12 research outputs found
Scale up of pluripotent stem cell cultivation and directed differentiation in a novel single-use bioreactor system
No full textScale up of pluripotent stem cell cultivation and differentiation in a novel single-use bioreactor system
No full textThe granulocytic inducer C/EBPalpha inactivates the myeloid master regulator PU.1
No full textVerschiedene Transkriptionsfaktoren spielen eine Rolle in der Entwicklung myeloischer Zellen. PU.1, ein Transkriptionsfaktor aus der ETS-Familie, ist sowohl für die Entwicklung lymphatischer als auch für die Entwicklung myeloischer Zellen von Bedeutung. Der Transkriptions
faktor C/EBPalpha, ein an den CCAAT-Enhancer bindendes Protein, ist hingegen wesentlich verantwortlich für die Entwicklung von Granulozyten. Wir stellen hier den ersten Nachweis dafür vor, dass C/EBPalpha die Funktion von PU.1 blockiert. PU.1 und C/EBPalpha können einander binden und sind in myeloischen Zellen kolokalisiert. Wenn C/EBPalpha PU.1 bindet, kann PU.1 einen minimalen Promotor mit Bindungsstelle für PU.1 nicht mehr aktivieren. Wir zeigen, dass der Leuzin-Zipper in der DNA-bindenden Domäne von C/EBPalpha mit der beta3/beta4-Region in der DNA-bindenden Domäne von PU.1 interagieren kann. Dadurch wird der Koaktivator von PU.1, c-jun, aus seiner Bindung mit PU.1 verdrängt. C/EBPalpha hemmt PU.1 nicht, indem es Korepressoren rekrutiert. Vielmehr vermindert C/EBPalpha die Expression von PU.1 in U-937-Zellen mit induzierbarem C/EBPalpha, indem es den autoregulatorischen Effekt PU.1 auf den PU.1-Promotor hemmt.
Ausserdem blockiert C/EBPalpha die durch PU.1 bedingte Entwicklung dendritischer Zellen aus CD34+ menschlichen Nabel blutzellen. Diese funktionelle Blockade von PU.1 durch C/EBPalpha könnte einer der Mechanismen sein, mit denen C/EBPalpha den durch PU.1 determinierten Weg der Zelldifferenzierung hemmt und sich Zellen unter dem Einfluss von C/EBPalpha zu Granulozyten entwickeln.Several transcription factors have been shown to play a role in myelopoiesis. PU.1, an ets-family transcription factor, is required for the development of both myeloid and lymphoid lineages while the transcription factor CCAAT/enhancer binding protein family member C/EBPalpha is essential for granulocytic development. We present here the first evidence that C/EBPalpha blocks the function of PU.1. PU.1 and C/EBPalpha interact physically and co-localize in myeloid cells. As a consequence of this interaction C/EBPalpha can inhibit the function of PU.1 to activate a minimal promoter containing only PU.1 DNA binding sites. We further demonstrate that the leucine zipper in the DNA binding domain of C/EBPalpha interacts with the beta3/beta4 region in the DNA binding domain of PU.1, and as a result displaces the PU.1 co-activator c-Jun. Finally, C/EBPalpha blocks PU.1 induced dendritic cell development from CD34+ human cord blood cells. The functional blocking of PU.1 by C/EBPalpha could be the mechanism by which C/EBPalpha inhibits the cell fates specified by PU.1, and directs cell development to the granulocytic lineage
Human pluripotent stem cell expansion in vertical-wheel bioreactors
No full textHuman induced pluripotent stem cells (hiPSC) have been regarded as an enormous breakthrough for medicine, since they can be derived from patients and be used to generate virtually all types of cells in the human body. One of the great bottlenecks in the usage of these cells for regenerative medicine or drug discovery applications is their expansion to relevant quantities. The Vertical-Wheel Bioreactors (PBS Biotech) present a novel scalable bioreactor configuration, whose agitation mechanism allows for homogeneous mixing conditions inside the single-use vessel, while conveying less shear stress to the cells when compared to traditional alternatives. These characteristics are advantageous for hiPSC expansion and thus, in this work, hiPSC were expanded in the Vertical-Wheel Bioreactor using different strategies, namely culturing the cells 1) on microcarriers and 2) as floating aggregates.
In the first approach, cells were cultured under xeno-free conditions, using the Essential 8 medium together with microcarriers and coatings devoid of any animal-derived products [1]. The culture conditions were optimized in terms of initial cell/microcarrier ratio, inoculation method and agitation rate, in the PBS 0.1 vessel (working volume: 80 mL). The cells were successfully expanded, maintaining a normal karyotype, up to a 6.7-fold increase in cell number, after 6 days. These optimized culture conditions were successfully repeated in a larger vessel, the PBS 0.5 (300 mL working volume) demonstrating the scalability of the Vertical-Wheel system.
In the second approach, hiPSC were expanded as floating aggregates, a methodology which does not require a separation step at the end of culture, to remove microcarriers, facilitating the downstream processing and Good Manufacturing Practice-compliance of the process. Cells were cultured in the PBS 0.1 (working volume: 60 mL), using mTeSR1, a serum-free medium and were monitored throughout culture regarding growth kinetics, aggregate size distribution and expression of pluripotency markers. The Vertical-Wheel Bioreactors were shown to efficiently keep the cell aggregates in suspension, under lower linear agitation speeds than an equivalent volume spinner flask (7 cm/s vs. 13 cm/s). Following 7 days of culture, cells were expanded up to a 5.2 ± 0.6-fold increase in cell number. The hiPSC aggregates increased in size over time, from an average diameter of 135 ± 61 µm to 397 ± 119 µm after 7 days. Pluripotency was maintained throughout time, as assessed by sustained high (\u3e 80%) expression of pluripotency markers OCT4, SOX2 and TRA-1-60, and low (\u3c 10%) expression of early differentiation marker SSEA-1. The results were validated using a second hiPSC line.
This study revealed that the Vertical-Wheel Bioreactor allows hiPSC growth either on microcarriers and as aggregates and suggested it to have advantages versus other configurations. These results make the Vertical-Wheel Bioreactor a promising platform for hiPSC expansion and, prospectively, differentiation approaches, contributing for the generation of bona fide cells for various biomedical applications, namely drug screening, disease modelling, and, ultimately, for Regenerative Medicine.
[1] Rodrigues CAV, Silva TP, Nogueira DES, Fernandes TG, Hashimura Y, Wesselschmidt R, Diogo MM, Lee B, Cabral JMS (2018), “Scalable Culture Of Human Induced Pluripotent Cells On Microcarriers Under Xeno‐Free Conditions Using Single‐Use Vertical‐Wheel™ Bioreactors”, Journal of Chemical Technology and Biotechnology, DOI: 10.1002/jctb.573
The zinc finger transcription factor Early Growth Response 2 (Egr-2) is an intrinsic regulator of T cell tolerance and homeostasis
No full textPhDTolerance of T cells to self-antigen is crucial to prevent the development of autoimmune
disease. How self-tolerance is controlled at the transcriptional level is, however,
unknown. We discovered that the transcription factor Early Growth Response 2 (Egr-2)
was expressed by tolerant T cells, and by CD4+CD44high T cells in the absence of overt
antigen stimulation, in vivo. To investigate the roles of Egr-2 in T cells, we generated
CD2 cell specific Egr-2 deficient (Egr-2 cKO) mice. The proliferation of Egr-2 cKO
CD44high T cells in vivo was markedly increased leading to progressive accumulation as
the mice aged. By 15 months of age CD4+CD44high cells constituted the predominant T
cell population in the peripheral lymphoid organs of Egr-2 cKO mice and expressed
high levels of the activation markers CD25 and CD69. In addition to this
lymphoproliferative disorder, 15 month old Egr-2 cKO mice showed signs of lupus-like
autoimmune disease. This autoimmune syndrome was characterised by
glomerulonephritis and proteinuria, infiltration of T cells into internal organs and,
crucially, auto-antibodies directed against nuclear components; the hallmark of lupus.
We observed decreased expression of the cyclin-dependent kinase inhibitor p21cip1 in
Egr-2 cKO CD4+CD44high T cells while TCR stimulation induced IFN-γ, and, in
particular, IL-17A and IL-17F expression was markedly increased. Consistent with
these findings, we observed increased numbers of IFN-γ and IL-17 producing CD4+ T
cells in Egr-2 cKO mice. The numbers of IFN-γ and IL-17 producing CD4+ T cells
further increased as the mice aged in parallel with the gradual development of
symptoms of lupus-like disease. These results demonstrate that Egr-2 is an intrinsic
regulator of both T cell homeostasis and T cell tolerance
The effect of in vitro culture on the stability, expansion and neuronal differentiation of human pluripotent cell lines
No full textPluripotent cells are defined by their ability to both self-renew and to differentiation into any cell type within the human body. As such, pluripotent cell lines are of great interest as starting material for drug screening and cell therapies for regenerative treatment of diseased tissues. Pluripotent cell lines were originally derived from germ cell tumors (embryonal carcinoma cells; EC), but have since been isolated and expanded from the inner cell mass of an early embryo (human embryonic stem cells; hESCs).
This project set out to investigate the relative ability of the pluripotent NTERA2 (EC) cell line and hESC lines: Shef3, HUES7 and RH5, to differentiate into neurons, using mechanical and enzymatic culture methods. Focus was placed on monitoring differentiation efficiency and function between the different lines.
The tumour origin, in addition to the poor reproducibility, low yield and reduced functionality of NTERA2 derived neurons, compared to primary neurons, makes their incorporation into regenerative therapies unlikely. As such, an enhanced neuronal differentiation protocol was developed for use in hESCs. Cell populations were monitored for relative changes in gene and protein expression at selected time points throughout differentiation using standard RT-PCR, Q-PCR and immuno fluorescence analysis. End stage neurons were screened for functionality using patch clamping and calcium imaging techniques. Monitoring of cellular behavior through differentiation was aided by the concurrent development of a portable microscope incubator stage in collaboration with Linkam scientific Ltd.
These data demonstrate a variation in the ability to generate neurons from pluripotent cell lines, and suggests a predetermined, preferential cell fate within each line, even at the level of pluripotency. This study also characterises in detail neuronal differentiation from pluripotent cells, adding to the understanding which is essential for translation into therapies for neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and Huntingdon’s disease
The function and origin of the CD4+ T cell in the classical Hodgkin lymphoma microenvironment
No full textPhDClassical Hodgkin lymphoma (CHL) is a germinal centre B cell malignancy where the bulk of the tumour comprises a non-clonal immune infiltrate enriched for CD4+ T cells. The role of these cells in the pathophysiology of CHL is poorly understood. Biomarkers predictive of clinical outcome in CHL are limited. This thesis examines microenvironment biomarkers with the goal of identifying the 10-20% of patients who are not cured by conventional therapy, and also investigates the function of the CD4+ T cell in CHL.
The prognostic power of FOXP3, a marker of regulatory T cells, CD68, a macrophage marker and CD20, a B cell marker, is validated in a new patient cohort and for the first time CD68 and FOXP3 are combined in a statistically robust scoring system. The data presented challenge the assumption that the microenvironment is Th2-polarised or senescent and demonstrates relative over-expression of T-BET, a Th1 marker and under-expression of PD1, a marker of senescence/exhaustion, with little evidence for Th2 marker expression. A cytokine-enriched in vitro culture system was developed demonstrating superior proliferation and longevity of CHL-derived T cells compared to non-malignant tissue-derived controls. These cells sustain expression of markers associated with proliferation and longevity (e.g. CD27, CD28) and remain functional (express cytokines) for many weeks. A panel of CD4+ T cell-specific markers was determined capable of differentiating CHL-derived from non-malignant or non-Hodgkin lymphoma-derived CD4+ T cells, in which markers of central memory (CD62L and CCR7) and early activation (CD69) are over-represented and markers of senescence (CD57 and PD1) are under-represented. Cytokine profiles were found to resemble Th1 (expression of IL2, IFN- and TNF expression) rather than Th2 (IL4, IL13, IL21, IL10 and IL6) responses.
The data presented confirm a new prognostic biomarker signature and show a Th1 rather than Th2-dominated microenvironment enriched for cytokine-secreting functional effector CD4+ T cells and long-lived, proliferative cells resembling central memory cells rather than hypoproliferative, anergic, non-functional T cells
The clinical and biological consequences of different FLT3 mutations in patients with AML
No full textCharacterisation of pathogenic markers in acute myeloid leukaemia (AML) may benefit
patients through refinement of risk stratification, application of molecularly targeted
therapy and improved understanding of AML biology. Whilst the presence of an
internal tandem duplication (ITD) within the fms-like tyrosine kinase-3 (FLT3) gene is
known to predict adverse outcome in young adults with AML, the clinical significance
of activating mutations in the tyrosine kinase domain (TKD) of FLT3 is unclear.
Therefore, a highly sensitive and specific denaturing-HPLC technique was developed to
screen for FLT3/TKDs in 1339 young adult patients with AML. Mutations were
detected in 161 (12%) cases, with a high incidence in patients with inv(16) (24%;
P=.009), a group in which FLT3/ITDs are uncommon. Unlike FLT3/ITDs, FLT3/TKDs
were associated with a favourable long-term outcome with a 10-year overall survival
(OS) of 36% for FLT3 WT, 51% for FLT3/ITD-TKD+ and 24% for FLT3/ITD+TKDpatients
(P<.001). The relative FLT3/TKD mutant level was highly variable with the
favourable prognosis residing in those patients with greater than 25% mutant alleles
(10-year OS of 59%), possibly reflecting the stage at which the mutation is acquired.
The mechanism of FLT3 activation also influenced sensitivity to FLT3-inhibitor
induced cytotoxicity, with FLT3/ITD+ blast cells more sensitive than FLT3/TKD+ cells.
Following lentiviral transduction, FLT3/ITD-transduced 32Dcl3 and Ba/F3 cells
demonstrated more rapid proliferation than FLT3/TKD-transduced cells. In an NB4 cell
line model of ATRA-induced myeloid differentiation, the presence of a FLT3/ITD
inhibited differentiation unlike a FLT3/TKD mutation which increased differentiation.
Furthermore, FLT3/ITD-transduced CD34 positive haematopoietic stem cells showed
greater cytokine-free survival of colony forming cells than FLT3/TKD-transduced cells.
Signalling studies also revealed that a FLT3/ITD induced stronger STAT5 activation
than a FLT3/TKD mutation. This unexpected genotype-phenotype relationship is of
direct relevance to current clinical decision making in AML, and may also provide
insights into mechanisms of chemoresistance
Methods affecting neuronal differentiation of human adult and pluripotent stem cells
No full textStem cells have significant potential to treat many age-related degenerative disorders that affect increasing numbers of people globally. This thesis investigated the capacity for omnicytes and human pluripotent stem cells (hPSC) to undergo directed differentiation towards neuronal cell types for the treatment of ischemic stroke and Parkinson’s disease respectively. Omnicytes express a range of markers related to pluripotency and plasticity; however they are a challenging cell source to use in the development of cell therapies. Variability in omnicyte quality was associated with patient source, disease type and cryopreservation, all of which affected the reproducibility of data. Successful generation of dopaminergic neurons was achieved using a suspension-based hPSC culture system, with modified culture medium designed to replicate endogenous signalling during development. Neurons expressing key markers of dopaminergic neurons were generated and were capable of producing dopamine in response to KCl challenge. The work also showed that transfection of saRNA could enhance the expression of key genes i.e. foxa2, lmx1a and TH, relative to mock transfected cultures, although not significantly. Results also showed that the specific hESC line used (Shef6) had a greater propensity for differentiation toward dopaminergic neurons than MSUH001 hiPSC. This work successfully used saRNA to enhance gene expression, but shows that transfection efficiency is a limiting factor to its use. However, if transfection efficiency can be addressed, saRNA will become a powerful tool in the generation of cell therapies, particularly if it can be applied to suspension cell cultures
Angiopoietin-2 and platelet-derived growth-BB factor cooperatively affect peripheral blood monocyte fibrinolysis
No full textIncludes bibliographical references
