Guinea pig models for translation of the developmental origins of health and disease hypothesis into the clinic.

Over 30 years ago Professor David Barker first proposed the theory that events in early life could explain an individual's risk of non-communicable disease in later life: the developmental origins of health and disease (DOHaD) hypothesis. During the 1990s the validity of the DOHaD hypothesis was extensively tested in a number of human populations and the mechanisms underpinning it characterised in a range of experimental animal models. Over the past decade, researchers have sought to use this mechanistic understanding of DOHaD to develop therapeutic interventions during pregnancy and early life to improve adult health. A variety of animal models have been used to develop and evaluate interventions, each with strengths and limitations. It is becoming apparent that effective translational research requires that the animal paradigm selected mirrors the tempo of human fetal growth and development as closely as possible so that the effect of a perinatal insult and/or therapeutic intervention can be fully assessed. The guinea pig is one such animal model that over the past two decades has demonstrated itself to be a very useful platform for these important reproductive studies. This review highlights similarities in the in utero development between humans and guinea pigs, the strengths and limitations of the guinea pig as an experimental model of DOHaD and the guinea pig's potential to enhance clinical therapeutic innovation to improve human health.

Probing function and structure of trehalose-6-phosphate phosphatases from pathogenic organisms suggests distinct molecular groupings.

The trehalose biosynthetic pathway is of great interest for the development of novel therapeutics because trehalose is an essential disaccharide in many pathogens but is neither required nor synthesized in mammalian hosts. As such, trehalose-6-phosphate phosphatase (TPP), a key enzyme in trehalose biosynthesis, is likely an attractive target for novel chemotherapeutics. Based on a survey of genomes from a panel of parasitic nematodes and bacterial organisms and by way of a structure-based amino acid sequence alignment, we derive the topological structure of monoenzyme TPPs and classify them into 3 groups. Comparison of the functional roles of amino acid residues located in the active site for TPPs belonging to different groups reveal nuanced variations. Because current literature on this enzyme family shows a tendency to infer functional roles for individual amino acid residues, we investigated the roles of the strictly conserved aspartate tetrad in TPPs of the nematode Brugia malayi by using a conservative mutation approach. In contrast to aspartate-213, the residue inferred to carry out the nucleophilic attack on the substrate, we found that aspartate-215 and aspartate-428 of BmTPP are involved in the chemistry steps of enzymatic hydrolysis of the substrate. Therefore, we suggest that homology-based inference of functionally important amino acids by sequence comparison for monoenzyme TPPs should only be carried out for each of the 3 groups.-Cross, M., Lepage, R., Rajan, S., Biberacher, S., Young, N. D., Kim, B.-N., Coster, M. J., Gasser, R. B., Kim, J.-S., Hofmann, A. Probing function and structure of trehalose-6-phosphate phosphatases from pathogenic organisms suggests distinct molecular groupings.

Free-standing nitrogen-doped reduced graphene oxide anode for lithium-ion batteries.

Graphenes have been considered suitable candidate materials for electrodes of energy storage devices such as lithium-ion batteries (LIBs) because of their outstanding mechanical, thermal and electrical properties. However, there are problems when using these carbon materials for electrodes because of their low electrochemical performance. In this work, to improve the electrochemical performances of graphenes, free-standing nitrogen-doped reduced graphene oxides (FNRGOs) were prepared as an anode for LIBs using a facile vacuum filtration method and thermal annealing at different temperatures. X-ray diffraction and X-ray photoelectron spectroscopy were employed to characterize the prepared samples, and then their electrochemical performance was investigated by galvanostatic charge/discharge (GCD) tests. GCD tests revealed that FNRGO prepared from thermal annealing at 500 degrees C exhibited good initial reversible capacity (502 mA h/g at 50 mA/g (0.14 C)) and enhanced cycle stability (capacity retention of 90.5% after 50th cycles at 100 mA/g (0.27 C), which demonstrated that FNRGOs were suitable candidates as anodes for LIBs.

Silver nanowire catalysts on carbon nanotubes-incorporated bacterial cellulose membrane electrodes for oxygen reduction reaction.

Silver nanowires have unique electrical, thermal and optical properties, which support their potential application in numerous fields including catalysis, electronics, optoelectronics, sensing, and surface-enhanced spectroscopy. Especially, their application such as catalysts for alkaline fuel cells (AFCs) have attracted much interest because of their superior electrical conductivity over that of any metal and their lower cost compared to Pt. In this study, multiwalled carbon nanotubes (MWCNTs)-incorporated bacterial cellulose (BC) membrane electrode with silver nanowire catalyst was prepared. First, acid-treated MWCNTs were incorporated into BC membranes and then freeze-dried after solvent exchange to tert-butanol in order to maintain the 3D-network macroporous structure. Second, silver nanowires synthesized by polyol process were introduced onto the surface of the MWCNTs-incorporated BC membrane through easy vacuum filtration. Finally, thermal treatment was carried out to confirm the effect of the PVP on the silver nanowire catalysts toward oxygen reduction reaction. The electrode with thermally treated silver nanowire had great electrocatalytic activity compared with non-treated one. These results suggest that the MWCNTs-incorporated BC electrode with silver nanowire catalysts after thermal treatment could be potentially used in cathodes of AFCs.

Amorphous carbon nanotube/MnO2/graphene oxide ternary composite electrodes for electrochemical capacitors.

Ternary composites of amorphous carbon nanotube/MnO2/graphene oxide (a-CNT/MnO2/GO) were synthesized by a facile direct redox reaction between potassium permanganate and a-CNT, which was prepared by anodic aluminum oxide template method following co-filtration with GO. Needle-like, 100-nm-thick, MnO2 crystals were homogeneously coated on the a-CNT surface, which was then covered with GO. The electrochemical performance of the resulting MnO2-coated a-CNTs exhibited a specific capacitance of 473 F/g at a scan rate of 5 mV/s, and excellent charge/discharge stability after 500 cycles.

Deep learning-based imaging reconstruction for MRI after neoadjuvant chemoradiotherapy for rectal cancer: effects on image quality and assessment of treatment response.

PURPOSE: To investigate the effects of deep learning-based imaging reconstruction (DLR) on the image quality of MRI of rectal cancer after chemoradiotherapy (CRT), and its accuracy in diagnosing pathological complete responses (pCR). METHODS: We included 39 patients (men: women, 21:18; mean age ± standard deviation, 59.1 ± 9.7 years) with mid-to-lower rectal cancer who underwent a long-course of CRT and high-resolution rectal MRIs between January 2020 and April 2021. Axial T2WI was reconstructed using the conventional method (MRIconv) and DLR with two different noise reduction factors (MRIDLR30 and MRIDLR50). The signal-to-noise ratio (SNR) of the tumor was measured. Two experienced radiologists independently made a blind assessment of the complete response on MRI. The sensitivity and specificity for pCR were analyzed using a multivariable logistic regression analysis with generalized estimating equations. RESULTS: Thirty-four patients did not have a pCR whereas five (12.8%) had pCR. Compared with the SNR of MRIconv (mean ± SD, 7.94 ± 1.92), MRIDLR30 and MRIDLR50 showed higher SNR (9.44 ± 2.31 and 11.83 ± 3.07, respectively) (p < 0.001). Compared to MRIconv, MRIDLR30 and MRIDLR50 showed significantly higher specificity values (p < 0.036) while the sensitivity values were not significantly different (p > 0.301). The sensitivity and specificity for pCR were 48.9% and 80.8% for MRIconv; 48.9% and 88.2% for MRIDLR30; and 38.8% and 86.7% for MRIDLR50, respectively. CONCLUSION: DLR produced MR images with higher resolution and SNR. The specificity of MRI for identification of pCR was significantly higher with DLR than with conventional MRI.

DNA methylation signatures in human neonatal blood following maternal antenatal corticosteroid treatment.

Antenatal corticosteroids (ACS) are used to treat women at risk of preterm birth to improve neonatal survival. Though affected children may be at long-term risk of neurobehavioural disorders, the driving mechanisms remain unknown. Animal studies have shown that ACS exposure can lead to overlapping changes in DNA methylation between the blood and the brain, identifying gene pathways for neurodevelopment, which highlights the potential to examine peripheral blood as a surrogate for inaccessible human brain tissue. We hypothesized that differential methylation will be identified in blood of term-born neonates following ACS. Mother-infant dyads that received ACS were retrospectively identified through the Ontario Birth Study at Sinai Health Complex and matched to untreated controls for maternal age, BMI, parity and foetal sex (n = 14/group). Genome-wide methylation differences were examined at single-nucleotide resolution in DNA extracted from dried bloodspot cards using reduced representative bisulfite sequencing approaches. 505 differentially methylated CpG sites (DMCs) were identified, wherein 231 were hypermethylated and 274 were hypomethylated. These sites were annotated to 219 genes, of which USP48, SH3PXD2A, NTM, CAMK2N2, MAP6D1 were five of the top ten genes with known neurological function. Collectively, the set of hypermethylated genes were enriched for pathways of transcription regulation, while pathways of proteasome activity were enriched among the set of hypomethylated genes. This study is the first to identify DNA methylation changes in human neonatal blood following ACS. Understanding the epigenetic changes that occur in response to ACS will support future investigations to delineate the effects of prenatal glucocorticoid exposure on human development.

Clinical Impact of a Quality Improvement Program Including Dedicated Emergency Radiology Personnel on Emergency Surgical Management: A Propensity Score-Matching Study.

OBJECTIVE: To investigate the clinical impact of a quality improvement program including dedicated emergency radiology personnel (QIP-DERP) on the management of emergency surgical patients in the emergency department (ED). MATERIALS AND METHODS: This retrospective study identified all adult patients (n = 3667) who underwent preoperative body CT, for which written radiology reports were generated, and who subsequently underwent non-elective surgery between 2007 and 2018 in the ED of a single urban academic tertiary medical institution. The study cohort was divided into periods before and after the initiation of QIP-DERP. We matched the control group patients (i.e., before QIP-DERP) to the QIP-DERP group patients using propensity score (PS), with a 1:2 matching ratio for the main analysis and a 1:1 ratio for sub-analyses separately for daytime (8:00 AM to 5:00 PM on weekdays) and after-hours. The primary outcome was timing of emergency surgery (TES), which was defined as the time from ED arrival to surgical intervention. The secondary outcomes included ED length of stay (LOS) and intensive care unit (ICU) admission rate. RESULTS: According to the PS-matched analysis, compared with the control group, QIP-DERP significantly decreased the median TES from 16.7 hours (interquartile range, 9.4-27.5 hours) to 11.6 hours (6.6-21.9 hours) (p < 0.001) and the ICU admission rate from 33.3% (205/616) to 23.9% (295/1232) (p < 0.001). During after-hours, the QIP-DERP significantly reduced median TES from 19.9 hours (12.5-30.1 hours) to 9.6 hours (5.7-19.1 hours) (p < 0.001), median ED LOS from 9.1 hours (5.6-16.5 hours) to 6.7 hours (4.9-11.3 hours) (p < 0.001), and ICU admission rate from 35.5% (108/304) to 22.0% (67/304) (p < 0.001). CONCLUSION: QIP-DERP implementation improved the quality of emergency surgical management in the ED by reducing TES, ED LOS, and ICU admission rate, particularly during after-hours.

Multimodal Assessment of Schizophrenia and Depression Utilizing Video, Acoustic, Locomotor, Electroencephalographic, and Heart Rate Technology: Protocol for an Observational Study.

BACKGROUND: Current standards of psychiatric assessment and diagnostic evaluation rely primarily on the clinical subjective interpretation of a patient's outward manifestations of their internal state. While psychometric tools can help to evaluate these behaviors more systematically, the tools still rely on the clinician's interpretation of what are frequently nuanced speech and behavior patterns. With advances in computing power, increased availability of clinical data, and improving resolution of recording and sensor hardware (including acoustic, video, accelerometer, infrared, and other modalities), researchers have begun to demonstrate the feasibility of cutting-edge technologies in aiding the assessment of psychiatric disorders. OBJECTIVE: We present a research protocol that utilizes facial expression, eye gaze, voice and speech, locomotor, heart rate, and electroencephalography monitoring to assess schizophrenia symptoms and to distinguish patients with schizophrenia from those with other psychiatric disorders and control subjects. METHODS: We plan to recruit three outpatient groups: (1) 50 patients with schizophrenia, (2) 50 patients with unipolar major depressive disorder, and (3) 50 individuals with no psychiatric history. Using an internally developed semistructured interview, psychometrically validated clinical outcome measures, and a multimodal sensing system utilizing video, acoustic, actigraphic, heart rate, and electroencephalographic sensors, we aim to evaluate the system's capacity in classifying subjects (schizophrenia, depression, or control), to evaluate the system's sensitivity to within-group symptom severity, and to determine if such a system can further classify variations in disorder subtypes. RESULTS: Data collection began in July 2020 and is expected to continue through December 2022. CONCLUSIONS: If successful, this study will help advance current progress in developing state-of-the-art technology to aid clinical psychiatric assessment and treatment. If our findings suggest that these technologies are capable of resolving diagnoses and symptoms to the level of current psychometric testing and clinician judgment, we would be among the first to develop a system that can eventually be used by clinicians to more objectively diagnose and assess schizophrenia and depression with the possibility of less risk of bias. Such a tool has the potential to improve accessibility to care; to aid clinicians in objectively evaluating diagnoses, severity of symptoms, and treatment efficacy through time; and to reduce treatment-related morbidity. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/36417.

Exoscopic En Bloc Carotid-Sparing Total Temporal Bone Resection: Feasibility Study and Operative Technique.

OBJECTIVE: To delineate the steps of exoscopic en bloc carotid artery-sparing total temporal bone resection for malignancies involving the temporal bone in a cadaveric model. METHODS: Dissections were performed on 3 right-sided (3 sides) formalin-fixed, latex-injected cadaveric specimens. An exoscopic en bloc carotid artery-sparing total temporal bone resection was performed on each cadaver. In the past 4 years, 8 patients have undergone exoscope-assisted internal carotid artery-sparing total temporal bone resection with the technique described in this report. As an example, we present a representative case of a patient in whom this technique was used. RESULTS: Exoscope-assisted en bloc total temporal bone resections were performed on 3 right-sided cadaveric specimens. The following steps were described to circumferentially expose the petrous temporal bone: infratemporal fossa exposure, temporal craniotomy for subtemporal middle fossa approach to the petrous bone, retrosigmoid craniotomy, and transjugular approach. Finally, 3 skull base osteotomies were performed to liberate anterior, medial, posterior attachments of the petrous bone for en bloc removal. Possible extensions of these dissections as indicated by tumor pathology were described. A case illustration and operative video utilizing these techniques is presented. CONCLUSIONS: Exoscope-assisted en bloc carotid artery-sparing total temporal bone resection is a feasible technique for management of malignancies with temporal bone invasion.

Examination of the reliability and feasibility of two smartphone applications to assess executive functioning in racially diverse older adults.

Inclusion of Black participants in clinical research is a national priority. Mobile applications and remote data collection may increase study access for diverse populations. This study examined the reliability and feasibility of two mobile smartphone application-based cognitive measures in a diverse middle aged and older adult sample. Black (n = 44; Mage = 59.93) and non-Hispanic white (NHW; n = 50; Mage = 61.06) participants completed traditional paper-based neuropsychological testing and two app-based measures, Arrows and Number Match. Intraclass correlations demonstrated poor to moderate reliability (range: .417-.569) between performance on the app-based versions and performance on the traditional versions. Performance score differences by racial group were not statistically significant. Both Black and NHW participants rated the app-based measures as feasible and acceptable, though Black participants endorsed a stronger likelihood of future use. These findings add to the growing literature on remote cognitive testing .

Impact of ex vivo Sample Handling on DNA Methylation Profiles in Human Cord Blood and Neonatal Dried Blood Spots.

The profiling of DNA methylation modifications in peripheral blood has significant potential to determine risk factors for human disease. Little is known concerning the sensitivity of DNA methylation profiles to ex vivo sample handling. Here, we studied typical conditions prior to sample storage associated with cord blood samples obtained from clinical investigations using reduced representation bisulfite sequencing. We examined both whole blood collected shortly after birth and dried blood spots, a potentially important source of neonatal blood for investigation of the DNA methylome and the Developmental Origins of Health and Disease in human cohorts because they are routinely collected during clinical care. Samples were matched across different time conditions, as they were from the same cord blood samples obtained from the same individuals. Maintaining whole blood ex vivo up to 24 h (4°C) or dried blood spots up to 7 days (room temp.) had little effect on DNA methylation profiles. Minimal differences were detected between cord blood immediately frozen and dried blood spots. Our results indicate that DNA methylation profiles are resilient to ex vivo sample handling conditions prior to storage. These data will help guide future human studies focused toward determination of DNA methylation modifications in whole blood.

Probiotic Lactobacillus rhamnosus GR-1 is a unique prophylactic agent that suppresses infection-induced myometrial cell responses.

Preterm birth (PTB) is a multifactorial syndrome affecting millions of neonates worldwide. Intrauterine infection can induce PTB through the secretion of pro-inflammatory cytokines and untimely activation of uterine contractions. In pregnant mice, prophylactic administration of probiotic Lactobacillus rhamnosus GR-1 supernatant (GR1SN) prevented lipopolysaccharide (LPS)-induced PTB and reduced cytokine expression in the uterine muscle (myometrium). In this study we sought to delineate the mechanisms by which GR1SN suppressed cytokine secretion in the myometrium. We observed that L. rhamnosus GR-1 uniquely secretes heat-resistant but trypsin-sensitive factors, which significantly suppressed LPS-induced secretion of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 in the human myometrial cell line, hTERT-HM. This effect was unique to GR1SN and could not be replicated using supernatant derived from non-GR-1 commensal lactobacilli species: L. rhamnosus GG, L. lactis, L. casei, or L. reuteri RC-14. Furthermore, pre-incubation of hTERT-HM cells with low-dose Pam3CSK (a TLR1/2 synthetic agonist which mimics LPS action) prior to LPS administration also significantly decreased LPS-induced cytokine secretion. This study highlights the distinct capacity of protein-like moieties secreted by L. rhamnosus GR-1 to inhibit pro-inflammatory cytokine production by human myometrial cells, potentially through a TLR1/2-mediated mechanism.

Induction of neural stem cell-like cells (NSCLCs) from mouse astrocytes by Bmi1.

Recently, Bmi1 was shown to control the proliferation and self-renewal of neural stem cells (NSCs). In this study, we demonstrated the induction of NSC-like cells (NSCLCs) from mouse astrocytes by Bmi1 under NSC culture conditions. These NSCLCs exhibited the morphology and growth properties of NSCs, and expressed NSC marker genes, including nestin, CD133, and Sox2. In vitro differentiation of NSCLCs resulted in differentiated cell populations containing astrocytes, neurons, and oligodendrocytes. Following treatment with histone deacetylase inhibitors (trichostatin A and valproic acid), the potential of NSCLCs for proliferation, dedifferentiation, and self-renewal was significantly inhibited. Our data indicate that multipotent NSCLCs can be generated directly from astrocytes by the addition of Bmi1.

Induction of serum amyloid A genes is associated with growth and apoptosis of HC11 mammary epithelial cells.

In this study, we examined the expression and functions of serum amyloid A (SAA) isoforms during apoptosis of HC11 mammary gland epithelial cells. Expression of SAA mRNAs and apoptosis were increased in HC11 cells by serum withdrawal and gradually decreased upon the addition of serum, or epidermal growth factor (EGF). TNFalpha treatment of HC11 cells also induced expression of SAA genes, and the effect on SAA1 and SAA2 expression was suppressed by treatment with MG132, and in cells transfected with a dominant negative mutant form of IkappaBalpha. Similar results were observed in response to interleukin-1 (IL-1), IL-6 and interferon gamma (IFNgamma). Furthermore, overexpression of the SAA1 and SAA2 isoforms suppressed growth and accelerated apoptosis of HC11 cells by increasing caspase 3/7 and caspase 8 activities, but the apoptotic effect of tumor necrosis factor alpha (TNFalpha) on HC11 cells was not enhanced. We found that expression of SAA1 and SAA2, but not SAA3, was regulated by an NFkappaB-dependent pathway, and that overexpression of SAA isoforms accelerated the apoptosis of HC11 cells.

Crystal structure of an ASCH protein from Zymomonas mobilis and its ribonuclease activity specific for single-stranded RNA.

Activating signal cointegrator-1 homology (ASCH) domains were initially reported in human as a part of the ASC-1 transcriptional regulator, a component of a putative RNA-interacting protein complex; their presence has now been confirmed in a wide range of organisms. Here, we have determined the trigonal and monoclinic crystal structures of an ASCH domain-containing protein from Zymomonas mobilis (ZmASCH), and analyzed the structural determinants of its nucleic acid processing activity. The protein has a central ß-barrel structure with several nearby α-helices. Positively charged surface patches form a cleft that runs through the pocket formed between the ß-barrel and the surrounding α-helices. We further demonstrate by means of in vitro assays that ZmASCH binds nucleic acids, and degrades single-stranded RNAs in a magnesium ion-dependent manner with a cleavage preference for the phosphodiester bond between the pyrimidine and adenine nucleotides. ZmASCH also removes a nucleotide at the 5'-end. Mutagenesis studies, guided by molecular dynamics simulations, confirmed that three residues (Tyr47, Lys53, and Ser128) situated in the cleft contribute to nucleic acid-binding and RNA cleavage activities. These structural and biochemical studies imply that prokaryotic ASCH may function to control the cellular RNA amount.

Preclinical Analysis of Fetal Human Mesencephalic Neural Progenitor Cell Lines: Characterization and Safety In Vitro and In Vivo.

We have developed a good manufacturing practice for long-term cultivation of fetal human midbrain-derived neural progenitor cells. The generation of human dopaminergic neurons may serve as a tool of either restorative cell therapies or cellular models, particularly as a reference for phenotyping region-specific human neural stem cell lines such as human embryonic stem cells and human inducible pluripotent stem cells. We cultivated 3 different midbrain neural progenitor lines at 10, 12, and 14 weeks of gestation for more than a year and characterized them in great detail, as well as in comparison with Lund mesencephalic cells. The whole cultivation process of tissue preparation, cultivation, and cryopreservation was developed using strict serum-free conditions and standardized operating protocols under clean-room conditions. Long-term-cultivated midbrain-derived neural progenitor cells retained stemness, midbrain fate specificity, and floorplate markers. The potential to differentiate into authentic A9-specific dopaminergic neurons was markedly elevated after prolonged expansion, resulting in large quantities of functional dopaminergic neurons without genetic modification. In restorative cell therapeutic approaches, midbrain-derived neural progenitor cells reversed impaired motor function in rodents, survived well, and did not exhibit tumor formation in immunodeficient nude mice in the short or long term (8 and 30 weeks, respectively). We conclude that midbrain-derived neural progenitor cells are a promising source for human dopaminergic neurons and suitable for long-term expansion under good manufacturing practice, thus opening the avenue for restorative clinical applications or robust cellular models such as high-content or high-throughput screening. Stem Cells Translational Medicine 2017;6:576-588.

Nylon 610/graphene oxide composites prepared by in-situ interfacial polymerization.

Nylon 610/nylon 610-grafted graphene oxide (nylon 610/GO-g-nylon 610) composites were fabricated using acyl chloride-functionalized graphene oxide by in-situ interfacial polymerization. GO-g-nylon 610 was synthesized by the condensation reaction between the acyl chloride groups of GO and the amino groups at the nylon 610 chains during the in-situ polymerization. Nylon 610/GO composites without grafting nylon 610 onto GO were also prepared to investigate the influence of grafting nylon 610 on the interfacial adhesion between GO and the nylon 610 matrix. The thermal properties of the nylon 610/GO-g-nylon 610 composites were enhanced with increasing GO-g-nylon 610 content in the nylon 610 matrix. The degradation temperature and thermal conductivity of the nylon 610/GO-g-nylon 610-10 composite were increased to 72.2 °C and 36.9%, respectively, compared with those of pure nylon 610. The crystallinity of the nylon 610/GO-g-nylon 610-10 composite was significantly lower than that of pure nylon 610 due to the hindered mobility of the nylon 610 chains by the strong interfacial adhesion between the GO-g-nylon 610 and the nylon 610 matrix.

Considerations during crown reattachment procedure over the pulpal exposure: case report.

Crown reattachment is the most conservative treatment which can be used to restore fractured tooth, presumably with sufficient strength, while maintaining original contour, incisal translucency, and reducing chair time and cost.However, in case of crown fracture with pin-point pulp exposure, we should cautiously minimize the irritation to the pulp and consider pre-treatment pulpal status, choice of pulp capping materials, choice of bonding system and treatment sequence during crown reattachment procedures. This case reports the considerations while crown reattachment with direct pulp capping using calcium hydroxide (Dycal, Dentsply Caulk).

Differentiation of human labia minora dermis-derived fibroblasts into insulin-producing cells.

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin- producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis- derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal- endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic ß-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin- induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.