In Vitro Differentiation of Hpscs by Standardised Methods

In vitro differentiation of hPSCs by standardised methods Orla O’ Shea1, Shalinee Khadun1,2 , Lyn Healy1, Anwar Baydoun2, Glyn Stacey1 1 UKSCB, NIBSC, Blanche Lane, South Mimms, Potters Bar, Herts, EN6 3QG 2 School of Life Sciences, University of Hertfordshire, Hatfield, Herts, AL10 9AB

INTRODUCTION

Human pluripotent stem cells (hPSCs) are valuable models to study disease and drug development, as they can be cultured indefinitely in vitro, and have the potential to form almost any cell types in the body. Cells used for regenerative medicine and clinical purposes must be processed, stored and tested to appropriate quality standards. The UK Stem Cell Bank (UKSCB) produces research grade ‘seed stocks’ of human embryonic stem cells (hESCs) under a HTA licence. It also assures the quality of banked cells through release QC testing. As the interest in using stem cells for clinical application continues to rise, the UKSCB has investigated and devel-

oped robust methods to suitably characterise pluripotency of banked hESCs. As part of our aim to support Figure 1: H9 Figure 2: NIBSC5 the wider stem cell community, the UKSCB has collaborated with the EU Framework 7 project 'Embryonic Stem cell-based Novel Alternative Testing Strategies' (ESNATS), on methods for toxicology screening of Figure 1-3: Shows hESC lines H9, NIBSC5 and NCL5 day 7: compounds using differentiated hESCs and has led to the development and standardisation of neuronal dif- brightfield A -Endoderm FP; B -Mesoderm FP: C-Ectoderm FP; ferentiation toxicology assays. This work has been further extended to qualify assays for the directed differ- D-Endoderm EB; E- Mesoderm EB; F-Ectoderm EB; entiation of hESCs to both primitive mesoderm and endoderm lineages. G-Sox17 IF Endoderm: H-Hand1 IF Mesoderm; I-(Pax6 NIBSC5 only) Nestin IF Ectoderm. Pluripotency is a key characteristic of hESCs and is defined as the ability to give rise to cells from all three All brightfield images taken at x4 germ layers of the body .This capability can be assessed in vivo using the teratoma assay, or in vitro, by the All IF images taken at x20 directed differentiation of the cells into all three germ layers. Many protocols have been published that ex- plore the use of small molecules and growth factors to induce differentiation Herein, we compare two different protocols; one EB generating and one monolayer adherent (flat plate), to demonstrate the pluripotency of two hESC lines and one iPSC line in vitro.

Figure 3: NCL5 MATERIALS AND METHODS

Human ESC and iPSC Culture and In Vitro Differentiation

All cell cultures were maintained at 37°C, 5% CO2. The hESC line H9 (WiCell) was propagated on mitomy- cin-C inactivated mouse embryonic fibroblast (iMEF) feeders layers with HES media [Knockout DMEM (Gibco), 20% knockout serum replacement media (Gibco), 1% Glutamax (Gibco), 1% non-essential amino acids (Gibco), 0.1% mercaptoethanol (Gibco), and 10ng/ml bFGF (Invitrogen). The iPSC line NIBSC5 and the hESC line NCL5 (Newcastle) were propagated on Matrigel (BD Sciences) coated 6 well plates using mTeSR1 medium (StemCell Technologies). Undifferentiated cells were passaged every 4-5 days by TrypLE Express (Gibco). Figure 4: Gene expression changes for endoderm for all cell lines for both EB Figure 5: Gene expression changes for mesoderm for all cell lines for both EB Two protocols were used to assess differentiation potential, based on methods of D’Amour 2005, Sa 2011, and FP methods. and FP. methods Chambers 2009. 1: Embryoid Bodies (EB) protocol: EB's containing 3000 cells were generated using Ag- grewells (StemCell Technologies) according to manufacturer's instructions. 2: Flat plate protocol (FP): hESCs were dissociated into single cells and seeded into a 6 well plate, coated with Matrigel, at 2x105 cells per well. In both protocols differentiation was induced by treating the hESCs with Knockout DMEM [Knockout replacement media ,(20% knockout replacement media (1% Glutamax, 1% nonessential amino acids, and 0.1% mercaptoethanol, with the following to induce Endoderm: 100ng/ml Activin A (R&D Systems), 0.1ng/ml Wnt3 (Day1 only) (R&D Systems), Mesoderm: 20ng/ml BMP4(R&D Systems), 20ng/ml Activin A (R&D Systems), Ectoderm: 10M SB 431542 (R&D Systems), 600ng/ml Dorsomorphin (R&D Systems), 35ng/ml Noggin (R&D Systems). EB's were placed in 10ml of differentiation media in non-tissue cultured treated petri dishes and left for 7 days. For the flat plate based assay 2mls of differentiation media was add- Figure 6: Gene expression changes for endoderm for all cell lines for both EB ed and the media was changed every 2 days for 7 days. EBs were also left to spontaneously differentiate Figure 7: Heat map comparison of the three germ layers demonstrates the simi- and FP methods. for 7 days in KRS. hESCs were then harvested for RNA extraction using Qiagen Rneasy Mini Kit according larities in gene expression fold change between the two differentiation methods. An increase in fold change is shown in red and decrease shown in green. to manufacturer's instructions. Comparative Real-Time PCR cDNA was produced using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer’s instructions and analysed with gene-specific probes (Applied Biosystems) by stand- ard methods and run on Quantstudio thermocycler. Each differentiated sample was compared against its undifferentiated counterpart as a normalised control using the Ct method, to give relative quantitation (RQ) values using GAPDH and ACTB as reference genes. Immunofluorescence Staining (IF) using TissueFaxs™ imaging system H9, NCL5, and NIBSC5 stem cell lines were cultured on Matrigel (BD) coated 24 well plates at 5x104 cells per well, in varying differentiation media's (see above) for 7 days. The cells were fixed and permeabilised with 1:1 ice-cold acetone/Methanol for 10 minutes. The cells were blocked with 10% FCS for 10 minutes. Figure 8: Spontaneous differentiation of EB’s fro each cell line. Primary antibodies (Abcam) were diluted 1/50 and l was applied to each well and incubated at room temperature for 4 hours. Secondary antibodies were diluted 1/200 (Abcam) and 300ul was applied to each well and incubated at room temperature for 1 hour. Cell nuclei were counter-stained with DAPI. Images CONCLUSION were acquired using TissueFaxs™. Statistical analysis

Geometric mean and Log2 was determined for each RQ value from each stem cell line and displayed as a  Both methods were successfully able to induce specific germ layer differentiation. heat map, to demonstrate method comparison of gene expression fold changes. Analysis was performed  Successful on both hESCs and iPSCs. RESULTS  Confirmed by Real time PCR and IF staining using TissueFaxs imaging system  Successful from hESCs cultured on iMEF or Matrigel.

Results demonstrate that both methods were successfully able to induce germ layer specific differentiation  Consistency was demonstrated from both methods across a number of germ layer specific genes. in all 3 stem cell lines.  Cost/time are the same for each protocol. Clear changes in morphology were seen on D7 FP ectoderm, mesoderm and ectoderm ,from all 3 stem cell  Strongest fold gene expression changes were seen in mesoderm. lines (A-C Figures 1,2,3). EBs also showed changes in morphology, both in shape ands size (D-F Figures 1,2,3). Tissuefaxs imaging confirmed fluorescence stained for a specific germ layer marker from each cell  Spontaneous differentiation as shown by KRS data was consistent for ectoderm gene upregulation from line G-I Figures (1,2,3). IF staining of EBs was unsuccessful due to high cell density and 3D structure caus- all three hPSC lines. ing imaging difficulties. RT-PCR showed increased expression of gene specific markers for each germ layer after FP differentiation ACKNOWLEDGEMENTS with upregulation of mesoderm specific genes HAND1, FGB and PiTX1, ectoderm specific genes Hes5 and FOXG1 and endoderm specific gene AFP, from most cell lines. Consistency was demonstrated by upregulation of the following genes from both methods, endo- Special thanks to Harsharon Bahia, Lesley Young, Pamela Pipkin and Alan Heath derm:SOX17, FOXA2; mesoderm: HAND1, BMP4; ectoderm: FOXG1, Hes5 (Figure 7). The UKSCB is funded by the MRC and BBSRC. Orla ‘O’ Shea is funded by EC FP7 Grant no.201619 Differentiation of EB’s from 3 cell lines in KRS for 7 days demonstrated down regulation of Oct4/Nanog and The UKSCB would like to thank the depositors of NCL5 (University of Newcastle) and H9 (WiCell, Wiscon- up regulation of markers from each germ layer, with NIBSC5 showing the highest expression of differentia- sin). tion genes, for all germ layer. H9 and NCL5 appear to be favouring ectoderm differentiation, demonstrated by upregulation of NEUROD1, SOX1 and HES5 (Figure 8).