Hematopoietic stem cells

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.

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.

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.

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.