Human Cardiac Progenitor Cells Divide Symmetrically and Asymmetrically
Silvana Bardelli, Claudia Bearzi, Roberto Rizzi, Cynthia Carrillo-Infante, Adriana Bastos
Carvalho,Domenico D’Amario, Arantxa Gonzalez, Roberto Bolli, Federico Quaini, Toru Hosoda,
Gianni Soldati, Tiziano Moccetti, Piero Anversa, Jan Kajstura
Departments of Anesthesia and Medicine and Division of Cardiology, Brigham and Women's Hospital, Harvard Medical School, Boston;
Swiss Stem Cells Bank Research Unit, Cardiocentro Ticino, Lugano, Switzerland
Introduction:
Fig.2. Three-dimentional
confocal
microscopy
reconstruction of mitotic
hCPCs. In a second group
of
experiments,
mitotic
hCPCs were identified and
BrdU distribution (green) in
the chromosomes (PI, red)
was analyzed.
A stem cell can divide asymmetrically, producing one
stem cell and one differentiating cell to maintain tissue
homeostasis, or symmetrically producing two stem cells.
Recent studies have documented that asymmetrically
dividing stem cells can selectively retain chromosomes
containing the “old” template DNA to prevent
accumulation of mutations during DNA replication.
C-kit BrdU PI
The aim of this study was to determine whether human ckit-positive cardiac progenitor cells (hCPCs) divide
asymmetrically by 1. Random DNA template segregation
or 2. Selective retention of the old template DNA strand.
BrdU distribution is uneven
and confined to half of the
chromosomes in the mitotic
figure.
C-kit BrdU PI
.Methods:
Fig.4 (A-C). Qdots cell loading in presence of BrdU. In a third set of experiments, hCPCs were loaded with Quantum Dots, cultured for 36 hours in the presence of BrdU and
examined 96 hours later. Quantum dots are progressively diluted by cell division independently from the modality of DNA template segregation. Thus, hCPCs with minimal levels of
Quantum Dots and bright BrdU localization were interpreted as replicating cells which retained the old DNA strand. By this approach, 5% of hCPCs displayed these two critical
properties.A: BrdU staining; B: Qdots staining; C: BrdU and Qdots staining after 36h culture
Human c-kit positive CPCs were labeled with BrdU. The
analysis was performed in three different sets of
experiments: limited cell dilution to obtain single cellderived clones; unequal BrdU distribution during mitosis
of hCPCs; and Qdots/BrdU differential dilution in
replicating hCPCs.
C-kit BrdU PI
100000,00
Qdots Fluorescence
C-kit BrdU PI
BrdU Fluorescence
1000000,00
10000,00
1000,00
100,00
10,00
Graph 1. BrdU distribution
1000000,00
100000,00
10000,00
1000,00
100,00
10,00
1,00
Results:
Graph 2. Qdots distribution
1,00
0
20
40
60
80
100
120
0
20
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120
140
Graphs 1 and 2. Qdots and BrdU distribution in cultured cells before chasing period. In graph 1 and 2 the distribution of BrdU and Qdots in cultured
hCPCs is shown. The distribution is represented in log scale. Before the chasing period a uniform distribution of both markers is apparent.
Fig.3. Dividing hCPCs. hCPCs in late stage of mitosis are
shown. A and B: The uneven distribution of the cell fate
determinant alpha-adaptin (blue) confirmed that hCPCs
underwent asymmetric division. C: The staining for GATA4
(white) suggested a commitment of a daughter cell to the
cardiomyogenic lineage.
Tab.3 BrdU/Qdots distribution
400000,00
350000,00
A
300000,00
Qdots
Fig.1. Clones of c-kit positive
hCPCs.
Human
cardiac
samples were enzymatically
dissociated and hCPCs were
sorted for c-kit. During in vitro
expantion,
hCPCs
were
exposed to BrdU for 36 hours to
reach a 90% degree of labelling.
BrdU-tagged
hCPCs
were
plated at limited dilution to
obtain single-cell derived clones.
A-C: hCPCs single-cell derived
clone showing a single BrdU
positive cell (circle); C: BrdU
positive
cell
at
higher
magnification;D-E: hCPCs single
cell derived clone showing a uniform BrdU labeling. Sixty clones comprising 10-125 c-kit-positive hCPCs
developed in 7-10 days. In 6% of the clones, one single BrdU-bright hCPC was identified. This cell was
surrounded by clonogenic daughter cells which were negative for BrdU. The number of BrdU-negative hCPCs
ranged from 25 to 111. In 94% of the clones, BrdU was uniformly distributed among clonogenic hCPCs.
Fig.5. BrdU-retaining cells. After 96 hours
chasing period some hCPCs still show a
bright staining for BrdU and very low Qdots
staining (arrow). These BrdU-retaining cells
represent about 5% of the entire population
Table 3. Qdots/BrdU distribution in cultured hCPCs after
96h chase period. After the chasing period Qdots and BrdU
are progressively diluted in cultured cells. A small
percentage of hCPCs dilutes Qdots staining but keeps
retaining BrdU.
250000,00
200000,00
150000,00
100000,00
50000,00
0,00
25000,00
35000,00
45000,00
55000,00
65000,00
75000,00
85000,00
95000,00
BrdU
Conclusion:
B
C
Immortal DNA strand cosegregation participates in
asymmetric division of hCPCs although random
segregation of DNA template is the prevailing
mechanism of hCPC growth.