Survival, migration and differentiation of retinal progenitor cells
Survival, migration and differentiation of retinal progenitor cells
transplanted on micro-machined poly(methyl methacrylate) scaffolds to the
subretinal space
Sarah Tao, Conan Young, Stephen Redenti, Yiqin Zhang, Henry Klassen, Tejal Desai and
Michael J. Young
Age related macular degeneration or AMD is the degeneration of the macula, a highly specialized region of the ocular retina. It leads to the loss of central vision and in severe cases it leads to bleeding in the retina and eventually blindness. It is one of the leading causes of blindness in the elderly and current therapies only slow down the progression of the disease but do not restore the lost vision.
Possible treatment therapies include transplantation of retinal progenitor cells (RPC) to the sub retinal space which would eventually differentiate into some of the lost photoreceptor cells of the retina. However there are many obstacles in delivering RPCs to the sub retinal space which is less than 100µm in width. Injection of RPCs to the sub retinal space is not effective as it leads to cell death and reflux of large amount of injected cells. Introduction of cells using scaffolds is more effective than standard bolus injection as it provides greater control over the microenvironment of the cell but due to the physical constraints provided by the ocular structure of the eye it is critical to develop a scaffold thin enough to be accommodated in the sub retinal space which would not induce any trauma. Not only should the scaffold be small enough it should have an optimum surface chemistry and topographic cues to induce differentiation.
This study looks into the survival, migration and differentiation of RPCs in the sub retinal space delivered via an ultra thin PMMA poly (methyl methacrylate) scaffold. The study also looks at the porosity of the scaffold and how it influences the adherence of RPCs to the scaffold and later differentiation. The PMMA scaffolds were fabricated using a two step photolithographic technique and ion etching process; they measured 6µm in width and contained pores 11µm in diameter, 63µm apart. PMMA has been used extensively in contact and intraocular lenses and is biocompatible. The scaffolds were coated with lysine and laminin after which RPCS were cultured on both porous and non-porous scaffolds for 7 days. The scaffolds with adherent RPCs were than surgically transplanted into the sub retinal space of mice. After 4 weeks the mice were sacrificed and retinal tissue sections were prepared for immunohistochemical characterization and microscopy.
Characterization of the sectioned tissue revealed that RPCs cultured on porous scaffolds had significantly greater morphological integration – migration and process extension with the host retinal layer. In four out of five transplant recipients that received RPCs from porous scaffolds had developed cell processes as compared to non porous scaffolds where only one out of five had such processes. Moreover RPCs from porous scaffolds revealed morphologic differentiation features consistent with retinal neurons and some even spanned the radial extent of the retina similar to meuller cells. They also expressed cell surface markers specific for retinal cells – recoverin, glial cell marker (GFAP) and neural marker – NF 200. RPCs cultured on non-porous scaffolds either died or failed to express the differentiation factors observed in the RPCs cultured on the porous counterparts.
In conclusion, the use of porous PMMA scaffolds not only increases the survival of cells during the delivery of RPCs to the sub retinal space in comparison to bolus injection but it also provides favorable micro environment for cell migration and differentiation into the retinal layer. I chose this paper as this has a major therapeutic application and is an elegant example of how a scaffold with specific properties can be used to reengineer a tissue.
Possible treatment therapies include transplantation of retinal progenitor cells (RPC) to the sub retinal space which would eventually differentiate into some of the lost photoreceptor cells of the retina. However there are many obstacles in delivering RPCs to the sub retinal space which is less than 100µm in width. Injection of RPCs to the sub retinal space is not effective as it leads to cell death and reflux of large amount of injected cells. Introduction of cells using scaffolds is more effective than standard bolus injection as it provides greater control over the microenvironment of the cell but due to the physical constraints provided by the ocular structure of the eye it is critical to develop a scaffold thin enough to be accommodated in the sub retinal space which would not induce any trauma. Not only should the scaffold be small enough it should have an optimum surface chemistry and topographic cues to induce differentiation.
This study looks into the survival, migration and differentiation of RPCs in the sub retinal space delivered via an ultra thin PMMA poly (methyl methacrylate) scaffold. The study also looks at the porosity of the scaffold and how it influences the adherence of RPCs to the scaffold and later differentiation. The PMMA scaffolds were fabricated using a two step photolithographic technique and ion etching process; they measured 6µm in width and contained pores 11µm in diameter, 63µm apart. PMMA has been used extensively in contact and intraocular lenses and is biocompatible. The scaffolds were coated with lysine and laminin after which RPCS were cultured on both porous and non-porous scaffolds for 7 days. The scaffolds with adherent RPCs were than surgically transplanted into the sub retinal space of mice. After 4 weeks the mice were sacrificed and retinal tissue sections were prepared for immunohistochemical characterization and microscopy.
Characterization of the sectioned tissue revealed that RPCs cultured on porous scaffolds had significantly greater morphological integration – migration and process extension with the host retinal layer. In four out of five transplant recipients that received RPCs from porous scaffolds had developed cell processes as compared to non porous scaffolds where only one out of five had such processes. Moreover RPCs from porous scaffolds revealed morphologic differentiation features consistent with retinal neurons and some even spanned the radial extent of the retina similar to meuller cells. They also expressed cell surface markers specific for retinal cells – recoverin, glial cell marker (GFAP) and neural marker – NF 200. RPCs cultured on non-porous scaffolds either died or failed to express the differentiation factors observed in the RPCs cultured on the porous counterparts.
In conclusion, the use of porous PMMA scaffolds not only increases the survival of cells during the delivery of RPCs to the sub retinal space in comparison to bolus injection but it also provides favorable micro environment for cell migration and differentiation into the retinal layer. I chose this paper as this has a major therapeutic application and is an elegant example of how a scaffold with specific properties can be used to reengineer a tissue.
11 comments:
Most researches on eye interest me since they are not as common as the researches on other organs; yet, eye problems occur in daily life. Regarding this paper, it seems to me that the research obtains a good result from using the PMMA scaffold. Do you know if there were prior researches using these PMMA scaffold? I wonder how the scientists decided to use the PMMA scaffold from so many other choices.
I am likewise interested in much of the research regarding to the reconstruction of damaged eye tissue since there isn’t a lot of research done in this field (especially when compared to myocardial infarctions, etc.). Your statement that injecting RPCs can lead to cell death is worthy of note and is something I wouldn’t have initially supposed. And, understandably, scaffold design for optical insertion is extremely difficult. Because of this, I liked your focus on scaffold design. This scaffold is particularly unique given its optical application and necessary limitation of being very thin. One quick thing, though: do you know why they coated the scaffold with lysine?? (Wikipedia didn't have anything about it being useful to scaffold design; I guess I can look into it later, though, if you aren't sure either...)
I liked this paper a lot. In my group project, we have decided to look at how rigidity affects cell behavior. Clearly, this research showed that a porous scaffold was much more beneficial for the survival, migration, and differentiation of the progenitor cells than compared to a less porous scaffold. I am excited to see in my project how porousness affects cell proliferation. Do you or the authors have an idea as of how exactly a more porous scaffold produces a more successful differentiation of progenitor cells?
Did they discuss about the about the aging factors that degenerate the macula? If yes, do these aging factors affect the new RPC introduced by scaffolds into body? What is the longevity of new RPC in body?
Merline Hidayat wrote:
"Do you know if there were prior researches using these PMMA scaffold? I wonder how the scientists decided to use the PMMA scaffold from so many other choices."
According to the paper they decided on using PMMA as a scaffold because it is easier for fabrication purposes using a photoresist in MEMS fabrication, its incorporation of a functional methyl ester group for potential modification and, "most importantly, its biocompatibility
with ocular tissue, which has been established through its long use as a material for contact and intraocular lenses"
Angelle : "do you know why they coated the scaffold with lysine?? "
I think the purpose of coating the slides with poly-L-Lysine is for adhering the tissue section on to the slide for further histological analysis. It has been used quite often for such purposes. I found this technical data sheet from the company and they provide a little bit of background, hope that helps.
http://www.emsdiasum.com/microscopy/technical/datasheet/19320.aspx
I see the link on lysine info has been truncated while posting so here it is again.
http://www.emsdiasum.com/microscopy
/technical/datasheet/19320.aspx
alvinpchan :"Do you or the authors have an idea as of how exactly a more porous scaffold produces a more successful differentiation of progenitor cells?"
Alvinpchan, I don't have a definite answer for our question. This is a big area of research and I know surface topography plays a big role in most aspects of cell behavior such as migration, proliferation and differentiation but how does that exactly influence the cell behavior would be hard to comment on
Nguyen Trinh: "Did they discuss about the about the aging factors that degenerate the macula? If yes, do these aging factors affect the new RPC introduced by scaffolds into body? What is the longevity of new RPC in body?"
They did not discuss specifically the factors causing macular degeneration but there are several factors which lead up to it. Wikipedia has an article on it and hopefully it will answer your question.
http://en.wikipedia.org/wiki/Macula
r_degeneration#Risk_factors
Regarding the factors that might affect these transplanted RPCs, it could be possible that they might degenrate again but it also depends how soon. Lot of things would have to be considered before this procedure is actually used for therapeutic purposes.
Longevity of transplanted RPCs will have to be determined with more in vivo trials since in this trial they sacraficed the mice after 4 weeks. That would be a question for further research.
Sorry for the late reply to all the comments posted on my article. I should have subscribed to the RSS feeds for the comments but anyways I hope I have answered your questions
I wonder if the porosity (not pore size) of the PMMA scaffold had an effect on the cell growth. I find their choice of using a PMMA scaffold very interesting because usually PMMA incites a large fibrous capsule when implanted into the body, which probably wouldn't be very good for your eye; perhaps the ultrathin design minimized this wound healing response.
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