搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here,we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the beta-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD,with 24.5 ± 7.6{\%} efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells,with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9{\%}. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16-19 weeks post-transplantation. We show that,by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification,the off-target effects including chromosomal rearrangements were significantly reduced. Taken together,our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs,a significant reduction in sickling of red blood cells,engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice. View Publication

Microfluidic systems create significant opportunities to establish highly controlled microenvironmental conditions for screening pluripotent stem cell fate. However,since cell fate is crucially dependent on this microenvironment,it remains unclear as to whether continual perfusion of culture medium supports pluripotent stem cell maintenance in feeder-free,chemically defined conditions,and further,whether optimum perfusion conditions exist for subsequent use of human embryonic stem cell (hESCs) in other microfludic systems. To investigate this,we designed microbioreactors based on resistive flow to screen hESCs under a linear range of flowrates. We report that at low rates (conditions where glucose transport is convection-limited with Péclet number textless1),cells are affected by apparent nutrient depletion and waste accumulation,evidenced by reduced cell expansion and altered morphology. At higher rates,cells are spontaneously washed out,and display morphological changes which may be indicative of early-stage differentiation. However,between these thresholds exists a narrow range of flowrates in which hESCs expand comparably to the equivalent static culture system,with regular morphology and maintenance of the pluripotency marker TG30 in textgreater95% of cells over 7 days. For MEL1 hESCs the optimum flowrate also coincided with the time-averaged medium exchange rate in static cultures,which may therefore provide a good first estimate of appropriate perfusion rates. Overall,we demonstrate hESCs can be maintained in microbioreactors under continual flow for up to 7 days,a critical outcome for the future development of microbioreactor-based screening systems and assays for hESC culture. View Publication

Regulatory T cell (Treg) therapy has the potential to induce transplantation tolerance so that immunosuppression and associated morbidity can be minimized. Alloantigen-reactive Tregs (arTregs) are more effective at preventing graft rejection than polyclonally expanded Tregs (PolyTregs) in murine models. We have developed a manufacturing process to expand human arTregs in short-term cultures using good manufacturing practice-compliant reagents. This process uses CD40L-activated allogeneic B cells to selectively expand arTregs followed by polyclonal restimulation to increase yield. Tregs expanded 100- to 1600-fold were highly alloantigen reactive and expressed the phenotype of stable Tregs. The alloantigen-expanded Tregs had a diverse TCR repertoire. They were more potent than PolyTregs in vitro and more effective at controlling allograft injuries in vivo in a humanized mouse model. View Publication

Pluripotent human embryonic stem cells (hESCs) are a potential renewable cell source for regenerative medicine and drug testing. To obtain adequate cell numbers for these applications,there is a need to develop scalable cell culture platforms to propagate hESCs. In this study,we encapsulated hESCs in calcium alginate microfibers as single cells,for expansion and differentiation under chemically defined conditions. hESCs were suspended in 1% (w/v) alginate solution at high cell density (textgreater10(7) cells/mL) and extruded at 5 m/min into a low calcium concentration bath (10 mM) for gelation. Mild citrate buffer (2.5 mM),which did not affect hESCs viability,was used to release the cells from the calcium alginate hydrogel. Encapsulation as single cells was critical,as this allowed the hESCs to grow in the form of relatively small and uniform aggregates. This alginate microfiber system allowed for expansion of an hESC line,HUES7,for up to five passages while maintaining pluripotency. Immunohistochemistry,polymerase chain reaction,and other analyses showed that passage 5 (P5) HUES7 cells expressed proteins and genes characteristic of pluripotent stem cells,possessed normal karyotype,and were able to form representative tissues of the three embryonic germ layers in vitro and in vivo. Encapsulated HUES7 cells at P5 could also be induced to directly differentiate into liver-like cells. Collectively,our experiments show that the alginate microfiber system can be used as a three-dimensional cell culture platform for long-term expansion and differentiation of hESCs under defined conditions. View Publication

The atypical cannabinoid Abn-CBD improves the inflammatory status in preclinical models of several pathologies,including autoimmune diseases. However,its potential for modulating inflammation in autoimmune type 1 diabetes (T1D) is unknown. Herein we investigate whether Abn-CBD can modulate the inflammatory response during T1D onset using a mouse model of T1D (non-obese diabetic- (NOD)-mice) and of beta cell damage (streptozotocin (STZ)-injected mice). Six-week-old female NOD mice were treated with Abn-CBD (0.1-1 mg/kg) or vehicle during 12 weeks and then euthanized. Eight-to-ten-week-old male C57Bl6/J mice were pre-treated with Abn-CBD (1 mg/kg of body weight) or vehicle for 1 week,following STZ challenge,and euthanized 1 week later. Blood,pancreas,pancreatic lymph nodes (PLNs) and T cells were collected and processed for analysis. Glycemia was also monitored. In NOD mice,treatment with Abn-CBD significantly reduced the severity of insulitis and reduced the pro-inflammatory profile of CD4+ T cells compared to vehicle. Concomitantly,Abn-CBD significantly reduced islet cell apoptosis and improved glucose tolerance. In STZ-injected mice,Abn-CBD decreased circulating proinflammatory cytokines and ameliorated islet inflammation reducing intra-islet phospho-NF-$\kappa$B and TXNIP. Abn-CBD significantly reduced 2 folds intra-islet CD8+ T cells and reduced Th1/non-Th1 ratio in PLNs of STZ-injected mice. Islet cell apoptosis and intra-islet fibrosis were also significantly reduced in Abn-CBD pre-treated mice compared to vehicle. Altogether,Abn-CBD reduces circulating and intra-islet inflammation,preserving islets,thus delaying the progression of insulitis. Hence,Abn-CBD and related compounds emerge as new candidates to develop pharmacological strategies to treat the early stages of T1D. View Publication

Activation of ErbB4 receptor signaling is instrumental in heart development,lack of which results in embryonic lethality. However,mechanism governing its intracellular signaling remains elusive. Using human pluripotent stem cells,we show that ErbB4 is critical for cardiogenesis whereby its genetic knockdown results in loss of cardiomyocytes. Phospho-proteome profiling and Western blot studies attribute this loss to inactivation of p38$\$ isoform which physically interacts with NKx2.5 and GATA4 transcription factors. Post-cardiomyocyte formation p38$\$/NKx2.5 downregulation is followed by p38$\$/MEF2c upregulation suggesting stage-specific developmental roles of p38 MAPK isoforms. Knockdown of p38$\$ similarly disrupts cardiomyocyte formation in spite of the presence of NKx2.5. Cell fractionation and NKx2.5 phosphorylation studies suggest inhibition of ErbB4-p38$\$ hinders NKx2.5 nuclear translocation during early cardiogenesis. This study reveals a novel pathway that directly links ErbB4 and p38$\$ the transcriptional machinery of NKx2.5-GATA4 complex which is critical for cardiomyocyte formation during mammalian heart development. View Publication

Lupus glomerulonephritis (GN) is an autoimmune disease characterized by immune complex-deposition,complement activation and glomerular inflammation. In lupus-prone NZM2328 mice,the occurrence of lupus GN was accompanied by a decrease in Treg cells and an increase in proinflammatory cytokine-producing T cells. Because IL-33 in addition to IL-2 has been shown to be important for Treg cell proliferation and ST2 (IL-33 receptor) positive Treg cells are more potent in suppressor activity,a hybrid cytokine with active domains of IL-2 and IL-33 was generated to target the ST2+ Treg cells as a therapeutic agent to treat lupus GN. Three mouse models were used: spontaneous and Ad-IFNalpha- accelerated lupus GN in NZM2328 and the lymphoproliferative autoimmune GN in MRL/lpr mice. Daily injections of IL233 for 5 days prevented Ad-IFNalpha-induced lupus GN and induced remission of spontaneous lupus GN. The remission was permanent in that no relapses were detected. The remission was accompanied by persistent elevation of Treg cells in the renal lymph nodes. IL233 is more potent than IL-2 and IL-33 either singly or in combination in the treatment of lupus GN. The results of this study support the thesis that IL233 should be considered as a novel agent for treating lupus GN. View Publication

BACKGROUND: ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors,whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS: Frozen UCB units were thawed,washed,immunomagnetically depleted of cells expressing glycophorin A and CD14,reacted for flow cytometric detection of ALDH,and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS: ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H,10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION: ALDH(br) populations recovered from thawed,banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid,myeloid and megakaryocytic blood elements. View Publication

The therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biologic properties. Although HSCs can be transduced by HIV-derived lentiviral vectors (LVs) in short ex vivo culture,they display low permissivity to the vector,requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation,we show that early-acting cytokines synergistically enhanced human HSC gene transfer by LVs without impairing engraftment and repopulation capacity. Using S-phase suicide assays,we show that transduction enhancement by cytokines was not dependent on cell cycle progression and that LVs can transduce quiescent HSCs. Pharmacologic inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer,allowing the reach of very high levels of vector integration in their progeny in vivo. Thus,LVs are effectively restricted at a postentry step by the activity of this proteolytic complex. Unexpectedly,cytokine stimulation rapidly and substantially down-regulated proteasome activity in hematopoietic progenitors,highlighting one mechanism by which cytokines may enhance permissiveness to LV gene transfer. These findings demonstrate that antiviral responses ultimately mediated by proteasomes strongly limit the efficiency of HSC transduction by LVs and establish improved conditions for HSC-based gene therapy. View Publication

The Fas receptor and its ligand have been implicated in mediating the bone marrow (BM) suppression observed in graft-versus-host disease and a number of other BM-failure syndromes. However,previous studies have suggested that Fas is probably not expressed on human hematopoietic stem cells (HSCs),but up-regulated as a consequence of their commitment and differentiation,suggesting that progenitors or differentiated blood cells,rather than HSCs,are the targets of Fas-mediated suppression. The present studies confirm that candidate HSCs in human cord blood and BM lack constitutive expression of Fas,but demonstrate that Fas expression on CD34+ progenitor and stem cells is correlated to their cell cycle and activation status. With the use of recently developed in vitro conditions promoting HSC self-renewing divisions,Fas was up-regulated on virtually all HSCs capable of multilineage reconstituting nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice in vivo,as well as on long-term culture-initiating cells (LTC-ICs). Similarly,in vivo cycling of NOD-SCID repopulating cells upon transplantation,resulted in up-regulation of Fas expression. However,repopulating HSCs expressing high levels of Fas remained highly resistant to Fas-mediated suppression,and HSC function was compromised only upon coactivation with tumor necrosis factor. Thus,reconstituting human HSCs up-regulate Fas expression upon active cycling,demonstrating that HSCs could be targets for Fas-mediated BM suppression. View Publication

Recent reports have indicated that human immunodeficiency virus (HIV) and murine leukemia virus (MLV) vectors preferentially integrate into active genes. Here,we used a novel approach based on genetic trapping to rapidly score several thousand integration sites and found that MLV vectors trapped cellular promoters more efficiently than HIV vectors. Remarkably,1 in 5 MLV integrations trapped an active promoter in different cell lines and primary hematopoietic cells. Such frequency was even higher in growth-stimulated lymphocytes. We show that the different behavior of MLV and HIV vectors was dependent on a different integration pattern within transcribed genes. Whereas MLV-based traps showed a strong bias for promoter-proximal integration leading to efficient reporter expression,HIV-based traps integrated throughout transcriptional units and were limited for expression by the distance from the promoter and the reading frame of the targeted gene. Our results indicate a strong propensity of MLV to establish transcriptional interactions with cellular promoters,a behavior that may have evolved to enhance proviral expression and may increase the insertional mutagenesis risk. Promoter trapping efficiency provides a convenient readout to assess transcriptional interactions between the vector and its flanking genes at the integration site and to compare integration site selection among different cell types and in different growth conditions. View Publication

The cellular reservoir for Kaposi sarcoma-associated herpesvirus (KSHV) infection in the hematopoietic compartment and mechanisms governing latent infection and reactivation remain undefined. To determine susceptibility of human CD34+ hematopoietic progenitor cells (HPCs) to infection with KSHV,purified HPCs were exposed to KSHV,and cells were differentiated in vitro and in vivo. Clonogenic colony-forming activity was significantly suppressed in KSHV-infected CD34+ cells,and viral DNA was predominantly localized to granulocyte-macrophage colonies differentiated in vitro. rKSHV.219 is a recombinant KSHV construct that expresses green fluorescent protein from a cellular promoter active during latency and red fluorescent protein from a viral lytic promoter. Infection of CD34+ HPCs with rKSHV.219 showed similar patterns of infection,persistence,and hematopoietic suppression in vitro in comparison with KSHV. rKSHV.219 infection was detected in human CD14+ and CD19+ cells recovered from NOD/SCID mouse bone marrow and spleen following reconstitution with rKSHV.219-infected CD34+ HPCs. These results suggest that rKSHV.219 establishes persistent infection in NOD/SCID mice and that virus may be disseminated following differentiation of infected HPCs into the B-cell and monocyte lineages. CD34+ HPCs may be a reservoir for KSHV infection and may provide a continuous source of virally infected cells in vivo. View Publication