Differentiation Of Human Stem Cells Research Articles

Forced LMX1A expression induces dorsal neural fates and disrupts patterning of human embryonic stem cells into ventral midbrain dopaminergic neurons.

The differentiation of human pluripotent stem cells into ventral mesencephalic dopaminergic (DA) fate is relevant for the treatment of Parkinson's disease. Shortcuts to obtaining DA cells through direct reprogramming often include forced expression of the transcription factor LMX1A. Although reprogramming with LMX1A can generate tyrosine hydroxylase (TH)-positive cells, their regional identity remains elusive. Using an invitro model of early human neural tube patterning, we report that forced LMX1A expression induced a ventral-to-dorsal fate shift along the entire neuroaxis with the emergence of roof plate fates despite the presence of ventralizing molecules. The LMX1A-expressing progenitors gave rise to grafts containing roof plate-derived choroid plexus cysts as well as ectopically induced TH-positive neurons of a forebrain identity. Early activation of LMX1A prior to floor plate specification was necessary for the dorsalizing effect. Our work suggests using caution in employing LMX1A for the induction of DA fate, as this factor may generate roof plate rather than midbrain fates.

Study of proanthocyanidin promotes osteogenic differentiation of human periodontal ligament stem cells through the transcription factor EB-induced autophagy-lysosome pathway

Objective: To investigate the mechanism of proanthocyanidin (PA) in regulating the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs), and to explore the effects of PA on the expression and nuclear translocation of transcription factor EB (TFEB) and on the autophagy-lysosome pathway. Methods: PDLSCs were divided into control group and PA group, which were subjected to RNA sequencing analysis (RNA Seq) to detect differentially expressed genes. The osteogenic differentiation ability and autophagy level were observed by real-time fluorescence quantitative PCR (RT-qPCR) analysis, alkaline phosphatase (ALP) staining and transmission electron microscope (TEM), respectively. Scratch assay and Transwell assay were used to detect the migration ability of PDLSCs. Lysotracker and immunofluorescence staining were used to detect the biogenesis of lysosomes. The total protein expression of transcription factor EB (TFEB) as well as that in cytoplasm and nucleus were detected by Western blotting. Confocal laser scanning microscope (CLSM) was used to observe the nuclear translocation of TFEB. The PDLSCs were treated with small interfering RNA (siRNA) technology to knock down the expression levels of TFEB gene with or without PA treatment. Western blotting was used to analyze the expressions of autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3 (LC3B), as well as osteogenic-related proteins runt-related transcription factor 2 (RUNX2), ALP, and osteocalcin in PDLSCs. Results: Compared with the control group, the osteogenic-related and autophagy-related genes showed differential expression in PDLSCs after PA treatment (P<0.05). The mRNA expression levels of osteogenic-related genes RUNX2 (2.32±0.15) and collagen type Ⅰ alpha 1 (COL1α1) (1.80±0.18), as well as the autophagy related genes LC3B (1.87±0.08) and Beclin1 (1.63±0.08) were significantly increased in the PA group, compared with the control group (1.01±0.16, 1.00±0.10, 1.00±0.07, 1.00±0.06, respectively, all P<0.01). Compared with the control group, the PA group had higher ALP activity, and more autophagosomes and autophagolysosomes observed by TEM. PA promoted the migration of PDLSCs (P<0.05) and the increased number of lysosomes and the expression of lysosomal associated membrane protein 1 (LAMP1). In the PA group, the relative expression level of total TFEB protein (1.49±0.07) and the nuclear/cytoplasmic expression of TFEB protein (1.52±0.12) were significantly higher than the control group (1.00±0.11, 1.00±0.13, respectively) (t=6.43, P<0.01; t=5.07, P<0.01). The relative nuclear/cytoplasmic fluorescence intensity of TFEB in the PA group (0.79±0.09) was increased compared with the control group (0.11±0.08) (t=8.32, P<0.01). Knocking down TFEB significantly reduced the expression of TFEB (1.00±0.15 vs 0.64±0.04), LAMP1 (1.00±0.10 vs 0.69±0.09), Beclin1 (1.00±0.05 vs 0.60±0.05), and LC3B Ⅱ/Ⅰ (1.00±0.06 vs 0.73±0.07) in PDLSCs (P<0.05, P<0.05, P<0.01, P<0.01). When TFEB gene was knocked down, the expression levels of Beclin1 (1.05±0.11), LC3B Ⅱ/Ⅰ (1.02±0.09), RUNX2 (1.04±0.10), ALP (1.04±0.16), and osteocalcin (1.03±0.15) proteins were significantly decreased in the PA group compared with the pre-knockdown period (1.28±0.03, 1.44±0.11, 1.38±0.11, 1.62±0.11, 1.65±0.17, respectively) (P<0.05, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). Conclusions: PA promotes the osteogenic differentiation of PDLSCs through inducing the expression and nuclear translocation of TFEB and activating the autophagy-lysosome pathway.

MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects

MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects

Spontaneous differentiation of human induced pluripotent stem cells to odorant-responsive olfactory sensory neurons

Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells (iPSCs), can differentiate into almost all cell types and are anticipated to have significant applications in the field of regenerative medicine. However, there are no reports of successfully directing iPSCs to become functional olfactory sensory neurons (OSNs) capable of selectively receiving odorant compounds. In this study, we employed dual SMAD inhibition and fibroblast growth factor 8 (FGF-8, reported to dictate olfactory fates) along with N-2 and B-27 supplements in the culture medium to efficiently induce the differentiation of iPSCs into neuronal cells with olfactory function through olfactory placode. Temporal gene expression and expression of OSN-specific markers during differentiation indicated that the expression of olfactory marker proteins and various olfactory receptors (ORs), which are markers of mature OSNs, was observed after approximately one month of differentiation culture, irrespective of the differentiation cues, suggesting differentiation into OSNs. Cells that exhibited specific responses to odorant compounds were identified after administering odorant compounds to differentiated iPSC-derived OSNs. This suggests the spontaneous generation of functional OSNs expressing diverse ORs that respond to odorant compounds from iPSCs.

Role of Na/K-ATPase α1 Caveolin-Binding Motif in Adipogenesis.

Deficiencies in mice and in humans have brought to the fore the importance of the caveolar network in key aspects of adipocyte biology. The conserved N-terminal caveolin-binding motif (CBM) of the ubiquitous Na/K-ATPase (NKA) α1 isoform, which allows NKA/caveolin-1 (Cav1) interaction, influences NKA signaling and caveolar distribution. It has been shown to be critical for animal development and ontogenesis, as well as lineage-specific differentiation of human induced pluripotent stem cells (hiPSC). However, its role in postnatal adipogenesis has not been fully examined. Using a genetic approach to alter CBM in hiPSC-derived adipocytes (iAdi-mCBM) and in mice (mCBM), we investigated the regulatory function of NKA CBM signaling in adipogenesis. Seahorse XF cell metabolism analyses revealed impaired glycolysis and decreased ATP synthesis-coupled respiration in iAdi-mCBM. These metabolic dysfunctions were accompanied by evidence of extensive remodeling of the extracellular matrix (ECM), including increased collagen staining, overexpression of ECM marker genes, and heightened TGF-β signaling uncovered by RNAseq analysis. Rescue of mCBM by lentiviral delivery of WT NKA α1 or treatment of mCBM hiPSC with the TGF-β inhibitor SB431542 normalized ECM, suggesting that NKA CBM signaling integrity is required for adequate control of TGF-β signaling and ECM stiffness during adipogenesis. The physiological impact was revealed in mCBM male mice with reduced fat mass accompanied by histological and transcriptional evidence of elevated adipose fibrosis and decreased adipocyte size. Based on these findings, we propose that the genetic alteration of the NKA/Cav1 regulatory path uncovered in human iAdi leads to lipodystrophy in mice.

Vaspin facilitates the proliferation and osteogenic differentiation of periodontal ligament stem cells.

To investigate whether visceral adipose tissue-derived serine protease inhibitor (vaspin) can alleviate the inhibitory effect of high-glucose (HG) culture on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) and to preliminarily explore the underlying mechanisms. High glucose produces damage to the regeneration of periodontal tissue of PDLSCs. The expression level of vaspin in periodontal tissue is high in periodontitis patients and effectively reduced after initial therapy of periodontal diseases. However, the effect of vaspin on PDLSCs remains unknown. PDLSCs were cultured in media augmented with 5.5 or 25.0 mM concentrations of glucose to elucidate the impact and mechanism of vaspin on PDLSCs under high glucose invitro. Proliferation was measured by Cell Counting Kit-8 (CCK8) assay. Osteogenesis of PDLSCs was assessed by alkaline phosphatase (ALP) staining, ALP activity, and Alizarin Red staining. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) were used to investigate the osteo-specific markers. Then, the molecular impact of vaspin in the presence/absence of HG on PDLSCs physiology was determined with TGF-β1/Smad signaling pathway as the main focus. It was revealed that the proliferation and osteogenic differentiation (OD) of PDLSCs under HG was reduced, and by adding vaspin the anti-osteogenic impact of HG was relieved. Moreover, vaspin enhanced TGF-β1/Smad signaling pathway activity. Pretreatment with TGF-β1 inhibitor blocked vaspin-triggered TGF-β1/Smad signal activation and minimized the vaspin-induced protective effect against HG-inhibited growth and OD. In summary, vaspin observably reduces HG-mediated inhibition of PDLSCs OD by modulating the TGF-β1/Smad signaling pathway. Vaspin may be a potential therapeutic for periodontal tissue regeneration in diabetic patients.

A novel miR-466l-3p/FGF23 axis promotes osteogenic differentiation of human bone marrow mesenchymal stem cells

BackgroundMicroRNAs (miRNAs) regulate osteogenic differentiation processes and influence the development of osteoporosis (OP). This study aimed to investigate the potential role of miR-466 l-3p in OP. MethodsThe expression levels of miR-466 l-3p and fibroblast growth factor 23 (FGF23) were quantified in the trabeculae of the femoral neck of 40 individuals with or without OP using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The impact of miR-466 l-3p or FGF23 expression on cell proliferation and the expression levels of runt-related transcription factor 2 (RUNX2), type I collagen (Col1), osteocalcin (OCN), osterix (OSX) and dentin matrix protein 1 (DMP1) was quantified in human bone marrow mesenchymal stem cells (hBMSCs) overexpressing miR-466 l-3p. Furthermore, alkaline phosphatase (ALP) staining and alizarin red staining were performed to measure ALP activity and the levels of calcium deposition, respectively. In addition, bioinformatics analysis, luciferase reporter assays, and RNA pull-down assays were conducted to explore the molecular mechanisms underlying the effects of miR-466 l-3p and FGF23 in osteogenic differentiation of hBMSCs. ResultsThe expression levels of miR-466 l-3p were significantly lower in femoral neck trabeculae of patients with OP than in the control cohort, whereas FGF23 levels exhibited the opposite trend. Furthermore, miR-466 l-3p levels were upregulated and FGF23 levels were downregulated in hBMSCs during osteogenic differentiation. Moreover, the high miR-466 l-3p expression enhanced the mRNA expression of RUNX2, Col1, OCN, OSX and DMP1, as well as cell proliferation, ALP activity, and calcium deposition in hBMSCs. FGF23 was found to be a direct target of miR-466 l-3p. FGF23 overexpression downregulated the expression of osteoblast markers and inhibited the osteogenic differentiation induced by miR-466 l-3p overexpression. qRT-PCR and Western blot assays showed that miR-466 l-3p overexpression decreased the expression levels of mRNAs and proteins associated with the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, whereas FGF23 upregulation exhibited the opposite trend. ConclusionIn conclusion, these findings suggest that miR-466 l-3p enhances the osteogenic differentiation of hBMSCs by suppressing FGF23 expression, ultimately preventing OP.

An injectable dental pulp-derived decellularized matrix hydrogel promotes dentin repair through modulation of macrophage response

Maintaining the viability of damaged pulp is critical in clinical dentistry. Pulp capping, by placing dental material over the exposed pulp, is a main approach to promote pulp-dentin healing and mineralized tissue formation. The dental materials are desired to impact on intricate physiological mechanisms in the healing process, including early regulation of inflammation, immunity, and cellular events. In this study, we developed an injectable dental pulp-derived decellularized matrix (DPM) hydrogel to modulate macrophage responses and promote dentin repair. The DPM derived from porcine dental pulp has high collagen retention and low DNA content. The DPM was solubilized by pepsin digestion (named p-DPM) and subsequently injected through a 25G needle to form hydrogel facilely at 37 °C. In vitro results demonstrated that the p-DPM induced the M2-polarization of macrophages and the migration, proliferation, and dentin differentiation of human dental pulp stem cells from deciduous teeth (SHEDs). In a mouse subcutaneous injection test, the p-DPM hydrogel was found to facilitate cell recruitment and M2 polarization during the early phase of implantation. Additionally, the acute pulp restoration in rat models proved that injectable p-DPM hydrogel as a pulp-capping agent had excellent efficacy in dentin regeneration. This study demonstrates that the DPM promotes dentin repair by modulating macrophage responses, and has a potential for pulp-capping applications in dental practice.

PGC-1α inhibits NLRP3 signaling through transcriptional activation of POP1 to alleviate inflammation and strengthen osteogenic differentiation of lipopolysaccharide-induced human periodontal stem cells

Periodontitis is a chronic infectious disease that affects the oral health of adults. Periodontal stem cells (PDLSCs) have good self-renewal and multipotential differentiation abilities to maintain the integrity of periodontal support structure and repair defects. This study aimed to elucidate the role of peroxisome proliferator activated receptor-γ co-activator 1-α (PGC-1α) in lipopolysaccharide (LPS)-induced PDLSCs and the underlying mechanisms related to predicated that pyrin domain (PYD)-only protein 1 (POP1). Notably downregulated PGC-1α and POP1 expression was observed in LPS-induced PDLSCs. PGC-1α or POP1 overexpression significantly reduced the inflammation and enhanced the osteogenic differentiation of LPS-treated PDLSCs. Particularly, PGC-1 bound to POP1 promoter region and upregulated POP1 expression. Moreover, POP1 knockdown ameliorated the impacts of PGC-1α overexpression on the inflammation and osteogenic differentiation in LPS-induced PDLSCs. Besides, PGC-1α inactivated NLRP3 signaling in LPS-treated PDLSCs, which was reversed by POP1 knockdown. Taken together, PGC-1α inhibits NLRP3 signaling through transcriptional activation of POP1, thereby alleviating inflammation and strengthening osteogenic differentiation of LPS-induced PDLSCs.

Low Initial Cell Density Promotes the Differentiation and Maturation of Human Pluripotent Stem Cells into Erythrocytes.

Human pluripotent stem cell (hPSC)-derived red blood cells (RBCs) possess great potential for compensating shortages in transfusion medicine. For better RBC generation from hPSCs, we compared the cell seeding density in the embryoid body formation-based hPSC induction protocol. In the selection of low- and high-density inoculation conditions, we found that low-density culture performed better in the final RBC product with more cell output and increased average cellular hemoglobin content. An elaborate study using flow cytometry demonstrated that low inoculation density promoted endothelial-to-hematopoietic transition, followed by improved hematopoietic progenitor formation and erythrocyte generation. The improved transformation from glycolysis to mitochondrial oxidation and reduced apoptosis might be responsible for this effect. Hints from RNA sequencing suggested that molecules involved in microenvironment interaction and metabolic regulation might respond for the different developmental potential. The possible mediators between outer message and intracellular response could be the nutrition sensors FOXO, PRKAA1 (AMPK), and MTOR genes. It is possible that low inoculation density triggered metabolic regulation signals, promoted mitochondrial oxidation, and resulted in enhanced cell amplification and hematopoietic differentiation. The low cell culture density will improve RBC generation from hPSCs.

Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids.

Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide an unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using colon organoids generated from healthy tissue of colorectal cancer patients. Culture of these organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1, FABP2) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the γ-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 showing only weak signals of cell differentiation, and were unresponsive to DBZ. We also investigated the effects of 1α,25-dihydroxyvitamin D3 (calcitriol) on differentiation. Calcitriol attenuated the effects of BMP4 and DBZ on colon normal organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features.

PAI-1 transfected-conditioned media promotes osteogenic differentiation of hBMSCs.

Reconstruction of injured bone remains challenging in the clinic owing to the lack of suitable bone grafts. The utilization of PAI-1 transfected-conditioned media (P-CM) has demonstrated its ability to facilitate the differentiation process of mesenchymal stem cells (MSCs), potentially serving as a crucial mediator in tissue regeneration. This research endeavored to explore the therapeutic potential of P-CM concerning the differentiation of human bone marrow mesenchymal stem cells (hBMSCs). To assess new bone formation, a rat calvaria critical defect model was employed, while in vitro experiments involved the use of the alizarin Red-S mineral induction test. In the rat calvaria critical defect model, P-CM treatment resulted in significan new bone formation. In vitro, P-CM treated hBMSCs displayed robust osteogenesis compared to the control group, as demonstrated by the mineral induction test using alizarin Red-S. P-CM with hydroxyapatite/β-tricalcium phosphate/fibrin gel treatment significantly exhibited new bone formation, and the expression of osteogenic associated markers was enhanced in the P-CM-treated group. In conclusion, results demonstrate that P-CM treatment significantly enhanced the osteogenic differantiation efficiency and new bone formation, thus could be used as an ideal therapeutic biomolecule for constructing bone-specific implants, especially for orthopedic and dental applications.

Evaluation puramatrix as a 3D microenvironment for neural differentiation of human breastmilk stem cells

The extracellular matrix is recognized as an efficient and determining component in the growth, proliferation, and differentiation of cells due to its ability to perceive and respond to environmental signals. Applying three-dimensional scaffolds can create conditions similar to the extracellular matrix and provide an opportunity to investigate cell fate. In this study, we employed the PuraMatrix hydrogel scaffold as an advanced cell culture platform for the neural differentiation of stem cells derived from human breastmilk to design an opportune model for tissue engineering.Isolated stem cells from breastmilk were cultured and differentiated into neural-like cells on PuraMatrix peptide hydrogel and in the two-dimensional system. The compatibility of breastmilk-derived stem cells with PuraMatrix and cell viability was evaluated by scanning electron microscopy and MTT assay, respectively. Induction of differentiation was achieved by exposing cells to the neurogenic medium. After 21 days of the initial differentiation process, the expression levels of glial fibrillary acidic protein (GFAP), microtubule-associated protein (MAP2), β-tubulin III, and neuronal nuclear antigen (NeuN) were analyzed using the immunostaining technique.The results illustrated a notable expression of MAP2, β-tubulin-III, and NeuN in the three-dimensional cell culture in comparison to the two-dimensional system, indicating the beneficial effect of PuraMatrix scaffolds in the process of differentiating breastmilk-derived stem cells into neural-like cells.In view of the obtained results, the combination of breastmilk-derived stem cells and PuraMatrix hydrogel scaffold could be an advisable preference for neural tissue regeneration and cell therapy.

A Non-Apoptotic Pattern of Caspase-9/Caspase-3 Activation During Differentiation of Human Embryonic Stem Cells into Cardiomyocytes.

In vitro studies have demonstrated that the differentiation of embryonic stem cells (ESCs) into cardiomyocytes requires activation of caspases through the mitochondrial pathway. These studies have relied on synthetic substrates for activity measurements, which can be misleading due to potential none-specific hydrolysis of these substrates by proteases other than caspases. Hence, caspase-9 and caspase-3 activation are investigated during the differentiation of human ESCs (hESCs) by directly assessing caspase-9 and -3 cleavage. Western blot reveals the presence of the cleaved caspase-9 prior to and during the differentiation of human ESCs (hESCs) into cardiomyocytes at early stages, which diminishes as the differentiation progresses, without cleavage and activation of endogenous procaspase-3. Activation of exogenous procaspase-3 by endogenous caspase-9 and subsequent cleavage of chromogenic caspase-3 substrate i.e. DEVD-pNA during the course of differentiation confirmes that endogenous caspase-9 has the potency to recognize and activate procaspase-3, but for reasons that are unknown to us fails to do so. These observations suggest the existence of distinct mechanisms of caspase regulation in differentiation as compared to apoptosis. Bioinformatics analysis suggests the presence of caspase-9 regulators, which may influence proteolytic function under specific conditions.

Increased Intracellular Insulin from Differentiated Stem Cells to Insulin- Producing Cells Incorporating Β-Carotene

Analyzes the effect of β-Carotene (BC) in the processes of differentiation of adipose tissue Human Mesenchymal Stem Cells (hADMSCs) and dental pulp Human Mesenchymal Stem Cells (hDPMSCs) for obtaining Insulin-Producing Cells (IPCs). The BC present in the methanolic extract was analyzed and quantified by UV-VIS, TLC and HPLC methods. The cell viability test was done by flow cytometry, a concentration of 1.5 μM of BC or AP extract no cytotoxic effect on stem cells, so this concentration was used for the Human Mesenchymal Stem Cells (hMSCs) differentiation protocols for obtaining IPCs. The differentiation tests were done with the AP extract or β-carotene standard addition which demonstrates the increased cell differentiation percentages by the presence of intracellular insulin. This was more outstanding in the hMSCs that were stimulated with the AP in stage 3 and applied at all stages mainly in hDPMSCs, reaching up to 74% of the insulin-positive population respect to 11% where the molecules were not added. RT-PCR was performed for the level of gene expression like MAFA, PDX1 and NKX6.1 and those are found well expressed on differentiated cells. This could be relevant in cell therapy in diabetes, where inclusion of AP extracts or BC may improve yield of IPCs.

Biomechanical Characterization of Retinal Pigment Epitheliums Derived from hPSCs Using Atomic Force Microscopy

AbstractThe retinal pigment epithelium (RPE), a multifunctional cell monolayer located at the back of the eye, plays a crucial role in the survival and homeostasis of photoreceptors. Dysfunction or death of RPE cells leads to retinal degeneration and subsequent vision loss, such as in Age-related macular degeneration and some forms of Retinitis Pigmentosa. Therefore, regenerative medicine that aims to replace RPE cells by new cells obtained from the differentiation of human pluripotent stem cells, is the focus of intensive research. However, despite their critical interest in therapy, there is a lack of biomechanical RPE surface description. Such biomechanical properties are tightly related to their functions. Herein, we used atomic force microscopy (AFM) to analyze both the structural and mechanical properties of RPEs obtained from four cell lines and at different stages of epithelial formation. To characterize epitheliums, we used apical markers in immunofluorescence and showed the increase of transepithelial resistance, as well as the ability to secrete cytokines with an apico-basal polarity. Then, we used AFM to scan the apical surface of living or fixed RPE cells. We show that RPE monolayers underwent softening of apical cell center as well as stiffening of cell borders over epithelial formation. We also observed apical protrusions that depend on actin network, suggesting the formation of microvilli at the surface of RPE epitheliums. These RPE cell characteristics are essential for their functions into the retina and AFM studies may improve the characterization of the RPE epithelium suitable for cell therapy. Graphical Abstract

Locally released H2 and O2 from an artificial periosteum modulate the survival and reparative activities of stem cells derived from the aged human nasal mucosa after transplantation

Stem cell therapies are limited by poor cell survival and engraftment. Elderly diabetic patients (EDPs) have a greater risk of bone graft failure. Herein, a g-C3N4/polydopamine (PDA@g-C3N4) composite nanosheet was successfully fabricated via in situ polymerization of PDA on a g-C3N4 surface. Then, the PDA/g-C3N4 composite was crosslinked with a fibrin hydrogel to prepare an artificial periosteum for EDPs. With the help of NIR, we showed that the synthetic artificial periosteum released H2 and O2 and enhanced the survival and osteogenic differentiation of aged human nasal mucosa-derived ectodermal stem cells (hEMSCs) in microenvironments characterized by oxidative stress and hypoxia. The artificial periosteum had pleiotropic effects on aged hEMSC reparative activities, modulating cytokine secretion and the protein expression of effectors associated with vascularization, and bone formation in vivo. Our findings imply that the developed artificial periosteum loaded with hEMSCs has great promise for the repair of bone defects in EDPs.

Cytoskeletal rearrangement precedes nucleolar remodeling during adipogenesis

Differentiation of adipose progenitor cells into mature adipocytes entails a dramatic reorganization of the cellular architecture to accommodate lipid storage into cytoplasmic lipid droplets. Lipid droplets occupy most of the adipocyte volume, compressing the nucleus beneath the plasma membrane. How this cellular remodeling affects sub-nuclear structure, including size and number of nucleoli, remains unclear. We describe the morphological remodeling of the nucleus and the nucleolus during in vitro adipogenic differentiation of primary human adipose stem cells. We find that cell cycle arrest elicits a remodeling of nucleolar structure which correlates with a decrease in protein synthesis. Strikingly, triggering cytoskeletal rearrangements mimics the nucleolar remodeling observed during adipogenesis. Our results point to nucleolar remodeling as an active, mechano-regulated mechanism during adipogenic differentiation and demonstrate a key role of the actin cytoskeleton in defining nuclear and nucleolar architecture in differentiating human adipose stem cells.

Long noncoding RNAs heat shock RNA omega nucleates TBPH and promotes intestinal stem cell differentiation upon heat shock

In Drosophila, long noncoding RNA Hsrω rapidly assembles membraneless organelle omega speckles under heat shock with unknown biological function. Here, we identified the distribution of omega speckles in multiple tissues of adult Drosophila melanogaster and found that they were selectively distributed in differentiated enterocytes but not in the intestinal stem cells of the midgut. We mimicked the high expression level of Hsrω via overexpression or intense heat shock and demonstrated that the assembly of omega speckles nucleates TBPH for the induction of ISC differentiation. Additionally, we found that heat shock stress promoted cell differentiation, which is conserved in mammalian cells through paraspeckles, resulting in large puncta of TDP-43 (a homolog of TBPH) with less mobility and the differentiation of human induced pluripotent stem cells. Overall, our findings confirm the role of Hsrω and omega speckles in the development of intestinal cells and provide new prospects for the establishment of stem cell differentiation strategies.

Human Placenta Decellularized Extracellular Matrix Hydrogel Promotes the Generation of Human Spinal Cord Organoids with Dorsoventral Organization from Human Induced Pluripotent Stem Cells.

Spinal cord organoids are of significant value in the research of spinal cord-related diseases by simulating disease states, thereby facilitating the development of novel therapies. However, the complexity of spinal cord structure and physiological functions, along with the lack of human-derived inducing components, presents challenges in the in vitro construction of human spinal cord organoids. Here, we introduce a novel human decellularized placenta-derived extracellular matrix hydrogel (DPECMH) and, combined with a new induction protocol, successfully construct human spinal cord organoids. The human placenta-sourced decellularized extracellular matrix (dECM), verified through hematoxylin and eosin staining, DNA quantification, and immunofluorescence staining, retained essential ECM components such as elastin, fibronectin, type I collagen, laminin, and so forth. The temperature-sensitive hydrogel made from human placenta dECM demonstrated good biocompatibility and promoted the differentiation of human induced pluripotent stem cell (hiPSCs)-derived spinal cord organoids into neurons. It displayed enhanced expression of laminar markers in comparison to Matrigel and showed higher expression of laminar markers compared to Matrigel, accelerating the maturation process of spinal cord organoids and demonstrating its potential as an organoid culture substrate. DPECMH has the potential to replace Matrigel as the standard additive for human spinal cord organoids, thus advancing the development of spinal cord organoid culture protocols and their application in the in vitro modeling of spinal cord-related diseases.