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  • During development extrinsic signals in a progenitor s

    2018-10-20

    During development, extrinsic signals in a progenitor’s microenvironment provide cues for self-renewal and lineage commitment. Although several growth factors, including fibroblast growth factors (FGFs) 2 (Perantoni et al., 1995), 8 (Perantoni et al., 2005), 9, and 20 (Barak et al., 2012) and epidermal growth factor (EGF)/transforming growth factor α (TGF-α) (Rogers et al., 1992), support the survival of MM KU0059436 and facilitate the limited expansion of this population in culture, they have proven to be insufficient for long-term propagation of progenitors with stem-like properties and nephronic potential. In this study, we optimize the niche for rat progenitors using growth factors, extracellular matrix, and Rho kinase inhibitor, which, in combination, sustain SIX2 and YAP nuclear expression. Moreover, we demonstrate that these factors contribute to the preferential propagation and partial stabilization of MM progenitors with the preservation of stem cell markers and a capacity for differentiation.
    Results
    Discussion Previously we reported the propagation of rMM progenitors with FT medium and short-term retention of functionality (Tanigawa et al., 2011), but, here, we demonstrate that cells lose Six2 and Pax2 expression in primary culture and become unresponsive to inductive signals. SIX2+/PAX2− mMMs were passaged successfully as nephrospheres, but the cells failed to epithelialize at any passage (Lusis et al., 2010). PAX2 facilitates competency in mMM progenitors, allowing them to undergo MET (Brophy et al., 2001), and is likely required for the long-term inducibility of these cells. As shown here, the combination of LIF and Y27632 sustained Pax2 expression in rMMs and stimulated nuclear levels of pPAX2. Therefore, both SIX2 and PAX2 may be functional determinants for the successful culture of nephronic stem cells. FTLY preferentially expanded the SIX2+ population in our mixed MM cultures. The exact basis for this is unknown but may be due to the elaboration of the LIF receptor on cap mesenchyme cells (Plisov et al., 2001). In embryonic rat kidney, LIFR antibody labeled the membranes of cap cells, whereas membrane staining of the interstitial stroma was not apparent. By whatever mechanism, the selectivity complicates our findings because many of our experiments were performed over a 7- to 10-day period, during which selection had occurred. Regardless, the use of an internal low-affinity binding site as the control should have partially mitigated any selectivity in our ChIP experiments. Moreover, the combination of STAT redundancy, partial retention of SIX2, and 3-day time points should have precluded the selective loss of SIX2+ cells in the Stat knockdown studies. Still, further study of the direct role of LIF on Six2 expression in these cells is clearly warranted. We demonstrate that LIF affects signaling in MM progenitors in a concentration-dependent manner (Figure 7; Figure S7). At 1 ng/ml, it induces phosphorylation primarily of STATs 1, 3, and 5 as well as the expression of several progenitor markers, whereas, at 50 ng/ml, it additionally activates both PLCγ and JNK. Because the higher concentration of LIF is associated with the induction of MET and nephronic tubule formation (Barasch et al., 1999; Plisov et al., 2001), it is possible that PLCγ and JNK are involved in those morphogenetic processes. The PLCγ metabolite IP3 activates PROTEIN KINASE C, which initiates an intracellular calcium cascade. Because knockdown of PLCγ upregulated the expression of the stem markers SIX2 and YAP, it is likely that LIF-activated PLCγ helps override the stemness program to direct cells to form tubules. This is supported by the fact that PLCγ-deficient chimeric mice develop cystic kidneys, a symptom of aberrant tubular proliferation (Shirane et al., 2001). In this study, LIF also induced JNK phosphorylation and upregulated the expression of the differentiation markers Lim1 and E-cadherin. This induction was attenuated by treatment with a JNK inhibitor. Furthermore, both the JNK and ROCK inhibitors facilitated expression of the progenitor marker Slug but, interestingly, only in concert with LIF. These findings suggest that the role of Y27632 may be to limit the LIF-induced, JNK-mediated differentiation of rMMs and, instead, help sustain cells in a more primitive state.