Abstract
Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34+ CD133+ cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg−/− (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34+ CD133+ fraction of expanded cells and that CD34+ CD133+ cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.
Highlights
Human hematopoietic stem cells (HSCs) were the first clinically important class of stem cells and are frequently used in autologous and heterologous transplantation to treat a range of hematologic malignancies and congenital immunological defects [1,2]
We have previously shown that stem cell factor (SCF), thrombopoietin (TPO) and fibroblast growth factor 1 (FGF1) in combination with angiopoietin-like proteins or insulin-like growth factor binding protein 2 (IGFBP2) support a significant expansion of murine HSCs in a feeder-cell-free, serum-free culture [12,13]
Human CD133+ cells were purified from cord blood and either injected into NOD-scid Il2rg2/2 (NSG) mice or cultured for 10 days with SCF, TPO, FGF, IGFBP2 and angiopoietin-like 5 as previously described [14]
Summary
Human hematopoietic stem cells (HSCs) were the first clinically important class of stem cells and are frequently used in autologous and heterologous transplantation to treat a range of hematologic malignancies and congenital immunological defects [1,2]. Efficient methods are needed for expanding HSCs rather than allowing cells to proliferate, differentiate and eventually die. There is a need for surrogate markers for rapid assessment of HSC expansion. This is because the functional verification by engrafting HSCs in immunodeficient mice takes months. By a combination of fractionation and in vivo reconstitution, studies have identified cell surface markers that differentiate freshly isolated HSCs from more committed progenitors in both humans and mice [7]. Identification of HSCs following expansion is complicated by changes in both surface marker expression and stem cell activity during the culture. Surface markers that closely correlate with SCID repopulating activity both before and after HSC culture have not been reported
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