human ips cell-derived motor neurons for modeling ... · motor neuron differentiation and purity 3d...
TRANSCRIPT
Human iPS Cell-derived Motor Neurons for Modeling Neurological Disease
Carrie Chavez, Chris McMahon, Chris Cain, Ben Meline, Brian Jarecki, Susan DeLaura and Eugenia JonesCellular Dynamics International, Inc., A FUJIFILM Company, Madison, WI USA
Abstract
Conclusion
iPSC-derived differentiated cells from apparently normal donor backgrounds were used to develop motor neurons using a modified 3D protocol. The resultant differentiated cells were > 90% neurons and >50% motor based on cell staining with b-tubulin and Isl1, respectively.
The resultant motor neurons, plated directly without the addition of glia show electrically active over extended time in culture.
When co-cultured with Astrocytes the motor neuron electrical activity increases.
We have developed a differentiation protocol that produces motor neuron from donor-derived iPSCs, that can be used to elucidate the mechanism of diseases, such as ALS.
Electrical ActivityMulti Electrode Array (MEA)
Motor Neurons Characterization Motor Neuron/Astrocyte Co-CultureElectrical ActivityThe aim of this study was to produce motor neurons from human induced pluripotent
stem cells (iPSC) with sufficient purity for use in a multitude of downstream assaysincluding electrophysiological recordings using a multi-electrode array (MEA) system. Inparticular we wanted to produce motor neurons that could be cultured in definedconditions, over long periods of time, without being hampered by outgrowth fromproliferative cell types or the need to plate on primary glia. Using a 3D differentiationprotocol, adapted from published methods, we were able to produce motor neuronsfrom iPSC, at greater than 50% purity as measured by Isl 1/2 and Tuj1 positive staining.These cells can be stored frozen, thawed, and cultured in media without glia forextended periods, simplifying experimental design and data interpretation. Using iPSClines from multiple donors we demonstrated the protocol is robust, that motor neuronproduction is independent of the donor iPSC line. We collected ICC, qPCR and MEAdata to characterize the motor neuron cells we produced. Taken together these datashow the characteristics and utilization of iPSC-derived motor neurons.
Human Neural Cell Types Currently Available iPSC-derived Neurons and Astrocytes
iCell Neurons
MyCell Products
iCell Astrocytes
iCell DopaNeurons
Cryopreserved
Human
Neural Cells
Human
Donor
Induced Pluripotent
Stem Cells
Motor Neuron Differentiation and Purity3D method adapted from published protocols
iPS cell
expansion
Motor Neuron Differentiation Cryo
Days 0 16
Neuron purity (b3-tubulin) >90%Motor Neurons (MNC) express
marker Olig2 (>50% at day 14)Post-thaw MNC express
characteristic markers, Isl1 (>50%) and Hb9 (>25%)
10 DIV
Isl1
Hb
9
Nestin
b3
-tu
bu
lin
Isl1
FSC FSC
b3
-tu
bu
lin
1.00E-05
1.00E-04
1.00E-03
1.00E-02
1.00E-01
1.00E+00
ISL1 HB9 CHAT LHX3 FOXP1 TBX20 PHOX2B HOXA2 HOXA4 HOXA5 HOXC4 HOXC5 HOXC6 HOXC9
CDI MN
iCell Dopa
Spinal cord
MN RegionalizationMN Pool Specification
Motor Neuron Identification
Expression of MNC markers at 8 DIV relative to GAPDH. Control RNA: Human spinal cord; iCell DopaNeurons
Cell Staining
Smi-32Isl1DAPI
ChATIsl1DAPI
Smi-32 selectively stains non-phosphorylated neurofilaments, ChAT stains cholinergic neurons, Isl1 stains motor neuron culture
00.25
0.50.75
11.25
9dp 12dp 14dp 17dp 21dp 24dp
mn
FR H
zDays Post Plating
Mean Firing Rate
0
1
2
3
4
9dp 12dp 14dp 17dp 21dp 24dp
BP
Ms
Days Post Plating
Bursting
0
100
200
300
400
500
9dp 12dp 14dp 17dp 21dp 24dp
Bu
rst
Inte
nsi
ty (
Hz)
Days Post Plating
Connectivity
9 DIV 16 DIV
MEA recordings of Motor Neurons show electrical activity starting at 14 DIV, reaching steady levels at 28-30 DIV.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
10dp 12dp 17dp 19dp 24dp 26dp 28dp 31dp 35dp 38dp 42dp 49dp
Mean Firing Rate
CDI 3D 5K 10K 20K
0
1
2
3
4
5
6
7
10dp 12dp 17dp 19dp 24dp 26dp 28dp 31dp 35dp 38dp 42dp 49dp
Bursts Per Minute
CDI 3D 5K 10K 20K
0
50
100
150
200
250
300
350
10dp 12dp 17dp 19dp 24dp 26dp 28dp 31dp 35dp 38dp 42dp 49dp
Connectivity
CDI 3D 5K 10K 20K
Cells plated at 60k/well on 48 well MEA, Astrocytes were overlaid at 5k, 10k, and 20k per well. After 3 weeks in culture, cells plated with astrocytes have a significantly higher mean firing rate than those plated without glia. The CDI 3D protocol produces motor neurons without glia cells.