Sarah Ohlemacher - Academia.edu (original) (raw)

Papers by Sarah Ohlemacher

Investigative Ophthalmology & Visual Science, Jun 11, 2015

Investigative Ophthalmology & Visual Science, Jul 13, 2018

Investigative Ophthalmology & Visual Science, Sep 26, 2016

Investigative Ophthalmology & Visual Science, Sep 26, 2016

Investigative Ophthalmology & Visual Science, Apr 30, 2014

Investigative Ophthalmology & Visual Science, Apr 30, 2014

Investigative Ophthalmology & Visual Science, Jun 11, 2015

Investigative Ophthalmology & Visual Science, Jun 23, 2017

Investigative Ophthalmology & Visual Science, Sep 26, 2016

Stem Cells Translational Medicine, Mar 1, 2016

The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has... more The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risks of genomic integration and potential constitutive expression of pluripotency factors and often exhibit low reprogramming efficiencies. The recent description of cellular reprogramming using synthetic mRNA molecules might eliminate these shortcomings; however, the ability of mRNA-reprogrammed hiPSCs to effectively give rise to retinal cell lineages has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNAreprogrammed hiPSCs to yield retinal cell types in a directed, stepwise manner. hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retroviral delivery of reprogramming factors. New lines of mRNA-reprogrammed hiPSCs were established and were subsequently differentiated into a retinal fate using established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation from these lines was compared with retroviral-derived cell lines at various stages of development. On differentiation, mRNAreprogrammed hiPSCs were capable of robust differentiation to a retinal fate, including the derivation of photoreceptors and retinal ganglion cells, at efficiencies often equal to or greater than their retroviralderived hiPSC counterparts. Thus, given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes, such methods likely represent a promising new approach for retinal stem cell research, in particular, those for translational applications.

Current protocols in stem cell biology, Feb 1, 2015

The procedure to efficiently and reproducibly differentiate retinal cells from human pluripotent ... more The procedure to efficiently and reproducibly differentiate retinal cells from human pluripotent stem cells (hPSCs) is described below. Cells are taken through a stepwise protocol to direct them toward a neural fate by treatment with neural induction medium (NIM), then to a retinal fate by exposure to retinal differentiation medium (RDM). Undifferentiated hPSCs are enzymatically lifted from matrigel-coated plates and exposed to NIM in suspension. Differentiation in suspension allows the cells to form 3 dimensional aggregates. At 7 days of differentiation, aggregates are plated and attach to 6 well plates, where a neuroepithelial fate begins to be established. Upon 16 days of differentiation, neurospheres are lifted and maintained in RDM to create a three-dimensional optic vesicle-like structure. This procedure allows for the efficient and timely generation of a variety of retinal cell types, including ganglion cells, retinal pigment epithelium, as well as cone and rod photoreceptors. The use of this protocol to generate a myriad of retinal cell types facilitates in vitro studies of human retinogenesis, and will enable retinal dysfunction to be more easily studied in vitro, as well as providing a large population of cells with which to aid in drug development and patient specific therapies.

PubMed, Dec 1, 2021

In partnership with the National Institute on Aging (NIA), the National Centralized Repository fo... more In partnership with the National Institute on Aging (NIA), the National Centralized Repository for Alzheimer’s Disease and Related Dementias (NCRAD) has been funded to establish a centralized repository of induced pluripotent stem cells (iPSCs). Investigators deposit iPSCs derived from patients with Alzheimer's disease and related dementias (ADRD), as well as iPSCs from healthy patients. However, much variability exists between iPSC lines, which is compounded by the diversity of culture conditions used by laboratories. Additionally, many labs do not have the resources to perform thorough quality control measures or to distribute high demand lines to other investigators. NCRAD strives to ease the distribution burden for investigators and provide standardized, quality controlled iPSCs to the ADRD community.

Stem cell reports, Apr 1, 2018

Retinal ganglion cells (RGCs) are the projection neurons of the retina and transmit visual inform... more Retinal ganglion cells (RGCs) are the projection neurons of the retina and transmit visual information to postsynaptic targets in the brain. While this function is shared among nearly all RGCs, this class of cell is remarkably diverse, comprised of multiple subtypes. Previous efforts have identified numerous RGC subtypes in animal models, but less attention has been paid to human RGCs. Thus, efforts of this study examined the diversity of RGCs differentiated from human pluripotent stem cells (hPSCs) and characterized defined subtypes through the expression of subtype-specific markers. Further investigation of these subtypes was achieved using single-cell transcriptomics, confirming the combinatorial expression of molecular markers associated with these subtypes, and also provided insight into more subtype-specific markers. Thus, the results of this study describe the derivation of RGC subtypes from hPSCs and will support the future exploration of phenotypic and functional diversity within human RGCs.

Stem Cells, Mar 21, 2016

Human pluripotent stem cells (hPSCs), including both embryonic and induced pluripotent stem cells... more Human pluripotent stem cells (hPSCs), including both embryonic and induced pluripotent stem cells, possess the unique ability to readily differentiate into any cell type of the body, including cells of the retina. Although previous studies have demonstrated the ability to differentiate hPSCs to a retinal lineage, the ability to derive retinal ganglion cells (RGCs) from hPSCs has been complicated by the lack of specific markers with which to identify these cells from a pluripotent source. In the current study, the definitive identification of hPSC-derived RGCs was accomplished by their directed, stepwise differentiation through an enriched retinal progenitor intermediary, with resultant RGCs expressing a full complement of associated features and proper functional characteristics. These results served as the basis for the establishment of induced pluripotent stem cells (iPSCs) from a patient with a genetically inherited form of glaucoma, which results in damage and loss of RGCs. Patient-derived RGCs specifically exhibited a dramatic increase in apoptosis, similar to the targeted loss of RGCs in glaucoma, which was significantly rescued by the addition of candidate neuroprotective factors. Thus, the current study serves to establish a method by which to definitively acquire and identify RGCs from hPSCs and demonstrates the ability of hPSCs to serve as an effective in vitro model of disease progression. Moreover, iPSC-derived RGCs can be utilized for future drug screening approaches to identify targets for the treatment of glaucoma and other optic neuropathies. STEM CELLS 2016;34:1553-1562 SIGNIFICANCE STATEMENT The current study represents the most comprehensive analysis of the ability to differentiate retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) to date. Due to a lack of truly specific features, definitive identification of RGCs from other cell types with similar features has proven difficult. The data presented details the ability to conclusively assign presumptive RGCs to the retinal lineage, with subsequent confirmation by morphological, phenotypic, and functional approaches. Utilizing this ability to generate RGCs, induced pluripotent stem cells were then derived from a patient with a genetically inherited form of glaucoma. These cells exhibited an increased susceptibility to apoptosis specifically upon the acquisition of an RGC fate, with subsequent approaches significantly rescuing this phenotype, underscoring the potential for disease modeling and pharmacological screening applications.

Scientific Reports, Sep 28, 2018

Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPS... more Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, serving as effective in vitro models of retinal development. However, a lack of emphasis has been placed upon the development and organization of retinal ganglion cells (RGCs) within retinal organoids. Thus, initial efforts were made to characterize RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a complement of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful for cellular replacement in which extensive axonal outgrowth is necessary to reach post-synaptic targets. Organoid-derived RGCs could help to elucidate factors promoting axonal outgrowth, thereby identifying approaches to circumvent a formidable obstacle to RGC replacement. As such, additional efforts demonstrated significant enhancement of neurite outgrowth through modulation of both substrate composition and growth factor signaling. Additionally, organoid-derived RGCs exhibited diverse phenotypes, extending elaborate growth cones and expressing numerous guidance receptors. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing neurite outgrowth from organoid-derived RGCs. Retinal ganglion cells (RGCs) play a critical role in the transmission of visual information between the eye and the brain, with many retinal degenerative diseases leading to the damage and loss of RGC axons 1-3. As RGCs have a limited capacity for regeneration following damage 4,5 , previous efforts to restore RGC connections have been limited by numerous obstacles, including an inability to regrow long-distance connections. Additionally, at later

Fundamental biomedical technologies, 2018

Human pluripotent stem cells (hPSCs) provide unprecedented access to the earliest stages of retin... more Human pluripotent stem cells (hPSCs) provide unprecedented access to the earliest stages of retinogenesis that remain inaccessible to investigation, thereby serving as powerful tools for studies of retinal development. Additionally, the ability to derive hPSCs from patient sources allows for the modeling of retinal degenerative diseases in vitro, with the potential to facilitate cell replacement strategies in advanced stages of disease. For these purposes, many studies over the past several years have directed the differentiation of hPSCs to generate retinal cells using stochastic methods of differentiation, yielding all major cell types of the retina. In particular, these studies have favored the derivation of RPE, photoreceptors, and more recently retinal ganglion cells for disease modeling, drug screening as well as cell replacement purposes. More recently, advances in retinal differentiation methods have led to the generation of three-dimensional retinal organoids that recapitulate key developmental and morphological features of the retina, including the stratified organization of retinal cells into a tissue-like structure. This review provides an overview of retinal differentiation from hPSCs and their potential use for studies of retinogenesis as well as diseases that affect the retina.

Investigative Ophthalmology & Visual Science, 2016

Microporous and Mesoporous Materials, 2020

Abstract ZIF-8 nanoporous films of variable thicknesses, which are proposed to be very low-k diel... more Abstract ZIF-8 nanoporous films of variable thicknesses, which are proposed to be very low-k dielectric films for chip industries, have been deposited on Si substrate using simple solution chemistry method. Contrary to the earlier reports, ZIF-8 crystals number density on the substrate in early deposition cycles increases leading to higher coverage of the substrate. The growth of crystal size or film thickness occurs in later deposition cycles. The deposition of ZIF-8 crystals is highly random, however a preferential growth in 002 orientation is observed. Synchrotron based X-ray photoelectron spectroscopy has confirmed Zn enrichment at surface of the films compared to imidazole linkers. Depth dependent Doppler broadening spectroscopy measurements utilizing the positronium diffusion process have unequivocally confirmed that the pores in ZIF-8 films are interconnected at micrometer scale leading to their accessibility from the outer surface. This type of pore architecture along with the surface enrichment might be responsible for adsorbate selectivity and very high adsorption capacity of these films as reported in literature. On thermal annealing, the pore architecture of these films begins to collapse at a much lower temperature (~373 K) than the decomposition temperature (~700 K) of ZIF-8 powder.

Investigative Ophthalmology & Visual Science, Jun 11, 2015

Investigative Ophthalmology & Visual Science, Jul 13, 2018

Investigative Ophthalmology & Visual Science, Sep 26, 2016

Investigative Ophthalmology & Visual Science, Sep 26, 2016

Investigative Ophthalmology & Visual Science, Apr 30, 2014

Investigative Ophthalmology & Visual Science, Apr 30, 2014

Investigative Ophthalmology & Visual Science, Jun 11, 2015

Investigative Ophthalmology & Visual Science, Jun 23, 2017

Investigative Ophthalmology & Visual Science, Sep 26, 2016

Stem Cells Translational Medicine, Mar 1, 2016

The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has... more The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risks of genomic integration and potential constitutive expression of pluripotency factors and often exhibit low reprogramming efficiencies. The recent description of cellular reprogramming using synthetic mRNA molecules might eliminate these shortcomings; however, the ability of mRNA-reprogrammed hiPSCs to effectively give rise to retinal cell lineages has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNAreprogrammed hiPSCs to yield retinal cell types in a directed, stepwise manner. hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retroviral delivery of reprogramming factors. New lines of mRNA-reprogrammed hiPSCs were established and were subsequently differentiated into a retinal fate using established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation from these lines was compared with retroviral-derived cell lines at various stages of development. On differentiation, mRNAreprogrammed hiPSCs were capable of robust differentiation to a retinal fate, including the derivation of photoreceptors and retinal ganglion cells, at efficiencies often equal to or greater than their retroviralderived hiPSC counterparts. Thus, given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes, such methods likely represent a promising new approach for retinal stem cell research, in particular, those for translational applications.

Current protocols in stem cell biology, Feb 1, 2015

The procedure to efficiently and reproducibly differentiate retinal cells from human pluripotent ... more The procedure to efficiently and reproducibly differentiate retinal cells from human pluripotent stem cells (hPSCs) is described below. Cells are taken through a stepwise protocol to direct them toward a neural fate by treatment with neural induction medium (NIM), then to a retinal fate by exposure to retinal differentiation medium (RDM). Undifferentiated hPSCs are enzymatically lifted from matrigel-coated plates and exposed to NIM in suspension. Differentiation in suspension allows the cells to form 3 dimensional aggregates. At 7 days of differentiation, aggregates are plated and attach to 6 well plates, where a neuroepithelial fate begins to be established. Upon 16 days of differentiation, neurospheres are lifted and maintained in RDM to create a three-dimensional optic vesicle-like structure. This procedure allows for the efficient and timely generation of a variety of retinal cell types, including ganglion cells, retinal pigment epithelium, as well as cone and rod photoreceptors. The use of this protocol to generate a myriad of retinal cell types facilitates in vitro studies of human retinogenesis, and will enable retinal dysfunction to be more easily studied in vitro, as well as providing a large population of cells with which to aid in drug development and patient specific therapies.

PubMed, Dec 1, 2021

In partnership with the National Institute on Aging (NIA), the National Centralized Repository fo... more In partnership with the National Institute on Aging (NIA), the National Centralized Repository for Alzheimer’s Disease and Related Dementias (NCRAD) has been funded to establish a centralized repository of induced pluripotent stem cells (iPSCs). Investigators deposit iPSCs derived from patients with Alzheimer's disease and related dementias (ADRD), as well as iPSCs from healthy patients. However, much variability exists between iPSC lines, which is compounded by the diversity of culture conditions used by laboratories. Additionally, many labs do not have the resources to perform thorough quality control measures or to distribute high demand lines to other investigators. NCRAD strives to ease the distribution burden for investigators and provide standardized, quality controlled iPSCs to the ADRD community.

Stem cell reports, Apr 1, 2018

Retinal ganglion cells (RGCs) are the projection neurons of the retina and transmit visual inform... more Retinal ganglion cells (RGCs) are the projection neurons of the retina and transmit visual information to postsynaptic targets in the brain. While this function is shared among nearly all RGCs, this class of cell is remarkably diverse, comprised of multiple subtypes. Previous efforts have identified numerous RGC subtypes in animal models, but less attention has been paid to human RGCs. Thus, efforts of this study examined the diversity of RGCs differentiated from human pluripotent stem cells (hPSCs) and characterized defined subtypes through the expression of subtype-specific markers. Further investigation of these subtypes was achieved using single-cell transcriptomics, confirming the combinatorial expression of molecular markers associated with these subtypes, and also provided insight into more subtype-specific markers. Thus, the results of this study describe the derivation of RGC subtypes from hPSCs and will support the future exploration of phenotypic and functional diversity within human RGCs.

Stem Cells, Mar 21, 2016

Human pluripotent stem cells (hPSCs), including both embryonic and induced pluripotent stem cells... more Human pluripotent stem cells (hPSCs), including both embryonic and induced pluripotent stem cells, possess the unique ability to readily differentiate into any cell type of the body, including cells of the retina. Although previous studies have demonstrated the ability to differentiate hPSCs to a retinal lineage, the ability to derive retinal ganglion cells (RGCs) from hPSCs has been complicated by the lack of specific markers with which to identify these cells from a pluripotent source. In the current study, the definitive identification of hPSC-derived RGCs was accomplished by their directed, stepwise differentiation through an enriched retinal progenitor intermediary, with resultant RGCs expressing a full complement of associated features and proper functional characteristics. These results served as the basis for the establishment of induced pluripotent stem cells (iPSCs) from a patient with a genetically inherited form of glaucoma, which results in damage and loss of RGCs. Patient-derived RGCs specifically exhibited a dramatic increase in apoptosis, similar to the targeted loss of RGCs in glaucoma, which was significantly rescued by the addition of candidate neuroprotective factors. Thus, the current study serves to establish a method by which to definitively acquire and identify RGCs from hPSCs and demonstrates the ability of hPSCs to serve as an effective in vitro model of disease progression. Moreover, iPSC-derived RGCs can be utilized for future drug screening approaches to identify targets for the treatment of glaucoma and other optic neuropathies. STEM CELLS 2016;34:1553-1562 SIGNIFICANCE STATEMENT The current study represents the most comprehensive analysis of the ability to differentiate retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) to date. Due to a lack of truly specific features, definitive identification of RGCs from other cell types with similar features has proven difficult. The data presented details the ability to conclusively assign presumptive RGCs to the retinal lineage, with subsequent confirmation by morphological, phenotypic, and functional approaches. Utilizing this ability to generate RGCs, induced pluripotent stem cells were then derived from a patient with a genetically inherited form of glaucoma. These cells exhibited an increased susceptibility to apoptosis specifically upon the acquisition of an RGC fate, with subsequent approaches significantly rescuing this phenotype, underscoring the potential for disease modeling and pharmacological screening applications.

Scientific Reports, Sep 28, 2018

Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPS... more Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, serving as effective in vitro models of retinal development. However, a lack of emphasis has been placed upon the development and organization of retinal ganglion cells (RGCs) within retinal organoids. Thus, initial efforts were made to characterize RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a complement of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful for cellular replacement in which extensive axonal outgrowth is necessary to reach post-synaptic targets. Organoid-derived RGCs could help to elucidate factors promoting axonal outgrowth, thereby identifying approaches to circumvent a formidable obstacle to RGC replacement. As such, additional efforts demonstrated significant enhancement of neurite outgrowth through modulation of both substrate composition and growth factor signaling. Additionally, organoid-derived RGCs exhibited diverse phenotypes, extending elaborate growth cones and expressing numerous guidance receptors. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing neurite outgrowth from organoid-derived RGCs. Retinal ganglion cells (RGCs) play a critical role in the transmission of visual information between the eye and the brain, with many retinal degenerative diseases leading to the damage and loss of RGC axons 1-3. As RGCs have a limited capacity for regeneration following damage 4,5 , previous efforts to restore RGC connections have been limited by numerous obstacles, including an inability to regrow long-distance connections. Additionally, at later

Fundamental biomedical technologies, 2018

Human pluripotent stem cells (hPSCs) provide unprecedented access to the earliest stages of retin... more Human pluripotent stem cells (hPSCs) provide unprecedented access to the earliest stages of retinogenesis that remain inaccessible to investigation, thereby serving as powerful tools for studies of retinal development. Additionally, the ability to derive hPSCs from patient sources allows for the modeling of retinal degenerative diseases in vitro, with the potential to facilitate cell replacement strategies in advanced stages of disease. For these purposes, many studies over the past several years have directed the differentiation of hPSCs to generate retinal cells using stochastic methods of differentiation, yielding all major cell types of the retina. In particular, these studies have favored the derivation of RPE, photoreceptors, and more recently retinal ganglion cells for disease modeling, drug screening as well as cell replacement purposes. More recently, advances in retinal differentiation methods have led to the generation of three-dimensional retinal organoids that recapitulate key developmental and morphological features of the retina, including the stratified organization of retinal cells into a tissue-like structure. This review provides an overview of retinal differentiation from hPSCs and their potential use for studies of retinogenesis as well as diseases that affect the retina.

Investigative Ophthalmology & Visual Science, 2016

Microporous and Mesoporous Materials, 2020

Abstract ZIF-8 nanoporous films of variable thicknesses, which are proposed to be very low-k diel... more Abstract ZIF-8 nanoporous films of variable thicknesses, which are proposed to be very low-k dielectric films for chip industries, have been deposited on Si substrate using simple solution chemistry method. Contrary to the earlier reports, ZIF-8 crystals number density on the substrate in early deposition cycles increases leading to higher coverage of the substrate. The growth of crystal size or film thickness occurs in later deposition cycles. The deposition of ZIF-8 crystals is highly random, however a preferential growth in 002 orientation is observed. Synchrotron based X-ray photoelectron spectroscopy has confirmed Zn enrichment at surface of the films compared to imidazole linkers. Depth dependent Doppler broadening spectroscopy measurements utilizing the positronium diffusion process have unequivocally confirmed that the pores in ZIF-8 films are interconnected at micrometer scale leading to their accessibility from the outer surface. This type of pore architecture along with the surface enrichment might be responsible for adsorbate selectivity and very high adsorption capacity of these films as reported in literature. On thermal annealing, the pore architecture of these films begins to collapse at a much lower temperature (~373 K) than the decomposition temperature (~700 K) of ZIF-8 powder.