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Objective The influence of treatment with granulocyte-colony stimulating factor (G-CSF) on the development of in-stent intimal hyperplasia is not known. We aimed to study this phenomenon in patients who had stents implanted in the course of an acute ST-elevation infarction and successively were treated with G-CSF. Method We performed angiography and intracoronary ultrasound follow-up after 5 months in 41 consecutive patients in the STEMMI trial, which is a randomized double-blind placebo-controlled study on the effect of G-CSF injections on the myocardial function following acute myocardial infarction in patients treated with primary percutaneous coronary stent implantation. The intracoronary ultrasound images were analyzed by a blinded and independent core laboratory. Results There were no differences in in-stent neo-intimal hyperplasia determined by intracoronary ultrasound between patients treated with G-CSF compared to patients treated with placebo. Neo-intimal hyperplasia per mm of stent was 1.87 (±1.41) and 1.89 (±1.39), respectively ( p =0.97). Angiographic in-segment restenosis (>50% diameter stenosis) was found in 28% of patients (24% in the G-CSF group and 33% in the placebo group; p =0.55). Conclusion G-CSF treatment following coronary stent implantation in primary PCI treated AMI patients does not increase in-stent restenosis excessively and it does not seem warranted to limit further study of effects of G-CSF for that reason.

Objectives A phase I safety and efficacy study with granulocyte colony stimulating factor (G-CSF) mobilization of bone marrow stem cells to induce vasculogenesis in patients with severe ischemic heart disease (IHD) was conducted. Design, patients and results 29 patients with IHD participated in the study. Thirteen patients were treated with G-CSF for 6 days and 16 patients served as controls. G-CSF treatment was without any serious adverse events. Four patients were "poor mobilizers" with a maximal increase in CD34+ cells to 5,000±700/mL blood (mean±S.D.) compared to 28,900±5,100/mL blood in "mobilizers". At the follow-up, G-CSF treated had improved in CCS classification, NTG consumption and angina attacks, but the controls only in CCS classification. No difference was seen between the two groups. The decline in NTG consumption tended to be significant in "mobilizers" compared to controls. Myocardial perfusion was unchanged at adenosine stress single photon emission computerized tomography (SPECT) or magnetic resonance images (MRI). Left ventricular ejection fraction decreased from 57% to 52% ( p p =0.07, SPECT) in G-CSF treated, but was unchanged measured with echocardiography. Conclusions Treatment by G-CSF improved symptoms but not signs of myocardial ischemia in patients with severe IHD. The effects seemed related to mobilization of stem cells. An adverse effect on ejection fraction could not be excluded.

Objective To develop a real-time PCR method, based on the 5′nuclease TaqMan technology, for quantitation of clonal cells in multiple myeloma (MM). Materials and Methods The real-time quantitative PCR method incorporates both an allele-specific oligonucleotides (ASO) primer and an ASO dual-labeled fluorogenic probe (ASO TaqMan probe). The ASO primer and probe corresponded to the complementary determining region 3 (CDR3) of the rearranged immunoglobulin heavy chain gene (IgH). With the use of a sequence detector, PCR product accumulation was measured through the ASO TaqMan probe. The real-time PCR method was compared with flow cytometric quantitation of myeloma plasma cells. Results The application of the real-time quantitative ASO IgH PCR method is illustrated by a sequential analysis of minimal residual disease (MRD) in bone marrow (BM) samples from myeloma patients undergoing peripheral blood stem cell (PBSC) transplantation. The real-time PCR method was able to quantitate residual malignant cells in BM samples from patients who were considered to be in complete remission. Further, it was illustrated that a potential problem in determining tumor cell content in myeloma BM samples is the heterogeneous infiltration of the marrow. Conclusion The application of the real-time PCR method provides a sensitive, highly specific, and reproducible quantitation of myeloma cells.

Introduction The most commonly-used method to assess progenitors and hematopoietic stem cells (HSC) present in a graft is the enumeration of phenotypically-defined CD34þ cells. A number of manuscripts, editorials and letters published recently in the Journal of Hematotherapy have addressed standardization methods for CD34þ cell determination [1– 7]. Even though higher numbers of CD34þ cells in the graft correlate with earlier white blood cell (WBC) engraftment and platelet engraftment [8,9] the CD34þ cell population is still extremely heterogeneous and contains a wide range of progenitors, from HSC to single-lineage committed progenitors. There is mounting interest in ex vivo manipulation of CD34þ cells, from ex vivo expansion of primitive progenitors and production of precursors, to the introduction of novel genetic material in HSC [10–14]. A number of culture systems have been described in which progenitors can be expanded and in which retroviral vectors can be introduced in CD34þ cells. However, transplantation of ex vivo manipulated progenitors in humans, as well as xenogeneic immunodeficient animals, has taught us that, although phenotypically-defined CD34þ cells or phenotypicallydefined primitive subsets of the CD34þ cell population can be maintained/expanded for several weeks, ex vivo manipulation for more than 4–7 days results in a dramatic loss of stem-cell capacity [15]. Although there is in vitro evidence that retroviruses can be introduced in > 50% of CD34þ cells, transplantation of such retrovirally-marked CD34þ cells in humans indicates that few, if any, vector-containing cells are detected after transplantation [16]. Therefore, the phenotypic characterization of primitive cells as CD34þ is far from sufficient and assays that can more accurately assess the fraction of progenitors with stem-cell characteristics within the CD34þ population are needed. A number of assays that evaluate CD34þ subpopulations which have more immature characteristics than the majority of the CD34þ population, are currently being used in research laboratories. In this overview, we will describe a number of assays developed in individual investigators’ laboratories that assess primitive human progenitors and, possibly, HSC. We have purposefully chosen to keep this review descriptive and not to recommend one or more assays to be adopted in the clinical setting for the following reasons: n First, assessment of the absolute frequency of primitive progenitors in a given cell population using similar assays in individual laboratories differs considerably. This may, in part, be due to minor differences in culture conditions and, in part, to differences in the interpretation of colony growth. Unless these culture assays are standardized, as has been done for the phenotypic determination of CD34þ cell number, comparison between different laboratories will remain difficult. n Second, it still remains to be determined which assays currently available accurately assess the number of repopulating HSC in a graft. n Third, the inability of the majority of the assays to provide a ‘real time’ answer to the question as to whether HSC are present or preserved, detracts from their clinical usefulness. However, comparisons between ‘stem cell content’ in a given cell population obtained in the research setting and clinical-transplantation results will eventually lead to improved clinical therapies.

Methods in Molecular Medicine: Hematopoietic Stem Cell Protocolsedited by C.A. Klug and C.T. Jordan, Humana Press, 2002. $99.50 (hardback) (332 pages) ISBN 0-89603-812-2It is a widely accepted assumption that there are two major classes of stem cells: embryonic stem cells derived from early embryos, able to replenish all cell types in the animal, and stem cells located in various organs of the body, dedicated to the replenishment of specific tissues such as blood. Clinical transplantations have demonstrated that stem cells exist in the haematopoietic system and stimulated research on methods for the identification and isolation of stem and progenitor cells. This has created a new discipline in medicine, ‘The Stem Cell Laboratory’, focusing on stem cell research and applications, for example, including cell substitution in management of diseases. Such laboratories and their specialists have an insatiable need for protocols describing the exact handling of techniques used for the identification, isolation and characterization of haematopoietic stem cells. The actual status of standardized techniques in mouse and man has recently been published in a comprehensive form by a series of active stem cell researchers.Haematopoietic Stem Cell Protocols presents classical technical protocols for ‘stem cell’ identification and isolation. These techniques include flow cytometry, selection or depletion techniques, cell-cycle analyses, semisolid medium culture of colony-forming units and early progenitors detected as long-term culture-initiating cells, in the ‘cobblestone-area-forming assay’, or as spleen colony-forming units. Quantification of long-term repopulating stem cells by limiting-dilution analysis in competitively repopulated hosts is described well and appears as an easily understandable protocol for human cell assays. Gene transfer and gene expression analyses are also covered with very informative protocols.However, much of what we know might be wrong. This was pointed out by Pottenand Loeffler, who realized that a stem cell's characteristics relate to its future potential and can only be studied effectively by allowing the cell to express that potential. Therefore, to characterize a cell as a stem cell, allowing it to differentiate, the original cell is lost; at the same time one might only see a limited range of responses. They continued this paradox by drawing analogy to Heisenberg's uncertainty principle. Originally formulated in quantum physics theory, it says that ‘the very act of measuring the properties of a certain body inevitably alters the characteristics of that body, thus giving rise to uncertainty in the evaluation of its properties’. Stem cell assays all observe the response after a perturbation to the system. So, the haematopoietic stem cell has not been identified and it is still an open question how it should be defined. While awaiting the answer, we have to accept well-studied and described protocols as a part of our bias.