Hematopoiesis

Studies in athletes have shown that carnitine supplementation may foster exercise performance. As reported in the majority of studies, an increase in maximal oxygen consumption and a lowering of the respiratory quotient indicate that dietary carnitine has the potential to stimulate lipid metabolism. Treatment with L-carnitine also has been shown to induce a significant postexercise decrease in plasma lactate, which is formed and used continuously under fully aerobic conditions. Data from preliminary studies have indicated that L-carnitine supplementation can attenuate the deleterious effects of hypoxic training and speed up recovery from exercise stress. Recent data have indicated that L-carnitine plays a decisive role in the prevention of cellular damage and favorably affects recovery from exercise stress. Uptake of L-carnitine by blood cells may induce at least three mechanisms: 1) stimulation of hematopoiesis, 2) a dose-dependent inhibition of collagen-induced platelet aggregatio...

Myelodysplastic syndromes and related disorders (myelodysplasia) are a heterogeneous group of myeloid neoplasms showing deregulated blood cell production with evidence of myeloid dysplasia and a predisposition to acute myeloid leukaemia, whose pathogenesis is only incompletely understood. Here we report whole-exome sequencing of 29 myelodysplasia specimens, which unexpectedly revealed novel pathway mutations involving multiple components of the RNA splicing machinery, including U2AF35, ZRSR2, SRSF2 and SF3B1. In a large series analysis, these splicing pathway mutations were frequent ( 45 to 85%) in, and highly specific to, myeloid neoplasms showing features of myelodysplasia. Conspicuously, most of the mutations, which occurred in a mutually exclusive manner, affected genes involved in the 39-splice site recognition during pre-mRNA processing, inducing abnormal RNA splicing and compromised haematopoiesis. Our results provide the first evidence indicating that genetic alterations of the major splicing components could be involved in human pathogenesis, also implicating a novel therapeutic possibility for myelodysplasia.

HEMATOPOIESIS Hematopoiesis merupakan proses produksi (mengganti sel yang mati) dan perkembangan sel darah dari sel induk / asal / stem sel, dimana terjadi proliferasi, maturasi dan diferensiasi sel yang terjadi secara serentak. Proliferasi sel menyebabkan peningkatan atau pelipat gandaan jumlah sel, dari satu sel hematopoietik pluripotent menghasilkan sejumlah sel darah. Maturasi merupakan proses pematangan sel darah, sedangkan diferensiasi menyebabkan beberapa sel darah yang terbentuk memiliki sifat khusus yang berbeda-beda (1) Tempat terjadinya hematopoiesis pada manusia : 1. Embrio dan Fetus a. Stadium Mesoblastik, Minggu ke 3-6 s/d 3-4 bulan kehamilan : Sel-sel mesenchym di yolk sac. Minggu ke 6 kehamilan produksi menurun diganti organ-organ lain. b. Stadium Hepatik, Minggu ke 6 s/d 5-10 bulan kehamilan : Menurun dalam waktu relatif singkat. Terjadi di Limpa, hati, kelenjar limfe c. Stadium Mieloid, Bulan ke 6 kehamilan sampai dengan lahir, pembentukan di sumsum tulang : Eritrosit, leukosit, megakariosit. (2) 2. Bayi sampai dengan dewasa Hematopoiesis terjadi pada sumsum tulang, normal tidak diproduksi di hepar dan limpa, keadaan abnormal dibantu organ lain. a. Hematopoiesis Meduler (N) Lahir sampai dengan 20 tahun : sel sel darah → sumsum tulang. Lebih dari 20 tahun : corpus tulang panjang berangsur-angsur diganti oleh jaringan lemak karena produksi menurun. (2) b. Hematopoiesis Ekstrameduler (AbN) Dapat terjadi pada keadaan tertentu,

Background: The abundance of monoclonal antibodies (mAb) and the routine use of quadruple stainings in flow cytometry allow stepwise analysis of bone marrow (BM) samples that are suspected for abnormal hematopoiesis. A screening phase that precedes lineage-specific classification phases should be sufficient to assess whether the BM has a normal or abnormal composition, as well as to identify the abnormal differentiation lineage.

In the very early stages of embryonic development, cells have the capability of dividing indefinately and then differentiating into any type of cell in the body. Recent studies have revealed that much of this remarkable developmental potential of embryonic stem cells is retained by small populations of cells within most tissues in the adult. Intercellular signals that control the proliferation, differentiation and survival of stem cells are being identified and include a diverse array of growth factors, cytokines and cell adhesion molecules. Intracellular mechanisms that regulate stem cell fate are also emerging and include established second messenger pathways, novel transcription factors and telomerase. The possibility that a decline in the numbers or plasticity of stem cell populations contributes to aging and age-related disease is suggested by recent findings. The remarkable plasticity of stem cells suggests that endogenous or transplanted stem cells can be 'tweaked' in ways that will allow them to replace lost or dysfunctional cell populations in diseases ranging from neurodegenerative and hematopoietic disorders to diabetes and cardiovascular disease.

Aplastic anemia, an unusual hematologic disease, is the paradigm of the human bone marrow failure syndromes. Almost universally fatal just a few decades ago, aplastic anemia can now be cured or ameliorated by stem-cell transplantation or immunosuppressive drug therapy. The pathophysiology is immune mediated in most cases, with activated type 1 cytotoxic T cells implicated. The molecular basis of the aberrant immune response and deficiencies in hematopoietic cells is now being defined genetically; examples are telomere repair gene mutations in the target cells and dysregulated T-cell activation pathways. Immunosuppression with antithymocyte globulins and cyclosporine is effective at restoring blood-cell production in the majority of patients, but relapse and especially evolution of clonal hematologic diseases remain problematic. Allogeneic stem-cell transplant from histocompatible sibling donors is curative in the great majority of young patients with severe aplastic anemia; the major challenges are extending the ben-efits of transplantation to patients who are older or who lack family donors. Recent results with alternative sources of stem cells and a variety of conditioning regimens to achieve their engraftment have been promising, with survival in small pediatric case series rivaling conventional transplantation results. (Blood.

In contrast to higher vertebrates, most fish species hatch at the embryonic stage of life. Consequently, they have to defend against a variety of micro-organisms living in their aquatic environment. This paper is focussed on the development of leucocytes functioning within this early innate system and later on in the acquired immune system (B and T cells). Most of the

ABBREVIATIONS: AT = adipose tissue; BM = bone marrow; CFU-F(s) = colony-forming unit fibroblast precursor(s); GMP = Good Manufacturing Practices; HSC(s) = hematopoietic stem cell(s); IGF = insulin-like growth factor; MSC(s) = mesenchymal stem cell(s); PDGF(s) = platelet-derived growth factor(s); UCB = umbilical cord blood. From the * The data were searched on the website of ClinicalTrials.gov (http://www.clinicaltrials.gov) on October 22, 2012. The following key words including "mesenchymal stem cells,"

Hematopoietic stem cells (HSCs) possess a distinct ability to perpetuate through self-renewal and to generate progeny that differentiate into mature cells of myeloid and lymphoid lineages. A better understanding of the molecular mechanisms by which HSCs replicate and differentiate from the perspective of developing new approaches for HSC transplantation is necessary for further advances. The interaction of the receptor tyrosine kinase-c-KIT-with its ligand stem cell factor plays a key role in HSC survival, mitogenesis, proliferation, differentiation, adhesion, homing, migration, and functional activation. Evidence that activating site-directed point mutations in the c-KIT gene contributes to its ligand-independent constitutive activation, which induces enhanced proliferation of HSCs, is accumulating. Similarly, and equally important, self-renewal is a process by which HSCs generate daughter cells via division. Self-renewal is necessary for retaining the HSC pool. Therefore, elucidating the molecular machinery that governs self-renewal is of key importance. The transcription factor, HOXB4 is a key molecule that has been reported to induce the in vitro expansion of HSCs via self-renewal. However, critical downstream effector molecules of HOXB4 remain to be determined. This concisely reviewed information on c-KIT and HOXB4 helps us to update our understanding of their function and mechanism of action in self-renewal, proliferation, and differentiation of HSCs, particularly modulation by c-KIT mutant interactions, and HOXB4 overexpression showing certain therapeutic implications. 755

Erythropoietin (EPO) increases the number of circulating erythrocytes primarily by preventing apoptosis of erythroid progenitors. In addition to this proerythroid action, results of recent studies show that systemically administered EPO is protective in vivo, in several animal models of neuronal injury. In vitro, EPO prevents neuronal apoptosis induced by a variety of stimuli. This review summarizes the neuroprotective actions of EPO and discusses the underlying mechanisms in terms of signal transduction pathways involved. The understanding of these mechanisms will help differentiate the neuroprotective actions of EPO from its role in the bone marrow.

The liver plays an important role in the production of haemopoietic hormones. It acts as the primary site of synthesis of erythropoietin (EPO) in the fetal stage, and it is the predominant thrombopoietin (TPO)-producing organ for life. In contrast to that of EPO and other liver proteins, the hepatic synthesis of TPO is influenced little by external signals. Hepatocytes express the TPO gene in a constitutive way, i.e. irrespective of the level of platelets in blood. Megakaryocytes and platelets remove the hormone from blood by means of their high-affinity TPO receptors. Normally, the plasma level of TPO is relatively low ( approximately 10(-12) mol/l). However, in thrombocytopenic states due to marrow failure or bleeding, the concentration of circulating TPO may increase greatly. The simple feedback regulation by TPO and its target cells is efficient in maintaining constant platelet numbers in healthy people. Persisting thrombocytopenia develops only in severe liver or marrow failure...

A review of published data on the in vitro, ex vivo, in vivo and clinical effects of fluoroquinolones on the synthesis of cytokines is provided. Fluoroquinolones (FQs) were found to affect both cellular and humoral immunity. In general, FQs exert their modulating effects only when used together with a co-stimulant. The in vitro studies generated heterogeneous data because of inhomogeneous effects triggered by different types of co-stimulants and differing responses of various cell lines on the stimuli. However, there is the general trend that FQs decrease the synthesis of pro-inflammatory cytokines. Studies in experimental animals generated homogenous data. All the FQs studied exerted significant clinical effects by attenuating cytokine responses in vivo. The FQs were found to be effective in vivo either in infections caused by organisms against which these are inactive or when dosed suboptimally, so that serum levels were lower than the susceptibilities of the causative pathogens. These in vivo effects were correlated with a significant decrease in pro-inflammatory cytokines like Il-1 and TNF. In addition, FQs were found to upregulate hematopoiesis. These immunomodulatory effects can be attributed in particular to those FQs with a cyclopropyl-moiety at the position N1 of the quinolone core structure, i. e. ciprofloxacin, moxifloxacin, grepafloxacin, sparfloxacin. The immunomodulatory effects of the FQs are due to their effects on intracellular cyclic AMP and phosphodiesterases, on transcription factors such as NF-kappa B, activitor protein 1 and a triggering effect on the eucaryotic equivalent of bacterial SOS response. All these studies indicate that FQs exert immunomodulatory activities in particular in latent or chronic infections.

Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications. We focus on the generation of cytometrycompatible data and on approaches to unsupervised multivariate clustering analysis. Finally, we provide a highlevel review of some recent benchmark reports that highlight the potential for massively multi-parameter mass cytometry.

The presence of cells or tissues from two individuals, chimeras, or the presence of cells and tissues that include the gonads, tetragametic chimerism can be detected by the analysis of cytogenetics and analysis of polymorphic genetic markers, using patterns of pedigree inheritance. These methodologies include determination of sex chromosomes, major histocompatibility complex (MHC) polymorphisms and panels of short tandem repeats (STRs) that include mitochondrial DNA markers. Studies routinely involve cases of temporal chimerism in blood transfusion, or following allotransplantation to measure the outcome of the organ, lymphopoietic tissues or bone marrow grafts. Demonstration of persistent chimerism is usually discovered in cases of inter-sexuality due to fusion of fraternal twins or in cases of fusion of embryos with demonstrable allogeneic monoclonality of blood which, excluded maternity or paternity when blood alone is used as the source of DNA. In single pregnancies it is possible to produce two kinds of microchimerism: feto-maternal and materno-fetal, but in cases of fraternal twin pregnancies it is Presented at the First Robert A Good Society Symposium, St. Petersburg, FL 2006.

Signal changes within the bone marrow adjacent to osteoarthritic joints are commonly seen on magnetic resonance (MR) images in humans and in dogs. The histological nature of these lesions is poorly known. In this study, we describe the MR imaging of bone marrow lesions adjacent to the stifle joints of dogs with experimental osteoarthritis over 13 months. Histology of the proximal tibia at the end of the study was compared with the last MR imaging findings. In five adult dogs, the left cranial cruciate ligament was transected. Postoperatively, MR imaging was performed at 1, 2, 3, 4, 6, 8, and 13 months. Dogs were euthanised after 13 months and histological specimen of the proximal tibia were evaluated. Bone marrow edema like MR imaging signal changes were seen in every MR examination of all dogs in one or more locations of the proximal tibia and the distal femur. Lesions varied in size and location throughout the whole study with the exception of constantly seen lesions in the epiphyseal and metaphyseal region at the level of the tibial eminence. On histology, hematopoiesis and myxomatous transformation of the bone marrow and/or intertrabecular fibrosis without signs of bone marrow edema were consistent findings in the areas corresponding to the MR imaging signal changes. We conclude that within the bone marrow, zones of increased signal intensity on fat suppressed MR images do not necessarily represent edema but can be due to cellular infiltration. Contrary to humans, hematopoiesis is seen in bone marrow edema-like lesions in this canine model of osteoarthritis.

DNA methylation is extensively reprogrammed during the early phases of mammalian development, yet genomic targets of this process are largely unknown. We optimized methylated DNA immunoprecipitation for low numbers of cells and profiled DNA methylation during early development of the mouse embryonic lineage in vivo. We observed a major epigenetic switch during implantation at the transition from the blastocyst to the postimplantation epiblast. During this period, DNA methylation is primarily targeted to repress the germline expression program. DNA methylation in the epiblast is also targeted to promoters of lineage-specific genes such as hematopoietic genes, which are subsequently demethylated during terminal differentiation.

Zeolites are natural or synthetic crystalline alumosilicates with ion exchanging properties. Supplied in fodder, they promote biomass production and animal health. Our aim was to assess the eects of the natural zeolite, clinoptilolite, on hematopoiesis, serum electrolytes and essential biochemical indicators of kidney and liver function in mice. Two preparations diering in particle size were tested: a powderized form obtained by countercurrent mechanical treatment of the clinoptilolite (MTCp) and normally ground clinoptilolite (NGCp). Young adult mice were supplied with food containing 12.5, 25 or 50% clinoptilolite powder. Control animals received the same food ration without the clinoptilolite. After 10, 20, 30 and 40 days, six animals from each group were exsanguinated to obtain blood for hematological and serum for biochemical measurements as well as to collect femoral bone marrow for determination of hematopoietic activity. Clinoptilolite ingestion was well tolerated, as judged by comparable body masses of treated and control animals. A 20% increase of the potassium level was detected in mice receiving the zeolite-rich diet, without other changes in serum chemistry. Erythrocyte, hemoglobin and platelet levels in peripheral blood were not materially aected. NGCp caused leukocytosis, with concomitant decline of the GM±CFU content in the bone marrow, which was attributed to intestinal irritation by rough zeolite particles. The mechanically treated clinoptilolite preparation caused similar, albeit less pronounced, changes. In a limited experiment, mice having transplanted mammary carcinoma in the terminal stage showed increased potassium and decreased sodium and chloride levels, severe anemia and leukocytosis, decreased bone marrow cellularity and diminished content of hematopoietic progenitor cells in the marrow. The clinoptilolite preparations ameliorated the sodium and chloride decline, whereas the eects on hematopoiesis were erratic. #

We identified nicotinamide phosphoribosyltransferase (NAMPT), also known as pre-B cell colony enhancing factor (PBEF), as an essential enzyme mediating granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis in healthy individuals and in individuals with severe congenital neutropenia. Intracellular NAMPT and NAD + amounts in myeloid cells, as well as plasma NAMPT and NAD + levels, were increased by G-CSF treatment of both healthy volunteers and individuals with congenital neutropenia. NAMPT administered both extracellularly and intracellularly induced granulocytic differentiation of CD34 + hematopoietic progenitor cells and of the promyelocytic leukemia cell line HL-60. Treatment of healthy individuals with high doses of vitamin B3 (nicotinamide), a substrate of NAMPT, induced neutrophilic granulocyte differentiation. The molecular events triggered by NAMPT include NAD + -dependent sirtuin-1 activation, subsequent induction of CCAAT/enhancer binding protein-a and CCAAT/enhancer binding protein-b, and, ultimately, upregulation of G-CSF synthesis and G-CSF receptor expression. G-CSF, in turn, further increases NAMPT levels. These results reveal a decisive role of the NAD + metabolic pathway in G-CSF-triggered myelopoiesis.

Strategies that increase homing to the bone marrow and engraftment efficacy of ex vivo expended CD34 + cells are expected to enhance their clinical utility. Here we report that nicotinamide (NAM), a form of vitamin B-3, delayed differentiation and increased engraftment efficacy of cord blood-derived human CD34 + cells cultured with cytokines. In the presence of NAM, the fraction of CD34 + CD38 L cells increased and the fraction of differentiated cells (CD14 + , CD11b + , and CD11c + ) decreased. CD34 + cells cultured with NAM displayed increased migration toward stromal cell derived factor-1 and homed to the bone marrow with higher efficacy, thus contributing to their increased engraftment efficacy, which was maintained in competitive transplants with noncultured competitor cells. NAM is a known potent inhibitor of several classes of ribosylase enzymes that require NAD for their activity, as well as sirtuin (SIRT1), class III NAD + -dependent-histone-deacetylase. We demonstrated that EX-527, a specific inhibitor of SIRT1 catalytic activity, inhibited differentiation of CD34 + cells similar to NAM, while specific inhibitors of NAD-ribosylase enzymes did not inhibit differentiation, suggesting that the NAM effect is SIRT1-specific. Our findings suggest a critical function of SIRT1 in the regulation of hematopoietic stem cell activity and imply the clinical utility of NAM for ex vivo expansion of functional CD34 + cells.

Impaired balance between mature and immature myeloid cells is one of the hallmarks of cancer. In cancer patients as well as in mouse models there is increasing evidence that progressive tumor growth is associated with an accumulation of immature myeloid cells, monocytes/macrophages, and with a decreased number and function of dendritic cells (DC). This review examines recent findings on the contribution of immature myeloid cells (ImC) to cancer-induced immune suppression.

Bacterial DNA and immunostimulatory (i.s.) synthetic CpG-oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as "nature's adjuvant" since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG-ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigenpresenting cell (APC) function. Unfractionated murine bone marrow-derived DC and FACS ®fractionated MHC class II low (termed immature DC) or MHC class II high populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG-ODN. Similar to lipopolysaccharide, i.s. CpG-ODN caused up-regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL-12, IL-6 and TNF- § . CpG-ODN-activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B-driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG-ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.

ABBREVIATIONS: AT = adipose tissue; BM = bone marrow; CFU-F(s) = colony-forming unit fibroblast precursor(s); GMP = Good Manufacturing Practices; HSC(s) = hematopoietic stem cell(s); IGF = insulin-like growth factor; MSC(s) = mesenchymal stem cell(s); PDGF(s) = platelet-derived growth factor(s); UCB = umbilical cord blood. From the * The data were searched on the website of ClinicalTrials.gov (http://www.clinicaltrials.gov) on October 22, 2012. The following key words including "mesenchymal stem cells,"

Abstract: The stem cell paradigm was first demonstrated in hematopoietic stem cells.

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Zeolites are natural or synthetic crystalline alumosilicates with ion exchanging properties. Supplied in fodder, they promote biomass production and animal health. Our aim was to assess the eects of the natural zeolite, clinoptilolite, on hematopoiesis, serum electrolytes and essential biochemical indicators of kidney and liver function in mice. Two preparations diering in particle size were tested: a powderized form obtained by countercurrent mechanical treatment of the clinoptilolite (MTCp) and normally ground clinoptilolite (NGCp). Young adult mice were supplied with food containing 12.5, 25 or 50% clinoptilolite powder. Control animals received the same food ration without the clinoptilolite. After 10, 20, 30 and 40 days, six animals from each group were exsanguinated to obtain blood for hematological and serum for biochemical measurements as well as to collect femoral bone marrow for determination of hematopoietic activity. Clinoptilolite ingestion was well tolerated, as judged by comparable body masses of treated and control animals. A 20% increase of the potassium level was detected in mice receiving the zeolite-rich diet, without other changes in serum chemistry. Erythrocyte, hemoglobin and platelet levels in peripheral blood were not materially aected. NGCp caused leukocytosis, with concomitant decline of the GM±CFU content in the bone marrow, which was attributed to intestinal irritation by rough zeolite particles. The mechanically treated clinoptilolite preparation caused similar, albeit less pronounced, changes. In a limited experiment, mice having transplanted mammary carcinoma in the terminal stage showed increased potassium and decreased sodium and chloride levels, severe anemia and leukocytosis, decreased bone marrow cellularity and diminished content of hematopoietic progenitor cells in the marrow. The clinoptilolite preparations ameliorated the sodium and chloride decline, whereas the eects on hematopoiesis were erratic. #

The HOM-C clustered prototype homeobox genes of Drosophila, and their counterparts, the HOX genes in humans, are highly conserved at the genomic level. These master regulators of development continue to be expressed throughout adulthood in various tissues and organs. The physiological and patho-physiological functions of this network of genes are being avidly pursued within the scientific community, but defined roles for them remain elusive. The order of expression of HOX genes within a cluster is co-ordinated during development, so that the 3 genes are expressed more anteriorly and earlier than the 5 genes. Mutations in HOXA13 and HOXD13 are associated with disorders of limb formation such as hand-foot-genital syndrome (HFGS), synpolydactyly (SPD), and brachydactyly. Haematopoietic progenitors express HOX genes in a pattern characteristic of the lineage and stage of differentiation of the cells. In leukaemia, dysregulated HOX gene expression can occur due to chromosomal translocations involving upstream regulators such as the MLL gene, or the fusion of a HOX gene to another gene such as the nucleoporin, NUP98. Recent investigations of HOX gene expression in leukaemia are providing important insights into disease classification and prediction of clinical outcome. Whereas the oncogenic potential of certain HOX genes in leukaemia has already been defined, their role in other neoplasms is currently being studied. Progress has been hampered by the experimental approach used in many studies in which the expression of small subsets of HOX genes was analysed, and complicated by the functional redundancy implicit in the HOX gene system. Attempts to elucidate the function of HOX genes in malignant transformation will be enhanced by a better understanding of their upstream regulators and downstream target genes.

Results. USSC constitutively produced SCF, LIF, TGF-1b, M-CSF, GM-CSF, VEGF, IL-1b, IL-6, IL-8, IL-11, IL-12, IL-15, SDF-1a, and HGF. When USSC were stimulated with IL-1b, G-CSF was released. Production of SCF and LIF were significantly higher in USSC compared to BMMSC. At 1, 2, 3, and 4 weeks, cocultivation of CD34 ϩ cells on the USSC layer resulted in a 14.6-fold ± 1.1-fold, 110.1-fold ± 17.9-fold, 151.8-fold ± 39.7-fold, and 183.6-fold ± 40.4fold amplification of total cells and in a 30.6-fold ± 4.4-fold, 101.4-fold ± 27.5-fold, 64.7-fold ± 15.8-fold, and 29.4-fold ± 3.1-fold amplification of CFC, respectively. LTC-IC expansion at 1 and 2 weeks was, with 2.0-fold ± 0.1-fold and 2.5-fold ± 0.3-fold, significantly higher for USSC than BMMSC (1.1-fold ± 0.03-fold and 1.1-fold ± 0.1-fold), but declined after day 21. Transwell cocultures of USSC did not significantly alter total cell or CFC expansion. Conclusions. USSC produce functionally significant amounts of hematopoiesis-supporting cytokines and are superior to BMMSC in expansion of CD34 ϩ cells from CB. USSC is therefore a suitable candidate for stroma-driven ex vivo expansion of hematopoietic CB cells for shortterm reconstitution. Ć

Pharmacological agents such as hydroxyurea (HU) have been known to cause induction of fetal hemoglobin and possibly may alleviate the symptoms in thalassemia intermedia patients. Thirty-seven patients with β-thalassemia intermedia were enrolled to assess response to HU therapy. Major response was defined as transfusion independence or hemoglobin rise of more than 20 g/l and minor response as rise in hemoglobin of 10–20 g/l or reduction in transfusion frequency by 50%. The median age was 10 years (range: 4–50 years) and median follow-up was 12 months (range: 4–36 months). Twenty-six patients (70.2%) showed response to HU therapy. Seventeen patients (45.9%) were major responders, and nine patients (24.3%) showed minor response. There was no correlation of response with β-thalassemia mutation or XmnI polymorphism; however, the presence of α3.7 deletion was associated with major response in three patients. Mean fetal hemoglobin (HbF) levels rose on HU therapy. Older age, low baseline F cell percent, and low baseline HbF levels (below 10%) were predictors of poor response. Response was evident within 1 month of starting HU therapy in the majority of responders. Thus, a short trial of HU therapy can predict durable response.

The role of hematopoietic stem cells in blood cell development is reasonably understood, whereas the identity and the function of bone marrow stromal cells are much less clear. Using stromal cells in bone marrow cultures of the Dexter type, a favorite medium for the study of hematopoiesis, we show that stromal cells actually represent a unique cell type. Conventional wisdom has held that stromal cells in Dexter cultures comprise a mixture of macrophages, hematopoietic cells, adipocytes, osteoblasts, fibroblasts, muscle cells, and endothelial cells. Our findings demonstrate that Dexter cultures consist of three cell types: macrophages (ϳ35%), hematopoietic cells (ϳ5%), and nonhematopoietic cells (ϳ60%). We have purified the nonhematopoietic cells free of macrophages and hematopoietic cells to produce compelling evidence that they in fact represent a single cell type (multidifferentiated mesenchymal progenitor cell, MPC) which coexpresses genes specific for various mesenchymal cell lineages including adipocytes, osteoblasts, fibroblasts, and muscle cells. We further show that these multi-or pluridifferentiated MPCs are capable of supporting hematopoiesis by demonstrating the expression of several hematopoietic growth factors and extracellular matrix receptors including G-CSF, SCF, VCAM-1, ICAM-1, and ALCAM. Since the MPCs can be easily purified to near homogeneity (95%), they can be of value in enhancing engraftment of hematopoietic stem cells. Also, this new understanding of bone marrow stromal cells as "one cell with many different faces" promises to advance our knowledge of regulatory cellular interactions within bone marrow.

This manuscript is intended to provide a best practice approach to accurately and consistently assess toxicant-induced bone marrow effects of test articles. In nonclinical toxicity studies, complete blood count data in conjunction with the histological examination of the bone marrow are recommended as the foundation for assessing the effect of test articles on the hematopoietic system. This approach alone can be used successfully in many studies. However, in some situations it may be necessary to further characterize effects on the different hematopoietic lineages, either by cytological or flow cytometric evaluation of the bone marrow. Both modalities can be used successfully, and which one is selected will depend on the expertise, preference of the facility, and the nature of the change in the bone marrow. Other specialized techniques such as clonogenic assays or electron microscopy are used rarely to further characterize hematotoxicity. The indications and techniques to successfully employ histological, cytological, or flow cytometric evaluation as well as clonogenic assays and electron microscopy are reviewed.

Factors such as cytokines, extra cellular matrix components, and small molecules, as well as the temporal association and concentration of these factors were tested on seven different human ES and iPS lines. We report the differentiation of up to 84% huCD45+ cells (average 41% ± 16, from 7 pluripotent lines) from the differentiation culture, including significant numbers of primitive CD45+/CD34+ and CD45+/CD34+/CD38hematopoietic progenitors. Moreover, the numbers of hematopoietic progenitor cells generated, as measured by colony forming unit assays were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. Our approach demonstrates highly efficient generation of multipotent hematopoietic progenitors with the highest efficiencies reported to date (CD45+/CD34+) using a single standardized differentiation protocol on several human ES and iPS lines. Our data add to the cumulating evidence for the existence of an in vitro derived precursor to the hematopoietic stem cell (HSC) with limited engrafting ability in transplanted mice, but with multipotent hematopoietic potential. Because this protocol efficiently expands the pre-blood precursors and hematopoietic progenitors, it is ideal for testing novel factors for the generation and expansion of definitive HSCs with long-term repopulating ability.