技术资料

Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in organ transplant recipients. The use of granulocyte-colony stimulating factor (G-CSF)-mobilized stem cells from HCMV seropositive donors is suggested to double the risk of late-onset HCMV disease and chronic graft-versus-host disease in recipients when compared to conventional bone marrow transplantation with HCMV seropositive donors,although the etiology of the increased risk is unknown. To understand mechanisms of HCMV transmission in patients receiving G-CSF-mobilized blood products,we generated a NOD-scid IL2Rγ(c)(null)-humanized mouse model in which HCMV establishes latent infection in human hematopoietic cells. In this model,G-CSF induces the reactivation of latent HCMV in monocytes/macrophages that have migrated into organ tissues. In addition to establishing a humanized mouse model for systemic and latent HCMV infection,these results suggest that the use of G-CSF mobilized blood products from seropositive donors pose an elevated risk for HCMV transmission to recipients. View Publication

AIMS: Macrophage scavenger receptor A (SR-A) and class B scavenger receptor CD36 (CD36) contribute to foam cell formation and atherogenesis via uptake of modified lipoproteins. So far,the role of these scavenger receptors has been studied mainly using knockout models totally lacking these receptors. We studied the role of SR-A and CD36 in foam cell formation and atherogenesis by RNA interference (RNAi)-mediated silencing,which is a clinically feasible method to down-regulate the expression of these receptors. METHODS AND RESULTS: We constructed lentivirus vectors encoding short hairpin RNAs (shRNAs) against mouse SR-A and CD36. Decreased SR-A but not CD36 expression led to reduced foam cell formation caused by acetylated low-density lipoprotein (LDL) in mouse macrophages,whereas the uptake of oxidized LDL was not altered. More importantly,silencing of SR-A upregulates CD36 and vice versa. In LDL receptor-deficient apolipoprotein B100 (LDLR(-/-)ApoB(100/100)) mice kept on a western diet,silencing of either SR-A or CD36 in bone marrow cells led to a marked decrease (37.4 and 34.2%,respectively) in cross-sectional lesion area,whereas simultaneous silencing of both receptors was not effective. CONCLUSION: Our results suggest that silencing of either SR-A or CD36 alone reduces atherogenesis in mice. However,due to reciprocal upregulation,silencing of both SR-A and CD36 is not effective. View Publication

Autosomal recessive osteopetrosis (ARO) is a group of genetic disorders that involve defects that preclude the normal function of osteoclasts,which differentiate from hematopoietic precursors. In half of human cases,ARO is the result of mutations in the TCIRG1 gene,which codes for a subunit of the vacuolar proton pump that plays a fundamental role in the acidification of the cell-bone interface. Functional mutations of this pump severely impair the resorption of bone mineral. Although postnatal hematopoietic stem cell transplantation can partially rescue the hematological phenotype of ARO,other stigmata of the disease,such as secondary neurological and growth defects,are not reversed. For this reason,ARO is a paradigm for genetic diseases that would benefit from effective prenatal treatment. Using the oc/oc mutant mouse,a murine model whose osteopetrotic phenotype closely recapitulates human TCIRG1-dependent ARO,we report that in utero transplantation of adult bone marrow hematopoietic stem cells can correct the ARO phenotype in a limited number of mice. Here we report that in utero injection of allogeneic fetal liver cells,which include hematopoietic stem cells,into oc/oc mouse fetuses at 13.5 days post coitum produces a high level of engraftment,and the oc/oc phenotype is completely rescued in a high percentage of these mice. Therefore,oc/oc pathology appears to be particularly sensitive to this form of early treatment of the ARO genetic disorder. View Publication

BACKGROUND: Conventional gene-therapy applications of hematopoietic stem cells (HSCs) involve purification of CD34+ progenitor cells from the mobilized peripheral blood,ex vivo transduction of the gene of interest into them,and reinfusion of the transduced CD34+ progenitor cells into patients. Eliminating the process of purification would save labor,time and money,while enhancing HSCs viability,transplantability and pluripotency. Lentiviral vectors have been widely used in gene therapy because they infect both dividing and nondividing cells and provide sustained transgene expression. One of the exceptions to this rule is quiescent primary lymphocytes,in which reverse transcription of viral DNA is not completed. METHODS: In the present study,we tested the possibility of targeting CD34+ progenitor cells within nonpurified human mobilized peripheral blood mononuclear cells (mPBMCs) utilizing vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vectors,based on the assumption that the CD34+ progenitor cells would be preferentially transduced. To further enhance the specificity of vector transduction,we also examined utilizing a modified Sindbis virus envelope (2.2) pseudotyped lentiviral vector,developed in our laboratory,that allows targeted transduction to specific cell receptors via antibody recognition. RESULTS: Both the VSV-G and 2.2 pseudotyped vectors achieved measurable results when they were used to target CD34+ progenitor cells in nonpurified mPBMCs. CONCLUSIONS: Overall,the data obtained demonstrate the potential of ex vivo targeting of CD34+ progenitor cells without purification. View Publication

Beta glucans are cell wall constituents of yeast,fungi and bacteria,as well as mushrooms and barley. Glucans are not expressed on mammalian cells and are recognized as pathogen-associated molecular patterns (PAMPS) by pattern recognition receptors (PRR). Beta glucans have potential activity as biological response modifiers for hematopoiesis and enhancement of bone marrow recovery after injury. We have reported that Maitake beta glucan (MBG) enhanced mouse bone marrow (BMC) and human umbilical cord blood (CB) cell granulocyte-monocyte colony forming unit (GM-CFU) activity in vitro and protected GM-CFU forming stem cells from doxorubicin (DOX) toxicity. The objective of this study was to determine the effects of MBG on expansion of phenotypically distinct subpopulations of progenitor and stem cells in CB from full-term infants cultured ex vivo and on homing and engraftment in vivo in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse. MBG promoted a greater expansion of CD34+CD33+CD38- human committed hematopoietic progenitor (HPC) cells compared to the conventional stem cell culture medium (P = 0.002 by ANOVA). CD34+CXCR4+CD38- early,uncommitted human hematopoietic stem cell (HSC) numbers showed a trend towards increase in response to MBG. The fate of CD34+ enriched CB cells after injection into the sublethally irradiated NOS/SCID mouse was evaluated after retrieval of xenografted human CB from marrow and spleen by flow cytometric analysis. Oral administration of MBG to recipient NOS/SCID mice led to enhanced homing at 3 days and engraftment at 6 days in mouse bone marrow (P = 0.002 and P = 0.0005,respectively) compared to control mice. More CD34+ human CB cells were also retrieved from mouse spleen in MBG treated mice at 6 days after transplantation. The studies suggest that MBG promotes hematopoiesis through effects on CD34+ progenitor cell expansion ex vivo and when given to the transplant recipient could enhance CD34+ precursor cell homing and support engraftment. View Publication

We developed a real-time copy number polymerase chain reaction assay for deletions on chromosome 20q (del20q),screened peripheral blood granulocytes from 664 patients with myeloproliferative disorders,and identified 19 patients with del20q (2.9%),of which 14 (74%) were also positive for JAK2-V617F. To examine the temporal relationship between the occurrence of del20q and JAK2-V617F,we performed colony assays in methylcellulose,picked individual burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte (CFU-G) colonies,and genotyped each colony individually for del20q and JAK2-V617F. In 2 of 9 patients,we found that some colonies with del20q carried only wild-type JAK2,whereas other del20q colonies were JAK2-V617F positive,indicating that del20q occurred before the acquisition of JAK2-V617F. However,in colonies from 3 of 9 patients,we observed the opposite order of events. The lack of a strict temporal order of occurrence makes it doubtful that del20q represents a predisposing event for JAK2-V617F. In 2 patients with JAK2-V617F and 1 patient with MPL-W515L,microsatellite analysis revealed that del20q affected chromosomes of different parental origin and/or 9pLOH occurred at least twice. The fact that rare somatic events,such as del20q or 9pLOH,occurred more than once in subclones from the same patients suggests that the myeloproliferative disorder clone carries a predisposition to acquiring such genetic alterations. View Publication

Homing of hematopoietic stem cells (HSCs) into the bone marrow (BM) is a prerequisite for establishment of hematopoiesis during development and following transplantation. However,the molecular interactions that control homing of HSCs,in particular,of fetal HSCs,are not well understood. Herein,we studied the role of the alpha6 and alpha4 integrin receptors for homing and engraftment of fetal liver (FL) HSCs and hematopoietic progenitor cells (HPCs) to adult BM by using integrin alpha6 gene-deleted mice and function-blocking antibodies. Both integrins were ubiquitously expressed in FL Lin(-)Sca-1(+)Kit(+) (LSK) cells. Deletion of integrin alpha6 receptor or inhibition by a function-blocking antibody inhibited FL LSK cell adhesion to its extracellular ligands,laminins-411 and -511 in vitro,and significantly reduced homing of HPCs to BM. In contrast,the anti-integrin alpha6 antibody did not inhibit BM homing of HSCs. In agreement with this,integrin alpha6 gene-deleted FL HSCs did not display any homing or engraftment defect compared with wild-type littermates. In contrast,inhibition of integrin alpha4 receptor by a function-blocking antibody virtually abrogated homing of both FL HSCs and HPCs to BM,indicating distinct functions for integrin alpha6 and alpha4 receptors during homing of fetal HSCs and HPCs. View Publication

Parthenogenetic embryonic stem (ES) cells with two oocyte-derived genomes (uniparental) have been proposed as a source of autologous tissue for transplantation. The therapeutic applicability of any uniparental cell type is uncertain due to the consequences of genomic imprinting that in mammalian uniparental tissues causes unbalanced expression of imprinted genes. We transplanted uniparental fetal liver cells into lethally irradiated adult mice to test their capacity to replace adult hematopoietic tissue. Both maternal (gynogenetic) and paternal (androgenetic) derived cells conveyed long-term,multilineage reconstitution of hematopoiesis in recipients,with no associated pathologies. We also establish that uniparental ES cells can differentiate into transplantable hematopoietic progenitors in vitro that contribute to long-term hematopoiesis in recipients. Hematopoietic tissue in recipients maintained fidelity of parent-of-origin methylation marks at the Igf2/H19 locus; however,variability occurred in the maintenance of parental-specific methylation marks at other loci. In summary,despite genomic imprinting and its consequences on development that are particularly evident in the androgenetic phenotype,uniparental cells of both parental origins can form adult-transplantable stem cells and can repopulate an adult organ. View Publication

OBJECTIVE: To investigate the kinetics of myocardial engraftment of bone marrow-derived mononuclear cells (BMNCs) after intracoronary injection using 99mTc-d,l-hexamethylpropylene amine oxime (99mTc-HMPAO) nuclear imaging in patients with acute and chronic anterior myocardial infarction. DESIGN: Nuclear imaging-derived tracking of BMNCs at 2 and 20 h after injection in the left anterior descending (LAD) coronary artery. SETTING: Academical cardiocentre. PATIENTS: Five patients with acute (mean (SD) age 58 (11) years; ejection fraction range 33-45%) and five patients with chronic (mean (SD) age 50 (6) years; ejection fraction range 28-34%) anterior myocardial infarction. INTERVENTIONS: A total of 24.2 x 10(8)-57.0 x 10(8) BMNCs (20% labelled with 700-1000 MBq 99mTc-HMPAO) were injected in the LAD coronary artery. RESULTS: At 2 h after BMNC injection,myocardial activity was observed in all patients with acute (range 1.31-5.10%) and in all but one patient with chronic infarction (range 1.10-3.0%). At 20 h,myocardial engraftment was noted only in three patients with acute myocardial infarction,whereas no myocardial activity was noted in any patient with chronic infarction. CONCLUSIONS: Engraftment of BMNCs shows dynamic changes within the first 20 h after intracoronary injection. Persistent myocardial engraftment was noted only in a subset of patients with acute myocardial infarction. View Publication

AIM: The recently published REPAIR-AMI and ASTAMI trial showed differences in contractile recovery of left ventricular function after infusion of bone marrow-derived cells in acute myocardial infarction. Since the trials used different protocols for cell isolation and storage (REPAIR-AMI: Ficoll,storage in X-vivo 10 medium plus serum; ASTAMI: Lymphoprep,storage in NaCl plus plasma),we compared the functional activity of BMC isolated by the two different protocols. METHODS AND RESULTS: The recovery of total cell number,colony-forming units (CFU),and the number of mesenchymal stem cells were significantly reduced to 77 +/- 4%,83 +/- 16%,and 65 +/- 15%,respectively,when using the ASTAMI protocol compared with the REPAIR protocol. The capacity of the isolated BMC to migrate in response to stromal cell-derived factor 1 (SDF-1) was profoundly reduced when using the ASTAMI cell isolation procedure (42 +/- 8% and 78 +/- 3% reduction in healthy and CAD-patient cells,respectively). Finally,infusion of BMC into a hindlimb ischaemia model demonstrated a significantly blunted blood-flow-recovery by BMC isolated with the ASTAMI protocol (54 +/- 6% of the effect obtained by REPAIR cells). Comparison of the individual steps identified the use of NaCl and plasma for cell storage as major factors for functional impairment of the BMC. CONCLUSION: Cell isolation protocols have a major impact on the functional activity of bone marrow-derived progenitor cells. The assessment of cell number and viability may not entirely reflect the functional capacity of cells in vivo. Additional functional testing appears to be mandatory to assure proper cell function before embarking on clinical cell therapy trials. View Publication

Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology,gene therapy,and clinical transplantation. Here,we demonstrate efficient ex vivo expansion of HSCs measured by long-term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133-sorted cells using a soluble form of Delta1. After a 3-week culture on immobilized Delta1 supplemented with stem cell factor,thrombopoietin,Flt-3 ligand,interleukin (IL)-3,and IL-6/soluble IL-6 receptor chimeric protein (FP6) in a serum- and stromal cell-free condition,we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self-renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/gammac(null) mice,which showed myeloid,B,T,and natural killer cells as well as CD133(+)CD34(+) cells,and hematopoietic reconstitution in the secondary recipients. Interestingly,the CD133-sorted cells contained approximately 4.5 times more SRCs than the CD34-sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation. View Publication

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell (HSC) transplantation alleviates complications such as graft-versus-host disease,leading to a speedy recovery of hematopoiesis. To meet this clinical demand,a fast MSC expansion method is required. In the present study,we examined the feasibility of using a rotary bioreactor system to expand MSCs from isolated bone marrow mononuclear cells. The cells were cultured in a rotary bioreactor with Myelocult medium containing a combination of supplementary factors,including stem cell factor and interleukin-3 and -6. After 8 days of culture,total cell numbers,Stro-1(+)CD44(+)CD34(-) MSCs,and CD34(+)CD44(+)Stro-1(-) HSCs were increased 9-,29-,and 8-fold,respectively. Colony-forming efficiency-fibroblast per day of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSC markers endoglin (SH2) and vimentin,whereas markers associated with lineage differentiation,including osteocalcin (osteogenesis),type II collagen (chondrogenesis),and C/EBP-alpha (CCAAT/enhancer-binding protein-alpha) (adipogenesis),were not detected. Upon induction,the bioreactor-expanded MSCs were able to differentiate into osteoblasts,chondrocytes,and adipocytes. We conclude that the rotary bioreactor with the modified Myelocult medium reported in this study may be used to rapidly expand MSCs. View Publication