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FGF signaling in specification of hESC-derived definitive endoderm

Ameri, Jacqueline LU (2010) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2010:6.
Abstract (Swedish)
Popular Abstract in Swedish

Diabetes är en snabbt växande folksjukdom som redan har drabbat 200 miljoner

världen runt. Det finns två typer av sjukdomen: typ I samt typ II diabetes. Den

förstnämnda drabbar vanligtvis de unga och beror på en s.k. autoimmun reaktion

där kroppens egna immunförsvar av okänd anledning plötsligt inte känner igen

insulincellerna och börjar attackera dem. Typ II diabetes uppstår däremot pga. en

kombination av insulinresistens, samt otillräcklig sekretion av insulin, och bryter

ut i vuxen ålder. Hälsosam kost och motion är av stor vikt för att förhindra typ

II diabetes. Det finns i dagsläget inget botemedel för diabetes och båda... (More)
Popular Abstract in Swedish

Diabetes är en snabbt växande folksjukdom som redan har drabbat 200 miljoner

världen runt. Det finns två typer av sjukdomen: typ I samt typ II diabetes. Den

förstnämnda drabbar vanligtvis de unga och beror på en s.k. autoimmun reaktion

där kroppens egna immunförsvar av okänd anledning plötsligt inte känner igen

insulincellerna och börjar attackera dem. Typ II diabetes uppstår däremot pga. en

kombination av insulinresistens, samt otillräcklig sekretion av insulin, och bryter

ut i vuxen ålder. Hälsosam kost och motion är av stor vikt för att förhindra typ

II diabetes. Det finns i dagsläget inget botemedel för diabetes och båda typerna

av sjukdomen behandlas med insulin som antingen administreras i tablettform

eller som injektioner. För att bota diabetes måste forskarna dels komma på vad

som utlöser den autoimmuna förstörelsen av insulincellerna, dels kunna komma

på ett sätt att bilda nya insulinceller som kan transplanteras till de drabbade. Den

mest framgångsrika behandlingen av typ I diabetes involverar transplantation av

öar med insulinceller. Det finns studier som visar att patienter som genomgått denna

transplantation har kunnat leva utan insulinbehandling i flera år. Men tyvärr har denna

behandling begränsningar, såsom att det krävs flera donatorer till en transplantation,

då ett stort antal av insulinöar krävs för lyckad behandling, och att det krävs livslång

behandling med immunosuppressiva medel för att de transplanterade cellerna inte

ska stötas bort. Det behövs därmed dels alternativa källor till insulinceller, dels

alternativa behandlingsstrategier.

Humana embryonala stamceller har förmågan att bilda vilka celltyper som helst

i människans kropp. Av denna anledning är de väldigt intressanta vid behandling

av degenerativa sjukdomar såsom diabetes, Alzheimers, Parkinson och MS, där de

skulle kunna ersätta den skadade vävnaden. För att kunna styra stamcellerna mot

specifika öden eller celltyper krävs det dock kunskap om exakt vilka signalvägar

som är av betydelse vid bildandet av de specifika cellerna och hur dessa aktiveras.

Genom olika djurmodeller har forskarna lyckats få en inblick i vilka gener och

stimuli som är av betydelse för pankreasutvecklingen, men det finns fortfarande

många luckor innan vi vet exakt hur en insulincell bildas i människan. Genom att

arbeta med humana embryonala stamceller har forskarna även en möjlighet att lära

sig mer om människan och hennes utveckling.

Många olika signaleringsvägar är involverade i att styra de omogna stamcellerna till

insulinproducerande celler. Idag kan vi genom tillsats av olika tillväxtfaktorer, som

har visat sig ha en betydande roll under pankreasutvecklingen i musmodeller, styra

utvecklingen av de omogna stamcellerna till definitivt endoderm, det grodlager som

ger upphov till pankreas, lever, lunga och tarmarna. Vår fortsatta strategi involverar

att hitta nya signaleringsvägar som är av betydelse för att ombilda dessa celler mot

10

ett förstadium till pankreas.

I denna avhandling presenteras två olika protokoll för att styra stamcellerna mot ett

förstadium till pankreas och insulinceller. I det ena protokollet lyckas vi även få ett

förstadium till lever, tarm och lungceller. Dessa nya kunskaper kommer att kunna

användas i vårt fortsatta arbete för att styra stamcellerna mot insulinproducerande

celler, som i framtiden kan användas för transplantation i diabetespatienter. Slutligen

beskrivs även en metod där vi genom genetisk modifiering av stamcellerna lyckas

introducera en ny gen som gör att stamcellerna fluorescerar grönt vid uttryck av

specifika gener. Dessa nya stamcellslinjer är oerhört viktiga i vårt fortsatta arbete

där vi försöker lära oss mer om hur de olika pankreasspecifika generna regleras. (Less)
Abstract
Diabetes affects around 200 million people worldwide. Curing diabetes would

require the prevention of autoimmune destruction of beta cells and restoration of

the beta cell mass restored either through regeneration or transplantation of the

insulin producing cells. Islet transplantation according to the Edmonton protocol

have been the most promising therapeutic option for Diabetes type I patients,

however, lack of cadaveric donor islets is a major obstacle and new strategies need

to be established. Human embryonic stem cells (hESCs) not only offer an excellent

source for establishment of strategies for future regenerative cell therapies and drug

discovery but also... (More)
Diabetes affects around 200 million people worldwide. Curing diabetes would

require the prevention of autoimmune destruction of beta cells and restoration of

the beta cell mass restored either through regeneration or transplantation of the

insulin producing cells. Islet transplantation according to the Edmonton protocol

have been the most promising therapeutic option for Diabetes type I patients,

however, lack of cadaveric donor islets is a major obstacle and new strategies need

to be established. Human embryonic stem cells (hESCs) not only offer an excellent

source for establishment of strategies for future regenerative cell therapies and drug

discovery but also offers en excellent experimental assay for understanding human

pancreas development.

Pancreas originates from the definitive endoderm (DE), one of the three germ

layers. The path from definitive endoderm to pancreatic progenitor cells and finally

the insulin producing beta cells involves sequential cell-fate decisions characterized

by the expression of multiple transcription factors. Governed by the developmental

biological principles that normally control foregut endoderm and pancreas

specification, numerous multifactor protocols for directing mature foregut-derived

cells from hESCs have been reported. However, to establish less complex and more

robust protocols there is a need to further understand the mechanism of action of

individual growth and differentiation factors in specification of human pluripotent

stem cells towards foregut derived cell lineages.

Here, we describe two studies where we have investigated the role of fibroblast

growth factor (FGF) signaling, specifically FGF2 and FGF4, and Retinoic acid (RA)

in specification of the hESC-derived DE development. Studies in lower vertebrates

have demonstrated that FGF2 acts in a restricted manner primarily patterning the

ventral foregut endoderm into liver and lung, whereas FGF4 exhibits broad anteriorposterior

and left-right patterning activities. Furthermore, whereas FGF2 is not

required for ventral pancreas development an inductive role of FGF2 has been

shown during dorsal pancreas formation. Whether FGF2 and FGF4 play a similar

role during human endoderm development remained however unknown. In contrast,

RA has frequently been employed (together with other growth factors) for directed

differentiation of hESCs to pancreatic endoderm.

In the first study we show that FGF2 in a dosage-dependent manner specifies

hESC-derived DE into different foregut lineages such as liver, lung, pancreatic

and intestinal cells. Furthermore, by dissecting the FGF receptor intracellular

pathway that regulates pancreas specification, we demonstrate for the first time to

our knowledge that induction of PDX1+ pancreatic progenitors in part relies on

8

FGF2-mediated activation of the MAPK signaling pathway. In the second study, we

show that FGF4 alone is not sufficient for induction of foregut endoderm but that

in combination with RA it efficiently induces PDX1+ cells from hESC-derived DE.

Specifically, FGF4 promoted cell survival in the differentiating hESCs. Hence, in

contrast to studies in lower vertebrates we demonstrate that FGF4 neither patterns

hESC-derived DE, nor induces PDX1+ pancreatic progenitors suggesting that FGF4

is not responsible for anterior-posterior patterning of the primitive gut during human

development.. Altogether, these observations suggest a broader gut endodermal

patterning activity of FGF2 that corresponds to what has previously been advocated

for FGF4, implying a functional switch from FGF4 to FGF2 during evolution. Thus,

our results provide new knowledge of how cell fate specification of human DE is

controlled – facts that will be of great value for future regenerative cell therapies.

Finally, we present a method for efficient gene targeting in hESCs, which allows the

monitoring of gene expression in living cells. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • PhD Grapin-Botton, Anne, Swiss Institute for Experimental Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne (EPFL)
organization
publishing date
type
Thesis
publication status
published
subject
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
volume
2010:6
pages
91 pages
publisher
Stem Cells and Pancreas Development Biology
defense location
Segerfalksalen, BMC
defense date
2010-01-29 13:00
ISSN
1652-8220
ISBN
978-91-86443-20-7
language
English
LU publication?
yes
id
39535ae5-4b91-41a0-97f5-2cf8256009a1 (old id 1527477)
date added to LUP
2010-01-11 14:39:18
date last changed
2018-05-29 10:19:01
@phdthesis{39535ae5-4b91-41a0-97f5-2cf8256009a1,
  abstract     = {Diabetes affects around 200 million people worldwide. Curing diabetes would<br/><br>
require the prevention of autoimmune destruction of beta cells and restoration of<br/><br>
the beta cell mass restored either through regeneration or transplantation of the<br/><br>
insulin producing cells. Islet transplantation according to the Edmonton protocol<br/><br>
have been the most promising therapeutic option for Diabetes type I patients,<br/><br>
however, lack of cadaveric donor islets is a major obstacle and new strategies need<br/><br>
to be established. Human embryonic stem cells (hESCs) not only offer an excellent<br/><br>
source for establishment of strategies for future regenerative cell therapies and drug<br/><br>
discovery but also offers en excellent experimental assay for understanding human<br/><br>
pancreas development.<br/><br>
Pancreas originates from the definitive endoderm (DE), one of the three germ<br/><br>
layers. The path from definitive endoderm to pancreatic progenitor cells and finally<br/><br>
the insulin producing beta cells involves sequential cell-fate decisions characterized<br/><br>
by the expression of multiple transcription factors. Governed by the developmental<br/><br>
biological principles that normally control foregut endoderm and pancreas<br/><br>
specification, numerous multifactor protocols for directing mature foregut-derived<br/><br>
cells from hESCs have been reported. However, to establish less complex and more<br/><br>
robust protocols there is a need to further understand the mechanism of action of<br/><br>
individual growth and differentiation factors in specification of human pluripotent<br/><br>
stem cells towards foregut derived cell lineages.<br/><br>
Here, we describe two studies where we have investigated the role of fibroblast<br/><br>
growth factor (FGF) signaling, specifically FGF2 and FGF4, and Retinoic acid (RA)<br/><br>
in specification of the hESC-derived DE development. Studies in lower vertebrates<br/><br>
have demonstrated that FGF2 acts in a restricted manner primarily patterning the<br/><br>
ventral foregut endoderm into liver and lung, whereas FGF4 exhibits broad anteriorposterior<br/><br>
and left-right patterning activities. Furthermore, whereas FGF2 is not<br/><br>
required for ventral pancreas development an inductive role of FGF2 has been<br/><br>
shown during dorsal pancreas formation. Whether FGF2 and FGF4 play a similar<br/><br>
role during human endoderm development remained however unknown. In contrast,<br/><br>
RA has frequently been employed (together with other growth factors) for directed<br/><br>
differentiation of hESCs to pancreatic endoderm.<br/><br>
In the first study we show that FGF2 in a dosage-dependent manner specifies<br/><br>
hESC-derived DE into different foregut lineages such as liver, lung, pancreatic<br/><br>
and intestinal cells. Furthermore, by dissecting the FGF receptor intracellular<br/><br>
pathway that regulates pancreas specification, we demonstrate for the first time to<br/><br>
our knowledge that induction of PDX1+ pancreatic progenitors in part relies on<br/><br>
8<br/><br>
FGF2-mediated activation of the MAPK signaling pathway. In the second study, we<br/><br>
show that FGF4 alone is not sufficient for induction of foregut endoderm but that<br/><br>
in combination with RA it efficiently induces PDX1+ cells from hESC-derived DE.<br/><br>
Specifically, FGF4 promoted cell survival in the differentiating hESCs. Hence, in<br/><br>
contrast to studies in lower vertebrates we demonstrate that FGF4 neither patterns<br/><br>
hESC-derived DE, nor induces PDX1+ pancreatic progenitors suggesting that FGF4<br/><br>
is not responsible for anterior-posterior patterning of the primitive gut during human<br/><br>
development.. Altogether, these observations suggest a broader gut endodermal<br/><br>
patterning activity of FGF2 that corresponds to what has previously been advocated<br/><br>
for FGF4, implying a functional switch from FGF4 to FGF2 during evolution. Thus,<br/><br>
our results provide new knowledge of how cell fate specification of human DE is<br/><br>
controlled – facts that will be of great value for future regenerative cell therapies.<br/><br>
Finally, we present a method for efficient gene targeting in hESCs, which allows the<br/><br>
monitoring of gene expression in living cells.},
  author       = {Ameri, Jacqueline},
  isbn         = {978-91-86443-20-7},
  issn         = {1652-8220},
  language     = {eng},
  pages        = {91},
  publisher    = {Stem Cells and Pancreas Development Biology},
  school       = {Lund University},
  series       = {Lund University, Faculty of Medicine Doctoral Dissertation Series},
  title        = {FGF signaling in specification of hESC-derived definitive endoderm},
  volume       = {2010:6},
  year         = {2010},
}