Cell Reprogramming, IPS Limitations, and Overcoming Strategies in Dental Bioengineering

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Uso de células iPS en terapia regenerativa renal: obtención de células iPS mediante vector no integrativo y diferenciación hacia podocitos.

[ES]En las sociedades modernas existe una creciente preocupación por el aumento de la incidencia de la enfermedad renal crónica. Debido a la deficiencia de donantes de órganos y al elevado coste del tratamiento de diálisis, existe la necesidad de desarrollar nuevos tratamientos para estos pacientes. La medicina regenerativa basada en la aplicación de células iPS es una opción prometedora para el tratamiento de esta enfermedad. Sin embargo, la falta de conocimientos sobre el estado pluripotencial de las células y sobre su proceso de diferenciación, así como las limitaciones derivadas del propio procedimiento de reprogramación, impiden su aplicación clínica en un futuro inmediato. Para que se convierta en realidad, numerosas investigaciones se están llevando a cabo con el objetivo de mejorar el procedimiento y hacerlo adecuado para su aplicación clínica. En este trabajo se propone un método que permitiría obtener células iPS a partir de células mesangiales mediante la transfección con un vector no integrativo, el virus Sendai, portador de los genes Oct3/4, Sox2, Klf4 y c-Myc. Al tratarse de un vector no integrativo, se minimizaría el efecto del proceso de reprogramación sobre la estabilidad del genoma celular. Además, en este proyecto se estudiará la capacidad de las células iPS obtenidas para diferenciarse en células progenitoras de podocitos que puedan ser aplicadas específicamente en terapias regenerativas para enfermos renales crónicos.

Direct reprogramming of fibroblasts into endothelial cells capable of angiogenesis and reendothelialization in tissue-engineered vessels

The generation of induced pluripotent stem (iPS) cells is an important tool for regenerative medicine. However, the main restriction is the risk of tumor development. In this study we found that during the early stages of somatic cell reprogramming toward a pluripotent state, specific gene expression patterns are altered. Therefore, we developed a method to generate partial-iPS (PiPS) cells by transferring four reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) to human fibroblasts for 4 d. PiPS cells did not form tumors in vivo and clearly displayed the potential to differentiate into endothelial cells (ECs) in response to defined media and culture conditions. To clarify the mechanism of PiPS cell differentiation into ECs, SET translocation (myeloid leukemia-associated) (SET) similar protein (SETSIP) was indentified to be induced during somatic cell reprogramming. Importantly, when PiPS cells were treated with VEGF, SETSIP was translocated to the cell nucleus, directly bound to the VE-cadherin promoter, increasing vascular endothelial-cadherin (VE-cadherin) expression levels and EC differentiation. Functionally, PiPS-ECs improved neovascularization and blood flow recovery in a hindlimb ischemic model. Furthermore, PiPS-ECs displayed good attachment, stabilization, patency, and typical vascular structure when seeded on decellularized vessel scaffolds. These findings indicate that reprogramming of fibroblasts into ECs via SETSIP and VEGF has a potential clinical application.

MicroRNA-199b modulates vascular cell fate during iPS cell differentiation by targeting the notch ligand jagged1 and enhancing VEGF signaling

Aims: Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalised medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation towards ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration.

Methods and Results: In this study we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen-coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR-199b is involved in EC differentiation. A step-wise increase in expression of miR-199 was detected during EC differentiation. Notably, miR-199b targeted the Notch ligand JAG1, resulting in VEGF transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA-mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR-199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR-199b-mediated inhibition of iPS cell differentiation towards SMCs. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR-199b also regulated VEGF expression and angiogenesis.

Conclusions: This study indicates a novel role for miR-199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses.

MicroRNA-199a induces differentiation of induced pluripotent stem cells into endothelial cells by targeting sirtuin 1

The ability to reprogram induced pluripotent stem (iPS) cells from somatic cells may facilitate significant advances in regenerative medicine. MicroRNAs (miRNAs) are involved in a number of core biological processes, including cardiogenesis, hematopoietic lineage differentiation and oncogenesis. An improved understanding of the complex molecular signals that are required for the differentiation of iPS cells into endothelial cells (ECs) may allow specific targeting of their activity in order to enhance cell differentiation and promote tissue regeneration. The present study reports that miR‑199a is involved in EC differentiation from iPS cells. Augmented expression of miR‑199a was detected during EC differentiation, and reached higher levels during the later stages of this process. Furthermore, miR‑199a inhibited the differentiation of iPS cells into smooth muscle cells. Notably, sirtuin 1 was identified as a target of miR‑199a . Finally, the ability of miR‑199a to induce angiogenesis was evaluated in vitro, using Matrigel plugs assays. This may indicate a novel function for miR‑199a as a regulator of the phenotypic switch during vascular cell differentiation. The present study provides support to the notion that with an understanding of the molecular mechanisms underlying vascular cell differentiation, stem cell regenerative therapy may ultimately be developed as an effective treatment for cardiovascular disease.

INFLUENCE OF NOX4 NADPH OXIDASE ON GENERATION AND ANGIOGENIC FUNCTION OF INDUCED PLURIPOTENT STEM CELL-DERIVED ENDOTHELIAL CELLS

Human induced pluripotent stem (iPS) cell-derived endothelial cells (ECs) hold clear potential for therapeutic angiogenesis as a novel strategy for ischaemic disease. Recently, we have developed a novel method for direct reprogramming of partial iPS (PiPS) cells, which unlike iPS cells, are generated before pluripotency so do not form tumours, and may be differentiated into ECs with characteristic morphology and pro-angiogenic actions. Our previous work showed that PiPS-derived ECs are capable of forming vascular-like tubes both in vitro and in vivo and promoting re-endothelialisation of ischemic tissue, with greater effectiveness versus mature ECs.

Interestingly, our preliminary data demonstrate that Nox NADPH oxidases, which are reported to influence stem cell function, are progressively induced during PiPs/PiPS-EC differentiation and in response to hypoxia, with Nox4 demonstrating highest expression. As this isoform is an established regulator of angiogenesis, we hypothesize that Nox4 plays a key role in modulating PiPS-EC generation and angiogenic function.

The aim of this project is therefore to investigate: (1) the specific role of Nox4 in direct reprogramming of PiPS cells and differentiation to PiPS-ECs; (2) whether genetic manipulation of Nox4 influences in vitro function of PiPs-ECs and their ability to promote in vivo angiogenesis. This will be achieved by employing established in vitro functional assays and an experimental model of hindlimb ischaemia with assessment of relevant end-points. Identification of a key role for Nox4 in regulating PiPS-EC generation/function may inform selective targeting of this isoform to enhance the efficiency of PiPS-EC differentiation and their capacity to treat ischemic disease.

Geração de blastocisto quimérico pela agregação de trofectoderme com a massa celular interna ou com células iPS

Pós-graduação em Ciências Biológicas (Farmacologia) - IBB

Kinin-B2 Receptor Activity Determines the Differentiation Fate of Neural Stem Cells

Bradykinin is not only important for inflammation and blood pressure regulation, but also involved in neuromodulation and neuroprotection. Here we describe novel functions for bradykinin and the kinin-B2 receptor (B2BkR) in differentiation of neural stem cells. In the presence of the B2BkR antagonist HOE-140 during rat neurosphere differentiation, neuron-specific beta 3-tubulin and enolase expression was reduced together with an increase in glial protein expression, indicating that bradykinin- induced receptor activity contributes to neurogenesis. In agreement, HOE-140 affected in the same way expression levels of neural markers during neural differentiation of murine P19 and human iPS cells. Kinin-B1 receptor agonists and antagonists did not affect expression levels of neural markers, suggesting that bradykinin-mediated effects are exclusively mediated via B2BkR. Neurogenesis was augmented by bradykinin in the middle and late stages of the differentiation process. Chronic treatment with HOE-140 diminished eNOS and nNOS as well as M1-M4 muscarinic receptor expression and also affected purinergic receptor expression and activity. Neurogenesis, gliogenesis, and neural migration were altered during differentiation of neurospheres isolated from B2BkR knock-out mice. Whole mount in situ hybridization revealed the presence of B2BkR mRNA throughout the nervous system in mouse embryos, and less beta 3-tubulin and more glial proteins were expressed in developing and adult B2BkR knock-out mice brains. As a underlying transcriptional mechanism for neural fate determination, HOE-140 induced up-regulation of Notch1 and Stat3 gene expression. Because pharmacological treatments did not affect cell viability and proliferation, we conclude that bradykinin-induced signaling provides a switch for neural fate determination and specification of neurotransmitter receptor expression.

Reprogramming of prostate cancer-associated stromal cells to embryonic stem-like

BACKGROUND CD90+ prostate cancer-associated (CP) stromal cells represent a diseased cell type found only in tumor tissue. They differ from their normal counterpart in gene expression and inductive signaling. Genetic reprogramming by induced pluripotent stem (iPS) cell technology can effectively change adult cells into stem-like cells through wholesale alteration of the gene expression program. This technology might be used to erase the abnormal gene expression of diseased cells. The resultant iPS cells would no longer express the disease phenotype, and behave like stem cells. METHODS CP stromal cells, isolated from tumor tissue of a surgically resected prostate by anti-CD90-mediated sorting and cultured in vitro, were transfected with in vitro packaged lentiviral expression vectors containing stem cell transcription factor genes POU5F1, LIN28, NANOG, and SOX2. RESULTS Alkaline phosphatase-positive iPS cells were obtained in about 3 weeks post-transfection at a frequency of 10-4. Their colony morphology was indistinguishable from that of human embryonic stem (ES) cells. Transcriptome analysis showed a virtually complete match in gene expression between the iPS and ES cells. CONCLUSIONS Genes of CP stromal cells could be fully inactivated by genetic reprogramming. As a consequence, the disease phenotype was cured. Prostate 72:14531463, 2012. (c) 2012 Wiley Periodicals, Inc.

INDUCED-PLURIPOTENT STEM-DERIVED NEURONAL PROGENITOR CELLS AS A NOVEL TREATMENT FOR NEURODEGENERATIVE DISEASES

Cellular therapies, as neuronal progenitor (NP) cells grafting, are promising therapies for patients affected with neurodegenerative diseases like Creutzfeldt-Jakob Disease (CJD). At this time there is no effective treatment or cure for CJD. The disease is inevitably fatal and affected people usually die within months of the appearance of the first clinical symptoms. Compelling evidence indicate that the hallmark event in the disease is the conversion of the normal prion protein (termed PrPC) into the disease-associated, misfolded form (called PrPSc). Thus, a reasonable therapeutic target would be to prevent PrP misfolding and prion replication. This strategy has been applied with poor results since at the time of clinical intervention substantial brain damage has been done. It seems that a more effective treatment aimed at patients with established symptoms of CJD would need to stop further brain degeneration or even recover some of the previously lost brain tissue. The most promising possibility to recover brain tissue is the use of NPs that have the potential to replenish the nerve cells lost during the early stages of the disease. Advanced cellular therapies, beside their potential for cell replacement, might be used as biomaterials for drug delivery in order to stimulate cell survival or the resolution the disease. Also, implanted cells can be genetically manipulated to correct abnormalities causing disease or to make them more resistant to the toxic microenvironments present in damaged tissue. In recent years cell engineering has been within the scope of the scientific and general community after the development of technologies able to “de-differentiate” somatic cells into induced-pluripotent stem (IPS) cells. This new tool permits the use of easy-to-reach cells like skin or blood cells as a primary material to obtain embryonic stem-like cells for cellular therapies, evading all ethical issues regarding the use of human embryos as a source of embryonic stem cells. The complete work proposes to implant IPS-derived NP cells into the brain of prion-infected animals to evaluate their therapeutic potential. Since it is well known that the expression of prion protein in the cell membrane is necessary for PrPSc mediated toxicity, we also want to determine if NPs lacking the prion protein have better survival rates once implanted into sick animals. The main objective of this work is to develop implantable neural precursor from IPS coming from animals lacking the prion protein. Specific aim 1: To develop and characterize cellular cultures of IPS cells from prp-/- mice. Fibroblasts from prp-/- animals will be reprogrammed using the four Yamanaka factors. IPS colonies will be selected and characterized by immunohistochemistry for markers of pluripotency. Their developmental capabilities will be evaluated by teratoma and embryoid body formation assays. Specific aim 2: To differentiate IPS cells to a neuronal lineage. IPS cells will be differentiated to a NP stage by the use of defined media culture conditions. NP cells will be characterized by their immunohistochemical profile as well as by their ability to differentiate into neuronal cells. Specific aim 3: Cellular labeling of neuronal progenitors cells for in vitro traceability. In order to track the cells once implanted in the host brain, they will be tagged with different methods such as lipophilic fluorescent tracers and transduction with GFP protein expression.

Evolution of emerging iPS cell-based therapies for age-related macular degeneration (AMD)

Stem cell-based regenerative medicine is poised to revolutionize the way diseases are treated. In recent years, induced pluripotent stem (iPS) cells, a newly stem cell species, has attracted significant attention. This paper seeks to understand the pathways along which emerging clinical research efforts in the field of iPS cells is evolved. In particular, the empirical case of age-related macular degeneration (AMD) is used, which is the world-pioneering clinical application of iPS cells. In line with the literature, this study explores the interrelations between three different pathways, such as biomedical scientific understanding, development of medical technologies, and learning in clinical practice. For this, a techmining approach is used including co-term, co-citation, and direct citation methods. Scientific publications indexed in the Thomson Reuters' Web of Science and Elsevier's Scopus databases form the basis of the study. This research first explores the iPS cell research landscape through the construction of a co-term map, particularly stressing the location and intensity of disease-tackling efforts; then focus on the evolution of scientific knowledge on AMD through co-citation networks and the main path algorithm on direct citations. At the researcher level, the development of four different research groups working on cell therapies for AMD is evaluated through the software CitNetExplorer. By integrating these approaches, the result shows a wider picture of the complexities inherent in the translation of knowledge into revolutionary clinical methods.

Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.

The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.

Cellular reprogramming strategies for degenerative disorders involving the retinal pigment epithelium

RESUMO: A reprogramação celular permite que uma célula somática seja reprogramada para outra célula diferente através da expressão forçada de factores de transcrição (FTs) específicos de determinada linhagem celular, e constitui uma área de investigação emergente nos últimos anos. As células somáticas podem ser experimentalmente manipuladas de modo a obter células estaminais pluripotentes induzidas (CEPi), ou convertidas directamente noutro tipo de célula somática. Estas descobertas inovadoras oferecem oportunidades promissoras para o desenvolvimento de novas terapias de substituição celular e modelos de doença, funcionando também como ferramentas valiosas para o estudo dos mecanismos moleculares que estabelecem a identidade celular e regulam os processos de desenvolvimento. Existem várias doenças degenerativas hereditárias e adquiridas da retina que causam deficiência visual devido a uma disfunção no tecido de suporte da retina, o epitélio pigmentar da retina (EPR). Uma destas doenças é a Coroideremia (CHM), uma doença hereditária monogénica ligada ao cromossoma X causada por mutações que implicam a perda de função duma proteína com funções importantes na regulação do tráfico intracelular. A CHM é caracterizada pela degenerescência progressiva do EPR, assim como dos foto-receptores e da coróide. Resultados experimentais sugerem que o EPR desempenha um papel importante na patogénese da CHM, o que parece indicar uma possível vantagem terapêutica na substituição do EPR nos doentes com CHM. Por outro lado, existe uma lacuna em termos de modelos in vitro de EPR para estudar a CHM, o que pode explicar o ainda desconhecimento dos mecanismos moleculares que explicam a patogénese desta doença. Assim, este trabalho focou-se principalmente na exploração das potencialidades das técnicas de reprogramação celular no contexto das doenças de degenerescência da retina, em particular no caso da CHM. Células de murganho de estirpe selvagem, bem como células derivadas de um ratinho modelo de knockout condicional de Chm, foram convertidos com sucesso em CEPi recorrendo a um sistema lentiviral induzido que permite a expressão forçada dos 4 factores clássicos de reprogramação, a saber Oct4, Sox2, Klf4 e c-Myc. Estas células mostraram ter equivalência morfológica, molecular e funcional a células estaminais embrionárias (CES). As CEPi obtidas foram seguidamente submetidas a protocolos de diferenciação com o objectivo final de obter células do EPR. Os resultados promissores obtidos revelam a possibilidade de gerar um valioso modelo de EPR-CHM para estudos in vitro. Em alternativa, a conversão directa de linhagens partindo de fibroblastos para obter células do EPR foi também abordada. Uma vasta gama de ferramentas moleculares foi gerada de modo a implementar uma estratégia mediada por FTs-chave, seleccionados devido ao seu papel fundamental no desenvolvimento embrionário e especificação do EPR. Conjuntos de 10 ou menos FTs foram usados para transduzir fibroblastos, que adquiriram morfologia pigmentada e expressão de alguns marcadores específicos do EPR. Adicionalmente, observou-se a activação de regiões promotoras de genes específicos de EPR, indicando que a identidade transcricional das células foi alterada no sentido pretendido. Em conclusão, avanços significativos foram atingidos no sentido da implementação de tecnologias de reprogramação celular já estabelecidas, bem como na concepção de novas estratégias inovadoras. Metodologias de reprogramação, quer para pluripotência, quer via conversão directa, foram aplicadas com o objectivo final de gerar células do EPR. O trabalho aqui descrito abre novos caminhos para o estabelecimento de terapias de substituição celular e, de uma maneira mais directa, levanta a possibilidade de modelar doenças degenerativas da retina com disfunção do EPR numa placa de petri, em particular no caso da CHM.---------------ABSTRACT: Cellular reprogramming is an emerging research field in which a somatic cell is reprogrammed into a different cell type by forcing the expression of lineage-specific transcription factors (TFs). Cellular identities can be manipulated using experimental techniques with the attainment of pluripotency properties and the generation of induced Pluripotent Stem (iPS) cells, or the direct conversion of one somatic cell into another somatic cell type. These pioneering discoveries offer new unprecedented opportunities for the establishment of novel cell-based therapies and disease models, as well as serving as valuable tools for the study of molecular mechanisms governing cell fate establishment and developmental processes. Several retinal degenerative disorders, inherited and acquired, lead to visual impairment due to an underlying dysfunction of the support cells of the retina, the retinal pigment epithelium (RPE). Choroideremia (CHM), an X-linked monogenic disease caused by a loss of function mutation in a key regulator of intracellular trafficking, is characterized by a progressive degeneration of the RPE and other components of the retina, such as the photoreceptors and the choroid. Evidence suggest that RPE plays an important role in CHM pathogenesis, thus implying that regenerative approaches aiming at rescuing RPE function may be of great benefit for CHM patients. Additionally, lack of appropriate in vitro models has contributed to the still poorly-characterized molecular events in the base of CHM degenerative process. Therefore, the main focus of this work was to explore the potential applications of cellular reprogramming technology in the context of RPE-related retinal degenerations. The generation of mouse iPS cells was established and optimized using an inducible lentiviral system to force the expression of the classic set of TFs, namely Oct4, Sox2, Klf4 and c-Myc. Wild-type cells, as well as cells derived from a conditional knockout (KO) mouse model of Chm, were successfully converted into a pluripotent state, that displayed morphology, molecular and functional equivalence to Embryonic Stem (ES) cells. Generated iPS cells were then subjected to differentiation protocols towards the attainment of a RPE cell fate, with promising results highlighting the possibility of generating a valuable Chm-RPE in vitro model. In alternative, direct lineage conversion of fibroblasts into RPE-like cells was also tackled. A TF-mediated approach was implemented after the generation of a panoply of molecular tools needed for such studies. After transduction with pools of 10 or less TFs, selected for their key role on RPE developmental process and specification, fibroblasts acquired a pigmented morphology and expression of some RPE-specific markers. Additionally, promoter regions of RPE-specific genes were activated indicating that the transcriptional identity of the cells was being altered into the pursued cell fate. In conclusion, highly significant progress was made towards the implementation of already established cellular reprogramming technologies, as well as the designing of new innovative ones. Reprogramming into pluripotency and lineage conversion methodologies were applied to ultimately generate RPE cells. These studies open new avenues for the establishment of cell replacement therapies and, more straightforwardly,raise the possibility of modelling retinal degenerations with underlying RPE defects in apetri dish, particularly CHM.

Dérivation de cellules souches pluripotentes induites autologues à partir du clonage somatique équin

Le recours aux cellules souches pour améliorer la réparation et guérison des blessures et maladies musculosquelettiques chez le cheval est de plus en plus fréquent. Les développements récents dans la reprogrammation cellulaire ont permis le développement de nouvelles sources de cellules souches pour ces thérapies régénératives. Des cellules souches pluripotentes induites (iPS) autologues peuvent être dérivées de cellules adultes par la reprogrammation directe à travers l'expression induite des gènes de pluripotence. Le clonage par transfert nucléaire (SCNT) suivi de la dérivation de cellules souches embryonnaires (ES) permet la reprogrammation indirecte des cellules adultes. Cependant, l’efficacité de ces deux méthodes pour la dérivation de cellules pluripotentes génétiquement stables est faible. Nous avons donc combiné les techniques SCNT et iPS dans le but de développer un protocole efficace de dérivation de cellules iPS autologues à partir de fibroblastes de la peau équine. Quatre facteurs de reprogrammation ont été introduits dans les cellules fibroblastes de fœtus clonés (ntFF) ainsi que les cellules ES provenant d’embryons clonés (ntES) pour induire leur reprogrammation en cellules iPS autologues. Les cellules ntFF-iPS et ntES-iPS ont des capacités prolifératives avancées et expriment des marqueurs de pluripotence importants. Par contre, les cellules ntES ont une efficacité de reprogrammation significativement supérieure aux cellules nt-FF et forment des colonies trois fois plus rapidement. Contrairement aux cellules ntES, les cellules ntES-iPS démontrent une augmentation de l’expression des marqueurs de pluripotence et survivent à la culture cellulaire prolongée. Les résultats présentés dans ce mémoire attestent que l’utilisation de la reprogrammation secondaire de cellules FF et ES clonées permet la production de cellules souches pluripotentes autologues stables chez le cheval.