Prospectively Isolated Tetraspanin+ Neoblasts Are Adult Pluripotent Stem Cells Underlying Planaria Regeneration.

Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.

The complexities of ligand/receptor interactions: Exploring the role of molecular vibrations and quantum tunnelling.

Molecular vibrations and quantum tunneling may link ligand binding to the function of pharmacological receptors. The well-established lock-and-key model explains a ligand's binding and recognition by a receptor; however, a general mechanism by which receptors translate binding into activation, inactivation, or modulation remains elusive. The Vibration Theory of Olfaction was proposed in the 1930s to explain this subset of receptor-mediated phenomena by correlating odorant molecular vibrations to smell, but a mechanism was lacking. In the 1990s, inelastic electron tunneling was proposed as a plausible mechanism for translating molecular vibration to odorant physiology. More recently, studies of ligands' vibrational spectra and the use of deuterated ligand analogs have provided helpful information to study this admittedly controversial hypothesis in metabotropic receptors other than olfactory receptors. In the present work, based in part on published experiments from our laboratory using planarians as an experimental organism, I will present a rationale and possible experimental approach for extending this idea to ligand-gated ion channels.

The DNA methyltransferase DMAP1 is required for tissue maintenance and planarian regeneration.

The precise regulation of transcription is required for embryonic development, adult tissue turnover, and regeneration. Epigenetic modifications play a crucial role in orchestrating and regulating the transcription of genes. These modifications are important in the transition of pluripotent stem cells and their progeny. Methylation, a key epigenetic modification, influences gene expression through changes in DNA methylation. Work in different organisms has shown that the DNA methyltransferase-1-associated protein (DMAP1) may associate with other molecules to repress transcription through DNA methylation. Thus, DMAP1 is a versatile protein implicated in a myriad of events, including pluripotency maintenance, DNA damage repair, and tumor suppression. While DMAP1 has been extensively studied in vitro, its complex regulation in the context of the adult organism remains unclear. To gain insights into the possible roles of DMAP1 at the organismal level, we used planarian flatworms that possess remarkable regenerative capabilities driven by pluripotent stem cells called neoblast. Our findings demonstrate the evolutionary conservation of DMAP1 in the planarian Schmidtea mediterranea. Functional disruption of DMAP1 through RNA interference revealed its critical role in tissue maintenance, neoblast differentiation, and regeneration in S. mediterranea. Moreover, our analysis unveiled a novel function for DMAP1 in regulating cell death in response to DNA damage and influencing the expression of axial polarity markers. Our findings provide a simplified paradigm for studying DMAP1's function in adult tissues.

Restoration of DNA integrity and the cell cycle by electric stimulation in planarian tissues damaged by ionizing radiation.

Exposure to high levels of ionizing γ radiation leads to irreversible DNA damage and cell death. Here, we establish that exogenous application of electric stimulation enables cellular plasticity and the re-establishment of stem cell activity in tissues damaged by ionizing radiation. We show that subthreshold direct current stimulation (DCS) rapidly restores pluripotent stem cell populations previously eliminated by lethally γ-irradiated tissues of the planarian flatworm Schmidtea mediterranea. Our findings reveal that DCS enhances DNA repair, transcriptional activity, and cell cycle entry in post-mitotic cells. These responses involve rapid increases in cytosolic Ca2+ concentration through the activation of L-type Cav channels and intracellular Ca2+ stores, leading to the activation of immediate early genes and ectopic expression of stem cell markers in post-mitotic cells. Overall, we show the potential of electric current stimulation to reverse the damaging effects of high-dose γ radiation in adult tissues. Furthermore, our results provide mechanistic insights describing how electric stimulation effectively translates into molecular responses capable of regulating fundamental cellular functions without the need for genetic or pharmacological intervention.

Non-senescent species are not immortal: Stress and decline in two planaria species.

Potential immortality is observed in several species (e.g. prickly pear cactus, hydra and flatworms) and is indicative of their negligible or even negative senescence rates. Unlike in senescent species, which experience reduced individual performance with age due to physiological degradation, species with negligible or negative senescence display mortality rates that remain constant or decline with age, respectively. These rates vary across taxa and are correlated with life history traits. Yet, the extent to which variable resource availability, a key driver of variation in life history traits, impacts species that show negligible or negative senescence is currently unknown. Here, we examine whether and how variation in the quantity, quality and feeding interval of resources impact population structure, population performance and life history trait trade-offs in two long-lived planaria that do not senesce: Schmidtea mediterranea and Dugesia tahitiensis. In a full factorial design, different combinations of resource quantity (reduced intake, standard intake and high intake) and quality (high and low quality) were provided in two different feeding intervals (7-day and 14-day intervals) for 19 weeks. We show that variability in resource availability, via decreases in quantity, quality and frequency of resources, does not diminish population viability in either species but does result in suboptimal conditions of stress in S. mediterranea. The high population viability we report can be attributed to two different mechanisms: increased reproduction or increased investment into maintenance at the expense of reproduction. Moreover, which mechanism was responsible for said high population viability was context-dependent and modulated by the specific life history strategy of the two planaria species. We show that suboptimal conditions can cause stress responses that have significant impacts on non-senescent species. The context-dependent response we observe suggests that species that do not senesce but are subject to suboptimal conditions of stress may ultimately exhibit declines in performance and ultimately die. A clearer understanding of the impact of suboptimal conditions of resource availability on non-senescent species is needed to determine the extent of stress experienced and ultimately whether a species can truly be immortal.

Cysteinyl-specialized proresolving mediators link resolution of infectious inflammation and tissue regeneration via TRAF3 activation.

The recently elucidated proresolving conjugates in tissue regeneration (CTR) maresin-CTR (MCTR), protectin-CTR (PCTR), and resolvin-CTR (RCTR), termed cysteinyl-specialized proresolving mediators (cys-SPMs) each promotes regeneration, controls infection, and accelerates resolution of inflammation. Here, we sought evidence for cys-SPM activation of primordial pathways in planaria (Dugesia japonica) regeneration that might link resolution of inflammation and regeneration. On surgical resection, planaria regeneration was enhanced with MCTR3, PCTR3, or RCTR3 (10 nM), each used for RNA sequencing. The three cys-SPMs shared up-regulation of 175 known transcripts with fold-change > 1.25 and combined false discovery rate (FDR) < 0.002, and 199 canonical pathways (FDR < 0.25), including NF-κB pathways and an ortholog of human TRAF3 (TNFR-associated factor 3). Three separate pathway analyses converged on TRAF3 up-regulation by cys-SPMs. With human macrophages, three cys-SPMs each dose-dependently increased TRAF3 expression in a cAMP-PKA-dependent manner. TRAF3 overexpression in macrophages enhanced Interleukin-10 (IL-10) and phagocytosis of Escherichia coli IL-10 also increased phagocytosis in a dose-dependent manner. Silencing of mouse TRAF3 in vivo significantly reduced IL-10 and macrophage phagocytosis. TRAF3 silencing in vivo also relieved cys-SPMs' actions in limiting polymorphonuclear neutrophil in E. coli exudates. These results identify cys-SPM-regulated pathways in planaria regeneration, uncovering a role for TRAF3/IL-10 in regulating mammalian phagocyte functions in resolution. Cys-SPM activation of TRAF3 signaling is a molecular component of both regeneration and resolution of infectious inflammation.

Extracellular matrix-regulator MMPA is required for the orderly proliferation of neoblasts and differentiation of ectodermal progenitor cells in the planarian Dugesia japonica.

Matrix metalloproteinases (MMPs) are members of a family of zinc-dependent metallopeptidase proteins that are widely found in plants, animals, and microorganisms. As the regulators of the extracellular matrix and basement membrane, MMPs play an important role in embryogenesis, development, innate immunity, and regeneration. However, the function of MMP family in planarian, a model for regeneration research, is still ambiguous. Here, we cloned 5 MMPs genes from Dugesia japonica and found that DjMMPA was associated with the process of regeneration, neoblasts cell maintenance confusion and destruction. Loss of DjMMPA led to homeostasis confusion and eventually death, owing to neoblasts proliferation disorder. Additionally, DjMMPA RNAi-treated animals had impaired regeneration after amputation. Furthermore, knockdown of DjMMPA had noticeable defects in cell differentiation of ectoderm, especially in eyes and neural progenitor cells, possibly by inhibiting Wnt signaling. Our results suggest that extracellular matrix-regulator MMPA is required for the orderly proliferation of neoblasts and differentiation of ectodermal progenitor cells in the planarian, which provide valuable information for further explorations into the molecular mechanism of MMPS, stem cells, and regeneration.

Comprehensive annotation and characterization of planarian tRNA and tRNA-derived fragments (tRFs).

tRNA-derived fragments (tRFs) have recently gained a lot of scientific interest due to their diverse regulatory roles in several cellular processes. However, their function in dynamic biological processes such as development and regeneration remains unexplored. Here, we show that tRFs are dynamically expressed during planarian regeneration, suggesting a possible role for these small RNAs in the regulation of regeneration. In order to characterize planarian tRFs, we first annotated 457 tRNAs in S. mediterranea combining two tRNA prediction algorithms. Annotation of tRNAs facilitated the identification of three main species of tRFs in planarians-the shorter tRF-5s and itRFs, and the abundantly expressed 5'-tsRNAs. Spatial profiling of tRFs in sequential transverse sections of planarians revealed diverse expression patterns of these small RNAs, including those that are enriched in the head and pharyngeal regions. Expression analysis of these tRF species revealed dynamic expression of these small RNAs over the course of regeneration suggesting an important role in planarian anterior and posterior regeneration. Finally, we show that 5'-tsRNA in planaria interact with all three SMEDWI proteins and an involvement of AGO1 in the processing of itRFs. In summary, our findings implicate a novel role for tRFs in planarian regeneration, highlighting their importance in regulating complex systemic processes. Our study adds to the catalog of posttranscriptional regulatory systems in planaria, providing valuable insights on the biogenesis and the function of tRFs in neoblasts and planarian regeneration.

Phylotranscriptomics interrogation uncovers a complex evolutionary history for the planarian genus Dugesia (Platyhelminthes, Tricladida) in the Western Mediterranean.

The Mediterranean is one of the most biodiverse areas of the Paleartic region. Here, basing on large data sets of single copy orthologs obtained from transcriptomic data, we investigated the evolutionary history of the genus Dugesia in the Western Mediterranean area. The results corroborated that the complex paleogeological history of the region was an important driver of diversification for the genus, speciating as microplates and islands were forming. These processes led to the differentiation of three main biogeographic clades: Iberia-Apennines-Alps, Corsica-Sardinia, and Iberia-Africa. The internal relationships of these major clades were analysed with several representative samples per species. The use of large data sets regarding the number of loci and samples, as well as state-of-the-art phylogenomic inference methods allowed us to answer different unresolved questions about the evolution of particular groups, such as the diversification path of D. subtentaculata in the Iberian Peninsula and its colonization of Africa. Additionally, our results support the differentiation of D. benazzii in two lineages which could represent two species. Finally, we analysed here for the first time a comprehensive number of samples from several asexual Iberian populations whose assignment at the species level has been an enigma through the years. The phylogenies obtained with different inference methods showed a branching topology of asexual individuals at the base of sexual clades. We hypothesize that this unexpected topology is related to long-term asexuality. This work represents the first phylotranscriptomic analysis of Tricladida, laying the first stone of the genomic era in phylogenetic studies on this taxonomic group.

Transplantation of fragments from different planaria: A bioelectrical model for head regeneration.

Head-tail planaria morphologies are influenced by the electric potential differences across the animal's primary axis, as evidenced e.g. by voltage-sensitive dyes and functional experiments that create permanent lines of 2-headed but genetically wild-type animals. However, bioelectrical and biochemical models that make predictions on what would happen in the case of spatial chimeras made by tissue transplantation from different planaria (different species and head shapes) are lacking. Here, we use a bioelectrical model to qualitatively describe the effects of tissue transplantation on the shape of the regenerated head. To this end, we assume that the cells may have distinct sets of ion channels and ascribe the system outcome to the axial distributions of average cell potentials over morphologically relevant regions. Our rationale is that the distributions of signaling ions and molecules are spatially coupled with multicellular electric potentials. Thus, long-time downstream transcriptional events should be triggered by short-time bioelectrical processes. We show that relatively small differences between the ion channel characteristics of the cells could eventually give noticeable changes in the electric potential profiles and the expected morphological deviations, which suggests that small but timely bioelectrical actions may have significant morphological effects. Our approach is based on the observed relationships between bioelectrical regionalization and biochemical gradients in body-plan studies. Such models are relevant to regenerative, developmental, and cancer biology in which cells with distinct properties and morphogenetic target states confront each other in the same tissue.

CREB-binding protein (CBP) gene family regulates planarian survival and stem cell differentiation.

In developmental biology, the regulation of stem cell plasticity and differentiation remains an open question. CBP(CREB-binding protein)/p300 is a conserved gene family that functions as a transcriptional co-activator and plays important roles in a wide range of cellular processes, including cell death, the DNA damage response, and tumorigenesis. The acetyl transferase activity of CBPs is particularly important, as histone and non-histone acetylation results in changes in chromatin architecture and protein activity that affect gene expression. Many studies have described the conserved functions of CBP/p300 in stem cell proliferation and differentiation. The planarian Schmidtea mediterranea is an excellent model for the in vivo study of the molecular mechanisms underlying stem cell differentiation during regeneration. However, how this process is regulated genetically and epigenetically is not well-understood yet. We identified 5 distinct Smed-cbp genes in S. mediterranea that show different expression patterns. Functional analyses revealed that Smed-cbp-2 appears to be essential for stem cell maintenance. On the other hand, the silencing of Smed-cbp-3 resulted in the growth of blastemas that were apparently normal, but remained largely unpigmented and undifferentiated. Smed-cbp-3 silencing also affected the differentiation of several cell lineages including neural, epidermal, digestive, and excretory cell types. Finally, we analysed the predicted interactomes of CBP-2 and CBP-3 as an initial step to better understand their functions in planarian stem cell biology. Our results indicate that planarian cbp genes play key roles in stem cell maintenance and differentiation.

Metabolic cost of development, regeneration, and reproduction in the planarian Schmidtea mediterranea.

Planaria are known for their ability to completely regenerate upon fissioning or experimental amputation. Yet, metabolic costs of regeneration have not been directly measured in planaria. Our goal was to establish the relationships between oxygen consumption (V̇O2), regeneration, and reproductive mode for asexual and sexual strains of Schmidtea mediterranea. We hypothesized that V̇O2 would vary by regeneration day for both sexual and asexual S. mediterranea, reflecting different costs of tissue reconstruction, but with an additional cost for regenerating sexual organs. Testes regeneration and body mass, as indicators of regeneration progress, and routine mass-specific V̇O2 as a function of maturity, regeneration, and reproductive mode, were measured over a 22-day regeneration period. Testes growth was highest in sexually mature adults, ~1/2 that in 14-day post-amputation sexual adults, and not detectable in juveniles and hatchlings. Mass-specific routine V̇O2 in sexuals was highest in mature controls at ~23 µl O2/g/h, but only half that in juveniles, hatchlings, and 14 day post-amputation adults. Both intact and 14-day post-amputation asexuals had a mass-specific routine V̇O2 of ~10-12 µl O2/g/h. The sum of V̇O2 of all amputated sections was ~100% higher than pre-amputation levels in the first 6 days of regeneration in asexuals, but not sexuals. There was no significant difference in V̇O2 of head, middle, and tail sections during regeneration. Overall, the highest metabolic costs associated with regeneration occurred during the initial 1-6 days of regeneration in both strains, but regeneration costs for sexual structures were not reflected in major V̇O2 differences between sexual and asexual strains.

Metabolites of Cannabis Induce Cardiac Toxicity and Morphological Alterations in Cardiac Myocytes.

Cannabis is one of the most commonly used recreational drugs worldwide. Rrecent epidemiology studies have linked increased cardiac complications to cannabis use. However, this literature is predominantly based on case incidents and post-mortem investigations. This study elucidates the molecular mechanism of Δ9-tetrahydrocannabinol (THC), and its primary metabolites 11-Hydroxy-Δ9-THC (THC-OH) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH). Treatment of cardiac myocytes with THC-OH and THC-COOH increased cell migration and proliferation (p < 0.05), with no effect on cell adhesion, with higher doses (250-100 ng/mL) resulting in increased cell death and significant deterioration in cellular architecture. Conversely, no changes in cell morphology or viability were observed in response to THC. Expression of key ECM proteins α-SMA and collagen were up-regulated in response to THC-OH and THC-COOH treatments with concomitant modulation of PI3K and MAPK signalling. Investigations in the planarian animal model Polycelis nigra demonstrated that treatments with cannabinoid metabolites resulted in increased protein deposition at transection sites while higher doses resulted in significant lethality and decline in regeneration. These results highlight that the key metabolites of cannabis elicit toxic effects independent of the parent and psychoactive compound, with implications for cardiotoxicity relating to hypertrophy and fibrogenesis.

Planarian regeneration between 1960s and 1990s: From skilful baffled ancestors to bold integrative descendants. A personal account.

Scientific fields grow by accretion of knowledge brought up by succesive generations of scientists. With the field of planarian regeneration as a general background, here I give a personal account of it from the late 1960s until the late 1990s when new research groups, namely Americans, broke into it. After a short historical summary, I report how I got into regeneration, why I choose planarians, and most especially the finding and description of the current model organism Schmidtea mediterranea. Next, I concentrate on the quantitative cellular approaches to regeneration, growth, and degrowth undertaken in our lab in Barcelona, and the long struggle to set neoblasts as a totipotent/pluripotent heterogeneous population of mitotic and non-mitotic cells for homeostatic somatic cell renewal, reproduction, and blastema formation during regeneration. Finally, the first attempts by several labs to analyze regeneration in molecular terms (RNAs, proteins, mAbs, gene detection, cloning and expression) are also covered. Along this essay, I am honoured to pay due tribute to our forebears (ancestors) from Morgan and Child to the French School researchers. In spite of being often baffled by planarian 'tricks', they struggled hard to find new techniques, new ways, and new ideas to tackle the main problems of regeneration, paving the way to those (descendants) that followed suit.

Planarian regeneration as a model of anatomical homeostasis: Recent progress in biophysical and computational approaches.

Planarian behavior, physiology, and pattern control offer profound lessons for regenerative medicine, evolutionary biology, morphogenetic engineering, robotics, and unconventional computation. Despite recent advances in the molecular genetics of stem cell differentiation, this model organism's remarkable anatomical homeostasis provokes us with truly fundamental puzzles about the origin of large-scale shape and its relationship to the genome. In this review article, we first highlight several deep mysteries about planarian regeneration in the context of the current paradigm in this field. We then review recent progress in understanding of the physiological control of an endogenous, bioelectric pattern memory that guides regeneration, and how modulating this memory can permanently alter the flatworm's target morphology. Finally, we focus on computational approaches that complement reductive pathway analysis with synthetic, systems-level understanding of morphological decision-making. We analyze existing models of planarian pattern control and highlight recent successes and remaining knowledge gaps in this interdisciplinary frontier field.

Staying in shape: Planarians as a model for understanding regenerative morphology.

A key requirement of tissue/organ regeneration is the ability to induce appropriate shape in situ. Regenerated structures need to be integrated with pre-existing ones, through the combined regulation of new tissue growth and the scaling of surrounding tissues. This requires a tightly coordinated control of individual cell functions such as proliferation and stem cell differentiation. While great strides have been made in elucidating cell growth and differentiation mechanisms, how overall shape is generated during regeneration remains unknown. This is because a significant gap remains in our understanding of how cell behaviors are coordinated at the level of tissues and organs. The highly regenerative planarian flatworm has emerged as an important model for defining and understanding regenerative shape mechanisms. This review provides an overview of the main processes known to regulate tissue and animal shape during planarian regeneration: adult stem cell regulation, the reestablishment of body axes, tissue remodeling in pre-existing structures, organ scaling and the maintenance of body proportion, and the bioelectrical regulation of animal morphology. In order for the field to move forward, it will be necessary to identify shape mutants as a means to uncover the molecular mechanisms that synchronize all these separate processes to produce the worm's final regenerative shape. This knowledge will also aid efforts to define the mechanisms that control the termination of regenerative processes.

DNA damage and tissue repair: What we can learn from planaria.

Faithful renewal of aging and damaged tissues is central to organismal lifespan. Stem cells (SCs) generate the cellular progeny that replenish adult tissues across the body but this task becomes increasingly compromised over time. The age related decline in SC-mediated tissue maintenance is a multifactorial event that commonly affects genome integrity. The presence of DNA damage in SCs that are under continuous demand to divide poses a great risk for age-related disorders such as cancer. However, performing analysis of SCs with genomic instability and the DNA damage response during tissue renewal present significant challenges. Here we introduce an alternative experimental system based on the planaria flatworm Schmidtea mediterranea to address at the organismal level studies intersecting SC-mediated tissue renewal in the presence of genomic instability. Planaria have abundant SCs (neoblasts) that maintain high rates of cellular turnover and a variety of molecular tools have been developed to induce DNA damage and dissect how neoblasts respond to this stressor. S. mediterranea displays high evolutionary conservation of DNA repair mechanisms and signaling pathways regulating adult SCs. We describe genetically induced-DNA damage models and highlight body-wide signals affecting cellular decisions such as survival, proliferation, and death in the presence of genomic instability. We also discuss transcriptomic changes in the DNA damage response during injury repair and propose DNA repair as key component of tissue regeneration. Additional studies using planaria will provide insights about mechanisms regulating survival and growth of cells with DNA damage during tissue renewal and regeneration.

Nervous system and tissue polarity dynamically adapt to new morphologies in planaria.

The coordination of tissue-level polarity with organism-level polarity is crucial in development, disease, and regeneration. Here, we characterize a new example of large-scale control of dynamic remodeling of body polarity. Exploiting the flexibility of the body plan in regenerating planarians, we used mirror duplication of the primary axis to show how established tissue-level polarity adapts to new organism-level polarity. Characterization of epithelial planar cell polarity revealed a remarkable reorientation of tissue polarity in double-headed planarians. This reorientation of cilia occurs even following irradiation-induced loss of all stem cells, suggesting independence of the polarity change from the formation of new cells. The presence of the two heads plays an important role in regulating the rate of change in overall polarity. We further present data that suggest that the nervous system itself adapts its polarity to match the new organismal anatomy as revealed by changes in nerve transport driving distinct regenerative outcomes. Thus, in planaria tissue-level polarity can dynamically reorient to match the organism-level anatomical configuration.

Sex-Inducing Activities of the Land Planarian Bipalium nobile Extract Fractions, Obtained Using Bioassay-Guided Fractionation, in the Freshwater Planarian Dugesia ryukyuensis.

Sexually mature planarians produce sex-inducing substances that induce postembryonic development of hermaphroditic reproductive organs in asexual freshwater planarians. Although the sex-inducing substances may be useful for elucidating the mechanism underlying this reproductive switch, the available information is limited. The potency of sex-inducing activity is conserved, at least at the order level. Recently, we showed that the sex-inducing activity in the land planarian Bipalium nobile was much higher than that in freshwater planarians. In the present study, we performed bioassay-guided fractionation of the sex-inducing substances produced by B. nobile and propose that crucial sex-inducing activity that triggers complete sexualization for asexual worms of the freshwater planarian Dugesia ryukyuensis is produced by additive and/or synergetic effects of various sex-inducing substances involved in ovarian development. The current study provided an isolation scheme for the minimum-required combination of sex-inducing substances for producing crucial sex-inducing activity.

A Comprehensive Conceptual and Computational Dynamics Framework for Autonomous Regeneration Systems.

Many biological organisms regenerate structure and function after damage. Despite the long history of research on molecular mechanisms, many questions remain about algorithms by which cells can cooperate towards the same invariant morphogenetic outcomes. Therefore, conceptual frameworks are needed not only for motivating hypotheses for advancing the understanding of regeneration processes in living organisms, but also for regenerative medicine and synthetic biology. Inspired by planarian regeneration, this study offers a novel generic conceptual framework that hypothesizes mechanisms and algorithms by which cell collectives may internally represent an anatomical target morphology towards which they build after damage. Further, the framework contributes a novel nature-inspired computing method for self-repair in engineering and robotics. Our framework, based on past in vivo and in silico studies on planaria, hypothesizes efficient novel mechanisms and algorithms to achieve complete and accurate regeneration of a simple in silico flatwormlike organism from any damage, much like the body-wide immortality of planaria, with minimal information and algorithmic complexity. This framework that extends our previous circular tissue repair model integrates two levels of organization: tissue and organism. In Level 1, three individual in silico tissues (head, body, and tail-each with a large number of tissue cells and a single stem cell at the centre) repair themselves through efficient local communications. Here, the contribution extends our circular tissue model to other shapes and invests them with tissue-wide immortality through an information field holding the minimum body plan. In Level 2, individual tissues combine to form a simple organism. Specifically, the three stem cells form a network that coordinates organism-wide regeneration with the help of Level 1. Here we contribute novel concepts for collective decision-making by stem cells for stem cell regeneration and large-scale recovery. Both levels (tissue cells and stem cells) represent networks that perform simple neural computations and form a feedback control system. With simple and limited cellular computations, our framework minimises computation and algorithmic complexity to achieve complete recovery. We report results from computer simulations of the framework to demonstrate its robustness in recovering the organism after any injury. This comprehensive hypothetical framework that significantly extends the existing biological regeneration models offers a new way to conceptualise the information-processing aspects of regeneration, which may also help design living and non-living self-repairing agents.