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Efficient generation of retinal progenitor cells from human embryonic stem cells
(Proc Natl Acad Sci USA 2006;103: 12769-12774)

Advance  Stem Cell Class, Fall 2006
Summarized by Krista Bono and Nidhi Shah

Lay Summary

The human body is prone to multiple diseases and insults throughout its lifetime. Science has progressed to a degree, where there are improved treatment and preventative options available for almost any human affliction. However, the treatment options have major limits. The field of stem cell biology as therapy has expanded immensely in the last decade. Ethical and scientific concerns dampen the use of human embryonic stem cells for treating retinal disorders such as, retinitis pigmentosa, age-related macular degeneration, and glaucoma. Embryonic stem cells are derived from 5-day old human blastocyts. These stem cells are intended to generate cells of all tissues. The focus of the referenced paper was to generate retinal cells from embryonic stem cells through attempts to use chemical signals that mimic the developing internal environment.

President Bush in August of 2001 made it illegal for investigators to conduct research on new embryonic stem cells with federal funds. Therefore, researchers could either use the existing embryonic stem cells or use private funds with newly obtained embryonic stem cells. The researchers of this study used federally approved embryonic stem cells to develop retinal cells in the laboratory. Cells were analyzed for their morphology and functionality. The growth and features of the cells were examined through staining using dyes and tagging the cells through antibodies specific for various types of retinal cells. The genetics of the cells was also examined through Polymerase Chain Reaction (PCR), which mapped out the genes typical of retinal cells. Finally, the cultured cells were grown again, with dissected retinas from mice that had retinal degeneration, to further examine morphology and growth. The functional analysis of the obtained retinal cells showed evidence of success since the cells sent signals as expected.

The types of cells derived from the embryonic stem cells were ganglion cells, amacrine cells, photoreceptors, and bipolar cells. The study also compared the experimentally obtained retinal cells to the naturally developed retinal cells in aborted fetuses, proving substantial similarity between both types of cells. The developmental timing of the genes was also conserved in the derived retinal progenitors.

Personal Comments:
Such manipulation of the microenvironment of a human body could be used to treat various retinal disorders, as well as other disorders such as, Parkinson’s disease, Alzheimer’s disease, among others. Lamba et al, propose that the cultured retinal progenitors can be produced quickly and effectively, as opposed to the prolonged period for development in a natural setting. This leaves the question of the type of cells that were generated. The investigators should have used other techniques to further characterize the generated derived cells. The combination of signaling molecules used and the sequence of use was probably a successful result of numerous trial and error attempts, since the developmental timing of the cells was similar to what is found in nature. In conclusion, the discussed study is in the right direction, since it provides a novel method to derive retinal cells from developing embryonic stem cells. Future studies are needed to determine if there are untoward effects of the embryonic nature of the cells, and also to show that these stem cells are considerably more efficient than adult cells.

Scientific Summary
Human embryonic stem cells (hESC) shows promise to efficiently generate retinal progenitors, as demonstrated in the summarized publication by Lamba et. al.  The research is significant due to blindness caused by difficulty to replace adult mammalian retinal neurons.  However in studying vertebrate embryogenesis, Lamba and colleagues developed a novel in vitro microenvironment that skewed hESC into retinal progenitor fate by combining noggin, Dickkopf-1 (Dkk-1) and insulin-like growth factor-1 (IGF-1).   

Material & Methods: The H-1 (WA-01) hESC line was maintained by standard procedure irradiated mouse embryonic fibroblast feeder cells.  The hESCs were treated with type IV collagenase and then plated in six-well ultra-low attachment plate to generate embryoid bodies.  The embryoid bodies were developed in 3-day cultures in low concentration of mouse noggin, human recombinant Dkk-1 and human recombinant IGF-1.  On the fourth day, embryoid bodies were transferred to poly(D-lysine)-Matrigel coated 35-mm plates to allow attachment and 3D-growth.  At this time, the culture media, referred to as retinal determination conditions, contained 10-fold increase of noggin, Dkk-1 and IGF-1 plus B-27 supplement for long-term viability of neurons, N-2 supplement for growth and expression of post-mitotic neurons of neuronal phenotype, and human recombinant basic fibroblast growth factor (bFGF).  Analyses for embryoid bodies were done at week one, two and three following the initiation of culture  (Figure 1)  Primary cell cultures were also analyzed and compared using eyes from 78-95-days-postconception fetuses from unidentied therapeutic abortions, obtained from the fetal tissue bank at University of Washington. 

Characterization techniques were immunofluorescence (IF), quantitative PCR (qPCR) and functionality studies were done by calcium imaging of glutamate responses or in vitro explant co-culture experiments from global GFP wild-type and Aipl1-/- mice.

Results:
Characterization:  Studies at the levels of protein and mRNA for eye field transcription factor (EFTF) have determined efficient induction of hESCs to induce cells consistent for retinal progenitor lineage. There was no significant gene expression for non-neural tissues and also for other regions of the central nervous system. The combination of Noggin, Dkk-1 and especially IGF-1 were essential for lineage differentiation from ESC to retinal progenitors. The latter was shown to differentiate in ganglion, amacrine, immature photoreceptor, bipolar and horizontal cells, indicating multilineage phenotype of the retinal progenitors.  In characterizing ganglion and amacrine cells, Pax6High was validated not to be an artifact since similar results were displayed in primary cell culture from 78-day human fetal retinas.  Moreover, studies pertaining to vertebrate embryogenesis using time-course studies and gene expression, the authors concluded that 3-week ESC-derived retinal progenitors could be analogous to 91-day human fetal retinas. This indicates that the in vitro assays show accelerated developmental by three-four weeks over normal human embryological development.   

Functionality:  Some of the ESC-derived retinal progenitor cells responded to glutamate and NMDA with the display of calcium fluxes, with concomitant synaptic development.  Due to the majority of the photoreceptor cells expressing immature markers, the authors co-cultured their cells with retinal explants from global GFP wild-type or Aipl1-/- mice. The results showed an increase of recoverin-immunoreactive cells only for null mice, while an increase in the population of cells expressing rod- and pan-photorector markers (Rho-4D2 and Nrl, respectively).

Conclusion:
Lamba et al. derived a novel microenvironment with high efficiency to induce hES cell lines into retinal progenitor lineage.  They discussed the differences among species in the response(s) to various factor(s) that might not always lead to the same efficiency or result(s). In their paper, they especially emphasize noggin.  Moreover, the accelerated developmental time, the comparable gene expression profile and the integration of immature photoreceptors to increase their expression of photoreceptor-specific markers of the hESC-derived retinal progenitors raises the question:  Could this methodology be a source for new neurons and photoreceptors for retinal repair?

Personal Comments: 
The characterization of the retinal progenitors was convincing. However, expanded functional studies are warranted to determine if the hESC-derived retinal progenitors can really be utilized for retinal repair.  Moreover, markers and gene expression do not always equal function.  Therefore, utilizing a retinal degenerating condition mouse model is proposed to answer how these cells will respond in a non-controlled, natural/diseased microenvironment, and if they are indeed functional.   

Overall, the paper was novel and intriguing; however, I would have liked to have read their interpretation for the results of the functional maturation analysis like they so elegantly did in determining the proper protocol and results for characterizing the cells.