Skeletal muscle has a remarkable capacity for regeneration, mediated by muscle stem cells that can self-renew or differentiate to form the mature myofibers of the tissue. Several human diseases are characterized by a loss of function and strength in skeletal muscle, with impairments in the ability to regenerate and consequent decreases in quality of life and increases in mortality. The work in this dissertation has focused on developing methods for combating muscle disease. This goal has been approached through attempts at cell replacement therapy - by generating muscle cells that can be engrafted in vivo. I also investigated the influence on regeneration of the skeletal muscle microenvironment (skeletal muscle-resident fibroblasts), and systemic environment (inflammation in myogenic and non-myogenic tissues), both of which were found to affect skeletal muscle stem cell behavior and the efficiency of myogenic regeneration. Ultimately, these studies identified novel factors that impair or improve skeletal muscle differentiation, and that offer the potential to modulate the process of muscle regeneration. In the process of investigating if induced pluripotent stem cells from skeletal muscle retain an epigenetic memory conducive to myogenic differentiation, I discovered that precursor cells in skeletal muscle reprogram to a pluripotent state more efficiently. However, these induced pluripotent stem cells, like embryonic stem cells, retain strong barriers to skeletal muscle differentiation. Together, these findings offer insights into the process of muscle regeneration, and suggest new potential pathways towards treatment of muscle disease.

Medulloblastoma, the most common pediatric malignant brain tumor, is comprised of a heterogeneous group of tumors with distinct molecular subtypes and clinical outcomes. In particular, tumors with a cancer stem cell (CSC) population have been observed to be more resistant to conventional therapies, necessitating the elucidation of pathways important in this population. Work in our lab has shown that neurosphere culture-enriched cells from Ptch1LacZ/+;Trp53-/- mouse medulloblastomas exhibit properties of self-renewal, expression of neural stem cell (NSC) markers and potent tumor-initiation. The pathway dependencies and mechanisms of self-renewal in these medulloblastoma neurospheres (MBNS) have not yet been characterized.

Stem cell biologists creating cells and tissues for therapies, disease modeling, and drug screening have observed that differentiating pluripotent stem cells (PSCs) tend to produce cells at an embryonic stage of development but have difficulty maturing into adult definitive cells. A better understanding of developmental maturation will provide insights into embryogenesis and permit more accurate disease modeling. In the hematopoietic system, primitive and definitive cells are distinguished by functional transplantation assays, well characterized cell surface antigens, and gene expression signatures. We examined the transition in vivo in transplanted murine hematopoietic stem cells (HSCs) and in vitro in human PSC (hPSC) derived red blood cells (RBCs). We found that the hematopoietic microenvironment of the recipient significantly affects the outcome of HSC transplantation. The earliest embryonic HSCs perform better in neonatal recipients, whereas more mature adult-like HSCs perform better in adult recipients. The preference may be related to different active hematopoietic niches in neonates and adults, as we observed adult HSCs homing to different tissues in neonatal and adult recipients. Additionally, we found that proliferation may enhance the neonatal engraftment potential of adult-like HSCs. Our data highlight the importance of the host environment on transplantation outcomes, and point to the neonatal transplant model as a tool to functionally examine the earliest HSCs and primitive derivatives of PSCs.

Colony-stimulating factor 1 receptor (CSF1R, Fms) is an integral transmembrane glycoprotein with tyrosine specific protein kinase activity that it is found on the mononuclear phagocytes to promote their survival, proliferation and differentiation. Colony-stimulating factor 1 (CSF-1), also known as M-CSF, is a protein ligand that acts on the CSF1R. There is a variable association of Fms with the stem cell marker CD34 on acute myeloid leukemia (AML) cells and this suggests different structures of the AML hierarchy in different patients. Mouse stromal cells (MS-5) were transduced with a plasmid containing human CSF-1 because mouse CSF-1 is inactive on human CSF1R. Results show that AML cells cultured with CSF-1-expressing stroma had a much better growth and survival than the control stroma, suggesting that CSF-1 might be a stimulating factor for the growth of leukemic stem cells.

Pluripotent stem cells provide the opportunity to study human cardiogenesis in vitro, and are a renewable source of tissue for drug testing and disease models, including replacement cardiomyocytes that may be a useful treatment for heart failure. Typically, differentiation is initiated by forming spherical cell aggregates wherein an extraembryonic endoderm (ExE) layer develops on the surface. Given that interactions between endoderm and mesoderm influence embryonic cardiogenesis, we examined the impact of human embryonic stem cell (hESC) aggregate size on endoderm and cardiac development. We first demonstrated aggregate size control by micropatterning hESC colonies at defined diameters and transferring the colonies to suspension. The ratio of endoderm (GATA-6) to neural (PAX6) gene and protein expression increased with decreasing colony size. Subsequently, maximum mesoderm and cardiac induction occurred in larger aggregates when initiated with endoderm-biased hESCs (high GATA-6:PAX6), and in smaller aggregates when initiated with neural-biased hESCs (low GATA-6:PAX6). Additionally, incorporating micropatterned aggregates in a stirred suspension bioreactor increased cell yields and contracting aggregate frequency. We next interrogated the relationship between aggregate size and endoderm and cardiac differentiation efficiency in size-controlled aggregates, generated using forced aggregation, in defined cardiogenic medium. An inverse relationship between endoderm cell frequency (FoxA2+ and GATA6+) and aggregate size was observed, and cardiogenesis was maximized in mid-size aggregates (1000 cells) based on frequency of cardiac progenitors (~50% KDRlow/C-KITneg) on day 5 and cardiomyocytes (~24% cTnT+) on day 16. To elucidate a relationship between endoderm frequency and cardiac differentiation efficiency, aggregates were initiated with varying frequencies of ExE progenitors (SOX7-overexpressing hESCs). Maximum cardiomyocyte frequencies (~27%) occurred in aggregates formed with 10 to 25% ExE progenitors. These findings suggest a geometric relationship between aggregate size and ExE differentiation efficiency subsequently impacts cardiomyocyte yield, elucidating a mechanism for endogenous control of cell fate through cell-cell interactions in the aggregate.

Pluripotent stem cells provide the opportunity to study human cardiogenesis in vitro, and are a renewable source of tissue for drug testing and disease models, including replacement cardiomyocytes that may be a useful treatment for heart failure. Typically, differentiation is initiated by forming spherical cell aggregates wherein an extraembryonic endoderm (ExE) layer develops on the surface. Given that interactions between endoderm and mesoderm influence embryonic cardiogenesis, we examined the impact of human embryonic stem cell (hESC) aggregate size on endoderm and cardiac development. We first demonstrated aggregate size control by micropatterning hESC colonies at defined diameters and transferring the colonies to suspension. The ratio of endoderm (GATA-6) to neural (PAX6) gene and protein expression increased with decreasing colony size. Subsequently, maximum mesoderm and cardiac induction occurred in larger aggregates when initiated with endoderm-biased hESCs (high GATA-6:PAX6), and in smaller aggregates when initiated with neural-biased hESCs (low GATA-6:PAX6). Additionally, incorporating micropatterned aggregates in a stirred suspension bioreactor increased cell yields and contracting aggregate frequency. We next interrogated the relationship between aggregate size and endoderm and cardiac differentiation efficiency in size-controlled aggregates, generated using forced aggregation, in defined cardiogenic medium. An inverse relationship between endoderm cell frequency (FoxA2+ and GATA6+) and aggregate size was observed, and cardiogenesis was maximized in mid-size aggregates (1000 cells) based on frequency of cardiac progenitors (~50% KDRlow/C-KITneg) on day 5 and cardiomyocytes (~24% cTnT+) on day 16. To elucidate a relationship between endoderm frequency and cardiac differentiation efficiency, aggregates were initiated with varying frequencies of ExE progenitors (SOX7-overexpressing hESCs). Maximum cardiomyocyte frequencies (~27%) occurred in aggregates formed with 10 to 25% ExE progenitors. These findings suggest a geometric relationship between aggregate size and ExE differentiation efficiency subsequently impacts cardiomyocyte yield, elucidating a mechanism for endogenous control of cell fate through cell-cell interactions in the aggregate.

The loss of regenerative capacity of bone, from fetal to adult to aged animals, has been attributed not only to a decline in the function of cells involved in bone formation but also to alterations in the bone microenvironment that occur through development and aging, including extracellular matrix (ECM) composition and growth/trophic factor content. In the development of novel treatments for bone repair, one potential therapeutic goal is the restoration of a more regenerative microenvironment, as found during embryonic development. One approach to creating such a microenvironment is through the use of stem cells. In addition to serving as a differentiated cell source, pluripotent stem cells, such as embryonic stem cells (ESCs), may possess the unique potential to modulate tissue environments via local production of ECM and growth factors. ESC-produced factors may be harnessed and delivered to promote functional tissue regeneration. Such an approach to generate a naturally derived, acelluar therapy has been employed successfully to deliver osteoinductive factors found within adult bone, in the form of demineralized bone matrix (DBM), but the development of treatments derived instead from developing, more regenerative tissues or cells remains attractive. Furthermore, the derivation of regenerative materials from an ESC source also presents the added benefit of eliminating donor to donor variability of adult, cadaveric tissue derived materials, such as DBM. Thus, the objective of this project was to examine the osteoinductive potential harbored within the embryonic microenvironment, in vitro and in vivo. The osteogenic differentiation of mouse ESCs as embryoid bodies (EBs) was evaluated in response to phosphate treatment, in vitro, including osteoinductive growth factor production. The osteoinductivity of EB-derived material (EBM) was then compared to that of adult tissue-derived DBM, in vivo. Phosphate treatment enhanced osteogenic differentiation of EBs. EBM derived from phosphate treated EBs retained bioactive, osteoinductive factors and induced new bone formation, demonstrating that the microenvironment within osteogenic EBs can be harnessed in an acellular material to yield in vivo osteoinductivity. This work not only provides new insights into the dynamic microenvironments of differentiating stem cells but also establishes an approach for the development of an ESC-derived, tissue specific therapy.

We sought to determine whether activation of the Wnt signaling pathway altered the function of hNSCs in vitro. We took three approaches to activate Wnt signaling: Wnt3a, constitutively stabilized β-catenin (ΔN90), and the GSK3 inhibitor BIO. While Wnt3a and ΔN90 had no effect on proliferation in both stem cell (+EGF/FGF) and differentiating (-EGF/FGF) conditions, BIO reduced proliferation in both. All methods of Wnt signaling activation promoted neuronal lineage commitment during hNSC differentiation. Furthermore, BIO was able to induce mild neuronal differentiation in stem cell conditions, suggesting that GSK3-inhibition interferes with several pathways to regulate hNSC fate decisions. We also probed BTSC function using BIO-mediated GSK3 inhibition. We found that in stem cell conditions, BIO was able to induce neuronal differentiation, decrease proliferation, and induce cell cycle arrest. Together this data suggests that GSK3-inhibition, possibly through activation of Wnt signaling, may offer a novel mechanism for the differentiation treatment of glioblastomas.

We sought to determine whether activation of the Wnt signaling pathway altered the function of hNSCs in vitro. We took three approaches to activate Wnt signaling: Wnt3a, constitutively stabilized β-catenin (ΔN90), and the GSK3 inhibitor BIO. While Wnt3a and ΔN90 had no effect on proliferation in both stem cell (+EGF/FGF) and differentiating (-EGF/FGF) conditions, BIO reduced proliferation in both. All methods of Wnt signaling activation promoted neuronal lineage commitment during hNSC differentiation. Furthermore, BIO was able to induce mild neuronal differentiation in stem cell conditions, suggesting that GSK3-inhibition interferes with several pathways to regulate hNSC fate decisions. We also probed BTSC function using BIO-mediated GSK3 inhibition. We found that in stem cell conditions, BIO was able to induce neuronal differentiation, decrease proliferation, and induce cell cycle arrest. Together this data suggests that GSK3-inhibition, possibly through activation of Wnt signaling, may offer a novel mechanism for the differentiation treatment of glioblastomas.

Derivation of lung epithelial cells from stem cells remains a challenging task, due in part to a lack of understanding of the molecular mediators driving commitment of endoderm to an early lung lineage. Reciprocal signalling between the lung mesenchyme and epithelium is crucial for proper differentiation and branching morphogenesis to occur. We hypothesized that the combination of signalling pathways comprising early epithelial-mesenchymal interactions and the 3-D spatial environment are required for induction of embryonic and induced pluripotent stem cells (ESC and iPSC, respectively) into a lung cell phenotype with the hallmarks of the distal niche. Aggregating early lung mesenchyme with endoderm-induced ESC and iPSC resulted in differentiation to an NKX2.1 and pro-SFTPC positive lineage. The differentiating cells organized into tubular structures and became polarized epithelial cells. Ultrastructure analysis revealed precursors of lamellar bodies, and Sftpb mRNA expression was detected. Quantification of the differentiation using an Nkx2.1-reporter ESC line revealed that 80% were committed to an early lung lineage, a vast improvement over what has previously been published. The FGF growth factor family comprises well-known mediators of growth and differentiation during the development of many organs, including the lung. We found that FGF2 signalling through the FGFR2iiic receptor isoform was mediating the commitment of the stem cells to an early lung epithelial phenotype, as defined by NKX2.1/proSFTPC expression. FGF7 signalling through the FGFR2iiib receptor was found to be important for the maturation and morphogenesis of the NKX2.1/proSFTPC positive lineage, but did not play a role in the initial commitment. The addition of FGF2 to endoderm-induced ESC or iPSC in the absence of mesenchyme was able to commit the cells to an NKX2.1-positive lineage, but no proSFTPC was detected. Furthermore,the cells did not become polarized and no longer organized into tubular structures. These findings suggest that while FGF2 is important for initial commitment, additional mesenchyme components including matrix proteins, supporting cell lineages and other growth factors are crucial for an efficient differentiation to an early lung epithelial cell lineage.