Hematopoietic stem cells

The Lineage Cell Depletion Kit, mouse and the Direct Lineage Cell Depletion Kit, mouse enable fast pre-enrichment of mouse HSCs from bone marrow by depleting cells expressing lineage markers. Designed to magnetically label mature cells (T cells, B cells, NK cells, dendritic cells, monocytes, granulocytes, and erythroid cells) and their differentiation-committed precursors, the kits generate an enriched population of untouched HSCs and progenitor cells. The Direct Lineage Cell Depletion Kit, mouse is especially formulated for a quick one-step pre-enrichment process, whereas the Lineage Cell Depletion Kit, mouse enables the most stringent depletion of Lin⁺ cells from bone marrow.

HSCs can be further separated from lineage negative (Lin^–) cells using specific MACS MicroBeads, like the CD117 MicroBeads, mouse or the Anti-Sca-1 MicroBead Kit.

CD117 MicroBeads isolate murine progenitor cells. CD117 (also known as c-kit, steel factor receptor, or stem cell factor receptor) encodes a 145 kDa cell surface glycoprotein belonging to the class III receptor tyrosine kinase family. It is expressed on the majority of hematopoietic progenitor cells, including multipotent HSCs and committed myeloid, erythroid, and lymphoid precursor cells. In addition to their hematopoietic cell differentiation potential, CD117⁺ stem cells from murine bone marrow are reportedly capable of differentiating into smooth muscle cells, myocytes, and endothelial cells in vivo. CD117 is also expressed on a few mature hematopoietic cells such as mast cells.

Isolated mouse HSCs can be further expanded in cell culture with HSC-relevant growth factors to obtain a larger number of cells or to elucidate mechanisms and signaling pathways driving self-renewal or differentiation. Early acting cytokines – mouse stem cell factor (SCF), Flt3-Ligand, and interleukin-6 (IL-6) – expand primitive HSCs, although current HSC culture systems allow only limited expansion and maintenance of primitive HSCs that can engraft and completely restore the entire blood system.

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.