Figure 3.

Adherent Enrichment and Expansion of Primary Human Basal PESCs In Vitro

(A–C) Combinatorial testing of factors for optimal expansion and enrichment of human PESCs. Starting with the defined MPM composition, the effect of different factors was determined by changes in the cell numbers after 120 hr (proliferation index 120 hr), n = 8 independent PESC preparations each. Statistical significance was evaluated by one-way ANOVA followed by Bonferroni post hoc tests, p < 0.05.

(A) Initial cell population after EPCAM enrichment and plating with ROCK inhibitor; additional supplementation of trace elements A/B/C, N2 supplement, BSA, BME, and lipid mix.

(B) Addition of N2 supplement to the MPM formulation is the main driver of proliferation for human basal PESCs.

(C) Progesterone and sodium selenite are the N2 constituents necessary for proliferation (HPM conditions).

(D) Micrographs, IHC, and immunofluorescence of cultured human basal PESCs, basal marker TP63, the luminal marker CK8, and the prostate cancer marker AMACR. Scale bars, 20 μm and 100 μM (for the immunofluorescence pictures). Flow-cytometric analyses of human basal PESCs at passage 5 for expression of CD49f, TROP2, EPCAM, and CD31.

(E) Amplification (cell numbers) of CD49f⁺/TROP2^high cells using the HPM culture method; n = 3 independent PESC preparations.

(F) Cloning efficiency as determined by in vitro limiting dilution and Matrigel-sphere-forming capacity of human PESCs cultured under the final optimal conditions (Primaria surface and HPM); n = 5 independent PESC preparations, p < 0.01 as determined by Student’s two-tailed t test.

(G) Morphology and expression of TROP2 and CD49f of differentiated human prostaspheres.

See also Figure S3 and Tables S1, S2, and S3.