Description

A peer-reviewed journal covering developments in the rapidly expanding area of ex vivo manipulation of cells of the hematopoietic system for therapeutic purposes. Includes engineering of stem cell grafts, immunotherapy, ex vivo expansion and hematopoiesis, cell targeting, gene therapy, cell cryopreservation and storage, graft evaluation, laboratory management, and regulatory compliance. Emphasis is on the transition of technologies from basic research to early clinical applications to demonstrate efficacy. Discontinued - now Stem Cells and Development.

Publisher details

Mary Ann Liebert

Publications in this journal

• Neural stem cells: one of the keys to everything.

  Authors: Marc A Williams

  Journal of hematotherapy & stem cell research. 12(6):591-4.

• Multiorgan engraftment and multilineage differentiation by human fetal bone marrow Flk1+/CD31-/CD34- Progenitors.

  Authors: Baijun Fang, Mingxia Shi, Lianming Liao, Shaoguang Yang, Yuhao Liu, Robert Chunhua Zhao

  Journal of hematotherapy & stem cell research. 12(6):603-13.

  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 weWe 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.

• Embryonic stem cells for neural replacement therapy: prospects and challenges.

  Authors: Su-Chun Zhang

  Journal of hematotherapy & stem cell research. 12(6):625-34.

  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 throughInjury 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.

• A new concept of stem cell disorders and their new therapy.

  Authors: Susumu Ikehara

  Journal of hematotherapy & stem cell research. 12(6):643-53.

  The author previously proposed a new concept of categorizing stem cell disorders as: (1) stem cell aplasia (aplastic anemia), (2) monoclonal hematopoietic stem cell proliferative syndrome (leukemiaThe author previously proposed a new concept of categorizing stem cell disorders as: (1) stem cell aplasia (aplastic anemia), (2) monoclonal hematopoietic stem cell proliferative syndrome (leukemia and myelodysplastic syndrome), and (3) polyclonal hemopoietic stem cell proliferative syndrome (systemic and organ-specific autoimmune diseases). This review includes the following two stem cell disorders: mesenchymal stem cell disorders and organ-specific stem cell disorders. Age-associated diseases such as Alzheimer's disease, osteoporosis, and lung fibrosis belong to the former, whereas carcinosarcoma in the lung and adeno-endocrine cell carcinoma in the stomach belong to the latter. Recently, we have established a new method for allogeneic bone marrow transplantation using chimerism-resistant autoimmune-prone MRL/lpr mice. In this method, whole bone marrow cells containing a small number of T cells and mesenchymal stem cells are directly injected into the bone marrow cavity. MRL/lpr mice treated by injection of stem cells survived more than 2 years without showing symptoms of autoimmune disease. To apply this method to humans, we established a new method for bone marrow cell harvesting using cynomolgus monkeys. In this method, cells are harvested from the long bones using a perfusion method and are then injected directly into the bone marrow cavity of recipients. In this review, we show that this new method may become a powerful strategy for the treatment of various intractable diseases.

• Adult neural stem cells and repair of the adult central nervous system.

  Authors: Eyleen Lay Keow Goh, Dengke Ma, Guo-Li Ming, Hongjun Song

  Journal of hematotherapy & stem cell research. 12(6):671-9.

  Neural stem cells are present not only in the developing nervous systems, but also in the adult central nervous system of mammals, including humans. The mature central nervous system has beenNeural stem cells are present not only in the developing nervous systems, but also in the adult central nervous system of mammals, including humans. The mature central nervous system has been traditionally regarded as an unfavorable environment for the regeneration of damaged axons of mature neurons and the generation of new neurons. In the adult central nervous system, however, newly generated neurons from adult neural stem cells in specific regions exhibit a striking ability to migrate, send out long axonal and dendritic projections, integrate into pre-existing neuronal circuits, and contribute to normal brain functions. Adult stem cells with potential neural capacity recently have been isolated from various neural and nonneural sources. Rapid advances in the stem cell biology have raised exciting possibilities of replacing damaged or lost neurons by activation of endogenous neural stem cells and/or transplantation of in vitro-expanded stem cells and/or their neuronal progeny. Before the full potential of adult stem cells can be realized for regenerative medicine, we need to identify the sources of stem cells, to understand mechanisms regulating their proliferation, fate specification, and, most importantly in the case of neuronal lineages, to characterize their functional properties. Equally important, we need to understand the neural development processes in the normal and diseased adult central nervous system environment, which is quite different from the embryonic central nervous system, where neural development has been traditionally investigated. Here we will review some recent progress of adult neural stem cell research that is applicable to developmental neurobiology and also has potential implications in clinical neuroscience.

• Artificial niches for human adult neural stem cells: possibility for autologous transplantation therapy.

  Authors: Charles Y Liu, Ulf Westerlund, Mikael Svensson, Morten C Moe, Mercy Varghese, Jon Berg-Johnsen, Michael L J Apuzzo, David A Tirrell, Iver A Langmoen

  Journal of hematotherapy & stem cell research. 12(6):689-99.

  Cellular transplantation therapy is thought to play a central role in the concept of restorative neurosurgery, which aims to restore function to the damaged nervous system. Stem cells represent aCellular transplantation therapy is thought to play a central role in the concept of restorative neurosurgery, which aims to restore function to the damaged nervous system. Stem cells represent a potentially renewable source of transplantable cells. However, control of the behavior of these cells, both in the process of clonogenic expansion and post-transplantation, represents formidable challenges. Stem cell behavior is thought to be directed by extracellular signals in their in vivo niches, many of which are protein or peptide based. As only one example, activation of Notch plays an important role in normal development and is the strongest known signal for stem cells to choose glial over neuronal fates. Therefore, artificial extracellular matrix proteins represent a potentially powerful tool to custom design artificial niches to strategically control stem cell behavior. We have developed a family of aECM proteins that incorporate the active domains of the DSL ligands to the Notch receptor into an elastin-based backbone. The development of our DSL-elastin artificial proteins demonstrates the design strategy and methodology for the production of bioactive artificial extracellular matrix proteins aimed at modulating stem cell behavior, and this method can be used to design other bioactive aECM proteins. In addition, we have developed a method for the isolation and characterization of adult human neural stem cells from periventricular tissue harvested from living patients. This paper reviews cellular transplantation therapy from the clinical perspective and summarizes ongoing work aimed at exploring the intriguing possibility of autologous transplantation, whereby neural stem cells can be harvested from adult patients, expanded or modified in vitro in artificial niches, and retransplanted into the original patient.

• Embryonic neural progenitor cells: the effects of species, region, and culture conditions on long-term proliferation and neuronal differentiation.

  Authors: R Smith, V Bagga, R A Fricker-Gates

  Journal of hematotherapy & stem cell research. 12(6):713-25.

  One of the major obstacles to the use of neural stem/progenitor cells in neuronal replacement therapy is the limited ability of these cells to generate sufficient numbers of specific neuronalOne of the major obstacles to the use of neural stem/progenitor cells in neuronal replacement therapy is the limited ability of these cells to generate sufficient numbers of specific neuronal phenotypes either in the culture dish or after transplantation in animal models of neurodegenerative disease. It is not yet fully understood whether embryonic neural stem and progenitor cells show species-specific or regional identities, or if current culture paradigms select for a particular subset of stem cells/progenitors with similar proliferation and differentiation capacities. To investigate this issue, we isolated embryonic neural progenitors derived from the developing rat and mouse central nervous system for in vitro culture to assess the regional, species-specific, and temporal effects on both cell proliferation and generation of neurons. Neurosphere cultures were derived from E13-15 mouse or rat developing striatum (medial, lateral, or whole ganglionic eminence), ventral mesencephalon, and cortex. We compared basic fibroblast growth factor and epidermal growth factor for their influence on cell proliferation and neuronal differentiation under defined differentiation paradigms. Seeding density and conditioned media were also tested for their effects on maintenance of cell proliferation over protracted time periods. Results showed that embryonic neural stem/progenitor cells maintained defined patterns of proliferation and neuronal differentiation, with both declining with time in vitro. Proliferation rate was more dependent on species and region than the neurotrophins or conditions used for culture. These results suggest that the appropriate selection of embryonic neural stem cells and culture conditions may be crucial for the optimization of their neurogenic potential.

• Growth factor deprivation induces an alternative non-apoptotic death mechanism that is inhibited by Bcl2 in cells derived from neural precursor cells.

  Authors: María del Carmen Cárdenas-Aguayo, Jesús Santa-Olalla, José-Manuel Baizabal, Luis-Miguel Salgado, Luis Covarrubias

  Journal of hematotherapy & stem cell research. 12(6):735-48.

  Although apoptosis has been considered the typical mechanism for physiological cell death, presently alternative mechanisms need to be considered. We previously showed that fibroblast growth factor-2Although apoptosis has been considered the typical mechanism for physiological cell death, presently alternative mechanisms need to be considered. We previously showed that fibroblast growth factor-2 (FGF2) could act as a survival factor for neural precursor cells. To study the death mechanism activated by the absence of this growth factor, we followed the changes in cell morphology and determined cell viability by staining with several dyes after FGF2 removal from mesencephalic neural-progenitor-cell cultures. The changes observed did not correspond to those associated with apoptosis. After 48 h in the absence of FGF2, cells began to develop vacuoles in their cytoplasm, a phenotype that became very obvious 3-5 days later. Double-membrane vacuoles containing cell debris were observed. Vacuolated cells did not stain with either ethidium bromide or trypan Blue, and did not show chromatin condensations. Nonetheless, during the course of culture, vacuolated cells formed aggregates with highly condensed chromatin and detached from the plate. Neural progenitor cells grown in the presence of FGF2 did not display any of those characteristics. The vacuolated phenotype could be reversed by the addition of FGF2. Typical autophagy inhibitors such as 3-MA and LY294002 inhibited vacuole development, whereas a broad-spectrum caspase inhibitor did not. Interestingly, Bcl-2 overexpression retarded vacuole development. In conclusion, we identified a death autophagy-like mechanism activated by the lack of a specific survival factor that can be inhibited by Bcl2. We propose that anti-apoptotic Bcl2 family members are key molecules controlling death activation independently of the cell degeneration mechanism used.

• Neural stem cell therapy in the aging brain: pitfalls and possibilities.

  Authors: Tobi L Limke, Mahendra S Rao

  Journal of hematotherapy & stem cell research. 12(6):615-23.

  As aging progresses, there is a decline in the brain's capacity to produce new neurons in the two neurogenic regions, the subventricular zone surrounding the lateral ventricles and the subgranularAs aging progresses, there is a decline in the brain's capacity to produce new neurons in the two neurogenic regions, the subventricular zone surrounding the lateral ventricles and the subgranular layer of the hippocampal dentate gyrus. The underlying cause of the declining neurogenesis is unknown, but is presumably related to age-related changes that occur during normal aging of the brain. It is exacerbated by age-related neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Stem cell-based therapy to replace lost and/or damaged cells in the aging brain is currently the focus of intense research. The two most promising approaches involve transplantation of exogenous tissue and promoting proliferation of endogenous cells. However, age-related changes in the brain environment, including elevated oxidative stress and accumulation of protein and lipid by-products, present several unique challenges that must be addressed before cell-based therapy can be used as a viable option. Although progress has been made toward replacement of lost cells and recovery of lost function, there are fundamental issues that need to be addressed for stem cell therapy to be successful in the aging brain. In this review, we focus on recent progresses made toward understand the biology of neural stem cells in the aging brain, as well as progress toward using stem cells to replace cells lost during disease.

• Adventures in clinical and basic science.

  Authors: Daniel B Drachman

  Journal of hematotherapy & stem cell research. 12(6):595-601.

• The future of cell-based transplantation therapies for neurodegenerative disorders.

  Authors: Stanley E Lazic, Roger A Barker

  Journal of hematotherapy & stem cell research. 12(6):635-42.

  Parkinson's disease is a common neurodegenerative disease with a lifetime incidence of 2.5% and a prevalence of at least 2% in individuals over 70 years old. Patients can be effectively treated withParkinson's disease is a common neurodegenerative disease with a lifetime incidence of 2.5% and a prevalence of at least 2% in individuals over 70 years old. Patients can be effectively treated with drugs that target the dopaminergic nigro-striatal pathway, but over time the efficacy of these medications is limited by the development of profound motor fluctuations and dyskinesias. This has prompted the search for alternative treatments, including the use of cell replacement therapies. Over the last decade, human fetal nigral transplants have demonstrated that dopaminergic neurons can survive and provide clinical benefit for patients with Parkinson's disease. However, there are clearly ethical concerns and a limit to the supply of this tissue as well as more recently anxieties over side effects. As a result, alternative sources of tissue have been investigated, and one such source are stem cells, which provide an attractive renewable tissue supply. In this review, we will discuss the current state-of-the-art and the characteristics of Parkinson's disease that increase its attraction as a target of stem cell therapy against results of current clinical trials using fetal neural grafts. Then we will discuss the various types and sources of stem cells, and some early transplantation results in animal models of Parkinson's disease. Finally we will discuss the prospect of using stem cells to deliver drugs and neurotrophic factors involved in neuroprotective and neuroreparative strategies in Parkinson's disease and other neurodegenerative conditions.

• Neural stem cells in the adult nervous system.

  Authors: Daniele Bottai, Roberta Fiocco, Fabrizio Gelain, Lidia Defilippis, Rossella Galli, Angela Gritti, L Angelo Vescovi

  Journal of hematotherapy & stem cell research. 12(6):655-70.

  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 systemThe 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.

• Postnatal astrocytes promote neural induction from adult human bone marrow-derived stem cells.

  Authors: Alexis Joannides, Phil Gaughwin, Mike Scott, Suzanne Watt, Alastair Compston, Siddharthan Chandran

  Journal of hematotherapy & stem cell research. 12(6):681-8.

  Neural stem cells (NSCs) have generated considerable interest because of their potential as a source of defined cells for drug screening or cell-based therapies for neurodegenerative diseases.Neural stem cells (NSCs) have generated considerable interest because of their potential as a source of defined cells for drug screening or cell-based therapies for neurodegenerative diseases. Ethical and practical considerations limit the availability of human fetal-derived neural tissue and highlight the need to consider alternative sources of human NSCs. Because of their ready availability, their ability to be easily expanded, and reports of neural potential, bone marrow-derived populations have become the focus of intense study with regard to their potential clinical utility. However, recent identification of spontaneous cell fusion and limited neuronal differentiation has tempered initial optimism. In this study, we demonstrate the monoclonal neural and mesodermal potential of adult human bone marrow mesenchymal cells. Critically, we show that sequential treatment with the mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) followed by postnatal hippocampal astrocyte conditioned medium significantly promotes the generation of neurofilament(+)/beta-tubulin(+) cells from bone marrow precursors. The ability to generate almost limitless numbers of neural precursors from a readily accessible autologous adult human source provides a platform for further studies and potentially has important therapeutic implications.

• High-dose immunosuppression and autologous hematopoietic stem cell rescue for severe multiple sclerosis.

  Authors: Athanasios Fassas, Aristide Kazis

  Journal of hematotherapy & stem cell research. 12(6):701-11.

  Multiple sclerosis is a relatively common and seriously disabling disease of autoimmune pathogenesis, for which there is currently no cure. Available therapies include immunomodulating agents andMultiple sclerosis is a relatively common and seriously disabling disease of autoimmune pathogenesis, for which there is currently no cure. Available therapies include immunomodulating agents and standard-dose immunosuppressants, which may be helpful but are not curative. Recently, studies in animal models have indicated that control of autoimmune disease can be obtained by high-dose immunosuppression followed by hematopoietic stem cell transplantation (rescue). Autologous transplants for severe and refractory multiple sclerosis were proposed in 1997 and have been performed ever since in selected patients and in the context of phase I/II trials. To date, more than 200 patients have been treated worldwide, and similar results were obtained in different centers: high-dose therapy suppresses inflammation in the brain to a degree superior to any other conventional therapy and seems to delay significantly clinical disease progression. There is, however, a procedure-related mortality risk of 1.5-5%, requiring careful patient selection before transplant. The treatment should be reserved for patients having high chance of response, i.e., young patients with low disability scores but rapidly progressing disease, having inflammatory rather than neurodegenerative changes in the central nervous system. The mechanism of action of transplantation is unclear. The initial concept of immune ablation by high-dose therapy and reconstitution of normal immunity from transplant-derived lymphocyte progenitors has given way to the concept of "resetting" the immune system and of bringing the disease to a lower level of activity. One could also speculate on a tissue repair effect, given the ability of human hematopoietic stem cells to migrate also into the central nervous system. The clinical effect of transplantation remains to be demonstrated in a randomized study. The Autoimmune Disease Working Party of the European Group for Blood and Marrow Transplantation has launched such a trial, comparing transplantation to the currently best available therapy, i.e., mitoxantrone, and in about 5 years we should know whether transplantation offers more than the benefit of a transient immunosuppressive effect.

• Neuronal differentiation of murine bone marrow Thy-1- and Sca-1-positive cells.

  Authors: F Locatelli, S Corti, C Donadoni, M Guglieri, F Capra, S Strazzer, S Salani, R Del Bo, F Fortunato, A Bordoni, G P Comi

  Journal of hematotherapy & stem cell research. 12(6):727-34.

  Recent evidence suggests that cells from bone marrow can acquire neuroectodermal phenotypes in cell culture or after transplantation in animal models and in the human brain. However, isolation of theRecent evidence suggests that cells from bone marrow can acquire neuroectodermal phenotypes in cell culture or after transplantation in animal models and in the human brain. However, isolation of the bone marrow cell subpopulation with neuronal differentiation potential remains a challenge. To isolate and expand neural progenitors from whole murine bone marrow, bone marrow was obtained from hind limb bone of C57BL6 mice and plated in culture with neuronal medium with basic fibroblast growth factor and epidermal growth factor. After 5-7 days in culture, cellular spheres similar to brain neurospheres appeared either floating or attached to culture dishes. These spheres were collected, dissociated, and expanded. The bone marrow-derived spheres were positive for nestin as assessed by immunocytochemistry and by reverse transcriptase polymerase chain reaction. Thy-1- and Sca-1-positive bone marrow cells selected by magnetic cell sorting resulted in a higher yield of nestin-positive spheres. After exposure to neuronal differentiative medium retinoic acid with and without Sonic hedgehog, cells positive for neuronal markers tubulin III (TuJ-1) and neurofilament (NF) were detected. The mRNA profile of these cells included the expression of TuJ-1, neuronal-specific enolase (NSE), and NF-light chain. To evaluate the in vivo behavior of these cells, spheres derived from bone marrow-derived cells of transgenic green fluorescent protein (GFP) mice were transplanted into newborn mouse brain. Two months later, the mouse neural cortex contained a minor proportion of GFP(+) cells co-expressing neuronal markers (TuJ-1, NF, MAP-2, NeuN). Although cell fusion phenomena with the host cells could not be ruled out, bone marrow-derived neurosphere transplantation could be a strategy for cellular mediated gene therapy.

• The potential role of HLA-G polymorphism in maternal tolerance to the developing fetus.

  Authors: Suraksha Agrawal, Manoj Kumar Pandey

  Journal of hematotherapy & stem cell research. 12(6):749-56.

  The mechanism by which the developing fetus survives the maternal immunity has eluded investigators and remains a central paradox in the field of stem cell research. If the mechanism can be defined,The mechanism by which the developing fetus survives the maternal immunity has eluded investigators and remains a central paradox in the field of stem cell research. If the mechanism can be defined, allogenic stem cells may find increased utility after transplantation. While several theories have been advanced, the differential expression of HLA antigens on trophoblasts has been the focus of many studies. Interestingly, an inverse relationship between HLA-G (nonclassical class I molecules) and class I MHC gene expression exists early in pregnancy. HLA-G transcripts are present in quite significant amounts in first-trimester placental tissue, particularly in the extravillous membranes, while the opposite occurs at term. This kind of expression is consistent with the theory that HLA-G might play a role in fetal protection. This could be consequent to either nonimmune (structural) or immune functions at the maternal-fetal interface. Current evidence suggest an immune function wherein HLA-G protects fetal cells from maternal uterine natural killer (NK) cells, which are found in large numbers within cells invading the trophoblasts. This effect has been attributed to maternal NK receptor alterations as well as inhibition of maternal NK cell traffic across the placenta. The recent identification of HLA-G polymorphism brings into play the potential role of these isoforms in fetal protection. Polymorphism may be associated with differential function or may effect linkage disequilibria with other HLA variants, providing fetal protection.

• Could the functional expression of HLA-G be exploited for successful stem cell transplantation and engraftment?

  Authors: Marc A Williams

  Journal of hematotherapy & stem cell research. 12(6):757-8.

• The therapeutic promise of nonembryonic stem cells--where's the beef?

  Authors: Denis English, Marc A Williams

  Journal of hematotherapy & stem cell research. 12(5):465-6.

• CD34+ cells and CD34+CD38- subset from mobilized blood show different patterns of adhesion molecules compared to those from steady-state blood, bone marrow, and cord blood.

  Authors: H Sovalat, E Racadot, M Ojeda, H Lewandowski, V Chabouté, P Hénon

  Journal of hematotherapy & stem cell research. 12(5):473-89.

  As suggested previously, a down-regulation of some cellular adhesion molecules (CAMs) on CD34(+) hematopoietic progenitor cells (HPC) may contribute to their egress from bone marrow (BM) toAs suggested previously, a down-regulation of some cellular adhesion molecules (CAMs) on CD34(+) hematopoietic progenitor cells (HPC) may contribute to their egress from bone marrow (BM) to peripheral blood (PB) by decreasing their adhesion to BM stromal cells. Besides counting the percentage of CAM-positive cells, we decided to define clearly the antigen density (AgD) of the CAM on mobilized- and steady-state CD34(+) HPC using QIFIKIT calibration beads. Five sources of cells were compared: PB and BM from normal donors (nPB, nBM) cord blood (CB), mobilized PB obtained from leukapheresis products (LKP), and mobilized BM (mBM) samples. In our study the CAM-AgD was the lowest on CD34(+) cells in LKP which, on the contrary, contained the highest percentage of CD117(+), CD54(+), CD58(+) cell subsets. As for CB, a greater proportion of CD44(+) and CD62L(+) cells was observed in LKP than in other products. The LKP-CD34(+) cell population contained a greater percentage of CD11a(+) cells when compared to mBM, but the lowest percentage of CD49d(+) and CD49e(+) cells when compared to all products. The proportion of the CD34(+)CD38(-) immature subset expressing CD11a, CD44, CD54, or CD62L was greater in LKP than in mBM; the CD62L-AgD was higher in LKP than in mBM. This quantitative analysis clearly showed a downregulation of all CAM on LKP-CD34(+). The CD44, CD62L, CD11a, and CD54 AgD decrease appears to be specifically involved in the egress of the CD34(+) subsets into PB. The control of antigen density of these adhesion molecules is likely to be clinically important for effective mobilization of HPC as well as for rapid engraftment following HPC transplant.

• Reactivation of fetal hemoglobin in adult stem cell erythropoiesis by transforming growth factor-beta.

  Authors: Ralph M Böhmer

  Journal of hematotherapy & stem cell research. 12(5):499-504.

  Treatment of adult blood-derived stem cells with transforming growth factor (TGF-beta) during the first 3-4 days in culture increases the proportions and absolute numbers of erythroid cellsTreatment of adult blood-derived stem cells with transforming growth factor (TGF-beta) during the first 3-4 days in culture increases the proportions and absolute numbers of erythroid cells subsequently expressing fetal hemoglobin (F+ cells). The change in F+ cell proportions may be due to globin switching or to selective effects on the expansion of stem cell subpopulations with different globin expression programs. To distinguish between the two mechanisms, we compared the effects of TGF-beta on proliferation and globin expression with the effects of well-researched agents known to increase fetal hemoglobin (HbF) in sickle cell patients. Hydroxyurea suppressed F+ and F- erythroid cells equally and thus did not affect the F+ proportions. Aza-cytidine and sodium butyrate, known reactivators of gamma-globin expression, suppressed F+ and F- cells differentially and increased F+ cell proportions with a dependence on treatment timing similar to that of TGF-beta. In contrast to TGF-beta, these agents had no superimposed stimulatory effect. The data suggest that TGF-beta reactivates gamma-globin expression, combined with a sequential stimulation and suppression of erythropoiesis. The similarities between the actions of TGF-beta and therapeutic reactivators of fetal hemoglobin make it conceivable that TGF-beta may have the potential to increase HbF in patients with beta-hemoglobin disorders.

• Large-scale immunomagnetic selection of CD14+ monocytes to generate dendritic cells for cancer immunotherapy: a phase I study.

  Authors: J Babatz, C Röllig, U Oelschlägel, S Zhao, G Ehninger, M Schmitz, M Bornhäuser

  Journal of hematotherapy & stem cell research. 12(5):515-23.

  Dendritic cells (DC) are professional antigen-presenting cells that are widely used in the experimental immunotherapy of cancer. For clinical use GMP-like protocols for the preparation ofDendritic cells (DC) are professional antigen-presenting cells that are widely used in the experimental immunotherapy of cancer. For clinical use GMP-like protocols for the preparation of functionally active dendritic cells (DC) in large numbers and at high purity are needed. However, the currently available protocols have certain disadvantages. In this study we tested the generation and clinical applicability of DC from monocyte preparations produced by immunomagnetic CD14(+) selection using a semiautomated clinical scale immunomagnetic column. Peripheral blood mononuclear cells (PBMC) of 10 patients with metastatic solid tumors were used. With the immunomagnetic separation, we obtained a cell suspension of high CD14(+) purity (median 97.4%, range 94.9-99.0) with a high monocyte yield (median 82.3%, range 63.9-100.0). Differentiation of CD14(+) cells into mature monocyte-derived DC was induced by incubation with IL-4, GM-CSF, TNF-alpha, PGE(2), IL-1 beta, and IL-6. Mature DC showed a high expression of CD83, HLA-DR, and the co-stimulatory molecules CD80 and CD86. Overall CD83(+) yield was 12.1% (range 4.0-29.4). Allogeneic T stimulatory capacity could be demonstrated for all DC preparations in proliferation assays. No significant differences in marker expression or T cell stimulation was detected between fresh DC and those derived from cryopreserved immature DC. Clinical administration of autologous DC by three different parenteral routes was tolerated by all 10 patients without systemic signs of toxicity. Our results indicate that immunomagnetic isolation of CD14(+) monocytes using the CliniMACS device is a suitable method for clinical-scale generation of functional DC under GMP-grade conditions. The selection can be performed in a closed system. Therefore, immunomagnetic CD14(+) selection can be seen as an alternative way to generate DC for clinical tumor vaccination protocols.

• Low efficiency of a newly introduced high-density microparticles method for B cell depletion in multiple myeloma patients undergoing autologous hematopoietic stem cell transplantation.

  Authors: P Perseghin, M Dassi, D Belotti, P Pioltelli, E M Pogliani

  Journal of hematotherapy & stem cell research. 12(5):537-41.

  Autologous peripheral blood stem cell (PBSC) transplantation proved to increase complete remission (CR) and DFS in multiple myeloma (MM) patients. CD34(+) cell selection has been used to reduceAutologous peripheral blood stem cell (PBSC) transplantation proved to increase complete remission (CR) and DFS in multiple myeloma (MM) patients. CD34(+) cell selection has been used to reduce possible myeloma cell contamination in the graft, but it has not been showed to offer substantial advantages when compared to unpurged grafts; on the contrary, an increase of infectious complications was observed. We investigated the feasibility of a new negative-selection method in this setting. B cell negative selection was performed by using Eligix B cell HDM method. B cell contamination in the yield and in the final product was investigated by flow cytometry. Three patients with newly diagnosed MM entered the study. CD34(+) cell recovery in the three procedures was 73, 97, and 106%, and CD3(+) cell recovery was 88, 86, and 102%, respectively. CD20(+) cell depletion was 100% in all procedures, while CD19(+) cell depletion was 0.37, 1.21, and 0.07 log, respectively. We found an unexpected unreliability and a low efficiency in this B cell depletion method and suggest the need for further extensive testing before its introduction in the preclinical and clinical settings, at least in MM patients. In fact, reasons of such unsatisfactory results are still controversial: platelet contamination/activation in the preselection product, plasma protein interference, reduced CD19 antigen expression on immature B cells, lack of specificity of anti-CD19 monoclonal antibodies, instable binding between anti-CD19-coated high-density microparticles (HDM) and CD19 antigen may, alone or in combination, be involved in the system's low performance.

• Supplementation of conventional freezing medium with a combination of catalase and trehalose results in better protection of surface molecules and functionality of hematopoietic cells.

  Authors: Lalita M Sasnoor, Vaijayanti P Kale, Lalita S Limaye

  Journal of hematotherapy & stem cell research. 12(5):553-64.

  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 toOur 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.

• Immunostimulatory oligonucleotides: ready for immunotherapy prime time!

  Authors: Ying K Tam

  Journal of hematotherapy & stem cell research. 12(5):467-71.

• Colony-stimulating activity of fetal liver cells: synergistic role of stem cell factor in bone marrow recovery from aplastic anemia.

  Authors: G M K Raju, S Guha, A Mukhopadhyay, L Kumar, V P Kale, S Mittal, D Deka, S Mohanty, V Kochupillai

  Journal of hematotherapy & stem cell research. 12(5):491-7.

  Previously, we and others have shown that fetal liver infusion (FLI) leads to autologous hematopoietic improvement in 40-54% of patients with aplastic anemia. However, whether this recovery wasPreviously, we and others have shown that fetal liver infusion (FLI) leads to autologous hematopoietic improvement in 40-54% of patients with aplastic anemia. However, whether this recovery was spontaneous or the effect of the infused liver cells was not clear. To dissect the role of FLI in autologous hematopoietic recovery, the colony-supporting potential of fetal liver-conditioned medium (FLCM) was evaluated in bone marrow (BM) cells of normal adult and aplastic anemia patients. In both cases, each sample of FLCM supported the growth of colony-forming cells in semi solid culture medium. The FLCM was assayed for the presence of four principal colony-stimulating cytokines, namely stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and erythropoietin (Epo). While GM-CSF, IL-3, and Epo were present in insignificant amounts or were altogether absent, 50-635 pg/ml of SCF was found in 8 of the 13 FLCM samples tested. Preliminary results of bioneutralization assay indicated the possible role of SCF, secreted by the FL cells, in colony-supporting activity of aplastic anemia and normal BM cells. Overall, our in vitro study implicates the paracrine role of infused FL cells in regenerating autologous hematopoiesis in aplastic anemia patients.

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