Skin keratinocytes pre-treated with embryonic stem cell-conditioned medium or BMP4 can be directed to an alternative cell lineage

Abstract

Objectives: 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.

Materials and 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.

Figure 1

Expression of Oct‐4 in mouse keratinocytes in non‐contact co‐culture with mouse ESC or in cultures exposed to CM. (a–c) 24 h transwell cultures of P0 keratinocytes in 1 : 1 DKSFM : uCM above P22 W4 ESC. (a) DAPI (4′,6‐diamidino‐2‐phenylindole); (b) Anti‐Oct‐4; (c) Merged. Note, punctate nuclear Oct‐4 staining in ~4% of keratinocytes. (d–f) 48 h transwell cultures. (d) DAPI; (e) anti‐Oct‐4; (f) merged. Note, nuclear Oct‐4 in ~4.5% of keratinocytes. (g–i) Keratinocytes treated with CM for 48 h. (g) DAPI; (h) anti‐Oct‐4; (i) merged. Note, nuclear Oct‐4 in ~4.2% of keratinocytes. (j–l) Control P0 neonatal mouse keratinocytes grown in DKSFM without mouse ESC. (j) DAPI; (k) anti‐Oct‐4; (l) merged. (m–o) Control P0 keratinocytes grown in 1 : 1 DKSFM : uCM without mouse ESC. (m) DAPI; (n) anti‐Oct‐4; (o) merged. Note absence of Oct‐4 in control cultures. ×40 magnification. Scale bar = 50 µm.

Figure 2

Embryonic gene expression in keratinocytes exposed to CM. (a) P0 neonatal keratinocytes expressed Oct‐4 (800 bp), Nanog (360 bp), Utf1 (154 bp), Rex‐1 (232 bp) and Sox‐2 (340 bp) after 24 h exposure to CM. (b) Keratinocytes, after 24 h exposure to uCM, did not express embryonic transcripts. (c) Control RT‐PCR without reverse transcriptase (RT).

Figure 3

Bone morphogenetic factor‐4 (BMP4) in CM and effects of recombinant BMP4 on keratinocyte gene expression. (a) Immunoblot analysis of conditioned (CM) and unconditioned (uCM) media show BMP4 in the CM (24.6 ng/mL). LIF was 2.2‐fold higher in uCM than in CM. Various concentrations of recombinant BMP4 were used as standards by which to quantify the amount of BMP4 present in the CM. 3 mL lyophilized medium loaded per well. (b–g) RT‐PCR; (b) no activation of Oct‐4 or its target transcripts in keratinocytes exposed to 50 ng/mL BMP4 in the presence of FBS. (c) 100 ng/mL of BMP4 activated Oct‐4, Nanog, Utf1, Rex‐1 and Sox‐2 in the presence and (d) absence of FBS. (e) After 5 days exposure to 100 ng/mL BMP4, keratinocytes still expressed Oct‐4, Nanog, Utf1, Rex‐1 and Sox‐2. (f) Keratinocytes cultured in 1 : 1 DKSFM : uCM for 5 days without BMP4 expressed K14. (g) In keratinocytes cultured in 1 : 1 DKSFM : uCM, plus 100 ng/mL BMP4 (renewed daily), for 5 days, K14 was down‐regulated by Day 3, and extinguished by day 5. (f–g) Lane 1 = GAPDH (135 bp); Lane 2 = Keratin 14 (K14) (513 bp).

Figure 4

Embryonic gene expression in keratinocytes following BMP4 removal. P0 neonatal keratinocytes were exposed to 100 ng/mL BMP4 (in 1 : 1 DKSFM : uCM) for 24 h. After 24 h, BMP4 was removed and expression of Oct‐4 and its target genes examined over 5 days. (a) 24 h after removal of BMP4, Oct‐4, Nanog, Utf1, Rex‐1 and Sox‐2 were still expressed. (b) 48 h post‐BMP4, Oct‐4 was no longer expressed, but Nanog, Utf1, Rex‐1 and Sox‐2 persisted. (c) 72 h post‐BMP4, Oct‐4 remained off; Nanog and Sox‐2 were extinguished; Utf1 and Rex‐1 were still present. (d) 96 h post‐BMP4, Utf1 was extinguished, while Rex‐1 was barely detectable. (e) 120 h post‐BMP4, no embryonic transcripts were expressed. (f) K14 transcript level is restored to control by 120 h post‐BMP4.

Figure 5

Expression of BMPR1A and Oct‐4 in keratinocytes following exposure to CM or recombinant BMP4. (a–e) Keratinocytes cultured in 1 : 1 DKSFM : CM for 24 h. (a) DAPI (4′,6‐diamidino‐2‐phenylindole); (b) anti‐BMPR1A; (c) anti‐Oct‐4; (d) merge BMPR1A and Oct‐4; (e) merge BMPR1A, Oct‐4 and DAPI. (f–j) Keratinocytes cultured in 1 : 1 DKSFM : uCM +100 ng/mL rBMP4, for 24 h. (f) DAPI; (g) anti‐BMPR1A; (h) anti‐Oct‐4; (i) merge BMPR1A and Oct‐4; (j) merged BMPR1A, Oct‐4 md DAPI. In a–j, note co‐staining of membrane BMPR1A and Oct‐4, and ESC‐like rounded morphology. Additionally, note small, rounded cells with bright cytoplasmic BMPR1A, but no Oct‐4, suggesting BMPR1A is expressed before Oct‐4. (k–o) Control 24 h DKSFM cultures. (k) DAPI; (l) anti‐BMPR1A; (m) anti‐Oct‐4; (n) merge BMPR1A and Oct‐4; (o) merge BMPR1A, Oct‐4 and DAPI. (p–t) Control 24 h 1 : 1 DKSFM : uCM cultures. (p) DAPI; (q) anti‐BMPR1A; (r) anti‐Oct‐4; (s) merge BMPR1A and Oct‐4; (t) merge BMPR1A, Oct‐4 and DAPI. Note, no Oct‐4 or membrane BMPR1A staining in controls. All images, ×40. Scale bar = 50 µm.

Figure 6

Neuronal morphology exhibited by keratinocytes exposed to CM or recombinant BMP4. Neonatal mouse skin keratinocytes were plated in 100% DKSFM for 24 h and cultured under the following conditions for 24 additional hours, then all cells exposed to neuronal differentiation medium for 10 days (note, days below refer to days in differentiation medium). (a–e) Keratinocytes treated with 1 : 1 DKSFM : CM. (a) Day 1; (b) day 3; (c) day 7; (d) day 10; (e) day 10, ×20 magnification. Insert represents enlargement of boxed area in e. (f–j) Keratinocytes treated with 1 : 1 DKSFM : uCM + BMP4 (f) Day 1; (g) day 3; (h) day 7; (i) day 10; (j) day 10, ×40 magnification. (k–n) Control keratinocytes cultured in 100% DKSFM for 10 days. (k) Day 1; (l) day 3; (m) day 7; (n) day 10. (o–r) Control, keratinocytes cultured in 1 : 1 DKSFM : uCM for 24 h, then cultured in neuronal differentiation medium for 10 days. (o) Day 1; (p) day 3; (q) day 7; (r) day 10. All images (except d and j) ×10 magnification. Scale bars = 200 µm.

Figure 7

Expression of neuronal proteins and receptors in cells exposure of CM or BMP4, then cultured in neuronal differentiation medium. Western blot analysis of nestin, neuronal nuclear protein (NeuN), NMDA receptor 1 (NR1), glutamate receptor 1 (GluR1), tyrosine hydroxylase (TH) and actin. Lane 1 = Control, keratinocytes exposed to 1 : 1 DKSFM : uCM for 24 h, then neuronal differentiation medium for 10 days. Lane 2 = Keratinocytes exposed to 1 : 1 DKSFM : CM for 24 h, then neuronal differentiation medium for 10 days. Lane 3 = Keratinocytes exposed to 1 : 1 DKSFM : uCM +100 ng/mL BMP4, for 24 h, then neuronal differentiation medium for 10 days. Lane 4 = control, keratinocytes grown in 100% DKSFM for 10 days. Lane 5 = P22 W4 ESC grown in ESC medium for 24 h, then neuronal differentiation medium for 10 days. Lane 6 = Skin fibroblasts grown in DMEM for 10 days. 50 µg of protein loaded per lane.

Figure 8

Expression of nestin, NeuN and K14 in cells grown for 7 days in neuronal differentiation medium. Keratinocytes were exposed to CM for 24 h then cultured in neuronal differentiation medium for 7 days. Dashed line marks border between keratinocyte colony and emerging neuronal‐type cells. Arrows indicate direction of neuron‐like cell migration. (a–c) Cultures stained with antibody to nestin. ×20 magnification. (a) DAPI (4′,6‐diamidino‐2‐phenylindole); (b) anti‐nestin; (c) merged. (d–f) Cultures stained with antibody to NeuN. ×40 magnification. (d) DAPI; (e) anti‐NeuN; (f) merged. Arrowheads mark NeuN⁺ nuclei. Note, multilayered colony edges showed non‐specific accumulation of NeuN antibody. (g–i) Cultures stained with antibody to keratin 14 (K14). ×20 magnification. (g) DAPI; (h) anti‐K14; (i) merged. Note, in the few K14⁺ neuronal‐type cells, the K14 filaments appeared collapsed around the nuclei. (j) ×60 magnification of antinestin/DAPI. (k) ×60 magnification of anti‐K14/DAPI. Arrows point to perinuclear clumps of keratin in cells. Scale bars = 50 µm.