Maintaining moderate levels of hypochlorous acid promotes neural stem cell proliferation and differentiation in the recovery phase of stroke

It has been shown clinically that continuous removal of ischemia/reperfusion-induced reactive oxygen species is not conducive to the recovery of late stroke.Indeed,previous studies have shown that excessive increases in hypochlorous acid after stroke can cause severe damage to brain tissue.Our previous studies have found that a small amount of hypochlorous acid still exists in the later stage of stroke,but its specific role and mechanism are currently unclear.To simulate stroke in vivo,a middle cerebral artery occlusion rat model was established,with an oxygen-glucose deprivation/reoxygenation model established in vitro to mimic stroke.We found that in the early stage(within 24 hours)of ischemic stroke,neutrophils produced a large amount of hypochlorous acid,while in the recovery phase(10 days after stroke),microglia were activated and produced a small amount of hypochlorous acid.Further,in acute stroke in rats,hypochlorous acid production was prevented using a hypochlorous acid scavenger,taurine,or myeloperoxidase inhibitor,4-aminobenzoic acid hydrazide.Our results showed that high levels of hypochlorous acid(200μM)induced neuronal apoptosis after oxygen/glucose deprivation/reoxygenation.However,in the recovery phase of the middle cerebral artery occlusion model,a moderate level of hypochlorous acid promoted the proliferation and differentiation of neural stem cells into neurons and astrocytes.This suggests that hypochlorous acid plays different roles at different phases of cerebral ischemia/reperfusion injury.Lower levels of hypochlorous acid(5 and 100μM)promoted nuclear translocation ofβ-catenin.By transfection of single-site mutation plasmids,we found that hypochlorous acid induced chlorination of theβ-catenin tyrosine 30 residue,which promoted nuclear translocation.Altogether,our study indicates that maintaining low levels of hypochlorous acid plays a key role in the recovery of neurological function.

Photobiomodulation:a novel approach to promote trans-differentiation of adipose-derived stem cells into neuronal-like cells

Photobiomodulation,originally used red and near-infrared lasers,can alter cellular metabolism.It has been demonstrated that the visible spectrum at 451-540 nm does not necessarily increase cell proliferation,near-infrared light promotes adipose stem cell proliferation and affects adipose stem cell migration,which is necessary for the cells homing to the site of injury.In this in vitro study,we explored the potential of adipose-derived stem cells to differentiate into neurons for future translational regenerative treatments in neurodegenerative disorders and brain injuries.We investigated the effects of various biological and chemical inducers on trans-differentiation and evaluated the impact of photobiomodulation using 825 nm near-infrared and 525 nm green laser light at 5 J/cm2.As adipose-derived stem cells can be used in autologous grafting and photobiomodulation has been shown to have biostimulatory effects.Our findings reveal that adipose-derived stem cells can indeed trans-differentiate into neuronal cells when exposed to inducers,with pre-induced cells exhibiting higher rates of proliferation and trans-differentiation compared with the control group.Interestingly,green laser light stimulation led to notable morphological changes indicative of enhanced trans-differentiation,while near-infrared photobiomodulation notably increased the expression of neuronal markers.Through biochemical analysis and enzyme-linked immunosorbent assays,we observed marked improvements in viability,proliferation,membrane permeability,and mitochondrial membrane potential,as well as increased protein levels of neuron-specific enolase and ciliary neurotrophic factor.Overall,our results demonstrate the efficacy of photobiomodulation in enhancing the trans-differentiation ability of adipose-derived stem cells,offering promising prospects for their use in regenerative medicine for neurodegenerative disorders and brain injuries.

Cardiomyocyte-like differentiation of human bone marrow mesenchymal stem cells after exposure to 5-azacytidine in vitro

Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073g/mL Percoll and propagated in the right cell culturing medium as previously described. The phenotypes of hMSCs were characterized with the use of flow cytometry. The hMSCs were cultured in cell culture medium (as control) and medium mixed with 5-aza for cellular differentiation. We examined by immunohistochemistry at 21 days the inducement of desmin, cardiac-specific cardiac troponin I (cTnI), GATA 4 and connexin-43 respectively. Results The hMSCs are fibroblast-like morphology and express CD44+ CD29+ CD90+ / CD34- CD45- CD31- CD11a. After 5-aza treatment, 20-30% hMSCs connected with adjoining cells and coalesced into myotube structures after 14days. Twenty-one days after 5-aza treatment, immunofluorescence showed that some cells expressed desmin,GATA4, cTnI and connexin-43 in 5,10 μmol/L 5-aza groups, but no cardiac specific protein was found in neither 3μmol/L 5-aza group nor in the control group. The ratio of cTnI positively stained cells in 10 μmol/L group was higher than that in 5 μmol/L group (65.3 ± 4.7% vs 48.2 ± 5.4%, P ＜ 0.05). Electron microscopy revealed that myofilaments were formed. The induced cells expressed cardiac-myosin heavy chain (MyHC) gene by reverse transcription-polymerase chain reaction (RT-PCR). Conclusions Theses findings suggest that hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration. (J Geriatr Cardiol 2004;1(2) :101-107. )

Induced Differentiation of Human Cord Blood Mesenchymal Stem/Pro genitor Cells into Cardiomyocyte-like Cells In Vitro

The feasibility of using cord blood mesenchymal stem/progenitor cells (CB-MSPCs) to regenerate cardiomyocytes and the optimal inducing conditions were investigated. The CB mononuclear cells were cultured in low serum DMEM medium to produce an adherent layer. After expansion, the adherent cells were added into cardiomyocyte inducing medium supplemented with 5-azacytidine. Cardiogenic specific contractile protein troponin T staining was performed to identify the cardiomy-ocyte-like cells. The results showed that the frequency of CB-MSPCs clones in CB mononuclear cells was 0. 5×10-6 and about 1. 3×107-fold expansion was achieved within 20 sub-cultivation. After car-diogenic induction, 70 % CB-MSPCs was differentiated into cardiomyocyte-like cells. It was indicated that low serum culture could expand CB-MSPCs extensively and the expanded CB-MSPCs could be induced to differentiate into cardiomyocyte-like cells in high efficiency.

Tissue Extracts From Infarcted Myocardium of Rats in Promoting the Differentiation of Bone Marrow Stromal Cells Into Cardiomyocyte-like Cells

Objective To investigate whether cardiac tissue extracts from rats could mimic the cardiac microenvironment and act as a natural inducer in promoting the differentiation of bone marrow stromal cells （BMSCs） into cardiomyocytes. Methods Three kinds of tissue extract or cell lysate [infarcted myocardial tissue extract （IMTE）, normal myocardial tissue extract （NMTE） and cultured neonatal myocardial lysate （NML）] were employed to induce BMSCs into cardiomyocyte-like cells. The cells were harvested at each time point for reverse transcription-polymerase chain reaction （RT-PCR） detection, immunocytochemical analysis, and transmission electron microscopy. Results After a 7-day induction, BMSCs were enlarged and polygonal in morphology. Myofilaments, striated sarcomeres, Z-lines, and more mitochondia were observed under transmission electron microscope. Elevated expression levels of cardiac-specific genes and proteins were also confirmed by RT-PCR and immunocytochemistry. Moreover, IMTE showed a greater capacity of differentiating BMSCs into cardiomyocyte-like cells. Conclusions Cardiac tissue extracts, especially IMTE, can effectively differentiate BMSCs into cardiomyocyte-like cells.

Transdifferentiation of Fetal Liver-delivered Mesenchymal Stem Cells into Cardiomyocyte-like Cells

Objectives To explore the possibility to induce mesenchymal stem cells from human fetal livers （FMSCs） to differentiate along cardiac lineage and the way to obtain high rate of differentiation. Methods Cells from passage 6-9 were plated at the density of 1.5 × 10^4/cm^2 and were treated with the combination of 5-azacytine（5-aza）, retinoitic acid（RA） and Dimethylsulfoxide （DMSO） in different doses when near confluence. 24 hours later, the treatment was removed by changing into normal medium without inducers. Different culture conditions were tried, including temperature, oxygen content and medium. Results When FMSCs were treated with highdose combination （ 5-aza 50 μM ＋RA 10-1 μM ＋ DMSO 1%） and modified combination（5-aza 50 μM ＋RA 10-3 μM ＋ DMSO 0.8 %） in cardiac differentiation medium （CDM）, at 37℃ and 20% 02, the cardiac differentiation was induced. When near confluence, cells became round and tended to gather together to form ball-like structures. 3 weeks after treatment, the cells were harvested and stained with anti-desmin and cardiac troponin I antibodies, and about 40% of the cells were positively stained. No beating cells observed during observation. Conclusions FMSCs cardiac have lineage the potential to differentiate along , and the stimulus for the cardiac differentiation is different from those for MSCs from different species.

How mesenchymal stem cells transform into adipocytes:Overview of the current understanding of adipogenic differentiation

Mesenchymal stem cells(MSCs)are stem/progenitor cells capable of self-renewal and differentiation into osteoblasts,chondrocytes and adipocytes.The transformation of multipotent MSCs to adipocytes mainly involves two subsequent steps from MSCs to preadipocytes and further preadipocytes into adipocytes,in which the process MSCs are precisely controlled to commit to the adipogenic lineage and then mature into adipocytes.Previous studies have shown that the master transcription factors C/enhancer-binding protein alpha and peroxisome proliferation activator receptor gamma play vital roles in adipogenesis.However,the mechanism underlying the adipogenic differentiation of MSCs is not fully understood.Here,the current knowledge of adipogenic differentiation in MSCs is reviewed,focusing on signaling pathways,noncoding RNAs and epigenetic effects on DNA methylation and acetylation during MSC differentiation.Finally,the relationship between maladipogenic differentiation and diseases is briefly discussed.We hope that this review can broaden and deepen our understanding of how MSCs turn into adipocytes.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation

BACKGROUND Validation of the reference gene(RG)stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction(RT-qPCR)data normalisation.Commonly,in an unreliable way,several studies use genes involved in essential cellular functions[glyceraldehyde-3-phosphate dehydro-genase(GAPDH),18S rRNA,andβ-actin]without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes.Furthermore,such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recom-mend two or more genes.It impacts the credibility of these studies and causes dis-tortions in the gene expression findings.For tissue engineering,the accuracy of gene expression drives the best experimental or therapeutical approaches.We cultivated DPSCs under two conditions:Undifferentiated and osteogenic dif-ferentiation,both for 35 d.We evaluated the gene expression of 10 candidates for RGs[ribosomal protein,large,P0(RPLP0),TATA-binding protein(TBP),GAPDH,actin beta(ACTB),tubulin(TUB),aminolevulinic acid synthase 1(ALAS1),tyro-sine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,zeta(YWHAZ),eukaryotic translational elongation factor 1 alpha(EF1a),succinate dehydrogenase complex,subunit A,flavoprotein(SDHA),and beta-2-micro-globulin(B2M)]every 7 d(1,7,14,21,28,and 35 d)by RT-qPCR.The data were analysed by the four main algorithms,ΔCt method,geNorm,NormFinder,and BestKeeper and ranked by the RefFinder method.We subdivided the samples into eight subgroups.RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm.The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs.Either theΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes.However,geNorm analysis showed RPLP0/EF1αin the first place.These algorithms’two least stable RGs were B2M/GAPDH.For BestKeeper,ALAS1 was ranked as the most stable RG,and SDHA as the least stable RG.The pair RPLP0/TBP was detected in most subgroups as the most stable RGs,following the RefFinfer ranking.CONCLUSION For the first time,we show that RPLP0/TBP are the most stable RGs,whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

An overview of autophagy in the differentiation of dental stem cells

Dental stem cells(DSCs)have attracted significant interest as autologous stem cells since they are easily accessible and give a minimal immune response.These properties and their ability to both maintain self-renewal and undergo multi-lineage differentiation establish them as key players in regenerative medicine.While many regulatory factors determine the differentiation trajectory of DSCs,prior research has predominantly been based on genetic,epigenetic,and molecular aspects.Recent evidence suggests that DSC differentiation can also be influenced by autophagy,a highly conserved cellular process responsible for maintaining cellular and tissue homeostasis under various stress conditions.This comprehensive review endeavors to elucidate the intricate regulatory mechanism and relationship between autophagy and DSC differentiation.To achieve this goal,we dissect the intricacies of autophagy and its mechanisms.Subsequently,we elucidate its pivotal roles in impacting DSC differentiation,including osteo/odontogenic,neurogenic,and angiogenic trajectories.Furthermore,we reveal the regulatory factors that govern autophagy in DSC lineage commitment,including scaffold materials,pharmaceutical cues,and the extrinsic milieu.The implications of this review are far-reaching,underpinning the potential to wield autophagy as a regulatory tool to expedite DSC-directed differentiation and thereby promote the application of DSCs within the realm of regenerative medicine.

Patient-derived induced pluripotent stem cells with a MERTK mutation exhibit cell junction abnormalities and aberrant cellular differentiation potential

BACKGROUND Human induced pluripotent stem cell(hiPSC)technology is a valuable tool for generating patient-specific stem cells,facilitating disease modeling,and invest-igating disease mechanisms.However,iPSCs carrying specific mutations may limit their clinical applications due to certain inherent characteristics.AIM To investigate the impact of MERTK mutations on hiPSCs and determine whether hiPSC-derived extracellular vesicles(EVs)influence anomalous cell junction and differentiation potential.METHODS We employed a non-integrating reprogramming technique to generate peripheral blood-derived hiPSCs with and hiPSCs without a MERTK mutation.Chromo-somal karyotype analysis,flow cytometry,and immunofluorescent staining were utilized for hiPSC identification.Transcriptomics and proteomics were employed to elucidate the expression patterns associated with cell junction abnormalities and cellular differentiation potential.Additionally,EVs were isolated from the supernatant,and their RNA and protein cargos were examined to investigate the involvement of hiPSC-derived EVs in stem cell junction and differentiation.RESULTS The generated hiPSCs,both with and without a MERTK mutation,exhibited normal karyotype and expressed pluripotency markers;however,hiPSCs with a MERTK mutation demonstrated anomalous adhesion capability and differentiation potential,as confirmed by transcriptomic and proteomic profiling.Furthermore,hiPSC-derived EVs were involved in various biological processes,including cell junction and differentiation.CONCLUSION HiPSCs with a MERTK mutation displayed altered junction characteristics and aberrant differentiation potential.Furthermore,hiPSC-derived EVs played a regulatory role in various biological processes,including cell junction and differentiation.

O-linkedβ-N-acetylglucosaminylation may be a key regulatory factor in promoting osteogenic differentiation of bone marrow mesenchymal stromal cells

Cumulative evidence suggests that O-linkedβ-N-acetylglucosaminylation(OGlcNAcylation)plays an important regulatory role in pathophysiological processes.Although the regulatory mechanisms of O-GlcNAcylation in tumors have been gradually elucidated,the potential mechanisms of O-GlcNAcylation in bone metabolism,particularly,in the osteogenic differentiation of bone marrow mesenchymal stromal cells(BMSCs)remains unexplored.In this study,the literature related to O-GlcNAcylation and BMSC osteogenic differentiation was reviewed,assuming that it could trigger more scholars to focus on research related to OGlcNAcylation and bone metabolism and provide insights into the development of novel therapeutic targets for bone metabolism disorders such as osteoporosis.

Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells

BACKGROUND Embryonic stem cells(ESCs)serve as a crucial ex vivo model,representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos.ESCs exhibit a unique combination of self-renewal potency,unlimited proliferation,and pluripotency.The latter is evident by the ability of the isolated cells to differ-entiate spontaneously into multiple cell lineages,representing the three primary embryonic germ layers.Multiple regulatory networks guide ESCs,directing their self-renewal and lineage-specific differentiation.Apoptosis,or programmed cell death,emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development.How-ever,the molecular mechanisms underlying the dynamic interplay between diffe-rentiation and apoptosis remain poorly understood.AIM To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells,using mouse ESC(mESC)models-mESC-B-cell lym-phoma 2(BCL-2),mESC-PIM-2,and mESC-metallothionein-1(MET-1)-which overexpress the anti-apoptotic genes Bcl-2,Pim-2,and Met-1,respectively.METHODS mESC-T2(wild-type),mESC-BCL-2,mESC-PIM-2,and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation.The hanging drop method was adopted to generate embryoid bodies(EBs)and induce terminal differentiation of mESCs.The size of the generated EBs was measured in each condition compared to the wild type.At the functional level,the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control.At the molecular level,quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers:Troponin T,GATA4,and NKX2.5.Additionally,troponin T protein expression was evaluated through immunofluorescence and western blot assays.RESULTS Our findings showed that the upregulation of Bcl-2,Pim-2,and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs,in comparison with their wild-type counterpart.Additionally,a decrease in the count of beating cardiomyocytes among differentiated cells was observed.Furthermore,the mRNA expression of three cardiac markers-troponin T,GATA4,and NKX2.5-was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line.Moreover,the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression.CONCLUSION Our findings revealed that the upregulation of Bcl-2,Pim-2,and Met-1 genes altered cardiac differentiation,providing insight into the intricate interplay between apoptosis and ESC fate determination.

A Cell Screening Algorithm Integrating Genetic and Numerical Differentiation

The consistency of the cell has a significant impact on battery capacity,endurance,overall performance,safety,and service life extension.However,it is challenging to identify cells with high consistency and no loss of battery energy.This paper presents a cell screening algorithm that integrates genetic and numerical differentiation techniques.Initially,a mathematical model for battery consistency is established,and a multi-step charging strategy is proposed to satisfy the demands of fast charging technology.Subsequently,the genetic algorithm simulates biological evolution to efficiently search for superior cell combinations within a short time while evaluating capacity,voltage consistency,and charge/discharge efficiency.Finally,through experimental validation and comparative analysis with similar algorithms,our proposed method demonstrates notable advantages in terms of both search efficiency and performance.

Experimental study on the induction of bone marrow stromal cells differentiating into cardiomyocyte-like cells with cardiomyocytes in vitro

Objective：To investigate the feasibility of bone marrow stromal cells （BMSCs） differenti ating into cardiomyocyte like cells in heterogeneous cardiomyocytes microenvironment in vitro. Methods： Mouse GFP-BMSCs were isolated by centrifugation through a Ficoll step gradient and purified by plating culture and depletion of the non-adherent cells. Neonatal rat cardiomyocytes （CMs） were isolated by enzymatic dissociation from hearts of 1-to 2-day old Sprague-Dawley （SD） rats and differentially plated to remove fibroblasts. Mouse GFP-BMSCs were cocuhured with neonatal rat CMs through direct and indirect contact, respectively. Cardiomyogenic differentiation of BMSCs was evaluated by immunostaining with an- ti-a-actin monoclonal antibody and observing synchronous contraction with adjacent CMs by phase contrast microphotography. Results： On day 7 of cocuhure, GFP-BMSCs （CMs ： BMSCs：4 ： 1）attached to nonfluorescent contracting cells （rat-derived CMs） showed myotube-like formation and started to contract synchronously with adjacent cardiomyocytes. About 10% of the fluorescent GFP-BMSCs were cardiomyocyte-like cells as determined by cell morphology and positive actin staining. Conclusion;Direct cell to-cell interaction with CMs is crucial for cardiomyogenic differentiation of BMSCs in heterogeneous CMs microenvironment in vitro. This provides a novel inducing pathway for directional differentiation of cardiovascular tissue engineering seed cells.

Dynamic Changes in Distribution of Lignin and Hemicelluloses in Cell Walls During Differentiation of Secondary Xylem in Eucommia ulmoides

The dynamic changes in the distribution of lignin and hemicelluloses (xylans and xyloglucans) in cell walls during the differentiation of secondary xylem in Eucommia ulmoides Oliv. were studied by means of ultraviolet light microscopy and transmission electron microscopy combined with immunogold labelling. In the cambial zone and cell expansion zone, xyloglucans were localized both in the tangential and radial walls, but no xylans or lignin were found in these regions. With the formation of secondary wall S-1 layer, lignin occurred in the cell corners and middle lamella, while xylans appeared in S-1 layer, and xyloglucans were localized in the primary walls and middle lamella. In pace with the formation of secondary wall S-2 and S-3 layer, lignification extended to S-1, S-2 and S-3 layer in sequence, showing a patchy style of lignin deposition. Concurrently, xylans distributed in the whole secondary walls and xyloglucans, on the other hand, still localized in the primary walls and middle lamella. The results indicated that along with the formation and lignification of the secondary wall, great changes had taken place in the cell walls. Different parts of cell walls, such as cell corners, middle lamella, primary walls and various layers of secondary walls, had different kinds of hemicelluloses, which formed various cell wall architecture combined with lignin and other cell wall components.

Targeted migration and differentiation of engrafted neural precursor cells in amyloid β-treated hippocampus in rats

Objective To observe the migration and differentiation of the neural precursor cells （NPCs） that derived from murine embryonic stem cells （ESCs） when they were transplanted into amyloid β （Aβ）-treated rat hippocampus. Methods MESPU35, a murine ESC cell line that express the enhanced green fluorescent protein （EGFP）, was induced differentiation into nestin-positive NPCs by modified serum-free methods. The Aβ plaques and the differentiation of the grafted cells were observed by immunofluorescent staining. Results Comparing 16 weeks with 4 weeks post-transplantation, the migration distance increased about 5 times; the rate of migratory NPCs differentiating into glial fibrillary acidic protein （GFAP）-positive cells kept rising from （30.41 ± 1.45）% to （49.25± 1.23）%, and the rate of NPCs differentiating into neurofilament 200 （NF200） positive cells increased from （16.68±0.95）% to （27.94± 1.21）%. Meanwhile, the GFAP-positive cells targeting to the ipsilateral side of Aβ plaques increased from 60.2% to 81.3 %, while the NF200-positive cells increased from 61.3% to 84.1%. The migration distance had significant positive linear correlations to the neuronal differentiation rate （r = 0.991） and to the astrocytic differentiation rate （r = 0.953）. Conclusion Engrafted NPCs migrate targetedly to the Aβ injection site and differentiate into neurons and astrocytes.

Programmed Cell Death During Secondary Xylem Differentiation in Eucommia ulmoides

Programmed cell death (PCD) during secondary xylem differentiation in Eucommia ulmoides Oliv. was examined using electron microscopy and by investigation of DNA fragmentation and degradation of caspase-like proteases (CLPs). DNA ladders were detected in developing secondary xylem by gel electrophoresis. DNA fragmentation was further confirmed by using the TdT-mediated dUTP nick-end labeling (TUNEL) method. Western blotting analysis showed that CLPs (caspase-8- and caspase-3-like proteases) and PARP (poly (ADP-ribose) polymerase) were degraded during secondary xylem differentiation. The results thus indicated that secondary xylem differentiation in E ulmoides was a typical process of PCD and the degradation of CLPs might be a constitutive PCD event during secondary xylem differentiation.

Regulatory genes controlling neural stem cells differentiation into neurons

The recent progress in neural stem cells （NSCs） research has shed lights on possibility of repair and restoration of neuronal function in neurodegenerative diseases using stem cells. Induction of stem cells differentiate into mature neurons is critical to achieve the clinical applications of NSCs. At present, molecular mechanisms modulating NSC differentiation are not fully understood. Differentiation of stem cells into neuronal and glial cells involves an array of changes in expression of transcription factors. Transcription factors then trigger the expression of a variety of central nervous system （CNS） genes that lead NSCs to differentiate towards different cell types. In this paper, we summarized the recent findings on the gene regulation of NSCs differentiation into neuronal cells.

Polymethylenebis [acetamides] Analogues.Synthesis and Differentiation-Inducing Activity on HL-60 Cells

报导了一系列多亚甲基双［酰胺］类化合物的合成，由－摩尔多亚甲基二甲酰氯分别和二摩尔２－氨基噻唑啉及５－氨基－１－甲基吡啶酮反应制得。测定了其体外对ＨＬ－６０人早幼粒白血病细胞的分化诱导活性，初步结果表明：Ｎ，Ｎ｀－双吡啶酮基六二甲酰胺和Ｎ，Ｎ｀－双噻唑啉基八亚甲基二甲酰胺分别在０．１ｍｍｏｌ／Ｌ和０．５ｍｍｏｌ／Ｌ浓度时，诱导分化百分率可达６０％。此浓度下细胞存活率分别为２６％及２２％，其有效诱导浓度比ＨＭＢＡ低十倍。

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.