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Hematopoietic stem cells (HSCs) are tissue-specific adult stem cells capable of differentiating into all blood cell types to ensure homeostasis of blood throughout life. The first signs of hematopoiesis (blood cell formation) occur during embryonic development in blood islands within the yolk sack and in the aorta-gonad-mesonephros region. These early HSCs migrate in later developmental stages into the placenta and fetal liver, which serve as the main compartments of prenatal hematopoiesis. Shortly before birth, HSCs migrate to bone marrow where adult hematopoiesis takes place throughout life.HSCs were the first somatic stem cells to be discovered. Research on HSCs dates back to their discovery in the 1960s, and has led to the development of fundamental concepts in stem cell biology, such as the stem cell niche, stem cell self-renewal, and asymmetric cell division. HSCs are used as a model to understand hematopoiesis regulation and to elucidate mechanisms driving stem cell self-renewal or the directed differentiation towards specific blood cell types. Clinically, HSCs or HSC-enriched fractions are used to treat blood related diseases, like leukemia, sickle cell disease, or the immune defect severe combined immune deficiency (SCID).
At a glance: HSCs in peripheral blood, blood products, and bone marrow
|Unmobilized apheresis||>0.1%||CD34, CD59, CD90, CD117||Hematopoiesis|
|Mobilized apheresis||0.5–2%||CD34, CD59, CD90, CD117||Hematopoiesis|
|Native bone marrow||0.5–1.5%||CD34, CD59, CD90, CD117||Hematopoiesis|
|Umbilical cord blood||1–2.5%||CD34, CD59, CD90, CD117||Hematopoiesis|
|Buffy coat||0.5%||CD34, CD59, CD90, CD117||Hematopoiesis|
|Peripheral blood||<0.1%||CD34, CD59, CD90, CD117||Hematopoiesis|
Miltenyi Biotec has created dedicated application protocols to work with and analyze HSCs.
Miltenyi Biotec has developed numerous products for the straightforward magnetic separation of human HSCs. Positive selection enables the direct labeling and separation of human HSCs, whereas depletion strategies effectively remove non-HSCs to isolate untouched HSCs.
For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic cell separation.
At a glance: Kits and reagents for the separation of HSCs from blood, blood products, and bone marrow
|Starting material||Isolation strategy||Comments||Automation||Product|
|Peripheral blood, umbilical cord blood, apheresis harvest, differentiated ES and iPS cells, bone marrow||Positive selection of target cells||Use for debris-rich starting materials or samples with low HSC content||Yes||CD34 MicroBead Kit UltraPure, human|
|Peripheral blood, umbilical cord blood, apheresis harvest, differentiated ES and iPS cells, bone marrow||Positive selection of target cells||Yes||CD34 MicroBead Kit, human|
|Umbilical cord blood, bone marrow, apheresis product||Two-step positive selection of target cells||Use to isolate primitive CD38– subset of CD34+ for further phenotypic or functional characterization||Yes||CD34+CD38– Cell Isolation Kit, human|
|Umbilical cord blood, bone marrow, apheresis product||Two-step positive selection of target cells||Use to isolate highly pure Lin– CD34+ HSCs||Yes||Diamond CD34 Isolation Kit, human|
|Umbilical cord blood, bone marrow, apheresis product||Positive selection of target cells||For positive selection or depletion of CD133+ cells of hematopoietic or endothelial origin||Yes||CD133 MicroBead Kit Hematopoietic Tissue, human|
|Umbilical cord blood, bone marrow, apheresis product||Depletion of non-target cells||Depletes cells expressing lineage markers||Yes||Lineage Cell Depletion Kit, human|
HSC-containing samples are routinely processed over a density gradient centrifugation to pre-enrich mononuclear cells as starting material for subsequent HSC isolation.
CD34 MicroBeads and related isolation kits allow positive selection of human HSCs and progenitor cells from various starting materials. CD34 is a well-established marker for these cells, and is also expressed on hemangioblasts, endothelial progenitor cells and mature endothelial cells. The CD34 MicroBead Kit UltraPure, human is especially formulated to handle debris-rich samples. Similarly, the CD133 MicroBead Kit – Hematopoietic Tissue, human enables positive selection of HSCs and progenitor cells, optimized for hematopoietic tissues.
CD34+ cells isolated from a debris-rich PBMC sample. The CD34 MicroBead Kit UltraPure, human was used to separate HSCs from a sample of PBMCs. Cells were stained with CD34-PE, CD45-FITC, and propidium iodide solution. Flow cytometry analysis shows high purity after enrichment.
BM-MNCs before separation
CD34+ cells in the lineage cell–depleted fraction.
Analysis of a bone marrow mononuclear cell (BM-MNC) population before and after lineage cell depletion. Lineage-negative cells were isolated from BM-MNCs using the Lineage Cell Depletion Kit, human and an LS Column. Cells were fluorescently stained with CD34-FITC and all fractions were analyzed by flow cytometry.
At a glance: Dedicated antibody cocktails for flow cytometry analysis of HSCs
|Markers in cocktail||Isolation kit or reagent used||Comment||Product|
|CD133/2-PE, CD34-APC, CD45-FITC||CD133 MicroBead Kit – Hematopoietic Tissue, human||Includes a control cocktail. Both treatment and control cocktails contain CD45-VioBlue (which binds a different epitope than CD45-FITC) to restrict analysis to leukocytes.||MC CD34/CD133 Stem Cell Cocktail, human|
|CD34-PE, CD45-FITC||CD34 MicroBead Kit, human, CD34 MicroBead Kit UltraPure, human, or Indirect CD34 MicroBead Kit, human||Includes a control cocktail. Both treatment and control cocktails contain CD45-VioBlue (which binds a different epitope than CD45-FITC) to restrict analysis to leukocytes||MC CD34 Stem Cell Cocktail, human|
HSCs are routinely analyzed by flow cytometry based on various extracellular and intracellular markers. The MC Antibody Cocktails simplify the flow cytometry analysis of cells separated by MACS Technology. All antibodies are optimally titrated to be simply added to an aliquot of a cell fraction before analysis.
Miltenyi Biotec also offers a wide range of unique and standard monoclonal antibodies for research of HSCs and progenitor cells, including CD34, CD38, CD133, CD117, and Anti-Sca-1. Fluorochrome-conjugated MACS Antibodies are perfectly suited for the identification, enumeration, and characterization of HSCs, which can be combined into panels tailored to specific research needs. An online tool to quickly construct the right multicolor flow cytometry panel for each research project can be accessed via the Related documents panel to the right.
At a glance: Kits and reagents for the expansion of HSCs
The number of HSCs available in human samples is often insufficient for downstream applications. HSCs can be expanded in culture with HSC-relevant growth factors to obtain higher cell numbers or to elucidate mechanisms and signaling pathways driving self-renewal or differentiation. Early-acting cytokines – human stem cell factor (SCF), Flt3-Ligand, and thrombopoietin (TPO) – expand primitive HSCs, although current HSC culture systems allow only limited expansion and maintenance of primitive HSCs that are able to engraft and completely restore the entire blood system.StemMACS™ HSC Expansion Media XF is specifically formulated for the robust expansion of HSCs from umbilical cord blood, bone marrow, or peripheral blood. The StemMACS HSC Expansion Cocktail, human, an optimized set of cytokines, can be added to the medium to create a xeno-free culture environment that preserves high levels of primitive CD34+CD133+ HSCs.
At a glance: Kits and reagents for the expansion and CFU analysis of HSCs
|Expansion and CFU analysis||Available in different formulations, with or without erythropoietin||StemMACS HSC-CFU Media, human|
HSC-CFU assays are used to examine the proliferative and differentiation potential of HSCs in vitro. Cells are cultured in a semi-solid culture medium supplemented with growth factors that drive differentiation into the myeloid lineage, resulting in outgrowth of distinct colonies from a single cell.
An online tutorial describing how to set up and conduct an HSC-CFU assay, plus pitfalls to avoid, can be accessed in the Related documents panel to the right.
Miltenyi Biotec offers different formulations of StemMACS HSC-CFU Media, human, our standardized, semi-solid medium based on methylcellulose in IMDM, supplemented with FBS, BSA, and different growth factors. The StemMACS HSC-CFU media are designed to maximize growth and differentiation of progenitor cells and allow the clonal progeny of a single cell to grow in a distinct cluster or colony. They are produced under tightly controlled manufacturing conditions and use highly qualified raw materials to provide a consistent and optimally performing colony assay.
A comparison of StemMACS HSC-CFU Media formulations and uses
|HSC-CFU complete with Epo||HSC-CFU complete w/o Epo||HSC-CFU lite with Epo||HSC-CFU basic*|
|The formulation supports growth of:|
|CFU-G, CFU-M, CFU-GM||Yes||Yes||Yes||Yes/No**|
|BFU-E, CFU-E, CFU-GEMM||Yes||No||Yes||Yes/No**|
|Methylcellulose in IMDM||1%||1%||1%||1%|
|Fetal bovine serum (FBS)||30%||30%||30%||30%|
|Bovine serum albumin (BSA)||1%||1%||1%||1%|
|L-glutamine||2 mM||2 mM||2 mM||2 mM|
|2-mercaptoethanol||0.1 nM||0.1 nM||0.1 nM||0.1 nM|
|Stem cell factor (SCF)||50 ng/mL||50 ng/mL||50 ng/mL||–|
|GM-CSF||20 ng/mL||20 ng/mL||10 ng/mL||–|
|G-CSF||20 ng/mL||20 ng/mL||–||–|
|IL-3||20 ng/mL||20 ng/mL||10 ng/mL||–|
|IL-6||20 ng/mL||20 ng/mL||–||–|
|Erythropoietin (Epo)||3 U/mL||–||3 U/mL||–|
* End formulation after dilution of 4 parts of HSC-CFU basic medium with 1 part diluent
** Depending on supplements added
At a glance: Kits and reagents for viral transduction of HSCs
|Viral transduction||Does not have to be pre-coated on cell culture surfaces||Vectofusin-1|
Viral transduction is a fast and efficient method to study gene function or modulate gene expression. It is also a potential approach for gene modification in the context of gene therapies. Transduction of HSCs is especially interesting because of the cells' potential to eliminate hematopoietic defects. The modification of HSCs with retroviral vectors often requires the presence of a transduction-enhancing reagent. Polycationic reagents induce aggregation of vector particles and facilitate binding of the vectors to target cells via electrostatic interaction. Bridging molecules, such as recombinant fibronectin, interact with both vector particles and cell membrane. Transduction performance can be enhanced by centrifugation.
Miltenyi Biotec offers the novel transduction enhancer Vectofusin-1®, a fully synthetic non-toxic cationic amphipathic peptide that supports high transduction levels with small amounts of retroviral vector. When added to culture medium, Vectofusin-1 promotes the entry of several retroviral pseudo-types into target cells.
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