Human pluripotent stem cell-derived extracellular vesicles:From now to the future

Extracellular vesicles(EVs)are nanometric particles that enclose cell-derived bioactive molecules in a lipid bilayer and serve as intercellular communication tools.Accordingly,in various biological contexts,EVs are reported to engage in immune modulation,senescence,and cell proliferation and differentiation.Therefore,EVs could be key elements for potential off-the-shelf cell-free therapy.Little has been studied regarding EVs derived from human pluripotent stem cells(hPSC-EVs),even though hPSCs offer good opportunities for induction of tissue regeneration and unlimited proliferative ability.In this review article,we provide an overview of studies using hPSC-EVs,focusing on identifying the conditions in which the cells are cultivated for the isolation of EVs,how they are characterized,and applications already demonstrated.The topics reported in this article highlight the incipient status of the studies in the field and the significance of hPSC-EVs’prospective applications as PSC-derived cell-free therapy products.

Influence of donor age on the differentiation and division capacity of human adipose-derived stem cells

BACKGROUND Human adipose-derived stromal/stem cells(hASCs)are one of the most useful types of mesenchymal stromal/stem cells,which are adult multipotent cells with great therapeutic potential for the treatment of several diseases.However,for successful clinical application,it is critical that high-quality cells can be obtained.Diverse factors seem to be able to influence cell quality and performance,especially factors related to donors’intrinsic characteristics,such as age.Nevertheless,there is no consensus regarding this characteristic,and there is conflicting information in the literature.AIM To investigate the growth kinetics and differentiation potential of adipose-derived stem cells isolated from the lipoaspirates of elderly and young donors.METHODS hASCs were harvested from liposuctioned adipose tissue obtained from female donors(aged 20-70 years).Cells were distributed into two groups according to age range:old hASCs(oASCs,≥55 years,n=9)and young hASCs(yASCs,≤35 years,n=9).For each group,immunophenotypic characterization was performed by flow cytometry.Population doubling time was assessed over seven days.For adipogenic potential evaluation,lipid deposits were assessed after 7 d,14 d and 21 d of adipogenic induction.Osteogenic potential was verified by analyzing cell mineralization after 14 d,21 d and 28 d of osteogenic induction.mRNA expression of PPARγ2,CEBPA and Runx2 were detected by quantitative reverse transcription polymerase chain reaction.RESULTS hASCs were successfully obtained,cultured,and grouped according to their age:yASCs(26.33±4.66 years old)and oASCs(64.78±4.58 years old).After maintenance of the cells in culture,there were no differences in morphology between cells from the young and old donors.Additionally,both groups showed classical immunophenotypic characteristics of mesenchymal stem/stromal cells.The average doubling time indicated that yASCs(4.09±0.94 d)did not significantly differ from oASCs(4.19±1.29 d).Concerning differentiation potential,after adipogenic and osteogenic induction,yASCs and oASCs were able to differentiate to greater levels than the noninduced control cells.However,no differences were found in the differentiation efficiency of yASCs and oASCs in adipogenesis or osteogenesis.Additionally,the mRNA expression of PPARγ2,CEBPA and Runx2 were similar in yASCs and oASCs.CONCLUSION Our findings suggest that age does not seem to significantly affect the cell division or adipogenic or osteogenic differentiation ability of adipose-derived stem cells isolated from lipoaspirates.

Barriers in contribution of human mesenchymal stem cells to murine muscle regeneration

AIM: To study regeneration of damaged human and murine muscle implants and the contribution of added xenogeneic mesenchymal stem cells(MSCs).METHODS: Minced human or mouse skeletal muscle tissues were implanted together with human or mouse MSCs subcutaneously on the back of non-obese diabetic/severe combined immunodeficient mice. The muscle tissues(both human and murine) were minced with scalpels into small pieces(< 1 mm3) and aliquoted in portions of 200 mm3. These portions were either cryopreserved in 10% dimethylsulfoxide or freshly implanted. Syngeneic or xenogeneic MSCs were added to the minced muscles directly before implantation. Implants were collected at 7, 14, 30 or 45 d after transplantation and processed for(immuno)histological analysis. The progression of muscle regeneration was assessed using a standard histological staining(hematoxylin-phloxinsaffron). Antibodies recognizing Pax7 and von Willebrand factor were used to detect the presence of satellite cells and blood vessels, respectively. To enable detection of the bone marrow-derived MSCs or their derivatives we used MSCs previously transduced with lentiviral vectors expressing a cytoplasmic LacZ gene. X-gal staining of the fixed tissues was used to detect β-galactosidase-positive cells and myofibers.RESULTS: Myoregeneration in implants of fresh murine muscle was evident as early as day 7, and progressed with time to occupy 50% to 70% of the implants. Regeneration of fresh human muscle was slower. These observations of fresh muscle implants were in contrast to the regeneration of cryopreserved murine muscle that proceeded similarly to that of fresh tissue except for day 45(P < 0.05). Cryopreserved human muscle showed minimal regeneration, suggesting that the freezing procedure was detrimental to human satellite cells. In fresh and cryopreserved mouse muscle supplemented with LacZ-tagged mouse MSCs, β-galactosidase-positive myofibers were identified early after grafting at the wellvascularized periphery of the implants. The contribution of human MSCs to murine myofiber formation was, however, restricted to the cryopreserved mouse muscle implants. This suggests that fresh murine muscle tissue provides a suboptimal environment for maintenance of human MSCs. A detailed analysis of the histological sections of the various muscle implants revealed the presence of cellular structures with a deviating morphology. Additional stainings with alizarin red and alcian blue showed myofiber calcification in 50 of 66 human muscle implants, and encapsulated cartilage in 10 of 81 of murine muscle implants, respectively.CONCLUSION: In mouse models the engagement of human MSCs in myoregeneration might be underestimated. Furthermore, our model permits the dissection of speciesspecific factors in the microenvironment.

Reprogramming mouse ear mesenchymal stem cells (EMSC) expressing the Dlk1-Dio3 imprinted gene cluster

The identification of a single, early marker for full developmental potential of induced pluripotent stem (iPS) cells has proven elusive. Recently, however, activation of the imprinted gene cluster, Dlk1-Dio3 has emerged as a viable candidate in the mouse. To explore the relationship between Dlk1-Dio3 expression and developmental potential more fully, we used murine ear mesenchymal stem cells (mEMSC) for iPS cell induction. Mouse EMSC are easily obtained and share functional characteristics with embryonic stem (ES) cells and therefore, may be a reliable non-embryonic source for iPS cell production. We report that mEMSC express high levels of Gtl2, a maternally expressed gene within the Dlk1-Dio3 imprinted cluster. Moreover, mEMSC produce Gtl2 expressing (Gtl2on) iPSC clones that share functional characteristics with ES cell clones. The production of Gtl2on iPS cell clones from mEMSC provides a new model with which to investigate the regulation of Dlk1-Dio3 cluster activity during direct cell reprogramming.

Temporal epigenetic modifications differentially regulate ES cell-like colony formation and maturation

Human somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells by forced expression of the transcription factors Oct4, Sox2, and either Klf4 and cMyc or Nanog and Lin28, using virus-based systems. However, low reprogramming efficiency and the potential for deleterious virus-induced genomic modification limit the clinical potential of this technology. Recent reports indicate, however, that the generation of iPS cells can be enhanced by the addition of synthetic small molecules, including epigenetic modulators. In this report, we demonstrate that the epigenetic modifiers Valproic Acid (VPA) and 5-azacytidine activate the reciprocal transcriptional regulation of endogenous pluripotency transcription factor genes in human dermal fibroblasts and that VPA alone can directly activate endogenous Oct4 in the absence of transgenes. Moreover, using human adipose cells, we demonstrate that histone deacetylase inhibition, prior to reprogramming factor transfection, increases embryonic stem (ES) cell-like colony formation ~2 - 3 fold. In addition, DNA methyltransferase (DNMT) inhibition during human ES cell culture promotes maturation of reprogrammed somatic cells, increasing the yield ~4 fold. These data provide proof of principle that reprogramming efficiency can be improved by inhibiting specific repressive epigenetic regulatory components at the levels of ES cell-like colony formation and maturation. In addition, these studies raise the interesting possibility that a more efficient small molecule-based reprogramming system may provide a superior alternative to current virus-based approaches.

Rif1 interacts with non-canonical polycomb repressive complex PRC1.6 to regulate mouse embryonic stem cells fate potential

Mouse embryonic stem cells(mESCs)cycle in and out of a transient 2-cell(2C)-like totipotent state,driven by a com-plex genetic circuit involves both the coding and repetitive sections of the genome.While a vast array of regulators,including the multi-functional protein Rif1,has been reported to influence the switch of fate potential,how they act in concert to achieve this cellular plasticity remains elusive.Here,by modularizing the known totipotency regulatory factors,we identify an unprecedented functional connection between Rif1 and the non-canonical polycomb repres-sive complex PRC1.6.Downregulation of the expression of either Rif1 or PRC1.6 subunits imposes similar impacts on the transcriptome of mESCs.The LacO-LacI induced ectopic colocalization assay detects a specific interaction between Rif1 and Pcgf6,bolstering the intactness of the PRC1.6 complex.Chromatin immunoprecipitation followed by sequencing(ChIP-seq)analysis further reveals that Rif1 is required for the accurate targeting of Pcgf6 to a group of genomic loci encompassing many genes involved in the regulation of the 2C-like state.Depletion of Rif1 or Pcgf6 not only activates 2C genes such as Zscan4 and Zfp352,but also derepresses a group of the endogenous retroviral element MERVL,a key marker for totipotency.Collectively,our findings discover that Rif1 can serve as a novel auxiliary component in the PRC1.6 complex to restrain the genetic circuit underlying totipotent fate potential,shedding new mechanistic insights into its function in regulating the cellular plasticity of embryonic stem cells.

培养角膜缘上皮移植后穿透性角膜移植术的早期结果

目的：描述曾行培养角膜缘上皮移植的患者再行穿透性角膜移植（PKP）的早期结果。 方法：对因化学烧伤造成角膜缘干细胞缺乏而行培养的角膜缘上皮移植后又进行穿透性角膜移植术的患者有关人口统计学、原发病因、角膜缘移植类型、眼表稳定性、视力、移植片透明度和并发症方面的资料进行回顾和复习。对受体角膜片组织病理学特征进行研究，重点观察上皮状态。 结果：在患有角膜缘干细胞缺乏而进行培养的角膜缘上皮移植术的125例患者中，15例接受了穿透性角膜移植术（PKP）。角膜缘上皮移植术后（自体移植组n=11，同种异体移植组n=4）平均间隔时间为7个月（范围2—12个月）。同种异体移植组的4例患者均接受了免疫抑制疗法。15只眼中14只眼（93％）获得了含有稳定的角膜上皮的健全的角膜植片。15只眼中14只眼术前最佳矫正视力低于20／200。PKP后平均随访8．3±5．0个月，8只眼最佳矫正视力高于20／60，5只眼最佳矫正视力20／200至20／60。2只眼低于20／200。15只眼中3只眼发生异体角膜植片的排斥反应但被成功治疗。1只眼发生植片排斥的同时发生了青光眼。同种异体角膜缘上皮移植无一例显示排斥表现。 结论：培养的角膜缘上皮移植后，在形成稳定的眼表后行PKP的早期结果是良好的。为了避免排斥反应，培养的同种异体角膜缘上皮移植者行充分的免疫抑制治疗是必需的。角膜同种异体移植能够单独排斥同种异体角膜缘移植物。

Human embryos in a dish - modeling early embryonic development with pluripotent stem cells

Stem cell-based embryo models present new opportunities to study early embryonic development.In a recent study,Kagawa et al.identified an approach to create human pluripotent stem cell-based blastoids that resemble the human blastocysts.These blastoids efficiently generated analogs of the EPI,TE,PrE lineages with transcriptomes highly similar to those found in vivo.Furthermore,the formation of these lineages followed the same sequence and pace of blas-tocyst development,and was also dependent on the same pathways required for lineage specification.Finally,the blastoids were capable of attaching to stimulated endometrial cells to mimic the process of implantation.While more comprehensive analysis is needed to confirm its validity and usefulness,this new blastoid system presents the latest development in the attempt to model early human embryogenesis in vitro.

IP3R-mediated Ca2＋ signals govern hematopoietic and cardiac divergence of Flk1＋ cells via the calcineurin-N FATc3-Etv2 pathway

Ca2＋ signals participate in various cellular processes with spatial and temporal dynamics, among which, inositol 1,4,5-trisphosphate receptors （IP3Rs）-mediated Ca2＋ signals are essential for early development. However, the underlying mechanisms of IP3R- regulated cell fate decision remain largely unknown. Here we report that IP3Rs are required for the hematopoietic and cardiac fate divergence of mouse embryonic stem cells （mESCs）. Deletion of IP3Rs （IP3R-tKO） reduced FIkl＋/PDGFRα- hematopoietic mesoderm, c-Kit＋/CD41＋ hematopoietic progenitor ceil population, and the colony-forming unit activity, but increased cardiac progenitor markers as well as cardiomyocytes. Concomitantly, the expression of a key regulator of hematopoiesis, Ely2, was reduced in IP3R-tKO cells, which could be rescued by the activation of Ca2＋ signals and calcineurin or overexpression of constitutively active form of NFATc3. Furthermore, IP3R-tKO impaired specific targeting of Ely2 by NFATc3 via its evolutionarily conserved cis-element in differentiating ESCs. Importantly, the activation of Ca2＋-calcineurin-NFAT pathway reversed the phenotype of IP3R-tKO cells. These findings reveal an unrecognized governing role of IP3Rs in hematopoietic and cardiac fate commitment via IP3Rs-Ca2＋-calcineurin-NFATc3- Etv2 pathway.

Enzyme-triggered deep tumor penetration of a dual-drug nanomedicine enables an enhanced cancer combination therapy

Cancer cells could be eradicated by promoting generation of excessive intracellular reactive oxygen species(ROS)via emerging nanomedicines.However,tumor heterogeneity and poor penetration of nanomedicines often lead to diverse levels of ROS production in the tumor site,and ROS at a low level promote tumor cell growth,thus diminishing the therapeutic effect of these nanomedicines.Herein,we construct an amphiphilic and block polymer-dendron conjugate-derived nanomedicine(Lap@pOEGMA-b-p(GFLG-Dendron-Ppa),GFLG-DP/Lap NPs)that incorporates a photosensitizer,Pyropheophorbide a(Ppa),for ROS therapy and Lapatinib(Lap)for molecular targeted therapy.Lap,an epidermal growth factor receptor(EGFR)inhibitor that plays a role in inhibiting cell growth and proliferation,is hypothesized to synergize with ROS therapy for effectively killing cancer cells.Our results suggest that the enzyme-sensitive polymeric conjugate,pOEGMA-b-p(GFLG-Dendron-Ppa)(GFLG-DP),releases in response to cathepsin B(CTSB)after entering the tumor tissue.Dendritic-Ppa has a strong adsorption capacity to tumor cell membranes,which promotes efficient penetration and long-term retention.Lap can also be efficiently delivered to internal tumor cells to play its role due to the increased vesicle activity.Laser irradiation of Ppa-containing tumor cells results in production of intracellular ROS that is sufficient for inducing cell apoptosis.Meanwhile,Lap efficiently inhibits proliferation of remaining viable cells even in deep tumor regions,thus generating a significant synergistic anti-tumor therapeutic effect.This novel strategy can be extended to the development of efficient membrane lipid-based therapies to effectively combat tumors.

NF-κB Signaling Pathway, Inflammation and Colorectal Cancer

There is growing evidence for a connection between inflammation and tumor development, and the nuclear factor kappa B （NF-κB）, a proinflammatory transcription factor, is hypothesized to promote tumorigenesis. Although the genetic evidence for the hypothesis has been lacking, recent papers have lent credence to this hypothesis. It has been reported that constitutive NF-κB activation in inflammatory bowel diseases （IBDs） increases risk of colorectal cancer （CRC） in the patients with the number of years of active disease. NF-κB activation might induce cellular transformation, mediate cellular proliferation, prevent the elimination of pre-neoplastic and fully malignant cells by up-regulating the anti-apoptosis proteins. Furthermore, NF-κB may contribute to the progression of CRC by regulating the expression of diverse target genes that are involved in cell proliferation （Cyclin D1）, angiogenesis （VEGF, IL-8, COX2）, and metastasis （MMP9）. These findings implicate NF-κB inhibition as an important therapeutic target in CRC. However, due to lack of knowledge about the specific roles of different NF-r,B subunits in different stage of carcinogenesis, and compounds to block specific subunits of NF-κB family, it will be a long time before the coming of targeting NF-κB in CRC therapy.