Regenerative Program Research Articles

Evidence for lung barrier regeneration by differentiation prior to binucleated and stem cell division.

With each breath, oxygen diffuses across remarkably thin alveolar type I (AT1) cells into underlying capillaries. Interspersed cuboidal AT2 cells produce surfactant and act as stem cells. Even transient disruption of this delicate barrier can promote capillary leak. Here, we selectively ablated AT1 cells, which uncovered rapid AT2 cell flattening with near-continuous barrier preservation, culminating in AT1 differentiation. Proliferation subsequently restored depleted AT2 cells in two phases, mitosis of binucleated AT2 cells followed by replication of mononucleated AT2 cells. M phase entry of binucleated and S phase entry of mononucleated cells were both triggered by AT1-produced hbEGF signaling via EGFR to Wnt-active AT2 cells. Repeated AT1 cell killing elicited exuberant AT2 proliferation, generating aberrant daughter cells that ceased surfactant function yet failed to achieve AT1 differentiation. This hyperplasia eventually resolved, yielding normal-appearing alveoli. Overall, this specialized regenerative program confers a delicate simple epithelium with functional resiliency on par with the physical durability of thicker, pseudostratified, or stratified epithelia.

End binding-3 inhibitor activates regenerative program in age-related macular degeneration

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.

One Step Double Augmentation with Human Dermis Allograft and Homologous PRP in Misdiagnosed and or Chronic Achilles Tendon Ruptures.

Misdiagnosed/chronic Achilles tendon injuries are rare and disabling for patients. The surgical treatment of these rare injuries aims to ensure the tendon heals mechanically and biologically. This is the prerequisite for a good clinical and functional outcome and reduces recurrences. The main aim of the study is to present a surgical technique that has proven to be original, reproducible, and capable of guaranteeing solid tendon repair and optimal tissue regeneration. We treated five patients, four males and one female, with the one-step double augmentation technique. All patients of this study complained of pain, but above all severe functional limitation that Achilles tendon injury had been causing for more than a month. In this study, we widely described the surgical technique, original and not found in the literature, which provides a biological graft (allograft of decellularized dermis) and homologous, thrombin-activated, platelet-rich plasma (H-PRP) in a single step. Surgical approach, always used by the first author, respected predefined steps: careful dissection and preparation of the peritendinous tissues from suture to the end of the procedure, tenorrhaphy, and augmentation with allopatch to obtain a mechanically effective repair to avoid recurrences, and finally "biological" augmentation with a unit of homologous, thrombin activated, PRP. We offered to all patients a regenerative rehabilitation program post-operatively. All patients were evaluated clinically (functional clinical tests and questionnaires) and instrumentally (elastic-sonography and perfusion MRI). The obtained results have been evaluated at a minimum follow-up of 18 months and a maximum of 24 months. In all patients pain was resolved, and district function and kinetic chains improved with resumption of daily activities, work, and sports. The present study confirmed the regenerative potential of decellularized dermis allograft and PRP (homologous and thrombin-activated). The same approach can also be exploited in cases of severe tendon destructuring and limited "intrinsic" regenerative potential at any age. The proposed one-step surgical technique of a double augmentation therefore appears useful, safe, reproducible, and applicable in all chronic tendon lesions with low regenerative potential.

Colitis-induced upregulation of tumor necrosis factor receptor-2 (TNFR2) terminates epithelial regenerative signaling to restore homeostasis

Colonic epithelial repair is a key determinant of health. Repair involves changes in epithelial differentiation, an extensive proliferative response, and upregulation of regeneration-associated "fetal-like" transcripts, including Ly6a (Sca-1), that represent Yap1 and interferon targets. However, little is known about how this regenerative program terminates and how homeostasis is restored during injury and inflammation. Here we show that, after the initial entry into the regenerative state, the subsequent upregulation of tumor necrosis factor (TNF) receptor 2 (R2, TNFR2, Tnfrsf1b) clears the regenerative signaling and restores homeostatic patterns of epithelial differentiation. Targeted deletion of epithelial TNFR2 invivo and in colonoid cultures revealed persistent expression of Ly6a, hyperproliferation, and reduced secretory differentiation. Moreover, mice lacking epithelial TNFR2 also failed to complete colon ulcer healing, suggesting that partial resolution of regenerative signaling is essential for the completion of the repair process. These results demonstrate how epithelial cells dynamically leverage a colitis-associated cytokine to choreograph repair.

Circadian clock regulator Bmal1 gates axon regeneration via Tet3 epigenetics in mouse sensory neurons

Axon regeneration of dorsal root ganglia (DRG) neurons after peripheral axotomy involves reconfiguration of gene regulatory circuits to establish regenerative gene programs. However, the underlying mechanisms remain unclear. Here, through an unbiased survey, we show that the binding motif of Bmal1, a central transcription factor of the circadian clock, is enriched in differentially hydroxymethylated regions (DhMRs) of mouse DRG after peripheral lesion. By applying conditional deletion of Bmal1 in neurons, in vitro and in vivo neurite outgrowth assays, as well as transcriptomic profiling, we demonstrate that Bmal1 inhibits axon regeneration, in part through a functional link with the epigenetic factor Tet3. Mechanistically, we reveal that Bmal1 acts as a gatekeeper of neuroepigenetic responses to axonal injury by limiting Tet3 expression and restricting 5hmC modifications. Bmal1-regulated genes not only concern axon growth, but also stress responses and energy homeostasis. Furthermore, we uncover an epigenetic rhythm of diurnal oscillation of Tet3 and 5hmC levels in DRG neurons, corresponding to time-of-day effect on axon growth potential. Collectively, our studies demonstrate that targeting Bmal1 enhances axon regeneration.

Endothelial FoxM1 reactivates aging-impaired endothelial regeneration for vascular repair and resolution of inflammatory lung injury.

Aging is a major risk factor of high incidence and increased mortality of acute respiratory distress syndrome (ARDS). Here, we demonstrated that persistent lung injury and high mortality in aged mice after sepsis challenge were attributable to impaired endothelial regeneration and vascular repair. Genetic lineage tracing study showed that endothelial regeneration after sepsis-induced vascular injury was mediated by lung resident endothelial proliferation in young adult mice, whereas this intrinsic regenerative program was impaired in aged mice. Expression of forkhead box M1 (FoxM1), an important mediator of endothelial regeneration in young mice, was not induced in lungs of aged mice. Transgenic FOXM1 expression or in vivo endothelium-targeted nanoparticle delivery of the FOXM1 gene driven by an endothelial cell (EC)-specific promoter reactivated endothelial regeneration, normalized vascular repair and resolution of inflammation, and promoted survival in aged mice after sepsis challenge. In addition, treatment with the FDA-approved DNA demethylating agent decitabine was sufficient to reactivate FoxM1-dependent endothelial regeneration in aged mice, reverse aging-impaired resolution of inflammatory injury, and promote survival. Mechanistically, aging-induced Foxm1 promoter hypermethylation in mice, which could be inhibited by decitabine treatment, inhibited Foxm1 induction after sepsis challenge. In COVID-19 lung autopsy samples, FOXM1 was not induced in vascular ECs of elderly patients in their 80s, in contrast with middle-aged patients (aged 50 to 60 years). Thus, reactivation of FoxM1-mediated endothelial regeneration and vascular repair may represent a potential therapy for elderly patients with ARDS.

Short-term PM2.5 exposure induces transient lung injury and repair

Exposure to fine atmospheric particulate matter (PM) is known to induce lung inflammation and injury; however, the way in which sophisticated endogenous lung repair and regenerative programs respond to this exposure remains unknown. In this study, we established a whole-body mouse exposure model to mimic real scenarios. Exposure to fine PM (PM with an aerodynamic diameter ≤ 2.5 µm [PM2.5]; mean 1.05 mg/m3) for 1-month elicited inflammatory infiltration and epithelial alterations in the lung, which were resolved 6 months after cessation of exposure. Immune cells that responded to PM2.5 exposure mainly included macrophages and neutrophils. During PM2.5 exposure, alveolar epithelial type 2 cells initiated rapid repair of alveolar epithelial mucosa through proliferation. However, the reparative capacity of airway progenitor cells (club cells) was impaired, which may have been related to the oxidative production of neutrophils or macrophages, as suggested in organoid co-cultures. These data suggested that the pulmonary toxic effects of short-term exposure to fine atmospheric PM at a certain dosage could be overcome through tissue reparative mechanisms.

Evaluation of Three Deworming Methods and their Long-Term Effect on the Weight of Buffaloes Raised in Silvopastoral Systems

Purpose of the Study: The present work aimed to evaluate the effect of three deworming methods on body weight gains in buffaloes raised in silvopastoral systems (SPS).
 Methods: Thirty buffaloes were divided into three groups (ten per group) as follows: 1) Albendazole 10 g + 1.3 g of cobalt sulfate at one week of age and 14 days later; 2) Albendazole 10 g + 1.3 g of cobalt sulfate at one week of age and 14 days later, then at 6 months and 14 days later; 3) Ivermectin 1 g at 30 days of age and then at six months of age. Birth, weaning (9 months), and final (18 months) weight were registered, and daily weight gains were calculated. The McMaster technique was used to evaluate the presence of parasites.
 Results: The overall prevalence of parasites in the animals evaluated was 93.33%. The most common parasites were: Strongylus sp., Neoascaris sp., Moniezia sp., and Eimeria sp. Before the application of the dewormers, initial values in G1, G2, and G3 were 360, 350, and 210 hpg/opg; after the application of the treatments, 60, 25, and 20 hpg/opg were obtained, respectively. Buffaloes in G2 showed significantly (P=0.046) high final weights (415.10±23.76 kg) compared to G1 (354.80±46.71 kg) but showed no difference with G3 (374.80±43.60k).
 Conclusion: It is concluded that albendazole at 10 g and a repeat at six months of age can be used in buffalo breeding in SPS, which could help to implement regenerative livestock programs, decreasing the use of Ivermectin.

Wnt3a-Loaded Extracellular Vesicles Promote Alveolar Epithelial Regeneration after Lung Injury.

Compromised regeneration resulting from the deactivation of Wnt/β-catenin signaling contributes to the progression of chronic obstructive pulmonary disease (COPD) with limited therapeutic options. Extracellular cytokine-induced Wnt-based signaling provides an alternative option for COPD treatment. However, the hydrophobic nature of Wnt proteins limits their purification and use. This study devises a strategy to deliver the membrane-bound wingless-type MMTV integration site family, member 3A (Wnt3a) over a long distance by anchoring it to the surface of extracellular vesicles (EVs). The newly engineered Wnt3aWG EVs are generated by co-expressing Wnt3a with two genes encoding the membrane protein, WLS, and an engineered glypican, GPC6ΔGPI -C1C2. The bioactivity of Wnt3aWG EVs is validated using a TOPFlash assay and a mesoderm differentiation model of human pluripotent stem cells. Wnt3aWG EVs activate Wnt signaling and promote cell growth following human alveolar epithelial cell injury. In an elastase-induced emphysema model, impaired pulmonary function and enlarged airspace are greatly restored by the intravenous delivery of Wnt3aWG EVs. Single-cell RNA sequencing-based analyses further highlight that Wnt3aWG EV-activated regenerative programs are responsible for its beneficial effects. These findings suggest that EV-based Wnt3a delivery represents a novel therapeutic strategy for lung repair and regeneration after injury.

The regeneration-responsive element careg monitors activation of Müller glia after MNU-induced damage of photoreceptors in the zebrafish retina.

In contrast to mammals, zebrafish can regenerate their damaged photoreceptors. This capacity depends on the intrinsic plasticity of Müller glia (MG). Here, we identified that the transgenic reporter careg, a marker of regenerating fin and heart, also participates in retina restoration in zebrafish. After methylnitrosourea (MNU) treatment, the retina became deteriorated and contained damaged cell types including rods, UV-sensitive cones and the outer plexiform layer. This phenotype was associated with the induction of careg expression in a subset of MG until the reconstruction of the photoreceptor synaptic layer. Single-cell RNA sequencing (scRNAseq) analysis of regenerating retinas revealed a population of immature rods, defined by high expression of rhodopsin and the ciliogenesis gene meig1, but low expression of phototransduction genes. Furthermore, cones displayed deregulation of metabolic and visual perception genes in response to retina injury. Comparison between careg:EGFP expressing and non-expressing MG demonstrated that these two subpopulations are characterized by distinct molecular signatures, suggesting their heterogenous responsiveness to the regenerative program. Dynamics of ribosomal protein S6 phosphorylation showed that TOR signaling became progressively switched from MG to progenitors. Inhibition of TOR with rapamycin reduced the cell cycle activity, but neither affected careg:EGFP expression in MG, nor prevented restoration of the retina structure. This indicates that MG reprogramming, and progenitor cell proliferation might be regulated by distinct mechanisms. In conclusion, the careg reporter detects activated MG, and provides a common marker of regeneration-competent cells in diverse zebrafish organs, including the retina.

Gliotransmission and adenosine signaling promote axon regeneration.

How glia control axon regeneration remains incompletely understood. Here, we investigate glial regulation of regenerative ability differences of closely related Drosophila larval sensory neuron subtypes. Axotomy elicits Ca2+ signals in ensheathing glia, which activates regenerative neurons through the gliotransmitter adenosine and mounts axon regenerative programs. However, non-regenerative neurons do not respond to glial stimulation or adenosine. Such neuronal subtype-specific responses result from specific expressions of adenosine receptors in regenerative neurons. Disrupting gliotransmission impedes axon regeneration of regenerative neurons, and ectopic adenosine receptor expression in non-regenerative neurons suffices to activate regenerative programs and induce axon regeneration. Furthermore, stimulating gliotransmission or activating the mammalian ortholog of Drosophila adenosine receptors in retinal ganglion cells (RGCs) promotes axon regrowth after optic nerve crush in adult mice. Altogether, our findings demonstrate that gliotransmission orchestrates neuronal subtype-specific axon regeneration in Drosophila and suggest that targeting gliotransmission or adenosine signaling is a strategy for mammalian central nervous system repair.

Aberrant cell state plasticity mediated by developmental reprogramming precedes colorectal cancer initiation.

Cell state plasticity is carefully regulated in adult epithelia to prevent cancer. The aberrant expansion of the normally restricted capability for cell state plasticity in neoplasia is poorly defined. Using genetically engineered and carcinogen-induced mouse models of intestinal neoplasia, we observed that impaired differentiation is a conserved event preceding cancer development. Single-cell RNA sequencing (scRNA-seq) of premalignant lesions from mouse models and a patient with hereditary polyposis revealed that cancer initiates by adopting an aberrant transcriptional state characterized by regenerative activity, marked by Ly6a (Sca-1), and reactivation of fetal intestinal genes, including Tacstd2 (Trop2). Genetic inactivation of Sox9 prevented adenoma formation, obstructed the emergence of regenerative and fetal programs, and restored multilineage differentiation by scRNA-seq. Expanded chromatin accessibility at regeneration and fetal genes upon Apc inactivation was reduced by concomitant Sox9 suppression. These studies indicate that aberrant cell state plasticity mediated by unabated regenerative activity and developmental reprogramming precedes cancer development.

Return of the Tbx5; lineage-tracing reveals ventricular cardiomyocyte-like precursors in the injured adult mammalian heart

The single curative measure for heart failure patients is a heart transplantation, which is limited due to a shortage of donors, the need for immunosuppression and economic costs. Therefore, there is an urgent unmet need for identifying cell populations capable of cardiac regeneration that we will be able to trace and monitor. Injury to the adult mammalian cardiac muscle, often leads to a heart attack through the irreversible loss of a large number of cardiomyocytes, due to an idle regenerative capability. Recent reports in zebrafish indicate that Tbx5a is a vital transcription factor for cardiomyocyte regeneration. Preclinical data underscore the cardioprotective role of Tbx5 upon heart failure. Data from our earlier murine developmental studies have identified a prominent unipotent Tbx5-expressing embryonic cardiac precursor cell population able to form cardiomyocytes, in vivo, in vitro and ex vivo. Using a developmental approach to an adult heart injury model and by employing a lineage-tracing mouse model as well as the use of single-cell RNA-seq technology, we identify a Tbx5-expressing ventricular cardiomyocyte-like precursor population, in the injured adult mammalian heart. The transcriptional profile of that precursor cell population is closer to that of neonatal than embryonic cardiomyocyte precursors. Tbx5, a cardinal cardiac development transcription factor, lies in the center of a ventricular adult precursor cell population, which seems to be affected by neurohormonal spatiotemporal cues. The identification of a Tbx5-specific cardiomyocyte precursor-like cell population, which is capable of dedifferentiating and potentially deploying a cardiomyocyte regenerative program, provides a clear target cell population for translationally-relevant heart interventional studies.

Semaphorins: Missing Signals in Age-dependent Alteration of Neuromuscular Junctions and Skeletal Muscle Regeneration.

Skeletal muscle is characterized by a remarkable capacity to rearrange after physiological changes and efficiently regenerate. However, during aging, extensive injury, or pathological conditions, the complete regenerative program is severely affected, with a progressive loss of muscle mass and function, a condition known as sarcopenia. The compromised tissue repair program is attributable to the gradual depletion of stem cells and to altered regulatory signals. Defective muscle regeneration can severely affect re-innervation by motor axons, and neuromuscular junctions (NMJs) development, ultimately leading to skeletal muscle atrophy. Defects in NMJ formation and maintenance occur physiologically during aging and are responsible for the pathogenesis of several neuromuscular disorders. However, it is still largely unknown how neuromuscular connections are restored on regenerating fibers. It has been suggested that attractive and repelling signals used for axon guidance could be implicated in this process; in particular, guidance molecules called semaphorins play a key role. Semaphorins are a wide family of extracellular regulatory signals with a multifaceted role in cell-cell communication. Originally discovered as axon guidance factors, they have been implicated in cancer progression, embryonal organogenesis, skeletal muscle innervation, and other physiological and developmental functions in different tissues. In particular, in skeletal muscle, specific semaphorin molecules are involved in the restoration and remodeling of the nerve-muscle connections, thus emphasizing their plausible role to ensure the success of muscle regeneration. This review article aims to discuss the impact of aging on skeletal muscle regeneration and NMJs remodeling and will highlight the most recent insights about the role of semaphorins in this context.

Distinct signaling signatures drive compensatory proliferation via S-phase acceleration.

Regeneration relies on cell proliferation to restore damaged tissues. Multiple signaling pathways activated by local or paracrine cues have been identified to promote regenerative proliferation. How different types of tissue damage may activate distinct signaling pathways and how these differences converge on regenerative proliferation is less well defined. To better understand how tissue damage and proliferative signals are integrated during regeneration, we investigate models of compensatory proliferation in Drosophila imaginal discs. We find that compensatory proliferation is associated with a unique cell cycle profile, which is characterized by short G1 and G2 phases and, surprisingly, by acceleration of the S-phase. S-phase acceleration can be induced by two distinct signaling signatures, aligning with inflammatory and non-inflammatory tissue damage. Specifically, non-autonomous activation of JAK/STAT and Myc in response to inflammatory damage, or local activation of Ras/ERK and Hippo/Yki in response to elevated cell death, promote accelerated nucleotide incorporation during S-phase. This previously unappreciated convergence of different damaging insults on the same regenerative cell cycle program reconciles previous conflicting observations on proliferative signaling in different tissue regeneration and tumor models.

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin β4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4+ CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling.

Cyclin‐dependent kinase 12‐mediated super‐enhancers as promising targets in cancer therapy

Cyclin‐dependent kinase 12‐mediated super‐enhancers as promising targets in cancer therapy

Considering Global Development? Insights from Applications for FDA Breakthrough Therapy and EMA PRIME Designations

The United States Food and Drug Administration and the European Medicines Agency (EMA) each have programs to expedite development of products identified as having potential to address unmet medical needs: the Breakthrough therapy (BT) and Regenerative Medicines Advanced Therapies designation programs in the US and the Priority Medicines (PRIME) scheme at EMA. We reviewed commonalities and differences in requests submitted and products designated through these programs, with the intent to explore ways to better support global development. During the period from PRIME’s launch in April 2016 to 31 December 2020, 151 requests were made to both BT and PRIME programs and the agencies reached concordant outcomes to grant or deny requests for almost two thirds of the cases (93/151, 62%), suggesting similar perspectives across international regulators on the potential of the products under study. Forty-two (42/151, 28%) products were granted both BT and PRIME, thus found by both Agencies to have the potential to address an unmet need for a serious condition, and thereby products for which efficient development would be highly desirable. Working toward better engagement on global development strategies is in the best interests of patients and public health. With this in mind, Agencies and sponsors should take advantage of existing collaborative opportunities, such as parallel scientific advice, and work to identify fresh approaches to support global development of products for unmet medical needs.

Elk-1 regulates retinal ganglion cell axon regeneration after injury

Adult central nervous system (CNS) axons fail to regenerate after injury, and master regulators of the regenerative program remain to be identified. We analyzed the transcriptomes of retinal ganglion cells (RGCs) at 1 and 5 days after optic nerve injury with and without a cocktail of strongly pro-regenerative factors to discover genes that regulate survival and regeneration. We used advanced bioinformatic analysis to identify the top transcriptional regulators of upstream genes and cross-referenced these with the regulators upstream of genes differentially expressed between embryonic RGCs that exhibit robust axon growth vs. postnatal RGCs where this potential has been lost. We established the transcriptional activator Elk-1 as the top regulator of RGC gene expression associated with axon outgrowth in both models. We demonstrate that Elk-1 is necessary and sufficient to promote RGC neuroprotection and regeneration in vivo, and is enhanced by manipulating specific phosphorylation sites. Finally, we co-manipulated Elk-1, PTEN, and REST, another transcription factor discovered in our analysis, and found Elk-1 to be downstream of PTEN and inhibited by REST in the survival and axon regenerative pathway in RGCs. These results uncover the basic mechanisms of regulation of survival and axon growth and reveal a novel, potent therapeutic strategy to promote neuroprotection and regeneration in the adult CNS.

An interview with Tyler Huycke.

Tyler Huycke is a postdoctoral researcher in Zev Gartner's and Ophir Klein's labs at the University of California San Francisco (UCSF), where he studies the relationship between mechanics and tissue patterning during gut morphogenesis. Tyler served as an editor for the Journal of Emerging Investigators and as a student co-leader of the developmental and regenerative biology graduate programme at Harvard University. He was awarded the Young Investigator's Award at the 2022 Santa Cruz Developmental Biology (SCDB) meeting, which celebrates outstanding early career researchers. I caught up with Tyler at the SCDB meeting to discuss the award, his research and his career.