Biomedical Engineering Reference
In-Depth Information
In another example, Stringari
et al
. utilized fluorescence lifetime microscopy (FLIM)
to image and discriminate undifferentiated human ESCs from differentiated progenies
by monitoring their metabolic activity [42]. In particular, FLIM is an imaging technique
that produces an image based on the differences in the exponential decay rate of fluores-
cence and can be used to measure the time-decay characteristics of the cell and tissue
microenvironment, thereby allowing for the molecular localization and identification of
intrinsic fluorophores or endogenous proteins [43]. By using cluster analysis of the pha-
sor distribution of FLIM images [44], this technique provides a powerful technique with
which to obtain a unique signature that can be used to monitor the metabolism of
intrinsic fluorophores, like nicotinamide adenine dinucleotide (NADH) [45] and lipid-
droplet-associated-granules (LDAG), which were found to correlate with the state of
stem-cell differentiation [46]. Specifically, by monitoring NADH and LDAGs, ESCs
that were induced to differentiate using BMP4 or retinoic acid, phasor FLIM could dis-
criminate between undifferentiated ESCs from differentiating ESCs (Figure 19.9A-D).
Moreover, this correlated well with measurements of the ESC marker OCT4, where
undifferentiated ESCs have a high expression of OCT4, while differentiating ESCs have
a characteristically low expression of OCT4 (Figure 19.9E-G). In addition, it was
reported that the FLIM phasor distribution, which is characteristic of undifferentiated
ESCs, was dominated by LDAG while differentiating ESCs treated with BMP4 were
shifted toward the central region of the phasor plot, representing an increase in NADH.
In a similar study, Stringari and co-workers also demonstrated that NADH metabolism
could be used to monitor stem-cell differentiation of
Caenorhabditis elegans
germ line
cells [44]. Overall, there are a number of microscopy-based screening methods that are
now available, which can be utilized for the noninvasive, label-free, and real-time moni-
toring of stem cell self-renewal and differentiation.
Conclusions
Given the tremendous potential that stem cells hold in the clinic as well as the increasing
interest in using nanomaterials [47], there is an urgent need for high-throughput methods
to screen stem cell self-renewal and differentiation in a simple, noninvasive, real-time,
and label-free fashion. To this end, recent efforts have been placed in developing and
adapting techniques from other fields, including electrical cell-substrate impedance,
microfluidic flow cytometry, electrochemical, Raman, and microscopy-based methods.
Each technique has its own individual advantages; for example, microfluidic flow
cytometry allows for the high-throughput screening of individual stem cells whereas
Raman and microscopy-based techniques can be used to monitor the unique chemical
structure and metabolism of stem cells, respectively. However, when compared to con-
ventional techniques used for the characterization of stem cells, including RT-PCR,
Northern and Western blotting, immunofluorescence, and flow cytometry, the methods
described in this chapter hold a number of general advantages. First, these methods are
simple to perform. Second, these methods can provide real-time monitoring of stem cell
self-renewal and differentiation, whereas conventional methods tend to be end-point
assays that can give only a snapshot of what is occurring in the stem cells. Third, and
most importantly, the described methods are noninvasive and, as such, stem cells can be
used even after being screened. Despite the fact that the field of high-throughput screen-
ing of stem cell self-renewal and differentiation is still in its infancy, advances are rapidly
being made in this field and we are moving ever closer to having the ability to screen the
