Huu S. TIEU Request for FDA to Establish Regenerating Human Cells as Law on December 13, 2016 President Barack H. Obama Signed the 21st Century CURES Act into FDA Regulation and Law

Background and Aims: On November 24, 2009, Huu S. TIEU and Golden Sunrise Pharmaceutical, Inc. (Golden Sunrise) applied for the Technology and Innovation to be reviewed and evaluated by the U.S. Food and Drug Administration (FDA). In the review and evaluation, it was requested by Golden Sunrise designated the new indications for this application under Serious or Life-threatening conditions or diseases. Discussions followed with the FDA, Huu S. TIEU, and Golden Sunrise for FDA approval on new products and new indications on existing new Medical Technology and Innovation. It was agreed in Year-2015 that the FDA would take the request for new indications to the United States Congress to establish into FDA regulation and law. At that time the following was the FDA Guidance—“Emergency Use of a Test Article” is exempt from prior Institutional Review Board or Advisory Committee evaluation and approval, provided that such emergency use is reported to the Institutional Review Board within five working days after use. Expedited Institutional Review Board or Advisory Committee approval is not permitted in emergency use. There has been no funding to the authors for the writing or publication of this article. Methods: It was requested by Huu S. TIEU and Golden Sunrise in documents given to the FDA to have Serious or Life-threatening conditions or diseases indication be recognized by law. On August 08, 2015, the FDA responding to this request took the documentation produced by Golden Sunrise to the United States Congress on behalf of Golden Sunrise and Huu S. TIEU. This article encompasses the FDA regulatory method as well as the discussion and results of the establishment of the FDA and the 21st Century Cures Act. Results: On December 13, 2016, H.R.34—114th United States Congress (2015-2016) 21st Century CURES Act was signed into law by President Barack H. Obama which included the Serious or Life-threatening indication to be written into the CURES Act. In summary, the 21st Century Cures Act is a landmark piece of legislation that enjoyed broad bipartisan support in United States Congress. The main goals of the Act are impactful and should transform future cancer, neurologic, and precision medicine or drug research as well as aid individuals with mental health is intended to facilitate the prompt approval of new agents and devices, clinicians should be aware of the types of data behind an approval and take this into consideration when developing illnesses and opioid dependence. However, some of the wording within the CURES Act regarding the drug and device approval process may bring pause to health care providers including pharmacists. Although this wording and implementing care plans and counseling patients. The 21st Century Cures Act was incorporated into laws and regulations by the FDA under § 3072 of the Act grants the Commissioner of Food and Drugs the authority to appoint and set the annual rate of pay for outstanding and qualified candidates to scientific, technical, or professional positions that support the development, review, and regulation of medical products.

Phenotypic changes of human cells in human-rat liver during partial hepatectomy-induced regeneration

AIM： To examine the human hepatic parenchymal and stromal components in rat liver and the phenotypic changes of human cells in liver of human-rat chimera （HRC） generated by in utero transplantation of human cells during partial hepatectomy （PHx）-induced liver regeneration. METHODS： Human hepatic parenchymal and stromal components and phenotypic changes of human cells during liver regeneration were examined by flow oytometry, in situ hybridization and immunohistochemistry. RESULTS： ISH analysis positive cells in hepatic demonstrated human Aluparenchyma and stroma of recipient liver. Functional human hepatocytes generated in this model potentially constituted human hepatic functional units with the presence of donor-derived human endothelial and biliary duct cells in host liver. Alpha fetoprotein （AFP）^＋, CD34^＋ and CD45^＋ cells were observed in the chimeric liver on day 10 after PHxinduced liver regeneration and then disappeared in PHx group, but not in non-PHx group, suggesting that dynamic phenotypic changes of human cells expressing AFP, CD34 and CD45 cells may occur during the chimeric liver regeneration. Additionally, immunostaining for human proliferating cell nuclear antigen （PCNA） showed that the number of PCNA-positive cells in the chimeric liver of PHx group was markedly increased, as compared to that of control group, indicating that donor-derived human cells are actively proliferated during PHx-induced regeneration of HRC liver. CONCLUSION： HRC liver provides a tool for investigating human liver regeneration in a humanized animal model.

Could hydrogen gas be produced using human cells?

Although fossil fuels are widely used to meet energy needs,intensive research has been carried out in recent years on hydrogen production from renewable sources due to their decrease over time and environmental pollution concerns.Biofuel cell technology is one of the promising current technologies.It has been proven that various microorganisms produce energy through their natural metabolism,and that energy production is produced in biofuel cells by exoelectrogenic microorganisms that can transfer electrons to an electrode surface.Although it has been stated that employing human cells to generate energy is feasible,it is unknown whether doing so would enable the production of hydrogen.Within the scope of this perspective article,the issue of hydrogen production in bioelectrolysis cells using human cells will be discussed for the first time.Optimizing hydrogen production in bioelectrolysis cells using human cells is important in terms of contributing to hydrogen technologies.Within the scope of the article,promising human cell lines for hydrogen production are emphasized and hydrogen production potentials in bioelectrolysis cells using these cell lines are discussed.In conclusion,some human cells can be used for hydrogen gas production in bioelectrolysis cells due to their bioelectrochemical and metabolic properties.

Transplantation of human placental chorionic plate-derived mesenchymal stem cells for repair of neurological damage in neonatal hypoxic-ischemic encephalopathy

Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy,neurosensory impairments,and cognitive deficits,and there is no effective treatment for complications related to hypoxic-ischemic encephalopathy.The therapeutic potential of human placental chorionic plate-derived mesenchymal stem cells for various diseases has been explored.However,the potential use of human placental chorionic plate-derived mesenchymal stem cells for the treatment of neonatal hypoxic-ischemic encephalopathy has not yet been investigated.In this study,we injected human placental chorionic plate-derived mesenchymal stem cells into the lateral ventricle of a neonatal hypoxic-ischemic encephalopathy rat model and observed significant improvements in both cognitive and motor function.Protein chip analysis showed that interleukin-3 expression was significantly elevated in neonatal hypoxic-ischemic encephalopathy model rats.Following transplantation of human placental chorionic plate-derived mesenchymal stem cells,interleukin-3 expression was downregulated.To further investigate the role of interleukin-3 in neonatal hypoxic-ischemic encephalopathy,we established an in vitro SH-SY5Y cell model of hypoxic-ischemic injury through oxygen-glucose deprivation and silenced interleukin-3 expression using small interfering RNA.We found that the activity and proliferation of SH-SY5Y cells subjected to oxygen-glucose deprivation were further suppressed by interleukin-3 knockdown.Furthermore,interleukin-3 knockout exacerbated neuronal damage and cognitive and motor function impairment in rat models of hypoxic-ischemic encephalopathy.The findings suggest that transplantation of hpcMSCs ameliorated behavioral impairments in a rat model of hypoxic-ischemic encephalopathy,and this effect was mediated by interleukin-3-dependent neurological function.

Inhibition of viability of human retinal microvascular endothelial cells by vialinin A under high glucose condition

AIM:To investigate the effects of vialinin A on viability of human retinal endothelial cells(HRECs)under high glucose condition and its potential mechanism.METHODS:The HRECs were divided into four groups:normal glucose control group(NG,5 mmol/L D-glucose),high glucose group(HG,30 mmol/L D-glucose),HG+1μmol/L vialinin A group,and HG+5μmol/L vialinin A group.The cell viabilities were measured with cell counting kit-8(CCK-8)assay for proliferation,with scratch assay for migration,and tube formation,for evaluation of the impact of vialinin A on cellular behaviour.Real-time PCR and Western blotting were used to determine the expression level of vascular endothelial growth factor(VEGF).RESULTS:The proliferative capacity and migration of HRECs was reduced by 5μmol/L vialinin A in high glucose environment(both P<0.05).Vialinin A also inhibited highglucose-induced tube formation of HRECs.The expression level of VEGF and PI3K in HRECs was also significantly decreased by vialinin A(P<0.05).CONCLUSION:Vialinin A inhibits the cell viability of HRECs.It may serve as a potential target for anti-angiogenic therapy.

SIRT1 inhibits apoptosis of human lens epithelial cells through suppressing endoplasmic reticulum stress in vitro and in vivo

AIM:To explore the effect of silent information regulator factor 2-related enzyme 1(SIRT1)on modulating apoptosis of human lens epithelial cells(HLECs)and alleviating lens opacification of rats through suppressing endoplasmic reticulum(ER)stress.METHODS:HLECs(SRA01/04)were treated with varying concentrations of tunicamycin(TM)for 24h,and the expression of SIRT1 and C/EBP homologous protein(CHOP)was assessed using real-time quantitative polymerase chain reaction(RT-PCR),Western blotting,and immunofluorescence.Cell morphology and proliferation was evaluated using an inverted microscope and cell counting kit-8(CCK-8)assay,respectively.In the SRA01/04 cell apoptosis model,which underwent siRNA transfection for SIRT1 knockdown and SRT1720 treatment for its activation,the expression levels of SIRT1,CHOP,glucose regulated protein 78(GRP78),and activating transcription factor 4(ATF4)were examined.The potential reversal of SIRT1 knockdown effects by 4-phenyl butyric acid(4-PBA;an ER stress inhibitor)was investigated.In vivo,age-related cataract(ARC)rat models were induced by sodium selenite injection,and the protective role of SIRT1,activated by SRT1720 intraperitoneal injections,was evaluated through morphology observation,hematoxylin and eosin(H&E)staining,Western blotting,and RT-PCR.RESULTS:SIRT1 expression was downregulated in TMinduced SRA01/04 cells.Besides,in SRA01/04 cells,both cell apoptosis and CHOP expression increased with the rising doses of TM.ER stress was stimulated by TM,as evidenced by the increased GRP78 and ATF4 in the SRA01/04 cell apoptosis model.Inhibition of SIRT1 by siRNA knockdown increased ER stress activation,whereas SRT1720 treatment had opposite results.4-PBA partly reverse the adverse effect of SIRT1 knockdown on apoptosis.In vivo,SRT1720 attenuated the lens opacification and weakened the ER stress activation in ARC rat models.CONCLUSION:SIRT1 plays a protective role against TM-induced apoptosis in HLECs and slows the progression of cataract in rats by inhibiting ER stress.These findings suggest a novel strategy for cataract treatment focused on targeting ER stress,highlighting the therapeutic potential of SIRT1 modulation in ARC development.

Efficacy of serum-free cultured human umbilical cord mesenchymal stem cells in the treatment of knee osteoarthritis in mice

BACKGROUND We investigated the efficacy of intra-articular injection of human umbilical cord mesenchymal stem cells(hUC-MSCs)for the treatment of osteoarthritis(OA)progression in the knee joint.Although many experimental studies of hUC-MSCs have been published,these studies have mainly used fetal bovine serumcontaining cultures of hUC-MSCs;serum-free cultures generally avoid the shortcomings of serum-containing cultures and are not subject to ethical limitations,have a wide range of prospects for clinical application,and provide a basis or animal experimentation for clinical experiments.AIM To study the therapeutic effects of serum-free hUC-MSCs(N-hUCMSCs)in a mouse model of knee OA.METHODS Fifty-five male C57BL/6 mice were randomly divided into six groups:The blank control group,model control group,serum-containing hUC-MSCs(S-hUCMSC)group,N-hUCMSC group and hyaluronic acid(HA)group.After 9 weeks of modeling,the serum levels of interleukin(IL)-1β and IL-1 were determined.Hematoxylin-eosin staining was used to observe the cartilage tissue,and the Mankin score was determined.Immunohistochemistry and western blotting were used to determine the expression of collagen type II,matrix metalloproteinase(MMP)-1 and MMP-13.RESULTS The Mankin score and serum IL-1 and IL-1β and cartilage tissue MMP-1 and MMP-13 expression were significantly greater in the experimental group than in the blank control group(P<0.05).Collagen II expression in the experimental group was significantly lower than that in the blank control group(P<0.05).The Mankin score and serum IL-1 and IL-1β and cartilage tissue MMP-1 and MMP-13 levels the experimental group were lower than those in the model control group(P<0.05).Collagen II expression in the experimental group was significantly greater than that in the model control group(P<0.05).CONCLUSION N-hUCMSC treatment significantly alleviate the pathological damage caused by OA.The treatment effects of the ShUCMSC group and HA group were similar.

Self-assembly of differentiated dental pulp stem cells facilitates spheroid human dental organoid formation and prevascularization

BACKGROUND The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine.Stem cells can self-organise into microsized organ units,partially modelling tissue function and regeneration.Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases.However,the lack of vasculature limits the utility of dental pulp organoids.AIM To improve survival and aid in recovery after stem cell transplantation,we demonstrated the three-dimensional(3D)self-assembly of adult stem cell-human dental pulp stem cells(hDPSCs)and endothelial cells(ECs)into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium(CM).METHODS During culture,primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM.The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids.The biological characteristics of the organoids were analysed,and the regulatory pathways associated with angiogenesis were studied.RESULTS The combination of these two agents resulted in prevascularized human dental pulp organoids(Vorganoids)that more closely resembled dental pulp tissue in terms of morphology and function.Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis.The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids.CONCLUSION In this innovative study,we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration,facilitating the development of clinical treatment strategies.

Preliminary study on the preparation of lyophilized acellular nerve scaffold complexes from rabbit sciatic nerves with human umbilical cord mesenchymal stem cells

BACKGROUND The gold standard of care for patients with severe peripheral nerve injury is autologous nerve grafting;however,autologous nerve grafts are usually limited for patients because of the limited number of autologous nerve sources and the loss of neurosensory sensation in the donor area,whereas allogeneic or xenografts are even more limited by immune rejection.Tissue-engineered peripheral nerve scaffolds,with the morphology and structure of natural nerves and complex biological signals,hold the most promise as ideal peripheral nerve“replacements”.AIM To prepare allogenic peripheral nerve scaffolds using a low-toxicity decellularization method,and use human umbilical cord mesenchymal stem cells(hUCMSCs)as seed cells to cultivate scaffold-cell complexes for the repair of injured peripheral nerves.METHODS After obtaining sciatic nerves from New Zealand rabbits,an optimal acellular scaffold preparation scheme was established by mechanical separation,varying lyophilization cycles,and trypsin and DNase digestion at different times.The scaffolds were evaluated by hematoxylin and eosin(HE)and luxol fast blue(LFB)staining.The maximum load,durability,and elastic modulus of the acellular scaffolds were assessed using a universal material testing machine.The acellular scaffolds were implanted into the dorsal erector spinae muscle of SD rats and the scaffold degradation and systemic inflammatory reactions were observed at 3 days,1 week,3 weeks,and 6 weeks following surgery to determine the histocompatibility between xenografts.The effect of acellular scaffold extracts on fibroblast proliferation was assessed using an MTT assay to measure the cytotoxicity of the scaffold residual reagents.In addition,the umbilical cord from cesarean section fetuses was collected,and the Wharton’s jelly(WJ)was separated into culture cells and confirm the osteogenic and adipogenic differentiation of mesenchymal stem cells(MSCs)and hUC-MSCs.The cultured cells were induced to differentiate into Schwann cells by the antioxidant-growth factor induction method,and the differentiated cells and the myelinogenic properties were identified.RESULTS The experiments effectively decellularized the sciatic nerve of the New Zealand rabbits.After comparing the completed acellular scaffolds among the groups,the optimal decellularization preparation steps were established as follows:Mechanical separation of the epineurium,two cycles of lyophilization-rewarming,trypsin digestion for 5 hours,and DNase digestion for 10 hours.After HE staining,no residual nuclear components were evident on the scaffold,whereas the extracellular matrix remained intact.LFB staining showed a significant decrease in myelin sheath composition of the scaffold compared with that before preparation.Biomechanical testing revealed that the maximum tensile strength,elastic modulus,and durability of the acellular scaffold were reduced compared with normal peripheral nerves.Based on the histocompatibility test,the immune response of the recipient SD rats to the scaffold New Zealand rabbits began to decline3 weeks following surgery,and there was no significant rejection after 6 weeks.The MTT assay revealed that the acellular reagent extract had no obvious effects on cell proliferation.The cells were successfully isolated,cultured,and passaged from human umbilical cord WJ by MSC medium,and their ability to differentiate into Schwann-like cells was demonstrated by morphological and immunohistochemical identification.The differentiated cells could also myelinate in vitro.CONCLUSION The acellular peripheral nerve scaffold with complete cell removal and intact matrix may be prepared by combining lyophilization and enzyme digestion.The resulting scaffold exhibited good histocompatibility and low cytotoxicity.In addition,hUC-MSCs have the potential to differentiate into Schwann-like cells with myelinogenic ability following in vitro induction.

Human umbilical cord mesenchymal stem cells derivedexosomes on VEGF-A in hypoxic-induced mice retinal astrocytes and mice model of retinopathy of prematurity

AIM:To observe the effect of human umbilical cord mesenchymal stem cells(hUCMSCs)secretions on the relevant factors in mouse retinal astrocytes,and to investigate the effect of hUCMSCs on the expression of vascular endothelial growth factor-A(VEGF-A)and to observe the therapeutic effect on the mouse model of retinopathy of prematurity(ROP).METHODS:Cultured hUCMSCs and extracted exosomes from them and then retinal astrocytes were divided into control group and hypoxia group.MTT assay,flow cytometry,reverse transcription-polymerase chain reaction(RT-PCR)and Western blot were used to detect related indicators.Possible mechanisms by which hUCMSCs exosomes affect VEGF-A expression in hypoxia-induced mouse retinal astrocytes were explored.At last,the efficacy of exosomes of UCMSCs in a mouse ROP model was explored.Graphpad6 was used to comprehensively process data information.RESULTS:The secretion was successfully extracted from the culture supernatant of hUCMSCs by gradient ultracentrifugation.Reactive oxygen species(ROS)and hypoxia inducible factor-1α(HIF-1α)of mice retinal astrocytes under different hypoxia time and the expression level of VEGF-A protein and VEGF-A mRNA increased,and the ROP cell model was established after 6h of hypoxia.The secretions of medium and high concentrations of hUCMSCs can reduce ROS and HIF-1α,the expression levels of VEGF-A protein and VEGF-A mRNA are statistically significant and concentration dependent.Compared with the ROP cell model group,the expression of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signal pathway related factors in the hUCMSCs exocrine group is significantly decreased.The intravitreal injection of the secretions of medium and high concentrations of hUCMSCs can reduce VEGF-A and HIF-1αin ROP model tissues.HE staining shows that the number of retinal neovascularization in ROP mice decreases with the increase of the dose of hUCMSCs secretion.CONCLUSION:In a hypoxia induced mouse retinal astrocyte model,hUCMSCs exosomes are found to effectively reduce the expression of HIF-1αand VEGF-A,which are positively correlated with the concentration of hUCMSCs exosomes.HUCMSCs exosomes can effectively reduce the number of retinal neovascularization and the expression of HIF-1αand VEGF-A proteins in ROP mice,and are positively correlated with drug dosage.Besides,they can reduce the related factors on the PI3K/AKT/mTOR signaling pathway.

Effects of miR-214-5p and miR-21-5p in hypoxic endometrial epithelial-cell-derived exosomes on human umbilical cord mesenchymal stem cells

BACKGROUND Thin endometrium seriously affects endometrial receptivity,resulting in a significant reduction in embryo implantation,and clinical pregnancy and live birth rates,and there is no gold standard for treatment.The main pathophysiological characteristics of thin endometrium are increased uterine arterial blood flow resistance,angiodysplasia,slow growth of the glandular epithelium,and low expression of vascular endothelial growth factor,resulting in endometrial epithelial cell(EEC)hypoxia and endometrial tissue aplasia.Human umbilical cord mesenchymal stem cells(HucMSCs)promote repair and regeneration of damaged endometrium by secreting microRNA(miRNA)-carrying exosomes.However,the initiation mechanism of HucMSCs to repair thin endometrium has not yet been clarified.AIM To determine the role of hypoxic-EEC-derived exosomes in function of HucMSCs and explore the potential mechanism.METHODS Exosomes were isolated from normal EECs(EEC-exs)and hypoxia-damaged EECs(EECD-exs),before characterization using Western blotting,nanoparticletracking analysis,and transmission electron microscopy.HucMSCs were cocultured with EEC-exs or EECD-exs and differentially expressed miRNAs were determined using sequencing.MiR-21-5p or miR-214-5p inhibitors or miR-21-3p or miR-214-5p mimics were transfected into HucMSCs and treated with a signal transducer and activator of transcription 3(STAT3)activator or STAT3 inhibitor.HucMSC migration was assessed by Transwell and wound healing assays.Differentiation of HucMSCs into EECs was assessed by detecting markers of stromal lineage(Vimentin and CD13)and epithelial cell lineage(CK19 and CD9)using Western blotting and immunofluorescence.The binding of the miRNAs to potential targets was validated by dual-luciferase reporter assay.RESULTS MiR-21-5p and miR-214-5p were lowly expressed in EECD-ex-pretreated HucMSCs.MiR-214-5p and miR-21-5p inhibitors facilitated the migratory and differentiative potentials of HucMSCs.MiR-21-5p and miR-214-5p targeted STAT3 and protein inhibitor of activated STAT3,respectively,and negatively regulated phospho-STAT3.MiR-21-5p-and miR-214-5p-inhibitor-induced promotive effects on HucMSC function were reversed by STAT3 inhibition.MiR-21-5p and miR-214-5p overexpression repressed HucMSC migration and differentiation,while STAT3 activation reversed these effects.CONCLUSION Low expression of miR-21-5p/miR-214-5p in hypoxic-EEC-derived exosomes promotes migration and differentiation of HucMSCs into EECs via STAT3 signaling.Exosomal miR-214-5p/miR-21-5p may function as valuable targets for thin endometrium.

Effects of P301L-TAU on post-translational modifications of microtubules in human iPSC-derived cortical neurons and TAU transgenic mice

TAU is a microtubule-associated protein that promotes microtubule assembly and stability in the axon.TAU is missorted and aggregated in an array of diseases known as tauopathies.Microtubules are essential for neuronal function and regulated via a complex set of post-translational modifications,changes of which affect microtubule stability and dynamics,microtubule interaction with other proteins and cellular structures,and mediate recruitment of microtubule-severing enzymes.As impairment of microtubule dynamics causes neuronal dysfunction,we hypothesize cognitive impairment in human disease to be impacted by impairment of microtubule dynamics.We therefore aimed to study the effects of a disease-causing mutation of TAU(P301L)on the levels and localization of microtubule post-translational modifications indicative of microtubule stability and dynamics,to assess whether P301L-TAU causes stability-changing modifications to microtubules.To investigate TAU localization,phosphorylation,and effects on tubulin post-translational modifications,we expressed wild-type or P301L-TAU in human MAPT-KO induced pluripotent stem cell-derived neurons(i Neurons)and studied TAU in neurons in the hippocampus of mice transgenic for human P301L-TAU(p R5 mice).Human neurons expressing the longest TAU isoform(2N4R)with the P301L mutation showed increased TAU phosphorylation at the AT8,but not the p-Ser-262 epitope,and increased polyglutamylation and acetylation of microtubules compared with endogenous TAU-expressing neurons.P301L-TAU showed pronounced somatodendritic presence,but also successful axonal enrichment and a similar axodendritic distribution comparable to exogenously expressed 2N4R-wildtype-TAU.P301L-TAU-expressing hippocampal neurons in transgenic mice showed prominent missorting and tauopathy-typical AT8-phosphorylation of TAU and increased polyglutamylation,but reduced acetylation,of microtubules compared with non-transgenic littermates.In sum,P301L-TAU results in changes in microtubule PTMs,suggestive of impairment of microtubule stability.This is accompanied by missorting and aggregation of TAU in mice but not in i Neurons.Microtubule PTMs/impairment may be of key importance in tauopathies.

Abnormal Expression of Eukaryotic Translation Factors in Malignant Transformed Human Bronchial Epithelial Cells Induced by Crystalline Nickel Sulfide

Objective To study the oncogenic potential of mouse translation initiation factor 3 （TIF3） and elongation factor-1δ （TEF-1δ） in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide （NiS）. Methods Abnormal expressions of human TIF3 and TEF-1δ genes in two kinds of NiS-transformed cells and NiS-tumorigenic cell lines were investigated and analyzed by the reverse transcript polymerase chain reaction （RT-PCR） and fluorescent quantitative polymerase chain reaction （FQ-PCR）, respectively. Results RT-PCR analysis primarily showed that both human TIF3 and TEF-1δ mRNA expressions in two kinds of NiS-transformed cells and NiS-tumorigenic cell lines were increased as compared with controls. FQ-PCR assay showed that the levels of TIF3 expressions in the transformed cells and tumorigenic cells were 3 and 4 times higher respectively, and the elevated expressions of TEF-16 eDNA copies were 2.7- to 3.5-fold in transformed cells and 4.1- to 5.2-fold in tumorigenic cells when compared with non-transformed cells, indicating that the over-expressions of human TIF3 and TEF-1δ genes were related to malignant degree of the cells induced by nickel. Conclusions These findings demonstrate that there are markedly abnormal expressions of TIF3 and TEF-1δ genes during malignant transformation of human bronchial epithelial cell lines induced by crystalline NiS. They seem to be the molecular mechanisms potentially responsible for human carcinogensis due to nickel.

Mitochondrial transport of catalytic RNAs and targeting of the organellar transcriptome in human cells

Mutations in the small genome present in mitochondria often result in severe pathologies.Different genetic strategies have been explored,aiming to rescue such mutations.A number of these strategies were based on the capacity of human mitochondria to import RNAs from the cytosol and designed to repress the replication of the mutated genomes or to provide the organelles with wild-type versions of mutant transcripts.However,the mutant RNAs present in mitochondria turned out to be an obstacle to therapy and little attention has been devoted so far to their elimination.Here,we present the development of a strategy to knockdown mitochondrial RNAs in human cells using the transfer RNA-like structure of Brome mosaic virus or Tobacco mosaic virus as a shuttle to drive trans-cleaving ribozymes into the organelles in human cell lines.We obtained a specific knockdown of the targeted mitochondrial ATP6 mRNA,followed by a deep drop in ATP6 protein and a functional impairment of the oxidative phosphorylation chain.Our strategy provides a powerful approach to eliminate mutant organellar transcripts and to analyse the control and communication of the human organellar genetic system.

Functional non-homologous end joining patterns triggered by CRISPR/Cas9 in human cells

CRISPR/Cas9-mediated genome engineering technologies are now widely applied in various organisms,including mouse and human cells（Cong et al.,2013;Mali et al.,2013;Yang et al.,2013;Hsu et al.,2014）.The most widely used customized CRISPR/Cas9（Sp Cas9）is derived from Streptococcus pyogenes（Cong et al.,2013）.

Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells

Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells

Human pluripotent stem cells represent a potentially unlimited source of functional pancreatic endocrine lineage cells. Here we report a highly efficient approach to induce human embryonic stem （ES） cells and induced pluripo- tent stem （iPS） cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. The differentiated human ES cells obtained by this approach comprised nearly 25% insulin-positive cells as assayed by flow cytometry analysis, which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these insulin-producing cells co-expressed mature β cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet β cells in vivo. In this study, we also demonstrated that EGF facilitates the expansion of PDXl-positive pancreatic progenitors. Moreover, our protocol also succeeded in efficiently inducing human iPS cells to differentiate into insuIin-producing ceils. Therefore, this work not only provides a new model to study the mechanism of human pancreatic specialization and maturation in vitro, but also enhances the possibility of utilizing patient-specific iPS cells for the treatment of diabetes.

In vitro derivation of functional insulin-producing cells from human embryonic stem cells

The capacity for self-renewal and differentiation of human embryonic stem （ES） cells makes them a potential source for generation of pancreatic beta cells for treating type I diabetes mellitus. Here, we report a newly developed and effective method, carried out in a serum-free system, which induced human ES cells to differentiate into insulin-producing cells. Activin A was used in the initial stage to induce definitive endoderm differentiation from human ES cells, as detected by the expression of the definitive endoderm markers Sox17 and Brachyury. Further, all-trans retinoic acid （RA） was used to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors pdxl and hlxb9. After maturation in DMEM/F12 serum-free medium with bFGF and nicotinamide, the differentiated cells expressed islet specific markers such as C-peptide, insulin, glucagon and glut2. The percentage of C-peptide-positive cells exceeded 15%. The secretion of insulin and C-peptide by these cells corresponded to the variations in glucose levels. When transplanted into renal capsules of Streptozotocin （STZ）-treated nude mice, these differentiated human ES cells survived and maintained the expression of beta cell marker genes, including C-peptide, pdxl, glucokinase, nkx6.1, lAPP, pax6 and Tcfl. Thirty percent of the transplanted nude mice exhibited apparent restoration of stable euglycemia; and the corrected phenotype was sustained for more than six weeks. Our new method provides a promising in vitro differentiation model for studying the mechanisms of human pancreas development and illustrates the potential of using human ES cells for the treatment of type I diabetes mellitus.

Using a non-radioisotopic, quantitative TRAP-based method detecting telomerase activities in human hepatoma cells

A non-radioisotopic, quantitative TRAP-based telomerase activity assay was established mainly by using SYBR Green-I staining instead of radioisotope. Comparing with conventional radioisotope based method, it was better in reproducibility and accuracy. Using this method, we found telomerase activities were absent in normal human liver cells, while detected in ail of four human hepatoma cell lines (BEL-7404, SMMC-7721, QGY-7903 and HCCM) without significant differences.

Expression of Bcl-2 inhibited Fas-mediated apoptosis in human hepatocellular carcinoma BEL-7404 cells

Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and "Death factor" family) are involved in regulating apoptosis. In order to know about the effect of Bcl-2 on apoptosis induced by Fas, a typical member of "Death factor" family, the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells, a human hepatocellular carcinoma cell line which expresses endogenous Fas, but not FasL and Bcl-2. The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells. However, the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis. Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis. These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.