A: 3D NEP DESIGN
1
CCNA2 Deletion Decreases Induced Neurons through 3D Nanochannel Electroporation Cristina Ortiz 1 , Catherine Czeisler 2 , Daniel Gallego-Perez 3 , Patrick Gygli 2 ,Aaron Cowgill 4 and José Otero 2 1 OSU College of Arts and Science, Department of Biology 2 OSU College of Medicine, Department of Pathology 3 College of Engineering, Department of Biomedical Engineering, 4 College of Arts and Science, Department of Neuroscience Results of NEP • NEP can uniquely control the plasmid ratio during reprograming Significance • Regenerative medicine is a clinical application of research that replaces human cells or organs to restore normal function, often with stem cells • Researchers can regenerate damaged tissues and organs or stimulate the body's own repair mechanisms • Directed reprogramming into specific cell lineages has results directly applicable to clinical studies with regenerative medicine • Future work: since Ascl1 had the greatest effect on neuronal reprogramming of the three transcription factors, perhaps Ascl1 requires CCNA2 to function Acknowledgements • Funded by: Research Supplement to the NIH Grant 3R21EB017539- 01A1S1 of Dr. José Otero • Dr. Otero as the advisor • Dr. Catherine Czeisler for continual support and guidance • The Ohio State University Wexner Medical Center who made this research possible • Fellow members of the team who helped in executing the project Background and Methods: NEP • Cell reprogramming can be done by transfection (introducing DNA into cells) traditionally done by bulk electroporation (BEP) • Nanochannel electroporation (NEP) is a novel method to transfect • NEP is optimal for gene delivery due to the capability of introducing complex combinations of DNA into a large number of individual cells • Conditions can be adjusted to control the amount of molecules delivered: high transfection efficiencies and low cell-to-cell variability • Achieved by applying a focused electric field through a nanochannel, which electrophoretically nanoporates and delivers precisely charged molecules into a cell Background: Cell Reprogramming • Cell reprogramming is a switch in gene expression of a specialized cell into an entirely different cell type Cell Reprogramming: Pathway of nuclear development and including a specific nuclear reprogramming pathway (Gallego-Perez et al., 2014) Results of CCNA2 Background and Methods: CCNA2 • Cyclins are proteins that regulate the cell cycle by activating cyclin- dependent kinases (CDKs) • CDKs then phosphorylate proteins necessary for the cell cycle • Cyclin A2, from the gene CCNA2, has been shown in previous research studies to be necessary to begin DNA replication, particularly the S-phase CDK-Cyclin Complex: When cyclin binds to CDK, the kinase is activated and can phosphorylate the target protein (Alberts et al., 2002) • Deletion of the CCNA2 gene locus was done by cre-mediated recombination of a CCNA2-floxed allele. • It’s effect on nuclear repro- gramming can be measured by the amount of TUJ1-positive cells Patterned Resist Coating Photoresist Coating on Apical/ Basal Surface -6 -4 -2 0 2 4 6 8 10 12 14 0 12 8 4 0 50 100 150 200 250 V X (μm) Y (μm) NEP Modified Transwell Plasmid Reservoir Bottom Electrode E (V/μm) WILD TYPE EMBRYOS WILD TYPE MEFs Dissociate & Expand NEP BAM +cre-GFP CCNA2 FL/FL EMBRYOS CCNA2 FL/FL MEFs Dissociate & Expand NEP BAM Adenovirus cre-GFP CCNA2 FL/FL EMBRYOS CCNA2 FL/FL MEFs Dissociate & Expand NEP BAM +cre-GFP CRE RFP TUJ1 DAPI/TUJ1/RFP DAPI/TUJ1/RFP TUJ1 RFP CRE DAPI/TUJ1/RFP TUJ1 RFP CRE Fixation and Analysis 14 days post-NEP of BAM D1 D2 D3 D4 E1 E2 E3 E4 F4 F3 F2 F1 3D NEP: (A) Transwell modification steps with scanning electron micrographs of the microwell array. (B) Circuit illustration of the resistance distribution (C) Schematic diagram illus- trating the NEP process, with image of a transfected/green cell in direct contact with an open Transwell nanochannel. Gallego-Perez et al., to be published) Induced Neuron Formation by NEP: (A-D) Epifluorscent photomicrographs of TUJ1 stained cells 7 days post BEP or NEP using differing ratios of BAM. (E-H) Phenotypic characterization of induced neurons post-NEP at 14 days. (I) Neuronal reprograming efficiency is plotted as a function of electroporation condition. (J) Neuronal complexity index of induced neurons by Sholl Radius analysis. (Gallego-Perez et al., to be published) Early deletion of CCNA2 blocks neuronal reprogramming: Experimental schematics are illustrated to the left of each panel. Molecular markers are denoted on the left of each panel. Images are epifluorescent photomicrographs. • CCNA2 is required for BAM-mediated induced neuronal formation A B C
Transcript of A: 3D NEP DESIGN
CCNA2 Deletion Decreases InducedNeurons through 3D Nanochannel ElectroporationCristina Ortiz1, Catherine Czeisler2, Daniel Gallego-Perez3, Patrick Gygli2,Aaron Cowgill4 and José Otero2
1OSU College of Arts and Science, Department of Biology 2OSU College of Medicine, Department of Pathology3College of Engineering, Department of Biomedical Engineering, 4College of Arts and Science, Department of Neuroscience
Results of NEP
• NEP can uniquely control the plasmid ratio during reprograming
Significance
• Regenerative medicine is a clinical application of research that
replaces human cells or organs to restore normal function, often
with stem cells
• Researchers can regenerate damaged tissues and organs or
stimulate the body's own repair mechanisms
• Directed reprogramming into specific cell lineages has results
directly applicable to clinical studies with regenerative medicine
• Future work: since Ascl1 had the greatest effect on neuronal
reprogramming of the three transcription factors, perhaps Ascl1
requires CCNA2 to function
Acknowledgements
• Funded by: Research Supplement to the NIH Grant 3R21EB017539-
01A1S1 of Dr. José Otero
• Dr. Otero as the advisor
• Dr. Catherine Czeisler for continual support and guidance
• The Ohio State University Wexner Medical Center who made this
research possible
• Fellow members of the team who helped in executing the project
Background and Methods: NEP
• Cell reprogramming can be done by transfection (introducing DNA
into cells) traditionally done by bulk electroporation (BEP)
• Nanochannel electroporation (NEP) is a novel method to transfect
• NEP is optimal for gene delivery due to the capability of introducing
complex combinations of DNA into a large number of individual cells
• Conditions can be adjusted to control the amount of molecules
delivered: high transfection efficiencies and low cell-to-cell variability
• Achieved by applying a focused electric field through a nanochannel,
which electrophoretically nanoporates and delivers precisely charged
molecules into a cell
Background: Cell Reprogramming
• Cell reprogramming is a switch in gene expression of a specialized cell
into an entirely different cell type
Cell Reprogramming: Pathway of nuclear
development and including a specific nuclear
reprogramming pathway (Gallego-Perez et
al., 2014)
Results of CCNA2Background and Methods: CCNA2
• Cyclins are proteins that regulate the cell cycle by activating cyclin-
dependent kinases (CDKs)
• CDKs then phosphorylate proteins necessary for the cell cycle
• Cyclin A2, from the gene CCNA2, has been shown in previous
research studies to be necessary to begin DNA replication, particularly
the S-phase
CDK-Cyclin Complex: When cyclin binds to CDK, the kinase is activated and can phosphorylate the
target protein (Alberts et al., 2002)
• Deletion of the CCNA2 gene
locus was done by cre-mediated
recombination of a CCNA2-floxed
allele.
• It’s effect on nuclear repro-
gramming can be measured by
the amount of TUJ1-positive cells
Patterned Resist
Coating
Photoresist
Coating on
Apical/
Basal Surface
-6
-4
-2
02
4
6
8
10
12
14
0 12840
50
100
150
200
250 V
X (mm)
Y (
mm
)
NEP
Modified Transwell
Plasmid Reservoir
Bottom Electrode
Y (mm)
E (
V/m
m)
00-2 2 4 6 8 10
10
5
15
20
25
Nanochannel
Microwell/Nanochannel
Interface
Nanoporating array circuit diagram
A: 3D NEP DESIGN
WILD TYPE
EMBRYOS
WILD TYPE
MEFs
Dissociate &
Expand
NEP BAM
+cre-GFP
CCNA2FL/FL
EMBRYOS
CCNA2FL/FL
MEFs
Dissociate &
Expand
NEP BAM
Adenovirus
cre-GFP
CCNA2FL/FL
EMBRYOS
CCNA2FL/FL
MEFs
Dissociate &
Expand
NEP BAM
+cre-GFP
CR
E
RF
P
TU
J1
DA
PI/
TU
J1
/RF
PD
AP
I/T
UJ
1/R
FP
TU
J1
RF
P
CR
E
DA
PI/T
UJ
1/R
FP
TU
J1
RF
P
CR
E
Fixation and Analysis 14 days post-NEP of BAM
D1 D2 D3 D4
E1 E2 E3 E4
F4F3F2F1
3D NEP:
(A) Transwell modification
steps with scanning electron
micrographs of the microwell
array.
(B) Circuit illustration of the
resistance distribution
(C) Schematic diagram illus-
trating the NEP process, with
image of a transfected/green
cell in direct contact with an
open Transwell nanochannel.
Gallego-Perez et al., to be
published)
Induced Neuron Formation by NEP: (A-D) Epifluorscent photomicrographs of TUJ1 stained cells
7 days post BEP or NEP using differing ratios of BAM. (E-H) Phenotypic characterization of
induced neurons post-NEP at 14 days. (I) Neuronal reprograming efficiency is plotted as a function
of electroporation condition. (J) Neuronal complexity index of induced neurons by Sholl Radius
analysis. (Gallego-Perez et al., to be published)
Early deletion of CCNA2 blocks neuronal reprogramming: Experimental schematics are illustrated to the left of
each panel. Molecular markers are denoted on the left of each panel. Images are epifluorescent photomicrographs.
• CCNA2 is required for BAM-mediated induced neuronal formation
A B
C
