Germ Layers Research Articles

Generation of an induced pluripotent stem cell line (HMSCATi004-A) from an early onset Parkinson’s disease patient with PRKN gene mutation

Parkin (PRKN) is recognized as causative gene in early-onset Parkinson’s disease (PD). Induced pluripotent stem cells (iPSCs) were derived from a 29-year-old PD patient carrying a heterozygous c.823C > T (p.R275W) variant and an exon 2–4 deletion in PRKN. The Generated iPSCs maintain a normal karyotype, express pluripotency markers, and retain the ability to differentiate into the three germ layers. This iPSC line serves as a valuable cellular model for investigating the pathogenesis of early-onset PD and development of potential therapeutic interventions.

Microscopic Anatomy of Orbital Septum.

To study the microscopic anatomy of the orbital septum and evaluate its relationships with the adjoined structures. Histological evaluation of 11 upper eyelids (5 right, 6 left) from 10 Japanese cadavers (age range: 36-94-year-old, average: 71.1) was performed. The specimens were fixed in 10% formalin and stained with Elastica Van Gieson. Specimens were microscopically analyzed using different magnifications (20×, 40×, and 100×). The orbital septum was the single fibrous layer in all the specimens, anteriorly separated from the retro-orbicularis fat pad capsule and posteriorly from the preaponeurotic fat pad capsule. At the junction of the orbital septum with the levator aponeurosis, the orbital septum proceeded towards the tarsal plate, and the preaponeurotic fat pad capsule reflected posteriorly. The orbital septum is constituted of a single layer of fibrous tissue anteriorly separated from the retro-orbicularis fat pad capsule and posteriorly from the preaponeurotic fat pad capsule by the corresponding virtual spaces.

Enhancing EEG data quality and precision for cloud-based clinical applications: an evaluation of the SLOG framework

Automation is revamping our preprocessing pipelines, and accelerating the delivery of personalized digital medicine. It improves efficiency, reduces costs, and allows clinicians to treat patients without significant delays. However, the influx of multimodal data highlights the need to protect sensitive information, such as clinical data, and safeguard data fidelity. One of the neuroimaging modalities that produces large amounts of time-series data is Electroencephalography (EEG). It captures the neural dynamics in a task or resting brain state with high temporal resolution. EEG electrodes placed on the scalp acquire electrical activity from the brain. These electrical potentials attenuate as they cross multiple layers of brain tissue and fluid yielding relatively weaker signals than noise-low signal-to-noise ratio. EEG signals are further distorted by internal physiological artifacts, such as eye movements (EOG) or heartbeat (ECG), and external noise, such as line noise (50 Hz). EOG artifacts, due to their proximity to the frontal brain regions, are particularly challenging to eliminate. Therefore, a widely used EOG rejection method, independent component analysis (ICA), demands manual inspection of the marked EOG components before they are rejected from the EEG data. We underscore the inaccuracy of automatized ICA rejection and provide an auxiliary algorithm-Second Layer Inspection for EOG (SLOG) in the clinical environment. SLOG based on spatial and temporal patterns of eye movements, re-examines the already marked EOG artifacts and confirms no EEG-related activity is mistakenly eliminated in this artifact rejection step. SLOG achieved a 99% precision rate on the simulated dataset while 85% precision on the real EEG dataset. One of the primary considerations for cloud-based applications is operational costs, including computing power. Algorithms like SLOG allow us to maintain data fidelity and precision without overloading the cloud platforms and maxing out our budgets.

Ovum Pick-Up and Ovaries Characterization of Black Bengal Goat from Slaughterhouse

Black Bengal goat is the only indigenous breed in Bangladesh. These are small-sized goats renowned for their high-quality meat, skin, and adaptability to adverse environmental conditions. To conserve the originality of this species, it is mandatory to develop an effective methodology for their germ cells to be harvested after death. Therefore, the study was conducted to assess the ovarian status and evaluate the most suitable method of ovum pick-up as well as the characterization of ovum from slaughterhouse specimens. A total of 182 ovaries were collected from local slaughterhouses from June 2022 to July 2023 and grossly examined to assess their status. Two methods were used to retrieve oocytes from the ovaries: aspiration and slicing. The grading of the ovum was done based on the germ layer present surrounding the oocytes. From 182 ovaries, the rates of corpus luteum (CL), tumor, and cyst were found to be 14.29%, 6.59%, and 27.47%, respectively. In the aspiration method, the significantly highest percentage of recovery rate (38.89%) and grade A oocytes (51.06%) were found within 1–3 hours of slaughtering. In the slicing method, the significantly highest percentage of recovery rate (19.57%) and grade A (60.61%) oocytes were found within 1–3 hours of slaughtering. The recovery rate was significantly higher (34.1%) in the aspiration method than in the slicing method (17.1%). There was no significant relationship in oocyte quality between the two methods. The recovery rate was not significantly dependent on the presence of CL. In conclusion, the findings from this study may be useful for assessing the quality of oocytes to preserve the germ cells and utilize them for in vitro fertilization for conserving Black Bengal goat.

Vasa vasorum of the no-touch saphenous vein graft observed using frequency-domain optical coherence tomography.

One possible reason for the long-term patency of no-touch saphenous vein grafts is the preservation of the vasa vasorum in the adventitia/perivascular adipose tissue. In this study, we investigated the vasa vasorum of the no-touch saphenous vein graft in vivo using frequency-domain optical coherence tomography, performed qualitative and quantitative analyses, and compared them with the conventional saphenous vein graft. A frequency-domain optical coherence tomography study was performed on 14 saphenous vein grafts at the postoperative coronary angiography 1-2 weeks after surgery (no-touch group, n = 9; conventional group, n = 5). Many signal-poor tubular lumen structures that can be recognised in the cross-sectional and longitudinal profiles, which indicates the vasa vasorum, were observed in the adventitial/perivascular adipose tissue layer in the no-touch saphenous vein grafts. In contrast, the vasa vasorum was less abundant in the conventional saphenous vein grafts. The vasa vasorum volume per millimetre of graft in the no- touch group was significantly higher than in the conventional group (0.0020 [0.0017, 0.0043] mm3 and 0.0003 [0.0000, 0.0006] mm3, P = 0.023). Frequency-domain optical coherence tomography showed abundant vasa vasorum in the thick adventitia/perivascular adipose tissue layer of no-touch saphenous veins in vivo. In contrast, few vasa vasorum was observed in the conventional saphenous vein grafts.

Establishment of an induced pluripotent cell line (ABCRIi001-A) from an elderly female for ageing research

Human induced pluripotent stem cells (hiPSCs) hold promises to model and understand human diseases, including those associated with ageing. Here, we describe ABCRIi001-A, a hiPSC line generated from peripheral blood mononuclear cells (PBMCs) of a 79-year-old female enrolled in a study for development of an ageing score (ALFA Score). PBMCs were reprogrammed using three Sendai virus-based reprogramming vectors (hKOS, hc-Myc, and hKlf4). ABCRIi001-A showed normal morphology and karyotype, viral clearance, absence of genomic aberrations, and their pluripotency was confirmed by expression of pluripotency-related markers and their ability to differentiate into the three germ layers. ABCRIi001-A is valuable for ageing-related studies.

Generation of patient-derived induced pluripotent stem cell line UJSi004-A from ultra-treatment-resistant schizophrenia

Schizophrenia is a severe mental illness with disabling effects. Induced pluripotent stem cells (iPSCs) are generated by reprogramming peripheral blood mononuclear cells (PBMCs) from patients with ultra-treatment-resistant schizophrenia (UTRS). iPSCs have normal karyotype, express pluripotency markers and differentiate into three germ layers in vivo. This iPSC cell line carries the genetic information of the patient and is a good model for studying disease mechanisms and developing new therapies.

Synthetic Hydrogel Substrate for Human Induced Pluripotent Stem Cell Definitive Endoderm Differentiation

Human induced pluripotent stem cells (hiPSCs) can give rise to multiple lineages derived from three germ layers, endoderm, mesoderm and ectoderm. Definitive endoderm (DE) cell types and tissues have great potential for regenerative medicine applications. Current hiPSC differentiation protocols focus on the addition of soluble factors; however, extracellular matrix properties are known to also play a role in dictating cell fate. Matrigel™ is the gold standard for DE differentiation, but this xenogeneic, poorly defined basement membrane extract limits the clinical translatability of DE-derived tissues. Here we present a fully defined PEG-based hydrogel substrate to support hiPSC-derived DE differentiation. We screened hydrogel formulations presenting different adhesive peptides and matrix stiffness. Our results demonstrate that presenting a short peptide, cyclic RGD, on the engineered PEG hydrogel supports the transition from undifferentiated hiPSCs to DE using a serum-free, commercially available kit. We show that increasing substrate stiffness (G’=1.0-4.0 kPa) results in an increased linear response in DE differentiation efficiency. We also include a temporal analysis of the expression of integrin and syndecan receptors as the hiPSCs undergo specification towards DE lineage. Finally, we show that focal adhesion kinase activity regulates hiPSC growth and DE differentiation efficiency. Overall, we present a fully defined matrix as a synthetic alternative for Matrigel™ supporting DE differentiation.

Generation of an integration-free induced pluripotent stem cell line, FJMAi001-A, from a Marfan syndrome patient with a heterozygous mutation c.2777G > A (p.Cys926Tyr) in FBN1

Marfan syndrome (MFS) is a heritable dominant disorder of fibrous connective tissue, caused by mutations in the gene encoding fibrillin-1 on chromosome 15. Here, we report an induced pluripotent stem cell (iPSC) line generated from a patient with MFS who carries a heterozygous mutation of c.2777G > A(p.Cys926Tyr) in the FBN1 gene using an episomal method. The hiPS-MFS cell line has normal karyotype, expresses pluripotency markers, and has the ability to form three germ layers in vivo.This MFS-specific iPSC line can be used as a cell disease model to study the pathogenesis of Marfan syndrome.

Derivation of induced pluripotent stem cell from a Baraitser-Winter Cerebrofrontofacial syndrome with ACTB mutation

Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF, OMIM: 243310) is a rare congenital developmental disorder marked by distinct facial dysmorphisms, coloboma, diminutive stature, and cognitive impairment, as initially described by Baraitser and Winter in 1988. Here, we derived human induced pluripotent stem cells (hiPSCs) from a 4-year-old male patient diagnosed with Baraitser-Winter Cerebrofrontofacial syndrome and harbouring a mutation in the ACTB gene. The newly established hiPSC line exhibited normal karyotypes and demonstrated the capacity to differentiate into all three germ layers. Additionally, these hiPSCs maintained their original genotype and expressed markers of pluripotency. Patient-derived hiPSCs would serve as a valuable tool for in vitro modelling of Baraitser-Winter Cerebrofrontofacial syndrome and reveal the potential pathogenesis induced by ACTB gene mutations

Establishment of a human induced pluripotent stem cell (iPSC) line (JUCTCi018-A) from a patient with Charcot-Marie-Tooth disease type 2EE (CMT2EE) due to a homozygous c.122G > A p.(Arg41Gln) mutation in the MPV17 gene

(Charcot-Marie-Tooth disease (CMT) is a genetic disorder affecting peripheral nerves. The human induced pluripotent stem cell (iPSC) line JUCTCi018-A was created using dermal fibroblasts from a Charcot-Marie-Tooth disease type 2EE (CMT2EE) patient with a homozygous missense mutation in the MPV17 gene (c. 122G > A, p.Arg41Gln). These fibroblasts were reprogrammed using Sendai viruses that encoded OCT4, SOX2, KLF4, and c-MYC reprogramming factors. The iPSCs demonstrated normal morphology and karyotype, expressed pluripotency markers, and the ability to differentiate into the three germ layers. This iPSC line is valuable for investigating the mechanisms underlying CMT2EE.

Differential growth is an emergent property of mechanochemical feedback mechanisms in curved plant organs.

Differential growth is central to eukaryotic morphogenesis. We showed using cellular imaging, simulations, and perturbations that light-induced differential growth in a curved organ, the Arabidopsis thaliana apical hook, emerges from the longitudinal expansion of subepidermal cells, acting in parallel with a differential in the material properties of epidermal cell walls that resist expansion. The greater expansion of inner hook cells that results in apical hook opening is gated by wall alkalinity and auxin, both of which are depleted upon illumination. We further identified mechanochemical feedback from wall mechanics to light stimulated auxin depletion, which may contribute to gating hook opening under mechanical restraint. These results highlight how plant cells coordinate growth among tissue layers by linking mechanics and hormonal gradients with the cell wall remodeling required for differential growth.

Omental Lipoma in a Slaughtered Lamb

Lipoma is a benign tumor of well-differentiated adipocytes that has been reported in some domestic animals. Omental lipomas in human and domestic animals are rare and reported as a case report. This study aimed to report an omental lipoma in a slaughtered sheep. In the observational examination of a sheep in a slaughterhouse in Tehran Province, Iran, and during the inspection of the abdominal area, a mass was seen on the greater omentum. The mass was sampled and placed in 10% formalin for histopathology. The mass was single, soft, dense, capsuled, oval, weighing 150 g, and 4.5×3.5 cm in size. Microscopic examination showed omental mass composed of uniform and mature lipocytes encased within a thick layer of immature connective tissue. The final diagnosis was primary benign lipoma. Lipoma can occur anywhere in the body where there are fat cells, and probably obesity and trauma are important risk factors for its occurrence. The size of the lipoma mass may help predict the age of the mass.

Computing whole embryo strain maps during gastrulation

Gastrulation is a critical process during embryonic development that transforms a single-layered blastula into a multilayered embryo with distinct germ layers, which eventually give rise to all the tissues and organs of the organism. Studies across species have uncovered the mechanisms underlying the building blocks of gastrulation movements, such as localized in-plane and out-of-plane epithelial deformations. The next challenge is to understand dynamics on the scale of the embryo: this requires quantifying strain tensors, which rigorously describe the differences between the deformed configurations taken on by local clusters of cells at time instants of observation and their reference configuration at an initial time. We present a systematic strategy for computing such tensors from the local dynamics of cell clusters, which are chosen across the embryo from several regions whose morphogenetic fate is central to viable gastrulation. As an application of our approach, we demonstrate a strategy of identifying distinct Drosophila morphological domains using strain tensors.

A Prospective Randomized Controlled Study of Stratafix versus Standard-of-Care for Deep Tissue Closure in Orthopedic Surgery.

Orthopedic deep surgical incisions require the approximation of 1 or multiple tissue layers. This prospective randomized controlled study aimed to assess the usefulness and effectiveness of a barbed suture technique (Stratafix symmetric PDS plus) versus the conventional interrupted knotted suture technique for deep tissue closure in orthopedic surgery by comparing deep fascia suture time, relative cost, and wound-related complications. A total of 254 patients with deep surgical incisions who underwent orthopedic surgery between October 1, 2020, and June 30, 2021, were recruited. Their general characteristics (age, sex, weight, height, body mass index, American Society of Anesthesiologists physical status score, total operation time, and length of deep incision) and factors related to deep incision wounds (suture type and number, wound closure time, and operation site outcomes) were collected. The general characteristics did not differ between the Stratafix and conventional groups. There were no between-group differences observed in total operation time or total anesthesia time. The wound suture times differed significantly. In the conventional group, the suture time per unit length was lower in the group with the length of deep incision under 20 cm but did not differ significantly for each wound size. In the Stratafix group, the suture time per unit length was lower in the group under 15 cm, with the shortest time observed for 10-14.9 cm, followed by 5.0-9.9 cm and the group under 5 cm. The conventional group developed 4 cases of superficial wound infection or surgical wound necrosis. One case of protruded suture tap occurred in the Stratafix group. The average suture time per unit length increased for lengths under 5 cm as barbed sutures required more time from the start of the first suture to finish of the last suture. There was no significant benefit for very short suture length. One barbed suture material allows a suture of approximately 10-12 cm; sutures beyond that require more time because the surgeon has to start again. The Stratafix group used less suture material than the conventional group.

POST-MASTECTOMY SURGICAL SITE INFECTION RATES IN FEMALES WITH BREAST CARCINOMA IN PREMENOPAUSAL AGE: A SINGLE CENTER STUDY

Breast surgery, like mastectomy, is generally considered a low-risk procedure for surgical site infections (SSIs), but when SSIs do occur, they can lead to significant complications. Identifying risk factors to improve patient outcomes and minimize complications is essential. Objective: This study aimed to determine the frequency of post-mastectomy surgical site infections (SSI) in premenopausal females with breast carcinoma and to identify the associated risk factors. Methods: This descriptive case study included 100 premenopausal females diagnosed with breast carcinoma who underwent mastectomy at a tertiary care hospital, Shaikh Zayed Hospital Lahore, from April 2024 to August 2024. The age range of participants was 25-45 years, with a mean age of 39 ± 5.3 years. Demographic data, including age, gender, body mass index (BMI), comorbidities, and American Society of Anesthesiologists (ASA) score, were collected. Intraoperative variables such as blood loss (mean: 350 ± 50 ml) and surgery duration (mean: 120 ± 15 minutes) were also recorded. All patients received standard postoperative antibiotic prophylaxis, including cefazolin or clindamycin, for those allergic to penicillin. The surgical site was assessed 30 days postoperatively for signs of infection, including erythema, pus discharge, or fever exceeding 38°C. SSIs were categorized based on the Centers for Disease Control and Prevention (CDC) guidelines into superficial, deep, and organ/space infections. Results: The overall infection rate was 7.5% (n=7). Of these, 4 cases were superficial infections, while 3 involved deeper tissue layers, requiring drainage and further antibiotics. Factors significantly associated with an increased risk of disease in the multivariate logistic regression model included a high wound class (contaminated or dirty; OR: 3.2, p=0.01), elevated ASA score (ASA III-IV; OR: 2.8, p=0.03), high BMI >30 (OR: 3.5, p=0.002), diabetes (OR: 4.1, p=0.001), use of surgical drains (OR: 2.9, p=0.02), and reoperation (OR: 4.3, p=0.005). The mean hospital stay was extended by 4.5 days (from 5.2 ± 1.5 days in non-infected patients to 9.7 ± 3.1 days in infected cases). Conclusion: The study found a post-mastectomy surgical site infection rate of 7.5% in premenopausal females, with significant risk factors including high BMI, diabetes, use of surgical drains, and the need for reoperation. A high wound class and elevated ASA score also predicted increased infection risk. Addressing these modifiable risk factors through optimized perioperative care, such as better glycemic control, judicious use of surgical drains, and minimizing reoperations, may help reduce SSI rates and improve outcomes for breast cancer patients.

Evaluating Strategies to Assess the Differentiation Potential of Human Pluripotent Stem Cells: A Review, Analysis and Call for Innovation.

Pluripotent stem cells have the ability to differentiate into all cells and tissues within the human body, and as a result they are attractive resources for use in basic research, drug discovery and regenerative medicine. In order to successfully achieve this application, starting cell sources ideally require in-depth characterisation to confirm their pluripotent status and their ability to differentiate into tissues representative of the three developmental germ layers. Many different methods to assess potency are employed, each having its own distinct advantages and limitations. Some aspects of this characterisation process are not always well standardised, particularly techniques used to assess pluripotency as a function. In this article, we consider the methods used to establish cellular pluripotency and subsequently analyse characterisation data for over 1590 human pluripotent cell lines from publicly available repositories in the UK and USA. In particular, we focus on the teratoma xenograft assay, its use and protocols, demonstrating the level of variation and the frequency with which it is used. Finally, we reflect on the implications of the findings, and suggest in vitro alternatives using modern innovative technology as a way forward.

Nanodosimetric investigation of the track structure of therapeutic carbon ion radiation part 1: measurement of ionization cluster size distributions

At the Heidelberg Ion-Beam Therapy Center, the track structure of carbon ions of therapeutic energy after penetrating layers of simulated tissue was investigated for the first time. Measurements were conducted with carbon ion beams of different energies and polymethyl methacrylate (PMMA) absorbers of different thicknesses to realize different depths in the phantom along the pristine Bragg peak. Ionization cluster size (ICS) distributions resulting from the mixed radiation field behind the PMMA absorbers were measured using an ion-counting nanodosimeter. Two different measurements were carried out: (i) variation of the PMMA absorber thickness with constant carbon ion beam energy and (ii) combined variation of PMMA absorber thickness and carbon ion beam energy such that the kinetic energy of the carbon ions in the target volume is constant. The data analysis revealed unexpectedly high mean ICS values compared to stopping power calculations and the data measured at lower energies in earlier work. This suggests that in the measurements the carbon ion kinetic energies behind the PMMA absorber may have deviated considerably from the expected values obtained by the calculations. In addition, the results indicate the presence of a marked contribution of nuclear fragments to the measured ICS distributions, especially if the carbon ion does not cross the target volume.

In Vivo Lifetime Imaging of the Internal O2 Dynamics in Corals with near-Infrared-Emitting Sensor Nanoparticles.

Mapping of O2 with luminescent sensors within intact animals is challenging due to attenuation of excitation and emission light caused by tissue absorption and scattering as well as interfering background fluorescence. Here we show the application of luminescent O2 sensor nanoparticles (∼50-70 nm) composed of the O2 indicator platinum(II) tetra(4-fluoro)phenyltetrabenzoporphyrin (PtTPTBPF) immobilized in poly(methyl methacrylate-co-methacrylic acid) (PMMA-MA). We injected the sensor nanoparticles into the gastrovascular system of intact colony fractions of reef-building tropical corals that harbor photosynthetic microalgae in their tissues. The sensor nanoparticles are excited by red LED light (617 nm) and emit in the near-infrared (780 nm), which enhances the transmission of excitation and emission light through biological materials. This enabled us to map the internal O2 concentration via time-domain luminescence lifetime imaging through the outer tissue layers across several coral polyps in flowing seawater. After injection, nanoparticles dispersed within the coral tissue for several hours. While luminescence intensity imaging showed some local aggregation of sensor particles, lifetime imaging showed a more homogeneous O2 distribution across a larger area of the coral colony. Local stimulation of symbiont photosynthesis in corals induced oxygenation of illuminated tissue areas and formation of lateral O2 gradients toward surrounding respiring tissues, which were dissipated rapidly after the onset of darkness. Such measurements are key to improving our understanding of how corals regulate their internal chemical microenvironment and metabolic activity, and how they are affected by environmental stress such as ocean warming, acidification, and deoxygenation. Our experimental approach can also be adapted for in vivo O2 imaging in other natural systems such as biofilms, plant and animal tissues, as well as in organoids and other cell constructs, where imaging internal O2 conditions are relevant and challenging due to high optical density and background fluorescence.

A geometric-tension-dynamics model of epithelial convergent extension

Convergent extension of epithelial tissue is a key motif of animal morphogenesis. On a coarse scale, cell motion resembles laminar fluid flow; yet in contrast to a fluid, epithelial cells adhere to each other and maintain the tissue layer under actively generated internal tension. To resolve this apparent paradox, we formulate a model in which tissue flow in the tension-dominated regime occurs through adiabatic remodeling of force balance in the network of adherens junctions. We propose that the slow dynamics within the manifold of force-balanced configurations is driven by positive feedback on myosin-generated cytoskeletal tension. Shifting force balance within a tension network causes active cell rearrangements (T1 transitions) resulting in net tissue deformation oriented by initial tension anisotropy. Strikingly, we find that the total extent of tissue deformation depends on the initial cellular packing order. T1s degrade this order so that tissue flow is self-limiting. We explain these findings by showing that coordination of T1s depends on coherence in local tension configurations, quantified by a geometric order parameter in tension space. Our model reproduces the salient tissue- and cell-scale features of germ band elongation during Drosophila gastrulation, in particular the slowdown of tissue flow after approximately twofold elongation concomitant with a loss of order in tension configurations. This suggests local cell geometry contains morphogenetic information and yields experimentally testable predictions. Defining biologically controlled active tension dynamics on the manifold of force-balanced states may provide a general approach to the description of morphogenetic flow.