Browsing by Subject "Regeneration"

Tissue-engineered skeletal muscle presents promising opportunities for developing high-fidelity in vitro models for investigating human muscle biology in the areas of regeneration and disease. In muscle regeneration, satellite cells (SCs) are essential for new muscle fiber formation; however, they lose their native quiescent state upon isolation, making in vitro studies of human SC function challenging. To optimally promote SC quiescence and enable exploration of SC dynamics in vitro, engineered muscle needs to recapitulate the native muscle microenvironment, which is comprised of muscle fibers, extracellular matrix, and other biochemical and mechanical cues. In disease modeling, mechanistic studies and therapeutic development are still extensively evaluated in animal models, which have limited translational relevance to patients. Specifically, Pompe disease is caused by a variety of mutations in the lysosomal enzyme acid alpha-glucosidase (GAA) that varyingly affect residual GAA activity and cannot be captured in the current GAA-/- mouse model. Therefore, human in vitro models are needed to enhance our mechanistic understanding of diseases and stimulate the development of effective therapies. To overcome these limitations, we set the dissertation goals to: 1) generate a pool of quiescent SCs and explore the mechanisms governing their formation and activation using an engineered skeletal muscle microenvironment and 2) develop a high-fidelity tissue-engineered skeletal muscle model for Pompe disease to investigate pathological mechanisms and test candidate therapies. To achieve these goals, we first compared methods for primary human myoblast expansion and found that p38 inhibition significantly increases the formation of Pax7+ cells in engineered 3D skeletal muscle tissues (“myobundles”). Gene expression analysis suggested that within the myobundle environment the Pax7+ cells adopt a quiescent phenotype (3D SCs), characterized by increased Pax7 expression, cell cycle exit, and Notch signaling activation relative to the original 2D expanded myoblasts. We then compared 3D SCs to previously described satellite-like cells that form alongside myotubes in 2D culture, termed reserve cells (RCs). Compared to RCs, 3D SCs showed an advanced quiescent phenotype characterized by a higher Pax7, Spry1, and Notch3 expression, as well as increased functional myogenesis demonstrated by formation of myobundles with higher contractile strength. To examine 3D SC activation, we tested several myobundle injury methods and identified treatment with a bee toxin, melittin, to robustly induce myofiber fragmentation, functional decline, and 3D SC proliferation. To further investigate the transcriptional processes describing how 2D myoblasts acquire 3D SC phenotype (i.e. deactivate) and how 3D SCs respond to injury (i.e. reactivate), we applied single cell RNA-sequencing (scRNA-seq) from which we discovered the existence of two subpools of 3D SCs—“quiescent” (qSC) and “activated” (aSC). The qSC subpool possessed greater expression of quiescence genes Pax7, Spry1, and Hey1, whereas the aSC subpool exhibited increased expression of inflammatory and differentiation markers. Furthermore, we performed trajectory inference along the deactivation process from 2D myoblasts to qSCs and identified deactivation-associated genes, included downregulated genes for proliferation, cytoskeletal reorganization, and myogenic differentiation. In response to tissue injury, we observed a decrease in the proportion of qSCs and an increase in the proportion of aSCs and committed myogenic progenitor cells suggestive of myogenic differentiation. In addition, we observed transcriptional changes within the aSC population reflective of SC activation in vivo, namely increased TNF- signaling, proliferation, and glycolytic and oxidative metabolism. These results strongly suggested that 3D SC heterogeneity and function recapitulate several aspects of native human SCs and could be applied to study human muscle regeneration and disease-associated SC dysfunction. To evaluate the myobundle system in the context of disease modeling, we developed the first 3D tissue-engineered skeletal muscle model of infantile onset Pompe disease (IOPD), the most severe form of Pompe disease. Diseased myobundles demonstrated characteristic GAA enzyme deficiency, accumulation of the GAA target glycogen, and lysosome enlargement. Despite exhibiting these key biochemical and structural hallmarks of disease, IOPD myobundles did not show deficits in contractile force generation or autophagic buildup. We therefore identified metabolic stress conditions that acutely targeted disease-associated abnormalities in the lysosomes and glycogen metabolism, which revealed impairments in contractile function and glycogen mobilization. To further elucidate the biological mechanisms underlying the phenotype of IOPD myobundles, we applied RNA sequencing (RNA-seq) and observed enrichment for terms consistent with Pompe disease phenotype including downregulation of gene sets involved in muscle contraction, increased endoplasmic reticulum stress, and reduced utilization of specific metabolic pathways. We then compared the transcriptomic profiles of GAA-/-¬ and wild-type mice to identify a Pompe disease signature and confirmed the presence of this signature in IOPD myobundles. Finally, treating IOPD myobundles with clinically used recombinant protein (rhGAA) therapy resulted in increased GAA activity, glycogen clearance, and a partial reversal of the disease signature, further confirming the utility of the myobundle system for studies of Pompe disease and therapy. In summary, this dissertation describes novel strategies for the formation and characterization of quiescent human SCs using the myobundle system. We present first-time application of scRNA-seq to engineered skeletal muscle, and uncover transcriptional descriptors of human myoblast deactivation and SC heterogeneity and activation. When utilizing human myobundles as a novel model of Pompe disease, we identified disease hallmarks and responses to therapy consistent with observations in Pompe patients. We anticipate that the findings and methods developed in this work will serve as a useful framework for the future engineering of regenerative human muscle for therapeutic and disease modeling applications.

The homeostatic hematopoietic stem cell compartment is comprised of quiescent long term self renewing stem cells and cycling short term stem cells with finite renewal potential. To study the molecular mechanisms governing self renewal of hematopoietic cells we must force them to enter the cell cycle and proliferate. One approach to accomplish this goal is to damage the hematopoietic compartment with ionizing radiation or cytotoxic chemotherapy. Such injuries ablate mature blood cells and drive the primitive stem cells into cycle. I have elected to use a simple model of hematopoietic damage and regeneration to study the molecular mechanisms controlling self renewal in hematopoietic stem cells. At the beginning of this project it was unclear whether the signaling pathways which homeostatically control self renewal are utilized during injury repair. In particular, there is very little understanding of the signals required for regeneration after radiation damage. We hypothesized extracellular signal transduction pathways provided by the microenvironment are critical mediators of the stem cell repair process. To address these topics and extend the previous work generated in our laboratory, I chose to pursue a candidate approach focusing on the Wnt and Notch developmental signaling pathways.

In order to examine the activation and requirement for each signaling cascade after radiation and chemotherapy damage we used a combination of loss of function and reporter mouse models. To this end, we have conducted the majority of experiments for the Wnt project in animals deficient in beta-catenin, the key transcription factor required in the pathway. Our investigations revealed the Wnt pathway is turned on within regenerating stem cells and loss of beta-catenin impairs regeneration of the stem cell compartment after both radiation and chemotherapy injury.

Using a Transgenic Notch Reporter mouse to investigate the role of Notch signaling following hematopoietic damage we determined the Notch pathway is also activated during regeneration. Furthermore, using a live imaging approach we discovered Notch activated cells change their fate choice during regeneration. To determine if Notch gain of function provides radio-protection we infected stem cells with an active form of Notch prior to radiation and then scored self renewal potential in vitro. This led us to the conclusion that Notch gain of function can provide a self renewal benefit to irradiated hematopoietic stem cells.

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Lung epithelium, the lining that covers the inner surface of the respiratory tract, is directly exposed to the environment and thus susceptible to airborne toxins, irritants, and pathogen-induced damages. In adult mammalian lungs, epithelial cells are generally quiescent but can respond rapidly to repair damaged tissues. Evidence from experimental injury models in rodents and human clinical samples has led to the identification of these regenerative cells, as well as pathological metaplastic states specifically associated with different forms of damages. The primary alveolar stem cell, alveolar type-2 cells (AT2s) are sparsely distributed and make up only 5% of the surface area. Despite this organization, AT2s are still able to maintain tissue homeostasis and achieve efficient repair after injury. However, the underlying mechanisms of stem cell activation, injury response, and subsequent cell-cell communication signals mediating resolution of injury and restoration of alveolar homeostasis remain elusive. Additionally, modulation of these regenerative cells for therapeutic potential has not been well established, primarily due to a lack of viable gene editing tools and vehicles for gene delivery to the alveolar stem cells, and neighboring niche. First, to better target the lung alveolus, we screened and identified cell-type specific adeno-associated virus (AAV) serotypes, enabling efficient targeting and gene expression of exogenous genes in alveolar stem cells as well neighboring mesenchyme. These tools were also capable for both in vitro and in vivo gene editing, forgoing the need for development of complex genetic mouse models as well as enabling diverse, viral-based screens. Second, using 3-dimensional, thick tissue imaging we reveal that a single AT2 cell can enface multiple alveolar compartments by virtue of a unique, multi-apical domain architecture. Lineage tracing and live imaging coupled with genetic and AAV-mediated selective ablation of AT2s was used to show robust recovery of AT2 numbers and distribution via clonal proliferation and migration, even after three successive rounds of ablation. Clonal tracing revealed that a single AT2 can differentiate to cover multiple alveolar cups. During injury repair, AT2s dynamically reorganize their apical domains to facilitate either migration or differentiation. Single- cell transcriptome profiling, genetic and pharmacologic disruption of actin dynamics, and evaluation of multiple physiologically relevant disease states identified the roles of actin cytoskeleton, cell migration, and multi-apical domains in AT2 recovery and regenerative potency. Lastly, using cell-type specific ablation of alveolar type 1 cells (AT1s), we identified novel mechanisms of epithelium-mediated signaling to mesenchymal fibroblasts, thereby uncovering novel mechanisms of fibrosis initiation and progression. Modulation of AT1 ablation dynamics preferentially drives fibrosis or, in contrast, emphysema, both at histological and physiological levels, as assessed by whole body plethysmography. Single-cell sequencing identified the epithelial and mesenchymal cell identities involved in regenerative processes, as well as identification of a PDGFA signaling axis between AT1s and resident alveolar fibroblasts necessary for fibroblast maintenance. We demonstrate that modulation of these signaling pathways during lung regeneration could enhance fibrosis or convert fibrosis to emphysema. In sum, the work presented herein both develops functional tools for perturbation of alveolar stem cells, as well as an improved understanding of alveolar architecture, stem cell dynamics during injury repair, homeostatic intercellular signaling, and mechanisms of disease progression.

Regeneration is a remarkable feat of developmental regrowth and patterning. The blastema is a mass of progenitor cells that enables complete regeneration of amputated salamander limbs or fish fins. Despite years of study, methodologies to identify and track blastemal cell progenies have been deficient, restricting our understanding of appendage regeneration at a cellular and molecular level. To bridge this knowledge gap, gene expression analysis, the generation of transgenic and mutant zebrafish, qualitative and quantitative analyses, morphological measurements, and chemical treatments were used to assess molecular and cellular processes involved in fin regeneration. Two main findings arose from these methods. The first provides evidence that connective tissue progenitors are rapidly organized into a scalable blueprint of lost structures, and that amputation stimulates resident cells to reset proximodistal positional information. The second identifies a fibroblast subpopulation near uninjured fin joints that contributes to the blastemal progenitor population. These findings reveal insights on cellular and molecular mechanisms of appendage regeneration and provide a basis for work exploring how cells in an adult vertebrate bone appendage coordinately rebuild a new structure.

The ability to regenerate after injury is quite astonishing, yet not all organisms share this ability. Mammalian genomes likely encode all gene products required to regenerate an amputated limb, yet they lack the correct instructions for strategically modulating those gene products to accomplish limb regeneration. While the catalogue of defined cell dynamics and molecular factors in tissue regeneration is expanding, we know comparatively little of how genes involved in regenerative events are engaged upon injury, despite decades of research. Certain non-mammalian vertebrates like salamanders and zebrafish possess these instructions, which exist as cis-regulatory elements that can direct expression of their target genes during regeneration. To identify candidate tissue regeneration enhancer elements (TREEs) important for zebrafish fin regeneration, we performed ATAC-seq from bulk tissue or purified fibroblasts of uninjured and regenerating caudal fins. We identified tens of thousands of DNA regions from each sample type with dynamic accessibility during regeneration, and assigned these regions to proximal genes with corresponding changes in expression by RNA-seq. To determine the extent to which these profiles reveal bona fide TREEs, we tested the sufficiency and requirements of several sequences in stable transgenic lines and mutant lines with homozygous deletions. Our study generates a new resource for dissecting the regulatory mechanisms of appendage generation and reveals a range of requirements for individual TREEs in control of regeneration programs.

The success of muscle development and regeneration requires cooperation from both myogenic and their supportive niche cells. The muscular niche is complex. At the cellular level it is composed of a broad number of cell types including: endothelial vessels, nerve and nerve-supporting cells, resident immune populations, and a heterogenous group of non-myogenic mesenchymal cells. The non-myogenic mesenchymal cells include pericytes, vascular smooth muscle cells, interstitial tenocyte-like cells, and fibro-adipogenic progenitors (FAPs). Like all members of the muscular niche, this fraction is vital to muscle development and regeneration. Despite their importance to muscle development, regeneration, and homeostasis, detailed identities within non-myogenic mesenchymal cells remain elusive. By understanding the distinct makeup of this population, we can provide a foundation to examine their important regulatory roles in the processes of muscle development, homeostasis, injury and disease.

This thesis utilizes single cell RNA sequencing to establish the populations of non-myogenic mesenchymal cells in developing muscle. Our analysis identified pericytes, vascular smooth muscle cells, and tenocyte-like cell populations while uncovering a new level of heterogeneity in FAPs that not previously appreciated. Despite classical understanding of FAPs as one group, this work found that FAPs were sub-divided into five distinct populations, which compose two trajectories spawning from a common progenitor. This thesis defines the functional differences of each FAP population through a series of experiments including: fluorescence activated cell sorting of various FAP groups, studying their spatial localization on immunofluorescence, and testing the response of different FAPs to multiple injury and disease models.

Separate preliminary work examines the impact of NOTCH signaling in FAPs and the broader non-myogenic mesenchymal cell groups. These studies discovered that NOTCH signaling in the non-myogenic mesenchymal group, but not FAPs specifically, regulates muscular growth and intramuscular adipogenesis. Altogether this thesis advances our understanding of the identity, and role of, non-myogenic mesenchymal cells, in muscle development and regeneration.

Regeneration occurs throughout the animal kingdom and is a well-studied

feature of many model organisms, yet the field lacks a fundamental understanding of

the real-time dynamics of cell behavior during regeneration. I discuss how existing

knowledge of regeneration may be used to inform efforts to translate these remarkable

feats of animals to human regeneration and present research that uses live imaging to

improve understanding of cell origins and diversification during regeneration in the

scale, focusing specifically on osteoblasts the matrix-depositing cells that divide and heal

bone injuries. I developed an imaging platform to monitor and quantify individual and

collective behaviors of osteoblasts in adult zebrafish scales. I show that a founder pool

of osteoblasts emerges through de novo differentiation within one day of scale plucking,

then diversifies across the primordium by two days after injury, with region-specific

changes in proliferation, cell shape, and cell death rates coincident with acquisition of

mature scale morphology. I also demonstrate a role for Fgf signaling in scale

regeneration and present tools for high resolution imaging studies of basal epidermal

cells during skin and scale injury. These findings demonstrate the value of live imaging

in revealing novel biology and gaining a more complete picture of the many complex

processes that must be elegantly choreographed to achieve tissue regeneration.

Zebrafish possess the remarkable capacity to regenerate from spinal cord injuries that would leave mammals such as humans permanently paralyzed. Much research into zebrafish spinal cord regeneration has focused on identifying extracellular growth factors and matrix components which create a pro-regenerative environment; however, it is just as important to identify and understand the transcription factors which control pro-regenerative transcriptional responses within the resident stem cell population of the spinal cord, and the signaling cascades which translate the known extracellular ligands into cellular responses. Using CRISPR/Cas9, we generated two novel transcription factor knockout zebrafish lines which we tested for spinal cord regeneration defects and found no difference in regenerative capacity. Using a chemical screen, we identify JNK signaling as a necessary regulator of glial cell cycling and tissue bridging during spinal cord regeneration in larval zebrafish. With a kinase translocation reporter, we visualize and quantify JNK signaling dynamics at single-cell resolution in glial cell populations in developing larvae and during injury-induced regeneration. Glial JNK signaling is patterned in time and space during development and regeneration, decreasing globally as the tissue matures and increasing in the rostral cord stump upon transection injury. Thus, we present a tool to visualize signaling activity in the larval zebrafish spinal cord and demonstrate that dynamic JNK activity after spinal cord injury directs a proliferative response of glial cells during spinal cord regeneration.

Zebrafish regenerate amputated caudal fins, restoring the size and shape of the original appendage. Regeneration requires generation of diverse cell types comprising the adult fin tissue. Knowledge of the cellular source of new cells and the molecules involved is fundamental to our understanding of regenerative responses. In this dissertation, the contribution made by the bone cells towards fin regeneration is investigated. Fate mapping of osteoblasts revealed that spared osteoblasts contribute only to regenerating osteoblasts and not to other cell types, thereby suggesting lineage restriction during fin regeneration. The functional significance of osteoblast contribution to fin regeneration is tested by developing an osteoblast ablation tool capable of drug induced loss of bone cells. Normal fin regeneration in the absence of resident osteoblast population suggests that the osteoblast contribution is dispensable and provides evidence for cellular plasticity during fin regeneration. To uncover the genes involved in proliferation of osteoblasts within the fin regenerate, a candidate in-situ screen was carried out and revealed bone specific expression of fgfr4 and twist3. Transgenic tools for visualization of gene expression confirmed the screen results. Knockdown of twist3 by morpholino antisense technology impedes fin regeneration. Mutant heterozygotes for twist3 were generated using genome editing reagents, which will enable loss-of-function study in future.