Fig. 3: Gene expression and transcriptomic analyses of naïve and primed hPSCs.

a Depiction of naïve- (KLF2, KLF4, STELLA, and KLF5) and primed- (CD24, OTX2, ZIC2, and DUSP6) hPSC-associated gene expression levels for H1 and H9 hESC lines analyzed by RT-qPCR. The aliquots of statically cultured, cells for each naïve (P4) and primed hPSC sample were collected for gene expression analysis before inoculating them into stirred suspension bioreactors. Those cells were counted as “NStat” and “PStat”. The aggregates of day 4 post-inoculation were collected for each naïve and primed hPSC sample for gene expression analysis. Expression was quantified relative to the housekeeping gene GAPDH and was normalized to statically cultured, primed hPSC (PStat) level (=1). All the cultures underwent fed-batch condition (60% media change, 48-h post-inoculation) for bioreactor cultures. The data presented are generated from inoculation density of 50,000 cells/mL. The data are represented as mean ± SEM (n = 3). **** = Adj-P < 0.0001, *** = Adj-P = 0.0002 and 0.0004, ** = Adj-P = 0.0021, * = Adj-P = 0.0291 using GraphPad Prism. Adj-P = Adjusted p value. RQ relative quantification. PStat = primed hPSCs cultured under static condition. PBior = primed hPSCs cultured under stirred suspension bioreactor condition. NStat = naïve hPSCs cultured under static condition. NBior = naïve hPSCs cultured under stirred suspension bioreactor condition. b RNA-sequencing analysis of naïve H9 hPSCs cultured under static suspension and static conditions. The violin plots show the enriched canonical pathways in static suspension compared to static culture (selected as a control reference) based on Ingenuity Pathway Analysis (IPA). The IPA z-score algorithm was used to identify enriched canonical pathways that are more active (positive z-score) or less active (negative z-score) according to the IPA database and observed gene expression in our RNA-seq dataset. Activated = genes expected to be upregulated (positive log2FC) if the pathway is activated according to IPA canonical pathway database. Inactivated = genes expected to be downregulated (negative log2FC) if the pathway is activated according to IPA canonical pathway database. No-expect = no direction of change expectation is available in the IPA canonical pathway database. Filter criteria for pathway enrichment significance was expected direction of change Z-score ≥ 2 or ≤−2, and –log10 (Benjamini–Hochberg multiple testing corrected p value of differential expression) ≥2. The suspension culture underwent fed-batch condition (60% media change, 48-h post-inoculation). Aggregates of day 4 post-culture were collected for the analysis. The data are representative of three replicates.