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STAR Protocols Jun 2024Adult humans cannot regenerate the enamel-forming cell type, ameloblasts. Hence, human induced pluripotent stem cell (hiPSC)-derived ameloblasts are valuable for...
Adult humans cannot regenerate the enamel-forming cell type, ameloblasts. Hence, human induced pluripotent stem cell (hiPSC)-derived ameloblasts are valuable for investigating tooth development and regeneration. Here, we present a protocol for generating three-dimensional induced early ameloblasts (ieAMs) utilizing serum-free media and growth factors. We describe steps for directing hiPSCs toward oral epithelium and then toward ameloblast fate. These cells can form suspended early ameloblast organoids. This approach is critical for understanding, treating, and promoting regeneration in diseases like amelogenesis imperfecta. For complete details on the use and execution of this protocol, please refer to Alghadeer et al..
PubMed: 38824640
DOI: 10.1016/j.xpro.2024.103100 -
The Plant Journal : For Cell and... Jun 2024Plants possess an outstanding capacity to regenerate enabling them to repair damages caused by suboptimal environmental conditions, biotic attacks, or mechanical damages... (Review)
Review
Plants possess an outstanding capacity to regenerate enabling them to repair damages caused by suboptimal environmental conditions, biotic attacks, or mechanical damages impacting the survival of these sessile organisms. Although the extent of regeneration varies greatly between localized cell damage and whole organ recovery, the process of regeneration can be subdivided into a similar sequence of interlinked regulatory processes. That is, competence to regenerate, cell fate reprogramming, and the repatterning of the tissue. Here, using root tip regeneration as a paradigm system to study plant regeneration, we provide a synthesis of the molecular responses that underlie both regeneration competence and the repatterning of the root stump. Regarding regeneration competence, we discuss the role of wound signaling, hormone responses and synthesis, and rapid changes in gene expression observed in the cells close to the cut. Then, we consider how this rapid response is followed by the tissue repatterning phase, where cells experience cell fate changes in a spatial and temporal order to recreate the lost stem cell niche and columella. Lastly, we argue that a multi-scale modeling approach is fundamental to uncovering the mechanisms underlying root regeneration, as it allows to integrate knowledge of cell-level gene expression, cell-to-cell transport of hormones and transcription factors, and tissue-level growth dynamics to reveal how the bi-directional feedbacks between these processes enable self-organized repatterning of the root apex.
PubMed: 38824611
DOI: 10.1111/tpj.16860 -
Stem Cell Research & Therapy Jun 2024Nerve guide conduits are a promising strategy for reconstructing peripheral nerve defects. Improving the survival rate of seed cells in nerve conduits is still a...
BACKGROUND
Nerve guide conduits are a promising strategy for reconstructing peripheral nerve defects. Improving the survival rate of seed cells in nerve conduits is still a challenge and microcarriers are an excellent three-dimensional (3D) culture scaffold. Here, we investigate the effect of the 3D culture of microcarriers on the biological characteristics of adipose mesenchymal stem cells (ADSCs) and to evaluate the efficacy of chitosan nerve conduits filled with microcarriers loaded with ADSCs in repairing nerve defects.
METHODS
In vitro, we prepared porous chitosan microspheres by a modified emulsion cross-linking method for loading ADSCs and evaluated the growth status and function of ADSCs. In vivo, ADSCs-loaded microcarriers were injected into chitosan nerve conduits to repair a 12 mm sciatic nerve defect in rats.
RESULTS
Compared to the conventional two-dimensional (2D) culture, the prepared microcarriers were more conducive to the proliferation, migration, and secretion of trophic factors of ADSCs. In addition, gait analysis, neuro-electrophysiology, and histological evaluation of nerves and muscles showed that the ADSC microcarrier-loaded nerve conduits were more effective in improving nerve regeneration.
CONCLUSIONS
The ADSCs-loaded chitosan porous microcarrier prepared in this study has a high cell engraftment rate and good potential for peripheral nerve repair.
Topics: Chitosan; Nerve Regeneration; Animals; Microspheres; Rats; Mesenchymal Stem Cells; Adipose Tissue; Rats, Sprague-Dawley; Sciatic Nerve; Porosity; Tissue Scaffolds; Male; Mesenchymal Stem Cell Transplantation; Cell Proliferation; Cells, Cultured
PubMed: 38824568
DOI: 10.1186/s13287-024-03753-w -
Alzheimer's & Dementia : the Journal of... Jun 2024Familial Alzheimer's disease (fAD) is heterogeneous in terms of age at onset and clinical presentation. A greater understanding of the pathogenicity of fAD variants and...
INTRODUCTION
Familial Alzheimer's disease (fAD) is heterogeneous in terms of age at onset and clinical presentation. A greater understanding of the pathogenicity of fAD variants and how these contribute to heterogeneity will enhance our understanding of the mechanisms of AD more widely.
METHODS
To determine the pathogenicity of the unclassified PSEN1 P436S mutation, we studied an expanded kindred of eight affected individuals, with magnetic resonance imaging (MRI) (two individuals), patient-derived induced pluripotent stem cell (iPSC) models (two donors), and post-mortem histology (one donor).
RESULTS
An autosomal dominant pattern of inheritance of fAD was seen, with an average age at symptom onset of 46 years and atypical features. iPSC models and post-mortem tissue supported high production of amyloid beta 43 (Aβ43). PSEN1 peptide maturation was unimpaired.
DISCUSSION
We confirm that the P436S mutation in PSEN1 causes atypical fAD. The location of the mutation in the critical PSEN1 proline-alanine-leucine-proline (PALP) motif may explain the early age at onset despite appropriate protein maturation.
HIGHLIGHTS
PSEN1 P436S mutations cause familial Alzheimer's disease. This mutation is associated with atypical clinical presentation. Induced pluripotent stem cells (iPSCs) and post-mortem studies support increased amyloid beta (Aβ43) production. Early age at onset highlights the importance of the PALP motif in PSEN1 function.
PubMed: 38824433
DOI: 10.1002/alz.13904 -
Natural Product Research Jun 2024Regenerative effects of sea anemone-derived exosomes on human foreskin fibroblasts (HFFs) were investigated. Water-based extracts from regenerating tissue were...
Regenerative effects of sea anemone-derived exosomes on human foreskin fibroblasts (HFFs) were investigated. Water-based extracts from regenerating tissue were collected at various time points, and exosomes were extracted after inducing wounds. Both the extract and exosomes were tested on HFF for proliferation and wound healing. analysis explored protein-protein docking between regenerative exosome proteins and HFF receptors. The MTT (3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazolium bromide proliferation assay and wound healing test of aquatic extract showed proliferative effects on HFF cell lines, with the 60 μg/mL concentration significantly enhancing cell migration. Exosomes were characterised. Exosomes showed a significantly positive effect on cell proliferation and migration at the 50 µg/mL concentration 48 h post-wound induction. analysis revealed potential binding affinities between exosome proteins and HFF receptors. In conclusion, optimised concentrations of -derived exosomes exhibited positive effects on HFF regeneration and migration, suggesting their potential in accelerating wound healing.
PubMed: 38824422
DOI: 10.1080/14786419.2024.2352144 -
Molecular Biology Reports Jun 2024Promoting the balance between bone formation and bone resorption is the main therapeutic goal for postmenopausal osteoporosis (PMOP), and bone marrow mesenchymal stem...
BACKGROUND
Promoting the balance between bone formation and bone resorption is the main therapeutic goal for postmenopausal osteoporosis (PMOP), and bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation plays an important regulatory role in this process. Recently, several long non-coding RNAs (lncRNAs) have been reported to play an important regulatory role in the occurrence and development of OP and participates in a variety of physiological and pathological processes. However, the role of lncRNA tissue inhibitor of metalloproteinases 3 (lncTIMP3) remains to be investigated.
METHODS
The characteristics of BMSCs isolated from the PMOP rat model were verified by flow cytometry assay, alkaline phosphatase (ALP), alizarin red and Oil Red O staining assays. Micro-CT and HE staining assays were performed to examine histological changes of the vertebral trabeculae of the rats. RT-qPCR and western blotting assays were carried out to measure the RNA and protein expression levels. The subcellular location of lncTIMP3 was analyzed by FISH assay. The targeting relationships were verified by luciferase reporter assay and RNA pull-down assay.
RESULTS
The trabecular spacing was increased in the PMOP rats, while ALP activity and the expression levels of Runx2, Col1a1 and Ocn were all markedly decreased. Among the RNA sequencing results of the clinical samples, lncTIMP3 was the most downregulated differentially expressed lncRNA, also its level was significantly reduced in the OVX rats. Knockdown of lncTIMP3 inhibited osteogenesis of BMSCs, whereas overexpression of lncTIMP3 exhibited the reverse results. Subsequently, lncTIMP3 was confirmed to be located in the cytoplasm of BMSCs, implying its potential as a competing endogenous RNA for miRNAs. Finally, the negative targeting correlations of miR-214 between lncTIMP3 and Smad4 were elucidated in vitro.
CONCLUSION
lncTIMP3 may delay the progress of PMOP by promoting the activity of BMSC, the level of osteogenic differentiation marker gene and the formation of calcium nodules by acting on the miR-214/Smad4 axis. This finding may offer valuable insights into the possible management of PMOP.
Topics: Mesenchymal Stem Cells; MicroRNAs; Osteogenesis; Animals; RNA, Long Noncoding; Osteoporosis, Postmenopausal; Female; Cell Differentiation; Rats; Smad4 Protein; Humans; Disease Models, Animal; Rats, Sprague-Dawley; Bone Marrow Cells
PubMed: 38824271
DOI: 10.1007/s11033-024-09652-w -
Molecular Biology Reports Jun 2024Circular RNA (circRNA) is a key player in regulating the multidirectional differentiation of stem cells. Previous research by our group found that the blue...
BACKGROUND
Circular RNA (circRNA) is a key player in regulating the multidirectional differentiation of stem cells. Previous research by our group found that the blue light-emitting diode (LED) had a promoting effect on the osteogenic/odontogenic differentiation of human stem cells from apical papilla (SCAPs). This research aimed to investigate the differential expression of circRNAs during the osteogenic/odontogenic differentiation of SCAPs regulated by blue LED.
MATERIALS AND METHODS
SCAPs were divided into the irradiation group (4 J/cm) and the control group (0 J/cm), and cultivated in an osteogenic/odontogenic environment. The differentially expressed circRNAs during osteogenic/odontogenic differentiation of SCAPs promoted by blue LED were detected by high-throughput sequencing, and preliminarily verified by qRT-PCR. Functional prediction of these circRNAs was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the circRNA-miRNA-mRNA networks were also constructed.
RESULTS
It showed 301 circRNAs were differentially expressed. GO and KEGG analyses suggested that these circRNAs were associated with some signaling pathways related to osteogenic/odontogenic differentiation. And the circRNA-miRNA-mRNA networks were also successfully constructed.
CONCLUSION
CircRNAs were involved in the osteogenic/odontogenic differentiation of SCAPs promoted by blue LED. In this biological process, circRNA-miRNA-mRNA networks served an important purpose, and circRNAs regulated this process through certain signaling pathways.
Topics: RNA, Circular; Humans; Osteogenesis; Cell Differentiation; Stem Cells; Odontogenesis; Dental Papilla; Light; MicroRNAs; Gene Ontology; Cells, Cultured; Gene Expression Profiling; RNA, Messenger; Gene Regulatory Networks; High-Throughput Nucleotide Sequencing; Gene Expression Regulation; Blue Light
PubMed: 38824241
DOI: 10.1007/s11033-024-09621-3 -
Molecular Biology Reports Jun 2024Tumor modeling using organoids holds potential in studies of cancer development, enlightening both the intracellular and extracellular molecular mechanisms behind...
BACKGROUND
Tumor modeling using organoids holds potential in studies of cancer development, enlightening both the intracellular and extracellular molecular mechanisms behind different cancer types, biobanking, and drug screening. Intestinal organoids can be generated in vitro using a unique type of adult stem cells which are found at the base of crypts and are characterized by their high Lgr5 expression levels.
METHODS AND RESULTS
In this study, we successfully established intestinal cancer organoid models by using both the BALB/c derived and mouse embryonic stem cells (mESCs)-derived intestinal organoids. In both cases, carcinogenesis-like model was developed by using azoxymethane (AOM) treatment. Carcinogenesis-like model was verified by H&E staining, immunostaining, relative mRNA expression analysis, and LC/MS analysis. The morphologic analysis demonstrated that the number of generated organoids, the number of crypts, and the intensity of the organoids were significantly augmented in AOM-treated intestinal organoids compared to non-AOM-treated ones. Relative mRNA expression data revealed that there was a significant increase in both Wnt signaling pathway-related genes and pluripotency transcription factors in the AOM-induced intestinal organoids.
CONCLUSION
We successfully developed simple carcinogenesis-like models using mESC-based and Lgr5 + stem cell-based intestinal organoids. Intestinal organoid based carcinogenesi models might be used for personalized cancer therapy in the future.
Topics: Animals; Organoids; Mice; Azoxymethane; Carcinogenesis; Mouse Embryonic Stem Cells; Wnt Signaling Pathway; Receptors, G-Protein-Coupled; Mice, Inbred BALB C; Intestines; Intestinal Neoplasms; Disease Models, Animal; Intestinal Mucosa
PubMed: 38824233
DOI: 10.1007/s11033-024-09660-w -
Peripheral apoptosis and limited clonal deletion during physiologic murine B lymphocyte development.Nature Communications Jun 2024Self-reactive and polyreactive B cells generated during B cell development are silenced by either apoptosis, clonal deletion, receptor editing or anergy to avoid...
Self-reactive and polyreactive B cells generated during B cell development are silenced by either apoptosis, clonal deletion, receptor editing or anergy to avoid autoimmunity. The specific contribution of apoptosis to normal B cell development and self-tolerance is incompletely understood. Here, we quantify self-reactivity, polyreactivity and apoptosis during physiologic B lymphocyte development. Self-reactivity and polyreactivity are most abundant in early immature B cells and diminish significantly during maturation within the bone marrow. Minimal apoptosis still occurs at this site, however B cell receptors cloned from apoptotic B cells show comparable self-reactivity to that of viable cells. Apoptosis increases dramatically only following immature B cells leaving the bone marrow sinusoids, but above 90% of cloned apoptotic transitional B cells are not self-reactive/polyreactive. Our data suggests that an apoptosis-independent mechanism, such as receptor editing, removes most self-reactive B cells in the bone marrow. Mechanistically, lack of survival signaling rather than clonal deletion appears to be the underpinning cause of apoptosis in most transitional B cells in the periphery.
Topics: Animals; Apoptosis; Clonal Deletion; B-Lymphocytes; Mice; Mice, Inbred C57BL; Receptors, Antigen, B-Cell; Cell Differentiation; Bone Marrow; Female; Precursor Cells, B-Lymphoid
PubMed: 38824171
DOI: 10.1038/s41467-024-49062-x -
Cell Death & Disease Jun 2024Obesity exacerbates tissue degeneration and compromises the integrity and reparative potential of mesenchymal stem/stromal cells (MSCs), but the underlying mechanisms...
Obesity exacerbates tissue degeneration and compromises the integrity and reparative potential of mesenchymal stem/stromal cells (MSCs), but the underlying mechanisms have not been sufficiently elucidated. Mitochondria modulate the viability, plasticity, proliferative capacity, and differentiation potential of MSCs. We hypothesized that alterations in the 5-hydroxymethylcytosine (5hmC) profile of mitochondria-related genes may mediate obesity-driven dysfunction of human adipose-derived MSCs. MSCs were harvested from abdominal subcutaneous fat of obese and age/sex-matched non-obese subjects (n = 5 each). The 5hmC profile and expression of nuclear-encoded mitochondrial genes were examined by hydroxymethylated DNA immunoprecipitation sequencing (h MeDIP-seq) and mRNA-seq, respectively. MSC mitochondrial structure (electron microscopy) and function, metabolomics, proliferation, and neurogenic differentiation were evaluated in vitro, before and after epigenetic modulation. hMeDIP-seq identified 99 peaks of hyper-hydroxymethylation and 150 peaks of hypo-hydroxymethylation in nuclear-encoded mitochondrial genes from Obese- versus Non-obese-MSCs. Integrated hMeDIP-seq/mRNA-seq analysis identified a select group of overlapping (altered levels of both 5hmC and mRNA) nuclear-encoded mitochondrial genes involved in ATP production, redox activity, cell proliferation, migration, fatty acid metabolism, and neuronal development. Furthermore, Obese-MSCs exhibited decreased mitochondrial matrix density, membrane potential, and levels of fatty acid metabolites, increased superoxide production, and impaired neuronal differentiation, which improved with epigenetic modulation. Obesity elicits epigenetic changes in mitochondria-related genes in human adipose-derived MSCs, accompanied by structural and functional changes in their mitochondria and impaired fatty acid metabolism and neurogenic differentiation capacity. These observations may assist in developing novel therapies to preserve the potential of MSCs for tissue repair and regeneration in obese individuals.
Topics: Humans; Mesenchymal Stem Cells; Obesity; Mitochondria; Epigenesis, Genetic; Adipose Tissue; Cell Differentiation; Female; Male; 5-Methylcytosine; Adult; Middle Aged; Cell Proliferation
PubMed: 38824145
DOI: 10.1038/s41419-024-06774-8