Generating iPSC-Derived Choroidal Endothelial Cells to Study Age-Related Macular Degeneration

Songstad, Allison E.; Wiley, Luke A; Duong, Khahn; Kaalberg, Emily E.; Flamme-Wiese, Miles J.; Cranston, Cathryn M.; Riker, Megan; Levasseur, Dana N.; Stone, Edwin M.; Mullins, Robert F.; Tucker, Budd A.

doi:10.1167/iovs.15-17073

Cited by 42 publications

(35 citation statements)

References 30 publications

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“…Furthermore, because the choriocapillaris provides oxygen and nutrients for both the RPE and photoreceptor cells, cell replacement strategies for either of these cell types should take the health of CECs into consideration. To date, we have successfully generated CECs with iPSCs derived from mouse fibroblasts [34] and are adapting these methods toward the development of EC progenitors and CECs using human fibroblast-derived iPSCs. Using this iPSC technology in combination with our decellularization protocol, we can evaluate and optimize methods for rebuilding the choroid using iPSC-induced CECs or EC progenitors.…”

Section: Discussionmentioning

confidence: 99%

Preparation and evaluation of human choroid extracellular matrix scaffolds for the study of cell replacement strategies

et al. 2017

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Endothelial cells (ECs) of the choriocapillaris are one of the first cell types lost during age-related macular degeneration (AMD), and cell replacement therapy is currently a very promising option for patients with advanced AMD. We sought to develop a reliable method for the production of human choroidal extracellular matrix (ECM) scaffolds, which will allow for the study of choroidal EC (CEC) replacement strategies in an environment that closely resembles the native tissue. Human RPE/choroid tissue was treated sequentially with Triton X-100, SDS, and DNase to remove all native cells. While all cells were successfully removed from the tissue, collagen IV, elastin, and laminin remained, with preserved architecture of the acellular vascular tubes. The ECM scaffolds were then co-cultured with exogenous ECs to determine if the tissue can support cell growth and allow EC reintegration into the decellularized choroidal vasculature. Both monkey and human ECs took up residence in the choriocapillary tubes of the decellularized tissue. Together, these data suggest that our decellularization methods are sufficient to remove all cellular material yet gentle enough to preserve tissue structure and allow for the optimization of cell replacement strategies.

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Section: Discussionmentioning

confidence: 99%

Preparation and evaluation of human choroid extracellular matrix scaffolds for the study of cell replacement strategies

et al. 2017

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“…As a metalloenzyme associated with both luminal and abluminal choriocapillary endothelial cell membranes, carbonic anhydrase IV (CA4) acts to regulate local pH and is thought to assist in the transport of CO 2 and ions into and out of capillaries [50]. Compared with other endothelial cell populations, expression of CA4 is enriched in mature choroidal endothelial cells [51]. Therefore, reduced CA4 gene expression, which is eventually followed by protein loss in these experiments, could indicate endothelial cell dedifferentiation or altered function, which, if mCRP exposure persists, may lead to severe endothelial cell dysfunction or loss altogether.…”

Section: Discussionmentioning

confidence: 99%

Monomeric C-reactive protein and inflammation in age-related macular degeneration

et al. 2016

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Age-related macular degeneration (AMD) is a devastating disease characterized by central vision loss in elderly individuals. Previous studies have suggested a link between elevated levels of total C-reactive protein (CRP) in the choroid, CFH genotype, and AMD status; however, the structural form of CRP present in the choroid, its relationship to CFH genotype, and its functional consequences have not been assessed. In this report, we studied genotyped human donor eyes (n = 60) and found that eyes homozygous for the high-risk CFH (Y402H) allele had elevated monomeric CRP (mCRP) within the choriocapillaris and Bruch’s membrane, compared to those with the low-risk genotype. Treatment of choroidal endothelial cells in vitro with mCRP increased migration rate and monolayer permeability compared to treatment with pentameric CRP (pCRP) or medium alone. Organ cultures treated with mCRP exhibited dramatically altered expression of inflammatory genes as assessed by RNA sequencing, including ICAM-1 and CA4, both of which were confirmed at the protein level. Our data indicate that mCRP is the more abundant form of CRP in human choroid, and that mCRP levels are elevated in individuals with the high-risk CFH genotype. Moreover, pro-inflammatory mCRP significantly affects endothelial cell phenotypes in vitro and ex vivo, suggesting a role for mCRP in choroidal vascular dysfunction in AMD.

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“…The differentiated cells ( Figure 17H) expressed CA4 and formed capillary tube-like structures that were morphologically indistinguishable from primary CECs (Figure 17G). The human iPSCs also differentiated into CECs via co-culture with the RF/6A monkey CEC line ( Figure 17I), a method that we recently reported for differentiation of mouse iPSC-derived CECs (168). The iPSC-derived CECs from this co-culture experiment also formed CA4 + tubes similar to the tubes observed in the human primary CEC co-culture experiment ( Figure 17J).…”

Section: Differentiating Human Ipscs Into Cecs Via Co-culture With Rfsupporting

confidence: 70%

Generating patient-specific induced pluripotent stem cell-derived choroirdal endothelium to study and treat macular degeneration

Songstad¹

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thank you for the amazing parties, getting me out of my shell, and teaching me how to be a successful graduate student. Thank you to Emily Malcolm-Cushing for the movie dates and always being there for me. Katie Chirco, thank you for the fun and encouraging lab chats and Java runs. Tasneem Sharma, thank you for being my fellow minion pal, candy supplier, and best bay buddy ever. Thank you to Jon Scholte for choosing to share your life with me and being my rock. Meeting you was one of the best things to happen to me in graduate school. And last, but definitely not least, I want to thank my incredible family: my grandfather, my grandmother, my sister, Nicole, and my parents, David and Aseema. Grandpa, thank you for your encouragement and always reminding me that quitters never win and winners never quit. Grandma Ruth, I miss you every day. You ignited my passion for AMD research. Nickie, thank you for teaching me to always see the bright side of every situation. I'm so proud of the strong woman you have become. Mom and Dad, thank you for encouraging me to always pursue my dreams, even if that meant being a perpetual student. You both are my role models and you inspire me everyday. I love all of you.

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Generating iPSC-Derived Choroidal Endothelial Cells to Study Age-Related Macular Degeneration

Abstract: Induced pluripotent stem cell-derived CECs will be a valuable tool for modeling of choriocapillaris-specific insults in AMD and for use in future choroidal endothelial cell replacement approaches.

Cited by 42 publications

References 30 publications

Preparation and evaluation of human choroid extracellular matrix scaffolds for the study of cell replacement strategies

Preparation and evaluation of human choroid extracellular matrix scaffolds for the study of cell replacement strategies

Monomeric C-reactive protein and inflammation in age-related macular degeneration

Generating patient-specific induced pluripotent stem cell-derived choroirdal endothelium to study and treat macular degeneration

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