This document discusses neural progenitor cells (NPCs), which are potent models for studying normal and diseased neurobiology. The author describes how ATCC has developed NPC lines from various sources including induced pluripotent stem cells. NPCs can be expanded and differentiated into neurons, astrocytes and oligodendrocytes. ATCC has optimized culture conditions for NPC growth and differentiation. Reporter NPC lines expressing GFP, NanoLuc, or HaloTag have also been generated. Initial NPC offerings from ATCC are outlined. In summary, ATCC provides a complete solution of NPCs and culture media for disease modeling and drug screening applications.

1. Neural progenitor cells - potent models of
normal and disease neurobiology
Brian Shapiro, Ph.D.
Technical Writer, Cell Biology, ATCC
November 19, 2015

2. Established
partner to
global
researchers
and
scientists
About ATCC
 Founded in 1925, ATCC is a non-profit
organization with headquarters in Manassas,
VA
 World’s premiere biological materials
resource and standards development
organization
 ATCC collaborates with, and supports, the
scientific community with industry-standard
biological products and innovative solutions
 Strong team of 400+ employees; over one-
third with advanced degrees
2

3. Brain cell type ATCC cell lines
Astrocyte √
Oligodendrocyte √
Microglia √
Neural progenitors √
Stanford Medicine, 2009
Region of Study ATCC cell lines
Cortex √
Cerebellum √
Hippocampus √
Locus Cereulus √
Medullary Raphe √
Mesencephalon √
Pituitary √
Retina/Visual System √
ATCC neuroscience cell lines
3

4. Brain cell type ATCC cell lines
Astrocyte √
Oligodendrocyte √
Microglia √
Neural progenitors √
Stanford Medicine, 2009
Neuro-Disorder ATCC cell lines
Alzheimer’s √
Cushing’s √
Epilepsy √
Gigantism/Acromegaly √
Neurooncology √
Parkinson’s √
Retinoblastoma √
Pituitary √
ATCC neuroscience cell lines
4

5. Overview
Introduction
 Neural differentiation
 Neural progenitor cells (NPCs)
Expanding and differentiating neural
progenitors
 Fibroblast- and CD34+-derived NPCs
 Lineage-specific GFP or NanoLuc®-HaloTag®
reporter NPCs
 The advantages of our complete NPC system
ATCC NPC availability and summary
5

6. Why use NPCs?
6
Human NPCs are widely used:
 Drug discovery
 Toxicological assessment
 Preclinical studies
Advantages of using NPCs:
 Human models with no donor variation
• Biologically relevant results/predictive system
 Cells exhibit full differentiation spectrum
• Neurons
• Astrocytes
• Oligodendrocytes
 Easy to use
• Complete system of cells and media will be
available
• Live imaging is possible
• Markers allow for easy endpoint readout
 Saves time
Neural progenitor cells

7. iPSCs for generating NPCs
Adapted from Li M et al. J. Biol. Chem. 289:4594-4599, 2014 and Deng W et al. EMBO Reports 11(3):161-5, 2010
Gene editing to correct
inherited diseases
or to insert reporters
7
Drug screening/toxicity testing

8. 8
iPSCs for generating NPCs
ATCC® No. Designation Gender Ethnicity
ACS-1024™ ATCC-BYS0110 Male
African
American
ACS-1028™ ATCC-BYS0114 Female
African
American
ACS-1025™ ATCC-BYS0111 Male Hispanic
ACS-1029™ ATCC-BXS0115 Female Hispanic
ACS-1026™ ATCC-BYS0112 Male Caucasian
ACS-1030™ ATCC-BXS0116 Female Caucasian
ACS-1027™ ATCC-BYS0113 Male Asian
ACS-1031™ ATCC-BXS0117 Female Asian

9. Neuronal differentiation of iPSCs
99
Astrocytes
(GFAP+)
Neurons
(Tuj1+ , MAP2+, DCX+, TH+)
Oligodendrocytes
(O4+, MBP+)
NPCs/NSCs
(Nestin+, Pax-6+)
iPSCs
(Tra-I-60+)
Embryoid bodies
4 to 8 weeks

10. iPSC-derived NPCs are useful for disease modeling
 Wren and colleagues created iPSCs from
microtubule-associated protein tau (MAPT)
N279K Parkinson’s disease patients
 NPCs were created
 N279K causes increases in 4 repeat to 3
repeat tau domains in MAPT
 Since MAPT binds microtubules vesicle
trafficking was investigated
 NPCs derived from patients with mutation
displayed impaired endocytic trafficking
10
Wren MC, et al. Mol Neurodeg 10:46, 2015.

11. NPCs for toxicological studies
 NPCs can be used to develop high
throughput toxicological studies
 Embryonic mouse brains NPCs
were isolated and differentiated
 Calcium ion flux
 Multi-electrode array experiments
 Cells were sensitive to
neurotransmitters
• Acetylcholine
• Dopamine
• Serotonin
• GABA
11
Martje W G, et al. Toxicol. Sci. 2014;137:428-435.

12. Overview
Introduction
 Neural differentiation
 Neural progenitor cells (NPCs)
Expanding and differentiating neural
progenitors
 Fibroblast- and CD34+-derived NPCs
 Lineage-specific GFP or NanoLuc®-HaloTag®
Reporter NPCs
 The advantages of our complete NPC system
ATCC NPC availability and summary
12

13. QC testing of ATCC® NPCs
13
Post-thaw cell viability: >80%
Post-thaw viable cell number: >1x106 cells/vial
Longevity: >15 PDLs or 5 passages
NPC marker expression: Nestin+, Pax-6+, and Tra-I-60-
Differentiation potential:
>70% Tuj1+ early neurons and
>10% TH+ dopaminergic neurons
Identity: STR profile matching parental iPSC line
Sterility, Mycoplasma, and viral panel testing: None detected

14. Morphology and growth curves of NPCs derived
from CD34+, HFF-1, and Parkinson’s iPSC lines
14
400 μm 400 μm 400 μm

15. NPCs derived from CD34+, HFF-1, and Parkinson’s
iPSC lines expressed Nestin and Pax-6 NPC markers
15

16. Dopaminergic neuron differentiation of NPCs
16

17. Astrocyte and oligodendrocyte differentiation of
NPCs
17
Astrocyte differentiation
Oligodendrocyte differentiation
O4

18. NPC Reporter lines and constructs
 DCXp-GFP: Mature neuron reporter line
Stop codon ZFN cutting
 GFAP-Nanoluc-Halotag: Astrocyte reporter line
 MAP2-Nanoluc-Halotag: Early neuron reporter line
ATCC® No. Designation
ACS-5005™
DCXp-GFP
Neural Progenitor Cells,
Human, Normal
Origin: XCL-1 hiPSCs
ACS-5006™
GFAP-Nanoluc-Halotag
Neural Progenitor Cells,
Human, Normal
Origin: XCL-1 hiPSCs
ACS-5007™
MAP2-Nanoluc-Halotag
Neural Progenitor Cells,
Human, Normal
Origin: XCL-1 hiPSCs
18
DCX ORF P2A GFP

19. 19Nestin + DAPI Pax-6 + DAPI Tra-I-60 + DAPI
DCXp-GFP
GFAP-Nanoluc-Halotag
MAP2-Nanoluc-Halotag
NPC marker expression in reporter NPC lines

20. Dopaminergic neuron differentiation of NPC reporter
lines
20
MAP2-Nanoluc-Halotag DCXp-GFP GFAP-Nanoluc-Halotag

21. Expression of the luciferase reporter during
dopaminergic neuron or astrocyte differentiation
21
Luciferase secretion during dopaminergic
neuron differentiation of MAP2-Nanoluc-
Halotag NPCs
Luciferase secretion during astrocyte
differentiation of GFAP-Nanoluc-Halotag
NPCs
MAP2-Nanoluc-Halotag

22. 22
Expression of the GFP or Halotag reporter during
dopaminergic neuron or astrocyte differentiationDCX-GFP
(Liveimaging)
GFAP-Nanoluc-
Halotag(ICC)
MAP2-Nanoluc-
Halotag(ICC)
MAP2 MAP2+HalotagHalotag

23. 23
0
2
4
6
8
10
12
14
16
18
20
3 4 5 6 7 8 9 10 11 12 13
AccumulativePDLs
0
2
4
6
8
10
12
14
16
18
20
3 4 5 6 7 8 9 10 11 12 13
AccumulativePDLs
ATCCNPC
growthmedia
CompanyBNPC
growthmedia
P7; Day 3 Passage #
Development of ATCC NPC growth media

24. Marker expression and astrocyte differentiation of
NPCs cultured in ATCC growth media
NPCs(P3)Astrocytes
Nestin + DAPI
PhaseGFAP + DAPIGFAP
Tra-I-60 + DAPIPax-6 + DAPI
24

25. Development of ATCC dopaminergic neuron
differentiation media (ATCC® ACS-3004™)
25
ATCCNPC
growthmedia
CompanyANPC
growthmedia
TH TH + DAPI Tuj1 + DAPI

26. Overview
Introduction
 Neural differentiation
 Neural progenitor cells (NPCs)
Expanding and differentiating neural
progenitors
 Fibroblast- and CD34+-derived NPCs
 Lineage-specific GFP or NanoLuc®-HaloTag®
Reporter NPCs
 The advantages of our complete NPC system
ATCC NPC availability and summary
26

27. ATCC initial NPC offerings
27
ATCC® No. Designation Availability
ACS-3003™ Neural Progenitor Cell Growth Kit Late 2015
ACS-3004™ Dopaminergic Differentiation Kit Late 2015
ACS-5001™
Neural Progenitor Cells, Parkinson’s
Origin: ATCC-DYS0530 (ACS-1013™) hiPSCs
In development
ACS-5003™
Neural Progenitor Cells, Normal
Origin: ATCC-BXS0117 (ACS-1031™) hiPSCs
Late 2015
ACS-5004™
Neural Progenitor Cells, Normal
Origin: ATCC-BYS0112 (ACS-1026™) hiPSCs
Late 2015
ACS-5005™
DCXp-GFP
Neural Progenitor Cells, Normal
Origin: XCL-1 hiPSCs
Late 2015
ACS-5006™
GFAP-Nanoluc-Halotag
Neural Progenitor Cells, Normal
Origin: XCL-1 hiPSCs
Late 2015
ACS-5007™
MAP2-Nanoluc-Halotag
Neural Progenitor Cells, Normal
Origin: XCL-1 hiPSCs
Late 2015

28. Summary
 Developed a process enabling generation of
unlimited supply of neural progenitor cells
 Optimized culture conditions for the expansion
and tri-lineage differentiation of NPCs
 Starting iPSC lines played an important role in
morphology, proliferative capacity, and
differentiation potential of NPCs
 CD34+-derived NPCs exhibited a better
proliferative capacity and greater efficiency of
tri-lineage differentiation
 Three gene-edited NPC reporter lines expressed
GFP, NanoLuc®, or HaloTag® during lineage
specific differentiation
 ATCC complete solution of NPC products
including NPCs and culture media provides a
powerful tool for disease modeling and drug
screening
28

29. Acknowledgments
29
Project team:
Leelamma Jacob, M.S., Ph.D.
Michelle Spencer, M.S.
Dezhong Yin, Ph.D.
Trademarks
NanoLuc®-HaloTag® are registered trademarks
of the Promega Corporation

30. Thank you
Questions?