Publications from researchers at the Buck Institute for Research on Aging http://www.buckinstitute.org/lambaLab © 2011 Buck Institute, All Rights Reserved Over the last few years, numerous studies have introduced strategies for the generation of neuronal populations from embryonic stem cells. These techniques are valuable both in the study of early neurogenesis and in the generation of an unlimited source of donor cells for replacement therapies. We have developed a protocol to direct mouse and human embryonic stem cells to retinal fates by using the current model of eye specification. Our method is a multistep protocol in which the cultures are treated with IGF1 and a combination of BMP and Wnt inhibitors to promote the expression of key retinal progenitor genes, as assayed by RTPCR and immunofluorescence microscopy. The retinal progenitor population spontaneously undergoes differentiation towards various types of retinal neurons, including photoreceptors. http://www.ncbi.nlm.nih.gov/pubmed/22688710 Sat, 31 Dec 2011 00:00:00 -0800 Previous studies have shown that Mller glia are closely related to retinal progenitors these two cell types express many of the same genes and after damage to the retina, Mller glia can serve as a source for new neurons, particularly in nonmammalian vertebrates. We investigated the period of postnatal retinal development when progenitors are differentiating into Mller glia to better understand this transition. FACS purified retinal progenitors and Mller glia from various ages of Hes5GFP mice were analyzed by Affymetrix cDNA microarrays. We found that genes known to be enriched/expressed by Mller glia steadily increase over the first three postnatal weeks, while genes associated with the mitotic cell cycle are rapidly downregulated from P0 to P7. Interestingly, progenitor genes not directly associated with the mitotic cell cycle, like the proneural genes Ascl1 and Neurog2, decline more slowly over the first 1014 days of postnatal development, and there is a peak in Notch signaling several days after the presumptive Mller glia have been generated. To confirm that Notch signaling continues in the postmitotic Mller glia, we performed in situ hybridization, immunolocalization for the active form of Notch, and immunofluorescence for BrdU. Using genetic and pharmacological approaches, we found that sustained Notch signaling in the postmitotic Mller glia is necessary for their maturation and the stabilization of the glial identity for almost a week after the cells have exited the mitotic cell cycle. http://www.ncbi.nlm.nih.gov/pubmed/21829655 Fri, 31 Dec 2010 00:00:00 -0800 A number of protocols have been published to induce retinal determination from human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC). Although all these studies have shown some degree of expression of markers of retinal cells, fewer than 30 markers are typically used to characterize the ESCderived retinal cells. Hence, it is not known whether they express all the genes present in normal developing retinal cells. To assess the efficiency of their retinal determination protocol at the transcriptome level and to understand the changes in human retinal gene expression patterns during development, the authors conducted a microarraybased analysis comparing human retina to hESCderived retinal cells. http://www.ncbi.nlm.nih.gov/pubmed/21345990 Fri, 31 Dec 2010 00:00:00 -0800 Embryonic stem (ES) cellderived photoreceptors are a promising cell source for enhanced invitro models of retinal degenerative diseases, but the more differentiated characteristics of retinal cells do not typically develop in dissociated cell cultures. Therefore, we have reconstructed organized retinal tissue by seeding dissociated cells into an array of aligned units that more faithfully mimics the retina. We solventprocessed poly(lacticcoglycolic acid) (PLGA) into a microchannel scaffold format to achieve this geometric constraint. We compared the effect of PLGA concentration on channel morphology and, along with other culture conditions, on the infiltration of dissociated newborn mouse retinal cells into the channels. Culturing scaffolds at the gasliquid interface with low serum media increased infiltrated rod photoreceptor viability 18fold over submerged, high serum cultures when evaluated after seven days. Rod photoreceptors and Mller glia aligned processes parallel to the microchannel walls. Otx2 and Pax6 subpopulations recapitulated lamination behavior. Further, we constructed scaffold/retinal pigment epithelium (RPE) cocultures and observed rods extending rhodopsinpositive processes toward RPE cells, mimicking normal rod polarization and morphology. Finally, human embryonic stem cellderived photoreceptors exhibited infiltration and morphological characteristics similar to mouse retinal cells inside the scaffolds. These findings constitute an important advance in generating tissuelevel retinal models from dissociated cells for use as drug screening platforms and in regenerative medicine. http://www.ncbi.nlm.nih.gov/pubmed/22115999 Fri, 31 Dec 2010 00:00:00 -0800 RNA transfection methods have not proven to be as popular as DNA methods due to the highly transient nature of the RNA inside the cell. However, there are many advantages in using RNA for gene overexpression, such as the rapidity of expression, the ability to express in all cell types without the need for celltypespecific promoters, and the ability to analyze the effects of gene overexpression in a transient manner. Therefore, we have developed a method (StabiLizingUtr: SLU) to stabilize the RNA for varying durations, using specific sequences from the 3'UTR of the Venezuelan equine encephalitis virus (VEEV). We have designed a plasmid for cloning genes upstream from repeated stabilizing sequences to generate mRNA with one or more VEEVstabilizing sequence motifs. We demonstrate this method in several cell and tissue types, including the mammalian cochlea, a tissue that has been difficult to transfect with other methods. http://www.ncbi.nlm.nih.gov/pubmed/20549727 Thu, 31 Dec 2009 00:00:00 -0800 Substantial progress has been made in the development of methods to direct embryonic stem cells to differentiate into various regions of the central nervous system (CNS). We have used the current model of eye specification to develop a protocol for directing human embryonic stem cells to generate retinal progenitors and various types of retinal neurons. Our method uses a multistep protocol in which embryoid bodies are treated with inhibitors of both BMP signaling and canonical Wnt signaling to promote expression of key eye field transcription factors (EFTFs), as assayed by RTPCR and immunofluorescence microscopy. The retinal progenitor cells spontaneously undergo differentiation into various types of retinal neurons, including photoreceptors, and this can be promoted by treatment with small molecule inhibitors of the Notch pathway. http://www.ncbi.nlm.nih.gov/pubmed/20336521 Thu, 31 Dec 2009 00:00:00 -0800 Photoreceptors, rods and cones are the most abundant cell type in the mammalian retina. However, the molecules that control their development are not fully understood. In studies of photoreceptor fate determination, we found that Blimp1 (Prdm1) is expressed transiently in developing photoreceptors. We analyzed the function of Blimp1 in the mouse retina using a conditional deletion approach. Developmental analysis of mutants showed that Otx2() photoreceptor precursors ectopically express the bipolar cell markers Chx10 (Vsx2) and Vsx1, adopting bipolar instead of photoreceptor fate. However, this fate shift did not occur until the time when bipolar cells are normally specified during development. Most of the excess bipolar cells died around the time of bipolar cell maturation. Our results suggest that Blimp1 expression stabilizes immature photoreceptors by preventing bipolar cell induction. We conclude that Blimp1 regulates the decision between photoreceptor and bipolar cell fates in the Otx2() cell population during retinal development. http://www.ncbi.nlm.nih.gov/pubmed/20110327 Thu, 31 Dec 2009 00:00:00 -0800 BACKGROUND: Inherited and acquired retinal degenerations are frequent causes of visual impairment and photoreceptor cell replacement therapy may restore visual function to these individuals. To provide a source of new retinal neurons for cell based therapies, we developed methods to derive retinal progenitors from human ES cells. METHODOLOGY/PHYSICAL FINDINGS: In this report we have used a similar method to direct induced pluripotent stem cells (iPS) from human fibroblasts to a retinal progenitor fate, competent to generate photoreceptors. We also found we could purify the photoreceptors derived from the iPS cells using fluorescence activated cell sorting (FACS) after labeling photoreceptors with a lentivirus driving GFP from the IRBP cisregulatory sequences. Moreover, we found that when we transplanted the FACS purified iPSC derived photoreceptors, they were able to integrate into a normal mouse retina and express photoreceptor markers. CONCLUSIONS: This report provides evidence that enriched populations of human photoreceptors can be derived from iPS cells. http://www.ncbi.nlm.nih.gov/pubmed/20098701 Thu, 31 Dec 2009 00:00:00 -0800 The retina, like most other regions of the central nervous system, is subject to degeneration from both genetic and acquired causes. Once the photoreceptors or inner retinal neurons have degenerated, they are not spontaneously replaced in mammals. In this review, we provide an overview of retinal development and regeneration with emphasis on endogenous repair and replacement seen in lower vertebrates and recent work on induced mammalian retinal regeneration from Mller glia. Additionally, recent studies demonstrating the potential for cellular replacement using postmitotic photoreceptors and embryonic stem cells are also reviewed. http://www.ncbi.nlm.nih.gov/pubmed/19660646 Wed, 31 Dec 2008 00:00:00 -0800 Recent evidence indicates that mouse and human embryonic stem cells (ESCs) are fixed at different developmental stages, with the former positioned earlier. We show that a narrow concentration of the naturally occurring shortchain fatty acid, sodium butyrate, supports the extensive selfrenewal of mouse and human ESCs, while promoting their convergence toward an intermediate stem cell state. In response to butyrate, human ESCs regress to an earlier developmental stage characterized by a gene expression profile resembling that of mouse ESCs, preventing precocious Xist expression while retaining the ability to form complex teratomas in vivo. Other histone deacetylase inhibitors (HDACi) also support human ESC selfrenewal. Our results indicate that HDACi can promote ESC selfrenewal across species, and demonstrate that ESCs can toggle between alternative states in response to environmental factors. http://www.ncbi.nlm.nih.gov/pubmed/19341625 Wed, 31 Dec 2008 00:00:00 -0800 Some of the most common causes of blindness involve the degeneration of photoreceptors in the neural retina photoreceptor replacement therapy might restore some vision in these individuals. Embryonic stem cells (ESCs) could, in principle, provide a source of photoreceptors to repair the retina. We have previously shown that retinal progenitors can be efficiently derived from human ESCs. We now show that retinal cells derived from human ESCs will migrate into mouse retinas following intraocular injection, settle into the appropriate layers, and express markers for differentiated cells, including both rod and cone photoreceptor cells. After transplantation of the cells into the subretinal space of adult Crx(/) mice (a model of Leber's Congenital Amaurosis), the hESCderived retinal cells differentiate into functional photoreceptors and restore light responses to the animals. These results demonstrate that hESCs can, in principle, be used for photoreceptor replacement therapies. http://www.ncbi.nlm.nih.gov/pubmed/19128794 Wed, 31 Dec 2008 00:00:00 -0800 Remodeling of the chromatin network plays an important role regulating embryonic development as well as differentiation. The SWI/SNF complex is an ATPdependent chromatinremodeling complex. It consists of several proteins, including an ATPase subunit, either Brg1 or Brm. Two subunits of this complex, Baf53a and Baf45, have been previously identified as being neural progenitorspecific. In this study, we show that Baf60c, another important part of this large complex, acts in a similar neural progenitorspecific manner. We show that during development Baf60c is expressed in neural progenitors in human retinas as well as mouse retina, cortex and spinal cord. Baf60c expression is lost during neural differentiation and its overexpression keeps the progenitors in a proliferative state through its interaction with the Notch pathway. Finally, we show that Baf60c is reexpressed in the Mller glial cells that reenter the cell cycle after neurotoxic damage. http://www.ncbi.nlm.nih.gov/pubmed/18816825 Mon, 31 Dec 2007 00:00:00 -0800 Neuronal degenerations in the retina are leading causes of blindness. Like most other areas of the CNS, the neurons of the mammalian retina are not replaced following degeneration. However, in nonmammalian vertebrates, endogenous repair processes restore neurons very efficiently, even after complete loss of the retina. We describe the phenomenon of retinal regeneration in nonmammalian vertebrates and attempts made in recent years to stimulate similar regenerative processes in the mammalian retina. In addition, we review the various strategies employed to replace lost neurons in the retina and the recent use of stem cell technologies to address problems of retinal repair. http://www.ncbi.nlm.nih.gov/pubmed/18522847 Mon, 31 Dec 2007 00:00:00 -0800 The retina is subject to degenerative conditions, leading to blindness. Although retinal regeneration is robust in lower vertebrates, regeneration does not occur in the adult mammalian retina. Thus, we have developed efficient methods for deriving retinal neurons from human embryonic stem (hES) cells. Under appropriate culture conditions, up to 80 of the H1 line can be directed to the retinal progenitor fate, and express a gene expression profile similar to progenitors derived from human fetal retina. The hES cellderived progenitors differentiate primarily into inner retinal neurons (ganglion and amacrine cells), with functional glutamate receptors. Upon coculture with retinas derived from a mouse model of retinal degeneration, the hES cell derived retinal progenitors integrate with the degenerated mouse retina and increase in their expression of photoreceptorspecific markers. These results demonstrate that human ES cells can be selectively directed to a neural retinal cell fate and thus may be useful in the treatment of retinal degenerations. http://www.ncbi.nlm.nih.gov/pubmed/16908856 Sat, 31 Dec 2005 00:00:00 -0800