Breathing is essential for human life, yet tens of millions of people in the U.S. alone suffer from lung diseases. With each breath, lungs are exposed to the external environment. Inhaled air first passes through the trachea, bronchi, and finally the bronchioles before it reaches the alveoli where gases are exchanged. A barrier of epithelial cells protects the airways. In addition, epithelial glands also secrete protein-rich fluids onto the airway surfaces to help maintain sterility. Injury, disease, or other factors can damage these cells, and regiospecific stem cells (SCs) can divide to replace them. However, many important details about lung SCs are still unknown. For example, what processes control SC division? How do region-specific SCs differ from one another? And how does disease or injury impact SC biology? We found that some processes that regulate lung development also control adult SC division following injury. We show that SCs from airway glands give rise to surface epithelial cell types and glandular cell types. In contrast, surface SCs only generated surface cell types. Finally, we identify a type of cell in the glands that can regenerate surface cell types after severe injury. These studies provide new insights into the neighborhoods in which SCs reside in the large airways and processes that control their contribution to airway repair following injury. Overall, this research provides important new insights into adult SC biology and conditions affecting lung health.

Adult stem cells are a small population present within several tissues of an individual, possessing two unique properties: one, the ability to differentiate to give rise to all the cell types of the tissue, and second, the ability to self-renew and make more of their own kind. Owing to these two properties, stem cells underlie the process of organogenesis during development and tissue homeostasis in adult life. In the past decade a small sub-population of cells having phenotypic and functional properties similar to normal stem cells have been identified within several tumors. Only this sub-population of cancer cells seems to have the ability to both initiate and maintain tumors. These cells have been termed as ‘cancer stem cells’ (CSCs) owing to their striking similarities with the normal stem cells of the tissue. It is therefore of fundamental importance to understand normal stem cell biology in order to understand tumorigenesis. The rarity of normal stem cells within adult tissues, the absence of specific cell surface markers to identify and isolate them, and the absence of suitable culture conditions to maintain them has marred our understanding of stem cell behaviour. Recently, growth of mammary cells in serum free suspension cultures resulted in the generation of floating spheroids termed “mammospheres” that were shown to be enriched in stem/progenitor cell population. We established the mammosphere system in our laboratory using mastectomy samples obtained from the Kidwai Memorial Institute of Oncology. In order to understand the composition of the spheres, the stem cell characteristics within them, and the long term self renewal potential of human mammary epithelial stem cells, a detailed phenotypic and functional characterization of the mammospheres was carried out. Phenotypic Characterization: Confocal microscopy of propidium iodide stained mammospheres demonstrated that these spheres are cellular and not hollow structures. Immunostaining revealed that primary mammospheres expressed the epithelial markers like E Cadherin, ESA, CK14, CK18 and CK 19, but failed to express nestin or CD34, indicating their epithelial origin, devoid of contamination from haematopoeitic or neural stem cells. The sizes of mammospheres ranged from 40 to 110 μm, while that of the cells within them ranged from 9-15 μm. Although the sizes of the largest and smallest spheres through subsequent passages remained consistent, the proportion of small spheres increased in later passages. These results indicate the difference in the sphere initiating cells. While a large sphere might be generated by a stem cell, a smaller sphere might be originating from a progenitor. Thus, heterogeneity exists within mammospheres, with respect to size and composition. Unique cell surface markers coupled with flow cytometry serves as useful tools to isolate stem cells. However, no specific marker profile has been reported for normal human breast stem cells. In several tissues, like blood, brain etc, markers of normal stem cells have been successfully used to isolate cancer stem cells within that tissue. Since breast cancer stem cells have already been identified as CD24low/-44high cells, we explored if the same marker profile would hold true to identify normal breast stem cells as well. Two-colour based flow cytometry revealed that only the CD24low/-44high subpopulation of mammospheres could re-generate mammospheres, as well as give rise to all the other cellular fractions. These data demonstrated that normal and cancerous breast stem cells share identical marker profile. Functional Characterization: In addition to cell surface markers, a Hoechst dye based strategy used to isolate stem cells, exploits their unique property to efflux certain lipophilic drugs and small molecules due to the overexpression of ABC family of cell surface transporters. Cells effluxing Hoechst appear as a low fluorescing ‘Side population’ (SP) in a bivariate FACS plot. We detected a small, but distinct SP in human breast cells, which had a CD24low44low profile, and failed to initiate new mammospheres. Thus, the SP cells in mammospheres failed to correspond to the stem cell subpopulation. The hallmark feature of a stem cell is its long term self renewal ability, given that it is the longest lived cell in the body. Long term culture of mammospheres was carried out by passaging the spheres every week. We failed to observe mammosphere formation beyond four passages though there were live, proliferating and undifferentiated cells in fourth passage spheres. These results suggested that either the mammopsheres didn’t contain stem cells to begin with, or their stemness is restricted to four in vitro passages. In order to assess if mammospheres contained stem cells to begin with, we assayed for telomerase activity, since in the adult tissue, only stem cells retain telomerase activity. Telomerase, an enzyme that maintains the length of telomeres through multiple rounds of cell division, is not active in somatic cells. We detected the expression and activity of this enzyme in primary mammospheres, suggesting that the spheres may contain stem cells withinthem Another unique property of a stem cell is its ‘quiescence’, owing to their infrequent divisions. This property is studied by chasing a label (like BrdU or H3-Thymidine), which is taken up by the cells at an earlier time point and retained within the cell after prolonged periods, like weeks or months. In long term culture of mammospheres, using BrdU as the label, 1-2 distinct cells could be detected within late passage spheres which had retained the label, indicating that stem cells may be present within the fourth passage mammospheres as well. Staining for β-Galactosidase activity revealed that almost 70% cells derived from fourth passage spheres were senescent. We speculated that this senescent environment might be one of the inhibitory reasons for further mammosphere formation. Alteration of mammosphere culture conditions for long term maintenance of stem cells. A high level of atmospheric O2 is known to be one of the reasons for inducing senescence in cells. Culturing cells in conventional tissue culture conditions exposes them to high levels of O2 (21%) as against the physiological levels of 1-3% O2. Therefore, to assess the effects of lowered, or physiologically relevant levels of O2 on mammosphere stem cell biology, the mammospheres were cultured in 3% O2. Under this altered condition, a close to 3-fold increase was observed in the number of mammospheres formed coupled with a significant increase in their survival and proliferation. In order to understand the molecular basis of this observation, a microarray based global gene expression profiling was carried out. We observed a significant upregulation of VEGF, a gene responsive to hypoxia; three growth factor related genes, namely adrenomedullin, cMET and osteopontin. Upregulation of β Catenin, the downstream effector of the Wnt signaling pathway was also observed, indicating a possible mechanism for the increase in self renewal seen in 3% O2. We also observed downregulation of the cell cycle inhibitor, Chk1, which in part might explain the observed increase in proliferation. The increase in the number of proliferating cells might be one of the reasons for an increase in the number of spheres, as observed in 3% O2. Even though a significant decrease in the number of senescent cells was detected at 3% O2, mammosphere formation was not seen beyond four passages. It is therefore possible that there are other physico-chemical parameters, comprising the niche of the mammospheres, coupled to the O2 level, which need to be improvised for long term culture of human mammary epithelial stem cells. To summarize, this work reports for the first time that human mammary epithelial stem cells have an identical marker profile as breast cancer stem cells, which is CD24low/-CD44high. It has also been demonstrated for the first time that in long term mammosphere culture, the number of self renewal divisions of human mammary stem cells is restricted to four in vitro passages, at which most of the cells undergo senescence. Altering one of the parameters of the niche, by culturing mammospheres at physiological O2 level failed to prolong the in vitro lifespan of the spheres, although cell survival, proliferation and sphere formation increased, indicating that the niche requirements of human mammary epithelial stem cells for their long term self renewal needs to be further characterized.

<strong>Osteoarthritis</strong>, the most prevalent joint disease in the United Kingdom, is a progressive condition that results in end-stage full-thickness cartilage loss and has important social and economic impacts on society. Since cartilage lacks regenerative capabilities, it is essential to develop approaches to initiate and enhance cartilage regeneration. In this context, tissue engineering is emerging as an attractive approach for the regeneration of cartilage tissue damaged due to disease or trauma. A scaffold-free cartilage construct, analogous to those found during embryonic precartilage condensation, has received much attention as an alternative novel modality for cartilage <strong>tissue engineering</strong>. Cartilage repair with <strong>scaffold-free</strong> tissue more closely resembles the natural situation and mimics the features of the original tissue. Moreover, scaffold-free cartilage implants can overcome the complications caused by the use of suboptimal scaffolds by avoiding the need for a foreign scaffold at all. Culturing cells into tissue patches without the requirement for a scaffold can be achieved through <strong>cell sheet engineering</strong>, which uses thermo-responsive culture dishes. However, the high costs of the tissue culture consumables, and the relatively low cellular yield, makes this process less attractive. This thesis presents a novel method for generating shape-, size- and thickness-adjustable 3-dimensional scaffold-free cell pellet sheets for use as implantable biological cell patches for cartilage tissue engineering. This new technique of bioengineering scaffold-free cell pellet sheets proves to be reproducible, easily applicable, sizable and thickness adjustable. <strong>Stem cells</strong> have added a new thrust to tissue engineering. Their distinctive self-renewal and plasticity have not only optimized many tissue engineering developments, but also rendered feasible some applications which would otherwise be unattainable with somatic cells. Human mesenchymal stem cells (HMSCs) were used to examine the optimal condition for generating cell pellet sheets with this new method. Furthermore, the resultant differentiated pellet sheets were compared directly with HMSCs, human chondrocytes and human fibroblasts alone to evaluate the feasibility of using this cell pellet sheet for clinical applications in terms of their biological and mechanical properties. The results of this thesis suggest that the engineered scaffold-free, chondrogenic, differentiated MSC pellet sheet not only exhibits desirable biologic features similar to chondrocytes, but also demonstrates good integrative and viscoelastic potential that might offer exciting possibilities for the development of novel biologically-based clinical therapies. In summary the data presented herein indicate the following points: <table><ul style="list-style-type:square"><li>The differentiation of human MSCs into chondrogenic cells was achieved.</li> <li>A novel approach of centrifugal seeding on a PDMS surface was shown to effectively generate chondrogenic-differentiated cell pellet sheets without impairing the biological functions of chondrocytes.</li> <li>Various cell types such as human MSCs, human chondrocytes and human fibroblasts were found to respond well to the novel methodology and generated viable, cohesive, less shrinkable, and readily-detachable cell pellet sheets, the size and thickness of which could be adapted as required. The results obtained were superior to those obtained using the conventional thermo-responsive culture dish method.</li></table> This new methodology developed in this thesis provides an approach to in vitro cell pellet sheet generation which is closer to the physiological process of cartilage development and which proved valuable for the study of in vitro generation of scaffold-free cell patches as an important adjunct to many traditional cartilage restorative procedures. Future research on in vivo assessment of the cell sheet and the functional role of these sheets in repairing damaged cartilage is recommended.

The haematopoietic stem cell (HSC) is found in the adult human bone marrow, where it gives rise to all the circulating blood cells throughout adulthood. Understanding the signalling events that programme these cells during development will improve HSC in vitro culture, their generation from embryonic stem cells or induced pluripotent stem cells, and their potential therapeutic application. HSCs bud from the floor of the dorsal aorta and seed the bone marrow via circulation. The precursors to the dorsal aorta and HSCs are called haemangioblasts, which are found in the dorsal lateral plate mesoderm in Xenopus. The knowledge of the location of these precursors allows their programming to be studied in detail during embryonic development. A key pathway implicated in the programming of HSCs is the BMP signalling pathway. Here, using both a small molecule inhibitor and a transgenic Xenopus line, BMP signalling has been inhibited post-gastrulation without perturbing the gross morphology of the embryo. This has shown that BMP signalling is required for HSC programming in the dorsal lateral plate mesoderm via the expression of a critical haematopoietic transcription factor, gata2. Morpholino knockdown of evi3has revealed it to be essential for HSC programming in the dorsal lateral plate mesoderm, where it is required for the expression of gata2. Furthermore, as evi3 is known to bind to the active BMP signalling complex, and as evi3 knockdown phenocopies post-gastrulation BMP inhibition, evi3 appears to be required for BMP signalling to initiate gata2 expression in the DLP. Taken together, the findings presented here demonstrate an essential post-gastrulation role of BMP signalling and Evi3 for programming HSCs in Xenopus.

Embryonic stem cells (ESCs) are a unique cell population that can differentiate into all three embryonic germ layers (endoderm, mesoderm, and ectoderm), rendering them an invaluable cell source for studying the molecular mechanisms of embryogenesis. Signaling molecules that direct tissue patterning during embryonic development are secreted by ESC aggregates, known as embryoid bodies (EBs). As many of these signaling proteins interact with the extracellular matrix (ECM), manipulation of the ESC extracellular environment provides a means to direct differentiation. ECM components, such as glycosaminoglycans (GAGs), play crucial roles in cell signaling and regulation of morphogen gradients during early development through binding and concentration of secreted growth factors. Thus, engineered biomaterials fabricated from highly sulfated GAGs, such as heparin, provide matrices for manipulation and efficient capture of ESC morphogens via reversible electrostatic and affinity interactions. Ultimately, biomaterials designed to efficiently capture and retain morphogenic factors offer an attractive platform to enhance the differentiation of ESCs toward defined cell types. The overall objective of this work was to examine the ability of microparticles synthesized from both synthetic and naturally-derived materials to enhance the local presentation of morphogens to direct ESC differentiation. The overall hypothesis was that microparticles that mimic the ECM can modulate ESC differentiation through sequestration of endogenous morphogens present within the EB microenvironment.

Mucopolysaccharidosis type IIIA (MPS IIIA) is caused by mutations in the N-sulphoglucosamine sulphohydrolase (SGSH) gene, leading to cellular accumulation of heparan sulphate and progressive neurodegeneration in patients. One of the proposed treatment methods is haematopoietic stem cell (HSC) gene therapy, which should result in an excess of SGSH produced in the peripheral organs and brain. The pre-clinical feasibility of this approach was demonstrated by our group in a mouse model of MPS IIIA. However, the overall efficiency of this method was limited and a number of approaches to solving these issues were addressed in this project in order to bring this therapy closer to clinical application. Our first aim was to optimise transduction of HSCs using cytokines, bovine serum albumin (BSA), and chemicals, such as MG132, genistein and valproic acid. Addition of BSA with cytokines improved cell viability, addition of MG132/ BSA/ cytokines improved transduction, but also caused cellular toxicity, while addition of genistein was inefficient. Addition of valproic acid with cytokines resulted in increased number of colony forming units. Next, we generated clinically applicable third generation pCCL lentiviral vector backbones with the eGFP reporter gene driven by one of ubiquitous hPGK or myeloid specific hCD11b and hCD18 internal human promoters, and optimised production of lentiviral vectors to increase titre and reduce production cost. These lentiviral vectors were used to transduce lineage depleted HSCs and transplanted into WT mice. Full chimerism and over 80% transduction were achieved with an average of 5 vector copy numbers/ cell. The hCD11b promoter resulted in the highest eGFP expression in monocytes and B cells in blood, but was weaker than the hPGK in T cells. The hCD18 promoter was more monocyte-specific but weak. Significant numbers of GFP-positive microglial cells were present in the brain from all groups, with an average of 25% transduced CD11b-positive cells in perfused mice. We subsequently codon-optimised (CO) the SGSH gene significantly improving enzyme activity, and transduced lineage depleted WT cells with one of hCD18.SGSH-CO, hCD11b.SGSH-CO, or hPGK.SGSH-CO lentiviral vectors, or MPS IIIA cells with either hCD11b.SGSH-CO or hPGK.SGSH-CO lentiviral vectors. These transduced cells were transplanted into MPS IIIA mice and outcomes were measured 6 months later. Only treatment with the hCD11b.SGSH-CO-LV transduced WT or MPS IIIA HSCs corrected abnormal behaviour of MPS IIIA mice. However, all treatments resulted in complete GAG storage clearance in the periphery and brain, and significantly elevated enzyme activity in the brain, liver and spleen to 7-11%, 60-75%, and 170-250% of WT enzyme activity respectively. A fine threshold of over 8.6% brain enzyme activity appeared to be required for behavioural correction in MPS IIIA mice. Further assessment of treated mice for the amount of secondary storage, HS sulphation patterning, neuroinflammation and longevity are still required for complete therapeutic assessment. However, it appears that neurological correction of the MPS IIIA mouse using MPS IIIA cells is feasible using a clinically-relevant pCCL vector with the hCD11b promoter and the codon-optimised SGSH gene.

No description available.

Despite the normally robust regenerative capacity of muscle tissue, extensive soft tissue damage often results in a functional loss that cannot be restored using classic reconstruction techniques. Although implanted biomaterials are capable of mechanically transmitting force generated from the remaining tissue, cellular repopulation, reinnervation and revascularization of the injured area is necessary to achieve complete functional restoration. Using an in vivo tissue engineering model, a 1.0 x 1.0 cm portion of the lateral gastrocnemius (LGAS) of Lewis rats was removed and replaced with a muscle-derived extracellular matrix (ECM). Constructs were seeded with bone marrow-derived (BMSCs) or adipose-derived stem cells (ADSCs) and the peroneal nerve was relocated over the implanted ECM. Creation of the defect resulted in a functional impairment of the LGAS, only capable of producing 85.1 ± 4.1% of the force generated in the contralateral LGAS following ECM implantation. A significant increase in specific tension (SPo) was seen in all groups following the nerve relocation procedure when compared to their corresponding cellular treatment without nerve relocation (p < 0.05). Histological quantification revealed significant increases in cellular content and blood vessel density in the top and bottom regions of ECM implants seeded with BMSCs (p < 0.05). The nerve relocation procedure significantly increased these same variables within the middle region of the ECM when compared to all groups lacking this treatment (p < 0.05). The presence of regenerating myofibers was immunofluorescently confirmed using antibodies against desmin, myosin heavy chain and laminin, while their developmental state was substantiated by the presence of central nuclei. These data corroborate a therapeutic effect of BMSCs on skeletal muscle regeneration within the ECM implant that was not seen following ADSC injection. Furthermore, the nerve relocation procedure stimulated an increased cellular and vascular growth within the middle region of the construct, likely the cause of improved functional output. / text

The fields of tissue engineering, regenerative medicine, and stem cell engineering are rapidly growing. However, these fields must overcome several obstacles before they can make a significant impact on treating cellular disorders. Two major hurdles that must be addressed are: determining how to control the pluripotency of stem cells and developing systems for high-throughput culture of stem cells. The prospect of using a cell source capable of differentiating into cells of any tissue in the body (embryonic stem cells) has received enormous interest in recent years. The pluripotent attribute of embryonic stem cells seems ideal but developing methods to drive embryonic stem cells to specific lineages, including the hematopoietic lineage, is a complex process dependent on multiple intrinsic and extrinsic factors including chemical, cellular, and environmental signaling. With regards to environmental signaling, the use of three-dimensional culture systems such as scaffolds and hydrogels, have been utilized in an attempt to drive lineage-specific differentiation in a synthetic, biomimetic microenvironment. To determine specific environmental factors responsible for hematopoietic differentiation a systematic biological and engineering process must be implemented. A biodegradable hydrogel composed of the hyaluronic acid, a polysaccharide abundant in the bone marrow microenvironment, and the synthetic polymer, poly(ethylene glycol) was formulated to culture mouse embryonic stem cells (mESCs). Photoencapsulation of mESCs did not significantly decrease cellular viability or proliferation. The FACS data was inconclusive however, from gene expression studies, it was determined that the hydrogel culture system promoted differentiation of mESCs as evidenced by a down-regulation of the gene encoding for stem cell maintenance transcription factor, Oct-3/4. Furthermore, embryoid bodies, necessary for in vitro differentiation were observed in the hydrogel systems. Although an increase in the gene encoding for the cell surface marker, c-kit was up-regulated, the surface marker, sca-1 was not up-regulated. Up-regulation of both c-kit and sca-1 is necessary for the development of hematopoietic progenitor cells. Results indicate that the differentiation of mESCs into the hematopoietic lineage was unsuccessful but differentiation in these hydrogel systems did occur. Future cell marker and gene expression studies are necessary to determine which cell lineage the encapsulated mESCs are differentiating into before the effects of incorporating other environmental, cellular, and chemical factors can be investigated. / text

No description available.