Challenges of mesenchymal stem cells in the clinical treatment of COVID-19.

The 2019 coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has brought an enormous public health burden to the global society. The duration of the epidemic, the number of infected people, and the widespread of the epidemic are extremely rare in modern society. In the initial stage of infection, people generally show fever, cough, and dyspnea, which can lead to pneumonia, acute respiratory syndrome, kidney failure, and even death in severe cases. The strong infectivity and pathogenicity of SARS-CoV-2 make it more urgent to find an effective treatment. Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells with the potential for self-renewal and multi-directional differentiation. They are widely used in clinical experiments because of their low immunogenicity and immunomodulatory function. Mesenchymal stem cell-derived exosomes (MSC-Exo) can play a physiological role similar to that of stem cells. Since the COVID-19 pandemic, a series of clinical trials based on MSC therapy have been carried out. The results show that MSCs are safe and can significantly improve patients' respiratory function and prognosis of COVID-19. Here, the effects of MSCs and MSC-Exo in the treatment of COVID-19 are reviewed, and the clinical challenges that may be faced in the future are clarified.

A Network Pharmacology-based Study on the Anti-aging Properties of Traditional Chinese Medicine Sisheng Bulao Elixir.

BACKGROUND: Traditional Chinese Medicine (TCM) has a rich history of use in preventing senescence for millennia in China. Nonetheless, a systematic method to study the antiaging properties and the underlying molecular mechanism of TCM remains absent. OBJECTIVE: The objective of this study is to decipher the anti-aging targets and mechanisms of Sisheng Bulao Elixir (SBE) using a systematic approach based on a novel aging database and network pharmacology. METHODS: Bioactive compounds and target proteins in SBE were identified via the Traditional Chinese Medicine System Pharmacology (TCMSP) database. Aging-related proteins were uncovered through alignment with the Ageing Alta database. A compound-target (CT) protein network analysis highlighted key flavonoids targeting aging. Core aging-related proteins were extracted through protein-protein interaction (PPI) network analysis. Molecular docking validated binding activities between core compounds and aging-related proteins. The antioxidant activity of SBE was confirmed using an in vitro senescent cells model. RESULTS: A total of 39 active compounds were extracted from a pool of 639 compounds in SBE. Through a matching process with the Aging Alta, 88 target proteins associated with the aging process were identified. Impressively, 80 out of these 88 proteins were found to be targeted by flavonoids. Subsequently, an analysis using CT methodology highlighted 11 top bioactive flavonoids. Notably, core aging-related proteins, including AKT1, MAPK3, TP53, VEGFA, IL6, and HSP90AA1, emerged through the PPI network analysis. Moreover, three flavonoids, namely quercetin, kaempferol, and luteolin, exhibited interactions with over 100 aging-related proteins. Molecular docking studies were conducted on these flavonoids with their shared three target proteins, namely AKT1, HSP90AA1, and IL6, to assess their binding activities. Finally, the antioxidant properties of SBE were validated using an in vitro model of senescent cells. CONCLUSION: This study offers novel insights into SBE's anti-aging attributes, providing evidence of its molecular mechanisms. It enhances our understanding of traditional remedies in anti-aging research.

Advances in microfluidic chips targeting toxic aggregation proteins for neurodegenerative diseases.

Neurodegenerative diseases (NDs) are characterized by nervous system damage, often influenced by genetic and aging factors. Pathological analysis frequently reveals the presence of aggregated toxic proteins. The intricate and poorly understood origins of these diseases have hindered progress in early diagnosis and drug development. The development of novel in-vitro and in-vivo models could enhance our comprehension of ND mechanisms and facilitate clinical treatment advancements. Microfluidic chips are employed to establish three-dimensional culture conditions, replicating the human ecological niche and creating a microenvironment conducive to neuronal cell survival. The incorporation of mechatronic controls unifies the chip, cells, and culture medium optimizing living conditions for the cells. This study provides a comprehensive overview of microfluidic chip applications in drug and biomarker screening for neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, and amyotrophic lateral sclerosis. Our Lab-on-a-Chip system releases toxic proteins to simulate the pathological characteristics of neurodegenerative diseases, encompassing ß-amyloid, α-synuclein, huntingtin, TAR DNA-binding protein 43, and Myelin Basic Protein. Investigating molecular and cellular interactions in vitro can enhance our understanding of disease mechanisms while minimizing harmful protein levels and can aid in screening potential therapeutic agents. We anticipate that our research will promote the utilization of microfluidic chips in both fundamental research and clinical applications for neurodegenerative diseases.

Stem cells from human exfoliated deciduous teeth rejuvenate the liver in naturally aged mice by improving ribosomal and mitochondrial proteins.

BACKGROUND AIMS: Aging is accompanied by a decline in cellular proteome homeostasis, mitochondrial, and metabolic function. Mesenchymal stromal cell (MSC) therapies have been reported to extend lifespan and delay some age-related pathologies, yet the anti-aging rate and mechanisms remain unclear. Here, we investigated the effects and mechanism by transplantation of stem cells from human exfoliated deciduous teeth (SHED) into the naturally aged mice model. METHODS: SHED were cultured in vitro and injected into mice by caudal vein. The in vivo imaging uncovered that SHED labeled by DiR dye mainly migrated to the liver, spleen, and lung organs of wild-type mice. As the main metabolic organ and SHED homing place, the liver was selected for proteomics and aging clock algorithm (LiverClock) analysis, which was constructed to estimate the proteomic pattern related to liver age state. RESULTS: After 6 months of continuous SHED injections, the liver proteomic pattern was reversed from senescent (∼30 months) to a youthful state (∼3 months), accompanied with upregulation of hepatocytes marker genes, anti-aging protein Klotho, a global improvement of liver functional pathways proteins, and a dramatic regulation of ribosomal and mitochondrial proteins, including upregulation of translation elongation and ribosome-sparing proteins Rpsa and Rplp0; elongation factors Eif4a1, Eef1b2, Eif5a; protein-folding chaperones Hsp90aa and Hspe1; ATP synthesis proteins Atp5b, Atp5o, Atp5j; and downregulation of most ribosomal proteins, suggesting that the proteome homeostasis destruction and mitochondria dysfunction in the aged mice liver might be relieved after SHED treatment. CONCLUSIONS: SHED treatment could dramatically relieve the senescent state of the aged liver, affect ribosome component proteins and upregulate the ribosomal biogenesis proteins in the aged mice liver. These results may help understand the improvements and mechanisms of SHED treatment in anti-aging.

The blood-brain barrier, a key bridge to treat neurodegenerative diseases.

As a highly selective and semi-permeable barrier that separates the circulating blood from the brain and central nervous system (CNS), the blood-brain barrier (BBB) plays a critical role in the onset and treatment of neurodegenerative diseases (NDs). To delay or reverse the NDs progression, the dysfunction of BBB should be improved to protect the brain from harmful substances. Simultaneously, a highly efficient drug delivery across the BBB is indispensable. Here, we summarized several methods to improve BBB dysfunction in NDs, including knocking out risk geneAPOE4, regulating circadian rhythms, restoring the gut microenvironment, and activating the Wnt/ß-catenin signaling pathway. Then we discussed the advances in BBB penetration techniques, such as transient BBB opening, carrier-mediated drug delivery, and nasal administration, which facilitates drug delivery across the BBB. Furthermore, various in vivo and in vitro BBB models and research methods related to NDs are reviewed. Based on the current research progress, the treatment of NDs in the long term should prioritize the integrity of the BBB. However, a treatment approach that combines precise control of transient BBB permeability and non-invasive targeted BBB drug delivery holds profound significance in improving treatment effectiveness, safety, and clinical feasibility during drug therapy. This review involves the cross application of biology, materials science, imaging, engineering and other disciplines in the field of BBB, aiming to provide multi-dimensional research directions and clinical ideas for the treating NDs.

Advances in mesenchymal stromal cells and nanomaterials for diabetic wound healing.

The delayed healing of diabetic wounds (DBW) will not only afflict patients, but also lead to amputation and even death in serious cases, which will create many negative effects on society and the country. Mesenchymal stromal cells (MSCs) have attracted extensive attention for their applications in chronic wound healing due to their excellent cytokine secretion ability and regulation of inflammation. Mesenchymal stromal cells can be used alone or wrapped with carrier/matrix and applied to enhance healing in the wound area. In this review, the etiology of the slow healing of DBW and the mechanism by which MSCs promote the healing of DBW were explored. In addition, we summarised the current research progress on the use of stromal cell-loaded biomaterials for wound healing in diabetic animal models. At the end of the article, we pointed out the challenges and improvement strategies for stromal cell therapy applied to DBW in future clinical practice.

Potential application of heat shock proteins as therapeutic targets in Parkinson's disease.

Parkinson's disease (PD) is a common chronic neurodegenerative disease, and the heat shock proteins (HSPs) are proved to be of great value for PD. In addition, HSPs can maintain protein homeostasis, degrade and inhibit protein aggregation by properly folding and activating intracellular proteins in PD. This study mainly summarizes the important roles of HSPs in PD and explores their feasibility as targets. We introduced the structural and functional characteristics of HSPs and the physiological functions of HSPs in PD. HSPs can protect neurons from damage by degrading aggregates with three mechanisms, including the aggregation and removing α-Synuclein (α-Syn) aggregates, promotion the autophagy of abnormal proteins, and inhibition the apoptosis of degenerated neurons. This study underscores the importance of HSPs as targets in PD and helps to expand new mechanisms in PD treatment strategies.

Induction of autophagy via the PI3K/Akt/mTOR signaling pathway by Pueraria flavonoids improves non-alcoholic fatty liver disease in obese mice.

Non-alcoholic fatty liver disease (NAFLD) is the most common among lipid metabolism disorders. Autophagy plays an important role in lipid metabolism in NAFLD. Pueraria flavonoids, the main active ingredients of Pueraria lobata, exert antioxidant and anti-inflammatory effects. Herein, we report the potential lipid-lowering and anti-inflammatory effects of Pueraria flavonoids on NAFLD induced by a high-fat diet. In vivo and in vitro experiments showed that Pueraria flavonoids reduced intracellular lipid deposition by inhibiting lipid synthesis and the release of pro-inflammatory cytokines. We analyzed the autophagy flux by mRFP-GFP-LC3 plasmid transfection to assess the role of autophagy in intracellular scavenging. After treating mice fed on high fat and HepG2 cells with Pueraria flavonoids, the number of autophagosomes increased significantly, along with the level of autophagy. The autophagy loss after siRNA transfection aggravated lipid deposition and the release of inflammatory cytokines. Mechanistically, Pueraria flavonoids trigger autophagy through PI3K/Akt/mTOR signaling pathway to reduce lipid deposition and inflammation. In summary, our results showed that Pueraria flavonoids stimulated autophagy by inhibiting the PI3K/Akt/mTOR signaling pathway, thereby reducing intracellular lipid accumulation and inflammation levels and alleviating NAFLD.

Potential of astrocytes in targeting therapy for Alzheimer's disease.

As the most common neurodegenerative disease, Alzheimer's disease (AD) exhibits an incomprehensible pathogenesis, which has led to the continuous failure of drug development in recent years. Although neuronal damage is considered a pathological feature of AD, treatment strategies targeting ß-amyloid (Aß) have not achieved beneficial effects. In-depth research on glial cells has revealed the strong importance and application prospects of astrocytes in the recovery of cognitive functions. This review summarizes the role of astrocytes in AD and the possibility of therapeutic strategies targeting astrocytes. Astrocytes are involved in brain lipid metabolism and can regulate the synthesis and degradation of Aß to affect the pathology of AD. The tau protein is phosphorylated by astrocytes, and this phosphorylation leads to the formation of neurofibrillary tangles (NFTs). Astrocytes can express a variety of receptors and inflammatory factors and participate in the neuroinflammatory process and the release of proinflammatory mediators. When the glutamate produced by the neurons is not cleared by astrocytes, neurons undergo apoptosis due to blocked cell metabolism. Therapies for astrocytes are highly efficient, and these include stem cell therapy, gene editing technology, astrocyte transformation and chemical drugs. Here, we discuss the advantages and disadvantages of animal and cell models applied to the study of targeted astrocyte therapies. This study helps elucidate the mechanism of astrocytes in AD and promotes the clinical application of potential therapeutic strategies targeting astrocytes.

Regulatory factor identification for nodal genes in zebrafish by causal inference.

Activation of nodal genes is critical for mesoderm and endoderm induction. Our previous study reported that zebrafish nodal genes ndr1/squint and ndr2/cyclops are coordinately regulated by maternal Eomesa, Hwa-activated ß-catenin (Hwa/ß-catenin) signaling, and Nodal autoregulation (Nodal/Smad2) signaling. However, the exact contribution and underlying mechanisms are still elusive. Here, we applied "causal inference" to evaluate the causal between the independent and dependent variables, and we found that Hwa/ß-catenin and Smad2 are the cause of ndr1 activation, while Eomesa is the cause of ndr2 activation. Mechanistically, the different cis-regulatory regions of ndr1 and ndr2 bound by Eomesa, ß-catenin, and Smad2 were screened out via ChIP-qPCR and verified by the transgene constructs. The marginal GFP expression driven by ndr1 transgenesis could be diminished without both maternal Eomesa and Hwa/ß-catenin, while Eomesa, not ß-catenin, could bind and activate ndr2 demonstrated by ndr2 transgenesis. Thus, the distinct regulation of ndr1/ndr2 relies on different cis-regulatory regions.

Targeting small heat shock proteins to degrade aggregates as a potential strategy in neurodegenerative diseases.

Neurodegenerative diseases (NDs) are aging-related diseases that involve the death of neurons in the brain. Dysregulation of protein homeostasis leads to the production of toxic proteins or the formation of aggregates, which is the pathological basis of NDs. Small heat shock proteins (HSPB) is involved in the establishment of a protein quality control (PQC) system to maintain cellular homeostasis. HSPB can be secreted into the extracellular space and delivered by various routes, especially extracellular vehicles (EVs). HSPB plays an important role in influencing the aggregation phase of toxic proteins involved in heat shock transcription factor (HSF) regulation, oxidative stress, autophagy and apoptosis pathways. HSPB conferred neuroprotective effects by resisting toxic protein aggregation, reducing autophagy and reducing neuronal apoptosis. The HSPB treatment strategies, including targeted PQC system therapy and delivery of EVs-HSPB, can improve disease manifestations for NDs. This review aims to provide a comprehensive insight into the impact of HSPB in NDs and the feasibility of new technology to enhance HSPB expression and EVs-HSPB delivery for neurodegenerative disease.

Intestinal Flora Affect Alzheimer's Disease by Regulating Endogenous Hormones.

Alzheimer's disease (AD) is a central nervous system disease that can lead to cognitive impairment and progressive memory loss. An increasing number of studies have shown that intestinal flora play a crucial role in regulating the brain-gut axis. Short-chain fatty acids are metabolites of intestinal flora that regulate hormone synthesis and play an essential role in microbial-intestinal-brain communication. An imbalance of intestinal flora can promote microglia to secrete proinflammatory factors, cause nerve inflammation, and then affect cognitive and learning ability. However, the mechanism is not clear. From this, we infer that endogenous hormones may be the medium for intestinal flora to affect the process of AD. This review of the relationships among AD, endogenous hormones, and intestinal flora expounds on the critical role of various hormones in the brain-gut axis. It discusses intervention measures aimed at intestinal flora to prevent or delay AD occurrence. Finally, the potential development prospects of fecal microbiota transplantation in treating AD are put forward, which provide potential ideas for future AD research.

Metformin pre-treatment of stem cells from human exfoliated deciduous teeth promotes migration and angiogenesis of human umbilical vein endothelial cells for tissue engineering.

BACKGROUND AIMS: Stem cells from human exfoliated deciduous teeth (SHED) play a significant role in tissue engineering and regenerative medicine. Angiogenesis is crucial in tissue regeneration and a primary target of regenerative medicine. As a first-line anti-diabetic drug, metformin demonstrates numerous valuable impacts on stem cells. This study aimed to explore metformin's impact and mechanism of action on SHED-mediated angiogenesis. METHODS: First, cell proliferation; flow cytometry; osteogenic, adipogenic and chondrogenic induction; and proteomics analyses were conducted to explore the role of metformin in SHED. Subsequently, migration and tube formation assays were used to evaluate chemotaxis and angiogenesis enhancement by SHED pre-treated with metformin under co-culture conditions in vitro, and relative messenger RNA expression levels were determined by quantitative reverse transcription polymerase chain reaction. Finally, nude mice were used for in vivo tube formation assay, and sections were analyzed through immunohistochemistry staining with anti-human CD31 antibody. RESULTS: Metformin significantly promoted SHED proliferation as well as osteogenic, adipogenic and chondrogenic differentiation. Proteomics showed that metformin significantly upregulated 124 differentially abundant proteins involved in intracellular processes, including various proteins involved in cell migration and angiogenesis, such as MAPK1. The co-culture system demonstrated that SHED pre-treated with metformin significantly improved the migration and angiogenesis of human umbilical vein endothelial cells. In addition, SHED pre-treated with metformin possessed greater ability to promote angiogenesis in vivo. CONCLUSIONS: In summary, the authors' findings illustrate metformin's mechanism of action on SHED and confirm that SHED pre-treated with metformin exhibits a strong capacity for promoting angiogenesis. This helps in promoting the application of dental pulp-derived stem cells pre-treated with metformin in regeneration engineering.

Human gingival mesenchymal stem cells improve movement disorders and tyrosine hydroxylase neuronal damage in Parkinson disease rats.

BACKGROUND AIMS: Gingival mesenchymal stem cells (GMSCs) demonstrate high proliferation, trilineage differentiation and immunomodulatory properties. Parkinson disease (PD) is the second most common type of neurodegenerative disease. This study aimed to explore the effect and mechanism of GMSC-based therapy in 6-hydroxydopamine-induced PD rats. METHODS: RNA sequencing and quantitative proteomics technology was used to validate the neuroprotective role of GMSCs therapeutic in 6-Hydroxydopamine -induced PD model in vitro and in vivo. Western blotting, immunofluorescence and real-time quantitative PCR verified the molecular mechanism of GMSCs treatment. RESULTS: Intravenous injection of GMSCs improved rotation and forelimb misalignment behavior, enhanced the anti-apoptotic B-cell lymphoma 2/B-cell lymphoma 2-associated X axis, protected tyrosine hydroxylase neurons, decreased the activation of astrocytes and reduced the astrocyte marker glial fibrillary acidic protein and microglia marker ionized calcium-binding adaptor molecule 1 in the substantia nigra and striatum of PD rats. The authors found that GMSCs upregulated nerve regeneration-related molecules and inhibited metabolic disorders and the activation of signal transducer and activator of transcription 3. GMSCs showed a strong ability to protect neurons and reduce mitochondrial membrane potential damage and reactive oxygen species accumulation. The safety of GMSC transplantation was confirmed by the lack of tumor formation following subcutaneous transplantation into nude mice for up to 8 weeks. CONCLUSIONS: The authors' research helps to explain the mechanism of GMSC-based therapeutic strategies and promote potential clinical application in Parkinson disease.

A porphyrin-triazatruxene dyad for ratiometric two-photon fluorescent sensing of intracellular viscosity.

By combining an electron-rich triazatruxene unit (TAT) to an electron-deficient zinc porphyrin fluorophore (ZnPor) via an ethynyl bridge, a new two-photon fluorescent viscosity rotor (TAT-ZnPor) with typical donor-π-acceptor (D-π-A) electronic configuration was developed for the ratiometric two-photon fluorescent detection of intracellular viscosity. The TAT-ZnPor dyad exhibited highly improved fluorescence quantum yield (Φem = 0.40) and two-photon absorption cross-section (δTPA = 811 GM) in comparison to the individual components. In the methanol/glycerol system, TAT-ZnPor showed sensitive fluorescence responses toward the change of viscosity. Upon elevating the viscosity from 0.59 to 947 cp, the blue emission band around 410 nm gradually enhanced, while the red band at 647 nm concomitantly quenched, leading to a remarkable intensity ratio (I410/I647) change from 0.70 to 81 (116-fold). TAT-ZnPor also displayed good cell imaging performance under one- and two-photon excitation, and strong mitochondria targeting ability in living cells, thus was successfully applied in detecting the change of mitochondrial viscosity during the nystatin-induced degeneration.

Inflammatory bowel disease: A potential pathogenic factor of Alzheimer's disease.

Alzheimer's disease (AD) is a central nervous system disease characterised by degenerative cognitive dysfunction and memory loss. In a society where the global population is gradually ageing, the health threats and financial burdens caused by AD are becoming increasingly severe since AD often occurs in old age. With the in-depth study of AD, many new pathogenic mechanisms have been proposed, among which bidirectional communication between intestinal microbes and the brain has attracted widespread attention. The aetiology of inflammatory bowel disease (IBD) is related to the imbalance of the gut microbiota. Epidemiological investigations have shown that patients with IBD are more likely to suffer from AD. Targeting IBD as a potential AD treatment target has attracted considerable interest. Here, we reviewed the link between chronic intestinal inflammation and central nervous system inflammation and found that IBD patients had a higher risk of AD than non-IBD patients. Preclinical models based on AD also showed that IBD aggravated the condition of AD. We discussed possible biological links between AD and IBD, including the gut-brain axis, autoimmunity, and the gut microbiota. In addition, IBD-induced changes in intestinal microbial metabolites, such as short-chain fatty acids, bile acids, and tryptophan, which aggravate the development of AD, were also discussed.

Stem cells from human exfoliated deciduous teeth relieves Alzheimer's disease symptoms in SAMP8 mice by up-regulating the PPARγ pathway.

The pathology of Alzheimer's disease (AD) is complex and heterogeneous, and there are currently no drugs that can stop its progression. The failure of traditional chemical small-molecule drug development showed the weakness of single target and made researchers look to cell therapy with multiple regulatory effects. Stem cells from human exfoliated deciduous teeth (SHED) are a kind of neural crest-derived mesenchymal stem cells which have broad prospects in the treatment of neurodegenerative diseases. In this study, we demonstrated the therapeutic effects of SHED in AD mice, including behavioral improvement, neuronal protection, and alleviation of neuroinflammation. Tracking experiments on SHED showed that some of the transplanted cells could enter the brain. To elucidate the role played by the majority of cells transplanted into veins, blood proteomic assays were performed. Data are available via ProteomeXchange with identifier PXD030313. Among the altered proteins, the PPAR pathway related to energy metabolism was considered to be an important signaling pathway involved in regulation through gene ontology analysis and pathway analysis. Western blot showed that the transplantation of SHED improved the glucose metabolism in AD mice by increasing the PPARγ signaling pathway. These results suggested that SHED have a potential in relieving AD pathological symptoms and improving behavioral cognition. The therapeutic mechanism of SHED is related to up-regulating PPARγ signaling pathway and reducing neuronal damage.

The combined therapy of mesenchymal stem cell transplantation and resveratrol for diabetes: Future applications and challenges.

Diabetes mellitus (DM) is a worldwide chronic epidemic disease of impaired glucose metabolism. Transplantation of mesenchymal stem cells (MSCs) is considered a promising emerging treatment strategy for diabetes. However, the harsh internal environment of DM patients can inhibit the treatment effects of transplanted MSCs. Fortunately, this adverse effect can be reversed by resveratrol (Res). Therefore, we investigated and summarized relevant studies on the combined treatment of diabetes with MSCs and resveratrol. This review presents the therapeutic effects of this combination therapy strategy on DM in glycemic control, anti-inflammatory, anti-oxidative stress and anti-fibrotic. Moreover, this review explained the mechanisms of MSCs and resveratrol in diabetes treatment from 3 aspects, including promoting cell survival and inhibiting apoptosis, inhibiting histiocyte fibrosis, and improving glucose metabolism. These findings help to understand in-depth mechanisms of the treatment of DM and help to propose a potential treatment strategy for DM and its complications.

Hydrogel supplemented with human platelet lysate enhances multi-lineage differentiation of mesenchymal stem cells.

Stem cells from human exfoliated deciduous teeth (SHED) can be used as a potential clinical material. But the use of xenogeneic ingredients will increase the risk of zoonotic disease transmission. Human platelet lysate (HPL) is a potential surrogate and used in human cell expansion with reliability in clinical applications. In this study, we synthesized chitosan/gelatin/gellan gum hydrogel supplemented with HPL and investigated the effect of 3D culture for SHED. TMT-tagged proteomics was used to decipher the secretome protein profiles of SHEDs and a total of 3209 proteins were identified, of which 23 were up-regulated and 192 were down-regulated. The results showed that hydrogel supplemented with HPL promoted SHED proliferation. After induction, the hydrogel coating contributed to osteogenic differentiation, adipogenic differentiation and differentiation into neural-like cells of SHED. SHED encapsulated in a hydrogel promotes migration and angiogenesis of HUVEC. In conclusion, our research found that hydrogel supplemented with HPL can be used as a method for SHED in standardized production and can contribute to the clinical application of SHED in cell therapy.

Chitosan Hydrogel Supplemented with Metformin Promotes Neuron-like Cell Differentiation of Gingival Mesenchymal Stem Cells.

Human gingival mesenchymal stem cells (GMSCs) are derived from migratory neural crest stem cells and have the potential to differentiate into neurons. Metformin can inhibit stem-cell aging and promotes the regeneration and development of neurons. In this study, we investigated the potential of metformin as an enhancer on neuronal differentiation of GMSCs in the growth environment of chitosan hydrogel. The crosslinked chitosan/ß-glycerophosphate hydrogel can form a perforated microporous structure that is suitable for cell growth and channels to transport water and macromolecules. GMSCs have powerful osteogenic, adipogenic and chondrogenic abilities in the induction medium supplemented with metformin. After induction in an induction medium supplemented with metformin, Western blot and immunofluorescence results showed that GMSCs differentiated into neuron-like cells with a significantly enhanced expression of neuro-related markers, including Nestin (NES) and ß-Tubulin (TUJ1). Proteomics was used to construct protein profiles in neural differentiation, and the results showed that chitosan hydrogels containing metformin promoted the upregulation of neural regeneration-related proteins, including ATP5F1, ATP5J, NADH dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3), and Glutamate Dehydrogenase 1 (GLUD1). Our results help to promote the clinical application of stem-cell neural regeneration.