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Objectives: To generate non-haematopoietic tissues from mobilized haematopoietic CD133+ stem cells. Materials and methods: Mobilized peripheral blood CD133+ cells from adult healthy donors were used. In vitro ability of highly enriched CD133+ cells from mobilized peripheral blood to generate multipotent cells, and their potential to give rise to cells with characteristics of neuroectoderm, endoderm and mesoderm layers was investigated. Results: We found that a recently identified population of CD45+ adherent cells generated in vitro after culture of highly purified CD133+ cells for 3–5 weeks with Flt3/Flk2 ligand and interleukin-6 can, in presence of the appropriate microenvironmental cues, differentiate into neural progenitor-like cells (NPLCs), hepatocyte-like cells and skeletal muscle-like cells. We have termed them to be adult multipotent haematopoietic cells (AMHCs). AMHC-derived NPLCs expressed morphological, phenotypic and molecular markers associated with primary neural progenitor cells. They can differentiate into astrocyte-like cells, neuronal-like cells and oligodendrocyte-like cells. Moreover, AMHC-derived NPLCs produced 3,4-dihydrophenylalanine and dopamine and expressed voltage-activated ion channels, suggesting their functional maturation. In addition, AMHC-derived hepatocyte-like cells and skeletal muscle-like cells, showed typical morphological features and expressed primary tissue-associated proteins. Conclusion: Our data demonstrate that AMHCs may therefore serve as a novel source of adult multipotent cells for autologous replacement cell therapies. [ABSTRACT FROM AUTHOR]

Abstract: A consistent and reproducible depletion technique is crucial for the successful transplantation of an ex vivo depleted graft. Our aim was to evaluate the efficacy of an ex vivo technique for depletion of αβ+ T cells using a biotinylated anti‐TCRαβ monoclonal antibody, which was performed by one clinical nurse specialist. Between 2012 and 2017, 119 depletion procedures from 216 apheresis using the anti‐TCRαβ monoclonal antibody were performed on 105 pediatric patients. The median log depletion of αβ+ T cells was 4.0 (range, 2.5‐5.0). The median recovery rates of CD34+, NK, and γδ+ T cells were 90.4%, 74.9%, and 75.9%, respectively. The efficacy of depletion of αβ+ T cells significantly improved over time and the duration of the depletion procedure significantly decreased over time. Our study demonstrated that this procedure for depletion of αβ+ T cells by skilled staff is highly effective at depleting target cells and obtaining CD34+ progenitor cells. [ABSTRACT FROM AUTHOR]

Abstract: Background: T‐cell depletion (TCD) effectively reduces severe graft‐versus‐host disease in recipients of HLA‐mismatched allografts. However, TCD is associated with delayed immune recovery and increased infections. We hypothesized that specific depletion of CD45RA+ naive T cells, rather than broad depletion of CD3+ T cells, can preserve memory‐immunity in the allografts and confer protection against important viral infections in the early post‐transplant period. Methods: Sixty‐seven patients who received TCD haploidentical donor transplantation for hematologic malignancy on 3 consecutive trials were analyzed. Results: Patients receiving CD45RA‐depleted donor grafts had 2000‐fold more donor T cells infused, significantly higher T‐cell counts at Day +30 post transplant (550/μL vs 10/μL; P < .001), and higher T‐cell diversity by Vbeta spectratyping at Day +100 (P < .001). Importantly, these recipients experienced a significant reduction in both the incidence (P = .002) and duration (P = .02) of any viremia (cytomegalovirus, Epstein‐Barr virus, or adenovirus) in the first 6 months post transplant. Specifically, recipients of CD3‐depleted grafts were more likely to experience adenovirus viremia (27% vs 4%, P = .02). Conclusion: CD45RA‐depletion provided a large number of donor memory T cells to the recipients and was associated with enhanced early T‐cell recovery and protection against viremia. [ABSTRACT FROM AUTHOR]

Background: Excessive T-cell depletion (TCD) is a prerequisite for graft manufacturing in haploidentical stem cell (SC) transplantation by using either CD34 selection or direct TCD such as CD3/CD19 depletion.Study Design and Methods: To optimize graft composition we compared 1) direct or indirect TCD only, 2) a combination of CD3/CD19-depleted with CD34-selected grafts, or 3) TCD twice for depletion improvement based on our 10-year experience with 320 separations in graft manufacturing and quality control.Results: SC recovery was significantly higher (85%, n = 187 vs. 73%, n = 115; p < 0.0001), but TCD was inferior (median log depletion, -3.6 vs. -5.2) for CD3/CD19 depletion compared to CD34 selection, respectively. For end products with less than -2.5 log TCD, a second depletion step led to a successful improvement in TCD. Thawing of grafts showed a high viability and recovery of SCs, but low NK-cell yield. To optimize individualized graft engineering, a calculator was developed to estimate the results of the final graft based on the content of CD34+ and CD3+ cells in the leukapheresis product.Conclusion: Finally, calculated splitting of the starting product followed by CD3/19 depletion together with CD34+ graft manipulation may enable the composition of optimized grafts with high CD34+-cell and minimal T-cell content. [ABSTRACT FROM AUTHOR]

Alloreactivity triggered by interaction between killer cell immunoglobulin-like receptors ( KIRs) and natural killer ( NK) cells plays a role in the graft-versus-tumor effect after hematopoietic stem cell transplantation ( SCT). Our aim in this study was to evaluate this role in the setting of T-cell-repleted haploidentical SCT with postinfusion high-dose cyclophosphamide ( PT-Cy). We included 33 patients. Among patient-donor pairs with at least 1 inhibitory KIR ( iKIR) gene mismatch, event-free survival ( EFS) and cumulative incidence of relapse 1 year after transplant were significantly better (85% vs. 37% [ P = 0.008] and 18% vs. 46% [ P = 0.041], respectively). A subanalysis in 12 patients with Hodgkin's lymphoma ( HL) showed an improvement in EFS 1 year after transplant in those patients with KIR ligand mismatch (100% vs. 25%, P = 0.012), although overall survival ( OS) was not affected (85% vs. 80%, P = 0.2). Eight of 12 patient-donors pairs presented iKIR mismatches. Of note, this outcome was better in the small subgroup, both for EFS (100% vs. 25%, P = 0.012) and for OS (100% vs. 37%, P = 0.004). Our data suggest that in the setting of T-cell-repleted haploidentical SCT with PT-Cy, iKIR mismatch is associated with improved survival, with particularly good results for both iKIR and KIR ligand mismatches in patients with HL. [ABSTRACT FROM AUTHOR]

Background As morbidity and mortality from liver disease continues to rise, new strategies are necessary. Liver transplantation is not only an expensive resource committing the patient to lifelong immunosuppression but also suitable donor organs are in short supply. Against this background, autologous stem cell therapy has emerged as a potential treatment option. Aim To evaluate if it is possible to make a judgement on the safety, feasibility and effect of autologous stem cell therapy for patients with liver disease. Methods MEDLINE and EMBASE were searched up until July 2013 to identify studies where autologous stem cell therapy was administered to patients with liver disease. Results Of 1668 studies identified, 33 were eligible for inclusion evaluating a median sample size of 10 patients for a median follow-up of 6 months. Although there was marked heterogeneity between studies with regards to type, dose and route of delivery of stem cell, the treatment was shown to be safe and feasible largely when a peripheral route of administration was used. Of the studies which also looked at biochemical outcome, statistically significant improvement in liver function tests was seen in 16 studies post-treatment. Conclusion Although autologous stem cell therapy is a much needed possibility in the treatment of liver disease, further robust clinical trials and collaborative protocols are required. [ABSTRACT FROM AUTHOR]

Adult haematopoietic stem/progenitor cells ( HSPCs) constitute the lifespan reserve for the generation of all the cellular lineages in the blood. Although massive progress in identifying the cluster of master genes controlling self-renewal and multipotency has been achieved in the past decade, some aspects of the physiology of HSPCs still need to be clarified. In particular, there is growing interest in the metabolic profile of HSPCs in view of their emerging role as determinants of cell fate. Indeed, stem cells and progenitors have distinct metabolic profiles, and the transition from stem to progenitor cell corresponds to a critical metabolic change, from glycolysis to oxidative phosphorylation. In this review, we summarize evidence, reported in the literature and provided by our group, highlighting the peculiar ability of HSPCs to adapt their mitochondrial oxidative/bioenergetic metabolism to survive in the hypoxic microenvironment of the endoblastic niche and to exploit redox signalling in controlling the balance between quiescence versus active cycling and differentiation. Especial prominence is given to the interplay between hypoxia inducible factor-1, globins and NADPH oxidases in managing the mitochondrial dioxygen-related metabolism and biogenesis in HSPCs under different ambient conditions. A mechanistic model is proposed whereby 'mitochondrial differentiation' is a prerequisite in uncommitted stem cells, paving the way for growth/differentiation factor-dependent processes. Advancing the understanding of stem cell metabolism will, hopefully, help to (i) improve efforts to maintain, expand and manipulate HSPCs ex vivo and realize their potential therapeutic benefits in regenerative medicine; (ii) reprogramme somatic cells to generate stem cells; and (iii) eliminate, selectively, malignant stem cells. Linked Articles This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8 [ABSTRACT FROM AUTHOR]

Haematopoeitic stem cell transplantation ( HSCT) is a curative procedure for children with malignant and non-malignant haematological disease as well as an expanding number of inherited disorders. Most patients lack a human leucocyte antigen-matched related donor, making alternative donors, such as closely matched unrelated donors, unrelated umbilical cord blood donations and haploidentical donors, necessary choices. T cell depletion ( TCD) has been employed for over 30 years to reduce the risk of graft-versus-host disease ( Gv HD) associated with non-genoidentical HSCT. However, until recently overall survival had not improved with TCD due to increased rates of graft failure, disease relapse and delayed immune reconstitution. Recent advances in graft manipulation and reduced toxicity conditioning regimens have offered renewed hope, particularly for children undergoing haploidentical HSCT, where encouraging results have been achieved using negative depletion techniques to retain beneficial accessory cells, which speed immune reconstitution and reduce disease relapse. Translational work building on megadose CD34+ selected grafts, including pathogen-specific immunotherapy, suicide gene therapy and other adoptive cellular immunotherapies, has also offered improved outcomes for such patients. [ABSTRACT FROM AUTHOR]

An introduction is presented in which the editor discusses various reports within the issue on topics including applications of natural killer (NK) cell therapy, NK-cell preparation and phase one of clinical trial by using a NK-cell system.

The article discuses the production and clinical utility of natural killer (NK) cells. It informs that preclinical NK-cell studies have been carried out on a clinical-scale by using current good manufacturing processes techniques and clinical-grade reagents. It also informs that NK-cell therapy products have been applied to patients having a range of malignancies including carcinomas, melanoma and leukemia.

Single cell analysis and cell sorting has enabled the study of development, growth, differentiation, repair and maintenance of 'liquid' tissues and their cancers. The application of these methods to solid tissues is equally promising, but several unique technical challenges must be addressed. This report illustrates the application of multidimensional flow cytometry to the identification of candidate stem/progenitor populations in non-small cell lung cancer and paired normal lung tissue. Seventeen paired tumor/normal lung samples were collected at the time of surgical excision and processed immediately. Tissues were mechanically and enzymatically dissociated into single cell suspension and stained with a panel of antibodies used for negative gating (CD45, CD14, CD33, glycophorin A), identification of epithelial cells (intracellular cytokeratin), and detection of stem/progenitor markers (CD44, CD90, CD117, CD133). DAPI was added to measure DNA content. Formalin fixed paraffin embedded tissue samples were stained with key markers (cytokeratin, CD117, DAPI) for immunofluorescent tissue localization of populations detected by flow cytometry. Disaggregated tumor and lung preparations contained a high proportion of events that would interfere with analysis, were they not eliminated by logical gating. We demonstrate how inclusion of doublets, events with hypodiploid DNA, and cytokeratin+ events also staining for hematopoietic markers reduces the ability to quantify epithelial cells and their precursors. Using the lung cancer/normal lung data set, we present an approach to multidimensional data analysis that consists of artifact removal, identification of classes of cells to be studied further (classifiers) and the measurement of outcome variables on these cell classes. The results of bivariate analysis show a striking similarity between the expression of stem/progenitor markers on lung tumor and adjacent tumor-free lung. © 2012 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

The cancer stem cell (CSC) hypothesis proposes that tumour growth is maintained by a distinct subpopulation of 'CSC'. This study applied flow cytometric methods, reported to detect CSC in both primary and cultured cancer cells of other species, to identify candidate canine subpopulations. Cell lines representing diverse canine malignancies, and cells derived from spontaneous canine tumours, were evaluated for expression of stem cell-associated surface markers (CD34, CD44, CD117 and CD133) and functional properties [Hoecsht 33342 efflux, aldehyde dehydrogenase (ALDH) activity]. No discrete marker-defined subsets were identified within established cell lines; cells derived directly from spontaneous tumours demonstrated more heterogeneity, although this diminished upon in vitro culture. Functional assays produced variable results, suggesting context-dependency. Flow cytometric methods may be adopted to identify putative canine CSC. Whilst cell lines are valuable in assay development, primary cells may provide a more rewarding model for studying tumour heterogeneity in the context of CSC. However, it will be essential to fully characterize any candidate subpopulations to ensure that they meet CSC criteria. [ABSTRACT FROM AUTHOR]

Although acute myeloid leukaemia ( AML) has long been recognized for its morphological and cytogenetic heterogeneity, recent high-resolution genomic profiling has demonstrated a complexity even greater than previously imagined. This complexity can be seen in the number and diversity of genetic alterations, epigenetic modifications, and characteristics of the leukaemic stem cells. The broad range of abnormalities across different AML subtypes suggests that improvements in clinical outcome will require the development of targeted therapies for each subtype of disease and the design of novel clinical trials to test these strategies. It is highly unlikely that further gains in long-term survival rates will be possible by mere intensification of conventional chemotherapy. In this review, we summarize recent studies that provide new insight into the genetics and biology of AML, discuss risk stratification and therapy for this disease, and profile some of the therapeutic agents currently under investigation. [ABSTRACT FROM AUTHOR]

The current review focuses on pathophysiology, animal models and molecular analysis of stroke and retinal ischemia, and the role of stem cells in recovery of these disease conditions. Research findings associated with ischemic stroke and retinal ischemia have been discussed, and efforts towards prevention and limiting the recurrence of ischemic diseases, as well as emerging treatment possibilities with endothelial progenitor cells (EPCs) in ischemic diseases, are presented. Although most neurological diseases are still not completely understood and reliable treatment is lacking, animal models provide a major step in validating novel therapies. Stem cell approaches constitute an emerging form of cell-based therapy to treat ischemic diseases since it is an attractive source for regenerative therapy in the ischemic diseases. In this review, we highlight the advantages and limitations of this approach with a focus on key observations from preclinical animal studies and clinical trials. Further research, especially on treatment with EPCs is warranted. J. Cell. Physiol. 227: 1269-1279, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Summary Analogous to T cells, Natural Killer (NK) cells may facilitate engraftment, combat infection, and control cancer in bone marrow or haematopoietic stem cell transplantation (HSCT); however, NK cells do not cause graft-versus-host disease. Killer immunoglobulin-like receptors (KIRs) regulate NK cell function, and recent data suggest that KIR is as important as its ligand (human leucocyte antigen; HLA) in HSCT for both malignant and non-malignant conditions. Because there is substantial variability in KIR gene content, allelic polymorphism, and cell-surface expression among people, careful selection of donors based on HLA and KIR is essential to optimize HSCT outcomes. Furthermore, NK cells may be used for adoptive immunotherapy after HSCT in place of conventional donor lymphocyte infusion, as part of pre-transplant cytoreductive therapy, or as an independent therapeutic agent in high-risk leukaemia in place of sibling donor HSCT. [ABSTRACT FROM AUTHOR]

As our understanding of the molecular mechanisms governing natural killer (NK) cell activity increases, their potential in cancer immunotherapy is growing increasingly prominent. This review analyses the currently available preclinical and clinical data regarding NK cell-based immunotherapeutic approaches in cancer starting from a historical background and an overview of molecular mechanisms taking part in NK cell responses. The status of NK cells in cancer patients, currently investigated clinical applications such as in vivo modulation of NK cell activity, ex vivo purification/expansion and adoptive transfer as well as future possibilities such as genetic modifications are discussed in detail. [ABSTRACT FROM AUTHOR]

Background The success of gene therapy for inherited neurodegenerative diseases such as metachromatic leukodystrophy (MLD) depends on the development of efficient gene delivery throughout the brain guarded by the blood-brain barrier and achieves distribution of the deficient enzyme throughout the brain. Direct injection of viral vector into the brain parenchyma is too invasive and may not be sufficient to treat the entire brain. As an alternative approach, we examined the feasibility of intrathecal (IT) injection of adeno-associated viral vector serotype 1 (AAV1). Methods AAV1 vector expressing arylsulfatase A (ASA) and green fluorescence protein (GFP) was intrathecally injected into ASA knockout MLD model mice. Expression of GFP was assessed by fluorescence microscopy and immunohistochemical methods, whereas the concentration of ASA was determined by a quantitative enzyme-linked immunosorbent assay. Results Broad distribution of GFP expression was seen throughout the brain after IT injection of AAV1 vector. In addition, a large number of nerve fibers in the dorsal spinal cord and many neural cell bodies in the dorsal root ganglia were efficiently transduced. Widespread distribution of ASA activity and a significant reduction of sulfatide content were confirmed in treated MLD model mice. Conclusions IT injection of AAV1 vector is a useful and non-invasive method for widespread gene delivery to the brain and dorsal root ganglia. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

BACKGROUND: Quantification of CD34+ mononuclear cells is the most important quality control measure for hematopoietic stem cell (HSC) transplantation. A fraction of CD34+ cells also express the CD133 antigen. These cells constitute a group of earlier, less-differentiated HSCs with a potentially higher capacity for engraftment. The correlation between total CD34+ peripheral HSCs and the fraction of these cells that coexpress CD133 was determined before and after automated collection by leukapheresis, as well as the effect of HSC CD133+ dose on hematopoiesis recovery. STUDY DESIGN AND METHODS: Granulocyte–colony-stimulating factor mobilization of HSCs from the marrow to the peripheral blood (PB) of allogeneic and autologous donors was followed by automated collection through leukapheresis on the fifth day. Quantification of CD34+ and CD133+ cells was performed on PB before collection and in the hematopoietic graft (HG) by flow cytometry. RESULTS: There was a significant correlation between CD133+ and CD34+ HSCs in the PB before collection and in the final product for grafting (r = 0.62 and 0.64; p < 0.01). CD34+ HSCs per µL in PB and the HG was the only variable that did not correlate (r = 0.18). CD34+/CD133+ correlation increased from 0.33 on PB to 0.94 on the leukapheresis product (p < 0.01). Time to recovery was not related to CD133+ HSCs infused. CONCLUSION: There was a significant correlation of both number per µL and percentage of CD34+/CD133+ HSCs before and after collection for transplantation; number of CD133+ cells had no apparent clinical impact on time to hematopoiesis regeneration. [ABSTRACT FROM AUTHOR]

BACKGROUND: Because of a high risk of graft-versus-host disease (GVHD), donor lymphocyte infusions with unmodified lymphapheresis products are not used after haploidentical hematopoietic stem cell transplantation. Natural killer (NK) cells have antitumor activity and may consolidate engraftment without inducing GVHD. Production of NK cells under good manufacturing practice (GMP) conditions in a sufficient number is difficult. STUDY DESIGN AND METHODS: Twenty-four apheresis procedures and subsequent NK-cell enrichment from 14 haploidentical donors were performed. NK-cell enrichment was performed using a GMP suitable immunomagnetic procedure. Factors influencing the NK-cell recovery, purity, and NK-cell dose were analyzed. RESULTS: A median number of 4.9 × 108 NK cells were obtained and median NK-cell recovery was 58 percent. Median T-cell depletion was 4.32 log. The absolute NK-cell number in the final product after processing significantly correlated with the preharvest NK-cell content of the peripheral blood (p = 0.002, r = 0.867). The NK-cell recovery was inversely correlated to the absolute NK-cell number in the apheresis product (p = 0.01, r = −0.51). The NK-cell dose per kg of body weight of the patient was inversely correlated to the weight of the patient (p = 0.007, r = −0.533). CONCLUSION: Donors with a high NK-cell count in peripheral blood are likely to provide NK-cell products with the highest cell number. However, maximal NK-cell dose is limited and high NK-cell doses may only be obtained for patients with a low body weight, making children and young adults the best candidates for NK-cell therapy. [ABSTRACT FROM AUTHOR]

CD133 antigen is an integral membrane glycoprotein that can bind with different cells. Originally, however, this cellular surface antigen was expressed in human stem cells and in various cellular progenitors of the haematopoietic system. Human cord blood has been described as an excellent source of CD133+ haematopoietic progenitor cells with a large application potential. One of the main objectives of the present study is to describe for the first time the ultrastructural characteristics of CD133+ stem cells using transmission electronic microscopy. Another objective of the manuscript is to demonstrate through transmission electronic microscopy the molecular image of magnetic nanoparticles connected to the stem cells of great biotechnological importance, as well as demonstrating the value of this finding for electronic paramagnetic resonance and its related nanobioscientific value. Ultrastructural results showed the monoclonal antibody anti-CD133 bound to the superparamagnetic nanoparticles by the presence of electrondense granules in cell membrane, as well as in the cytoplasm, revealing the ultrastructural characteristics of CD133+ cells, exhibiting a round morphology with discrete cytoplasmic projections, having an active nucleus that follows this morphology. The cellular cytoplasm was filled up with mitochondrias, as well as microtubules and vesicles pinocitic, characterizing the process as being related to internalization of the magnetic nanoparticles that were endocyted by the cells in question. Electronic paramagnetic resonance analysis of the CD133+ stem cells detected that the signal (spectrum) generated by the labelled cells comes from the superparamagnetic nanoparticles that are bound to them. These results strongly suggest that these CD133+ cells can be used in nanobiotechnology applications, with benefits in different biomedical areas. [ABSTRACT FROM AUTHOR]

CD133 (prominin-1) was the first in a class of novel pentaspan membrane proteins to be identified in both humans and mice, and was originally classified as a marker of primitive haematopoietic and neural stem cells. Due to the highly restricted expression of CD133 family molecules on plasma membrane protrusions of epithelial and other cell types, in association with membrane cholesterol, a role in the organization of plasma membrane topology has also recently been assigned to this family. Studies have now confirmed the utility of CD133 as a marker of haematopoietic stem cells for human allogeneic transplantation. In addition, CD133 represents a marker of tumour-initiating cells in a number of human cancers, and therefore it may be possible to develop future therapies towards targeting cancer stem cells via this marker. The development of such therapies will be aided by a clearer understanding of the molecular mechanisms and signalling pathways that regulate the behaviour of CD133-expressing cells, and new data outlining the role of Wnt, Notch, and bone morphogenetic protein (BMP) signalling in CD133+ cancer stem cell regulation are discussed within. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

BACKGROUND: Effective T-cell depletion is a prerequisite for haploidentical peripheral blood progenitor cell (PBPC) transplantation. This study was performed to investigate the performance of magnetic cell sorting–based direct large-scale T-cell depletion, which is an attractive alternative to standard PBPC enrichment procedures. STUDY DESIGN AND METHODS: PBPCs were harvested from 11 human leukocyte antigen (HLA)-haploidentical donors. T cells labeled with anti-CD3–coated beads were depleted with a commercially available magnetic separation unit (CliniMACS, Miltenyi Biotec) with either the Depletion 2.1 (D2.1, n = 11) or the novel Depletion 3.1 (D3.1, n = 12) program. If indicated, additional CD34+ selections were performed (n = 6). Eleven patients received T-cell-depleted grafts after reduced-intensity conditioning. RESULTS: The median log T-cell depletion was better with the D2.1 compared to the D3.1 (log 3.6 vs. log 2.3, p < 0.05) and was further improved by introducing an immunoglobulin G (IgG)-blocking step (log 4.5 and log 3.4, respectively). The D3.1 was superior to the D2.1 (p < 0.05) in median recovery of CD34+ cells (90% vs. 78%) and in median recovery of CD3– cells (87% vs. 76%). The median processing times per 1010 total cells were 0.90 hours (D2.1) and 0.35 hours (D3.1). The transplanted grafts (directly T-cell–depleted products with or without positively selected CD34+ cells) contained a median of 10.5 × 106 per kg CD34+, 0.93 × 105 per kg CD3+, and 11.6 × 106 per kg CD56+. Rapid engraftment was achieved in 10 patients. The incidences of acute graft-versus-host disease were less than 10 percent (Grade I/II) and 0 percent (Grade III/IV). CONCLUSION: The novel D3.1 program with IgG blocking enables highly effective, time-saving large-scale T-cell depletion. Combining direct depletion techniques with standard CD34+ selection enables the composition of grafts optimized to the specific requirements of the patients. [ABSTRACT FROM AUTHOR]

The impact of human leucocyte antigen (HLA) class I expression intensity and killer-cell immunoglobulin-like receptor (KIR)-mismatch was investigated on natural killer (NK)-cell mediated lysis of B-lineage leukaemic blasts from 21 paediatric patients in vitro. Blast susceptibility to standardised NK-activity and HLA-expression differed widely. A clear association between HLA-molecules/cell (range 442 000–13 000) and specific lysis (mean 59%, range 13–98%) was observed ( r2 = 0·68). A compound model incorporating HLA-expression and KIR-ligand-mismatch provided an even stronger association ( r2 = 0·87), whereas KIR-ligand-mismatch alone was not significant. Assessment of these factors might identify patients who could benefit from NK-mediated graft-versus-leukaemia effects after mismatched stem cell transplantation. [ABSTRACT FROM AUTHOR]

In recent years, endothelial progenitor cells (EPCs), gave rise to increasing interest because of their possible use as a therapeutic tool in the treatment of vascular lesions in ischemic tissues or as a target for anti neoplastic therapy. It has been shown that several drugs can increase the number of EPCs into the peripheral blood (PB). However, there is insufficient data concerning the mobilization and collection of EPCs during CD34+ cell mobilization. In this study, we have evaluated EPC mobilization and collection in a series of 47 patients affected by lymphoid neoplasms [31 non Hodgkin lymphoma and 16 multiple myeloma] undergoing CD34+ cell mobilization with cyclophosphamide (4000 mg/m2) and Filgrastim (5 μg/kg). PB EPCs identified by flow cytometry as CD34+/VEGFR2+/CD133+ cells showed a peak on day +10. This peak paralleled that of PB CD34+/CD45+ cells. A direct correlation was observed between CD34+ and CD34+/VEGFR2+/CD133+ cells ( r = 0.99 P < 0.0001). An average of 23.7 × 10e6 CD34+/VEGFR2+CD133+ cells have been collected (range 12.1–41.76 × 10e6). These findings showed that in hematological diseases, cyclophosphamide in combination with filgrastim allows the mobilization and collection of large numbers of EPCs which may be used for reparative medicine studies in these patients. [ABSTRACT FROM AUTHOR]

The main obstacles to successful haploidentical haematopoietic stem cell transplantation from a mismatched family member donor are delayed immune reconstitution, vulnerability to infections and severe graft- versus-host disease (GvHD). We designed a reduced-intensity conditioning regimen that excluded total body irradiation and anti-thymocyte globulin in order to expedite immune reconstitution after a CD3-depleted haploidentical stem cell transplant. This protocol was used to treat 22 paediatric patients with refractory haematological malignancies. After transplantation, 91% of the patients achieved full donor chimaerism. They also showed rapid recovery of CD3+ T-cells, T-cell receptor (TCR) excision circle counts, TCR β repertoire diversity and natural killer (NK)-cells during the first 4 months post-transplantation, compared with those results from a group of patients treated with a myeloablative conditioning regimen. The incidence and extent of viremia were limited and no lethal infection was seen. Only 9% of patients had grade 3 acute GvHD, while 27% patients had grade 1 and another 27% had grade 2 acute GvHD. This well-tolerated regimen appears to accelerate immune recovery and shorten the duration of early post-transplant immunodeficiency, thereby reducing susceptibility to viral infections. Rapid T-cell reconstitution, retention of NK-cells in the graft and induction of low grade GvHD may also enhance the potential anti-cancer immune effect. [ABSTRACT FROM AUTHOR]

A novel stem cell marker prominin-1 (CD133) has been shown to be expressed on a subpopulation of CD34+ haematopoietic stem and progenitor cells. The aim of this study was to compare in parallel commercially available CD34+ and CD133+ isolation methods based on paramagnetic bead-coupled antibodies using clinical-grade samples of mobilized peripheral blood from 10 individual healthy donors under identical conditions. The CD133 negative fraction from the first selection was used for CD34+ enrichment to obtain an additional CD34+/CD133− population. Although no significant difference in total cell expansion between cells isolated from the three procedures was observed in a 7-day cytokine-driven suspension culture, the long-term culture-initiating cell assay demonstrated that cells derived by CD34+ isolation contain less primitive progenitors than those isolated based on CD133+ selection. Interestingly, CD34+-enriched progenitors, especially the CD34+/CD133− fraction, contained a significantly higher proportion of erythroid colony-forming cells, whereas the highest content of myeloid colony-forming cells was concentrated in the CD133+ selected cells. These subtle differences between CD34+ and CD133+ immunomagnetic selection will have to be explored for their potential clinical relevance. [ABSTRACT FROM AUTHOR]

The in vitro culture of porcine bone marrow-derived mesenchymal stem cells (MSCs) was used for the investigation of adult stem cell biology. Isolated porcine MSCs possessed the ability to proliferate extensively in an antioxidants-rich medium containing 5% fetal bovine serum (FBS). Greater than 40 serial MSC passages and 100 cell population doublings have been recorded for some MSC batches. Early and late passage MSCs were defined here as those cultures receiving less than 5 trypsin passages and more than 15 trypsin passages, respectively. Consistent with their robust ability to proliferate, both the early and late passage MSCs expressed the cell-cycle promoting enzyme p34cdc2 kinase. Late MSCs, however, exhibited certain features reminiscent of cellular aging such as actin accumulation, reduced substrate adherence, and increased activity of lysosomal acid β-galactosidase. Early MSCs retained the multipotentiality capable of chondrogenic, osteogenic, and adipogenic differentiation upon induction in vitro. In contrast, late MSCs were only capable of adipogenic differentiation, which was greatly enhanced at the expense of the osteochondrogenic potential. Along with these changes in multipotentiality, late MSCs expressed decreased levels of the bone morphogenic protein (BMP-7) and reduced activity of alkaline phosphatase. Late MSCs also exhibited attenuated synthesis of the hematopoietic cytokines granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), and stem cell factor (SCF). The long-term porcine MSC culture, thus, provides a model system to study the molecular interplay between multiple MSC differentiation cascades in the context of cellular aging. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Background Gaucher disease is a lysosomal storage disorder resulting from a deficiency of glucocerebrosidase (GC). Recently, lentivirus vectors have been developed for efficient gene transfer into hematopoietic stem cells (HSCs). A recombinant lentivirus vector was used to evaluate the transduction of the human GC gene into murine bone-marrow-derived HSCs and its expression in their progeny. Methods Murine HSCs were transduced with lentivirus vector (lenti-EF-GC; MOI = 10-100). We transplanted female wild-type C57BL/6J mice with genetically modified male HSCs via the tail vein. Results We show that intravenous transplantation of transduced HSCs has therapeutic potential. Enzyme activity was increased two- to three-fold in various tissues, especially in the hematopoietic system. Numerous transplanted HSCs survived for 6 months and were shown by PCR to contain the provirus genes; the Y chromosome was identified by FISH analysis in the cells of female mouse recipients. Conclusions The recombinant lentiviral vector transduces HSCs that are capable of long-term gene expression in vivo. This approach is potentially useful for the treatment of patents with Gaucher disease and other lysosomal storage disorders. Copyright © 2005 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

There are more than 40 different forms of inherited lysosomal storage diseases (LSDs) known to occur in humans and the aggregate incidence has been estimated to approach 1 in 7000 live births. Most LSDs are associated with high morbidity and mortality and represent a significant burden on patients, their families, and health care providers. Except for symptomatic therapies, many LSDs remain untreatable, and gene therapy is among the only viable treatment options potentially available. Therapies for some LSDs do exist, or are under evaluation, including heterologous bone marrow transplantation (BMT), enzyme replacement therapy (ERT), and substrate reduction therapy (SRT), but these treatment options are associated with significant concerns, including high morbidity and mortality (BMT), limited positive outcomes (BMT), incomplete response to therapy (BMT, ERT, and SRT), life-long therapy (ERT, SRT), and cost (BMT, ERT, SRT). Gene therapy represents a potential alternative therapy, albeit a therapy with its own attendant concerns. Animal models of LSDs play a critical role in evaluating the efficacy and safety of therapy for many of these conditions. Naturally occurring animal homologs of LSDs have been described in the mouse, rat, dog, cat, guinea pig, emu, quail, goat, cattle, sheep, and pig. In this review we discuss those animal models that have been used in gene therapy experiments and those with promise for future evaluations. Copyright © 2004 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Neoangiogenesis is an integral component of bone marrow myeloproliferation in patients with myelofibrosis with myeloid metaplasia (MMM). As extramedullary haematopoiesis is a constitutive feature of MMM, we studied spleen neoangiogenesis by a computerized image analysis in MMM patients. Compared with five normal subjects, spleen CD34-staining capillary vascular density (CVD) was 2·1–3·03 times higher than the upper range of normal in six of the 15 (40%) MMM patients. CD8-staining sinusoidal vascular density (SVD) was constantly normal or lesser than normal and was inversely correlated with CVD ( R = −0·53; P = 0·04). In MMM patients who did not receive cytoreductive or radiation therapy in the month before splenectomy ( n = 9), the CVD was a significant determinant of spleen size ( R = 0·88; P = 0·04). In MMM patients, the number of spleen CD34+ haematopoietic stem cells was increased from 1·2 to 98 times the upper limit of normal, and predicted the expansion of CVD ( R = 0·57; P = 0·03). A population of cells expressing the CD34+/CD133+/VEGFR-2+ angiopoietic phenotype was present in the blood and spleen of five of seven patients. These results document that neoangiogenesis is an integral component of spleen re-localization of haematopoietic stem cells and suggest a cellular mechanism for spleen neoangiogenesis. [ABSTRACT FROM AUTHOR]

Investigates the clinical feasibility and safety of CD133-based selection and transplantation in a small number of pediatric patients. Marker of more primitive hematopoietic progenitors; Proportion of CD133 selected cells; Evidence of a rapid platelet recovery; T-cell regeneration.

The broader application of stem cell transplantation (SCT) for paediatric diseases has been limited by a lack of human leucocyte antigen (HLA)-matched donors. Virtually all children, however have at least one haploidentical parent who could serve as a donor. Such a donor is immediately available and the considerable costs of additional HLA typing, registry and banking expenditures that are necessary to procure an unrelated donor, could be reduced. Recent technological advances appear to have overcome the historical problems of graft rejection and severe graft versus host disease in the haploidentical setting, and in the latest studies the overall survival for children undergoing haploidentical SCT for leukaemia is now comparable with that following unrelated donor bone marrow or cord blood transplantation. Post-transplant infectious complications and leukaemia relapse remain the most important barriers yet to overcome, and new directions in the use of adoptive cellular immunity appear to be promising in this respect. Haploidentical SCT is now a viable option for those children who do not have an HLA compatible sibling or fully matched unrelated donor. The relative merits of a haploidentical family donor versus mismatched unrelated bone marrow or cord blood donation needs to be assessed in prospective, randomized clinical trials. [ABSTRACT FROM AUTHOR]

Abstract: The transplantation of megadoses of haploidentical hematopoietic stem cells is for a number of children with malignant or nonmalignant diseases the only curative approach. In order to prevent severe GvHD, the removal of T lymphocytes from the stem cell graft either by positive selection of CD34+ stem cells or by negative depletion of CD3+ T lymphocytes is necessary. We present the results obtained so far by CD34+ positive selection and discuss new techniques of graft engineering which might hopefully further improve the outcome of haploidentical stem cell transplantation. [ABSTRACT FROM AUTHOR]

Cases of Burkitt's lymphoma (BL) from north-western Iran were investigated for the usage and somatic mutational pattern of their immunoglobulin variable region genes. Potentially functional VH genes were amplified from 6/12 of the tumour masses and all of these were derived from the VH3 family, with 4/6 being derived from the most commonly used VH3 family member, V3-23. All of the tumour sequences were mutated from their germline counterparts, to varying degrees, with a mean level of 5.8%, indicating that the cell of origin had encountered the germinal centre. Intraclonal sequence heterogeneity was also evident in 4/6 of the lymphomas, showing that the tumour cells had undergone further somatic mutation following neoplastic transformation. Analysis of the five potentially functional mutated VH sequences showed a significant clustering of replacement mutations in the complementarity-determining region 2, consistent with a role for antigen in selection of tumour cell sequences. The pattern of extensive somatic mutation, and intraclonal variation, in these mainly EBV+ve tumours, was similar to that previously reported in VH sequences of EBV+ve endemic BL (eBL) and EBV-ve sporadic BL (sBL), with the mean level of somatic mutation lying between those reported for eBL (7.7%) and sBL (4.0%). However, VH gene bias and the distribution of mutations in the Iranian cases showed features which differed from those reported for endemic or sporadic BL. [ABSTRACT FROM AUTHOR]