Development of in-vitro in-silico technologies for modelling and analysis of haematological malignancies

Worldwide, haematological malignancies are responsible for roughly 6% of all the cancer-related deaths. Leukaemias are one of the most severe types of cancer, as only about 40% of the patients have an overall survival of 10 years or more. Myelodysplastic Syndrome (MDS), a pre-leukaemic condition, is a blood disorder characterized by the presence of dysplastic, irregular, immature cells, or blasts, in the peripheral blood (PB) and in the bone marrow (BM), as well as multi-lineage cytopenias. We have created a detailed, lineage-specific, high-fidelity in-silico erythroid model that incorporates known biological stimuli (cytokines and hormones) and a competing diseased haematopoietic population, correctly capturing crucial biological checkpoints (EPO-dependent CFU-E differentiation) and replicating the in-vivo erythroid differentiation dynamics. In parallel, we have also proposed a long-term, cytokine-free 3D cell culture system for primary MDS cells, which was firstly optimized using easily-accessible healthy controls. This system enabled long-term (24-day) maintenance in culture with high (>75%) cell viability, promoting spontaneous expansion of erythroid phenotypes (CD71+/CD235a+) without the addition of any exogenous cytokines. Lastly, we have proposed a novel in-vitro in-silico framework using GC-MS metabolomics for the metabolic profiling of BM and PB plasma, aiming not only to discretize between haematological conditions but also to sub-classify MDS patients, potentially based on candidate biomarkers. Unsupervised multivariate statistical analysis showed clear intra- and inter-disease separation of samples of 5 distinct haematological malignancies, demonstrating the potential of this approach for disease characterization. The work herein presented paves the way for the development of in-vitro in-silico technologies to better, characterize, diagnose, model and target haematological malignancies such as MDS and AML.Open Acces

Microencapsulación de células con fines terapeúticos: avances en la biocompatibilidad

233 p.: il.En la actualidad, los avances realizados en biología, genética y tecnología farmacéutica han permitido redefinir el concepto y las aplicaciones terapéuticas de la tecnología de microencapsulación de células. Hoy en día es posible inmovilizar células de distinto origen como células primarias, células genéticamente modificadas o incluso células madre en matrices poliméricas recubiertas por una membrana semipermeable que permite el tránsito de nutrientes, oxígeno, desechos celulares y productos terapéuticos secretados por las células,mientras que impide la entrada de moléculas y células inmunocompetentes. Sin embargo, una de las principales limitaciones que presenta este tipo de terapia es el rechazo por parte del sistema inmunológico del huésped receptor a las células implantadas, generalmente más acentuado cuando se lleva a cabo una aproximación xenogénica. Es un hecho conocido los importantes efectos secundarios producidos por los tratamientos crónicos con agentes inmunosupresores, por lo que cualquier avance que permita reducir la administración de éstos fármacos supondrá una importante mejora terapéutica. Además de esto, en la actualidad, para poder asegurar un correcto intercambio de material de investigación entre grupos colaboradores, la posibilidad de criopreservar además de células, estructuras biocompatibles desarrolladas para inmovilizar células, está siendo considerada por muchos grupos de investigación. En la presente memoria experimental se ha llevado a cabo una completa caracterización in vitro e in vivo de las microcápsulas APA (alginato-poli-l-lisina-alginato) que contenían mioblastos modificados genéticamente para liberar Epo, tras implante subcutáneo en un modelo murino alogénico. Además, la administración subcutánea de las microcápsulas criopreservadas durante 45 días, empleando un protocolo de enfriamiento lento y un 10% de DMSO, originó niveles sostenidos de Epo durante periodos prolongados de tiempo, en un modelo murino alogénico. La evaluación de los sistemas microencapsulados en un modelo xenogénico de rata, empleando una inmunosupresión transitoria basadoa en Tacrolimus (intramuscular), puso de manifiesto la importancia de realizar un periodo mínimo de inmunosupresión para mantener la viabilidad celular (4 semanas). Finalmente, se evaluó la administración conjunta de microesferas de PLGA de dexametasona junto a mioblastos secretores de Epo microencapsulados. La liberación de dexametasona a partir de las microesferas de PLGA proporcionó una herramienta farmacológica eficaz para prevenir la respuesta inflamatoria aguda provocada tanto por los biomateriales empleados en la generación de los sistemas como por los procedimientos quirúrgicos utilizados durante el proceso de implante en modelo murino. Los resultados obtenidos sugieren que la tecnología de microencapsulación de células puede ser una alternativa prometedora como sistema de liberación de productos terapéuticos,incluso en ambientes xenogénicos, empleando agentes inmunosupresores o anti-inflamatorios de forma transitoria que alivien la respuesta inmunológica al implante

Single and Dual Growth Factor Delivery from Poly-E-caprolactone Scaffolds for Pre-Fabricated Bone Flap Engineering.

Autografts are utilized to reconstruct large craniofacial bone defects; however, they result in donor site morbidity and defect geometry mismatch. Pre-fabricating a bone flap overcomes these drawbacks by integrating a patient specific scaffold with biologics, implanting it in the latissimus dorsi for a period of time and then transplanting it to the defect site as a partially remodeled construct. Polycaprolactone (PCL) is a biocompatible polymer that has mechanical properties suitable for bone tissue engineering. It must be integrated with biologics, however, to stimulate bone formation. The purpose of this work was to investigate bone regeneration using PCL and dual protein delivery. Bone morphogenetic protein-2 (BMP2) was adsorbed or conjugated onto a PCL scaffold in a clinically applicable setting (1 hour exposure at room temperature). Adsorbed BMP2 had a small burst release and was bioactive as indicated by C2C12 alkaline phosphatase expression. Interestingly, conjugated BMP2 had a sustained release but was not bioactive. When implanted subcutaneously, adsorbed BMP2 had increased bone volume (BV), elastic modulus, and ingrowth when compared to conjugation. Next, a collagen sponge was fabricated inside of a BMP2-adsorbed PCL scaffold to deliver vascular endothelial growth factor (VEGF). In addition, a modular PCL scaffold was developed in which the inner and outer modular portions were adsorbed with BMP2 and VEGF, respectively. In both systems, the VEGF was bioactive as indicated by increased endothelial cell proliferation. Dual delivery of BMP2+VEGF significantly increased BV from 4 to 8 weeks in an ectopic location, whereas, BMP2 alone did not. Finally, erythropoietin (EPO) and BMP2 were delivered from the outer and inner portions of the modular scaffold, respectively. The adsorbed EPO was bioactive as indicated by increased endothelial cell proliferation. At 4 weeks, dual EPO+BMP2 delivery significantly increased BV and ingrowth when compared to BMP2 alone. In conclusion, adsorbing BMP2 onto PCL may be optimal for clinical use. Delivering VEGF with BMP2 increases the bone regeneration rate from 4 to 8 weeks, and delivering EPO with BMP2 increases the BV at 4 weeks when compared to BMP2 alone, making multiple biologics delivery a promising method to increase the regenerated bone for pre-fabricated flaps.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111451/1/jankip_1.pd

Modeling of sickle cell anemia utilizing disease-specific induced pluripotent stem cells

Sickle cell anemia, caused by a point mutation that affects the HBB gene, is one of the most common human genetic disorders world-wide and has a high morbidity and mortality. A single FDA approved drug, hydroxyurea, is available for its ability to induce fetal hemoglobin expression, a major modulator of disease severity. Not every patient responds to treatment and additional HbF-inducing drugs are needed. In this thesis, I outline an induced pluripotent stem cell-based approach to the study of sickle cell disease (SCD). In the lab, we are currently building a library of SCD-induced pluripotent stem cell (iPSC) lines from a cohort of SCD patients with different genetic backgrounds and fetal hemoglobin levels. Utilizing a directed-differentiation approach, iPSC can give rise to hematopoietic progenitors that are similar to megakaryocyte-erythroid progenitors and can be further specified to become cells of either lineage. I examined the hypothesis that an iPSC-based system would be capable of producing fully functional erythroid cells and also recapitulate the variation in fetal hemoglobin levels seen in SCD patients. Directed-differentiation of iPSCs produced erythroid-lineage cells that were responsive to oxygen levels and erythropoietin, and were capable of further maturation and increased hemoglobin production. A humanized mouse model demonstrated the ability of these cells to localize to the bone marrow, contribute to the peripheral blood, and survive in vivo for over two weeks. The maturation capability of SCD-specific iPSC-derived erythroid lineage cells was correlated with hemoglobin expression and compared to control cells. Characterization of in vitro and in vivo differences between control and SCD-specific iPSC-derived erythroid-lineage cells demonstrated variation amongst individuals, similar to the variation seen in patients. Both of these patient-specific iPSC-based in vitro and in vivo models allow for the examination of the effect of genetic variability on fetal hemoglobin expression and also for the modeling of patient-specific responses to drug treatment. This information will facilitate better clinical treatment of the disease

Expression of growth factor receptors by haematopoietic stem and progenitor cells

The mechanisms that govern the lineage commitment of haematopoietic stem and progenitor cells (HSPCs) have been a topic of debate since the 1960s. Two models of lineage commitment have been described; a permissive model, in which haematopoietic growth factors (HGFs) stimulate proliferation and survival of distinct HSPC subpopulations to permit stochastic lineage-specification, and a deterministic model, which proposes that HGFs instruct HSPCs to differentiate towards a specific cell lineage. To provide further insight into whether HGFs provide instructive cues or act in a selective manner, this study has investigated the expression of fms-like tyrosine kinase 3 (Flt3), and the receptors for erythropoietin (EpoR) and macrophage-colony stimulating factor (M-CSFR) by single HSPCs within the bone marrow. Using single-cell qRT-PCR and flow cytometry, a large number of novel HSPC subpopulations have been identified based on receptor expression. Importantly, multiplex analysis of protein and mRNA expression revealed that the above receptors are rarely co-expressed during the early stages of haematopoiesis. Furthermore, Flt3 expression was identified within the haematopoietic stem cell compartment and in vitro analysis demonstrated that Flt3 ligand primarily acts on a subpopulation of downstream progenitors. These findings suggest that Flt3, EpoR and M-CSFR differentially regulate distinct early HSPC subpopulations

Development of a bio-inspired in silico-in vitro platform: towards personalised healthcare through optimisation of a bone-marrow mimicry bioreactor

Human red blood cell production, or erythropoiesis, occurs within bone marrow. Living animal and human cadaver models have demonstrated the marrow production of red blood cells is a spatially-complex process, where cells replicate, mature, and migrate between distinct niches defined by biochemical nutrient access, supportive neighboring cells, and environmental structure. Unfortunately, current research in understanding normal and abnormal human production of blood takes place in petri dishes and t-flasks as 2D liquid suspension cultures, neglecting the role of the marrow environment for blood production. The culture of blood on marrow-mimetic 3D biomaterials has been used as a laboratory model of physiological blood production, but lacks characterization. In this work, a 3D biomaterial platform is developed and to capture the in vivo blood production process and manufacture red blood cells from human umbilical cord blood. First ceramic hollow fibres were designed and tested to be incorporated and perfused in a 3D porous scaffold bioreactor to mimic marrow structure, provide a better expansion of cell numbers, a better diffusion of nutrients, and allow for the continuous, non-invasive harvest of small cells in comparison to static, unperfused biomaterials. Quantitative 3D image analysis tools were developed to spatially assess bioreactor distributions and associations of and between different cell types. Using these tools, the bioreactor distribution of red blood cell production were characterized within niches in collaboration with supportive, non-blood cell types and designed miniaturised, parallelised mini-bioreactors to further explore bioreactor capabilities. This thesis presents a hollow fibre bioreactor able to produce blood cells alongside supportive cells at 1,000-fold higher cell densities with 10-fold fewer supplemented factor than flask cultures, without serum, with one cell source, and continuously harvest enucleate red blood cell product to provide a physiologically-relevant model for cell expansion protocols.Open Acces

GM-CSF, IL-3 and G-CSF receptors on acute myeloid leukemia cells : function, regulation of expression, and ligand binding characteristics

IL-3, GM-CSF and G-CSF stimulate proliferation of human acute myeloid leukemia in vitro, but patterns of response among clinical cases are diverse. As described in Chapters 2 and 3, numbers and affinity of IL-3, GM-CSF and G-CSF receptors on cells of patients with AML were assessed and correlated with the proliferative response of the cells to IL-3, GM-CSF and G-CSF. In 13 of 15 cases of primary AML high affinity receptors for IL-3 were demonstrable on the cells. The average numbers of IL-3 receptors ranged from 21-145 receptors per cell. Normal white blood cells showed IL-3 receptors on their surface at similar densities. IL-3 receptor positivity often correlated with GM-CSF receptor positivity of AML, GMCSF receptors were demonstrated on the cells of 11 of 15 cases although average numbers of GM-CSF receptors were 10 times greater. The binding of G-CSF to normal and human AML cells was investigated in a series of 14 cases of primary AML. In all 14 cases specific receptors for G-CSF were demonstrated on purified blast cells. The average numbers of G-CSF receptors ranged from very low (specific binding scarcely detectable) to 428 receptors per cell. Normal granulocytes showed G-CSF binding sites on their surface at higher densities (703 to 1,296 sites/cell). GCSF receptors appeared of a single affinity type with a dissociation constant (Kct) ranging between 214 to 378 pM for AML blasts and 405 to 648 pM for normal peripheral blood granulocytes. The in vitro response of the cells to exogenous IL-3, GM-CSF or G-CSF was examined by measuring thymidine uptake. IL-3 and GM-CSF were potent inducers of DNA synthesis in vitro. In 12 of 14 cases including those with relatively low specific binding, G-CSF was a potent inducer of DNA synthesis of blasts in vitro; apparently relatively few receptors permit activation of AML cell growth. In a minority of cases however, the cells were unable to respond to IL-3 (4 of 15 cases), GM-CSF (4 of 15 cases) or G-CSF (2 of 14 cases) in spite of normal receptor availability on the cell surface. The inability of the cells to respond to stimulation might be caused by the inability of the receptors to transduce a secondary signal into the cells. The results from these experiments taken together did not provide evidence for overexpression or gross changes in receptor affinity as an explanation for AML growt

Neurexophilin1 suppresses the proliferation of hematopoietic progenitor cells

Indiana University-Purdue University Indianapolis (IUPUI)Neurexin I alpha (NRXN1α) and Dystroglycan (DAG1) are membrane receptors which serve as mutual ligands in the neuronal system. Neurexophilins (NXPHs) bind NRXN1α. Both NRXN1α and DAG1 were expressed in primitive populations in human cord blood (huCB) and murine bone marrow (muBM), with high concentrations of NXPHs in huCB plasma. We evaluated effects of these molecules on huCB and muBM hematopoietic progenitor (HPC) and stem (HSC) cells. At both a single and population level in vitro, we found that NXPH1 is a potent inhibitor of HPC proliferation acting through NRXN1α, an effect antagonized by DAG1. Injection of recombinant NXPH1 in vivo resulted in myelo- and lymphosuppression, with absolute numbers and cycling status of functional and phenotypically defined HPCs dose- and time-dependently decreased, and absolute numbers and cycling status of phenotypically defined longer-term repopulation HSCs increased. Competitive transplants showed an initial decrease in engraftment of NXPH1-treated cells, with an intermediate stage increase in engraftment. The increase in HSCs is at least partially mediated by the mTOR pathway and is thought to be homeostatic in nature. These results demonstrate the presence and function of a regulated signaling axis in hematopoiesis centered on NRXN1α and its modulation by DAG1 and NXPH1

The physiology and pathophysiology of hepcidin

Hepcidin, the critical iron regulatory factor, is a small peptide produced by the hepatocytes in response to increased body iron and inflammation. Circulating hepcidin controls both intestinal iron absorption and the release of iron from macrophages into plasma via a negative iron feedback system. I developed a novel competitive immunoassay for hepcidin using a polyclonal antibody produced against synthetic hepcidin. I validated the immunoassay and determined it was able to discriminate between healthy controls and selected disease groups. I compared the immunoassay against another established method of measuring hepcidin. I established that plasma hepcidin has a diurnal rhythm and that plasma hepcidin increases in response to intravenous iron in anaemic patients. Elevated levels of hepcidin in renal failure may have a role in the erythropoietin resistance observed in renal anaemia. In haemodialysis patients, hepcidin levels were significantly elevated, but there was no correlation with inflammatory markers. Elevated hepcidin was associated with anemia, but erythropoietin dose was negatively correlated with hepcidin, suggesting that erythropoietin suppresses hepcidin levels. This was confirmed in patients when hepcidin levels significantly decreased after erythropoietin treatment. The association between plasma hepcidin and other iron parameters were also examined in healthy controls after erythropoietin administration and venesection. Profound hepcidin suppression was observed after an erythropoietin dose, with peak levels reduced by 73.2%, and then gradually recovering over the following two weeks. A similar but more gradual change in hepcidin was observed after reducing hematocrit by removal of 250 mL blood. The studies suggested that the marrow–hepcidin axis is regulated by factors other than those specifically investigated. In summary, I have developed and validated a novel immunoassay for hepcidin which will allow further investigation of the vital role of this peptide in iron homeostasis and human physiology