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Generation of early human neuroepithelial progenitors from primary cells for biomedical applications
(2018)
Patient-specific induced pluripotent stem cells (iPSCs) emerged as a promising cell source for disease modeling and drug screening as well as a virtually unlimited source for restorative therapy. The thesis deals with three major topics to help realizing biomedical applications with neural stem cells.
To enable the generation of transgene-free iPSCs, alternatives to retroviral reprogramming were developed. Hence, the adaptation and evaluation of reprogramming using excisable lentiviral constructs, Sendai virus (SeV) and synthetic mRNA-based methods was assessed in the first part of this thesis. hiPSCs exhibit the pluripotency markers OCT4, SSEA-4, TRA1-60 which were confirmed by immunofluorescence and flow cytometry. Besides, the potential to differentiate in cell types of all three germ layers was detected, confirming pluripotent identity of proliferating colonies resulting from various reprogramming strategies. However, major differences such as high efficiency with SeV in contrast to a relatively low efficiency with mRNA in regard to passage number and the phenotype of starting fibroblasts were observed. Furthermore, a prolonged clone- and passage-dependent residual presence of viral RNA genes was identified in SeV-iPSCs for up to 23 passages using RT-PCR underlining the importance of careful monitoring of clone selection. In contrast, viral-free reprogramming by synthetic mRNA represents a fully non-integrative approach but requires further refinement to be efficiently applicable to all fibroblasts.
The second part of this thesis deals with the establishment of a rapid monolayer approach to differentiate neural progenitor cells from iPSCs. To achieve this, a two-step protocol was developed allowing first the formation of a stable, primitive NPC line within 7 days which was expanded for 2-3 passages. In a second step, a subsequent adaptation to conditions yielding neural rosette-like NPCs followed. Both neural lines were demonstrated to be expandable, cryopreservable and negative for the pluripotency marker OCT4. Furthermore, a neural precursor identity including SOX1, SOX2, PAX6, Nestin was confirmed by immunofluorescence and quantitative RT-PCR. Moreover, the differentiation resulted in TUJ1-positive neurons and GFAP-positive astrocytes. Nonetheless, the outcome of glial differentiation from primitive NSCs remained low, whereas FGF/EGF-NPCs were efficiently differentiated into GFAP-positive astrocytes which were implicated in a cellular model of the blood brain barrier.
The third and major objective of this study was to generate human early neural progenitor cells from fetal brain tissue with a wide neural differentiation capacity. Therefore, a defined medium composition including small molecules and growth factors capable of modulation of crucial signaling pathways orchestrating early human development such as SHH and FGF was assessed. Indeed, specific culture conditions containing TGFβ inhibitor SB431542, SHH agonist Purmorphamine, GSK3β inhibitor CHIR99021 and basic FGF, but no EGF enabled robust formation of early neuroepithelial progenitor (eNEP) colonies displaying a homogeneous morphology and a high proliferation rate. Moreover, primary eNEPs exhibit a relatively high clonogenicity of more than 23 % and can be monoclonally expanded for more than 45 passages carrying a normal karyotype. Characterization by immunofluorescence, flow cytometry and quantitative RT-PCR revealed a distinct NPC profile including SOX1, PAX6, Nestin and SOX2 and Prominin. Furthermore, primary eNEPs show NOTCH and HES5 activation in combination with non-polarized morphology, indicative of an early neuroepithelial identity. Microarray analysis unraveled SOX11, BRN2 and other HES-genes as characteristic upregulated genes. Interestingly, eNEPs were detected to display ventral midbrain/hindbrain regional identity. The validation of yielded cell types upon differentiation indicates a strong neurogenic potential with more than 90 % of TUJ1-positive neurons. Moreover, astrocytes marked by GFAP and putative myelin structures indicating oligodendrocytes were identified. Electrophysiological recordings revealed functionally active neurons and immunofluorescence indicate GABAergic, glutamatergic, dopaminergic and serotonergic subtypes. Additionally, putative physiological synapse formation was observed by the presence of Synapsin and PSD-95 as well as by ultrastructural examination. Notably, rare neurons stained positive for the peripheral neuronal marker Peripherin suggesting the potential of eNEPS to give rise to cells of neural tube and neural crest origin. By the application of specific differentiation protocols an increase of TH-positive neurons or neural crest-derivatives such as putative A- and C-sensory neurons and mesenchymal cells was identified. Taken together, primary eNEPs might help to elucidate mechanisms of early human neurodevelopment and will serve as a novel source for cell replacement and further biomedical applications.
The pancreas and the small intestine are pivotal organs acting in close synergism to regulate glucose metabolism. After absorption and processing of dietary glucose within the small intestine, insulin and glucagon are released from pancreatic islet cells to maintain blood glucose homeostasis. Malfunctions affecting either individual, organ-specific functions or the sophisticated interplay of both organs can result in massive complications and pathologic conditions. One of the most serious metabolic diseases of our society is diabetes mellitus (DM) that is hallmarked by a disturbance of blood glucose homeostasis. Type 1 (T1DM) and type 2 (T2DM) are the main forms of the disease and both are characterized by chronic hyperglycemia, a condition that evokes severe comorbidities in the long-term. In the past, several standard treatment options allowed a more or less adequate therapy for diabetic patients. Albeit there is much effort to develop new therapeutic interventions to treat diabetic patients in a more efficient way, no cure is available so far. In view of the urgent need for alternative treatment options, a more systemic look on whole organ systems, their biological relation and complex interplay is needed when developing new therapeutic strategies for DM.
T1DM is hallmarked by an autoimmune-mediated destruction of the pancreatic β-cell mass resulting in a complete lack of insulin that is in most patients restored by applying a life-long recombinant insulin therapy. Therefore, novel regenerative medicine-based concepts focus on the derivation of bioartificial β-like cells from diverse stem cell sources in vitro that survive and sustain to secrete insulin after implantation in vivo. In this context, the first part of this thesis analyzed multipotent intestinal stem cells (ISCs) as alternative cell source to derive bioartificial, pancreatic β-like cells in vitro. From a translational perspective, intestinal stem cells pose a particularly attractive cell source since intestinal donor tissues could be obtained via minimal invasive endoscopy in an autologous way. Furthermore, intestinal and pancreatic cells both derive from the same developmental origin, the endodermal gut tube, favoring the differentiation process towards functional β-like cells. In this study, pancreas-specific differentiation of ISCs was induced by the ectopic expression of the pancreatic transcription factor 1 alpha (Ptf1a), a pioneer transcriptional regulator of pancreatic fate. Furthermore, pancreatic lineage-specific culture media were applied to support the differentiation process. In general, ISCs grow in vitro in a 3D Matrigel®-based environment. Therefore, a 2D culture platform for ISCs was established to allow delivery and ectopic expression of Ptf1a with high efficiency. Next, several molecular tools were applied and compared with each other to identify the most suitable technology for Ptf1a delivery and expression within ISCs as well as their survival under the new established 2D conditions. Success of differentiation was investigated by monitoring changes in cellular morphology and induction of pancreatic differentiation-specific gene expression profiles. In summary, the data of this project part suggest that Ptf1a harbors the potential to induce pancreatic differentiation of ISCs when applying an adequate differentiation media. However, gene expression analysis indicated rather an acinar lineage-determination than a pancreatic β-cell-like specification. Nevertheless, this study proved ISCs not only as interesting stem cell source for the generation of pancreatic cell types with a potential use in the treatment of T1DM but alsoPtf1a as pioneer factor for pancreatic differentiation of ISCs in general.
Compared to T1DM, T2DM patients suffer from hyperglycemia due to insulin resistance. In T2DM management, the maintenance of blood glucose homeostasis has highest priority and can be achieved by drugs affecting the stabilization of blood glucose levels. Recent therapeutic concepts are aiming at the inhibition of the intestinal glucose transporter Na+-D-Glucose cotransporter 1 (SGLT1). Pharmacological inhibition of SGLT1 results in reduced postprandial blood glucose levels combined with a sustained and increased Glucagon-like peptide 1 (GLP-1) secretion. So far, systemic side effects of this medication have not been addressed in detail. Of note, besides intestinal localization, SGLT1 is also expressed in various other tissues including the pancreas. In context of having a closer look also on the interplay of organs when developing new therapeutic approaches for DM, the second part of this thesis addressed the effects on pancreatic islet integrity after loss of SGLT1. The analyses comprised the investigation of pancreatic islet size, cytomorphology and function by the use of a global SGLT1 knockout (SGLT1-/-) mouse model. As SGLT1-/- mice develop the glucose-galactose malabsorption syndrome when fed a standard laboratory chow, these animals derived a glucose-deficient, fat-enriched (GDFE) diet. Wildtype mice on either standard chow (WTSC) or GDFE (WTDC) allowed the discrimination between diet- and knockout-dependent effects. Notably, GDFE fed mice showed decreased expression and function of intestinal SGLT1, while pancreatic SGLT1 mRNA levels were unaffected. Further, the findings revealed increased isled sizes, reduced proliferation- and apoptosis rates as well as an increased α-cell and reduced β-cell proportion accompanied by a disturbed cytomorphology in islets when SGLT1 function is lost or impaired. In addition, pancreatic islets were dysfunctional in terms of insulin- and glucagon-secretion. Moreover, the release of intestinal GLP-1, an incretin hormone that stimulates insulin-secretion in the islet, was abnormal after glucose stimulatory conditions. In summary, these data show that intestinal SGLT1 expression and function is nutrient dependent. The data obtained from the islet studies revealed an additional and new role of SGLT1 for maintaining pancreatic islet integrity in the context of structural, cytomorphological and functional aspects. With special emphasis on SGLT1 inhibition in diabetic patients, the data of this project indicate an urgent need for analyzing systemic side effects in other relevant organs to prove pharmacological SGLT1 inhibition as beneficial and safe.
Altogether, the findings of both project parts of this thesis demonstrate that focusing on the molecular and cellular relationship and interplay of the small intestine and the pancreas could be of high importance in context of developing new therapeutic strategies for future applications in DM patients.