One-step cell biomanufacturing platform:porous gelatin microcarrier beads promote human embryonic stem cell-derived midbrain dopaminergic progenitor cell differentiation in vitro and survival after transplantation in vivo

Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.

In vitro assay system for primordial germ cell development

In mammalian embryos, primordial germ cells （PGCs） are induced in proximal epiblast by BMP signals expressed in extraembryonic tissues , and one of the most important downstream key players is Blimpl . Blimpl is known to act as a sup- pressor of somatic genes＇ expression in PGC specification process. It is assumed that there are still many unidentified molecules involved in PGC development. However, to analyze gene functions in mammalian embryos by generating knockout animals is labor-intensive, and a more concise way to efficiently identify genes regulating PGC development is desirable.

Immunity-and-matrix-regulatory cells enhance cartilage regeneration for meniscus injuries: a phase I dose-escalation trial

Immunity-and-matrix-regulatory cells(IMRCs)derived from human embryonic stem cells have unique abilities in modulating immunity and regulating the extracellular matrix,which could be mass-produced with stable biological properties.Despite resemblance to mesenchymal stem cells(MSCs)in terms of self-renew and tri-lineage differentiation,the ability of IMRCs to repair the meniscus and the underlying mechanism remains undetermined.Here,we showed that IMRCs demonstrated stronger immunomodulatory and pro-regenerative potential than umbilical cord MSCs when stimulated by synovial fluid from patients with meniscus injury.Following injection into the knees of rabbits with meniscal injury,IMRCs enhanced endogenous fibrocartilage regeneration.In the dose-escalating phase I clinical trial(NCT03839238)with eighteen patients recruited,we found that intra-articular IMRCs injection in patients was safe over 12 months post-grafting.Furthermore,the effective results of magnetic resonance imaging(MRI)of meniscus repair and knee functional scores suggested that 5×107 cells are optimal for meniscus injury treatment.In summary,we present the first report of a phase I clinical trial using IMRCs to treat meniscus injury.Our results demonstrated that intra-articular injection of IMRCs is a safe and effective therapy by providing a permissive niche for cartilage regeneration.

Combination of Quercetin, Hirudin and Cinnamaldehyde Promotes Schwann Cell Differentiation and Myelination against High Glucose by Inhibiting ERK Signaling Pathway

Objective:To investigate the therapeutic and synergistic effects of QHC(combination of quercetin(Q),hirudin(H)and cinnamaldehyd(C))on Schwann cell differentiation and myelination against high glucose(HG)induced injury.Methods:Primary-culture Schwann cells exposed to HG(50 mmol/L)for 72 h and Schwann cell–dorsal root ganglion(DRG)neuron cocultures exposed to HG(50 mmol/L)for 7 days were employed as in vitro model of diabetic neuropathy.The cells were randomly divided into 10 groups:control(CON,25 mmol/L glucose),HG(50 mmol/L glucose),HG plus 10μmol/L quercetin(Q),HG plus 0.04 IU/mL hirudin(H),HG plus 100 nmol/L cinnamaldehyd(C),HG plus 10μmol/L quercetin and 0.04 IU/mL hirudin(QH),HG plus 10μmol/L quercetin and 50 nmol/L cinnamaldehyd(QC),HG plus 0.04 IU/mL hirudin and 50 nmol/L cinnamaldehyd(HC),HG plus 10μmol/L quercetin,0.04 IU/mL hirudin and 50 nmol/L cinnamaldehyd(QHC)or 10μmol/L U0126.Cell differentiation was evaluated by periaxin immunofluorescence staining.The protein expression levels of myelin protein zero(P0),myelin basic protein(MBP),myelin-associated glycoprotein(MAG),extracellular signal-regulated kinase(ERK),p-ERK,p-c-Jun,c-Jun,notch intracellular domain(NICD)and the mRNA expression levels of P0,MBP,MAG,Krox-20,Notch1 and Jagged1 were detected by Western blotting and real-time quantitative PCR analysis.The secretion of ciliary neurotrophic factor(CNTF)was determined by enzyme-linked immunosorbent assay(ELISA).The number and length of the myelin segments were evaluated by MBP immunofluorescence staining.The expression and the location of p-ERK in cocultures were detected by MAG and p-ERK immunofluorescence double staining.Results:Co-treatment with Q,C,H and their combination promoted Schwann cell differentiation,increased CNTF secretion,up-regulated the protein and m RNA expressions of myelin,and increased the number and length of the myelin segments(P<0.01 or P<0.05).In particular,the combination therapy of Q,H and C was superior to the respective monotherapy(P<0.01).Combination therapy of QHC exhibited higher inhibitory activities for ERK signaling related molecules than each monomer or the combination of the two monomers(P<0.01).Conclusions:QHC combination yielded synergy in promoting Schwann cell differentiation and myelination and the protective effect may involve in the inhibition of ERK signaling pathway,providing scientific evidence for better understanding of combination of Q,H and C in clinical applications.

Diagnosis and Treatment Guidelines for Mesenchymal Stem Cell Therapy for Coronavirus Disease 2019(Beijing,2021)

The coronavirus disease 2019(COVID-19)can be caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection,and has led to millions of deaths among more than 100 million infected people around the world according to the declaration from World Health Organization.Dysregulated immune response of both the innate and adaptive immune systems is subsistent on COVID-19 patients,of which the degree are associated with disease severity,lung damage and long term functional disability.Current treatment options have included antiretroviral drugs,anti-inflammatory factors,antibodies,immune checkpoint inhibitors,and convalescent plasma therapy.More recently,mesenchymal stem cell(MSC)therapy has been explored for the management and control of COVID-19,particularly with the aim of preventing or at least mitigating respiratory co-morbidities.Though the safety and efficacy of stem cell therapy have been validated in multiple phase I–III clinical trials,to date,no standardized stem cell preparation,administration dosage or interval,product QA/QC testing,storage,transportation,or disposal protocols have been established.The present paper proposes a systematic methodology that addresses all the foregoing process steps and evaluation criteria for the efficacious and safe administration of MSCs in the treatment of patients infected with COVID-19.

Standard:Human intestinal organoids

Organoids have attracted great interest for disease modelling,drug discovery and development,and tissue growth and homeostasis investigations.However,lack of standards for quality control has become a prominent obstacle to limit their translation into clinic and other applications.“Human intestinal organoids”is the first guideline on human intestinal organoids in China,jointly drafted and agreed by the experts from the Chinese Society for Cell Biology and its branch society:the Chinese Society for Stem Cell Research.This standard specifies terms and definitions,technical requirements,test methods,inspection rules for human intestinal organoids,which is applicable to quality control during the process of manufacturing and testing of human intestinal organoids.It was originally released by the Chinese Society for Cell Biology on 24 September 2022.We hope that the publication of this standard will guide institutional establishment,acceptance and execution of proper practical protocols and accelerate the international standardization of human intestinal organoids for applications.

Standard:Human intestinal cancer organoids

Intestinal cancer is one of the most frequent and lethal types of cancer.Modeling intestinal cancer using organoids has emerged in the last decade.Human intestinal cancer organoids are physiologically relevant in vitro models,which provides an unprecedented opportunity for fundamental and applied research in colorectal cancer.“Human intestinal cancer organoids”is the first set of guidelines on human intestinal organoids in China,jointly drafted and agreed by the experts from the Chinese Society for Cell Biology and its branch society:the Chinese Society for Stem Cell Research.This standard specifies terms and definitions,technical requirements,test methods for human intestinal cancer organoids,which apply to the production and quality control during the process of manufacturing and testing of human intestinal cancer organoids.It was released by the Chinese Society for Cell Biology on 24 September 2022.We hope that the publication of this standard will guide institutional establishment,acceptance and execution of proper practocal protocols,and accelerate the international standardization of human intestinal cancer organoids for clinical development and therapeutic applications.

Anti-Oxidative and Anti-Inflammatory Effects of Cinnamaldehyde on Protecting High Glucose-Induced Damage in Cultured Dorsal Root Ganglion Neurons of Rats

Objective： To examine the mechanism underlying the beneficial role of cinnamaldehyde on oxidative damage and apoptosis in high glucose （HG）-induced dorsal root ganglion （DRG） neurons in vitro. Methods： HG-treated DRG neurons were developed as an in vitro model of diabetic neuropathy. The neurons were randomly divided into five groups： the control group, the HG group and the HG groups treated with 25, 50 and 100 nmol/L cinnamaldehyde, respectively. Cell viability was examined by 3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyltetrazolium bromide （MTT） assay and apoptosis rate was evaluated by the in situ TdT-mediated dUTP nick end labeling （TUNEL） assay. The intracellular level of reactive oxygen species （ROS） was measured with flow cytometry. Expression of nuclear factor-kappa B （NF-κ B）, inhibitor of κ B （I κ B）, phosphorylated I κ B （p-IκB）, tumor necrosis factor （TNF）-α, interleukin-6 （IL-6） and caspase-3 were determined by western blotting and real-time quantitative reverse transcription polymerase chain reaction （RT-PCR）. Expression of nuclear factor erythroid 2-related factor 2 （Nrf2） and hemeoxygenase-1 （HO-1） were also measured by western blotting. Results： Cinnamaldehyde reduced HG-induced loss of viability, apoptosis and intracellular generation of ROS in the DRG neurons via inhibiting NF- K B activity. The western blot assay results showed that the HG-induced elevated expressions of NF- κB, I κ B and p-I κ B were remarkably reduced by cinnamaldehyde treatment in a dose-dependent manner （P〈0.01）. The HG-induced over-expression of NF-κ B p65 mRNA was remarkably attenuated after cinnamaldehyde treatment in a dose-dependent manner （P〈0.01）. However, the expressions of Nrf2 and HO-1 were not upregulated. Treatment with cinnamaldehyde not only attenuated caspase-3 activation and the caspase cleavage cascade in DRG neurons, but also lowered the elevated IL-6, TNF-α, cyclo-oxygenase and inducible nitric oxide synthase levels, indicating a reduction in inflammatory damage. Conclusions： Cinnamaldehyde protected DRG neurons from the deleterious effects of HG through inactivation of NF- κB pathway but not through activation of Nrf2/HO-1. And thus cinnamaldehyde may have potential application as a treatment for DPN.

Nao-Fu-Cong ameliorates diabetic cognitive dysfunction by inhibition of JNK/CHOP/Bcl2-mediated apoptosis in vivo and in vitro

Chinese herbal compound Nao-Fu-Cong(NFC) has been mainly used to treat cognitive disorders in Traditional Chinese Medicine(TCM). The present study aimed to investigate whether its neuroprotective effects might be related to the inhibition of JNK/CHOP/Bcl2-mediated apoptosis pathway or not. We randomly assigned STZ(60 mg·k^(-1))-induced diabetic rats into control group, diabetic model group and NFC groups(low-dose, medium-dose and high-dose). The primary culture of hippocampal neurons were transferred into different culture media on the third day. The cells were then divided into control group, high-glucose group, NFC(low-dose, medium-dose and high-dose) groups, CHOP si-RNA intervention group, JNK pathway inhibitor SP600125 group and oxidative stress inhibitor N-acetylcysteine(NAC) group. NFC significantly improved the cognitive function of diabetic rats, and had neuroprotective effect on hippocampal neurons cultured in high glucose. Further research results showed that NFC could reduce the apoptosis of hippocampal neurons in rats with diabetic cognitive dysfunction. NFC had inhibitory effects on CHOP/JNK apoptosis pathway induced by high glucose, and also decreased the levels of ROS and increased the mitochondrial membrane potential. These suggested that the neuroprotective effect of NFC might be related to the inhibition of CHOP and JNK apoptotic signaling pathways, and the cross pathway between oxidative stress and mitochondrial damage pathway.

In vivo longitudinal and multimodal imaging of hypoxia-inducible factor 1a and angiogenesis in breast cancer

Background:Angiogenesis and hypoxia-inducible factor 1a(HIF-1a)play major roles in solid tumors.This study aimed to establish a longitudinal and multimodal imaging model for in vivo evaluation of HIF1a and angiogenesis in breast cancer.Methods:By transfection of a 5 hypoxia-responsive element(HRE)/green fluorescent protein(GFP)plasmid,the cell line Ca761-hregfp was established,which emitted green fluorescence triggered by HIF-1a under hypoxia.The cells were subjected to CoCl2-simulated hypoxia to confirm the imaging strategy.We grew Ca761-hre-gfp cells in the left rear flanks of twelve 615 mice.Experiments were conducted on days 4,9,15,and 19.For in vivo analysis,Ca761-hre-gfp subcutaneous allografted tumors were imaged in vivo using contrast-enhanced ultrasound(CEUS)and fluorescence imaging(FLI)during tumor development.The tumor size,CEUS peak intensity,and FLI photons were measured to evaluate tumor growth,angiogenesis,and HIF-1a activity,respectively.After each experiment,three mice were randomly sacrificed and tumor specimens were collected to examine HIF-1a activity and the microvessel density(MVD).Results:In vitro,both green fluorescence and HIF-1a expression were detected in Ca761-hre-gfp cells treated with CoCl2,indicating the suitability of the cells to detect HIF-1a activity.In vivo,HIF-1a activity first increased and then decreased,which was significantly correlated with angiogenic changes(r=0.803,P=0.005).These changes were confirmed by immunohistochemical staining of HIF-1a and MVD.Conclusions:The findings validated the Ca761-hre-gfp murine allograft model for reliable evaluation of HIF-1a activity and angiogenesis longitudinally using both molecular and pre-clinical non-invasive imaging modalities.The cell line may be useful for studies of anti-HIF pathway therapies.

Autologous Transplantation for Parkinson's Disease Patients:Feasibility and Challenge

Parkinson’s Disease(PD),second only to Alzheimer’s disease,is a neurodegenerative disease,most commonly occurring in people over the age of 65 years and is mostly caused by loss of dopamine neurons[1].Clinically,motor symptoms such as resting tremor,motor retardation,muscular rigidity,and disturbance of postural balance are the main symptoms,followed by non-motor symptoms such as cognitive impairment,autonomic nervous system dysfunction,depression,and sleep disorder[2].In 2016,>6.1 million people were affected with PD globally,2.4 times the number in 1990.The large number of affected people,coupled with the high mortality and disability rates,has placed a great burden on society[3].Traditional treatment methods mainly include drugs and surgery and are supplemented by physical therapy.