Hematopoietic Stem Cell Protocols: Hematopoietic stem cell analysis ‘step by step’

Abstract

Hematopoietic Stem Cell Protocols (Methods in Molecular Medicine)edited by Christopher A. Klug and Craig T. Jordan. Humana Press, 2001.The past decade has produced remarkable advances in our ability to characterize the various cell lineages of human hematopoietic stem cells (HSCs). HSCs are self-renewing progenitors that give rise to all lineages of blood cells. They are found in all hematopoietic organs, from para-aortic mesoderm and yolk sac in fetuses to the bone marrow, blood and spleens of adults. Our knowledge of HSCs can be applied successfully in clinical practice. The most obvious immediate benefit is the possibility of increasing the chemotherapeutic dose in cancer and hematological malignancies by way of hematopoietic stem cell transplantation. This technique has potential in many clinical settings, including the treatment of other non-malignant diseases.Medical knowledge is constantly renewed. This is particularly true for hematopoietic stem-cell transplantation, hematopoietic gene therapy, solid organ transplantation and somatic tissue regeneration. The technology involved is developing rapidly and in this context the selection of stem cells and their characterization and expansion in vitro are vital processes. Perhaps the most exciting potential of cell therapy is our ability to manipulate cells by modifying their physiological properties before returning them to a patient. Cell therapy is evolving quickly, drawing on cell biology, molecular biology, virology, immunology and cell quantification techniques and also on biomedical engineering.These advances require well defined, reproducible and firmly established laboratory methods for investigating HSCs. The hematopoietic system comprises a concentrated series of stem and transit progenitor cell compartments of progressively restricted potentiality and proliferative capacity. In this setting, Hematopoietic Stem Cell Protocols, edited by Christopher A. Klug and Craig T. Jordan, is an essential handbook for novice and experienced investigators alike, which gives a wide variety of step-by-step instructions for the study of mouse and human HSCs of both embryonic and adult origin.The 20 chapters present techniques for stem cell analysis, some of which have become standardized over the past few years. Many chapters contain contributions by the researchers who first developed and introduced the procedures. Each chapter begins with a short general introduction referring to most of the main publications in the field, then the major part precisely reports the materials and methods, giving technical laboratory details or even ‘home-made cooking secrets’. Methods are described in the following areas: aorta–gonad–mesonephros and yolk sac HSCs; isolation of mouse HSCs; flow cytometric analysis and immunoselection of HSCs; purification of human HSCs by flow cytometry; cycling and turnover of HSC; cell-cycle analysis of primitive stem cells; hematopoietic colony-forming cells; long-term culture-initiating cell (LTC-IC) assays for human and murine cells; assays for cobblestone area-forming cells; colony forming unit on the spleen (CFU-S) assays; T-cell progenitor activity and competitive repopulation assays for HSCs in the NOD and SCID models. Protocols for stem cell expansion and the hematopoietic maturation of ES cells are included, as are detailed methods for the genetic modification of stem cells, for example, retroviral infection of murine HSCs, retroviral transduction of purified HSCs, retroviral-mediated transduction and HIV-based vectors. The last section deals with gene expression in HSCs and the use of two-dimensional gene-expression fingerprinting.This is a comprehensive compendium of practical techniques, a state-of-the-art handbook. It will be a useful tool for teams involved in either basic hematopoiesis research or progenitoror stem cell quantification for cell therapy protocols in the context of patient care. Thanks to a multidisciplinary approach, from fundamental to practical aspects, this book reports advances made in the field of hematopoiesis, which could subsequently be applied for diagnostic and therapeutic purposes.