Anatomy; Curriculum; Epithelial Cells; Ophthalmology; Pigment Epithelium of Eye; Retina; Models, Educational
Stem Cell Center, Yale: Tissue Specific Stem Cells
Surgery: Rizzolo Lab
Retinal degenerations are major causes of visual impairment, especially in aging and diabetic populations. My laboratory uses human embryonic and induced pluripotent stem cells to study and treat retinal disease. The retinal pigment epithelium (RPE) separates photoreceptors from their blood supply in the choroid, and therefore, is responsible for their health. We demonstrated how RPE functions are regulated by interactions with the retina and the choroid. Our goal is to bioengineer a three-dimensional culture model that includes RPE and retinal neurons derived from human stem cells. We will use this platform to screen potential therapeutic agents and determine their mechanism of action. In mouse models, early transplantation of RPE or retinal progenitors slows retinal degeneration, but later transplantation does not reverse degeneration that has already occurred. We are examining whether the engineered tissue restores vision in late stage disease. In a second approach, we are examining how the process of autophagy may be exploited to reverse degeneration of the RPE.
Extensive Research Description
The retinal pigment epithelium (RPE) plays a central role in retinal physiology by forming the outer blood-retinal barrier and supporting the function of the photoreceptors. Many retinopathies involve a disruption of the epithelium's interactions with the neural retina or its uncontrolled proliferation. Surgical interventions limit the progression of disease, but fail to restore function. Although encouraging progress has been made with RPE transplantation, its effectiveness is limited to the earliest stages of disease when patients would be reluctant to have surgery. Our goal is to expand that window of opportunity by understanding the interactions of the RPE with its neighbors, the choroid and the neural portion of the retina. Our early studies with chick RPE demonstrated that: 1) As the neural retina matures, it secretes factors that induce the RPE to form the outer blood-retinal barrier by decreasing the permeability of RPE junctions. 2) At the RPE/neural retina interface, extracellular matrix or cell-cell interactions regulate the distribution of certain integrins. These integrins are redistributed when the neural retina and its extracellular matrix mature. 3) Initially, diffusible factors produced by the neural retina maintain the apical polarity of the Na,K-ATPase. These retinal factors differ from those that decrease the permeability of the monolayer, and may act indirectly through effects on the structure of the apical microvilli. 4) Gene array studies demonstrate that 40% of the RPE transcriptome changes in parallel with retinal development. Retinal secretions regulate many of these changes.
Our current research asks whether the chick studies are relevant to human biology. Surprisingly, we found that the tight junctions of human RPE differ significantly from those of non-primate vertebrates -- surprising because tight junctions serve a conserved function. Tight junctions are an integral part of any blood-tissue barrier, because they regulate diffusion across the paracellular spaces of an epithelial monolayer. Tight junctions form a network of anastomosing strands that encircles each cell and binds it to its neighbors in the monolayer. They regulate the permeability and selectivity of the paracellular path and are matched with the ion channels and transporters that regulate the transcellular movement of solutes. Claudins are a family of at least 24 proteins that determine the properties of tight junctions and each epithelium expresses a subset that reflects the physiology of the organ. Human RPE expresses a different set of claudins that non-primate vertebrates which implies differences in retinal physiology.
In both cultures of human fetal RPE (hfRPE) and human embryonic stem cell (hESC)-derived RPE, we found that we could make the barrier function more in vivo-like by using a serum free medium that we call SFM-1. Studies of the transcriptome demonstrate that SFM-1 affects many genes and that adaptation of the cultures to SFM-1 furthers the maturation of hESC-RPE. This was demonstrated in two cells, H1 and H9. Examining function and the transcriptome leads us to believe that even after SFM-1, hESC-RPE remains less mature than hfRPE isolated from 16 week-gestation fetal eyes. The study identifies 25 marker genes that can be used to monitor the maturation of RPE that we predict will occur when hESC-RPE is co-cultured with hESC-derived retinal precursors (RPC).
To test this hypothesis, RPE and RPC need to be culture in the same media. It appears that SFM-1 furthers the maturation of RPC so that co-culture is feasible. We have found that a scaffold of gelatin decorated with glycoproteins provides a way to culture RPC as a flat sheet that can be layered atop a sheet of RPE. Preliminary data indicate that gene expression is altered in both the RPE and RPC layers following co-culture.
I believe this co-culture model will provide a superior platform to explore the efficacy of drugs that might treat retinal degenerations or improve the efficacy of transplantation. Further, the model itself may prove to be a suitable tissue for transplantation into patients with advanced retinal degeneration.
The international community of medical educators struggles with how to decompress an overcrowded curriculum. The questions have become what to teach, when to teach it and how to teach it in less time. The problem is especially acute for anatomy. Even though the classical anatomy course is a large component of medical school, residency programs believe their residents come ill-prepared. Further, the pool of qualified instructors is shrinking. To address these issues of content, efficiency and instructors, I investigated what students need, how they learn and how instructors teach. I call the resulting method “Clinically-Engaged Anatomy”. Clinically-engaged anatomy engages students in professional behaviors to learn the anatomy that prepares them for clerkships. Students learn how to draw inferences from skillful observation to form testable hypotheses, test them and teach others about the process. The coursework requires students to develop the teamwork skills that characterize modern medical practice. The clinical cases that drive the curriculum are cases commonly encountered in Yale affiliated hospitals. Students study the anatomy that underlies the patient’s history, physical exam, imaging studies and medical or surgical resolution. The cadaver becomes a simulated patient whereby anatomy is explored by performing surgical procedures. This approach fosters integration of anatomy with clinical training and has attracted large numbers of clinic faculty to participate. Despite a 30% reduction in course hours, we demonstrated that students recall more when they enter clerkships. Clinically-engaged anatomy merges advanced web resources with laboratory dissection. My “Anatomy Clinic” website attracts more than 17,000 non-Yale page viewings per year from around the world and is used in Great Britain for their “Basic Training Programme for Anatomy Professionals”. The anesthesiology and otolaryngology residency training programs adapted these methods to their laboratory sessions. Therefore, clinically engaged anatomy has identified important anatomy to teach, conveys that knowledge effectively in less time, and attracts a large number of faculty who would not participate in the old course.
Current research asks how the new Medical School Curriculum impacts student outcomes in the new anatomy course. All of the basic science courses were integrated with the aim to shortened courses by reducing redundancy, leveraging integration to teach more efficiently, and using innovation.Ongoing projects focus on the effectiveness of new innovations and whether integration has achieved its goals.
A second project investigates how clinical students incorporate interactive computer activities into their daily work and how that information might guide curricular development.
- We use fetal and stem cell-derived culture models of human retinal pigment epithelium RPE to study mechanisms of retinal degeneration and examine putative therapeutic agents.
- We are investigating the role of autophagy in young and aged RPE, and how changes in autophagy might relate to age-related macular degeneration.
- We use embryonic stem cells to study the interactions of retinal and RPE progenetors with the goal of developing superior engineered tissues for drug testing and transplantation.
- We are investigating how the new Medical School Curriculum impacts the anatomy course. Pedagogical innovation and integration with other courses are being used to deliver a shortened, yet more effective anatomy course.
The student's dilemma, liver edition: Incorporating the sonographer's language into clinical anatomy education.
Hall MK, Mirjalili SA, Moore CL, Rizzolo LJ. (2015) Anat. Sci. Educ. 8, 283-288.
TRP Channels Localize to Subdomains of the Apical Plasma Membrane in Human Fetal Retinal Pigment Epithelium.
Zhao, P.Y., Gan, G., Peng, S., Wang, S.B., Chen, B., Adelman, R.A., Rizzolo, L.J. (2015) Invest. Ophthalmol. Vis. Sci. 56, 1916-1923.
Engineering a blood-retinal barrier with human embryonic stem cell-derived retinal pigment epithelium: Transcriptome and functional analysis.
Peng,S., Gan,G., Qiu, C., Zhong, M., An, H., Adelman, R.A., and Rizzolo, L.J. (2013) Stem Cell Trans. Med. 2:534-544
Effects of proinflammatory cytokines on the claudin-19 rich tight junctions of human retinal pigment epithelium (RPE).
Peng,S., Gan,G., Rao, V.S., Adelman, R.A., and Rizzolo, L.J. (2012) Invest. Ophthalmol. Vis. Sci. 53, 5016-5028.
Integration of tight junctions and claudins with the barrier functions of the retinal pigment epithelium.
Rizzolo, L.J., Peng, S., Luo, Y, and Xiao, W., (2011) Prog. Retin. Eye Res. 30:296-323.
Design, implementation and evaluation of an innovative anatomy course. Anat. Sci. Educ.
Rizzolo, L.J., Rando, W.C., O’Brien, M.K.. Haims, A.H., Abrahams, J.J, and Stewart W.B. (2010) 3:109-120
Human Adult Retinal Pigment Epithelial Stem Cell-Derived RPE Monolayers Exhibit Key Physiological Characteristics of Native Tissue.
Blenkinsop TA, Saini JS, Maminishkis A, Bharti K, Wan Q, Banzon T, Lotfi M, Davis J, Singh D, Rizzolo LJ, Miller S, Temple S, Stern JH (2015) Invest Ophthalmol Vis Sci. 56:7085-99
Full List of PubMed Publications
- Singh D, Wang SB, Xia T, Tainsh L, Ghiassi-Nejad M, Xu T, Peng S, Adelman RA, Rizzolo LJ: A biodegradable scaffold enhances differentiation of embryonic stem cells into a thick sheet of retinal cells. Biomaterials. 2018 Feb; 2017 Oct 31. PMID: 29128844
- Xu T, Singh D, Liu J, Li H, Peng S, Rizzolo LJ, Wang SB: Neferine, is not inducer but blocker for macroautophagic flux targeting on lysosome malfunction. Biochem Biophys Res Commun. 2018 Jan 1; 2017 Nov 29. PMID: 29197576
- Xia T, Rizzolo LJ: Effects of diabetic retinopathy on the barrier functions of the retinal pigment epithelium. Vision Res. 2017 Oct; 2017 Mar 30. PMID: 28347688
- Peng S, Wang SB, Singh D, Zhao PY, Davis K, Chen B, Adelman RA, Rizzolo LJ: Claudin-3 and claudin-19 partially restore native phenotype to ARPE-19 cells via effects on tight junctions and gene expression. Exp Eye Res. 2016 Oct; 2016 Sep 1. PMID: 27593915
- Blenkinsop TA, Saini JS, Maminishkis A, Bharti K, Wan Q, Banzon T, Lotfi M, Davis J, Singh D, Rizzolo LJ, Miller S, Temple S, Stern JH: Human Adult Retinal Pigment Epithelial Stem Cell-Derived RPE Monolayers Exhibit Key Physiological Characteristics of Native Tissue. Invest Ophthalmol Vis Sci. 2015 Nov. PMID: 26540654
- Hall MK, Mirjalili SA, Moore CL, Rizzolo LJ: The student's dilemma, liver edition: incorporating the sonographer's language into clinical anatomy education. Anat Sci Educ. 2015 May-Jun; 2015 Jan 8. PMID: 25573229
- Zhao PY, Gan G, Peng S, Wang SB, Chen B, Adelman RA, Rizzolo LJ: TRP Channels Localize to Subdomains of the Apical Plasma Membrane in Human Fetal Retinal Pigment Epithelium. Invest Ophthalmol Vis Sci. 2015 Mar 3; 2015 Mar 3. PMID: 25736794
- Rizzolo LJ: Barrier properties of cultured retinal pigment epithelium. Exp Eye Res. 2014 Sep; 2014 Apr 14. PMID: 24731966
- Peng S, Gan G, Qiu C, Zhong M, An H, Adelman RA, Rizzolo LJ: Engineering a blood-retinal barrier with human embryonic stem cell-derived retinal pigment epithelium: transcriptome and functional analysis. Stem Cells Transl Med. 2013 Jul; 2013 Jun 3. PMID: 23734062
- Peng S, Gan G, Rao VS, Adelman RA, Rizzolo LJ: Effects of proinflammatory cytokines on the claudin-19 rich tight junctions of human retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2012 Jul 27; 2012 Jul 27. PMID: 22761260
- Rizzolo LJ, Peng S, Luo Y, Xiao W: Integration of tight junctions and claudins with the barrier functions of the retinal pigment epithelium. Prog Retin Eye Res. 2011 Sep; 2011 Jun 17. PMID: 21704180
- Peng S, Rao VS, Adelman RA, Rizzolo LJ: Claudin-19 and the barrier properties of the human retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2011 Mar; 2011 Mar 14. PMID: 21071746
- Rizzolo LJ, Rando WC, O'Brien MK, Garino A, Stewart WB: Effectiveness of a shortened, clinically engaged anatomy course for physician assistant students. Anat Sci Educ. 2011 Mar-Apr; 2011 Feb 18. PMID: 21337718
- Peng S, Adelman RA, Rizzolo LJ: Minimal effects of VEGF and anti-VEGF drugs on the permeability or selectivity of RPE tight junctions. Invest Ophthalmol Vis Sci. 2010 Jun; 2009 Dec 30. PMID: 20042644
- Rizzolo LJ, Rando WC, O'Brien MK, Haims AH, Abrahams JJ, Stewart WB: Design, implementation, and evaluation of an innovative anatomy course. Anat Sci Educ. 2010 May-Jun. PMID: 20496432
- Elansary M, Goldberg B, Qian T, Rizzolo LJ: The 2008 anatomy ceremony: essays. Yale J Biol Med. 2009 Mar. PMID: 19325944
- Rizzolo LJ, Drake RL: Anatomists debate the value of a teaching credential. Anat Sci Educ. 2008 Mar-Apr. PMID: 19177383
- Sun R, Peng S, Chen X, Zhang H, Rizzolo LJ: Diffusible retinal secretions regulate the expression of tight junctions and other diverse functions of the retinal pigment epithelium. Mol Vis. 2008; 2008 Dec 8. PMID: 19057659
- Rizzolo LJ, Chen X, Weitzman M, Sun R, Zhang H: Analysis of the RPE transcriptome reveals dynamic changes during the development of the outer blood-retinal barrier. Mol Vis. 2007 Jul 23; 2007 Jul 23. PMID: 17679949
- Rizzolo LJ: Development and role of tight junctions in the retinal pigment epithelium. Int Rev Cytol. 2007. PMID: 17338922
- Rizzolo LJ, Stewart WB: Should we continue teaching anatomy by dissection when ...? Anat Rec B New Anat. 2006 Nov. PMID: 17109419
- Luo Y, Fukuhara M, Weitzman M, Rizzolo LJ: Expression of JAM-A, AF-6, PAR-3 and PAR-6 during the assembly and remodeling of RPE tight junctions. Brain Res. 2006 Sep 19; 2006 Jul 21. PMID: 16859655
- Luo Y, Zhuo Y, Fukuhara M, Rizzolo LJ: Effects of culture conditions on heterogeneity and the apical junctional complex of the ARPE-19 cell line. Invest Ophthalmol Vis Sci. 2006 Aug. PMID: 16877439
- Warner JH, Rizzolo LJ: Anatomical instruction and training for professionalism from the 19th to the 21st centuries. Clin Anat. 2006 Jul. PMID: 16617459
- Rizzolo LJ, Stewart WB, O'Brien M, Haims A, Rando W, Abrahams J, Dunne S, Wang S, Aden M: Design principles for developing an efficient clinical anatomy course. Med Teach. 2006 Mar. PMID: 16707295
- Rahner C, Fukuhara M, Peng S, Kojima S, Rizzolo LJ: The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development. J Cell Sci. 2004 Jul 1. PMID: 15226402
- Peng S, Rahner C, Rizzolo LJ: Apical and basal regulation of the permeability of the retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2003 Feb. PMID: 12556417
- Rizzolo LJ: Human dissection: an approach to interweaving the traditional and humanistic goals of medical education. Anat Rec. 2002 Dec 15. PMID: 12467081
- Rizzolo LJ, Aden M, Stewart WB: Correlation of Web usage and exam performance in a human anatomy and development course. Clin Anat. 2002 Aug. PMID: 12203379