Culturing of human pluripotent stem cells in a mesoderm biased state

Here we present a new culturing system, PRIMO Plus, to generate mesoderm biased human pluripotent stem cells by cross antagonism of pro-differentiation and pro-pluripotency factors in a fully dened medium. We differentiation


Introduction
The differentiation of human pluripotent stem cells (hPSC) towards clinically relevant cells for regenerative medicine has been hampered by poor differentiation e ciencies. This may be a result, in part, of the heterogeneity that exists within stem cell cultures. We have generated a new stem cell medium PRIMO Plus, which biases cells towards mesodermal differentiation without losing pluripotency. The cells can be maintained over multiple passaged and when cultured in this medium appear to be trapped at a relevant development stage on a mesoderm differentiation trajectory that impact upon their differentiation outcome. We hope that this medium could be used as a tool in both improving the differentiation of hPSCs towards mesodermal derivatives but also to investigate the basic biological principles behind biased sub-states, the exit from pluripotency and the commitment to differentiation. The basal medium of PRIMO Plus is E8. Our E8 was made in house with a recipe adapted from Chen et al, 2011, although commercially available version can be used instead. One key difference in the in-house media from the recipe published by Chen et al, 2011 is the replacement of standard glutamine with GlutaMax (Thermo sher). Glutamax is a thermostable form of glutamine, which is bound to alanine to increase stability. Large batches of 50X E8 supplements were prepared and frozen as 10ml aliquots at -20°C. Defrosted 10ml aliquots were added to 490ml of DMEM/F12 without glutamine and ltered using a CytoOne Bottle Top Filtration Unit 0.2µM lter.
1. Dilute 5X stock 1 in 5 with E8 media, ie 10ml of stock with 40ml of E8. 3. Coat the surface of a Tissue Culture ask with Vitronectin (3ml per T25, 2ml per T12.5) and leave at room temperature for minimum of 1 hour.
Following incubation at room temperature, asks are now ready for use or alternatively can be stored in the fridge and used within 2 weeks. If stored in the fridge prior to use asks must be left to return to room temperature for a minimum of 1 hour.
6. Add 1ml ReleSR to the bottom of ask, rock the ask back and forth to ensure all cells have been coated, incubate for ~20 seconds at RT. 7. Tilt the ask su ciently that ReleSR pools in one of the bottom corners of the ask and aspirate away the ReleSR.
8. Incubate cells for 2-4 minutes at room temperature -Incubation period is dependent on cells line and colony size and should be adjusted appropriately. For large colonies the incubation time will have to be increased. Cultures on Geltrex usually require longer incubation. 9. Inactivate ReleSR by adding 2ml of PRIMO PLUS medium and slowly rocking back and forth, cells can be encouraged to detach by tapping the ask -avoid very aggressive tapping of the ask which will encourage any differentiated cells to also detach.
10. Check cells under the microscope to assess colony size 11. Resuspend pellet in appropriate volume of PRIMO Plus medium and distribute into prepared asks as relevant for the desired splitting ratio. Normally passaged as small clumps using ReleSR at a ratio of 1:6, sometimes 1:12, every 3-4 days (optimal split ratios and growth periods will have to be established experimentally for different PSC lines).
12. Final volume of media in asks or plates should be kept to a low volume, for a T25 this should be3 ml. Higher volumes result in poor plating e ciency.
14. Medium must be replaced daily because of LPAs short half-life.
15. Cells should be passaged again before reaching high con uency (over 90%), dense areas will lead to differentiation.
Important Notes: The medium is delicately balanced, over feeding can push the cells towards differentiation but under feeding can do the same as the cells degrade the LPA. We suggest to have cells growing in E8 rst and allow them to reach high con uency (~80%) then passage with standard ReleSR technique into PRIMO Plus medium. Feed so that you see the visible colour change in the media the following day. For T25s we would normally passage into 3-4ml, then over the days increasing the volume by 1-2mls up to 10ml the day before passage.

Troubleshooting
Increased Differentiation in cultures: Batches of reagents, particularly LPA and CHIR99021 may effect results, either increasing the concentration of LPA or lowering the concentration of CHIR99021 might alleviate these issues. In our hands the optimal concentrations worked well as long as cells were fed with the appropriate volume of medium daily, this might require optimisation per cell line.

Anticipated Results
After initial seeding in PRIMO Plus morphology changes might become apparent in cultures. This can appear as at, spread and distended colonies, these should compact over the days of culture, with colonies appearing rounder and with shinier edges under the microscope. After the line has adapted to the medium, colonies favour the more compact morphology post-passage but the at colonies can sometimes still be seen. When differentiation occurs, it has appeared as small, spiky, broblast like cells that do not maintain a colony morphology. This can normally be removed with accurate ReleSR passaging to leave the differentiated cells in the ask. Decreasing the incubation time of ReleSR can be helpful to achieve this clean-up.
Assessing cells in mesoderm biased state: Surface marker analysis: Cells can be harvested and assessed for pluripotency associated surface marker expression by ow cytometry. In our hands, the marker, SSEA-3 (Kannagi et al., 1983) , proved the most sensitive marker. Cultures should maintain high SSEA-3 expression between 80-100% and should be compared to the same line grown in E8 medium.

Gene expression analysis:
Gene expression for pluripotency and early mesendodermal markers can be assessed by qPCR. Example pluripotency genes to assess can include: POU5F1, NANOG, SOX2, and DNMT3B. Example mesendodermal genes to assess can include: T (TBXT), EOMES, GATA4, GATA6 and HAND1. Samples should be compared to hPSC growing in E8, in general expression of pluripotency genes should not deviate too far from E8 expression levels but mesendodermal genes should demonstrate strong upregulation.