Reprogramming postnatal human epidermal keratinocytes toward functional neural crest fates

Running Title: Human keratinocyte derived neural crest cells

Vivek K. Bajpai1, #, Laura Kerosuo 2,*, Georgios Tseropoulos1,*, Kirstie A. Cummings3,*, Xiaoyan Wang1, Pedro Lei1, Biao Liu 4,5, Song Liu4, 5, Gabriela Popescu3, Marianne E. Bronner2 and Stelios T. Andreadis1, 6,7, §

1Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, NY 14260

2Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125

3Department of Biochemistry, Neuroscience Program, School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14214

4Center for Personalized Medicine, and 5Dept. of Biostatistics and Bioinformatics Roswell Park Cancer Institute, Buffalo, NY 14263

6Department of Biomedical Engineering, University at Buffalo, NY 14260

7Center of Excellence in Bioinformatics and Life Sciences, Buffalo, NY 14263

Key words: Neural crest, Epidermal keratinocytes, Neural plate border, Neural crest induction, Reprogramming

# Current Address:Department of Chemical and Systems Biology, School of Medicine, Stanford University, Stanford, CA, 94305

* These authors contributed equally to the work.

§ Address for all Correspondence:

Stelios Andreadis, Ph.D.

Professor

Bioengineering Laboratory, 908 Furnas Hall

Department of Chemical and Biological Engineering,

Department of Biomedical Engineering, and

Center of Excellence in Bioinformatics and Life Sciences

University at Buffalo, The State University of New York

Amherst, NY 14260-4200, USA

Tel: (716) 645-1202

Fax: (716) 645-3822

Email:

SUPPLEMENTARY FIGURE LEGENDS

Figure S1: Starting KC population is devoid of melanocytes. A human epidermal KC colony morphology after removal of the fibroblast feeder layer (A). Flow cytometry (B) and immunofluorescence (C) of KC shows lack of melanocyte specific marker, PMEL (SILV) and uniform expression of KC marker, KRT14. Primary human melanocytes lack KRT14 and express PMEL and served as positive control(Representative data from 2 of 3 donors are shown).

Figure S2: Neonatal basal epidermal KC display neural plate border characteristics and are induced into NC fate in response to FGF2 and IGF1 treatment. Human epidermal KCexpress basal KC proteins (KRT14, ITGB1, TP63) (A). Neonatal KCexpress neural plate border specific genes (MYC, SOX9, SNAI2, MSX2, IRX2, DLX3, TFAP2A, and KLF4)(A) and proteins (SNAI2, TFAP2A, MYC,KLF4) (B). KC to NC induction by FGF2 is potentiated by IGF1 as shown by qRT-PCR for key NC genes (NES, PAX3, FOXD3, TWIST1, NGFR, SOX10, B3GAT1) and proliferation marker KI67 (C). Collagen type I, fibronectin and lamininECM coatings supported expression of NC genes to a similar extent (D). Collagen type I and fibronectin increase KC to NC induction as shown by increased number of SOX+NES+ cells ((E). Proliferation kinetics and clonogenic ability of KC-NC (F,G).KC clones give rise to SOX10 and NESpositive NC cells (H). Scale bars, 50μm. All values are meanSD (*p<0.001, #p<0.01).

Figure S3: Adult KC possess neural plate border characteristics. Adult KC express neural plate border specific genes (MYC, SOX9, SNAI2, MSX2, IRX2, DLX3, TFAP2A, and KLF4) (A). Adult KC express the basal KC marker KRT14 as well as neural plate border specific proteins (SNAI2, KLF4, and TFAP2A)(B).Scale bars, 50μm.

Figure S4: Molecular and genome-wide characteristics of KC-NC. KC-NC express NC lineage proteins (SOX10, FOXD3, PAX3, NGFR and NES) as determined by measurements of immunofluorescence intensity using ImageJ(A,B). KC-NC undergo EMT as seen by upregulation of CDH2, downregulation of CDH1 (C,D,E), expression of VIM (Vimentin) (F) and several ECM genes (I). As CDH1 is repressed during EMT, β-catenin (CTNNB1) translocates to the nucleus of KC-NC (E) as compared to the cell border in KC (D). Transcriptome (RNA-Seq) analysis was performed on KC and respective KC-NC and was in good agreement with qRT-PCR results as examined for NC markers (n=3donors) (G). KC expressed several neural plate border genes (H) and their induction into NC fate was marked by changes in the expression of several signal transduction (J), epigenetic and cell cycle related genes (K,L) (n=3donors). Scale bars, 50μm. All values are meanSD.

Figure S5: KC-NC derived mesenchymal cells show similar immunophenotype as bone marrow derived mesenchymal stem cells. KC-NC derived mesenchymal cells expressed CD73, CD90, CD44, CD105 and CD49b and lacked pan-leukocyte marker, CD45 as determined by flow cytometry (n=3donors) (A). Compared to ASMC, KC-NC derived SMC significantly remodeled fibrin hydrogels as evidenced by increased ultimate tensile stress of KC-NC-SMC based tissue constructs (n=3donors) (B). $, p<0.05.

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