IU Indianapolis ScholarWorks :: Browsing by Subject "Stem cell"

Browsing by Subject "Stem cell"

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Astrocytes modulate neurodegenerative phenotypes associated with glaucoma in OPTN(E50K) human stem cell-derived retinal ganglion cells
(Elsevier, 2022) Gomes, Cátia; VanderWall, Kirstin B.; Pan, Yanling; Lu, Xiaoyu; Lavekar, Sailee S.; Huang, Kang-Chieh; Fligor, Clarisse M.; Harkin, Jade; Zhang, Chi; Cummins, Theodore R.; Meyer, Jason S.; Medical and Molecular Genetics, School of Medicine
Although the degeneration of retinal ganglion cells (RGCs) is a primary characteristic of glaucoma, astrocytes also contribute to their neurodegeneration in disease states. Although studies often explore cell-autonomous aspects of RGC neurodegeneration, a more comprehensive model of glaucoma should take into consideration interactions between astrocytes and RGCs. To explore this concept, RGCs and astrocytes were differentiated from human pluripotent stem cells (hPSCs) with a glaucoma-associated OPTN(E50K) mutation along with corresponding isogenic controls. Initial results indicated significant changes in OPTN(E50K) astrocytes, including evidence of autophagy dysfunction. Subsequently, co-culture experiments demonstrated that OPTN(E50K) astrocytes led to neurodegenerative properties in otherwise healthy RGCs, while healthy astrocytes rescued some neurodegenerative features in OPTN(E50K) RGCs. These results are the first to identify disease phenotypes in OPTN(E50K) astrocytes, including how their modulation of RGCs is affected. Moreover, these results support the concept that astrocytes could offer a promising target for therapeutic intervention in glaucoma.
Astrocytes Regulate the Development and Maturation of Retinal Ganglion Cells Derived from Human Pluripotent Stem Cells
(Elsevier, 2019-02-12) VanderWall, Kirstin B.; Vij, Ridhima; Ohlemacher, Sarah K.; Sridhar, Akshayalakshmi; Fligor, Clarisse M.; Feder, Elyse M.; Edler, Michael C.; Baucum, Anthony J.; Cummins, Theodore R.; Meyer, Jason S.; Biology, School of Science
Retinal ganglion cells (RGCs) form the connection between the eye and the brain, with this connectivity disrupted in numerous blinding disorders. Previous studies have demonstrated the ability to derive RGCs from human pluripotent stem cells (hPSCs); however, these cells exhibited some characteristics that indicated a limited state of maturation. Among the many factors known to influence RGC development in the retina, astrocytes are known to play a significant role in their functional maturation. Thus, efforts of the current study examined the functional maturation of hPSC-derived RGCs, including the ability of astrocytes to modulate this developmental timeline. Morphological and functional properties of RGCs were found to increase over time, with astrocytes significantly accelerating the functional maturation of hPSC-derived RGCs. The results of this study clearly demonstrate the functional and morphological maturation of RGCs in vitro, including the effects of astrocytes on the maturation of hPSC-derived RGCs.
Circadian rhythms in bipolar disorder patient-derived neurons predict lithium response: Preliminary studies
(Springer Nature, 2021) Mishra, Himanshu K.; Ying, Noelle M.; Luis, Angelica; Wei, Heather; Nguyen, Metta; Nakhla, Timothy; Vandenburgh, Sara; Alda, Martin; Berrettini, Wade H.; Brennand, Kristen J.; Calabrese, Joseph R.; Coryell, William H.; Frye, Mark A.; Gage, Fred H.; Gershon, Elliot S.; McInnis, Melvin G.; Nievergelt, Caroline M.; Nurnberger, John I.; Shilling, Paul D.; Oedegaard, Ketil J.; Zandi, Peter P.; The Pharmacogenomics of Bipolar Disorder Study; Kelsoe, John R.; Welsh, David K.; McCarthy, Michael J.; Psychiatry, School of Medicine
Bipolar disorder (BD) is a neuropsychiatric illness defined by recurrent episodes of mania/hypomania, depression and circadian rhythm abnormalities. Lithium is an effective drug for BD, but 30–40% of patients fail to respond adequately to treatment. Previous work has demonstrated that lithium affects the expression of “clock genes” and that lithium responders (Li-R) can be distinguished from non-responders (Li-NR) by differences in circadian rhythms. However, circadian rhythms have not been evaluated in BD patient neurons from Li-R and Li-NR. We used induced pluripotent stem cells (iPSCs) to culture neuronal precursor cells (NPC) and glutamatergic neurons from BD patients characterized for lithium responsiveness and matched controls. We identified strong circadian rhythms in Per2-luc expression in NPCs and neurons from controls and Li-R, but NPC rhythms in Li-R had a shorter circadian period. Li-NR rhythms were low-amplitude and profoundly weakened. In NPCs and neurons, expression of PER2 was higher in both BD groups compared to controls. In neurons, PER2 protein levels were higher in BD than controls, especially in Li-NR samples. In single cells, NPC and neuron rhythms in both BD groups were desynchronized compared to controls. Lithium lengthened period in Li-R and control neurons but failed to alter rhythms in Li-NR. In contrast, temperature entrainment increased amplitude across all groups, and partly restored rhythms in Li-NR neurons. We conclude that neuronal circadian rhythm abnormalities are present in BD and most pronounced in Li-NR. Rhythm deficits in BD may be partly reversible through stimulation of entrainment pathways.
Damaging effects of cigarette smoke on organs and stem/progenitor cells and the restorative potential of cell therapy
(2017-06-23) Barwinska, Daria; March, Keith L.; Basile, David P.; Broxmeyer, Hal; Clauss, Matthias; Traktuev, Dmitry O.
Cigarette smoking (CS) continues to be a significant modifiable factor contributing to a variety of diseases including cardiovascular, pulmonary and renal pathologies. It was suggested that smoking have detrimental effect of the body’s progenitor cells of bone marrow and peripheral organs. Since the concept of cell therapy that utilizes adipose stem/stromal cells (ASC) is gaining momentum it becomes critical to assess the therapeutic activities of the progenitors isolated from smokers. This study has revealed that CS negatively impacts the vasculogenic potential of ASC, in vitro, as well as weakening their therapeutic activity in vivo when tested in mouse model of hindlimb ischemia. We hypothesized that the decrease in vasculogenic activity of ASC is attributed to a higher level of expression of an angiostatic factor Activin A by ASC from CS donors. These findings clearly suggest that smokers should be evaluated for potential exclusion from early clinical trials of autologous cell therapies, or assessed as a separate cohort. The donor’s health status should be considered when choosing between autologous vs allogeneic cell therapies. We then examined the effect of CS on development of kidney pathology in mice. CS exposure led to decrease in kidney weights, capillary rarefaction, and cortical blood perfusion, and in parallel led to increase in kidney fibrosis and iron deposition. Interestingly, infusion of healthy ASC to the mice following CSexposure reversed CS-induced damages. This strongly support the notion that ASC-based therapy may provide rejuvenation effect. In the other subset of studies, we hypothesized that CS-induced lung emphysematous changes are preceded by suppression of bone marrow (BM) hematopoietic progenitor cells (HPC). We have revealed that intermittent BM mobilization with AMD3100 may mitigate the CS-induced myelo-suppression and deterioration of lung function and morphology. We observed that treatment of mice with AMD3100, while exposed to CS, preserves HPC at the levels of healthy control mice. Furthermore, AMD3100 treatment preserved lung parenchyma from pathological changes. These data suggest that while CS has a myelo-suppressive effect, administration of AMD3100 preserved BM-HPC and ameliorated lung damage.
Early Wnt Signaling Activation Promotes Inner Ear Differentiation via Cell Caudalization in Mouse Stem Cell-Derived Organoids
(Oxford University Press, 2023) Tang, Pei-Ciao; Chen, Li; Singh, Sunita; Groves, Andrew K.; Koehler, Karl R.; Liu, Xue Zhong; Nelson, Rick F.; Otolaryngology -- Head and Neck Surgery, School of Medicine
The inner ear is derived from the otic placode, one of the numerous cranial sensory placodes that emerges from the pre-placodal ectoderm (PPE) along its anterior-posterior axis. However, the molecular dynamics underlying how the PPE is regionalized are poorly resolved. We used stem cell-derived organoids to investigate the effects of Wnt signaling on early PPE differentiation and found that modulating Wnt signaling significantly increased inner ear organoid induction efficiency and reproducibility. Alongside single-cell RNA sequencing, our data reveal that the canonical Wnt signaling pathway leads to PPE regionalization and, more specifically, medium Wnt levels during the early stage induce (1) expansion of the caudal neural plate border (NPB), which serves as a precursor for the posterior PPE, and (2) a caudal microenvironment that is required for otic specification. Our data further demonstrate Wnt-mediated induction of rostral and caudal cells in organoids and more broadly suggest that Wnt signaling is critical for anterior-posterior patterning in the PPE.
Elucidating Cellular Mechanisms Underlying Retinal Ganglion Cell Neurodegeneration in a Human Pluripotent Stem Cell-Derived Model
(2022-12) Huang, Kang-Chieh; Cummins, Theodore R.; Meyer, Jason S.; Marrs, James A.; Perrin, Benjamin J.; Lasagna Reeves, Cristian A.
Glaucoma is a leading cause of blindness characterized by the progressive loss of retinal ganglion cells (RGCs), essentially severing the connection between the eye and the brain. Among many underlying causes of the disease, mutations in the Optineurin (OPTN) gene result in severe RGC neurodegeneration in the absence of elevated intraocular pressure, providing a novel opportunity to study molecular mechanisms that lead to RGC neurodegeneration associated with glaucoma. Efforts of this study establishing a human pluripotent stem cell (hPSC)-derived in vitro disease model by inserting OPTN(E50K) mutation via CRISPR/Cas9 genome editing and investigate the cellular mechanisms of RGC neurodegeneration associated with glaucoma. OPTN(E50K) RGCs revealed neurodegeneration phenotypes, including downregulation of RGCs transcription factors, neurite retraction, and hyperexcitability, suggesting that OPTN(E50K) RGCs can serve as an appropriate disease model to study glaucoma-associated neurodegeneration. Since OPTN serves a primary role as an autophagy receptor, we further hypothesized that the OPTN(E50K) mutation disrupts autophagy in RGCs, and modulation of autophagy by mammalian target of rapamycin (mTOR)-independent pathways can preserve RGC phenotypes by maintaining mTOR signaling. OPTN(E50K) RGCs exhibited a higher number of OPTN puncta along with an overall reduced expression of OPTN protein, indicating a gain of toxic protein accumulation or loss of protein function. Furthermore, OPTN(E50K) RGCs revealed an accumulation of the autophagosome protein LC3 in a punctal manner as well as increased expression of lysosomal proteins, suggesting a disruption of degradation pathway in autophagosome and lysosome fusion. As mTOR complex 1 (mTORC1) signaling serves as a negative regulator of autophagy, a downregulation of mTORC1 signaling via activation of stress sensor adenosine monophosphate-activated protein kinase (AMPK) was observed as a possible compensatory mechanism for autophagy deficits in OPTN(E50K) RGCs. Pharmacological inhibition of mTOR in wild-type hRGCs resulted in similar disease-related phenotypes, while preservation of the mTOR pathway in OPTN(E50K) RGCs by treatment with the mTOR-independent autophagy modulator trehalose cleared OPTN accumulated puncta, preserving mTORC1 signaling, as well as rescuing neurodegenerative phenotypes. To further validate these associations in an animal model, the microbead occlusion mouse model was established by injection of magnetic microbeads in the anterior chamber to block aqueous outflow resulting ocular hypertension. In agreement with our findings in hRGCs, a decrease in mTOR signaling associated with an increase in the expression of autophagy-associated proteins was observed in RGCs in the microbead occlusion model. Additionally, these disease-related phenotypes were observed specifically within RGCs but not cortical neurons with an underlying OPTN(E50K) mutation, demonstrating that autophagy represents an essential pathway in RGCs to maintain homeostasis, and selective disrupt of autophagy in RGCs leads to neurodegeneration. Taken together, the results of this study highlight an essential balance between autophagy and mTORC1 signaling that is essential for the homeostasis of RGCs, while disruption to these signaling pathways contributes to neurodegenerative features in glaucoma. These results also demonstrated the ability to pharmacologically intervene to experimentally manipulate these pathways and rescue neurodegenerative phenotypes, providing a potential therapeutic target to prevent glaucoma-associated neurodegeneration.
Emerging Landscape of Preclinical Models for Studying COVID-19 Neurologic Diseases
(American Chemical Society, 2023-09-06) Li, Jason; Wang, Jing; Wang, Hu; Neurology, School of Medicine
COVID-19 (Coronavirus Disease 2019) is an infectious disease caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and has globally infected 768 million people and caused over 6 million deaths. COVID-19 primarily affects the respiratory system but increasing reports of neurologic symptoms associated with COVID-19 have been reported in the literature. The exact mechanism behind COVID-19 neurologic pathophysiology remains poorly understood due to difficulty quantifying clinical neurologic symptoms in humans and correlating them to findings in human post-mortem samples and animal models. Thus, robust preclinical experimental models for COVID-19 neurologic manifestations are urgently needed. Here, we review recent advances in in vitro, in vivo, and other models and technologies for studying COVID-19 including primary cell cultures, pluripotent stem cell-derived neurons and organoids, rodents, nonhuman primates, 3D bioprinting, artificial intelligence, and multiomics. We specifically focus our discussion on the contribution, recent advancements, and limitations these preclinical models have on furthering our understanding of COVID-19's neuropathic physiology. We also discuss these models' roles in the screening and development of therapeutics, vaccines, antiviral drugs, and herbal medicine, and on future opportunities for COVID-19 neurologic research and clinical management.
Extension of retinofugal projections in an assembled model of human pluripotent stem cell-derived organoids
(Cell Press, 2021-09-14) Fligor, Clarisse M.; Lavekar, Sailee S.; Harkin, Jade; Shields, Priya K.; VanderWall, Kirstin B.; Huang, Kang-Chieh; Gomes, Cátia; Meyer, Jason S.; Biology, School of Science
The development of the visual system involves the coordination of spatial and temporal events to specify the organization of varied cell types, including the elongation of axons from retinal ganglion cells (RGCs) to post-synaptic targets in the brain. Retinal organoids recapitulate many features of retinal development, yet have lacked downstream targets into which RGC axons extend, limiting the ability to model projections of the human visual system. To address these issues, retinal organoids were generated and organized into an in vitro assembloid model of the visual system with cortical and thalamic organoids. RGCs responded to environmental cues and extended axons deep into assembloids, modeling the projections of the visual system. In addition, RGC survival was enhanced in long-term assembloids, overcoming prior limitations of retinal organoids in which RGCs are lost. Overall, these approaches will facilitate studies of human visual system development, as well as diseases or injuries to this critical pathway.
Functional Differences Between Placental Micro- and Macrovascular Endothelial Colony-Forming Cells
(Wiley, 2016-03) Solomon, Ioana; O’Reilly, Megan; Ionescu, Lavinia; Alphonse, Rajesh S.; Rajabali, Saima; Zhong, Shumei; Vadivel, Arul; Shelley, W. Chris; Yoder, Mervin C.; Thébaud, Bernard; Department of Pediatrics, IU School of Medicine
Alterations in the development of the placental vasculature can lead to pregnancy complications, such as preeclampsia. Currently, the cause of preeclampsia is unknown, and there are no specific prevention or treatment strategies. Further insight into the placental vasculature may aid in identifying causal factors. Endothelial colony-forming cells (ECFCs) are a subset of endothelial progenitor cells capable of self-renewal and de novo vessel formation in vitro. We hypothesized that ECFCs exist in the micro- and macrovasculature of the normal, term human placenta. Human placentas were collected from term pregnancies delivered by cesarean section (n = 16). Placental micro- and macrovasculature was collected from the maternal and fetal side of the placenta, respectively, and ECFCs were isolated and characterized. ECFCs were CD31(+), CD105(+), CD144(+), CD146(+), CD14(-), and CD45(-), took up 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein, and bound Ulex europaeus agglutinin 1. In vitro, macrovascular ECFCs had a greater potential to generate high-proliferative colonies and formed more complex capillary-like networks on Matrigel compared with microvascular ECFCs. In contrast, in vivo assessment demonstrated that microvascular ECFCs had a greater potential to form vessels. Macrovascular ECFCs were of fetal origin, whereas microvascular ECFCs were of maternal origin. ECFCs exist in the micro- and macrovasculature of the normal, term human placenta. Although macrovascular ECFCs demonstrated greater vessel and colony-forming potency in vitro, this did not translate in vivo, where microvascular ECFCs exhibited a greater vessel-forming ability. These important findings contribute to the current understanding of normal placental vascular development and may aid in identifying factors involved in preeclampsia and other pregnancy complications.
Generating Inner Ear Organoids from Mouse Embryonic Stem Cells
(Springer, 2016) Longworth-Mills, Emma; Koehler, Karl R.; Hashino, Eri; Otolaryngology -- Head and Neck Surgery, School of Medicine
This protocol describes a three-dimensional culture method for generating inner ear sensory epithelia, which comprises sensory hair cells and a concurrently arising neuronal population. Mouse embryonic stem cells are initially plated in 96-well plates with differentiation media; following aggregation, Matrigel is added in order to promote epithelialization. A series of small molecule applications is then used over the first 14 days of culture to guide differentiation towards an otic lineage. After 16-20 days, vesicles containing inner ear sensory hair cells and supporting cells arise from the cultured aggregates. Aggregates may be analyzed using immunohistochemistry and electrophysiology techniques. This system serves as a simple and relatively inexpensive in vitro model of inner ear development.

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