Regeneration Pat Barker Epub Download
Mazie Wingeier
Small populations of adult stem cells are responsible for the remarkable ability of the epithelial lining of the intestine to be efficiently renewed and repaired throughout life. The recent discovery of specific markers for these stem cells, together with the development of new technologies to track endogenous stem cell activity in vivo and to exploit their ability to generate new epithelia ex vivo, has greatly improved our understanding of stem cell-driven homeostasis, regeneration and cancer in the intestine. These exciting new insights into the biology of intestinal stem cells have the potential to accelerate the development of stem cell-based therapies and ameliorate cancer treatments.
B) Venn diagram depicting significant overlap between genes associated with high levels of follicle regeneration in mouse skin (in vivo) and human keratinocytes treated with poly (I:C) in vitro published by Karim et al., 2011 under "type":"entrez-geo","attrs":"text":"GSE21260","term_id":"21260"GSE21260.
The hair follicle (HF) is a unique miniorgan, which self-renews for a lifetime. Stem cell populations of multiple lineages reside within human HF and enable its regeneration. In addition to resident HF stem/progenitor cells (HFSPCs), the cells with similar biological properties can be induced from human-induced pluripotent stem cells (hiPSCs). As approaches to regenerate HF by combining HF-derived cells have been established in rodents and a huge demand exists to treat hair loss diseases, attempts have been made to bioengineer human HF using HFSPCs or hiPSCs.
The aim of this review is to comprehensively summarize the strategies to regenerate human HF using HFSPCs or hiPSCs. HF morphogenesis and regeneration are enabled by well-orchestrated epithelial-mesenchymal interactions (EMIs). In rodents, various combinations of keratinocytes with mesenchymal (dermal) cells with trichogenic capacity, which were transplanted into in vivo environment, have successfully generated HF structures. The regeneration efficiency was higher, when epithelial or dermal HFSPCs were adopted. The success in HF formation most likely depended on high receptivity to trichogenic dermal signals and/or potent hair inductive capacity of HFSPCs. In theory, the use of epithelial HFSPCs in the bulge area and dermal papilla cells, their precursor cells in the dermal sheath, or trichogenic neonatal dermal cells should elicit intense EMI sufficient for HF formation. However, technical hurdles, represented by the limitation in starting materials and the loss of intrinsic properties during in vitro expansion, hamper the stable reconstitution of human HFs with this approach. Several strategies, including the amelioration of culture condition or compartmentalization of cells to strengthen EMI, can be conceived to overcome this obstacle. Obviously, use of hiPSCs can resolve the shortage of the materials once reliable protocols to induce wanted HFSPC subsets have been developed, which is in progress. Taking advantage of their pluripotency, hiPSCs may facilitate previously unthinkable approaches to regenerate human HFs, for instance, via bioengineering of 3D integumentary organ system, which can also be applied for the treatment of other diseases.
In fact, human HF regeneration for treating non-autoimmune-mediated hair loss diseases, such as androgenetic alopecia or female pattern hair loss, may serve as an ideal model to probe the feasibility of regenerative medicine approaches for several reasons: (1) HF is easily accessible and observable; (2) HF morphogenesis, biology, and physiology have been well understood; (3) in vitro maintenance and cultivation of HF or HF-derived cells have been established; (4) at least in rodents, the methodologies to reconstitute HFs in vivo have been established; and (5) autologous transplantation of HFs in bald area has been widely conducted, etc. [3,4,5,6]. Of note, HF is a periodically self-renewing miniorgan harboring multiple stem/progenitor cell populations represented by epithelial HF stem cells (HFSCs) at the bulge area (Fig. 1c) and DP or its precursors in the dermal sheath (DS) (Fig. 1d), which serve as ideal cell sources for HF regeneration and, potentially for hiPSC generation [4,5,6]. Ultimately, HF-derived hiPSCs can be converted into HFSCs and unlimitedly supply materials for human HF regeneration [7].
HF morphogenesis and regeneration depends on intensive and well-orchestrated interactions between receptive epithelial and inductive mesenchymal components (Fig. 2) [3, 5, 8]. In the past attempts to bioengineer HF, variously prepared epithelial and mesenchymal components were combined and grafted into a permissive in vivo environment to elicit intercompartmental interactions [5, 6]. Theoretically, less-committed and highly proliferative HFSCs or progenitor cells could efficiently yield HFs in those conventional assays. In line with this hypothesis, HFSCs were shown to be favorable materials for HF regeneration, at least, in rodents [9, 10]. However, the use of human HFSCs or progenitor cells for such application was hampered by the limitation in collectable cells and the loss of their intrinsic properties during in vitro expansion [4]. Improvement of culture condition to maintain/restore their intrinsic property is pivotal.
The aim of this review is to summarize the strategies for using human HFSCs and progenitor cells or hiPSCs for HF regeneration with a particular emphasis on the enhancement of epithelial-mesenchymal interactions (EMIs).
Various approaches have been attempted to elicit EMIs sufficient for HF regeneration [4]. However, all assays are based on the same principle; combining responder epithelial cells with inducer mesenchymal cells that are placed into a neutral permissive environment [5]. A two-step approach consisting of in vitro experimentations to establish a condition to maximize EMIs and in vivo HF reconstitution assays adopting materials prepared in the condition optimized in vitro study would be beneficial [4, 5].
Considering that HF regeneration efficiency should correlate well with the intensity of EMI, in vivo HF reconstitution assays adopting cell transplantation into immunodeficient mice would be the most optimal way to trigger EMI (Fig. 4) [5]. In the chamber assay, mixture of KCs and trichogenic dermal cells (mostly DP, DS, or neonatal dermal fibroblasts) were transplanted into a silicon chamber grafted on to the dorsal fascial surface (Fig. 4). The chamber keeps the humidity and space enabling HF reconstitution. Newly generated HFs can be visible from the outside of the body. However, larger number of the cells and longer period is necessary for this assay, when compared to the path assay, in which epithelial and dermal cell mixture is directly injected into the hypodermis of mice (Fig. 4). The former requires 10 million or more cells and around 35 days to see HFs, while the latter requires around 1 million cells and around 10 days (summarized in [5]). In addition, a large regenerated HF-bearing area can be observed in the chamber assay, but several patches can be generated in a single mouse in the patch assay (Fig. 4). Because of the limitation in human samples usable for experimentations and the technical simplicity, the patch technique may be favorable for attempts to regenerate HF using human-derived cells [11]. The sandwich assay is also used to evaluate hair inductive capacity of dermal cells [5, 18]. In this assay, tested dermal cell aggregates were implanted between the epidermis and dermis of globular skin piece, which is subsequently grafted in the subcutaneous space. When HF or similar structure is formed, the transplanted dermal cells can be considered to possess trichogenic activity [5, 18]. In addition to those assays, some derivatives with minor modifications have been reported with varying levels of success (summarized in [5]).
Approaches for HF regeneration in vivo. In the chamber assay, mixture of keratinocytes and trichogenic dermal cells are transplanted into a silicone chamber on the back of immunodeficient mice. In the patch assay, cell mixture is directly injected into the hypodermis of immunodeficient mice
A possible approach to overcome this issue would be to increase the receptivity of KCs to trichogenic dermal signals by predisposing them to follicular fate. Activation of Wnt/β-catenin pathway may be a promising approach [36,37,38] as forced expression of β-catenin in the epidermis resulted in ectopic expression of hair keratins or de novo hair follicle formation in mice [39, 40]. Modulation of p63 expression in KCs may also enhance the response to trichogenic dermal message to the level analogous to that in HFSCs [41]. Yet, an extreme caution needs to be paid for adopting these strategies for human HF regeneration, as aberrant expression of such genes may result in tumor formation. For instance, overactivation of β-catenin could give rise to pilomatricoma [42].
An alternative approach to enhance KC receptivity to dermal signal is to use neonatal or embryonic KCs. Past in vivo grafting studies demonstrated that neonatal or fetal KCs were able to regenerate HF or HF-like structures [24, 44, 45]. Some HF-forming capacity could still be observed after cultivation of fetal cells. Apparently, this strategy cannot be directory adopted for clinical applications; however, these observations can drop a hint for enhancing EMIs for HF regeneration. Human adult KCs can reacquire some juvenile properties by basic fibroblast growth factors treatment [46]. Likewise, exposure of KC to major factors playing key roles in the early phase of HF morphogenesis may allow KCs to exhibit HF forming cell (e.g., hair placode cell) phenotype. WNT, Ectodysplasin-A (EDA), BMP, and sonic hedgehog (SHH) signaling pathways are involved in HF placode formation [3, 8]. Either activation or suppression of these pathways in cultured KCs by supplementation of ligands could endow the cells with some HFSC properties. Feasibility of this approach is under investigation using human 3D skin equivalents and preliminary data suggested upregulation of several hair placode signature genes could be achieved.
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