We herein show that simple supplementation with

We herein show that simple supplementation with doxycycline greatly improves both hESC/iPSC viability and self-renewal. In practice, doxycycline dramatically enhances hESC/iPSC expandability, and the effects continue for long time periods. The effects of doxycycline are mediated by direct activation of the PI3K-AKT intracellular pathway, which has recently been reported as the most crucial signal for hESC/iPSC self-renewal (Bendall et al., 2007; Singh et al., 2012).
Results We recently showed that hNSCs are extremely sensitive to insulin, an indispensable culture supplement, and thus survival of these cells is ensured only within a narrow range of low insulin concentrations (Rhee et al., 2013). In addition, survival of hNSC derived from hESC/iPSCs is greatly dependent on the cultured cell density. Doxycycline supplementation (1 μg/ml) strikingly prevented apoptotic cell death induced by high insulin concentration or low cell density in H9 hESC-derived NSC cultures, as revealed by estimations of total numbers of viable cells and cells positive for activated caspase-3 (Figures 1A and 1B) and Annexin V/propidium iodide (PI) (data not shown). We then tested the effects of doxycycline on differentiated neurons. Dopamine, serotonin, and gamma-aminobutyric glutamate receptor antagonist (GABA)-secreting neurons are derived by terminal differentiation of hES/iPS-NSCs. The neurons underwent apoptosis if the neurotrophic factors supplemented were withdrawn, which substantially reduced the numbers of each neuronal subtype. Doxycycline largely prevented such apoptosis and neuronal loss (Figure 1C). Doxycycline from different sources (companies) showed similar effects on hNSC survival (Figures S1A and S1B available online). The doxycycline analogs tetracycline and minocycline also promoted cell survival, but other antibacterial agents did not, indicating that doxycycline-mediated cell survival is not associated with its antibiotic effect. NSCs derived from hESCs (hES-NSCs) are highly expandable for prolonged periods of proliferation through multiple cell passages (Hong et al., 2008; Ko et al., 2007). Doxycycline increased cell yields attained during expansion of H9 hES-NSCs through eight passages (Figure 1D). Neuronal differentiation of the multipassaged hNSCs treated with doxycycline was not altered (data not shown). In contrast to hES-NSCs, we have recently shown that NSCs derived from hiPSCs established using viral vector-mediated gene delivery (viral hiPSCs) exhibited early senescence and apoptotic cell death during passaging (Rhee et al., 2011). Thus, cultures of NSCs derived from viral hiPSCs (viral hiPS-NSCs) were barely maintained for two to four cell passages; no definite cell expansion took place and at the end of this period, none were viable (Rhee et al., 2011) (Figures 1E and 1F). Hemangioblasts derived from viral hiPSCs versus hESCs likewise exhibited differences in expansion potential and senescence (Feng et al., 2010). Doxycycline supplementation strikingly prevented senescence of retroviral- and lentiviral-based hiPS-NSCs during passaging (Figure 1H; data not shown). Indeed, doxycycline addition allowed the viral hiPS-NSC cultures to be maintained for at least seven cell passages and to undergo discrete cell number increases (Figures 1E and 1F). These findings strongly indicate that doxycycline supplementation circumvents the limitation of viral hiPS-NSCs as a stable cell source. Based on doxycycline-mediated effects on cell survival in neural lineages at different developmental stages, we postulated that the effects of doxycycline and its analogs would extend to cells at earlier developmental stages, such as pluripotent stem cells. Culturing human pluripotent hESC/hiPSCs is technically difficult, with problems in maintaining self-renewal and pluripotency for long periods of culture and massive cell death occurring, particularly after cell dissociation. To examine the effect of doxycycline on human pluripotent stem cell survival, hESC (H9) clusters were dissociated into one to five cells and plated on Matrigel in mTeSR1 medium in the presence or absence of doxycycline (1 μg/ml). Doxycycline supplementation strikingly increased formation of colonies that were positive for alkaline phosphatase (AP), an undifferentiated hES/iPSC marker, 5 days after plating (22.5 ± 13.5 versus 99.3 ± 11.2 colonies/cm2 without versus with doxycycline, respectively). Enhanced AP+ colony formation in the presence of doxycycline was also apparent in all other hESC (HSF6) and hiPSC lines tested (Rv-hiPS 01-1 and Rv-hiPS 02-3 (retroviral-hiPSCs); IMR90-1 and IMR90-4 (lentiviral-hiPSC); piPS-#1 and piPS-#2 (protein based-hiPSC)) (Figure 2A). The doxycycline effect was further confirmed by counting colonies expressing the other pluripotent cell markers NANOG and TRA-1-60 in cultures of the commonly used hESC lines H1, H7, H9, HUES6, and HSF6 (Figure 2B; data not shown) and in different culture conditions (Figure 2C). In gene expression arrays, expression of 35 of 39 apoptosis-related genes was lower in doxycycline-treated versus untreated hESCs, whereas the opposite pattern occurred for antiapoptotic gene expression (Figure 2D).