Review Accepted after revision: July 23, 2014 Published online: March 4, 2015
Cells Tissues Organs DOI: 10.1159/000366098
Nestin-Expressing Hair Follicle-Accessible Pluripotent Stem Cells for Nerve and Spinal Cord Repair Robert M. Hoffman AntiCancer Inc., and Department of Surgery, University of California San Diego, San Diego, Calif., USA
Abstract Nestin-expressing stem cells of the hair follicle, discovered by our laboratory, have been shown to be able to form neurons and other nonfollicle cell types. We have shown that the nestin-expressing stem cells from the hair follicle can effect the repair of peripheral nerve and spinal cord injury. The hair follicle stem cells differentiate into neuronal and glial cells after transplantation to the injured peripheral nerve and spinal cord, and enhance injury repair and locomotor recovery. We have termed these cells hair follicle-accessible pluripotent (HAP) stem cells. When the excised hair follicle with its nerve stump was placed in Gelfoam® 3D histoculture, HAP stem cells grew and extended the hair follicle nerve which consisted of βIII-tubulin-positive fibers with F-actin expression at the tip. These findings indicate that βIII-tubulinpositive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in HAP stem cells, which appeared to play a major role in its elongation and interaction with other nerves in 3D Gelfoam® histoculture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve
This paper is dedicated to the memory of A.R. Moossa, MD.
© 2015 S. Karger AG, Basel 1422–6405/15/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/cto
ganglion. Our results suggest that a major function of the HAP stem cells in the hair follicle is for growth of the follicle sensory nerve. HAP stem cells have critical advantages over embryonic stem cells and induced pluripotent stem cells in that they are highly accessible, require no genetic manipulation, are nontumorigenic, and do not present ethical issues for regenerative medicine. © 2015 S. Karger AG, Basel
Introduction: Properties of the Hair Follicle
The hair follicle comprises concentric cylinders with many cell types that produce highly specialized proteins, especially keratin, which is a major component of hair. Follicle formation is regulated by the adjacent mesenchymal dermal papilla (DP) [Hardy, 1992]. The hair follicle continuously cycles through the anagen (hair growth), catagen (involution), and telogen (resting) phases throughout the life of the mammal (fig. 1) [Li et al., 1991; Hardy, 1992; Paus and Cotsarelis, 1999; Hoffman, 2000; Uchu-
Abbreviations used in this paper
BA DP GFP HAP ND-GFP
bulge area dermal papilla green fluorescent protein hair follicle-accessible pluripotent nestin-driven GFP
Robert M. Hoffman, PhD AntiCancer Inc. 7917 Ostrow Street San Diego, CA 92111 (USA) E-Mail all @ anticancer.com
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Key Words Hair follicle · Bulge · Nestin · Stem cells · Pluripotent · Green fluorescent protein · Schwann cells · Sciatic nerve · Repair · Spinal cord repair
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Anagen day 2 Anagen day 4
Catagen day 18
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Fig. 1. The hair-growth cycle: the position of nestin-GFP stem cells at each phase of the hair follicle cycle [Li et al., 2003]. SG = Sebaceous
gonova et al., 2011a]. The bulge area (BA) of the hair follicle has been shown to be a niche for hair follicle stem cells [Cotsarelis et al., 1990; Li et al., 2003; Liu et al., 2011; Uchugonova et al., 2011a].
Hair-Follicle Accessible Pluripotent Stem Cells
Our laboratory discovered the existence of stem cells in the hair follicle that express nestin, a stem cell marker in the central nervous system. This discovery resulted in a paradigm change in the concept of hair follicle stem cells and in the field of adult stem cells itself [Li et al., 2003]. The surprising discovery of nestin-expressing cells in the hair follicle occurred with the use of transgenic mice 2
Cells Tissues Organs DOI: 10.1159/000366098
in which the expression of green fluorescent protein (GFP) is driven by the promoter of the neural stem cell marker, nestin (ND-GFP). The nestin-expressing hair follicle stem cells expressed GFP in the transgenic mice, which enabled their discovery. The nestin-expressing stem cells are relatively small, oval shaped, surround the hair shaft, and are interconnected by short dendrites. In the mid- and late-anagen phase, the nestin-expressing cells are located in the upper outer-root sheath as well as in the BA but not in the hair matrix bulb, suggesting that they form the outer-root sheath (fig. 1, 2). At the very beginning of the anagen phase, the ND-GFP cells can be seen forming the hair follicle (fig. 3). Mignone et al. [2007] have confirmed these results. Yu et al. [2006] showed that nestin was present in human hair follicle Hoffman
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gland; EP = epidermis.
EP
SG
SG c a
d
b
Fig. 2. Nestin-GFP-expressing stem cells in the telogen phase of the hair cycle of nestin-driven GFP transgenic mouse skin. a Diagram of a telogen hair follicle shows the position of the nestin-GFP-expressing hair follicle stem cells. b–d The telogen nestin-GFP-expressing hair follicle stem cells are shown from low to high magnification under fluorescence microscopy [Li et al., 2003]. SG = Sebaceous gland; EP = epidermis.
stem cells, thus confirming our original observation in mice [Li et al., 2003]. Yu et al. [2006] suggested that the nestin-expressing stem cells they isolated from the human hair follicle are different from the keratinocyte stem cells and melanocyte stem cells also present in the hair follicle. This indicates that the hair follicle probably contains several distinct populations of stem cells [Nishimura et al., 2002; Morris et al., 2004; Tumbar et al., 2004; Rhee et al., 2006]. We have defined the nestin-expressing hair-follicle accessible pluripotent (HAP) stem cells based on the characteristics described below.
Nestin-GFP-expressing hair follicle stem cells
SG
SG
Telogen hair follicle Nascent anagen hair follicle forming from stem cells
Fig. 3. A new anagen hair follicle is being formed by the nestinGFP stem cells (right hair follicle, white arrows) [Li et al., 2003]. SG = Sebaceous gland. Original magnification. ×400.
Human Hair Follicles Also Have HAP Stem Cells HAP stem cells can be readily isolated from the human scalp [Amoh et al., 2009b]. In the intact human hair fol-
licle dissected from the scalp, the cells immediately below the sebaceous glands just above the BA were observed to be nestin positive and K15 negative. In contrast, the hair follicle BA itself contained nestin-negative, K15-positive cells. The human nestin-expressing HAP follicle stem cells could differentiate into other cell types, including βIII-tubulin-positive neurons, S-100-positive and GFAPpositive glial cells, K15-positive keratinocytes, and smooth muscle antigen-positive smooth muscle cells similar to mouse HAP cells [Amoh et al., 2005a, 2009a]. Yu et al. [2006, 2010] characterized human nestin-expressing HAP stem cells. These cells expressed Nanog and Oct4, which are neural crest and neuron stem cell
HAP Stem Cells for Nerve and Spinal Cord Repair
Cells Tissues Organs DOI: 10.1159/000366098
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HAP Stem Cells Differentiate to Many Types of Cells We originally predicted the hair follicle nestin-expressing stem cells could differentiate to neurons and possibly other cell types [Li et al., 2003]. Subsequently, it was demonstrated that nestin-expressing HAP stem cells could differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro [Sieber-Blum et al., 2004; Amoh et al., 2005a; Hoffman, 2006; Sieber-Blum et al., 2006; Amoh et al., 2009a; Biernaskie et al., 2009]. HAP stem cells were positive for the stem cell marker CD34 and negative for keratin 15. HAP stem cells differentiated into neurons after transplantation to the subcutis of nude mice [Amoh et al., 2005a]. It has been confirmed that HAP cells are pluripotent stem cells by at least six laboratories: Li et al. [2003]; Amoh et al. [2004, 2005a, 2008, 2009a, b, 2010]; Sieber-Blum et al. [2004, 2006]; Yu et al. [2006, 2010]; Mignone et al. [2007], and Liu et al. [2014].
BA
Hair shaft
markers, as well as embryonic stem cell transcription factors. The human HAP cells formed spheres in vitro and differentiated into myogenic, melanocytic, and neuronal cell lineages in single-cell culture. The human nestin-expressing HAP stem cells also differentiated into adipocyte, chondrocyte, and osteocyte lineages. HAP Stem Cells Can Effect Nerve Repair After transplantation of HAP stem cells from the mouse hair follicle to severed peripheral nerves in mice, the cells differentiated mostly into glial fibrillary-acidicprotein (GFAP)-expressing Schwann cells, which support neuron regrowth. The transplanted mice recovered the ability to walk normally, as the transplanted nerve could stimulate gastrocnemius muscle contraction [Amoh et al., 2005b]. Human HAP stem cells could also repair severed peripheral nerves of the mouse [Amoh et al., 2005b, 2009b, 2010]. HAP Stem Cells Can Effect Spinal Cord Repair The nestin-expressing mouse hair follicle stem cells were injected into the injured area of the spinal cord in mice. The HAP stem cells promoted the recovery of the injured spinal cord by differentiating into glial-like cells and enabled significant locomotor improvement [Amoh et al., 2008].
Location of HAP Stem Cells in the BA and DP
Our laboratory compared the nestin-expressing HAP stem cells in the BA and the DP and found that these cells were present in the BA throughout the hair cycle, but in the DP only in the early anagen phase. The nestin-ex4
Cells Tissues Organs DOI: 10.1159/000366098
80 μm
20 μm
pressing HAP stem cells from both regions had a similar morphology. The cells from both regions had very long processes extending from them, as shown by confocal microscopy (fig. 4) [Liu et al., 2011; Uchugonova et al., 2011a, b]. Nestin-expressing HAP cells from both the BA and the DP could differentiate into neurons and other cell types in vitro. The nestin-expressing HAP stem cells from both the BA and DP had an equal ability to affect spinal cord repair and locomotor recovery, subsequent to transplantation, by differentiating into Schwann and neuraltype cells [Liu et al., 2011].
The BA Is the Source of HAP Stem Cells
The nestin-expressing HAP stem cells from the BA trafficked to the DP as well as into the epidermis, including during wound healing. These results suggest that the BA is a source of skin stem cells, including perhaps those identified as skin-derived precursor cells [Biernaskie et al., 2009; Uchugonova et al., 2011a]. Nestin-expressing mouse hair follicle HAP stem cells expressing red fluorescent protein were induced by retinoic acid and fetal bovine serum to differentiate, and were then transplanted together with Matrigel into the transected distal sciatic or tibial nerve stump of transgenic nude mice ubiquitously expressing GFP. The transplanted cells differentiated into neurons with large round nuclei and long extensions. Transplanted cells in the distal nerve stump expressed the neuron marker Tuji1 as well as the motor neuron markers Isl 1/2 and EN1. Muscle fiber areas in the nestin-expressing HAP stem cell plus Matrigel-transplanted animals were much larger than that in the Matrigel-only transplanted animals after 4 Hoffman
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Fig. 4. Confocal fluorescence microscopy images of nestin-GFP hair follicle stem cells in the BA [Uchugonova et al., 2011a]. Left panel = low magnification; right panel = high magnification.
Growth of Nerves in Histoculture from HAP Stem Cells
Our laboratory established the technique of long-term Gelfoam® histoculture of skin [Li et al., 1991, 1992]. Subsequently, whiskers isolated from ND-GFP mice were cultured on Gelfoam®. Confocal imaging was used to monitor HAP stem cells trafficking in real time between the BA and DP to determine the fate of the stem cells. It was observed over a 2-week period that the HAP stem cells trafficked from the BA toward the DP area and extensively grew out onto Gelfoam®, forming nerve-like structures [Duong et al., 2012]. In a following study, mouse vibrissa hair follicles, including their sensory nerve stump, were excised from ND-GFP mice and placed in Gelfoam® histoculture. βIIItubulin-positive fibers, comprising HAP stem cells, extended up to 500 mm from the whisker nerve stump in histoculture. Growing fibers had growth cones on their tips expressing F-actin. These findings indicate that βIIItubulin-positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in HAP stem cells, which appeared to play a major role in its elongation and interaction with other nerves in 3D culture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion [Mii et al., 2013a]. The sciatic nerve was excised from the ND-GFP mice. The sciatic nerve was found to contain HAP-like cells. When the HAP-like cells in the mouse sciatic nerve were histocultured on Gelfoam®, they formed fibers extending the nerve. The fibers consisted of ND-GFP-expressing spindle cells, which co-expressed the neuron marker βIIItubulin, the immature Schwann cell marker p75NTR and TrkB, which is associated with neurons. The βIII-tubulinpositive fibers had growth cones on their tips expressing F-actin, indicating that they were growing axons. Proliferating HAP-like stem cells in the injured sciatic nerve were also observed in vivo. These stem cells were also seen in posterior nerves of the spine, but not in the spinal cord itself, when placed in Gelfoam® histoculture [Mii et al., 2013b]. We also observed HAP-like stem cells in the fungiform papilla in the tongue. The nestin-expressing HAP-like HAP Stem Cells for Nerve and Spinal Cord Repair
stem cells in the fungiform papilla are located around a peripheral sensory nerve immediately below the taste bud. These HAP-like cells co-express the neural crest cell marker p75NTR. The fungiform papilla HAP-like stem cells formed spheres in suspension culture in DMEM-F12 medium supplemented with basic fibroblast growth factor-2. The spheres consisted of nestin-expressing HAP-like cells that co-expressed the neural crest marker p75NTR and developed expression of the stem cell marker CD34. The HAP-like stem cells of these spheres differentiated into the following cell types: βIII-tubulinexpressing nerve cells; GFAP-expressing glial cells; K15expressing keratinocytes, and smooth muscle antigenexpressing cells. These results suggest the HAP-like stem cells of the fungiform papilla differentiated into multiple cell types [Mii et al., 2014].
Conclusion
ND-GFP mice enabled us to make the observation that hair follicles contain nestin-expressing HAP stem cells. HAP stem cells originate in the BA [Li et al., 2003], migrate to the DP [Uchugonova et al., 2011a], and can form the outer-root sheath and other parts of the follicle [Li et al., 2003] as well as the hair follicle-associated nerve. The HAP stem cells express stem cell markers other than nestin, such as CD34. The HAP stem cells can differentiate into many cell types, including neurons and glial cells in vitro. HAP stem cells are also pluripotent in vivo and have been shown to effect peripheral nerve and spinal cord repair by differentiating into neural- and glial-type cells. HAP stem cells have also been found in the human scalp, and these stem cells have been shown to be pluripotent and can affect the repair of peripheral nerves. The sciatic nerve and tongue fungiform papilla also contain HAP-like stem cells [Mii et al., 2013b, 2014]. The ability of the HAP stem cells to effect nerve and spinal cord repair, as well as their easy access and autologous use, suggest the potential of these cells for regenerative medicine. Compared to embryonic stem cells or induced pluripotent stem (iPS) cells [Keirstead et al., 2005; Nori et al., 2011; Okano et al., 2013], HAP stem cells have important advantages in that they can be used autologously, are non-oncogenic, and do not pose ethical issues. Furthermore, they can be readily expanded in culture after isolation from the patient’s hair follicle.
Cells Tissues Organs DOI: 10.1159/000366098
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weeks. Thus, transplanted nestin-expressing HAP stem cells can differentiate into motor neurons and reduce muscle atrophy after sciatic nerve transection [Liu et al., 2014].
References
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Cells Tissues Organs DOI: 10.1159/000366098
Hoffman, R.M. (2000) The hair follicle as a gene therapy target. Nat Biotechnol 18: 20–21. Hoffman, R.M. (2006) The pluripotency of hair follicle stem cells. Cell Cycle 5: 232–233. Keirstead, H.S., G. Nistor, G. Bernal, M. Totoiu, F. Cloutier, K. Sharp, O. Steward (2005) Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci 25: 4694–4705. Li, L., L.B. Margolis, R.M. Hoffman (1991) Skin toxicity determined in vitro by three-dimensional, native-state histoculture. Proc Natl Acad Sci USA 88: 1908–1912. Li, L., J. Mignone, M. Yang, M. Matic, S. Penman, G. Enikolopov, R.M. Hoffman (2003) Nestin expression in hair follicle sheath progenitor cells. Proc Natl Acad Sci USA 100: 9958–9961. Li, L., R. Paus, L.B. Margolis, R.M. Hoffman (1992) Hair shaft elongation, follicle growth, and spontaneous regression in long-term, gelatin sponge-supported histoculture of human scalp skin. Proc Natl Acad Sci USA 89: 8764–8768. Liu, F., A. Uchugonova, H. Kimura, C. Zhang, M. Zhao, L. Zhang, K. Koenig, J. Duong, R. Aki, N. Saito, S. Mii, Y. Amoh, K. Katsuoka, R.M. Hoffman (2011) The bulge area is the major hair follicle source of nestin-expressing multipotent stem cells which can repair the spinal cord compared to the dermal papilla. Cell Cycle 10: 830–839. Liu, F., C. Zhang, R.M. Hoffman (2014) Nestinexpressing stem cells from the hair follicle can differentiate into motor neurons and reduce muscle atrophy after transplantation to injured nerves. Tissue Eng Part A 20: 656–662. Mignone, J.L., J.L. Roig-Lopez, N. Fedtsova, D.E. Schones, L.N. Manganas, M. Maletic-Savatic, W.M. Keyes, A.A. Mills, A. Gleiberman, M.Q. Zhang, G. Enikolopov (2007) Neural potential of a stem cell population in the hair follicle. Cell Cycle 6: 2161–2170. Mii, S., Y. Amoh, K. Katsuoka, R.M. Hoffman (2014) Comparison of nestin-expressing multipotent stem cells in the tongue fungiform papilla and vibrissa hair follicle. J Cell Biochem 115: 1070–1076. Mii, S., J. Duong, Y. Tome, A. Uchugonova, F. Liu, Y. Amoh, N. Saito, K. Katsuoka, R.M. Hoffman (2013a) The role of hair follicle nestinexpressing stem cells during whisker sensorynerve growth in long-term 3D culture. J Cell Biochem 114: 1674–1684. Mii, S., F. Uehara, S. Yano, B. Tran, S. Miwa, Y. Hiroshima, Y. Amoh, K. Katsuoka, R.M. Hoffman (2013b) Nestin-expressing stem cells promote nerve growth in long-term 3-dimensional Gelfoam®-supported histoculture. PLoS One 8: e67153.
Morris, R.J., Y. Liu, L. Marles, Z. Yang, C. Trempus, S. Li, J.S. Lin, J.A. Sawicki, G. Cotsarelis (2004) Capturing and profiling adult hair follicle stem cells. Nat Biotechnol 22: 411–417. Nishimura, E.K., S.A. Jordan, H. Oshima, H. Yoshida, M. Osawa, M. Moriyama, I.J. Jackson, Y. Barrandon, Y. Miyachi, S. Nishikawa (2002) Dominant role of the niche in melanocyte stem-cell fate determination. Nature 416: 854–860. Nori, S., Y. Okada, A. Yasuda, O. Tsuji, Y. Takahashi, Y. Kobayashi, K. Fujiyoshi, M. Koike, Y. Uchiyama, E. Ikeda, Y. Toyama, S. Yamanaka, M. Nakamura, H. Okano (2011) Grafted human-induced pluripotent stemcell-derived neurospheres promote motor functional recovery after spinal cord injury in mice. Proc Natl Acad Sci USA 108: 16825– 16830. Okano, H., M. Nakamura, K. Yoshida, Y. Okada, O. Tsuji, S. Nori, E. Ikeda, S. Yamanaka, K. Miura (2013) Steps toward safe cell therapy using induced pluripotent stem cells. Circ Res 112: 523–533. Paus, R., G.N. Cotsarelis (1999) The biology of hair follicles. N Engl J Med 341: 491–497. Rhee, H., L. Polak, E. Fuchs (2006) Lhx2 maintains stem cell character in hair follicles. Science 312: 1946–1949. Sieber-Blum, M., M. Grim, Y.F. Hu, V. Szeder (2004) Multipotent neural crest stem cells in the adult hair follicle. Dev Dyn 231: 258–269. Sieber-Blum, M., L. Schnell, M. Grim, Y.F. Hu, R. Schneider, M.E. Schwab (2006) Characterization of epidermal neural crest stem cell (EPINCSC) grafts in the lesioned spinal cord. Mol Cell Neurosci 32: 67–81. Tumbar, T., G. Guasch, V. Greco, C. Blanpain, W.E. Lowry, M. Rendl, E. Fuchs (2004) Defining the epithelial stem cell niche in skin. Science 303: 359–363. Uchugonova, A., J. Duong, N. Zhang, K. König, R.M. Hoffman (2011a) The bulge area is the origin of nestin-expressing multipotent stem cells of the hair follicle. J Cell Biochem 112: 2046–2050. Uchugonova, A., R.M. Hoffman, M. Weinigel, K. Koenig (2011b) Watching stem cells in the skin of living mice noninvasively. Cell Cycle 10: 2017–2020. Yu, H., D. Fang, S.M. Kumar, L. Li, T.K. Nguyen, G. Acs, M. Herlyn, X. Xu (2006) Isolation of a novel population of multipotent adult stem cells from human hair follicles. Am J Pathol 168: 1879–1888. Yu, H., S.M. Kumar, A.V. Kossenkov, L. Showe, X. Xu (2010) Stem cells with neural crest characteristics derived from the bulge region of cultured human hair follicles. J Invest Derm 130: 1227–1236.
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Amoh, Y., Y. Hamada, R. Aki, K. Kawahara, R.M. Hoffman, K. Katsuoka (2010) Direct transplantation of uncultured hair-follicle multipotent stem (hfPS) cells promotes the recovery of peripheral nerve injury. J Cell Biochem 110: 272–277. Amoh, Y., M. Kanoh, S. Niiyama, Y. Hamada, K. Kawahara, Y. Sato, R.M. Hoffman, K. Katsuoka (2009b) Human hair follicle multipotent stem (hfPS) cells promote regeneration of peripheral-nerve injury: an advantageous alternative to ES and iPS cells. J Cell Biochem 107: 1016–1020. Amoh, Y., M. Kanoh, S. Niiyama, K. Kawahara, Y. Satoh, K. Katsuoka, R.M. Hoffman (2009a) Human and mouse hair follicles contain both multipotent and monopotent stem cells. Cell Cycle 8: 176–177. Amoh, Y., L. Li, R. Campillo, K. Kawahara, K. Katsuoka, S. Penman, R.M. Hoffman (2005b) Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves. Proc Natl Acad Sci USA 102: 17734–17738. Amoh, Y., L. Li, K. Katsuoka, R.M. Hoffman (2008) Multipotent hair follicle stem cells promote repair of spinal cord injury and recovery of walking function. Cell Cycle 7: 1865–1869. Amoh, Y., L. Li, K. Katsuoka, S. Penman, R.M. Hoffman (2005a) Multipotent nestin-positive, keratin-negative hair-follicle-bulge stem cells can form neurons. Proc Natl Acad Sci USA 102: 5530–5534. Amoh, Y., L. Li, M. Yang, A.R. Moossa, K. Katsuoka, S. Penman, R.M. Hoffman (2004) Nascent blood vessels in the skin arise from nestin-expressing hair follicle cells. Proc Natl Acad Sci USA 101: 13291–13295. Biernaskie, J., M. Paris, O. Morozova, B.M. Fagan, M. Marra, L. Pevny, F.D. Miller (2009) SKPs derive from hair follicle precursors and exhibit properties of adult dermal stem cells. Cell Stem Cell 5: 610–623. Cotsarelis, G., T.T. Sun, R.M. Lavker (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61: 1329–1337. Duong, J., S. Mii, A. Uchugonova, F. Liu, A.R. Moossa, R.M. Hoffman (2012) Real-time confocal imaging of trafficking of nestin-expressing multipotent stem cells in mouse whiskers in long-term 3-D histoculture. In Vitro Cell Dev Biol Anim 48: 301–305. Hardy, M.H. (1992) The secret life of the hair follicle. Trends Genet 8: 55–61.
Cells Tissues Organs DOI: 10.1159/000366098
Nestin-Expressing Hair Follicle-Accessible Pluripotent Stem Cells for Nerve and Spinal Cord Repair Robert M. Hoffman AntiCancer Inc., and Department of Surgery, University of California San Diego, San Diego, Calif., USA
Abstract Nestin-expressing stem cells of the hair follicle, discovered by our laboratory, have been shown to be able to form neurons and other nonfollicle cell types. We have shown that the nestin-expressing stem cells from the hair follicle can effect the repair of peripheral nerve and spinal cord injury. The hair follicle stem cells differentiate into neuronal and glial cells after transplantation to the injured peripheral nerve and spinal cord, and enhance injury repair and locomotor recovery. We have termed these cells hair follicle-accessible pluripotent (HAP) stem cells. When the excised hair follicle with its nerve stump was placed in Gelfoam® 3D histoculture, HAP stem cells grew and extended the hair follicle nerve which consisted of βIII-tubulin-positive fibers with F-actin expression at the tip. These findings indicate that βIII-tubulinpositive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in HAP stem cells, which appeared to play a major role in its elongation and interaction with other nerves in 3D Gelfoam® histoculture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve
This paper is dedicated to the memory of A.R. Moossa, MD.
© 2015 S. Karger AG, Basel 1422–6405/15/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/cto
ganglion. Our results suggest that a major function of the HAP stem cells in the hair follicle is for growth of the follicle sensory nerve. HAP stem cells have critical advantages over embryonic stem cells and induced pluripotent stem cells in that they are highly accessible, require no genetic manipulation, are nontumorigenic, and do not present ethical issues for regenerative medicine. © 2015 S. Karger AG, Basel
Introduction: Properties of the Hair Follicle
The hair follicle comprises concentric cylinders with many cell types that produce highly specialized proteins, especially keratin, which is a major component of hair. Follicle formation is regulated by the adjacent mesenchymal dermal papilla (DP) [Hardy, 1992]. The hair follicle continuously cycles through the anagen (hair growth), catagen (involution), and telogen (resting) phases throughout the life of the mammal (fig. 1) [Li et al., 1991; Hardy, 1992; Paus and Cotsarelis, 1999; Hoffman, 2000; Uchu-
Abbreviations used in this paper
BA DP GFP HAP ND-GFP
bulge area dermal papilla green fluorescent protein hair follicle-accessible pluripotent nestin-driven GFP
Robert M. Hoffman, PhD AntiCancer Inc. 7917 Ostrow Street San Diego, CA 92111 (USA) E-Mail all @ anticancer.com
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Key Words Hair follicle · Bulge · Nestin · Stem cells · Pluripotent · Green fluorescent protein · Schwann cells · Sciatic nerve · Repair · Spinal cord repair
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Anagen day 2 Anagen day 4
Catagen day 18
Catagen day 20
Anagen day 6
Anagen day 10
Fig. 1. The hair-growth cycle: the position of nestin-GFP stem cells at each phase of the hair follicle cycle [Li et al., 2003]. SG = Sebaceous
gonova et al., 2011a]. The bulge area (BA) of the hair follicle has been shown to be a niche for hair follicle stem cells [Cotsarelis et al., 1990; Li et al., 2003; Liu et al., 2011; Uchugonova et al., 2011a].
Hair-Follicle Accessible Pluripotent Stem Cells
Our laboratory discovered the existence of stem cells in the hair follicle that express nestin, a stem cell marker in the central nervous system. This discovery resulted in a paradigm change in the concept of hair follicle stem cells and in the field of adult stem cells itself [Li et al., 2003]. The surprising discovery of nestin-expressing cells in the hair follicle occurred with the use of transgenic mice 2
Cells Tissues Organs DOI: 10.1159/000366098
in which the expression of green fluorescent protein (GFP) is driven by the promoter of the neural stem cell marker, nestin (ND-GFP). The nestin-expressing hair follicle stem cells expressed GFP in the transgenic mice, which enabled their discovery. The nestin-expressing stem cells are relatively small, oval shaped, surround the hair shaft, and are interconnected by short dendrites. In the mid- and late-anagen phase, the nestin-expressing cells are located in the upper outer-root sheath as well as in the BA but not in the hair matrix bulb, suggesting that they form the outer-root sheath (fig. 1, 2). At the very beginning of the anagen phase, the ND-GFP cells can be seen forming the hair follicle (fig. 3). Mignone et al. [2007] have confirmed these results. Yu et al. [2006] showed that nestin was present in human hair follicle Hoffman
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gland; EP = epidermis.
EP
SG
SG c a
d
b
Fig. 2. Nestin-GFP-expressing stem cells in the telogen phase of the hair cycle of nestin-driven GFP transgenic mouse skin. a Diagram of a telogen hair follicle shows the position of the nestin-GFP-expressing hair follicle stem cells. b–d The telogen nestin-GFP-expressing hair follicle stem cells are shown from low to high magnification under fluorescence microscopy [Li et al., 2003]. SG = Sebaceous gland; EP = epidermis.
stem cells, thus confirming our original observation in mice [Li et al., 2003]. Yu et al. [2006] suggested that the nestin-expressing stem cells they isolated from the human hair follicle are different from the keratinocyte stem cells and melanocyte stem cells also present in the hair follicle. This indicates that the hair follicle probably contains several distinct populations of stem cells [Nishimura et al., 2002; Morris et al., 2004; Tumbar et al., 2004; Rhee et al., 2006]. We have defined the nestin-expressing hair-follicle accessible pluripotent (HAP) stem cells based on the characteristics described below.
Nestin-GFP-expressing hair follicle stem cells
SG
SG
Telogen hair follicle Nascent anagen hair follicle forming from stem cells
Fig. 3. A new anagen hair follicle is being formed by the nestinGFP stem cells (right hair follicle, white arrows) [Li et al., 2003]. SG = Sebaceous gland. Original magnification. ×400.
Human Hair Follicles Also Have HAP Stem Cells HAP stem cells can be readily isolated from the human scalp [Amoh et al., 2009b]. In the intact human hair fol-
licle dissected from the scalp, the cells immediately below the sebaceous glands just above the BA were observed to be nestin positive and K15 negative. In contrast, the hair follicle BA itself contained nestin-negative, K15-positive cells. The human nestin-expressing HAP follicle stem cells could differentiate into other cell types, including βIII-tubulin-positive neurons, S-100-positive and GFAPpositive glial cells, K15-positive keratinocytes, and smooth muscle antigen-positive smooth muscle cells similar to mouse HAP cells [Amoh et al., 2005a, 2009a]. Yu et al. [2006, 2010] characterized human nestin-expressing HAP stem cells. These cells expressed Nanog and Oct4, which are neural crest and neuron stem cell
HAP Stem Cells for Nerve and Spinal Cord Repair
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HAP Stem Cells Differentiate to Many Types of Cells We originally predicted the hair follicle nestin-expressing stem cells could differentiate to neurons and possibly other cell types [Li et al., 2003]. Subsequently, it was demonstrated that nestin-expressing HAP stem cells could differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro [Sieber-Blum et al., 2004; Amoh et al., 2005a; Hoffman, 2006; Sieber-Blum et al., 2006; Amoh et al., 2009a; Biernaskie et al., 2009]. HAP stem cells were positive for the stem cell marker CD34 and negative for keratin 15. HAP stem cells differentiated into neurons after transplantation to the subcutis of nude mice [Amoh et al., 2005a]. It has been confirmed that HAP cells are pluripotent stem cells by at least six laboratories: Li et al. [2003]; Amoh et al. [2004, 2005a, 2008, 2009a, b, 2010]; Sieber-Blum et al. [2004, 2006]; Yu et al. [2006, 2010]; Mignone et al. [2007], and Liu et al. [2014].
BA
Hair shaft
markers, as well as embryonic stem cell transcription factors. The human HAP cells formed spheres in vitro and differentiated into myogenic, melanocytic, and neuronal cell lineages in single-cell culture. The human nestin-expressing HAP stem cells also differentiated into adipocyte, chondrocyte, and osteocyte lineages. HAP Stem Cells Can Effect Nerve Repair After transplantation of HAP stem cells from the mouse hair follicle to severed peripheral nerves in mice, the cells differentiated mostly into glial fibrillary-acidicprotein (GFAP)-expressing Schwann cells, which support neuron regrowth. The transplanted mice recovered the ability to walk normally, as the transplanted nerve could stimulate gastrocnemius muscle contraction [Amoh et al., 2005b]. Human HAP stem cells could also repair severed peripheral nerves of the mouse [Amoh et al., 2005b, 2009b, 2010]. HAP Stem Cells Can Effect Spinal Cord Repair The nestin-expressing mouse hair follicle stem cells were injected into the injured area of the spinal cord in mice. The HAP stem cells promoted the recovery of the injured spinal cord by differentiating into glial-like cells and enabled significant locomotor improvement [Amoh et al., 2008].
Location of HAP Stem Cells in the BA and DP
Our laboratory compared the nestin-expressing HAP stem cells in the BA and the DP and found that these cells were present in the BA throughout the hair cycle, but in the DP only in the early anagen phase. The nestin-ex4
Cells Tissues Organs DOI: 10.1159/000366098
80 μm
20 μm
pressing HAP stem cells from both regions had a similar morphology. The cells from both regions had very long processes extending from them, as shown by confocal microscopy (fig. 4) [Liu et al., 2011; Uchugonova et al., 2011a, b]. Nestin-expressing HAP cells from both the BA and the DP could differentiate into neurons and other cell types in vitro. The nestin-expressing HAP stem cells from both the BA and DP had an equal ability to affect spinal cord repair and locomotor recovery, subsequent to transplantation, by differentiating into Schwann and neuraltype cells [Liu et al., 2011].
The BA Is the Source of HAP Stem Cells
The nestin-expressing HAP stem cells from the BA trafficked to the DP as well as into the epidermis, including during wound healing. These results suggest that the BA is a source of skin stem cells, including perhaps those identified as skin-derived precursor cells [Biernaskie et al., 2009; Uchugonova et al., 2011a]. Nestin-expressing mouse hair follicle HAP stem cells expressing red fluorescent protein were induced by retinoic acid and fetal bovine serum to differentiate, and were then transplanted together with Matrigel into the transected distal sciatic or tibial nerve stump of transgenic nude mice ubiquitously expressing GFP. The transplanted cells differentiated into neurons with large round nuclei and long extensions. Transplanted cells in the distal nerve stump expressed the neuron marker Tuji1 as well as the motor neuron markers Isl 1/2 and EN1. Muscle fiber areas in the nestin-expressing HAP stem cell plus Matrigel-transplanted animals were much larger than that in the Matrigel-only transplanted animals after 4 Hoffman
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Fig. 4. Confocal fluorescence microscopy images of nestin-GFP hair follicle stem cells in the BA [Uchugonova et al., 2011a]. Left panel = low magnification; right panel = high magnification.
Growth of Nerves in Histoculture from HAP Stem Cells
Our laboratory established the technique of long-term Gelfoam® histoculture of skin [Li et al., 1991, 1992]. Subsequently, whiskers isolated from ND-GFP mice were cultured on Gelfoam®. Confocal imaging was used to monitor HAP stem cells trafficking in real time between the BA and DP to determine the fate of the stem cells. It was observed over a 2-week period that the HAP stem cells trafficked from the BA toward the DP area and extensively grew out onto Gelfoam®, forming nerve-like structures [Duong et al., 2012]. In a following study, mouse vibrissa hair follicles, including their sensory nerve stump, were excised from ND-GFP mice and placed in Gelfoam® histoculture. βIIItubulin-positive fibers, comprising HAP stem cells, extended up to 500 mm from the whisker nerve stump in histoculture. Growing fibers had growth cones on their tips expressing F-actin. These findings indicate that βIIItubulin-positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in HAP stem cells, which appeared to play a major role in its elongation and interaction with other nerves in 3D culture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion [Mii et al., 2013a]. The sciatic nerve was excised from the ND-GFP mice. The sciatic nerve was found to contain HAP-like cells. When the HAP-like cells in the mouse sciatic nerve were histocultured on Gelfoam®, they formed fibers extending the nerve. The fibers consisted of ND-GFP-expressing spindle cells, which co-expressed the neuron marker βIIItubulin, the immature Schwann cell marker p75NTR and TrkB, which is associated with neurons. The βIII-tubulinpositive fibers had growth cones on their tips expressing F-actin, indicating that they were growing axons. Proliferating HAP-like stem cells in the injured sciatic nerve were also observed in vivo. These stem cells were also seen in posterior nerves of the spine, but not in the spinal cord itself, when placed in Gelfoam® histoculture [Mii et al., 2013b]. We also observed HAP-like stem cells in the fungiform papilla in the tongue. The nestin-expressing HAP-like HAP Stem Cells for Nerve and Spinal Cord Repair
stem cells in the fungiform papilla are located around a peripheral sensory nerve immediately below the taste bud. These HAP-like cells co-express the neural crest cell marker p75NTR. The fungiform papilla HAP-like stem cells formed spheres in suspension culture in DMEM-F12 medium supplemented with basic fibroblast growth factor-2. The spheres consisted of nestin-expressing HAP-like cells that co-expressed the neural crest marker p75NTR and developed expression of the stem cell marker CD34. The HAP-like stem cells of these spheres differentiated into the following cell types: βIII-tubulinexpressing nerve cells; GFAP-expressing glial cells; K15expressing keratinocytes, and smooth muscle antigenexpressing cells. These results suggest the HAP-like stem cells of the fungiform papilla differentiated into multiple cell types [Mii et al., 2014].
Conclusion
ND-GFP mice enabled us to make the observation that hair follicles contain nestin-expressing HAP stem cells. HAP stem cells originate in the BA [Li et al., 2003], migrate to the DP [Uchugonova et al., 2011a], and can form the outer-root sheath and other parts of the follicle [Li et al., 2003] as well as the hair follicle-associated nerve. The HAP stem cells express stem cell markers other than nestin, such as CD34. The HAP stem cells can differentiate into many cell types, including neurons and glial cells in vitro. HAP stem cells are also pluripotent in vivo and have been shown to effect peripheral nerve and spinal cord repair by differentiating into neural- and glial-type cells. HAP stem cells have also been found in the human scalp, and these stem cells have been shown to be pluripotent and can affect the repair of peripheral nerves. The sciatic nerve and tongue fungiform papilla also contain HAP-like stem cells [Mii et al., 2013b, 2014]. The ability of the HAP stem cells to effect nerve and spinal cord repair, as well as their easy access and autologous use, suggest the potential of these cells for regenerative medicine. Compared to embryonic stem cells or induced pluripotent stem (iPS) cells [Keirstead et al., 2005; Nori et al., 2011; Okano et al., 2013], HAP stem cells have important advantages in that they can be used autologously, are non-oncogenic, and do not pose ethical issues. Furthermore, they can be readily expanded in culture after isolation from the patient’s hair follicle.
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weeks. Thus, transplanted nestin-expressing HAP stem cells can differentiate into motor neurons and reduce muscle atrophy after sciatic nerve transection [Liu et al., 2014].
References
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