Multipotent skin-derived precursors: adult neural crest-related precursors with therapeutic potential

We previously made the surprising finding that cultures of multipotent precursors can be grown from the dermis of neonatal and adult mammalian skin. These skin-derived precursors (SKPs) display multi-lineage differentiation potential, producing both neural and mesodermal progeny in vitro, and are an apparently novel precursor cell type that is distinct from other known precursors within the skin. In this review, we begin by placing these findings within the context of the rapidly evolving stem cell field. We then describe our recent efforts focused on understanding the developmental biology of SKPs, discussing the idea that SKPs are neural crest-related precursors that (i) migrate into the skin during embryogenesis, (ii) persist within a specific dermal niche, and (iii) play a key role in the normal physiology, and potentially pathology, of the skin. We conclude by highlighting some of the therapeutic implications and unresolved questions raised by these studies.     

Isolation of multipotent adult stem cells from the dermis of mammalian skin

We describe here the isolation of stem cells from juvenile and adult rodent skin. These cells derive from the dermis, and clones of individual cells can proliferate and differentiate in culture to produce neurons, glia, smooth muscle cells and adipocytes. Similar precursors that produce neuron-specific proteins upon differentiation can be isolated from adult human scalp. Because these cells (termed SKPs for skin-derived precursors) generate both neural and mesodermal progeny, we propose that they represent a novel multipotent adult stem cell and suggest that skin may provide an accessible, autologous source of stem cells for transplantation.     

Isolation and Characterization of Two Kinds of Stem Cells from the Same Human Skin Back Sample with Therapeutic Potential in Spinal Cord Injury

Backgrounds and Objective

Spinal cord injury remains to be a challenge to clinicians and it is attractive to employ autologous adult stem cell transplantation in its treatment, however, how to harvest cells with therapeutic potential easily and how to get enough number of cells for transplantation are challenging issues. In the present study, we aimed to isolate skin-derived precursors (SKPs) and dermal multipotent stem cells (dMSCs) simultaneously from single human skin samples from patients with paraplegia.

Methods

Dissociated cells were initially generated from the dermal layer of skin samples from patients with paraplegia and cultured in SKPs proliferation medium. Four hours later, many cells adhered to the base of the flask. The suspended cells were then transferred to another flask for further culture as SKPs, while the adherent cells were cultured in dMSCs proliferation medium. Twenty-four hours later, the adherent cells were harvested and single-cell colonies were generated using serial dilution method. [³H]thymidine incorporation assay, microchemotaxis Transwell chambers assay, RT-PCR and fluorescent immunocytochemistry were employed to examine the characterizations of the isolated cells.

Results

SKPs and dMSCs were isolated simultaneously from a single skin sample. SKPs and dMSCs differed in several respects, including in terms of intermediate protein expression, proliferation capacities, and differentiation tendencies towards mesodermal and neural progenies. However, both SKPs and dMSCs showed high rates of differentiation into neurons and Schwann cells under appropriate inducing conditions. dMSCs isolated by this method showed no overt differences from dMSCs isolated by routine methods.

Conclusions

Two kinds of stem cells, namely SKPs and dMSCs, can be isolated simultaneously from individual human skin sample from paraplegia patients. Both of them show ability to differentiate into neural cells under proper inducing conditions, indicating their potential for the treatment of spinal cord injury patients by autologous cell transplantation.     

Morphogenesis and renewal of hair follicles from adult multipotent stem cells

The upper region of the outer root sheath of vibrissal follicles of adult mice contains multipotent stem cells that respond to morphogenetic signals to generate multiple hair follicles, sebaceous glands, and epidermis, i.e., all the lineages of the hairy skin. At the time when hair production ceases and when the lower region of the follicle undergoes major structural changes, the lower region contains a significant number of clonogenic keratinocytes, and can then respond to morphogenetic signals. This demonstrates that multipotent stem cells migrate to the root of the follicle to produce whisker growth. Moreover, our results indicate that the clonogenic keratinocytes are closely related, if not identical, to the multipotent stem cells, and that the regulation of whisker growth necessitates a precise control of stem cell trafficking.     

The bulge area is the origin of nestin-expressing pluripotent stem cells of the hair follicle

Nestin-expressing pluripotent stem cells have been found both in the bulge area (BA) as well as the dermal papilla (DP). Nestin-expressing stem cells of both the BA and DP have been previously shown to be able to form neurons and other non-follicle cell types. The nestin-expressing stem cells from the DP have been termed skin precursor or SKP cells. Both nestin-expressing DP and BA cells have been previously shown to effect repair of the injured spinal cord and peripheral nerve, with the BA being the greater and more constant source of the stem cells. The BA contains nestin-expressing stem cells throughout the hair cycle, whereas nestin-expressing dermal papillae stem cells were found in early and mid-anagen only. Our previous studies have shown that the nestin-expressing stem cells in the BA and DP have similar morphological features. The cells from both regions have a small body diameter of approximately 7 μm with long extrusions, as shown by 2-photon imaging. In the present study, using 2-photon imaging of whisker follicles from transgenic mice expressing nestin-driven green fluorescent protein (ND-GFP), we demonstrate that the BA is the source of the nestin-expressing stem cells of the hair follicle. The nestin-expressing stem cells migrate from the BA to the DP as well as into the surrounding skin tissues including the epidermis, and during wound healing, suggesting that the BA may be the source of the stem cells of the skin itself.     

Real-time confocal imaging of trafficking of nestin-expressing multipotent stem cells in mouse whiskers in long-term 3-D histoculture

We have previously demonstrated that nestin-expressing multipotent hair follicle stem cells are located above the hair follicle bulge and can differentiate into neurons and other cell types in vitro. The nestin-expressing hair follicle stem cells promoted the recovery of pre-existing axons when they were transplanted to the severed sciatic nerve or injured spinal cord. We have also previously demonstrated that the whisker hair follicle contains nestin-expressing stem cells in the dermal papilla (DP) as well as in the bulge area (BA), but that their origin is in the BA. In the present study, we established the technique of long-term Gelfoam? histoculture of whiskers isolated from transgenic mice in which nestin drives green fluorescent protein (ND-GFP). Confocal imaging was used to monitor ND-GFP-expressing 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 stem cells trafficked from the BA toward the DP area and extensively grew out onto Gelfoam? forming nerve-like structures. This new method of long-term histoculture of whiskers from ND-GFP mice will enable the extensive study of the behavior of nestin-expressing multipotent stem cells of the hair follicle.     

The adult hair follicle: cradle for pluripotent neural crest stem cells

This review focuses on the recent identification of two novel neural crest-derived cells in the adult mammalian hair follicle, pluripotent stem cells, and Merkel cells. Wnt1-cre/R26R compound transgenic mice, which in the periphery express beta-galactosidase in a neural crest-specific manner, were used to trace neural crest cells. Neural crest cells invade the facial epidermis as early as embryonic day 9.5. Neural crest-derived cells are present along the entire extent of the whisker follicle. This includes the bulge area, an epidermal niche for keratinocyte stem cells, as well as the matrix at the base of the hair follicle. We have determined by in vitro clonal analysis that the bulge area of the adult whisker follicle contains pluripotent neural crest stem cells. In culture, beta-galactosidase-positive cells emigrate from bulge explants, identifying them as neural crest-derived cells. When these cells are resuspended and grown in clonal culture, they give rise to colonies that contain multiple differentiated cell types, including neurons, Schwann cells, smooth muscle cells, pigment cells, chondrocytes, and possibly other types of cells. This result provides evidence for the pluripotentiality of the clone-forming cell. Serial cloning showed that bulge-derived neural crest cells undergo self-renewal, which identifies them as stem cells. Pluripotent neural crest cells are also localized in the back skin hair of adult mice. The bulge area of the whisker follicle is surrounded by numerous Merkel cells, which together with innervating nerve endings form slowly adapting mechanoreceptors that transduce steady skin indentation. Merkel cells express beta-galactosidase in double transgenic mice, which confirms their neural crest origin. Taken together, our data indicate that the epidermis of the adult hair follicle contains pluripotent neural crest stem cells, termed epidermal neural crest stem cells (eNCSCs), and one newly identified neural crest derivative, the Merkel cell. The intrinsic high degree of plasticity of eNCSCs and the fact that they are easily accessible in the skin make them attractive candidates for diverse autologous cell therapy strategies.     

Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin

Given their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell-like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and display extensive self-renewal capacity in sphere cultures. To determine the origin of these cells, we genetically mapped the fate of neural crest cells in face and trunk skin of mouse. In whisker follicles of the face, many mesenchymal structures are neural crest derived and appear to contain cells with sphere-forming potential. In the trunk skin, however, sphere-forming neural crest-derived cells are restricted to the glial and melanocyte lineages. Thus, self-renewing cells in the adult skin can be obtained from several neural crest derivatives, and these are of distinct nature in face and trunk skin. These findings are relevant for the design of therapeutic strategies because the potential of stem and progenitor cells in vivo likely depends on their nature and origin.     

Characterization of multipotent adult stem cells from the skin: transforming growth factor-beta (TGF-beta) facilitates cell growth

Recently, adult stem cells have been isolated from the skin and designated as skin-derived precursors (SKPs). These SKPs, cultured in vitro, can give rise to neurons, glia, smooth muscle cells, and adipocytes. In the current study, we confirmed the clonal expansion of SKPs using a sphere-forming culture system in a medium containing methylcellulose. Among the growth factors, only transforming growth factor-beta (TGF-beta) was revealed to uniquely facilitate the sphere formation and proliferation of the SKPs in combination with EGF and bFGF. In addition, TGF-beta did not alter phenotypical characteristics of the SKPs under sphere-forming conditions. The effect of TGF-beta on sphere formation was not observed in neural stem cells, which expressed a different set of cell surface markers from SKPs, suggesting that SKPs have distinct features. Although the number of SKPs decreased with age, TGF-beta increased the sphere colony formation and proliferation in all ages. These results suggest that SKPs maintained in the presence of TGF-beta during culture are of potential use in cell-replacement therapies employing adult tissue sources.     

NG2 proteoglycan expression in mouse skin: altered postnatal skin development in the NG2 null mouse.

In early postnatal mouse skin, the NG2 proteoglycan is expressed in the subcutis, the dermis, the outer root sheath of hair follicles, and the basal keratinocyte layer of the epidermis. With further development, NG2 is most prominently expressed by stem cells in the hair follicle bulge region, as also observed in adult human skin. During telogen and anagen phases of the adult hair cycle, NG2 is also found in stem cell populations that reside in dermal papillae and the outer root sheaths of hair follicles. Ablation of NG2 produces alterations in both the epidermis and subcutis layers of neonatal skin. Compared with wild type, the NG2 null epidermis does not achieve its full thickness due to reduced proliferation of basal keratinocytes that serve as the stem cell population in this layer. Thickening of the subcutis is also delayed in NG2 null skin due to deficiencies in the adipocyte population.     

CCN2 modulates hair follicle cycling in mice

It is critical to understand how stem cell activity is regulated during regeneration. Hair follicles constitute an important model for organ regeneration because, throughout adult life, they undergo cyclical regeneration. Hair follicle stem cells—epithelial cells located in the follicle bulge—are activated by periodic β-catenin activity, which is regulated not only by epithelial-derived Wnt, but also, through as-yet-undefined mechanisms, the surrounding dermal microenvironment. The matricellular protein connective tissue growth factor (CCN2) is secreted into the microenvironment and acts as a multifunctional signaling modifier. In adult skin, CCN2 is largely absent but is unexpectedly restricted to the dermal papillae and outer root sheath. Deletion of CCN2 in dermal papillae and the outer root sheath results in a shortened telogen-phase length and elevated number of hair follicles. Recombinant CCN2 causes decreased β-catenin stability in keratinocytes. In vivo, loss of CCN2 results in elevated numbers of K15-positive epidermal stem cells that possess elevated β-catenin levels and β-catenin–dependent reporter gene expression. These results indicate that CCN2 expression by dermal papillae cells is a physiologically relevant suppressor of hair follicle formation by destabilization of β-catenin and suggest that CCN2 normally acts to maintain stem cell quiescence.     

The multipotency of adult vibrissa follicle stem cells

Several studies focused on the characterization of bulge keratinocytes have proved that they are multipotent stem cells, being recruited not only to regenerate the hair follicle itself, but also the sebaceous gland and the epidermis. However, due to the difficulty in preparing transplantable cell sheets harvested with conventional enzymatic digestion, there is still no direct evidence of the bulge stem cells’ multipotency. Whether they can respond to adult dermal papilla (DP) signals in recombination experiments also remains unclear. In this study, we addressed this problem by culturing and detaching intact bulge keratinocyte sheets from thermo-responsive culture dishes, only by reducing its temperature. When sheets of mass cultured bulge keratinocytes isolated from rat vibrissa follicles were recombined with fresh adult DPs and sole skin dermis in vivo, regeneration of epidermis and sebaceous gland-like structures, and formation of hair bulb with differentiating inner root sheath and hair cuticle were observed within 3 weeks. However, regardless the expression of stem cells markers like CD34, SA1004 and SA1006, no structures were observed when cloned bulge keratinocytes were used to prepare cell sheets and recombinants, revealing the possible existence of monoclonal stem cells within the bulge region. This report is the first to succeed in harvesting adult bulge keratinocyte sheets. Using these sheets it is demonstrated that bulge stem cells directly respond to adult DP signals to induce hair bulb formation in vivo.     

The PI3K-Akt pathway inhibits senescence and promotes self-renewal of human skin-derived precursors in vitro

Skin-derived precursors (SKPs) are embryonic neural crest- or somite-derived multipotent progenitor cells with properties of dermal stem cells. Although a large number of studies deal with their differentiation ability and potential applications in tissue damage repair, only a few studies have concentrated on the regulation of SKP self-renewal. Here, we found that after separation from their physiological microenvironment, human foreskin-derived SKPs (hSKPs) quickly senesced and lost their self-renewal ability. We observed a sharp decrease in Akt activity during this process, suggesting a possible role of the PI3K-Akt pathway in hSKP maintenance in vitro. Blocking this pathway with several inhibitors inhibited hSKP proliferation and sphere formation and increased hSKP senescence. In contrast, activating this pathway with PDGF-AA and a PTEN inhibitor, bpV(pic), promoted proliferation, improved sphere formation, and alleviated senescence of hSKPs, without altering their differentiation potential. Data also implied that this effect was associated with altered actions of FoxO3 and GSK-3β. Our results suggest an important role of the PI3K-Akt pathway in the senescence and self-renewal of hSKPs. These findings also provide a better understanding of the cellular mechanisms underlying hSKP self-renewal and stem cell senescence to allow more efficient expansion of hSKPs for regenerative medical applications.     

Embryonic dermal condensation and adult dermal papilla induce hair follicles in adult glabrous epidermis through different mechanisms

Hair induction in the adult glabrous epidermis by the embryonic dermis was compared with that by the adult dermis. Recombinant skin, composed of the adult sole epidermis and the embryonic dermis containing dermal condensations (DC), was transplanted onto the back of nude mice. The epidermis of transplants formed hairs. Histology on the induction process demonstrated the formation of placode-like tissues, indicating that the transplant produces hair follicles through a mechanism similar to that underlying hair follicle development in the embryonic skin. An isolated adult rat sole skin piece, inserted with either an aggregate of cultured dermal papilla (DP) cells or an intact DP between its epidermis and dermis, was similarly transplanted. The transplant produced hair follicles. Histology showed that the epidermis in both cases surrounded the aggregates of DP cells. The epidermis never formed placode-like tissues. Thus, it was concluded that the adult epidermal cells recapitulate the embryonic process of hair follicle development when exposed to DC, whereas they get directly into the anagen of the hair cycle when exposed to DP. The expression pattern of Edar and Shh genes, and P-cadherin protein during the hair follicle development in the two types of transplants supported the above conclusion.     

Giant Panda (Ailuropoda melanoleuca) Buccal Mucosa Tissue as a Source of Multipotent Progenitor Cells

Since the first mammal was cloned, the idea of using this technique to help endangered species has aroused considerable interest. However, several issues limit this possibility, including the relatively low success rate at every stage of the cloning process, and the dearth of usable tissues from these rare animals. iPS cells have been produced from cells from a number of rare mammalian species and this is the method of choice for strategies to improve cloning efficiency and create new gametes by directed differentiation. Nevertheless information about other stem cell/progenitor capabilities of cells from endangered species could prove important for future conservation approaches and adds to the knowledge base about cellular material that can be extremely limited. Multipotent progenitor cells, termed skin-derived precursor (SKP) cells, can be isolated directly from mammalian skin dermis, and human cheek tissue has also been shown to be a good source of SKP-like cells. Recently we showed that structures identical to SKPs termed m-SKPs could be obtained from monolayer/ two dimensional (2D) skin fibroblast cultures. Here we aimed to isolate m-SKPs from cultured cells of three endangered species; giant panda (Ailuropoda melanoleuca); red panda (Ailurus fulgens); and Asiatic lion (Panthera leo persica). m-SKP-like spheres were formed from the giant panda buccal mucosa fibroblasts; whereas dermal fibroblast (DF) cells cultured from abdominal skin of the other two species were unable to generate spheres. Under specific differentiation culture conditions giant panda spheres expressed neural, Schwann, adipogenic and osteogenic cell markers. Furthermore, these buccal mucosa derived spheres were shown to maintain expression of SKP markers: nestin, versican, fibronectin, and P75 and switch on expression of the stem cell marker ABCG2. These results demonstrate that giant panda cheek skin can be a useful source of m-SKP multipotent progenitors. At present lack of sample numbers means that we can only postulate why we were unable to obtain m-SKPs from the lion and red panda cultures. However the giant panda observations point to the value of archiving cells from rare species, and the possibilities for later progenitor cell derivation.