Our objective was to address current cell source limitations in engineering pancreatic â-cells for the treatment of type 1 diabetes by investigating retroviral genetic modification of murine embryonic stem cells (mESC) with a murine stem cell virus (MSCV) encoding proendocrine transcription factor Neurogenin 3 (Ngn3). We found that expression of Ngn3 and the enhanced green fluorescent protein (eGFP) reporter gene were both significantly silenced in genetically modified mESCs. To overcome this obstacle and enhance the efficiency of retroviral gene transfer to mESCs in general, we employed a virus-polymer complexation method to deliver more transgenes to mESCs. Despite increased transgene delivery and integration in mESCs, transgene expression did not increase. Results suggest mESCs may be restricted in several steps of retrovirus transduction. We then investigated which steps of the virus lifecycle restrict efficient transduction of mESCs by using a recombinant MMuLV-derived retrovirus and a recombinant HIV-1-derived lentivirus to compare three major steps in the transduction of mESCs and NIH 3T3 cells - virus binding, virus integration, and transgene expression. We found that retroviruses and lentiviruses similarly bind 3 or 4-fold less efficiently to R1 mES cells than to NIH 3T3 fibroblasts. We also detected 3-fold fewer integrated retrovirus transgenes and 11-fold lower expression levels in NIH 3T3 cells, suggesting the primary limitation to retrovirus transduction may be low levels of transgene expression. In contrast we detected 10-fold fewer integrated lentivirus transgenes and 8-fold lower expression levels, suggesting lentivirus transduction may be limited by inefficient intracellular post-binding steps of transduction. We then investigated whether depletion of linker histone 1 in mESCs would alleviate silencing of retrovirus transgenes and improve gene transfer by transducing histone H1c, H1d, H1e triple null mESCs with different recombinant vectors. We found this did not improve viral gene transfer. This research is significant for improving protocols for gene transfer to ES cells and facilitating the use of modified ES cells in regenerative medicine.

Indiana University-Purdue University Indianapolis (IUPUI) / Endothelial Colony Forming Cells (ECFCs) are highly proliferative endothelial progenitor cells with clonal proliferative potential and in vivo vessel forming ability. While endothelial cells have been derived from human induced pluripotent stem cells (hiPS) or human embryonic stem cells (hES), they are not highly proliferative and require ectopic expression of a TGFβ inhibitor to restrict plasticity. Neuropilin-1 (NRP-1) has been reported to identify the emergence of endothelial precursor cells from human and mouse ES cells undergoing endothelial differentiation. However, the protocol used in that study was not well defined, used uncharacterized neuronal induction reagents in the culture medium, and failed to fully characterize the endothelial cells derived. We hypothesize that NRP-1 expression is critical for the emergence of stable endothelial cells with ECFC properties from hES cells. We developed a novel serum and feeder free defined endothelial differentiation protocol to induce stable endothelial cells possessing cells with cord blood ECFC-like properties from hES cells. We have shown that Day 12 hES cell-derived endothelial cells express the endothelial markers CD31+ NRP-1+, exhibit high proliferative potential at a single cell level, and display robust in vivo vessel forming ability similar to that of cord blood-derived ECFCs. The efficient production of the ECFCs from hES cells is 6 logs higher with this protocol than any previously published method. These results demonstrate progress towards differentiating ECFC from hES and may provide patients with stable autologous cells capable of repairing injured, dysfunctional, or senescent vasculature if these findings can be repeated with hiPS.

Indiana University-Purdue University Indianapolis (IUPUI) / To cope with the high metabolic demands of the body during pregnancy, the maternal liver adapts by increasing its mass and size. This increase is proportional to the increase in total body weight during the course of gestation. The pregnancy-induced maternal liver growth is a result of both hepatocyte hypertrophy and hyperplasia. Microarray analysis of pregnant maternal livers shows markedly different gene expression profiles when compared to a non-pregnant state. Most interesting was the 2,500-fold up-regulation in the mRNA expression of Ascl1, a transcription factor responsible for the differentiation of neural progenitor cells into various neuronal types, during the second half of pregnancy. Our investigation aimed at (1) characterizing the identity of maternal hepatic Ascl1-expressing cells and (2) tracing the fate of Ascl1-expressing cells in the maternal liver during pregnancy. Timed pregnancies were generated and non-pregnant (NP) and pregnant maternal livers were harvested and analysed. To identify the maternal hepatic Ascl1-expressing cells we used the Ascl1GFP/+ reporter mouse line. NP and gestation day 15 (D15) maternal livers were immunostained for green fluorescent protein (GFP). The result shows that GFP-positive, Ascl1-expressing cells are hepatocyte-like cells, which are present in D15 maternal livers, but absent in NP livers. The Rosa26floxstopLacZ/ floxstopLacZ;Ascl1CreERT2/+ mouse line was used to trace the fate of Ascl1-expressing cells during pregnancy. LacZ staining of gestation day 13 (D13) and 18 (D18) maternal livers demonstrates that D13 hepatic Ascl1-expressing cells (labeled with LacZ) undergo hyperplasia to repopulate a large portion of D18 maternal livers. Furthermore, LacZ and HNF4α co-staining of D13 and D18 maternal livers shows the presence of two populations of LacZ-expressing cells: HNF4α+ population and HNF4α- population. HNF4α+ LacZ-expressing cells represent hepatocyte lineage cells that are derived from Ascl1-expressing cells. We observe that, towards the end of pregnancy, a considerable portion of the maternal liver is comprised of hepatocytes derived from Ascl1-expressing cells. Taken together, our preliminary study suggests that pregnancy induces maternal liver turnover via Ascl1-expressing cells.

Thesis (PhD (Physiology (Human and animal))-- University of Stellenbosch, 2007. / Definition: Stem cells are unspecialised cells with the capacity for long-term self-renewal and the ability to differentiate into multiple cell-lineages. The potential for the application of stem cells in clinical settings has had a profound effect on the future of regenerative medicine. However, to be of greater therapeutic use, selection of the most appropriate cell type, as well as optimisation of stem cell incorporation into the damaged tissue is required. In adult skeletal muscle, satellite cells are the primary stem cell population which mediate postnatal muscle growth. Following injury or in diseased conditions, these cells are activated and recruited for new muscle formation. In contrast, the potential of resident adult stem cell incorporation into the myocardium has been challenged and the response of cardiac tissue, especially to ischaemic injury, is scar formation. Following muscle damage, various growth factors and cytokines are released in the afflicted area which influences the recruitment and incorporation of stem cells into the injured tissue. Transforming Growth Factor-β (TGF-β) is a member of the TGF-β-superfamily of cytokines and has at least three isoforms, TGF-β1, -β2, and -β3, which play essential roles in the regulation of cell growth and regeneration following activation and stimulation of receptor-signalling pathways. By improving the understanding of how TGF-β affects these processes, it is possible to gain insight into how the intercellular environment can be manipulated to improve stem cell-mediated repair following muscle injury. Therefore, the main aims of this thesis were to determine the effect of the three TGF-β isoforms on proliferation, differentiation, migration and fusion of muscle progenitor cells (skeletal and cardiac) and relate this to possible improved mechanisms for muscle repair. The effect of short- and long-term treatment with all three TGF-β isoforms were investigated on muscle progenitor cell proliferation and differentiation using the C2C12 skeletal muscle satellite and P19 multipotent embryonal carcinoma cell-lineages as in vitro model systems. Cells were treated with 5 ng/mℓ TGF-β isoforms unless where stated otherwise. In C2C12 cells, proliferating cell nuclear antigen (PCNA) expression and localisation were analysed, and together with total nuclear counts, used to assess the effect of TGF-β on myoblast proliferation (Chapter 5). The myogenic regulatory factors MyoD and myogenin, and structural protein myosin heavy chain (MHC) were used as protein markers to assess early and terminal differentiation, respectively. To establish possible mechanisms by which TGF-β isoforms regulate differentiation, further analysis included determination of MyoD localisation and the rate of MyoD degradation in C2C12 cells. To assess the effect of TGF-β isoforms on P19 cell differentiation, protein expression levels of connexin-43 and MHC were analysed, together with the determination of embryoid body numbers in differentiating P19 cells (Chapter 6). Furthermore, assays were developed to analyse the effect of TGF-β isoforms on both C2C12 and P19 cell migration (Chapter 7), as well as fusion of C2C12 cells (Chapter 8). Whereas all three isoforms of TGF-β significantly increased proliferation of C2C12 cells, differentiation results, however, indicated that especially following long-term incubation, TGF-β isoforms delayed both early and terminal differentiation of C2C12 cells into myotubes. Similarly, myocyte migration and fusion were also negatively regulated following TGF-β treatment. In the P19 cell-lineage, results demonstrated that isoform-specific treatment with TGF-β1 could potentially enhance differentiation. Further research is however required in this area, especially since migration was greatly reduced in these cells. Taken together, results demonstrated variable effects following TGF-β treatment depending on the cell type and the duration of TGF-β application. Circulating and/or treatment concentrations of this growth factor could therefore be manipulated depending on the area of injury to improve regenerative processes. Alternatively, when selecting appropriate stem or progenitor cells for therapeutic application, the effect of the immediate environment and subsequent interaction between the two should be taken into consideration for optimal beneficial results.

Thesis (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Problem statement For utilitarians, human beings have intrinsic moral significance based on only two acquired characteristics: sentience, or the ability to suffer, and psychological personhood. Sentience is the entrance-requirement for moral significance, but does not justify a "right to life" claim; at most a "right" not to suffer. Personhood, described as some sort of self-conscious awareness with a concept of the future, may justify a "right to life" claim. However, since personhood is absent in prenatal beings, and only develops some time after birth, the implication is that such beings have little moral significance and may, for instance, be killed "at will". The moral problem that I address in this dissertation is to investigate, assess and evaluate the utilitarian position on the moral status or value of prenatal life. Methodology and results I firstly, on the basis of an extensive literature study, make a detailed analysis of the utilitarian position with reference to a number of themes that I have identified in their argument. This is followed by a critical philosophical evaluation of the utilitarian position, based on six particular arguments: • Utilitarianism is philosophically incoherent. It over-simplifies the moral argument in claiming that consequences are all that matter morally. Its underlying moral theory is at odds with moral claims contained in contemporary notions of human rights and individual justice. It ignores the moral significance of special obligations to special groups. • Utilitarianism potentially has unacceptable consequences. It IS inherently discriminatory and may lead to legitimate "slippery slope" fears. • Utilitarianism clashes with our fundamental moral intuitions on the value of prenatal life. These intuitions are cherished in most world religions. • Contrary to the utilitarian position, speciesism is inevitable to the human condition, especially argued from a position of existential phenomenology. Self- constitution, simultaneous constitution of the world as we know it, and the very possibility of morality are possible only within a particular notion of speciesism. • The potentiality of pre-persons to develop into persons cannot be as convincingly ignored as is done by the utilitarian. • There is a basic and underlying need and intuition to protect vulnerable human beings, of which pre-persons are exemplars. These notions clash with utilitarian theory. As an alternative, I introduce, set out and evaluate a two-phased position on the moral significance of pre-personal human life, a position of respectfulness of prenatal and pre-personal human life based upon its humanity, potentiality and separation-viability. This leads, firstly, to the conclusion of a graded, sliding scale conception of human prepersonal moral significance in line with the level of development and with the actuation of potentiality. Secondly, it leads to the conclusion that the advent of separationsurvivability (viability) is a morally significant cut-off point beyond which the human fetus may "normally" have a justifiable right to the continuation of its life. In as far as the application of my argument is concerned, I develop a "moderate" position with reference to the abortion debate. Whilst I recognize that all human prenatal beings of which it can be argued that they have a reasonable chance to develop their intrinsic potentiality, i.e., to become full-fledged persons, should have the opportunity to do so, I also recognize that neither this position, nor the complexities of life make it possible to hold "absolute" positions on the justifiability of abortion. I explore this extremely problematic notion in the text. That having been said, the advent of separation-survivability may imply a "moral cut-off point", beyond which termination is only rarely justified. I argue that I find no moral hindrance to wellmotivated research on human pre-embryos and stem cells. / AFRIKAANSE OPSOMMING: Probleemstelling Utilitariste huldig sterk omlynde standpunte oor die waarde van lewe. Hulle redeneer dat menslike (inderwaarheid, alle lewende) wesens slegs op grond van twee eienskappe intrinsieke morele waarde kan verwerf: sentiëntisme, d.i. die vermoë om lyding te ervaar, en persoonstatus. Sentiëntisme is 'n bepalende vereiste vir morele status, maar regverdig nie 'n "reg op lewe"-aanspraak nie. Persoonsyn, verstaan as 'n vorm van selfbewustheid tesame met 'n bewuste belang by die voortsetting van eie bestaan, mag wel so 'n aanspraak regverdig. Voorgeboortelike (en "voorpersoonlike") wesens is egter nie persone nie; hulle word eers (aansienlik) ná geboorte volwaardige persone. Die implikasie is dat sulke wesens weinig morele status het, en byvoorbeeld, na willekeur gedood mag word. Die morele probleem wat ek in hierdie dissertasie aanspreek is om die utilitaristiese beskouing ten opsigte van die morale status of waarde van voorgeboortelike lewe krities-filosofies te ondersoek en te evalueer. Metodologie en gevolgtrekkings Eerstens maak ek na aanleiding van 'n gedetaileerde literatuurstudie 'n in-diepte analise van van die utilitaristiese posisie aan die hand van 'n aantal temas wat ek in hul argument geïdentifiseer het. Daarna volg 'n krities-filosofiese evaluasie van die utilitaristiese posisie, aan die hand van ses argumente: • Utilitarisme is filosofies onsamehangend. Dit oorvereenvoudig die morele argument deur voor te gee dat gevolge al is wat moreel saakmaak. Die onderliggende utilitaristiese teorie bots met die morele eise vervat in kontemporêre sienings van menseregte en geregtigheid. Dit negeer die morele belangrikheid van spesiale verpligtinge teenoor spesiale belangegroepe. • Utilitarisme het potensieelonaanvaarbare gevolge. Dit IS inherent diskriminerend en kan lei tot onkeerbare glybaan ("slippery slope")-argumente. Utilitarisme bots met ons fundamentele morele intuïsies betreffende die waarde van voorgeboortelike lewe. Hierdie intuïsies word onder meer ondersteun deur die meeste hoofstroom godsdienste. • Spesiësisme is, in kontras met die utilitaristiese beskouing, onafwendbaar vir ons selfverstaan as mense, soos aangetoon kan word met 'n beroep op die eksistensiële fenomenologie. Self-konstituering, gelyktydige konstituering van die wêreld van die mens, en selfs die moontlikheid van moraliteit is slegs moontlik vanuit' n bepaalde spesiësistiese beskouing. • Die potensialiteit van "pre-persone" om tot volwaardige persone te ontwikkel kan nie, soos die utilitaris doen, sonder meer geïgnoreer word nie. • Daar is 'n basiese en onderliggende morele eis om swak en weerlose menslike wesens te beskerm. Hierdie idees bots lynreg met utilitaristiese teorie. As 'n alternatief tot die utilitaristiese beskouing, ontwikkel ek 'n twee-fase posisie betreffende die morele waarde van voorgeboortelike menslike lewe. Ek noem hierdie posisie agting vir voorgeboortelike en voor-persoonlike menslike lewe gebaseer op die menslikheid, potensialiteit en oorleefbaarheid van prenatale mense. Dit lei, eerstens, tot die gevolgtrekking van 'n gegradeerde glyskaal konsepsie van voor-persoonlike menslike morele waarde, min of meer parallel aan die vlak van ontwikkeling en die ontwikkeling van potensialiteit. Tweedens lei dit tot die gevolgtrekking dat die ontwikkeling van lewensvatbaarheid 'n moreel-beduidende afsnypunt is waarna die menslike fetus "normaalweg" aanspraak kan maak op 'n reg dat sy lewe voortgesit moet word. In soverre dit die toepassing van my argument betref, ontwikkel ek 'n "gematigde" posisie vis-á-vis aborsie. Ek redeneer dat alle menslike voorgeboortelike wesens wat 'n redelike kans het dat hul intrinsieke potensialiteit verder sal ontwikkel, die geleentheid daartoe gegun behoort te word. Ek aanvaar ook dat nog hierdie beskouing, nog die kompleksiteit van die menslike bestaan "absolute" posisies moreel regverdig. Die problematiek en inherente spanning tussen hierdie oënskynlik-opponerende posisies word in die teks bespreek. Nogtans beskou ek die ontwikkeling van lewensvatbaarheid as 'n moreel insiggewende afsnypunt waarna terminasie net in buitengewone omstandighede moreel regverdigbaar is.

This dissertation is the compilation of three separate works of research revolving around the theme of regulation of biomedical technologies that are either emerging or that have undergone significant developments over the past decade or so. Each of these three research works examines a legal response to a technological development in the areas of biotechnology and/or medicine and addresses one or more challenges - ethical, constitutional, legal or one that is related to public policy - created by that response. The first work of research, which was published in the Administrative Law Review in March 2008, examines the legality of the restrictions imposed by the administration of President George W. Bush on the funding of research involving human embryonic stem cells. Reaching the conclusion that the Bush Administration's actions were outright illegal in more than one way, the research highlights existing tensions in the division of decision-making power between the President and executive agencies and between Congress and the President. The second work of research, which was published in the Columbia Science and Technology Law Review in August 2010, reviews the regulation of genetic screening and testing of donated reproductive tissue in the United States. Analyzing the regulation in the federal, state and industry level, the research highlights significant shortcomings of the regulation of this area and, drawing on the experience of other countries, advocates the regulation of this area by the FDA. The third and last work of research of which this dissertation consists is dedicated to the examination of the newly created regime of statutory exclusivities afforded to biological pharmaceuticals under the Biologics Price Competition and Innovation Act (BPCIA) as it compares to the protection afforded to such products under patent law. The research concludes that allowing biological pharmaceuticals to benefits from parallel protection under both patent law and the statutory exclusivities regime established under BPCIA does not contribute to incentivizing innovation and might have undesirable ramifications from a public policy perspective. Hence, the research proposes limiting the protection afforded to biological pharmaceutical products, namely to the protection under either patent law or BPCIA, by suspending the ability to enforce patents covering biological pharmaceuticals against generic applicants under BPCIA. In addition, the research examines the proposition that under some circumstances it would be possible to substitute patent protection for statutory exclusivities.

Thesis (MA (Journalism))--University of Stellenbosch, 2006. / Moral questions arising from advances in science and technology are proliferating exponentially. Much controversy surrounds the ways in which biotechnology is used to eradicate a vast range of diseases and injuries. Stem cell research is one such way. Throughout the world stem cell research has been met with varying responses that range from opposition and criticism to approval and advocacy. As a result, it has attracted significant attention from the news media. The media have been accused of bias by focusing only on the controversial aspects of the research as opposed to reporting fully and fairly on the remarkable scientific advances. In this study I look at the patterns of media attention paid to stem cell research in the international weekly magazine Time between November 1998 and September 2005 inclusive. Contrary to the results expected on the basis of my literature study which pointed out the notion that the media tend to focus on sensational news more than non-controversial issues, I found that Time did a fair job in reporting on the scientific aspects of stem cell research. The percentage content of articles by year, focusing on scientific information of stem cells, dominated other news frames. The two years following the 2000 and 2004 American presidential elections, are however marked by the dominance of policy frames. This study found that Time covered controversial issues like embryonic stem cell research, public funding debates and political policy development in direct relation to their rise and fall on the political agenda in the United States.

Indiana University-Purdue University Indianapolis (IUPUI) / The maternal liver shows marked growth during pregnancy to accommodate the development and metabolic needs of the placenta and fetus. Previous study has shown that the maternal liver grows proportionally to the increase in body weight during gestation by hyperplasia and hypertrophy of hepatocytes. As the maternal liver is enlarged, the transcript level of Ascl1, a transcription factor essential to progenitor cells of the central nervous system and peripheral nervous system, is highly upregulated. The aims of the study were to (1) identify hepatic Ascl1-expressing cells, and (2) study the functions of Ascl1 in maternal liver during pregnancy. In situ hybridization shows that most cell types (parenchymal, nonparenchymal, and mesothelial cells) express Ascl1 mRNA in maternal livers during gestation and in male regenerating livers. Notably, hepatic mesothelial cells abundantly express Ascl1 during pregnancy and liver regeneration. Inducible ablation of Ascl1 gene during pregnancy results in maternal liver enlargement, litter size reduction, and fetal growth retardation. In addition, maternal hepatocytes deficient in Ascl1 gene lack majority of their cytosols and exhibit β-catenin nuclear translocation, while maintaining their cellular boundary and identity. In summary, in both maternal liver during pregnancy and regenerating liver, the expression of Ascl1 is induced in most cell types. Mesothelial cells are potential origin of Ascl1-expressing cells. Ascl1 gene is essential for the progression of normal pregnancy

Indiana University-Purdue University Indianapolis (IUPUI) / Photoreceptors are a class of sensory neuronal cells that are deleteriously affected in many disorders and injuries of the visual system. Significant injury or loss of these cells often results in a partial or complete loss of vision. While previous studies have determined many necessary components of the gene regulatory network governing the establishment, development, and maintenance of these cells, the necessary and sufficient profile and timecourse of gene expression and/or silencing has yet to be elucidated. Arduous protocols do exist to derive photoreceptors in vitro utilizing pluripotent stem cells, but only recently have been able to yield cells that are disease- and/or patient-specific. The discovery that mammalian somatic cells can be directly reprogrammed to another terminally-differentiated cell phenotype has inspired an explosion of research demonstrating the successful genetic reprogramming of one cell type to another, a process which is typically both more timely and efficient than those used to derive the same cells from pluripotent stem cell sources. Therefore, the emphasis of this study was to establish a novel system to be used to determine a minimal transcriptional network capable of directly reprogramming mouse embryonic fibroblasts (MEFs) to rod photoreceptors. The tools, assays, and experimental design chosen and established herein were designed and characterized to facilitate this determination, and preliminary data demonstrated the utility of this approach for accomplishing this aim.

Indiana University-Purdue University Indianapolis (IUPUI) / Human induced pluripotent stem (iPS) cells have the unique ability to differentiate into 200 or so somatic cell types that make up the adult human being. The use of human iPS cells to study development and disease is a highly exciting and interdependent field that holds great promise in understanding and elucidating mechanisms behind cellular differentiation with future applications in drug screening and cell replacement studies for complex and currently incurable cellular degenerative disorders. The recent advent of iPS cell technology allows for the generation of patient-specific cell lines that enable us to model the progression of a disease phenotype in a human in vitro model. Differentiation of iPS cells toward the affected cell type provides an unlimited source of diseased cells for examination, and to further study the developmental progression of the disease in vitro, also called the “disease-in-a-dish” model. In this study, efforts were undertaken to recapitulate the differentiation of distinct retinal cell affected in two highly prevalent retinal diseases, Usher syndrome and glaucoma. Using a line of Type III Usher Syndrome patient derived iPS cells efforts were undertaken to develop such an approach as an effective in vitro model for studies of Usher Syndrome, the most commonly inherited disorder affecting both vision and hearing. Using existing lines of iPS cells, studies were also aimed at differentiation and characterization of the more complex retinal cell types, retinal ganglion cells (RGCs) and astrocytes, the cell types affected in glaucoma, a severe neurodegenerative disease of the retina leading to eventual irreversible blindness. Using a previously described protocol, the iPS cells were directed to differentiate toward a retinal fate through a step-wise process that proceeds through all of the major stages of neuroretinal development. The differentiation process was monitored for a period of 70 days for the differentiation of retinal cell types and 150 days for astrocyte development. The different stages of differentiation and the individually derived somatic cell types were characterized by the expression of developmentally associated transcription factors specific to each cell type. Further approaches were undertaken to characterize the morphological differences between RGCs and other neuroretinal cell types derived in the process. The results of this study successfully demonstrated that Usher syndrome patient derived iPS cells differentiated to the affected photoreceptors of Usher syndrome along with other mature retinal cell types, chronologically analogous to the development of the cell types in a mature human retina. This study also established a robust method for the in vitro derivation of RGCs and astrocytes from human iPS cells and provided novel methodologies and evidence to characterize these individual somatic cell types. Overall, this study provides a unique insight into the application of human pluripotent stem cell biology by establishing a novel platform for future studies of in vitro disease modeling of the retinal degenerative diseases: Usher syndrome and glaucoma. In downstream applications of this study, the disease relevant cell types derived from human iPS cells can be used as tools to further study disease progression, drug screening and cell replacement strategies.