N-Cadherin is expressed by putative stem/progenitor cells and melanocytes in the human limbal epithelial stem cell niche

Corneal epithelial stem cells are known to be localized to the basal layer of the limbal epithelium, providing a model system for epithelial stem cell biology; however, the mechanisms regarding the maintenance of these stem cells in their specialized niche remain poorly understood. N-cadherin is a member of the classic cadherin family and has previously been demonstrated to be expressed by hematopoietic stem cells. In the present study, we demonstrate that N-cadherin is expressed by putative stem/progenitor cells, as well as melanocytes, in the human limbal epithelial stem cell niche. In addition, we demonstrate that upon in vitro culture using 3T3 feeder layers, loss of N-cadherin expression occurs with cell proliferation. These results indicate that N-cadherin may be a critical cell-to-cell adhesion molecule between corneal epithelial stem/progenitor cells and their corresponding niche cells in the limbal epithelium.     

Differentiation of human embryonic stem cells into corneal epithelial-like cells by in vitro replication of the corneal epithelial stem cell niche

Human embryonic stem cells (hESCs) are pluripotent cells capable of differentiating into any cell type of the body. It has long been known that the adult stem cell niche is vital for the maintenance of adult stem cells. The cornea at the front of the eye is covered by a stratified epithelium that is renewed by stem cells located at its periphery in a region known as the limbus. These so-called limbal stem cells are maintained by factors within the limbal microenvironment, including collagen IV in basement membrane and limbal fibroblasts in the stroma. Because this niche is very specific to the stem cells (rather than to the more differentiated cells) of the corneal epithelium, it was hypothesized that replication of these factors in vitro would result in hESC differentiation into corneal epithelial-like cells. Indeed, here we show that culturing of hESC on collagen IV using medium conditioned by the limbal fibroblasts results in the loss of pluripotency and differentiation into epithelial-like cells. Further differentiation results in the formation of terminally differentiated epithelial-like cells not only of the cornea but also of skin. Scanning electron microscopy shows that some differences exist between hESC-derived and adult limbal epithelial-like cells, necessitating further investigation using in vivo animal models of limbal stem cell deficiency. Such a model of hESC differentiation is useful for understanding the early events of epithelial lineage specification and to the eventual potential application of epithelium differentiated from hESC for clinical conditions of epithelial stem cell loss. Disclosure of potential conflicts of interest is found at the end of this article.     

Characterisation of Human Limbal Side Population Cells Isolated Using an Optimised Protocol From an Immortalised Epithelial Cell Line and Primary Limbal Cultures

The challenges in limbal stem cell biology largely remain in the process of identification, isolation and expansion of these adult corneal epithelial stem cells of the eye. Due to the absence of specific limbal stem cell markers, identification and isolation of putative limbal stem cells is a complicated task. The side population assay is an isolation method that utilises the ability of stem cells to efflux the DNA-binding dye Hoechst 33342 (or other vital dyes) combined with dual wavelength flow cytometry and is a valuable strategy to enrich for limbal stem cells. This assay has been used to successfully identify stem/ progenitor cell populations in a variety of tissues and cell lines. Here we optimise this assay to identify SP cell populations in both primary human limbal epithelial cultures and in an established human corneal epithelial cell line. The limbal SP fraction showed higher expression of ATP-binding cassette sub-family G member 2 (ABCG2), ΔNp63—a common limbal stem cell marker and the stem cell marker Sox2 compared to non-SP cells (NSP).     

Characterization of the limbal epithelial stem cell niche: novel imaging techniques permit in vivo observation and targeted biopsy of limbal epithelial stem cells

It is anticipated that stem cell (SC) therapy will enable the regeneration of diseased tissues and organs. Understanding SC niches is an essential step toward realizing this goal. By virtue of its optical transparency and physical separation of SC and transient amplifying cell compartments, the human cornea provides a unique opportunity to visualize and observe a population of adult stem cells, limbal epithelial stem cells (LESCs), in their niche environment. To date, the characteristics of the LESC niche have remained unclear. State-of-the-art imaging techniques were used to construct a three-dimensional (3D) view of the entire human corneal limbus and identify the structural characteristics of the LESC niche. Two distinct candidate LESC niche structures were identified. Cells within these structures express high levels of the putative limbal stem cell markers p63alpha and ABCG2; however, current methods cannot identify for certain which exact cells within this cell population are truly LESCs. These structures could be located and observed in vivo in normal human subjects, but not in patients with clinically diagnosed corneal LESC deficiency. The distribution of these structures around the corneal circumference is not uniform. Biopsies targeted to limbal regions rich in LESC niche structures yielded significantly higher numbers of LESCs in culture. Our findings demonstrate how adult stem cell niches can be identified and observed in vivo in humans and provide new biological insight into the importance of LESC niche structures in maintaining normal LESC function. Finally, the concept of targeted biopsy of adult SC niches improves stem cell yield and may prove to be essential for the successful development of novel adult stem cell therapies. Disclosure of potential conflicts of interest is found at the end of this article.     

The gastrointestinal tract stem cell niche

The gastrointestinal epithelium is unique in that cell proliferation, differentiation, and apoptosis occur in an orderly fashion along the crypt-villus axis. The intestinal crypt is mainly a proliferative compartment, is monoclonal and is maintained by stem cells. The villus represents the differentiated compartment, and is polyclonal as it receives cells from multiple crypts. In the small intestine, cell migration begins near the base of the crypt, and cells migrate from here emerging onto the villi. The basal crypt cells at position 5 are candidate stem cells. As the function of stem cells is to maintain the integrity of the intestinal epithelium, it must self-renew, proliferate, and differentiate within a protective niche. This niche is made up of proliferating and differentiating epithelial cells and surrounding mesenchymal cells. These mesenchymal cells promote the epithelial-mesenchymal crosstalk required to maintain the niche. A stochastic model of cell division has been proposed to explain how a single common ancestral stem cell exists from which all stem cells in a niche are descended. Our group has argued that these crypts then clonally expand by crypt fission, forming two daughters’ crypts, and that this is the mechanism by which mutated stem cells or even cancer stem cell clones expand in the colon and in the entire gastrointestinal tract. Until recently, the differentiation potential of stem cells into adult tissues has been thought to be limited to cell lineages in the organ from which they were derived. Bone marrow cells are rare among adult stem cells regarding their abundance and role in the continuous, lifelong, physiological replenishment of circulating cells. In human and mice experiments, we have shown that bone marrow can contribute to the regeneration of intestinal myofibroblasts and thereby after epithelium following damage, through replacing the cells, which maintain the stem cells niche. Little is known about the markers characterizing the stem and transit amplifying populations of the gastrointestinal tract, although musashi-1 and hairy and enhancer of split homolog-1 have been proposed. As the mammalian gastrointestinal tract develops from the embryonic gut, it is made up of an endodermally-derived epithelium surrounded by cells of mesoderm origin. Cell signaling between these two tissue layers plays a critical role in coordinating patterning and organogenesis of the gut and its derivatives. Many lines of evidence have revealed that Wnt signaling is the most dominant force in controlling cell proliferation, differentiation, and apoptosis along the crypt-villus axis. We have found Wnt messenger RNAs expression in intestinal subepithelial myofibroblasts and frizzled messenger RNAs expression in both myofibroblasts and crypt epithelium. Moreover, there are many other factors, for example, bone morphogenetic protein, homeobox, forkhead, hedgehog, homeodomain, and platelet-derived growth factor that are also important to stem cell signaling in the gastrointestinal tract.     

The porcine limbal epithelial stem cell niche as a new model for the study of transplanted tissue-engineered human limbal epithelial cells

Transparency of the human cornea is dependent upon the integrity of its epithelium and hence a population of limbal epithelial stem cells (LESCs). We have previously shown that LESCs reside in limbal epithelial crypts at the periphery of the human cornea. In this study the anatomy and functionality of the porcine limbus was evaluated for the first time as a novel model of the human limbus. Scanning electron microscopy, confocal microscopy, and histology revealed common structures in the porcine and human limbus in terms of the location and topography of palisades of Vogt and limbal epithelial crypts. Epithelial cells harvested from crypt regions achieved higher colony forming efficiency than cultures established from the noncrypt regions and central cornea. Also, expression of the putative SC markers p63α and integrin β1 brightness was higher in the basal layer of the crypt regions, as shown by immunocytochemistry. De-epithelialized porcine corneas were used as an in vitro organ culture model to study the fate of transplanted human epithelium cultured from the limbus. Multilayered epithelium was observed after ～1 week. Subsequently, wounds were inflicted on the central corneal epithelium. The wounded tissue healed within 5-7 days, and multilayering of the central corneal epithelium was re-established. The transplanted epithelia were repeatedly wounded at least four times and the wounds healed by 1 week. Putative SC marker expression of the transplanted epithelia was confirmed using immunohistochemistry. These results demonstrate that the porcine limbus shares features with the human limbus and as such provides a suitable model for the study of cultured limbal epithelial cell transplantation. These data have significant clinical value as this model can provide information on LESC fate post-transplantation and their ability to respond to injury, which is not possible to study in patients.     

Intrastromal invasion by limbal epithelial cells is mediated by epithelial-mesenchymal transition activated by air exposure

Corneal epithelial stem cells are located in the basal layer of the limbus between the cornea and the conjunctiva. Regulation of these limbal epithelial progenitor cells by the stromal niche dictates corneal surface health. To further characterize this process, limbal explants were cultured at the air-fluid interface, termed air-lifting, to stimulate the niche. As compared to submerged cultures, air-lifting significantly promoted epithelial stratification, migration, proliferation, and intrastromal invasion by limbal epithelial cells. Epithelial intrastromal invasion was noted when the limbal, but not corneal, epithelium was recombined with the limbal stroma containing live, but not dead, cells. Invading limbal basal cells displayed up-regulated nuclear expression of p63 and Ki67, down-regulated E-cadherin and cornea-specific keratin 3, and switched expression of beta-catenin from intercellular junctions to the nucleus and cytoplasm, indicating the activation of the Wnt/beta-catenin pathway. Invaded cells isolated by collagenase from the stroma of air-lifted, but not submerged, explants showed vivid clonal growth on 3T3 fibroblast feeder layers and complete epithelial-mesenchymal transition by expressing nuclear p63 and cytoplasmic S100A4. These findings collectively suggest that epithelial-mesenchymal transition via the Wnt/beta-catenin pathway influences the fate of limbal epithelial cells, likely to be progenitor cells, between regeneration and fibrosis when the stromal niche is activated.     

Limbal epithelial side-population cells have stem cell-like properties, including quiescent state

Corneal epithelial (CE) stem cells are believed to reside in the basal layer of the limbal epithelium but remain poorly understood due to the lack of an accepted in vivo reconstitution assay as well as definitive markers for epithelial stem cells. It has been reported that side-population (SP) cells with the ability to efflux the DNA-binding dye Hoechst 33342 have stem cell-like properties and that the SP phenotype accurately represents a quiescent and immature stem cell population in the adult bone marrow. In the present study, we investigated whether SP cells isolated from the limbal epithelium have stem cell-like properties. SP cells, separated by fluorescence-activated cell sorting, comprise approximately 0.4% of all limbal epithelial cells and have markedly higher expression of the stem cell markers ABCG2, Bmi-1, and nestin but no expression of markers for differentiated CE cells compared with non-SP cells. Cell-cycle and telomerase activity analyses revealed that SP cells are growth arrested and reside in the quiescent state. Moreover, limbal epithelial SP cells did not demonstrate proliferative capabilities under typical in vitro epithelial cell culture conditions using 3T3 feeder layers. These findings present the possibility that quiescent limbal epithelial SP cells may represent an extremely immature stem cell population compared with currently defined epithelial stem or progenitor cells.     

Comparative Analysis of Human-Derived Feeder Layers with 3T3 Fibroblasts for the Ex Vivo Expansion of Human Limbal and Oral Epithelium

Corneal transplantation with cultivated limbal or oral epithelium is a feasible treatment option for limbal stem cell deficiency (LSCD). Currently utilized co-culture of stem cells with murine 3T3 feeder layer renders the epithelial constructs as xenografts. To overcome the potential risks involved with xenotransplantation, we investigated the use of human-derived feeder layers for the ex vivo expansion of epithelial (stem) cells. Human limbal and oral epithelium was co-cultured with mouse 3T3 fibroblasts, human dermal fibroblasts (DF), human mesenchymal stem cells (MSC), and with no feeder cells (NF). Cell morphology was monitored with phase-contrast microscopy, and stem cell characteristics were assessed by immunohistochemistry, real-time PCR for p63 and ABCG2, (stem cell markers), and by colony-forming efficiency (CFE) assay. Immunohistochemical analysis detected positive staining for CK3 (cornea specific marker) and Iβ1 and p63 (putative stem cell markers) in all culture conditions. The level of Iβ1 and p63 was significantly higher in both limbal and oral cells cultured on the 3T3 feeder, as compared to the MSC or NF group (p<0.01). This level was comparable to the cells cultured on DF. Expression of p63 and ABCG2 in limbal and oral epithelial cells in the 3T3 and DF groups was significantly higher than that in the MSC or NF group (p<0.01). No statistical difference was detected between 3T3 and DF groups. The CFE of both limbal and oral cells co-cultured on 3T3 fibroblasts was comparable to cells grown on DF, and was significantly higher than that of cells co-cultured with MSC or NF (p<0.01). Epithelial cells grown on a DF feeder layer maintained a stem cell-like phenotype, comparable to cells grown on a 3T3 feeder layer. In conclusion, DF provides a promising substitute for 3T3 feeder cells during cultivation of xenobiotic-free corneal equivalents.     

From hair to cornea: toward the therapeutic use of hair follicle-derived stem cells in the treatment of limbal stem cell deficiency

Limbal stem cell deficiency (LSCD) leads to severe ocular surface abnormalities that can result in the loss of vision. The most successful therapy currently being used is transplantation of limbal epithelial cell sheets cultivated from a limbal biopsy obtained from the patient's healthy, contralateral eye or cadaveric tissue. In this study, we investigated the therapeutic potential of murine vibrissae hair follicle bulge-derived stem cells (HFSCs) as an autologous stem cell (SC) source for ocular surface reconstruction in patients bilaterally affected by LSCD. This study is an expansion of our previously published work showing transdifferentiation of HFSCs into cells of a corneal epithelial phenotype in an in vitro system. In this study, we used a transgenic mouse model, K12(rtTA/rtTA) /tetO-cre/ROSA(mTmG) , which allows for HFSCs to change color, from red to green, once differentiation to corneal epithelial cells occurs and Krt12, the corneal epithelial-specific differentiation marker, is expressed. HFSCs were isolated from transgenic mice, amplified by clonal expansion on a 3T3 feeder layer, and transplanted on a fibrin carrier to the eye of LSCD wild-type mice (n = 31). The HFSC transplant was able to reconstruct the ocular surface in 80% of the transplanted animals; differentiating into cells with a corneal epithelial phenotype, expressing Krt12, and repopulating the corneal SC pool while suppressing vascularization and conjunctival ingrowth. These data highlight the therapeutic properties of using HFSC to treat LSCD in a mouse model while demonstrating a strong translational potential and points to the niche as a key factor for determining stem cell differentiation.     

改良组织块培养法体外培养人角膜上皮干细胞

文题释义：角膜上皮干细胞：属于单能干细胞,具有细胞周期长、低分化状态、增殖潜力大、不对称分裂等特点,定位于角膜缘基底细胞层,又称之为角膜缘干细胞,对角膜上皮细胞更新及维持角膜透明起着重要作用。角膜缘干细胞的体外培养方法：主要包括酶消化培养法和组织块培养法。酶消化培养法是利用DispaseⅡ酶破坏角膜缘上皮细胞与基底膜之间的半桥粒连接,然后剥取角膜缘上皮层,再使用胰酶将其消化为单个细胞进行培养。组织块培养法没有经过酶的双重消化,将剖取的角膜缘组织块进行贴壁,细胞游离出组织块进行贴壁生长,需要一个漫长的过程。  摘要背景：角膜上皮干细胞定位于角膜缘,又称之为角膜缘干细胞,临床上由于眼表严重热烧伤、化学性烧伤、慢性炎症等原因引起的角膜缘干细胞缺乏或功能障碍治疗较为棘手。目前利用组织工程技术体外培养角膜上皮干细胞并进行临床移植成为新型有效的治疗方向。目的：探讨在无血清培养条件下采用改良组织块培养法培养人角膜上皮干细胞的可行性。方法：人角膜缘组织来自河南省眼库,植片直径小于8 mm角膜移植术后的供者剩余眼球材料,手术显微镜下剖取角膜缘上皮层外2/3区域,采用2种方法培养人角膜上皮干细胞,常规组织块培养组是将组织块上皮面向上贴壁,加入K-SFM培养液后置于 37 ℃、体积分数为5%CO₂细胞培养箱中培养;改良组织块培养组是先将组织块浸泡于K-SFM培养液中,置于细胞培养箱中孵育12 h,然后组织块上皮面向下贴壁培养。组织块周边有细胞游离出贴壁生长记作“培养第1天”,每日相差显微镜下观察细胞生长变化。利用免疫荧光染色技术检测改良组织块培养第5,10,14天时原代细胞中p63及K3的表达。结果与结论：①改良组织块培养组出膜时间明显短于常规组织块培养组(P < 0.05),出膜率明显高于常规组织块培养组(P < 0.05);②改良组织块培养组细胞生长状态良好,培养第10天可见小体积细胞较多,聚集成灶状分布;培养第14天可见细胞克隆灶,克隆灶内细胞体积较小,形态均一;③培养第5天,K3表达量较多,p63表达量较少;培养第10天,K3和p63表达量均增多;培养第14天,K3表达量未见明显增多,p63表达量明显增多;④在无血清培养条件下,改良组织块培养法能显著促进角膜上皮干细胞的游离,提高体外培养细胞数量,为人角膜缘上皮组织片的构建提供种子细胞。ORCID: 0000-0001-8370-174X(许中中) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4 signaling to prevent differentiation

Corneal epithelial stem cells (SCs) are an ideal model for investigating how adult lineage-committed epithelial SCs are regulated by an anatomically defined and accessible niche, that is, limbal palisades of Vogt, located between the cornea and the conjunctiva. We have used collagenase digestion to isolate the entire limbal epithelial SCs and subjacent mesenchymal cells, and we have demonstrated that their close association is crucial for promoting epithelial clonal growth, implying that the latter serves as niche cells (NCs). After their close association was disrupted by trypsin/EDTA, single SCs and NCs could reunite to generate sphere growth in three-dimensional Matrigel in the embryonic SC medium, and that such sphere growth initiated by SC-NC reunion was mediated by SDF-1 uniquely expressed by limbal epithelial progenitor cells and its receptor CXCR4, but not CXCR7, strongly expressed by limbal stromal NCs. Inhibition of CXCR4 by AMD3100 or a blocking antibody to CXCR4 but not CXCR7 disrupted their reunion and yielded separate spheres with a reduced size, while resultant epithelial spheres exhibited more corneal differentiation and a notable loss of holoclones. For the first time, these results provide strong evidence supporting that limbal SC function depends on close physical association with their native NCs via SDF-1/CXCR4 signaling. This novel in vitro model of sphere growth with NCs can be used for investigating how limbal SC self-renewal and fate decision might be regulated in the limbal niche.     

Umbilical Cord Lining Stem Cells as a Novel and Promising Source for Ocular Surface Regeneration

The stem cells involved in renewal of the corneal epithelium are located in the basal region of the limbus, a narrow transition zone surrounding the cornea. In many ocular surface disorders loss of these stem cells results in partial or complete vision loss. Conventional corneal transplant in these patients is associated with dismal results. Stem cell transplantation offers new hope to such patients. The umbilical cord is emerging as an important source of stem cells that may have potential clinical applications. There are advantages to the use of umbilical cord stem cells as these cells are less immunogenic, non-tumorigenic, highly proliferative and ethically acceptable. In this study, we have confirmed the expression of several putative limbal stem cell markers such as HES1, ABCG2, BMI1, CK15 as well as cell adhesion-associated molecules INTEGRIN-α6, -α9, -β1, COLLAGEN-IV and LAMININ in our recently characterized CLEC-muc population derived from human umbilical cord. Ex vivo expansion of these cells on a human amniotic membrane substrate formed a stratified cell sheet that similarly expresses some of these molecules as well as cornea-specific cytokeratins, CK3 and CK12. Transplantation of a bioengineered CLEC-muc sheet in limbal stem cell-deficient rabbit eyes resulted in regeneration of a smooth, clear corneal surface with phenotypic expression of the normal corneal-specific epithelial markers CK3, CK12 but not CK4 or CK1/10. Our results suggest that CLEC-muc is a novel stem cell that can be ex vivo expanded for corneal epithelial regeneration in the treatment of various eye diseases.     

Gremlin 1 depletion in vivo causes severe enteropathy and bone marrow failure

The intestinal epithelium is perpetually renewed from a stem cell niche in the base of crypts to maintain a healthy bowel mucosa. Exit from this niche and maturation of epithelial cells requires tightly controlled gradients in BMP signalling, progressing from low BMP signalling at the crypt base to high signalling at the luminal surface. The BMP antagonist gremlin 1 (Grem1) is highly expressed by subepithelial myofibroblasts adjacent to the intestinal crypts but its role in regulating the stem cell niche and epithelial renewal in vivo has not been explored. To explore the effects of Grem1 loss in adulthood following normal growth and development, we bred mice (ROSA26CreER-Grem1 ^flx/flx) in which Grem1 could be deleted by tamoxifen administration. While Grem1 remained intact, these mice were healthy, grew normally, and reproduced successfully. Following Grem1 depletion, the mice became unwell and were euthanised (at 7–13 days). Post-mortem examination revealed extensive mucosal abnormalities throughout the small and large intestines with failure of epithelial cell replication and maturation, villous atrophy, and features of malabsorption. Bone marrow hypoplasia was also observed with associated early haematopoietic failure. These results demonstrate an essential homeostatic role for gremlin 1 in maintaining normal bowel epithelial function in adulthood, suggesting that abnormalities in gremlin 1 expression can contribute to enteropathies. We also identified a previously unsuspected requirement for gremlin 1 in normal haematopoiesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

The Limbal Epithelial Progenitors in the Limbal Niche Environment

Limbal epithelial progenitors are stem cells located in limbal palisades of vogt. In this review, we present the audience with recent evidence that limbal epithelial progenitors may be a powerful stem cell resource for the cure of human corneal stem cell deficiency. Further understanding of their mechanism may shed lights to the future successful application of stem cell therapy not only to the eye tissue, but also to the other tissues in the human body.     

活体及体外条件下对角膜上皮干细胞的定位

背景：眼表存在两种形式的上皮干细胞即角膜上皮干细胞和结膜上皮干细胞,角膜上皮干细胞在角膜上皮细胞更新和角膜透明的维持方面起着重要作用。 目的：采用活体激光扫描角膜共焦显微镜和免疫荧光染色技术相结合的方法,从活体和体外层面上对角膜上皮干细胞进行定位研究。 方法：收集2009年9月至2012年9月来河南省眼科研究所就诊的单侧角膜缘干细胞缺乏患者,使用活体激光扫描角膜共焦显微镜检查患者双眼,健侧眼为对照。扫描方位依次为中央角膜及上、下、左、右方的角膜缘,记录扫描图像并分析。眼球材料来自于河南省眼库,切取角膜中央和角膜缘组织,组织包埋剂包被、冰冻切片,切片厚度5-7 μm;免疫荧光染色技术检测p63、ABCG2、K3和Connexin 43在角膜中央及角膜缘上皮层的表达。 结果与结论：共有24例患者确诊为单侧角膜缘干细胞缺乏,活体激光扫描角膜共焦显微镜下患侧眼角膜病变区可见结膜细胞及杯状细胞;角膜缘区域Vogt栅栏状结构消失,色素细胞消失,取而代之的是大量纤维瘢痕化组织。免疫荧光染色示表达ABCG2和p63的细胞主要在角膜缘上皮基底层,尤其在近结膜侧的角膜缘及角膜缘中间部表达相对较高,而中央角膜上皮层细胞不表达;K3及Connexin43在角膜缘上皮基底细胞层不表达,中央角膜上皮全层表达。通过活体激光扫描角膜共焦显微镜观察及干细胞标记物检测显示角膜上皮干细胞主要存在于角膜缘外2/3区域的Vogt栅栏基底部及钉突结构中。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Analysis of the clonal architecture of the human small intestinal epithelium establishes a common stem cell for all lineages and reveals a mechanism for the fixation and spread of mutations

Little is known about the clonal structure or stem cell architecture of the human small intestinal crypt/villus unit, or how mutations spread and become fixed. Using mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion of stem cell progeny, we aimed to provide answers to these questions. Enzyme histochemistry (for cytochrome c oxidase and succinate dehydrogenase) was performed on frozen sections of normal human duodenum. Laser‐capture microdissected cells were taken from crypts/villi. The entire mitochondrial genome was amplified using a nested PCR protocol; sequencing identified mutations and immunohistochemistry demonstrated specific cell lineages. Cytochrome c oxidase‐deficient small bowel crypts were observed within all sections: negative crypts contained the same clonal mutation and all differentiated epithelial lineages were present, indicating a common stem cell origin. Mixed crypts were also detected, confirming the existence of multiple stem cells. We observed crypts where Paneth cells were positive but the rest of the crypt was deficient. We have demonstrated patches of deficient crypts that shared a common mutation, suggesting that they have divided by fission. We have shown that all cells within a small intestinal crypt are derived from one common stem cell. Partially‐mutated crypts revealed some novel features of Paneth cell biology, suggesting that either they are long‐lived or a committed Paneth cell‐specific long‐lived progenitor was present. We have demonstrated that mutations are fixed in the small bowel by fission and this has important implications for adenoma development. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.