Isolation of multipotent nestin-expressing stem cells derived from the epidermis of elderly humans and TAT-VHL peptide-mediated neuronal differentiation of these cells

Abstract

A specialized population of cells residing in the hair follicle is quiescent but shows pluripotency for differentiating into epithelial-mesenchymal lineage cells. Therefore, such cells are hoped to be useful as implantable donor cells for regenerative therapy. Recently, it was reported that intracellular delivery of TAT-VHL peptide induces neuronal differentiation of skin-derived precursors. In the present study, we successfully isolated multipotent stem cells derived from the epidermis of elderly humans, characterized these cells as being capable of sphere formation and strong expression of nestin, fibronectin, and CD34 but not of keratin 15, and identified the niche of these cells as being the outer root sheath of the hair follicles. In addition, we showed that TAT-VHL peptide induced their neuronal differentiation in vitro, and confirmed by fluorescence immunohistochemistry the neuronal differentiation of such peptide-treated cells implanted into rodent brains. These multipotent nestin-expressing stem cells derived from human epidermis are easily accessible and should be useful as donor cells for neuronal regenerative cell therapy.

Figure 1

(A) One week after the start of a primary culture of cells derived from the epidermis from an elderly human. Sphere formation was recognized; (B) Four-week-old primary culture. Numerous floating spheres were observed; (C) Results from a sphere-forming assay. After 4 weeks, the rate of sphere formation became slow. Scale bars = 50 μm.

Figure 2

Fluorescence immunocytochemistry of the cultured stem cells after dissociation of the spheres into single cells. The spheres at 5 weeks in primary culture were dissociated by pipetting. (A) Triple fluorescence immunocytochemistry for expressions of CD34 (green) and fibronectin (red) and visualization of nuclei (blue); (B) Triple fluorescence immunocytochemistry for nestin (green), NGFR p75 (red), and nuclei (blue); (C) Triple fluorescence immunocytochemistry for nestin (green), keratin 15 (red), and nuclei (blue); (D) Percentages of the cells showing expression of the five proteins examined. High expressions of nestin, fibronectin, and CD34 were found, whereas those of keratin 15 and NGFR p75 were low. DAPI, nuclear marker; Merge, all three images superimposed; (E–G) Fluorescence immunocytochemical study on differentiation of stem cells into neurons expressing microtubule-associated protein (MAP)-2 (green, E), astrocytes expressing glial fibrillary acidic protein (GFAP; green, F), and smooth muscle cells expressing smooth muscle actin (SMA; green, G), with the nucleus in blue. Scale bars = 50 μm.

Figure 2

Fluorescence immunocytochemistry of the cultured stem cells after dissociation of the spheres into single cells. The spheres at 5 weeks in primary culture were dissociated by pipetting. (A) Triple fluorescence immunocytochemistry for expressions of CD34 (green) and fibronectin (red) and visualization of nuclei (blue); (B) Triple fluorescence immunocytochemistry for nestin (green), NGFR p75 (red), and nuclei (blue); (C) Triple fluorescence immunocytochemistry for nestin (green), keratin 15 (red), and nuclei (blue); (D) Percentages of the cells showing expression of the five proteins examined. High expressions of nestin, fibronectin, and CD34 were found, whereas those of keratin 15 and NGFR p75 were low. DAPI, nuclear marker; Merge, all three images superimposed; (E–G) Fluorescence immunocytochemical study on differentiation of stem cells into neurons expressing microtubule-associated protein (MAP)-2 (green, E), astrocytes expressing glial fibrillary acidic protein (GFAP; green, F), and smooth muscle cells expressing smooth muscle actin (SMA; green, G), with the nucleus in blue. Scale bars = 50 μm.

Figure 3

Fluorescence immunohistochemistry for skin, including epidermis and dermis, and epidermis only. (A) Triple fluorescence immunohistochemistry for expressions of nestin (green) and CD34 (red), and visualization of nuclei (blue) in the skin. Both nestin and CD34-co-expressing cells (arrow) are shown at the outer root sheath of hair follicles; (B) Triple fluorescence immunohistochemistry for expressions of nestin (green) and fibronectin (red), and visualization of nuclei (blue) in the skin. Both nestin and fibronectin-co-expressing cells (double arrows) are seen at the outer root sheath of hair follicles; (C) Double fluorescence immunohistochemistry for expression of keratin 15 (green) and visualization of nuclei (blue). Keratin 15 was identified in the hair follicle and keratinocyte layer; (D) Triple fluorescence immunohistochemistry of epidermis alone for nestin (green) and keratin 15 (red) and visualization of nuclei (blue). A nestin-expressing cell population was identified in the hair follicles. Keratin 15 was expressed in the outer layer. Scale bars = 50 μm.

Figure 4

Fluorescence immunocytochemistry of the cultured stem cells after intracellular delivery of TAT-VHL peptide. (A) Triple fluorescence immunocytochemistry for expressions of MAP2 (green) and neurofilament-M (NFM, red) and visualization of nuclei (blue); (B) Triple fluorescence immunocytochemistry for NeuN (green) and neurofilament-H (NFH, red) and visualization of nuclei (blue); (C) Percentages of the TAT-VHL peptide- or TAT peptide-containing cells expressing MAP2, NFM, NFH or NeuN. The TAT-VHL peptide cells showed significantly higher expression of all 4 proteins (** p < 0.001). Scale bars = 30 μm.

Figure 5

Fluorescence immunohistochemistry for rodent brain tissues implanted with peptide-transferred nestin-expressing stem cells pre-stained with red-fluorescent PKH26-PCL. Confocal immunohistochemical images showed expression of neuronal marker Tuj-1 (green) or NeuN (green). The nuclei were stained with DAPI (blue). PKH-pre-stained TAT-VHL peptide-containing cells showed significantly more expression of Tuj-1 and NeuN than did the pre-stained TAT peptide-containing ones (p < 0.01). Scale bar = 20 μm.