Skin keratinocytes pre-treated with embryonic stem cell-conditioned medium or BMP4 can be directed to an alternative cell lineage.
OBJECTIVE
In this study, we have investigated whether secreted factors from embryonic stem cells (ESCs) could reprogramme keratinocytes and increase their potential to be directed into alternative cell lineages.


METHODS
Contact and non-contact co-cultures of skin keratinocytes and murine ESCs were used initially to confirm any reprogramming ability of ESC-conditioned medium (CM). Immunofluoresence was used to assess nuclear expression of octamer-4 (Oct-4), as well as to confirm neuronal protein expression in neuroectodermally directed keratinocytes. Transcript expression changes were evaluated using semiquantitative reverse transcription-polymerase chain reaction. Western blotting, accompanied by densitometry analysis, was used to evaluate protein expression following morphology changes.


RESULTS
We found that keratinocytes treated with ESC-CM changed their morphology and were stimulated to express the pluripotency regulator, Oct-4, and its target transcripts, Sox-2, Nanog, Utf1 and Rex-1. We demonstrate that at least one of the reprogramming factors is bone morphogenetic factor-4 (BMP4). Pre-treated keratinocytes could be specifically directed to differentiate into cells of the neuronal lineage. The majority of responsive keratinocytes were the epidermal stem cell population, with a small percentage of transit-amplifying cells also being affected.


CONCLUSIONS
Our results suggest that ESC-CM contains a number of factors, including BMP4, which are capable of reprogramming mouse skin keratinocytes to make them more developmentally potent, as evidenced by their ability to be re-differentiated into cells of the neuronal lineage. Our findings also imply a continuum of differentiation within the basal keratinocyte population. An increase in developmental potential combined with directed differentiation could increase the therapeutic relevancy of somatic cells.
Grinnell
K L
KL
Department of Anatomy and Cell Biology, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA.
Bickenbach
J R
JR
D000818:Animals; D001483:Base Sequence; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D052005:Bone Morphogenetic Protein Receptors, Type I / Q000235:genetics; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D017931:DNA Primers / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D053547:Keratin-14 / Q000235:genetics; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000494:pharmacology
C525718:BMP2 protein, human; C525809:BMP4 protein, human; C525719:Bmp2 protein, mouse; C525810:Bmp4 protein, mouse; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D017077:Culture Media, Conditioned; D017931:DNA Primers; D053547:Keratin-14; C507446:Krt14 protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C498212:Bmpr1a protein, mouse; D052005:Bone Morphogenetic Protein Receptors, Type I