Isolating a pure population of epidermal stem cells for use in tissue engineering

Continuously renewing tissues, such as the epidermis, are maintained by stem cells that slowly proliferate and remain in the tissue for life. Although it has been known for decades that epithelial stem cells can be identified as label-retaining cells (LRCs) by long term retention of a nuclear label, isolating a pure population of stem cells has been problematic. Using a Hoechst and propidium iodide dye combination and specifically defined gating, we sorted mouse epidermal basal cells into three fractions, which we have now identified as stem, transient amplifying (TA), and non-proliferative basal cells. More than 90% of freshly isolated stem cells showed a G0/G1 cell cycle profile, while greater than 20% of the TA cells were actively dividing. Both stem and TA cells retained proliferative capacity, but the stem cells formed larger, more expandable colonies in culture. Both populations could be transduced with a retroviral vector and used to bioengineer an epidermis. However, only the epidermis from the stem cell population continued to grow and express the reporter gene for 6 months in organotypic culture. The epidermis from the transient amplifying cell fraction completely differentiated by 2 months. This novel sorting method yields pure viable epithelial stem cells that can be used to bioengineer a tissue and to test permanent recombinant gene expression.     

Keratinocyte stem cells, label-retaining cells and possible genome protection mechanisms

Stem cells are the crucial cells upon which the entire tissue is dependent. Here we define and discuss what is meant by and known about keratinocyte stem cells. One way in which these cells have been studied is by their ability to retain radioactivity labelled thymidine for long periods of time (label retaining cells, LRCs). The underlying mechanism has been assumed in the past to be slow cycling but a more likely explanation is the selective segregation of old and new DNA strands (Cairns's hypothesis). Experiments in the small intestine indicate that the stem cells here are selectively sorting their DNA and becoming LRCs. A possible role for p53 in stem cell biology is presented.     

Investigating human keratinocyte stem cell identity

Studies of the regulatory networks controlling intrinsic properties and fate of adult stem cells are in a large part performed in animal models. Epidermis is one of the most accessible human tissues for researchers, which is a critical parameter for conducting programs dedicated to this knowledge in human stem cell systems. Keratinocyte stem cells constitute a particularly valuable model, because of this practical aspect, but more importantly because their existence is for decades validated by the clinical demonstration of their impressive capacity for epidermis regeneration. For the fundamentalist, human keratinocyte stem cells represent a unique system to dissect the genetic and epigenetic controls of "stemness" and self-renewal. For this purpose, a highly limiting point is our current inability of obtaining a cellular material corresponding to keratinocyte stem cells with homogeneous phenotypic and functional characteristics. The search for tools suitable for the prospective selection of keratinocyte stem cells will benefit from studies conducted at the broad level of the global stem cell field, as well as from more specifically targeted approaches. Advances in that direction are tightly linked to the development of functional assays allowing reliable assessment and modeling of the different stem cell-associated functional characteristics.     

Epidermal stem cells have the potential to assist in healing damaged tissues

Homeostasis of continuously renewing tissues, such as the epidermis, is maintained by somatic undifferentiated, self-renewing stem cells, which are thought to persist throughout life. Through a series of labeling experiments, we previously showed that stem cells from mouse skin did not divide often, but they did divide at a steady rate in vivo. Using our recently redefined sorting method, we isolated epidermal stem and transit amplifying (TA) cells from mouse skin. When injected into a developing blastocyst or into damaged tissues, the stem cells, but not the TA cells, could participate in the formation of new tissues. We hypothesize that all tissues contain reserved undifferentiated stem cells that are primed to react if needed. These reserve stem cells could restore the tissue in which they reside or they could be called upon to help restore another tissue that was severely damage.     

The bone marrow is akin to skin: HCELL and the biology of hematopoietic stem cell homing

The recent findings that adult stem cells are capable of generating new blood vessels and parenchymal cells within tissues they have colonized has raised immense optimism that these cells may provide functional recovery of damaged organs. The use of adult stem cells for regenerative therapy poses the challenging task of getting these cells into the requisite sites with minimum morbidity and maximum efficiency. Ideally, tissue-specific colonization could be achieved by introducing the stem cells intravascularly and exploiting the native physiologic processes governing cell trafficking. Critical to the success of this approach is the use of stem cells bearing appropriate membrane molecules that mediate homing from vascular to tissue compartments. Hematopoietic stem cells (HSC) express a novel glycoform of CD44 known as hematopoietic cell E-/L-selectin ligand (HCELL). This molecule is the most potent E-selectin ligand natively expressed on any human cell. This article reviews our current understanding of the molecular basis of HSC homing and will describe the fundamental "roll" of HCELL in opening the avenues for efficient HSC trafficking to the bone marrow, the skin and other extramedullary sites.     

Hematopoietic stem cell transplantation for auto immune rheumatic diseases

Stem cells have their origins in the embryo and during the process of organogenesis, these differentiate into specialized cells which mature to form tissues. In addition, stem cell are characterized by an ability to indefinitely self renew. Stem cells are broadly classified into embryonic stem cells and adult stem cells. Adult stem cells can be genetically reprogrammed to form pluripotent stem cells and exist in an embroyonic like state. In the early phase of embryogenesis, human embryonic stem cells only exist transiently. Adult stem cells are omnipresent in the body and function to regenerate during the process of apoptosis or tissue repair. Hematopoietic stem cells (HSC) are adult stem cells that form blood and immune cells. Autoimmune responses are sustained due to the perennial persistence of tissue self autoantigens and/or auto reactive lymphocytes. Immune reset is a process leading to generation of fresh self-tolerant lymphocytes after chemotherapy induced elimination of self or autoreactive lymphocytes. This forms the basis for autologous HSC transplantation (HSCT). In the beginning HSCT had been limited to refractory autoimmune rheumatic diseases (AIRD) due to concern about transplant related mortality and morbidity. However HSCT for AIRD has come a long way with better understanding of patient selection, conditioning regime and supportive care. In this narrative review we have examined the available literature regarding the HSCT use in AIRD.     

The bone marrow is akin to skin: HCELL and the biology of hematopoietic stem cell homing

The recent findings that adult stem cells are capable of generating new blood vessels and parenchymal cells within tissues they have colonized has raised immense optimism that these cells may provide functional recovery of damaged organs. The use of adult stem cells for regenerative therapy poses the challenging task of getting these cells into the requisite sites with minimum morbidity and maximum efficiency. Ideally, tissue-specific colonization could be achieved by introducing the stem cells intravascularly and exploiting the native physiologic processes governing cell trafficking. Critical to the success of this approach is the use of stem cells bearing appropriate membrane molecules that mediate homing from vascular to tissue compartments. Hematopoietic stem cells (HSC) express a novel glycoform of CD44 known as hematopoietic cell E-/L-selectin ligand (HCELL). This molecule is the most potent E-selectin ligand natively expressed on any human cell. This article reviews our current understanding of the molecular basis of HSC homing and will describe the fundamental "roll" of HCELL in opening the avenues for efficient HSC trafficking to the bone marrow, the skin and other extramedullary sites.     

Regulation of intestinal stem cells

Stem cells are essential for maintaining the tissue integrity of all adult tissues. The manipulation of adult stem cells has the potential for cell regeneration and in curing diseases; however, the intestinal stem cell remains enigmatic. Although much work has focused on characterization of the intestinal stem cell within its in vivo niche, the lack of reliable markers complicates its isolation and therefore its in vitro manipulation. Understanding what regulates the intestinal stem cell within its niche will provide valuable insight into how these cells can be manipulated in culture. Comparing the regulation of this niche in the developing and mature intestine is a valuable untapped resource. A small number of signaling pathways are functionally conserved during development. These pathways are beginning to emerge as critical regulators of the stem cell niche. This review focuses on the regulation of the intestinal stem cell niche.     

The interfollicular epidermal stem cell saga: sensationalism versus reality check

Adult stem cells in rapidly renewing tissues have been classically defined as rare, relatively quiescent cells with the unique capacity to constantly self-renew and regenerate tissues during homeostasis. Although this view remains firmly embedded in the skin field, particularly in the area of hair follicle stem cell biology, it has been challenged by a number of notable publications in 2007. These papers leave an uncomfortable feeling with the reader if one believes that stem cells and transit amplifying cells are two polar opposites and 'never the twain shall meet.' Even if you do not subscribe to this extreme view, the implications appear to be far-reaching given that the majority of techniques devised for stem cell identification have used the fundamental tenet that the proliferating compartment is comprised of two distinct, mutually exclusive compartments, i.e. a minor proportion of long-lived quiescent stem cells with unlimited self-renewal and a large pool of rapidly cycling, short-lived transient amplifying cells with limited or no self-renewal capacity in normal steady-state conditions. However, these recent findings have resulted in papers that could be described as sensationalistic because they make little or no attempt to reconcile their observations with the large bulk of historical data with direct bearing on the interpretation of stem cell activity in normal steady-state conditions. Here, we offer some explanations that may help to integrate all of the data while presenting a case that both quiescent stem cells and cycling 'transit amplifying' cells contribute to epidermal replacement.     

Optimization of a transplant model to assess skin reconstitution from stem cell-enriched primary human keratinocyte populations

Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated; and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.     

Isolation and identification of stem cells from adult cashmere goat skin

Background  Continuously renewing epithelia are maintained by stem cells that slowly proliferate and remain in the tissues for life. It has been known for decades that mouse epithelial stem cells can be selected by adherence to specific integrins.
Methods  The adherence of cashmere goat epidermal cells to collagen type IV for 10 min was used to obtain enriched epidermal stem cells. The characteristics of the rapidly adherent epidermal cells were determined.
Results  The rapidly adherent epidermal cells exhibited the stem cell characteristics of immaturity, were quiescent, showed a high colony formation efficiency, and expressed candidate surface markers for epidermal stem cells (keratin 15, keratin 19, p63, CD34, and β1-integrin).
Conclusions  The rapidly adherent epidermal cells represented the epidermal stem cell population.     

Senescent dermal fibroblasts enhance stem cell migration through CCL2/CCR2 axis

During aging, increases in the number of senescent cells are seen in various tissues. On the other hand, stem cells play crucial roles in tissue repair and homeostasis. Therefore, it is likely that stem cells give rise to new cells that replace senescent cells. However, how stem cells contribute to homeostasis in the dermis has not been elucidated. Here, we investigated the effects of factors secreted from senescent fibroblasts on stem cells. We found that senescent human dermal fibroblast (HDF) conditioned medium (CM) significantly enhanced stem cell migration compared with young HDF CM. The senescent HDF CM strongly secreted chemokine ligand 2 (CCL2). Furthermore, CCL2 was found to enhance stem cell migration, and the inhibition of CCR2, a receptor for CCL2, reduced stem cell migration. These results suggest that senescent fibroblasts recruit stem cells by secreting various factors and that the CCL2/CCR2 axis is one of the mechanisms underlying this phenomenon.     

Murine epidermal label-retaining cells isolated by flow cytometry do not express the stem cell markers CD34, Sca-1, or Flk-1.

Keratinocyte stem cells are present in the murine epidermis, based on both in vitro and in vivo evidence, and better characterization of these cells remains an active goal. Because keratinocyte stem cells are believed to cycle slowly, a good method for identification is based on their ability to retain nucleoside analog, such as bromodeoxyuridine. Adult stem cells have been identified in other tissues, including hematopoietic, neural, and skeletal muscle, and stem cell surface markers have been characterized. We wanted to determine if cell-surface markers present on both hematopoietic and skeletal muscle stem cells (CD34, Sca-1, and Flk-1) were also present on keratinocyte stem cells, and could be used to identify them. The cell-surface expression of cells that retained bromodeoxyuridine label for at least 21 d was compared with that of nonlabel-retaining cells. Double-labeling for flow cytometric analysis was employed, and label-retaining cells were found to lack expression of the tested markers. Beta1 integrin levels were also evaluated, and although high expression was found on label-retaining cells, it was not specific for these cells.     

Mitotic rates of rat epidermis. During growth, maturity, senility, and regeneration.

Adult epidermal tissues are renewing cell populations. Mitosis continuously provides new cells replacing those that became keratinized and ultimately shed. It seems plausible to assume that in the epidermis of growing individuals mitosis supplies cells both for cell addition (growth) and renewal. During growth, more cells are presumably retained in the tissue than become lost through desquamation. It is feasible by cutaneous incision to temporarily revert adult epidermis into a growing cell population. Presumably also during healing, cell production exceeds cell loss. What is the magnitude of cell production for renewal of adult epidermis, for simultaneous cell addition and renewal during growth, as well as during epidermal regeneration? To elucidate these problems, the mitotic rates of three epidermal cell populations (ear, plantar, and abdominal epidermis) were determined in the growing, adult, and senile rat. Further, the adult ear and back skin epidermis was converted by cutaneous incision into regenerating cell populations, and the mitotic rates were determined during healing at intervals from six hours to nine days.     

Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells

Very primitive hematopoietic stem cells have been identified as side population cells based on their ability to efflux a fluorescent vital dye, Hoechst 33342. In this study we show that keratinocytes with the same side population phenotype are also present in the human epidermis. Although side population keratinocytes have the same dye-effluxing phenotype as bone marrow side population cells and can be blocked by verapamil, they do not express increased levels of the ABCG2 transporter that is believed to be responsible for the bone marrow side population phenotype. Because bone marrow side population cells have stem cell characteristics, we sought to determine if side population keratinocytes represent a keratinocyte stem cell population by comparing side population keratinocytes with a traditional keratinocyte stem cell candidate, label-retaining keratinocytes. Flow cytometric analyses demonstrated that side population keratinocytes have a different cell surface phenotype (low beta1 integrin and low alpha6 integrin expression) than label-retaining keratinocytes and represent a unique population of keratinocytes distinctly different from the traditional keratinocyte stem cell candidate. Future in vivo studies will be required to analyze the function of side population keratinocytes in epidermal homeostasis and to determine if side population keratinocytes have characteristics of keratinocyte stem cells.     

Bone morphogenetic protein 5 regulates the number of keratinocyte stem cells from the skin of mice

Understanding keratinocyte stem cell regulation is important in understanding the pathogenesis of wound healing and nonmelanoma skin cancer. We previously used a sensitive and quantitative assay for in vitro keratinocyte colony formation and mapped the keratinocyte stem cell locus (Ksc1) on mouse chromosome 9. Examination of the candidate genes in this locus disclosed a sequence variant in the gene for bone morphogenetic protein 5 (Bmp5). In this report, we used a naturally occurring mouse with a null mutation in this gene to probe stem cell properties in mouse epidermis. We found that the mutant keratinocytes had a significant reduction in the size and number of clonogenic keratinocytes. The mutant mice had a 50% reduction in the number of label-retaining cells when compared with their littermates. Addition of exogenous Bmp5 protein increased the number and size of keratinocyte colonies in the mutant as well as their wild-type littermates. Surprisingly, the mutant mice showed at least a 2-fold increase in skin tumor susceptibility over their littermates. We conclude that a naturally occurring mutation in Bmp5 affects keratinocyte stem cell proliferation, and skin tumor susceptibility, and is a candidate stem cell regulatory gene in the Ksc1 locus.     

非节段型白癜风患者皮损单细胞转录图谱的初步研究

【摘要】 目的 采用单细胞RNA测序技术鉴定和区分白癜风皮损真表皮细胞亚群，并研究它们之间的关系。方法 2019年9月在杭州市第三人民医院皮肤科门诊收集2例健康人（无免疫及系统性疾病）正常皮肤和2例非节段型稳定期白癜风患者皮损样本，采用10 × Genomics单细胞RNA-Seq技术检测，对所有样本的11 000个细胞进行单细胞转录组测序。通过Seurat软件分析、筛选和统计细胞亚群。结果 对2例正常皮肤基因表达的聚类分析发现包括角质形成细胞、成纤维细胞、神经及黑素细胞、内皮细胞、组织干细胞和以树突细胞及T细胞为主的免疫细胞群。2例白癜风皮损中分化和数量异常的有成纤维细胞和4类角质形成细胞亚群，其中，成纤维细胞占比为0，低于正常皮肤（0.4%），角质形成细胞亚群5、6、10、12占比（8.03%、7.36%、3.52%、0.91%）均显著高于正常皮肤（4.47%、3.53%、2.69%、0.28%，均P＜0.01）。上述亚群的角质形成细胞处于细胞分化的末端，同时还具有极显著且特异的标记基因，分析显示，标记基因主要与细胞间相互作用和细胞稳态密切有关，GO和KEGG分析显示，角质形成细胞亚群5、6主要与细胞间连接和细胞黏附及细胞骨架功能相关，角质形成细胞亚群10与细胞稳态紧密相关。结论 国内首次报道通过单细胞测序手段研究白癜风皮损的转录表达谱，初步发现4群数量及功能差异的角质形成细胞，提示角质形成细胞亚群的异常分化与功能异常可能影响白癜风的发生发展。     

Survivin: a dual player in healthy and diseased skin

Survivin belongs to the inhibitor of apoptosis (IAP) protein family, and, in addition to the antiapoptotic functions, it also regulates the cell cycle. The survivin gene generates five major isoforms with diverse and opposite functions. Survivin is highly expressed in cancer and in few normal adult tissues, including skin. It is mostly detected in the nucleus of keratinocyte stem cells (KSCs), but it is also expressed in melanocytes and fibroblasts. Survivin isoforms are differentially detected in subpopulations of human keratinocytes, exerting contrasting activities. Survivin has an important role in the regulation of cell cycle in keratinocytes, and it protects these cells from anoikis and UV-induced apoptosis. In melanoma, survivin is abundantly expressed, and its subcellular localization varies depending upon tumor thickness and invasiveness. Survivin overexpression has been shown in squamous cell carcinoma (SCC), and it is also involved in UVB-induced carcinogenesis. The presence of survivin both in the nucleus and in the cytoplasm throughout the epidermal layers of psoriatic lesions suggests the involvement of this protein in the keratinocyte alterations typical of this disease. Additional studies on the expression of survivin isoforms and their subcellular localization in relation to function will confirm the key role of survivin in the skin and will open the field to new therapeutic strategies for many cutaneous conditions.     

Keratinocyte stem cell assays: an evolving science

Although the existence of epithelial stem cells in the skin has been known for some decades from cell kinetic studies performed in vivo, attempts to prospectively isolate these cells for further biological characterization have been made possible relatively recently facilitated by the availability of antibodies that detect cell surface markers on epidermal cells. Elegant gene marking studies in vivo have provided confirmation of the patterns of epithelial tissue replacement predicted by classical cell turnover studies. But, the identification of candidate epidermal stem cells ex vivo remains an area of great controversy, requiring the re-evaluation of current experimental approaches that rely of necessity on predicted epidermal stem cell behavior in culture. Here we review the diverse experimental approaches utilized to identify keratinocyte stem cells and their underlying assumptions. We conclude that hair follicles and interfollicular epidermis each have their own self-renewing stem cell populations, contributing to distinct regions of the epithelium during homeostasis, although this is perturbed during wound healing. The need for the development of more rigorous assays for stem cell activity is highlighted given our recent observations using current assays and the discovery of new surface markers that identify putative epidermal stem cells.