Microgel-based carriers enhance skeletal stem cell reprogramming towards immunomodulatory phenotype in osteoarthritic therapy.

Skeletal stem cells (SSC) have gained attentions as candidates for the treatment of osteoarthritis due to their osteochondrogenic capacity. However, the immunomodulatory properties of SSC, especially under delivery operations, have been largely ignored. In the study, we found that Pdpn+ and Grem1+ SSC subpopulations owned immunoregulatory potential, and the single-cell RNA sequencing (scRNA-seq) data suggested that the mechanical activation of microgel carriers on SSC induced the generation of Pdpn+Grem1+Ptgs2+ SSC subpopulation, which was potent at suppressing macrophage inflammation. The microgel carriers promoted the YAP nuclear translocation, and the activated YAP protein was necessary for the increased expression of Ptgs2 and PGE2 in microgels-delivered SSC, which further suppressed the expression of TNF-ɑ, IL-1ß and promoted the expression of IL-10 in macrophages. SSC delivered with microgels yielded better preventive effects on articular lesions and macrophage activation in osteoarthritic rats than SSC without microgels. Chemically blocking the YAP and Ptgs2 in microgels-delivered SSC partially abolished the enhanced protection on articular tissues and suppression on osteoarthritic macrophages. Moreover, microgel carriers significantly prolonged SSC retention time in vivo without increasing SSC implanting into osteoarthritic joints. Together, our study demonstrated that microgel carriers enhanced SSC reprogramming towards immunomodulatory phenotype to regulate macrophage phenotype transformation for effectively osteoarthritic therapy by promoting YAP protein translocation into nucleus. The study not only complement and perfect the immunological mechanisms of SSC-based therapy at the single-cell level, but also provide new insight for microgel carriers in stem cell-based therapy.

Co-overexpression of OPN, IGF-1 and CNTF augment the therapeutic effect of DPSC on spinal cord injury.

Background aims: Spinal cord injury (SCI) is one of the most complex and destructive diseases of the nervous system, which can lead to permanent loss of tactile perception. But existing treatment methods have limited effects. To establish a novel method that may be therapeutic in repairing the injured spinal cord, gene-modified dental pulp stem cells (DPSCs) were injected in situ. Methods: Adenovirus carrying osteopontin (OPN), Insulin-like growth factor 1 (IGF-1) and cailiary-derived neurotrophic factor (CNTF) (Ad-OIC) was constructed. After modified with Ad-OIC, supernatant of DPSC were co-cultured with HT-22 cells and the effect of DPSC-OIC on the HT-22 cells were evaluated via Cell Counting Kit-8 (CCK-8) assay, Real-Time polymerase chain reaction (PCR) analysis, laser confocal microscopy and fluorescence activating cell sorter (FACS). DPSC-OIC were injected in the lesion area of injured spinal cord and the survival time of transplanted cells were measured by bioluminescence imaging system. The recovery of the injured spinal cord was evaluated by behavioral score, radiological evaluation and immunopathological analysis. Results: DPSC-OIC could enhance the proliferation and axon growth of HT-22 cells, and protect HT-22 cells from H2O2 induced apoptosis. The transplanted DPSC-Null or DPSC-OIC could survive for more than two weeks in local injection site. DPSC-OIC treatment could increase Basso-Mouse Scale (BMS) scores, improve Magnetic Resonance Imaging (MRI) manifestation and promote bladder function recovery. Less apoptotic neurons and more proliferative cells were found in the lesion area of DPSC-OIC treated spinal cord. Nestin+ cells and neural stem cell marker (Sox2) were both up-regulated after DPSC-OIC treatment. Additionally, inhibitory extracellular matrix proteoglycan Neural/Glial Antigen 2 (NG2) was down-regulated and axon growth promotive factor fibronectin was up-regulated after both DPSC-Null (DPSCs infected with Ad-Null) and DPSC-OIC treatments. Conclusions: DPSC-OIC could be a novel effective method for treating SCI.

Recombinant Neorudin for the Prevention of Deep-Vein Thrombosis After Spinal-Cord Injury.

Background: Whether anticoagulant therapy should be used after spinal-cord injury (SCI) surgery was controversial. The anticoagulation characteristics of a newly developed anticoagulant, recombinant neorudin (EPR-hirudin (EH)), were explored using a rat model of SCI to provide a basis for clinical anticoagulation therapy of SCI. Methods: A rat model of SCI was developed by Allen's method. Then, thrombosis in the inferior vena cava was induced by ligation. The low-bleeding characteristics of EH were explored by investigating dose-response and time-effect relationships, as well as multiple administration of EH, on thrombus formation complicated with SCI. Results: EH inhibited thrombosis in a dose-dependent manner by reducing the wet weight and dry weight of the thrombus. An inhibiting action of EH on thrombosis was most evident in the group given EH 2 h after SCI. After multiple intravenous doses of EH, thrombosis inhibition was improved to that observed with low molecular weight heparin (LMWH) (87% vs 90%). EH administration after SCI neither increased bleeding in the injured spine nor damaged to nerve function. Bleeding duration and activated partial thromboplastin time were increased in the high-dose EH group compared with that in the normal-saline group, but were lower than those in the LMWH group. Conclusion: EH can reduce thrombus formation in a rat model of SCI, and bleeding is decreased significantly compared with that using LMWH. EH may prevent thrombosis after SCI or spinal surgery.

The toxicity assessment of neorudin in cynomolgus monkeys.

In this study, the toxicity effects on circulatory system and respiratory system, and the acute toxicity test of recombinant neorudin (EPR-hirudin, EH) in cynomolgus monkeys were evaluated to provide reference information for clinical studies. Eighteen cynomolgus monkeys were randomly divided into three groups for single intravenous administration of 3, 30 mg/kg EH and normal saline, respectively. The changes of respiratory frequency, respiratory intensity, blood pressure and electrocardiogram before and after administration were recorded. In acute toxicity test, six cynomolgus monkeys were intravenously received EH at a single dose of 171, 257, 385, 578, 867 and 1300 mg/kg respectively. The vital signs, hematology, serum biochemistry, coagulation indexes and electrocardiogram indexes of the animals were determined before administration and on the 7th and 14th day after administration. As the results showed that there were no significant abnormal changes in respiratory frequency, respiratory intensity, blood pressure or electrocardiogram in cynomolgus monkeys after receiving EH at 3 mg/kg and 30 mg/kg, and there was no statistical difference between the treated groups and normal saline group. In the acute toxicity test, no significant abnormalities were observed in vital signs, hematology, serum biochemistry, coagulation indexes and electrocardiogram indexes of six cynomolgus monkeys at day 7 and 14 after EH administration. Furthermore, autopsies of all cynomolgus monkeys showed no abnormalities. The results of toxicokinetics showed that AUClast of the drug increased in proportion to the EH dose in the range of 171-578 mg/kg, and increased in over proportion to the EH dose in the range of 578-1300 mg/kg. The variation of Cmax was basically consistent with AUClast. In a sum, A single intravenous injection of 3 and 30 mg/kg of EH did not affect the circulatory system and respiratory system in cynomolgus monkeys and the maximum tolerated dose of EH in cynomolgus monkey is over 1300 mg/kg (equivalent to 619-1300 times of the proposed clinical equivalent dose).

CD146+ Umbilical Cord Mesenchymal Stem Cells Exhibit High Immunomodulatory Activity and Therapeutic Efficacy in Septic Mice.

Background: Several studies have shown that MSCs can significantly improve the survival of sepsis animals. CD146+ mesenchymal stem cells (MSCs) correlate with high therapeutic potency. However, their therapeutic effect on sepsis and detail mechanisms have not been explored. Methods: The effect of CD146±MSCs on differentiation of Treg, Th1, Th17 subsets was evaluated by flow cytometry. The effects of CD146±MSCs on RAW264.7 phagocytosis and LPS-stimulated polarization were studied using a co-culture protocol. Luminex bead array and RNA sequencing were employed to determine the mechanisms of MSCs on LPS-stimulated RAW264.7. The Arg1 protein was detected by Western blot. CD146±MSCs were injected into LPS-induced sepsis mice by tail vein. The therapeutic effect was assessed by organ HE staining, T-cell subsets, cytokine in plasma, peritoneal macrophages, infiltrating monocytes subpopulations. Results: In vitro, CD146+MSCs could significantly increase the proportion of Treg cells. Co-culture with CD146+MSCs increase the phagocytic rate of RAW264.7. CD146+MSCs regulate M2-type macrophages production more rapidly. The transcript profile differences between the CD146+MSCs and CD146-MSCs groups were clustered in arginine metabolism pathways. CD146+MSCs decreased NO production and increased ARG1 expression. CD146+MSCs secreted higher level of IL15,IFNγ, VEGF and lower level of IL1ß, IL8 under LPS stimuli. In vivo, The level of IL10 at 24h and CXCL1, IFNγ at 12h in CD146+MSCs group was the highest. CD146+MSCs treatment enhances the phagocytic capacity of peritoneal macrophages. CD146+MSCs also increases the ratios of CD11b+Ly6Clo reparative monocytes and CD11b+Ly6Chi inflammatory monocytes until 24h. Conclusion: Compared with CD146-MSCs, CD146+MSCs can accelerate the end of the inflammatory response and have robust anti-inflammatory effects, by increasing the Treg cells, promoting macrophage phagocytosis, enhancing the reparative macrophage, secreting more VEGF, etc.

DPSCs Protect Architectural Integrity and Alleviate Intervertebral Disc Degeneration by Regulating Nucleus Pulposus Immune Status.

Intervertebral disc (IVD) degeneration is the primary cause for low back pain that has a high prevalence in modern society and poses enormous economic burden on patients. Few effective therapeutic strategies are available for IVD degeneration treatment. To understand the biological effects of dental pulp stem cells (DPSCs) on nucleus pulposus (NP) cells, we carried out RNA sequencing, bioinformatic analysis which unveiled gene expression differences, and pathway variation in primarily isolated patients' NP cells after treatment with DPSCs supernatant. Western blot and immunofluorescence were used to verify these molecular alterations. Besides, to evaluate the therapeutic effect of DPSCs in IVD degeneration treatment, DPSCs were injected into a degeneration rat model in situ, with treatment outcome measured by micro-CT and histological analysis. RNA sequencing and in vitro experiments demonstrated that DPSCs supernatant could downregulate NP cells' inflammation-related NF-κB and JAK-STAT pathways, reduce IL-6 production, increase collagen II expression, and mitigate apoptosis. In vivo results showed that DPSCs treatment protected the integrity of the disc structure, alleviated extracellular matrix degradation, and increased collagen fiber expression. In this study, we verified the therapeutic effect of DPSCs in an IVD degeneration rat model and elucidated the underlying molecular mechanism of DPSCs treatment, which provides a foundation for the application of DPSCs in IVD degeneration treatment.

Recent advances in the therapeutic efficacy of hepatocyte growth factor gene-modified mesenchymal stem cells in multiple disease settings.

Mesenchymal stem cell (MSC) therapy is considered a new treatment for a wide range of diseases and injuries, but challenges remain, such as poor survival, homing and engraftment rates, thus limiting the therapeutic efficacy of the transplanted MSCs. Many strategies have been developed to enhance the therapeutic efficacy of MSCs, such as preconditioning, co-transplantation with graft materials and gene modification. Hepatocyte growth factor (HGF) is secreted by MSCs, which plays an important role in MSC therapy. It has been reported that the modification of the HGF gene is beneficial to the therapeutic efficacy of MSCs, including diseases of the heart, lung, liver, urinary system, bone and skin, lower limb ischaemia and immune-related diseases. This review focused on studies involving HGF/MSCs both in vitro and in vivo. The characteristics of HGF/MSCs were summarized, and the mechanisms of their improved therapeutic efficacy were analysed. Furthermore, some insights are provided for HGF/MSCs' clinical application based on our understanding of the HGF gene and MSC therapy.

Comparison of CD146 +/- mesenchymal stem cells in improving premature ovarian failure.

BACKGROUND: Mesenchymal stem cells (MSCs) are a heterogeneous group of subpopulations with differentially expressed surface markers. CD146 + MSCs correlate with high therapeutic and secretory potency. However, their therapeutic efficacy and mechanisms in premature ovarian failure (POF) have not been explored. METHODS: The umbilical cord (UC)-derived CD146 +/- MSCs were sorted using magnetic beads. The proliferation of MSCs was assayed by dye670 staining and flow cytometry. A mouse POF model was established by injection of cyclophosphamide and busulfan, followed by treatment with CD146 +/- MSCs. The therapeutic effect of CD146 +/- MSCs was evaluated based on body weight, hormone levels, follicle count and reproductive ability. Differential gene expression was identified by mRNA sequencing and validated by RT-PCR. The lymphocyte percentage was detected by flow cytometry. RESULTS: CD146 +/- MSCs had similar morphology and surface marker expression. However, CD146 + MSCs exhibited a significantly stronger proliferation ability. Gene profiles revealed that CD146 + MSCs had a lower levels of immunoregulatory factor expression. CD146 + MSCs exhibited a stronger ability to inhibit T cell proliferation. CD146 +/- MSCs treatment markedly restored FSH and E2 hormone secretion level, reduced follicular atresia, and increased sinus follicle numbers in a mouse POF model. The recovery function of CD146 + MSCs in a reproductive assay was slightly improved than that of CD146 - MSCs. Ovary mRNA sequencing data indicated that UC-MSCs therapy improved ovarian endocrine locally, which was through PPAR and cholesterol metabolism pathways. The percentages of CD3, CD4, and CD8 lymphocytes were significantly reduced in the POF group compared to the control group. CD146 + MSCs treatment significantly reversed the changes in lymphocyte percentages. Meanwhile, CD146 - MSCs could not improve the decrease in CD4/8 ratio induced by chemotherapy. CONCLUSION: UC-MSCs therapy improved premature ovarian failure significantly. CD146 +/- MSCs both had similar therapeutic effects in repairing reproductive ability. CD146 + MSCs had advantages in modulating immunology and cell proliferation characteristics.

The Antithrombotic Effect of Recombinant Neorudin on Thrombi.

Introduction: Recombinant neorudin (EPR-hirudin, EH) was developed through the addition of an EPR (Glu-Pro-Arg) peptide to the amino terminus of hirudin, which can be recognized and cut by coagulation factors XIa (FXIa) and/or Xa (FXa). In this study, the low-bleeding antithrombotic effects of EH were evaluated utilizing experimental models of thrombosis in rabbits and rats to provide a test basis for clinical trials. Methods: The bleeding risks of EH and hirudin were first compared in mice by the tail-clipping method, and then the antithrombotic activity of EH was investigated in a rabbit model of arteriovenous bypass thrombosis and a rat model of thrombotic cerebral infarction. Results: In mice, intravenous administration of EH at 1.5 mg/kg and 3 mg/kg did not affect the bleeding time compared with normal saline, while the administration of hirudin at 1.5 mg/kg prolonged the bleeding time by over 3 times the administration of normal saline. Furthermore, intravenous administration of EH had a significant dose-dependent inhibitory effect on the formation and development of arteriovenous bypass thrombosis and thrombotic cerebral infarction. Compared with an equimolar dose of hirudin, the antithrombotic effect of EH was similar, while the bleeding side effects were significantly attenuated. Moreover, when the antithrombotic effects were similar, EH had a shorter bleeding time and was associated with less bleeding than low molecular weight heparin (LMWH). EH had a therapeutic effect on thrombotic cerebral infarction without increasing the occurrence of cerebral hemorrhage. Conclusion: The findings from the preclinical animal models used in this study showed that EH could not only effectively inhibit thrombus formation but also reduce the risk of bleeding.

Inhibitory role of recombinant neorudin on canine coronary artery thrombosis.

The anticoagulant application is an effective treatment modality for cardiovascular diseases such as coronary heart disease, unstable angina pectoris, and myocardial infarction. In this study, the antithrombotic effect of recombinant neorudin (EPR-hirudin, EH) was evaluated using a canine model of coronary artery thrombosis. A canine model with platelet thrombosis in the left circumferent branch of the coronary artery was designed using Folt's method, and the anti-thrombus activity of EH was investigated. Femoral administration of EH intravenously had a significant dose-dependent inhibitory effect on canine coronary artery thrombosis and the effective rates were 66.7% (p < .05), 83.3% (p < .05), and 100% (p < .01) after injection of 0.3, 1.0, and 3.0 mg/kg EH, respectively. Furthermore, EH demonstrated lower bleeding, with shorter bleeding time and less bleeding loss than low molecular weight heparin (LMWH). Under the similar effect intensity of EH and LMWH (85 IU/kg), the bleeding time of the EH group at 30 min was shorter, and the blood loss at 30-120 min was less than that of LMWH (p < .05 and p < .05-.001, respectively). EH had a significant dose-dependent inhibitory effect in the dose range of 0.3-3.0 mg/kg on the coronary artery thrombosis and lower bleeding side effects than LMWH with a similar antithrombosis effect.

Long-term hypoxia inhibits the passage-dependent stemness decrease and senescence increase of human dental pulp stem cells.

Dental pulp stem cells (DPSCs) derived from discarded orthodontic teeth are easily obtained and have become a promising source for mesenchymal stem cell-based therapy. However, the pulp tissue is limited, and long-term culture induces cell senescence. Hypoxic culture was expected to be suitable for DPSC expansion, but the results have been contradictory. The aim of this study was to verify the effect of hypoxic culture on human DPSCs (hDPSCs). hDPSCs were isolated and cultured in normoxic (ambient O2 concentration) and hypoxic (5% O2) environments from passage 3 (P3) to P6. The biological characteristics of the cells at P4 (short-term culture) and P6 (long-term culture) were evaluated, including the expression of surface markers, cellular proliferation activity, cellular senescence, and spontaneous and induced differentiation. The results showed that the morphology, phenotype, and proliferation activity of hDPSCs were not affected by hypoxic culture. Long-term normoxic culture of hDPSCs induced cell stemness loss and cell senescence, while hypoxic culture could alleviate these effects. The expression of the stemness markers STRO-1 and OCT4 was increased and the number of senescent cells and the expression of the senescence-related genes P53 and TGF-ß were reduced by long-term hypoxic culture. Spontaneous osteogenic and adipogenic differentiation did not occur during long-term normoxic culture. However, hypoxic culture suppressed the expression of the osteogenic markers ALP and RUNX-2 and the adipogenic markers PPAR-γ and FABP4. The induced osteogenic and adipogenic differentiation was apparently reduced by hypoxic culture as well. Our findings indicate that long-term hypoxia culture is beneficial to the maintenance of hDPSCs' biological characteristics and provide some insights into their large-scale expansion.

Dental Pulp Mesenchymal Stem Cells Attenuate Limb Ischemia via Promoting Capillary Proliferation and Collateral Development in a Preclinical Model.

Critical limb ischemia (CLI), an end-stage manifestation of peripheral artery disease (PAD), still lacks effective therapeutic strategies. Recently, dental pulp-derived mesenchymal stem cells (DP-MSCs) have been attracting more and more attentions in therapeutic applications due to their high proliferation ability, powerful osteogenic differentiation potential, and effective anti-inflammatory effects. In this study, we compared the therapeutic effects of MSCs derived from different sources in a femoral artery-ligated preclinical ischemic model. We found that treatments with MSCs, including bone marrow- (BM-), adipose- (AD-), dental pulp- (DP-), and umbilical cord- (UC-) derived MSCs, improved limb functions, reduced inflammatory responses, increased angiogenesis, and promoted regeneration of muscle fiber. Among them, DP-MSCs and BM-MSCs produced much more impressive effects in restoring limb functions and promoting angiogenesis. The flow velocity restored to nearly 20% of the normal level at 3 weeks after treatments with DP-MSCs and BM-MSCs, and obvious capillary proliferation and collateral development could be observed. Although neovascularization was induced in the ischemic limb after ligation, MSCs, especially DP-MSCs, significantly enhanced the angiogenesis. In vitro experiments showed that serum deprivation improved the expression of angiogenic factors, growth factors, and chemokines in DP-MSCs and UC-MSCs, but not in BM-MSCs and AD-MSCs. However, DP-MSCs produced stronger therapeutic responses than UC-MSCs, which might be due to the higher expression of hepatocyte growth factor (HGF) and hypoxia-inducible factor-1 α (HIF-1α). We speculated that DP-MSCs might stimulate angiogenesis and promote tissue repair via expressing and secreting angiogenic factors, growth factors, and chemokines, especially HGF and HIF-1α. In conclusion, DP-MSCs might be a promising approach for treating CLI.

Overexpression of Hepatocyte Growth Factor in Dental Pulp Stem Cells Ameliorates the Severity of Psoriasis by Reducing Inflammatory Responses.

Psoriasis is an autoimmune disease still lacking standard treatment, and it has been demonstrated that mesenchymal stem cells (MSCs) are capable of immunoregulation. The underlying mechanism might involve the secretion of soluble cytokines, such as hepatocyte growth factor (HGF). This study aims to investigate the therapeutic effect of HGF-overexpressed dental pulp stem cells (DPSCs) [DPSCs; HGF overexpressed DPSCs (HGF-DPSCs)] on imiquimod-induced psoriasis. DPSCs were isolated and transfected by adenovirus vector carrying HGF gene (Ad-HGF). The immunoregulatry abilities of DPSCs and HGF-DPSCs were investigated by coculture of the MSCs with peripheral blood mononuclear cells (PBMCs) under appropriated stimulation. The psoriatic mice were treated with saline control, DPSCs, or HGF-DPSCs. Then the mice spleens were collected and weighted. The psoriatic skin lesions were analyzed by Hematoxylin/Eosin and immunohistochemical staining for histopathological changes, and quantitative real-time polymerase chain reaction to detect the expression levels of CD4+ T cell-related transcription factors and cytokines. The mice blood serum was measured by MILLIPLEX analysis and enzyme-linked immunosorbent assay to evaluate the expression levels of inflammation cytokines. The coculture experiments showed HGF overexpression enhanced the immunoregulation abilities of DPSCs not by suppressing PBMCs' proliferation, but by downregulating T helper 1 (Th1), Th17 cells, and upregulating regulatory T (Treg) cells. In psoriatic skin lesions, the psoriasis-like erythema, scaling, and thickening were ameliorated; and the expression of cytokeratin 6 (CK6), and cytokeratin 17 (CK17) were downregulated by DPSCs and HGF-DPSCs treatment. HGF overexpression enhanced the decrease of spleen masses; enhanced the downregulation of the expression levels of interferon-gamma (IFN-γ), tumor necrosis factor-α, and interleukin (IL)-17A in the blood serums; enhanced the downregulation of T-box transcription factor 21 (T-bet), IFN-γ, retinoic acid-related orphan receptor-γt (RORγt), IL-17A, IL-17F, IL-23, and upregulation of Foxp3 and IL-10 in the psoriatic skin lesions. Therefore, HGF overexpression enhanced DPSCs' treatment effect on psoriasis mainly by reducing inflammatory responses. These findings might provide new immunoregulation strategies for psoriasis treatment.

A phase I, single and continuous dose administration study on the safety, tolerability, and pharmacokinetics of neorudin, a novel recombinant anticoagulant protein, in healthy subjects.

The aim of this study was to evaluate the tolerability, safety, and pharmacokinetics of single and continuous dose administration of recombinant neorudin (EPR-hirudin, EH) by intravenous administration in healthy subjects, and to provide a safe dosage range for phase II clinical research. Forty-four subjects received EH as a single dose of between 0.2 and 2.0 mg/kg by intravenous bolus and drip infusion. In addition, 18 healthy subjects were randomly divided into three dose groups (0.15, 0.30, and 0.45 mg/kg/h) with 6 subjects in each group for the continuous administration trial. Single or continuous doses of neorudin were generally well tolerated by healthy adult subjects. There were no serious adverse events (SAEs), and all adverse events (AEs) were mild to moderate. Moreover, no subjects withdrew from the trial because of AEs. There were no clinically relevant changes in physical examination results, clinical chemistry, urinalysis, or vital signs. The incidence of adverse events was not significantly related to drug dose or systemic exposure. After single-dose and continuous administration, the serum EH concentration reached its peak at 5 min, and the exposure increased with the increase in the administered dose. The mean half-life (T1/2 ), clearance (Cl), and apparent volume of distribution (Vd) of EH ranged from 1.7 to 2.5 h, 123.9 to 179.7 ml/h/kg, and 402.7 to 615.2 ml/kg, respectively. The demonstrated safety, tolerability, and pharmacokinetic characteristics of EH can be used to guide rational drug dosing and choose therapeutic regimens in subsequent clinical studies. Clinical trial registration: Chinadrugtrials.org identifier: CTR20160444.

Clinical-grade human dental pulp stem cells suppressed the activation of osteoarthritic macrophages and attenuated cartilaginous damage in a rabbit osteoarthritis model.

BACKGROUND: Although increasing evidence has demonstrated that human dental pulp stem cells (hDPSCs) are efficacious for the clinical treatment of skeletal disorders, the underlying mechanisms remain incompletely understood. Osteoarthritis (OA) is one of the most common degenerative disorders in joints and is characterized by gradual and irreversible cartilaginous tissue damage. Notably, immune factors were newly identified to be closely related to OA development. In this study, we explored the modulatory effects of clinical-grade hDPSCs on osteoarthritic macrophages and their protective effects on cartilaginous tissues in OA joints. METHODS: The cell morphology, immunophenotype, and inflammatory factor expression of osteoarthritic macrophages were explored by phase contrast microscope, transmission electron microscopy, immunostaining, flow cytometry, quantitative polymerase chain reaction, and enzyme linked immunosorbent assay, respectively. Additionally, the factors and signaling pathways that suppressed macrophage activation by hDPSCs were determined by enzyme-linked immunosorbent assay and western-blotting. Furthermore, hDPSCs were administered to a rabbit knee OA model via intra-articular injection. Macrophage activation in vivo and cartilaginous tissue damage were also evaluated by pathological analysis. RESULTS: We found that hDPSCs markedly inhibited osteoarthritic macrophage activation in vitro. The cell morphology, immunophenotype, and inflammatory factor expression of osteoarthritic macrophages changed into less inflammatory status in the presence of hDPSCs. Mechanistically, we observed that hDPSC-derived hepatocyte growth factor and transforming growth factor ß1 mediated the suppressive effects on osteoarthritic macrophages. Moreover, phosphorylation of MAPK pathway proteins contributed to osteoarthritic macrophage activation, and hDPSCs suppressed their activation by partially inactivating those pathways. Most importantly, injected hDPSCs inhibited macrophage activation in osteochondral tissues in a rabbit knee OA model in vivo. Further histological analysis showed that hDPSCs alleviated cartilaginous damage to knee joints. CONCLUSIONS: In summary, our findings reveal a novel function for hDPSCs in suppressing osteoarthritic macrophages and suggest that macrophages are efficient cellular targets of hDPSCs for alleviation of cartilaginous damage in OA. hDPSCs treat OA via an osteoarthritic macrophages-dependent mechanisms. hDPSCs suppress the activation of osteoarthritic macrophages in vitro and in vivo and alleviate cartilaginous lesions in OA models.

Proteome Profiling Identified Amyloid-ß Protein Precursor as a Novel Binding Partner and Modulator of VGLUT1.

BACKGROUND: The toxicity of excessive glutamate release has been implicated in various acute and chronic neurodegenerative conditions. Vesicular glutamate transporters (VGLUTs) are the major mediators for the uptake of glutamate into synaptic vesicles. However, the dynamics and mechanism of this process in glutamatergic neurons are still largely unknown. OBJECTIVE: This study aimed to investigate the candidate protein partners of VGLUT1 and their regulatory roles in the vesicles in rat brain. METHODS: Pull down assay, co-immunoprecipitation assay, or split-ubiquitin membrane yeast two hybrid screening coupled with nanoRPLC-MS/MS were used to identify the candidate protein partners of VGLUT1 in the vesicles in rat brain. The in vitro and in vivo models were used to test effects of AßPP, Atp6ap2, Gja1, and Synataxin on VGLUT1 expression. RESULTS: A total of 255 and 225 proteins and 172 known genes were identified in the pull down assay, co-immunoprecipitation assay, or split-ubiquitin yeast two-hybrid screening respectively. The physiological interactions of SV2A, Syntaxin 12, Gja1, AßPP, and Atp6ap2 to VGLUT1 were further confirmed. Knockdown of Atp6ap2, Gja1, and Synataxin increased VGLUT1 mRNA expression and only knockdown of AßPP increased both mRNA and protein levels of VGLUT1 in PC12 cells. The regulatory function of AßPP on VGLUT1 expression was further confirmed in the in vitro and in vivo models. CONCLUSION: These results elucidate that the AßPP and VGLUT1 interacts at vesicular level and AßPP plays a role in the regulation of VGLUT1 expression which is essential for maintaining vesicular activities.

HGF-Modified Dental Pulp Stem Cells Mitigate the Inflammatory and Fibrotic Responses in Paraquat-Induced Acute Respiratory Distress Syndrome.

Paraquat (PQ) poisoning can cause acute lung injury and progress to pulmonary fibrosis and eventually death without effective therapy. Mesenchymal stem cells (MSCs) and hepatocyte growth factor (HGF) have been shown to partially reverse this damage. MSCs can be derived from bone marrow (BM-MSCs), adipose tissue (AD-MSCs), umbilical cord (UC-MSCs), dental pulp (DPSCs), and other sources. The biological characteristics of MSCs are specific to the tissue source. To develop an effective treatment for PQ poisoning, we compared the anti-inflammatory and antifibrotic effects of UC-MSCs and DPSCs and chose and modified a suitable source with HGF to investigate their therapeutic effects in vitro and in vivo. In this study, MSCs' supernatant was beneficial to the viability and proliferation of human lung epithelial cell BEAS-2B. Inflammatory and fibrosis-related cytokines were analyzed by real-time PCR. The results showed that MSCs' supernatant could suppress the expression of proinflammatory and profibrotic cytokines and increase the expression of anti-inflammatory and antifibrotic cytokines in BEAS-2B cells and human pulmonary fibroblast MRC-5. Extracellular vesicles (EVs) derived from MSCs performed more effectively than MSCs' supernatant. The effect of DPSCs was stronger than that of UC-MSCs and was further strengthened by HGF modification. PQ-poisoned mice were established, and UC-MSCs, DPSCs, and DPSCs-HGF were administered. Histopathological assessments revealed that DPSCs-HGF mitigated lung inflammation and collagen accumulation more effectively than the other treatments. DPSCs-HGF reduced lung permeability and increased the survival rate of PQ mice from 20% to 50%. Taken together, these results indicated that DPSCs can suppress inflammation and fibrosis in human lung cells better than UC-MSCs. The anti-inflammatory and antifibrotic effects were significantly enhanced by HGF modification. DPSCs-HGF ameliorated pulmonitis and pulmonary fibrosis in PQ mice, effectively improving the survival rate, which might be mediated by paracrine mechanisms. The results suggested that DPSCs-HGF transplantation was a potential therapeutic approach for PQ poisoning.

Dental Pulp Stem Cell-Derived Extracellular Vesicles Mitigate Haematopoietic Damage after Radiation.

Radiation therapy can cause haematopoietic damage, and mesenchymal stem cells (MSCs) derived extracellular vesicles (EVs) have been shown to reverse this damage. Our previous research showed that dental pulp stem cells (DPSCs) have a strong proliferation capacity and can produce abundant amounts of EVs to meet the requirements for use in vitro and in vivo. DPSCs derived EVs (DPSCs-EVs) are evaluated for their effect on reducing haematopoietic damage. Haematopoietic stem cell (HSC) numbers and function were assessed by flow cytometry, peripheral blood cell counts, histology and bone marrow transplantation. Epidermal growth factor (EGF) was used as a reference for evaluating the efficiency of EVs. miRNA microarray was employed to find out the changes of miRNA expression after cells being irradiated in vivo and the role they may play in mitigation the radiation caused injury. We observed the effect of DPSCs-EVs on promoting proliferation and inhibiting apoptosis of human umbilical vein endothelial cells (HUVECs) and FDC-P1 cells in vitro. We found that DPSCs-EVs and EGF could comparably inhibit the decrease in WBC, CFU count and KSL cells in vivo. We also verified that EVs could accelerate the recovery of long-term HSCs. In summary, DPSCs-EVs showed an apoptosis resistant effect on HUVECs and FDC-P1 cells after radiation injury in vitro. EVs from DPSCs were comparable to EGF in their ability to regulate haematopoietic regeneration after radiation injury in vivo. Radiation could alter the expression of some miRNAs in bone marrow cells, and EVs could correct these changes to some extent. Graphical abstract.

Safety and efficacy assessment of allogeneic human dental pulp stem cells to treat patients with severe COVID-19: structured summary of a study protocol for a randomized controlled trial (Phase I / II).

OBJECTIVES: To assess the safety and therapeutic effects of allogeneic human dental pulp stem cells (DPSCs) in treating severe pneumonia caused by COVID-19. TRIAL DESIGN: This is a single centre, two arm ratio 1:1, triple blinded, randomized, placebo-controlled, parallel group, clinical trial. PARTICIPANTS: Twenty serious COVID-19 cases will be enrolled in the trial from April 6th to December 31st 2020. INCLUSION CRITERIA: hospitalised patients at Renmin Hospital of Wuhan University satisfy all criteria as below: 1)Adults aged 18-65 years;2)Voluntarily participate in this clinical trial and sign the "informed consent form" or have consent from a legal representative.3)Diagnosed with severe pneumonia of COVID-19: nucleic acid test SARS-CoV-2 positive; respiratory distress (respiratory rate > 30 times / min); hypoxia (resting oxygen saturation < 93% or arterial partial pressure of oxygen / oxygen concentration < 300 mmHg).4)COVID-19 featured lung lesions in chest X-ray image. EXCLUSION CRITERIA: Patients will be excluded from the study if they meet any of the following criteria. 1.Patients have received other experimental treatment for COVID-19 within the last 30 days;2.Patients have severe liver condition (e.g., Child Pugh score >=C or AST> 5 times of the upper limit);3.Patients with severe renal insufficiency (estimated glomerular filtration rate <=30mL / min/1.73 m2) or patients receiving continuous renal replacement therapy, hemodialysis, peritoneal dialysis;4.Patients who are co-infected with HIV, hepatitis B, tuberculosis, influenza virus, adenovirus or other respiratory infection viruses;5.Female patients who have no sexual protection in the last 30 days prior to the screening assessment;6.Pregnant or lactating women or women using estrogen contraception;7.Patients who are planning to become pregnant during the study period or within 6 months after the end of the study period;8.Other conditions that the researchers consider not suitable for participating in this clinical trial. INTERVENTION AND COMPARATOR: There will be two study groups: experimental and control. Both will receive all necessary routine treatment for COVID-19. The experimental group will receive an intravenous injection of dental pulp stem cells suspension (3.0x107 human DPSCs in 30ml saline solution) on day 1, 4 and 7; The control group will receive an equal amount of saline (placebo) on the same days. Clinical and laboratory observations will be performed for analysis during a period of 28 days for each case since the commencement of the study. MAIN OUTCOMES: 1. Primary outcome The primary outcome is Time To Clinical Improvement (TTCI). By definition, TTCI is the time (days) it takes to downgrade two levels from the following six ordered grades [(grade 1) discharge to (grade 6) death] in the clinical state of admission to the start of study treatments (hDPSCs or placebo). Six grades of ordered variables: GradeDescriptionGrade 1:Discharged of patient;Grade 2:Hospitalized without oxygen supplement;Grade 3:Hospitalized, oxygen supplement is required, but NIV / HFNC is not required;Grade 4:Hospitalized in intensive care unit, and NIV / HFNC treatment is required;Grade 5:Hospitalized in intensive care unit, requiring ECMO and/or IMV;Grade 6:Death. ABBREVIATIONS: NIV, non-invasive mechanical ventilation; HFNC, high-flow nasal catheter; IMV, invasive mechanical ventilation. 2. Secondary outcomes 2.1 vital signs: heart rate, blood pressure (systolic blood pressure, diastolic blood pressure). During the screening period, hospitalization every day (additional time points of D1, D4, D7 30min before injection, 2h ± 30min, 24h ± 30min after the injection) and follow-up period D90 ± 3 days. 2.2 Laboratory examinations: during the screening period, 30 minutes before D1, D4, D7 infusion, 2h ± 30min, 24h ± 30min after the end of infusion, D10, D14, D28 during hospitalization or discharge day and follow-up period D90 ± 3 days. 2.3 Blood routine: white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, neutrophils, lymphocytes, monocytes, eosinophils Acidic granulocyte count, basophil count, red blood cell, hemoglobin, hematocrit, average volume of red blood cells, average red blood cell Hb content, average red blood cell Hb concentration, RDW standard deviation, RDW coefficient of variation, platelet count, platelet specific platelet average Volume, platelet distribution width,% of large platelets; 2.4 Liver and kidney function tests: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transferase, prealbumin, total protein, albumin, globulin, white / globule ratio , Total bilirubin, direct bilirubin, cholinesterase, urea, creatinine, total carbon dioxide, uric acid glucose, potassium, sodium, chlorine, calcium, corrected calcium, magnesium, phosphorus, calcium and phosphorus product, anion gap, penetration Pressure, total cholesterol, triacylglycerol, high density lipoprotein cholesterol, Low density lipoprotein cholesterol, lipoprotein a, creatine kinase, lactate dehydrogenase, estimated glomerular filtration rate. 2.5 Inflammation indicators: hypersensitive C-reactive protein, serum amyloid (SAA); 2.6 Infectious disease testing: Hepatitis B (HBsAg, HBsAb, HBeAg, HBeAb, HBcAb), Hepatitis C (Anti-HCV), AIDS (HIVcombin), syphilis (Anti-TP), cytomegalovirus CMV-IgM, cytomegalovirus CMV-IgG; only during the screening period and follow-up period D90 ± 3. 2.7 Immunological testing: Collect peripheral blood to detect the phenotype of T lymphocyte, B lymphocyte, natural killer cell, Macrophage and neutrophil by using flow cytometry. Collect peripheral blood to detect the gene profile of mononuclear cells by using single-cell analyses. Collect peripheral blood serum to detect various immunoglobulin changes: IgA, IgG, IgM, total IgE; Collect peripheral blood serum to explore the changes of cytokines, Th1 cytokines (IL-1 ß, IL-2, TNF-a, ITN-γ), Th2 cytokines (IL-4, IL-6, IL -10). 2.8 Pregnancy test: blood ß-HCG, female subjects before menopause are examined during the screening period and follow-up period D90 ± 3. 2.9 Urine routine: color, clarity, urine sugar, bilirubin, ketone bodies, specific gravity, pH, urobilinogen, nitrite, protein, occult blood, leukocyte enzymes, red blood cells, white blood cells, epithelial cells, non-squamous epithelial cells , Transparent cast, pathological cast, crystal, fungus; 2.10 Stool Routine: color, traits, white blood cells, red blood cells, fat globules, eggs of parasites, fungi, occult blood (chemical method), occult blood (immune method), transferrin (2h ± 30min after the injection and not detected after discharge). RANDOMIZATION: Block randomization method will be applied by computer to allocate the participants into experimental and control groups. The random ratio is 1:1. BLINDING (MASKING): Participants, outcomes assessors and investigators (including personnel in laboratory and imaging department who issue the sample report or image observations) will be blinded. Injections of cell suspension and saline will be coded in accordance with the patient's randomisation group. The blind strategy is kept by an investigator who does not deliver the medical care or assess primary outcome results. NUMBERS TO BE RANDOMIZED (SAMPLE SIZE): Twenty participants will be randomized to the experimental and control groups (10 per group). TRIAL STATUS: Protocol version number, hDPSC-CoVID-2019-02-2020 Version 2.0, March 13, 2020. Patients screening commenced on 16th April and an estimated date of the recruitment of the final participants will be around end of July. . TRIAL REGISTRATION: Registration: World Health Organization Trial Registry: ChiCTR2000031319; March 27,2020. ClinicalTrials.gov Identifier: NCT04336254; April 7, 2020 Other Study ID Numbers: hDPSC-CoVID-2019-02-2020 FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Pulp stem cells with hepatocyte growth factor overexpression exhibit dual effects in rheumatoid arthritis.

BACKGROUND: To investigate the therapeutic effect of human dental pulp stem cells (DPSCs) transfected with adenovirus expressing hepatocyte growth factor (HGF) in a mouse model of collagen-induced arthritis (CIA). METHODS: DPSCs were modified with Ad-HGF to produce HGF-overexpressing DPSCs, DPSCs-HGF. In experimental mouse CIA model, DPSCs-HGF and DPSCs-Null (modified with Ad-Null) were engrafted via intravenously after disease onset, which was determined by the presence of joint swelling. The therapeutic effects on joints were evaluated at 49 days after collagen injection by histopathological analysis and microcomputed tomography imaging. The inflammatory cytokines were analyzed both in sera and joints via MILLIPLEX kit and immunohistochemical staining, respectively, and the regulatory T cells (Tregs) were analyzed in peripheral blood by using flow cytometry. Furthermore, primary fibroblast-like synoviocytes were isolated, colony formation analysis and FACS were performed to evaluate the effect of HGF on the proliferation and cell cycle of FLSs. Western blot assay was carried out to clarify the signal pathway of HGF-cMet. RESULTS: We found that without HGF modification, DPSC transfusion was helpful in controlling autoimmune status, local synovitis, and bone erosion after intravenous administration. However, HGF-modified DPSCs have dual role in rheumatoid arthritis (RA). In the early phase, HGF overexpression inhibited RA progression by its immunosuppressive effects, while in the late phase, HGF promoted synovitis by activating fibroblast-like synoviocytes to produce pathogenic IL-6, accelerating cell proliferation and inducing apoptosis resistance via phosphorylating the c-Met/Akt pathway. The overall effect of HGF modification attenuated the therapeutic effect of DPSCs. CONCLUSIONS: Our study provides a comprehensive evaluation of the therapeutic effect of DPSCs in the mouse model and a primary answer to the divergence of whether HGF is harmful or helpful in RA.