Download A Novel, Multifactorial Approach for hiPSC Differentiation

2015 World Stem Cell Summit
A Novel, Multifactorial Approach for hiPSC Differentiation and
Reprogramming Using an Automated Cell Culture System
®
T. Guo3, M. Watson3, N. Devaraju3, S.C. Boutet3, J. Davila2, J. Gibson1, H.W.Chung1, L. Szpankowski3, B. Fowler3, K. Hukari3, M. Norris3, D. Phan3, M. Thu3, M. Wong3,
H. Choi3, G. Harris3, Y. Lu3, M. Lam3, C. Johnson3, A. Leyrat3, X. Wang, G. Sun3, J. West3, M. Unger3, R.C. Jones3, M. Wernig2, C. Nelson1, N. Li3
University of Connecticut Department of Molecular and Cell biology, Storrs, CT, USA
2 Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
3Fluidigm Corporation, R&D, South San Francisco, CA, USA
1
A major challenge in the stem cell field is to define the optimal condition for cell expansion,
differentiation and reprogramming. Because multiple intracellular and extracellular
signaling pathways are involved in each cellular process, a combinatorial approach to
screen multiple factors is highly desirable. To facilitate the exploratory processes, we have
developed Callisto™, an automated cell culture system for cell manipulation and
environmental control. The system consists of an integrated fluidic circuit (IFC), an
electropneumatic controller instrument, experimental designer software and automated
run-time control software. Each IFC has 32 culture microchambers and 16 reagent inlets.
Each microchamber can be dosed separately with different combinations and ratios of the
16 reagents at various predefined time points. Callisto enables long-term cell culture (more
than three weeks) with three-day hands-off operation. Previously using this system we have
developed a novel nonintegrating method for direct conversion of human BJ fibroblasts to
neurons, and also demonstrated the reprogrammation of human fibroblasts into human
induced pluripotent cells (hiPSCs). Here we demonstrate an efficient transfection protocol
of siRNAs and mRNAs in fibroblasts and hiPSCs. We have also demonstrated using
lentivirus and retrovirus to differentiate hiPSCs to neurons and reprogramming human
fibroblasts to hiPSCs. In summary, the automated microfluidic platform employs precise
control of the microenvironment of cells, facilitates studies of multifactorial combinations
and enables development of robust, reproducible and chemically defined cell culture and
manipulation. Figure 6. Differentiation of hiPSCs into neurons using
NGN2 lentivirus
Figure 4. hiPSCs cultured on IFC are indistinguishable from
cells cultured in standard well plates.
A
A
rtTA, NGN2, GFP (100%)
rtTA, NGN2, GFP (85%)
D
GFP
rtTA, NGN2, GFP (15%)
TRA-1-60
rtTA, NGN2, GFP (5%)
C
B
GFP
Phase
GFP
MAP2
β-III tubulin
DAPI
Phase
GFP
MAP2
β-III tubulin
DAPI
IFC
C
The Callisto system allows easy setup of regular culture experiments using virus expressing rtTA
and a virus expressing eGFP, NGN2 and puromycin resistance gene as a fusion protein linked by
P2A and T2A sequences and driven by a TetO promoter2. hiPS cells were infected with different
doses of lentivirus stocks as indicated in (A) and the expression of NGN2 was induced for 24
hours, selected with puromycin for an additional three days and, finally, cultured in neuronal
medium to allow for maturation of the induced neurons for three days. The culture could easily be
monitored after infection via live imaging of GFP at 10x (A) (scale bar = 490 µm) and 20x (B) (scale
bar = 40 µm). At the end of the experiment, the cells were fixed and stained on IFC using MAP2
and βIII-tubulin antibodies with DAPI as nuclear staining (scale bar = 20 µm). Plate
C
GFP
rtTA, NGN2, GFP (30%)
Figure 1. Callisto system components
The major components of the
Callisto system include: (A) an
IFC to provide fluidic paths and
cell culture microchambers for
cell seeding and treatment, (B)
an instrument to provide thermal,
pneumatic and environmental
(gas, humidity) control of the IFC
to enable long-term cell culture
and dosing, (C) software to
design, monitor and record
experiments, and (D) a reagent
kit to support cell loading, live
harvest and lyse and harvest. B
Replicate 3
rtTA, NGN2, GFP (55%)
Results
B
Replicate 2
Control (no virus)
A
Replicate 1
Phase
Introduction
hiPSCs were seeded and cultured on IFC for three days and then live-stained with fluorescently
labeled TRA-1-60 antibody. In a standard well plate experiment, cells stained with the same
method demonstrated the same TRA1-60 expression on IFC (scale bar = 490 µm) (A). To determine
if indeed hiPSCs cultured on IFC and on standard well plates were indistinguishable at the
population level, we harvested single cells and bulk lysate from IFC and from standard well plates,
and used the C1 and the Biomark HD for single-cell and bulk analysis (B). Pairwise comparison of
single-cell and bulk populations of standard well plate and IFC hiPSCs shows that iPSC population
cultured on standard well plates and on IFC have the same gene expression profiles using a
pluripotency gene panel1 (C). Chamber_SC: single cells from IFC chamber, plate_SC: single cell
from standard well plate culture, chip_lysis: bulk lysate directly harvested from IFC chambers. Figure 7. Retrovirus-mediated reprogramming of human
fibroblasts into hiPSCs
7 days
A
Phase
Figure 5. The Callisto system provides precise reagent delivery
and active mixing for easy setup of multifactorial treatment and
dose response.
14 days
Phase
SSEA-4
SSEA-4/Tra-1-60
5% OKSM
A
% Dye 1
50
0
100
90
75
50
25
10
0
% Dye 2
50
0
0
10
25
50
75
90
100
50
0
100
90
75
50
25
10
0
50
50
75
90
100
50
0
100
90
75
50
25
10
0
50
100
0
0
50
0
10
25
50
75
90
100
50
0
100
0
50
10% OKSM
50
0
0
10
25
0
Figure 2. Principle of the cell culture IFC
20% OKSM
Medium control
nGFP 100%
The IFC features 32 cell culture microchambers, which can be individually treated with
any combination of 16 input factors—simultaneously or on different schedule. The
multiplexer, a fully programmable microfluidic delivery system, can input cells, media and
reagents to individual culture chambers (1 mm2 footprint, 100 nL volume) which can output
into separate outlets supernatants, cells or lysates.
nGFP 50%
nGFP 25%
Medium control Oct4 DAPI Medium control Dose Dissociate DNA or RNA analysis with Biomark or NGS Cell culture on Callisto Fix Stain Harvest Barcode Single-­‐cell protein detecIon on CyTOF® Imaging by microscopy Plate and expand fluidigm.com
Nanog
Sox2
Oct4
Otx2
1,000
100
10
H9 hESC
10% OKSM IFC
Standard plate
10% OKSM IFC
Standard plate
14 days
Human BJ fibroblasts were infected with different amounts of a retrovirus mediating the
expression of Oct4, Klf4, Sox2 and c-Myc (OKSM)3. Seven and 14 days post infection, cells were
live-stained respectively with antibody against SSEA-4 and antibodies against SSEA-4 and
TRA-1-60 (scale bar = 50 µm) (A). Increased amounts of retroviral particles resulted in increased
reprogramming efficiency. Partially reprogrammed cells can also be live-harvested four days
post infection and expanded and matured in standard 6-well plates 10 and 14 days post
reseeding (scale bar = 40 µm) (B). Analysis of pluripotency gene expression demonstrates an
efficient reprogramming of human fibroblasts on IFC similar to the one in the standard well
plate (C).
D
Conclusion
Control siRNA Oct4 siRNA The Callisto system allows easy setup for combinatorial dosing as demonstrated by delivery of two
different fluorescent dyes at various ratios into individual chambers. Fluorescence intensities were
represented in pseudocolor in (A) (scale bar = 490 µm). Using this system, we demonstrated a
simple way to perform dose response. Examples shown here are nGFP mRNA transfection in human
BJ fibroblasts cultured on IFC (scale bar = 30 µm) (B) and knockdown of Oct4 with siRNA
transfection in hiPSCs (scale bar = 240 µm) (C). Immunostaining with Oct4 antibody revealed
significant decrease in Oct4-positive cells after knockdown with Oct4 siRNA. Cells were lysed and
harvested on IFC and lysates were used for gene expression analysis through qPCR. Gene
expression data correlated with immunostaining (D).
•  The Callisto system enables long-term culturing of different cell types and automated
dosing of cells with combinations of miRNAs, mRNAs and small molecules at predefined
times. •  We have developed a streamlined workflow to characterize cells by immunostaining and
by single- or bulk-cell genomic analysis. •  The flexibility of the Callisto system supports complex and time-consuming applications
including cell maintenance, RNA transfection, reprogramming and differentiation.
References
1.  Guo, G. et al. Developmental Cell 18 (2010): 675–685.
2.  Zhang, Y. et al. Neuron 78 (2013): 785–798.
3.  Chung et al., PLOS One 9 (2014): e95304.
Acknowledgments and notes
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Bulk gene expression on Biomark™ HD Single-­‐cell genomics with the C1™ system 14 days
Control siRNA nGFP 12.5%
Pretreat IFC Seed cells Feed cells 10 days
C
10,000
Figure 3. Callisto general workflow
Lyse B
Gene expression level
(relative to BJ)
C
Oct4 siRNA B
For Research Use Only. Not for use in diagnostic procedures.
© 2015 Fluidigm Corporation. All rights reserved. Fluidigm, the Fluidigm logo, Biomark, C1, Callisto, and CyTOF are trademarks or registered trademarks of Fluidigm Corporation in the United States and/or other countries. 12/2015 This work is supported in part by a California Institute for Regenerative Medicine (CIRM) Tools and
Technologies Grant (RT2-02052, Development and Application of Versatile, Automated, Microfluidic
Cell Culture System). We thank Shuyuan Yao, Ph.D. for insightful discussions, and Guangwen Wang,
Ph.D. from Stanford University for kindly providing the hiPSCs.