Browsing by Subject "Differentiation"

The intervertebral discs (IVD) contribute to structural stability of the spinal column, attenuate the impact of compressive loads, and enable a wide spectrum of motions. As a consequence of aging, the majority of the adult population experiences painful symptoms associated with IVD degeneration - a condition characterized by diminished integrity of tissue components. Current treatment options unfortunately cannot restore IVD structure and function. At the present, an avenue of great interest involves autologous or allogeneic cell delivery to the degenerated IVD. Induced pluripotent stem cells (iPSCs) have demonstrated their capacity to differentiate into various cell types. A posited strategy for regenerative medicine applications entails deriving iPSCs from a patient's own somatic cells and directing them toward a specific lineage.

The overall objective of this study is to assess the potential of mouse iPSCs to regenerate nucleus pulposus (NP) cells of the IVD. Previous work identified CD24 as an NP marker, while recent data from our lab noted its expression in mouse iPSCs. The first portion of this thesis employed magnetic activated cell sorting (MACS) to isolate a CD24high iPSC population. Notochordal gene expression was analyzed in this undifferentiated cell fraction via real time RT-PCR. Mouse iPSCs were then cultured in a laminin-rich, 3D culture system for up to 28 days, and NP phenotype was assessed by immunostaining.

The latter half of this work focused on producing a more conducive environment for NP differentiation of mouse iPSCs. This involved the addition of low oxygen tension and notochordal conditioned medium (NCCM) to the culture platform. Mouse iPSCs were evaluated for ability to adopt an NP-like phenotype through a combination of immunostaining and biochemical assays. Furthermore, they were compared to NIH 3T3 mouse embryonic fibroblasts cultured under the same conditions.

Results demonstrated that a CD24high fraction of mouse iPSCs could be successfully retrieved and differentiated into a population that could synthesize matrix components similar to that in native NP. Likewise, the addition of hypoxia and NCCM generated similar phenotypic results. 3T3 fibroblasts unexpectedly exhibited transdifferentiation potential as well. Altogether, these studies conclude that mouse iPSCs do have potential to differentiate into NP-like cells and may be applied to future cell-based therapies for restoration of the degenerated IVD.

Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the embyronal tumor neuroblastoma, where differentiation is a validated treatment strategy, remain unclear. The neuroblastoma tumor stroma is thought to suppress neuroblast growth via release of soluble differentiating factors. Here we identify critical components of the differentiating stroma secretome and describe preclinical testing of a novel therapeutic strategy based on their mechanism of action.

Expression of heparan sulfate proteoglycans (HSPGs), including TβRIII, GPC1, GPC3, SDC3, and SDC4, is decreased in neuroblastoma, high in the stroma, and suppresses tumor growth. High expression of TβRIII, GPC1, and SDC3 is associated with improved patient prognosis. HSPGs signal via heparan sulfate binding to FGFR1 and FGF2, which leads to phosphorylation of FGFR1 and Erk MAPK, and upregulation of the transcription factor inhibitor of DNA binding 1 (Id1). Surface expression and treatment with soluble HSPGs promotes neuroblast differentiation via this signaling complex. Expression of individual HSPGs positively correlates with Id1 expression in neuroblastoma patient samples and multivariate regression demonstrates that expression of HSPGs as a group positively correlates with Id1 expression, underscoring the clinical relevance of this pathway. HSPGs also enhance differentiation from FGF2 released by the stroma and FGF2 is identified as a potential serum prognostic biomarker in neuroblastoma patients.

The anticoagulant heparin has similar differentiating effects to HSPGs, decreasing neuroblast proliferation and reducing tumor growth while extending survival in an orthotopic xenograft model of neuroblastoma. Dissection of individual sulfation sites identifies 2-O-, 3-O-de-sulfated heparin (ODSH) as a differentiating agent that suppresses orthotopic xenograft growth and metastasis in two models while avoiding anticoagulation. These studies form the preclinical rationale for a multicenter clinical trial currently being proposed.

In conclusion, these studies translate mechanistic insights in neuroblast HSPG function to identify heparins as differentiating agents for clinical development in neuroblastoma, while demonstrating that tumor stroma biology can inform design of targeted molecular therapeutics.

As a major component of the adaptive immune system, CD4+ T cells play a vital role in host defense and immune tolerance. The potency and accuracy of CD4+ T cell-mediated protection lie in their ability to differentiate into distinct subsets that could carry out unique duties. In this dissertation, we dissected the roles and interplays between two emerging mechanisms, miRNAs and epigenetic processes, in regulating CD4+ T cell-mediated responses. Using both gain- and loss-of-function genetic tools, we demonstrated that a miRNA cluster, miR-17-92, is critical to promote Th1 responses and suppress inducible Treg differentiation. Mechanistically, we found that through targeting Pten, miR-17-92 promotes PI3K activation. Strong TCR-PI3K activation leads to the accumulation of DNMT1, elevated CpG methylation in the foxp3 promoter, and suppression of foxp3 transcription. Furthermore, we demonstrated that an epigenetic regulator, methyl CpG binding protein 2 (MeCP2), is critical to sustain Foxp3 expression in Tregs, and to support Th1 and Th17 differentiation in conventional CD4+ T cells (Tcons). In Tregs, MeCP2 directly binds to the CNS2 region of foxp3 locus to promote its local histone H3 acetylation; while in Tcons, MeCP2 enhances the locus accessibility and transcription of miR-124, which negatively controls SOCS5 translation to support STAT1, STAT3 activation and Th1, Th17 differentiation. Overall, miRNAs and epigenetic processes may crosstalk to control CD4+ T cell differentiation and function.

A cell’s trajectory from stem cell to differentiation, while often portrayed as a linear progression, is best described as a network that produces a mature state through several pathways acting together. There are few examples that describe gene regulatory network changes during the entire trajectory of cell differentiation. The goal of my project was to define the gene regulatory network required for a stem cell to become a differentiated cell in the Arabidopsis thaliana root. The root is a powerful model for identifying basic principles of differentiation. Plant cells do not migrate therefore entire lineages from stem cell to mature progeny are spatially confined. Furthermore, the root displays indeterminate growth, facilitating the study of many different developmental stages at a single time. One cell type of the root, the endodermis, is particularly suitable for study because the molecular components required for its formation and terminal differentiation are established. In order to understand the path from stem cell to differentiated cell in the endodermis, we asked what transcription factors are sufficient to program a non-native cell-type into endodermis. Our results show the transcription factors SHORTROOT and MYB36 have limited ability to reprogram a non-native cell-type (the epidermis) and that this reprogramming is reversible in the absence of additional cues. The stele-derived signaling peptide CIF2 stabilizes SHORTROOT-induced reprogramming. The outcome is a partially impermeable barrier deposited in the sub-epidermal cell layer that has a transcriptional signature similar to endodermis. The induction mechanism depends on MYB36 and CIF2’s receptor, but may be independent of the transcription factor SCARECROW. These results highlight a non cell-autonomous induction mechanism for endodermis that resembles differentiation in many animal systems.

Changes in gene transcription driven by the activation of intracellular calcium signaling pathways play an important role in neural development and plasticity. A growing body of evidence suggests that stimulus-driven modulation of histone modifications play an important role in the regulation of neuronal activity-regulated gene transcription. However, the histone modifying enzymes that are targets of activity-regulated signaling cascades in neurons remain to be identified. The histone demethylases (HDMs) are a large family of enzymes that have selective catalytic activity against specific sites of histone methylation. To identify HDMs that may be important for activity-regulated gene transcription in neurons, we induced seizures in mice and screened for HDMs whose expression is induced in the hippocampus. Among the few HDMs that changed expression, Kdm6b showed the highest induction. Kdm6B is a histone H3K27-specific HDM whose enzymatic activity leads to transcriptionally permissive chromatin environments. In situ hybridization analysis revealed that Kdm6b is highly induced in post-mitotic neurons of the dentate gyrus region of the hippocampus. We can recapitulate the activity-dependent induction of Kdm6b expression in cultured hippocampal neurons by application of Bicuculline, a GABAA receptor antagonist that leads to synaptic NMDA receptor activation and calcium influx. Kdm6b expression is also induced following application of BDNF, a neurotrophic factor that is upregulated in the seized hippocampus. To investigate possible functions of Kdm6b in neuronal development, we performed in situ hybridization analysis that allows for the identification of regions with high Kdm6b expression that could be sites of potential function in the developing mouse brain. We found high levels of Kdm6b expression in the inner layer of the external granule layer of the cerebellum, a region where pre-migratory immature neurons reside and a site of significant apoptosis. On the basis of this data and the fact that intracellular calcium signaling arising from synaptic firing supports neuronal survival, we explored the necessity for Kdm6b in the survival of cultured cerebellar granule cells. Knock down of Kdm6b by RNAi increases cell death, demonstrating that Kdm6b contributes to neuronal survival. Ongoing experiments are addressing the role of Kdm6b in neuronal differentiation. Overall these data raise the possibility that stimulus-dependent regulation of Kdm6b, and perhaps regulation of H3K27 methylation mediated by Kdm6b, may contribute to the regulation of gene expression in neurons and thus to their proper development and plasticity.

Over 1.5 billion individuals are infected with intestinal helminths worldwide, with a majority of those infected living in developing nations. In developed nations intestinal helminth infections are very rare, while incidences of allergy and asthma are common. The incidence of allergic afflictions is growing rapidly every year. Both clearance of helminth infections and propagation of allergic disease are mediated by type 2 immune responses. The cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13) play major roles in the propagation of type 2 immune responses. IL-4 and IL-13 are produced by a number of immune cells, within both the innate and adaptive arms of immunity, that are important in driving allergic responses.

CD4+ T follicular helper (Tfh) cells reside in the B-cell follicle and specialize in aiding the maturation of germinal center (GC) B cells. IL-4 produced by Tfh cells is required for GC B cell Immunoglobulin (Ig) class switching to type-2 isotypes, IgE and IgG1. IgE serves as a critical mediator of type-2 immune responses. CD4+ T helper 2 (Th2) cells localize to the periphery at sites of infection and damage. Th2 cells make both IL-4 and IL-13 cytokines. Th2 cells, along with multiple innate cell types, are critical for driving the peripheral hallmarks of type-2 immune responses, including mucus production and smooth muscle contractility.

Elucidating the pathways that regulate the differentiation, function, and maintenance of Tfh and Th2 cells is critically important for discovering potential therapies for allergic disease and helminth infections. Notch signaling is capable of driving Th2 IL-4 production and differentiation in vitro. However, the in vivo role for Notch signaling in Th2 populations remains unclear. The mechanisms controlling Tfh IL-4 production are largely unknown. Given that Notch signaling is required for the differentiation of Tfh cells and is known to influence cytokine production in T cells, we hypothesized that Notch signaling also plays an important role in regulating the function of Tfh cells. Nippostrongylus brasiliensis infection drives a robust type-2 immune response and allows for analysis of both Tfh and Th2 cells. Here, infection with N. brasiliensis was used to characterize whether Notch signaling is required for Th2 and Tfh differentiation and function in vivo.

Deletion of Notch receptors on T cells of infected mice results in reduced IL-4 producing Tfh, but not Th2 cells. As a result, we saw impairments in overall Tfh functionality while peripheral Th2 immunity remained intact. Notch deficient T cells had major impairments in Tfh, but not Th2, cell differentiation. Overexpression of Notch signaling in CD4+ T cells leads to increased IL-4 production by Tfh cells, but not Th2 cells. Furthermore, we identified that conventional dendritic cells (cDCs) do play a role as sources for Notch ligand early during the immune response. However, neither cDCs or follicular dendritic cells (FDCs) are essential sources of Notch ligand to drive Tfh cell differentiation.

While Notch signaling is critical for Tfh differentiation, it is not known if Notch signaling plays a continued role beyond Tfh differentiation. We used pharmacologic inhibition of Notch signaling to assess a role for Notch in Tfh maintenance. Here, we show that inhibition of Notch signaling after Tfh differentiation results in altered expression and activity of important trafficking receptors. This change was accompanied by aberrant localization of IL-4 expressing T cells in the lymph node. Additionally, late Notch inhibition resulted in an altered transcriptional program in Tfh cells. These findings suggest that Notch signaling plays a critical role in Tfh, but not Th2 driven immunity. In total, the data shown here demonstrate that Notch signaling is not only important for Tfh differentiation, but also for regulating Tfh cell fate, function, and maintenance.

Neuroblastoma is a pediatric cancer arising from undifferentiated neural crest-derived precursor cells. Treatment strategies for neuroblastoma aim to promote neuroblast differentiation, however current therapies available are only modestly effective. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that act to promote neuroblast differentiation, though the precise factors released and their mechanism of action in neuroblastoma remains unclear. Here, we identify a novel component of the differentiating stroma secretome and harness stroma biology to inform the use of a combination therapy for neuroblastoma treatment.

HBEGF expression is decreased in neuroblastoma compared to benign disease, correlating to an increase in mortality. HBEGF protein is expressed only in stromal compartments of tumor specimens, with tissue from late-stage disease containing very little stroma or HBEGF. Addition of soluble HBEGF to neuroblastoma cell lines leads to increased neuroblast differentiation and decreased proliferation. Heparan sulfate proteoglycans (HSPGs) and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor (EGFR), leading to activation of the ERK1/2 and STAT3 pathways and upregulation of the inhibitor of DNA binding 1 transcription factor.

Expression of the type III TGF-β receptor (TβRIII), an HSPG, is epigenetically regulated in neuroblastoma cells via direct binding of the N-Myc transcription factor to Sp-1 sites on the TβRIII promoter. Analysis of patient microarray data demonstrate that other members of the differentiating stroma secretome, including HBEGF and EGFR, are positively correlated with TβRIII expression, suggesting that these proteins may be co-regulated. Treatment with inhibitors aimed at blocking N-Myc function, including inhibitors of histone deacetylases, DNA methyltransferases (DNMTs), and aurora kinase A (AurkA) can promote neuroblast differentiation and decrease proliferation. The combination of the DNMT inhibitor decitabine with the AurkA inhibitor MLN8237 enhances differentiation and reduces proliferation compared to either agent alone. Importantly, the combination of clinically achievable doses of these targeted agents dramatically reduces tumor growth in orthotopic xenograft models of neuroblastoma, identifying a novel combination therapy that may benefit children with this disease.

In conclusion, these studies dissect the tumor microenvironment to identify an important member of the differentiating stroma secretome, while also revealing a combination therapy for clinical development that has the potential to decrease adverse side effects and increase effectiveness of neuroblastoma treatment.