Person Recognition Based on Deep Gait: A Survey.

Gait recognition, also known as walking pattern recognition, has expressed deep interest in the computer vision and biometrics community due to its potential to identify individuals from a distance. It has attracted increasing attention due to its potential applications and non-invasive nature. Since 2014, deep learning approaches have shown promising results in gait recognition by automatically extracting features. However, recognizing gait accurately is challenging due to the covariate factors, complexity and variability of environments, and human body representations. This paper provides a comprehensive overview of the advancements made in this field along with the challenges and limitations associated with deep learning methods. For that, it initially examines the various gait datasets used in the literature review and analyzes the performance of state-of-the-art techniques. After that, a taxonomy of deep learning methods is presented to characterize and organize the research landscape in this field. Furthermore, the taxonomy highlights the basic limitations of deep learning methods in the context of gait recognition. The paper is concluded by focusing on the present challenges and suggesting several research directions to improve the performance of gait recognition in the future.

Classification of Indoor Human Fall Events Using Deep Learning.

Human fall identification can play a significant role in generating sensor based alarm systems, assisting physical therapists not only to reduce after fall effects but also to save human lives. Usually, elderly people suffer from various kinds of diseases and fall action is a very frequently occurring circumstance at this time for them. In this regard, this paper represents an architecture to classify fall events from others indoor natural activities of human beings. Video frame generator is applied to extract frame from video clips. Initially, a two dimensional convolutional neural network (2DCNN) model is proposed to extract features from video frames. Afterward, gated recurrent unit (GRU) network finds the temporal dependency of human movement. Binary cross-entropy loss function is calculated to update the attributes of the network like weights, learning rate to minimize the losses. Finally, sigmoid classifier is used for binary classification to detect human fall events. Experimental result shows that the proposed model obtains an accuracy of 99%, which outperforms other state-of-the-art models.

CricShotClassify: An Approach to Classifying Batting Shots from Cricket Videos Using a Convolutional Neural Network and Gated Recurrent Unit.

Recognizing the sport of cricket on the basis of different batting shots can be a significant part of context-based advertisement to users watching cricket, generating sensor-based commentary systems and coaching assistants. Due to the similarity between different batting shots, manual feature extraction from video frames is tedious. This paper proposes a hybrid deep-neural-network architecture for classifying 10 different cricket batting shots from offline videos. We composed a novel dataset, CricShot10, comprising uneven lengths of batting shots and unpredictable illumination conditions. Impelled by the enormous success of deep-learning models, we utilized a convolutional neural network (CNN) for automatic feature extraction, and a gated recurrent unit (GRU) to deal with long temporal dependency. Initially, conventional CNN and dilated CNN-based architectures were developed. Following that, different transfer-learning models were investigated-namely, VGG16, InceptionV3, Xception, and DenseNet169-which freeze all the layers. Experiment results demonstrated that the VGG16-GRU model outperformed the other models by attaining 86% accuracy. We further explored VGG16 and two models were developed, one by freezing all but the final 4 VGG16 layers, and another by freezing all but the final 8 VGG16 layers. On our CricShot10 dataset, these two models were 93% accurate. These results verify the effectiveness of our proposed architecture compared with other methods in terms of accuracy.

Image Defogging Framework Using Segmentation and the Dark Channel Prior.

Foggy images suffer from low contrast and poor visibility problem along with little color information of the scene. It is imperative to remove fog from images as a pre-processing step in computer vision. The Dark Channel Prior (DCP) technique is a very promising defogging technique due to excellent restoring results for images containing no homogeneous region. However, having a large homogeneous region such as sky region, the restored images suffer from color distortion and block effects. Thus, to overcome the limitation of DCP method, we introduce a framework which is based on sky and non-sky region segmentation and restoring sky and non-sky parts separately. Here, isolation of the sky and non-sky part is done by using a binary mask formulated by floodfill algorithm. The foggy sky part is restored by using Contrast Limited Adaptive Histogram Equalization (CLAHE) and non-sky part by modified DCP. The restored parts are blended together for the resultant image. The proposed method is evaluated using both synthetic and real world foggy images against state of the art techniques. The experimental result shows that our proposed method provides better entropy value than other stated techniques along with have better natural visual effects while consuming much lower processing time.

Study on the Efficacy of Platelet-rich Fibrin Matrix in Hair Follicular Unit Transplantation in Androgenetic Alopecia Patients.

Objective: Hair loss is a significant problem worldwide. The most common cause of hair loss in men is male androgenetic alopecia, male pattern baldness, which is primarily due to the presence of nonfunctional or dead hair follicles in the scalp. Hair follicular unit transplantation has been a widely used technique to transplant hair follicles into bald areas. Although follicular unit transplantation generally gives satisfactory hair transplantation, efforts have been made to further increase the efficacy of follicular unit transplantation in hair regeneration. The crucial discovery of platelet-derived growth factors has resulted in the development of novel autologous therapeutic methods. Platelet-rich fibrin matrix represents a revolutionary step in the platelet gel therapeutic concept. This technique is fast and involves minimal in vitro manipulations. In this paper, the authors studied the efficacy of platelet-rich fibrin matrix in conjunction with follicular unit transplantation for regeneration of new hair in bald areas in male androgenetic alopecia patients. Design: Ten male subjects between 18 and 50 years of age with Norwood Alopecia from Grade 4 to 6 were chosen for the study. Setting: The study was performed at Derma Solutions clinic, Bengaluru, Karnataka, India. Participants: Patients with thyroid disorders, bleeding disorders, or other co-existing morbidities were excluded. Results: The number of hair follicles began to increase progressively after platelet-rich fibrin matrix treatment was performed on the right side of the scalp and the effect was very distinct after six months of platelet-rich fibrin matrix treatment. Conclusion: This study clearly indicates that platelet-rich fibrin matrix plays a key role in hair regeneration using follicular unit transplantation techniques. Further studies are needed to determine how platelet-rich fibrin matrix helps improve hair retention and regeneration. Additionally, it would be interesting to know how long the effect of platelet-rich fibrin matrix lasts after the termination of therapy. Thus, a future longitudinal study would be very useful. (J ClinAesthetDermatol. 2016;9(9):29-35.).

Platelet-Rich Plasma Growth Factor Concentrated Spray (Keratogrow®) as a Potential Treatment for Androgenic Alopecia.

The objective is to investigate the safety and clinical efficacy of Autologous Platelet Rich Plasma Concentrated Spray (Keratogrow®), for hair loss. Autologous -PRP spray, prepared from a small volume of blood, was applied on the selected patients' scalps at least twice daily. Three months treatments were given for each patient. The effectiveness of the medication was measured by changes in hair regrowth after 3 months determined by physical exam and digital photography. At the end of the 3 cycles of treatment, the patients presented clinical improvement in the mean number of hairs, with a mean increase of hairs in the target area, and a mean increase in total hair density compared with baseline values.

Efficacy of Activated 3X Platelet-Rich Plasma in the Treatment of Androgenic Alopecia.

BACKGROUND: Platelet-rich plasma (PRP) has shown remarkable beneficial effects without any major adverse reactions in the treatment of androgenic alopecia .The growth factors in activated autologous PRP induces the proliferation of dermal papilla cells. OBJECTIVES: To investigate the clinical efficacy of Platelet Rich Plasma prepared using Merisis One Step Gel Separation Technology in treatment of androgenic alopecia. METHODS: Five patients were given autologous PRP injections on the affected area of alopecia over a period of three months at interval of two - three weeks and results were assessed. RESULTS: Three months after the treatment, the patients presented clinical improvement in the hair counts, hair thickness, hair root strength and overall alopecia. CONCLUSION: PRP appears to be a cheap, effective and promising therapy for androgenic alopecia with no major adverse effects.

Treatment of AVN Using Autologous BM Stem Cells and Activated Platelet-Derived Growth Factor Concentrates.

BACKGROUND: Avascular Necrosis (AVN) of hip is a devastating condition seen in younger individuals. It is the ischemic death of the constituents of the bone cartilage of the hip. The femoral head (FH) is the most common site for AVN. It results from interruption of the normal blood flow to the FH that fits into the hip socket. Earlier studies using autologous bone marrow stem cell concentrate injections have shown encouraging results with average success rates. The current study was designed to improve significantly the cartilage regeneration and clinical outcome. METHODS: Total of 48 patients underwent autologous bone marrow stem cell and activated platelet-rich plasma derived growth factor concentrate (PRP-GFC) therapy for early and advanced stages AVN of femoral head in a single multi-specialty center. The total treatment was divided into three phases. In the phase I, all the clinical diagnostic measurements such as magnetic resonance imaging (MRI), computed tomography (CT) etc. with respect to the AVN patients and bone marrow aspiration from posterior iliac spine from the patients were carried out. In the phase II, isolation of stem cells and preparation from the patients were performed. Subsequently, in phase III, the stem cells and PRP- GFCs were transplanted in the enrolled patients. RESULTS: Ninety three percent of the enrolled AVN patients showed marked enhancement in the hip bone joint space (more than 3mm) after combined stem cells and PRP-GFC treatment as evidenced by comparison of the pre- and post-treatment MRI data thus indicative of regeneration of cartilage. The treated patients showed significant improvement in their motor function, cartilage regrowth (3 to 10mm), and high satisfaction in the two-year follow-up. CONCLUSION: Combination of stem cell and PRP-GFC therapy has shown promising cartilage regeneration in 45 out of 48 patients of AVN. This study clearly demonstrates the safety and efficacy of this treatment. Larger numbers of patients need to be evaluated to better understand the efficacy of the combined stem cell and PRP-GFC therapy on AVN patients.

Antiaging Effects of an Intensive Mind and Body Therapeutic Program through Enhancement of Telomerase Activity and Adult Stem Cell Counts.

OBJECTIVE: Key modalities of integrative medicine known to rejuvenate the mind and body are meditation, yoga, and controlled diet. It has been shown previously that intensive or prolonged mind and body therapies (MBT) may have beneficial effects on the well-being of healthy people and in patients. Telomerase activity and levels of peripheral blood adult pluripotent stem cells (PB-APSC) are reliable markers of long-term well-being that are known to decrease with age. The objective of this study is to understand the effect of our MBT program on telomerase activity and stem cells in blood collected from the participants. DESIGN: Here, we have investigated the effects of an intensive three weeks MBT retreat on telomerase activity and the peripheral blood stem cells in participants before and after the MBT. A total of 108 people were enrolled in the study; 38 men and 70 women (aged 18-90) randomly assigned for the study. RESULTS: Telomerase activity was greater in retreat participants at the end of the MBT retreat. About 45% of people showed more than one-fold increase of telomerase activity after our MBT program. Furthermore, about 27% of people showed more pronounced fold increase (2-fold) in telomerase activity after the MBT. In addition, a substantial percentage of people (about 90%) exhibited increased stem cell counts after the MBT. CONCLUSIONS: The data suggest increased telomerase activity and stem cells count in peripheral blood from MBT retreat participants that may lead to increased longevity and better quality of life at latter age.

Human Umbilical Cord Mesenchymal Stem Cells in the Treatment of Duchenne Muscular Dystrophy: Safety and Feasibility Study in India.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a musculo-degenerative disease characterized by lack of dystrophin production with no definite cure available currently. Discarded umbilical cord is a potential source of mesenchymal stem cells which are non-immunogenic and can be used for transplantation in allogenic set ups. Given the regenerative and anti-inflammatory properties of mesenchymal stem cells (MSCs), here we investigated its role in the cellular therapy of DMD patients. DESIGN: This is a single-blinded study conducted in various hospitals of India situated in Mumbai, Delhi, and Lucknow. Inclusion criteria for enrolling the patients in the study were boys aged between 5 to 18 years, absence of dystrophin in the immunohistochemistry of muscle biopsy and mutation in dystrophin gene in cytogenetic analysis. The exclusion criteria were presence of dystrophin in the muscle biopsy, patients on corticosteroids etc. UC-MSCs (2 millions/kg body weight) were administered through IV and IM injection. Muscle power in muscles of proximal upper limb, distal upper limb, proximal lower limb, distal lower limb, hip flexors, hip extensors, hip abductors, and paraspinal muscles were measured in 11 DMD patients after UC-MSCs transplantation and were followed for up to 3 years (average follow up 1.5 years). 5 DMD patients did not receive any UC-MSCs transplantation and served as the control group. RESULTS: The treatment group (N = 11 at baseline) had a pretransplantation strength of 3.45 ± 1.0357 and 4.090 ± 0.8312 in muscles of proximal upper limb and distal upper limb respectively. After 1 year (N = 9) these strengths remained stable with an average of 3.78 (1.03) and 4.22 (0.83). In contrast, the control group (N = 5) has a pre-transplantation strength of 3.6 (0.54) and 4 (1) in the proximal and distal upper limb respectively. After 1 year, (N = 5) 3/5 subjects had a slight but not statistically significant decrease in the proximal upper limb, mean 3.0 (1.0) and 5/5 had a lunit decrease in strength, mean 3.0 (1.0). The treatment group had a pre-transplantation strength of 2.0909 ± 0.8312 and 3.1181 ± 0.8738 in muscles of distal and proximal lower limbs respectively. At 1 year (N = 9), 4/9 subjects had a 1 unit increase in strength in the distal lower limb (mean 3.78 (0.97)) and 8/9 subjects had a lunit increase in strength in the proximal lower limb, mean 3.11 (1.05). The control group has a mean of 3.41 (0.54) and 3.0 (1.0) at baseline in the distal and proximal lower limb respectively. By 1 year, 3/5 subjects had a 1 unit decrease (mean 2.8 (0.45)) and 5/5 had a lunit decrease, mean 2.0 (1.0) in distal and proximal lower limb strength. Stability in muscle function was also achieved in muscles of hip flexors, hip extensors, hip abductors, and paraspinal muscles at one year as compared to untreated group. CONCLUSION: UC-MSCs administration not only resulted in the stabilization of muscle power but also did not show GVHD or any deleterious effects on the patients and thus may be considered as safe option for treatment of DMD as compared to control untreated group although further larger double-blinded studies are needed.

Chondroprotection using naturally occurring mineral supplementation formula in degenerative osteoarthritis of the knees.

OBJECTIVE: To evaluate the therapeutic and safety efficacy of a naturally occurring mineral supplementation in the treatment of symptomatic knee osteoarthritis (OA). PATIENTS AND METHODS: A prospective, single centre, study of 50 patients aged 50 years and above with painful and radiological Osteoarthritis of knees was carried out for one year. Patients received 40 drops of naturally occurring commercially available mineral supplement concentrate mineral drops purportedly derived from the Great Salt Lake in Utah. Efficacy was objectively confirmed by evaluating changes in the thickness of articular cartilage, joint space width, synovial fluid analysis and subjectively by changes in WOMAC scores and 6 Minute pain-free Walking Distance. RESULTS: The composite WOMAC scores were significantly improved by 17.2 points from a mean of 52 at baseline by year end. 18 (41%) patients showed improvement of more than 100 feet for the pain free distance covered during a 6 minute walk at one year follow-up. Ultrasonologicaly, at one year cartilage thickness improved by at least 0.01 mm in 9 (21%) patients. Though radiologicallynone of patient showed increase in joint space it was noticed that only 2(4.6%) patients had decline of joint space width of more than 0.5 mm. Average cell count reduced to 205/microlitre from a value of 520/microlitre at the start of study suggesting that the mineral supplement used had structural efficacy. CONCLUSION: Clinically relevant, statistically significant symptomatic and statistically insignificant structural improvement occurred over 1 year period in patients receiving the naturally occurring mineral supplement. The protection of the joint cartilages from progressive degeneration during osteoarthritis by these supplements indicates towards a chondrocyte regenerative potential of this supplement. Such regeneration may occur through activation of tissue specific adult chondrocyte precursors or stem cells.

Clinical safety in using unmatched allogeneic umbilical cord blood mononuclear cells transplantations in non-haematopoietic degenerative conditions.

AIM: Evaluation of safety in using unmatched human allogeneic umbilical cord blood cells for therapeutic use in individuals with non-haematopoietic degenerative conditions. BACKGROUND: The historical data and several recent immunological arguments suggest the therapeutic use of allogeneic Cord Blood Mononuclear Cells (CBMNCs), as these cells do not elicit immune response. Customarily, HLA matched cord blood MNCs are used along with prolonged immunosuppression in treatment of haematological conditions. Lately, unmatched CBMNCs are widely used in case of unavailability of HLA matched cord blood. There have been suggestions for using unmatched allogeneic cord blood MNCs for degenerative conditions without an immunoconditioning regimen. METHOD: 49 patients with non-haematopoietic degenerative conditions were treated with HLA-unmatched allogeneic hUCB MNCs. Intrathecal/I.V injections (1-2 million cells/kg body weight) were given. Clinical, biochemical and haematological adverse events were evaluated. RESULTS: The haematological and biochemical parameters showed no major deviation from the normal. Clinically, no acute adverse effects or GVHD were observed with the used dosage. CONCLUSION: This study supports/suggests clinical safety in therapeutic medical use of unmatched allogeneic CBMNCs when used at low dosage in non-haematopoietic degenerative conditions.

Regenerative potential of dental pulp mesenchymal stem cells harvested from high caries patient's teeth.

Dental pulp are known to contains stem cells or dentinogenic progenitors that are responsible for dentin repair. Dental pulp Stem cells from Human Exfoliated Deciduous teeth (SHED) represent a population of postnatal stem cells capable of extensive proliferation and multipotential or multilineage differentiations. This potential for tissue regeneration has become the current basis for dental pulp stem cell banking. Here, we have attempted to develop a protocol for harvesting stem cells from patients with High Caries tooth, which are most often electively discarded. We have characterized the stem cells with mesenchymal stem cell markers and have compared their potential to grow in culture, doubling times, and differentiate into different lineages, with normal bone marrow mesenchymal stem cells (MSCs). We observed that the MSCs from dental pulp grew faster, with lower doubling time, and had equal efficiency in differentiating to various lineages, when subjected to standard directed differentiation protocols. This paper establishes that discarded High Carries Tooth can be a good source for regenerative medicine and also could be a potential source for MSCs and dental pulp MSC banking.

Nanotechnology in stem cells research: advances and applications.

Human beings suffer from a myriad of disorders caused by biochemical or biophysical alteration of physiological systems leading to organ failure. For a number of these conditions, stem cells and their enormous reparative potential may be the last hope for restoring function to these failing organ or tissue systems. To harness the potential of stem cells for biotherapeutic applications, we need to work at the size scale of molecules and processes that govern stem cells fate. Nanotechnology provides us with such capacity. Therefore, effective amalgamation of nanotechnology and stem cells - medical nanoscience or nanomedicine - offers immense benefits to the human race. The aim of this paper is to discuss the role and importance of nanotechnology in stem cell research by focusing on several important areas such as stem cell visualization and imaging, genetic modifications and reprogramming by gene delivery systems, creating stem cell niche, and similar therapeutic applications.

Nature vs. nurture: gold perpetuates "stemness".

Adult tissues contain quiescent reservoirs of multipotent somatic stem cells and pluripotent embryonic-like stem cells (ELSCs). Credited with regenerative properties gold is used across both -contemporary and -ancient medicines. Here, we show that gold exerted these effects by enhancing the pool of pluripotent ELSC while improving their stemness. We used hESCs as an in-vitro model to understand if gold could enhance self-renewal and pluripotency. Swarna-bhasma (SB), an ancient Indian gold microparticulate (41.1 nm), preparation, reduced spontaneous-differentiation, improved self-renewal, pluripotency and proliferation of hESCs. Colloidal gold-nanoparticles (GNP) (15.59 nm) were tested to confirm that the observations were attributable to nanoparticulate-gold. SB and GNP exposure: maintained -stemness, -karyotypic stability, enhanced pluripotency till day-12, increased average colony-sizes, and reduced the number of autonomously-derived differentiated FGFR1 positive fibroblast-niche-cells/colony. Particulate-gold induced upregulation of FGFR1 and IGF2 expression, and decrease in IGF1 secretion indicates IGF1/2 mediated support for enhanced pluripotency and self-renewal in hESCs.

Y-27632 enhances differentiation of blastocyst like cystic human embryoid bodies to endocrinologically active trophoblast cells on a biomimetic platform.

Trophoblast differentiation and formation of the placenta are important events linked to post-implantation embryonic development. Models mimicking the biology of trophoblast differentiation in a post-implantation maternal microenvironment are needed for understanding disorders like placental-ischemia or for applications in drug-screening, and would help in overcoming the ethical impasse on using human embryos for such research. Here we attempt to create such a model by using embryoid bodies (EBs) and a biomimetic platform composed of a bilayer of fibronectin and gelatin on top of low-melting agarose. Using this model we test the hypothesis that cystic-EBs (day 30) that resemble blastocysts morphologically, are better sources as compared to noncytic EBs (day 10), for functional trophoblast differentiation; and that the Rho kinases inhibitor Y27632 can enhance this differentiation. Non/cytic EBs with/out Y27632 were grown on this platform for 28 days, and screened from secretion and expression of trophoblast and other lineage markers using ECLIA, RT-PCR, and Immunofluorescence. All EBs attached on this surface and rapidly proliferated into hCG and progesterone (P2) secreting functional trophoblast cells. However, the cells derived from cytic-EBs and cytic-EBs+ Y27632 showed the maximum secretion of these hormones and expressed IGF2, supporting our hypothesis. Also Y27632 reduced extraembryonic endoderm and trophoblast lineage differentiation from early noncystic-EBs, whereas, it specifically enhanced the induction of trophoblast and multinucleated syncitiotrophoblast differentiation from late cystic-EBs. In vivo trophoblast differentiation can be replicated in fibronectin based biomaterials, using cytic-EBs and by maneuvering the Rho-ROCK pathways. Response of EBs to a compound may vary temporally, and determination of their right stage is crucial for applications in directed-differentiation or drug-screening.

A sweet potion to put embryonic stem cells to sleep.

Human embryonic stem cells (hESCs) are rapidly revolutionizing the areas of drug screening and therapy. In view of their applications and high operational costs at global multicentric setups, the ability to store and transport hESCs and derivatives under ambient temperatures, and their cryopreservation without compromising the stemness, function, and viability, is becoming imperative. Here we discuss the need for a natural cryoprotectant and biopreservative with a potential to improve cryopreservation, ambient temperature storage, and shipping of hESCs and derivatives. Trehalose, a naturally occurring disaccharide with therapeutic properties, protects the integrity of cells against desiccation, dehydration, and extreme heat or cold, and has been successfully tested for some somatic stem cell types. However, the biggest setback is the inability of mammalian cells to internalize trehalose. Here we review the methods being developed at different laboratories to facilitate its intercellular transport and advocate the need for similar advances in hESCs.

FGF2 secreting human fibroblast feeder cells: a novel culture system for human embryonic stem cells.

Human embryonic stem cell (hESC) lines are traditionally derived and maintained on mouse embryonic fibroblasts (MEF) which are xenogeneic and enter senescence rapidly. In view of the clinical implications of hESCs, the use of human fibroblast as feeders has been suggested as a plausible alternative. However, use of fibroblast cells from varying sources leads to culture variations along with the need to add FGF2 in cultures to sustain ES cell pluripotency. In this study we report the derivation of FGF2 expressing germ layer derived fibroblast cells (GLDF) from hESC lines. These feeders could support the pluripotency, karyotypes and proliferation of hESCs with or without FGF2 in prolonged cultures as efficiently as that on MEF. GLDF cells were derived from embryoid bodies and characterized for expression of fibroblast markers by RT-PCR, Immunofluorescence and by flow cytometry for CD marker expression. The expression and secretion of FGF2 was confirmed by RT-PCR, Western blot, and ELISA. The hESC lines cultured on MEF and GLDF were analyzed for various stemness markers. These feeder cells with fibroblast cells like properties maintained the properties of hESCs in prolonged culture over 30 passages. Proliferation and pluripotency of hESCs on GLDF was comparable to that on mouse feeders. Further we discovered that these GLDF cells could secrete FGF2 and maintained pluripotency of hESC cultures even in the absence of supplemental FGF2. To our knowledge, this is the first study reporting a novel hESC culture system which does not warrant FGF2 supplementation, thereby reducing the cost of hESC cultures.

Human embryonic stem cells: preclinical perspectives.

Human embryonic stem cells (hESCs) have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic.

Endotoxin-induced silencing of mesoderm induction and functional differentiation: role of HMGB1 in pluripotency and infection.

OBJECTIVES: Mechanisms underpinning Gram-negative bacterial vaginosis-induced birth anomalies are obscure. Ethical issues limit such studies on peri-implantation-stage human embryos. Here we have used embryoid bodies (EBs) as an in vitro model to examine the effect of Gram-negative bacterial endotoxins/lipopolysaccharides (LPS) on the faithful induction of germ lineages during embryogenesis. The role of LPS-inducible cytokine and pluripotency-related DNA-binding protein HMGB1 was also studied in these EBs. METHODS: EBs derived from the human embryonic stem cell line HUES9 were exposed to 12.5 pg/ml of LPS for 48 h. The expression profile of the ectoderm, endoderm, mesoderm and trophectoderm lineage markers, such as beta III-tubulin, GATA4, BMP2, Brachury and beta-hCG, were studied, by RT-PCR and immunofluorescence. Inhibition of mesoderm induction was confirmed by RT-PCR analysis for hANP, cTnT, ABCG2, GATA2, BMP4 and HAND1. Osteoblast differentiation was induced in the EBs, and confirmed by von Kosa and Alizarin red staining. A comet assay was also carried out to assess the degree of apoptosis in these EBs. RESULTS AND CONCLUSIONS: We found that the LPS-treated EBs were selectively silenced for mesoderm markers and failed to differentiate into functional osteoblasts. HMGB1 expression was absent in the normal EBs and was found to be localized in the cytoplasm of the LPS-treated EBs. Overall, our data indicate that endotoxin-induced HMGB1 expression in the peri-implantation-stage embryos can bring about severe birth defects of, for example, the bone and heart. This study also indicates that HMGB1 could be involved in maintenance of pluripotency in the human embryonic stem cells by impeding their differentiation.