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ReferencePhetfong J et al. (JUL 2016) Cell and Tissue Research 365 1 101--112
Cell type of origin influences iPSC generation and differentiation to cells of the hematoendothelial lineage
The use of induced pluripotent stem cells (iPSCs) as a source of cells for cell-based therapy in regenerative medicine is hampered by the limited efficiency and safety of the reprogramming procedure and the low efficiency of iPSC differentiation to specialized cell types. Evidence suggests that iPSCs retain an epigenetic memory of their parental cells with a possible influence on their differentiation capacity in vitro. We reprogramme three cell types, namely human umbilical cord vein endothelial cells (HUVECs), endothelial progenitor cells (EPCs) and human dermal fibroblasts (HDFs), to iPSCs and compare their hematoendothelial differentiation capacity. HUVECs and EPCs were at least two-fold more efficient in iPSC reprogramming than HDFs. Both HUVEC- and EPC-derived iPSCs exhibited high potentiality toward endothelial cell differentiation compared with HDF-derived iPSCs. However, only HUVEC-derived iPSCs showed efficient differentiation to hematopoietic stem/progenitor cells. Examination of DNA methylation at promoters of hematopoietic and endothelial genes revealed evidence for the existence of epigenetic memory at the endothelial genes but not the hematopoietic genes in iPSCs derived from HUVECs and EPCs indicating that epigenetic memory involves an endothelial differentiation bias. Our findings suggest that endothelial cells and EPCs are better sources for iPSC derivation regarding their reprogramming efficiency and that the somatic cell type used for iPSC generation toward specific cell lineage differentiation is of importance. View PublicationCatalog #:Product Name:05850mTeSR™107923Dispase (1 U/mL)04435MethoCult™ H4435 Enriched85850mTeSR™118551EasySep™ Human PE Positive Selection Kit -
ReferenceKarpinski J et al. (APR 2016) Nature Biotechnology 34 4 401--9
Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity.
Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells, we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently, precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo, including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy. View PublicationCatalog #:Product Name:02697StemSpan™ CC11017896EasySep™ Human Cord Blood CD34 Positive Selection Kit II17952EasySep™ Human CD4+ T Cell Isolation Kit21000RoboSep™-S04435MethoCult™ H4435 Enriched -
ReferencePhondeechareon T et al. (OCT 2016) Annals of hematology 95 10 1617--1625
Generation of induced pluripotent stem cells as a potential source of hematopoietic stem cells for transplant in PNH patients.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by lack of CD55 and CD59 on blood cell membrane leading to increased sensitivity of blood cells to complement. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for PNH, however, lack of HLA-matched donors and post-transplant complications are major concerns. Induced pluripotent stem cells (iPSCs) derived from patients are an attractive source for generating autologous HSCs to avoid adverse effects resulting from allogeneic HSCT. The disease involves only HSCs and their progeny; therefore, other tissues are not affected by the mutation and may be used to produce disease-free autologous HSCs. This study aimed to derive PNH patient-specific iPSCs from human dermal fibroblasts (HDFs), characterize and differentiate to hematopoietic cells using a feeder-free protocol. Analysis of CD55 and CD59 expression was performed before and after reprogramming, and hematopoietic differentiation. Patients' dermal fibroblasts expressed CD55 and CD59 at normal levels and the normal expression remained after reprogramming. The iPSCs derived from PNH patients had typical pluripotent properties and differentiation capacities with normal karyotype. After hematopoietic differentiation, the differentiated cells expressed early hematopoietic markers (CD34 and CD43) with normal CD59 expression. The iPSCs derived from HDFs of PNH patients have normal levels of CD55 and CD59 expression and hold promise as a potential source of HSCs for autologous transplantation to cure PNH patients. View PublicationCatalog #:Product Name:05850mTeSR™107923Dispase (1 U/mL)07920ACCUTASE™04435MethoCult™ H4435 Enriched85850mTeSR™1 -
ReferenceGribaldo L et al. (NOV 1999) Experimental hematology 27 11 1593--8
Comparison of in vitro drug-sensitivity of human granulocyte-macrophage progenitors from two different origins: umbilical cord blood and bone marrow.
Predictive in vitro hematotoxicity assays using human cells will provide estimation of tolerable level and aid considerably the development of agents with greater therapeutic activity and less toxicity. Human hematopoietic cells can be derived from three sources: human bone marrow by sternal or femoral aspiration, mobilized peripheral blood, or umbilical cord blood samples collected from placentas after deliveries. Because of the difficulties to have a continuous supply of bone marrow cells from normal human donors and the related ethical problems, we performed a study to compare the sensitivity of human bone marrow cells (h-BMC) and human cord blood cells (h-CBC) to chemicals in order to confirm if h-CBC can readily replace bone marrow cells in checking the sensitivity of GM-CFU progenitors to drugs as preliminarily reported in literature. Our results showed that the prediction of IC50 values in human model is quite similar by using h-BMC or h-CBC. On the contrary, the type of medium influenced in a significant way the ICs determination of some drugs. View PublicationCatalog #:Product Name:04436MethoCult™ SF H443604064Starter Kit for MethoCult™ H4034 Optimum04100MethoCult™ H410004230MethoCult™ H423004236MethoCult™ SF H423604431MethoCult™ H443104434MethoCult™ H4434 Classic04464Starter Kit for MethoCult™ H4434 Classic04531MethoCult™ H453104535MethoCult™ H4535 Enriched Without EPO04536MethoCult™ SF H453604564Starter Kit for MethoCult™ H4534 Classic Without EPO04035MethoCult™ H4035 Optimum Without EPO04330MethoCult™ H433004034MethoCult™ H4034 Optimum04435MethoCult™ H4435 Enriched04534MethoCult™ H4534 Classic Without EPO -
ReferenceDonahue RE et al. (JAN 2000) Blood 95 2 445--52
High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34(+) cells.
We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells, up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking, however, decreased to 0.1% or lower within 2 weeks. In contrast, EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks, the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis, and EGFP expression was observed in CD4(+), CD8(+), CD20(+), and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452) View PublicationCatalog #:Product Name:04436MethoCult™ SF H443604064Starter Kit for MethoCult™ H4034 Optimum04100MethoCult™ H410004230MethoCult™ H423004236MethoCult™ SF H423604431MethoCult™ H443104434MethoCult™ H4434 Classic04464Starter Kit for MethoCult™ H4434 Classic04531MethoCult™ H453104535MethoCult™ H4535 Enriched Without EPO04536MethoCult™ SF H453604564Starter Kit for MethoCult™ H4534 Classic Without EPO04035MethoCult™ H4035 Optimum Without EPO04330MethoCult™ H433004034MethoCult™ H4034 Optimum04435MethoCult™ H4435 Enriched04534MethoCult™ H4534 Classic Without EPO -
ReferenceMayani H et al. (JUN 1993) Blood 81 12 3252--8
Cytokine-induced selective expansion and maturation of erythroid versus myeloid progenitors from purified cord blood precursor cells.
To study the role of different cytokine combinations on the proliferation and differentiation of highly purified primitive progenitor cells, a serum-free liquid culture system was used in combination with phenotypic and functional analysis of the cells produced in culture. CD34+ CD45RAlo CD71lo cells, purified from umbilical cord blood by flow cytometry and cell sorting, were selected for this study because of their high content of clonogenic cells (34%), particularly multipotent progenitors (CFU-MIX, 12% of all cells). Four cytokine combinations were tested: (1) mast cell growth factor (MGF; a c-kit ligand) and interleukin-6 (IL-6); (2) MGF, IL-6, IL-3, and erythropoietin (Epo); (3) MGF, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 fusion protein (FP), macrophage colony-stimulating factor (M-CSF), and granulocyte-CSF (G-CSF); and (4) MGF, IL-6, FP, M-CSF, G-CSF, and Epo. Maximum numbers of erythroid progenitors (BFU-E, up to 55-fold increase) and mature erythroid cells were observed in the presence of MGF, IL-6, IL-3, and Epo, whereas maximum levels of myeloid progenitors (CFU-C, up to 70-fold increase) and mature myeloid cells were found in cultures supplemented with MGF, IL-6, FP, M-CSF, and G-CSF. When MGF, IL-6, FP, M-CSF, G-CSF, and Epo were present, maximum levels of both erythroid and myeloid progenitors and their progeny were observed. These results indicate that specific cytokine combinations can act directly on primitive hematopoietic cells resulting in significant expansion of progenitor cell numbers and influencing their overall patterns of proliferation and differentiation. Furthermore, the observations presented in this study suggest that the cytokine combinations used were unable to bias lineage commitment of multipotent progenitors, but rather had a permissive effect on the development of lineage-restricted clonogenic cells. View PublicationCatalog #:Product Name:04436MethoCult™ SF H443604064Starter Kit for MethoCult™ H4034 Optimum04100MethoCult™ H410004230MethoCult™ H423004236MethoCult™ SF H423604431MethoCult™ H443104434MethoCult™ H4434 Classic04464Starter Kit for MethoCult™ H4434 Classic04531MethoCult™ H453104535MethoCult™ H4535 Enriched Without EPO04536MethoCult™ SF H453604564Starter Kit for MethoCult™ H4534 Classic Without EPO04035MethoCult™ H4035 Optimum Without EPO04330MethoCult™ H433004034MethoCult™ H4034 Optimum04435MethoCult™ H4435 Enriched04534MethoCult™ H4534 Classic Without EPO -
ReferenceConneally E et al. (JAN 1996) Blood 87 2 456--64
Rapid and efficient selection of human hematopoietic cells expressing murine heat-stable antigen as an indicator of retroviral-mediated gene transfer.
Recombinant retroviruses offer many advantages for the genetic modification of human hematopoietic cells, although their use in clinical protocols has thus far given disappointing results. There is therefore an important need to develop new strategies that will allow effectively transduced primitive hematopoietic target populations to be both rapidly characterized and isolated free of residual nontransduced but biologically equivalent cells. To address this need, we constructed a murine stem cell virus (MSCV)-based retroviral vector containing the 228-bp coding sequence of the murine heat-stable antigen (HSA) and generated helper virus-free amphotropic MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells. Light density and, in some cases, lineage marker-negative (lin-) normal human marrow or mobilized peripheral blood cells preactivated by exposure to interleukin-3 (IL-3), IL-6, and Steel factor in vitro for 48 hours were then infected by cocultivation with these MSCV-HSA producer cells for a further 48 hours in the presence of the same cytokines. Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours later showed 21% to 41% (mean, 27%) of those that were still CD34+ to have acquired the ability to express HSA. The extent of gene transfer to erythroid and granulopoietic progenitors (burst-forming unit-erythroid and colony-forming unit-granulocyte-macrophage), as assessed by the ability of these cells to form colonies of mature progeny in the presence of normally toxic concentrations of G418, averaged 11% and 12%, respectively, in 6 experiments. These values could be increased to 100% and 77%, respectively, by prior isolation of the CD34+HSA+ cell fraction and were correspondingly decreased to an average of 2% and 5%, respectively, in the CD34+HSA- cells. In addition, the extent of gene transfer to long-term culture-initiating cells (LTC-IC) was assessed by G418 resistance. The average gene transfer to LTC-IC-derived colony-forming cells in the unsorted population was textless or = 7% in 4 experiments. FACS selection of the initially CD34+HSA+ cells increased this value to 86% and decreased it to 3% for the LTC-IC plated from the CD34+HSA- cells. Transfer of HSA gene expression to a phenotypically defined more primitive subpopulation of CD34+ cells, ie, those expressing little or no CD38, could also be shown by FACS analysis of infected populations 24 hours after infection. These findings underscore the potential use of retroviral vectors encoding HSA for the specific identification and non-toxic selection immediately after infection of retrovirally transduced populations of primitive human hematopoietic cells. In addition, such vectors should facilitate the subsequent tracking of their marked progeny using multiparameter flow cytometry. View PublicationCatalog #:Product Name:04436MethoCult™ SF H443604064Starter Kit for MethoCult™ H4034 Optimum04100MethoCult™ H410004230MethoCult™ H423004236MethoCult™ SF H423604431MethoCult™ H443104434MethoCult™ H4434 Classic04464Starter Kit for MethoCult™ H4434 Classic04531MethoCult™ H453104535MethoCult™ H4535 Enriched Without EPO04536MethoCult™ SF H453604564Starter Kit for MethoCult™ H4534 Classic Without EPO04035MethoCult™ H4035 Optimum Without EPO04330MethoCult™ H433004034MethoCult™ H4034 Optimum04435MethoCult™ H4435 Enriched04534MethoCult™ H4534 Classic Without EPO04437MethoCult™ Express -
ReferenceFarese AM et al. (JAN 1996) Blood 87 2 581--91
Acceleration of hematopoietic reconstitution with a synthetic cytokine (SC-55494) after radiation-induced bone marrow aplasia.
The synthetic cytokine (Synthokine) SC-55494 is a high-affinity interleukin-3 (IL-3) receptor ligand that stimulates greater in vitro multilineage hematopoietic activity than native IL-3, while inducing no significant increase in inflammatory activity relative to native IL-3. The aim of this study was to investigate the in vivo hematopoietic response of rhesus monkeys receiving Synthokine after radiation-induced marrow aplasia. Administration schedule and dose of Synthokine were evaluated. All animals were total-body irradiated (TBI) with 700 cGy 60Co gamma radiation on day 0. Beginning on day 1, cohorts of animals (n = 5) received Synthokine subcutaneously (SC) twice daily with 25 micrograms/kg/d or 100 micrograms/kg/d for 23 days or 100 micrograms/kg/d for 14 days. Control animals (n = 9) received human serum albumin SC once daily at 15 micrograms/kg/d for 23 days. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (NEUT; absolute neutrophil count [ANC] textless 500/microL) and thrombocytopenia (THROM; platelet count textless 20,000/microL) were assessed. Synthokine significantly (P textless .05) reduced the duration of THROM versus the HSA-treated animals regardless of dose or protocol length. The most striking reduction was obtained in the animals receiving 100 micrograms/kg/d for 23 days (THROM = 3.5 v 12.5 days in HSA control animals). Although the duration of NEUT was not significantly altered, the depth of the nadir was significantly lessened in all animal cohorts treated with Synthokine regardless of dose versus schedule length. Bone marrow progenitor cell cultures indicated a beneficial effect of Synthokine on the recovery of granulocyte-macrophage colony-forming units that was significantly higher at day 24 post-TBI in both cohorts treated at 25 and 100 micrograms/kg/d for 23 days relative to the control animals. Plasma pharmacokinetic parameters were evaluated in both normal and irradiated animals. Pharmacokinetic analysis performed in irradiated animals after 1 week of treatment suggests an effect of repetitive Synthokine schedule and/or TBI on distribution and/or elimination of Synthokine. These data show that the Synthokine, SC55 94, administered therapeutically post-TBI, significantly enhanced platelet recovery and modulated neutrophil nadir and may be clinically useful in the treatment of the myeloablated host. View PublicationCatalog #:Product Name:04436MethoCult™ SF H443604064Starter Kit for MethoCult™ H4034 Optimum04100MethoCult™ H410004230MethoCult™ H423004236MethoCult™ SF H423604431MethoCult™ H443104434MethoCult™ H4434 Classic04464Starter Kit for MethoCult™ H4434 Classic04531MethoCult™ H453104535MethoCult™ H4535 Enriched Without EPO04536MethoCult™ SF H453604564Starter Kit for MethoCult™ H4534 Classic Without EPO04035MethoCult™ H4035 Optimum Without EPO04330MethoCult™ H433004034MethoCult™ H4034 Optimum04435MethoCult™ H4435 Enriched04534MethoCult™ H4534 Classic Without EPO04437MethoCult™ Express -
ReferencePetzer AL et al. (FEB 1996) Proceedings of the National Academy of Sciences of the United States of America 93 4 1470--4
Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium.
A major goal of experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. In normal marrow, such cells appear to be identical to (or represent a subset of) a population referred to as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for textgreater or = 5 weeks when cocultured with stromal fibroblasts. Some expansion of LTC-ICs in vitro has recently been described, but identification of the factors required and whether LTC-IC self-renewal divisions are involved have remained unresolved issues. To address these issues, we examined the maintenance and/or generation of LTC-ICs from single CD34+ CD38- cells cultured for variable periods under different culture conditions. Analysis of the progeny obtained from cultures containing a feeder layer of murine fibroblasts engineered to produce steel factor, interleukin (IL)-3, and granulocyte colony-stimulating factor showed that approximately 20% of the input LTC-ICs (representing approximately 2% of the original CD34+ CD38- cells) executed self-renewal divisions within a 6-week period. Incubation of the same CD34+ CD38- starting populations as single cells in a defined (serum free) liquid medium supplemented with Flt-3 ligand, steel factor, IL-3, IL-6, granulocyte colony-stimulating factor, and nerve growth factor resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days, approximately 40% of which included textgreater or = 1 LTC-IC. In contrast, in similar cultures containing methylcellulose, input LTC-ICs appeared to persist but not divide. Overall the LTC-IC expansion in the liquid cultures was 30-fold in the first 10 days and 50-fold by the end of another 1-3 weeks. Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplification should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic applications. View PublicationCatalog #:Product Name:04436MethoCult™ SF H443604064Starter Kit for MethoCult™ H4034 Optimum04100MethoCult™ H410004230MethoCult™ H423004236MethoCult™ SF H423604431MethoCult™ H443104434MethoCult™ H4434 Classic05100MyeloCult™ H510004464Starter Kit for MethoCult™ H4434 Classic04531MethoCult™ H453104535MethoCult™ H4535 Enriched Without EPO04536MethoCult™ SF H453604564Starter Kit for MethoCult™ H4534 Classic Without EPO04035MethoCult™ H4035 Optimum Without EPO04330MethoCult™ H433004034MethoCult™ H4034 Optimum04435MethoCult™ H4435 Enriched04534MethoCult™ H4534 Classic Without EPO