Deciphering "Immaturity-Stemness" in Human Epidermal Stem Cells at the Levels of Protein-Coding and Non-Coding Genomes: A Prospective Computational Approach.

The epidermis hosts populations of epithelial stem cells endowed with well-documented renewal and regenerative functions. This tissue thus constitutes a model for exploring the molecular characteristics of stem cells, which remain to date partially characterized at the molecular level in human skin. Our group has investigated the regulatory functions of the KLF4/TGFB1 and the MAD4/MAX/MYC signaling pathways in the control of the immaturity-stemness versus differentiation fate of keratinocyte stem and precursor cells from human interfollicular epidermis. We described that down-modulation of either KLF4 or MXD4/MAD4 using RNA interference tools promoted an augmented stemness cellular status; an effect which was associated with significant transcriptional changes, as assessed by RNA-sequencing. Here, we have implemented a computational approach aimed at integrating the level of the coding genome, comprising the transcripts encoding conventional proteins, and the non-coding genome, with a focus on long non-coding RNAs (lncRNAs). In addition, datasets of micro-RNAs (miRNAs) with validated functions were interrogated in view of identifying miRNAs that could make the link between protein-coding and non-coding transcripts. Putative regulons comprising both coding and long non-coding transcripts were built, which are expected to contain original pro-stemness candidate effectors available for functional validation approaches. In summary, interpretation of our basic functional data together with in silico biomodeling gave rise to a prospective picture of the complex constellation of transcripts regulating the keratinocyte stemness status.

In vivo quantitative MRI: T1 and T2 measurements of the human brain at 0.064 T.

OBJECTIVE: To measure healthy brain [Formula: see text] and [Formula: see text] relaxation times at 0.064 T. MATERIALS AND METHODS: [Formula: see text] and [Formula: see text] relaxation times were measured in vivo for 10 healthy volunteers using a 0.064 T magnetic resonance imaging (MRI) system and for 10 test samples on both the MRI and a separate 0.064 T nuclear magnetic resonance (NMR) system. In vivo [Formula: see text] and [Formula: see text] values are reported for white matter (WM), gray matter (GM), and cerebrospinal fluid (CSF) for automatic segmentation regions and manual regions of interest (ROIs). RESULTS: [Formula: see text] sample measurements on the MRI system were within 10% of the NMR measurement for 9 samples, and one sample was within 11%. Eight [Formula: see text] sample MRI measurements were within 25% of the NMR measurement, and the two longest [Formula: see text] samples had more than 25% variation. Automatic segmentations generally resulted in larger [Formula: see text] and [Formula: see text] estimates than manual ROIs. DISCUSSION: [Formula: see text] and [Formula: see text] times for brain tissue were measured at 0.064 T. Test samples demonstrated accuracy in WM and GM ranges of values but underestimated long [Formula: see text] in the CSF range. This work contributes to measuring quantitative MRI properties of the human body at a range of field strengths.

Relaxation measurements of an MRI system phantom at low magnetic field strengths.

OBJECTIVE: Temperature controlled T1 and T2 relaxation times are measured on NiCl2 and MnCl2 solutions from the ISMRM/NIST system phantom at low magnetic field strengths of 6.5 mT, 64 mT and 550 mT. MATERIALS AND METHODS: The T1 and T2 were measured of five samples with increasing concentrations of NiCl2 and five samples with increasing concentrations of MnCl2. All samples were scanned at 6.5 mT, 64 mT and 550 mT, at sample temperatures ranging from 10 °C to 37 °C. RESULTS: The NiCl2 solutions showed little change in T1 and T2 with magnetic field strength, and both relaxation times decreased with increasing temperature. The MnCl2 solutions showed an increase in T1 and a decrease in T2 with increasing magnetic field strength, and both T1 and T2 increased with increasing temperature. DISCUSSION: The low field relaxation rates of the NiCl2 and MnCl2 arrays in the ISMRM/NIST system phantom are investigated and compared to results from clinical field strengths of 1.5 T and 3.0 T. The measurements can be used as a benchmark for MRI system functionality and stability, especially when MRI systems are taken out of the radiology suite or laboratory and into less traditional environments.

MXD4/MAD4 Regulates Human Keratinocyte Precursor Fate.

Deciphering the pathways that regulate human epidermal precursor cell fate is necessary for future developments in skin repair and graft bioengineering. Among them, characterization of pathways regulating the keratinocyte (KC) precursor immaturity versus differentiation balance is required for improving the efficiency of KC precursor ex vivo expansion. In this study, we show that the transcription factor MXD4/MAD4 is expressed at a higher level in quiescent KC stem/progenitor cells located in the basal layer of human epidermis than in cycling progenitors. In holoclone KCs, stable short hairpin-RNA‒mediated decreased expression of MXD4/MAD4 increases MYC expression, whose modulation increases the proliferation of KC precursors and maintenance of their clonogenic potential and preserves the functionality of these precursors in three-dimensional epidermis organoid generation. Altogether, these results characterize MXD4/MAD4 as a major piece of the stemness puzzle in the human epidermis KC lineage and pinpoint an original avenue for ex vivo expansion of human KC precursors.

Heterozygote Dopamine Transporter Knockout Rats Display Enhanced Cocaine Locomotion in Adolescent Females.

Cocaine is a powerful psychostimulant that is one of the most widely used illicit addictive. The dopamine transporter (DAT) plays a major role in mediating cocaine's reward effect. Decreases in DAT expression increase rates of drug abuse and vulnerability to comorbid psychiatric disorders. We used the novel DAT transgenic rat model to study the effects of cocaine on locomotor behaviors in adolescent rats, with an emphasis on sex. Female rats showed higher response rates to cocaine at lower acute and chronic doses, highlighting a higher vulnerability and perceived gender effects. In contrast, locomotor responses to an acute high dose of cocaine were more marked and sustained in male DAT heterozygous (HET) adolescents. The results demonstrate the augmented effects of chronic cocaine in HET DAT adolescent female rats. Knockout (KO) DAT led to a level of hyperdopaminergia which caused a marked basal hyperactivity that was unchanged, consistent with a possible ceiling effect. We suggest a role of alpha synuclein (α-syn) and PICK 1 protein expressions to the increased vulnerability in female rats. These proteins showed a lower expression in female HET and KO rats. This study highlights gender differences associated with mutations which affect DAT expression and can increase susceptibility to cocaine abuse in adolescence.

Immunosuppressive Properties of Epidermal Keratinocytes Differ According to Their Immaturity Status.

Preservation of a functional keratinocyte stem cell pool is essential to ensure the long-term maintenance of epidermis integrity, through continuous physiological renewal and regeneration in case of injury. Protecting stem cells from inflammation and immune reactions is thus a critical issue that needs to be explored. Here, we show that the immature CD49fhigh precursor cell fraction from interfollicular epidermis keratinocytes, comprising stem cells and progenitors, is able to inhibit CD4 + T-cell proliferation. Of note, both the stem cell-enriched CD49fhigh/EGFRlow subpopulation and the less immature CD49fhigh/EGFRhigh progenitors ensure this effect. Moreover, we show that HLA-G and PD-L1 immune checkpoints are overexpressed in CD49fhigh precursors, as compared to CD49flow differentiated keratinocytes. This potency may limit immune reactions against immature precursors including stem cells, and protect them from exacerbated inflammation. Further exploring this correlation between immuno-modulation and immaturity may open perspectives in allogenic cell therapies.

Skin Immunity and Tolerance: Focus on Epidermal Keratinocytes Expressing HLA-G.

Although the role of epidermal cells in skin regeneration has been extensively documented, their functions in immunity and tolerance mechanisms are largely underestimated. The aim of the present review was to outline the state of knowledge on resident immune cells of hematopoietic origin hosted in the epidermis, and then to focus on the involvement of keratinocytes in the complex skin immune networks acting in homeostasis and regeneration conditions. Based on this knowledge, the mechanisms of immune tolerance are reviewed. In particular, strategies based on immunosuppression mediated by HLA-G are highlighted, as recent advances in this field open up perspectives in epidermis-substitute bioengineering for temporary and permanent skin replacement strategies.

Human Keratinocytes Inhibit CD4+ T-Cell Proliferation through TGFB1 Secretion and Surface Expression of HLA-G1 and PD-L1 Immune Checkpoints.

Human skin protects the body against infection and injury. This protection involves immune and epithelial cells, but their interactions remain largely unknown. Here, we show that cultured epidermal keratinocytes inhibit allogenic CD4+ T-cell proliferation under both normal and inflammatory conditions. Inhibition occurs through the secretion of soluble factors, including TGFB1 and the cell-surface expression of HLA-G1 and PD-L1 immune checkpoints. For the first time, we here describe the expression of the HLA-G1 protein in healthy human skin and its role in keratinocyte-driven tissue immunomodulation. The overexpression of HLA-G1 with an inducible vector increased the immunosuppressive properties of keratinocytes, opening up perspectives for their use in allogeneic settings for cell therapy.

The ADIPS Pilot National Diabetes in Pregnancy Benchmarking Programme.

BACKGROUND: To test the feasibility of benchmarking the care of women with pregnancies complicated by hyperglycaemia. METHODS: A retrospective audit of volunteer diabetes services in Australia and New Zealand involving singleton pregnancies resulting in live births between 2014 and 2020. Ranges are shown and compared across services. RESULTS: The audit included 10,144 pregnancies (gestational diabetes mellitus (GDM) = 8696; type 1 diabetes (T1D) = 435; type 2 diabetes (T2D) = 1013) from 11 diabetes services. Among women with GDM, diet alone was used in 39.4% (ranging among centres from 28.8-57.3%), metformin alone in 18.8% (0.4-43.7%), and metformin and insulin in 10.1% (1.5-23.4%); when compared between sites, all p < 0.001. Birth was by elective caesarean in 12.1% (3.6-23.7%) or emergency caesarean in 9.5% (3.5-21.2%) (all p < 0.001). Preterm births (<37 weeks) ranged from 3.7% to 9.4% (p < 0.05), large for gestational age 10.3-26.7% (p < 0.001), admission to special care nursery 16.7-25.0% (p < 0.001), and neonatal hypoglycaemia (<2.6 mmol/L) 6.0-27.0% (p < 0.001). Many women with T1D and T2D had limited pregnancy planning including first trimester hyperglycaemia (HbA1c > 6.5% (48 mmol/mol)), 78.4% and 54.6%, respectively (p < 0.001). CONCLUSION: Management of maternal hyperglycaemia and pregnancy outcomes varied significantly. The maintenance and extension of this benchmarking service provides opportunities to identify policy and clinical approaches to improve pregnancy outcomes among women with hyperglycaemia in pregnancy.

A standard system phantom for magnetic resonance imaging.

PURPOSE: A standard MRI system phantom has been designed and fabricated to assess scanner performance, stability, comparability and assess the accuracy of quantitative relaxation time imaging. The phantom is unique in having traceability to the International System of Units, a high level of precision, and monitoring by a national metrology institute. Here, we describe the phantom design, construction, imaging protocols, and measurement of geometric distortion, resolution, slice profile, signal-to-noise ratio (SNR), proton-spin relaxation times, image uniformity and proton density. METHODS: The system phantom, designed by the International Society of Magnetic Resonance in Medicine ad hoc committee on Standards for Quantitative MR, is a 200 mm spherical structure that contains a 57-element fiducial array; two relaxation time arrays; a proton density/SNR array; resolution and slice-profile insets. Standard imaging protocols are presented, which provide rapid assessment of geometric distortion, image uniformity, T1 and T2 mapping, image resolution, slice profile, and SNR. RESULTS: Fiducial array analysis gives assessment of intrinsic geometric distortions, which can vary considerably between scanners and correction techniques. This analysis also measures scanner/coil image uniformity, spatial calibration accuracy, and local volume distortion. An advanced resolution analysis gives both scanner and protocol contributions. SNR analysis gives both temporal and spatial contributions. CONCLUSIONS: A standard system phantom is useful for characterization of scanner performance, monitoring a scanner over time, and to compare different scanners. This type of calibration structure is useful for quality assurance, benchmarking quantitative MRI protocols, and to transition MRI from a qualitative imaging technique to a precise metrology with documented accuracy and uncertainty.

Gestational diabetes - Major problems with post-partum testing.

BACKGROUND: Post-partum follow up testing of women with gestational diabetes mellitus (GDM) is important. All women, and their family doctors, receive written reminders. There are no recent major Australian reviews of the efficacy and compliance with this advice conducted in an ethnically representative population and using the current diagnostic criteria. AIM: The aim was to examine a cohort of women with recently diagnosed GDM and a completed pregnancy to determine what proportion had been tested and what were the difficulties in having testing carried out. METHODS: Women who were diagnosed with gestational diabetes and attended the Diabetes Service in 2017 were followed up in 2019. Attempted contact was made using an unidentified land line, an identifiable mobile phone and a postal survey. Compliance with testing advice was the major parameter considered. RESULTS: There were 714 women with GDM, 75 were excluded: 64 after pass one and 11 after pass two. In total, only 339/639 (53.1%) could be contacted. Of these women, 334 agreed to be surveyed; 207 (62.0%) had a post-partum test. Of the 127 women who had not had a test, 113 agreed to have an HbA1c. Only 13/113 (11.5%) had this done within a month. CONCLUSION: Contacting women, even within a short time after the pregnancy, is difficult. The number of post-partum tests carried out is suboptimal. Written advice to all women and their doctors does not appear to be working. A review of the cost effectiveness of this approach and development of new methods may be worthwhile.

When the Search for Stemness Genes Meets the Skin Substitute Bioengineering Field: KLF4 Transcription Factor under the Light.

The transcription factor "Kruppel-like factor 4" (KLF4) is a central player in the field of pluripotent stem cell biology. In particular, it was put under the spotlight as one of the four factors of the cocktail originally described for reprogramming into induced pluripotent stem cells (iPSCs). In contrast, its possible functions in native tissue stem cells remain largely unexplored. We recently published that KLF4 is a regulator of "stemness" in human keratinocytes. We show that reducing the level of expression of this transcription factor by RNA interference or pharmacological repression promotes the ex vivo amplification and regenerative capacity of two types of cells of interest for cutaneous cell therapy: native keratinocyte stem and progenitor cells from adult epidermis, which have been used for more than three decades in skin graft bioengineering, and keratinocytes generated by the lineage-oriented differentiation of embryonic stem cells (ESCs), which have potential for the development of skin bio-bandages. At the mechanistic level, KLF4 repression alters the expression of a large set of genes involved in TGF-ß1 and WNT signaling pathways. Major regulators of TGF-ß bioavailability and different TGF-ß receptors were targeted, notably modulating the ALK1/Smad1/5/9 axis. At a functional level, KLF4 repression produced an antagonist effect on TGFß1-induced keratinocyte differentiation.

Exposure of Human Skin Organoids to Low Genotoxic Stress Can Promote Epithelial-to-Mesenchymal Transition in Regenerating Keratinocyte Precursor Cells.

For the general population, medical diagnosis is a major cause of exposure to low genotoxic stress, as various imaging techniques deliver low doses of ionizing radiation. Our study investigated the consequences of low genotoxic stress on a keratinocyte precursor fraction that includes stem and progenitor cells, which are at risk for carcinoma development. Human skin organoids were bioengineered according to a clinically-relevant model, exposed to a single 50 mGy dose of γ rays, and then xeno-transplanted in nude mice to follow full epidermis generation in an in vivo context. Twenty days post-xenografting, mature skin grafts were sampled and analyzed by semi-quantitative immuno-histochemical methods. Pre-transplantation exposure to 50 mGy of immature human skin organoids did not compromise engraftment, but half of xenografts generated from irradiated precursors exhibited areas displaying focal dysplasia, originating from the basal layer of the epidermis. Characteristics of epithelial-to-mesenchymal transition (EMT) were documented in these dysplastic areas, including loss of basal cell polarity and cohesiveness, epithelial marker decreases, ectopic expression of the mesenchymal marker α-SMA and expression of the EMT promoter ZEB1. Taken together, these data show that a very low level of radiative stress in regenerating keratinocyte stem and precursor cells can induce a micro-environment that may constitute a favorable context for long-term carcinogenesis.

Impairment of Base Excision Repair in Dermal Fibroblasts Isolated From Nevoid Basal Cell Carcinoma Patients.

The nevoid basal cell carcinoma syndrome (NBCCS), also called Gorlin syndrome is an autosomal dominant disorder whose incidence is estimated at about 1 per 55,600-256,000 individuals. It is characterized by several developmental abnormalities and an increased predisposition to the development of basal cell carcinomas (BCCs). Cutaneous fibroblasts from Gorlin patients have been shown to exhibit an increased sensitivity to ionizing radiations. Mutations in the tumor suppressor gene PTCH1, which is part of the Sonic Hedgehog (SHH) signaling pathway, are responsible for these clinical manifestations. As several genetic mutations in the DNA repair genes are responsible of photo or radiosensitivity and high predisposition to cancers, we hypothesized that these effects in Gorlin syndrome might be due to a defect in the DNA damage response (DDR) and/or the DNA repair capacities. Therefore, the objective of this work was to investigate the sensitivity of skin fibroblasts from NBCCS patients to different DNA damaging agents and to determine the ability of these agents to modulate the DNA repair capacities. Gorlin fibroblasts showed high radiosensitivity and also less resistance to oxidative stress-inducing agents when compared to control fibroblasts obtained from healthy individuals. Gorlin fibroblasts harboring PTCH1 mutations were more sensitive to the exposure to ionizing radiation and to UVA. However, no difference in cell viability was shown after exposure to UVB or bleomycin. As BER is responsible for the repair of oxidative DNA damage, we decided to assess the BER pathway efficacy in Gorlin fibroblasts. Interestingly, a concomitant decrease of both BER gene expression and BER protein activity was observed in Gorlin fibroblasts when compared to control. Our results suggest that low levels of DNA repair within Gorlin cells may lead to an accumulation of oxidative DNA damage that could participate and partly explain the radiosensitivity and the BCC-prone phenotype in Gorlin syndrome.

NFATC2 Modulates Radiation Sensitivity in Dermal Fibroblasts From Patients With Severe Side Effects of Radiotherapy.

Although it is well established that 5 to 15% of radiotherapy patients exhibit severe side-effects in non-cancerous tissues, the molecular mechanisms involved are still poorly known, and the links between cellular and tissue radiosensitivity are still debated. We here studied fibroblasts from non-irradiated skin of patients with severe sequelae of radiotherapy, to determine whether specific basal cell activities might be involved in susceptibility to side-effects in normal tissues. Compared to control cells, patient fibroblasts exhibited higher radiosensitivity together with defects in DNA repair. Transcriptome profiling of dermal fibroblasts from 16 radiotherapy patients with severe side-effects and 8 healthy individuals identified 540 genes specifically deregulated in the patients. Nuclear factor of activated T cells 2 (NFATC2) was the most differentially expressed gene, poorly expressed at both transcript and protein level, whereas the NFATC2 gene region was hypermethylated. Furthermore, NFATC2 expression correlated with cell survival after irradiation. Finally, silencing NFATC2 in normal cells by RNA interference led to increased cellular radiosensitivity and defects in DNA repair. This study demonstrates that patients with clinical hypersensitivity also exhibit intrinsic cellular radiosensitivity in their normal skin cells. It further reveals a new role for NFATC2 as a potential regulator of cellular sensitivity to ionizing radiation.

Magnetic resonance fingerprinting Part 1: Potential uses, current challenges, and recommendations.

Magnetic resonance fingerprinting (MRF) is a powerful quantitative MRI technique capable of acquiring multiple property maps simultaneously in a short timeframe. The MRF framework has been adapted to a wide variety of clinical applications, but faces challenges in technical development, and to date has only demonstrated repeatability and reproducibility in small studies. In this review, we discuss the current implementations of MRF and their use in a clinical setting. Based on this analysis, we highlight areas of need that must be addressed before MRF can be fully adopted into the clinic and make recommendations to the MRF community on standardization and validation strategies of MRF techniques. Level of Evidence: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2020;51:675-692.

Iterative Three-Dimensional Epidermis Bioengineering and Xenografting to Assess Long-Term Regenerative Potential in Human Keratinocyte Precursor Cells.

The functional definition of somatic adult stem cells is based on their regenerative capacity, which allows tissue regeneration throughout life. Thus, refining methodologies to characterize this capacity is of great importance for progress in the fundamental knowledge of specific keratinocyte subpopulations but also for preclinical and clinical research, considering the high potential of keratinocytes in cell therapy. We present here a methodology which we define as iterative xenografting, which originates in the classical model of human skin substitute xenografts onto immunodeficient recipient mice. The principle of this functional assay is first to perform primary xenografts to assess graft take and the quality of epidermal differentiation. Then, human keratinocytes are extracted from primary graft samples to perform secondary xenografts, to assess the presence and preservation of functional keratinocyte stem cells with long-term regenerative potential. In the example of experiments shown, iterative skin xenografting was used to document the high regenerative potential of epidermal holoclone keratinocytes.

KLF4 inhibition promotes the expansion of keratinocyte precursors from adult human skin and of embryonic-stem-cell-derived keratinocytes.

Expanded autologous skin keratinocytes are currently used in cutaneous cell therapy, and embryonic-stem-cell-derived keratinocytes could become a complementary alternative. Regardless of keratinocyte provenance, for efficient therapy it is necessary to preserve immature keratinocyte precursors during cell expansion and graft processing. Here, we show that stable and transient downregulation of the transcription factor Krüppel-like factor 4 (KLF4) in keratinocyte precursors from adult skin, using anti-KLF4 RNA interference or kenpaullone, promotes keratinocyte immaturity and keratinocyte self-renewal in vitro, and enhances the capacity for epidermal regeneration in mice. Both stable and transient KLF4 downregulation had no impact on the genomic integrity of adult keratinocytes. Moreover, transient KLF4 downregulation in human-embryonic-stem-cell-derived keratinocytes increased the efficiency of skin-orientated differentiation and of keratinocyte immaturity, and was associated with improved generation of epidermis. As a regulator of the cell fate of keratinocyte precursors, KLF4 could be used for promoting the ex vivo expansion and maintenance of functional immature keratinocyte precursors.

Noninvasive, Nondestructive Measurement of Tomato Concentrate Spoilage in Large-Volume Aseptic Packages.

Low frequency nuclear magnetic resonance (NMR) is used to noninvasively and nondestructively detect spoiled tomato concentrate stored in >200 L metal-lined containers. It is shown that longitudinal and transverse NMR relaxation times change as the tomato concentrate spoils. A rapid, viscosity-dependent spoilage detection method that takes advantage of the inherent inhomogeneity in single-sided NMR instruments is proposed. Here, the effective transverse magnetization decay rate is used as a parameter to determine tomato concentrate spoilage. Three different low frequency, single-sided NMR instruments are described and compared to determine the optimum sensor for spoiled tomato concentrate detection in large-format, metal-lined, aseptic containers. The most effective NMR sensor for this application is temperature stable and has large magnetic field gradients and a homogeneous magnetic field region offset >0.5 cm from the magnet surface. PRACTICAL APPLICATION: This manuscript describes a noninvasive and nondestructive tomato concentrate spoilage detector for application to large-format, sealed, commercial storage bins.