induced pluripoten (ips) cells: an emerging technology...
TRANSCRIPT
Induced pluripoten (iPS) cells: an emerging technology platformMaria J. Barrero
Centre de Medicina Regenerativa de Barcelona
Our body is composed by more than 200 different cell types…
…among them some are considered stem cells
5‐7 day embryoSingle cellembryo
6 week embryo
Embryonic Embryonic Adult Adult
TotipotentTotipotent OligopotentOligopotent
Embryonic Stem (ES) cellsPluripotent
Embryonic germ (EG) cellsPluripotent
Cord blood Stem cellsMultipotent
Adultstem cellsMultipotent
Embryonic vs. Adult Stem Cells
•Ethical issues/iPS
•Known how to isolate them
•Easy to expand in vitro
•Give rise to many tissues
•Difficult to control differentiation
•First clinical trials just started
•Difficult to identify and isolate
•Difficult to expand in vitro
•Give rise to limited tissues
•Easier to control differentiation
•Currently used to treat deseases
Embryonic Adult
Other sources of embryonic stem cells
Somatic cell REPROGRAMMING
Embryonic stem cell programmSkin cell programm
In vitro manipulation
1956 1981 1988 1992 1996
1998 2002 2004
First bone
marrow
transplant
mouseESisolated
HSC from
mouse
isolated
SC in human
brain
identified
human ESisolated
first animalcreated by
nuclear
transfer
pancreatic
cells from mouse ES
cure diabetic
mice
dopaminergic
neurons from
human ES
Stem cell time line
2006 2007
mouse iPS human iPS
Skin cells culture
iPS cells= induced pluripotent stem cells
Reprogramming using defined factors
Reprogramming
Oct4Sox2Klf‐4c‐myc
• Self‐renewal• Pluripotent
Generation of Induced pluripoten stem (iPS)cells.
1. Goal: therapeutic, basic research, screenings.
2. Choice of somatic cell.
3. Choice of reprogramming factors.
4. Delivery of reprogramming factors.
5. Selection and expansion of reprogrammed cells
6. Characterization of iPS cells
7. Set up of drug screening.
Generation of Induced pluripoten stem (iPS)cells.
Seeding
somatic
cells
Delivery
of
factors
Splitand
change to
ES media
appearance
of first
colonies
1‐2 days 2‐5 days 7‐30 days
expansion and
characterization
of colonies
3 months
Choice of somatic cellChoice of somatic cell
Amniotic fluid
Endometrium
Fetal lung fibroblasts
Umbilical vein endothelial cells
Cord Blood
Menstrual blood
Neural adult stem cells
Astrocytes
Keratinocytes
Adult dermal fibroblast
B‐lymphocytes
Reprogramming efficiency
Disease type Disease name Genetic cause N. of lines Cell type Control line Phenotype Drug test
Neurological Parkinson's disease Polygenic 23 Dopaminergic neurons hiPSC No obvious defect ND
Polygenic (with
LRRK2
mutation)4 Dopaminergic neurons hiPSC Neuronal death with chemicals Yes
Amyotrophic lateral sclerosis Polygenic 3 Motor neurons hESC ND ND
Spinal muscular atrophy Monogenic 2 Motor neurons hiPSC Loss of neuron formation, loss of SMN
gene expression
Yes
Familial dysautonomia Monogenic 2 Neural crest cells hiPSC, hESC Loss of neural crest cells Yes
RETT syndrome Monogenic 4 Neurons hiPSC Loss of synapses, reduced spine density,
smaller soma size
Yes
Huntington's disease Monogenic 2 ND hiPSC, hESC ND ND
Friedreich ataxia Monogenic 6+ ND hESC Changes GAA‐TTC repeat ND
Blood Fanconi anaemia Monogenic 19 Blood cell hiPSC, hESC Corrected loss of FANCA
function ND
Fragile X syndrome Monogenic 11 ND hiPSC, hESC Loss of FMR1 expression ND
Cardiac and
vascular
Long QT 1 syndrome Monogenic 6 Cardiomyocytes hiPSC Increased cardiomyocyte depolarization Yes
Long QT 2 syndrome Monogenic Not reported Cardiomyocytes hiPSC Increased cardiomyocyte depolarization Yes
LEOPARD syndrome Monogenic 6 Cardiomyocytes hiPSC, hESC Increased cardiomyocyte size, decreased
MAPK signaling
ND
Timothy syndrome Monogenic 16 Cardiomyocytes hiPSC Increased cardiomyocyte depolarization Yes
Hutchinson Gilford Progeria Monogenic 4 Smooth muscle cells,
mesenchymal stem cells
hiPSC, hESC Smooth muscle and mes‐enchymal cell
apoptosis
ND
Monogenic 6 Smooth muscle cells hiPSC Smooth muscle cell nuclear morphology
and ageing phenotype
ND
Duchenne muscular dystrophy Monogenic 2 ND hiPSC, hESC ND ND
Pancreatic Type 1 diabetes Polygenic 4 Insulin‐
and glucagon‐
producing cells
hESC ND ND
Hepatic A1‐antitrypsin deficiency Monogenic 19 Hepatocytes hiPSC Loss of A1‐antitrypsin expression Yes
Others Prader‐Willi syndrome Monogenic 4 Neurons hiPSC, hESC Imprint disorder ND
Angelman and Prader‐
Willi
syndrome
Monogenic 13 Neurons hiPSC, hESC Loss of paternal UBE3A expression ND
Down syndrome Monogenic 2 ND hiPSC, hESC ND ND
Choice of Reprogramming Factors.
Sox2
c‐mycKlf4
Oct4 Nanog Lin28
1. Proteins
2. miRNAs
3. ChemicalsVPA
UTF Sall4
BIX‐01294
5′‐azacytidine SAHA TSA
miR302/367
mir‐200c /302 s /369 s
miR‐302/372
Virus: lentivirus,Retrovirus, adenovirus
DNA: transfection
Protein: transfection,PTD
RNA: transfection
Advantage Disadvantage
Delivery methods
CheapEasy to produceefficient
integrative
CheapEasy to producenon integrative
Inefficient
Integrative
Expensiveineficient
Non integrative
CheapEasy to producenon integrative
ineficient
iPS cell time line
2006 2007 2008 2009
2010 2011
mouse iPS OSKM Retrovirus
human iPS OSKM retrovirus
iPS without myc
iPS from NSC with two factors
iPS with two factors+ small molecules
Non integrative iPS (adenovirus)
Non integrative iPS (excisable lenti)
Desease specific iPS cells
Non integrative iPS (episomal)
Non integrative iPS (proteins)
Non integrative iPS (RNA)
iPS using miRNAs
iPS cells have a high rate of mutations
Selection and expansion of coloniesSelection and expansion of colonies
Seeding
somatic
cells
Delivery
of
factors
Splitand
change to
ES media
appearance
of first
colonies
1‐2 days 2‐5 days 7‐30 days
expansion and
characterization
of colonies
3 months
ES/iPS cell culture conditions
matrigelHES media: Serum replacement
FGFFibroblast conditioned
Passage: Enzymatic
Feeder:
Human irradiated
fibroblast
HES media: Serum replacementFGF
Passage: Mechanical
matrigelDefined media (mTeSR)Passage: Enzymatic
Selection and expansion of coloniesSelection and expansion of colonies
Partially Reprogrammed Cells
•Selfrenewal•Downregulation of cell
specific expression programs•Expression of transgenes
Fibroblast
Oct4Sox2Klf4c‐Myc
Fully Reprogrammed Cells
•Pluripotent•Activation of pluripotency
genes.•Silencing of transgenes•Bivalent modifications
Stress/Senescence
Chromatin
Tumor Teratoma
iPS
Characterization of iPS lines coloniesCharacterization of iPS lines colonies
Characterization of iPS lines coloniesCharacterization of iPS lines colonies
1. Molecular characterization:• Silencing of transgenes
expression of transgenes
0
0.2
0.4
0.6
0.8
1
1.2KE
RATO
S
keratos 4F
shRD
#1 P4
shRD
#2 p4
shRD
#3 p4
shRD
#4 p4
shRD
#5 p4
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shM1#2 p
shM1 #3
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shM2#1 p4
shM2 #2
P4
shM2#3 p4
shM2 #4
P4
ES4 p1
4
Trans‐Oct4Trans‐Sox2Trans‐Klf4Trans‐Myc
Levels to
GAPD
H
Characterization of iPS lines coloniesCharacterization of iPS lines colonies
1. Molecular characterization:• Induction of pluripotency factors
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
keratos
ES4 p1
4
shRD
#1 P4
shRD
#2 p4
shRD
#3 p4
shRD
#4 p4
shRD
#5 p4
shM1#1 p4
shM1#2 p4
shM1 #3
P4
shM2#1 p4
shM2 #2
P4
shM2#3 p4
shM2 #4
P4
KLF4cMYCOct4SOX2NanogCRIPTORex1
expression of endogenous genes
Levels to
GAPD
H
Characterization of iPS lines coloniesCharacterization of iPS lines colonies
1. Molecular characterization:• Induction of pluripotency factors
Characterization of iPS lines coloniesCharacterization of iPS lines colonies
2. Functional characterization:• In vitro differentiation: formation of embryoid bodies
FGFSerum replacement
Fetal bovine serumAscorbic acidActivine A
Characterization of iPS linesCharacterization of iPS lines
2. Functional characterization:• In vivo differentiation: teratoma formation
Characterization of mouse iPS linesCharacterization of mouse iPS lines
2. Functional characterization:• Germline contribution in chimaeras
"We can differentiate the disease‐specific iPS cells into certain lineages, and we can
screen for a drug that can alleviate the phenotype of the disease"
Advantage:Unlimited source of human cell types
Challenges:
Drug companies require large and consistent numbers of differentiated cells at high
purity to be able to use them in their discovery and toxicity screening systems.→
Improvement of differentiation protocols
Easy readout of phenotypes. (reporter genes, colorimetric assays...)
Drug screeningsDrug screenings
La ley españolaLa ley española
Para generar o trabajar con "human
ES/iPS cell lines":
1. Obtener el informe favorable del Comitè
d’Ètica d’Investigació
Clínica (CEIC):
•Valorar els projectes de recerca (PR) i protocols d’assaigs clínics (AC) i les seves modificacions, en relació
amb els objectius definits, l’evidència científica i la justificació
dels
riscs i molèsties previsibles per als participants en funció
dels potencials beneficis.
•
Avaluar la preparació
dels investigadors per al projecte o l’assaig proposat, així
com
també
la idoneïtat de les instal∙lacions i equipaments disponibles en el centre on es pretén
dur a terme el PR o AC
2. Con el informe favorable del comité
ético solicitar la aprobación al Departamento de
Salud. (Banco Nacional de Líneas Celulares o a la Comisión de Garantías de la donación y
utilización de células y tejidos humanos)
3. El investigador principal se compromete a informar a la comisión de los progresos del
proyecto y a proporcionar al Banco Nacional de Líneas Celulares las líneas obtenidas.
La ley españolaLa ley española
Es obligatorio registrar las "human
ES/iPS cell lines" generadas en el Banco Nacional de
Líneas Celulares
Copia de la autorización de derivación de la línea celular, junto con informe del Comité
Ético.
Copia de cualquier publicación científica relacionada con la derivación y/o caracterización de
la línea.C. V. del investigador principal
Descripción de las características morfológicas de la línea en cultivoControles microbiológicos realizados (micoplasma)Marcadores pluripotenciaCapacidad de diferenciación in vitro (embryoid bodies)Capacidad de diferenciación in vivo (teratoma)Consistencia celular tras 6 pases de congelación y descongelaciónPase en el momento del registro