The current gold standard of therapy for treatment of tibial fracture nonunion is iliac crest bone graft. However, this intervention is associated with significant morbidity to the donor site. Research into alternative interventions highlights the role of mesenchymal stem cells (MSCs). MSCs are capable of differentiating into mature, organized osseous tissue as well as recruiting local vascular cells. However, there are few prospective studies demonstrating the therapeutic potential of MSCs in fracture nonunion. The proposed study is a multicenter single-blinded controlled study of MSC application compared to iliac crest bone graft in the setting of fracture nonunion of the tibia. The study subjects will be evaluated at each return to care with mRUST radiographic scoring as well as Short-Form 12 evaluation of general health. These results will be correlated with MSC concentrations as assessed by FACS analysis. The data from this study will help to characterize MSCs as a possible therapeutic intervention in fracture nonunion.

Myelodysplastic syndromes (MDS) encompass a heterogeneous group of age-related hematopoietic disorders characterized by ineffective and incomplete hematopoiesis leading to an increased risk of acute myeloid leukemia (AML). The development of accurate and easily used in vitro models is crucial for understanding the pathogenesis of MDS and identifying potential therapeutic targets. Induced pluripotent stem cells (iPSCs) can be used to study MDS due to their ability to differentiate into any cell type depending on the environment. The main limitation is that the blood progenitors produced by iPSCs are of a fetal state, which hinders modeling of MDS, a disease of older adulthood. This study aimed to optimize the maturation state of blood progenitors derived from iPSCs by induction of the micro-RNA let-7, which, we hypothesize will increase the maturation and adult phenotypic state of hematopoietic progenitors. iPSC lines were generated from healthy controls and samples containing the SRSF2 mutation, a common mutation in MDS, containing a doxycycline-inducible, stabilized let-7 transgene. A stepwise differentiation efficiently drove the iPSCs toward hematopoietic progenitors and, subsequently, other mature lineages. The hematopoietic progenitors were characterized by assessing the expression of specific cell surface markers and functional properties using flow cytometry, colony-forming assays, and multi-lineage differentiation abilities. These findings demonstrate the potential of using iPSC engineering to create a novel model for MDS and other age-biased disorders by inducing let-7 expression in iPSCs and, when differentiating them, exposing them to doxycycline to promote an adult cell phenotype. This approach offers a valuable potential tool for elucidating the molecular mechanisms underlying these disorders and exploring potential therapeutic interventions. / 2026-03-13T00:00:00Z

No description available.

abstract: Induced pluripotent stem cells (iPSCs) are an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. We describe the establishment of primary dermal fibroblasts cells lines from 28 autopsy donors. These fibroblasts were used to examine the proliferative effects of establishment protocol, tissue amount, biopsy site, and donor age. As proof-of-principle, iPSCs were generated from fibroblasts from a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. To our knowledge, this is the first study describing autopsy donor-derived somatic cells being used for iPSC generation and subsequent neural differentiation. This unique approach also enables us to compare iPSC-derived cell cultures to endogenous tissues from the same donor. We utilized RNA sequencing (RNA-Seq) to evaluate the transcriptional progression of in vitro-differentiated neural cells (over a timecourse of 0, 35, 70, 105 and 140 days), and compared this with donor-identical temporal lobe tissue. We observed in vitro progression towards the reference brain tissue, supported by (i) a significant increasing monotonic correlation between the days of our timecourse and the number of actively transcribed protein-coding genes and long intergenic non-coding RNAs (lincRNAs) (P < 0.05), consistent with the transcriptional complexity of the brain, (ii) an increase in CpG methylation after neural differentiation that resembled the epigenomic signature of the endogenous tissue, and (iii) a significant decreasing monotonic correlation between the days of our timecourse and the percent of in vitro to brain-tissue differences (P < 0.05) for tissue-specific protein-coding genes and all putative lincRNAs. These studies support the utility of autopsy donors' somatic cells for iPSC-based neurological disease models, and provide evidence that in vitro neural differentiation can result in physiologically progression. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2013

The cancer stem cell (CSC) model of carcinogenesis and progression posits that within a tumor lies a subpopulation of cells that solely possess the ability to initiate a tumor and to differentiate into tumor cell lineages. Although the behavior of such cells is known, the challenge is to identify factors that characterize the CSC subpopulation. In this thesis, cell lines were identified that, when grown in three-dimensions, gave rise to organized colonies containing lumens originating from differentiating cells (“lumen lines”) and to densely-packed, spherical colonies originating from non-differentiating cells (“dense lines”). A microarray comparison of the pair identified genes upregulated in dense lines, including CD55 and BMI1, and in lumen lines, including CDX1 (Chapter 3). CD55 was used to isolate CD55high CSCs via flow cytometry that are able to self-renew, differentiate, initiate more colonies, proliferate more rapidly and exhibit an increased G2/M cell cycle population as opposed to unfractionated cells. Furthermore, the CD55high cells were able to give rise to more differentiated, lumen colonies in vitro, indicating that CD55 enriches for cells possessing a capacity to differentiate, and were able to enrich the CD24highCD44high putative CSC population further (Chapter 4). CDNA induction of BMI1 and CDX1 expression led to increased clonogenicity/proliferation and decreased clonogenicity/proliferation, respectively, and incorporation of a CDX1 reporter construct into the SW1222 cell line identified CDX1+ cells as a low-expressing population of CD55 (Chapter 5). Finally, co-culture of cell lines in an in vivo-like environment with intestinal myofibroblasts promoted the CSC population by enhancing clonogenicity, proliferation and expression of CD55 (Chapter 6). The results of this thesis implicate CD55 as a potent marker of colorectal cancer stemness, link the expression of BMI1 and CDX1 to cancer stemness and differentiation, respectively, and identify a role for the in vivo stem cell niche in maintaining the CSC population.

Scientific research is propelled by two objectives: Understanding and recognizing the essential biology of life, and deciphering this to uncover possible therapeutics in order to improve quality of life as well as relieve pain from disease. The aim of the work described in this thesis was to dissect the fundamental requirements of induced pluripotent stem cells both in pluripotency and differentiation with a particular focus on atrial specificity. Drug targeting of atrial-specific ion channels has been difficult because of lack of availability of appropriate cardiac cells, and preclinical testing studies have been carried out in non-cardiac cell lines, heterogeneous cardiac populations or animal models that have been unable to accurately represent human cardiomyocyte physiology. Therefore, we sought out to develop a preparation of cardiomyocytes showing an atrial phenotype with adult characteristics from human induced-pluripotent stem cells. A culture programme involving the use of Gremlin 2 allowed differentiation of cardiomyocytes with an atrial phenotype from human induced-pluripotent stem cells. When these differentiated cultures were dissociated into single myocytes a substantial fraction of cells showed a rod-shaped morphology with a single central nucleus that was broadly similar to that observed in cells isolated from atrial chambers of the heart. Immunolabelling of these myocytes for cardiac proteins (including RyR2 receptors, actinin-2, F-actin) showed striations with a sarcomere spacing of slightly less than 2um. The isolated rod-shaped cells were electrically quiescent unless stimulated to fire action potentials with an amplitude of 100 mV from a resting potential of approximately -70 mV. Proteins expressed included those for IK₁, IK_ur channels. Ca²⁺ Transients recorded from spontaneously beating cultures showed increases in amplitude in response to stimulation of adrenoceptors (both alpha and beta). With the aim of identifying key signaling mechanisms in directing cell fate, our new protocol allowed differentiation of human myocytes with an atrial phenotype and adult characteristics that show functional adrenoceptor signaling pathways and are suitable for investigation of drug effects.

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.

No description available.

In the work presented this thesis, two different conditions that are classified as protein misfolding diseases: Alzheimer's disease and lysozyme amyloidosis and proteins that could have a beneficial effect in these diseases, have been studied using Drosophila melanogaster, commonly known as the fruit fly. The fruit fly has been used for over 100 years to study and better understand fundamental biological processes. Although the fruit fly, unlike humans, is an invertebrate, many of its central biological mechanisms are very similar to ours. The first transgenic flies were designed in the early 1980s, and since then, the fruit fly has been one of the most widely used model organisms in studies on the effects of over-expressed human proteins in a biological system; one can regard the fly as a living, biological test tube. For  most proteins, it is necessary that they fold into a three-dimensional structure to function properly. But sometimes the folding goes wrong; this may be due to mutations that make the protein unstable and subject to misfolding. A misfolded protein molecule can then aggregate with other misfolded proteins. In Alzheimer's disease, which is the most common form of dementia, protein aggregates are present in the brains of patients. These aggregates are composed of the amyloid-β (Aβ) peptide, a small peptide of around 42 amino acids which is cleaved from the larger, membrane-bound, protein AβPP by two different enzymes, BACE1 and γ-secretase. In the first part of this thesis, two different fly models for Alzheimer’s disease were used: the Aβ fly model, which directly expresses the Aβ peptide, and the AβPP-BACE1 fly model, in which all the components necessary to produce the Aβ peptide in the fly are expressed in the fly central nervous system (CNS). The two different fly models were compared and the results show that a significantly smaller amount of the Aβ peptide is needed to achieve the same, or an even greater, toxic effect in the AβPP-BACE1 model compared to the Aβ model. In the second part of the thesis, these two fly models for Alzheimer’s disease were again used, but now to investigate whether lysozyme, a protein involved in our innate immune system, can counteract the toxic effect of Aβ generated in the fly models. And indeed, lysozyme is able to save the flies from Aβ-induced toxicity. Aβ and lysozyme were found to interact with each other in vivo. The second misfolding disease studied in this thesis is lysozyme amyloidosis. It is a rare, dominantly inherited amyloid disease in which mutant variants of lysozyme give rise to aggregates, weighing up to several kilograms, that accumulate around the kidneys and liver, eventually leading to organ failure. In the third part of this thesis, a fly model for lysozyme amyloidosis was used to study the effect of co-expressing the serum amyloid P component (SAP), a protein that is part of all protein aggregates found within this disease class. SAP is able to rescue the toxicity induced by expressing the mutant variant of lysozyme, F57I, in the fly's CNS. To further investigate how SAP was able to do this, double-expressing lysozyme flies, which exhibit stronger disease phenotypes than those of the single-expressing lysozyme flies previously studied, were used in the fourth part of this thesis. SAP was observed to reduce F57I toxicity and promote F57I to form aggregates with more distinct amyloid characteristics. In conclusion, the work included in this thesis demonstrates that: i) Aβ generated from AβPP processing in the fly CNS results in higher proteotoxicity compared with direct expression of Aβ from the transgene, ii) lysozyme can prevent Aβ proteotoxicity in Drosophila and could thus be a potential therapeutic molecule to treat Alzheimer’s disease and iii) in a Drosophila model of lysozyme amyloidosis, SAP can prevent toxicity from the disease-associated lysozyme variant F57I and promote formation of aggregated lysozyme morphotypes with amyloid properties; this is important to take into account when a reduced level of SAP is considered as a treatment strategy for lysozyme amyloidosis.

The aim of these studies was to increase our understanding of the non-structural proteins 3 and 5B (NS3 and NS5B) from the hepatitis C virus (HCV), and thereby contribute to the development of new and better drugs against HCV. By studying NS3 with substitutions identified to be associated with resistance to NS3 inhibitors in clinical trials (R155Q, A156T and D168V) it was found that not all inhibitors were affected, indicating that cross-resistance can be avoided. Substitutions at position 526 and 528 in the helicase domain of this bifunctional enzyme were introduced and the effect on the protease was investigated. These substitutions affected protease inhibition, showing that the helicase can influence the protease. This interplay between the two domains is also involved in the discovered activation of the enzyme at low inhibitor concentrations. Being a case of "enzyme memory", the phenomenon stresses the importance of using full-length NS3 for enzymatic assays. Inhibitors with novel designs, with presumed increased stability in vivo, were developed and, even though they were found to be of low potency, provide alternative ideas of how to design an inhibitor. Detailed information about the interaction between NS3 and its protein cofactor NS4A or several protease inhibitors were determined using a direct binding assay. The rate constants of the inhibitor interactions were affected by NS4A and it was also possible to visualize time-dependent binding inhibitors. A good correlation between interaction data (Kd or koff) and inhibition data (Ki) or replicon data (EC50) was also seen. The same approach was used for studying the interactions between NS5B and several non-nucleoside inhibitors, providing information of the chemodynamics and giving insights into inhibitor design.   Taken together, all these studies have resulted in new information about, and new tools with which to study, NS3 and NS5B. This is of great importance in the struggle to find new and potent drugs, leading to a cure for HCV infection.