Biomedical Engineering Reference
In-Depth Information
McCulloch 1961). In their seminal publication, again using marker chromo-
somes, they demonstrated that multi-lineage splenic colonies arising from a
single cell were formed after the injection of bone marrow into irradiated
murine recipients. Furthermore, these single cells (termed the spleen colony-
forming unit [CFU-S]) were capable of producing new colonies in secondary
recipients, demonstrating self-renewal capacity (Siminovitch et al. 1963). How-
ever, subsequent studies demonstrated that CFU-S were a heterogeneous cell
population and incapable of producing lymphocytes and thus, could not be
considered true pluripotent HSCs (reviewed by Iscove 1990). More recently, the
surface phenotype of pluripotent HSCs was described following advances in cell
purification techniques including fluorescence-activated cell sorting (FACS;
Spangrude et al. 1988) and the development of efficient retroviral gene transfer
techniques which permit clonal marking of the progeny of individual HSCs
(Guenechea et al. 2001).
1.2 Techniques for Studying Stem Cells
in the Hematopoietic System
The in vitro study of HSCs is difficult for a number of reasons. First, these are
rare cells with only relatively small numbers present in any individual. Second,
in vitro culture results in varying degrees of expansion and differentiation of
these cells depending on the culture conditions. This results in the loss of stem
cell phenotype as the cells mature and acquire lineage-specific markers, and the
stem cells become diluted by their more mature progeny. Third, despite exten-
sive studies into the functional and phenotypic properties of HSCs (Weissman
2000), the mechanisms which control self-renewal versus proliferation and
differentiation remain unexplained. The HSC achieves both these functions
via asymmetric cell division in which one new HSC is produced along with
one daughter which then undergoes symmetrical division and differentiates.
However, HSCs can also undergo symmetrical cell division to produce two
daughter cells. It is believed that stem cell fate is decided by, as yet unidentified,
factors in the stem cell niche (Wilson and Trumpp 2006).
Several combinations of cell surface markers have been used to identify and/
or purify murine and human HSC by FACS. Murine HSC have been identified
as Lin
, Thy1.1
lo
, Sca-1
+
, c-Kit
high
, rhodamine123
lo
, and CD34
/int
and
human HSC as Lin
-
, Thy1
+
,CD34
+
, and CD38
neg/lo
(Passegue et al. 2003).
However, all cells with these phenotypes are not true HSC; in murine bone
marrow, the frequency of true HSC is
0.02%, making the HSC a very rare cell
indeed. Very recently, in human cord blood, improved purification of HSC was
achieved by isolating low-density lineage-negative or CD34
+
cells with elevated
levels of aldehyde dehydrogenase activity (Christ et al. 2007). This gave a much
improved long-term repopulating cell frequency of 0.3%. In further studies to
characterize the murine HSC compartment, Kiel et al. compared the gene
