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Figure 1A. hPSCs Maintained in �ձ𳧸�™-������ with Daily and Restricted Feed Schedules Exhibit Comparable Colony Morphology

hPSCs were maintained on Vitronectin XF™ for five passages. Phase-contrast images were taken on day 7 after seeding. For restricted feeds, hPSCs were fed with a double volume (4 mL) of medium on day 2 after passage, followed by two consecutive skipped days of feeds, with a final single-volume feed (2 mL) on day 5, prior to passaging on day 6 or 7.

Figure 1B. hPSCs Maintained in �ձ𳧸�™-������ with Daily and Restricted Feed Schedules have Comparable Expansion Rates

hPSCs were maintained on Vitronectin XF™ for five passages. At the end of each passage cell counts were obtained using the Nucleocounter®️ NC-200 ChemoMetec automated cell counter to count DAPI-stained nuclei. The log2 transformed cumulative fold expansion was plotted against time in culture (days).

Figure 2. Native bFGF Levels are Stabilized at 37°C in �ձ𳧸�™-������

�ձ𳧸�™-������ and TeSR™-E8™ were incubated at 37°C for 24, 48, and 72 hours. FGF2 levels were measured by Meso Scale Discovery (MSD) immunoassay; data was normalized to t = 0 levels for TeSR™-E8™ and �ձ𳧸�™-������, respectively. FGF2 levels in �ձ𳧸�™-������ remain at 36.7 ± 5.61% of t = 0 levels at 72 hours when incubated at 37°C. Data representative of n = 3 biological replicates ± SD.

Figure 3. hPSCs Cultured in �ձ𳧸�™-������ with Restricted Feeding Maintain Demonstrate Classic hPSC Colony Morphology

hPSCs maintained in �ձ𳧸�™-������ were passaged as aggregates with ��𳢱𳧸�™ passaging reagent every 6-7 days for greater than 10 passages. hPSCs maintained in �ձ𳧸�™-������ exhibit hPSC-like morphology, forming densely packed, round colonies with smooth edge morphology. Homogeneous cell morphology characteristic of hPSCs are observed, including large nucleoli and scant cytoplasm.

Figure 4. hPSCs Maintained in �ձ𳧸�™-������ Have Improved Attachment and Higher Overall Expansion Compared to Low-Protein Medium

(A) hPSCs cultured in �ձ𳧸�™-������ demonstrate a higher plating efficiency compared to hPSCs maintained in low-protein medium (TeSR™-E8™). Plating efficiency is calculated by seeding a known number of aggregates and comparing to the number of established colonies on day 7. (B) hPSCs maintained in �ձ𳧸�™-������ exhibit a higher average fold expansion per passage compared to TeSR™-E8™. (C) hPSCs cultured in �ձ𳧸�™-������ demonstrate consistent expansion and minimal cell line-to-cell-line variability between ES and iPS cell lines assessed. Cumulative fold expansion was measured from passage 1 to 5. Data represented as mean plating efficiency or fold expansion across 10 passages ± SD. MG = Matrigel®; VN = Vitronectin XF™.

Figure 5. hPSCs Cultured in �ձ𳧸�™-������ with Restricted Feeding Maintain a Normal Karyotype

ES (H9 & H1) and iPS (STiPS-M001 & STiPS-F016) cell lines cultured in �ձ𳧸�™-������ were screened for chromosomal abnormalities using the hPSC Genetic Analysis Kit and by G-banding at ≥ 10 passages. Representative data are shown for (A) H1 ES and STiPS-F016 hiPS cell cultures at passage 10; no common chromosomal abnormalities were detected using the hPSC Genetic Analysis Kit, and (B) H1 ES cultures; these displayed a normal karyotype by G-banding at passage 11 and 13 respectively.

Figure 6. hPSCs Cultured in �ձ𳧸�™-������ Express Markers of the Undifferentiated State and Differentiate to the Three Germ Layers

(A) hPSCs maintained in �ձ𳧸�™-������ exhibit high levels of TRA-1-60 and OCT4 by flow cytometry at passage 5 and 10. Across n = 5 cell lines, the average TRA-1-60 expression was 92.8 ± 3.77 %, and percent OCT-4 positive cells were 98.1 ± 1.79%. Data shown represent an average of passage 5 and 10 flow results for each cell line. MG = Matrigel®; VN = Vitronectin XF™. (B) Efficient differentiation to the three germ layers was demonstrated in one hES and one hiPS cell line maintained for > 5 passages in �ձ𳧸�™-������. Cultures were processed for flow cytometry and assessed for CXCR4+/SOX17+ cells on day 5 following differentiation using the STEMdiff™ Definitive Endoderm Kit. Cultures were processed for flow cytometry and assessed for Brachyury (T)+/OCT4- cells on day 5 following differentiation in STEMdiff™ Mesoderm Induction Medium. Cultures were processed for flow cytometry and assessed for PAX6+/Nestin+ cells on day 7 following monolayer differentiation using STEMdiff™ Neural Induction Medium.

Figure 7. hPSCs Culture in �ձ𳧸�™-������ Differentiate to Cardiomyocytes with the STEMdiff™ Cardiomyocyte Differentiation Kit

Efficient differentiation to cardiomyocytes was demonstrated in 1 hES and 1 hiPS cell line maintained in �ձ𳧸�™-������ using the STEMdiff™ Cardiomyocyte Differentiation Kit. Expression of cardiac troponin T (cTnT) was assessed by immunocytochemistry (ICC) and by flow cytometry.