IU Indianapolis ScholarWorks :: Browsing by Author "Meyer, Jason"

Browsing by Author "Meyer, Jason"

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Analyses of the development and function of stem cell derived cells in neurodegenerative diseases
(2022-12) Lavekar, Sailee Sham; Meyer, Jason; Canfield, Scott; Belecky-Adams, Teri; Mastracci, Teresa; Perrin, Benjamin
Human pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some in vitro approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different in vitro systems that could help better mimic certain physiological systems that tend to not be mimicked by in vivo models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction. Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in in vivo models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using in vivo models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.
Axonal Outgrowth and Pathfinding of Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells
(2020-08) Fligor, Clarisse; Meyer, Jason; Marrs, James; Belecky-Adams, Teri; Jones, Kathryn; Baucum, AJ
Retinal ganglion cells (RGCs) serve as a vital connection between the eye and the brain with damage to their axons resulting in loss of vision and/or blindness. Reti- nal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, providing a valuable model of RGC development in vitro. The working hypothesis of these studies is that hPSC-derived RGCs are capable of extensive outgrowth and display target specificity and pathfinding abilities. Initial efforts focused on charac- terizing RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner express- ing a compliment of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful to investigate and model the extensive axonal outgrowth necessary to reach post-synaptic targets. As such, additional efforts aimed to elucidate factors promoting axonal outgrowth. Results demonstrated significant enhancement of axonal outgrowth through modulation of both substrate composi- tion and growth factor signaling. Furthermore, RGCs possessed guidance receptors that are essential in influencing outgrowth and pathfinding. Subsequently, to de- termine target specificity, aggregates of hPSC-derived RGCs were co-cultured with explants of mouse lateral geniculate nucleus (LGN), the primary post-synaptic target of RGCs. Axonal outgrowth was enhanced in the presence of LGN, and RGCs dis- played recognition of appropriate targets, with the longest neurites projecting towards LGN explants compared to control explants or RGCs grown alone. Generated from xvii the fusion of regionally-patterned organoids, assembloids model projections between distinct regions of the nervous system. Therefore, final efforts of these studies focused upon the generation of retinocortical assembloids in order to model the long-distance outgrowth characteristic of RGCs. RGCs displayed extensive axonal outgrowth into cortical organoids, with the ability to respond to environmental cues. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC develop- ment, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing outgrowth as well as modeling long-distance projections and pathfinding abilities.
Build your own retina: modeling retinogenesis and disease using human pluripotent stem cells
(2017) Sridhar, Akshayalakshmi; Meyer, Jason
Human pluripotent stem cells (hPSCs) allow for the unprecedented ability to recapitulate early stages of human development, which are otherwise inaccessible to investigation. This is especially true for one of the earliest events in human development, the establishment of a neuroretinal fate from an unspecified pluripotent population. To test the ability of hPSCs to serve in this capacity, hPSCs were generated using mRNA-reprogamming methods and maintained in xenogeneic-free differentiation conditions. These cells were directed to differentiate using a three-dimensional approach to analyze their ability to successfully recapitulate early events in human development in a temporal and developmentally-appropriate fashion. To do so, hPSCs were first directed to an anterior neural phenotype, which was confirmed by analysis of stage-specific neural transcription factors via immunocytochemistry and quantitative RT-PCR. Next, the cells were directed to an optic vesicle-like stage, where the presumptive retinal cells were identified by the expression of specific transcription factors. Finally, three-dimensional optic vesicle-like retinal organoids were identified, isolated, and further analyzed for the expression of markers associated with some of the differentiated cell types of the neural retina. Upon establishment of hPSC-derived retinal organoids, this system was further utilized to study the neurodegeneration in glaucoma and provide insights into the disease mechanisms. Overall, the results of this study help to demonstrate the suitability of hPSC-differentiation approaches as an effective tool to model retinal development and disease.
Characterization of Ethanol-induced Effects on Zebrafish Retinal Development: Mechanistic Perspective and Therapeutic Strategies
(2016) Muralidharan, Pooja; Marrs, James A.; Leung, Yuk Fai; Belecky-Adams, Teri; Meyer, Jason; Anderson, Ryan M.; Randall, Stephen K.
Fetal alcohol spectrum disorder (FASD) is a result of prenatal alcohol exposure, producing a wide range of defects including craniofacial, sensory, motor and cognitive deficits. Many ocular abnormalities are frequently associated with FASD including microphthalmia, optic nerve hypoplasia, and cataracts. FASD is highly prevalent in low socioeconomic populations, where it is also accompanied by higher rates of malnutrition and alcoholism. Using zebrafish as a model to study FASD retinal defects has been extremely insightful in understanding the ethanol-induced retinal defects at the cellular level. Zebrafish embryos treated with ethanol from mid-blastula transition through somitogenesis (2-24 hours post fertilization; hpf) showed defects similar to human ocular deficits including microphthalmia, optic nerve hypoplasia, and photoreceptor differentiation defects. Ethanol exposure altered critical transcription factor expression involved in retinal cell differentiation. Retinoic acid (RA) and folic acid (FA) nutrient co-supplementation rescued optic nerve and photoreceptor differentiation defects. Ethanol exposure during retinal morphogenesis stages (16-24 hpf), produced retinal defects like those seen with ethanol exposure between 2-24 hpf. Significantly, during ethanol-sensitive time window (16-24 hpf), RA co-supplementation moderately rescued these defects, whereas, FA cosupplementation showed significant rescue of optic nerve and photoreceptor differentiation. RA, but not FA, supplementation after ethanol exposure could restore ethanol-induced optic nerve and photoreceptor differentiation defects. Ethanol exposure did not affect timing of retinal cell differentiation induction, but later increased retinal cell death and proliferation. Ethanol-treated embryos showed increased retinal proliferation in the outer nuclear layer (ONL), inner nuclear layer (INL), and ciliary marginal zone (CMZ) at 48 hpf and 72 hpf. In order to identify the genesis of ethanol-induced persistent retinal defects, ethanol effects on retinal stem cell populations in the CMZ and the Müller glial cells (MGCs) were examined. Ethanol treated retinas had an expanded CMZ indicated by histology and Alcama, a retinal stem cell marker, immunolabeling, but reduced expression of rx1 and the cell cycle exit marker, cdkn1c. Ethanol treated retinas also showed reduced MGCs. At 72 hpf, ONL of ethanol exposed fish showed fewer photoreceptors expressing terminal differentiation markers. Importantly, these poorly differentiated photoreceptors co-expressed the basic helix-loop-helix (bHLH) proneural differentiation factor, neurod, indicating that ethanol exposure produced immature and undifferentiated photoreceptors. Reduced differentiation along with increased progenitor marker expression and proliferation suggest cell cycle exit failure due to ethanol exposure. These results suggested that ethanol exposure disrupted stem cell differentiation progression. Wnt, Notch and proneural gene expression regulate retinal stem cell proliferation and transition into progenitor cells. Ethanol exposure disrupted Wnt activity in the CMZ as well as Notch activity and neurod gene expression in the retina. RA and FA co-supplementation were able to rescue Wnt activity in the CMZ and rescue downstream Notch activity. To test whether the rescue of these Wnt-active cells could restore the retinal cell differentiation pathways, ethanol treated embryos were treated with Wnt agonist. This treatment could restore Wnt-active cells in the CMZ, Notch-active cells in the retina, proliferation, and photoreceptor terminal differentiation. We conclude that ethanol exposure produced persistent defects in the stem cell Wnt signaling, a critical pathway in retinal cell differentiation. Further analysis of underlying molecular mechanisms will provide insight into the embryonic origins of ethanol-induced retinal defects and potential therapeutic targets to cure this disorder.
DYRK1A-Related Trabecular Defects in Male Ts65Dn Mice Emerge During a Critical Developmental Window
(2021-08) LaCombe, Jonathan M.; Roper, Randall; Goodlett, Charles; Li, Jiliang; Wallace, Joseph; Meyer, Jason
Down syndrome (DS) is a complex genetic disorder caused by the triplication of human chromosome 21 (Hsa21). The presence of an extra copy of an entire chromosome greatly disrupts the copy number and expression of over 350 protein coding genes. This gene dosage imbalance has far-reaching effects on normal development and aging, leading to cognitive and skeletal defects that emerge earlier in life than the general population. The present study begins by characterizing skeletal development in young male Ts65Dn mice to test the hypothesis that skeletal defects in male Ts65Dn mice are developmental in nature.Femurs from young mice ranging from postnatal day 12- to 42-days of age (P12-42) were measured and analyzed by microcomputed tomography (μCT). Cortical defects were present generally throughout development, but trabecular defects emerged at P30 and persisted until P42. The gene Dual-specificity tyrosine-regulated kinase 1a (Dyrk1a) is triplicated in both DS and in Ts65Dn mice and has been implicated as a putative cause of both cognitive and skeletal defects. To test the hypothesis that trisomic Dyrk1a is related to the emergence of trabecular defects at P30, expression of Dyrk1a in the femurs of male Ts65Dn mice was quantified by qPCR. Expression was shown to fluctuate throughout development and overexpression generally aligned with the emergence of trabecular defects at P30. The growth rate in trabecular measures between male Ts65Dn and euploid littermates was similar between P30 and P42, suggesting a closer look into cellular mechanisms at P42. Assessment of proliferation of BMSCs, differentiation and activity of osteoblasts showed no significant differences between Ts65Dn and euploid cellular activity, suggesting that the cellular microenvironment has a greater influence on cellular activity than genetic background. These data led to the hypothesis that reduction of Dyrk1a gene expression and pharmacological inhibition of DYRK1A could be executed during a critical period to prevent the emergence of trabecular defects at P30. To tests this hypothesis, doxycycline-induced cre-lox recombination to reduce Dyrk1a gene copy number or the DYRK1A inhibitor CX-4945 began at P21. The results of both genetic and pharmacological interventions suggest that trisomic Dyrk1a does not influence the emergence of trabecular defects up to P30. Instead, data suggest that the critical window for the rescue of trabecular defects lies between P30 and P42.
Elucidating the Influence of Microglia on Retinal Ganglion Cells in a Human Pluripotent Stem Cell Model
(2024-06) Harkin, Jade; Meyer, Jason; Sheets, Patrick; Landreth, Gary; Block, Michelle; Sharma, Tasneem; Gomes, Catia
Glaucoma is a complex disease that leads to irreversible blindness, characterized by the loss of retinal ganglion cells (RGCs), which are the cells that transmit visual information from your eye into your brain. Evidence suggests that microglia, the resident immune cells in the central nervous system, may have a detrimental role in the onset and the progression of glaucoma. Microglia become activated in response to damage, pathogens and toxins and are initially thought to be beneficial to RGCs. However, when these cells are activated for excessive periods of time, they are thought to be harmful to RGCs. Thus, we sought to develop novel human pluripotent stem cell (hPSC)-derived microglia, astrocyte and RGC co-cultures to determine how microglia activation modulates RGC phenotypes in a human cellular model. Healthy and LPS-activated microglia were first co-cultured with RGCs for up to 3 weeks and the effects of microglia upon RGCs were assessed. Additionally, healthy and LPS-activated microglia were also co-cultured with astrocytes and RGCs for up to three weeks to assess if LPS-treated microglia can activate astrocytes and the effects this would have on RGCs. Results showed that when co-cultured with RGCs alone for 1 week, microglia activation is initially beneficial to RGCs. However, when co-cultured with RGCs for 3 weeks, microglia activation leads to RGC damage. Consequently, when astrocytes are present, microglia activation is harmful to RGCs in both short-term and long-term co-cultures, suggesting an additional role for microglia modulation of astrocytes, further leading to neurodegeneration. Taken together, our results have allowed for the precise study of how individual cell types are adversely affected in disease-relevant states, how microglia can directly influence RGCs, and how multiple co-cultures of human microglia, astrocytes and RGCs allows for a more sophisticated investigation of cellular interactions in disease states relevant to glaucoma.
Human Stem Cell Differentiated Retinal Ganglion Cells for Developing Glaucoma Neuroprotection and Cell Replacement Strategies
(2024-07) Anbarasu, Kavitha; Das, Arupratan; Corson, Timothy; Meyer, Jason; Graham, Brett; Janga, Sarath
Progressive loss of retinal ganglion cells (RGCs) leads to glaucoma. Early diagnosis offers an opportunity to protect existing RGCs. In advanced glaucoma, most RGCs are lost causing blindness and cell replacement therapy the only option. We used a human stem cell-based RGC differentiation model to develop neuroprotection by restoring mitochondrial homeostasis and enhancing RGC differentiation efficiency to increase the success of cell replacement therapy. Unmyelinated axons in RGCs require high levels of ATP, making disrupted mitochondria a risk factor in glaucoma. Our goal was to restore mitochondrial homeostasis through mitophagy (mitochondrial autophagy) and mitobiogenesis (mitochondrial biogenesis). Mutations in the mitophagy protein Optineurin (OPTNE50K) are found in patients with normal tension glaucoma and hence, we also used RGCs with the E50K mutation. We discovered that hRGCE50Ks suffer from mitobiogenesis issues, Parkin/Pink mediated mitophagy defects, and have OPTNE50K-Tank binding kinase-1 (TBK1) aggregates. hRGCE50Ks have lower mitochondrial mass and a higher mitochondrial load. We inhibited TBK1 to induce mitochondrial biogenesis and dissolve OPTNE50K-TBK1 aggregates. Our results show TBK1 inhibition triggered mitobiogenesis, dissolved aggregates, decreased mitochondrial ATP production load, and increased spare respiratory capacity, leading to neuroprotection. With complete RGC loss, enhancing differentiation to progenitor cells with lower cell division capacity can improve the success of cell replacement therapy and reduce teratoma formation and poor tissue integration. We observed that stem cells use proteasomes for mitochondrial degradation, while hRGCs use the lysosomal mitophagy pathway. Our results indicate that proteasomal activity declines during differentiation to hRGCs. Inhibition of proteasomal activity during early differentiation resulted in higher and faster RGC differentiation, with similar effects seen in motor neuron differentiation. We did not observe metabolic reprogramming in differentiating cells upon proteasomal activity inhibition but saw changes in cell cycle distribution, specifically an increase in the number of cells in the G1 phase. Proteomics analysis post-inhibitory treatment showed elevated neuronal differentiation proteins. Our results can be translated to minimize injection cell numbers and other risks of cell replacement therapy. In summary, my research identifies novel mechanisms for restoring mitochondrial homeostasis for neuroprotection in glaucomatous RGCs and develops an enhanced differentiation strategy to aid the success of cell replacement therapy.
Immunoreactivity of Pluripotent Markers SSEA-5 and L1CAM in Human Tumors, Teratomas, and Induced Pluripotent Stem Cells
(Hindawi Publishing Corporation, 2013) Cassidy, Linda; Choi, Meerim; Meyer, Jason; Chang, Rui; Seigel, Gail M.; Department of Biology, School of Science
Pluripotent stem cell markers can be useful for diagnostic evaluation of human tumors. The novel pluripotent marker stage-specific embryonic antigen-5 (SSEA-5) is expressed in undifferentiated human induced pluripotent cells (iPSCs), but little is known about SSEA-5 expression in other primitive tissues (e.g., human tumors). We evaluated SSEA-5 immunoreactivity patterns in human tumors, cell lines, teratomas, and iPS cells together with another pluripotent cell surface marker L1 cell adhesion molecule (L1CAM). We tested two hypotheses: (1) SSEA-5 and L1CAM would be immunoreactive and colocalized in human tumors; (2) SSEA-5 and L1CAM immunoreactivity would persist in iPSCs following retinal differentiating treatment. SSEA-5 immunofluorescence was most pronounced in primitive tumors, such as embryonal carcinoma. In tumor cell lines, SSEA-5 was highly immunoreactive in Capan-1 cells, while L1CAM was highly immunoreactive in U87MG cells. SSEA-5 and L1CAM showed colocalization in undifferentiated iPSCs, with immunopositive iPSCs remaining after 20 days of retinal differentiating treatment. This is the first demonstration of SSEA-5 immunoreactivity in human tumors and the first indication of SSEA-5 and L1CAM colocalization. SSEA-5 and L1CAM warrant further investigation as potentially useful tumor markers for histological evaluation or as markers to monitor the presence of undifferentiated cells in iPSC populations prior to therapeutic use.
The Impact of SBF2 on Taxane-Induced Peripheral Neuropathy
(2021-05) Cunningham, Geneva Mari; Schneider, Bryan P.; Radovich, Milan; Liu, Yunlong; Skaar, Todd; Meyer, Jason; Ivan, Mircea
The main focus of this study is to determine the impact of Set-Binding Factor 2 (SBF2) on human-derived neurons in the context of taxane-induced peripheral neuropathy. Taxane-induced peripheral neuropathy (TIPN) is a devastating survivorship issue for many cancer patients; SBF2 has been previously identified as a potential germline predictor that has been found to be significantly associated with severe TIPN in African American (AA) patients. The work described here provides ex vivo support for the use of SBF2 as a genotypic biomarker to identify a priori which patients are at a higher risk of manifesting severe TIPN. This study demonstrates that diminished expression of SBF2 exacerbated the effect of paclitaxel on viability and morphology and altered the functional response of a neuronal model exposed to paclitaxel treatment. Furthermore, transcriptomic work showed that reduced expression of SBF2 in a neuronal model treated with paclitaxel impacted the expression of genes that modulate stress-induced cell death and pain threshold. Altogether, these findings suggest that SBF2 plays a role in the development of TIPN. This work sheds light on the pathways potentially involving SBF2 that can be studied to further evaluate the function of this gene in neurons and its contribution to severe TIPN. Further functional approaches investigating these pathways will be pivotal in elucidating the underlying biological mechanism for this toxicity and identifying novel targeted therapeutic strategies to prevent or treat TIPN.
Neural Repair by Enhancing Endogenous Hippocampal Neurogenesis Following Traumatic Brain Injury
(2019-10) Wang, Xiaoting; Xu, Xiao-Ming; Chen, Jinhui; Jones, Kathryn J.; Meyer, Jason; Pollok, Karen E.
Traumatic brain injury (TBI) is a critical public health issue in the United States, affecting about 2.8 million people annually. Extensive cell death and neural degeneration directly and diffusively caused by the initial mechanical insult results in a wide range of neurological complications post-trauma. Learning and memory dysfunction is one of the most common complains. Hippocampal neuronal loss, together with other mechanisms, largely contributes to learning and memory impairment as well as other cognitive dysfunctions post-trauma. To date, no FDA-approved drug is available to target cell death or improve learning and memory following TBI. It is of great interest to develop alternative approaches targeting neural repair instead. Neural stem/progenitor cells (NSCs) in the adult hippocampus undergo life-long neurogenesis supporting learning and memory functions, thus hold great promise for post-traumatic neuronal replacement. The previous studies demonstrated that TBI transiently increase NSC proliferation. However, it is debated on whether TBI affects neurogenesis. The mechanism of TBI-enhanced NSC proliferation remains elusive. In the current studies, I have investigated post-traumatic neurogenesis after different injury severities, evaluated integration of post-injury born neurons, illustrated a molecular mechanism mediating TBI-enhanced NSC proliferation, proposed a de novo state of NSCs, and tested effects of a pharmacological approach on spatial learning and memory function recovery. My results demonstrated that post-traumatic neurogenesis is affected by injury severities, partially explained the pre-existing inconsistency among works from different groups. Post-injury born neurons integrate in neural network and receive local and distal inputs. TBI promotes functional recruitment of post-injury born neurons into neural circuits. Mechanistically, mechanistic target of rapamycin (mTOR) pathway is required primarily for TBI-enhanced NSC proliferation; NSCs feature a de novo alert state, in which NSCs are reversibly released from quiescence and primed for proliferation. Furthermore, my data demonstrated a beneficial role of ketamine in improving post-traumatic spatial learning possibly by activating mTOR signal in NSCs and/or promoting neuronal activity of post-injury born neurons. Together, my data support the feasibility of neurogenesis mediated neuronal replacement, provide a target for enhancing post-traumatic NSC proliferation and subsequent neurogenesis, and prove a potential pharmacological approach benefiting post-traumatic functional recovery in learning and memory.

Browsing by Author "Meyer, Jason"

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