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. 1997 Jun 10;94(12):6279-84.
doi: 10.1073/pnas.94.12.6279.

An in vitro tubulogenesis system using cell lines derived from the embryonic kidney shows dependence on multiple soluble growth factors

H Sakurai  1 , E J Barros, T Tsukamoto, J Barasch, S K Nigam
Affiliations

An in vitro tubulogenesis system using cell lines derived from the embryonic kidney shows dependence on multiple soluble growth factors

H Sakurai et al. Proc Natl Acad Sci U S A. .

Abstract

Interactions between the ureteric bud (UB) and metanephric mesenchyme are crucial for tubulogenesis during kidney development. Two immortalized cell lines derived from the day 11.5 embryonic kidney, UB cells, which appear to be epithelial (cytokeratin-positive, E-cadherin-positive, and ZO-1-positive by immunostaining) and BSN cells, which are largely mesenchymal (vimentin-positive, but negative for cytokeratin, cell surface E-cadherin, and cell surface ZO-1), were used to establish an in vitro tubulogenesis system. BSN cells expressed hepatocyte growth factor (HGF) and transforming growth factor-beta1 mRNAs, and its conditioned medium (BSN-CM) contained factors capable of activating the epidermal growth factor (EGF) receptor (EGFR). When UB cells were cultured in an extracellular matrix gel in the presence of the embryonic kidney or BSN-CM, the UB cells underwent morphogenetic changes characteristic of early in vitro branching tubulogenesis. These changes were largely inhibited by a combination of neutralizing anti-HGF antibodies and the EGFR inhibitor tyrphostin AG1478, suggesting that EGFR ligands, together with HGF, account for much of this early morphogenetic activity. Nevertheless, there was a significant fraction of tubulogenic activity that could not be inhibited, suggesting the existence of other soluble factors. Whereas HGF, EGF, transforming growth factor alpha, basic fibroblast growth factor (bFGF), and insulin-like growth factor 1 (IGF-1), or a mixture of these growth factors, induced epithelial processes for up to 3 days, only IGF-1, possibly bFGF, and the mixture were able to sustain morphogenesis for longer periods, though not nearly to the same degree as BSN-CM. Moreover, only BSN-CM induced branching tubular structures with clear lumens, consistent with the existence of other soluble factors crucial for the formation and/or maintenance of branching tubular structures with lumens in vitro.

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Figures

Figure 1
Figure 1
Immunocytochemical characterization of UB and BSN cells. Confluent monolayers of UB and BSN cells were stained for cytokeratin, vimentin, E-cadherin, ZO-1, and binding of Dolichos biflorus lectin. (×450.)
Figure 2
Figure 2
Northern blots of total RNA extracted from UB and BSN cell monolayers were probed for HGF, c-met, and TGF-β1. Ethidium bromide staining of 28S rRNA was used to estimate RNA loaded in each lane.
Figure 3
Figure 3
UB cells exhibit multicellular cords, tubules with lumens, and multicellular cysts in ECM gels in BSN-CM, depending on matrix composition. UB cells were suspended in 80% collagen type I/20% growth factor reduced Matrigel in the presence of BSN-CM containing 1% FCS. (A) After 7 days of culture, the cells grew to form multicellular cord-like structures. (B) After 15 days of culture, clear lumens could be seen in UB tubules. (C) When UB cells were suspended in 100% growth factor reduced Matrigel, even in the presence of BSN-CM and 1% FCS, they remained multicellular cysts even after 15 days of culture. (Bar = 50 μm.)
Figure 4
Figure 4
BSN-CM shows tubulogenic activity comparable to that elaborated by the embryonic kidney upon UB cells grown in three-dimensional ECM gels. The percentages of UB cells/colonies exhibiting processes (an early step in tubulogenesis) were used for semiquantitative measures of tubulogenic activity under each condition. The percentages were standardized to untreated control (= 100%) and presented as means ± SE. The results were representative of four separate experiments. (A) Day-13 embryonic kidneys were placed on top of the three-dimensional collagen gel culture system. UB cell tubulogenesis was evaluated at 24 hr and 5 days of culture. At 24 hr, a large portion of tubulogenic activity was inhibited by a combination of neutralizing anti-HGF antibodies (10 μg/ml) and the EGFR inhibitor tyrphostin AG 1478 (0.3 μM). However, at 5 days about 50% of tubulogenic activity could not be inhibited. (B) BSN-CM with 1% FCS was applied on top of UB cells in three-dimensional collagen gel culture. As was the case for embryonic kidney coculture, there were differences between 24 hr and 5 days in terms of tubulogenic inhibition profile by anti-HGF antibodies and tyrphostin AG 1478. (C) BSN-CM phosphorylated tyrosine residues of EGFR. A subconfluent mIMCD3 cell monolayer was stimulated with EGF (20 ng/ml) or BSN-CM in the presence or absence of tyrphostin AG 1478 (0.3 μM). The whole cell lysates were subjected to Western blotting with anti-phosphotyrosine antibodies. In the second (EGF) and fourth (BSN-CM) lanes, ≈190-kDa protein was phosphorylated. The phosphorylation was completely inhibited by tyrphostin AG 1478 (third and fifth lanes).
Figure 5
Figure 5
BSN-CM maintains UB cell morphogenesis better than any other purified growth factor or their combination. Purified growth factors, a combination of growth factors (GF-mix: HGF, EGF, bFGF, IGF-1, and GDNF), or BSN-CM was applied on top of UB cells suspended in collagen gels. Tubulogenic activity was semiquantified by evaluating percentage of UB cells/colonies with processes/cords/tubules. At 24 hr of serum-free culture, HGF, EGF, TGF-α, bFGF, and IGF-1 were as capable of inducing UB cell processes as were GF-mix or BSN-CM medium. TGF-β1, PDGF, and GDNF were not effective. At 5 days of culture (with 1% serum), only high concentrations of bFGF and IGF-1 could sustain the growth of UB cell processes into cord-like structures. BSN-CM was much more potent than any other growth factors listed or their combination. Statistical significance was determined and is presented in Table 1, where a qualitative analysis of structures is also presented.
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