Hematopoietic Stem Cells (HSCs)
Effectively determining viability & cell count for stem cell transplants
Hämatopoetische Stamm- / Progenitorzellen (HSPCs) werden häufig für allogene oder autologe Stammzelltransplantationen zur Behandlung von Blut- oder Knochenkrebs wie Leukämie und Myelomen verwendet. Hier werden HSCs für die Zellersatztherapie verwendet, wobei diese direkt von einem HLA-kompatiblen gesunden Spender entnommen und einem Leukämiepatienten zugeführt werden, um die erkrankten Zellen zu ersetzen.
HSPCs werden auch isoliert und in reifere Zelltypen der Blutlinie differenziert, als Alternative zu vom Patienten stammenden Materialien zu finden, z. B. für Blutinfusionen und Thrombozytenisolate, die während der Operation oder anderer Arten von Behandlungen verwendet werden. Beim Banking von Nabelschnurblut werden HSPCs aus der Nabelschnur von Neugeborenen isoliert und für eine mögliche zukünftige Verwendung für diese Person oder Verwandte, oder als Ressource für die Erforschung der möglichen Differenzierung und Verwendung der HSP-Zellen für neue Behandlungsmethoden aufbewahrt.
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.
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Verweise
- JE Till and EA McCulloch: A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 1961; 14, 213–222.
- 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.