Section 5 Cont.section 5-6

Front 1

in tet-on system, to find reporgramming duration, yamanaka factors expressed when___(part of tetoracycline antibotics) added to culture

Back 1

doxycycline

Front 2

When the factors were expressed for less than 10 days_____. At least_____days of factor expression were
necessary to derive iPSCs.

Back 2

no iPSCs emerged
12

Front 3

after 14-16 days iPSCs colonies could

Back 3

grow without doxycycline
this duration (2weeks) coincides with expression of endogenous oct4/nanog

Front 4

do factors need to be integrated permenantley into genome

Back 4

no, we can treat cells temporarily

Front 5

-Some iPSC lines carry epigenetic marks typical to the
-due to this, not all iPSCs may be

Back 5

cell lineage of their origin
equivalent
*they show epigenetic memory

Front 6

other than the epigenetic memory of iPSC these cells are entirely compatible with ___by most criteria
and show reprogramming can be used to establish the esc ___

Back 6

esc
phenotype

Front 7

iPSCs present a great promise to generate______ pluripotent stem cell lines.

Back 7

patient-specific

Front 8

reprogramming is__process

Back 8

gradual

Front 9

dna methylation/histone modification affected by

Back 9

cells of origin

Front 10

experimental manipulations difficult and highly inaccessible because

Back 10

all developmental processes in mammals occur in utero

Front 11

if we can direct ESCs to differentiate into a specific lineage at our will, then, we should be able to

Back 11

produce target cells for cell replacement therapy

Front 12

induction of directed differentiation in pluripotent stem cells is the essential step for

Back 12

clinical applications of ESCs

Front 13

can u use esc's/ipsc's for analyzing drug effects

Back 13

yes

Front 14

so induction of directed esc diff important but hard to do because

Back 14

proportion of cells made and absolute numbers are small

Front 15

type 1 diabetes due to

Back 15

loss of insulin secreting cells
can possibly use esc/ipsc's replacemnt therapy

Front 16

pancrease derived from___
pancreatic buds induced on__(2() sides of gut
develops from
has both (2) function
islets make___

Back 16

endoderm
dorsal and ventral
embryonic gut
endo/exocrine
endocrine hormones

Front 17

alpha cells (20%) make
beta cells (50%) make
delta (5%) make

Back 17

glucagon
insulin
somatostain
*endocrine hormones

Front 18

acini secrete

Back 18

digetsive enzymes (exocrine)

Front 19

During the pancreatic development, a series of transcription factors are expressed sequentially in a
_____manner

Back 19

timely

Front 20

in pancreas development (Shh) is expressed in definitive endoderm but its expression____as pancreas specified
Pdx1 is

Back 20

declines
upregulated

Front 21

delete pdx1=

Back 21

no pancreas

Front 22

Although Pdx1 is initially expressed only
temporarily at the time of pancreatic specification (~embryonic day 10), the gene is expressed again
later when

Back 22

pancreatic b-cells emerge

Front 23

pdx1 is ___TF

Back 23

homeodomain

Front 24

pax4 is homedomain TF that induces

Back 24

beta cells

Front 25

stz (streoptozotocin)

Back 25

kills beta cells

Front 26

transplanting beta cells derived from esc leads to

Back 26

reduced blood glucose
*implies beta cells from esc are functional
**this is proof of principle study for directed diff induced in esc's

Front 27

next slides looking at beta induction using GF's

Back 27

..

Front 28

since ESC's similar to ICM gastrulation like responses need to be induced, this done by(2)

Back 28

NODAL AND WNT

Front 29

nodal is part of___family

Back 29

TGFbeta

Front 30

cells destined to become endoderm located
near _____at the primitive-streak stage. It secretes a growth factor, called___. High level of that promotes differentiation into the _____, low level stimulates differentiation into the ____.

Back 30

node
“nodal”
endoderm
mesoderm
*ie location of cells, level of nodal, and endodermal differentiation seems to correlate each other

Front 31

activin (TGFbeta family GF) can mimic___in vitro

Back 31

nodal

Front 32

patterns of Shh expression in endoderm correlate with

Back 32

proximity of notochord
*shh exp in dorsal endo suppressed by notochord
shh expressed in notochord/ventral endo

Front 33

(2) factors in notochord are what suppress shh in dorsal endo

Back 33

activin, FGF2

Front 34

when activin is added to in vitro culture of the isolate gut, dose dependent induction of _____and ____is seen

Back 34

Pdx1 , insulin

Front 35

low dose of FGF2=
medium=
high=

Back 35

pdx1 exp
insulin exp
reduce pdx1/insulin

Front 36

effect of activin suppressed by

Back 36

shh

Front 37

neutralizing antibodies+shh=

Back 37

genes for pancreas development induced ie shh expression needs to be inhibited for pancreas development to happen

Front 38

notochord is__organ

Back 38

mesodermal

Front 39

singals between endoderm and mesoderm control (2) of pancreas

Back 39

identity and patterning

Front 40

researchers made 5step protocol for pancreatic induction from hESC. step 5 when generate mature insulin secreting beta cell in vitro BUT they got rid of step 5 why?

Back 40

-completing all diff processes made no sense
-had limited number of beta cells in vitro
-end of culture esc derived cells expressed pancreas genes and initiated endocrine lineage *didnt mature enough to make insulin/glucagon

Front 41

implanted human pancreas (embryonic) under kidney of mouse. before implanted had few cells stained by antiinsulin/glucagon antibodies-->confirms that
-after 2 months of implant saw
-after 21 weeks____cells seen
*results show that human beta cells can develop

Back 41

-embryonic pancreas had developed in donor BUT endocrine had not
-increase growth (200X weight, 5000x # beta cells)
-insulin expressing
*spont from immature pancreas

Front 42

primitive pancreatic cells divide and prolif but once they commit to diff into the____lineage they do not prolif or prolif much less

Back 42

endocrine cell
*pdx1(more primitive) incorp BrdU and ngn(more committed to endocrine lineage) dont

Front 43

to verify that have pancreatic cells you:
measure level of __in blood
inject___at dose that kills mice beta more selectively than human beta cells
found that __level didnt change after injection suggest human___effective in absence of mouse counterpart
if remove implanted ESC derived pancreas cells glucose levels___

Back 43

insulin
STZ
glucose
insulin
overshot

Front 44

4 practical issues about 5step

Back 44

effciency
tumorigenicity
transplantion site
complexity