Human Deciduous Teeth Stem Cells (SHED) Display Neural Crest Signature Characters

Abstract

Human dental tissues are sources of neural crest origin multipotent stem cells whose regenerative potential is a focus of extensive studies. Rational programming of clinical applications requires a more detailed knowledge of the characters inherited from neural crest. Investigation of neural crest cells generated from human pluripotent stem cells provided opportunity for their comparison with the postnatal dental cells. The purpose of this study was to investigate the role of the culture conditions in the expression by dental cells of neural crest characters. The results of the study demonstrate that specific neural crest cells requirements, serum-free, active WNT signaling and inactive SMAD 2/3, are needed for the activity of the neural crest characters in dental cells. Specifically, the decreasing concentration of fetal bovine serum (FBS) from regularly used for dental cells 10% to 2% and below, or using serum-free medium, led to emergence of a subset of epithelial-like cells expressing the two key neural crest markers, p75 and HNK-1. Further, the serum-free medium supplemented with neural crest signaling requirements (WNT inducer BIO and TGF-β inhibitor REPSOX), induced epithelial-like phenotype, upregulated the p75, Sox10 and E-Cadherin and downregulated the mesenchymal genes (SNAIL1, ZEB1, TWIST). An expansion medium containing 2% FBS allowed to obtain an epithelial/mesenchymal SHED population showing high proliferation, clonogenic, multi-lineage differentiation capacities. Future experiments will be required to determine the effects of these features on regenerative potential of this novel SHED population.

Fig 1. Morphological and gene expression characteristics of SHED cultured in 1%FBS and 0%FBS DentEpiMesMed.

(a)—bright field images of the cells in 1%FBS medium and in 0%FBS medium (2.5ng/ml of the TGFβ was added), respectively. Scale bars 50μm; (b)(c)—number of the cells expressing mesenchymal (CD90, CD105, CD73) and neural crest (p75, HNK-1) genes in medium containing 1% FBS and 0% FBS, respectively; (d)–number of the cells co-expressing CD73 and p75 genes in 1%FBS and 0% FBS medium. Hear and below see Material and Methods section for composition of DentEpiMesMed.

Fig 2. Phenotypic and p75 instability in the cells at serum-free and at low FBS concentrations.

(a)—SHED containing 4.1% p75⁺ cells in DentEpiMesMed (2%FBS) were fractionated by MACS using an anti-p75 antibody into p75-positive and p75-negative fractions; the p75-negative cells were cultured five days in the same medium and the amount of the cells with the marker were determined by flow cytometry. (b), (c)—SHED containing 17.8% p75⁺ cells in the 0%FBS DentEpiMesMed (2.5ng/ml of the TGFβ was added) were used to sort out the p75⁺ cells which were cultured five days under two conditions: 1%FBS (b) and 0%FBS (2.5ng/ml of the TGFβ was added) (c); the proportion of the p75⁺ cells were determined by flow cytometry.

Fig 3. Characteristics of SHED cultured in neural crest vs. 2%FBS conditions.

The cells were isolated and cultured for two passages in 2% FBS-containing DentEpiMesMed (B) and a sample of the cells were cultured as suspension for five days in serum-free medium containing 1μM BIO and 5 μM REPSOX then seeded in fibronectin-coated plate (A). The properties of the cells grown in the two conditions were determined. (a)–bright field images comparing the morphology of the cells on (A) and (B); (b)–flow cytometry evaluation of the SHED populations expressing mesenchymal (CD73), epithelialization (E-cadherin) and neural-crest (p75) genes. (c) immunofluoresence assay of Sox10 in (A) and (B); (d) RT-qPCR-determined relative transcription levels of E-cadherin, ZEB1, TWIST, ZO-1, SNAIL1 and SOX10 genes.

Fig 4. General characteristics of SHED population cultured in DentEpiMesMed 2%FBS.

A. RT-PCR analysis of expression of pluripotency (a) and neural crest migratory (b) genes; RNA of NCCIT, an embryocarcinoma line (ATCC), was included as a pluripotency control in (a); ACTB gene and (RT-) served as reaction controls. Immunofluorescence assay of pluripotency markers of SHED (c), cells from the human ESC line (ESI-BIO, US) were included as positive control (d). B. Proliferation rate of SHED measured during five passages (a), and clonogenicity (b, c) of the cells. C. Osteogenic and adipogenic differentiation potentials of the cells. Osteogenic potential: (a)- calcium deposits detected by staining with Alizarin Red S; (b)- quantitative evaluation of the Alizarin Red S intensity by spectrophotometry (Epoch, Biotek)) in control and in differentiated (diff.) cultures; (c)- RT-PCR analysis of the transcription of ALP and BGLAP genes. GAPDH and RT- were positive and negative controls, respectively. Adipogenic potential: (d)—lipid droplets in adipocytes; (e)- RT-PCR assay of the expression of FABP4 and PPAR genes. D. Neuronal lineage potential. (a)–morphological changes, expression of SOX2, MASH-1 at day 5 and βIII-tubulin at day 14 determined by immunofluorescence analysis; (b)—RT-PCR analysis of transcription of SOX2 and MASH-1 genes. GAPDH and RT- were positive and negative controls, respectively.