Table 2.

Considerations for PSC culture.

Source material	Study design	Reprogramming method	Replicates
In-house derived hPSCs: - Dependent on access to somatic cell sources (skin, blood, urine), ease and efficiency of reprogramming (e.g., urine is easily accessible, but more laborious than other sources to reprogram) - Age (younger cells reprogram more efficiently) - Access to patients with mutations of interest can be challenging Purchased cell lines (e.g., Coriell, EBISC, StemBANCC, HipSci, NINDS): - Dependent on price, availability of desired mutation, patient medical data - Assess restrictions on use (expansion, banking, differentiation, commercial use) - Verify appropriate patient consent and institutional review forms available (ICF and IRB review and approval)	- Biological replicates are critical: collect from multiple donors where possible - Non affected familial control or isogenic wild-type lines are necessary - For case-control study designs: multiple clones required - Consider genetic engineering if controls or lines with mutation of interest are unavailable	Integrating vs. non-integrating reprogramming: - Genomic integration of vector can lead to unwanted secondary mutations and reactivation of reprogramming factors - Certain vectors are more effective with certain cell types (Okita et al., 2013) - Labor cost: non-integrating methods often require specialized equipment (e.g., electroporator) - Financial cost: increasing efficiency typically comes with higher cost - Selection of reprogramming factors (e.g., OCT4, SOX2, c-MYC, KLF4) - Consider published protocols to increase reprogramming efficiency (Huangfu et al., 2008; Esteban et al., 2010) - Selection of high efficiency reprogramming medium	Accounting for inherent variability among hPSC lines: - Include hPSCs from at least 3 similarly affected subjects. If not possible, use at least 3 hPSC clones per donor - Ensure all experimental lines are of a similar age - Sex balance: use an equal number of female and male hPSC lines - Consider other potential mitigating health factors from sample (other health issues, diet, potential genetic modifiers)
Culture Considerations	Quality Control	Maintenance	Differentiation/Maturation Method
- Dedicated Biological Safety Cabinets and safety protocols for human tissue work - Separate space for human vs. other mammalian culture preferable - Asses incoming human samples for presence of infectious viruses - Mycoplasma testing of all new cell lines - Cryogenic storage essential for biobanking viable hPSCs	- Daily morphological assessment: visibly differentiated cells can be manually removed - Karyotype all new lines, genetic analysis (QPCR for common abnormalities) for new thaws and before important experiments - Assess pluripotency (teratoma or trilineage differentiation) of all new lines - Assess markers of pluripotent state (e.g., OCT4, TRA-1–60) for all new lines	- Choose between defined (feeder and serum free) or undefined (feeders and serum/serum replacement, sometimes more efficient for differentiation) culture conditions - Clump vs. single cell passaging (single cell passaging can increase chromosomal abnormalities) - Matrix: defined (vitronectin, laminin) or undefined (matrigel) - Low protein, robust or stabilized growth media - Regulatory compliance of hPSC growth media- Feed, passaging and banking schedule	- Developmental patterning to drive lineage induction vs. forward programming (e.g., NGN2 overexpression) - Pure populations or co-cultures - 2D (largely homogenous cell types) vs. 3D (e.g., heterogeneous organoids) architecture - Transdifferentiation (retain epigenetic signature of source by skipping PSC stage) - Identify best time-points to freeze and biobank cells, with high post-thaw viability - Maturation strategy (e.g., increased culture time, physiological maturation medium, addition of cell stressors)