C. T. Charlesworth et al. (SEP 2018)
Molecular therapy. Nucleic acids 12 89--104
Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting.
Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here,we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the $\beta$-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation,there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70{\%} of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting,HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules,UM171 and SR1,stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of $\beta$-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.
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Lam AC et al. (DEC 2001)
Transfusion 41 12 1567--76
Preclinical ex vivo expansion of cord blood hematopoietic stem and progenitor cells: duration of culture; the media, serum supplements, and growth factors used; and engraftment in NOD/SCID mice.
BACKGROUND: Ex vivo expansion of cord blood (CB) hematopoietic stem and progenitor cells increases cell dose and may reduce the severity and duration of neutropenia and thrombocytopenia after transplantation. This study's purpose was to establish a clinically applicable culture system by investigating the use of cytokines,serum-free media,and autologous plasma for the expansion of CB cells and the engraftment of expanded product in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. STUDY DESIGN AND METHODS: Enriched CB CD34+ cells were cultured in four media (Iscove's modified Dulbecco's medium with FCS,Gibco; X-Vivo-10,BioWhittaker; QBSF-60,Quality Biological; and StemSpan SFEM,Stem Cell Technologies) with four cytokine combinations (thrombopoietin [TPO],SCF,Flt-3 ligand [FL] with and without G-CSF,and/or IL-6). The effect of autologous CB plasma was also investigated. The read-out measures were evaluated on Days 8 and 12. After expansion at the optimized condition,cultured cells were transplanted into sublethally irradiated NOD/SCID mice. The engraftment of human CD45+ cells and subsets in the bone marrow,spleen,and peripheral blood was determined. RESULTS: QBSF-60 or StemSpan SFEM supported high yields of early progenitors (CD34+ cells,textlessor= 64.8-fold; CD34+CD38- cells,330-fold; CFU-granulocyte erythroid macrophage megakaryocyte [GEMM],248-fold) and CFUs of the myeloid (CFU-GM,407-fold) and erythroid (BFU/CFU-E,144-fold) lineages. The expansion of the megakaryocytic lineage was consistently higher in X-Vivo-10 (CFU-megakaryocyte,684-fold). Autologous plasma promoted colony formation but reduced CD34+ cells and CFU-GEMM. The addition of G-CSF or IL-6 improved cell yields; G-CSF was more effective for committed progenitors. Expansion products from cultures in QBSF-60 with the cytokines engrafted and differentiated into the myeloid and lymphoid lineages in NOD/SCID mice. CONCLUSION: The data supported the strategy of expansion. The optimized condition may be applicable to clinical expansion for the abrogation or reduction of posttransplant cytopenia.
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S. S. De Ravin et al. (APR 2016)
Nature biotechnology 34 4 424--9
Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease.
Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58{\%} Venus(+) HSPCs with 6-16{\%} human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD),caused by mutations in the gp91phox subunit of the NADPH oxidase,TI of a gp91phox transgene into AAVS1 resulted in ∼15{\%} gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs,4-11{\%} of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases.
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产品类型：
产品号#：
09600
09650
产品名：
StemSpan™ SFEM
StemSpan™ SFEM
文献
J. Yen et al. (NOV 2018)
Scientific reports 8 1 16304
TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells.
CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as $\beta$-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex,consisting of purified Cas9 protein and guide RNA,into HSPCs. Because $\beta$-hemoglobinopathies are most prevalent in developing countries,it is desirable to have a reliable,efficient,easy-to-use and cost effective delivery method. With this goal in mind,we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF),a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.
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产品类型：
产品号#：
22001
22005
22006
22007
22008
22009
22011
22012
产品名：
STEMvision™ 人脐带血7-天CFU分析包
STEMvision™ 彩色人脐带血14-天CFU分析包
STEMvision™ 彩色人骨髓14-天CFU分析包
STEMvision™ 彩色人动员外周血14-天CFU分析包
STEMvision™ 小鼠总CFU分析包
STEMvision™ 小鼠髓系CFU分析包
STEMvision™ 小鼠红系CFU分析包
STEMvision™ 小鼠CFU分析包（髓系和红系）
文献
Ali N et al. (APR 2009)
Blood 113 16 3690--5
Forward RNAi screens in primary human hematopoietic stem/progenitor cells.
The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation,we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex,pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion,respectively,thereby demonstrating the potential of this forward screening approach in primary human stem cell populations.
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A specific in vitro bioassay for measuring erythropoietin levels in human serum and plasma.
The accurate measurement of biologically active erythropoietin (Ep) in human serum and plasma using present in vivo and in vitro bioassays is difficult because of the presence of both inhibitors and non-Ep stimulators of erythropoiesis. We have developed a simple procedure to quantitatively purify Ep from serum and plasma for subsequent testing in the phenylhydrazine-treated mouse spleen cell assay. The method involves absorption of Ep to an immobilized high-affinity anti-Ep monoclonal antibody and acid elution of the antibody-bound material. After neutralization,the eluted EP is then tested directly in the in vitro bioassay without interference by other serum proteins. By using magnetic beads as a solid support for the antibody,washing and elution steps can be performed rapidly and efficiently. Recoveries of Ep after this procedure show very little sample-to-sample variation and are consistently between 45% and 55%,which is close to the maximum binding expected for the anti-Ep antibody. Coupled with the 7.4-fold concentration that this procedure affords,there is an overall increase in sensitivity of three- to fourfold,which makes this assay suitable for accurately measuring Ep levels in patients with below-average titers. Results with this magnetic bead assay indicate that accurate and reproducible estimates for Ep levels in the serum and plasma from healthy donors as well as from patients with hematologic disorders can be obtained. Titers of biologically active Ep in the sera from a group of patients with either leukemia or lymphoma were found to be elevated,and the values correlated well with titers of immunoreactive Ep measured in the Ep radioimmunoassay. Because of its specificity and high sensitivity,the magnetic bead assay is a valuable alternative to immunoassays for the measurement of elevated,normal,and even subnormal Ep levels in human serum and plasma.
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EasySep™小鼠TIL（CD45）正选试剂盒
文献
技术窍门Genome Editing of Human CD34+ Hematopoietic Stem and Progenitor Cells Using the ArciTect™ CRISPR-Cas9 System and StemSpan™ Media
1:06:52
New Tools for the Ex Vivo Expansion of Human Hematopoietic Stem and Progenitor Cells
UM171 & UM729: Novel Small Molecules for the Ex Vivo Expansion of Human Hematopoietic Stem Cells
BrochureUM171 & UM729: Novel Small Molecules for the Ex Vivo Expansion of Human Hematopoietic Stem Cells
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