Lu S-J et al. (JUL 2013)
Regenerative medicine 8 4 413--424
3D microcarrier system for efficient differentiation of human pluripotent stem cells into hematopoietic cells without feeders and serum [corrected].
BACKGROUND Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential,a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. MATERIALS & METHODS hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation,the medium for 3D microcarriers was directly switched to EB medium. RESULTS hESCs on 3D microcarriers maintained pluripotency and formed EBs,which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating,EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore,this 3D system can also be adapted to human induced pluripotent stem cells,which generate functional hemangioblasts with high efficiency. CONCLUSION This 3D serum- and stromal-free microcarrier system is important for future clinical applications,with the potential of developing to a GMP-compatible scalable system.
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High-titer retroviral vectors containing the enhanced green fluorescent protein gene for efficient expression in hematopoietic cells.
Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene,a codon humanized,red-shifted variant of the green fluorescent protein (GFP) gene,which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34(+) cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6,up to 16% of the cells expressed EGFP,as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers.
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04034
04044
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MethoCult™H4034 Optimum
MethoCult™H4034 Optimum
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Kunova M et al. (NOV 2010)
Reproductive biomedicine online 21 5 676--86
Development of humanized culture medium with plant-derived serum replacement for human pluripotent stem cells.
For human embryonic stem cells (ESC) to be used in cell replacement therapies,they must be grown under good manufacturing conditions in a chemically defined medium that lacks animal proteins. This study examined the ability of a newly designed medium containing the plant-derived serum replacement VegetaCell and other reagents of human origin to support undifferentiated growth and pluripotency of human ESC. This medium was tested in several culture systems,using human fibroblasts as a feeder layer or Matrigel in a feeder-free culture. Even under the most stringent feeder-free conditions without conditioned medium,human ESC exhibited an undifferentiated morphology,expressed markers of undifferentiated cells,demonstrated high alkaline phosphatase activity and multilineage differentiation and retained a normal karyotype. Compared with human ESC grown in standard culture conditions,human ESC maintained in humanized VegetaCell medium show longer cell cycles and decreased cell death. The availability of an animal protein-free medium supplemented with the low-cost VegetaCell reagent expands the repertoire of media for culturing human ESC as well as induced pluripotent stem cells for drug testing and cell replacement therapy.
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Niwa A et al. (JAN 2011)
PLoS ONE 6 7 e22261
A novel Serum-Free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors
Elucidating the in vitro differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose,a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study,we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover,single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new,robust,and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation.
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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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EasySep™小鼠TIL（CD45）正选试剂盒
产品手册MethoCult™ Media for Performing Hematopoietic Colony-Forming Unit (CFU) Assays![3D microcarrier system for efficient differentiation of human pluripotent stem cells into hematopoietic cells without feeders and serum [corrected].](https://www.stemcell.cn/static/version1759333448/frontend/Stemcell/default/zh_Hans_CN/Magento_Catalog/images/product/placeholder/small_image.jpg)
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技术公告MethoCult™ for Rat Hematopoietic Colony-Forming Unit (CFU) Assays
产品手册StemSpan™: Defined Media and Supplements for Hematopoietic Cell Expansion
技术公告Genome Editing of Human CD34+ Hematopoietic Stem and Progenitor Cells Using the ArciTect™ CRISPR-Cas9 System and StemSpan™ Media
科学海报Optimized Media and Workflow for the Expansion of Human Pluripotent Stem Cells as Aggregates in Suspension Cultures
