Human Keratinocyte Stem Cells Research Articles

Induction of human stem cells into ameloblasts by reaggregation strategy

BackgroundHuman epithelium-derived stem cells and induced pluripotent stem cells (hiPSCs) possess the capability to support tooth formation and differentiate into functional enamel-secreting ameloblasts, making them promising epithelial-component substitutes for future human tooth regeneration. However, current tissue recombination approaches are not only technically challenging, requiring precise induction procedures and sophisticated microsurgery, but also exhibit low success rates in achieving tooth formation and ameloblastic differentiation.MethodsSuspended human keratinocyte stem cells (hKSCs) or cells from three hiPSC lines were directly mixed with dissociated embryonic mouse dental mesenchymal cells (mDMCs) that possess odontogenic potential in different proportions and reaggregated them to construct bioengineered tooth germs. The success rates of tooth formation and ameloblastic differentiation were confirmed after subrenal culture. The sorting capability, sequential development, and ameloblastic differentiation of stem cells were examined via GFP tracing, RT-PCR, and histological analysis, respectively.ResultsOur reaggregation approach achieved an impressive success rate of more than 90% in tooth formation and 100% in ameloblastic differentiation when the chimeric tooth germs contained 1%~10% hKSCs or 5% hiPSCs. In addition, we observed that hiPSCs, upon exposure to mDMCs, initially transformed into epidermal cells, as indicated by KRT14 and CD29 expression, before progressing into dental epithelial cells, as indicated by SP6 and SHH expression. We also found that epithelial-derived hiPSCs, when reaggregated with mDMCs, were more favorable for tooth formation than their mesenchymal-derived counterparts.ConclusionsThis study establishes a simplified yet highly effective cell-cell reaggregation strategy for inducing stem cells to support tooth formation and differentiate into functional ameloblasts, paving the way for novel approaches for the development of stem cell-based tooth organoids and bioengineered tooth germs in vitro.

Low temperature and mTOR inhibition favor stem cell maintenance in human keratinocyte cultures

Adult autologous human epidermal stem cells can be extensively expanded ex vivo for cell and gene therapy. Identifying the mechanisms involved in stem cell maintenance and defining culture conditions to maintain stemness is critical, because an inadequate environment can result in the rapid conversion of stem cells into progenitors/transient amplifying cells (clonal conversion), with deleterious consequences on the quality of the transplants and their ability to engraft. Here, we demonstrate that cultured human epidermal stem cells respond to a small drop in temperature through thermoTRP channels via mTOR signaling. Exposure of cells to rapamycin or a small drop in temperature induces the nuclear translocation of mTOR with an impact on gene expression. We also demonstrate by single‐cell analysis that long‐term inhibition of mTORC1 reduces clonal conversion and favors the maintenance of stemness. Taken together, our results demonstrate that human keratinocyte stem cells can adapt to environmental changes (e.g., small variations in temperature) through mTOR signaling and constant inhibition of mTORC1 favors stem cell maintenance, a finding of high importance for regenerative medicine applications.

The impact of airborne ultrafine particulate matter on human keratinocyte stem cells.

Air pollution is today fully acknowledged to be a significant public health problem. Rapid urbanization exposed us to a variety of unhealthy ambient air pollutants at high concentrations. The emergence of airborne ultrafine particles has added an additional dimension to this already complex problem of air pollution. The skin has different functions, one of them being the protection against the deleterious effect of external agents. The aim of this study is to evaluate the impact of airborne ultrafine particles (UFP) pollution on skin aging and on keratinocyte differentiation. Ex vivo human skin biopsies and cultured keratinocytes stem cells (KSC) were submitted to diesel exhaust-derived UFP. Reactive oxygen species (ROS) production was assessed with the MitoSOX™ probe. Keratinocyte stemness potential was evaluated by the immunodetection of keratin 15 (K15) and p63 (∆N isoforms). Effect of UFP on the epithelial niche maintenance was evaluated by immunodetection of Sox9. Reconstructed epidermis model was used to assess the impact of UFP on keratinocyte differentiation and aging. UFP exposure induced ROS production and disturbed K15, ∆Np63 and Sox9 expression in KSC or ex vivo skin. Finally, investigations on reconstructed epidermis revealed a phenotype marked by impaired keratinocyte differentiation. These results indicate that UFP pollution is a potent extrinsic factor of skin aging, affecting the keratinocyte stem cell potential and the skin renewal process.

In vitro characterization of iridoid and phenylethanoid glycosides from Cistanche phelypaea for nutraceutical and pharmacological applications.

"Desert hyacinths" are a remarkable group of parasitic plants belonging to genus Cistanche, including more than 20 accepted species typically occurring in deserts or coastal dunes parasitizing roots of shrubs. Several Cistanche species have long been a source of traditional herbal medicine or food, being C.deserticola and C.tubulosa the most used in China. This manuscript reports the isolation and identification of some phenylethanoid and iridoid glycosides, obtained from the hydroalcoholic extract of C.phelypaea collected in Spain. The present study aims to characterize the antioxidant activity of C.phelypaea metabolites in the light of their application in nutraceutical and cosmeceutical industries and the effect of acetoside, the most abundant metabolite in C.phelypaea extract, on human keratinocyte and pluripotent stem cell proliferation and differentiation. Our study demonstrated that acetoside, besides its strong antioxidant potential, can preserve the proliferative potential of human basal keratinocytes and the stemness of mesenchymal progenitors necessary for tissue morphogenesis and renewal. Therefore, acetoside can be of practical relevance for the clinical application of human stem cell cultures in tissue engineering and regenerative medicine.

Hierarchically Structured Surfaces Prepared by Phase Separation: Tissue Mimicking Culture Substrate

The pseudo 3D hierarchical structure mimicking in vivo microenvironment was prepared by phase separation on tissue culture plastic. For surface treatment, time-sequenced dosing of the solvent mixture with various concentrations of polymer component was used. The experiments showed that hierarchically structured surfaces with macro, meso and micro pores can be prepared with multi-step phase separation processes. Changes in polystyrene surface topography were characterized by atomic force microscopy, scanning electron microscopy and contact profilometry. The cell proliferation and changes in cell morphology were tested on the prepared structured surfaces. Four types of cell lines were used for the determination of impact of the 3D architecture on the cell behavior, namely the mouse embryonic fibroblast, human lung carcinoma, primary human keratinocyte and mouse embryonic stem cells. The increase of proliferation of embryonic stem cells and mouse fibroblasts was the most remarkable. Moreover, the embryonic stem cells express different morphology when cultured on the structured surface. The acquired findings expand the current state of knowledge in the field of cell behavior on structured surfaces and bring new technological procedures leading to their preparation without the use of problematic temporary templates or additives.

Assaying Candidate Human Skin Keratinocyte Stem Cells by Determining Their Long-Term Serial Proliferative Output in Culture.

Stem cells are found in niches around the body, including the epidermis of the skin, and can be distinguished from their more committed progeny by their high long-term proliferative capacity in vitro. Here we describe a technique used to isolate three main epidermal cell fractions from human neonatal foreskin termed early differentiating (ED), transient amplifying (TA) and keratinocyte stem cells (KSC) based on their differential expression of two cell surface markers: CD49f and CD71. These three fractions were cultivated in parallel in a serial proliferation assay to determine their long-term proliferative output. This assay demonstrates that the KSC fraction had the highest proliferative output (total cell yield) over a long experimental timeframe of 2-3months, as well as a higher proliferative rate compared to the other two fractions (P>0.05). This assay can be utilized under similar conditions to determine the proliferative capacity of other putative stem cells using novel stem cell markers for epidermal or other stem cell populations.

Restoration of Immune Privilege in Human Dermal Papillae Controlling Epithelial-Mesenchymal Interactions in Hair Formation.

Hair follicles are among a handful of organs that exhibit immune privilege. Dysfunction of the hair follicle immune system underlies the development of inflammatory diseases, such as alopecia areata. Quantitative reverse transcription PCR and immunostaining was used to confirm the expression of major histocompatibility complex class I in human dermal papilla cells. Through transcriptomic analyses of human keratinocyte stem cells, major histocompatibility complex class I was identified as differentially expressed genes. Organ culture and patch assay were performed to assess the ability of WNT3a conditioned media to rescue immune privilege. Lastly, CD8+ T cells were detected near the hair bulb in alopecia areata patients through immunohistochemistry. Inflammatory factors such as tumor necrosis factor alpha and interferon gamma were verified to induce the expression of major histocompatibility complex class I proteins in dermal papilla cells. Additionally, loss of immune privilege of hair follicles was rescued following treatment with conditioned media from outer root sheath cells. Transcriptomic analyses found 58 up-regulated genes and 183 down-regulated genes related in MHC class I+ cells. Using newborn hairpatch assay, we demonstrated that WNT3a conditioned media with epidermal growth factor can restore hair growth. In alopecia areata patients, CD8+ T cells were increased during the transition from mid-anagen to late catagen. Identification of mechanisms governing epithelial and mesenchymal interactions of the hair follicle facilitates an improved understanding of the regulation of hair follicle immune privilege.

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.

Label-free quality control and identification of human keratinocyte stem cells by deep learning-based automated cell tracking.

Stem cell‐based products have clinical and industrial applications. Thus, there is a need to develop quality control methods to standardize stem cell manufacturing. Here, we report a deep learning‐based automated cell tracking (DeepACT) technology for noninvasive quality control and identification of cultured human stem cells. The combination of deep learning‐based cascading cell detection and Kalman filter algorithm‐based tracking successfully tracked the individual cells within the densely packed human epidermal keratinocyte colonies in the phase‐contrast images of the culture. DeepACT rapidly analyzed the motion of individual keratinocytes, which enabled the quantitative evaluation of keratinocyte dynamics in response to changes in culture conditions. Furthermore, DeepACT can distinguish keratinocyte stem cell colonies from non‐stem cell‐derived colonies by analyzing the spatial and velocity information of cells. This system can be widely applied to stem cell cultures used in regenerative medicine and provides a platform for developing reliable and noninvasive quality control technology.

Molecular Mapping of Hydrogen Sulfide Targets in Normal Human Keratinocytes.

Although sulfur-rich thermal waters have ancestrally been used in the context of dermatological conditions, a global mapping of the molecular effects exerted by H2S on human keratinocytes is still lacking. To fill this knowledge gap, we subjected cultured human keratinocytes to distinct amounts of the non-gaseous hydrogen sulfur donor NaHS. We first checked that H2S accumulated in the cytoplasm of keratinocytes under our experimental conditions andused a combination of proteomics, genomics and biochemical approaches to unravel functionally relevant H2S targets in human keratinocytes. We found that the identified targets fall into two main categories: (i) the oxidative stress response molecules superoxide dismutase 2 (SOD2), NAD(P)H quinone dehydrogenase 1 (NQO1) and culin 3 (CUL3) and (ii) the chemokines interleukin-8 (IL-8) and CXCL2. Interestingly, NaHS also stimulated the caspase-1 inflammasome pathway, leading to increased secretion of the pro-inflammatory molecule interleukin-18 (IL-18). Interestingly, the secretion of interleukin-1 beta (IL-1β) was only modestly impacted by NaHS exposure despite a significant accumulation of IL-1β pro-form. Finally, we observed that NaHS significantly hampered the growth of human keratinocyte progenitors and stem cells cultured under clonogenic conditions or as epidermal cell sheets. We conclude that H2S exerts specific molecular effects on normal human keratinocytes.

Loss of the Epigenetic Mark 5-hmC in Psoriasis: Implications for Epidermal Stem Cell Dysregulation

Epigenetic regulation has a profound influence on stem cell fate during normal development in maintenance of physiologic tissue homeostasis. Here we report diminished ten-eleven translocation (TET) methylcytosine dioxygenase expression and loss of the DNA hydroxymethylation mark 5-hydroxymethylcytosine (5-hmC) in keratinocyte stem cells and transit amplifying cells in human psoriasis and in imiquimod-induced murine psoriasis. Loss of 5-hmC was associated with dysregulated keratinocyte stem cell kinetics, resulting in accumulation of nestin and FABP5-expressing transit amplifying cells to produce classic psoriatic epidermal architecture. Moreover, 5-hmC loss was accompanied by diminished TET1 and TET2 mRNA expression. Genome-wide mapping of epidermal 5-hmC in murine psoriasis revealed loci-specific loss of 5-hmC in genes regulating stem cell homeostasis, including MBD1, RTN1, STRN4, PRKD2, AKT1, and MAPKAP2, as well as those associated with RAR and Wnt/β-catenin signaling pathways. Invitro restoration of TET expression by ascorbic acid was accomplished in cultured human keratinocyte stem cells to show similar Ca++-induced differentiation, resulting in increased 5-hmC levels and reduced nestin expression. To our knowledge, an epigenetic deficiency in psoriasis with relevance to stem cell dysregulation has not been previously reported. This observation raises the possibility that epigenetic modifiers that impact on the TET-5-hmC pathway may be a relevant approach of heretofore unappreciated therapeutic utility.

Automated collective motion analysis validates human keratinocyte stem cell cultures

Identification and quality assurance of stem cells cultured in heterogeneous cell populations are indispensable for successful stem cell therapy. Here we present an image-processing pipeline for automated identification and quality assessment of human keratinocyte stem cells. When cultivated under appropriate conditions, human epidermal keratinocyte stem cells give rise to colonies and exhibit higher locomotive capacity as well as significant proliferative potential. Image processing and kernel density estimation were used to automatically extract the area of keratinocyte colonies from phase-contrast images of cultures containing feeder cells. The DeepFlow algorithm was then used to calculate locomotion speed of the colony area by analyzing serial images. This image-processing pipeline successfully identified keratinocyte stem cell colonies by measuring cell locomotion speed, and also assessed the effect of oligotrophic culture conditions and chemical inhibitors on keratinocyte behavior. Therefore, this study provides automated procedures for image-based quality control of stem cell cultures and high-throughput screening of small molecules targeting stem cells.

IRF2 is a master regulator of human keratinocyte stem cell fate

Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to induce pluripotency and transdifferentiate cells. We investigate whether transcriptional circuitry also governs phenotypic changes within a given cell type by comparing human primary keratinocytes with intrinsically high versus low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate IRF2 as antagonistic to stemness and show that it binds and regulates active cis-regulatory elements at interferon response and antigen presentation genes. CRISPR-KD of IRF2 in keratinocytes with low stem cell potential increases self-renewal, migration and epidermis formation. These data demonstrate that transcription factor regulatory circuitries, in addition to maintaining cell identity, control plasticity within cell types and offer potential for therapeutic modulation of cell function.

593 Label-free identification of human keratinocyte stem cells by deep learning-based quantitative cell motion analysis

593 Label-free identification of human keratinocyte stem cells by deep learning-based quantitative cell motion analysis

Low temperature culture enhances ameloblastic differentiation of human keratinocyte stem cells.

Previous studies have demonstrated that several types of human stem cells of non-dental origin can be induced to differentiate into enamel-secreting ameloblasts after recombined with mouse embryonic dental mesenchyme. However, the successful rate of ameloblastic differentiation is about rather low, which presents a major obstacle for future stem cell-based whole tooth bioengineering. Previous studies have shown that cultures at reduced temperature could improve the differentiation capability of stem cells in tissue engineering. In this study, we systematically investigated the effects of low temperature on the viability, proliferation and stemness of human keratinocytes stem cells (hKSCs) in cell culture and further examined ameloblastic differentiation of the hKSCs in human-mouse recombinant chimeric tooth germs. Our results demonstrated that low temperature indeed reduces growth rate and maintains healthy undifferentiated morphology of hKSCs without any effects on cell viability. Moreover, examination of stemness makers revealed improved stemness of hKSCs cultured at low temperature with increased expression of stemness markers K15, CD29 and p63 and decreased expression differentiation marker K10, as compared to those cultured at 37°C. These low temperature treated hKSCs, when recombined with mouse embryonic dental mesenchyme, exhibited significantly increased rate (40%) of ameloblastic differentiation, as compared to that (17%) in tissue recombinants with those hKSCs treated at standard temperature. Our studies demonstrate that low temperature cell culture improves the stemness and plasticity of hKSCs, which in turn enhances ameloblastic differentiation capability of the stem cells in bioengineered teeth.

Nicotinamide Metabolism Modulates the Proliferation/Differentiation Balance and Senescence of Human Primary Keratinocytes

Nicotinamide (NAM) is the main precursor of nicotinamide adenine dinucleotide (NAD+), a coenzyme essential for DNA repair, glycolysis, and oxidative phosphorylation. NAM has anti-aging activity on human skin, but theunderlying mechanisms of action are unclear. Using 3-dimensional organotypic skin models, we show thatNAM inhibits differentiation of the upper epidermal layers and maintains proliferation in the basal layer. In2-dimensional culture, NAM reduces the expression of early and late epidermal differentiation markers andincreases the proliferative capacity of human primary keratinocytes. This effect is characterized by elevated clonogenicity and an increased proportion of human primary keratinocyte stem cell (holoclones) compared tocontrols. By contrast, preventing the conversion of NAM to NAD+ using FK866 leads to premature humanprimary keratinocyte differentiation and senescence, together with a dramatic drop in glycolysis and cellular adenosine triphosphate levels while oxidative phosphorylation is moderately affected. All these effectsare rescued by addition of NAM, known to compete with FK866, which suggests that conversion to NAD+ is part of the mechanistic response. These data provide insights into the control of differentiation, proliferation, and senescence by NAM and NAD+ in skin. They may lead to new therapeutic advances for skin conditions characterized by dysregulated epidermal homeostasis and premature skin aging, such as photoaging.

Production of progenitor cells from primary human epithelial cell monolayer cultures.

Primary keratinocytes derived from human epidermis are widely used in tissue engineering and regenerative medicine. An important aspect in clinical applications is the preservation of human skin keratinocyte stem cells. However, it is difficult to expand the number of human skin keratinocyte stem cells, which are undifferentiated and highly proliferative in culture by using standard cell culture methods. It is even more difficult to identify them, since universal specific markers for human skin keratinocyte stem cells have not been identified. In this paper, we show a method to produce a large number of primary progenitor human skin keratinocytes by using our novel culture techniques. Primary human skin keratinocyte monolayers are cultured using twice the volume of medium without serum and lacking essential fatty acids. Once the cells reach 70-80% confluence, they begin to float up into the overlying medium and are called "epithelial pop-up keratinocytes (ePUKs)" allowing the cells to be passaged without the use of trypsin. We analyzed the properties of ePUKs by cell size, cell viability, immunocytofluorescence biomarker staining, and cell cycle phase distribution by fluorescence-activated cell sorting (FACS). Our results showed that these ePUKs appear to be progenitor epithelial cells, which are small in size, undifferentiated, and have a high proliferative capacity. We believe that ePUKs are suitable for use in medical applications requiring a large number of primary human progenitor skin keratinocytes.

Efficient induction of functional ameloblasts from human keratinocyte stem cells

BackgroundAlthough adult human tissue-derived epidermal stem cells are capable of differentiating into enamel-secreting ameloblasts and forming teeth with regenerated enamel when recombined with mouse dental mesenchyme that possesses odontogenic potential, the induction rate is relatively low. In addition, whether the regenerated enamel retains a running pattern of prism identical to and acquires mechanical properties comparable with human enamel indeed warrants further study.MethodsCultured human keratinocyte stem cells (hKSCs) were treated with fibroblast growth factor 8 (FGF8) and Sonic hedgehog (SHH) for 18 h or 36 h prior to being recombined with E13.5 mouse dental mesenchyme with implantation of FGF8 and SHH-soaked agarose beads into reconstructed chimeric tooth germs. Recombinant tooth germs were subjected to kidney capsule culture in nude mice. Harvested samples at various time points were processed for histological, immunohistochemical, TUNEL, and western blot analysis. Scanning electronic microscopy and a nanoindentation test were further employed to analyze the prism running pattern and mechanical properties of the regenerated enamel.ResultsTreatment of hKSCs with both FGF8 and SHH prior to tissue recombination greatly enhanced the rate of tooth-like structure formation to about 70%. FGF8 and SHH dramatically enhanced stemness of cultured hKSCs. Scanning electron microscopic analysis revealed the running pattern of intact prisms of regenerated enamel is similar to that of human enamel. The nanoindentation test indicated that, although much softer than human child and adult mouse enamel, mechanical properties of the regenerated enamel improved as the culture time was extended.ConclusionsApplication of FGF8 and SHH proteins in cultured hKSCs improves stemness but does not facilitate odontogenic fate of hKSCs, resulting in an enhanced efficiency of ameloblastic differentiation of hKSCs and tooth formation in human–mouse chimeric tooth germs.

Dual Role of the Anaphase Promoting Complex/Cyclosome in Regulating Stemness and Differentiation in Human Primary Keratinocytes

Cdc20 and Cdh1 activate the anaphase-promoting complex/cyclosome, a master cell cycle regulator. Although cell cycle modifications occur during differentiation of stem cells, a role for the anaphase-promoting complex/cyclosome on stem cell fate has not been established in embryonic or adult human tissues. We found that differentiated human primary keratinocytes from the skin express extremely low levels of Cdc20 compared with human primary keratinocyte stem cells (holoclones). In agreement with this, staining of human skin biopsies showed that Cdc20 is expressed in occasional cells from the basal and epibasal layers of the epidermis and is absent from the differentiated layers. Conversely, Cdh1 is preferentially expressed in differentiated cells. Interestingly, partial silencing of Cdc20 enhanced differentiation, indicating that loss of Cdc20 might be a cause rather than a consequence of terminal differentiation. By contrast, Cdh1 silencing induced the opposite cellular phenotype, which was characterized by an increase in stemness, cellular proliferation, and loss of differentiation markers. These data pinpoint the anaphase-promoting complex/cyclosome as a key regulator of adult stem cell fate. They also demonstrate the critical and opposing roles of Cdc20 and Cdh1 in controlling the balance between human primary keratinocyte proliferation and differentiation, and therefore in regulating skin homeostasis.

685 Locomotive ability of human keratinocyte stem cells is an intrinsic property for stem cell expansion and epidermal reconstruction

685 Locomotive ability of human keratinocyte stem cells is an intrinsic property for stem cell expansion and epidermal reconstruction