Retinal stem cells and repair

Question 1

what is the retina?

Answer 1

extension of the brain. It is a layered structure that has 6 major neuronal types and 1 glial cells. There are rods, cones, bipolar cells, horizontal cells and amercing cells, retinal ganglion cells and muller glia

Question 2

what are the rod photoreceptor cells?

Answer 2

• responsible for dim light, black and white vision. Found mainly in the periphery of the retina

Question 3

what are the cone cells?

Answer 3

responsible for daytime vision, details and colour/ Concentrated in the region called the macula at the centre of the retina

Question 4

• what are the retinal epithelium

Answer 4

it is located between the light sensitive outer segments of the photoreceptors and good supply of the choroid. It is responsible for the maintenance and homeostasis of the neuroretina

Question 5

what is the main cause of irreversible blindness?

Answer 5

loss of photoreceptors - the cones- macular degeneration

Question 6

what is retinitis pigmentosa?

Answer 6

loss of rods

Question 7

do photoreceptors regenerate?

Answer 7

no

Question 8

what are the two main approached to repairing a diseased retina ?

Answer 8

• • endogenous repair approach: to reverse the cellular loss by encouraging the retina to repair itself
• transplantation of retinal cells (retinal progenitors, RPE and photoreceptors) generated from stem cells. Cell based therapies, replacing retinal cells by transplantation

Question 9

why in theory is the replacement of retinal cells like cones and rods seemingly quite straightforward?

Answer 9

• only one type of cell needs to be replaced and only on synapse needs to be formed

Question 10

wat are the pre-requisites of successful transplantation?

Answer 10

Identification of appropriate donor cells- need to find cell that will have the capacity to migrate and differentiate and also not form cancers
• Successful incorporation of transplanted cells into host retina:
• The transplanted cells need to migrate to the correct layer
• Transplanted cells need to mature
• The newly transplanted neurons need to integrate into the existing neural circuits
• Rescue vision

Question 11

where can you find stem cells to be used in transplantation?

Answer 11

adult stem cells: a pool of multipoint stem cells persists in several adult tissues able to generate the types of cells present in the tissue where they resides. e.g. retinal progenitor cells, CE and iris cells, Muller glia in the retina
- can also use iPSC or embryonic stem cells.

Question 12

what is the role of adult stem cells?

Answer 12

they help to replenish some of the boy’s cells when needed
- the are found in several tissues that need a constant supply of new cells such as the blood, skin, lining of the gut and brain

Question 13

what is the ciliary marginal zone?

Answer 13

• region in the eye which contains stem cells which will give rise to differentiated cells
• in the fish this happens throughout life
• mice have the ciliary epithelium bt doesn’t have this constant capacity to replenish that we see in the fish

Question 14

what are the 4 sources of stem cells in the mammal eye and what are their characteristics?

Answer 14

• ciliary epithelium-derived stem cells
• RPE-derived stem cells
• Iris-derived stem cells
  (all of these cells have the same embrylogical origin and are capable of limited de-differenitation into proliferative retinal progneitors in response to disease and activation of signalling pathway s
• the muller glia-derived stem cells can also be induced to proliferate in vivo in response to injury

Question 15

what is the profile of retinal stem cells in the mammal?

Answer 15

Retinal stem cells in ciliary body (CB), more specifically the pigmented ciliary epithelium (CE) of the CB and the pigmented iris.
• Mammalian CE cells maintain a phenotype analogous to undifferentiated retinal stem/progenitor cells in vitro.
• When induced to differentiate, these cells express markers of both neurons and glia, and some markers of mature retinal cells.

Question 16

what happens when you transplant of CE which contains retinal progenitor cells, into the sub retinal space?

Answer 16

• these cells survive well but didn’t migrate

- they differentiated into glial cells and remained in the sub retinal

Question 17

due to the fact transplantation of retinal progenitor cells doesn’t seem to work very well, what is the alternative?

Answer 17

• induce endogenous repair ro find a new source of cels

Question 18

why are muller cells a good alternative to Retinal progenitor cells?

Answer 18

In contrast to the CE, these cells also demonstrate greater potential for differentiation into mature retinal neurons in vivo, as well as migrating within the neural retina to occupy the correct laminar region appropriate for the newly generated cell type. after injury?

Question 19

how did they test whether photoreceptor persecutors could be used in the lab?

Answer 19

• they used cells from different points in development- nil GFP mice to label rod cells.
• when you transplant retinal progenitors cells, they do migrate into the outer nuclear layer and stay in the sub retinal space and form their own niche
• but when you take these cells at p4-p8 one of these cells will migrate to the outer nuclear layer
• the did it with mature differentiated neurons- some were able to integrate but not as ll et committed post mitotic precursors (those used before

Question 20

how do they carry out a transplant?

Answer 20

• they use a label and then use a syringe to take up the cells and then they inject them into the back of the eye and transpant inbeween the RPE ad the neural retina (rod specific marker) and you can see their migration ebecause they are labelled with GFP
• 3 weeks post translation they could see hey had integrated and formed synapses and extended processes

Question 21

how did they test the response of the integrated receptors?

Answer 21

• they used a mouse with rods that dont function properly
  -they looked at the functional connections by following synapse formation
• they looked at the appropriate responses to light (patch clamping)
• they visually evoked activity in visual cortex (intrinsic cortical imaging)
  they also performed behaviour tests:
• they saw improved visual responses in the mouse which the can watch from the mouse tracking the stripes
• they did water maze- the mouse escapes to the platform

Question 22

even though photoreceptor precursors seem to migrate and integrate really well, what is the problem with using these ofr human clinical trials?

Answer 22

the would need to be harvested from a foetus during the third trimester of pregnancy which ant be done and there is a need for a renewable source of cells for therapy. Therefore we need a better source - iPSC or ESC

Question 23

where can ESC be isolated from?

Answer 23

• the inner cell mass of the blaastocys

Question 24

from what organisms can ESC be isolated from?

Answer 24

humans, mice and monkeys

Question 25

what factors does a progenitor need to become a photoreceptor? explain the entire process that they carried out.

Answer 25

They cultured ESCs in a 3d floating culture. They added factors to induce anterior forebrain fate : Dkk, lefty, FCS and then activin in that order. Then they used a GFP viral label to pick out the rx+ cells. Then they placed these cells in the 3D culture and added DAPT (notch inhibitor) and FGF, Shh and RA. Then they stained for rhodopsin and they got the pic cup formation.

Question 26

how did they try to mimic eye development tin vitro using mouse ESCs?

Answer 26

they use a floating culture in which they use 3000 specifically ES cells and then they provided with an extracellular matrix called matrigel. These cells start stop form a neuroepithelial. By day 5 (they used rx GFP) they start to see vesicles poking out that were positive fro rx. Then they saw that these vesicles started to invaginate to form optic cps just like in retinal development,
- the were able to form a proper retina lamination (?)

Question 27

how did they label and purify the ESC-derived photoreceptor from the embryoid body that formed the eye field and then optic cup?

Answer 27

• they used an AAV viral vector- which labeled the cell sin green.These cells were ten isolated and transplanted into the eye of adult animals - they used GFP with a rhodopsin promoter

Question 28

how did they characterised the photoreeptor development?

Answer 28

• they used crx and rhodopsin darker- they compared the timing that day 36 is the same as p12 and day26-29 is the same as p4 and p6.
• In the adult retina photoreceptor segment structure is made up of an inner and outer segment (IS and OS) connected together by connecting cilium (CC) containing 9 microtubules. In the ESC-derived retinal cultures, inner segment (IS) and connecting cilia (CC)-like structures were observed.The connecting cilia were similar to those found in the retina and the cross- sectional image below shows the 9 pairs of microtubules. However, even in late stage cultures no outer-segment like structures were observed.

Question 29

what happened when she transplanted the ESC derived phorotrecetpors into the retina?

Answer 29

• they were able to migrate into the correct layer, form nice inner segments and form out segments. - rod transducin was expressed in the green cells in the mice lacking this protein.
• Integrated rods (green) extend processes that terminate as round, synapses-like structures, located in close proximity to the rod bipolar cells (h). These synapse-like structures expressed the rod synapse markers Dystrophin and Ribeye (i,j). 3D reconstruction of individual integrated green rod show the correct spatial alignment and morphology of the ribbon synapse in relation to the rod spherule.

Question 30

what was a problem they discovered when the transplanted the ESC derived photoreceptors?

Answer 30

• very low numbers of integration
• The number of integrated green photoreceptors from day 26 and day 29 cultures was significantly greater than that obtained from day 34 cultures- this is similar to what is seen in the photoreceptor precursor used

Question 31

can human ESC cells be used for retinal differentiation?

Answer 31

• yes can may embryo did bodies which contain human photoreceptors0 late with cone expressing reporter - labelled with opsin and sopsin

Question 32

what cell types are made from ESC cells?

Answer 32

• All retinal cell types, including photoreceptors, can be differentiated from human ESC cells

Question 33

can human ESCs be used to do the same as was seen in the mouse

Answer 33

yes- they have produced cones specifically too

Question 34

what are outer segment of photoreceptors?

Answer 34

they are formed from stacked membranous discs packed with visual pigment and enzmes required for phototransduction and are essential for mediating efficient light evoked responses

Question 35

what was the problem that the group was having with its culture of ESC derived photoreeptors?

Answer 35

they were using a 2D culture system but these cells were unable to integrate after transplantation

Question 36

did the optic cup like structures express apical basal polarity?

Answer 36

• yes- the apical side faces the interior of the embryoid body

Question 37

how did the investigate how closely ESC-derived photoreceptor development within the 3D system compared with normal photoreceptor development in vivo? what did they find

Answer 37

by analyzing the time-course of expres- sion of a number of photoreceptor-specific proteins in ESC-derived photoreceptors and photoreceptors from wild-type C57Bl/6J postnatal retinas. There is a peak of Crx expression from postnatal day (P) 3 to P6 in the early postnatal retina, which diminishes in more mature photoreceptors24–26. Similarly, in our wEB cultures the number of Crx+ photoreceptor precursors increased markedly between days 20 and 24 and decreased after day 26 . The reduction in Crx protein levels was accompanied by a substantial increase in the presence of Rhodopsin and Recoverin (Fig. 2a), placing cells at day 26 of culture at a stage similar to the P4–P6 stage of development

Question 38

how did they select the photorecetpors from the embed bodies?en did they show to be formed ?

Answer 38

used an adeno-associated viral vector (pseudotype 2/9) carrying a GFP reporter under the control of a Rhodopsin promoter

Question 39

how did they show that these ESC derived photoreceptors from embryoid bodies could integrate and form out segment s?

Answer 39

We transplanted ~200,000 Rhop.GFP+ FACS-sorted precur- sors (days 26–29) by means of subretinal injection into the adult Gnat1−/− mouse, a model of stationary night blindness, which lacks rod function because of the absence of rod α-Transducin phototrans- duction protein27. Three weeks after transplantation, Rhop.GFP+- sorted photoreceptor precursors had migrated and integrated into the recipient ONL (Fig. 3a). Integrated ESC-derived rods were correctly oriented within the ONL and were usually found in small clusters, a characteristic frequently seen in transplants using donor-derived photoreceptor precursors2,9. Moreover, integrated rods displayed morphological features typical of mature photoreceptors, including inner and outer segments projected toward the host retinal pigment epithelium

Question 40

in vivo, later stage photoreceptors precursors dont integrate as well as P3-P4. How did they test whether this was the case for the embryo by derived photoreceptors?

Answer 40

• they compared the integration rate of early postnatal to late postnatal (day 34)
• indeed the found that integration was lower

Question 41

how have human stem cells been used to prevent degeneration in ways other than transplantation?

Answer 41

• neural stem cells have been implanted and the growth factors that they secrete growth factors and have been shown to rescue models of degeneration in the meta model of retinitis pigments

Question 42

how can gene therapy be used to treat degeneration diseases and what is the problem with this approach

Answer 42

• can use gene therapy to correct the muted cells but gene therapy requires a genetic diagnosis and for the cells carrying the mutation to be alive at the time of therapy, often necessitating early inter- vention.

Question 43

how can optogenetics be used to treat degenerative disorders ?

Answer 43

to replace photoreceptor input by conferring light sensitivity to the remaining inner retinal neurons via the intro- duction of genetically encoded light sensitive proteins, using viral vectors

Question 44

in what way is the retina continuously added to in lower vertebrates?

Answer 44

in concentric rings

Question 45

how can retinal progenitor cells in the mine CMZ-like area be coaxed into proliferating and how does this change over time?

Answer 45

• they can be stimulated by ectopic activation go signalling pathways and y disease - insulin and FGF have been used in mice to do this- but this declines with age

Question 46

in what exciting animal have CMV-like cells been found? how has this been linked to humans?!

Answer 46

• peripheral region of the pirate eye

- interestingly, someone observed that in the presence of EGF, cultured human eyes upregulate a proliferation marker

Question 47

how does retinal regeneration occur in amphibians?

Answer 47

Retinal regeneration in amphibians occurs predominantly via RPE transdifferentiation. In salamanders and tadpole frogs, acute injury to the retina stimulates the cells of the RPE to de- differentiate and proliferate to become retinal neurogenic pre- cursors.

Question 48

how does retinal repair occur in zebrafish?

Answer 48

retinal
regeneration and repair in fish is achieved by Müller glial cell proliferation. Upon activation, these cells proliferate, albeit for a limited number of divisions, whereupon the progeny subse- quently migrate from the INL to the outer nuclear layer (ONL) and differentiate into rod photoreceptors.

Question 49

what was the seminal paper related to muller glial cells?

Answer 49

• muller cells could proliferate in response to neurotropic damage: Müller glia down-regulate the tumour suppressor gene, p27Kip1, within the first 24 h in response to ouabain and domoic acid-induced injury. This and subsequent studies by Ooto et al. showed for the first time that the mammalian retina is capable of some degree of regeneration when exposed to appropriate stimuli

Question 50

what does it mean for a neuron to be integrated?

Answer 50

(1) show appropriate location in the ONL, (2) one of the inner or outer pro- cesses are visible, as well as (3) inner or outer segments and/or (4) synaptic bouton-like structures with the host INL

Question 51

once the mebryoid bodies have been produced, how are the photoreceptors isolated?

Answer 51

he whole retina and/or ESC-derived EBs are dissociated and the photoreceptors are isolate and purified by Fluorescent Activated Cell sorting (FACs).

Question 52

what does FAC mean?

Answer 52

Fluorescent Activated Cell sorting

Question 53

what are ciliary epithelium derived stem cells and how can they used?

Answer 53

Dispersed throughout the pigmented epithelium, these proposed retinal stem cells represent a very rare population (approximately 0.2% of CE pigmented cells), and under normal conditions remain quiescent during adult life. However, when isolated from the eye and cultured in vitro, these cells demonstrate characteristics typical of neural stem cells, including self-renewal and multipotency.

Question 54

have iPSCs been used to create photoreceptors? were they successful?

Answer 54

a human iPS cell line towards photoreceptor cell fate. Approximately 12% of the differentiated cells were CRX-positive, with 1% of all cells in culture expressing late postnatal markers, RECOVERIN and RHODOPSIN. In order to purify and isolate photo- receptors only, the cells were labelled and FAC sorted for the human photoreceptor specific promoter IRBP driving GFP (IRBP-GFP), introduced by lentiviral vectors. Following transplantation into adult wild type eyes, approximately 50 cells per eye were shown to migrate into the ONL. However, no inner processes projecting to- wards the INL, or inner and outer segments were present

Question 55

what has takashi done with using sheets of photoreceptors?

Answer 55

• used 3D differentiation protocol to differentiate mouse ES or iPS cells onto neuroepithelium structures containing a defined ONL.