Dermal spheres express stem cell markers. (A) A dermal sphere (DS) and a human embryonic body (EB) stained with OCT4 antibody. Immunofluorescence revealed expression of OCT4 (green) within dermal spheres and EB cells, that is typically localized to nuclei (stained blue with DAPI). Scale bars: 100 μm (for EB) and 70 μm (DSC). (B) Dermal spheres are positive for the neural crest stem cell marker nestin (green). Scale bar: 100 μm. (C) The neural crest stem cell marker NGFRp75 (green, cytoplasmic staining) is expressed in dermal spheres. Nuclei are stained blue with DAPI. Scale bar: 100 μm. (D) Upper panel, co-staining dermal spheres for OCT4 (green) and NGFRp75 (red) shows distinctive cells that coexpress both markers. Scale bar: 40 μm. Lower panel, control staining with mouse IgG and rabbit IgG. Scale bar: 100 μm. (E) FACS analysis of the cell surface marker NGFRp75 in a human foreskin dermal cell suspension. Fibroblasts (FF) and the WM3248 melanoma cell line were used as negative and positive controls, respectively.

Further characterization of the stem cell properties of DSCs by induction of differentiation into several cell lineages. (A-D) DSC spheres were enzymatically dissociated into single cells and plated in tissue culture-grade chamber slides in differentiation medium. After 14 days, lineage-specific staining was performed. Scale bar: 50 μm. Neural differentiated cells become immunoreactive to β3 tubulin (A). Differentiated smooth muscle cells express smooth muscle actin (SMA) (B). Differentiated chondrocytes are positive for collagen II (C). Differentiated adipocytes are detected by Oil-red-O staining (D). (E-H) Melanocytic differentiation was performed in melanocyte differentiation medium. Immunofluorescent staining reveals that most differentiated cells are positive for the melanocyte markers MITF (E) DCT (F) and S100 (G), and the pigmentation marker HMB45 (H). Scale bar: 50 μm.

DSC-derived melanocytes gain E-cadherin expression upon contact with keratinocytes. (A) NGFRp75 (red)-positive DSCs are negative for E-cadherin (green). (B) NGFRp75 (red)-positive DSCs are negative for N-cadherin (green). (C) After 14 days of culture in the skin reconstructs, NGFRp75 (red)-positive cells in the dermis did not express E-cadherin (green). Epidermal keratinocytes show strong E-cadherin expression. (D) After 14 days of culture in the skin reconstructs, a few cells in dermis express N-cadherin (red), whereas NGFRp75 (red)-positive DSCs do not. (E) Differentiated melanocytes on basement membrane expressed melanocytic marker S100 (red) and E-cadherin (green). (F) S100 (red)-positive melanocytes on basement membrane do not express N-cadherin (green). White dotted lines in C-F indicate the epidermis-dermis junction. Nuclei are counterstained with DAPI (blue). Scale bar: 50 μm.

Isolation and expansion of primary dermal spheres in HESCM4 medium. (A) Day 0: a suspension of single cells is obtained from neonatal human foreskin dermis. (B) Day 7: small cell clusters form in HESCM4 media. (C) Day 14: cell clusters become non-adherent or loosely adherent to culture plates and form sphere-like structures. (D) A subcultured (third passage) dermal sphere shows the same morphology as the primary culture. (E) Epidermal melanocytes do not survive in HESCM4 medium. The micrograph shown was taken after 7 days of culture. (F) Fibroblasts grow well in HESCM4 medium but do not form spheres. The micrograph was taken after 7 days of culture. Scale bar: 100 μm.

DSCs are capable of self-renewal and proliferation. (A) Limiting dilution assays showed that a single cell derived from a DSC sphere re-formed a sphere. A typical sphere appeared after approximately 90 days. (B) Ki67 staining detected proliferating cells in an embryoid body (EB), a DSC sphere and a human hair follicle stem cell sphere (HFSC). Nuclei are stained blue with DAPI; red phalloidin (Pha) staining allows visualization of actin filaments. Scale bars: 100 μm (A) and 50 μm (B).

Melanocytic differentiation of DSCs in 3D culture. (A) Differentiated melanocytes from DSC spheres were embedded in organotypic skin reconstructs and stained for melanin, S100 and tyrosinase (TYR), respectively. Melanocytic-marker-positive cells localize to the epidermal-dermal junction in the same manner as normal epidermal melanocytes in skin reconstructs. Scale bar: 50 μm. (B) Single cells are capable of migrating out from DSC spheres in collagen I matrix. Scale bar: 150 μm. (C) Undifferentiated DSC spheres embedded into the dermis of skin reconstructs with fibroblasts (FF, transduced with green fluorescent protein). After 4 days, keratinocytes were seeded on top of the dermis to form an epidermis. After additional 14 days of culture, single cells migrate out from DSC spheres toward the basement membrane (white dotted line) at the epidermis-dermis junction. Melanocytes on basement membranes express only the melanocytic marker HMB45 (red), whereas dermal melanocytes coexpress NGFRp75 (purple) and HMB45 (red). Nuclei are counterstained with DAPI (blue). Differential interference contrast (DIC) image detected the position of the basement membrane based on the difference in cell density between the epidermis and the dermis. Scale bar: 50 μm. (D) DSCs were transduced with a GFP-expressing lentiviral vector and embedded in organotypic skin reconstructs. Skin reconstructs were serially harvested 5, 8, 10, 12, and 14 days after seeding keratinocytes, to monitor the migration of GFP-expressing cells. White dotted lines indicate the epidermis-dermis junction. Scale bar: 50 μm.

Cells positive for stem cell markers are located in human foreskin dermis. (A) OCT4-positive cells are found in human foreskin and are located in the dermis. The enlarged image (right panel) shows OCT4 expression in nuclei. Scale bars: 100 μm (left) and 30 μm (right). (B) Immunohistochemistry staining indicates that a number of NGFRp75-immunoreactive cells are located in the dermis. Most of the NGFRp75-positive cells are spindle shaped (arrows). Some small and rounded NGFRp75-positive cells are seen as single cells (arrowheads). Scale bar: 100 μm. (C) Immunofluorescent staining shows a few cells in the foreskin dermis that coexpress NGFRp75 and OCT4. The enlarged image (right panel) shows a single cell expressing both NGFRp75 (red) and OCT4 (green). Nuclei are stained with DAPI (blue). Scale bars: 100 μm (left) and 20 μm (right).