Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

Abstract

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

Keywords: FACS sorting; epidermis; human skin; ultrasound-guided grafting.

Fig. 1.

Isolated human foreskin keratinocytes before culture express KERATIN (K) 5 (A), K10 (B), K14 (C), and VIMENTIN (D). They were negative for NESTIN (E) and DECORIN (F). As a positive control for DECORIN, human fibroblast isolated from foreskin showed immunoreactivity (G). Cells incubated with only the secondary antibodies did not show unspecific staining (H). Scale bar = 15 μm. Keratinocytes selectively express K5, K10, and K14 compared with fibroblasts; fibroblasts presented exclusive positivity for DECORIN, compared with keratinocytes (I). **P < 0.01; ***P < 0.001.

Fig. 2.

Keratinocytes cultured as aggregates in UDMM-Epi serum-containing medium (A) or neural medium (B). After 4 days, aggregates were dissociated and replated on tissue culture plates either in UDMM-Epi (C) or in neural medium (D). Note that the aggregates in UDMM-Epi are larger and dissociated cells in UDMM-Epi show classical keratinocyte morphology. In contrast, dissociated cells incubated in neural medium present morphology reminiscent of neural precursors. Scale bar = 150 µm (A and B) and 50 µm (C and D).

Fig. 3.

Aggregates either in UDMM-Epi medium (A) or in neural medium (B) were fixed, sectioned, and stained for VIMENTIN and NESTIN. Scale bar = 50 μm. The neural medium significantly induced the presence of NESTIN-positive cells (C). ***P < 0.001.

Fig. 4.

Aggregates were dissociated, replated, and fixed to analyze the expression of several markers. Keratinocytes growing in UDMM-Epi medium (A) were positive for K5 and VIMENTIN, and negative for K10, K14, and NESTIN. Cells cultured in the neural medium (B) lost the expression of K5 and gained NESTIN immunoreactivity. In both panels, control pictures correspond to cells incubated only with secondary antibodies, to discard unspecific binding. Scale bar = 15 μm. Cells in control medium had a significantly higher proportion of K5+ cells and the neural medium significantly increased the presence of NESTIN-positive cells (C). ***P < 0.001.

Fig. 5.

Analysis of expression of transcripts for NESTIN (A), SOX2 (B), VIMENTIN (C), SOX1 (D), and MUSASHI1 (MSI1, E) by RT-qPCR in the serum containing (Control, ctl) or the neural medium in cells growing as aggregates or after dissociation and replating. Total RNA was isolated from the indicated conditions and retrotranscribed to obtain cDNA, which was amplified by qPCR with specific primers. These values were normalized by the housekeeping gene β-ACTIN. Bars are mean ± standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001. RT-qPCR: quantitative real-time polymerase chain reaction.

Fig. 6.

Isolation of GFP-expressing keratinocytes after lentiviral transduction. (A) Keratinocytes growing in UDMM-Epi medium after viral transduction. Cells were trypsinized and analyzed by flow cytometry. Individual cells were selected (B), cellular debris was omitted (C), and cells with a high value of GFP fluorescence were recovered by cell sorting (D). All cells were GFP-positive after sorting (E). These fluorescent cells were grown as aggregates in UDMM-Epi (F) or neural (G) media. For A, E, F, and G, the images on the left correspond to phase contrast, the middle panel to the fluorescent picture, and the right picture is the merge of the former 2. Scale bar = 50 µm (A and E) and 150 µm (F and G). GFP: green fluorescent protein.

Fig. 7.

Human keratinocytes differentiate to neuronal-like cells when grafted in the developing rat brain. Ultrasound-guided injections of dissociated aggregates cultured in UDMM-Epi medium (A) or neural medium (B). Grafting was performed in 12-day-old embryos and the analysis was performed 4 days later in the brain of the recovered embryos. GFP is shown in green, β-III TUBULIN (detected with the TUJ1 antibody) is presented in red, Hoechst staining of the nuclei is shown in blue in the merge images, and MAP2 in magenta. The top row of each panel corresponds to low-magnification confocal images, and the third picture corresponding to the merge image. A white square labeled 1 marks the area shown at higher magnification in the second row images of each panel, that include orthogonal projections. To better appreciate the neuronal-like morphology, zoom-in images labeled with white squares numbered 2 and 3 are presented in the fourth column for each neuronal marker. The GFP-positive processes of cells that coexpress β-III TUBULIN or MAP2 are indicated with white arrowheads. Scale bars = 50 μm.