Type 1 Diabetes (T1D) is an autoimmune disorder in which the pancreatic β-cells are destroyed by the body's immune system. The reduced number of β-cells leads to inadequate insulin secretion and high glucose levels in the body. The requirement of insulin injection throughout life and lack of donors for islet transplantations has prompted a search for more accessible and available sources of insulin producing cells that can be transplanted in T1D patients. To that end, the discovery of induced pluripotent stem (iPS) cells has provided a potential source of precursors for cell therapy for T1D. iPS cells are reprogrammed somatic cells which can be transplanted back into the patient from whom the somatic cells were initially derived, thus potentially avoiding immune rejection when transplanted. As a potential therapy for T1D, we aim to derive insulin producing cells (IPCs) from human iPS cells. In contrast to the conventional two dimensional (2D) cell culture systems used in many iPS derived IPC studies, the inner cell mass (ICM) from which various organs differentiate during embryogenesis is a cluster of cells that enables signaling crosstalk between cells of different types. Three dimensional (3D) cell culture systems allows cells to form cell clusters that promote cell - cell signaling. Hence, we hypothesized that 3D cell culture systems will yield better efficiency of differentiation to functional IPCs in vitro than 2D cultures. Initially, the synthetic polymers sodium alginate and matrigel were analyzed for their ability to enable cell clustering to establish 3D cell culture systems. The 3D cell environment established using matrigel was used for the differentiation of human iPS cells to Insulin Producing Cells (IPC). The cells were first converted to endodermal cells. A mixture of growth factors then induced the differentiation of endodermal cells to pancreatic cells. The pancreatic cells were converted to IPCs that resemble pancreatic β-cells. Our 3D differentiated IPCs strongly expressed pancreatic endocrine transcription factors and pancreatic hormones. The IPCs also produced insulin when exposed to a high glucose environment. But the number of IPCs obtained at the end of the differentiation was low. Hence, our results demonstrate that 3D differentiation generates functional IPCs in vitro unlike 2D differentiation. In the future we aim to improve the percentage of IPCs that we generate from the 3D differentiation. Our expectation is that these cells will be able to cure hyperglycemia in diabetic mice more rapidly compared to the 2D differentiated cells owing to their proven insulin production in the presence of a high glucose environment in vitro.

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.

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.

No description available.

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.

Conjugation of the Human Immunodeficiency Virus Transactivator of Transcription (TAT) to active proteins allows transport into the intracellular environment. This feature can be harnessed to deliver combinations of reprogramming factors (RFs) such as c-Myc, Oct4, Klf4 and Sox2 into somatic cells to derive induced pluripotent stem cells (iPSCs). For this project, TAT-fusion proteins including four TAT-conjugated RFs (TAT-RFs) have been produced and purified. All four TAT-RFs can bind specific DNA sequences. Bioactivity was tested in live cells using a novel assay based on an engineered fibroblast cell line that can be induced to express RFs by doxycycline and subsequently generate iPSCs. To test each TAT-RF, reprogramming was blocked by transient silencing of a single RF by siRNA and rescued by the corresponding TAT-RF. The results of this assay suggested that TAT-Klf4 was bioactive in cells; however, definitive evidence could not be obtained for other RFs.

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.

Conjugation of the Human Immunodeficiency Virus Transactivator of Transcription (TAT) to active proteins allows transport into the intracellular environment. This feature can be harnessed to deliver combinations of reprogramming factors (RFs) such as c-Myc, Oct4, Klf4 and Sox2 into somatic cells to derive induced pluripotent stem cells (iPSCs). For this project, TAT-fusion proteins including four TAT-conjugated RFs (TAT-RFs) have been produced and purified. All four TAT-RFs can bind specific DNA sequences. Bioactivity was tested in live cells using a novel assay based on an engineered fibroblast cell line that can be induced to express RFs by doxycycline and subsequently generate iPSCs. To test each TAT-RF, reprogramming was blocked by transient silencing of a single RF by siRNA and rescued by the corresponding TAT-RF. The results of this assay suggested that TAT-Klf4 was bioactive in cells; however, definitive evidence could not be obtained for other RFs.

<p>Massive loss of cardiac tissue as a result of myocardial infarction can create a poorly-conducting substrate with impaired contractility, ultimately leading to heart failure and lethal arrhythmias. Recent advances in pluripotent stem cell research have provided investigators with potent sources of cardiogenic cells that may be transplanted into failing hearts to provide electrical and mechanical support. Experiments in both small and large animal models have shown that standard cell delivery techniques suffer from poor retention and engraftment of cells. In contrast, the transplantation of engineered cardiac tissues may provide improved cell retention at the injury site, creating a more localized paracrine effect and yielding more efficient structural and functional repair. However, tissue engineering methodologies to assemble cardiomyocytes or cardiac progenitors into aligned, 3-dimensional (3D) myocardial tissues capable of physiologically relevant electrical conduction and force generation are lacking. The objective of this thesis was thus to develop a methodology to generate highly functional engineered cardiac tissues starting from pluripotent stem cells.</p><p>To accomplish this goal, we first derived purified populations of cardiac myocytes from mouse embryonic stem cells (mESC-CMs) by antibiotic selection driven by an &#945;-myosin heavy-chain promoter. Culture conditions that yielded robust mESC-CM electrical coupling and fast action potential propagation were optimized in confluent cell monolayers. We then developed a microfabrication-based tissue engineering approach to create engineered cardiac tissues ("patches") with uniform 3D cell alignment. We found that, unlike in monolayers, mESC-CMs required a population of supporting cardiac fibroblasts to enable the formation of 3D engineered tissues. Detailed structural, electrical and mechanical characterization demonstrated that engineered cardiac patches consisted of dense, uniformly aligned, highly differentiated and electromechanically coupled mESC-CMs and supported rapid action potential conduction velocities between 22 - 25cm/s and contractile force amplitudes of up to 2mN. </p><p>Next, we sought to circumvent the use of primary cardiac fibroblasts by utilizing a single pluripotent stem cell-derived source, multipotent cardiovascular progenitors (CVPs) capable of differentiating into vascular smooth muscle and endothelial cells in addition to cardiomyocytes. CVPs were derived from mouse embryonic stem cells and induced pluripotent stem (iPS) cells by antibiotic selection driven by an Nkx2-5 enhancer element. Similar to mESC-CMs, CVPs formed highly differentiated cell monolayers with electrophysiological properties that improved with time in culture to levels achieved with pure mESC-CMs. However, unlike mESC-CMs, CVPs formed highly functional 3D engineered cardiac tissues without the addition of cardiac fibroblasts, enabling engineered cardiac tissues to be formed from a single, entirely stem cell-derived source.</p><p>Finally, we explored mechanisms of synergistic cardiac fibroblast/myocyte signaling in 3D engineered tissues by using cardiac fibroblasts of different developmental stages in the settings of direct 3D co-culture as well as in conditioned media studies. When co-cultured with fetal cardiac fibroblasts, mESC-CMs were capable of two-fold faster action potential propagation and 1.5-fold higher maximum contractile force generation than when co-cultured with adult cardiac fibroblasts. These functional improvements were associated with enhanced mESC-CM spreading and upregulation of important ion channel, coupling, and contractile proteins. Conditioned medium studies revealed that compared to adult fibroblasts, fetal cardiac fibroblasts secreted distinct paracrine factors that promoted mESC-CM spreading and spontaneous contractility in 3D engineered tissues and acted via the MEK-ERK pathway. Quantitative gene expression analysis revealed paracrine factor candidates that may mediate this action.</p><p>In summary, this thesis presents methods and underlying mechanisms for generation of highly functional cardiac tissues from pluripotent stem cell sources. These techniques and findings provide foundation for future engineering of human ES and iPS cell-based cardiac tissues for therapeutic and drug screening applications.</p> / Dissertation

Recent advances in cellular reprogramming have enabled the generation of embryoniclike cells from virtually any cell of the body. These inducible pluripotent stem cells (iPSCs) are capable of indefinite self-renewal while maintaining the ability to differentiate into all cell types. Nowhere will this technology have a greater impact than in the ability to generate disease and patient-specific cell lines. Here we explore the capacity of human iPSCs reprogrammed from peripheral blood endothelial progenitor cells lines to differentiate into atrial myocytes for the study of patient specific atrial physiology. Methods and Results: Late outgrowth endothelial progenitor cells (EPCs) cultured from clinical blood samples provided a robust cell source for genetic reprogramming. Transcriptome analysis hinted that EPCs would be comparatively more amenable to pluripotent reprogramming than the traditional dermal fibroblast. After 6 passages, EPCs were transduced with a doxycycline inducible lentivirus system encoding human transcription factors OCT4, SOX2, KLF4 and Nanog to permit differentiation after removal of doxycycline. The high endogenous expression of key pluripotency transcripts enhanced the ease of iPSC generation as demonstrated by the rapid emergence of typical iPSC colonies. Following removal of doxycycline, genetically reprogrammed EPC-iPSC colonies displayed phenotypic characteristics identical to human embryonic stem cells and expressed high levels of the pluripotent markers SSEA-4, TRA1-60 and TRA1-81. After exposure to conditions known to favor atrial identity, EPC- iPSC differentiating into sheets of beating cardiomyocytes that expressed high levels of several atrial-specific expressed genes (CACNA1H, KCNA5, and MYL4). Conclusions: EPCs provide a stable platform for genetic reprogramming into a pluripotent state using a doxycycline conditional expression system that avoids reexpression of oncogenic/pluripotent factors. Human EPC-derived iPSC can be differentiated into functional cardiomyocytes that express characteristic markers of atrial identity.