Showing 1 - 12 of 34 results for "04435"
- ReferenceM. V. J. Braham et al. (apr 2019) Advanced healthcare materials e1801444
A Human Hematopoietic Niche Model Supporting Hematopoietic Stem and Progenitor Cells In Vitro.
Niches in the bone marrow regulate hematopoietic stem and progenitor cell (HSPC) fate and behavior through cell-cell interactions and soluble factor secretion. The niche-HSPC crosstalk is a very complex process not completely elucidated yet. To aid further investigation of this crosstalk, a functional in vitro 3D model that closely represents the main supportive compartments of the bone marrow is developed. Different combinations of human stromal cells and hydrogels are tested for their potential to maintain CD34+ HSPCs. Cell viability, clonogenic hematopoietic potential, and surface marker expression are assessed over time. Optimal HSPC support is obtained in presence of adipogenic and osteogenic cells, together with progenitor derived endothelial cells. When cultured in a bioactive hydrogel, the supportive cells self-assemble into a hypoxic stromal network, stimulating CD34+ CD38+ cell formation, while maintaining the pool of CD34+ 38- HSPCs. HSPC clusters colocalize with the stromal networks, in close proximity to sinusoidal clusters of CD31+ endothelial cells. Importantly, the primary in vitro niche model supports HSPCs with no cytokine addition. Overall, the engineered primary 3D bone marrow environment provides an easy and reliable model to further investigate interactions between HSPCs and their endosteal and perivascular niches, in the context of normal hematopoiesis or blood-related diseases. View PublicationCatalog #: Product Name: 04435 MethoCult™ H4435 Enriched Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched - ReferenceR. O. Bak et al. (FEB 2018) Nature protocols 13 2 358--376
CRISPR/Cas9 genome editing in human hematopoietic stem cells.
Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene-function relationships and potentially transform curative hematological gene and cell therapies. However, there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein, we provide a detailed protocol for the production, enrichment, and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol, researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines, researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks, and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis, as well as for the correction of genetic mutations in HSC transplantation-based therapies for diseases such as sickle cell disease, $\beta$-thalassemia, and primary immunodeficiencies. View PublicationCatalog #: Product Name: 09605 StemSpan™ SFEM II 04435 MethoCult™ H4435 Enriched Catalog #: 09605 Product Name: StemSpan™ SFEM II Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched - 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: 04436 MethoCult™ SF H4436 04064 Starter Kit for MethoCult™ H4034 Optimum 04100 MethoCult™ H4100 04230 MethoCult™ H4230 04236 MethoCult™ SF H4236 04431 MethoCult™ H4431 04434 MethoCult™ H4434 Classic 05100 MyeloCult™ H5100 04464 Starter Kit for MethoCult™ H4434 Classic 04531 MethoCult™ H4531 04535 MethoCult™ H4535 Enriched Without EPO 04536 MethoCult™ SF H4536 04564 Starter Kit for MethoCult™ H4534 Classic Without EPO 04035 MethoCult™ H4035 Optimum Without EPO 04330 MethoCult™ H4330 04034 MethoCult™ H4034 Optimum 04435 MethoCult™ H4435 Enriched 04534 MethoCult™ H4534 Classic Without EPO Catalog #: 04436 Product Name: MethoCult™ SF H4436 Catalog #: 04064 Product Name: Starter Kit for MethoCult™ H4034 Optimum Catalog #: 04100 Product Name: MethoCult™ H4100 Catalog #: 04230 Product Name: MethoCult™ H4230 Catalog #: 04236 Product Name: MethoCult™ SF H4236 Catalog #: 04431 Product Name: MethoCult™ H4431 Catalog #: 04434 Product Name: MethoCult™ H4434 Classic Catalog #: 05100 Product Name: MyeloCult™ H5100 Catalog #: 04464 Product Name: Starter Kit for MethoCult™ H4434 Classic Catalog #: 04531 Product Name: MethoCult™ H4531 Catalog #: 04535 Product Name: MethoCult™ H4535 Enriched Without EPO Catalog #: 04536 Product Name: MethoCult™ SF H4536 Catalog #: 04564 Product Name: Starter Kit for MethoCult™ H4534 Classic Without EPO Catalog #: 04035 Product Name: MethoCult™ H4035 Optimum Without EPO Catalog #: 04330 Product Name: MethoCult™ H4330 Catalog #: 04034 Product Name: MethoCult™ H4034 Optimum Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched Catalog #: 04534 Product Name: MethoCult™ H4534 Classic Without EPO - 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: 04436 MethoCult™ SF H4436 04064 Starter Kit for MethoCult™ H4034 Optimum 04100 MethoCult™ H4100 04230 MethoCult™ H4230 04236 MethoCult™ SF H4236 04431 MethoCult™ H4431 04434 MethoCult™ H4434 Classic 04464 Starter Kit for MethoCult™ H4434 Classic 04531 MethoCult™ H4531 04535 MethoCult™ H4535 Enriched Without EPO 04536 MethoCult™ SF H4536 04564 Starter Kit for MethoCult™ H4534 Classic Without EPO 04035 MethoCult™ H4035 Optimum Without EPO 04330 MethoCult™ H4330 04034 MethoCult™ H4034 Optimum 04435 MethoCult™ H4435 Enriched 04534 MethoCult™ H4534 Classic Without EPO 04437 MethoCult™ Express Catalog #: 04436 Product Name: MethoCult™ SF H4436 Catalog #: 04064 Product Name: Starter Kit for MethoCult™ H4034 Optimum Catalog #: 04100 Product Name: MethoCult™ H4100 Catalog #: 04230 Product Name: MethoCult™ H4230 Catalog #: 04236 Product Name: MethoCult™ SF H4236 Catalog #: 04431 Product Name: MethoCult™ H4431 Catalog #: 04434 Product Name: MethoCult™ H4434 Classic Catalog #: 04464 Product Name: Starter Kit for MethoCult™ H4434 Classic Catalog #: 04531 Product Name: MethoCult™ H4531 Catalog #: 04535 Product Name: MethoCult™ H4535 Enriched Without EPO Catalog #: 04536 Product Name: MethoCult™ SF H4536 Catalog #: 04564 Product Name: Starter Kit for MethoCult™ H4534 Classic Without EPO Catalog #: 04035 Product Name: MethoCult™ H4035 Optimum Without EPO Catalog #: 04330 Product Name: MethoCult™ H4330 Catalog #: 04034 Product Name: MethoCult™ H4034 Optimum Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched Catalog #: 04534 Product Name: MethoCult™ H4534 Classic Without EPO Catalog #: 04437 Product Name: MethoCult™ Express - 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: 04436 MethoCult™ SF H4436 04064 Starter Kit for MethoCult™ H4034 Optimum 04100 MethoCult™ H4100 04230 MethoCult™ H4230 04236 MethoCult™ SF H4236 04431 MethoCult™ H4431 04434 MethoCult™ H4434 Classic 04464 Starter Kit for MethoCult™ H4434 Classic 04531 MethoCult™ H4531 04535 MethoCult™ H4535 Enriched Without EPO 04536 MethoCult™ SF H4536 04564 Starter Kit for MethoCult™ H4534 Classic Without EPO 04035 MethoCult™ H4035 Optimum Without EPO 04330 MethoCult™ H4330 04034 MethoCult™ H4034 Optimum 04435 MethoCult™ H4435 Enriched 04534 MethoCult™ H4534 Classic Without EPO 04437 MethoCult™ Express Catalog #: 04436 Product Name: MethoCult™ SF H4436 Catalog #: 04064 Product Name: Starter Kit for MethoCult™ H4034 Optimum Catalog #: 04100 Product Name: MethoCult™ H4100 Catalog #: 04230 Product Name: MethoCult™ H4230 Catalog #: 04236 Product Name: MethoCult™ SF H4236 Catalog #: 04431 Product Name: MethoCult™ H4431 Catalog #: 04434 Product Name: MethoCult™ H4434 Classic Catalog #: 04464 Product Name: Starter Kit for MethoCult™ H4434 Classic Catalog #: 04531 Product Name: MethoCult™ H4531 Catalog #: 04535 Product Name: MethoCult™ H4535 Enriched Without EPO Catalog #: 04536 Product Name: MethoCult™ SF H4536 Catalog #: 04564 Product Name: Starter Kit for MethoCult™ H4534 Classic Without EPO Catalog #: 04035 Product Name: MethoCult™ H4035 Optimum Without EPO Catalog #: 04330 Product Name: MethoCult™ H4330 Catalog #: 04034 Product Name: MethoCult™ H4034 Optimum Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched Catalog #: 04534 Product Name: MethoCult™ H4534 Classic Without EPO Catalog #: 04437 Product Name: MethoCult™ Express - 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: 04436 MethoCult™ SF H4436 04064 Starter Kit for MethoCult™ H4034 Optimum 04100 MethoCult™ H4100 04230 MethoCult™ H4230 04236 MethoCult™ SF H4236 04431 MethoCult™ H4431 04434 MethoCult™ H4434 Classic 04464 Starter Kit for MethoCult™ H4434 Classic 04531 MethoCult™ H4531 04535 MethoCult™ H4535 Enriched Without EPO 04536 MethoCult™ SF H4536 04564 Starter Kit for MethoCult™ H4534 Classic Without EPO 04035 MethoCult™ H4035 Optimum Without EPO 04330 MethoCult™ H4330 04034 MethoCult™ H4034 Optimum 04435 MethoCult™ H4435 Enriched 04534 MethoCult™ H4534 Classic Without EPO Catalog #: 04436 Product Name: MethoCult™ SF H4436 Catalog #: 04064 Product Name: Starter Kit for MethoCult™ H4034 Optimum Catalog #: 04100 Product Name: MethoCult™ H4100 Catalog #: 04230 Product Name: MethoCult™ H4230 Catalog #: 04236 Product Name: MethoCult™ SF H4236 Catalog #: 04431 Product Name: MethoCult™ H4431 Catalog #: 04434 Product Name: MethoCult™ H4434 Classic Catalog #: 04464 Product Name: Starter Kit for MethoCult™ H4434 Classic Catalog #: 04531 Product Name: MethoCult™ H4531 Catalog #: 04535 Product Name: MethoCult™ H4535 Enriched Without EPO Catalog #: 04536 Product Name: MethoCult™ SF H4536 Catalog #: 04564 Product Name: Starter Kit for MethoCult™ H4534 Classic Without EPO Catalog #: 04035 Product Name: MethoCult™ H4035 Optimum Without EPO Catalog #: 04330 Product Name: MethoCult™ H4330 Catalog #: 04034 Product Name: MethoCult™ H4034 Optimum Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched Catalog #: 04534 Product Name: MethoCult™ H4534 Classic Without EPO - 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: 07923 Dispase (1 U/mL) 04435 MethoCult™ H4435 Enriched 85850 mTeSR™1 07920 ACCUTASE™ Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched Catalog #: 85850 Product Name: mTeSR™1 Catalog #: 07920 Product Name: ACCUTASE™ - 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: 17896 EasySep™ Human Cord Blood CD34 Positive Selection Kit II 17952 EasySep™ Human CD4+ T Cell Isolation Kit 21000 RoboSep™-S 04435 MethoCult™ H4435 Enriched 02697 StemSpan™ CC110 Catalog #: 17896 Product Name: EasySep™ Human Cord Blood CD34 Positive Selection Kit II Catalog #: 17952 Product Name: EasySep™ Human CD4+ T Cell Isolation Kit Catalog #: 21000 Product Name: RoboSep™-S Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched Catalog #: 02697 Product Name: StemSpan™ CC110 - 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: 07923 Dispase (1 U/mL) 04435 MethoCult™ H4435 Enriched 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched Catalog #: 85850 Product Name: mTeSR™1 - ReferenceMa N et al. (MAY 2015) Journal of Biological Chemistry 290 19 12079--12089
Factor-induced Reprogramming and Zinc Finger Nuclease-aided Gene Targeting Cause Different Genome Instability in $\$-Thalassemia Induced Pluripotent Stem Cells (iPSCs).
The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However, it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps, we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in beta-hemoglobin gene (HBB) that cause severe beta-thalassemia (beta-Thal), corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting, and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing, we uncovered seven copy number variations, five small insertions/deletions, and 64 single nucleotide variations (SNVs) in beta-Thal iPSCs before the gene targeting step and found a single small copy number variation, 19 insertions/deletions, and 340 single nucleotide variations in the final gene-corrected beta-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps, suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting. View PublicationCatalog #: Product Name: 04435 MethoCult™ H4435 Enriched 85850 mTeSR™1 Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched Catalog #: 85850 Product Name: mTeSR™1 - ReferenceYao Y et al. (FEB 2012) Human gene therapy 23 2 238--42
Generation of CD34+ cells from CCR5-disrupted human embryonic and induced pluripotent stem cells.
C-C chemokine receptor type 5 (CCR5) is a major co-receptor for the entry of human immunodeficiency virus type-1 (HIV-1) into target cells. Human hematopoietic stem cells (hHSCs) with naturally occurring CCR5 deletions (Δ32) or artificially disrupted CCR5 have shown potential for curing acquired immunodeficiency syndrome (AIDS). However, Δ32 donors are scarce, heterologous bone marrow transplantation is not exempt of risks, and genetic engineering of autologous hHSCs is not trivial. Here, we have disrupted the CCR5 locus of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) using specific zinc finger nucleases (ZFNs) combined with homologous recombination. The modified hESCs and hiPSCs retained pluripotent characteristics and could be differentiated in vitro into CD34(+) cells that formed all types of hematopoietic colonies. Our results suggest the potential of using patient-specific hHSCs derived from ZFN-modified hiPSCs for treating AIDS. View PublicationCatalog #: Product Name: 04435 MethoCult™ H4435 Enriched 85850 mTeSR™1 Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched Catalog #: 85850 Product Name: mTeSR™1 - ReferenceJan M et al. (MAR 2011) Proceedings of the National Academy of Sciences of the United States of America 108 12 5009--14
Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker.
Hematopoietic tissues in acute myeloid leukemia (AML) patients contain both leukemia stem cells (LSC) and residual normal hematopoietic stem cells (HSC). The ability to prospectively separate residual HSC from LSC would enable important scientific and clinical investigation including the possibility of purged autologous hematopoietic cell transplants. We report here the identification of TIM3 as an AML stem cell surface marker more highly expressed on multiple specimens of AML LSC than on normal bone marrow HSC. TIM3 expression was detected in all cytogenetic subgroups of AML, but was significantly higher in AML-associated with core binding factor translocations or mutations in CEBPA. By assessing engraftment in NOD/SCID/IL2Rγ-null mice, we determined that HSC function resides predominantly in the TIM3-negative fraction of normal bone marrow, whereas LSC function from multiple AML specimens resides predominantly in the TIM3-positive compartment. Significantly, differential TIM3 expression enabled the prospective separation of HSC from LSC in the majority of AML specimens with detectable residual HSC function. View PublicationCatalog #: Product Name: 04435 MethoCult™ H4435 Enriched 84435 MethoCult™ GF H84435 Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched Catalog #: 84435 Product Name: MethoCult™ GF H84435
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