Harnessing the potential of pluripotent stem cells to develop novel platforms to study human motor neurons in vitro

Tese de Doutoramento em Medicina.Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the capacity to differentiate into all three embryonic germ layers and give rise virtually to any cell type in the body. For this reason, they represent an exciting new approach to unravelling the mechanisms of human embryonic development, for drug discovery and disease modelling in vitro. The unique ability to generate relevant cell types from human pluripotent stem cells (hPSCs) opens the possibility of creating inexhaustible sources of cells that are otherwise not open to study in the human body, especially those from the central nervous system. In line with this, the successful specification of human motor neurons from hPSCs has opened new avenues for the study of motor neuron disorders like ALS, a fatal neurodegenerative disease characterized by progressive demise of motor neurons in the cortex, brainstem and anterior horn of the spinal cord. However, the motor neuron yields from existing differentiation protocols are suboptimal, leading to the generation of populations of mixed progenitor cells and postmitotic neurons. In addition, the current understanding on the survival requirements of human motor neurons remains limited. These are significant hurdles for the generation of neuronal cells in quantities that will allow the prosecution of innovative studies based on motor neurons generated from human pluripotent stem cells. Here, we initially used spinal motor neuron cultures specified from hESCs following an optimized protocol and demonstrated a remarkably high level of ongoing birth of new motor neurons during the ensuing 15 days after the regular 31-day period of motor neuron differentiation. Based on previous rodent studies this finding was unexpected and could represent a significant potential confound for some published studies using cultures differentiated in similar conditions to reveal determinants that alter motor neuron survival. We envisioned taking advantage of the ongoing genesis of motor neurons in two distinct ways. On one hand, to address the problem of insufficient motor neuron yields, we conducted a low-throughput screening study by testing a small collection of 160 bioactive molecules to discover small molecules capable of increasing motor neuron numbers in culture, either by enhancing neurogenesis and/or increasing survival. The Rho-kinase (ROCK) inhibitor Y-27632 was the only tested compound shown to be capable of robustly increasing motor neuron numbers up to four-fold after 9 days in culture, an effect which was evident in both hESC- and hiPSC-derived motor neuron cultures. The increase in motor neuron numbers was later demonstrated to be associated with the enhancement of progenitor proliferation and motor neuron survival. These effects were likely induced through an unknown ROCK-independent mechanism, since other seven small molecules in the family could not elicit comparable increases in motor neuron numbers. On the other hand, to overcome the potential confound effect of ongoing neurogenesis on motor neuron survival studies we used FACS sorting to purify human motor neurons derived from a HB9::GFP hESC reporter line, based on their cellular characteristics and GFP expression. The resulting nearly pure populations of human motor neurons were used to create an assay for agents with direct effects on motor neuron survival. Y-27632 was successfully applied to expand hESC-derived motor neuron cultures before the FACS sorting procedures, leading to a final increase in total motor neuron yields. Similarly to previous studies employing purified populations of chick and rodent embryonic motor neurons, the purified human motor neurons were demonstrated to be responsive to the survival-promoting actions of specific neurotrophic factors (GDNF, BDNF and CNTF), as well as Y-27362 itself. Therefore, we successfully developed original strategies that allow us to significantly increase motor neuron yields from hPSC-derived cultures and to create a robust survival assay using a pure population of human motor neurons specified from hPSCs. Our findings reveal that Y-27632 might constitute a new powerful tool with invaluable contributions to the study of pluripotent stem-cell derived human motor neurons. Human adipose tissue constitutes an appealing alternative source of stem cells which capture the genetic background of the person from which they are obtained. From fat tissue one can easily isolate adult human adipose-derived stem cells (hADSCs) which can be cultivated in vitro and differentiated to other cell types, though with a more restricted potential than hPSCs. The current knowledge on the survival and expansion requirements of hADSCs is limited and protocols to efficiently drive these cells towards a neuronal lineage still need to be developed. Here, we expanded the study of the effects of Y-27632 on human stem cells and were able to show that Y-27632 does not robustly increase the survival and proliferation of hADSCs, a novel finding which demonstrates that the effects of Y-27632 are likely to be cell-specific. In summary, the different methodological advances reported in this thesis should be of general interest for the preparation of human motor neurons on a large scale and for studies addressing the molecular processes underlying motor neuron genesis, survival and degeneration. The body of knowledge reported in this thesis should be of general importance for researchers using human stem cells to study other neuronal populations and other diseases.As células estaminais embrionárias humanas (hESCs) e as células estaminais pluripotentes induzidas humanas (hiPSCs) têm a capacidade de se diferenciar nos três folhetos germinativos embrionários e dar origem virtualmente a qualquer tipo celular existente no organismo. Dessa forma, constitutem uma nova abordagem na descoberta dos mecanismos que regulam o desenvolvimento embrionário humano, na descoberta de novos fármacos e na modelação de patologias in vitro. A capacidade única de gerar tipos celulares de interesse a partir destas células estaminais pluripotentes humanas (hPSCs) cria a possibilidade de serem geradas fontes ilimitadas de células que dificilmente estão acessíveis para estudo no corpo humano, sendo o caso das células do sistema nervoso central. Nesse sentido, a criação com sucesso de neurónios motores humanos a partir de hPSCs tem conduzido a novas avenidas no estudo de doenças do neurónio motor, sendo o caso da Esclerose Lateral Amiotrófica (ELA), uma doença neurodegenerative fatal caracterizada pela morte progressiva de neurónios motores no córtex cerebral, tronco cerebral e corno anterior da medula espinhal. No entanto, o resultado final dos protocolos de diferenciação existentes não são ainda os ideiais, dando origem à formação de populações mistas de células progenitoras neuronais e neurónios motores pós-mitóticos. Para além disso, o conhecimento sobre os requisitos destes neurónios motores para se manterem vivos é ainda escasso. Estes factos no seu conjunto constituem importantes barreiras para a produção de células neuronais em quantidades suficientes que permitam o desenvolvimento de estudos inovadores tendo por base os neurónios motores criados a partir de células estaminais pluripotentes humanas. Neste trabalho, começamos inicialmente por usar culturas de neurónios motores da espinhal medula criados a partir de hESCs, seguindo um protocolo optimizado; e demonstramos níveis consideráveis de génese contínua de neurónios motores durante 15 dias após o período habitual de 31 dias para a diferenciação de neurónios motores. Tendo por base os estudos anteriores usando modelos animais, este achado não era expectável e constituía, de facto, um importante factor de confundimento em estudos onde eram utilizadas culturas de neurónios motores geradas em condições semelhantes para estudar factores que possam afectar a sobrevivência dos neurónios motores. Consideramos então que seria possível tirar partido da criação contínua de neurónios motores através de duas abordagens distintas. Por um lado, no sentido de responder ao problema de produção de neurónios motores em quantidades insuficientes, conduzimos um teste de screening de moléculas em pequena escala, usando cerca de 160 compostos, para tentar encontrar alguma que fosse capaz de aumentar o número de neurónios motores em cultura, através da potenciação do efeito de neurogénese contínua e/ou aumento da sobrevivência neuronal. O inibidor da cinase Rho (ROCK) Y-27632 foi descrito como o único composto testado capaz de aumentar de forma significativa o número de neurónio motores em cultura, com efeito máximo de quadruplicação do número de neurónio motores ao fim de 9 dias em cultura, estando o efeito presente em culturas de neurónios motores geradas a partir de hESCs e hiPSCs. Este aumento no número de neurónios motores em cultura foi mais tarde demonstrado estar associado a uma potenciação da proliferação de células progenitoras, bem como, ao aumento da sobrevivência dos neurónios motores. Estes efeitos alegadamente ocorrem através de um mecanismo desconhecido, muito provavelmente independente da inibição de ROCK, na medida em que, outras sete moléculas da mesma família foram testadas e não foram capazes de originar aumentos semelhantes no número de neurónios motores. Por outro lado, de forma a contornar o potencial efeito de confundimento nos estudos de sobrevivência dos neurónios motores inerente à neurogénese contínua, usamos a tecnologia FACS para isolar neurónios motores humanos criados a partir de uma linha reporter HB9::GFP, com base nas suas características celulares e expressão de GFP. As populações de neurónios motores praticamente puras resultantes foram então usadas para desenvolver um ensaio com o objectivo de testar agentes com efeito directo na sobrevivência dos neurónios motores humanos. Antes do procedimento de FACS, o composto Y-27632 foi usado com sucesso na expansão das culturas de neurónios motores, levando a números mais elevados de neurónios motores obtidos com este procedimento. Em linha com estudos anteriores envolvendo o uso de populações purificadas de neurónios motores embrionários de pinto e roedor, os neurónios motores humanos purificados neste trabalho demonstraram ter capacidade de responder às acções pró-sobrevivência de factores neurotróficos específicos (GDNF, BDNF e CNTF), bem como ao composto Y-27632. Deste modo, consegui desenvolver com sucesso algumas estratégias que permitem aumentar de forma significativa a produção de neurónios motores a partir de culturas derivadas de hPSCs, bem como, criar um ensaio de sobrevivência neuronal robusto, com base em neurónios motores humanos purificados a partir de culturas de neurónios motores. Os nossos achados sugerem que o composto Y-27632 pode constituir uma nova ferramenta poderosa no estudo de neurónios motores humanos criados a partir de células estaminais pluripotentes. O tecido adiposo constitui uma fonte alternativa interessante de células estaminais que retêm o património genético da pessoa de onde são obtidas. A partir da gordura podemos isolar com facilidade células estaminais humanas adultas derivadas da gordura (hADSCs), que podem ser cultivadas in vitro e se diferenciar noutros tipos celulares, apesar do potencial mais restrito quando comparadas com as hPSCs. O conhecimento actual sobre os requisitos de sobrevivência e expansão destas células é ainda limitado e protocolos para a geração eficiente de neurónios a partir destas ainda não foram desenvolvidos. Neste trabalho, ampliamos o estudo dos efeitos da molécula Y-27632 nas células estaminais humanas e fomos capazes de demonstrar que a aplicação do composto Y-27632 não aumenta a proliferação e sobrevivência das hADSCs, um novo achado que ajuda a demonstrar que os efeitos de Y-27632 serão provavelmente específicos consoante o tipo celular em estudo. Em suma, os diferentes avanços metodológicos documentados nesta tese deverão ser de interesse geral na geração de neurónios motores humanos em grande quantidades, bem como, na condução de estudos com vista ao melhor conhecimento dos mecanismos da motoneurogénese, sobrevivência e degeneração. O conjunto de conhecimentos cristalizados nesta tese poderão vir a constituir-se como importantes para outros investigadores que usam células estaminais humanas para estudar outras populações neuronais e outras patologias.Portuguese Foundation for Science and Technology (FCT) through a PhD Studentship SFRH/BD/33421/2008.Luso-American Development Foundation (FLAD), Project ALS, P2ALS and NYSTEM

Failure of Y-27632 to improve the culture of adult human adipose-derived stem cells

Y-27632 is a well-known inhibitor of the Rho-associated coiled kinase (ROCK) and has been shown to significantly improve the culture of a variety of multipotent stem cell types. However, the effects of Y-27632 on the expansion of adult human adipose-derived stem cell (hADSC) cultures remain to be established. Here, we aimed to characterize the effects of Y-27632 on the culture of hADSCs. Adult hADSCs were isolated from subjects submitted to elective plastic surgery procedures and cultivated in vitro under optimized conditions. Our results show that the continuous supplementation of hADSC cultures with Y-27632 led to decreased numbers of cells and decreased global metabolic viability of hADSC cultures when compared with control conditions. This effect appeared to be dependent on the continuous presence of the drug and was shown to be concentration-dependent and significant for 10 muM and 20 muM of Y-27632. Moreover, the Y-27632-induced decrease in hADSC numbers was not linked to a block in global cell proliferation, as cell numbers consistently increased from the moment of plating until passaging. In addition, Y-27632 was not able to increase the number of hADSCs present in culture 24 hours after passaging. Taken together, our results suggest that, in contrast to other stem cell types, Y-27632 supplementation is not a suitable strategy to enhance hADSC culture expansion.We thank Jeffrey M Gimble (Center for Stem Cell Research and Regenerative Medicine, Tulane University and LaCell LLC, New Orleans, LA, USA) for kindly isolating, characterizing, and sharing the cellular lines of hADSCs used in the present study and for critical input on the manuscript. We also would very much like to thank Laurent Roybon (Stem Cell Laboratory for CNS Disease Modeling, Department of Experimental Medical Science, Lund University, Lund, Sweden) for the utilization of Metamorph NX software for automated cell quantification. We are grateful to Miguel Carvalho, Ana Pires, Eduardo Gomes, Fabio Teixeira and Nuno Silva for technical assistance. This work was supported by the Portuguese Foundation for Science and Technology (predoctoral fellowship to NJL [SFRH/BD/33421/2008]; FCT Investigator Program to AJS) and the Luso-American Development Foundation

Basaloid follicular hamartoma of the eyelid: a case report and literature review about an unusual lesion in the ocular region

Basaloid follicular hamartoma (BFH) is a normally benign, uncommon, malformative lesion involving the hair follicles, which usually poses challenges in the differential diagnosis with other benign and malignant tumours, especially basal cell carcinoma, due to significant clinical and morphological overlap. Here, we report the case of a 53-year-old male who presented with a mass in the upper left eyelid evolving for one year. The patient had a previous history of total colectomy and an abdominal desmoid tumour within the context of Familial Adenomatous Polyposis (FAP), with a documented germline mutation in the Adenomatous Polyposis Coli (APC) gene. The eyelid lesion was biopsied and the histological analysis of the three small tissue fragments received revealed fragments with cutaneous–conjunctival lining displaying a subepithelial proliferation of basaloid nests with peripheral palisading, compatible with primitive hair follicles. There were images of anastomosis between different basaloid nests, which had their connection to the epithelial lining preserved. The stroma had high cellularity and sometimes primitive mesenchymal papillae were evident. Pleomorphism was absent, mitotic figures were barely identified, and no necrosis was seen. The basaloid nests did not have epithelial–stromal retraction nor mucin deposits. A diagnosis of BFH was proposed, which was later confirmed after surgical excision of the whole eyelid lesion. No evidence of carcinoma was present. This case illustrates the main features of the rare benign eyelid BFH. The standard medical or surgical approach of these lesions remains to be firmly established. Nearly nine months after surgical excision our patient remains well without signs of disease recurrence

Abdominal Actinomycosis misdiagnosed as liposarcoma

Actinomycosis is an uncommon, endogenous, and chronic infection with varied and nonspecific clinical features such as abdominal, pelvic or cervical masses, ulcerative lesions, abscesses, draining fistula, fibrosis, and constitutional symptoms. The disease ensues when the bacteria disrupt the mucosal barrier, invade, and spread throughout interfascial planes. Currently, the diagnosis of actinomycosis is challenging because of its very low frequency and depending on the clinical presentation it may masquerade malignancies. Therapy consists initially in intravenous penicillin, followed by an oral regimen that may be extended until a year of treatment. A timely diagnosis is crucial to avoid extensive therapeutic attempt as surgery. However, a biopsy or drainage of abscesses and fistula’s tract may be required not only as a diagnostic procedure as part of the therapy. We report the case of a 72-year-old woman with an abdominal mass initially misdiagnosed as a liposarcoma. A second biopsy of a skin lesion of the abdominal wall made the diagnosis of actinomycosis, avoiding a major surgical procedure. The patient was treated with a long-term course of antibiotics with favorable outcome. Liposarcoma was ruled out after the patient’s full recovery with antibiotics and the misdiagnosis was credit to the overconfidence on the immunohistochemical positivity to MDM2

Prognostic biomarkers in uveal melanoma: the status quo, recent advances and future directions

Uveal melanoma (UM) is the most common malignant intraocular tumour in the adult population. It is a rare cancer with an incidence of nearly five cases per million inhabitants per year, which develops from the uncontrolled proliferation of melanocytes in the choroid (≈90%), ciliary body (≈6%) or iris (≈4%). Patients initially present either with symptoms like blurred vision or photopsia, or without symptoms, with the tumour being detected in routine eye exams. Over the course of the disease, metastases, which are initially dormant, develop in nearly 50% of patients, preferentially in the liver. Despite decades of intensive research, the only approach proven to mildly control disease spread are early treatments directed to ablate liver metastases, such as surgical excision or chemoembolization. However, most patients have a limited life expectancy once metastases are detected, since there are limited therapeutic approaches for the metastatic disease, including immunotherapy, which unlike in cutaneous melanoma, has been mostly ineffective for UM patients. Therefore, in order to offer the best care possible to these patients, there is an urgent need to find robust models that can accurately predict the prognosis of UM, as well as therapeutic strategies that effectively block and/or limit the spread of the metastatic disease. Here, we initially summarized the current knowledge about UM by compiling the most relevant epidemiological, clinical, pathological and molecular data. Then, we revisited the most important prognostic factors currently used for the evaluation and follow-up of primary UM cases. Afterwards, we addressed emerging prognostic biomarkers in UM, by comprehensively reviewing gene signatures, immunohistochemistry-based markers and proteomic markers resulting from research studies conducted over the past three years. Finally, we discussed the current hurdles in the field and anticipated the future challenges and novel avenues of research in UM.N.J.L. would like to thank all members of the Laboratory of Clinical and Experi-mental Pathology (LPCE), Centre Hospitalier Universitaire de Nice, Nice, France; and all members of the Anatomic Pathology Service, Pathology Department, Centro Hospitalar e Universitario do Porto,Porto, Portugal, especially to JoseRamon Vizcaino (Head of Service), Joana Raposo Alves (Advisor ofPathology Training), Andre Coelho, David Tente and Francisca Emanuel Costa for their continuous support and help in the developmen

The selective COX-2 inhibitor etoricoxib reduces acute inflammatory markers in a model of neurogenic laryngitis but loses its efficacy with prolonged treatment

The selective COX-2 inhibitor Etoricoxib reduces acute inflammatory markers in a model of neurogenic laryngitis but loses its efficacy with prolonged treatment.OBJECTIVE: A randomised experimental study was used to evaluate the therapeutic effect of a selective cyclooxygenase-2 (COX-2) inhibitor in neurogenic laryngitis. MATERIALS AND METHODS: Male Wistar Han rats were subjected to the nasogastric intubation model (NGI) of laryngitis for 1 and 2 weeks. The NGI animals were divided into three groups: (1) treated with COX-2 inhibitor Etoricoxib, (2) vehicle and (3) non-intubated animals. A fourth group of animals was submitted to NGI only. Laryngeal sections were immunostained for substance P (SP) and calcitonin gene-related peptide (CGRP) fibre-immunoreactivity (IR) and quantification of COX-2 positive cells through stereological analysis. The expression of COX-2, interleukins IL-1beta, IL-6, IL-10 and tumour necrosis factor-alpha (TNF-alpha) was determined by quantitative real time QRT-PCR. TREATMENT: Etoricoxib (6 mg/kg/day) was prepared in 0.9% sterile saline with 5% glucose (vehicle) and administered daily during 1 or 2 weeks. RESULTS: Treatment for 1 week with Etoricoxib attenuated the CGRP-IR fibre depletion, the COX-2-IR increased cell number and the TNF-alpha and COX-2 mRNA increased levels induced by NGI. Two weeks of treatment had no beneficial effect. CONCLUSIONS: Etoricoxib is effective in neurogenic laryngitis for limited periods of administration, indicating that selective COX-2 inhibitors should be evaluated in the future.This study was supported by Fundacao Calouste Gulbenkian Project No 74551 and Fundacao Grunenthal (Portugal)

A new model of laryngitis: neuropeptide, cyclooxygenase, and cytokine profile

Objectives/Hypothesis: To develop and characterize a new model of laryngeal inflammation by analyzing the presence of neurogenic peptides and expression of cyclooxygenases (COX) and cytokines in the mucosa.Study Design: Laryngitis induced by nasogastric intubation (NGI) was evaluated by histopathologic changes of the mucosa, alterations in calcitonin gene related peptide (CGRP) and substance P (SP) neuropeptides in sensory fibers, and COX-1,2, and cytokine (interleukin [IL]-1, IL-6, IL-10, tumor necrosis factor [TNF]-alpha) expression in the laryngeal mucosa.Methods: Rats submitted to NGI for 1 to 5 weeks were compared with controls. Laryngeal sections were immunostained for stereologic analysis of SP and CGRP fiber density and number of mucosal cells expressing COX-2. Alterations in inflammatory mediators were evaluated by quantitative reverse-transcriptase polymerase chain reaction.Results: NGI induced metaplasia of the epithelium and narrowing of the laryngeal lumen because of hypertrophy of laryngeal glandules and edema. An initial decrease in CGRP- and SP-immunoreactive fibers in the laryngeal mucosa (1-3 wk) was reverted with time (5 wk). COX-2 expression in mucosal cells increased progressively, reaching a maximum level at 5 weeks, and was observed in mononuclear immune cells, which is indicative of a chronic inflammatory process. In regard to mRNA expression levels of inflammatory mediators, TNF-alpha was increased during the 5 week NGI, and IL-10 decreased during the 5 weeks,whereas IL-beta, IL-6, and COX-2 increased in the first 1 to 2 weeks and returned to baseline at 5 weeks.Conclusions: This NGI model results in laryngeal chronic inflammation without direct mechanical aggression of the mucosa and may contribute to the study of future therapeutic approaches to this pathology.FEDERFundação para a Ciência e Tecnologia (FCT) - POCTI/NSE/46399/2002Fundação Calouste Gulbenkian - projecto nº 7455

A rare cecal subepithelial tumor in a Crohn´s Disease patient

Appendiceal tumors comprise a variety of histologic types, including appendiceal mucinous neoplasms, which can be grouped as premalignant lesions, tumors of uncertain malignant potential, and malignant lesions. The appendiceal mucinous neoplasms are characterized by mucinous epithelial proliferation with extracellular mucin and pushing tumor margins, commonly an incidental finding during operative exploration. We report the case of a low-grade appendiceal mucinous neoplasm presenting as a subepithelial lesion in Crohn´s Disease patient. The diagnosis was not straightforward, and only surgical resection allowed an accurate diagnosis. Although Inflammatory Bowel Disease is a risk factor for the development of colorectal neoplasms, the absolute risk for appendiceal tumors is uncertain. The frequency of progression to malignancy remains to be determined

Anatomy Teaching at the School of Health Sciences of University of Minho

info:eu-repo/semantics/publishedVersio

Neurotrophic requirements of human motor neurons defined using amplified and purified stem-cell derived cultures

Neurotrophic requirements of human motor neurons defined using amplified and purified stem-cell derived culturesHuman motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.This work was funded by Project A.L.S., P2ALS and NYSTEM grant number CO24415. The work of N.J.L. was supported by the Portuguese Foundation for Science and Technology SFRH/BD/33421/2008 and the Luso-American Development Foundation. B.J.-K. was supported by the National Institute of Neurological Disorders and Stroke (NINDS). L.R. was supported by the Swedish Brain Foundation/Hjarnfonden. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript