Methods to Identify and Isolate Quiescent Human Muscle Derived Stem Cells
1
+12Bissell, T A; 1,2 Chirieleison S C; 1,2Witt, M; 1,2Kilne, D; 1-3Deasy, B M
Live Cell Imaging Lab, Departments of Orthopaedic Surgery, 2 Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA,
3
McGowan Institute of Regenerative Medicine, University of Pittsburgh Medical Center
[email protected]
Introduction:
During muscle stem cell transplantation studies, it has been found
that only a small fraction of the cells survive the transplantation process
and contribute to regeneration. It is believed that this small subset of
cells is the quiescent subpopulation (1-3). We previously showed that
muscle stem cells are proliferatively heterogeneous (i.e. containing both
dividing and non-dividing cells (4)). Among the non-dividing fraction
are quiescent stem cells that are not currently dividing, but do have the
potential to re-enter the cell cycle and actively start dividing to give rise
to progeny which would participate in tissue repair. These cells are
considered to be the theoretical reserve of a population with the potential
to regenerate a population during times of stress or tissue injury.
Retrospective analysis of some of our studies shows that transplantation
of stem cell populations with more quiescent cells contribute more to
muscle regeneration (3-5).
However, a challenge to the study of this potent population is that no
definitive markers exist for quiescent cells that allow for direct
separation of the cells from the population. Here, we have developed a
unique process involving Live Cell Imaging that allows quiescent cells
to be identified and studied. This technology allows the cells to be
tracked so that their hierarchical lineage and divisional status can be
determined. Our goal is to 1) directly identify quiescent cells 2) isolate
the quiescent subpopulation and 3) verify that the quiescent subpopulation re-activates / re-enters the cycle to give rise to new progeny.
Methods:
Human muscle derived stem cells (hMDSC) were obtained from
Cook Myosite (Cook Myosite, Harmar, Pa). The cells were fluorescently
labeled with 5-chloromethylfluorescein diacetate (CMFDA), which
decreased in concentration and intensity with each subsequent division
because the signal is split between daughter cells (Fig 1). After seven
days in culture, the cells were sorted using flow cytometry in CMFDA
positive cells (non-dividing cells) and CMFDA negative cells (dividing
cells).The sorted cells were subsequently examined for long-term
expansion to determine longevity and were observed using live cell
imaging. PI cell cycle analysis was performed on the parent population.
The unique time lapsed imaging system allowed the separated cells to
be observed over a period of six days in a controlled environment.
Visible and fluorescent images were taken every ten minutes. The jpegs
were analyzed for behavioral parameters using image analysis software.
Quiescence, division time, population doubling time, and mitotic
fraction were determined. Cells were also tracked to determine the
lineage of the cells and for the construction of lineage trees.
A
B
Fig 1: A.) Over 5 days, the CFSE signal decreases to the control (D0=
day 0). B.) The CFSE signal decreases when a cell divides
Results:
The cells were sorted based on their fluorescence (Fig 2) and two
distinct populations could be obtained based on FITC expression. PI cell
cycle analysis showed that the
non-dividing population had
more cells in G1 phase (Fig 3).
In support of our hypothesis,
the percentage of dividing cells
was 51% for the non-dividing
cells and 73% for the dividing
cells. Following activation, the
non-dividing cells were able to
expand as well as the dividing
cells during an in vitro
expansion. At 40 days, the
dividing and non-diving cells
had equal longevity with 12.4
population doublings each.
Cell tracking and the construction of lineage trees demonstrated the
presence of a quiescent population and differences between the two
separated populations. In the non-dividing population, 61.1% of the cells
that divided did not divide until after three days. In the dividing
population, many division events were observed within the first three
days, and the cells continued to divide throughout the entire 6 day
period. Further, in the CMFDA[-] population, 50% of the cells showed
cell division activity within the first 6 hours , while only 22% of the
CMFDA[+] population cells showed this same activity.
Fig 3. PI cell cycle analysis showed more non-dividing cells in G1. The
dividing cells have a greater percentage of dividing cells. Both
populations have the same longevity at 40 days in an in vitro expansion.
Cells were directly tracked in order to determine their lineage and
establish their divisional status. The non-dividing population showed the
presence of re-activated quiescent cells. 4 generations of a cell are
shown in the lineage tree (Fig 4). More divisions and lineages with more
generations were observed in the dividing population.
A
B
Fig 4: A.) Tracked cell lineages B.) Associated lineage tree with 4
generations (G1= generation 1)
Conclusion:
The ability to identify the subpopulations such as quiescent cells
allows for a more in depth understanding of the heterogeneity of stem
cell populations. Stem cell therapies require using cell populations that
can survive the transplantation process and give rise to reparative
progeny. The younger quiescent cells may have the greatest proliferative
capacity, making them the cells most able to contribute to muscle
regeneration.
Here we showed that live cell imaging is a unique technology that
allows for cells to be observed over time in a controlled environment.
Direct observation allows the cells to be followed and lineage histories
established to demonstrate proliferative activity and quiescence. After
labeling with CMFDA and separating the cells using flow cytometry, the
quiescent cells were further identified and isolated. The cells retaining
the CMFDA were identified as non-dividing cells which allowed the
cells to be isolated. Using live cell imaging the quiescent cells were
validated. The cells did not initially divide, but they were able to be
activated and recruited into the cell cycle. These methods can be used to
study stem cell quiescence from any stem cell source and more
importantly, this provides a method to examine the relationship between
cell quiescence and cell therapy based tissue repair.
1. Baroffio, A et al, Differentiation 60 (1996). 2. Beauchamp, J, et al, J
Cell Biol 144 (1999). 3. B. M. Deasy et al., Mol Biol Cell 16, 3323 (Jul,
2005). 4.B. M. Deasy et al., J Cell Biol 177, 73 (Apr 9, 2007). 5. Z.
Qu-Petersen et al., J Cell Biol 157, 851 (May 28, 2002).
Poster No. 1674 • ORS 2011 Annual Meeting