Hematopoietic Stem Cells (HSCs)

Effectively Determining Viability & Cell Count for Stem Cell Transplants

Hematopoietic stem cells (HSCs) are multipotent cells that are isolated largely from bone marrow but are also found in umbilical cord blood and the peripheral blood mononuclear cell (PBMC) fraction. HSCs were the first cells to be recognized as stem cells and serve as progenitors for blood and immune cells1. CD34 is the main marker identifying them as multipotent and of hematopoietic origin.

Hematopoietic stem/progenitor cells (HSPCs) are often used for allogeneic or autologous stem cell transplantations for the treatment of blood or bone cancers such as leukemia and myeloma. Here, HSCs are used for cell-replacement therapy, taking HSCs directly from an HLA-matched healthy donor and giving them to a leukemia patient to replace the diseased cells.

HSPCs are also isolated and differentiated into more mature cell types of the blood lineage as an alternative to patient-derived materials e.g. for blood infusions and platelet isolates used during surgery or other types of treatments. In cord-blood banking, HSPCs are isolated from the umbilical cord of newborns and stored for potential future use for that person or relatives, or as a resource for research into the potential differentiation and use of the HSPCs for new treatment methods.

With the advances in gene editing, a well-known example being the CRISPR-Cas9 system, hematopoietic stem/progenitor cells (HSPCs) are used to treat and eliminate genetic diseases such as sickle cell anemia2, and especially for cell-based immunotherapy using B cells, T cells and NK cells are used to generate chimeric antigen receptors (CARs) to treat cancers.

For many purposes using HSPCs, the cells need to be isolated and a multipotent state should be maintained to allow them to proliferate without spontaneous differentiation occurring. Further differentiation to mature blood cells or other downstream processing, or manipulation of the cells, can be done following a multitude of protocols.

For all protocols, quantifying the starting or manipulated cell population, and identifying its genetic markers and potency state, are crucial to obtaining satisfactory results, validating treatment potentials, and predicting therapeutic outcomes.

Challenges of Counting & Viability of HSCs

Bone marrow and whole blood samples contain red blood cells (RBCs), platelets, and PBMCs or white blood cells (WBCs), a group of cells including leukocytes, mesenchymal stem cells (MSCs), and HSCs (bone marrow and peripheral blood only). Cell count and viability determination of freshly isolated PBMCs is challenging as the sample still contains RBCs and automated cell counters based on bright-field are limited in their ability to distinguish PBMCs from RBCs.

Solutions for HSC Work

To quantify and assess the viability of the total cell population from umbilical cord or bone marrow samples, the NucleoCounter® NC-202™ will only count PBMCs and exclude RBCs and platelets, as they are only weakly stained. For whole blood samples, the NucleoCounter® offers the Viability and Cell Count – Blood Assay to cope with a very high concentration of red blood cells.

By using a lysis solution, RBCs will lyse to minimize the quenching effect of the hemoglobin and to ensure robust staining of PBMCs with acridine orange (AO) and DAPI to detect total and dead cells, respectively. Due to the lack of nuclei and thereby weak staining, platelets are not detected. The accompanying NucleoView™ software allows the user to verify that all cells have been counted correctly.

If you require a full analysis of cell markers, as well as counting and viability data, the NucleoCounter® NC-3000™ has a flexible staining assay, FlexiCyte™, which enables you to stain up to two different samples at a time with multiple markers for user-defined protocols and provides adjustable analysis settings before and after data acquisition.

References

  1. JE Till and EA McCulloch: A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 1961; 14, 213–222.
  2. MA DeWitt, W Magis, NL Bray et al.: Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci Transl Med. 2016; 8(360):360ra134.

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