This study details our efforts to establish a sustainable cell culture of Macrobrachium rosenbergii, involving a 90-day longitudinal study on embryonic primary cell culture. Daily monitoring revealed changes from singular diverse-sized cells attaching on days 4-10 to interconnected cell clusters, transitioning from 2D to combined 2D and 3D structures by days 7-10, leading to spheroid-like cell masses. Metabolic activity peaked at day 26, with proliferation and increased cell numbers retained up to date 40 before gradually declining. Cell-cycle analyses by FACS showed an inverse relationship between G0/G1 and S phases. Populations sorted on days 3 and 24 revealed eight seeded populations expressing proliferation markers MrMYC and MrPCNA, with six also expressing stem-cell markers MrOct-4 and MrSox-2/3. With increased cell density, metabolic activity, and reduced G0/G1 distribution towards S, it was hypothesized that splitting cultures at high-proliferating mitotic ratio peaks could enable successful passages. Indeed, two consecutive passages were achieved after 8 days and 4 days in culture. Cells post-passages expressed species-specific Mr18S, MrMYC, MrPCNA, MrOct-4, and MrSox-2/3. After decades of striving for a crustacean cell line, this study presents M. rosenbergii cultures comprising putative proliferating/stem-cell sub-populations. These mixed-population prawn embryonic cell cultures have the potential to serve as a foundational platform for immortalization, contributing to the longstanding goal of establishing sustainable cell-culture lines.

Dilated cardiomyopathy (DCM) is a primary myocardial disease, leading to heart failure and excessive risk of sudden cardiac death with rather poorly understood pathophysiology. In 2015, Parvari's group identified a recessive mutation in the autophagy regulator, PLEKHM2 gene, in a family with severe recessive DCM and left ventricular non-compaction (LVNC). Fibroblasts isolated from these patients exhibited abnormal subcellular distribution of endosomes, Golgi apparatus, lysosomes and had impaired autophagy flux. To better understand the effect of mutated PLEKHM2 on cardiac tissue, we generated and characterized induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) from two patients and a healthy control from the same family. The patient iPSC-CMs showed low expression levels of genes encoding for contractile functional proteins (α and β-myosin heavy chains and 2v and 2a-myosin light chains), structural proteins integral to heart contraction (Troponin C, T and I) and proteins participating in Ca²⁺ pumping action (SERCA2 and Calsequestrin 2) compared to their levels in control iPSC-derived CMs. Furthermore, the sarcomeres of the patient iPSC-CMs were less oriented and aligned compared to control cells and generated slowly beating foci with lower intracellular calcium amplitude and abnormal calcium transient kinetics, measured by IonOptix system and MuscleMotion software. Autophagy in patient’s iPSC-CMs was impaired as determined from a decrease in the accumulation of autophagosomes in response to chloroquine and rapamycin treatment, compared to control iPSC-CMs. Impairment in autophagy together with the deficiency in the expression of NKX2.5, MHC, MLC, Troponins and CASQ2 genes, which are related to contraction-relaxation coupling and intracellular Ca²⁺ signaling, may contribute to the defective function of the patient CMs and possibly affect cell maturation and cardiac failure with time.

The Saint Catherine region of southern Sinai Peninsula’s drylands encompasses a unique combination of geophysical conditions. These features have formed highly diverse habitats, which support extremely complex plant communities. Throughout history, the local Bedouin population has used many of these plants for therapeutic purposes. The objectives of this study were to document the medicinal knowledge for as many as possible plant species, evaluate this ethnobotanic knowledge against a modern biomedical database, and identify links between the two disciplines. On-site gathering of information, through structured interviews with two local key informants, revealed the traditional use of 90 native vegetation species for a range of therapeutic purposes. Surveying the modern biomedical uses of these plant species in the PubMed database revealed medicinal use for 41 of them, as well as other plant species without known traditional therapeutic uses. Of the 41 plant species, in-depth integration of traditional and modern knowledge was implemented for 20 species, for which sufficient information was found in the PubMed. The results highlight the common ground between the two disciplines, and propose bridges between traditional and modern medicines. The study stresses the need for additional research in ethnobotany, which may assist in developing new plant-based medications.

More than 50% of pre-clinical studies fail despite a long and expensive journey of drug discovery using animal models [1]. Currently, there is a human-specific replacement: human patient induced pluripotent stem cells (hiPSCs)/human embryonic stem cells (hESCs) and their counterpart corrected cells (using Crispr methodology), which enable the establishment of disease-in-a-dish models encompassing patients’ genetics, which were eluted in this issue. This Editorial review contains three parts: cellular models, three-dimensional models and reviews. Piera Trionfini et al. describe how allogeneic iPSCs and hypoimmunogenic iPSCs serve as new cost-effective and off-the-shelf opportunities for cell therapy in liver diseases. They achieved this goal by employing the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to delete the β2-Microglobulin (B2M) and the Class II Major Histocompatibility Complex Transactivator (CIITA) genes [2]. LU PAN et al. employ a model of neural progenitor cell (NPCs) migration assay to answer the unsolved problem of the impacts of sevoflurane anesthesia on the impairment of NPC migration and, as such, on cognitive function. They were able to show that a sevoflurane-induced decrease in NPC migration is dependent on cyclophilin D (CypD) levels [3].

Human-induced pluripotent stem cells (hiPSCs) serve as a sustainable resource for studying the molecular foundation of disease development, including initiation and deterioration. Although the process of reprogramming adult cells is accompanied by the obliteration of part of the epigenetic signature, usually upon differentiation of hiPSCs into specific cells, such as brain, heart, or muscle cells, these cells are adequate models for studying disease pathology and for drug discovery, as described in this Special Issue. In considering iPSCs as models for mono/complex diseases and as a potential future replacement for animal studies, Costa et al. [1] suggest an original method to compensate for the immature electrophysiology of human-induced pluripotent-stem-cell-derived cardiomyocytes via co-culturing them with a human embryonic kidney (HEK) cell line expressing the Inward Rectifier Channel Kir2.1 (HEK-IK1). They tested the potential of this co-culture system to generate an electrical syncytium with an adult-like cardiac electrophysiology and concluded that it can be achieved.

Crustacean cell line immortalization has gained a great deal of attention in recent decades for both scientific and applied reasons. Our goal in this study was to advance the state of art towards establishing an immortalized cell line by improving the proliferation rates of primary cells isolated from embryos of the giant freshwater prawn Macrobrachium rosenbergii by using a lentivirus expressing the Ras oncogene. The choice of Ras derived from its involvement in various cellular pathways, such as cell growth, differentiation, and survival, and its use as a tool for in-vitro immortalization, e.g., a specific mutated Ras (RasV12) was used to generate an arthropod cell line. Complementarily, in-silico screening of M. rosenbergii transcriptomic libraries for Ras expression indicated that Ras is already expressed at very early stages of embryo development. In the current study, we transduced primary M. rosenbergii embryonic cells with a lentivirus expressing RasV12 by using the white spot syndrome virus (WSSV IE1) promoter. Expression and sequencing (as followed by sequencing cDNA, confocal microscopy and FACS analysis) of the mutated Ras in the transduced cells confirmed that the lentivirus was successfully integrated into the genome. The lenti-MrRas transduction rate was 23% in the total primary cell population and more than 80% in a sub-population of cells with high granularity. Proliferation of lenti-MrRas transfected cells was enhanced to almost 1200% of the seeding density by the end of our experiment (18 days), which was double that of the control. We were thus successful in enhancing the longevity of embryonic primary cell cultures by ectopic expression of the mutated Ras protein, but the improvement was not sufficient for immortalization.

Pleckstrin Homology And RUN Domain Containing M2 (PLEKHM2) [delAG] mutation causes dilated cardiomyopathy with left ventricular non-compaction (DCM-LVNC), resulting in a premature death of PLEKHM2[delAG] individuals due to heart failure. PLEKHM2 is a factor involved in autophagy, a master regulator of cellular homeostasis, decomposing pathogens, proteins and other cellular components. Autophagy is mainly carried out by the lysosome, containing degradation enzymes, and by the autophagosome, which engulfs substances marked for decomposition. PLEKHM2 promotes lysosomal movement toward the cell periphery. Autophagic dysregulation is associated with neurodegenerative diseases’ pathogenesis. Thus, modulation of autophagy holds considerable potential as a therapeutic target for such disorders. We hypothesized that PLEKHM2 is involved in neuronal development and function, and that mutated PLEKHM2 (PLEKHM2[delAG]) neurons will present impaired functions. Here, we studied PLEKHM2-related abnormalities in induced pluripotent stem cell (iPSC)-derived motor neurons (iMNs) as a neuronal model. PLEKHM2[delAG] iMN cultures had healthy control-like differentiation potential but exhibited reduced autophagic activity. Electrophysiological measurements revealed that PLEKHM2[delAG] iMN cultures displayed delayed functional maturation and more frequent and unsynchronized activity. This was associated with increased size and a more perinuclear lysosome cellular distribution. Thus, our results suggest that PLEKHM2 is involved in the functional development of neurons through the regulation of autophagic flux.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology provides the means for accurate genomic editing. It has been applied in many kinds of cells and animals for functional genomic studies and for precise selective breeding. Nonetheless, this method has not yet been applied in one of the most important – and well studied – decapod crustacean aquaculture species, the giant freshwater prawn Macrobrachium rosenbergii. We thus established two CRISPR platforms for M. rosenbergii—the first through direct injection into early-stage embryos (entire organism genome editing) and the second by electroporation of a primary embryonic cell culture. The systems were calibrated by optimizing Cas9 concentrations, delivery methods and editing efficiencies. Editing patterns utilizing multiple guides were examined through next generation sequencing. Our results showed a wide range of editing efficiencies in embryos, in some cases reaching as high as 100%. In contrast, in primary embryonic cell cultures, the highest editing efficiency obtained reached a maximum of 64%. In addition, there was a striking difference between the two platforms in terms of the pattern of deletions around the Cas9 cut site. This finding suggests distinct repair mechanisms in the two systems, which calls for further clarification. A phenotypic proof of concept was provided through the investigation of an early acting paired box protein 6 (Pax6) transcription factor, which showed clear effects on eye development in edited embryos and larvae. The current study lays down the foundations for precise functional genomic research and applications of genome editing in crustacean species for both aquaculture and sustainable biocontrol, opening opportunities for the creation of selected crustacean lines with distinct attributes.

Obstructive sleep apnea syndrome (OSAS) patients suffer from cardiovascular morbidity, which is the leading cause of death in this disease. Based on our previous work with transformed cell lines and primary rat cardiomyocytes, we determined that upon incubation with sera from pediatric OSAS patients, the cell’s morphology changes, NF-κB pathway is activated, and their beating rate and viability decreases. These results suggest an important link between OSAS, systemic inflammatory signals and end-organ cardiovascular diseases. In this work, we confirmed and expanded these observations on a new in vitro system of beating human cardiomyocytes (CM) differentiated from human embryonic stem cells (hES). Our results show that incubation with pediatric OSAS sera, in contrast to sera from healthy children, induces over-expression of NF-κB p50 and p65 subunits, marked reduction in CMs beating rate, contraction amplitude and a strong reduction in intracellular calcium signal. The use of human CM cells derived from embryonic stem cells has not been previously reported in OSAS research. The results further support the hypothesis that NF-κB dependent inflammatory pathways play an important role in the evolution of cardiovascular morbidity in OSAS. This study uncovers a new model to investigate molecular and functional aspects of cardiovascular pathology in OSAS.

Novel monosex biotechnologies in crustacean aquaculture involve the induction of sex reversal through manipulations of the androgenic gland (AG), and its most prominent factor, the insulin-like androgenic gland (IAG) hormone, during early developmental stages. In the giant freshwater prawn Macrobrachium rosenbergii, all-female populations are desirable for aquaculture, since the females can be stocked at higher densities and exhibit more uniform growth than males. Recently, a novel biotechnology for all-female aquaculture was developed based on injection into post larvae of suspended primary AG cells from mature M. rosenbergii males. However, this biotechnology depends on the availability of appropriate male donors and it requires delicate surgical procedures on the small endocrine AG to produce appropriate quantities of cells for the sex manipulation. We therefore established a new platform for the production of M. rosenbergii IAG (Mr-IAG) in hematopoietic cells. The method rests on the induction of ectopic Mr-IAG expression under the white spot syndrome virus (WSSV) immediate-early 1 (IE1) promoter via a novel lentiviral vector. Our results demonstrate that M. rosenbergii primary cells infected with the Mr-IAG lentiviruses are capable of transcription, translation and secretion of Mr-IAG in culture. Our new platform, which produces easy-to-harvest cells in abundance, could replace the AG cells used in the first step of the above-mentioned biotechnology for all-female aquaculture and, importantly, pave the way for producing monosex populations in other edible crustacean aquaculture species. In addition, a lentiviral system for crustacean cells provides a useful tool for basic and applied research in crustacean species.