Biomedical Engineering Reference
In-Depth Information
expression profiles of highly purified HSCs and non-self-renewing multipotent
progenitor cells (Kiel et al. 2005). Cell surface receptors of the Signaling
Lymphocyte Activation Molecule (SLAM) family, including CD150, CD244,
and CD48 were differentially expressed between different primitive progenitor
subpopulations. HSCs were CD150
+
CD244
CD48
, whereas multipotent
progenitors were CD150
CD244
+
CD48
and lineage-restricted progenitors
were CD150
CD244
+
CD48
+
. This was the first description of a receptor
family whose expression combination distinguished HSCs from progenitor
cells.
Several models have been developed to study HSC both in vitro and in vivo.
Primitive hematopoietic cells with proliferative potential can be maintained in
culture for extended periods of time, typically several months. These culture
conditions have been called long-term bone marrow culture (LTBMC; Cou-
lombel et al. 1983). Briefly, LTBMC requires the formation of a supportive
stromal layer which supplies the necessary microenvironment to allow the
primitive hematopoietic cells to proliferate over time. An application of
LTBMC is an assay that measures the number of LTC-IC (Sutherland et al.
1991; Hogge et al. 1996) and is the most stringent in vitro stem cell assay. In this
assay, the cells of interest are overlayed on pre-established, irradiated stromal
layers. After 5-8 weeks culture the contents of each plate are set up in a
committed progenitor assay called a colony-forming unit (CFU) assay for a
further 2 weeks. At the end of this time, the number of colonies formed is
counted and this allows the frequency of LTC-IC to be determined.
Models using SCID (lack B and T cells) and NOD-SCID (lack B, T, and NK
cells and have other immune deficiencies) mice have been extensively used to
study normal HSC and leukemic stem cells (LSCs) (Holyoake et al. 1999a,b).
The transplantation of HSC into SCID or NOD-SCID murine models and
reconstitution of hematopoiesis are used to define HSCs as SCID-repopulating
cells (SRC). In addition, SCID and NOD-SCID models have also been used to
identify SCID leukemia-initiating cells (SL-IC), which are human leukemia
progenitor cells with the ability to reconstitute leukemia in these murine xeno-
transplantation assays. In SCID or NOD-SCID models, following sublethal
total body irradiation, normal or leukemic human cells are injected intrave-
nously into the mice. Six weeks to 6 months later, the mice are sacrificed and the
bone marrow is harvested and stained with human-specific antibodies to iden-
tify the engraftment of human hematopoietic cells by FACS (Holyoake et al.
1999a). NOD-SCID mice are superior to SCID mice for functional assessment
of HSC and LSC as, in SCID models, large numbers of cells need to be
inoculated into the mice for engraftment to occur and serial transplantation is
not possible. Serial transplantation experiments in NOD-SCID mice have now
become the method of choice in many laboratories for assessing the engraft-
ment potential and self-renewal capacity of normal and malignant HSC.
Limiting dilution analysis is widely used both in vitro and in vivo to enumer-
ate LTC-IC or HSC and provide information on an individual cell's potential
(Coulombel 2004). This technique uses varying dilutions of cell suspensions
