Abstract

Purification and Expansion of Hematopoietic Stem Cells Based on Proteins Expressed by a Novel Stromal Cell Population

Harvey F. Lodish

Hematopoietic stem cells (HSCs) form the basis of bone marrow transplantation for treatment of leukemia and other cancers, and are also a promising cell target for developing gene therapies for treating a broad variety of human diseases. Development of these important clinical applications of HSCs are greatly hampered by a lack of understanding of the extracellular signals that govern their fates and the difficulty in expanding the numbers of these cells in culture. Several years ago we uncovered a novel fetal liver cell population that supports ex vivo expansion of HSCs. DNA array experiments showed that, among other proteins, insulin-like growth factor 2 (IGF-2), angiopoietin-like 3 (Angptl3), are specifically expressed in these cells but not in several cell types that do not support HSCs. We then developed a simple serum-free culture medium for mouse bone marrow HSCs, using low levels of Stem Cell Factor (SCF), thrombopoietin (Tpo), IGF-2, FGF-1, and Angptl3 that supported a greater than 25-fold increase in numbers of long-term repopulating HSCs after a 10-day culture. A similar set of growth factors supports ex vivo expansion of human cord blood HSCs in amounts potentially useful in clinical practice. More recently we showed that these supportive fetal liver cells express large amounts of many HSC-supportive cytokines additional to IGF-2 and Angptl3, and are likely hepatoblasts. We propose that these are the stromal cells that support HSC expansion during embryonic hematopoiesis; whether they also function in adult mice and humans is under intense investigation.

The Role of miRNAs in Leukemic Cells and Normal Hematopoetic Cells

Ai Kotani

Recent studies on microRNAs (miRNAs) in animals have revealed that they control many developmental and physiological processes. Mixed lineage leukemia (MLL) related acute lymphocytic leukemia (ALL) show poor prognosis, largely because the mechanisms by which these leukemias develop are not understood despite intensive research based on well-known concepts and methods. In MLL related ALL, expression of many miRNAs is downregulated, raising the possibility that downregulation of some miRNAs plays a critical role in the pathogenesis of this disease. We showed that the downregulation of miR-128 causes glucocorticoid resistance, whereas downregulation of miR-126 freezes of differentiation in MLL related ALL which is associated with poor prognosis of MLL related ALL. Moreover, we identified a novel DNA base change located on primary miR-128 and SNPs of primary miR-126 in the genome of MLL related ALL derived cell line. The base change significantly affects the processing of both miR-128 and miR-126. Northern blot analysis shows that processing of miR-128 which contains base change cannot be explained in canonical miRNA processing mechanism. These findings suggest that studying miRNA in leukemia not only reveals its critical functions in its pathogenesis but also a clue to possible novel mechanism of miRNA biogenesis.

Apolipoprotein E isoforms in Metabolic Syndrome and Atherosclerosis

Nobuyo Maeda

The risk of developing atherosclerosis in humans increases progressively in association with the three common alleles of APOE in the order APOE*2 < APOE*3 < APOE*4. To investigate the mechanism behind this association, we generated mice expressing human apoE2, apoE3 or apoE4 in place of mouse apoE. We found that apoE4 mice, but not apoE2 or apoE3 mice, develop hypercholesterolemia and atherosclerosis, when they are made to express high levels human LDLR. In contrast, apoE2 mice develop atherosclerosis only when LDLR expression is genetically low. Mice with humanized lipid metabolism have, therefore, uncovered an important interplay between the apoE isoforms and LDLR levels in a manner previously not suspected. We further observed that the body weight gain in response to the consumption of a Western type high fat diet was less in mice expressing apoE4 than those expressing apoE3. Mice expressing apoE2 gained weight most. This mirrors with observations in human populations that body mass index is associated with apoE genotypes in the order of APOE*2 > APOE*3 > APOE*4. However, although epidydimal fat tissues in apoE4 mice were smaller, they were composed of larger adipocytes and expressed PPARγ and adiponectin less than the fat tissues in apoE3 mice. These data suggest an impaired adipocyte differentiation. Indeed, apoE4 mice were less efficient in handling the glucose overload than apoE3 mice. Although both mice became equally glucose intolerant after a prolonged feeding of the high-fat diet, a treatment with rosiglitazone improved insulin sensitivity in apoE3 mice but not in apoE4 mice. Instead, rosiglitazone treatment caused severe fatty changes in the liver in association with reduced LDL receptor and apoE, but increased VLDL receptor expression in apoE4 mice compared to those in apoE3 mice. Thus the roles of apoE are not limited to the lipoprotein metabolism but also important for the total energy homeostasis, contributing to the development of metabolic syndrome in isoform-dependent manners.

Personalized Medicine: Predicting Antipsychotic Dosing Using PET

David C. Mamo

Currently dosing of medications, including antipsychotic drugs, rely on data derived from clinical trials. This has a number of limitations including not accounting for individual variability in age, gender, medical & medication co-morbidity, and individual pharmacokinetics. A further limitation of clinical trials is the necessity of including large samples of the population, making these trials costly and thereby decreasing their feasibility. Dosing of antipsychotic drugs have primarily been studied in younger patients with schizophrenia, and the dosing of these medications with advancing age remains relatively unexamined and subject to either maintenance of the same high doses used in younger patients or a matter of trial and error with risk of relapse. We have previously used neurochemical brain PET imaging to predict dosing of antipsychotics in younger patients using very small samples of patients, and have recently shown that this design can be safely applied to older patients. We present a model of individualized dosing of antipsychotic drugs across the life spectrum that seeks to predict for the individual patient the dose adjustment that is necessary to maintain wellness. This model incorporates data from population kinetic studies with neurochemical PET imaging data collected in older patients. This model has been subject to preliminary testing in a pilot study, demonstrating feasibility and predictive validity. Current larger clinical and PET studies are underway in Toronto and Tokyo to develop this model and translate it to the bedside for individualized dosing of antipsychotics in older patients relying solely on patients' demographic data and plasma antipsychotic level.