皮毛之道：以表皮器官为研究模型探讨再生生物学与再生医学的基础概念

再生医学是一个具有巨大潜力的新兴医学领域。该文以此为方向讨论了再生医学研究中的三个关键问题,并以非神经外胚层器官的干细胞行为为例做进一步的探讨。第一,如何获取干细胞,介绍了包括胚胎干细胞、组织干细胞和诱导性多能干细胞的获得途径,以及若干组织细胞重编程的成功范例;第二,如何将干细胞转化为组织和器官,这需要了解干细胞分化以及形态发生的机制,并以羽毛的形态发生为模型,引入了千细胞拓扑生物学的概念以及干细胞微环境调控塑造器官形态的机制;第三,如何将干细胞及其转化产物置于患者体内,并以鼠毛生长周期波为例,阐明了宏观环境因素如何调控干细胞的活性：最后,还分析了在器官发生中干细胞的自组织对于新生毛发组织工程的重要意义。该文的许多原则不仅限于皮肤,同时也适用于其它体内器官。通过对生物再生的过程的基础研究,我们可以受到生物再生之道的启发,逐渐理解组织修复及再生的机制,并提高分子和细胞水平上的干细胞操作技术,希望在不久的将来将干细胞研究成果应用于临床医学。     

Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling

In mammalian skin, multiple types of resident cells are required to create a functional tissue and support tissue homeostasis and regeneration. The cells that compose the epithelial stem cell niche for skin homeostasis and regeneration are not well defined. Here, we identify adipose precursor cells within the skin and demonstrate that their dynamic regeneration parallels the activation of skin stem cells. Functional analysis of adipocyte lineage cells in mice with defects in adipogenesis and in transplantation experiments revealed that intradermal adipocyte lineage cells are necessary and sufficient to drive follicular stem cell activation. Furthermore, we implicate PDGF expression by immature adipocyte cells in the regulation of follicular stem cell activity. These data highlight adipogenic cells as skin niche cells that positively regulate skin stem cell activity, and suggest that adipocyte lineage cells may alter epithelial stem cell function clinically.     

Participation of bone marrow derived cells in cutaneous wound healing

Bone marrow has long been known to be a source of stem cells capable of regeneration of the hematopoeitic system. Recent reports, however, have indicated that bone marrow might also contain early stem cells that can differentiate into other organ tissues such as skin. While these studies have illustrated that bone marrow stem cells could find their way to the skin, they have not addressed the dynamics of how bone marrow stem cells might participate in the homeostatis and regeneration of skin. In this report we followed green fluorescent protein (GFP) labeled bone marrow transplanted into non-GFP mice in order to determine the participation of bone marrow stem cells in cutaneous wounds. Our results indicate that there are a significant number of bone marrow cells that traffic through both wounded and non-wounded skin. Wounding stimulated the engraftment of bone marrow cells to the skin and induced bone marrow derived cells to incorporate into and differentiate into non-hematopoietic skin structures. This report thus illustrates that bone marrow might be a valuable source of stem cells for the skin and possibly other organs. Wounding could be a stimulus for bone marrow derived stem cells to travel to organs and aid in the regeneration of damaged tissue.     

Pathologic Bladder Microenvironment Attenuates Smooth Muscle Differentiation of Skin Derived Precursor Cells: Implications for Tissue Regeneration

Smooth muscle cell containing organs (bladder, heart, blood vessels) are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs) can easily be isolated from adult skin and can be differentiated in vitro into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. In vivo, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from ex vivo stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF) suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain) as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells in vitro, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in in vivo settings. These observations must be considered in drafting any regeneration strategies.     

E-cadherin regulates the behavior and fate of epithelial stem cells and their progeny in the mouse incisor

Stem cells are essential for the regeneration and homeostasis of many organs, such as tooth, hair, skin, and intestine. Although human tooth regeneration is limited, a number of animals have evolved continuously growing teeth that provide models of stem cell-based organ renewal. A well-studied model is the mouse incisor, which contains dental epithelial stem cells in structures known as cervical loops. These stem cells produce progeny that proliferate and migrate along the proximo-distal axis of the incisor and differentiate into enamel-forming ameloblasts. Here, we studied the role of E-cadherin in behavior of the stem cells and their progeny. Levels of E-cadherin are highly dynamic in the incisor, such that E-cadherin is expressed in the stem cells, downregulated in the transit-amplifying cells, re-expressed in the pre-ameloblasts and then downregulated again in the ameloblasts. Conditional inactivation of E-cadherin in the cervical loop led to decreased numbers of label-retaining stem cells, increased proliferation, and decreased cell migration in the mouse incisor. Using both genetic and pharmacological approaches, we showed that Fibroblast Growth Factors regulate E-cadherin expression, cell proliferation and migration in the incisor. Together, our data indicate that E-cadherin is an important regulator of stem cells and their progeny during growth of the mouse incisor.     

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.     

Potential applications of germline cell-derived pluripotent stem cells in organ regeneration

Impressive progress has been made since the turn of the century in the field of stem cells. Different types of stem cells have now been isolated from different types of tissues. Pluripotent stem cells are the most promising cell source for organ regeneration. One such cell type is the germline cell-derived pluripotent cell, which is derived from adult spermatogonial stem cells. The germline cell-derived pluripotent stem cells have been obtained from both human and mouse and, importantly, are adult stem cells with embryonic stem cell-like properties that do not require specific manipulations for pluripotency acquisition, hence bypassing problems related to induced pluripotent stem cells and embryonic stem cells. The germline cell-derived pluripotent stem cells have been induced to differentiate into cells deriving from the three germ layers and shown to be functional in vitro. This review will discuss the plasticity of the germline cell-derived pluripotent stem cells and their potential applications in human organ regeneration, with special emphasis on liver regeneration. Potential problems related to their use are also highlighted.     

Controlling skin morphogenesis: hope and despair

To master tissue and organ morphogenesis necessitates a thorough understanding of the cellular and molecular events involved in development, renewal, repair and regeneration. Skin reconstruction is the paradigm of tissue engineering. The transplantation of autologous adult epidermal stem cells is a life-saving procedure as it regenerates the indispensable barrier function of the skin, but the reconstruction of fully functional skin has been hampered by the complexity of the process. The recent identification of multipotent epithelial stem cells in adult hair follicles and of multipotent stem cells in dermis raises new hopes.     

Deer antler – A novel model for studying organ regeneration in mammals

Deer antler is the only mammalian organ that can fully grow back once lost from its pedicle – the base from which it grows. Therefore, antlers probably offer the most pertinent model for studying organ regeneration in mammals. This paper reviews our current understanding of the mechanisms underlying regeneration of antlers, and provides insights into the possible use for human regenerative medicine. Based on the definition, antler renewal belongs to a special type of regeneration termed epimorphic. However, histological examination failed to detect dedifferentiation of any cell type on the pedicle stump and the formation of a blastema, which are hallmark features of classic epimorphic regeneration. Instead, antler regeneration is achieved through the recruitment, proliferation and differentiation of the single cell type in the pedicle periosteum (PP). The PP cells are the direct derivatives of cells resident in the antlerogenic periosteum (AP), a tissue that exists in prepubertal deer calves and can induce ectopic antler formation when transplanted elsewhere on the deer body. Both the AP and PP cells express key embryonic stem cell markers and can be induced to differentiate into multiple cell lineages in vitro and, therefore, they are termed antler stem cells, and antler regeneration is a stem cell-based epimorphic regeneration. Comparisons between the healing process on the stumps from an amputated mouse limb and early regeneration of antlers suggest that the stump of a mouse limb cannot regenerate because of the limited potential of periosteal cells in long bones to proliferate. If we can impart a greater potential of these periosteal cells to proliferate, we might at least be able to partially regenerate limbs lost from humans. Taken together, a greater understanding of the mechanisms that regulate the regeneration of antlers may provide a valuable insight to aid the field of regenerative medicine.This article is part of a Directed Issue entitled: Regenerative Medicine: the challenge of translation.     

Role of epidermal stem cells in repair of partial-thickness burn injury after using Moist Exposed Burn Ointment (MEBO) histological and immunohistochemical study

Moist Exposed Burn Ointment (MEBO^®) is widely used topical agent applied on skin burn. This study investigated the effect of MEBO topical application on activation and proliferation of epidermal stem cells through the immunohistochemical localization of cytokeratin 19 (CK19) as a known marker expressed in epidermal stem cells. Biopsies from normal skin and burn wounds were taken from 21 patients with partial thickness burn 1, 4, 7, 14, 21, and 28 days after treatment with MEBO. Tissue sections were prepared for histological study and for CK19 immunohistochemical localization. In control skin, only few cells showed a positive CK19 immune-reaction. Burned skin showed necrosis of full thickness epidermis that extended to dermis. Gradual regeneration of skin accompanied with an enhancement in CK19 immune-reactivity was noted 4, 7, 14 and 21 days after treatment with MEBO. On day 28, a complete regeneration of skin was observed with a return of CK19 immune-reactivity to the basal pattern again. In conclusion, the enhancement of epidermal stem cell marker CK19 after treatment of partial thickness burn injuries with MEBO suggested the role of MEBO in promoting epidermal stem cell activation and proliferation during burn wound healing.     

Simultaneous Isolation of Three Different Stem Cell Populations from Murine Skin

The skin is a rich source of readily accessible stem cells. The level of plasticity afforded by these cells is becoming increasingly important as the potential of stem cells in Cell Therapy and Regenerative Medicine continues to be explored. Several protocols described single type stem cell isolation from skin; however, none of them afforded simultaneous isolation of more than one population. Herein, we describe the simultaneous isolation and characterization of three stem cell populations from the dermis and epidermis of murine skin, namely Epidermal Stem Cells (EpiSCs), Skin-derived Precursors (SKPs) and Mesenchymal Stem Cells (MSCs). The simultaneous isolation was possible through a simple protocol based on culture selection techniques. These cell populations are shown to be capable of generating chondrocytes, adipocytes, osteocytes, terminally differentiated keratinocytes, neurons and glia, rendering this protocol suitable for the isolation of cells for tissue replenishment and cell based therapies. The advantages of this procedure are far-reaching since the skin is not only the largest organ in the body, but also provides an easily accessible source of stem cells for autologous graft.     

Chemical modulation of cell fates: <Emphasis Type="Italic">in situ</Emphasis> regeneration

Chemical modulation of cell fates has been widely used to promote tissue and organ regeneration. Small molecules can target the self-renewal, expansion, differentiation, and survival of endogenous stem cells for enhancing their regenerative power or induce dedifferentiation or transdifferentiation of mature cells into proliferative progenitors or specialized cell types needed for regeneration. Here, we discuss current progress and potential using small molecules to promote in vivo regenerative processes by regulating the cell fate. Current studies of small molecules in regeneration will provide insights into developing safe and efficient chemical approaches for in situ tissue repair and regeneration.     

Establishment and characterization of hepatic stem-like cell lines from normal adult rat liver

The liver is a unique organ with the potential to regenerate from injury. Hepatic stem cells contribute to liver regeneration when surviving hepatocytes in injured liver are unable to proliferate. To investigate the mechanism of liver regeneration in vitro, we established hepatic stem cell lines named HY1, HY2 and HY3, derived from a healthy liver of adult rat. HY cells showed an expression pattern similar to oval cells, and efficiently induced hepatic differentiation following sequential treatment with type I collagen, transforming growth factor-beta1 (TGF-beta1), and hepatocyte growth factor (HGF) or oncostatin M (OSM). These results suggested that HY cells are liver stem cells representing an excellent tool for in vitro studies on liver regeneration.     

EGFR-mediated epidermal stem cell motility drives skin regeneration through COL17A1 proteolysis

Skin regenerative capacity declines with age, but the underlying mechanisms are largely unknown. Here we demonstrate a functional link between epidermal growth factor receptor (EGFR) signaling and type XVII collagen (COL17A1) proteolysis on age-associated alteration of keratinocyte stem cell dynamics in skin regeneration. Live-imaging and computer simulation experiments predicted that human keratinocyte stem cell motility is coupled with self-renewal and epidermal regeneration. Receptor tyrosine kinase array identified the age-associated decline of EGFR signaling in mouse skin wound healing. Culture experiments proved that EGFR activation drives human keratinocyte stem cell motility with increase of COL17A1 by inhibiting its proteolysis through the secretion of tissue inhibitor of metalloproteinases 1 (TIMP1). Intriguingly, COL17A1 directly regulated keratinocyte stem cell motility and collective cell migration by coordinating actin and keratin filament networks. We conclude that EGFR-COL17A1 axis–mediated keratinocyte stem cell motility drives epidermal regeneration, which provides a novel therapeutic approach for age-associated impaired skin regeneration.     

Epidermal stem cells - role in normal, wounded and pathological psoriatic and cancer skin

In this review we focus on epidermal stem cells in the normal regeneration of the skin as well as in wounded and psoriatic skin. Furthermore, we discuss current data supporting the idea of cancer stem cells in the pathogenesis of skin carcinoma and malignant melanoma. Epidermal stem cells present in the basal layer of the interfollicular epidermis and in the bulge region of the hair follicle play a critical role for normal tissue maintenance. In wound healing, multipotent epidermal stem cells contribute to re-epithelization. It is possible that defects in growth control of either epidermal stem cells or transit amplifying cells constitute a primary pathogenetic factor in the epidermal hyperproliferation seen in psoriasis. In cutaneous malignancies mounting evidence supports a stem cell origin in skin carcinoma and malignant melanoma and a possible existence of cancer stem cells.     

Unexpected encounter of the parasitic kind

Both parasitology and stem cell research are important disciplines in their own right. Parasites are a real threat to human health causing a broad spectrum of diseases and significant annual rates morbidity and mortality globally. Stem cell research, on the other hand, focuses on the potential for regenerative medicine for a range of diseases including cancer and regenerative therapies. Though these two topics might appear distant, there are some "unexpected encounters". In this review, we summarise the various links between parasites and stem cells. First,we discuss how parasites' own stem cells represent interesting models of regeneration that can be translated to human stem cell regeneration. Second, we explore the interactions between parasites and host stem cells during the course of infection. Third, we investigate from a clinical perspective, how stem cell regeneration can be exploited to help circumvent the damage induced by parasitic infection and its potential to serve as treatment options for parasitic diseases in the future. Finally, we discuss the importance of screening for pathogens during organ transplantation by presenting some clinical cases of parasitic infection following stem cell therapy.     

Chemical modulation of cell fates: in situ regeneration

Chemical modulation of cell fates has been widely used to promote tissue and organ regeneration. Small molecules can target the self-renewal, expansion, differentiation, and survival of endogenous stem cells for enhancing their regenerative power or induce dedifferentiation or transdifferentiation of mature cells into proliferative progenitors or specialized cell types needed for regeneration. Here, we discuss current progress and potential using small molecules to promote in vivo regenerative processes by regulating the cell fate. Current studies of small molecules in regeneration will provide insights into developing safe and efficient chemical approaches for in situ tissue repair and regeneration.