STEMdiff APEL Medium

Defined, Animal Component-Free Basal Medium for hPSC Differentiation

STEMdiff APEL Medium is a defined, serum-free and animal component-free medium specifically developed to support human pluripotent stem cell (hPSC) differentiation along ectoderm, mesoderm and endoderm lineages. Developed by STEMCELL Technologies and based on a published formulation, STEMdiff
1

FigurE 1. Differentiation of hPSCs into hematopoietic cells using STEMdiff APEL Medium

A
10 4
2.45%

hiPS-4D1 cells
5.63%

H9 cells
10 4
6.10% 5.30%

APEL Medium is a versatile, growth factor-free, basal medium

CD45 APC

Although optimized to be used following maintenance in either mTeSR1 or TeSR2 media and using the EB-based AggreWell system, this medium can also be used to differentiate cells in adherent cell-based systems. For examples of differentiation protocols in which STEMdiff APEL Medium has been used, please contact techsupport@stemcell.com.

102

CD45 APC
80.4% 11.5%

that is to be supplemented with specific lineage-inducing factors.

10 3

10 3

102

101

101

10 0

101

102

10 3

10 4

10 0

69.5%

19.1%

101

102

10 3

10 4

CD34 PE

CD34 PE

B
10 4
1.33% 8.48%

C
Percent Positive Cells (d13)

50 45 40 35 30 25 20 15 10 5

%CD34+

%CD34+ CD45+

10 3

CD31 FITC

102

101

10 0

81.7%

8.48%

0
1 2 3 4 5 6 7 8 9 10 11 12

101

102

10 3

10 4

CD34 PE

Experiment Number

(A) hiPS-4D1 cells (left) and H9 cells (right) were differentiated based on Ng et al.1 and Chadwick et al.,2 with the following changes: (1) STEMdiff APEL Medium was substituted for DMEM with 20% FBS as a basal medium; (2) cells were maintained prior to differentiation for at least 10 passages in mTeSR 1 Maintenance Medium and Matrigel; (3) the entire differentiation procedure was performed in adherent cell culture, on a Matrigel coated surface instead of an EB-based method. Cells were analyzed by flow cytometry on Day 13 of differentiation culture for expression of hematopoietic markers CD34 and CD45. (B) CD34hi cells co-expressed CD31, indicating that endothelial cells are also produced with this protocol. (C) In 12 experiments, an average of 28.0%

Advantages of STEMdiff

APEL

Medium

( 8.6 STDEV standard deviation) of cells expressed CD34 on Day 13, and 7.3% ( 5.1%) of the cells were CD34+CD45+ double positive.

Defined Animal component-free Versatile, growth factor-free formulation to allow for differentiation to multiple lineages

Product Information
ProDuCT NAME STEMdiff APEL Medium uNIT SIzE 100 mL CATALog # 05210

T H E C E L L E X P E RT S

W W W.S TEM C EL L . C O M

29156

V ER SI O N 1 .0.0 | JUNE 2011

TOLL- FREE T. 1 8 0 0 667 0322 ORDERS@STEMCELL .COM

T. +1 60 4 877 0713

TOLL- FREE F. 1 8 0 0 567 2899

F. +1 60 4 877 070 4

INFO@STEMCELL .COM

FOR FULL CONTAC T DETAILS WORLDWIDE VISIT OUR WEBSITE

F O R R E S E A R C H U S E O N LY. N O T F O R T H E R A P E U T I C O R D I A G N O S T I C U S E .

STEMdiff APEL Medium

Defined, Animal Component-Free Basal Medium for hPSC Differentiation

FigurE 2. Differentiation of hPSCs into cardiomyocytes using STEMdiff APEL Medium

FigurE 3. Differentiation of hPSCs into definitive endoderm using STEMdiff APEL Medium and published cytokines

T EAC H N I C A L B U L L E T I N
100 TM
TM

10 4

10 4

Percent Beating EBs

CXCr4-PE

CXCr4-PE

STEMdiff APEL Medium: Basal Medium for Differentiation 90 10 of Human PSCs to Multiple Lineages 80
3

10 3

70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9

102

102

101

101

10 0 10 0

101

102

10 3

10 4

10 0 10 0

101

102

10 3

10 4

SoX17-APC

SoX17-APC

Experiment Number

B
10 4
3.71%

10 3

H9 hESCs were differentiated based on Rezania et al.4 with the following changes: (1) STEMdiff APEL Medium was substituted for RPMI with 2% BSA as a basal medium; and (2) cells were maintained prior to the differentiation for at least 10 passages in mTeSR 1 Maintenance Medium and Matrigel. On Day 4, cells were highly positive for CXCR4 and SOX17, which together mark definitive endoderm. Left: cells cultured in STEMdiff APEL Medium alone (no cytokines added); Right: differentiated cells after culture in STEMdiff APEL Medium with inducing cytokines.

cTnT FITC

102

Support Products
ProDuCT
0 200 400 600 800 1000

101

CATALog #
05850 / 05870 05875 / 05857 BD Catalog #354277 04434 07920 36254 07923 07171 / 07172 27845 / 27945 27865 / 27965 27840 / 27940 27215 / 27250 18167 / 18147 02514 02524 02628 / 02828 02634 02630 / 02830 02503 / 02603 02506 / 02606 02615 / 02855 02640 / 02840 10413 10513 10613 10534 10417

10

mTeSR 1 Defined Maintenance Medium

TM

FSC

MatrigelTM MethoCultTM Classic H4434 AccutaseTM DMEM/F12 Dispase (1 mg/mL) Y-27632 AggreWellTM400 plates AggreWellTM800 plates AggreWellTM400Ex plates 37 m Reversible Cell Strainer, Small/Large EasySepTM hESC-Derived CD34 Positive Selection Kit rh Activin A Bone Morphogenetic Protein-4 (BMP-4) rh VEGF rh bFGF rh SCF rh IL-3 rh IL-6 rh G-CSF rh Flt-3/Flk-2 Ligand

hPSCs were differentiated into cardiomyocytes based on Yang et al. 3 with the following changes: STEMdiff APEL Medium was substituted for StemPro -34 SFM as a basal medium; (2) cells were maintained prior to differentiation for at least 10 passages in mTeSR 1 Maintenance Medium and Matrigel; (3) EBs were formed in AggreWell 400 plates, and (4) the entire procedure was carried out within the AggreWell 400 plate. (A) Beating EBs were counted on Day 16 of the culture, and varied between 5% and 95% of total EBs (n=9). (B) Expression of cTnT was measured on Day 16 of the culture by flow cytometry. Shown is a representative example of cTnT positive cells, from directed differentiation of H9 hESC.

references
1. 2. 3. 4. Ng ES et al. Nat Protoc. 3:768-776, 2008 Chadwick K et al. Blood. 102:906-915, 2003 Yang L et al. Nature. 453:524-528, 2008 Rezania A et al. Diabetes. 60:239-247, 2011

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CD34 Antibody, Clone 8G12, FITC-Conjugated CD34 Antibody, Clone 8G12, PE-Conjugated CD34 Antibody, Clone 8G12, APC-Conjugated CD34 Antibody, Clone 563, PE-Conjugated CD45 Antibody, Clone 2D1, FITC-Conjugated