The hair follicle is considered a mini-organ which makes it a useful model for studying regenerative processes and cross-tissue interactions due to its unique populations of cell types and specialized pools of adult stem cells. Hair itself is a defining feature of the skin organ and is critical for external protection, thermoregulation, sweat and pheromone relays, and social interactions. In the bulge niche, hair follicle stem cells (HFSCs) oscillate between activation and quiescence to create the hair cycle. This process is cyclically and dynamically maintained throughout a coated animal's lifetime, requiring precise temporal and spatial control of the HFSC niche. However, a complete inventory of the mechanisms underlying hair follicle homeostasis remains unclear. Herein I will briefly explain the current dogma of signaling pathways regulating HFSCs. But, the novelty of my work will go on to further describe the synergistic – yet understudied – roles of metabolic control and canonical G-protein coupled receptors (GPCR) and signaling in modulating downstream genes facilitating HFSC biology. The data out of these projects of course will yield new avenues for the development of metabolic/pharmacological compounds alike for regenerative medicine and equally important molecular management of adult stem cell homeostasis.

Pluripotent Stem Cells (PSCs) have the unique ability to divide indefinitely and self-renew in vitro, as well as differentiate into all cell types of the body. These cells present intriguing opportunities to study development and biology in novel ways, and can be utilized to produce large amounts of cells with therapeutic applications. In order to safely use PSCs to model difficult to study diseases, or to replace injured or diseased tissues in patients, we need to ensure that the in vitro differentiation process is effective. However, the field has encountered great difficulty in making fully differentiated, mature cells in vitro from PSCs. While it is currently possible to differentiate PSCs into many types of central nervous system (CNS) cells, we and others have shown that CNS cells derived from PSCs are immature and are most epigenetically and functionally similar to cells from the CNS at a mid-gestational time point. Since the developing nervous system is tightly temporally regulated in terms of division, differentiation, organization, and connection, this roadblock currently poses a challenge to the utility of PSC-derived CNS cells.

We have identified a molecular circuit consisting of LIN28 proteins and the let-7 microRNAs that regulates differentiation and maturation in the CNS and other organ systems. In this dissertation I present data to demonstrate that manipulation of the LIN28/let-7 circuit can induce functional maturation in CNS progenitors by acting through structural epigenetic protein HMGA2 and the Notch signaling pathway. Furthermore, by exploring the regulation of the let-7 miRNAs at the level of transcription, I propose that dynamic temporal regulation of some let-7 family members is an important driver of change in the LIN28/let-7 circuit. Finally, I describe efforts to generate PSC-derived inhibitory interneurons and to characterize their aging and maturation in vitro and in vivo, with the aim of understanding the mechanisms of post-mitotic neuronal functional maturation. These studies will pave the way for the generation of appropriately mature PSC-derived CNS cells, and may be broadly applicable toward generating mature cells from PSCs for the purposes of biological study or therapeutic utility.

Rett syndrome is a severe neurodevelopmental disorder that arises due to a mutation inmethyl-CpG-binding protein 2 (MECP2). Rett syndrome patients suffer delay in mental, physical and cognitive development. The mechanism underlying the onset of Rett syndrome remains poorly understood. We used Rett patient derived human induced pluripotent stem cells (hiPSCs) to model Rett syndrome in vitro in an attempt to elucidate the pathways implicated in Rett phenotype. We discovered that Rett neurons undergo neuronal stress resulting in an increased expression of OCT1 and P53 target genes. In addition, mutant neurons exhibit premature senescence accompanied by elevated levels of DNA damage. Analyzing neuronal phenotype revealed a significant decrease in the complexity of dendritic arborization. This led us to explore these neuronal stress signatures further by analyzing Rett patient brain samples with single-cell RNA sequencing. We discovered that lack of MECP2 leads to misregulated synaptic genes as iii well as abnormal metabolism. We confirmed faulty metabolism and mitochondrial respiration in our in vitro model, and showed that various types of neuronal stress lead to induction of OCT1 in vitro.

Human pluripotent stem cells (hPSCs) have opened up numerous avenues, including regenerative medicine and modeling development and disease. However, when compared to the fetal tissue-derived counterparts, in vitro derived hPSC progenies reflect very early human development (<6 weeks). Therefore, it is vital to understand the mechanisms of early human development to facilitate maturation of hPSCs in vitro. Low oxygen tension is important for maintaining the pluripotency of in vitro cultured human embryonic stem cells. However, the influence of oxygen tension on fidelity of PSC differentiation had not been studied extensively until now. In fact, embryogenesis before 10 weeks of gestation occurs under low oxygen (<2.5% O2). We hypothesized that manipulating oxygen tension to a physiological level in vitro would facilitate the transcriptional and functional maturity of hPSC derivatives by more faithfully mimicking in vivo conditions. We discovered that physiological oxygen tension (2% O2) promotes astrogliogenesis upon NPC differentiation and determined that hypoxia inducible factors (HIFs) are key transcription factors involved in this process. Activating HIF with small molecules facilitated astrogliogenesis, whereas silencing HIF delayed this process. Even transient changes in oxygen concentration affected cell fate through HIF by regulating the activity of MYC, a regulator of LIN28/let-7 that is critical for fate decisions in neural progenitors. Taking advantage of RNA-seq, metabolomics, and ChIP-seq techniques, we uncovered evidence of an early coherent response of neural progenitors to physiological oxygen at the transcriptional, metabolic and epigenetic levels. This response correlates with a persistent cell fate change, namely, the neuroepithelial cell (NEC) to radial glial cell (RGC) transition, which may contribute to astrogliogenesis upon differentiation. Taken together, physiological oxygen and HIF signaling play a key role in regulating cell fate decision in human neural progenitors. Furthermore, small molecules that activate HIF signaling can be simple tools to quickly and efficiently promote the development of mature cell types.

For an increasing number of cancers, the cell of origin has been demonstrated to be the

resident adult stem cell. One such cancer is squamous cell carcinoma, for which recent

studies in our lab traced its origin to the hair follicle stem cells. Malignant transformation

is thought to coincide with a dramatic shift towards the use of glycolysis and establishment

of a ‘Warburg’ state – increased metabolism of glucose to lactate. How the Warburg Effect

is established during tumor initiation and progression in vivo remains unclear. The current

consensus is that the bulk of the energy generated in most adult tissue cells is created by

oxidative phosphorylation, while more highly proliferative cells, such as activated immune

cells and cells transformed to make a tumor, mainly use glycolysis. Little is known

about how individual cell types generate energy in vivo, however, and how their

metabolism influences basic cell fate decisions such as cell division, migration or

differentiation. Using genetically engineered mouse models that allow the study of both tissue

homeostasis and the Warburg Effect in vivo, I have made important observations that

provide the basis for new investigations into the role of metabolism in key cell fate

decisions by adult stem cells. In this dissertation I present data indicating that hair follicle

stem cells possess a unique metabolic profile that may be critical for their maintenance

and for their response to oncogenic insults. Importantly, they suggest the possibility that

the “Warburg Effect” is the result of the expansion of an already glycolytic subpopulation,

namely the hair follicle stem cells.

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) are pluripotent stem cells (hPSCs) that have the capacity to differentiate into a panoply of various cell types and to serve as a model for human development. Nevertheless, it is unknown whether in vitro development of hPSCs mirrors in vivo human development. We performed comprehensive transcriptome profiling to compare hPSC-derived progeny with tissue-derived counterparts. While hESC and hiPSC make nearly identical progeny for the neural, hepatic and mesenchymal lineages, these hPSC-derived progeny maintained, regardless of the cell lineage, the expression of genes normally associated with early development, including LIN28A, LIN28B and DPPA4. These data and expression patterns in early human fetal tissue suggested the hPSC-derivatives were reflective of very early human development. The LIN28/let-7 pathway has been known to regulate development. We hypothesize that high Lin28 and low level let-7 activities may be inhibiting the developmental maturity in hPSC-derivatives. In addition, such a LIN28/let-7 expression pattern is also thought to contribute to tumor aggressiveness. We have established a human cell-based system to screen for small molecules that could modulate LIN28/let-7 activity. This screening could potentially unveil powerful small molecules that could modulate developmental maturity and therapeutically combat cancer. Previous reports and our own transcriptional profiling indicate that hPSCs are suitable for modeling early human development. Epithelial to mesenchymal transitions (EMTs) are thought to be essential to generate diversity of tissues during early fetal development, but these events are impossible to study at the molecular level in vivo in humans. The first EMT event in human development occurs just prior to generation of the primitive streak. Because hPSCs are thought to most closely resemble cells found in epiblast stage embryos prior to formation of the primitive streak, we sought to model this first human EMT in vitro with hPSCs. We have demonstrated the EMT progression in hPSCs with embryoid bodies. We also identified PTK7 as a marker of this EMT population, which was used to purify these cells for identification of novel markers of human germ layer development.

Human pluripotent stem cells (hPSCs) are derived from the developing blastocyst or through transcription factor based reprogramming. hPSCs have the capacity to self-renew and give rise to all cell types of the human body. These two defining characteristics make them attractive for a variety of uses including regenerative medicine and modeling human development and disease. While it is clear that hPSCs can differentiate into all cell types, it is unknown how similar hPSC derivatives are to cells that have undergone development in vivo. Using previously established differentiation protocols we demonstrated that both types of hPSCs could generate functional motor neurons that displayed repetitive action potentials characteristic of mature motor neurons. Furthermore, these motor neurons differentiated through a progressive and predictable induction of developmental stages, suggesting hPSC differentiation appears to obey normal developmental progression. In a comprehensive comparison of hPSC derivatives and tissue-derived counterparts, we determined that the progeny of hPSCs are most similar to a cell type found prior to 6 weeks of gestation. These hPSC derivatives continued to express genes normally found at 3-5 weeks of gestation, including LIN28; while tissue derived counterparts had already silenced these genes. In vitro function demonstrated that hPSC derivatives also acted like developmentally primitive cells. These findings suggest that developmental timing is conserved in vitro and perhaps even innate to the differentiating cell. Finally, we explored the role of the LIN28/let-7 pathway on the developmental maturity of hPSC-derived neural progenitor cells (hPSC-NPCs) as measured by their ability to generate neurons or glia. We determined that addition of let-7s into hPSC-NPCs can correct a significant number of the genes differentially expressed between hPSC derivatives and tissue-derived counterparts. Furthermore, the manipulation has a modest, but significant effect on the functional maturity of PSC-NPCs. Taken together, these data demonstrate that hPSCs can serve as an excellent and tractable model of human development.

Background

Aims

Materials and methods

Results

Conclusion