Bone marrow mesenchymal stem cell-derived exosomes shuttle microRNAs to endometrial stromal fibroblasts that promote tissue proliferation /regeneration/ and inhibit differentiation.

BACKGROUND: Human bone marrow-derived stem cells (hBMDSCs) are well characterized mediators of tissue repair and regeneration. An increasing body of evidence indicates that these cells exert their therapeutic effects largely through their paracrine actions rather than clonal expansion and differentiation. Here we studied the role of microRNAs (miRNAs) present in extracellular vesicles (EVs) from hBMDSCs in tissue regeneration and cell differentiation targeting endometrial stromal fibroblasts (eSF). METHODS: Extracellular vesicles (EVs) are isolated from hBMDSCs, characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) techniques. Extracted total RNA from EVs was subjected to RNA seq analysis. Transfection and decidualization studies were carried out in endometrial stromal fibroblasts (eSF). Gene expression was analyzed by qRTPCR. Unpaired t-test with Welch's correction was used for data analysis between two groups. RESULTS: We identified several microRNAs (miRNAs) that were highly expressed, including miR-21-5p, miR-100-5p, miR-143-3p and let7. MiR-21 is associated with several signaling pathways involved in tissue regeneration, quiescence, cellular senescence, and fibrosis. Both miR-100-5p and miR-143-3p promoted cell proliferation. MiR-100-5p specifically promoted regenerative processes by upregulating TGF-ß3, VEGFA, MMP7, and HGF. MiR-100-5p blocked differentiation or decidualization as evidenced by morphologic changes and downregulation of decidualization mediators including HOXA10, IGFBP1, PRL, PR-B, and PR. CONCLUSION: EVs delivered to tissues by hBMDSCs contain specific miRNAs that prevent terminal differentiation and drive repair and regeneration. Delivery of microRNAs is a novel treatment paradigm with the potential to replace BMDSCs in cell-free regenerative therapies.

Injury-induced cooperation of InhibinßA and JunB is essential for cell proliferation in Xenopus tadpole tail regeneration.

In animal species that have the capability of regenerating tissues and limbs, cell proliferation is enhanced after wound healing and is essential for the reconstruction of injured tissue. Although the ability to induce cell proliferation is a common feature of such species, the molecular mechanisms that regulate the transition from wound healing to regenerative cell proliferation remain unclear. Here, we show that upon injury, InhibinßA and JunB cooperatively function for this transition during Xenopus tadpole tail regeneration. We found that the expression of inhibin subunit beta A (inhba) and junB proto-oncogene (junb) is induced by injury-activated TGF-ß/Smad and MEK/ERK signaling in regenerating tails. Similarly to junb knockout (KO) tadpoles, inhba KO tadpoles show a delay in tail regeneration, and inhba/junb double KO (DKO) tadpoles exhibit severe impairment of tail regeneration compared with either inhba KO or junb KO tadpoles. Importantly, this impairment is associated with a significant reduction of cell proliferation in regenerating tissue. Moreover, JunB regulates tail regeneration via FGF signaling, while InhibinßA likely acts through different mechanisms. These results demonstrate that the cooperation of injury-induced InhibinßA and JunB is critical for regenerative cell proliferation, which is necessary for re-outgrowth of regenerating Xenopus tadpole tails.

Single-cell chromatin profiling reveals genetic programs activating proregenerative states in nonmyocyte cells.

Cardiac regeneration requires coordinated participation of multiple cell types whereby their communications result in transient activation of proregenerative cell states. Although the molecular characteristics and lineage origins of these activated cell states and their contribution to cardiac regeneration have been studied, the extracellular signaling and the intrinsic genetic program underlying the activation of the transient functional cell states remain largely unexplored. In this study, we delineated the chromatin landscapes of the noncardiomyocytes (nonCMs) of the regenerating heart at the single-cell level and inferred the cis-regulatory architectures and trans-acting factors that control cell type-specific gene expression programs. Moreover, further motif analysis and cell-specific genetic manipulations suggest that the macrophage-derived inflammatory signal tumor necrosis factor-α, acting via its downstream transcription factor complex activator protein-1, functions cooperatively with discrete transcription regulators to activate respective nonCM cell types critical for cardiac regeneration. Thus, our study defines the regulatory architectures and intercellular communication principles in zebrafish heart regeneration.

Circular RNA Cdr1as inhibits proliferation and delays injury-induced regeneration of the intestinal epithelium.

Circular RNAs (circRNAs) are highly expressed in the mammalian intestinal epithelium, but their functions remain largely unknown. Here, we identified the circRNA Cdr1as as a repressor of intestinal epithelial regeneration and defense. Cdr1as levels increased in mouse intestinal mucosa after colitis and septic stress, as well as in human intestinal mucosa from patients with inflammatory bowel disease and sepsis. Ablation of the Cdr1as locus from the mouse genome enhanced renewal of the intestinal mucosa, promoted injury-induced epithelial regeneration, and protected the mucosa against colitis. We found approximately 40 microRNAs, including miR-195, differentially expressed between intestinal mucosa of Cdr1as-knockout (Cdr1as-/-) versus littermate mice. Increasing the levels of Cdr1as inhibited intestinal epithelial repair after wounding in cultured cells and repressed growth of intestinal organoids cultured ex vivo, but this inhibition was abolished by miR-195 silencing. The reduction in miR-195 levels in the Cdr1as-/- intestinal epithelium was the result of reduced stability and processing of the precursor miR-195. These findings indicate that Cdr1as reduces proliferation and repair of the intestinal epithelium at least in part via interaction with miR-195 and highlight a role for induced Cdr1as in the pathogenesis of unhealed wounds and disrupted renewal of the intestinal mucosa.

Regenerative growth is constrained by brain tumor to ensure proper patterning in Drosophila.

Some animals respond to injury by inducing new growth to regenerate the lost structures. This regenerative growth must be carefully controlled and constrained to prevent aberrant growth and to allow correct organization of the regenerating tissue. However, the factors that restrict regenerative growth have not been identified. Using a genetic ablation system in the Drosophila wing imaginal disc, we have identified one mechanism that constrains regenerative growth, impairment of which also leads to erroneous patterning of the final appendage. Regenerating discs with reduced levels of the RNA-regulator Brain tumor (Brat) exhibit enhanced regeneration, but produce adult wings with disrupted margins that are missing extensive tracts of sensory bristles. In these mutants, aberrantly high expression of the pro-growth factor Myc and its downstream targets likely contributes to this loss of cell-fate specification. Thus, Brat constrains the expression of pro-regeneration genes and ensures that the regenerating tissue forms the proper final structure.

Spatial transcriptomics reveals light-induced chlorenchyma cells involved in promoting shoot regeneration in tomato callus.

Callus is a reprogrammed cell mass involved in plant regeneration and gene transformation in crop engineering. Pluripotent callus cells develop into fertile shoots through shoot regeneration. The molecular basis of the shoot regeneration process in crop callus remains largely elusive. This study pioneers the exploration of the spatial transcriptome of tomato callus during shoot regeneration. The findings reveal the presence of highly heterogeneous cell populations within the callus, including epidermis, vascular tissue, shoot primordia, inner callus, and outgrowth shoots. By characterizing the spatially resolved molecular features of shoot primordia and surrounding cells, specific factors essential for shoot primordia formation are identified. Notably, chlorenchyma cells, enriched in photosynthesis-related processes, play a crucial role in promoting shoot primordia formation and subsequent shoot regeneration. Light is shown to promote shoot regeneration by inducing chlorenchyma cell development and coordinating sugar signaling. These findings significantly advance our understanding of the cellular and molecular aspects of shoot regeneration in tomato callus and demonstrate the immense potential of spatial transcriptomics in plant biology.

miR-378 influences muscle satellite cells and enhances adipogenic potential of fibro-adipogenic progenitors but does not affect muscle regeneration in the glycerol-induced injury model.

Skeletal muscle regeneration relies on the reciprocal interaction between many types of cells. Regenerative capacity may be altered in different disorders. In our study, we investigated whether the deletion of miR-378a (miR-378) affects muscle regeneration. We subjected 6-week-old wild-type (WT) and miR-378 knockout (miR-378-/-) animals to the glycerol-induced muscle injury and performed analyses in various time-points. In miR-378-/- animals, an elevated abundance of muscle satellite cells (mSCs) on day 3 was found. Furthermore, fibro-adipogenic progenitors (FAPs) isolated from the muscle of miR-378-/- mice exhibited enhanced adipogenic potential. At the same time, lack of miR-378 did not affect inflammation, fibrosis, adipose tissue deposition, centrally nucleated fiber count, muscle fiber size, FAP abundance, and muscle contractility at any time point analyzed. To conclude, our study revealed that miR-378 deletion influences the abundance of mSCs and the adipogenic potential of FAPs, but does not affect overall regeneration upon acute, glycerol-induced muscle injury.

microRNA-501 controls myogenin+/CD74+ myogenic progenitor cells during muscle regeneration.

OBJECTIVE: Skeletal muscle regeneration is markedly impaired during aging. How adult muscle stem cells contribute to this decrease in regenerative capacity is incompletely understood. We investigated mechanisms of age-related changes in myogenic progenitor cells using the tissue-specific microRNA 501. METHODS: Young and old C57Bl/6 mice were used (3 months or 24 months of age, respectively) with or without global or tissue-specific genetic deletion of miR-501. Muscle regeneration was induced using intramuscular cardiotoxin injection or treadmill exercise and analysed using single cell and bulk RNA sequencing, qRT-PCR and immunofluorescence. Muscle fiber damage was assessed with Evan`s blue dye (EBD). In vitro analysis was performed in primary muscle cells obtained from mice and humans. RESULTS: Single cell sequencing revealed myogenic progenitor cells in miR-501 knockout mice at day 6 after muscle injury that are characterized by high levels of myogenin and CD74. In control mice these cells were less in number and already downregulated after day 3 of muscle injury. Muscle from knockout mice had reduced myofiber size and reduced myofiber resilience to injury and exercise. miR-501 elicits this effect by regulating sarcomeric gene expression through its target gene estrogen-related receptor gamma (Esrrg). Importantly, in aged skeletal muscle where miR-501 was significantly downregulated and its target Esrrg significantly upregulated, the number of myog+/CD74+ cells during regeneration was upregulated to similar levels as observed in 501 knockout mice. Moreover, myog+/CD74+-aged skeletal muscle exhibited a similar decrease in the size of newly formed myofibers and increased number of necrotic myofibers after injury as observed in mice lacking miR-501. CONCLUSIONS: miR-501 and Esrrg are regulated in muscle with decreased regenerative capacity and loss of miR-501 is permissive to the appearance of CD74+ myogenic progenitors. Our data uncover a novel link between the metabolic transcription factor Esrrg and sarcomere formation and demonstrate that stem cell heterogeneity in skeletal muscle during aging is under miRNA control. Targeting Esrrg or myog+/CD74+ progenitor cells might improve fiber size and myofiber resilience to exercise in aged skeletal muscle.

Malat1 deficiency prevents neonatal heart regeneration by inducing cardiomyocyte binucleation.

The adult mammalian heart has limited regenerative capacity, while the neonatal heart fully regenerates during the first week of life. Postnatal regeneration is mainly driven by proliferation of preexisting cardiomyocytes and supported by proregenerative macrophages and angiogenesis. Although the process of regeneration has been well studied in the neonatal mouse, the molecular mechanisms that define the switch between regenerative and nonregenerative cardiomyocytes are not well understood. Here, using in vivo and in vitro approaches, we identified the lncRNA Malat1 as a key player in postnatal cardiac regeneration. Malat1 deletion prevented heart regeneration in mice after myocardial infarction on postnatal day 3 associated with a decline in cardiomyocyte proliferation and reparative angiogenesis. Interestingly, Malat1 deficiency increased cardiomyocyte binucleation even in the absence of cardiac injury. Cardiomyocyte-specific deletion of Malat1 was sufficient to block regeneration, supporting a critical role of Malat1 in regulating cardiomyocyte proliferation and binucleation, a landmark of mature nonregenerative cardiomyocytes. In vitro, Malat1 deficiency induced binucleation and the expression of a maturation gene program. Finally, the loss of hnRNP U, an interaction partner of Malat1, induced similar features in vitro, suggesting that Malat1 regulates cardiomyocyte proliferation and binucleation by hnRNP U to control the regenerative window in the heart.

Unravelling the limb regeneration mechanisms of Polypedates maculatus, a sub-tropical frog, by transcriptomics.

BACKGROUND: Regeneration studies help to understand the strategies that replace a lost or damaged organ and provide insights into approaches followed in regenerative medicine and engineering. Amphibians regenerate their limbs effortlessly and are indispensable models to study limb regeneration. Xenopus and axolotl are the key models for studying limb regeneration but recent studies on non-model amphibians have revealed species specific differences in regeneration mechanisms. RESULTS: The present study describes the de novo transcriptome of intact limbs and three-day post-amputation blastemas of tadpoles and froglets of the Asian tree frog Polypedates maculatus, a non-model amphibian species commonly found in India. Differential gene expression analysis between early tadpole and froglet limb blastemas discovered species-specific novel regulators of limb regeneration. The present study reports upregulation of proteoglycans, such as epiphycan, chondroadherin, hyaluronan and proteoglycan link protein 1, collagens 2,5,6, 9 and 11, several tumour suppressors and methyltransferases in the P. maculatus tadpole blastemas. Differential gene expression analysis between tadpole and froglet limbs revealed that in addition to the expression of larval-specific haemoglobin and glycoproteins, an upregulation of cysteine and serine protease inhibitors and downregulation of serine proteases, antioxidants, collagenases and inflammatory genes in the tadpole limbs were essential for creating an environment that would support regeneration. Dermal myeloid cells were GAG+, EPYC+, INMT+, LEF1+ and SALL4+ and seemed to migrate from the unamputated regions of the tadpole limb to the blastema. On the other hand, the myeloid cells of the froglet limb blastemas were few and probably contributed to sustained inflammation resulting in healing. CONCLUSIONS: Studies on non-model amphibians give insights into alternate tactics for limb regeneration which can help devise a plethora of methods in regenerative medicine and engineering.

Transgenic skin biomechanical properties following first lifesaving epidermal regeneration using combined gene and cell therapy.

BACKGROUND: In 2017, we reported the first life-saving regeneration of virtually an entire epidermis by combined gene and stem cell therapy. Recently, we demonstrated excellent long-term stability of this transgenic epidermis. Skin quality in this experimental approach and its potential application in other conditions were elucidated here regarding long-term outcomes of biomechanical properties. PATIENTS AND METHODS: Analysis of biomechanical properties including skin elasticity, anisotropy and friction was performed on multiple body sites 24, 36 and 60 months following transplantation. Firstly, the sites were matched against and compared to remaining stable non-transgenic areas as well as to a control group of 13 healthy subjects. Parameters for skin elasticity, deformation and friction were assessed non-invasively. RESULTS: Biomechanical properties of the transgenic epidermis showed encouraging results in comparison to both the remaining stable non-transgenic skin as well as healthy controls. Skin elasticity was comparable to the controls. Skin friction showed some decrease in both transgenic and non-transgenic areas as compared to the controls. CONCLUSIONS: The excellent functional outcomes of the transgenic epidermis demonstrate stable long-term results of this novel combined gene and stem cell therapy for epidermal regeneration. Thus, other applications for this technology, such as treatment of specific burns, should be explored.

PTBP1 controls intestinal epithelial regeneration through post-transcriptional regulation of gene expression.

The intestinal epithelial regeneration is driven by intestinal stem cells under homeostatic conditions. Differentiated intestinal epithelial cells, such as Paneth cells, are capable of acquiring multipotency and contributing to regeneration upon the loss of intestinal stem cells. Paneth cells also support intestinal stem cell survival and regeneration. We report here that depletion of an RNA-binding protein named polypyrimidine tract binding protein 1 (PTBP1) in mouse intestinal epithelial cells causes intestinal stem cell death and epithelial regeneration failure. Mechanistically, we show that PTBP1 inhibits neuronal-like splicing programs in intestinal crypt cells, which is critical for maintaining intestinal stem cell stemness. This function is achieved at least in part through promoting the non-productive splicing of its paralog PTBP2. Moreover, PTBP1 inhibits the expression of an AKT inhibitor PHLDA3 in Paneth cells and permits AKT activation, which presumably maintains Paneth cell plasticity and function in supporting intestinal stem cell niche. We show that PTBP1 directly binds to a CU-rich region in the 3' UTR of Phlda3, which we demonstrate to be critical for downregulating the mRNA and protein levels of Phlda3. Our results thus reveal the multifaceted in vivo regulation of intestinal epithelial regeneration by PTBP1 at the post-transcriptional level.

tRNA-derived small RNA, 5'tiRNA-Gly-CCC, promotes skeletal muscle regeneration through the inflammatory response.

BACKGROUND: Increasing evidence shows that tRNA-derived small RNAs (tsRNAs) are not only by-products of transfer RNAs, but they participate in numerous cellular metabolic processes. However, the role of tsRNAs in skeletal muscle regeneration remains unknown. METHODS: Small RNA sequencing revealed the relationship between tsRNAs and skeletal muscle injury. The dynamic expression level of 5'tiRNA-Gly after muscle injury was confirmed by real-time quantitative PCR (q-PCR). In addition, q-PCR, flow cytometry, the 5-ethynyl-2'-deoxyuridine (Edu), cell counting kit-8, western blotting and immunofluorescence were used to explore the biological function of 5'tiRNA-Gly. Bioinformatics analysis and dual-luciferase reporter assay were used to further explore the mechanism of action under the biological function of 5'tiRNA-Gly. RESULTS: Transcriptome analysis revealed that tsRNAs were significantly enriched during inflammatory response immediately after muscle injury. Interestingly, we found that 5'tiRNA-Gly was significantly up-regulated after muscle injury (P < 0.0001) and had a strong positive correlation with inflammation in vivo. In vitro experiments showed that 5'tiRNA-Gly promoted the mRNA expression of proinflammatory cytokines (IL-1ß, P = 0.0468; IL-6, P = 0.0369) and the macrophages of M1 markers (TNF-α, P = 0.0102; CD80, P = 0.0056; MCP-1, P = 0.0002). On the contrary, 5'tiRNA-Gly inhibited the mRNA expression of anti-inflammatory cytokines (IL-4, P = 0.0009; IL-10, P = 0.0007; IL-13, P = 0.0008) and the mRNA expression of M2 markers (TGF-ß1, P = 0.0016; ARG1, P = 0.0083). Flow cytometry showed that 5'tiRNA-Gly promoted the percentage of CD86+ macrophages (16%, P = 0.011) but inhibited that of CD206+ macrophages (10.5%, P = 0.012). Immunofluorescence showed that knockdown of 5'tiRNA-Gly increased the infiltration of M2 macrophages to the skeletal muscles (13.9%, P = 0.0023) and inhibited the expression of Pax7 (P = 0.0089) in vivo. 5'tiRNA-Gly promoted myoblast the expression of myogenic differentiation marker genes (MyoD, P = 0.0002; MyoG, P = 0.0037) and myotube formation (21.3%, P = 0.0016) but inhibited the positive rate of Edu (27.7%, P = 0.0001), cell viability (22.6%, P = 0.003) and the number of myoblasts in the G2 phase (26.3%, P = 0.0016) in vitro. Mechanistically, we found that the Tgfbr1 gene is a direct target of 5'tiRNA-Gly mediated by AGO1 and AGO3. 5'tiRNA-Gly dysregulated the expression of downstream genes related to inflammatory response, activation of satellite cells and differentiation of myoblasts through the TGF-ß signalling pathway by targeting Tgfbr1. CONCLUSIONS: These results reveal that 5'tiRNA-Gly potentially regulated skeletal muscle regeneration by inducing inflammation via the TGF-ß signalling pathway. The findings of this study uncover a new potential target for skeletal muscle regeneration treatment.

DNA Methylation Dynamics During Esophageal Epithelial Regeneration Following Repair with Acellular Silk Fibroin Grafts in Rat.

Esophageal pathologies such as atresia and benign strictures often require surgical reconstruction with autologous tissues to restore organ continuity. Complications such as donor site morbidity and limited tissue availability have spurred the development of acellular grafts for esophageal tissue replacement. Acellular biomaterials for esophageal repair rely on the activation of intrinsic regenerative mechanisms to mediate de novo tissue formation at implantation sites. Previous research has identified signaling cascades involved in neoepithelial formation in a rat model of onlay esophagoplasty with acellular silk fibroin grafts, including phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt) signaling. However, it is currently unknown how these mechanisms are governed by DNA methylation (DNAme) during esophageal wound healing processes. Reduced-representation bisulfite sequencing is performed to characterize temporal DNAme dynamics in host and regenerated tissues up to 1 week postimplantation. Overall, global hypermethylation is observed at postreconstruction timepoints and an inverse correlation between promoter DNAme and the expression levels of differentially expressed proteins during regeneration. Site-specific hypomethylation targets genes associated with immune activation, while hypermethylation occurs within gene bodies encoding PI3K-Akt signaling components during the tissue remodeling period. The data provide insight into the epigenetic mechanisms during esophageal regeneration following surgical repair with acellular grafts.

Genome size and gas chromatography-mass spectrometry (GC-MS) analysis of field-grown and in vitro regenerated Pluchea lanceolata plants.

Pluchea lanceolata is a threatened pharmacologically important plant from the family Asteraceae. It is a source of immunologically active compounds; large-scale propagation may offer compounds with medicinal benefits. Traditional propagation method is ineffective as the seeds are not viable; and root sprout propagation is a slow process and produces less numbers of plants. Plant tissue culture technique is an alternative, efficient method for increasing mass propagation and it also facilitate genetic improvement. The present study investigated a three-way regeneration system in P. lanceolata using indirect shoot regeneration (ISR), direct shoot regeneration (DSR), and somatic embryo mediated regeneration (SER). Aseptic leaf and nodal explants were inoculated on Murashige and Skoog (MS) medium amended with plant growth regulators (PGRs), 2,4-dichlorophenoxy acetic acid (2,4-D), 1-naphthalene acetic acid (NAA), and 6-benzyl amino purine (BAP) either singly or in combinations. Compact, yellowish green callus was obtained from leaf explants in 1.0 mg/l BAP (89.10%) added medium; ISR percentage was high, i.e., 69.33% in 2.0 mg/l BAP + 0.5 mg/l NAA enriched MS with 4.02 mean number of shoots per callus mass. Highest DSR frequency (67.15%) with an average of 5.62 shoot numbers per explant was noted in 0.5 mg/l BAP added MS medium. Somatic embryos were produced in 1.0 mg/l NAA fortified medium with 4.1 mean numbers of somatic embryos per culture. On BAP (1.0 mg/l) + 0.5 mg/l gibberellic acid (GA3) amended medium, improved somatic embryo germination frequency (68.14%) was noted showing 12.18 mean numbers of shoots per culture. Histological and scanning electron microscopic (SEM) observation revealed different stages of embryos, confirming somatic embryogenesis in P. lanceolata. Best rooting frequency (83.95%) of in vitro raised shootlets was obtained in 1.0 mg/l IBA supplemented half MS medium with a maximum of 7.83 roots per shoot. The regenerated plantlets were transferred to the field with 87% survival rate. The 2C genome size of ISR, DSR, and SER plants was measured and noted to be 2.24, 2.25, and 2.22 pg respectively, which are similar to field-grown mother plant (2C = 2.26 pg). Oxidative and physiological events suggested upregulation of enzymatic activities in tissue culture regenerated plants compared to mother plants, so were photosynthetic pigments. Implementation of gas chromatography-mass spectrometry (GC-MS) technique on in vivo and in vitro raised plants revealed the presence of diverse phyto-chemicals. The yields of alpha amyrin and lupeol (medicinally important triterpenoids) were quantified using high-performance thin-layer chromatography (HPTLC) method and enhanced level of alpha amyrin (2.129 µg g-1 dry wt) and lupeol (1.232 µg g-1 dry wt) was noted in in vitro grown leaf tissues, suggesting in vitro conditions act as a potential trigger for augmenting secondary metabolite synthesis. The present protocol represents a reliable mass propagation technique in producing true-to-type plants of P. lanceolata, conserving 2C DNA and ploidy successfully without affecting genetic homogeneity.

Metabolic Changes Associated With Cardiomyocyte Dedifferentiation Enable Adult Mammalian Cardiac Regeneration.

BACKGROUND: Cardiac regeneration after injury is limited by the low proliferative capacity of adult mammalian cardiomyocytes (CMs). However, certain animals readily regenerate lost myocardium through a process involving dedifferentiation, which unlocks their proliferative capacities. METHODS: We bred mice with inducible, CM-specific expression of the Yamanaka factors, enabling adult CM reprogramming and dedifferentiation in vivo. RESULTS: Two days after induction, adult CMs presented a dedifferentiated phenotype and increased proliferation in vivo. Microarray analysis revealed that upregulation of ketogenesis was central to this process. Adeno-associated virus-driven HMGCS2 overexpression induced ketogenesis in adult CMs and recapitulated CM dedifferentiation and proliferation observed during partial reprogramming. This same phenomenon was found to occur after myocardial infarction, specifically in the border zone tissue, and HMGCS2 knockout mice showed impaired cardiac function and response to injury. Finally, we showed that exogenous HMGCS2 rescues cardiac function after ischemic injury. CONCLUSIONS: Our data demonstrate the importance of HMGCS2-induced ketogenesis as a means to regulate metabolic response to CM injury, thus allowing cell dedifferentiation and proliferation as a regenerative response.

TBX3 regulates the transcription of VEGFA to promote osteoblasts proliferation and microvascular regeneration.

Objective: Osteochondral decellularization can promote local vascular regeneration, but the exact mechanism is unknown. The aim of this study is to study osteogenic microvascular regeneration in single cells. Methods: The scRNA-seq dataset of human periosteal-derived cells (hPDCs) were analyzed by pySCENIC. To examine the role of TBX3 in osteogenesis and vascularization, cell transfection, qRT-PCR, western blot, and CCK-8 cell proliferation assays were performed. Results: TCF7L2, TBX3, FLI1, NFKB2, and EZH2 were found to be transcription factors (TFs) most closely associated with corresponding cells. The regulatory network of these TFs was then visualized. Our study knocked down the expression of TBX3 in human osteoblast cell lines. In the TBX3 knockdown group, we observed decreased expression of VEGFA, VEGFB, and VEGFC. Moreover, Western blot analysis showed that downregulating TBX3 resulted in a reduction of VEGFA expression. And TBX3 stimulated osteoblast proliferation in CCK-8 assays. Conclusion: TBX3 regulates VEGFA expression and promotes osteoblast proliferation in skeletal microvasculature formation. The findings provide a theoretical basis for investigating the role of TBX3 in promoting local vascular regeneration.

The Mechanism of Wnt Pathway Regulated by Telocytes to Promote the Regeneration and Repair of Intrauterine Adhesions.

Background: Telocytes (TCs), a novel interstitial cell type in the reproductive tract, participating in pathophysiology of intrauterine adhesions (IUA). This study further investigates the hypothesis that TCs, a source of Wnt, promote the regeneration and repair of IUA. Methods: RNA sequencing datasets of IUA patient (GSE160633) and mouse intestine mesenchymal cells (GSE94072) in GEO database were analyzed for differentially expressed genes (DEGs), and quantitative real-time PCR (qRT-PCR) measured indicated gene expression in TC-educated endometrial stromal cells (ESCs) and noneducated ESCs and verified the results of data mining from GEO database. Results: The expression levels of Wnt genes were downregulated in IUA compared to the control and were upregulated in TCs. In particular, the changes of Wnt5a expression level were the most significant (logFC = 4.0314 and adjusted P value = 0.0023), and the relative Wnt5a expression level was remarkably higher in TC-educated ESCs than noneducated ESCs verified by qRT-PCR (P = 0.0027). Conclusions: TCs may enhance the regeneration and repair of IUA through the Wnt signaling pathway.

Muscle regeneration affects Adeno Associated Virus 1 mediated transgene transcription.

Duchenne muscular dystrophy is a severe neuromuscular disease causing a progressive muscle wasting due to mutations in the DMD gene that lead to the absence of dystrophin protein. Adeno-associated virus (AAV)-based therapies aiming to restore dystrophin in muscles, by either exon skipping or microdystrophin expression, are very promising. However, the absence of dystrophin induces cellular perturbations that hinder AAV therapy efficiency. We focused here on the impact of the necrosis-regeneration process leading to nuclear centralization in myofiber, a common feature of human myopathies, on AAV transduction efficiency. We generated centronucleated myofibers by cardiotoxin injection in wild-type muscles prior to AAV injection. Intramuscular injections of AAV1 vectors show that transgene expression was drastically reduced in regenerated muscles, even when the AAV injection occurred 10 months post-regeneration. We show also that AAV genomes were not lost from cardiotoxin regenerated muscle and were properly localised in the myofiber nuclei but were less transcribed leading to muscle transduction defect. A similar defect was observed in muscles of the DMD mouse model mdx. Therefore, the regeneration process per se could participate to the AAV-mediated transduction defect observed in dystrophic muscles which may limit AAV-based therapies.

Stromal-vascular fraction and adipose-derived stem cell therapies improve cartilage regeneration in osteoarthritis-induced rats.

This study aimed to evaluate the effects of the stromal vascular fraction (SVF) and adipose-derived stem cells (ADSCs) on cartilage injury in an osteoarthritis (OA) rat model. Sodium iodoacetate (3 mg/50 µL) was used to induce OA in the left knee joint of rats. On day 14 after OA induction, 50 µL of SVF (5 × 106cells), ADSCs (1 × 106 cells), or 0.9% normal saline (NS) was injected into the left knee-joint cavity of each group. The macroscopic view and histological sections revealed that the articular cartilage in the NS group was damaged, inflamed, uneven and thin, and had hyperchromatic cell infiltration. Notably, the cartilage surface had recovered to nearly normal and appeared smooth and bright on day 14 in the SVF and ADSC groups. Additionally, the white blood cell counts in the SVF and ADSC groups were higher than those in the NS group on day 14. Plasma IL-1ß levels on days 7 and 14 were reduced in the SVF and ADSC groups. These results indicated that both SVF and ADSC treatments may assist in articular cartilage regeneration after cartilage injury. Cell therapy may benefit patients with OA. However, clinical trials with humans are required before the application of SVF and ADSC treatments in patients with OA.