Spelling suggestions: "subject:"STEM cells"" "subject:"STEM wells""
1101 |
Transplantace limbálních kmenových buněk a jejich využití k rekonstrukci povrchu oka / Limbal stem cell transplantation and their utilization for ocular surface reconstruction.Lenčová, Anna January 2015 (has links)
Aims: Limbal stem cell (LSC) deficiency is one of the most challenging ocular surface diseases. The aim of this thesis was to study damaged ocular surface reconstruction. Therefore, a mouse model of limbal transplantation was estab- lished. Furthermore, LSC isolation, transfer of LSCs and bone marrow-derived mesenchymal stem cells (MSCs) on nanofiber scaffolds were studied. Material and methods: Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were transplanted orthotopically into BALB/c mice. Graft survival, immune re- sponse and the effect of monoclonal antibodies (mAb) (anti-CD4 and anti-CD8 cells) were analyzed. Mouse LSCs were separated by Percoll gradient; subse- quently, they were analyzed for the presence of LSC and differentiation corneal epithelial cell markers and characteristics using real-time PCR and flow cytom- etry. Nanofiber scaffolds seeded with LSCs and MSCs were transferred onto the damaged ocular surface in mouse and rabbit models. Cell growth on scaffolds, post-operative inflammatory response and survival of transferred cell were ana- lyzed. Results: Limbal allografts were rejected promptly by the Th1-type of immune response (IL-2, IFN-γ) involving CD4+ cells and nitric oxide produced by macro- phages, contrary to the prevailing Th1 and Th2 immune responses (IL-4, IL-10) in...
|
1102 |
REGULATION AND FUNCTION OF HAM GENES AND MERISTEM DEVELOPMENT IN CERATOPTERIS RICHARDIIYuan Geng (12455814) 25 April 2022 (has links)
<p> </p>
<p>The growth of land plants depends on a group of pluripotent stem cells in a tissue called the meristem. Seed plants initiate and maintain different types of meristems at the asexual sporophyte stage, and they generate sexual gametophytes, which are dependent on their sporophytes and are devoid of a meristem. In contrast, aside from forming indeterminate meristems at the sporophyte stage, seedless vascular plants, including ferns, also develop meristems in their gametophytes to drive gametophyte development and formation of sexual organs. To date, compared to the well-characterized cell behaviors and regulatory pathways in the meristems of seed plants, the molecular and cellular basis of meristem development in seedless ferns is still poorly understood. </p>
<p>In several seed plants, the HAIRY MERISTEM (HAM) family transcription factors play important roles in maintaining the indeterminacy of shoot apical meristems and promoting the <em>de novo</em> formation of axillary meristems. In the first part of this dissertation, through constructing a comprehensive phylogeny, I found that HAM family members are widely present in land plants and duplicated in a common ancestor of flowering plants, leading to the formation of two distinct groups: type I and type II. In addition, HAM members from different seed plants and seedless plants are able to replace the roles of the Arabidopsis type-II <em>HAM</em> genes, maintaining established shoot apical meristems and promoting the initiation of new stem cell niches in Arabidopsis. Furthermore, preliminary functional studies of the <em>HAM </em>homolog (<em>CrHAM</em>) in the model fern<em> Ceratopteris richardii</em> suggest that CrHAM is required for maintaining the indeterminacy of multicellular meristems in Ceratopteris gametophytes. Collectively, these results indicate that HAM family members may serve as common regulators in control of meristem development in both seed plants and seedless vascular plants. </p>
<p>In the remaining chapter of this dissertation, long-term time-lapse confocal imaging was performed using Ceratopteris stable transgenic plants, in which each individual cell (nucleus) was labelled with a fluorescent marker. Real-time lineage, identity, and division activity of each single cell from meristem initiation to establishment in Ceratopteris gametophytes were then determined. Additionally, cell fate and lineage alterations during <em>de novo</em> formation of new meristems were examined by mechanical perturbations. These quantitative analyses lead to the conclusion that in Ceratopteris gametophytes, initiation and proliferation of multicellular meristems relies on a few marginal cell lineages. Once established, the meristem maintains an actively dividing zone during gametophyte development. Within the meristem, cell division is independent of cell lineages and marginal cells are more actively dividing than inner cells. The meristem also triggers differentiation of adjacent cells into egg-producing archegonia in a position-dependent manner. </p>
<p>In summary, this work provides insight into the evolution of key stem-cell regulators and advances the understanding of diversified meristem development in land plants. </p>
|
1103 |
Investigating the role of cell-autonomous ROS status in the regulation of hippocampal neural precursor cells in adult miceAdusumilli, Vijaya 16 November 2020 (has links)
Adult hippocampal neurogenesis entails a continued recruitment of neural precursor cells (NPCs) into active cell cycle and their progressive transition into post-mitotic granule cells. These adult born neurons integrate into the existing circuitry and confer structural plasticity, which aids in key hippocampal functions. For sustained neurogenesis, the cell cycle entry of the NPCs has to be tightly controlled. Environmental cues strongly, and differentially, regulate this checkpoint. Voluntary physical activity represents such an established strong stimulus that results in enhanced proliferation within the neurogenic niche. However, mechanistic insights into the maintenance and regulation of quiescence and the responsiveness of the NPCs to acute physical activity, as a form of adaptive neurogenesis, are yet to be elucidated. In my doctoral studies, we identified redox regulation as a key pathway regulating the cellular state equilibrium. I further explored the role of cellular oxidative stress in the neurogenic course and in adaptive neurogenic responses. Our results show that non-proliferative precursors within the hippocampal dentate gyrus, unlike in other stem cell systems, are marked by high levels of cellular reactive oxygen species (ROS). Using cytometric methodologies, ex vivo bioassays and transcriptional profiling, we revealed that classifying cells based on intracellular ROS content identified functionally defined sub-populations of adult NPCs. We propose that a drop in intracellular ROS content precedes the transition of cellular states, specifically from quiescence to active proliferation. Acute physical activity involves the activation of non- proliferating cells through a transient Nox2-dependent ROS surge in high-ROS, quiescent NPCs. In the absence of Nox2, baseline neurogenesis was unaffected, but the activity- dependent response was abolished. These findings shed new light on the discrete cellular events, which maintain the homeostasis between distinct cellular states of NPCs within the adult murine hippocampus.:Zusammenfassung 3
Summary 4
Acknowledgements 5
Index 8
List of figures 10
List of tables 11
Abbreviations 12
Publications 14
Introduction 15
Adult hippocampal neurogenesis 16
Adult subventricular neurogenesis 21
Methods to study adult neurogenesis 23
Environmental regulation of neurogenesis 26
Redox regulation in a stem cell 29
Working hypothesis 31
Specific aims 31
Materials and methods 32
Mice 34
Physical activity paradigm 35
Thymidine labelling and tissue preparation 35
Fluorescence immunohistochemistry 35
DG and SVZ dissection and dissociation 36
Flow cytometry 36
Gating for ROS classes 36
Neurosphere culture 37
Generation of monolayer culture 37
Inducing quiescence through BMP4 treatment 38
Next Generation sequencing (NGS) 38
RNA extraction 38
Quality control and differential expression 39
Functional enrichment and expression profiles 41
RNA isolation and quantitative RTPCR (qRT-PCR) 43
Ki67 immunochemistry and quantification of in vivo proliferation 45
Quantification and statistical analysis 46
Data and software availability 48
Results 49
Intracellular ROS content functionally delineates subpopulations of neural precursor cells 49
Resolution of ROS profiles of DG and SVZ and neurosphere bioassay 49
Distribution of Nes-GFP cells into different ROS classes 54
Neural precursors of the different ROS classes have distinct molecular profiles 55
Changes in intracellular ROS content precede cell fate changes 65
ROS profiling of other cell types within the DG 70
ROS profiling of Astrocytes and type-1 cells 70
ROS profiling of Doublecortin (Dcx)positive cells of the neurogenic lineage 74
ROS profiling of microglial cells within the DG 77
Resolving the response of Nes-GFP subpopulations to environmental stimulus 78
Nes-GFP+ cells of the hiROS class specifically respond to physical activity 81
Changes in ROS content are not driven by mitochondrial activity 83
In vitro monolayer culture of NPCs as an independent corroboration 86
Discussion 89
The organization of an active stem cell niche with respect to redox content 89
Cytometric classification of cells within the DG 91
Establishing the cellular states of redox defined subsets of Nes-GFP+ adult precursors within the DG 95
Timeline of baseline proliferation within precursors and identifying the subset of precursors responsive to de novo physical activity 97
Monolayer culture to study cellular states and redox regulation 100
Nox2 dependency as a discriminatory feature of adaptive neurogenesis 101
Conclusion 103
References 104
Declarations 122
Anlage 1 122
Anlage 2 124
|
1104 |
Novel techniques for engineering neural tissue using human induced pluripotent stem cellsDe la Vega Reyes, Laura 24 December 2019 (has links)
Tissue engineering (TE) uses a combination of biomaterial scaffolds, cells, and drug delivery systems (DDS) to create tissues that resemble the human physiology. Such engineered tissues could be used to treat, repair, replace, and augment damaged tissues or organs, for disease modeling, and drug screening purposes. This work describes the development and use of novel strategies for engineering neural tissue using a combination of drug delivery systems (DDS), human induced pluripotent stem cells (hiPSCs), and bioprinting technologies for the generation of a drug screening tool to be used in the process of drug discovery and development. The DDS consisted of purmorphamine (puro) loaded microspheres that were fabricated using an oil-in-water single emulsion with 84% encapsulation efficiency and showed the slow release of puro for up to 46 days in vitro. Puro and retinoic acid (RA)-loaded microspheres were combined with hiPSCs-derived neural aggregates (NAs) that differentiated into neural tissues expressing βT-III and showed increased neural extension. hiPCS-derived neural progenitor cells (NPCs) were bioprinted on a layer-by-layer using a fibrin based-bioink and extrusion based- bioprinting. The bioprinted structures showed >81% cellular viability after 7 days of culture in vitro and the expression of the mature motor neuron (MN) markers HB9 and CHAT. Lastly, hiPCS-derived NPCs were bioprinted in combination with puro and RA-loaded microspheres and cultured for 45 days in vitro. The microspheres slowly released the drug and after 30 and 45 days the tissues contained mature neurons, astrocytes and oligodendrocytes expressing CHAT, GFAP, and O4, respectively. Changes in membrane potential indicated tissue responsiveness to different types of treatments such as acetylcholine and gamma-aminobutyric acid (GABA). In the future the bioprinted tissues could contain localized regions of varied drug releasing microspheres using a concentration gradient to promote differentiation into specific cell types in order to create more complex tissues. Moreover, these tissues will benefit from the presence of a neurovascular unit (NVU). Upon validation, the engineered tissues could be used as preclinical tools to test potential drugs and be used for personalized medicine by using patient specific hiPSCs. / Graduate / 2020-11-19
|
1105 |
The Effects of Nano-Hydroxyapatite in a Double Antibiotic Paste-Loaded Methycellulose Carrier on Dental Pulp Stem CellsEverhart, Adam R. January 2019 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / The effects of hydroxyapatite in a DAP-loaded MC carrier on dental pulp stem cells
Introduction: Regenerative endodontic procedures (REP) require disinfection techniques to eliminate bacteria from the infected immature root canal system and promote new growth of the pulp-dentin complex. Double antibiotic paste (DAP), a mixture of ciprofloxacin and metronidazole, has shown efficacy in doing so while minimizing cytotoxicity on dental pulp stem cells (DPSC). Stem cells, scaffolding, and growth factors are necessary in the maturation, proliferation, and differentiation of mesenchymal stem cells into the root canal system. Nano-hydroxyapatite (n-HA) has a history of biocompatibility and, in addition, has shown promising effects as a tissue bioengineering material.
Objective: The aim of this in vitro study was to investigate the proliferation and mineralization of DPSC in the presence of 1% DAP and methylcellulose (MC) with varying concentrations of nano-hydroxyapatite.
Materials and Methods: DPSC were plated in 24-well plates containing culture media. The next day, semi-permeable 0.1 mm Transwell chambers were inserted into the wells to separate the reservoirs for medicaments. Treatment paste composed of methylcellulose containing 1% DAP with either 0.25%, 0.50%, or 1.0% nano-hydroxyapatite was added along with culture media. Methylcellulose alone and calcium hydroxide (Ultracal) were used as control groups. After 3 days, cells were evaluated for cytotoxic effects using an MTS proliferation assay (n = 10, in triplicate). DPSCs were also cultured with these medicaments for 7 days in osteogenic media and evaluated for alkaline phosphatase (ALP) activity and mineralization activity (n = 13, in triplicate). Comparisons between groups for differences in mineralization, BSA, and ALP activity were performed using analysis of variance (ANOVA), with different variances allowed for each group and a random effect included in the model to account for correlation within each of the three trials. A simulation-based model was used to adjust for multiple comparisons.
Results: Addition of n-HA treatment groups increased mineralization significantly greater than calcium hydroxide, with MC alone and MC+DAP+0.5% HA providing the greatest effect. Regarding ALP, all HA concentrations performed significantly greater than MC and DAP concentrations. Proliferation demonstrated similar metabolic activity in all experimental groups with few comparisons significant.
Conclusion: The challenge in REPs is to maintain survival, and preferably promote the proliferation and development of DPSCs into the pulp-dentin complex with a consistent treatment outcome. The combination of DAP with hydroxyapatite may allow for both disinfection and improved mineralization and cellular differentiation. This contribution has shown significant ability to increase stem cell differentiation into an osteogenic lineage as well as calcium deposition, indicating end goal results of regenerative procedures.
|
1106 |
The impact of hydroxyapatite on alkaline phosphatase activity and mineral deposition of dental pulp stem cells using a double antibiotic paste loaded methylcellulose carrierFischer, Benjamin I. January 2020 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic procedures (REPs) are a type of endodontic treatment aimed at replacing damaged tooth structures, including dentin and root structures, as well as cells of the pulp-dentin complex. Double antibiotic paste (DAP) has been shown to be efficacious in achieving disinfection of the root canal system while minimizing cytotoxicity to dental pulp stem cells (DPSCs). Hydroxyapatite (HA) is an extracellular, mineralized component of bone that has shown much promise as a scaffold in the field of regenerative medicine.
Objective: The objective of this study was to evaluate the effects of HA in a DAP loaded methylcellulose (MC) carrier on the differentiation and mineral deposition of DPSC over time.
Materials and Methods: DPSCs were plated in 24-well plates with culture media. The following day, semi-permeable 0.1 m chambers were inserted into the wells to separate the reservoirs and permit delivery of medicaments. 100 L treatment paste composed of MC with 1% DAP and either 0.5% or 1.0% nano-HA was added, followed by additional culture media. After 3 days of treatment, medicaments were removed and DPSCs were cultured for an additional 9 days with replacement of media every 3-4 days. At Day 12, DPSCs were evaluated for alkaline phosphatase (ALP) activity using a biochemical assay and mineral deposition using an Alizarin Red S Ca2+ staining assay (4 wells/group). Comparisons between groups were performed using one-way analysis of variance (ANOVA) with a 5% significance level used for all tests.
Results: A trend towards increased ALP and mineral deposition activity was noted among the groups with HA added to DAP with MC. Although these trends were not statistically significant, a trend towards increased ALP and mineral deposition was observed after 3-day medicament exposure. The results were similar to previous findings using 7-day medicament treatments.
Conclusion: The addition of HA showed a trend towards improved differentiation and mineral deposition of DPSCs compared to DAP with MC. Although additional studies are required, these results showed suggest that even with a shortened treatment time, increased differentiation and mineral deposition of DPSCs may be possible. This study provides additional support that low concentration DAP in a MC carrier has potential application in regenerative endodontic procedures. The novel addition of HA may provide additional osteogenic potential.
|
1107 |
Transcriptional characterization of osteogenic and adipogenic differentiation of human bone marrow derived mesenchymal stem cells in 2D and 3D peptide hydrogel culture system.Rony, R M Imtiaz Karim 17 December 2018 (has links)
No description available.
|
1108 |
Kultivace limbálních a mezenchymálních buněk na různých typech nosičů pro využití v oftalmologii. / The culture of limbal and mesenchymal cells on various feeders for their use in ophthalmology.Trošan, Peter January 2019 (has links)
P.Trošan Ph.D. Thesis Abstract Limbal stem cell deficiency (LSCD) is a disease characterized by the deficiency of stem cells in the limbus, which are responsible for the homeostasis and renewal of the corneal epithelium. This disorder results in corneal neovascularization, chronical inflammation and opacification, which may lead to loss of vision. The most successful treatment is the transplantation of limbal tissue or cultured limbal epithelial cells (LECs) onto the damaged ocular surface. The human amniotic membrane (HAM) is used as the feeder of the LECs culture, as well as for the LSCD treatment. HAM is also widely used in clinical practice, particularly for the treatment of chronic wounds. This dissertation is particularly concerned on cell therapy for LSCD, on preparation of cells suitable for grafting onto the ocular surface, on the improvement of the LECs culture conditions, and on the preparation of appropriate carrier for the transfer of cells onto the damaged cornea. During my work I have used a wide spectrum of methods, e.g. cell cultures (LECs, mesenchymal stem, amniotic epithelial, conjunctival epithelial, goblet and 3T3 cells), immunohisto- and immunocytochemistry, microscopy, proliferation and colony forming assays, reverse transcription and quantitative real-time PCRs and statistical...
|
1109 |
WNT7A and EGF Alter Myogenic Differentiation in hiPSCs Derived from Duchenne Muscular Dystrophy PatientsMadana, Maria 22 June 2023 (has links)
Duchenne Muscular Dystrophy (DMD) is a disorder caused by loss-of-function mutations in dystrophin, a critical protein that maintains muscle fiber integrity. Our lab discovered that dystrophin-deficient skeletal muscle stem cells, also known as satellite cells, cannot generate enough myogenic progenitors for proper muscle regeneration. Previously, we demonstrated that WNT7A, a protein expressed during muscle regeneration, stimulates symmetric division of satellite cells, and gives rise to two daughter satellite cells. Conversely, epidermal growth factor (EGF) induces asymmetric division, which generates one daughter satellite cell and one committed precursor cell. We aimed to investigate these satellite cell division mechanisms following WNT7A or EGF treatment in a human model using healthy and DMD-patient derived hiPSCs differentiated into the myogenic lineage. The presence of satellite-like cells was confirmed in both lines by their characteristic expression of PAX7 and other myogenic markers. Intriguingly, DMD-patient hiPSCs precociously differentiated compared to healthy control human induced pluripotent stem cells (hiPSCs). More notably, WNT7A treatment had a potent effect on the DMD differentiated cells. High content analysis revealed an expansion of the satellite-like cell pool as observed by a higher number of PAX7+ cells within the total population and gene expression analysis demonstrated a significant increase in global PAX7 expression. In contrast, EGF treatment reduced the number of PAX7+ cells and increased the proportion of MYOG+ cells within the myogenic population, indicating an increase in myogenic progenitors. Taken together, WNT7A and EGF can alter the myogenic differentiation program of healthy and DMD-patient derived hiPSCs by modulating the satellite-like cell division dynamics.
|
1110 |
Obstacles and Circumvention Strategies for Hematopoietic Stem Cell Transduction by Recombinant Adeno-associated Virus VectorsMaina, Caroline Njeri 18 March 2009 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / High-efficiency transduction of hematopoietic stem cells (HSCs) by recombinant adeno-associated virus serotype 2 (AAV2) vectors is limited by (i) inadequate expression of cellular receptor/co-receptors for AAV2; (ii) impaired intracellular trafficking and uncoating in the nucleus; (iii) failure of the genome to undergo second-strand DNA synthesis; and (iv) use of sub-optimal promoters. Systematic studies were undertaken to develop alternative strategies to achieve high-efficiency transduction of primary murine HSCs and lineage-restricted transgene expression in a bone marrow transplant model in vivo. These included the use of: (i) additional AAV serotype (AAV1, AAV7, AAV8, AAV10) vectors; (ii) self-complementary AAV (scAAV) vectors; and (iii) erythroid cell-specific promoters. scAAV1 and scAAV7 vectors containing an enhanced green-fluorescent protein (EGFP) reporter gene under the control of hematopoietic cell-specific enhancers/promoters allowed sustained transgene expression in an erythroid lineage-restricted manner in both primary and secondary transplant recipient mice.
Self complementary AAV vectors containing an anti-sickling human beta-globin gene under the control of either the beta-globin gene promoter/enhancer, or the human parvovirus B19 promoter at map-unit 6 (B19p6) were tested for their efficacy in a human erythroid cell line (K562), and in primary murine hematopoietic progenitor cells (c-kit+, lin-). These studies revealed that (i) scAAV2-beta-globin vectors containing only the HS2 enhancer are more efficient than ssAAV2-beta-globin vectors containing the HS2+HS3+HS4 enhancers; (ii) scAAV-beta-globin vectors containing only the B19p6 promoter are more efficient than their counterparts containing the HS2 enhancer/beta-globin promoter; and (iii) scAAV2-B19p6-beta-globin vectors in K562 cells, and scAAV1-B19p6-beta-globin vectors in murine c-kit+, lin- cells, yield efficient expression of the beta-globin protein. These studies suggest that the combined use of scAAV serotype vectors and the B19p6 promoter may lead to expression of therapeutic levels of beta-globin gene in human erythroid cells, which has implications in the potential gene therapy of beta-thalassemia and sickle cell disease.
|
Page generated in 0.0554 seconds