergic/ progenitors/without/aborted/fetuses?

A rostral caudal B 0 μM 2 μM GSK3i GSK3i FB MB HB DiEn SC dorsal A B C D E TelEn Nt ventral C F A B C D E 0 μM 2 μM FOXG10 μM 2 μM DAPI LHX2 A B C D E FOXG1TBR1 side view FEZF1LHX2 D top view TBR1SIX3 OTX2 FEZF1OTX2 Forebrain SIX3EN1 OTX2EN2 PAX8 WNT1EN1 LMX1AEN2 LMX1BWNT1 OTX2 PAX8 DAPI LMX1APAX2 LMX1BPAX5 PAX2PAX7 E Mibbrain side view PAX5PAX8 OTX2 OTX2 GBX2PAX7 DAPI PAX8IRX3 HOXA1GBX2 Agnete Kirkeby FB MB HB HOXA2IRX3 HOXA1 G HOXA3 HOXA2HOXA4 0 μM 4 μM HOXA3HOXA5 top view Hindbrain HOXA4 OTX2 HOXA5

A B C D E A B MBC D E FOXG1 OTX2 DAPI FOXG1 0 2525 5050 75 100100 LHX2 LHX2 TBR1 TBR1 I 25 50 75 100 H FEZF1 J FEZF1 + FGF8b SIX3 SIX3 80 15 LHX2 OTX2 OTX20 μM 4 μM OTX20 μM 2 μM A B C D E 60 A B C D E EN1 A B C D E 10 EN1 A B C D E FOXG1 FOXG1 EN2 40 EN2 LHX2 5 LHX2 WNT1 20 WNT1 Fold over H9 ESC FEZF1 Fold over H9 ESC FEZF1 LMX1A 0 0 LMX1A SIX3 SIX3 Forebrain LMX1B A B C D E A B C D E Forebrain LMX1B OTX2 Position in MISTR Position in MISTR OTX2 PAX2 8,000 15,000 WNT1 PAX2 EN1 EN1 EN1 PAX5 6,000 PAX5 WNT1 10,000 WNT1 PAX7 PAX7 LMX1A 4,000 LMX1A PAX8 5,000 PAX8 LMX1B 2,000 LMX1B Fold over H9 ESC Fold over H9 ESC

Mibbrain GBX2 Mibbrain GBX2 PAX2 PAX2 IRX3 0 0 IRX3 PAX7 A B C D E A B C D E PAX7 HOXA1 Position in MISTR Position in MISTR HOXA1 GBX2 60 1,000 GBX2 HOXA2 GBX2 HOXA3 HOXA2 IRX3 800 IRX3 HOXA3 HOXA3 40 HOXA1 600 HOXA1 HOXA4 HOXA4 HOXA2 400 HOXA2 HOXA5 20 HOXA5

Hindbrain 200 Hindbrain Fold over H9 ESC HOXA3 Fold over H9 ESC HOXA3 HOXA5 0 0 HOXA5 A B C D E A B C D E MB Position in MISTR Position in MISTR MB

0 25 50 75 100 0 25 50 75 100

0 25 50 75 100 0 25 50 75 100 How,to,produce,dopaminergic, progenitors,without,aborted,fetuses?

WNT(activation((GSK3i) Day,14,tissue (estimated,equivalent,to,4,wks pc)

Forebrain/midbrain, OTX2,,DAPI Hindbrain

Midbrain Hindbrain

OTX2,,NKX2.1 DAPI Telencephalon Diencephalon Midbrain Hindbrain

OTX2 FB MB HB SC HB SC FB MB PAX8 FB NKX2.1 FB HB SC FB MB FB Kirkeby(lab,(unpublished(data Agnete Kirkeby Contents

• Stem-cells-are-defined-by-self4renewal-and-differentiation-potency • Stem-cells-are-best-categorized-according-to-their-differentiation- potency-(pluripotent,-multipotent,-unipotent),-not-by-their-origin-in- embryos-or-adults • Induced-pluripotent-stem-cells-(iPSCs)-allow-for-patient4specific- disease-modeling • Numerous-clinical-trials-are-registered-on-multipotent-stem-cell- therapy • First-clinical-trials-are-registered-on-pluripotent-(ESC,-iPSC)-stem-cell- therapy BrainStem*collaborating* Financial*support partners • Innovation*Fund*Denmark • University*of* • EU*Marie*Curie*(STEMMAD) Copenhagen • Copenhagen*Consortium*for* • Danish*Dementia* Designer*Organisms Research*Center BrainStem • University*of* Stem%cell%center%of% Southern*Denmark excellence%in% • Aarhus*University Thanks for*your attention! neurology • Bioneer • Lundbeck • Innovative*Concepts* in*Drug*Development* (F) • Lund*University*(S) • Biotalentum (H)