Epithelial-mesenchymal transition-derived cells exhibit multilineage differentiation potential similar to mesenchymal stem cells

Abstract

The epithelial-to-mesenchymal transition (EMT) is an embryonic process that becomes latent in most normal adult tissues. Recently, we have shown that induction of EMT endows breast epithelial cells with stem cell traits. In this report, we have further characterized the EMT-derived cells and shown that these cells are similar to mesenchymal stem cells (MSCs) with the capacity to differentiate into multiple tissue lineages. For this purpose, we induced EMT by ectopic expression of Twist, Snail, or transforming growth factor-beta in immortalized human mammary epithelial cells. We found that the EMT-derived cells and MSCs share many properties including the antigenic profile typical of MSCs, that is, CD44(+), CD24(-), and CD45(-). Conversely, MSCs express EMT-associated genes, such as Twist, Snail, and mesenchyme forkhead 1 (FOXC2). Interestingly, CD140b (platelet-derived growth factor receptor-beta), a marker for naive MSCs, is exclusively expressed in EMT-derived cells and not in their epithelial counterparts. Moreover, functional analyses revealed that EMT-derived cells but not the control cells can differentiate into alizarin red S-positive mature osteoblasts, oil red O-positive adipocytes and alcian blue-positive chondrocytes similar to MSCs. We also observed that EMT-derived cells but not the control cells invade and migrate towards MDA-MB-231 breast cancer cells similar to MSCs. In vivo wound homing assays in nude mice revealed that the EMT-derived cells home to wound sites similar to MSCs. In conclusion, we have demonstrated that the EMT-derived cells are similar to MSCs in gene expression, multilineage differentiation, and ability to migrate towards tumor cells and wound sites.

Figure 1

Morphology and expression pattern of genes in EMT-derived HMECs and bone marrow MSCs. A. Light microscopic images (10X) of HMECs ectopically expressing Twist, Snail, TGF-β1 or the empty vector as well as bone marrow-derived MSCs. B&C. Quantitative RT-PCR analysis of EMT associated genes including E-cadherin, vimentin, fibronectin and N-cadherin (B) and EMT-regulating transcription factors, including FOXC2, Twist, Snail, and Zeb-1 in MSCs (C). D. The expression of cell surface markers associated with MSCs in EMT-derived HMECs expressing Twist, Snail, TGF-β or the control vector. Quantitative RT-PCR was performed in triplicate (mean +/− SD).

Figure 2

Osteoblastic differentiation of EMT-derived mesenchyme-like HMECs and MSCs. A. Following culture in osteoblastic differentiation media, HMECs expressing Twist or Snail and MSCs were positive for alkaline phosphatase activity while the vector control cells were not. B&C. Staining for calcium deposition in EMT-derived HMECs and MSCs after osteoblastic differentiation using 1% Alizarin Red S (B) or 1% silver nitrate (von Kossa staining) (C). D. Quantitative RT-PCR analysis for the expression of osteoblast markers osteocalcin (left panel) and alkaline phosphatase (right panel) in EMT-derived HMECs, control HMECs and MSCs subjected to osteoblast differentiation for different lengths of time (0, 5 and 10 days).

Figure 3

Adipogenic and chondrogenic differentiation of EMT-derived HMECs and MSCs. A. Following adipogenic differentiation, the EMT-derived HMECs and MSCs stained positive with Oil Red O dye (top) and fluorescent LipidTox^®, which stains oil droplets (bottom). Conversely, vector control HMECs did not stain using similar treatment (right panel). B. Quantitative RT-PCR analysis for the expression of the adipocyte marker PPARγ after 21 days in adipocyte differentiation condition. C. Quantitative RT-PCR analysis for the expression of LPL at different time points as indicated in the figure in EMT-derived HMECs, control HMECs and MSCs subjected to adipocyte differentiation. Quantitative RT-PCR was performed in triplicate (mean +/− SD). D. Chondrocytic nodules formed by EMT-derived HMECs or MSCs stained positive with Alcian Blue 8GX (top panel). These sections were counter stained with nuclear fast red solution. Immunohistochemistry was performed on chondrocyte sections using collagen-I antibody (middle panel) and collagen-II (bottom panel). The vector-infected HMECs did not form any chondrocytes nodules under identical conditions.

Figure 4

Matrigel invasion assay to demonstrate migration of EMT-derived cells towards MDA-MB-231 breast cancer cells. (A) Three ×10⁴ of Twist or Snail or vector alone expressing HMECs or MSCs were incubated in the upper well of the invasion chamber in the presence or absence of PDGF-bb ligand (10ng/ml) (B) or MDA-MB-231 cells (C) in the bottom well. After 36 hr of incubation, cells that had migrated to the bottom side of the 8 µm membrane were stained and counted as described in methods.

Figure 5

In vivo wound homing by EMT-derived HMECs and MSCs. (A) Bioluminescent imaging of nude mice harboring wounds and injected with 3 × 10⁵ adenoviral firefly luciferase labeled EMT-derived HMECs or MSCs via the tail vein. These experiments were performed in triplicate and repeated three times. (B) Quantitative measurement of the relative fold change of bioluminescence. Wound regions were manually drawn and the signal intensity was expressed as photon flux or photons/s/cm² (p/s/cm²) compared to vector control HMECs.