Nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells are located in the bulge area of the follicle. Previous studies have shown that HAP stem cells can differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. HAP stem cells effected nerve and spinal cord regeneration in mouse models. Recently, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. The differentiation potential to cardiac muscle cells was greatest in the upper part of the follicle. The beat rate of the cardiac muscle cells was stimulated by isoproterenol. In the present study, we observed that isoproterenol directs HAP stem cells to differentiate to cardiac muscle cells in large numbers in culture compared to HAP stem cells not supplemented with isoproterenol. The addition of activin A, bone morphogenetic protein 4, and basic fibroblast growth factor, along with isoproternal, induced the cardiac muscle cells to form tissue sheets of beating heart muscle cells. These results demonstrate that HAP stem cells have great potential to form beating cardiac muscle cells in tissue sheets.

Hair follicle-associated-pluripotent (HAP) stem cells are located in the bulge area of the hair follicle, express the stem-cell marker, nestin, and have been shown to differentiate to nerve cells, glial cells, keratinocytes, smooth muscle cells, cardiac muscle cells, and melanocytes. Transplanted HAP stem cells promote the recovery of peripheral nerve and spinal cord injuries and have the potential for heart regeneration as well. In the present study, we implanted mouse green fluorescent protein (GFP)-expressing HAP stem-cell spheres encapsulated in polyvinylidene fluoride (PVDF)-membrane cylinders into the severed sciatic nerve of immunocompetent and immunocompromised (nude) mice. Eight weeks after implantation, immunofluorescence staining showed that the HAP stem cells differentiated into neurons and glial cells. Fluorescence microscopy showed that the HAP stem cell hair spheres promoted rejoining of the sciatic nerve of both immunocompetent and immunodeficient mice. Hematoxylin and eosin (H&E) staining showed that the severed scatic nerves had regenerated. Quantitative walking analysis showed that the transplanted mice recovered the ability to walk normally. HAP stem cells are readily accessible from everyone, do not form tumors, and can be cryopreserved without loss of differentiation potential. These results suggest that HAP stem cells may have greater potential than iPS or ES cells for regenerative medicine.

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We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair-follicle stem cells located in the bulge area which are termed hair-follicle-associated pluripotent (HAP) stem cells. HAP stem cells from mouse and human could form spheres in culture, termed hair spheres, which are keratin 15-negative and nestin-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Subsequently, we demonstrated that nestin-expressing stem cells could effect nerve and spinal cord regeneration in mouse models. Recently, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. We recently observed that isoproterenol directs HAP stem cells to differentiate to cardiac-muscle cells in large numbers in culture compared to HAP stem cells not supplemented with isoproterenol. The addition of activin A, bone morphogenetic protein 4, and basic fibroblast growth factor, along with isoproternal, induced the cardiac muscle cells to form tissue sheets of beating heart muscle cells. In the present study, we report that, under hypoxic conditions, HAP stem cells differentiated to troponin-positive cardiac-muscle cells at a higher rate that under normoxic conditions. Hypoxia did not influence the differentiation to other cell types. For future use of HAP stem cells for cardiac muscle regeneration, hypoxia should enhance the rate of differentiation thereby providing patients more opportunities to use their own HAP stem cells which are easily accessible, for this purpose. J. Cell. Biochem. 118: 554-558, 2017. © 2016 Wiley Periodicals, Inc.

We have previously discovered nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells and have shown that they can differentiate to neurons, glia, and many other cell types. HAP stem cells can be used for nerve and spinal cord repair. We have recently shown the HAP stem cells can differentiate to beating heart-muscle cells and tissue sheets of beating heart-muscle cells. In the present study, we determined the efficiency of HAP stem cells from mouse vibrissa hair follicles of various ages to differentiate to beating heart-muscle cells. We observed that the whiskers located near the ear were more efficient to differentiate to cardiac-muscle cells compared to whiskers located near the nose. Differentiation to cardiac-muscle cells from HAP stem cells in cultured whiskers in 4-week-old mice was significantly greater than in 10-, 20-, and 40-week-old mice. There was a strong decrease in differentiation potential of HAP stem cells to cardiac-muscle cells by 10 weeks of age. In contrast, the differentiation potential of HAP stem cells to other cell types did not decrease with age. The possibility of rejuvenation of HAP stem cells to differentiate at high efficiency to cardiac-muscle cells is discussed.