This document summarizes a webinar presented by Andrew Gaffney and Erin Knock of STEMCELL Technologies. They discussed STEMCELL's development of the healthy control human iPSC line SCTi003-A and single-use iPSCdirect product, which were manufactured to high quality standards. They also discussed STEMCELL's neural progenitor cells derived from SCTi003-A, which can be expanded over multiple passages while maintaining their ability to differentiate into neurons and astrocytes. The neural progenitor cells demonstrate expected marker expression and can be cryopreserved and re-thawed while retaining their differentiation potential.

2. IMAGE
Fully Characterized Human
iPSCs and NPCs for
Downstream Differentiation
Applications
Presenters:
Andrew Gaffney, PhD - Director, Stem Cell Manufacturing and Commercialization
Erin Knock, PhD - Associate Director, Neural Biology
Date 2023-06-29

3. STEMCELL acknowledges that our work spans many territories and
that our head office is located on the traditional unceded territories of
the xʷməθkʷəy̓əm (Musqueam), Sḵwx̱wú7mesh (Squamish), and
səlilwətaɬ (Tsleil-Waututh) Nations.
Land Acknowledgement

4. IMAGE
Development of Human
iPSCs Under Stringent
Quality Standards
A Highly Characterized Human iPSC Line
for Stem Cell Research
Presenter
Andrew Gaffney, PhD
Date 2023-06-29

5. 5
Background
● BSc Biology at The University of Sheffield
● MSc Genetic Epidemiology at the University of Sheffield
● PhD Pediatric Oncology at the University of Leeds and the
University of Sheffield
○ Human ES cell model of Ewing sarcoma
● Nine years experience at STEMCELL Technologies
○ Field Applications Scientist
○ Product Management
○ Stem Cell Manufacturing and Commercialization
Andrew Gaffney
Director, Stem Cell Manufacturing
and Commercialization
STEMCELL Technologies
Director, Stem Cell Manufacturing and Commercialization
Speaker: Andrew Gaffney

6. 6
What iPSC Quality Standards Does STEMCELL Manufacture To?

7. 7
SCTi003-A Has Been Certified by hPSCreg® Based
on Community Standards
hPSCreg® Standardized nomenclature Certified
hpscreg.eu/cell-line/SCTi003-A
SCTi003-A
Institute Donor ID Clone #
iPSC / ESC

8. 8
Assessment Pre-Master Master Cell Bank
Working
Cell Bank
Commercial
Cell Bank
Viability - ✓ ✓ ✓
Recovery - ✓ ✓ ✓
Sterility - ✓ ✓ ✓
Viral screen - ✓ - -
Mycoplasma - ✓ ✓ ✓
Cell line identity - ✓ ✓ ✓
Residual vector ✓ - - -
T cell clonality assay ✓ - - -
Karyotype ✓ ✓ ✓ ✓
20q FISH - ✓ - -
Copy number variants - ✓ ✓ ✓
Whole exome/genome sequencing - ✓ - -
Undifferentiated marker analysis -
Yes, 5 passages
after thaw
Yes, 3 passages
after thaw
Yes, 3 passages
after thaw
Trilineage differentiation ✓ ✓ - -
STEMCELL’s Extensive iPSC Characterization Process

9. 9
SCTi003-A Donor Characteristics

10. 10
How to Thaw and Maintain SCTi003-A
Culture type Adherent
Maintenance medium mTeSR™ Plus (Cat no. 100-0276)
Substrate Corning® Matrigel® hESC-Qualified Matrix
Supplement Not required
Dissociation method Aggregate passaging
Dissociation reagent ReLeSR™ (Cat no. 05872)
Split ratio 1:30 - 1:60 every 5 - 7 days
Incubator atmosphere 37°C, 5% CO2, and 95% humidity
Cryopreservation reagent CryoStor® CS10 (Cat no. 07930)
Thaw recommendation
After thaw, pellet cells and resuspend in
1 mL mTeSR™ Plus. Aliquot into a
pre-prepared six-well plate at six different
densities: 150 μL, 100 μL, 75 μL, 50 μL,
25 μL, and 15 μL. Select the well with optimal
colony density for passaging at Day 7.
Note - Culture conditions are reflective of how the cell line was maintained
prior to cryopreservation.
150 μL 100 μL 75 μL
50 μL 25 μL 15 μL

11. 11
SCTi003-A Demonstrates High Quality
Undifferentiated hPSC Morphology
● SCTi003-A was thawed and maintained in mTeSR™ Plus on Corning® Matrigel® hESC-Qualified Matrix
● Colonies contain densely packed cells and develop multi-layering when ready to be passaged
● iPSCs retain prominent nucleoli and high nuclear-to-cytoplasmic ratio

12. 12
Optimal SCTi003-A Colony Density At Day 7 Post-Thaw
Optimal Density
Low Density
High Density
● SCTi003-A iPSCs were thawed at a range of concentrations and expanded in mTeSR™ Plus for seven days
● It is recommended that a culture is passaged once it has reached an optimal density consisting of large,
multilayered colonies that have just begun to merge

13. 20q FISH: Copy Number Variants:
EpiPluriScore:
Karyotype:
Morphology:
Undifferentiated Marker Expression:
TRA-1-60 OCT4
Pluripotency:
SCTi003-A Has Undergone Extensive Characterization

14. 14

15. 15
SCTi003-A Does Not Contain Any Pathogenic or
Likely Pathogenic Variants in TP53 or BCOR

16. 16
Determining SCTi003-A Donor Ancestry by
Whole Exome Sequencing
Ancestry: European
Type: Closest
Contribution:
African = 0% East Asian = 0%
European = 78.2% South Asian = 21.8%
● EthSEQ is an analytical pipeline for identifying conserved ancestral groups
● The donor profile is compared to a reference database containing thousands
of genotypes of known ancestries collated from the 1000 Genomes Project
● STEMCELL’s internal ancestry determination workflow has been validated
using whole exome sequencing data from the Simons Genome Diversity
Project, a database that contains genotype data from hundreds of individuals
with known ancestries
EthSEQ Analysis:

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SCTi003-A Is Compatible With a Multitude of STEMdiff™ Kits

18. 18
● SCTi003-A iPSCs were transitioned from 2D culture in mTeSR™ Plus to
expansion in suspension culture for eight serial passages in TeSR™-AOF 3D
with consistent expansion
● Cells were expanded using a 250 mL Nalgene Storage Bottle with suspension
culture volumes of 15 - 60 mL
● Cells were expanded for an additional three passages using the PBS MINI 0.1
(#100-1006) with a suspension culture volume of 100 mL
SCTi003-A Is Suitable for Scale-Up in Bioreactors
TeSR™-AOF 3D
(#100-0720)
PBS MINI 0.1
(#100-1006)

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Why Use iPSCdirect™?

20. 20
iPSCdirect™: Workflow
mTeSR™ Plus
CloneR™2

21. 21
iPSCdirect™ Robustly Generates Predetermined
Confluent Monolayers of Cells

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iPSCdirect™ Characterization And Quality Control
Copy Number Variants:
Karyotype:
Undifferentiated
Marker Expression:
Attachment:
TRA-1-60 OCT4

23. 23
● iPSCdirect™ was differentiated using the STEMdiff™
Trilineage Differentiation Kit (#05230), and subjected
to flow cytometry analysis
● Two markers from each embryonic germ layer were
assessed including PAX6 and NESTIN (ectoderm),
NCAM and BRACHYURY (mesoderm), and CXCR4
and SOX17 (endoderm)
● All lineage-specific markers were expressed
by >70% of differentiated cells
STEMdiff™ Trilineage
Differentiation Kit
#05320
iPSCdirect™ Demonstrates Trilineage Differentiation Potential

24. 24
iPSCdirect™ Differentiates to Neural Progenitor Cells,
Cardiomyocytes and Hepatocyte-Like Cells
Neural Progenitors Cells Cardiomyocytes Hepatocyte-like Cells
SOX1 PAX6 DAPI
STEMdiff™ SMADi Neural Induction
Kit
#08581
STEMdiff™ Ventricular
Cardiomyocyte Differentiation Kit
#05010
STEMdiff™ Hepatocyte Kit
#100-0520

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iPSCdirect™
● Transfected iPSCdirect™ cells at 50% confluency with eGFP mRNA post thaw and recovery
● eGFP was visualized 24 hours post-transfection
iPSCdirect™ Demonstrates Transfection compatibility
DAPI
(live)
GFP

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Conclusions
SCTi003-A and iPSCdirect™
● STEMCELL has developed the healthy control human iPSC line,
SCTi003-A, manufactured to stringent quality control standards
● SCTi003-A has been successfully differentiated to 24 cell types to
date, including cardiomyocytes, neurons, and microglia
● iPSCdirect™ is a single-use, high-density version of the SCTi003-A
iPSC line
● With iPSCdirect™, cells can be thawed and prepared for
differentiation to downstream cell types in as little as 24 hours
www.stemcell.com/scti003-a
LEARN MORE:
www.stemcell.com/ipscdirect
LEARN MORE:

27. IMAGE
The Potential of Human
Pluripotent Stem Cell-
Derived Neural Progenitor
Cells
Presenter
Erin Knock, PhD
Date 2023-06-29

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Background
● BSc Biology at Acadia University
● PhD Human Genetics at McGill University
○ Genetics and epigenetics of colon cancer formation
● 8 years of postdoctoral experience at Cambridge University
and University of Toronto
○ Developmental biology of neural stem cells
○ Pluripotent stem cell models of Alzheimer’s disease
Erin Knock
Associate Director of Neural Biology
STEMCELL Technologies
Associate Director of Neural Biology, Research and Development
Speaker: Erin Knock

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Neural Progenitor Cells (NPCs) Give Rise to Neurons and Glia
Albert and Huttner 2018 Front in Neurosci

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Human Neural Progenitor Cells for Development and Disease
From Subramamian et. al., Front. Cell. Neurosci. 2020 (13)

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Human Neural Progenitor Cells for Studying Repair
From Gilbert et. al., Cells 2022 11(5)

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Human Neural Progenitor Cells for High Throughput
Pegoraro and Misteli Trends in Genetics 2017 33(9)

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STEMdiff™ NIM +SMADi for Efficient Cell Differentiation
● Combines STEMdiff™ NIM with the STEMdiff™
SMADi Neural Induction Supplement for serum-
free, directed neural induction of hPSCs.
● Dual SMAD inhibition (SMADi) inhibits TGF-β-
and BMP-dependent SMAD protein signaling,
mimicking the action of neurodevelopmental
morphogens to strongly drive the neural program
(Chambers et al., 2009).
Monolayer protocol
DAPI PAX6 SOX1 Nestin

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Features of Frozen Human Neural Progenitor Cells
Express Expected
NPC Makers
DAPI GABA TuJ-1
Differentiate to
Downstream Cell Types
Expand Over
Multiple Passages
DAPI PAX6 SOX1 Nestin
Average = 2.8 ± 0.5-fold

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Expression of Expected Proteins by Immunochemistry

36. 36
Comparison of Gene Expression to hPSCs, hPSC-Derived NPCs
NPC experiment 2
NPC experiment 1
hiPSC
Time_Point_Experiment

37. 37
Greater Than 2 Fold Expansion of Neural Progenitor Cells
Yield: 220 million viable NPCs from a single vial of cryopreserved PSCs.

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Efficient Differentiation to Forebrain Neurons
DAPI
GFAP
βIII-TUB

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Retention of Primarily Neurogenic Capacity Up To Passage 5
2
77.1% βIIITUB
13.4% S100B
4
68.8% βIIITUB
30.4% S100B
6
34.0% βIIITUB
36.0% S100B
0 (direct from thaw)
95.0% βIIITUB
1.3% GFAP
500 µm

40. 40
Efficient Differentiation to Astrocytes

41. 41
Retention of Gliogenic Capacity Remains Up To Passage 10
6
85.1% GFAP
84.9% S100B
10
81.6% GFAP
83.6% S100B
0 (direct from thaw)
80.8% GFAP
82.4% S100B
500 µm

42. 42
Re-Cryopreservation of Neural Progenitor Cells is Possible
● Using STEMdiff™ Neural Progenitor Freezing Medium or CryoStor® CS10.
Forebrain Neurons from re-
cryopreserved p2 NPCs
Forebrain Neurons from fresh
p2 NPCs
βIIITUB
GFAP
500 µm

43. 43
Conclusions
Frozen Human Neural Progenitor Cells
● Retain expected marker expression post-thaw.
● Can be reliably passaged up to 5 passages with average
expansion rate of 2.8 fold, and maintain >50% βIIITUB
differentiation to neurons.
● Can be reliably passaged up to 10 passages while
maintaining >70% GFAP/S100β differentiation to
astrocytes.
● Can be expanded and re-cryopreserved if desired.
www.stemcell.com/npcs
LEARN MORE:

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Scientists Helping Scientists™
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please contact your sales representative or visit www.stemcell.com
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