Development of a Piggybac based direct reprogramming system for derivation of integration free induced pluripotent stem cells

Induced pluripotent stem cells (iPSc) have great potential for applications in regenerative medicine, disease modeling and basic research. Several methods have been developed for their derivation. The original method of Takahashi and Yamanaka involved the use of retroviral vectors which result in insertional mutagenesis, presence in the genome of potential oncogenes and effects of residual transgene expression on differentiation bias of each particular iPSc line. Other methods have been developed, using different viral vectors (adenovirus and Sendai virus), transient plasmid transfection, mRNA transduction, protein transduction and use of small molecules. However, these methods suffer from low efficiencies; can be extremely labor intensive, or both. An additional method makes use of the piggybac transposon, which has the advantage of inserting its payload into the host genome and being perfectly excised upon re-expression of the transposon transposase. Briefly, a policistronic cassette expressing Oct4, Sox2, Klf4 and C-Myc flanked by piggybac terminal repeats is delivered to the cells along with a plasmid transiently expressing piggybac transposase. Once reprogramming occurs, the cells are re-transfected with transposase and subclones free of tranposon integrations screened for. The procedure is therefore very labor intensive, requiring multiple manipulations and successive rounds of cloning and screening. The original method for reprogramming with the the PiggyBac transposon was created by Woltjen et al in 2009 (schematized here) and describes a process with which it is possible to obtain insert-free iPSc. Insert-free iPSc enables the establishment of better cellular models of iPS and adds a new level of security to the use of these cells in regenerative medicine. Due to the fact that it was based on several low efficiency steps, the overall efficiency of the method is very low (<1%). Moreover, the stochastic transfection, integration, excision and the inexistence of an active way of selection leaves this method in need of extensive characterization and screening of the final clones. In this work we aime to develop a non-integrative iPSc derivation system in which integration and excision of the transgenes can be controlled by simple media manipulations, avoiding labor intensive and potentially mutagenic procedures. To reach our goal we developed a two vector system which is simultaneously delivered to original population of fibroblasts. The first vector, Remo I, carries the reprogramming cassette and GFP under the regulation of a constitutive promoter (CAG). The second vector, Eneas, carries the piggybac transposase associated with an estrogen receptor fragment (ERT2), regulated in a TET-OFF fashion, and its equivalent reverse trans-activator associated with a positive-negative selection cassette under a constitutive promoter. We tested its functionality in HEK 293T cells. The protocol is divided in two the following steps: 1) Obtaining acceptable transfection efficiency into human fibroblasts. 2) Testing the functionality of the construct 3) Determining the ideal concentration of DOX for repressing mPB-ERT2 expression 4) Determining the ideal concentration of TM for transposition into the genome 5) Determining the ideal Windows of no DOX/TM pulse for transposition into the genome 6) 3, 4 and 5) for transposition out of the genome 7) Determination of the ideal concentration of GCV for negative selection We successfully demonstrated that ENEAS behaved as expected in terms of DOX regulation of the expression of mPB-ERT2. We also demonstrated that by delivering the plasmid into 293T HEK cells and manipulating the levels of DOX and TM in the medium, we could obtain puromycin resistant lines. The number of puromycin resistant colonies obtained was significantly higher when DOX as absent, suggesting that the colonies resulted from transposition events. Presence of TM added an extra layer of regulation, albeit weaker. Our PCR analysis, while not a clean as would be desired, suggested that transposition was indeed occurring, although a background level of random integration could not be ruled out. Finally, our attempt to determine whether we could use GVC to select clones that had successfully mobilized PB out of the genome was unsuccessful. Unexpectedly, 293T HEK cells that had been transfected with ENEAS and selected for puromycin resistance were insensitive to GCV.

Differentiation of human pluripotent stem cells to the neuronal dopaminergic fate

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2013

Using induced pluripotent stem cells to investigate the mechanistic link between Gaucher disease and Parkinson related synucleinopathies

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Establishment of a protocol for the cryopreservation of cell sheets of adipose tissue stem cells

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

The activity of mouse Cerberus like 2 during cardiogenesis - genetic and morphogenetic studies

Cardiogenesis is a delicate and complex process that requires the coordination of an intricate network of pathways and the different cell types. Therefore, understanding heart development at the morphogenetic level is an essential requirement to uncover the causes of congenital heart disease and to provide insight for disease therapies. Mouse Cerberus like 2 (Cerl2) has been defined as a Nodal antagonist in the node with an important role in the Left-Right (L/R) axis establishment, at the early embryonic development. As expected, Cerl2 knockout mice (Cerl2-/-) showed multiple laterality defects with associated cardiac failure. In order to identify the endogenous role of Cerl2 during heart formation independent of its described functions in the node, we accurately analyzed animals where laterality defects were not present. We thereby unravel the consequences of Cerl2 lossof- function in the heart, namely increased left ventricular thickness due to hyperplasia of cardiomyocytes and de-regulated expression of cardiac genes. Furthermore, the Cerl2 mutant neonates present impaired cardiac function. Once that the cardiac expression of Cerl2 is mostly observed in the left ventricle until around midgestration, this result suggest a specific regulatory role of Cerl2 during the formation of the left ventricular myoarchitecture. Here, we present two possible molecular mechanisms underlying the cardiac Cerl2 function, the regulation of Cerl2 antagonist in activation of the TGFßs/Nodal/Activin/Smad2 signaling identified by increased Smad2 phosphorilation in Cerl2-/- hearts and the negative feedback between Cerl2 and Wnt/ß-catenin signaling in heart formation. In this work and since embryonic stem cells derived from 129 mice strain is extensively used to produce targeted mutants, we also present echocardiographic reference values to progressive use of juveniles and young adult 129/Sv strain in cardiac studies. In addition, we investigate the cardiac physiology of the surviving Cerl2 mutants in 129/Sv background over time through a follow-up study using echocardiographic analysis. Our results revealed that Cerl2-/- mice are able to improve and maintain the diastolic and most of systolic cardiac physiologic parameters as analyzed until young adult age. Since Cerl2 is no longer expressed in the postnatal heart, we suggest that an intrinsic and compensatory mechanism of adaptation may be active for recovering the decreased cardiac function found in Cerl2 mutant neonates. Altogether, these data highlight the role of Cerl2 during embryonic heart development in mice. Furthermore, we also suggest that Cerl2-/- may be an interesting model to uncover the molecular, cellular and physiological mechanisms behind the improvement of the cardiac function, contributing to the development of therapeutic approaches to treat heart failures.

Differential regulation of hippocampal neurogenesis by nitric oxide following seizures

The fact that the adult brain is able to produce new neurons or glial cells from neural stem cells (NSC) became one of the most interesting and challenging fields of research in neuroscience. Endogenous adult neurogenesis occurs in two main regions of the brain: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) in the dentate gyrus. Brain injury may be accompanied by increased neurogenesis, although neuroinflammation promotes the activation of microglial cells that can be detrimental to the neurogenic process. Nitric oxide (NO) is one of the factors released by microglia that can be proneurogenic. The mechanism by which NO promotes the proliferation of NSCs has been intensively studied. However, little is known about the role of NO in migration, survival and differentiation of the newborn cells. The aim of this work was to investigate the role of NO from inflammatory origin in proliferation, migration, differentiation and survival of NSCs from the dentate gyrus in a mouse model of status epilepticus. We also assessed neuroinflammation in the same injury model. Our work showed that NO increased proliferation of the early-born cells after seizures, but is detrimental for their survival. NO also increased migration of neuroblasts. Moreover, NO was important to maintain long-term neuroinflammation. Taken together, these results show that NO may be a good target to promote proliferation and migration of NSCs following seizures, but compromises survival of early-born cells.