Highly efficient immunomagnetic purification of
cardiomyocytes derived from human pluripotent stem cells
Kristin Noack, Andreas Bosio, and Dominik Eckardt
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
3
Introduction
Pure cardiomyocytes (CMs) derived from human
pluripotent stem cells (hPSCs) are of high interest for
heart disease modeling, drug safety studies, and
development of cellular therapies. Although several
protocols for cardiac differentiation of hPSCs have
been developed, major limitations are, e.g., the high
variability in differentiation efficacy due to clone-toclone variations and the heterogeneity of generated
CM populations. For that reason we have developed
several tools to improve the workflow starting
from CM differentiation and monolayer dissociation
via MACS® Technology–based CM purification
to downstream analysis using flow cytometry,
immunofluorescence stainings, and calcium imaging.
Additionally, a novel cryopreservation method for
enriched CMs was established.
Purified CMs are functional
and express CM-specific markers
Regardless of the strategy chosen, magnetically
enriched CMs attached well, initiated contractions,
and could be stably maintained in culture. They showed
a typical morphology and sarcomeric structure.
Moreover, flow cytometry analysis demonstrated the
expression of CM-specific markers, such as α-Actinin,
Myosin Heavy Chain (MHC), MLC2v, MLC2a, and
Troponin T, indicating that the newly developed cell
separation strategy is suitable for enrichment
of CMs and different CM subtypes. Labeling with
the Ca2+-sensitive dye FLUO-8® indicated functionality
of purified CMs, as typical Ca2+ fluxes were observed.
hPSC cultivation and cardiac differentiation
Differentiated
cardiomyocytes
10²
10²
10¹
1
0
-1
DAPI
α-Actinin
10¹
10²
10¹
10³
Oct-4
α-Actinin
0
250
500
750 1000
Forward Scatter
Figure 1
2
Purification of hPSC-derived CMs
In order to identify antibodies suitable for enrichment
of CMs or depletion of non-myocytes, we performed
a surface marker screening with more than 400 antibodies
between days 10 and 20 of differentiation. Although no
exclusive CM markers were identified, we found several
markers that either labeled CMs and non-myocytes or
subpopulations of CMs. Based on these data we
developed a novel magnetic cell separation procedure
that consistently delivered CM purities of >90%,
regardless of the differentiation protocol, hPSC line
Strategy A Depletion of non-target cells
used, time point and efficiency of differentiation.
Depending on the hPSC differentiation efficiency
different magnetic cell sorting strategies for the
enrichment of hPSC-derived CMs were applicable.
For cell populations with low differentiation
efficiencies, e.g., at CM ratios of <50%, strategy A
(depletion of non-myocytes) could be combined
with strategy B (CM enrichment). For samples showing
a higher differentiation efficiency strategy A alone
was sufficient.
MHC
MLC2v
Figure 3
4
Cryopreserved CMs maintain high viability
Enriched CMs could be cryopreserved in StemMACS
Cryo-Brew medium. The optimal freezing density
was 5×106 cells/250 µL. Thawed CMs showed high
viabilities and were cultured most efficiently in
90
80
70
60
50
40
30
20
10
0
Cryo-Brew
Cryo-Brew
5×106 cells/250 µL
Differentiated hPSCs
Purified CMs
90% FCS
+ 10% DMSO
1×106 cells/mL
Conclusion
Troponin T
We developed novel tools supporting the
workflow for efficient generation, magnetic purification,
flow cytometry or immunofluorescence-based
characterization, and cryopreservation of hPSC-derived
CMs.
•The PSC-derived Cardiomyocyte Isolation Kit,
human enables the enrichment of hPSC-derived
CMs to high purities regardless of the differentiation
efficiency, stem cell clone, differentiation time
point, or differentiation protocol used.
Forward scatter
cTnT
DAPI
Figure 2
90% RPMI (B27)
+ 10% DMSO
Figure 4
Enrichment of CMs
Pre-enriched CM fraction
cTnT
DAPI
the presence of StemMACS Thiazovivin and at a cell
density of 3×105 cells/cm2. Cells initiated contractions
24–48 h after thawing.
100
Strategy B Enrichment of target cells
Depletion of non-myocytes
MLC2a
Day 1 after plating
10³
Day 3 after plating
10³
Troponin T
SSEA-4
hPSCs cultivated in
iPS-Brew XF medium
1
0
-1
-1 0 1
MLC2v
activation (CHIR99021) and inhibition (IWR-1) of Wnt
signaling. First contractions were observed at day 9
of differentiation and CMs showed a typical
morphology and sarcomeric structure (right).
Viability in %
hPSCs were maintained under xeno-free conditions
in StemMACS™ iPS-Brew XF medium (left) and
differentiated to CMs with efficiencies of up to
80% using a monolayer protocol1 with consecutive
FLUO-8 labeling
Troponin T
1
MLC2a
Results
cTnT
DAPI
•Purified CMs were analyzed using our newly
developed recombinant antibody conjugates
labeling intracellular cardiac muscle proteins,
such as α-Actinin, Troponin T, MHC, MLC2a, and
MLC2v for precise detection of CMs and distinction
between CM subtypes.
•
Magnetically purified CMs start rhythmic
contractions 24 h after plating, can be stably
maintained in culture, show regular Ca2+ fluxes
and can be cryopreserved with good viabilities.
Reference
1.Lian, X. et al. (2013) Nat. Protoc. 8: 162–175.
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