Journal of hematotherapy & stem cell research

Journal of Hematotherapy & Stem Cell ResearchVol. 12, No. 1 CorrespondenceLong-lasting Hypogammaglobulinemia Following Rituximab Administration for Epstein-Barr Virus-Related Post-Transplant Lymphoproliferative Disease Preemptive TherapyElio Castagnola, Sandro Dallorso, Maura Faraci, Giuseppe Morreale, Daniela Di Martino, Emilio Cristina, Lucia Scarso, and Edoardo LaninoElio Castagnola, Sandro Dallorso, Maura Faraci, Giuseppe Morreale, Daniela Di Martino, Emilio Cristina, Lucia Scarso, and Edoardo LaninoPublished Online:9 Jul 2004https://doi.org/10.1089/152581603321210082AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail "Long-lasting Hypogammaglobulinemia Following Rituximab Administration for Epstein-Barr Virus-Related Post-Transplant Lymphoproliferative Disease Preemptive Therapy." , 12(1), pp. 9–10FiguresReferencesRelatedDetailsCited byRituximab-associated Hypogammaglobulinemia in pediatric patients with autoimmune diseases28 August 2019 | Pediatric Rheumatology, Vol. 17, No. 1Transplant Infectious Disease, Vol. 21, No. 3Recurrent bacterial pneumonia due to immunoglobulin G2 subclass deficiency after allogeneic hematopoietic stem cell transplantation: Efficacy of immunoglobulin replacement25 March 2018 | Transplant Infectious Disease, Vol. 20, No. 3Juvenile-onset systemic lupus erythematosus (jSLE) – Pathophysiological concepts and treatment optionsBest Practice & Research Clinical Rheumatology, Vol. 31, No. 4A case of septic arthritis caused by a Mycoplasma salivarium strain resistant towards Ciprofloxacin and Clarithromycin in a patient with chronic lymphatic leukemiaDiagnostic Microbiology and Infectious Disease, Vol. 86, No. 1Immunreconstitution and Infectious Complications After Rituximab Treatment in Children and Adolescents: What Do We Know and What Can We Learn from Adults?29 January 2015 | Cancers, Vol. 7, No. 1Rituximab-associated hypogammaglobulinemia: Incidence, predictors and outcomes in patients with multi-system autoimmune diseaseJournal of Autoimmunity, Vol. 57Hypogammaglobulinaemia after rituximab treatment--incidence and outcomes28 April 2014 | QJM, Vol. 107, No. 10Pneumocystis jirovecii pneumonia in two patients with systemic lupus erythematosus after rituximab therapy9 January 2014 | Clinical Rheumatology, Vol. 33, No. 3Safety of rituximab in rheumatoid arthritis: A long-term prospective single-center study of gammaglobulin concentrations and infectionsJoint Bone Spine, Vol. 79, No. 4Tolérance du rituximab dans la polyarthrite rhumatoïde : expérience monocentrique du suivi au long cours des concentrations sériques de gammaglobulines et de la survenue d'infectionRevue du Rhumatisme, Vol. 79, No. 4Does rituximab increase the incidence of infectious complications? A narrative reviewInternational Journal of Infectious Diseases, Vol. 15, No. 1Viral-load and B-lymphocyte monitoring of EBV reactivation after allogeneic hemopoietic SCT in children26 October 2009 | Bone Marrow Transplantation, Vol. 45, No. 6Outcome of treatment of Epstein-Barr virus-related post-transplant lymphoproliferative disorder in hematopoietic stem cell recipients: a comprehensive review of reported casesTransplant Infectious Disease, Vol. 11, No. 5Delay in B-lymphocyte recovery and function following rituximab for EBV-associated lymphoproliferative disease early post-allogeneic hematopoietic SCT24 November 2008 | Bone Marrow Transplantation, Vol. 43, No. 9Rituximab Maintenance for the Treatment of Patients With Follicular Lymphoma: Systematic Review and Meta-analysis of Randomized Trials18 February 2009 | JNCI: Journal of the National Cancer Institute, Vol. 101, No. 4Risk factors for infection in haematology patients treated with rituximabEuropean Journal of Haematology, Vol. 82, No. 1Overview of Infections in the Immunocompromised Host17 September 2008Non-endocrine late complications in children after allogeneic haematopoietic SCT11 June 2008 | Bone Marrow Transplantation, Vol. 41, No. S2Hypogammaglobulinemia with a selective delayed recovery in memory B cells and an impaired isotype expression after rituximab administration as an adjuvant to autologous stem cell transplantation for non-Hodgkin lymphomaEuropean Journal of Haematology, Vol. 77, No. 3Rituximab for steroid-refractory chronic graft-versus-host diseaseBlood, Vol. 108, No. 2Persistent Panhypogammaglobulinemia After CHOP-Rituximab for HIV-Related LymphomaJournal of Clinical Oncology, Vol. 23, No. 1Aberrant Blood Grouping Results in a Patient with Splenomegaly and ThrombocytopeniaLaboratory Medicine, Vol. 35, No. 9Prolonged hypogammaglobulinemia following rituximab treatment for post transplant Epstein–Barr virus-associated lymphoproliferative disease1 December 2003 | Bone Marrow Transplantation, Vol. 33, No. 1 Volume 12Issue 1Feb 2003 To cite this article:Elio Castagnola, Sandro Dallorso, Maura Faraci, Giuseppe Morreale, Daniela Di Martino, Emilio Cristina, Lucia Scarso, and Edoardo Lanino.Long-lasting Hypogammaglobulinemia Following Rituximab Administration for Epstein-Barr Virus-Related Post-Transplant Lymphoproliferative Disease Preemptive Therapy.Journal of Hematotherapy & Stem Cell Research.Feb 2003.9-10.http://doi.org/10.1089/152581603321210082Published in Volume: 12 Issue 1: July 9, 2004PDF download

Injury or degeneration of the vertebrate central nervous system often disrupts neuronal circuitry that is built by projection neurons during early embryonic life. Repair of neural network through regeneration of these early-born projection neurons in adult life often fails since stem cells residing in the adult brain are generally programmed to give rise to late-born interneurons. Thus, exogenous cells are needed to rebuild the neural circuitry. Nevertheless, cell replacement in the brain remains a challenging goal because of the lack of safe and effective donor cells, as well as difficulty in remodeling the nonneurogenic adult CNS environment. Here I will concentrate on the donor side and discuss how recent advancement in stem cell technology offers hope for transplant therapy, with a focus on the potentials and hurdles of human embryonic stem cells as a sustainable source.

The development of culture systems that facilitate ex vivo maintenance and expansion of transplantable hematopoietic progenitor cells (HPC) is vital to stem cell transplantation. The use of a monolayer of stromal cells on which to grow HPC in direct contact allows high efficiency ex vivo expansion of HPC. Here, we report an establishment of three murine embryonic fibroblast stromal cell lines from adherent cells of day-12 mouse embryos. Among them, HYMEQ-5 was most efficient in supporting long-term maintenance of human umbilical cord blood (CB) CD34(+) cells. Human CB CD34(+) cells cultured on HYMEQ-5 in the presence of stem cell factor (SCF), thrombopoietin, and flk-ligand (FL) showed high expansion of CD34(+)CD38(-) cells and highly proliferative potential-colony forming cells (HPP-CFC). Direct cell-to-cell contact between CD34(+) cells and HYMEQ-5 was important for this expansion. RT-PCR analysis showed that HYMEQ-5 produced FL, SCF, interleukin-6, and macrophage colony-stimulating factor (M-CSF). Expanded CB CD34(+) cells efficiently reconstituted hematopoiesis in nonobese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. These findings suggest that HYMEQ-5 provides a milieu that supports long-term human hematopoiesis as well as ex vivo expansion of human CB CD34(+) HPC. This cell line may facilitate elucidation of the mechanism of cellular interactions between HPC and stromal cells.

The possibility of infection transmission by infusion of cryopreserved peripheral blood stem cells concentrates or bone marrow is well known. For this reason the European Blood and Marrow Transplantation Group (EBMT) and International Society for Haemotherapy and Graft Engineering (ISHAGE) standards include a panel of serological tests to be performed in donors and patients with the aim to lower the likelihood of infection transmission. The study was performed on a group of 71 patients and 22 donors. No laboratory signs of active infection were found in 15 donors ( 13 related, 2 unrelated), i.e., in 68.2% and in 55 patients (77.5%). The active infection from herpes viruses was the most common ( in patients 13, in donors 7). Hepatitis B was found in only one case. The cytomegalovirus (CMV) immunoglobulin G (IgG) test was the most common marker of previous infection, and it was found in 14 donors and 55 patients. We can conclude that the rate of clinically unsuspected infections in donors and patients, including cases requiring immediate treatment among the patient groups, is relatively high and fully justifies the practice of prophylactic serological testing using the whole palette of tests according to EBMT and ISHGE in both autologous and allogenous transplantations of hematopoietic stem cells.

Adoptive transfer of donor-derived cytomegalovirus (CMV)-specific cytotoxic T cell (CTL) clones can restore immunity in allogeneic stem cell transplant recipients, providing protection against CMV disease. Current methods for selecting and expanding CMV-specific T cell clones are technically difficult, making adoptive T cell therapy impractical for routine clinical use. In this study, we describe a method for ex vivo generation and expansion of high-purity CMV-specific CTL using peptide-pulsed dendritic cells as antigen-presenting cells. Generation of CMV-specific CTL in numbers sufficient for clinical use in the time span of 4 weeks was accomplished in 6 of 8 CMV-seropositive donors. Examination of pp65 specificity by HLA/peptide tetramer staining demonstrated that a purity of greater than 95% peptide-specific cells could be obtained after two weekly stimulations and retained after further expansion for 3-4 weeks. Median expansion of total cell number was greater than 500-fold and expansion of peptide-specific CTL by tetramer staining was greater than 1.7 x 10(5)-fold. Four weeks after initiating CTL culture, we were able to generate greater than 10(9) total cells that specifically lysed target cells loaded with CMV peptide and cells infected with CMV. This simple and rapid method for generating high-purity CMV-specific CTL for adoptive immunotherapy is currently being examined for routine clinical use for allogeneic stem cell transplantation.

Immunotherapy with monocyte-derived dendritic cells (Mo-DCs) is applied to an increasing number of patients requiring large-scale production of clinical-grade dendritic cells with standardized Mo-DC generation protocols. In many countries, e.g., in Germany, Mo-DCs are legally considered medicinal products, which must be produced under Good Manufacturing Practice (GMP) conditions by an institution holding an official production license. Plastic adherence, immunomagnetic selection of CD14(+) monocytes and depletion of CD2(+) and CD19(+) cells are used to enrich monocytes for Mo-DC culture. The latter two have received approval of the European Union (CE). However, enrichment by plastic adherence is well-established and commonly used for clinical and research Mo-DC applications. The various plastic materials, nevertheless, have not been officially approved for monocyte selection for clinical use. In the present study therefore, we compared three methods for enrichment of CD14(+) monocytes with regard to efficiency of enrichment, yield of monocyte-derived functional mature dendritic cells, cost effectiveness, and handling. We demonstrate that CD14 selection and CD2 and CD19 depletion yield similar results regarding purity of mature DEs MoDCs (97-99% vs. 64-97%) and their immunostimulatory capacity. However, cell preparations cultured after CD14 selection possessed 91% to 97% CD14(+) cells, whereas CD2-/and DC19-depleted preparations contained only 8% to 57% CD14(+) cells. Thus, positive selection requires smaller culture volumes to generate equal numbers of Mo-DCs. Both methods gave better results than plastic adherence. In conclusion, of the techniques examined, CD14 selection of monocytes gave the best results regarding reproducibility, yield, and purity of the resulting monocytes and mature Mo-DCs.

Our previous studies had shown that a combination of the bio-antioxidant catalase and the membrane stabilizer trehalose in the conventional freezing mixture affords better cryoprotection to hematopoietic cells as judged by clonogenic assays. In the present investigation, we extended these studies using several parameters like responsiveness to growth factors, expression of growth factor receptors, adhesion assays, adhesion molecule expression, and long-term culture-forming ability. Cells were frozen with ( test cells) or without additives ( control cells) in the conventional medium containing 10% dimethylsulfoxide ( DMSO). Experiments were done on mononuclear cells (MNC) from cord blood/fetal liver hematopoietic cells (CB/FL) and CD34(+) cells isolated from frozen MNC. Our results showed that the responsiveness of test cells to the two early-acting cytokines, viz. interleukin-3 (IL-3) and stem cell factor (SCF) in CFU assays was better than control cells as seen by higher colony formation at limiting concentrations of these cytokines. We, therefore, analyzed the expression of these two growth factor receptors by flow cytometry. We found that in cryopreserved test MNC, as well as CD34(+) cells isolated from them, the expression of both cytokine receptors was two- to three-fold higher than control MNC and CD34(+) cells isolated from them. Adhesion assays carried out with CB/FL-derived CD34(+) cells and KG1a cells showed significantly higher adherence of test cells to M210B4 than respective control cells. Cryopreserved test MNC as well as CD34(+) cells isolated from them showed increased expression of adhesion molecules like CD43, CD44, CD49d, and CD49e. On isolated CD34(+) cells and KG1a cells, there was a two- to three-fold increase in a double-positive population expressing CD34/L-selectin in test cells as compared to control cells. Long-term cultures (LTC) were set up with frozen MNC as well as with CD34(+) cells. Clonogenic cells from LTC were enumerated at the end of the fifth week. There was a significantly increased formation of CFU from test cells than from control cells, indicating better preservation of early progenitors in test cells. Our results suggest that use of a combination of catalase and trehalose as a supplement in the conventional freezing medium results in better protection of growth factor receptors, adhesion molecules, and functionality of hematopoietic cells, yielding a better graft quality.

Journal of Hematotherapy & Stem Cell ResearchVol. 12, No. 1 Highlights of Our Next IssueHighlights of Our Next IssueDenis EnglishDenis EnglishSearch for more papers by this authorPublished Online:9 Jul 2004https://doi.org/10.1089/152581603321210055AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail "Highlights of Our Next Issue." , 12(1), p. 1FiguresReferencesRelatedDetailsCited byDifferential Responses of Human Neural and Hematopoietic Stem Cells to Ethanol Exposure Hsiao-Nan Hao, Graham C. Parker, Jane Zhao, Kaveh Barami, and William D. Lyman9 July 2004 | Journal of Hematotherapy & Stem Cell Research, Vol. 12, No. 4 Volume 12Issue 1Feb 2003 To cite this article:Denis English.Highlights of Our Next Issue.Journal of Hematotherapy & Stem Cell Research.Feb 2003.1-1.http://doi.org/10.1089/152581603321210055Published in Volume: 12 Issue 1: July 9, 2004PDF download

In this methodological study, we describe an assay for analysis of proliferative T cell responses in patients with severe leukopenia. Severe treatment-induced cytopenia is observed in patients with malignant disorders who receive conventional intensive chemotherapy or autologous stem cell transplantation. The quantitative T cell defect can then be characterized by flow cytometric analysis of membrane molecule expression, whereas the functional status of the remaining T cell population is more difficult to evaluate. In the present study, we describe a standardized whole blood assay that requires small sample volumes and can be used for repeated analysis even in severely ill patients. The assay is based on the following strategy: (i) blood samples are diluted with the serum-free medium X-vivo 10, (ii) T cells are activated either with monoclonal immunoglobulin E (IgE) anti-CD3 or anti-CD3 plus anti-CD28; (iii) T cell proliferation is assayed by [(3)H]thymidine incorporation after 4 days of in vitro culture. These proliferative responses are not affected by the plasma levels of interleukin-2 (IL-2), sIL-2-R alpha, IL-7 and IL-15, and the kinetics of the response are not altered by the presence of exogenous cytokines. Detectable proliferation is observed for most patients with treatment-induced cytopenia. We conclude that the assay can be used for functional characterization of remaining T lymphocytes in patients with severe T lymphopenia.

The high proliferative potential of cord blood (CB) stem cells and the identification of the key factor of megakaryopoiesis, thrombopoietin (TPO), permit the ex vivo expansion of megakaryocytes (MKs) for possible use in early post-transplant support of patients and the production of functional platelets for transfusion. However, culture conditions for the generation of adequate MKs for this purpose are not yet optimized. Therefore, we sought to define the mixture of early-acting cytokines and TPO that would promote the expansion of MK progenitors over other lineages and result in overall better MK expansion and platelet yields. CB CD34(+)-enriched cells were cultured in serum-free medium for 17 days in presence of TPO alone or in various combinations with early-acting cytokines used at different concentrations and addition times. MK expansion and polyploidy and platelet production were monitored by flow cytometry analysis using specific surface markers (CD41 and CD42b) and propidium iodide labeling. Our results showed that the use of high concentrations of stem cell factor (SCF) and Flt-3 ligand (FL) in early CB TPO-supplemented cultures was more favorable to monocytic and granulocytic cell expansion. However, we observed that their presence in limiting amounts was required for the preferential expansion of MK progenitors. The addition of SCF, FL, TPO, and interleukin-6 (IL-6) at high concentrations in secondary cultures of these expanded MKs resulted in optimal MK proportion (approximately 25% of MKs) and expansion (>300 MK per seeded cell), highest proportions of polyploid MKs (22% of mature MKs > or = 8N), and best platelet yields. Our results indicate that TPO-induced MK progenitors are more sensitive to early-acting cytokines than non-MK cells. We propose that MKs generated in the optimized conditions, in combination with immature stem/progenitor cells, could prove useful for the short-term platelet recovery following CB transplantation.

The use of human umbilical cord blood (hUCB)-a rich source of nonembryonic or adult stem cells-has recently been reported to ameliorate behavioral consequences of stroke. In this study, we tested whether human cord blood leukocytes also ameliorate behavioral impairments of spinal cord injury. Rats were divided into five groups: (1) laminectomy (without spinal cord injury) only; (2) laminectomy + cord blood infusion; (3) spinal cord injury + cord blood infused 1 day post injury; (4) spinal cord injury + cord blood infused 5 days post injury; and (5) spinal cord injury only. Spinal cord injury was induced by compressing the spinal cord for 1 min with an aneurysm clip calibrated to a closing pressure of 55 g. Open-field behavior was assessed 1, 2, and 3 weeks after intravenous injection of prelabeled human cord blood cells. Open-field test scores of spinal cord injured rats treated with human cord blood at 5 days were significantly improved as compared to scores of rats similarly injured but treated at day 1 as well as the otherwise untreated injured group. The results suggest that cord blood stem cells are beneficial in reversing the behavioral effects of spinal cord injury, even when infused 5 days after injury. Human cord blood-derived cells were observed in injured areas, but not in noninjured areas, of rat spinal cords, and were never seen in corresponding areas of spinal cord of noninjured animals. The results are consistent with the hypothesis that cord blood-derived stem cells migrate to and participate in the healing of neurological defects caused by traumatic assault.

Sublethal cyclophosphamide treatment induces unique regeneration patterns in bone marrow and the spleen of a mouse. Colony-forming units spleen (CFU-S)(day 8), CFU-granulocyte-macrophage (GM), nucleated cell counts, and their differentials in bone marrow, spleen, and peripheral blood were determined in mice treated with a single dose of cyclophosphamide. To study further the mechanisms underlying the unique patterns of hematopoietic regeneration after cyclophosphamide, mRNA levels for stem cell factor (SCF), Flt-3 ligand, and macrophage inflammatory factor (MIP)-1 alpha cytokines were determined in bone marrow and spleen. Granulocyte precursor cells were less depleted by cyclophosphamide compared to erythroid nucleated cells and lymphocytes both in bone marrow and spleen. Rapid expansion of granulopoietic cells increased the granulocytic/erythroid ratio significantly during regeneration. CFU-S in the bone marrow and the spleen showed different sensitivity in vivo but not in vitro to cyclophosphamide; CFU-GM were equisensitive in both sites. In bone marrow, an initial fast recovery of CFU-S and CFU-GM on days 2 to 3 was followed by a secondary deep decline in their numbers occurring between days 5 and 7. This decline was accompanied with a depression of CFU-S proliferation and with significantly increased CFU-S numbers in the peripheral blood. In the spleen, absolute CFU-S and CFU-GM numbers were increased several-fold at this time. Seven days after cyclophosphamide, the spleen contained 69% of the total body CFU-S compared to 4% in controls. Splenectomy did not abolish the secondary disease of CFU-S in the bone marrow, but it led to a marked elevation of circulating leukocytes and CFU-S. There was an eight-fold increase in the SCF mRNA level in the bone marrow 2 days after cyclophosphamide, corresponding with a high proliferation rate of CFU-S. No significant changes in mRNAs for Flt-3 ligand and MIP-1 alpha have been found. This in-depth analysis of murine hematopoietic responses to cyclophosphamide provides evidence for the complexity of the involved local and systemic regulations. This represents a significant challenge to experimental hematology, which could now be tackled with methods allowing the study of changes in the gene expression during cyclophosphamide-induced hematopoietic damage.

We previously reported that Flk1+/CD31-/CD34- cells isolated from human fetal bone marrow can differentiate at the single cell level into endothelial and hematopoietic cells in vitro. Here we report that within this cell population reside cells that can differentiate into the epithelium of liver, lung, gut, as well as the cells of both hematopoietic and endothelial system after primary or secondary transplantation into irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Hence, Flk1+/CD31-/CD34- cells possess remarkable differentiation potential and may thereby provide an alternative to hematopoietic stem cells for transplantation. In addition, our results show this stem cell population effectively accelerated wound healing in NOD/SCID mice and thus holds therapeutic promise for treatment of genetic disorders, organ dysfunction, and tissue repair in humans.

An unusually high incidence of apoptosis in S-phase cells is characteristically found in the bone marrow (BM) of patients with myelodysplastic syndromes (MDS). Previously, E2F1, c-myc, and Cyclin D1 have been shown to bring about both S-phase changes and/or apoptotic changes. We have already found a stoichiometric imbalance between pRb and E2F1 causing deregulated E2F1 activity in these disorders. In the present study, we investigated the status of Cyclin D1 in relation to E2F1 and apoptosis in 19 patients with a confirmed diagnosis of MDS in comparison with 6 healthy donors. Cyclin D1 was localized immunohistochemically using a specific monoclonal antibody (1:150 dilution) in plastic-embedded BM sections. The nuclear localization of Cyclin D1 graded on a subjective rating scale of 0 (negligible staining) to 8+ (highest), demonstrated negligible levels in normal marrows (median 1+), and in 11/19 evaluable MDS marrows. In contrast, 8/19 MDS biopsies showed an almost four-fold increase in Cyclin D1 localization (p< or =0.001). A western blot analysis of E2F1 in corresponding bone marrow (BM) aspirate mononuclear cells (MNC) demonstrated that the MDS patients with elevated Cyclin D1 expression also had a significant increase in E2F1 protein (p< or =0.03). Additionally, these patients revealed higher levels of mRNA of one of the E2F1 transcriptional target genes, dihydrofolate reductase (DHFR, p=0.01). Subsequently, the relationship of Cyclin D1 with apoptosis was elucidated in a colocalization experiment in BM biopsy sections using immunohistochemistry for Cyclin D1 and in situ end labeling of DNA (ISEL) for apoptosis. The percentage of ISEL-positive apoptotic cells was several fold higher in MDS as compared to normal BMs (p=0.009). Interestingly, 7-41% (median 20%) of the apoptotic cells in different MDS BMs revealed co-localization of Cyclin D1 in their nucleus, whereas in normal BMs co-localization was virtually absent (p=0.008). Thus, it is possible that in a subset of MDS patients, apoptotic death of bone marrow cells may involve Cyclin D1/E2F1 pathway.

BM2 cells are chicken monoblasts transformed by the v-myb oncogene of avian myeloblastosis virus. The constitutively high v-myb expression interferes with the terminal differentiation of BM2 cells, but these cells can be induced to differentiate into macrophage-like cells by phorbol esters. Histone acetylation plays an important role in regulation of transcription and is particularly relevant to the regulation and pathology of hematopoiesis. In the present study, we examined the contribution of elevated histone acetylation to the differentiation of BM2 cells. Inhibition of the activity of endogenous histone deacetylases by trichostatin A (TSA) resulted in histone hyperacetylation causing cell cycle arrest and differentiation of BM2 cells into macrophage polykaryons. TSA did not affect the level of v-Myb protein in BM2 cells, but it downregulated its transcription activation capability. This suggests that chromatin remodeling can be significantly engaged in regulation of proliferation and differentiation of leukemic cells.

Homing and engraftment of hematopoietic stem cells (HSCs) to bone marrow ( BM) is a complex process that primarily depends on the cell-surface expression of adhesion molecules on stem and stromal cells. Here we report an in vitro model for homing of stem cells on pre-established stromal layer; the stroma-adhered cells were found to engraft, multiply, and differentiate in BM of age-matched mice. In vitro study revealed that initially the adhesion of BM cells on irradiated stroma was increased with time, and it attained a peak at 2 h of contacts. During that time, 44.1 +/- 6.5% (n = 8) cells were adhered, and this value was maintained up to 6 x 10(6) cells. The adhered cell fraction was enriched by 3.9-, 2.5-, and 1.7-fold Sca-1, colony forming cell (CFC), and cobblestone area forming cells (CAFC), respectively, as compared to the fresh BM cells. These adhered cells homed to BM with an engraftment efficiency of 11.8 +/- 2.5% (n = 6). The homed cells reconstituted BM of myeloablative mice by self-renewing and differentiating into myeloid cells. Overall, a simple in vitro model system has been described to study homing and grafting of HSCs that can be deployed to any possible experimental conditions to investigate the interactions between stromal and stem cells.

The concept of the immutability of the nervous tissue has recently been replaced with the new idea that a continuous neurogenic turnover does occur in some limited areas of the central nervous system (CNS). At least two neurogenic regions of the adult mammalian CNS are involved in this process: the subventricular zone of the forebrain and the dentate gyrus of the hippocampus, which are considered to be a reservoir of new neural cells. Neural stem cells (NSCs) are multipotential progenitors that have self-renewal capability. While in vivo endogenous NSCs seem able to produce almost exclusively neurons, a single NSC in vitro is competent to generate neurons, astrocytes, and oligodendrocytes. NSCs lack a specific morphology and unambiguous surface markers that could allow their identification. For this reason, one of the major difficulties in identifying stem cells is that they are defined in terms of their functional capabilities, the determination of which might alter the cells' nature. The purpose of this review is to describe the characteristics of the NSCs of the adult mammalian CNS, their potentiality in terms of proliferation and differentiation capabilities, as well as their stability in long-term culture, all attributes that make them a good tool for tissue replacement therapies.

We investigated whether plating a stable amount of CD34(+) cells improves the CFU-GM assay. Data of CFU-GM assays performed with leukaphereses products in two transplant centers using a commercial collagen-based medium and unified CFU-GM scoring criteria were pooled and analyzed according to the numbers of CD34(+) cells plated. A first series of 113 CFU-GM assays was performed with a fixed number of mononuclear cells (i.e., a variable number of CD34(+) cells). In these cultures the CFU-GM/CD34 ratio varied according to the number of CD34(+) cells plated: median CFUGM/CD34 ratios were 1/6.2 to 1/6.6 for grafts containing <2% CD34(+) cells, vs. 1/10.2 for grafts containing > or =2% CD34(+) cells. The median CFU-GM/CD34 ratio also varied depending on pathology: 1/9.3 for multiple myeloma (MM), 1/6.8 for Hodgkin's disease (HD), 1/6.5 for non-Hodgkin lymphoma (NHL), and 1/4.5 for solid tumors (ST). A second series of 95 CFU-GM assays was performed with a fixed number of CD34(+) cells (220/ml). The range of median CFU-GM/CD34 ratios was narrowed to 1/7.0 to 1/5.2, and coefficients of variation for CFU-GM counts decreased by half to 38.1% (NHL), 36.1% (MM), 49.9% (HD), and 22.4% (ST). In addition, CFU-GM scoring was facilitated as the percentages of cultures with >50 CFU/GM/ml decreased from 6.7% to 43.8% when a variable number of CD34(+) cells was plated, to 4.5% to 16.7% when 220 CD34(+) cells/ml were plated. Hence, plating a fixed number of CD34(+) cells in collagen gels improves the CFU-GM assay by eliminating cell number-related variability and reducing pathology-related variability in colony growth.

Positive CD34(+) selection to purge blood cell harvests is one way to attempt to reduce the high relapse risk after high-dose chemotherapy (HDT) supported by autologous blood cell transplantation (ABCT) in patients with multiple myeloma (MM). Until recently, however, the impact of CD34(+) selection, if any, on long-term clinical outcome in MM has remained obscure. We have analyzed engraftment kinetics, response to HDT, progression-free survival (PFS), and overall survival (OS) for 64 consecutive MM patients who have been treated with up-front HDT plus ABCT at our institution between 1993 and 1998. Nonrandomized comparisons were made between transplants with unselected (39 patients) and CD34(+)-selected (25 patients) grafts. The engraftment kinetics, need of blood product support, discharge time from hospital, and response to HDT were similar for both unselected and selected transplants. The median PFS was also similar (26 and 30 months, respectively) for the both groups. With a median follow-up time for the survivors of 67.5 months, the median OS (78 and 75 months, respectively) did not differ between transplants with unselected and selected grafts. In conclusion, this nonrandomized study suggests that positive CD34(+) selection has no beneficial impact on long-term outcome of patients with MM.