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ANAT 381 > Stem Cells 4 > Flashcards

Stem Cells 4 Flashcards

1
Q

Describe movie exp -Parkinson’s

A

Parkinson’s = bc of death of dopaminergic neurons
Aborted fetus cells - dopamingeric neurons in specific region of brain - fetal progenitor cells = inject into patient = 1 mil cells into where degenerated neurons are
= HE IMPROVEDDDD

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2
Q

Describe general embryonic dev of pancreas

A

ENDODERMal origin
Buds out from fore/mid gut, dorsal and ventral side
Common origin = same as liver, endodermal
Cancer = liver <—> pancreatic cells and vice versa (also cam induce this switch)
As gi tract turning = buds merge and make pancreas, 2 weeks merge together and make pancreas
Pancreas = sac of enzymes, Sanwhich between gi

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3
Q

Name and describe the functions of a pancreas

A

Acini = secrete digestive enzymes like trypsinogen, lipase, amylase (to gi tract = outside body) so EXOCRINE
Islet = secrete endocrine hormones - insulin from B cells (decrease glucose level in blood, small peptide), glucagon from alpha cells (opposite) ENDOCRINE
*homeostasis of whole body

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4
Q

What correlates with induction of B cells

A

PAX 4 expression

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5
Q

Describe expression of diff tfs during pancreas dev

A

Early pancreatic dev = shh shut down and PDX1 tf turned on (when mouse primitive streak and organ dev starts)
Endocrine cell precursors = no PDX1, but ngn3
B cell = PDX1 comes back and pax4 expressed only tentatively, when B cells appear from endocrine precursor cells, maybe acts as trigger to B cell dev in vivo

Tfs involved in differentiation of pancreatic B cells

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6
Q

Describe induction of pancreatic B cells from mouse escs - step 1

A

CMV promoter = cytomegalovirus, drives gene, exogenous promoter so hat spontaneously activates gene downstream = pax 4 gene
Transgene still autonomously express pax4 after incorporation into genome
To induce differentiation = scrape off es cells - off culture dish - to induce, physically scrape so sphere

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7
Q

Describe induction of pancreatic B cells from mouse escs - step 2

A

Hsitological sections, antibodies to visualize target protein expression - insulin, conjugate antibody with fluorescent dye = becomes red when exposed to uv
Number of cells express insulin - expressed at protein levels but must confirm if has biological action as insulin and not just protein expression here

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8
Q

Describe induction of pancreatic B cells from mouse escs - step 3

A

Exp = stz- antibiotic, streptozotocin = attackes and kills beta cells, rodents become miserable and have to Give them accomodations so they survive
Transplanted embryoid body into one group= normalizes glucose levels, cells can sense condition and work too hard, - too low glucose cells and then comes back up and maintain = cells capable of sensing what happens in body and assuring accordingly
Stz control another group= severe diabetic condition, v high glucose
and wild type control

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9
Q

Describe summary of pancreas initial exp - pax4 - mouse

A

Pax4 = tf known to be important for inductions of pancreatic B cells during embryonic dev
Forced expression in escs induce B cells

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10
Q

Describe summary of pancreas initial exp - transplantation of pax4 - mouse

A

Transplantation of b cels derived from escs leads to reduction of blood glucose levels, reversing diabetic conditions in mouse disease model = implies that B cells produced from escs are functional = can regulate secretion of insulin
Results give proof of principle for a directed differentiation induced in escs

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11
Q

Describe summary of pancreas initial exp - risks - mouse

A

—> icm cells but also teratoma cells So if exp went longer than only 2 weeks = could induce tertaomas (at around 1 monthg)
Genetic manipulation bears a level of safety risks

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12
Q

Describe induction of pancreatic B cells from human escs gen

A

Know factors expressed in embryos = timing and exactly were- parallel to mouse
Want to induce these steps = follow normal developmental cascade but in vitro using es cells

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13
Q

Describe culture scheme for B cell induction - gen =HUMAns

A

5 stages
Genes that should be expressed are shown
And growth factors used to induce these genes at each stage
Roughly following expression fo Morphogens during normal pancreatic dev
Empirical determination = of various parameters - dose, timing, duration,combination ect = trial and error- when does not work = hard

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14
Q

Describe culture scheme for B cell induction - all 5 stages

A

Stage 1 = definitive endoderm, activin + wnt and then just activin
Stage 2 = primitive gut tube = fgf10 + cyc
Stage 3 = posterior foregut, ra + cyc + fgf10
Stage 4 = pancreatic endoderm and endocrine precursor +/- dapt; ex4 PAX4 MARKER TO ENSURE PROPER PATH- CORRECTLY DOING IT
Stage 5= hormone expressing endocrine cell, +/- ex4, igf1, hgf, INS = GENE EXPRESSION MARKS END

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15
Q

Describe forced commitment of Hescs to endodermal lineage

A

To differentiate = induce es to endoderm
Needed = activin/nodal
High levels of nodal —> endoderm
Low levels of nodal —> mesoderm
Not tfs but soluble extra cellular molecules that can be applied in vitro
Nodal = tgfb fam mem, activin can mimic nodal actions to induce endoderm, used activin since nodal not available at time

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16
Q

Describe fate map of mouse

A

Results of study - 1991
Injected specific dye into epiblast little by little and see what dye incorporated to what tissues/organs
Cells that become endoderm emerge close to node= where nodal secreted
So high levels of nodal —> endoderm
Suggest that lower dose does not induce enddoerm in multi potent or almost pluripotent cells (Epiblast)
Dose dependent induction of endoderm and mesoderm confirmed in vitro and in vivo

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17
Q

Describe endoderm to pancreatic anlagen

A

Signals between endoderm and mesoderm govern identity and patterning fo pancreas
First = soluble factors, shut down shh before endocrine precursors induced
Want to shut down shh so use inhibitor = cyc (cylopamine, shh signalling inhibitors)
Shh, activin, Fgf

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18
Q

Describe shh expression - where

A

Notochord developing = derived by mesoderm eventually becomes some vertebrae
Blue cells = express shh
In sit hybridization - for cells expressing shh
Bottom of neural plate, endodermal cells at tip of embryo
Notochord does express shh but side facing notochord = no shh

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19
Q

Describe shhh expression - what it means

A

Notochord (mesoderm origin) represses shh expression in endoderm
May correspond to shutting down in area of gut where pancreas should be

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20
Q

What does notochord do in endoderm

A

Suppresses shh expression in endoderm

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21
Q

Describe when remove notochord

A

Surgically remove notochord then measure shh
Ectopic shh expression - before completion fo notochord enododermal signalling
Induces shh expression in dorsal enddoerm, where shh expression was suppressed
mRNA of shh appears after removal of notochord

22
Q

Describe when ectopic implant notochord

A

Reduces shh expression
Usually notochord on ventral side but put on dorsal side = mRNA shh declines, in expression levels

23
Q

Describe when ectopic implant of Somite

A

No effects
When transplant something else = no shut down

24
Q

Describe exp of seeing tf expression - how

A

Look at factors expressed in notochord
Take out endoderm and put in culture well (wrapped endoderm in collagen, so endoderm stable in medium), in vitro exp, add activin and fgf in diff doses , measure readout transcripts using pcr

25
Q

Describe exp of seeing tf expression - results for activin

A

Dose dependent increase of insulin
Positive influences differentiation of mesoderm to B cell that produces insulin

26
Q

Describe exp of seeing tf expression - results for fgf2

A

At low/med dose = induces insulin gene, but if too much = shut down

27
Q

Describe stage 4/ 5 ish

A

Established 5 step procedure
But found stage 4 proceeds by simply culturing for another 3 days without added factors = didn’t need to do anything’s
(Posterior foregut —> pancreatic endoderm and endocrine precursor)
Further omitted stage 5 bc of exp done - for clinical applications, es cells proliferate aggressively, differentiation efficiency low, needs many B cells for humans

28
Q

Describe human embryonic pancreas —> immunodeficient mice

A

Transplanted human embryonic pancreas (6-9 weeks gestation) into mouse - fetal pancreatic progenitor state, immature state of cells
Into kidney capsule of immunodeficient mice, nick embryos and make space between membrane and insert human tissues, fate grows here = human pancreas
Weight of implant increases 200 fold and number of humanbeta cells increases 500 fold

29
Q

Describe what happens to endocrine pancreas - cell dev gen

A

Antibody immuno fluorescent staining
Endocrine pancreas develops spontaneously
Primitive types of pancreatic cells proliferate bit those in lineage commitment do not

30
Q

Describe INSULIN GLUCAGON - before and after transplantation

A

Before = v small number insulin positive cells
After transplantation - 3 months = see many insulin secreting cells

31
Q

Describe INSULIN PDX1- before and after transplantation

A

Before transplantation = no insulin positive cells, PDX1 positive cells everywhere
4 weeks after transplantation = less PDX1
21 weeks after transplantation = 5 months, PDX1 expression back and insulin secreting cells

32
Q

Describe what Brdu is

A

Uridine conjugated with bromine = incorporated in genome when cells divide = prospective - if cells Brdu positive = cells did divide

33
Q

Describe BRDU PDX1 NGN3 - staining

A

PDX1 = everywhere, Brdu staining corresponds to PDX1, primitive enddoermal cells dividing a lot
NGN3 = growth factor, expressed at Time of endocrine precursors developing
Advanced cell stages do not proliferate = do not need final differentiated cells, more immature = divide more

34
Q

DESCRIBE EXP where transplant B cells into mouse - set up

A

Transplanted stage 4 cells into mouse
Immunodeficient mice with normal B cells - so implant didn’t affect them but then
Stz dose used = kills mouse beta cells and not human beta cells, so B cells of mouse idled but glucose level does not go up

35
Q

DESCRIBE EXP where transplant B cells into mouse - results

A

Stable blood glucose at first then explant= glucose shot up = induced human beta cells were working, opposite exp as first one but results prove same thing
roughly following expression patterns of Morphogens during normal pancreatic dev and apply to induce and derive desired cell types

36
Q

DESCRIBE EXP where transplant B cells into mouse - type of exp

A

Empirical determination of various parameters - dose, timing, duration, combination, etc

37
Q

Describe some practical issues to consider = efficiency

A

Two lines of hESCs were examined, and functional glucose-responsive grafts were produced with both lines = 100%
Functional grafts were found in 70 – 90% of animals used (insulin responsiveness restored)
Should work with hiPSCs

38
Q

Describe some practical issues to consider = tumorigenecity

A

One out of 46 grafts showed a teratoma = 2.2%
Six other grafts had teratomatous tissue elements = 13% —> Total ~15% (neoplasticism transformation taking plac,e protocol robots but not completely eliminating es stem cells, if transplanted into individual that genetically matches es cells = chance of causing cancer)
Expected to occur with iPSC-derived cells

39
Q

Describe some practical issues to consider = transplantation site

A

Current clinical practice is to transplant islets into the liver, because;
- most insulin from the pancreas is used in the liver, and
- techniques are well developed for transplantation into the liver.
Half of the b-cells die shortly after transplantation (happens in many organ transplantations), perhaps because of:
- low oxygen tension,
- an active immune response, and
- high levels of toxins and drugs in the liver.

40
Q

Describe some practical issues to consider = complexity

A

Many growth factors, complex culture protocols, costs, time consuming, expensive

41
Q

Describe eye development - gen

A

Inducing tissues using organoids
Fetal brain - neuroectoderm
Surface ectoderm = skin
Invagination = once neutral tissue touches skin = skin starts invaginating towards brain, (makes lens placode)
Makes cup of retina and skin bud
Skin bud becomes lens
Brain part becomes retina - photoreceptor cells at bottom
Light concentrated by lens —> retina at bottom = photoreceptors then (signalling neuron comes back to surface then goes to brain)

42
Q

Describe making an eye from mouse escs - embryonic body

A

Embryoid body based differentiation induction
Depends on size
Aggregate = make small embryoid body —> tends to become brain, neural tissues preferentially
Es cells = digest, single cell suspension and it into cup and spin down to make pellet
Put palette on medium - cell pellet becomes ball = embryoid body
Cut section and stain neuron specific proteins = pellet cells in embryoid body differentiate into neural tissues
Suspension culture of aggregates of ~3000 escs lead to organization of cells expression bf1 = telencephalon (embryonic brain precursor) expressed

43
Q

Describe making an eye from mouse escs - exp

A

Ebs made by quick aggregation were culture in an eco gel instead of complete suspension in a medium
Use embryoid body but put on gel, not floating state= cell aggregate put into eco, gave suppport to embryoid body = coated in medium, supported by gel and
Monitored daily

44
Q

Describe making an eye from mouse escs - results

A

Started spontaneous differentiation to eye cup
Evagination then invagination and makes cup
See retinal pigment epithelial cells towards bottom, neural retinal pointed to surface, photoreceptors cells, new neurons and ganglia = cell types almost identical to normal eye dev
But no lens in regenerated eye cup since no skin!

45
Q

What is organoid

A

Made in bioreactor from es cells
Reconstruction of tissue/organ ex vivo

46
Q

Describe brain organoid

A

From pluripotent stem cells

47
Q

Describe intestine organoid

A

From endogenous stem cells
Intestinal crypt, regenerates intestinal like structure

48
Q

Describe potential applications of organoids

A

Organ regeneration/replacement (Organoids = immature so cannot really replace organs)
Dug/toxin screening/safety tests = makes ipscs from patients with genetic diseases then convert to organoids and then researchers can investigate pathogenesis
Investigate pathogenesis research

49
Q

What did use of ipcs make possible

A

Patient specific therapy

50
Q

What is an advantage of ipscs

A

Can come from any cell types, not just aborted fetuses

51
Q

What is a good thing about ipscs

A

Robust proliferation of ipscs makes it possible to scale up the strategy or to overcome a potential inefficiency of differentiation induction

ANAT 381 (24 decks)

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