Central nervous system injuries often lead to devastating consequences due to an unfavourable environment created after the injury. Current treatments have yet to address the environment for improved prospects of functional recovery. Transplantation of Schwann cells into the lesion site could in part address the issue, promoting nerve regeneration and enhancing functional recovery. Bone marrow stromal cells (BMSCs) promise to be a viable, autologous source for Schwann cell derivation. Fate-committed Schwann cells derived from BMSCs through co-culture with purified dorsal root ganglia (DRG) neurons suggest that the DRG neurons present juxtacrine cues that direct commitment to the Schwann cell fate. We hypothesize that Neuregulin 1 type III (NRG1(III)) is one such juxtacrine cue to which BMSC-derived Schwann cell-like cells (SCLC) respond in the switch to fate commitment. In this study, NRG1(III) was found to be expressed on freshly isolated DRG neurons and that SCLCs expressed both the ErbB2 and 3 receptors. Western blot analysis for phosphorylated Akt and MAPK provided indicators of downstream signalling of NRG1/ErbB complexes. We then tested if both the soluble and membrane bound forms of NRG1 mediate SCLC differentiation towards fate commitment. In contrast to the membrane-bound form on DRG neurons, soluble NRG1 failed to direct the SCLCs towards the Schwann cell fate. HEK293T cells that stably overexpress NRG1(III) were generated and tested as a neuronal surrogate that presents NRG1(III) on the cell surface. In a 5-day co-culture system with HEK293TNrg1(III) cells, SCLCs were found to develop elongated processes, acquiring either unipolar or bipolar morphology that resembles that of Schwann cells. Screening for marker expression by RT-PCR suggested that at this stage of morphological transition, SCLCs were not yet committed to the Schwann cell fate. The co-culture system will be pursued to find ex vivo conditions that direct differentiation of BMSC-derived SCLCs to fate-committed Schwann cells. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Transient receptor potential (TRP) channels play important roles in cellular physiology and biology. The present PhD project investigated the functional expression of TRPV and TRPM channels in human preadipocytes and cardiac c-kit+ progenitor cells and their roles in regulating cell proliferation, adipogenic differentiation or migration. In addition, the role of store-operated Ca2+ entry (SOCE) channels in regulating cell proliferation and migration was also studied in human cardiac c-kit+ progenitor cells using multiple approaches including whole-cell patch voltage-clamp, confocal microscope, molecular biology, etc. We found that TRPV2, TRPV4 and TRPM7 channels were abundantly expressed in human preadipocytes. Activation of TRPV2 channels by probenecid caused a long-lasting intracellular Ca2+ transient, while activation of TRPV4 channels by 4-PDD induced Ca2+ oscillations. TRPM7 current was recorded with a Mg2+-free pipette solution, and inhibited by 2-aminoethyl diphenyl borate (2-APB). Silence of TRPV2 or TRPM7, but not TRPV4, with the specific shRNA, reduced cell proliferation via inhibiting cyclin D1, cyclin E, and p-ERK1/2. Individually silencing these three channels decreased adipogenic differentiation by reducing p-Akt kinase. The results indicate that TRPV2, TRPV4 and TRPM7 are involved in adipogenesis, while TRPV2 and TRPM7, but not TRPV4, regulate cell proliferation in human preadipocytes. In second part of the thesis, abundant expression of TRPV2, TRPV4, and TRPM7 channels was demonstrated in human cardiac c-kit+ progenitor cells. Similar to human preadipocytes, probenecid and 4-PDD activated Ca2+ signaling, and TRPM7 current recorded with a Mg2+-free pipette solution was inhibited by 2-APB. Silencing TRPV2 or TRPM7, but not TRPV4, inhibited cell proliferation by arresting cells at G0/G1 phase with a reduced cyclin D, cyclin E, and p-ERK1/2. Cell migration was decreased with silence of TRPV2, TRV4 or TRPM7 via inhibiting p-Akt kinase. The results show that TRPV2, TRPV4 and TRPM7 mediate cell migration, while TRPV2 and TRPM7, but not TRPV4 channels, participate in regulating cell proliferation. In third part of the thesis, we demonstrated that SOCE channels were composed of TRPC1, STIM1 and Orai1 by protein-protein interaction. Silence of TRPC1, STIM1, or Orai1 with specific siRNA reduced Ca2+ influx through SOCE channels, decreased cell proliferation by inhibiting cyclin D1 and cyclin E, and slowed down cell migration via reducing p-Akt kinase. These results suggest that TRPC1, STIM1 and Orai1 are the major components of SOCE channels in human cardiac c-kit+ cells. SOCE channels play an essential role in regulating cell proliferation and migration. Collectively, this PhD project has demonstrated for the first time that 1) TRPV2, TRPV4, and TRPM7 are abundantly expressed in human preadipocytes and cardiac c-kit+ progenitor cells. 2) These TRP channels regulate adipogenic differentiation in preadipocytes and migration in cardiac c-kit+ progenitor cells. 3) TRPV2 and TRPM7, but not TRPV4, are involved in cell proliferation of human preadipocytes and cardiac c-kit+ progenitor cells. 4) TRPC1, STIM1 and Orai1 are interacted to form SOCE channels and regulate cell proliferation and migration in human cardiac c-kit+ cells. 5) All the above physiological roles of TRPV2, TRPV4, TRPM7, and SOCE channels are mediated by cyclin D1, cyclin E, p-ERK1/2, and/or p-Akt. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

The potential of determining stem cell fate through mechanoregulation has been demonstrated recently. However, the underlying mechanism remains largely unknown. Previously, we developed a novel microencapsulation technique to entrap cells in a nanofibrous collagen meshwork and use the cell-collagen model to study mechanoregulation of human mesenchymal stem cells (hMSCs). Initially, hMSCs were randomly distributed within the construct. Upon cyclic compression, hMSCs reoriented towards a direction along the loading axis. Cytoskeleton, being the major sub-cellular machinery supporting cell shape and motility, should play crucial role in sensing and responding mechanical signals. Therefore, a better understanding in the change of cytoskeleton and associated molecules upon mechanical loading is a prerequisite to rationalizing the loading regimes for stem cell-based functional tissue engineering. In the current project, we hypothesize that hMSCs encapsulated in 3D collagen construct will respond to cyclic compression by remodeling the cytoskeleton structures and altering the interactions with collagen matrix. hMSCs collagen construct were cyclically compressed for 9 hours through micromanipulator based compression system. After compression, constructs were harvested either immediately after compression, 2 hours after compression and 24 hours compression, together with non-loading control group. Here, we report compression-induced novel changes in cytoskeleton. Firstly, omnidirectional filopodia-like structures together with stress fibers bucking were observed immediately after 9hrs of cyclic compression. Secondly, actin patches were observed shortly after removal of 9hrs compression before the actin fibers resumed. Apart from exhibiting similar morphology with filopodia, the omnidirectional filopodia-like structures may share a similar function in interacting with ECM. Co-localization of the major membrane-bound matrix metalloproteinases MT1-MMP with actin staining was found along the length of the filopodia-like structures. A local collagen digestion zone, characterized by the presence of collagenase cleaved collage, was found co-localizing at least partially with the filopodia-like structures around the cell. Whether creating pericellular collagen digestion zone was mediated by MT1-MMP along the compression-induced filopodia like structures and what functions the digestion zone serves are interesting question to answer in the future. Another interesting observation is the complete disassembly of pre-existing stress fibers followed by formation by numerous actin patches throughout the cell shortly after removal of the compression loading. Stress fibers reformed in 24 hours after removal of the loading. Quantitative measurement of F:G actin ratio agrees with such disassembly and reassembly dynamics. Colocalization of actin branching protein arp2/3 with the actin patches was found, suggesting that mechanically loaded hMSCs were re-establishing actin cytoskeleton network from these nucleation centers. Further studies are required to figure out the underlying significance of the loading-induced cytoskeleton dynamics in hMSCs and whether the actin patches Arp2/3 complex associates with endocytosis of cleaved collagen fragments. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Some stem-like cells that have received limited attention but may be of considerable clinical value reside in the transition zone between the corneal endothelium (CE) and trabecular meshwork (TM) at the posterior limbus of the eyes. A growing body of evidence has revealed that these cells may be able to rejuvenate the CE, TM or both. However, these stem-like cells have not been clearly defined and we have thus named them PET cells (Progenitor cells of the Endothelium and Trabeculum). Both the CE and TM cells are unique cell types in the eye that do not normally replace themselves once they are lost in ageing or diseases, such as Fuch’s endothelial dystrophy and primary open angle glaucoma. To date, no therapy exists that targets the rejuvenation of the compromised CE or TM in diseases. Therefore, the potential to repair or replace the diseased CE or TM through a cell repopulation approach is an important area that needs to be explored. The use of PET cells for tissue rejuvenation may represent an attractive therapeutic direction for the regeneration of the CE and/or TM. As a good animal model for PET cells is lacking, we sought to identify whether bovine eyes could serve as a good large tissue model for the studies of PET cells. The comparative anatomy of the human and bovine posterior limbus was studied using light, confocal and scanning electron microscopy. Immunohistochemical studies were performed to localize the stem cell niche for the PET cells. Sphere culture was used to isolate and amplify progenitor cells from the CD and TM respectively. A detailed characterization of the spheres and their progenies was performed with immunocytochemistry, quantitative reverse-transcription polymerase chain reaction and differentiation and functional assays. We showed the presence of stem or progenitor cells in the bovine CE, transition zone and TM in situ. The bovine TM insert region may house a stem cell niche that is comparable to that observed in humans. The progenitor cells isolated from the bovine CE and TM that grew as floating spheres demonstrated similar phenotypes in terms of stem cell marker expression. In addition, both the CE and TM spheres were bipotent, highly proliferative and had limited self-renewal capacity. However, they showed a high propensity to differentiate back into the cell type of their tissue of origin. We speculated that the PET cells become more tissues-specific as they migrate away from their niche towards the CE or TM. This may be a reasonable explanation why the CE and TM spheres respectively adopted their original lineage upon non-directed differentiation. Taken together, our results support the hypotheses that PET cells are present in the posterior limbus of bovine eyes and that bovine eyes may serve as a good large tissue model for the studies of these cells. The PET cells represent an attractive target for developing new treatments to regenerate both the CE and TM, thereby reducing the requirement for donor corneas and invasive treatments in glaucomatous patuents. / published_or_final_version / Ophthalmology / Doctoral / Doctor of Philosophy

Stem cell research provides a useful tool for unraveling the molecular mechanisms and shedding light on cell transplantation therapies. Traditional qualitative experimental observation lacks the rich dynamic information, and therefore prevents obtaining the insight into cellular processes such as stochastic cell fate control and biological pattern formation. Quantitative approach helps promote research of these challenges. As passive measurements, the available commercial solutions have their own drawbacks and need to be improved to fulfill specialized requirements of the cutting edge studies. The multidisciplinary technology developed in this study provides multidimensional observation of cellular dynamic processes, automatic extraction of spatial-temporal quantitative information, and precisely active optical modulation of cell. Here a novel RII autofocus system was developed to solve the focus drift problem by utilizing fiber collimated laser beam to reduce the beam size. The propagation properties of Gaussian beam indicate that high resolution could be achieved by minimizing focused Rayleigh length. The stability of live cell imaging system is improved by the RII system, which was demonstrated to have better focus resolution than the leading commercial solutions (1.3-5.3% of the objective DOF vs. 33-50%). When the home-built microscopy is incorporated with the RII system, it enables automatic and continuous observation of cellular dynamics to identify critical information, such as rare dedifferentiation and pattern formation mechanism. A novel fluorescent image processing algorithm was developed to automatically segment cells and extract dynamic information from vast observation data. The idea of mimicking human visualization was first applied in cell segmentation. A new criterion SILC was proposed for precise cell linking, and a novel evaluation of tracking results aiming at extracting effective information was firstly suggested in this study. This efficient algorithm was proved to have high average detection rate (94.5%) under different densities (100-2000 cells/〖mm〗^2), and high tracking result (at least 80%) of cells. Novel active light modulations were explored aiming at precisely modulating the exogenous gene expression of stem cells and quantitatively determining the motion properties of genetically programmed cells in extremely high density. The development of biophotonics described in this thesis covers all the methodology holes in quantitative study of stem cell differentiation and pattern formation. This multidisciplinary approach is expected to help unravel critical dynamic properties of stem cells, and eventually contribute to make stem cell therapy a safe and effective option. / published_or_final_version / Physics / Doctoral / Doctor of Philosophy

Human endometrium undergoes cyclic tissue breakdown and regeneration throughout a woman of reproductive life. It has been speculated that there is a subpopulation of stem/progenitor cells residing in the endometrium responsible to its remarkable capability. One key feature that distinguishes the adult stem cells is their quiescent state within the stem cell niche. Based on this unique stem cell property, slow-cycled cells from human endometrial tissues were examined and characterized. The first aim of this study was to examine label retaining cells (LRCs) in long-term engrafted human endometrial tissues using a mouse xenotransplantation model in vivo. After various initial approaches, the maximum initial labeling of LRCs was determined and, only stromal LRCs were detected after a 12-week chase. Mesenchymal stem cell (MSCs) and stem cell-like phenotypic appearances were detected in a small proportion of stromal LRCs. The second objective was to establish a novel method for identifying slow-cycled cells from cultivated endometrial epithelial and stromal cells in vitro. The method identified cells that retained fluorescence label after long-term culture. These cells were termed fluorescence retaining cells (FRCs). The variables and the different initial approaches leading to the establishment of the protocol for the identification of FRC were optimized. The third objective further characterized the endometrial stromal FRCs. Endometrial stromal FRCs were enriched with cells having higher colony forming and self-renewal abilities when compared with non-FRCs. At subsequent passage (P2) the adult stem cell characteristics of cells derived from FRCs was more prominent and differentiated into mesenchymal lineages. Clonally derived stromal FRCs expressed higher self-renewal and pluripotent genes (BMI-1, NANGOG, OCT4, and SOX2). They also expressed endometrial mesenchymal stem cell phenotypic surface markers: CD146/PDGFRβ and W5C5, suggesting these cells might be of MSC origin. The fourth objective was to examine the role of epithelial-stromal interactions on regulation of endometrial stem cells. Menstrual cycle day 2 epithelial conditioned medium significantly enhanced the colony forming ability of endometrial stromal cells. Protein expression analysis of the early menstrual phase conditioned medium showed two cytokines: angiogenin and interleukin 8, potentially involved in the regulation of endometrial stromal stem/progenitor cells. The fifth objective was to examine the reconstitution ability of the endometrial clonogenic cells in vivo using a mouse xenotransplantation model. Endometrial clonogenic stromal cells together with epithelial cells reconstituted muscle-like tissue. Although no endometrial-like tissue was detected, the findings provide the first in vivo evidence that stromal stem/progenitor cells are present within clonogenic cells. Overall, the thesis reports the existence of slow-cycled cells from human endometrium. By using various label retention techniques in vitro and in vivo a subpopulation of slow-cycled stromal cells which exhibit characteristics properties of adult stem cells was demonstrated for the first time. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

abstract / Biochemistry / Doctoral / Doctor of Philosophy

abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

MSCs are the most studied subtype of adult stem cells and have been derived from most postnatal organs and tissues. MSCs are defined as having the capacity to self-renew and to differentiate into both mesodermal and non-mesodermal lineages, and are immunosuppressive. For these properties, MSCs have been considered ideal candidates for regenerative medicine and have been used in several clinical trials. The difficulty, however, to preserve the potency of the cells during culture expansion and to monitor differentiation are obstacles in their use in the clinic and have emphasized the need to investigate molecular pathways underlying stem cell fate-decisions during differentiation in more detail. Hippo pathway was recently identified in Drosophila melanogaster and mammals, and controls organ size by regulating cell proliferation, apoptosis and differentiation. It is composed of a core of serine/threonine kinases and scaffold proteins that when activated, phosphorylate and inhibit yes-associated protein (YAP) transcriptional co-factor. Inactivation of YAP in some stem and progenitor cells by this pathway is required for their differentiation. In contrast, failure to inhibit YAP enhances proliferation and may cause oncogenic transformation. In the present study, the expression of multiple YAP variants was confirmed in human and mouse MSCs. In both human and mouse, YAP was inhibited in response to cell-contact inhibition and remained unchanged during in vitro chondrogenic differentiation. Overexpression of human (hYAP1) variant in C3H/10T1/2 cells did not appear to affect colony formation, cell cycle distribution or cell size, but increased cell proliferation, induced cell transformation and reduced the in vitro differentiation capacity of the cells towards the chondrogenic, adipogenic and osteogenic lineages. The effects of hYAP1 overexpression are hypothesized to be either a result of a nuclear co-factor function or indirectly via protein interactions in the cytoplasmic compartment. Hippo pathway and YAP are possible pharmacological targets for modulation of MSC function.

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