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. 2009 Feb;42(1):29-37.
doi: 10.1111/j.1365-2184.2008.00572.x.

Conditioned medium from renal tubular epithelial cells initiates differentiation of human mesenchymal stem cells

P C Baer  1 , J Bereiter-Hahn, C Missler, M Brzoska, R Schubert, S Gauer, H Geiger
Affiliations

Conditioned medium from renal tubular epithelial cells initiates differentiation of human mesenchymal stem cells

P C Baer et al. Cell Prolif. 2009 Feb.

Abstract

Objectives: Mesenchymal-epithelial interactions play a pivotal role in tubular morphogenesis and in maintaining the integrity of the kidney. During renal repair, similar mechanisms may regulate cellular reorganization and differentiation. We have hypothesized that soluble factors from proximal tubular epithelial cells (PTC) induce differentiation of adipose-derived adult mesenchymal stem cells (ASC). This hypothesis has been tested using cultured ASC and PTC.

Material and methods: Conditioned medium was prepared from injured PTC and transferred to ASC cultures. ASC proliferation was analysed by a fluorometric and photometric assay. Signal transduction was analysed by phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2). Grade of ASC differentiation was assessed by morphological analysis and cell expression of characteristic markers.

Results: Conditioned medium significantly induced proliferation and phosphorylation of ERK1/ERK2 of ASC. After 12 days of incubation, cell morphology changed to an epithelial-like monolayer. Expression of cytokeratin 18 was induced by conditioned medium, while alpha-smooth muscle actin, CD49a and CD90 expression decreased. These alterations strongly indicate onset of the differentiation process to the epithelial lineage. In summary, soluble factors from PTC induce signal transduction and differentiation of ASC.

Conclusions: Our study shows that conditioned medium from renal tubular epithelial cells provides a convenient source of inductive signals to initiate differentiation of ASC towards epithelial lineage. We deduce that these interactions may play an important role during renal repair mechanisms.

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Figures

Figure 1
Figure 1
Characteristic Western blot analysis. (a) Phospho‐ERK1/ERK2 (upper blot) and whole ERK (lower blot) in adipose‐derived adult mesenchymal stem cells (ASC) cultured in conditioned or control medium. Lane 1, medium 199 (M199); lane 2, M199 with 10% foetal calf serum (MF); lane 3, supplemented M199 (MS); lane 4, conditioned MS (pMS); lane 5, conditioned M199 (pM199). (b) Time‐dependent phosphorylation of ERK1/ERK2. Phospho‐ERK1/ERK2 (upper blot) and whole ERK (lower blot). Lane 1, M199 10 min; lane 2, pM199 10 min; lane 3, pM199 30 min; lane 4, pM199 60 min; lane 5, pM199 120 min. (c) Influence of PD98059 (PD) on ERK1/ERK2 phosphorylation. Phospho‐ERK1/ERK2 (upper blot) and whole ERK (lower blot). Lane 1, M199 + PD; lane 2, pM199 + PD; lane 3, pM199 10 min. Densitometric analysis is listed in Table 2.
Figure 2
Figure 2
Cell morphology. Six thousand five hundred adipose‐derived adult mesenchymal stem cells (ASC) per square centimetre were seeded and cultured for 12 days in conditioned or control medium. Cells were from the same isolation, trypsinized at the same time and seeded at the same cell density. Undifferentiated ASC displayed an elongated ‘fibroblastoid’ morphology (a), whereas conditioned medium 199 (pM199) (b) induced a change in cell morphology to a more epithelial‐like monolayer (bar: 100 µm).
Figure 3
Figure 3
Flow cytometry. Representative overlay histograms of the expression of adipose‐derived adult mesenchymal stem cell (ASC) markers after incubation with conditioned medium 199 (pM199) for 12 days (n = 5). Solid filled grey histogram (pM199), black histogram (M199), dashed‐line grey histogram (isotype control, only in the histograms on the left side). Dot blot shows forward and sideward scatter analysis of ASC.
Figure 4
Figure 4
Characteristic Western blot analysis. Expression of cytokeratin 18 (45 kDa, upper blot) after incubation in conditioned medium for 12 days (lane 1, M199; lane 2, pM199; lane 3, pMS). In contrast, α‐smooth muscle actin expression was lowered (42 kDa, middle blot), while vimentin expression was almost unaffected (58 kDa, lower blot).
Figure 5
Figure 5
Immunofluorescent staining of cytokeratin 18. Expression of the epithelial marker cytokeratin 18 could be demonstrated after incubation with conditioned medium for 12 days (b, c), whereas controls in standard medium were negative (a).
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