Academic literature on the topic 'StemCell'

During the process of care of patients who suffered acute myocardial infarction we observed how complex is this disease. Nowadays, stemcell therapy opens a new horizon and hope for these patients. We conducted this study to improve our knowledge as nurses in face of this new therapeutic model. This study is a case report of a patient who suffered an anterior acute myocardial infarction and was admitted in a coronary care unit and enrolled in stemcell transplantation study protocol. The nurse has a fundamental role in the process of care during the hospitalization period clarifying the patient’s doubts and perspectives. The data was collected in patient’s record.During the patient length of stay we emphasized our nursing care based on his individual value.

Background: Mutations in ELANE are a cause of cyclic and severe congenital neutropenia (SCN), predominantly autosomal dominant disorders. ELANE encodes neutrophil elastase (NE), a tissue specific serine protease. Neutropenia occurs because mutant NE impairs survival and maturation of myeloid cells. More than 130 disease causing mutations in ELANE have been identified and cell permeable inhibitors of NE can correct the defect in cell survival and maturation in cellular models. (Makaryan et al. J Leukoc Biol. 2017;102:1143). CRISPR/Cas9 knock-out (KO) of ELANE in patients' hematopoietic stem cells (HSCs) corrects myeloid cell differentiation, (Nasri et al Haematologica 2020:105:598), but this approach targets both alleles, eliminates normal NE and may impair innate immunity. Hypothesis: Selective, single allele editing is a preferred strategy for correcting ELANE associated neutropenia and other autosomal dominant disorders. Methods: Emendo Biotherapeutics developed CRISPR-associated allele specific KO based on single nucleotide polymorphisms (SNPs) located in the vicinity of ELANE. Three SNPs cover ~80% of the population. In the current study, editing of the mutated allele was achieved by targeting SNP rs1683564 located downstream to the 3'UTR of ELANE and mediating a biallelic break in intron 4, with excision of the 3'UTR, destabilized the mutated allele transcript. For editing we utilized RNPs assembled from Emendo's proprietary nuclease, OMNI-50, a guide targeting the SNP and a guide targeting a region in intron 4. OMNI-50 has low off-target activity and high allele specific editing when targeting either the reference or the alternative form of the SNPs. The efficacy of this composition was tested on normal and SCN patient-derived HSCs harboring the relevant SNP. (Figure 1) A patient's bone marrow CD34+ cells with the S126L mutation and rs1683564 were enriched using RosetteSep™ Progenitor Cell Enrichment Cocktail (Stemcell Technologies), purified by density gradient centrifugation using Lymphoprep (Stemcell Technologies). The purified CD34+ cells were expanded 4 days in StemSpan™ SFEM II media, supplemented with StemSpan™ CD34+ Expansion Supplement. Expanded CD34+ cells were further purified using StemSep™ Human CD34 Positive Cocktail and purity verified by FACS analysis using CD34 and CD45 antibodies. Purified CD34+ cells were cryopreserved using serum free CryoStor® CS10 media (Stemcell Technologies) and stored in Liquid Nitrogen Vapor Phase. Normal human blood CD34+ progenitor cells, harboring rs1683564 were similarly treated as the control. Allele specific excision was determined by ddPCR. To determine excision efficiency for the patient and control, we amplified two regions in ELANE, Exon 1 and Exon 5 (excised region), using two different probes labeled with FAM and HEX, respectively. We used OMNI-50 nuclease (Emendo Bio, Ness Ziona, Israel), electroporation (Lonza 4D nucleofector) and StemCell media (Stemcell Technologies, Vancouver, BC) to modify, grow and differentiate the CD34+ cells. We assessed myeloid cell proliferation and differentiation using daily cell counts, cytospins stained with Diff-Quik, and CD14, CD66b, CD15, CD16, CD11b labeling and flow cytometry. Results: Unedited patient cells demonstrated significant abnormalities in proliferation and differentiation. Cytospins from unedited patient CD34+ cells at 14 days showed block of myeloid differentiation and 4-fold greater monocytes compared to control, consistent with the hematopoietic defect in SCN patients. Single allele ELANE KO significantly improved these cellular abnormalities: total cell proliferation increased 41% and CD14/CD66b and CD15/CD11b positive cells increased by 107% and 65.5%, respectively. Studies are in progress for additional patients harboring different ELANE mutations. Conclusions: Single allele KO may resolve concerns about unwanted effects of total elimination of the normal gene products with gene editing for autosomal dominant diseases and be an effective strategy for treating ELANE associate neutropenia. Disclosures Sabo: Emendo BioTherapeutics: Research Funding. Makaryan:Emendo BioTherapeutics: Research Funding. Poulsen:Emendo BioTherapeutics: Research Funding. Povodovski:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Dicken:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Herman:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Emmanuel:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Dale:X4 Pharmaceuticals: Research Funding; Emendo BioTherapeutics: Consultancy; X4 Pharmaceuticals: Honoraria.

Abstract Abstract 4586 Allogeneic hematopoetic transplantation is the only curative treatment in patients with fanconi anemia indicated as soon transfusion dependency occurs and a matched family or unrelated donor is available. Overall survival for matched or nearly (5/6) matched unrelated donor transplantation improved since the introduction of fludarabine-based conditioning regimens from 30% to 53%. Transplantation after malignant transformation, in heavily pretransfused patients or from alternative donors is associated with a dismal outcome. We report the case of a 7 year old girl with fanconi anemia who developed AML-M4 (monosomy 7 and gain on chromosome 3) with 24% peripheral blasts during donor search for HCT. Remission was acchieved with a mild chemotherapeutic regimen consisting of a single dose of intravenous (300mg/m2) and intrathecal (40mg) cytarabine and daily oral thioguanine (cumulative dose: 930mg/m2). 4 weeks after diagnosis of AML, the girl was transferred for allogeneic PBSCT (CD3/19 depleted) from an unrelated mismatched (7/10) donor. Conditioning regimen consisted of Fludarabine, Busulfex, ATG and OKT-3. Unfortunately, mixed chimerism was observed and on day +21 graft rejection was diagnosed. The patient underwent a second PBSCT from the same donor after conditioning with Fludarabine, Alemtuzumab and Cyclophosphamide and 12 days later received a stem-cell-boost due to poor hematological regeneration. Prolonged aplasia under treatment with G-CSF was suspicious for non-engraftment. The indication for a third PBSCT with her haploidentical father as donor was set. Conditioning in the aplastic patient with elevated autologous T-Lymphocytes consisted of ATG only for 3 days. Full engraftment was achieved on day +11. CMV-reactivation was effectively treated with a combination therapy of valganciclovir and foscavir. One year after the third PBSCT, the patient is in continuing morphologic remission with normal Cytogenetics and complete donor chimerism. Mild Graft vs. Host Disease (Grade I, Skin) dissolved after short term therapy with Prednison. Still she suffers under minor restrictions in her every day life. We demonstrate here the case of a CMV-positive patient with fanconi anemia and AML, polytransfused (27 red-cell-packs and 94 platelet-transfusions) who survived mismatched PBSCT of her CMV-positive father after two failed PBSCT from an alternative 7/10-mismatched donor. To our knowledge this is the first patient with fanconi anemia and AML, successfully transplanted from an haploidentical donor. Disclosures: No relevant conflicts of interest to declare.

Due to the severe consequences of viral infection in allogeneic hematopoietic stem cell transplantation (allo-HSCT) patients, the routine use of polymerase chain reaction (PCR) screening of viruses is common in clinic, while the sensitivities of molecular methods are not always sufficient for blood samples. We aimed to increase the detection efficiency of viremia in allo-HSCT patients with M1 bead enrichment. Blood samples of allo-HSCT patients with fever were collected. Simultaneously with analyses of real-time PCR without enrichment, M1 bead enrichment followed by real-time PCR was applied to detect possible viruses in these samples, and metagenomic next-generation sequencing analyses were also applied in 10 samples negative with real-time PCR without enrichment. Various species of viruses were detected with M1 enrichment method. Significantly, some viruses that had not been found by real-time PCR without M1 enrichment were also detected by those with M1 enrichment and verified by metagenomic next-generation sequencing analyses. Furthermore, blood samples enriched with M1 beads had lower Ct values of real-time PCR assay than those that had not been treated. In conclusion, M1 bead enrichment increased the detection efficiency of most viruses in hematological malignancy patients.

Il meccanismo alla base della riparazione di un danno a cellule renali è ancora oggi oggetto di dibattito e potrebbe coinvolgere sia cellule terminalmente differenziate che l'esistenza di cellule staminali multipotenti quiescenti. Sfruttando il saggio di formazione di sfere e la tecnica del FACS sorting, il nostro gruppo ha isolato una popolazione di cellule marcate col clorante PKH26 che avessero caratteristiche di cellule staminali renali adulte (PKHhigh). In precedenza abbiamo valutato la capacità di queste cellule di differenziare in vitro in lineage epiteliale, podocitico ed endoteliale. Abbiamo anche dimostrato che la popolazione PKHhigh è eterogenea nella sua composizione e all'interno delle nefrosfere le cellule con capacità simil-staminali sono PKHhigh/CD133+/CD24- (RSC). Abbiamo recentemente pubblicato che le nostre cellule delle nefrosfere, che comprendono cellule PKHhigh e la loro progenie PKHlow/neg, sono in grado di ripopolare scaffold renali umani decellularizzati. Con questa ricerca, ora abbiamo come scopo: i) di dimostrare le capacità rigenerative delle cellule staminali PKHhigh, anche in assenza della loro progenie PKHlow/neg. ii) di trovare la signature molecolare delle RSC che, tra PKHhigh, sono le cellule con capacità staminali più ampie. Per raggiungere questi obiettivi, abbiamo messo in coltura cellule PKHhigh su scaffold acellulari per 30 giorni e le cellule delle strutture ripopolate sono state caratterizzate mediante immunofluorescenza sequenziale, utilizzando specifici marcatori di differenziamento. Alcune strutture hanno indicato un differenziamento terminale in lineage epiteliale tubulare prossimale o distale o in quello endoteliale. Solo poche strutture mostravano invece la coespressione di alcuni o di tutti i marcatori testati, suggerendo un fenotipo ancora immaturo. Per la comprensione della signature molecolare delle RSC, è stata eseguita l'analisi trascrittomica delle RSC stesse, della loro progenie PKHlow/neg e di colture primarie terminalmente differenziate e sono stati evidenziati geni differenzialmente espressi (DEG). L’analisi bioinfomatica mediante Gene Set Enrichment Analysis ha suggerito uno stato immaturo delle nostre RSC, ma comunque diverso da cellule staminali embrionali. Infine, incrociando le liste di DEG sono stati ottenuti potenziali marcatori e alcuni di essi sono stati selezionati e validati. In conclusione, abbiamo evidenziato le capacità differenziative in senso epiteliale sia prossimale che distale ed endoteliale delle cellule PKHhigh. Inoltre, abbiamo dimostrato che le cellule PKHhigh, completamente prive di qualsiasi marcatore endoteliale, sono in grado di dare origine a strutture simil-endoteliali. La coespressione occasionale di marcatori epiteliali ed endoteliali in strutture ripopolate potrebbe indicare uno stato precoce di transizione verso un differenziamento terminale. Il possibile ruolo della progenie PKHlow / neg nel velocizzare il differenziamento terminale delle cellule PKHhigh dovrà essere chiarito. Infine, la signature delle RSC ottenuta potrà aprire la possibilità di isolamento diretto di cellule staminali renali adulte da tessuto renale normale.<br>The mechanism underlying the recovery of renal cell injury is still a matter of debate that concerns the involvement of fully differentiated cells, or the existence of quiescent scattered multipotent stem cells. By sphere forming assay and sorting, our group isolated a population of PKH26 most fluorescent cells with characteristics of adult renal stem-like cells (PKHhigh cells). We previously assessed the ability of PKHhigh cells to differentiate in vitro along epithelial, podocytic and endothelial lineages. We also demonstrated that PKHhigh population is heterogeneous in composition and within nephrospheres the cells with stem capacities are PKHhigh/CD133+/CD24- (RSC). We recently published that our nephrosphere cells, comprising PKHhigh cells and their PKHlow/neg progeny, are able to repopulate human decellularized renal scaffolds. With this research, we now aim: I) to prove the regenerative capabilities of PKHhigh stem-like cells, even in absence of their PKHlow/neg progeny. II) to find the molecular signature of RSC that among PKHhigh cells are those with the wider stem capacities. To reach these aims, we cultured isolated PKHhigh cells on acellular scaffolds for 30 days and the cells of the repopulated structures were characterized by sequential immunofluorescence using specific markers of differentiation. Some structures indicated a specific lineage differentiation into proximal and distal tubular epithelium and endothelium. Only few structures coexpressed some or all markers tested, indicating still immature phenotypes. For the disclosure of RSC molecular signature, transcriptomic analysis of RSC, of their PKHlow/neg progeny and of terminally differentiated primary cell cultures (PCC) was performed and differentially expressed genes (DEG) were evidenced. Bioinformatic Gene Set Enrichment Analysis suggested a renal immature status of our RSC, but different from embryonic stem cells. Crossing DEG lists potential markers were obtained and some of them were selected and validated. In conclusion, we highlighted the proximal and distal tubular epithelial and endothelial differentiative and regenerative abilities of PKHhigh cells. Moreover, we showed that PKHhigh cells, completely lacking any endothelial marker, were able to give rise to endothelial-like structures. The occasional coexpression of epithelial and endothelial markers in repopulated structures may indicate a transitional early status toward cell differentiation. The eventual role of the PKHlow/neg progeny of PKHhigh cells in speeding up the complete PKHhigh differentiation will be clarified. Finally, the obtained RSC signature would open the possibility for the direct isolation of adult renal stem-like cells from normal kidney tissue.

Pax7 is essential for the function of muscle satellite cells. It was previously determined that Pax7 methylation is essential for its transcriptional activity and function in satellite cells. We investigated whether Pax7 displays other post-translational modifications playing a role in its function. By mass spectrometry using immunoprecipitated FLAG-Pax7, we identified two lysine residues (K105 and K193) within the Pax7 protein that are acetylated. Pax7 transcriptional activity was monitored using a luciferase reporter under the control of Myf5, a Pax7 target gene. Treatment with Trichostatin A, a histone deacetylase inhibitor, increased significantly luciferase activity, but this activity was progressively loss when the created Pax7 mutants (K105R, K193R) were introduced. This suggests that acetylation plays a role in Pax7 transcriptional activity. To identify the acetyltransferase modulating Pax7 activity, we used a candidate approach. Myst1 is expressed in muscle satellite cells. Myst1 is known for its interaction with Wdr5 and MLL1/2, a known Pax7 partner. We detected an interaction between Pax7 and Myst1 by co-immunoprecipitation in fibroblasts and in primary myoblasts. Myst1 knockdown decreases Pax7 acetylation status suggesting Myst1 as Pax7’s acetylase. Myst1 siRNA knockdown negatively impacts many Pax7’s target genes as well as primary myoblast proliferation. Moreover, primary myoblasts treated with siMyst1 express higher levels of MyoD. In satellite cells Myst1 reduction through siRNA knockdown significantly reduces satellite cells progenitor expansion as well as increases MyoD expression. In all, Myst1 modulating Pax7 activity through acetylation represents a novel mechanism in muscle stem cell biology.

The bottlenose dolphin, Tursiops truncatus, is a mammal completely adapted to marine life. As long-lived, long-term residents of bays, sounds, and estuaries, high trophic feeder with large blubber stores, acting as depots for anthropogenic toxins, dolphins can be used as important indicators of the health of the marine ecosystems. Dolphins consume the same food and share the coastal environment with humans thus serving also as effective sentinels for public health problems. In this dissertation, the latest new technologies in cell and molecular biology are applied to the study of bottlenose dolphin biology and health. Included in the new technologies presented are the most used (microarray) and the most current (RNA sequencing, RNAseq) transcriptomic approaches, and novel technologies in stem cell research for subsequent in vitro testing. In the first investigation, an ex vivo assay was applied to gene expression microarrays. Small slices of bioptic skin from the bottlenose dolphin were cultured and exposed to different concentrations of perfluorooctanoic acid (PFOA) and bisphenol A (BPA), to assess the variation in global gene expression induced by two contaminants of emerging concerns (CECs). Transcriptomic changes were analyzed using a species-specific microarray. The skin transcriptome hold information on contaminant exposure, potentially predictive of longterm effects on dolphin health. Moreover, the differentially expressed genes have shown to be a good resource for identification of contaminant-specific biomarkers of exposure. Although microarrays technology continues to advance generating high throughput transcriptomics, it can only be employed to detect known sequences, and can’t be used to discover novel RNA forms and variants. The transcriptomics dramatic expansion observed in the past few years is mainly due to the developments in RNA-seq. In the second investigation presented, we performed longitudinal analysis of healthy managed bottlenose dolphins across seasons to establish baseline data for blood transcriptome analysis using RNA-seq. Dolphin genes showed less seasonal variability than that reported in humans, but the majority of significant genes identified were shared. Besides the seasonal component to changes in blood gene expression, associations of gene co-expression modules with age, gender or hematological parameters were also found. The last study presents the development of a protocol to isolate and characterize stem cells for the creation of a cell bank for dolphin’s therapeutic use and of an unlimited source of cells for research and biomedical applications. The protocol is developed to be a lessinvasive alternative of current mesenchymal stem cells isolation methods (such as the isolation of stem cells from adipose tissue) and it is focused on the placental tissue and the umbilical cord. Quantitative metrics were used to confirm the morphology of the cells, and gene and protein markers of stemness were also examined. The mesenchymal stem cells characterized were viable, with a proper karyotype, and responded positively to the verification tests employed. The application of the latest technologies to non-model organisms research, such as in the dissertation here presented, reveled a significant contribution for a deeper understanding of the biology and health of the dolphin, establishing its relevance for future studies of the impact of environmental challenges on the marine inhabitants.<br>Il tursiope, Tursiops truncatus, è un mammifero completamente adattato alla vita in mare. E’ un animale longevo, predatore di apice, ha un’attitudine costiera e residente, e inoltre presenta uno deposito di grasso sottocutaneo, in cui si accumulano facilmente tossine di origine antropica. Queste caratteristiche lo rendono un ideale indicatore dello stato di salute dell’ecosistema marino. La sua condivisione con l’uomo degli ambienti costieri marini, e del nutrimento che ne deriva, ne fanno anche un ottimo organismo sentinella per problemi di salute pubblica. In questa tesi è presentata l'applicazione delle più innovative tecnologie nel campo della biologia cellulare e molecolare allo studio della biologia e dello stato di salute del tursiope. Tra le nuove tecnologie presentate ci sono sia gli ultimi approcci di trascrittomica, come microarray e RNA-sequencing, sia le ultime novità nel campo delle colture cellulari, come l’utilizzo di cellule staminali. Il primo studio descritto presenta l’accoppiamento di un saggio ex vivo ad analisi di espressione genica, utilizzando un microarray specie-specifico. Fettine di tessuto cutaneo di tursiope sono state messe in cultura e trattate con diverse concentrazioni di bisfenolo A e di acido perfluoroottanoico, per analizzare variazioni dell’espressione genica indotta da questi due contaminanti, appartenenti della categoria dei contaminanti emergenti. Il trascrittoma della pelle ha dimostrato di essere informativo di esposizioni a contaminanti e potenzialmente predittivo dello stato di salute del tursiope. Inoltre, i geni differenzialmente espressi identificati tramite questa analisi hanno mostrato essere una buona fonte da cui selezionare biomarcatori per la valutazione dell’esposizione a contaminanti antropici. Sebbene la tecnologia dei microarray continui ad avanzare, generando trascrittomi a sempre più alta risoluzione, essa può essere impiegata solo per analizzare sequenze già note, tralasciando nuove informazioni su RNA e loro varianti. Negli ultimi anni la trascrittomica ha subito un notevole incremento, principalmente grazie all’impiego di next generation sequencing in RNA-seq. Il secondo studio affrontato in questa dissertazione ha utilizzato RNA-seq per un’analisi longitudinale attraverso le stagioni, di un gruppo di delfini monitorati mensilmente in ottime condizioni di salute, al fine di generare dati di riferimento per analisi trascrittomiche di sangue del tursiope. I geni espressi nelle quattro stagioni sono, per la maggior parte, gli stessi evidenziati da studi analoghi sull’uomo, se pur in numero notevolmente inferiore. Oltre alla variabilità introdotta dalla stagionalità, sono stati evidenziati moduli di co-espressione genica associati con età, genere e valori ematologici degli animali. L’ultima sperimentazione presentata descrive la messa a punto di un protocollo sviluppato per isolare e caratterizzare cellule staminali mesenchimali con un duplice scopo di creare una banca cellulare per uso terapeutico per il tursiope stesso, e di avere una fonte illimitata di cellule per ricerca ed applicazioni biomediche. Il protocollo prevede l’utilizzo del tessuto placentale e del cordone ombelicale e offre un’alternativa meno invasiva al metodo attuale di isolamento delle cellule staminali mesenchimali da tessuto adiposo. Le cellule staminali mesenchimali isolate si mantengono stabili nelle condizioni di coltura testate, con cariotipo specie-specifico, e rispondono positivamente ad ogni test di verifica di identità e comportamento. L’applicazione di tecnologie di avanguardia allo studio di un organismo non-modello, come nel caso del tursiope evidenziato in questa tesi, ha mostrato un sostanziale e approfondito contributo alla comprensione della biologia e dello stato di salute del tursiope, stabilendo la sua rilevanza in studi futuri incentrati sugli effetti dei cambiamenti ambientali sugli abitanti dell’ecosistema marino costiero.

Hepatozyten sind von großer Bedeutung in Klinik und Forschung. Da sie derzeit nicht kultiviert werden können wird vielfach versucht sie aus Vorläuferzellen zu generieren. In dieser Arbeit wurde die Entwicklung von zwei Stammzelllinien in einem xenogenen Transplantationsmodell mit immundefizienten Mäusen untersucht. Verwendet wurden Stammzellen aus humanem Nabelschnurblut und vordifferenzierte humane Monozyten, sogenannte Neohepatozyten. Es wurden jeweils 750.000 dieser Zellen in die Leber von NOD/SCID-Mäusen transplantiert und die Lebern im Verlauf explantiert. Die Identifikation der transplantierten Zellen im Gewebeschnitt gelang durch den Membranfarbstoff CM-DiI. Zur genetischen Charakterisierung wurde eine in situ Hybridisierung mit human- bzw. mausspezifischen Gensonden etabliert. So konnten die transplantierten Zellen sicher in den Mauslebern detektiert werden. Im Weiteren wurde immunhistochemisch humanes Albumin in Mauslebern nachgewiesen. Humanes Albumin diente als Differenzierungsmarker und zeigt eine Entwicklung der transplantierten Zellen in Richtung Hepatozyten an. Interessanterweise wurden bei der Analyse beider Zelllinien zwei Typen Humanalbumin-positiver Zellen beobachtet: Typ I Zellen traten meist in Gruppen auf, waren kleiner als Hepatozyten und trugen einen dichten, dezentral gelegenen Kern. Sie lagen im Parenchym oder in kleineren Gefäßen und waren in der Lage, wie Hepatozyten, Glycogen und Eisen zu speichern. Typ II Zellen traten einzeln auf und glichen in ihrer Form exakt den Hepatozyten. Durch die Kombination von Immunhistochemie und in situ Hybridisierung konnten die Humanalbumin-positiven Zellen genetisch charakterisiert werden. Typ I Zellen trugen einen menschlichen Kern. Ihre Entstehung kann durch Teildifferenzierung der Stammzellen erklärt werden. Typ II Zellen wiesen einen Mauskern auf. Ein möglicher Entstehungsmechanismus ist der horizontale Gentransfer nach Zerfall der transplantierten Zellen im Zuge einer Immunreaktion. Der formale Nachweis eines solchen Phänomens steht noch aus.

Human pluripotent stem cells are increasingly used for cell-based regenerative therapies worldwide, with the use of embryonic and induced pluripotent stem cells as potential treatments for a range of debilitating and chronic conditions. However, with the level of chromosomal aneuploidies the cells may generate in culture, their safety for therapeutic use could be in question. This study aimed to develop sensitive and high-throughput assays for the detection and quantification of human pluripotent stem cell aneuploidies, to assess any changes in their positioning in nuclei, as well as investigate the possible roles of lamins in the accumulation of aneuploidies. Using Droplet Digital PCR™, we optimised the detection of aneuploid cells in a predominantly diploid background. An assay was established for the sensitive detection of up to 1% of mosaicism and was used for the monitoring of low-level chromosome copy number changes across different cell lines, conditions and passages in the human pluripotent stem cells. In addition, fluorescence in-situ hybridisation was used to map genes ALB and AMELX on chromosomes 4 and X, respectively, in karyotype-stable chromosome X aneuploid lymphoblastoid cell lines. Our results demonstrated significant alternations in the gene loci positioning in the chromosome X aneuploid cell lines. Using the same established method, the positioning of ALB and AMELX was monitored, alongside the genomic instability with ddPCR™, in the different human pluripotent stem cell lines, conditions and passage. We demonstrated a highly plastic nuclear organisation in the pluripotent stem cells with many changes occurring within a single passage. Furthermore, these results were not exclusive to a single cell line or condition, regardless of the presence or absence of feeder cells and of passage number, and the flexibility of the chromatin organisation remained throughout the duration of the study. We demonstrated high levels of genomic instability with recurrent gains and losses in the AMELX copy number in the human embryonic stem cells during the course of our study, however no significant changes in their gene loci positioning from these abnormalities were observed. xvi | P a g e Additionally, we observed reduced levels of lamin B2 in the aneuploid lymphoblastoid cell lines and complete loss in some hPSC samples. Our results support recent findings that suggest a link between lamin B2 loss and the formation of chromosome aneuploidies in cell culture. In conclusion, our data demonstrates several key novel findings. Firstly, we have established a sensitive technique for the detection of up to 1% mosaicism, which to our knowledge is the most sensitive assay currently available. Secondly, we showed significant changes in the gene loci positioning between aneuploid and diploid cell lines. Thirdly, utilising our novel ddPCR™ assay, we demonstrated the karyotypical instability of hPCSs with consistent gains and/or loses of gene copy numbers in a short period of time in culture. When studying the effects of different growth conditions, we showed that the karyotypical instability was not exclusive to a single condition or a combination of conditions, and what is more, the karyotypical abnormalities detected were not observed to change the gene positioning of hPSCs significantly, with the genome organisation remaining plastic. Finally, our results support a potential association of lamin B2 loss and karyotypical instability. We conclude that more sensitive and robust techniques need to be readily used by clinicians for the screening of potential therapeutic hPSCs.

This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.<br>Bachelors<br>Burnett College of Biomedical Sciences<br>Molecular Biology and Microbiology