REPROGRAMMING FIBROBLAST
EPITHELIAL TRANSITION
THROUGH
MESENCHYMAL-TO-
MALTA, T.M. [email protected],2, PICANÇO-CASTRO,
V. 3, RUSSO1,2
1,2
2
CARBOLANTE, E.M.S. , RODRIGUES, E.S. , PANEPUCCI, R.A. , PEREIRA, L.V. 4, COVAS,
D.T. 2,5, KASHIMA, S. 1,2.
(1) Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São
Paulo, Brazil.
(2) Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, University
of São Paulo, Ribeirão Preto, São Paulo, Brazil.
(3) Veritas Life Sciences LTDA, Incubadora SUPERA, Campus USP, Ribeirão Preto, São Paulo, Brazil.
(4) Department of Genetic and Evolutionary Biology, Institute of Bioscience, University of São Paulo,
São Paulo, São Paulo, Brazil.
(5) Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
1. Introduction
Overexpression of specific transcription factors in somatic cells give rise to induced pluripotent stem cells
(iPS) that exhibit morphologic, molecular and functional similarities to embryonic stem cells (ESC). iPS
cells provide an unprecedented platform to model human disease, to screen for pharmacological
molecules and as a source of patient-specific stem cell. The reprogramming mechanisms of iPS cells are
largely unknown and it would be important to elucidate them to improve the efficiency of iPS generation.
During reprogramming of fibroblasts, these cells gradually transit from a single layer of adherent cells to
become multilayered epithelial cells, a process suggestive of a mesenchymal-to-epithelial transition
(MET). With this in mind, the aim of this study was to analyze the global expression profile of iPS-TSM,
a reprogrammed human fibroblast generated with forced expression of TCL1A, SOX2 and c-MYC, in
order to investigate if genes involved in MET are modulated in iPS-TSM, comparing with the donor
fibroblast cell line and with ESCs. Moreover, we established cell lines with overexpression of TCL1A
individually in order to find out its contribution to the reprogramming process, as this gene had never
been used for reprogramming before.
2. Methods
We used a microarray platform composed by 44,000 human genes to perform the gene expression profile.
We further validated some gene transcripts levels by quantitative PCR (qPCR).
3. Results
Initially, we examined 44 genes involved with MET in the microarray libraries and found that these genes
had been modulated after the induction and have a similar pattern of expression to the ESCs. We
identified that important epithelial markers were activated in the iPS-TSM cells, including CDH1.
Moreover, COL1A2 and CDH2, which are mesenchymal markers, were downregulated in the iPS-TSM
cells. We also detected that SNAI1, TWIST1, and TGFB1, that are inhibitors of MET, were
downmodulated in iPS-TSM cells. The qPCR results were in agreement with the microarray data. Finally,
we analyzed the individual contribution of TCL1A, for which we expressed this gene in the fibroblast and
compared with the donor cell and with ESCs. Intriguingly, qPCR analysis revealed that TCL1A alone was
able to induce CDH1 mRNA expression and also to downregulate the MET repressors SNAI1, TWIST1
and TGFB1.
4. Conclusion
In summary, the analysis revealed that genes involved in MET were modulated in iPS-TSM cells and that
TCL1A may contribute to the initial steps of human reprogramming through downmodulation of MET
repressors.
Financial Support: CTC/INCTC/FAPESP, CNPq, FUNDHERP.