Monitoring the ex-vivo expansion of human mesenchymal stem/stromal cells in xeno-free microcarrier-based reactor systems by MIR spectroscopy

Human mesenchymal stem/stromal cells (MSCs) have received considerable attention in the field of cell-based therapies due to their high differentiation potential and ability to modulate immune responses. However, since these cells can only be isolated in very low quantities, successful realization of these therapies requires MSCs ex-vivo expansion to achieve relevant cell doses. The metabolic activity is one of the parameters often monitored during MSCs cultivation by using expensive multi-analytical methods, some of them time-consuming. The present work evaluates the use of mid-infrared (MIR) spectroscopy, through rapid and economic high-throughput analyses associated to multivariate data analysis, to monitor three different MSCs cultivation runs conducted in spinner flasks, under xeno-free culture conditions, which differ in the type of microcarriers used and the culture feeding strategy applied. After evaluating diverse spectral preprocessing techniques, the optimized partial least square (PLS) regression models based on the MIR spectra to estimate the glucose, lactate and ammonia concentrations yielded high coefficients of determination (R2 ≥ 0.98, ≥0.98, and ≥0.94, respectively) and low prediction errors (RMSECV ≤ 4.7%, ≤4.4% and ≤5.7%, respectively). Besides PLS models valid for specific expansion protocols, a robust model simultaneously valid for the three processes was also built for predicting glucose, lactate and ammonia, yielding a R2 of 0.95, 0.97 and 0.86, and a RMSECV of 0.33, 0.57, and 0.09 mM, respectively. Therefore, MIR spectroscopy combined with multivariate data analysis represents a promising tool for both optimization and control of MSCs expansion processes.

Immune reconstitution after autologous hematopoietic stem cell transplantation

Abstract The investigation of the web of relationships between the different elements of the immune system has proven instrumental to better understand this complex biological system. This is particularly true in the case of the interactions between B and T lymphocytes, both during cellular development and at the stage of cellular effectors functions. The understanding of the B–T cells interdependency and the possibility to manipulate this relationship may be directly applicable to situations where immunity is deficient, as is the case of cancer or immune suppression after radio and chemotherapy. The work presented here started with the development of a novel and accurate tool to directly assess the diversity of the cellular repertoire (Chapter III). Contractions of T cell receptor diversity have been related with a deficient immune status. This method uses gene chips platforms where nucleic acids coding for lymphocyte receptors are hybridized and is based on the fact that the frequency of hybridization of nucleic acids to the oligonucleotides on a gene chip varies in direct proportion to diversity. Subsequently, and using this new method and other techniques of cell quantification I examined, in an animal model, the role that polyclonal B cells and immunoglobulin exert upon T cell development in the thymus, specifically on the acquisition of a broader repertoire diversity by the T cell receptors (Chapter IV and V). The hypothesis tested was if the presence of more diverse peptides in the thymus, namely polyclonal immunoglobulin, would induce the generation of more diverse T cells precursors. The results obtained demonstrated that the diversity of the T cell compartment is increased by the presence of polyclonal immunoglobulin. Polyclonal immunoglobulin, and particularly the Fab fragments of the molecule, represent the most diverse self-molecules in the body and its peptides are presented by antigen presenting cells to precursor T cells in the thymus during its development. This probably contributes significantly to the generation of receptor diversity. Furthermore, we also demonstrated that a more diverse repertoire of T lymphocytes is associated with a more effective and robust T cell immune function in vivo, as mice with a more diverse T cell receptors reject minor histocompatiblility discordant skin grafts faster than mice with a shrunken T cell receptor repertoire (Chapter V). We believe that a broader T cell receptor diversity allows a more efficient recognition and rejection of a higher range of external and internal aggressions. In this work it is demonstrated that a reduction of TCR diversity by thymectomy in wild type mice significantly increased survival of H-Y incompatible skin grafts, indicating decrease on T cell function. In addiction reconstitution of T-cell diversity in mice with a decreased T cell repertoire diversity with immunoglobulin Fab fragments, lead to a increase on TCR diversity and to a significantly decreased survival of the skin grafts (Chapter V). These results strongly suggest that increases on T cell repertoire diversity contribute to improvement of T cell function. Our results may have important implications on therapy and immune reconstitution in the context of AIDS, cancer, autoimmunity and post myeloablative treatments. Based on the previous results, we tested the clinical hypothesis that patients with haematological malignancies subjected to stem cell transplantation who recovered a robust immune system would have a better survival compared to patients who did not recover such a robust immune system. This study was undertaken by the examination of the progression and overall survival of 42 patients with mantle cell non-Hodgkin lymphoma receiving autologous hematopoietic stem cell transplantation (Chapter VI). The results obtained show that patients who recovered higher numbers of lymphocytes soon after autologous transplantation had a statistically significantly longer progression free and overall survivals. These results demonstrate the positive impact that a more robust immune system reconstitution after stem cell transplantation may have upon the survival of patients with haematological malignancies. In a similar clinical research framework, this dissertation also includes the study of the impact of recovering normal serum levels of polyclonal immunoglobulin on the survival of patients with another B cell haematological malignancy, multiple myeloma, after autologous stem cell transplantation (Chapter VII). The relapse free survival of the 110 patients with multiple myeloma analysed was associated with their ability to recover normal serum levels of the polyclonal compartment of immunoglobulin. These results suggest again the important effect of polyclonal immunoglobulin for the (re)generation of the immune competence. We also studied the impact of a robust immunity for the response to treatment with the antibody anti CD20, rituximab, in patients with non- Hodgkin’s lymphoma (NHL) (Chapter VIII). Patients with higher absolute counts of CD4+ T lymphocytes respond better (in terms of longer progression free survival) to rituximab compared to patients with lower number of CD4+ T lymphocytes. These observations highlight again the fact that a competent immune system is required for the clinical benefit of rituximab therapy in NHL patients. In conclusion, the work presented in this dissertation demonstrates, for the first time, that diverse B cells and polyclonal immunoglobulin promote T cell diversification in the thymus and improve T lymphocyte function. Also, it shows that in the setting of immune reconstitution, as after autologous stem cell transplantation for mantle cell lymphoma and in the setting of immune therapy for NHL, the absolute lymphocyte counts are an independent factor predicting progression free and overall survival. These results can have an important application in the clinical practice since the majority of the current treatments for cancer are immunosuppressive and implicate a subsequent immune recovery. Also, the effects of a number of antineoplastic treatments, including biological agents, depend on the immune system activity. In this way, studies similar to the ones presented here, where methods to improve the immune reconstitution are examined, may prove to be instrumental for a better understanding of the immune system and to guide more efficient treatment options and the design of future clinical trials. Resumo O estudo da rede de inter-relações entre os diversos elementos do sistema immune tem-se mostrado um instrumento essencial para uma melhor compreensão deste complexo sistema biológico. Tal é particularmente verdade no caso das interacções entre os linfócitos B e T, quer durante o desenvolvimento celular, quer ao nível das funções celulares efectoras. A compreensão da interdependência entre linfócitos B e T e a possibilidade de manipular esta relação pode ser directamente aplicável a situações em que a imunidade está deficiente, como é o caso das doenças neoplásicas ou da imunossupressão após radio ou quimioterapia. O trabalho apresentado nesta dissertação iniciou-se com o desenvolvimento de um novo método laboratorial para medir directamente a diversidade do reportório celular (Capítulo III). Reduções da diversidade do reportório dos receptores de células T têm sido relacionadas com um estado de imunodeficiência. O método desenvolvido utiliza “gene chips”, aos quais hibridizam os ácidos nucleicos codificantes das cadeias proteicas dos receptores linfocitários. A diversidade é calculada com base na frequência de hibridização do ácido nucleico da amostra aos oligonucleótidos presentes no “gene chip”. De seguida, e utilizando este novo método e outras técnicas de quantificação celular examinei, num modelo animal, o papel que as células policlonais B e a imunoglobulina exercem sobre o desenvolvimento linfocitário T no timo, especificamente na aquisição de um reportório diverso de receptores T (Capítulos IV e V). Testei, então, a hipótese de que a presença no timo de péptidos mais diversos, como a imunoglobulna policlonal, induzisse a génese de precursores T mais diversos. Demonstrámos que a diversidade do compartimento T é aumentado pela presença de imunoglobulina policlonal. A imunoglobulina policlonal, e particularmente os fragmentos Fab desta molécula, representam as moléculas autólogas mais diversas presentes nos organismos vertebrados. Estes péptidos são apresentados por células apresentadoras de antigénio às células precursoras T no timo, durante o desenvolvimento celular T. Tal, provavelmente, contribui para a génese da diversidade dos receptores. Também demonstrámos que a presença de um reportório mais diverso de linfócitos T se associa a um incremento da função imunológica T in vivo. Uma diversidade de receptores T mais extensa parece permitir um reconhecimento e rejeição mais eficientes de um maior número de agressores internos e externos. Demonstrámos que ratinhos com receptores de células T (RCT) com maior diversidade rejeitam transplantes cutâneos discordantes para antigénios minor de histocompatibilidade mais rapidamente do que ratinhos com um menor reportório T (Capítulo V). Por outro lado, uma redução da diversidade do RCT, causada por timectomia de ratinhos de estirpes selvagens, mostrou aumentar significativamente a sobrevivência de transplantes cutâneos incompatíveis para o antigénio H-Y (antigénio minor de histocompatibilidade), indicando uma diminuição da função linfocitária T. Além disso, a reconstituição da diversidade dos linfócitos T em ratinhos com uma diversidade de reportório T diminuída, induzida pela administração de fragmentos Fab de imunoglobulina, conduz a um aumento da diversidade dos RCT e a uma diminuição significativa da sobrevivência dos enxertos cutâneos (Capítulo V). Estes resultados sugerem que o aumento do reportório de células T contribui para uma melhoria das funções celulares T e poderão ter implicações importantes na terapêutica e reconstitutição imunológica em contexto de SIDA, neoplasias, autoimunidade e após tratamentos mieloablativos. Baseado nos resultados anteriores, decidimos testar a hipótese clínica de que doentes com neoplasias hematológicas sujeitos a transplantação de precursores hematopoiéticos e com recuperação imunológica precoce após transplante teriam uma sobrevivência mais longa do que doentes que não recuperassem tão bem a sua imunidade. Analisámos a sobrevivência global e sobrevivência sem doença de 42 doentes com linfoma não Hodgkin de células do manto sujeitos a transplante autólogo de precursores hematopoiéticos (Capítulo VI). Os resultados obtidos mostraram que os doentes que recuperaram contagens mais elevadas de linfócitos imediatamente após o transplante autólogo, apresentaram uma sobrevivência global e sem progressão mais longa do que doentes que não recuperaram contagens linfocitárias tão precocemente. Estes resultados demonstram o efeito positivo de uma reconstitutição imunológica robusta após transplante de presursores hematopoiéticos, sobre a sobrevivência de doentes com neoplasias hematológicas. Do mesmo modo, estudámos o efeito que a recuperação de níveis séricos normais de imunoglobulina policlonal tem na sobrevivência de doentes com outras neoplasias hematológicas de linfócitos B, como o mieloma múltiplo,após transplante autólogo de precursos hematopoiéticos (Capítulo VII). A sobrevivência livre de doença dos 110 doentes com mieloma múltiplo analizados está associada com a sua capacidade de recuperar níveis séricos normais do compartmento policlonal de imunoglobulina. Estes resultados pioneiros indicam a importância da imunoglobulina policlonal para a génese de competência imunológica. Também estudámos o impacto de um sistema imunitário eficiente sobre a resposta ao tratamento com o anticorpo anti CD20, ituximab, em doentes com linfoma não Hodgkin (LNH) (Capítulo VIII). Os resultados mostram que doentes com valores mais elevados de linfócitos T CD4+ respondem melhor (em termos de maior sobrevida livre de doença) ao rituximab, do que doentes com valores mais baixos. Estas observações ilustram a necessidade de um sistema imunitário competente para o benefício clínico da terapêutica com rituximab em doentes com LNH. Em conclusão, o trabalho apresentado nesta dissertação demonstra que as células B e a imunoglobulina policlonal promovem a diversidade das células T no timo e melhoram a função linfocitária T periférica. Concomitantemente, também demonstrámos que, no contexto de reconstituição imune, por exemplo, após transplante autólogo de precursores hematopoiéticos em doentes com linfomas de células do manto, o número absoluto de linfócitos é uma factor independente da sobrevivência. Os resultados demonstram, também, a importância dos valores de linfocitos T na resposta ao tratamento com rituximab no caso de doentes com LNH. O mesmo princípio se prova pelo facto de que doentes com mieloma múltiplo sujeitos a transplante autólogo de precursores hematopoiéticos que recuperam valores normais séricos de imunoglobulinas policlonais, terem melhores taxas de resposta em comparação com doentes que não recuperam valores normais de imunoglobulinas policlonais. Estes resultados podem ter importantes aplicações na prática clínica dado que a maioria dos tratamentos de doenças neoplásicas implica imunossupressão e, subsequente, recuperação imunológica. Estes estudos podem ser um instrumento fundamental para uma melhor compreensão do sistema imune e guiar uma escolha mais eficiente de opções terapêuticas bem como contribuir para a concepção de futuros estudos clínicos.

Microencapsulation technology: a powerful tool for human embryonic stem cells expansion and cryopreservation

Dissertation presented to obtain a Master degree in Biotechnology at the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Towards a Mobile Three-dimensional Modelling System for Underground Structures

Robotica 2012: 12th International Conference on Autonomous Robot Systems and Competitions April 11, 2012, Guimarães, Portugal

Application of Visual-Inertial SLAM for 3D Mapping of Underground Environments

The underground scenarios are one of the most challenging environments for accurate and precise 3d mapping where hostile conditions like absence of Global Positioning Systems, extreme lighting variations and geometrically smooth surfaces may be expected. So far, the state-of-the-art methods in underground modelling remain restricted to environments in which pronounced geometric features are abundant. This limitation is a consequence of the scan matching algorithms used to solve the localization and registration problems. This paper contributes to the expansion of the modelling capabilities to structures characterized by uniform geometry and smooth surfaces, as is the case of road and train tunnels. To achieve that, we combine some state of the art techniques from mobile robotics, and propose a method for 6DOF platform positioning in such scenarios, that is latter used for the environment modelling. A visual monocular Simultaneous Localization and Mapping (MonoSLAM) approach based on the Extended Kalman Filter (EKF), complemented by the introduction of inertial measurements in the prediction step, allows our system to localize himself over long distances, using exclusively sensors carried on board a mobile platform. By feeding the Extended Kalman Filter with inertial data we were able to overcome the major problem related with MonoSLAM implementations, known as scale factor ambiguity. Despite extreme lighting variations, reliable visual features were extracted through the SIFT algorithm, and inserted directly in the EKF mechanism according to the Inverse Depth Parametrization. Through the 1-Point RANSAC (Random Sample Consensus) wrong frame-to-frame feature matches were rejected. The developed method was tested based on a dataset acquired inside a road tunnel and the navigation results compared with a ground truth obtained by post-processing a high grade Inertial Navigation System and L1/L2 RTK-GPS measurements acquired outside the tunnel. Results from the localization strategy are presented and analyzed.

Comparison of serology, antigenemia assay and the polymerase chain reaction for monitoring active cytomegalovirus infections in hematopoietic stem cell transplantation patients

Forty-six allogeneic hematopoietic stem cell transplantation (HSCT) patients were monitored for the presence of CMV antibodies, CMV-DNA and CMV antigens after transplantation. Immunoenzymatic serological tests were used to detect IgM and the increase in CMV IgG antibodies (increase IgG), a nested polymerase chain reaction (N-PCR) was used to detect CMV-DNA, and an antigenemia assay (AGM) was used to detect CMV antigens. The presence of CMV-IgM and/or CMV-increase IgG antibodies was detected in 12/46 (26.1%) patients, with a median time between HSCT and the detection of positive serology of 81.5 days. A positive AGM was detected in 24/46 (52.2%) patients, with a median time between HSCT and antigen detection of 62 days. Two or more consecutive positive N-PCR results were detected in 32/46 (69.5%) patients, with a median time between HSCT and the first positive PCR of 50.5 days. These results confirmed that AGM and mainly PCR are superior to serology for the early diagnosis of CMV infection. Six patients had CMV-IgM and/or CMV-increase IgG with a negative AGM (five cases) or N-PCR assay (one case). In five of these cases the serological markers were detected during the first 100 days after HSCT, the period of highest risk. These findings support the idea that serology may be useful for monitoring CMV infections in HSCT patients, especially when PCR is unavailable.

Mesenchymal Stem Cell Therapy in Heart Disease

Cardiovascular disease is among the main causes of mortality and morbidity worldwide. Despite significant advances in medical and interventional therapy, the prognosis of conditions such as ischemic heart disease is still dismal. There is thus a need to investigate new therapeutic tools, one of which is stem cell therapy. Hematopoietic stem cells are the most studied type, and the fact that their biology is relatively well understood has led to their being used in preclinical research and clinical trials. However, the results of some of these studies have been controversial, which has opened the way for studies on other cell types, such as mesenchymal stem cells. These cells have immunomodulatory properties which suggest that they have therapeutic potential in cardiology. In the present article, the authors review the state of the art regarding mesenchymal stem cells, from basic and translational research to their use in clinical trials on ischemic heart disease, heart failure and arrhythmias, and discuss possible future uses.

Serological monitoring of a Toxoplasma infection after hematopoietic stem cell transplantation

We report a primary response to Toxoplasma gondii following a hematopoietic stem cell transplantation in a patient with multiple myeloma. The primary response to T. gondii was supported by IgM, IgG and IgA seroconversion. The patient was promptly treated and there were no complications related to toxoplasmosis in the subsequent months.

The monitoring of hematopoietic stem cell transplant donors and recipients from endemic areas for malaria

Malaria is an unusual complication after hematopoietic stem cell transplantation in non-endemic countries. However, transplant candidates, recipients and donors living in endemic regions frequently report previous episodes of malaria. This fact could represent an important risk for immunosuppressed recipients that could develop severe malaria cases. We report a case of hematopoietic stem cell transplant (HSCT) in which the donor had a history of previous malaria, and close monitoring was performed before and after procedure by parasitological and molecular tests. The donor presented Plasmodium vivax in thick blood smears one month after transplant and was treated according to Brazilian Health Ministry guidelines. The polymerase chain reaction (PCR) was able to detect malaria infection in the donor one week earlier than thick blood film. Even without positive results, the recipient was pre-emptively treated with chloroquine in order to prevent the disease. We highlight the importance of monitoring recipients and donors in transplant procedures with the aim of reducing the risk of malaria transmission.

INCIDENCE OF DIARRHEA BY Clostridium difficile IN HEMATOLOGIC PATIENTS AND HEMATOPOIETIC STEM CELL TRANSPLANTATION PATIENTS: RISK FACTORS FOR SEVERE FORMS AND DEATH

We describe the rate of incidence of Clostridium difficile-associated diarrhea (CDAD) in hematologic and patients undergone stem cell transplant (HSCT) at HC-FMUSP, from January 2007 to June 2011, using two denominators 1,000 patient and 1,000 days of neutropenia and the risk factors associated with the severe form of the disease and death. The ELISA method (Ridascreen-Biopharm, Germany) for the detections of toxins A/B was used to identify C. difficile. A multivariate analysis was performed to evaluate potential factors associated with severe CDAD and death within 14 days after the diagnosis of CDAD, using multiple logistic regression. Sixty-six episodes were identified in 64 patients among 439 patients with diarrhea during the study period. CDA rate of incidence varied from 0.78 to 5.45 per 1,000 days of neutropenia and from 0.65 to 5.45 per 1,000 patient-days. The most common underlying disease was acute myeloid leukemia 30/64 (44%), 32/64 (46%) patients were neutropenic, 31/64 (45%) undergone allogeneic HSCT, 61/64 (88%) had previously used antibiotics and 9/64 (13%) have severe CDAD. Most of the patients (89%) received treatment with oral metronidazole and 19/64 (26%) died. The independent risk factors associated with death were the severe form of CDAD, and use of linezolid.

Intracoronary Delivery of Human Mesenchymal/Stromal Stem Cells: Insights from Coronary Microcirculation Invasive Assessment in a Swine Model

BACKGROUND: Mesenchymal stem/stromal cells have unique properties favorable to their use in clinical practice and have been studied for cardiac repair. However, these cells are larger than coronary microvessels and there is controversy about the risk of embolization and microinfarctions, which could jeopardize the safety and efficacy of intracoronary route for their delivery. The index of microcirculatory resistance (IMR) is an invasive method for quantitatively assessing the coronary microcirculation status. OBJECTIVES: To examine heart microcirculation after intracoronary injection of mesenchymal stem/stromal cells with the index of microcirculatory resistance. METHODS: Healthy swine were randomized to receive by intracoronary route either 30x106 MSC or the same solution with no cells (1% human albumin/PBS) (placebo). Blinded operators took coronary pressure and flow measurements, prior to intracoronary infusion and at 5 and 30 minutes post-delivery. Coronary flow reserve (CFR) and the IMR were compared between groups. RESULTS: CFR and IMR were done with a variance within the 3 transit time measurements of 6% at rest and 11% at maximal hyperemia. After intracoronary infusion there were no significant differences in CFR. The IMR was significantly higher in MSC-injected animals (at 30 minutes, 14.2U vs. 8.8U, p = 0.02) and intragroup analysis showed a significant increase of 112% from baseline to 30 minutes after cell infusion, although no electrocardiographic changes or clinical deterioration were noted. CONCLUSION: Overall, this study provides definitive evidence of microcirculatory disruption upon intracoronary administration of mesenchymal stem/stromal cells, in a large animal model closely resembling human cardiac physiology, function and anatomy.

Intravenous Busulfan for Autologous Stem Cell Transplantation in Adult Patients with Acute Myeloid Leukemia: a Survey of 952 Patients on Behalf of the Acute Leukemia Working Party of the European Group for Blood and Marrow Transplantation

Oral busulfan is the historical backbone of the busulfan+cyclophosphamide regimen for autologous stem cell transplantation. However intravenous busulfan has more predictable pharmacokinetics and less toxicity than oral busulfan; we, therefore, retrospectively analyzed data from 952 patients with acute myeloid leukemia who received intravenous busulfan for autologous stem cell transplantation. Most patients were male (n=531, 56%), and the median age at transplantation was 50.5 years. Two-year overall survival, leukemia-free survival, and relapse incidence were 67±2%, 53±2%, and 40±2%, respectively. The non-relapse mortality rate at 2 years was 7±1%. Five patients died from veno-occlusive disease. Overall leukemia-free survival and relapse incidence at 2 years did not differ significantly between the 815 patients transplanted in first complete remission (52±2% and 40±2%, respectively) and the 137 patients transplanted in second complete remission (58±5% and 35±5%, respectively). Cytogenetic risk classification and age were significant prognostic factors: the 2-year leukemia-free survival was 63±4% in patients with good risk cytogenetics, 52±3% in those with intermediate risk cytogenetics, and 37 ± 10% in those with poor risk cytogenetics (P=0.01); patients ≤50 years old had better overall survival (77±2% versus 56±3%; P<0.001), leukemia-free survival (61±3% versus 45±3%; P<0.001), relapse incidence (35±2% versus 45±3%; P<0.005), and non-relapse mortality (4±1% versus 10±2%; P<0.001) than older patients. The combination of intravenous busulfan and high-dose melphalan was associated with the best overall survival (75±4%). Our results suggest that the use of intravenous busulfan simplifies the autograft procedure and confirm the usefulness of autologous stem cell transplantation in acute myeloid leukemia. As in allogeneic transplantation, veno-occlusive disease is an uncommon complication after an autograft using intravenous busulfan.

Overexpressing BDNF in Neural Stem Cells using non-viral gene delivery strategies

Dissertação para obtenção do Grau de Mestre em Biotecnologia