Human Umbilical Cord Blood CD34 Research Articles

Effect of Human Umbilical Cord Blood Progenitor Cells Versus Mononuclear Cells on Acute Renal Failure Rat Model

Acute renal failure (ARF) resulting from ischemic or toxic insults remains a major health care problem because of its grave prognosis and the limited effectiveness of available treatment modalities. Current treatment options for ARF are limited to supportive measures and preventive strategies, none of which have been definitively shown to alter mortality. To assess the ability of human umbilical cord blood CD34(+) (HUCB CD34(+)) cells and mononuclear (HUCB MNC) cells to improve renal function of nephrotoxic kidney. ARF was induced in 30 rats by glycerol. After 24 hours, ARF was confirmed by increased blood urea nitrogen (BUN), serum urea and creatinine levels. The rats were divided into 3 groups, group one included 10 rats treated with HUCB CD34(+) cells, group two included 10 rats treated with HUCB MNC and group three included 10 rats treated with normal saline. Five rats were included in the study as a normal control group. Serial measurement of BUN, serum urea and creatinine levels were done every three days throughout the study. To proof homing of HUCB CD34(+) into renal tissue, Y chromosome detection in renal tissue was carried out using Real time polymerase chain reaction (PCR) technique. Four days after the therapy, the renal function of CD34(+) and MNC treated rats improved in comparison to saline treated rats. After 2 weeks of therapy and at the end of the study (28 days), ANOVA test revealed that, there was significant difference between the four studied groups (P=.000). Y chromosomes were detected in kidneys of CD34(+) treated rats and MNC treated rats. HUCB CD34(+) cells and HUCB MNC improve renal function of nephrotoxic kidney with superiority to the HUCB MNC.

The Role of Collagen Type I on Hematopoietic and Mesenchymal Stem Cells Expansion and Differentiation

The three dimensional scaffold of the bone marrow (BM) niches is composed of various elements including extracellular matrix proteins and cell types, such as collagen type I (Col I) and stroma cells. Interaction of stem cells with their microenvironment is important for their regulation. In the marrow, Col I is mostly localized in the endosteal regions. The objective of this work was to investigate the role of Col I in the regulation of Hematopoietic Stem Cells (HSC) and Mesenchymal Stem Cells (MSC) growth. Col I was extracted from rat tail tendons and its purity confirmed. Human BM MSCs and umbilical cord blood (UCB) CD34+cells were used as Stem Cell sources. MSCs were cultured in medium with serum while CB CD34+cells were cultured without serum with cytokines. The impact of increasing concentrations of Col I (0-50 µg mL-1for coating) on the growth of Hematopoietic Progenitor Cells (HPC) and MSCs was investigated by cytometry, microscopy and clonogenic progenitor assays. Only a minority of CD34+cells expressed the Col I receptor α2β1prior to culture, while the opposite was observed when hematopoietic cells were placed in culture. Col I coated surfaces reduced the expansion of hematopoietic cells by 25% compared to control, while expansions of myeloid and MK progenitors were either unchanged or negatively affected by Col I, respectively. The differentiation of HPCs was also affected on Col I as demonstrated by differences in the frequencies of various cell lineages, such as CD34+cells, megakaryocytes (MK), erythrocytes and others. In contrast to HPCs, Col I surfaces increased MSCs proliferation but had little impact on osteoblasts derived from MSCs. Taken together, this study provides new insights into the regulatory activities of Col I on Stem Cells residing in the marrow.

The cross-talk between ROS and p38MAPKα in the Ex Vivo expanded human umbilical cord blood CD133+ cells

This study investigated the correlation between and compared the effects of reactive oxygen species (ROS) and p38 mitogen-activated protein kinase α (p38MAPKα) in the ex vivo expanded umbilical cord blood (hUCB) CD133(+) cells. hUCB CD133(+) cells were cultured in the hematopoietic stem cells (HSCs) culture medium with N-acetylcysteine (NAC, an anti-oxidant), p38MAPKα-specific inhibitor (SB203580) or their combination. The levels of ROS and expression of phosphorylated p38MAPKα (p-p38) in CD133(+) cells were flow cytometrically detected. The efficacy of ex vivo expansion was evaluated by the density of CD133(+) cell sub-group colony-forming cells (CFC) and cobblestone area-forming cells (CAFC) assay. Our results showed decreased ROS levels in NAC, SB203580, and their combination treatment groups were almost 37%, 48%, and 85%, respectively. Furthermore, SB203580 abrogated the activation of p38MAPKα more obviously than NAC. Moreover, the CD133(+) cells in SB203580 treatment group had a 21.93±1.36-fold increase, and 14.50±1.19-fold increase in NAC treatment group, but only 10.13±0.57-fold increase in control group. In addition, SB203580 treatment led a higher level increase in the number of CFU and CAFC than NAC did. These findings suggested that, in expanded CD133(+) cells, ROS activates p38MAPKα, which, in turn, induces ROS production, and p38MAPKα might be the most suitable regulator in ROS-p38MAPKα pathway for the promotion of HSCs ex vivo expansion.

The effect of nanofibre surface amine density and conjugate structure on the adhesion and proliferation of human haematopoietic progenitor cells

Previous studies have shown that substrate surface chemistry and topography exhibit significant impact on haematopoietic progenitor cell adhesion, proliferation and differentiation. In the present study, the effect of surface amine density and structure of grafted polymer chains on the adhesion and expansion of haematopoietic progenitor cells was investigated. Cryopreserved human umbilical cord blood CD133(+) cells were expanded in cytokine-supplemented medium on ethylenediamine (EDA)- or 2-aminoethyl methacrylate hydrochloride (AEMA)-grafted polyethersulphone (PES) nanofibre scaffolds for 10 days. Although the percentage of CD34(+) cells among the expanded cells increased with the surface amine density, the maximum fold expansion of CD34(+) cells was obtained at a moderate amine density of 20-80 nmol cm(-2). When comparing nanofibre matrices with similar amine densities, but prepared with two different methods, cells cultured on the AEMA-grafted PES nanofibre matrix showed lower fold expansion in terms of total cell number (300 ± 84 fold) and CD34(+) cell number (68 ± 19-fold) in comparison with those cultured on EDA-grafted nanofibres (787 ± 84-fold and 185 ± 84-fold, respectively). These results indicate that the surface amine density and the conjugate structure are important determinants for the preservation of CD34 surface marker and expansion efficiency of CD34(+) cells.

Ex vivo differentiation of natural killer cells from human umbilical cord blood CD34+progenitor cells

Natural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies.

CD9 up-regulation on CD34+ cells with ingenol 3,20-dibenzoate does not improve homing in NSG mice

To the editor: Leung et al demonstrated the potential importance of the tetraspanin CD9[1][1] in regulating migration, adhesion, and homing of human umbilical cord blood (UCB) CD34+ hematopoietic stem and progenitor cells.[2][2] Using a neutralizing antibody to CD9 they inhibited marrow and splenic

Effect of human umbilical cord blood CD34+ progenitor cells transplantation in diabetic mice

Shortage of donor organs spurs research into alternative means of generating β cells. Stem cells might represent a potential source of tissues for cell therapy protocols, and diabetes is a candidate disease that may benefit from cell replacement protocols. We examined the effect of transplanted human umbilical cord blood CD34+ cells on some detailed parameters in streptozotocin- (STZ) induced diabetic mice. An experimental study was conducted in the departments of clinical pathology, physiology and pathology of Faculty of Medicine, Suez Canal University. Thirty male albino mice 8–12 weeks were included and subdivided into 3 groups, first group served as normal control group, second group as diabetic control after induction of diabetes with STZ and third group treated diabetic mice by injection of positively selected CD34 progenitor cells from human umbilical cord blood (HUCB) with a dose of one million cells/mouse. Blood glucose and serum insulin were measured at specific time interval and immunohistochemical (IHC) analysis and histopathology on pancreas were conduced. Data were analyzed using chi square between groups. Intravenous injection of CD34+ cells caused significant improvement in blood glucose level (277.9 ± 102.5 mg/dl in treated group vs 530.3 ± 99 mg/dl in untreated group, p < 0.01). Blood level of mouse insulin was higher in the treated group as compared with untreated diabetic mice (0.77 ± 0.2 ng/ml in treated group versus 0.26 ± 0.09 in untreated group, p < 0.001). IHC analysis for detection of human insulin producing cells in pancreas of treated mice revealed that 33.3% positive cellular staining and 55.6% positive sinusoidal staining were detected. In conclusion, Transplantation of HUCB-CD34+ cells appear to be a modality of stem cell therapy in diabetes mellitus.

Human and rhesus macaque hematopoietic stem cells cannot be purified based only on SLAM family markers

AbstractVarious combinations of antibodies directed to cell surface markers have been used to isolate human and rhesus macaque hematopoietic stem cells (HSCs). These protocols result in poor enrichment or require multiple complex steps. Recently, a simple phenotype for HSCs based on cell surface markers from the signaling lymphocyte activation molecule (SLAM) family of receptors has been reported in the mouse. We examined the possibility of using the SLAM markers to facilitate the isolation of highly enriched populations of HSCs in humans and rhesus macaques. We isolated SLAM (CD150+CD48−) and non-SLAM (not CD150+CD48−) cells from human umbilical cord blood CD34+ cells as well as from human and rhesus macaque mobilized peripheral blood CD34+ cells and compared their ability to form colonies in vitro and reconstitute immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 γc receptornull, NSG) mice. We found that the CD34+ SLAM population contributed equally or less to colony formation in vitro and to long-term reconstitution in NSG mice compared with the CD34+ non-SLAM population. Thus, SLAM family markers do not permit the same degree of HSC enrichment in humans and rhesus macaques as in mice.

Human UCB CD133+ Cell Infusion In NOD-SCID Mice with Acute Hind Limb Ischemia Improves Reperfusion of An Ischemic Bed

AbstractAbstract 4697Clinical trials have shown intracoronary or intramuscular injections of hematopoietic stem cells (HSC) ameliorate vascular ischemia. HSCs are isolated from bone marrow (BM), umbilical cord blood (UCB), and adult peripheral blood (AB). Critical limb ischemia patients, injected with autologous HSC into ischemic tissue had improved perfusion and reduced amputations. However, autologous HSC effectiveness declines with age. We hypothesized that UCB CD133+ HSCs' are more efficacious than autologous HSC at revascularization for an ischemic bed.Immune reactive surface antigens on UCB CD133+ cells were determined using multi-parameter flow cytometry. UCB CD133+ allogeneic immune responses from 72 h co-cultures were analyzed using a mixed lymphocyte culture (MLR) proliferation assays, and induced cytokine production found using TH1(inflammatory)/TH2(anti-inflammatory) cytometric bead assays (CBA) (BD Biosciences (San Jose, CA). In vivo studies were done using NOD/SCID mice treated with low-dose irradiation (200cGy) then femoral ligation. Study mice were divided into 3 treatment groups and underwent intracoronary infusions of; 1×106 UCB CD133+, 0.5×106 UCB mononuclear cells (MNCs), or EGM media alone. Day 3 post-ligation and cell infusion, quadriceps from 3 mice in each group were harvested, total RNA isolated using Trizol™ and RNA for each treatment group pooled. Pooled RNA was converted to cDNA then assayed using mouse ABI Affymetrix™ Gene chip. Computational pathway analyzes were run for significant transcriptome changes. Gene array data was confirmed on select genes using quantitative reverse transcriptase real time PCR (qPCR). Treated mice were followed for ischemic bed reperfusion by Laser Doppler imaging for 28 days post-ligation. UCB CD133+ cells express MHC class I and II, but lack CD80 and CD86. Allogeneic MLR assays using UCB and AB MNCs showed UCB CD133+ cells were stimulatory to MNC proliferation. The MNCs alloresponse to UCB CD133+ cells produced high concentration of interleukin 4 (IL-4) and IL-10 indicative of a TH2 response. These results show allogeneic UCB CD133+ cells induce a TH2 immune response due to an absence of co-stimulation of immune cells. To verify whether the results are recapitulated in vivo, we treated acute limb ischemia in NOD/SCID mice with human UCB CD133+ cells and monitored revascularization by blood flow and Affymetrix mouse gene array. Computational analysis of Affymetrix mouse gene arrays from comparing all 3 treatment protocols indicated that ischemic quadriceps treated with UCB CD133+ cells exhibited a significant enrichment of anti-angiogeneic, chemotaxic, cytokine, and immune response genes. UCB CD133+ treated mice had increase expression of anti-inflammatory, and anti-angiogeneic genes versus controls. Conversely media and UCB MNC treated mice had equal to or higher pro-inflammatory gene expression. The anti-inflammatory gene response was confirmed for 2 of the 3 genes, arginase 1, CXCL7, and tumor growth factor beta binding protein 2 (TGFβBP2) comparing CD133+ treated mice to UCB MNC treated mice. TGFβBP2 was not observed to change in vivo. UCB CD133+ cells treated mice also had improved blood perfusion of the ischemic bed at 28 days post-ligation versus control protocols. The in vivo mouse model supports the in vitro observation, but demonstrates improved reperfusion over controls.Currently there are 2 non-exclusive hypotheses for the function of CD133+ cells in revascularization. First the CD133+ cells integrate and differentiate into new vascular endothelial cells and the second is that the CD133+ cells augment revascularization via a paracrine effect which does not require integration into the host tissue. In vitro studies suggest that UCB CD133+ cells promote an allogeneic TH2 type response because of a lack of co-stimulatory immune surface antigens, resulting in high concentrations of IL-4 and IL-10. This skewed TH2 response inhibits inflammation, the subsequent tissue damage, and may augment revascularization. Although these studies cannot exclude a role for UCB CD133+ cellular integration, results support the paracrine hypothesis as infusion of selected CD133+ cells appears to promote an anti-inflammatory, anti-angiogenic profile at day 3 post-ligation and at 1 month improved reperfusion compared with non-selected MNC. Disclosures:No relevant conflicts of interest to declare.

The Neurexin I Alpha/Neurexophilin Axis as An Anti-Proliferative Factor In Human Cord Blood and Murine Bone Marrow Hematopoiesis.

AbstractAbstract 2627The membrane-bound receptors Neurexin I alpha (NRXN1α) and Dystroglycan (DAG1) serve as ligands for each other, and the Neurexophilins (NXPHs) are thought to function as natural antagonists for this interaction. This signaling axis centered on NRXN1α, DAG1 and the three highly homologous NXPHs, plays a well-defined role in neuronal development and synapse development. The failure of DAG1 knock-out mice to develop a mesoderm led us to hypothesize that this axis plays a role in hematopoiesis. Consistent with previous research, DAG1 is heavily (>95%) expressed in human (hu) umbilical cord blood (CB) CD34+ cells, including the primitive CD38- subpopulation. NRXN1α is expressed at similar levels in these populations as well. Hematopoietic cell populations are more precisely defined in the murine (mu) system, and adult mu bone marrow (BM) exhibits a similar trend to hu CB with NRXN1α expression being higher in the more primitive cells and decreasing as the cells differentiate. A high concentration of Neurexophilins is present in the hu CB plasma (900ng/ml), though it is absent in adult mu blood plasma and fetal bovine serum. Neither blocking DAG1 with a well-described blocking antibody, nor stimulation with recombinant NRXN1α has any observable effect on stimulation of hematopoietic progenitor cell (HPC) proliferation in vitro. This led us to investigate the other receptor present in the axis, NRXN1α. We first used alpha-latrotoxin, an excitatory toxin for NRXN1α. While no stimulatory or inhibitory proliferation effect was observed under normal growth conditions, alpha-latrotoxin did promote HPC survival under growth factor starved conditions as effectively as SDF1α/CXCL12 (a known HPC survival enhancing factor), suggesting a possible protective role for the DAG1-NRXN1α axis during stress. In contrast, we identified the Neurexophilins as anti-proliferative agents for immature hematopoietic cells. When injected in vivo, recombinant NXPH1 acts in a myelo- and lympho-suppressive manner. Absolute numbers of mu HPCs were suppressed in a dose- and time-dependent manner, with the maximal dosage decreasing HPC numbers by over 50%. The decrease in absolute colony number was mirrored by a decrease in the cycling population of HPCs in mice treated with NXPH1, as measured by the high specificity activated tritiated thymidine kill assay. Peripheral blood neutrophils, lymphocytes, monocytes and platelets, were also decreased in a time-dependant fashion after in vivo exposure to NXPH1. Intriguingly, the most primitive LTR-HSCs behave in the opposite manner, with a three-fold increase in cycling status induced via the Rapamycin sensitive mTOR pathway. During in vitro culture NXPH1 is only able to suppress colony forming ability of unseparated HPCs treated with both GM-CSF and SCF in combination in comparison to stimulation by other cytokines. This response is most likely due to increased NRXN1α expression in response to stimulation by GM-CSF and SCF. Given the high level of DAG1 expression on colony forming hematopoietic cells, we hypothesized that endogenous DAG1 may be competing with NXPH1 for NRXN1α binding. We thus exposed cells to a well studied DAG1 blocking antibody or plated CD34+ cells in single cell culture. The in vitro results were then able to recapitulate the results observed in vivo and a 50% decrease in colony forming ability is observed in combination with the following cytokines: GM-CSF, IL-3, GM-CSF+SCF, and IL-3+SCF as well as under maximally stimulatory conditions (IL-3, GM-CSF, SCF, and Epo for hu cells and SCF, PWMSCM, and Epo for mu cell). The ability of NXPH1 to inhibit proliferation of CD34+ cells in single cell culture suggests that NXPH1 acts directly on HPCs. The anti-proliferative function of the NXPHs are consistent with previous literature correlating NXPH expression in cancers with negative prognoses and these data may provide a mechanism for the immune system evasion of cancer. Additionally, the high concentration of NXPH in CB plasma may help explain the relatively long time to engraftment of transplanted CB cells. More broadly, these results demonstrate the presence and function of a tightly regulated signaling axis centered on NRXN1α in hematopoiesis. Disclosures:Broxmeyer:CordUse: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Intravenous infusion of GDNF gene-modified human umbilical cord blood CD34+ cells protects against cerebral ischemic injury in spontaneously hypertensive rats

This study assessed the potential of intravenous transplantation of human umbilical cord blood (HUCB) CD34+ cells transfected with glial cell line-derived neurotrophic factor (GDNF) gene to exert therapeutic benefits in spontaneous hypertensive rats (SHR) exposed to transient middle cerebral artery occlusion (MCAO). SHR with MCAO were randomly assigned to receive intravenously transplantation of vehicle, the plasmid containing the enhanced green fluorescent protein (pEGFP)-CD34+ cells or pEGFP-GDNF-CD34+ cells at 6 h after stroke. The CD34+ cells transfected with GDNF gene expressed higher levels of GDNF mRNA and protein than nontransfected HUCB CD34+ cells in vitro. At 28 days after transplantation of GDNF gene modified CD34+ cells, significantly more GFP positive cells, neurons, and astrocytes, likely derived from the grafted cells, populated the peri-infarct area compared to those injected with pEGFP-CD34+ cells or vehicle. Furthermore, the stroke animals transplanted with GDNF gene modified CD34+ cells showed a significant increase in GDNF level in the infarcted hemisphere, reduced brain infarction volume, and enhanced functional recovery compared with those that received pEGFP-CD34+ cells. This study supports the use of a combined gene and stem cell therapy for treating stroke.

Reversal of Hepatic Fibrosis by Human CD34(+) Stem/Progenitor Cell Transplantation in Rats.

Human umbilical cord blood (UCB) cells have many advantages as grafts for cell transplantation. Here, we transplant UCB cells into injured liver fibrosis, investigated the hepatic potential of UCB cells both in vitro and in vivo. a CCl4 rat model with liver fibrosis was prepared. Human (UCB) CD34(+) stem cell was separated with MACS (magnetic cell sorting). Cells were cultured with and without hepatic differentiation medium. Rats were divided into 3 groups; group (1): control healthy, group (2): CCl4 injected rats and group 3: CCl4/CD34(+)injected rats with human differentiated and undifferentiated cells through intrahepatic (IH) and intravenous (IV) routes. A significant elevation was detected in serum albumin in CCl4/CD34(+) compared to the CCl4 group (p<0.001). Serum ALT, had a significant decrease of its level after administration of stem cells compared to the CCl4 group (p<0.001). However, it was still significantly higher than control (p<0.001) with no significant difference between the groups that received stem cells. Histopathological examination of liver tissue showed that stem cells have a significant antifibrotic effect. Concerning gene expression, the collagen gene (rat) was highly expressed in the CCl4 group whereas its expression was significantly decreased after administration of stem cells. Human albumin and matrix metalloproteinase (MMP2) genes were expressed in liver tissues in the groups that received stem cells. Highest expression was in the group that received un-differentiated cells I.V. human UCB CD34(+) stem cells can ameliorate liver fibrosis in rats.

In vitro non-viral lipofectamine delivery of the gene for glial cell line-derived neurotrophic factor to human umbilical cord blood CD34+ cells

Using a lipofection technique, we explored a non-viral delivery of plasmid DNA encoding a rat pGDNF (glial cell line-derived neurotrophic factor) to CD34+ cells derived from human umbilical cord blood (HUCB) cells in order to obtain cells stably expressing the GDNF gene. The target gene GDNF was amplified from cortex cells of newborn Sprague–Dawley rats by reverse transcriptase polymerase chain reaction (RT-PCR) and inserted into vector pEGFP-N1 to construct the eukaryotic expression vector pEGFP/GDNF. The positive clones were identified by sequencing and endonuclease digestion. The expression of pEGFP/GDNF-transfected HUCB cells CD34+ was examined by ELISA. Single fragment of 640 bp was obtained after the rat GDNF cDNA was amplified by RT-PCR. Two fragments of about 4.3 kb and 640 pb were obtained after digestion of recombinant plasmid pEGFP/GDNF with XhoI/KpnI. The nucleic acid fragment of 640 bp was confirmed to agree well with the sequence of GDNF gene published by GenBank. The expression of GDNF mRNA and the level of GDNF from pEGFP/GDNF-transfected CD34+ cells were increased substantially, compared with pEGFP control plasmid transfected CD34+ cells ( P < 0.05). Moreover, co-culture of primary rat cells with the pEGFP/GDNF-transfected CD34+ cells promoted enhanced neuroprotection against oxygen-glucose deprivation induced cell dysfunctions. The present results support the use of the non-viral plasmid liposome for therapeutic gene expression for stem cell therapy.

Mesenchymal stem cells from bone marrow show a stronger stimulating effect on megakaryocyte progenitor expansion than those from non-hematopoietic tissues

In order to evaluate whether mesenchymal stem cells (MSCs) from non-hematopoietic tissues are able to regulate megakaryocytopoiesis, we identified human MSCs from adult bone marrow (ABM), fetal pancreas (FPan) and umbilical cord (UC), and their abilities to support megakaryocyte (MK) differentiation from CD34+ hematopoietic progenitor cells (HPCs) were comparatively studied. First, MSCs were isolated from ABM, FPan and UC then their growth kinetics, molecular characterization and mesodermal differentiation capacity were determined. ABM-MSCs, FPan-MSCs and UC-MSCs were irradiated and cocultured with human umbilical cord blood (UCB) CD34+ cells, and the expansion efficiency of MK progenitor cells and MK formation were analysed and compared. Finally, SCF, IL-6 and GM-CSF expression by the three types of MSCs were also examined. Our results showed that FPan-MSCs and UC-MSCs shared most of the characteristic of ABM-MSCs, including morphology, immunophenotype, adipogenic and osteogenic differentiation potentials. Compared with ABM-MSCs, fetal MSCs had higher proliferative capacity. After 7 days’ coculture, the maximal production of CD34+/CD41a+ cells was obtained in a group of CD34+ HPCs + ABM-MSCs. Furthermore, this group produced more MK colonies than other groups (p < 0.05). Surface antigen and ploidy analysis morphological observation demonstrated that a proportion of expanded cells in each group differentiated into mature MKs. ABM-MSCs, FPan-MSCs and UC-MSCs were revealed to express SCF, IL-6 and GM-CSF at mRNA level. We conclude that FPan-MSCs and UC-MSCs have the ability to promote megakaryocytopoiesis, while ABM-MSCs expand more MK progenitor cells from CD34+ HPCs than MSCs from non-hematopoietic tissues and CD34+ cells alone.

Constitutive Activation of Rac GTPases Is Induced by Cooperating Mutations in a Human Model of MLL-AF9 Leukemia.

Abstract 2959Poster Board II-935Retroviral transduction of primary human umbilical cord blood CD34+ cells with a gene encoding MLL-AF9 (MA9) efficiently promotes immortalization in vitro and development of leukemia in a xenotransplant model. MA9 cells remain cytokine dependent with a strict requirement for Flt3 signaling. Additionally, the cells are hypersensitive to inhibition of the RAC GTPases. In this current study, we have examined a potential link between these pathways. We found that MA9 cells proliferate in culture media containing Flt3 Ligand (FL) as the sole cytokine, indicating that Flt3 signaling is sufficient for growth and survival. Furthermore, introduction of a mutant Flt3-ITD gene in these cells (MA9-ITD cells) not only promotes cytokine independent growth, but also significantly decreases leukemia latency in xenotransplant experiments. Consistent with the persistent requirement for active Rac signaling, we found that MA9 cells stimulated with FL induce the activated form of Rac (Rac-GTP) while MA9-ITD cells contain constitutively high levels of Rac-GTP, even after serum starvation and in the absence of all cytokines. Interestingly, we found that expression of the activated mutant NRas-G12D in MA9 cells (MA9-NRas cells) also leads to constitutive Rac activation, cytokine independent growth, and decreased leukemia latency upon transplantation into immunodeficient mice. Taken together, these results indicate that RAC activation may be a common integrating pathway by which secondary mutations cooperate in the progression towards leukemia. Consistent with this notion, both MA9-ITD and MA9-NRas cells remain sensitive to Rac inhibition with a small molecule Rac inhibitor or shRNA knockdown of Rac1 or Rac2. These data indicate that Rac signaling remains critical for MA9 survival and proliferation even in the context of additional mutations and highlights the therapeutic potential for targeting Rac. Disclosures:No relevant conflicts of interest to declare.

Cyclooxygenase-2 Derived Prostaglandin E2 Is Required for Dendritic Cell Differentiation From Hematopoietic Progenitor Cells.

Abstract 1499Poster Board I-522Dendritic cells (DCs) are an attractive target for therapeutic manipulation of the immune system due to their potent antigen presentation capacity and ability to induce effective immune response. In steady-state conditions different DC subsets including myeloid DCs (CD11c+CD11b+B220neg) and plasmacytoid DCs (CD11c+CD11bnegB220+) are generated in bone-marrow (BM) from hematopoietic stem cells through a series of differentiation steps. We recently demonstrated that prostaglandin (PGE2), the predominant metabolite of arachidonic acid metabolism by cyclooxygenase (COX) enzymes, enhances the homing, survival and proliferation of hematopoietic stem cells (Hoggatt et. al., Blood 2009). In this study, we examined the requirement of prostaglandins in development of DCs from hematopoietic progenitor cells. In vivo treatment of mice for 4 days with the non-steroidal anti-inflammatory drug (NSAID) indomethacin (2.5 mg/kg/bid), a dual COX1/COX2 inhibitor, produced a 59.5±17.8 % (p<0.02) reduction in total bone-marrow DC number compared to control mice treated with vehicle alone. Interestingly, indomethacin selectively decreased marrow CD11c+CD11b+B220neg myeloid DCs without affecting CD11c+CD11bnegB220+ plasmacytoid DCs. To determine whether fewer DCs in the bone marrow of indomethacin treated mice was due to the impairment of DC differentiation from their hematopoietic progenitor cells, we stimulated differentiation of DCs from lineage depleted (CD5−,CD45R−, CD11b−, Anti-Gr-1−, 7-4− and Ter-119−) bone marrow cells using Flt-3 ligand for 9 days in vitro and treated cultures with indomethacin (1 microM), SC-560 (10 microM), a selective COX-1 inhibitor or NS-398 (10 microM), a selective COX-2 inhibitor. Indomethacin produced a 1.98±0.38 fold, (p<0.02) reduction and the COX-2 inhibitor NS-398 produced a 1.52±0.04 fold, (p<0.05) reduction in CD11c+CD11b+B220neg myeloid DC generation compared to control, while the COX-1 specific inhibitor SC-560 was without effect. As expected, Flt-3-ligand induced plasmacytoid DC (CD11c+CD11bnegB220+) differentiation was not affected by selective COX inhibitors. Indomethacin also impaired generation of CD11a+CD14neg Langerhans DC from human umbilical cord blood CD34+ cells. Measurement of PGE2 production in culture supernatants from DC-producing cultures demonstrated detectable PGE2 after 6 days of culture and DC generation from BM progenitors in these cultures was impaired when PGE2 synthesis was blocked on day 6 by indomethacin administration. Indomethacin treatment during the first 5 days of Flt3-ligand stimulated DC differentiation cultures did not decrease DC production. To identify mechanisms responsible for this impairment in Flt-3 ligand-induced DC generation from hematopoietic progenitor cells, we analyzed the effect of indomethacin on DC-committed precursor cell proliferation and survival. Survival of DC-committed precursors defined as CD11clow CD11bbrightMHCIIlow was reduced 35±2.5% (p<0.05) in indomethacin treated cultures compared to control. However, indomethacin did not affect DC precursor proliferation as measured by BrdU incorporation assay. To elucidate the signaling mechanisms by which indomethacin impaired the survival of DC precursors, we added selective receptor agonists to each of known PGE receptors, EP1-4, during Flt3-ligand induced DC differentiation. The EP1/EP3 agonist 17-phenyl trinor PGE2 rescued the DC precursors from indomethacin mediated death, whereas Butaprost, a specific EP2 agonist and L902688, a selective EP4 agonist, failed to rescue DC precursor death. DCs developed in the presence of prostaglandin inhibitors did not show any defect in LPS-induced activation and expressed CD40, CD80, CD86 and MHCII levels similar to control as measured by flow cytometry. In addition, DC developed in the absence of endogenous PGE2 production successfully induced T-cell activation as measured by mix lymphocyte reaction assay (MLR). In conclusion, COX-2 mediated prostaglandin production by DC-committed hematopoietic precursors confers resistance to cell death via signaling through EP1/EP3 receptors and promotes dendritic cell development. Disclosures:No relevant conflicts of interest to declare.

Interleukin-21 Induces the Differentiation of Human Umbilical Cord Blood CD34. −lineage− Cells Into Pseudomature Lytic NK Cells.

Abstract 1476Poster Board I-499 Background:Umbilical cord blood (UCB) is increasingly used as an alternative source of transplantable CD34+ haematopoietic stem cells (HSC) for neoplastic and non-neoplastic diseases. In addition to CD34-expressing HSC, human UCB contains a rare population of CD34−lineage− cells endowed with the ability to differentiate along the T/NK pathway in response to interleukin (IL)-15 and in the presence of a stromal cell support. IL-21 is a four-helix bundle cytokine released by activated CD4+ T cells and by NKT cells. IL-21 is a crucial regulator of NK cell function, whose influence on IL-15-induced differentiation of CD34−lineage− cells has not been investigated previously. The present study was designed and conducted to address whether IL-21 might replace the stromal cell requirements and foster the IL-15-induced NK differentiation of human UCB CD34−lineage− cells. Methods:CD34−lineage− cells were maintained in liquid culture with 10−6M hydrocortisone, 20 ng/ml Flt3-L and 20 ng/ml SCF, with the addition of 50 ng/ml IL-15 and 20 ng/ml IL-21, either alone or in combination. Cultures were established in the absence of feeder cells or serum supplementation. Cytokine-treated cells were used to evaluate the following parameters: a) cell surface phenotype; b) expression of molecular determinants of lymphoid/NK cell differentiation; c) secretion of IFN-γ, GM-CSF, TNF-α and CCL3/MIP-1α; d) cytolytic activity against NK-sensitive tumour cell targets and e) relative amount of Stat1 (Tyr701), Stat3 (Tyr705) and Stat5 (Tyr694) phosphorylation in response to IL-21. For all the above detailed experiments, control cultures were established with UCB-derived CD34+ HSC. Results:Freshly isolated CD34−lineage− cells stained negatively for stem cell-associated (CD34, CD133) and NK/lymphoid surface antigens (CD7, CD56, CD16, CD3, TCRαβ), and comprised 0.22% on average of UCB mononuclear cells (samples analyzed = 8). CD34−lineage− cells proliferated vigorously in response to IL-15 and IL-21 (average fold expansion at week +4 of culture = 42.5) but not to IL-21 alone, and up-regulated phosphorylated Stat1 and Stat3 proteins, in good agreement with previously published reports on the IL-21-induced activation of Stat signaling. CD34−lineage− cells expanded by IL-21 in combination with IL-15 acquired a peculiar lymphoid morphology with heavy cytoplasmic granules. When compared with CD34-derived NK cells, CD34−lineage− cells emerging from IL-15+IL-21-containing cultures expressed very low levels of CD16 and killer-cell immunoglobulin-like receptor (KIR), but high levels of CD56, NKG2D and IL-21 receptor, consistent with pseudo-mature NK cells. IL-21/IL-15-differentiated cells up-regulated mRNA signals for Bcl-2, GATA-3 and Id2, a master switch required for NK-cell development, and harboured un-rearranged TCRγ genes, suggesting that NK commitment under the experimental conditions here established occurs through a pathway that does not include TCR rearrangement. From a functional standpoint, IL-21/IL-15-treated cells secreted copious amounts of IFN-γ, GM-CSF and CCL3/MIP-1α, and expressed cell surface CD107a upon contact with NK-sensitive tumour targets, a measure of exocytosis of NK secretory granules. Specifically, an average 65±11% of CD56+ NK cells differentiated with IL-15+IL-21 stained positively for CD107a in co-cultures established with NK-sensitive K562 cells. NK cell degranulation occurred at significantly lower levels in co-cultures containing K562 cells and IL-15-differentiated CD34−lineage− cells (mean percentage of CD107a+CD56+ NK cells equal to 35±6 at E:T ratio = 1; p < 0.01 compared with cultures containing IL-15+IL-21-matured NK cells), suggesting that IL-15 and IL-21 exerted synergistic effects on NK activity. Finally, NK cells differentiated from CD34+ HSC with either IL-15 alone or IL-15+IL-21 manifested a similar cytotoxic activity to that of cytokine-differentiated CD34−lineage− cells. Conclusions:This study suggests that considerable numbers of highly pure, lytic CD56+CD16−/+ NK cells for adoptive immunotherapy can be obtained from UCB CD34−lineage− cells using a serum-free, feeder cell-free culture system. The findings highlighted herein also shed some light into the developmental intermediates of NK cells that can be differentiated after the exposure of CD34−lineage− cells to IL-21. Disclosures:No relevant conflicts of interest to declare.

A Modified Foamy Viral Envelope Enhances Gene Transfer Efficiency and Reduces Toxicity of Lentiviral FANCA Vectors in Fanca-/- HSCs.

Abstract 696Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow failure, genomic instability, and a predisposition to malignancies. Stem cell gene transfer technology is a potentially promising therapy, however, we have previously shown that prolonged ex vivo culture of cells using gamma retroviruses, results in a high incidence of apoptosis and predisposes the surviving reinfused cells to hematological malignancy in a murine model of FA. Here, we developed a lentiviral vector encoding the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSV-G or a modified foamy virus (FV) envelope to correct murine Fanca-/- stem and progenitor cells. An overnight transduction protocol was utilized to minimize genotoxic stress due to extended ex vivo manipulations. Transduction and expression of hFANCA was confirmed by three classical functional and biochemical measures in vitro: improved survival of clonogenic progenitors in the presence of mitomycin C (MMC), correction of MMC-induced G2/M arrest, and by the restoration of Fancd2 mono-ubiquitination. Furthermore, in vivo competitive repopulation experiments demonstrated that the repopulating ability of Fanca-/- stem cells transduced with the lentivirus encoding hFANCA was equivalent to that of wild-type stem cells, and the genetically-corrected reconstituting Fanca-/- cells were resistant to MMC and TNF-αa. Importantly, while a significant toxicity was observed using the VSV-G envelope, the toxicity of the FV envelope to murine c-kit+ cells was limited.In parallel experiments, human umbilical cord blood CD34+ cord blood cells were transduced with either a VSV-G- or FV envelope-pseudotyped lentivirus encoding the EGFP reporter gene. Transplantation of 4×105 cells into NOD/SCID/gamma-chainnull yielded a peripheral blood human chimerism comparable to the untransduced control cells (∼30%) regardless of the envelope. However, a much higher gene transfer efficiency of CD34+ cells was observed prior to transplantation when the FV envelope was employed (∼60%), as compared to the VSV-G envelope (∼15%). Furthermore, a similar 4 fold increase in transduction efficiency was observed in peripheral blood and in progenitors isolated from the bone marrow of both primary and secondary long term reconstituted mice. Collectively, these data indicate that the lentiviral construct pseudotyped with FV envelope can efficiently correct murine FA HSC/progenitor cells in a short transduction protocol and that the modified foamy envelope offers significantly greater transduction efficiency at comparable titers in long term reconstituting human cells in a xenograft model. This envelope may offer significant advantages compared to VSV-G in moving forward to phase 1 clinical trials. Disclosures:No relevant conflicts of interest to declare.

Lentiviral Short Hairpin RNA Silencing of DNM3Decreases Megakaryoycte Progenitor Proliferation and Inhibits the Formation of the Demarcation Membrane System.

Abstract 4594Dynamin 3 (DNM3) is a mechanochemical enzyme, which participates in membrane dynamics by hydrolyzing nucleotides to link cell membranes to the actin cytoskeleton. Recently, we identified DNM3 to be differentially up-regulated during megakaryocyte (MK) development and reported that DNM3 participates in the proliferation of MK progenitors. We report here that dynamin 3 not only participates in MK progenitor amplification, but is also involved in the formation of the demarcation membrane system (DMS). To explore the role of dynamin 3 during MK development, we used three different lentiviral DNM3 shRNA constructs to infect human umbilical cord blood (UCB) CD34+ cells that were then cultured to differentiate along the MK lineage. Relative to controls, CD34+ cells transfected with DNM3 shRNAs significantly decreased the total number of cells produced in culture as well as the number of colony forming unit-megakaryocytes (CFU-MKs). As a result, the total number of CD41+ and CD61+ progeny cells was also reduced. To investigate the role dynamin 3 plays during the maturation of MKs, a Src family kinase inhibitor, SU6656, was used to induce high polyploidization and the development of a complex DMS in the UT-7/mpl cell line. The styryl membrane dye di-8-ANEPPS was used to monitor the formation of the DMS. In the absence of SU6656 treatment, UT-7/mpl cells showed a peripheral staining pattern that was indicative of cells that did not have extensive plasma membrane invaginations. In contrast, the staining pattern of UT-7/mpl cells treated with SU6656 clearly showed a well-developed plasma membrane system with invaginations. When DNM3 was silenced in SU6656 treated UT-7/mpl cells the results showed a significant reduction in di-8-ANEPPS staining relative to empty vector and non-specific negative controls. These findings are consistent with the conclusion that dynamin 3 participates in the morphogenesis of the DMS. Disclosures:No relevant conflicts of interest to declare.

CD133+Endothelial Progenitor Cells as a Potential Cell Source for a Bioartificial Glomerulus

Development of a bioartificial glomerulus, a hemofilter in which the inner surface of hollow fibers is endothelialized, requires expandable, nonimmunogenic, antithrombogenic, and highly permeable endothelial cells. We used human umbilical cord blood CD133(+) endothelial progenitor cells (EPCs) to evaluate the feasibility of application of EPCs for bioartificial glomerulus. Numbers of adhered CD133(+) EPCs (adhered EPCs) was approximately 25 to 30 times as great in the expansion culture group as in the non-expansion group. Adhered EPCs had endothelial cell features, including the expression of CD31, Kinase domain region, von Willebrand factor, vascular endothelial-cadherin, positive for Ulex europeus agglutinin I staining, and up-take of acetylated low-density lipoprotein. Adhered EPCs secreted 6-keto-prostaglandin F(1alpha) identically to that secreted by human umbilical vein endothelial cells (HUVECs). The cells also expressed messenger RNA for phospholipase A(2), cyclooxygenase (COX)-1, COX-2, prostaglandin I(2) synthase, tissue plasminogen activator, and thrombomodulin (TM). TM protein in adhered EPCs properly activated protein C. Scanning electron microscopy revealed the suppression of platelet adhesion and aggregation on the surface of cell monolayer. Adhered EPCs treated with 50 microg/mL of cytochalasin B induced a larger diameter and a greater number of fenestrae, subsequently producing significantly more ultrafiltration than the non-treated cell. These results suggest that CD133(+) EPCs would potentially be applicable in bioartificial glomerulus.