Regeneration In Animals Research Articles

From regeneration to the evolution of development and philosophy: the work of professor Galina Korotkova (1924–2009), on the 100th anniversary of her birth

Evolutionary and developmental biology are among the most dynamically developing areas of modern biology. Both have a long and turbulent history, especially in Russia (USSR). However, any science develops thanks to the breakthrough research of individual scientists and scientific teams. In this paper, we briefly analyzed the main theoretical works of Leningrad State University professor G. P. Korotkova (1924–2009), who made a significant contribution to general biology at the end of the twentieth century. G. P. Korotkova is known for her pioneering work in the field of regeneration of invertebrate animals and, first of all, sponges, evolutionary and philosophical aspects of biology. In particular, her contribution to the theoretical aspects of regenerative biology, to the development of philosophical aspects of the problems of wholenessis considered. The principles of her original hypothesis of the origin and phase evolution of ontogenesis are briefly outlined, as well as her ideas regarding the theoretical aspects of the biology and organization of sponges (Porifera).

Cordycepin-Loaded Dental Pulp Stem Cell-Derived Exosomes Promote Aged Bone Repair by Rejuvenating Senescent Mesenchymal Stem Cells and Endothelial Cells.

Aging impairs bone marrow mesenchymal stem cell (BMSC) functions as well as associated angiogenesis which is critical for bone regeneration and repair. Hence, repairing bone defects in elderly patients poses a formidable challenge in regenerative medicine. Here, the engineered dental pulp stem cell-derived exosomes loaded with the natural derivative of adenosine Cordycepin (CY@D-exos) are fabricated by means of the intermittent ultrasonic shock, which dually rejuvenates both senescent BMSCs and endothelial cells and significantly improve bone regeneration and repair in aged animals. CY@D-exos can efficiently overcome the senescence of aged BMSCs and enhance their osteogenic differentiation by activating NRF2 signaling and maintaining heterochromatin stability. Importantly, CY@D-exos also potently overcomes the senescence of vascular endothelial cells and promotes angiogenesis. In vivo injectable gelatin methacryloyl (GelMA) hydrogels with sustained release of CY@D-exos can accelerate bone injury repair and promote new blood vessel formation in aged animals. Taken together, these results thus demonstrate that cordycepin-loaded dental pulp stem cell-derived exosomes display considerable potential to be developed as a next-generation therapeutic agent for promoting aged bone regeneration and repair.

An Updated Review on the Mechanisms, Pre‐Clinical and Clinical Comparisons of Nicotinamide Mononucleotide (NMN) and Nicotinamide Riboside (NR)

ABSTRACTThe study of nicotinamide adenine dinucleotide (NAD+) biology has gained popularity recently. Research discovered that two NAD+ biosynthesis intermediates, nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), are biosynthetic precursors of NAD+, an essential chemical for metabolism. Many studies show that NAD+ levels fall considerably with aging, with NAD+ regeneration in aging animals increasing longevity and enhancing health. According to research on the two molecules, NMN and NR, supplementation with either raises NAD+ levels during aging. The objective of this review is to highlight current developments in the biology of NAD+ in relation to NR or NMN supplement. This review highlightings significant discoveries about NR and NMN supplementation in relation to NAD+ biological activities and health benefits.

Generation of human expandable limb-bud-like progenitors via chemically induced dedifferentiation

In certain highly regenerative animals, cellular dedifferentiation occurs after injury, allowing specialized cells to become progenitor cells for regeneration. However, this capacity is restricted in human cells due to reduced plasticity. Here, we introduce a chemical-induced dedifferentiation approach that reverts the differentiated cells to a progenitor-like state, conferring the features of human limb bud cells from human adult somatic cells. These chemically induced human limb-bud-like progenitors (hCiLBP cells) show a high degree of transcriptomic similarity to human embryonic limb bud progenitors. Importantly, we established culture conditions that allow hCiLBP cells to undergo extensive expansion while maintaining population homogeneity and long-term self-renewal capacity. Moreover, hCiLBP cells exhibit increased osteochondrogenic differentiation ability, providing an innovative platform for generation of skeletal lineage cell types. These results highlight a potential therapeutic approach for repairing damaged human tissues through reversal of developmental pathways from mature cells to expandable progenitor cells.

Atomic Force Microscopy Measurements of Cartilage in Intact and Regenerating Axolotl Limbs.

Mechanical forces provide important signals for normal cell function and pattern formation in developing tissues, and their role has been widely studied during embryogenesis and pathogenesis. Comparatively, little is known of these signals during animal regeneration. The axolotl is an important model organism for the study of regeneration, given its ability to fully restore many organs and tissues after injury, including missing cartilage and bone. Due to its crucial role as the main supporting tissue in the vertebrate body, regaining skeletal function during regeneration requires both the restoration of the missing structures as well as their mechanical properties. This protocol describes a method for processing axolotl limb samples for atomic force microscopy (AFM), which is the gold standard for probing cell and tissue mechanical properties at high spatial resolution. Taking advantage of the regenerative capabilities of the axolotl, this study measured the stiffness of limb cartilage during homeostasis and two stages of limb regeneration: tissue histolysis and cartilage condensation. We show that AFM is a valuable tool for gaining insights into dynamic tissue restructuring and the mechanical changes that occur during regeneration.

A population of Vasa2 and Piwi1 expressing cells generates germ cells and neurons in a sea anemone

Germline segregation, essential for protecting germ cells against mutations, occurs during early embryogenesis in vertebrates, insects and nematodes. Highly regenerative animals (e.g., cnidarians), however, retain stem cells with both germinal and somatic potentials throughout adulthood, but their biology and evolution remain poorly understood. Among cnidarians (e.g., sea anemones, jellyfish), stem cells are only known in few hydrozoans (e.g., Hydra). Here, we identify and characterize a rare, multipotent population of stem and/or progenitor cells expressing the conserved germline and multipotency proteins Vasa2 and Piwi1 in the sea anemone Nematostella vectensis. Using piwi1 and vasa2 transgenic reporter lines, we reveal that the Vasa2+/Piwi1+ cell population generates not only gametes, but also a diversity of proliferative somatic cells, including neural progenitors, in juveniles and adults. Our work has uncovered a multipotent population of Vasa2+/Piwi1+ stem/progenitor cells that forms the cellular basis to understand body plasticity and regenerative capacities in sea anemones and corals.

SoxC and MmpReg promote blastema formation in whole-body regeneration of fragmenting potworms Enchytraeus japonensis

Regeneration in many animals involves the formation of a blastema, which differentiates and organizes into the appropriate missing body parts. Although the mechanisms underlying blastema formation are often fundamental to regeneration biology, information on the cellular and molecular basis of blastema formation remains limited. Here, we focus on a fragmenting potworm (Enchytraeus japonensis), which can regenerate its whole body from small fragments. We find soxC and mmpReg as upregulated genes in the blastema. RNAi of soxC and mmpReg reduce the number of blastema cells, indicating that soxC and mmpReg promote blastema formation. Expression analyses show that soxC-expressing cells appear to gradually accumulate in blastema and constitute a large part of the blastema. Additionally, similar expression dynamics of SoxC orthologue genes in frog (Xenopus laevis) are found in the regeneration blastema of tadpole tail. Our findings provide insights into the cellular and molecular mechanisms underlying blastema formation across species.

Nanos2 marks precursors of somatic lineages and is required for germline formation in the sea anemone Nematostella vectensis.

In animals, stem cell populations of varying potency facilitate regeneration and tissue homeostasis. Notably, germline stem cells in both vertebrates and invertebrates express highly conserved RNA binding proteins, such as nanos, vasa, and piwi. In highly regenerative animals, these genes are also expressed in somatic stem cells, which led to the proposal that they had an ancestral role in all stem cells. In cnidarians, multi- and pluripotent interstitial stem cells have only been identified in hydrozoans. Therefore, it is currently unclear if cnidarian stem cell systems share a common evolutionary origin. We, therefore, aimed to characterize conserved stem cell marker genes in the sea anemone Nematostella vectensis. Through transgenic reporter genes and single-cell transcriptomics, we identify cell populations expressing the germline-associated markers piwi1 and nanos2 in the soma and germline, and gene knockout shows that Nanos2 is indispensable for germline formation. This suggests that nanos and piwi genes have a conserved role in somatic and germline stem cells in cnidarians.

Core-shell structured nanomembrane with sequential immune and vascular isolation effects followed by chondrogenic induction to promote stable stem cell-based subcutaneous cartilage regeneration in large animals

Bone marrow stem cell (BMSC)-regenerated cartilage (BRC) shows promising prospects for clinically repairing cartilage defects. However, when subcutaneously implanted into immunocompetent large animals, BRC is prone to ossification due to vascular invasion and inflammatory infiltration, compromising its chondrogenic stability. To address this challenge, we developed a core–shell structured KGN/Gel@Cur/PLCL nanomembrane using coaxial electrospinning technology to enhance the chondrogenic stability of BRC in subcutaneous niche. The shell layer comprises curcumin (Cur), known for its potent immunomodulatory and anti-angiogenic effects, and poly(L-lactide-co-caprolactone) (PLCL). The core layer comprises Kartogenin (KGN), a recognized chondrogenic inducer, and gelatin (Gel). This nanomembrane encapsulated BRCs induced from goat BMSCs in vitro and was subcutaneously implanted into autologous goats. The nanomembrane’s physical barrier and Cur release from the shell layer prevented vascular invasion and inflammatory infiltration. Additionally, KGN release from the core layer maintained the BRC’s chondrogenic phenotype and promoted cartilage maturation. These combined mechanisms significantly inhibited BRC ossification and preserved its chondrogenic stability. chondrogenic phenotype and promoted cartilage maturation. These combined mechanisms significantly inhibited BRC ossification and maintained its chondrogenic stability. Overall, this study highlights the effectiveness of the core–shell structured KGN/Gel@Cur/PLCL nanomembrane in providing sequential immune and vascular isolation, followed with chondrogenic induction, to prevent BRC ossification and preserve the chondrogenic stability of subcutaneously implanted BRC in large animal. This is crucial for the clinical translation of stem cell therapy into subcutaneous cartilage repair.

Role and Regulatory Mechanism of circRNA_14820 in the Proliferation and Differentiation of Goat Skeletal Muscle Satellite Cells.

Skeletal muscle satellite cells (SMSCs), a type of myogenic stem cell, play a pivotal role in postnatal muscle regeneration and repair in animals. Circular RNAs (circRNAs) are a distinct class of non-coding RNA molecules capable of regulating muscle development by modulating gene expression, acting as microRNAs, or serving as protein decoys. In this study, we identified circ_14820, an exonic transcript derived from adenosine triphosphatase family protein 2 (ATAD2), through initial RNA-Seq analysis. Importantly, overexpression of circ_14820 markedly enhanced the proliferation of goat SMSCs while concomitantly suppressing their differentiation. Moreover, circ_14820 exhibited predominant localization in the cytoplasm of SMSCs. Subsequent small RNA and mRNA sequencing of circ_14820-overexpressing SMSCs systematically elucidated the molecular regulatory mechanisms associated with circ_14820. Our preliminary findings suggest that the circ_14820-miR-206-CCND2 regulatory axis may govern the development of goat SMSCs. These discoveries contribute to a deeper understanding of circRNA-mediated mechanisms in regulating skeletal muscle development, thereby advancing our knowledge of muscle biology.

Highly regenerative species-specific genes improve age-associated features in the adult Drosophila midgut

BackgroundThe remarkable regenerative abilities observed in planarians and cnidarians are closely linked to the active proliferation of adult stem cells and the precise differentiation of their progeny, both of which typically deteriorate during aging in low regenerative animals. While regeneration-specific genes conserved in highly regenerative organisms may confer regenerative abilities and long-term maintenance of tissue homeostasis, it remains unclear whether introducing these regenerative genes into low regenerative animals can improve their regeneration and aging processes.ResultsHere, we ectopically express highly regenerative species-specific JmjC domain-encoding genes (HRJDs) in Drosophila, a widely used low regenerative model organism. Surprisingly, HRJD expression impedes tissue regeneration in the developing wing disc but extends organismal lifespan when expressed in the intestinal stem cell lineages of the adult midgut under non-regenerative conditions. Notably, HRJDs enhance the proliferative activity of intestinal stem cells while maintaining their differentiation fidelity, ameliorating age-related decline in gut barrier functions.ConclusionsThese findings together suggest that the introduction of highly regenerative species-specific genes can improve stem cell functions and promote a healthy lifespan when expressed in aging animals.

A wound-induced differentiation trajectory for neurons

Animals capable of whole-body regeneration can replace any missing cell type and regenerate fully functional new organs, including new brains, de novo. The regeneration of a new brain requires the formation of diverse neural cell types and their assembly into an organized structure with correctly wired circuits. Recent work in various regenerative animals has revealed transcriptional programs required for the differentiation of distinct neural subpopulations, however, how these transcriptional programs are initiated in response to injury remains unknown. Here, we focused on the highly regenerative acoel worm, Hofstenia miamia, to study wound-induced transcriptional regulatory events that lead to the production of neurons and subsequently a functional brain. Footprinting analysis using chromatin accessibility data on a chromosome-scale genome assembly revealed that binding sites for the Nuclear Factor Y (NFY) transcription factor complex were significantly bound during regeneration, showing a dynamic increase in binding within one hour upon amputation specifically in tail fragments, which will regenerate a new brain. Strikingly, NFY targets were highly enriched for genes with neuronal function. Single-cell transcriptome analysis combined with functional studies identified soxC+ stem cells as a putative progenitor population for multiple neural subtypes. Further, we found that wound-induced soxC expression is likely under direct transcriptional control by NFY, uncovering a mechanism for the initiation of a neural differentiation pathway by early wound-induced binding of a transcriptional regulator.

Exploring the Function of Epicardial Cells Beyond the Surface.

The epicardium, previously viewed as a passive outer layer around the heart, is now recognized as an essential component in development, regeneration, and repair. In this review, we explore the cellular and molecular makeup of the epicardium, highlighting its roles in heart regeneration and repair in zebrafish and salamanders, as well as its activation in young and adult postnatal mammals. We also examine the latest technologies used to study the function of epicardial cells for therapeutic interventions. Analysis of highly regenerative animal models shows that the epicardium is essential in regulating cardiomyocyte proliferation, transient fibrosis, and neovascularization. However, despite the epicardium's unique cellular programs to resolve cardiac damage, it remains unclear how to replicate these processes in nonregenerative mammalian organisms. During myocardial infarction, epicardial cells secrete signaling factors that modulate fibrotic, vascular, and inflammatory remodeling, which differentially enhance or inhibit cardiac repair. Recent transcriptomic studies have validated the cellular and molecular heterogeneity of the epicardium across various species and developmental stages, shedding further light on its function under pathological conditions. These studies have also provided insights into the function of regulatory epicardial-derived signaling molecules in various diseases, which could lead to new therapies and advances in reparative cardiovascular medicine. Moreover, insights gained from investigating epicardial cell function have initiated the development of novel techniques, including using human pluripotent stem cells and cardiac organoids to model reparative processes within the cardiovascular system. This growing understanding of epicardial function holds the potential for developing innovative therapeutic strategies aimed at addressing developmental heart disorders, enhancing regenerative therapies, and mitigating cardiovascular disease progression.

Optical Switchers to Manipulate Intracellular Pathways and Boost Tissue Regeneration

AbstractThe possibility to remotely manipulate intracellular pathways in single cells is among the current goals of regenerative medicine, demanding new strategies to enhance tissue repair and reprogram stem cell activity. Plasmonic nanomaterials are addressing this need, due to improvements in the controlled synthesis allowing convenient regulation and precise thermal positioning. Leveraging on the thermal properties of gold nanoprisms (AuNPs) and on the unparalleled regenerating capabilities of the small invertebrate Hydra vulgaris, here the possibility to activate the molecular machinery underlying the animal regeneration by using AuNPs and applying regular pulses of near infrared irradiation (NIR) is shown. The efficiency of the head regeneration, reproductive capability, and stem cell proliferation rate are boosted by the AuNP photostimulation, indicating NIR triggered hyperthermia as new tool to enhance tissue regeneration. By transcriptional profiling of key developmental genes in animals exposed to external heat or irradiated an estimation of the heat developed in vivo by intracellular nanoheaters is obtained, revealing Hydra as a living thermometer to test performance of plasmonic materials. These results shed light on a novel function of heat emitting nanoparticles to control cell stemness through the activation of molecular pathways that can be targeted for regenerative medicine or wound healing strategies.

A Novel Approach to Comparative RNA-Seq Does Not Support a Conserved Set of Orthologs Underlying Animal Regeneration.

Molecular studies of animal regeneration typically focus on conserved genes and signaling pathways that underlie morphogenesis. To date, a holistic analysis of gene expression across animals has not been attempted, as it presents a suite of problems related to differences in experimental design and gene homology. By combining orthology analyses with a novel statistical method for testing gene enrichment across large data sets, we are able to test whether tissue regeneration across animals shares transcriptional regulation. We applied this method to a meta-analysis of six publicly available RNA-Seq data sets from diverse examples of animal regeneration. We recovered 160 conserved orthologous gene clusters, which are enriched in structural genes as opposed to those regulating morphogenesis. A breakdown of gene presence/absence provides limited support for the conservation of pathways typically implicated in regeneration, such as Wnt signaling and cell pluripotency pathways. Such pathways are only conserved if we permit large amounts of paralog switching through evolution. Overall, our analysis does not support the hypothesis that a shared set of ancestral genes underlie regeneration mechanisms in animals. After applying the same method to heat shock studies and getting similar results, we raise broader questions about the ability of comparative RNA-Seq to reveal conserved gene pathways across deep evolutionary relationships.

Plant regeneration in the new era: from molecular mechanisms to biotechnology applications.

Plants or tissues can be regenerated through various pathways. Like animal regeneration, cell totipotency and pluripotency are the molecular basis of plant regeneration. Detailed systematic studies on Arabidopsis thaliana gradually unravel the fundamental mechanisms and principles underlying plant regeneration. Specifically, plant hormones, cell division, epigenetic remodeling, and transcription factors play crucial roles in reprogramming somatic cells and reestablishing meristematic cells. Recent research on basal non-vascular plants and monocot crops has revealed that plant regeneration differs among species, with various plant species using distinct mechanisms and displaying significant differences in regenerative capacity. Conducting multi-omics studies at the single-cell level, tracking plant regeneration processes in real-time, and deciphering the natural variation in regenerative capacity will ultimately help understand the essence of plant regeneration, improve crop regeneration efficiency, and contribute to future crop design.

Comparative model of minimal spinal cord injury reveals a rather anti-inflammatory response in the lesion site as well as increased proliferation in the central canal lining in the neonates compared to the adult rats.

Spinal cord injury (SCI) resulting from trauma decreases the quality of human life. Numerous clues indicate that the limited endogenous regenerative potential is a result of the interplay between the inhibitory nature of mature nervous tissue and the inflammatory actions of immune and glial cells. Knowledge gained from comparing regeneration in adult and juvenile animals could draw attention to factors that should be removed or added for effective therapy in adults. Therefore, we generated a minimal SCI (mSCI) model with a comparable impact on the spinal cord of Wistar rats during adulthood, preadolescence, and the neonatal period. The mechanism of injury is based on unilateral incision with a 20ga needle tip according to stereotaxic coordinates into the dorsal horn of the L4 lumbar spinal segment. The incision should harm a similar amount of gray matter on a coronal section in each group of experimental animals. According to our results, the impact causes mild injury with minimal adverse effects on the neurological functions of animals but still has a remarkable effect on nervous tissue and its cellular and humoral components. Testing the mSCI model in adults, preadolescents, and neonates revealed a rather anti-inflammatory response of immune cells and astrocytes at the lesion site, as well as increased proliferation in the central canal lining in neonates compared with adult animals. Our results indicate that developing nervous tissue could possess superior reparative potential and confirm the importance of comparative studies to advance in the field of neuroregeneration.

Regeneration in the absence of canonical neoblasts in an early branching flatworm.

The remarkable regenerative abilities of flatworms are closely linked to neoblasts - adult pluripotent stem cells that are the only division-competent cell type outside of the reproductive system. Although the presence of neoblast-like cells and whole-body regeneration in other animals has led to the idea that these features may represent the ancestral metazoan state, the evolutionary origin of both remains unclear. Here we show that the catenulid Stenostomum brevipharyngium, a member of the earliest-branching flatworm lineage, lacks conventional neoblasts despite being capable of whole-body regeneration and asexual reproduction. Using a combination of single-nuclei transcriptomics, in situ gene expression analysis, and functional experiments, we find that cell divisions are not restricted to a single cell type and are associated with multiple fully differentiated somatic tissues. Furthermore, the cohort of germline multipotency genes, which are considered canonical neoblast markers, are not expressed in dividing cells, but in the germline instead, and we experimentally show that they are neither necessary for proliferation nor regeneration. Overall, our results challenge the notion that canonical neoblasts are necessary for flatworm regeneration and open up the possibility that neoblast-like cells may have evolved convergently in different animals, independent of their regenerative capacity.

Remodeling and Regenerative Properties of Fully Absorbable Meshes for Abdominal Wall Defect Repair: A Systematic Review and Meta-Analysis of Animal Studies.

Fully absorbable meshes can repair abdominal wall defects and effectively reduce the incidence of complications, but different types of fully absorbable meshes have different remodeling and regeneration effects. In order to investigate and compare the effects of different fully absorbable meshes on remodeling and regeneration in animals and reduce the biological risk of clinical translation, SYRCLE was adopted to evaluate the methodological quality of the included studies, and GRADE and ConQual were used to evaluate the quality of evidence. According to the inclusion and exclusion criteria, a total of 22 studies related to fully absorbable meshes were included in this systematic review. These results showed that fiber-based synthetic materials and fiber-based natural materials exhibited better restorative and regenerative effects indicated by infiltration and neovascularization, when compared with a porcine acellular dermal matrix. In addition, the human acellular dermal matrix was found to have a similar regenerative effect on the host extracellular matrix and scaffold degradation compared to the porcine acellular dermal matrix, porcine intestinal submucosa, and fiber-based natural materials, but it offered higher tensile strength than the other three. The quality of the evidence in this field was found to be poor. The reasons for downgrading were analyzed, and recommendations for future research included more rigor in study design, more transparency in result reporting, more standardization of animal models and follow-up time for better evaluation of the remodeling and regenerative performance of abdominal wall hernia repair meshes, and less biological risk in clinical translation.

A chromatin code for limb segment identity in axolotl limb regeneration

The salamander limb correctly regenerates missing limb segments because connective tissue cells have segment-specific identities, termed "positional information". How positional information is molecularly encoded at the chromatin level has been unknown. Here, we performed genome-wide chromatin profiling in mature and regenerating axolotl limb connective tissue cells. We find segment-specific levels of histone H3K27me3 as the major positional mark, especially at limb homeoprotein gene loci but not their upstream regulators, constituting an intrinsic segment information code. During regeneration, regeneration-specific regulatory elements became active prior to the re-appearance of developmental regulatory elements. In the hand, the permissive chromatin state of the homeoprotein gene HoxA13 engages with the regeneration program bypassing the upper limb program. Comparison of regeneration regulatory elements with those found in other regenerative animals identified a core shared set of transcription factors, supporting an ancient, conserved regeneration program.