Development of clinically

manufacturingcompatible
of CAR-T
induced pluripotent stem
cells from
cells (iPSC)

RATCHAPONG
1
NETSRITHONG
 2
Limitations of adoptive T cell therapy

No mature T cells – SCID-X1, RAG2-

SCID
Insufficient heathy T cells – immunosuppressive
drugs, conditioning regimen

Expand modified
Collect T cells from Modify T cells
• T cells
peripheral blood Chimeric antigen
Transfer cells to the patient
Patient receptors (CARs)
• Transgenic TCR
T cell exhaustion– long-term
ex vivo expansion
 3
Induced Pluripotent
Stem Cells (iPSCs)

• Generated from adult cells by Oct3/4, Sox2 , Klf4, c-Myc

introduction of reprogramming
factors.
• Have unlimited self-renewal
and can differentiate to any cell
type
• No ethical concern regarding
embryo destruction
http://learn.genetics.utah.edu/
 4
Combining iPSC technology with adoptive T
cell therapy
Unlimited self-renewal

Collect T cells from Differentiate to

Reprogram to
peripheral blood functional T cells Transfer cells to the patient
Healthy donor iPSC and modify
• Correct mutation
• Chimeric antigen
receptors (CARs)
• Transgenic TCR
• TCR and HLA KO

Off-the-shelf T cells
T cell development in vivo

Famili et al. Future science OA (2017)

5
 6

Generate iPSC-derived T cells in vitro

Mouse stromal cell line T cells

T-iPSCs CD34+ HSPCs Expressing Notch ligand
(OP9-DL4)
 7
HSPC differentiation
iPSCs ME HEP HSPCs

Plate iPSC single cells Re-plate Collect floating cells

Day : -3 0 2 3 5 8
Essential 8 RPMI + B27 + Ascorbic acid
CHIR9902 VEGF + FGF2
1
SB431542

Matrigel-coated plate
Netsrithong et al. Stem cell research & therapy, 2020
 8
Morphology and expression of mesodermal markers
Day 5 Day 5
Day 0

CD34
CD31
Day 8 Day 8

CD34
Scale bar = 100 μm CD43
 9
Induction of T Cells using OP9-DL4

Transfer HEPs
or HSPCs cells Collect suspension cells
Plate OP9-DL4 cells Transfer to a new
Transfer to
dish every 5 days
a new dish

Day : -2 OR -1 0 3 5 25
OP9 medium : α-MEM + 20 % FBS + 1% glutamine

10 ng/ml SCF + 10 ng/ml Flt-3L + 5 ng/ml hIL-7

OP9-DL4 cells
 10
Generation of iT Cells using OP9-DL4
CD45+ CD5+ CD7+ gated Day 25

Morphology of differentiated cells CD45+ CD5+ CD7+ gated Single positive induction
on OP9-DL4 on Day 25
 11
OP9-DL4 co-culture system
• Lack macrophage colony-
Bone marrow of stimulating factor
OP/OP MICE (MCSF/CSF1) which
would normally support
myelopoiesis
• Provide DL4, IL-7,
CXCL12 and SCF in
OP9 stromal cell line culture to support T cell
development
Overexpress
Delta-like 4 • Contamination of
xenogeneic antigen

OP9-DL4 stromal cell line

Zúñiga-Pflücker. Nature Reviews Immunology (2004)

 12

Generate iPSC-derived T cells in xenogeneic-

free culture conditions

OP9-DL4 co-culture
T-iPSCs HSPCs T cells

Feeder-free protocol

Culture well coated with T cells

rhDL4 and rhVCAM-1
12
 13
HSPC differentiation
iPSCs ME HEP HSPCs

Plate iPSC single cells Re-plate Collect floating cells

Day : -3 0 2 3 5 8
Essential 8 RPMI + B27 + Ascorbic acid
CHIR9902 VEGF + FGF2
1
SB431542 IL-3 + IL-6 + SCF +
FLT3L
Matrigel-coated plate
Expression of hematopoietic makers
-Cytokines +Cytokines on Day 5
Live cell gated

+SB on Day 5

CD34

+SB on Day 3

CD43
T Cell differentiation

15
 16
Induction of T Cells using StemSpan™ T
Cell Generation Kit
Transfer Day 8
Collect suspension cells
HSPCs
Coat plate Transfer to a new
Transfer to
dish every 14 days
a new dish

Day : -1 0 3 7 14 28
StemSpan™ Serum-Free Expansion Medium II

Supplement 1 Supplement 2

StemSpan™ Lymphoid Differentiation Coating Material

-Cytokines
+Cytokines T cell differentiation of Day 8 cells (added SB on Day 5)

17
 T cell differentiation of Day 8 SB cells (added SB on Day 3)
-Cytokines
+Cytokines

18
 19
HSPC differentiation
iPSCs ME HEP HSPCs

Plate iPSC single cells Re-plate Collect floating cells

Day : -3 0 2 3 5 8
Essential 8 RPMI + B27 + Ascorbic acid
CHIR9902 VEGF + FGF2
1
SB431542 IL-3 + IL-6 + SCF +
FLT3L
Vitronectin-coated plate
 T cell differentiation of Day 8 SB cells (added SB on Day 3)
-Cytokines
+Cytokines

20
 21
HSPC differentiation
iPSCs ME HEP HSPCs

Plate iPSC single cells Re-plate Collect floating cells

Day : -3 0 2 3 5 8
Essential 8 RPMI + B27 + Ascorbic acid
CHIR9902 VEGF + FGF2
1
SB431542 IL-3 + IL-6 + SCF +
FLT3L
Vitronectin-coated plate
Expression of hematopoietic makers

-Cytokines +Cytokines
Live cell gated

CD34

CD43
 23
Day 8
HSPCs
4 weeks

Tdiff medium in DL4+ VCAM1

4 weeks

Tdiff medium in StemSpan™ coating

4 weeks

StemSpan™ medium in DL4+VCAM1

4 weeks

StemSpan™ medium in StemSpan™ coating

 T cell differentiation of Day 8 SB cells (with cytokines)

Tdiff medium + DL4/VCAM1Tdiff medium + SS coating SS medium + DL4/VCAM1 SS medium + SS coating

 T cell differentiation of Day 8 SB cells (without cytokines)
Tdiff medium + DL4/VCAM1

Tdiff medium + SS coating

 T cell differentiation of Day 8 SB cells (without cytokines)
SS medium + DL4/VCAM1

SS medium + SS coating
 27
Day 8
HSPCs
4 weeks

StemSpan™ medium in DL4+VCAM1

4 weeks

Tdiff medium in DL4+VCAM1

4 weeks

Medium 1 in DL4+VCAM1
4 weeks

Medium 2 in DL4+VCAM1
 28
HSPC differentiation
iPSCs ME HEP HSPCs

Plate iPSC single cells Re-plate Collect floating cells

Day : -3 0 2 3 5 8
Essential 8 RPMI + B27 + Ascorbic acid
CHIR9902 VEGF + FGF2
1
SB431542

Vitronectin-coated plate
Expression of hematopoietic makers

Day 5 Day 8
Live cell gated

CD34

CD43
Contramination!!!
 31
HSPC differentiation
iPSCs ME HEP HSPCs

Plate iPSC single cells Re-plate Collect floating cells

Day : -3 0 2 3 5 8
Essential 8 RPMI + B27 + Ascorbic acid
CHIR9902 VEGF + FGF2
1
SB431542

Vitronectin-coated plate
 32
HSPC differentiation
iPSCs ME HEP HSPCs

Plate iPSC single cells Re-plate Collect floating cells

Day : -3 0 2 3 5 8
Essential 8 IMDM
CHIR9902 VEGF + FGF2
1
SB431542

Vitronectin-coated plate
Morphology of hematopoietic makers

Day 3 Day 5 Day 8

Expression of hematopoietic makers

Day 3 Day 5 Day 8

Live cell gated

KDR

CD235a
Expression of hematopoietic makers

Day 3 Day 5 Day 8

Live cell gated

CD34

CD43
36
 37
Day 8
HSPCs
4 weeks

StemSpan™ medium in DL4+VCAM1

4 weeks

Tdiff medium in DL4+VCAM1

4 weeks

Medium 1 in DL4+VCAM1