Indiana University-Purdue University Indianapolis (IUPUI) / Ethical considerations of the human embryo have involved heated dispute and seem always to result in the same interminable debate. A history of this debate, however, shows a shift in the language used to distinguish between degrees of moral status – while the debate once focused on the presence or absence of “human life,” now it is more likely to hear whether the qualifications for “personhood” have been met. In other words, any member of the human species may deserve some level of respect, but only the “persons” deserve full moral respect. This leaves open the possibility for a human being who is not actually a person – a “nonperson human being.” As an answer to the question of exactly what kind of respect to give the human embryo, Aristotelian moral philosophy offers a unique perspective, one which is distinctive from the familiar debate. Aristotle’s concept of ergon, or function, is a key to understanding what is essential in any human being, because it reveals the importance of potentiality to our nature as rational beings. A philosophical view of function, combined with the data of modern embryology, makes the case that our proper function is the vital part of who we are as human beings, and that a disruption of human function constitutes a true harm. This thesis contrasts Aristotelian proper human function with the modern understanding of a “nonperson human being,” especially as articulated within the ethical theory of Peter Singer. This understanding of function, revealing the essence of human potential and linked with human development, offers a sort of “common-sense morality” response to modern views on personhood.

Indiana University-Purdue University Indianapolis (IUPUI) / Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine neurotransmitter that is synthesized from the amino acid L-tryptophan and is reported to localize in mitochondria of embryonic stem cells. Even before its role as a neurotransmitter in mature brain was discovered, 5-HT has been shown to play an important role in regulating brain development. However, there is a lack of knowledge about the downstream target genes regulated by serotonin in embryonic stem (ES) cells. Towards this end, our study helps in understanding transcriptional regulatory mechanisms of 5-HT responsive genes in ES cells. By combining the gene expression data with motif prediction algorithms, literature validation and comparison with public domain data, gene targets specific to endogenous or exogenous 5-HT in ES cells were identified. By performing one-way ANOVA, and volcano plots using GeneSpring software, we identified 44 5-HT induced and 29 5-HT suppressed genes, likely to be transcriptionally regulated by 4 & 2 TFs respectively. Motif enrichment analysis on these target genes using MotifScanner revealed that the transcription factor TFAP2A plays a key role in regulating the expression of 5-HT responsive genes. Furthermore, by comparing our dataset with published expression profiles of ES cells, we observed a number of 5-HT responsive target genes showing enrichment in ES cells. Genes such as Nanog, Slc38a5, Hoxb1 and Eif2s1 from this analysis have been observed to be components of ‘stemness’ phenotypes reported in literature. Functional annotation of the 5-HT responsive genes identified gene ontologies such as regulation of translation in response to stress and energy derivation by oxidation, suggesting a regulatory role for 5-HT in mitochondrial functions of ES cells. Additionally, enrichment of other biological process terms such as development of various parts of nervous system, cell adhesion, and apoptosis suggests that 5-HT target genes may play an important role in ES cell differentiation. Our study implemented a new combinatorial approach for identifying gene regulatory mechanisms involved in 5-HT responsive genes and proposed potential mediatory role for serotonin in ES cell differentiation and growth. Thus, this study provides potential 5-HT target genes in ES cells for biological validation.

Adult stem cells are key components of animal biology, with the vital task of replenishing cells in numerous tissue types. This function hinges on the prior specification and arrangement of requisite numbers of stem cells and supportive niche cells during development. Here, I investigate the signaling pathways which act on adult Drosophila ovarian Follicle Stem Cells (FSCs) and their niche cells, Escort Cells (ECs) and Follicle Cells (FCs), during pupal development; location of a cell upon eclosion determines cell identity. Using lineage tracing approaches, we examine how the Wnt, JAK-STAT, Hippo, and Hedgehog pathways influence precursor behaviors, namely cell division, location, and survival, with a focus on the novel process underlying budding of the first egg chamber midway through pupation. We find that pupal Intermingled Cells (ICs), the precursors to adult ECs, FSCs, and FCs, are also the source of Extra Germarial Crown (EGC) cells and basal stalk cells which surround the first budded egg chamber. As in adults, Wnt primarily affects precursor location, with high Wnt corresponding to anterior cell fates and low Wnt producing a posterior bias. JAK-STAT has a similar, but reverse, effect on location, and appears to contribute to division in posterior locations. The Hippo pathway, mediated by its effector Yorkie (Yki), mainly affects precursor division and survival, with some effects on location. The role of the Hedgehog pathway in precursor division and posterior movement appears to be mediated via Yki activity. Understanding these developmental processes could contribute to generating organoids for therapeutic discovery.

In the prevailing model, R-spondin (Rspo)-dependent 𝘓𝘨𝘳5+ crypt base columnar (CBC) cells are the only dedicated intestinal stem cells (ISCs) that sustain epithelial regeneration during homeostasis by upward migration of their progeny through an elusive transit-amplifying (TA) intermediate in the upper crypt. Paradoxically, the intestinal epithelium is resilient to 𝘓𝘨𝘳5+ CBC cell loss. Elicited by intriguing R-spondin (Rspo) gain- and loss-of-function phenotypes that suggest regeneration emerges from a subset of 𝘓𝘨𝘳5- cells, here we combine single-cell RNA-sequencing (scRNA-seq) with time-resolved fate mapping to identify a proliferative population of multi-potent upper crypt cells in the putative location of TA cells. Distinct from the 𝘓𝘨𝘳5+ CBC cells and marked by expression of 𝘍𝘨𝘧𝘣𝘱1 - a gene which we demonstrate is essential for regeneration - these cells generate progeny that migrate bi-directionally along the crypt-villus axis and, unexpectedly, also serve as a source for the 𝘓𝘨𝘳5+ cells at the base. 𝘍𝘨𝘧𝘣𝘱1+ cells are resilient to Rspo signaling blockade and sustain epithelial homeostasis in the context of 𝘓𝘨𝘳5+ cell loss, suggesting functional independence. Consistent with their stem rather than TA cell function, our results point to the existence of a novel cellular hierarchy in the intestinal epithelium, contesting the regenerative capabilities of the 𝘓𝘨𝘳5+ CBC cell and helping reconcile many of the 𝘓𝘨𝘳5+ CBC model inconsistencies.

Tse, Chi Lok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 116-131). / Abstracts in English and Chinese. / Declaration --- p.i / Abstract --- p.iii / Abstract in Chinese --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.X / List of Tables --- p.xii / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- Regenerative Medicine --- p.1 / Chapter 1.2 --- Embryonic Stem Cells and Reprogramming --- p.3 / Chapter 1.3 --- Transdifferentiation --- p.6 / Chapter 1.4 --- The Cerebellum --- p.7 / Chapter 1.4.1 --- Functions of the cerebellum --- p.7 / Chapter 1.4.2 --- Structure and organization of the cerebellum --- p.8 / Chapter 1.4.3 --- Principle cellular components in the cerebellum --- p.12 / Chapter 1.4.3.1 --- Purkinje cells --- p.12 / Chapter 1.4.3.2 --- Granule cells --- p.12 / Chapter 1.4.3.3 --- Mossy fibres --- p.13 / Chapter 1.4.3.4 --- Climbing fibres --- p.13 / Chapter 1.4.3.5 --- Deep cerebellar nuclei --- p.13 / Chapter 1.4.3.6 --- Other cerebellar neurons --- p.14 / Chapter 1.4.3.7 --- Neuroglia of the cerebellum --- p.16 / Chapter 1.4.4 --- Circuitry of the cerebellum --- p.17 / Chapter 1.5 --- Development of the Cerebellum --- p.21 / Chapter 1.5.1 --- Anatomical changes during cerebellar development --- p.21 / Chapter 1.5.2 --- Molecular control of cerebellar development --- p.25 / Chapter 1.5.2.1 --- Specification of the cerebellar region --- p.25 / Chapter 1.5.2.2 --- Neurogenesis from the ventricular zone --- p.26 / Chapter 1.5.2.3 --- Neurogenesis from rhombic lip --- p.29 / Chapter 1.6 --- Scope of the Thesis --- p.33 / Chapter CHAPTER 2 --- Materials and General Methods / Chapter 2.1 --- Materials for Molecular Biological Work --- p.35 / Chapter 2.1.1 --- Enzymes --- p.35 / Chapter 2.1.2 --- Chemicals and others --- p.35 / Chapter 2.1.3 --- Plasmid vectors and plasmid --- p.36 / Chapter 2.1.4 --- Solutions and media --- p.36 / Chapter 2.2 --- Materials for Tissue/Cell Culture --- p.38 / Chapter 2.2.1 --- Chemicals --- p.38 / Chapter 2.2.2 --- Culture media and solutions --- p.38 / Chapter 2.2.3 --- Culture cells --- p.39 / Chapter 2.3 --- Animals --- p.40 / Chapter 2.4 --- Materials for Immunocytochemistry --- p.40 / Chapter 2.5 --- Oligonucleotide Primers --- p.41 / Chapter 2.6 --- RNA Extraction --- p.44 / Chapter 2.7 --- Generation of cDNA from mRNA --- p.44 / Chapter 2.8 --- Preparation of Recombinant Plasmid DNA --- p.45 / Chapter 2.8.1 --- Small scale preparation of DNA --- p.45 / Chapter 2.8.2 --- QLAGEN plasmid midiprep kit method --- p.46 / Chapter 2.9 --- Preparation of Specific DNA Fragment from Agarose Gel --- p.46 / Chapter 2.10 --- Subcloning of DNA Fragments --- p.47 / Chapter 2.10.1 --- Preparation of cloning vectors --- p.47 / Chapter 2.10.2 --- Subcloning of DNA fragment --- p.48 / Chapter 2.10.3 --- Transformation of DNA into competent cells --- p.48 / Chapter 2.11 --- Preparation of Competent Cells --- p.48 / Chapter CHAPTER 3 --- Generation and Characterization of Induced Neurons / Chapter 3.1 --- Introduction --- p.50 / Chapter 3.2 --- Experimental Procedures --- p.51 / Chapter 3.2.1 --- Construction of expression vector --- p.51 / Chapter 3.2.1.1 --- Preparation of insert DNA --- p.51 / Chapter 3.2.1.2 --- Construction of entry vector --- p.52 / Chapter 3.2.1.3 --- Construction of destination vector --- p.52 / Chapter 3.2.1.4 --- Construction of expression vector --- p.52 / Chapter 3.2.2 --- Generation of induced neural cells --- p.57 / Chapter 3.2.2.1 --- Culture of mouse embryonic fibroblasts (MEF) --- p.57 / Chapter 3.2.2.2 --- Production of expression vector containing retroviruses --- p.57 / Chapter 3.2.2.3 --- Transfection and induction of neural fate of MEF --- p.57 / Chapter 3.2.3 --- Immunocytochemcial analysis --- p.58 / Chapter 3.2.4 --- Efficiency calculation --- p.59 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- A screen for cerebellar Purkinje and granule cell fate-inducing factors --- p.62 / Chapter 3.3.2 --- Characterization of the induced neurons --- p.67 / Chapter 3.3.2.1 --- Granule cell induction --- p.67 / Chapter 3.3.2.2 --- Purkinje cell induction --- p.71 / Chapter 3.4 --- Discussion --- p.102 / Chapter 3.4.1 --- Roles of inducing factors in Purkinje cells and granule cells development --- p.102 / Chapter 3.4.2 --- Mechanism of neural transdifferentiation --- p.107 / Chapter CHAPTER 4 --- Future Directions / Chapter 4.1 --- Complete Induction of Purkinje Cell Fate --- p.111 / Chapter 4.2 --- Induced Neurons of Different Subtypes --- p.112 / Chapter 4.3 --- Mechanism of Transdifferentiation --- p.114 / Chapter 4.4 --- Transdifferentiation and Regenerative Medicine --- p.114 / Bibliography --- p.116

The derivation of embryonic stem cells (ESCs) has created an invaluable resource for scientific study and discovery. Further improvement in differentiation protocols is necessary to generate the large number of cells needed for clinical relevance. The goal of this work was to develop a method to incorporate biomaterial microparticles (MPs) within stem cell aggregates and to evaluate their use for local control of the cellular microenvironment for directed differentiation. The effects of unloaded MPs on ESC differentiation were first determined by controlled incorporation of poly(lactic-co-glycolic acid) (PLGA), agarose and gelatin MPs. Embryoid body (EB) formation, cell viability, and gross morphology were not affected by the presence of the MPs. Further analysis of gene expression and patterns of phenotypic marker expression revealed alterations in the differentiation profile in response to material incorporation. The ability of MPs to direct ESC differentiation was investigated by incorporation of growth factor loaded MPs within EBs. MPs were loaded with bone morphogenetic protein-4 (BMP-4). BMP-4 loaded MPs incorporated within EBs induced mesoderm gene expression while inhibiting expression of an ectoderm marker compared to untreated EBs. Finally, magnetic MPs (magMPs) were incorporated within EBs to induce magnetic sensitivity. The responsiveness of EBs to applied magnetic fields was controlled by the number of magMPs incorporated within the aggregates. Magnetic guidance was then used to control the precise location of single EBs or populations of EBs for bioreactor culture and for construction of heterogeneous cell constructs. Overall, the results indicated that PSC differentiation within spheroids is sensitive to various types of biomaterials. Incorporation of MPs within EBs can be used to direct ESC differentiation by control of the cellular environment from microscale interactions, by delivery of soluble factors, to macroscale interactions, by control of EB position in static and suspension cultures.

Stem and progenitor cells are an attractive cell source for the treatment of degenerative diseases due to their potential to differentiate into multiple cell types and provide large cell yields. Thus far, however, clinical applications have been limited due to inefficient differentiation into desired cell types with sufficient yields for adequate tissue repair and regeneration. The ability to spontaneously aggregate in suspension makes embryonic stem cells (ESCs) amenable to large-scale culture techniques for the production of large yields of differentiating cell spheroids (termed embryoid bodies or EBs); however, the introduction of hydrodynamic conditions may alter differentiation profiles within EBs and should be methodically examined. The work presented here employs a novel, laboratory-scale hydrodynamic culture model to systematically interrogate the effects of ESC culture hydrodynamics on cardiomyocyte differentiation through the modulation of a developmentally-relevant signaling pathway. The fluidic environment was defined using computational fluid dynamic modeling, and the effects of hydrodynamic conditions on EB formation, morphology and structure were assessed. Additionally, EB differentiation was examined through gene and protein expression, and indicated that hydrodynamic conditions modulate differentiation patterns, particularly cardiogenic lineage development. This work illustrates that mixing conditions can modulate common signaling pathways active in ESC differentiation and suggests that differentiation may be regulated via bioprocessing parameters and bioreactor design.

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Indiana University-Purdue University Indianapolis (IUPUI) / Embryonic stem cells (ESCs) are unique in that they have potential to give rise to every cell type of the body. Little is known about stimuli that promote mouse (m)ESC differentiation and proliferation. Therefore the purpose of this study was to determine the role of Toll Like Receptor (TLR) ligands in mESCs proliferation, survival, and differentiation in the presence of Leukemia Inhibitory Factor (LIF). We hypothesized that TLRs are expressed and functional, and when activated by their ligand will induce survival, proliferation, and prevent differentiation. In this study, mESC line E14 was used to determine the expression of TLRs at the mRNA level and three mESC lines, R1, CGR8, and E14, were used to determine cell surface protein levels. We found expression of TLRs 1, 2, 3, 5, and 6 at the mRNA level, but no expression of TLRs 4, 7, 8, and 9 in the E14 mESC line. We confirmed the presence of TLR-2 but not of TLR-4, protein on the cell surface using flow cytometric analysis for all three cell lines. We focused our studies mainly on TLR-2 using the E14 cell line. Pam3Cys, is a synthetic triacyl lipoprotein and a TLR-2 ligand, which induced a significant increase in mESC proliferation on Days 3, 4, and 5 and enhanced survival of mESC in a dose dependent manner in the context of delayed addition of serum. All the latter experiments were performed in triplicate and student T-test was performed to establish significant differences. Next, we demonstrated functionality of TLR-2 via the MyD88/IKK pathway, where MyD88 was expressed and IKKα/β phosphorylation was enhanced. This was associated with increased NF-κB nuclear translocation upon activation by Pam3Cys. Finally, we showed that there were no changes in expression of mESCs markers Oct-4, KLF-4, Sox-2, and SSEA-1, thus illustrating that the mESCs may have remained in a pluripotent state after activation with the TLR-2 ligand in the presence of LIF. These results demonstrate that mESCs can respond to microbial products, such as Pam3Cys, and can induce proliferation and survival of the mESCs. This finding expands the role of TLRs and has some implications in understanding embryonic stem cell biology.