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. Retinal 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 characterizing RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing 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 composition and growth factor signaling. Furthermore, RGCs possessed guidance receptors that are essential in influencing outgrowth and pathfinding. Subsequently, to determine 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 displayed recognition of appropriate targets, with the longest neurites projecting towards LGN explants compared to control explants or RGCs grown alone. Generated from 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 development, 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.

Aims: Limbal stem cell (LSC) deficiency is one of the most challenging ocular surface diseases. The aim of this thesis was to study damaged ocular surface reconstruction. Therefore, a mouse model of limbal transplantation was estab- lished. Furthermore, LSC isolation, transfer of LSCs and bone marrow-derived mesenchymal stem cells (MSCs) on nanofiber scaffolds were studied. Material and methods: Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were transplanted orthotopically into BALB/c mice. Graft survival, immune re- sponse and the effect of monoclonal antibodies (mAb) (anti-CD4 and anti-CD8 cells) were analyzed. Mouse LSCs were separated by Percoll gradient; subse- quently, they were analyzed for the presence of LSC and differentiation corneal epithelial cell markers and characteristics using real-time PCR and flow cytom- etry. Nanofiber scaffolds seeded with LSCs and MSCs were transferred onto the damaged ocular surface in mouse and rabbit models. Cell growth on scaffolds, post-operative inflammatory response and survival of transferred cell were ana- lyzed. Results: Limbal allografts were rejected promptly by the Th1-type of immune response (IL-2, IFN-γ) involving CD4+ cells and nitric oxide produced by macro- phages, contrary to the prevailing Th1 and Th2 immune responses (IL-4, IL-10) in...

<p>Adult prostate stem cells (PSC) are a rare epithelial progenitor population in the prostate. While essential for normal homeostasis, they have also been implicated in hyperplasia and cancer initiation. While studies have shown that inflammatory growth factors and cytokines can fuel stem cell expansion, the impact of inflammation on PSC is not well understood. To study the impact of inflammation on the prostate, the Ratliff laboratory developed the Prostate Ovalbumin Expressing Transgenic 3 (POET3), an inducible mouse model of abacterial T cell mediated prostate inflammation, which functions as a model for human autoimmune prostatitis. Previous studies using the POET3 demonstrated that inflammation increased proliferation and differentiation of PSC enrichments. Based on these findings, it was speculated that inflammation impacts prostate stem cells to enhance mechanisms of survival, possibly as a means of tissue protection.</p><p>Since androgen receptor (AR) signaling is the major driver of cellular differentiation and survival in the prostate, it was further hypothesized that inflammation promotes AR signaling in the PSC. To address this hypothesis, PSC and their resulting organoids from inflamed and non-inflamed (naïve) POET3 mice as well as human patient samples were assessed for AR and its signaling components.</p><p>These data were expanded by single cell mRNA sequencing using Fluidigm’s C1 platform, which revealed changes in stem cell populations, differential expression of interleukin 1 alpha (IL-1⍺) and its signaling components, and upregulation of various genes associated with immune regulation. Thus, experiments described herein probed the impacts of inflammation on AR, IL-1⍺, and T cell regulatory abilities in the PSC.</p>The results of these studies indicate that indeed, inflammation increases PSC survival. Inhibition of IL-1⍺ via inflammation-mediated up-regulation of IL-1 receptor antagonist (IL-1RA) promotes AR signaling, resulting in proliferation, differentiation, and AR target gene expression which can be modulated by Enzalutamide (a clinical AR inhibitor). Furthermore, PSC from inflamed mice are able to suppress cytotoxic T cell function in <i>ex vivo</i> assays. These studies set the foundation for new ways to treat proliferative diseases of the prostate by targeting IL-1⍺, AR, and immune regulation in the PSC.

Aims: Limbal stem cell (LSC) deficiency is one of the most challenging ocular surface diseases. The aim of this thesis was to study damaged ocular surface reconstruction. Therefore, a mouse model of limbal transplantation was estab- lished. Furthermore, LSC isolation, transfer of LSCs and bone marrow-derived mesenchymal stem cells (MSCs) on nanofiber scaffolds were studied. Material and methods: Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were transplanted orthotopically into BALB/c mice. Graft survival, immune re- sponse and the effect of monoclonal antibodies (mAb) (anti-CD4 and anti-CD8 cells) were analyzed. Mouse LSCs were separated by Percoll gradient; subse- quently, they were analyzed for the presence of LSC and differentiation corneal epithelial cell markers and characteristics using real-time PCR and flow cytom- etry. Nanofiber scaffolds seeded with LSCs and MSCs were transferred onto the damaged ocular surface in mouse and rabbit models. Cell growth on scaffolds, post-operative inflammatory response and survival of transferred cell were ana- lyzed. Results: Limbal allografts were rejected promptly by the Th1-type of immune response (IL-2, IFN-γ) involving CD4+ cells and nitric oxide produced by macro- phages, contrary to the prevailing Th1 and Th2 immune responses (IL-4, IL-10) in...

<p>  </p> <p>The growth of land plants depends on a group of pluripotent stem cells in a tissue called the meristem. Seed plants initiate and maintain different types of meristems at the asexual sporophyte stage, and they generate sexual gametophytes, which are dependent on their sporophytes and are devoid of a meristem. In contrast, aside from forming indeterminate meristems at the sporophyte stage, seedless vascular plants, including ferns, also develop meristems in their gametophytes to drive gametophyte development and formation of sexual organs. To date, compared to the well-characterized cell behaviors and regulatory pathways in the meristems of seed plants, the molecular and cellular basis of meristem development in seedless ferns is still poorly understood. </p> <p>In several seed plants, the HAIRY MERISTEM (HAM) family transcription factors play important roles in maintaining the indeterminacy of shoot apical meristems and promoting the <em>de novo</em> formation of axillary meristems. In the first part of this dissertation, through constructing a comprehensive phylogeny, I found that HAM family members are widely present in land plants and duplicated in a common ancestor of flowering plants, leading to the formation of two distinct groups: type I and type II. In addition, HAM members from different seed plants and seedless plants are able to replace the roles of the Arabidopsis type-II <em>HAM</em> genes, maintaining established shoot apical meristems and promoting the initiation of new stem cell niches in Arabidopsis. Furthermore, preliminary functional studies of the <em>HAM </em>homolog (<em>CrHAM</em>) in the model fern<em> Ceratopteris richardii</em> suggest that CrHAM is required for maintaining the indeterminacy of multicellular meristems in Ceratopteris gametophytes. Collectively, these results indicate that HAM family members may serve as common regulators in control of meristem development in both seed plants and seedless vascular plants. </p> <p>In the remaining chapter of this dissertation, long-term time-lapse confocal imaging was performed using Ceratopteris stable transgenic plants, in which each individual cell (nucleus) was labelled with a fluorescent marker. Real-time lineage, identity, and division activity of each single cell from meristem initiation to establishment in Ceratopteris gametophytes were then determined. Additionally, cell fate and lineage alterations during <em>de novo</em> formation of new meristems were examined by mechanical perturbations. These quantitative analyses lead to the conclusion that in Ceratopteris gametophytes, initiation and proliferation of multicellular meristems relies on a few marginal cell lineages. Once established, the meristem maintains an actively dividing zone during gametophyte development. Within the meristem, cell division is independent of cell lineages and marginal cells are more actively dividing than inner cells. The meristem also triggers differentiation of adjacent cells into egg-producing archegonia in a position-dependent manner. </p> <p>In summary, this work provides insight into the evolution of key stem-cell regulators and advances the understanding of diversified meristem development in land plants. </p>

Adult hippocampal neurogenesis entails a continued recruitment of neural precursor cells (NPCs) into active cell cycle and their progressive transition into post-mitotic granule cells. These adult born neurons integrate into the existing circuitry and confer structural plasticity, which aids in key hippocampal functions. For sustained neurogenesis, the cell cycle entry of the NPCs has to be tightly controlled. Environmental cues strongly, and differentially, regulate this checkpoint. Voluntary physical activity represents such an established strong stimulus that results in enhanced proliferation within the neurogenic niche. However, mechanistic insights into the maintenance and regulation of quiescence and the responsiveness of the NPCs to acute physical activity, as a form of adaptive neurogenesis, are yet to be elucidated. In my doctoral studies, we identified redox regulation as a key pathway regulating the cellular state equilibrium. I further explored the role of cellular oxidative stress in the neurogenic course and in adaptive neurogenic responses. Our results show that non-proliferative precursors within the hippocampal dentate gyrus, unlike in other stem cell systems, are marked by high levels of cellular reactive oxygen species (ROS). Using cytometric methodologies, ex vivo bioassays and transcriptional profiling, we revealed that classifying cells based on intracellular ROS content identified functionally defined sub-populations of adult NPCs. We propose that a drop in intracellular ROS content precedes the transition of cellular states, specifically from quiescence to active proliferation. Acute physical activity involves the activation of non- proliferating cells through a transient Nox2-dependent ROS surge in high-ROS, quiescent NPCs. In the absence of Nox2, baseline neurogenesis was unaffected, but the activity- dependent response was abolished. These findings shed new light on the discrete cellular events, which maintain the homeostasis between distinct cellular states of NPCs within the adult murine hippocampus.:Zusammenfassung 3 Summary 4 Acknowledgements 5 Index 8 List of figures 10 List of tables 11 Abbreviations 12 Publications 14 Introduction 15 Adult hippocampal neurogenesis 16 Adult subventricular neurogenesis 21 Methods to study adult neurogenesis 23 Environmental regulation of neurogenesis 26 Redox regulation in a stem cell 29 Working hypothesis 31 Specific aims 31 Materials and methods 32 Mice 34 Physical activity paradigm 35 Thymidine labelling and tissue preparation 35 Fluorescence immunohistochemistry 35 DG and SVZ dissection and dissociation 36 Flow cytometry 36 Gating for ROS classes 36 Neurosphere culture 37 Generation of monolayer culture 37 Inducing quiescence through BMP4 treatment 38 Next Generation sequencing (NGS) 38 RNA extraction 38 Quality control and differential expression 39 Functional enrichment and expression profiles 41 RNA isolation and quantitative RTPCR (qRT-PCR) 43 Ki67 immunochemistry and quantification of in vivo proliferation 45 Quantification and statistical analysis 46 Data and software availability 48 Results 49 Intracellular ROS content functionally delineates subpopulations of neural precursor cells 49 Resolution of ROS profiles of DG and SVZ and neurosphere bioassay 49 Distribution of Nes-GFP cells into different ROS classes 54 Neural precursors of the different ROS classes have distinct molecular profiles 55 Changes in intracellular ROS content precede cell fate changes 65 ROS profiling of other cell types within the DG 70 ROS profiling of Astrocytes and type-1 cells 70 ROS profiling of Doublecortin (Dcx)positive cells of the neurogenic lineage 74 ROS profiling of microglial cells within the DG 77 Resolving the response of Nes-GFP subpopulations to environmental stimulus 78 Nes-GFP+ cells of the hiROS class specifically respond to physical activity 81 Changes in ROS content are not driven by mitochondrial activity 83 In vitro monolayer culture of NPCs as an independent corroboration 86 Discussion 89 The organization of an active stem cell niche with respect to redox content 89 Cytometric classification of cells within the DG 91 Establishing the cellular states of redox defined subsets of Nes-GFP+ adult precursors within the DG 95 Timeline of baseline proliferation within precursors and identifying the subset of precursors responsive to de novo physical activity 97 Monolayer culture to study cellular states and redox regulation 100 Nox2 dependency as a discriminatory feature of adaptive neurogenesis 101 Conclusion 103 References 104 Declarations 122 Anlage 1 122 Anlage 2 124

Tissue engineering (TE) uses a combination of biomaterial scaffolds, cells, and drug delivery systems (DDS) to create tissues that resemble the human physiology. Such engineered tissues could be used to treat, repair, replace, and augment damaged tissues or organs, for disease modeling, and drug screening purposes. This work describes the development and use of novel strategies for engineering neural tissue using a combination of drug delivery systems (DDS), human induced pluripotent stem cells (hiPSCs), and bioprinting technologies for the generation of a drug screening tool to be used in the process of drug discovery and development. The DDS consisted of purmorphamine (puro) loaded microspheres that were fabricated using an oil-in-water single emulsion with 84% encapsulation efficiency and showed the slow release of puro for up to 46 days in vitro. Puro and retinoic acid (RA)-loaded microspheres were combined with hiPSCs-derived neural aggregates (NAs) that differentiated into neural tissues expressing βT-III and showed increased neural extension. hiPCS-derived neural progenitor cells (NPCs) were bioprinted on a layer-by-layer using a fibrin based-bioink and extrusion based- bioprinting. The bioprinted structures showed >81% cellular viability after 7 days of culture in vitro and the expression of the mature motor neuron (MN) markers HB9 and CHAT. Lastly, hiPCS-derived NPCs were bioprinted in combination with puro and RA-loaded microspheres and cultured for 45 days in vitro. The microspheres slowly released the drug and after 30 and 45 days the tissues contained mature neurons, astrocytes and oligodendrocytes expressing CHAT, GFAP, and O4, respectively. Changes in membrane potential indicated tissue responsiveness to different types of treatments such as acetylcholine and gamma-aminobutyric acid (GABA). In the future the bioprinted tissues could contain localized regions of varied drug releasing microspheres using a concentration gradient to promote differentiation into specific cell types in order to create more complex tissues. Moreover, these tissues will benefit from the presence of a neurovascular unit (NVU). Upon validation, the engineered tissues could be used as preclinical tools to test potential drugs and be used for personalized medicine by using patient specific hiPSCs. / Graduate / 2020-11-19

Indiana University-Purdue University Indianapolis (IUPUI) / The effects of hydroxyapatite in a DAP-loaded MC carrier on dental pulp stem cells Introduction: Regenerative endodontic procedures (REP) require disinfection techniques to eliminate bacteria from the infected immature root canal system and promote new growth of the pulp-dentin complex. Double antibiotic paste (DAP), a mixture of ciprofloxacin and metronidazole, has shown efficacy in doing so while minimizing cytotoxicity on dental pulp stem cells (DPSC). Stem cells, scaffolding, and growth factors are necessary in the maturation, proliferation, and differentiation of mesenchymal stem cells into the root canal system. Nano-hydroxyapatite (n-HA) has a history of biocompatibility and, in addition, has shown promising effects as a tissue bioengineering material. Objective: The aim of this in vitro study was to investigate the proliferation and mineralization of DPSC in the presence of 1% DAP and methylcellulose (MC) with varying concentrations of nano-hydroxyapatite. Materials and Methods: DPSC were plated in 24-well plates containing culture media. The next day, semi-permeable 0.1 mm Transwell chambers were inserted into the wells to separate the reservoirs for medicaments. Treatment paste composed of methylcellulose containing 1% DAP with either 0.25%, 0.50%, or 1.0% nano-hydroxyapatite was added along with culture media. Methylcellulose alone and calcium hydroxide (Ultracal) were used as control groups. After 3 days, cells were evaluated for cytotoxic effects using an MTS proliferation assay (n = 10, in triplicate). DPSCs were also cultured with these medicaments for 7 days in osteogenic media and evaluated for alkaline phosphatase (ALP) activity and mineralization activity (n = 13, in triplicate). Comparisons between groups for differences in mineralization, BSA, and ALP activity were performed using analysis of variance (ANOVA), with different variances allowed for each group and a random effect included in the model to account for correlation within each of the three trials. A simulation-based model was used to adjust for multiple comparisons. Results: Addition of n-HA treatment groups increased mineralization significantly greater than calcium hydroxide, with MC alone and MC+DAP+0.5% HA providing the greatest effect. Regarding ALP, all HA concentrations performed significantly greater than MC and DAP concentrations. Proliferation demonstrated similar metabolic activity in all experimental groups with few comparisons significant. Conclusion: The challenge in REPs is to maintain survival, and preferably promote the proliferation and development of DPSCs into the pulp-dentin complex with a consistent treatment outcome. The combination of DAP with hydroxyapatite may allow for both disinfection and improved mineralization and cellular differentiation. This contribution has shown significant ability to increase stem cell differentiation into an osteogenic lineage as well as calcium deposition, indicating end goal results of regenerative procedures.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic procedures (REPs) are a type of endodontic treatment aimed at replacing damaged tooth structures, including dentin and root structures, as well as cells of the pulp-dentin complex. Double antibiotic paste (DAP) has been shown to be efficacious in achieving disinfection of the root canal system while minimizing cytotoxicity to dental pulp stem cells (DPSCs). Hydroxyapatite (HA) is an extracellular, mineralized component of bone that has shown much promise as a scaffold in the field of regenerative medicine. Objective: The objective of this study was to evaluate the effects of HA in a DAP loaded methylcellulose (MC) carrier on the differentiation and mineral deposition of DPSC over time. Materials and Methods: DPSCs were plated in 24-well plates with culture media. The following day, semi-permeable 0.1 m chambers were inserted into the wells to separate the reservoirs and permit delivery of medicaments. 100 L treatment paste composed of MC with 1% DAP and either 0.5% or 1.0% nano-HA was added, followed by additional culture media. After 3 days of treatment, medicaments were removed and DPSCs were cultured for an additional 9 days with replacement of media every 3-4 days. At Day 12, DPSCs were evaluated for alkaline phosphatase (ALP) activity using a biochemical assay and mineral deposition using an Alizarin Red S Ca2+ staining assay (4 wells/group). Comparisons between groups were performed using one-way analysis of variance (ANOVA) with a 5% significance level used for all tests. Results: A trend towards increased ALP and mineral deposition activity was noted among the groups with HA added to DAP with MC. Although these trends were not statistically significant, a trend towards increased ALP and mineral deposition was observed after 3-day medicament exposure. The results were similar to previous findings using 7-day medicament treatments. Conclusion: The addition of HA showed a trend towards improved differentiation and mineral deposition of DPSCs compared to DAP with MC. Although additional studies are required, these results showed suggest that even with a shortened treatment time, increased differentiation and mineral deposition of DPSCs may be possible. This study provides additional support that low concentration DAP in a MC carrier has potential application in regenerative endodontic procedures. The novel addition of HA may provide additional osteogenic potential.

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