Induced Stem Cells Research Articles

Proteomic and functional comparison between human induced and embryonic stem cells.

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.

Endothelial Beta 1 Integrins are Necessary for Microvascular Function and Glucose Uptake.

Microvascular insulin delivery to myocytes is rate limiting for the onset of insulin-stimulated muscle glucose uptake. The structural integrity of capillaries of the microvasculature is regulated, in part, by a family of transmembrane adhesion receptors known as integrins, which are composed of an α and β subunit. The integrin β1 (itgb1) subunit is highly expressed in endothelial cells (EC). EC itgb1 is necessary for the formation of capillary networks during embryonic during development and its knockdown blunts the reactive hyperemia that manifests during ischemia reperfusion. We investigated the contribution of EC itgb1 in microcirculatory function and glucose uptake with emphasis in skeletal muscle. We hypothesized that loss of EC itgb1 would impair microvascular hemodynamics and glucose uptake during insulin stimulation, creating 'delivery'-mediated insulin resistance. An itgβ1 knockdown mouse model was developed to avoid lethality of embryonic gene knockout and the deteriorating health resulting from early post-natal inducible gene deletion. Mice with (itgb1fl/flSCLcre) and without (itgb1fl/fl) tamoxifen inducible stem cell leukemia cre recombinase (SLCcre) expression at 10 days post cre induction had comparable exercise tolerance and pulmonary and cardiac functions. Using robust in vivo experimental platforms (i.e., intravital microscopy and hyperinsulinemic-euglycemic clamp), we show that itgb1fl/flSCLcre mice compared to itgb1fl/fl littermates have, i) deficits in capillary flow rate, flow heterogeneity, and capillary density; ii) impaired insulin-stimulated glucose uptake despite sufficient transcapillary insulin efflux; and iii) reduced insulin-stimulated glucose uptake due to perfusion-limited glucose delivery. Thus, EC itgb1 is necessary for microcirculatory function and to meet the metabolic challenge of insulin stimulation.

β-catenin Orchestrates Gli1+ Cell Fate in Condylar Development and TMJOA

The fibrocartilage stem cells (FCSCs) on the surface of the condyle play an essential role in cartilage homeostasis and regeneration. However, few well-defined stem cell markers have been identified for the analysis of FCSCs’ cell fate and regulation mechanism. In this study, we first mapped the transcriptional landscape of the condylar cartilage and identified a Gli1+ subset. Label-retaining cells and our lineage-tracing study showed that Gli1 labeled a group of FCSCs. Conditional knockout β-catenin inhibited Gli1+ cells differentiating into hypertrophic chondrocytes. In discectomy-induced temporomandibular joint osteoarthritis (TMJOA), Gli1+ cells were further activated, and their differentiation into hypertrophic chondrocytes was accelerated, which induced stem cell pool depletion. The deletion of β-catenin in Gli1+ cells preserved the FCSC pool and alleviated TMJOA cartilage degeneration. Collectively, we uncovered that a Gli1+ FCSC subpopulation and Wnt/β-catenin signaling orchestrate the Gli1+ cell fate in condyle postnatal development and TMJOA.

Induction of human stem cells into ameloblasts by reaggregation strategy

BackgroundHuman epithelium-derived stem cells and induced pluripotent stem cells (hiPSCs) possess the capability to support tooth formation and differentiate into functional enamel-secreting ameloblasts, making them promising epithelial-component substitutes for future human tooth regeneration. However, current tissue recombination approaches are not only technically challenging, requiring precise induction procedures and sophisticated microsurgery, but also exhibit low success rates in achieving tooth formation and ameloblastic differentiation.MethodsSuspended human keratinocyte stem cells (hKSCs) or cells from three hiPSC lines were directly mixed with dissociated embryonic mouse dental mesenchymal cells (mDMCs) that possess odontogenic potential in different proportions and reaggregated them to construct bioengineered tooth germs. The success rates of tooth formation and ameloblastic differentiation were confirmed after subrenal culture. The sorting capability, sequential development, and ameloblastic differentiation of stem cells were examined via GFP tracing, RT-PCR, and histological analysis, respectively.ResultsOur reaggregation approach achieved an impressive success rate of more than 90% in tooth formation and 100% in ameloblastic differentiation when the chimeric tooth germs contained 1%~10% hKSCs or 5% hiPSCs. In addition, we observed that hiPSCs, upon exposure to mDMCs, initially transformed into epidermal cells, as indicated by KRT14 and CD29 expression, before progressing into dental epithelial cells, as indicated by SP6 and SHH expression. We also found that epithelial-derived hiPSCs, when reaggregated with mDMCs, were more favorable for tooth formation than their mesenchymal-derived counterparts.ConclusionsThis study establishes a simplified yet highly effective cell-cell reaggregation strategy for inducing stem cells to support tooth formation and differentiate into functional ameloblasts, paving the way for novel approaches for the development of stem cell-based tooth organoids and bioengineered tooth germs in vitro.

Improvement of androgenic alopecia by extracellular vesicles secreted from hyaluronic acid-stimulated induced mesenchymal stem cells

BackgroundAndrogenetic alopecia (AGA) is a common form of hair loss. Androgens, such as testosterone and dihydrotestosterone, are the main causes of AGA. Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) can reduce AGA. However, preparing therapeutic doses of MSCs for clinical use is challenging. Induced pluripotent stem cell-derived MSCs (iMSCs) are homogenous and easily expandable, enabling scalable production of EVs. Hyaluronic acid (HA) can exert various functions including free radical scavenging, immune regulation, and cell migration. Herein, we examined whether hyaluronic acid (HA) stimulation of iMSCs could produce EVs with enhanced therapeutic outcomes for AGA.MethodsEVs were collected from iMSCs primed with HA (HA–iMSC–EVs) or without HA (iMSC–EVs). The characteristics of EVs were examined using dynamic light scattering, cryo-transmission electron microscopy, immunoblotting, flow cytometry, and proteomic analysis. In vitro, we compared the potential of EVs in stimulating the survival of hair follicle dermal papilla cells undergoing testosterone-mediated AGA. Additionally, the expression of androgen receptor (AR) and relevant growth factors as well as key proteins of Wnt/β-catenin signaling pathway (β-catenin and phosphorylated GSK3β) was analyzed. Subsequently, AGA was induced in male C57/BL6 mice by testosterone administration, followed by repeated injections of iMSC–EVs, HA–iMSC–EVs, finasteride, or vehicle. Several parameters including hair growth, anagen phase ratio, reactivation of Wnt/β-catenin pathway, and AR expression was examined using qPCR, immunoblotting, and immunofluorescence analysis.ResultsBoth types of EVs showed typical characteristics for EVs, such as size distribution, markers, and surface protein expression. In hair follicle dermal papilla cells, the mRNA levels of AR, TGF-β, and IL-6 increased by testosterone was blocked by HA–iMSC–EVs, which also contributed to the augmented expression of trophic genes related to hair regrowth. However, no notable changes were observed in the iMSC–EVs. Re-activation of Wnt/β-catenin was observed in HA–iMSC–EVs but not in iMSC–EVs, as shown by β-catenin stabilization and an increase in phosphorylated GSK3β. Restoration of hair growth was more significant in HA–iMSC–EVs than in iMSC–EVs, and was comparable to that in mice treated with finasteride. Consistently, the decreased anagen ratio induced by testosterone was reversed by HA–iMSC–EVs, but not by iMSC–EVs. An increased expression of hair follicular β-catenin protein, as well as the reduction of AR was observed in the skin tissue of AGA mice receiving HA–iMSC–EVs, but not in those treated with iMSC–EVs.ConclusionsOur results suggest that HA–iMSC–EVs have potential to improve AGA by regulating growth factors/cytokines and stimulating AR-related Wnt/β-catenin signaling.

Induction of Pluripotent Stem Cells from Muscle Cells of Large Yellow Croaker (Larimichthys Crocea) Via Electrotransfection.

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cells reprogrammed from somatic cells back into an embryonic-like pluripotent state of stem cells to study development, disease and potential gene therapies. The induction and regulation mechanisms of iPSCs in fish are still unclear. By using the transfection technique, we investigated the crucial function of the OSKMNL factor co-expression for somatic reprogramming in the muscle cell line of large yellow croaker (Larimichthys crocea) (LYCMs) and successfully established a stable iPSCs line (Lc-OSNL-iPSCs). Stable culturing of iPSCs with high alkaline phosphatase activity and a stable karyotype was achieved. The qRT-PCR and immunofluorescence labeling results revealed that Lc-OSNL-iPSCs displayed a high expression level of pluripotent marker genes such as Nanog, Oct4, and Sox2. There were significant differences between Lc-OSNL-iPSCs, Lc-OSKMNL-iPSCs, and LYCMs, and the expression of several genes in maintaining cell pluripotency was up-regulated when the pluripotency signal pathway of stem cells was activated. The technical system for inducing iPSCs of Larimichthys crocea was constructed in this study. This system can serve as a basic model to understand germ cell differentiation mechanism, gender control, genetics, and breeding of large yellow croaker and a platform for studying iPSCs in fish. Interestingly, the acquired iPSCs serves as a useful material for the directional induction of muscle stem cells, thereby establishing the groundwork for obtaining "artificial fish" in the future.

Shear-thinning hydrogel for allograft cell transplantation and externally controlled transgene expression

This work establishes the design of a fully synthetic, shear-thinning hydrogel platform that is injectable and can isolate engineered, allogeneic cell therapies from the host. We utilized RAFT to generate a library of linear random copolymers of N,N-dimethylacrylamide (DMA) and 2-vinyl-4,4-dimethyl azlactone (VDMA) with variable mol% VDMA and degree of polymerization. Poly(DMA-co-VDMA) copolymers were subsequently modified with either adamantane (Ad) or β-cyclodextrin (Cd) through amine-reactive VDMA to prepare hydrogel precursor macromers containing complementary guest-host pairing pendant groups that, when mixed, form shear-thinning hydrogels. Rheometric evaluation of the hydrogel library enabled identification of lead macromer structures comprising 15 mol% pendants (Ad or Cd) and a degree of polymerization of 1000; mixing of these Ad and Cd functionalized precursors yielded hydrogels possessing storage modulus above 1000 Pa, tan(δ) values below 1 and high yield strain, which are target characteristics of robust but injectable shear-thinning gels. This modular system proved amenable to nanoparticle integration with surface-modified nanoparticles displaying Ad. The addition of the Ad-functionalized nanoparticles simultaneously improved mechanical properties of the hydrogels and enabled extended hydrogel retention of a model small molecule in vivo. In studies benchmarking against alginate, a material traditionally used for cell encapsulation, the lead hydrogel showed significantly less fibrous encapsulation in a subcutaneous implant site. Finally, this platform was utilized to encapsulate and extend in vivo longevity of inducible transgene-engineered mesenchymal stem cells in an allogeneic transplant model. The hydrogels remained intact and blocked infiltration by host cells, consequently extending the longevity of grafted cell function relative to a benchmark, shear-thinning hyaluronic acid-based gel. In sum, the new synthetic, shear-thinning hydrogel system presented here shows potential for further development as an injectable platform for delivery and in situ drug modulation of allograft and engineered cell therapies.

Transcription factor-based transdifferentiation of human embryonic to trophoblast stem cells.

During the first week of development, human embryos form a blastocyst composed of an inner cell mass and trophectoderm (TE) cells, the latter of which are progenitors of placental trophoblast. Here, we investigated the expression of transcripts in the human TE from early to late blastocyst stages. We identified enrichment of the transcription factors GATA2, GATA3, TFAP2C and KLF5 and characterised their protein expression dynamics across TE development. By inducible overexpression and mRNA transfection, we determined that these factors, together with MYC, are sufficient to establish induced trophoblast stem cells (iTSCs) from primed human embryonic stem cells. These iTSCs self-renew and recapitulate morphological characteristics, gene expression profiles, and directed differentiation potential, similar to existing human TSCs. Systematic omission of each, or combinations of factors, revealed the crucial importance of GATA2 and GATA3 for iTSC transdifferentiation. Altogether, these findings provide insights into the transcription factor network that may be operational in the human TE and broaden the methods for establishing cellular models of early human placental progenitor cells, which may be useful in the future to model placental-associated diseases.

Sequencing-based study of neural induction of human dental pulp stem cells.

Techniques for triggering neural differentiation of embryonic and induced pluripotent stem cells into neural stem cells and neurons have been established. However, neural induction of mesenchymal stem cells, including dental pulp stem cells (DPSCs), has been assessed primarily based on neural-related gene regulation, and detailed studies into the characteristics and differentiation status of cells are lacking. Therefore, this study was aimed at evaluating the cellular components and differentiation pathways of neural lineage cells obtained via neural induction of human DPSCs. Human DPSCs were induced to neural cells in monolayer culture and examined for gene expression and mechanisms underlying differentiation using microarray-based ingenuity pathway analysis. In addition, the neural lineage cells were subjected to single-cell RNA sequencing (scRNA-seq) to classify cell populations based on gene expression profiles and to elucidate their differentiation pathways. Ingenuity pathway analysis revealed that genes exhibiting marked overexpression, post-neuronal induction, such as FABP7 and ZIC1, were associated with neurogenesis. Furthermore, in canonical pathway analysis, axon guidance signals demonstrated maximum activation. The scRNA-seq and cell type annotations revealed the presence of neural progenitor cells, astrocytes, neurons, and a small number of non-neural lineage cells. Moreover, trajectory and pseudotime analyses demonstrated that the neural progenitor cells initially engendered neurons, which subsequently differentiated into astrocytes. This result indicates that the aforementioned neural induction strategy generated neural stem/progenitor cells from DPSCs, which might differentiate and proliferate to constitute neural lineage cells. Therefore, neural induction of DPSCs may present an alternative approach to pluripotent stem cell-based therapeutic interventions for nervous system disorders.

A conserved transcription factor regulatory program promotes tendon fate.

Tendons, which transmit force from muscles to bones, are highly prone to injury. Understanding the mechanisms driving tendon fate would impact efforts to improve tendon healing, yet this knowledge is limited. To find direct regulators of tendon progenitor emergence, we performed a zebrafish high-throughput chemical screen. We established forskolin as a tenogenic inducer across vertebrates, functioning through Creb1a, which is required and sufficient for tendon fate. Putative enhancers containing cyclic AMP (cAMP) response elements (CREs) in humans, mice, and fish drove specific expression in zebrafish cranial and fin tendons. Analysis of these genomic regions identified motifs for early B cell factor (Ebf/EBF) transcription factors. Mutation of CRE or Ebf/EBF motifs significantly disrupted enhancer activity and specificity in tendons. Zebrafish ebf1a/ebf3a mutants displayed defects in tendon formation. Notably, Creb1a/CREB1 and Ebf1a/Ebf3a/EBF1 overexpression facilitated tenogenic induction in zebrafish and human pluripotent stem cells. Together, our work identifies the functional conservation of two transcription factors in promoting tendon fate.

Endothelial β1 Integrins are Necessary for Microvascular Function and Glucose Uptake.

Microvascular insulin delivery to myocytes is rate limiting for the onset of insulin-stimulated muscle glucose uptake. The structural integrity of capillaries of the microvasculature is regulated, in part, by a family of transmembrane adhesion receptors known as integrins, which are composed of an α and β subunit. The integrin β1 (itgβ1) subunit is highly expressed in endothelial cells (EC). EC itgβ1 is necessary for the formation of capillary networks during embryonic during development and its knockdown in adult mice blunts the reactive hyperemia that manifests during ischemia reperfusion. In this study we investigated the contribution of skeletal muscle EC itgβ1 in microcirculatory function and glucose uptake. We hypothesized that loss of EC itgβ1 would impair microvascular hemodynamics and glucose uptake during insulin stimulation, creating 'delivery'-mediated insulin resistance. An itgβ1 knockdown mouse model was developed to avoid lethality of embryonic gene knockout and the deteriorating health resulting from early post-natal inducible gene deletion. We found that mice with (itgβ1fl/flSCLcre) and without (itgβ1fl/fl) inducible stem cell leukemia cre recombinase (SLCcre) expression at 10 days post cre induction have comparable exercise tolerance and pulmonary and cardiac functions. We quantified microcirculatory hemodynamics using intravital microscopy and the ability of mice to respond to the high metabolic demands of insulin-stimulated muscle using a hyperinsulinemic-euglycemia clamp. We show that itgβ1fl/flSCLcre mice compared to itgβ1fl/fl littermates have, i) deficits in capillary flow rate, flow heterogeneity, and capillary density; ii) impaired insulin-stimulated glucose uptake despite sufficient transcapillary insulin efflux; and iii) reduced insulin-stimulated glucose uptake due to perfusion-limited glucose delivery. Thus, EC itgβ1 is necessary for microcirculatory function and to meet the metabolic challenge of insulin stimulation.

Hydrogel microspheres for stem cell recruitment and induction of directed differentiation in osteoarthritis therapy

The utilization of stem cell biotechnology for osteoarthritis (OA) treatment is hindered by high operational costs, difficulties in maintaining stem cell viability, and limited efficacy of unguided stem cell differentiation. Magnesium ions (Mg2+), crucial for tissue regeneration and repair, hold significant potential in OA treatment. However, further investigation is required to elucidate the precise mechanisms of Mg2+ in OA treatment. Here, we investigated the optimal concentration of Mg2+ to induce directed differentiation of stem cells and synthesized bioactive hydrogel microspheres (MDGM-Ps) capable of precisely releasing this concentration using microfluidic technology. Synthesized via polymerization of dopamine methacrylamide (DMA) and methacrylated gelatin (GelMA), these microspheres could chelate Mg2+ and incorporate platelet-derived growth factor-BB into the hydrogel matrix for sustained release. In vitro, MDGM-Ps showed excellent biocompatibility and promoted stem cell recruitment. Importantly, Mg2+ released from MDGM-Ps could guide stem cell differentiation through the PI3K-AKT pathway. Moreover, in vivo experiments suggested that MDGM-Ps could maintain the cartilage matrix and mitigate the progression of OA. In summary, MDGM-Ps not only harnessed the advantages of stem cell therapy, but also expanded the therapeutic applications of metal ions, thereby presenting a promising candidate for OA therapy.

Forming Single-Cell-Derived Colon Cancer Organoid Arrays on a Microfluidic Chip for High Throughput Tumor Heterogeneity Analysis.

Single-cell-derived tumor organoids (STOs) possess a distinct genetic background, making them valuable tools for demonstrating tumor heterogeneity. In order to fulfill the high throughput demands of STO assays, we have developed a microfluidic chip containing 30 000 microwells, which is dedicated to a single cell culture approach for selective expansion and differential induction of cancer stem cells. The microwells are coated with a hydrophilic copolymer to eliminate cell adhesion, and the cell culture is supported by poly(ethylene glycol) (PEG) to establish a nonadhesive culture environment. By utilizing an input cell density of 7 × 103·mL-1, it is possible to construct a 4000 single cell culture system through stochastic cell occupation. We demonstrate that the addition of 15% PEG10000 in the cell culture medium effectively prevents cell loss while facilitating tumor stem cell expansion. As were demonstrated by HCT116, HT29, and SW480 colon cancer cells, the microfluidic approach achieved a STO formation rate of ∼20%, resulting in over 800 STOs generated from a single culture. Comprehensive analysis through histomorphology, immunohistochemistry, drug response evaluation, assessment of cell invasion, and biomarker detection reveals the heterogeneity among individual STOs. Specifically, the smaller STOs exhibited higher invasion and drug resistance capabilities compared with the larger ones. The developed microfluidic approach effectively facilitates STO formation and offers promising prospects for investigating tumor heterogeneity, as well as conducting personalized therapy-focused drug screening.

Collagen-based hydrogels induce stem cell chondrogenesis and hyaline cartilage regeneration: an in vivo study

Injectable, self-crosslinking collagen-based hydrogels are beneficial for chondrocytes to secrete matrix, positioning them as promising candidates for cartilage tissue engineering. However, previous studies lacked insight into the ability of cell-free collagen-based hydrogels to regenerate hyaline cartilage defect. Therefore, this study aimed to evaluate the potential of collagen-based hydrogels (Col and ColHA) to induce chondrogenic differentiation of stem cells and in situ hyaline cartilage regeneration. Both Col and ColHA hydrogels self-crosslinked in situ and exhibited similar physical properties. In vitro experiments showed they supported the survival, adhesion, spreading, and proliferation of bone marrow stem cells (BMSCs). Moreover, both hydrogels induced ectopic differentiation of BMSCs into chondrocytes when implanted subcutaneously into the back of nude mice. ColHA hydrogel notably enhanced type II collagen secretion. The results of repairing cartilage defects in situ revealed both hydrogels facilitated hyaline cartilage regeneration and maintained cartilage phenotype without exogenous BMSCs. Hydrogels encapsulating BMSCs expedited cartilage repair, and ColHA/BMSC constructs showed better mechanical properties, suggesting their potential for cartilage repair applications. This study implies that collagen-based hydrogels are good candidates for hyaline cartilage regeneration.

The Differential Developmental Neurotoxicity of Valproic Acid on Anterior and Posterior Neural Induction of Human Pluripotent Stem Cells.

Valproic acid (VPA), widely used as an antiepileptic drug, exhibits developmental neurotoxicity when exposure occurs during early or late pregnancy, resulting in various conditions ranging from neural tube defects to autism spectrum disorders. However, toxicity during the very early stages of neural development has not been addressed. Therefore, we investigated the effects of VPA in a model where human pluripotent stem cells differentiate into anterior or posterior neural tissues. Exposure to VPA during the induction of neural stem cells induced different developmental toxic effects in a dose-dependent manner. For instance, VPA induced cell death more profoundly during anteriorly guided neural progenitor induction, while inhibition of cell proliferation and enhanced differentiation were observed during posteriorly guided neural induction. Furthermore, acute exposure to VPA during the posterior induction step also retarded the subsequent neurulation-like tube morphogenesis process in neural organoid culture. These results suggest that VPA exposure during very early embryonic development might exhibit cytotoxicity and subsequently disrupt neural differentiation and morphogenesis processes.

Neural Progenitor Cell-Mediated Magnetic Nanoparticles for Magnetic Resonance Imaging and Photothermal Therapy of Glioma.

Glioma is the most common primary malignant tumor in the brain. The diagnostic accuracy and treatment efficiency of glioma are facing great challenges due to the presence of the blood-brain barrier (BBB) and the high infiltration of glioma. There is an urgent need to explore the combination of diagnostic and therapeutic approaches to achieve a more accurate diagnosis, as well as guidance before and after surgery. In this work, we induced human induction of pluripotent stem cell into neural progenitor cells (NPCs) and synthesized nanoprobes labeled with enhanced green fluorescent protein (EGFP, abbreviated as MFe3O4-labeled EGFP-NPCs) for photothermal therapy. Nanoprobes carried by NPCs can effectively penetrate the BBB and target glioma for the purpose of magnetic resonance imaging and guiding surgery. More importantly, MFe3O4-labeled EGFP-NPCs can effectively induce local photothermal therapy, conduct preoperative tumor therapy, and inhibit the recurrence of postoperative glioma. This work shows that MFe3O4-labeled EGFP-NPCs is a promising nanoplatform for glioma diagnosis, accurate imaging-guided surgery, and effective photothermal therapy.

Three‐dimensional bioprinting biphasic multicellular living scaffold facilitates osteochondral defect regeneration

AbstractDue to tissue lineage variances and the anisotropic physiological characteristics, regenerating complex osteochondral tissues (cartilage and subchondral bone) remains a great challenge, which is primarily due to the distinct requirements for cartilage and subchondral bone regeneration. For cartilage regeneration, a significant amount of newly generated chondrocytes is required while maintaining their phenotype. Conversely, bone regeneration necessitates inducing stem cells to differentiate into osteoblasts. Additionally, the construction of the osteochondral interface is crucial. In this study, we fabricated a biphasic multicellular bioprinted scaffold mimicking natural osteochondral tissue employing three‐dimensional (3D) bioprinting technology. Briefly, gelatin‐methacryloyl (GelMA) loaded with articular chondrocytes and bone marrow mesenchymal stem cells (ACs/BMSCs), serving as the cartilage layer, preserved the phenotype of ACs and promoted the differentiation of BMSCs into chondrocytes through the interaction between ACs and BMSCs, thereby facilitating cartilage regeneration. GelMA/strontium‐substituted xonotlite (Sr‐CSH) loaded with BMSCs, serving as the subchondral bone layer, regulated the differentiation of BMSCs into osteoblasts and enhanced the secretion of cartilage matrix by ACs in the cartilage layer through the slow release of bioactive ions from Sr‐CSH. Additionally, GelMA, serving as the matrix material, contributed to the reconstruction of the osteochondral interface. Ultimately, this biphasic multicellular bioprinted scaffold demonstrated satisfactory simultaneous regeneration of osteochondral defects. In this study, a promising strategy for the application of 3D bioprinting technology in complex tissue regeneration was proposed.

Viscoelastic cues to induce stem cell migration and neuronal differentiation in cell-free hydrogel-assisted TBI recovery

The concept of tissue-inducing biomaterials, such as osteoinductive biomaterials, has inspired the design of regenerative material with biomimetic cues to manipulate cell/tissue responses but has been little applied for neuroinduction in traumatic brain injury (TBI) treatment. Meanwhile, material design has typically focused on elasticity without viscoelasticity taken into consideration. Here, guided by the intrinsic viscoelasticity of brain tissue and the decisive role of viscoelasticity in cell-matrix interactions, we developed a family of brain-mimicking hydrogels, in which the viscoelasticity can be tuned over a wide range under the premise that hydrogels have a low modulus comparable to that of brain tissue. Then, we revealed the promoted migration of stem cells on the viscoelastic hydrogel resulted from the increased number of motor − clutch pairs and filopodia protrusions, as well as their enhanced neuronal differentiation. In a rat TBI model, the cell-free viscoelastic hydrogel successfully induced endogenous stem cells to migrate into lesions and differentiate into neurons, contributing to brain tissue regeneration and neurological function restoration. This study reveals the great promise of biomimetic viscoelastic matrices for TBI treatment, and simultaneously, provides intriguing insights for the design of tissue-inducing biomaterials.

Male-pronuclei-specific granulin facilitates somatic cells reprogramming via mitigating excessive cell proliferation and enhancing lysosomal function.

Critical reprogramming factors resided predominantly in the oocyte or male pronucleus can enhance the efficiency or the quality of induced pluripotent stem cells (iPSCs) induction. However, few reprogramming factors exist in the male pronucleus had been verified. Here, we demonstrated that granulin (Grn), a factor enriched specifically in male pronucleus, can significantly improve the generation of iPSCs from mouse fibroblasts. Grn is highly expressed on Day 1, Day 3, Day 14 of reprogramming induced by four Yamanaka factors and functions at the initial stage of reprogramming. Transcriptome analysis indicates that Grn can promote the expression of lysosome-related genes, while inhibit the expression of genes involved in DNA replication and cell cycle at the early reprogramming stage. Further verification determined that Grn suppressed cell proliferation due to the arrest of cell cycle at G2/M phase. Moreover, ectopic Grn can enhance the lysosomes abundance and rescue the efficiency reduction of reprogramming resulted from lysosomal protease inhibition. Taken together, we conclude that Grn serves as an activator for somatic cell reprogramming through mitigating cell hyperproliferation and promoting the function of lysosomes.

Human olfactory neurosphere-derived cells: A unified tool for neurological disease modelling and neurotherapeutic applications.

As one of the leading causes of global mortality and morbidity, various neurological diseases cause social and economic burdens. Despite significant advances in the treatment of neurological diseases, establishing a proper disease model, especially for degenerative and infectious diseases, remains a major challenging issue. For long, mice were the model of choice but suffered from serious drawbacks of differences in anatomical and functional aspects of the nervous system. Furthermore, the collection of post-mortem brain tissues limits their usage in cultured cell lines. Overcoming such limitations has prompted the usage of stem cells derived from the peripheral nervous system, such as the cells of the olfactory mucosa as a preferred choice. These cells can be easily cultured in vitro and retain the receptors of neuronal cells life-long. Such cells have various advantages over embryonic or induced stem cells, including homology, and ease of culture and can be conveniently obtained from diseased individuals through either biopsies or exfoliation. They have continuously helped in understanding the genetic and developmental mechanisms of degenerative diseases like Alzheimer's and Parkinson's disease. Moreover, the mode of infection of various viruses that can lead to post-viral olfactory dysfunction, such as the Zika virus can be monitored through these cells in vitro and their therapeutic development can be fastened.