Isolation and characterization of pluripotent human spermatogonial stem cell-derived cells

Several reports have documented the derivation of pluripotent cells (multipotent germline stem cells) from spermatogonial stem cells obtained from the adult mouse testis. These spermatogonia-derived stem cells express embryonic stem cell markers and differentiate to the three primary germ layers, as well as the germline. Data indicate that derivation may involve reprogramming of endogenous spermatogonia in culture. Here, we report the derivation of human multipotent germline stem cells (hMGSCs) from a testis biopsy. The cells express distinct markers of pluripotency, form embryoid bodies that contain derivatives of all three germ layers, maintain a normal XY karyotype, are hypomethylated at the H19 locus, and express high levels of telomerase. Teratoma assays indicate the presence of human cells 8 weeks post-transplantation but limited teratoma formation. Thus, these data suggest the potential to derive pluripotent cells from human testis biopsies but indicate a need for novel strategies to optimize hMGSC culture conditions and reprogramming.     

Protective effects of human induced pluripotent stem cell-derived exosomes on high glucose-induced injury in human endothelial cells

AIM: To investigate the effects of human induced pluripotent stem cell-derived exosomes( hi PSC-exo) on cell viability,capillarylike structure formation,and senescence in endothelial cells exposed to high glucose. METHODS: Exosomes were isolated from the conditional medium of hi PSCs and confirmed by transmission electron microscopy,nanoparticle tracking analysis,and Western blot analysis using Alix and CD63 as markers. hi PSC-exo were labeled with PKH26 for tracking. Cultured HUVECs were treated with high glucose(33 mmol / L) with or without hi PSC-exo(20 mg / L) for 48 h,and cell viability,capillary tube formation,and senescence were assessed. RESULTS: hi PSC-exo showed a typical cup shape and could be taken up by human umbilical vascular endothelial cells(HUVECs) in a concentration-dependent manner. When exposed to high glucose,viability and tube formation in HUVECs was significantly reduced,whereas the proportion of senescent cells was higher compared to that in control HUVECs( P 0. 01). Furthermore,hi PSC-exo restored cell viability and capillary-like structure formation,and reduced senescence in HUVECs exposed to high glucose(P 0. 01). However,hi PSC-exo had minimal effects on normal HUVECs. Therefore,stem cell-derived exosomes can promote cell proliferation, enhance capillary-like structure formation, and reduce senescence in endothelial cells exposed to high glucose.CONCLUSION: Our study highlights the role of exosomes derived from hi PSC and may provide a new strategy for maintaining vascular health,preventing vascular aging,and avoiding pathological vascular remodeling that occurs in many diseases.     

Characterisation of human induced pluripotent stem cell-derived endothelial cells under shear stress using an easy-to-use microfluidic cell culture system

Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) can contribute to elucidating the pathogenesis of heart and vascular diseases and developing their treatments. Their precise characteristics in fluid flow however remain unclear. Therefore, the aim of the present study is to characterise these features. We cultured three types of ECs in a microfluidic culture system: commercially available human iPS-ECs, human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs). We then examined the mRNA expression levels of endothelial marker gene cluster of differentiation 31 (CD31), fit-related receptor tyrosine kinase (Flk-1), and the smooth muscle marker gene smooth muscle alpha-actin, and investigated changes in plasminogen activator inhibitor-1 (PAI-1) secretion and intracellular F-actin arrangement following heat stress. We also compared expressions of the arterial and venous marker genes ephrinB2 and EphB4, and the endothelial gap junction genes connexin (Cx) 37, 40, and 43 under fluidic shear stress to determine their arterial or venous characteristics. We found that iPS-ECs had similar endothelial marker gene expressions and exhibited similar increases in PAI-1 secretion under heat stress as HUVECs and HUAECs. In addition, F-actin arrangement in iPSC-ECs also responded to heat stress, as previously reported. However, they had different expression patterns of arterial and venous marker genes and Cx genes under different fluidic shear stress levels, showing that iPSC-ECs exhibit different characteristics from arterial and venous ECs. This microfluidic culture system equipped with variable shear stress control will provide an easy-to-use assay tool to examine characteristics of iPS-ECs generated by different protocols in various laboratories and contribute to basic and applied biomedical researches on iPS-ECs.     

Efficient differentiation of human embryonic and induced pluripotent stem cells into functional astrocytes

Human high-grade gliomas (hHGG) remain a therapeutic challenge in neuro-oncology despite current multimodality treatments. We recently demonstrated that murine embryonic stem cell (mESC)-derived astrocytes conditionally expressing proapoptotic genes can successfully be used to induce apoptosis and tumor shrinkage of hHGG tumor in vitro and in an in vivo mouse model. The first step in the translation of these results to the clinical settings, however, requires availability of human embryonic stem cells (hESC)- and/or induced pluripotent cell (hiPSC)-derived astrocytes engineered to express proapoptotic genes. The potential for directed differentiation of hESCs and hiPSCs to functional postmitotic astrocytes is not fully characterized. In this study, we show that once specified to neuro-epithelial lineage, hiPSC could be differentiated to astrocytes with a similar efficiency as hESC. However, our analyses of 2?hESC and 2 hiPSC cell lines showed some variability in differentiation potential into astrocytic lineages. Both the hESC- and hiPSC-derived astrocytes appeared to follow the functional properties of mESC-derived astrocytes, namely, migration and tropism for hHGG. This work provides evidence that hESC- and hiPSC-derived cells are able to generate functionally active astrocytes. These results demonstrate the feasibility of using iPSC-derived astrocytes, a new potential source for therapeutic use for brain tumors and other neurological diseases.     

Rat embryonic fibroblasts improve reprogramming of human keratinocytes into induced pluripotent stem cells

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells.     

Induction of cardiomyogenesis in human embryonic stem cells by human embryonic stem cell-derived definitive endoderm

We previously reported that chick anterolateral endoderm (AL endoderm) induces cardiomyogenesis in mouse embryoid bodies. However, the requirement to micro-dissect AL endoderm from gastrulation-stage embryos precludes its use to identify novel cardiomyogenic factors, or to scale up cardiomyocyte numbers for therapeutic experiments. To circumvent this problem we have addressed whether human definitive endoderm (hDE) cells, which can be efficiently generated in large numbers from human embryonic stem cells (hESCs), can mimic the ability of AL endoderm to induce cardiac myogenesis. Results demonstrate that both hDE cells and medium conditioned by them induce cardiac myogenesis in pluripotent hESCs, as indicated by rhythmic beating and immunohistochemical/quantitative polymerase chain reaction monitoring of marker gene expression. The cardiomyogenic effect of hDE is enhanced when pluripotent hESCs are preinduced to the mes-endoderm state. Because this approach is tractable and scalable, it may facilitate identification of novel hDE-secreted factors for inclusion in defined cardiomyogenic cocktails.     

Production of erythroid cells from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPS)

Erythroid progenitors can be generated ex vivo from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPS). Development of laboratory scale culture conditions capable of generating mature functional erythrocytes from human embryonic stem cells or human induced pluripotent stem cells would open the possibility for manufacture of therapeutic quantities of red cells and thereby new clinical transfusion products. Current attempts to produce erythrocytes from human embryonic stem cells reveal the need for greater understanding of the process whereby primitive erythropoiesis switches to definitive fetal and adult erythropoiesis and the factors driving erythrocyte maturation. Studies with human embryonic stem cells have already yielded encouraging results but functional mature biconcave erythrocytes have yet to be generated from these cells.     

In vitro imaging of angiogenesis using embryonic stem cell-derived endothelial cells

Angiogenesis is an important event during developmental processes, and it plays a key role in neovascularization. The development of an in vitro model that can be used for live imaging of vessel growth will facilitate the study of molecular and cellular mechanisms for the growth of blood vessels. Embryonic stem cells (ESCs) are considered to be a novel renewable source for the derivation of genetically manipulable endothelial cells (ECs). To derive green fluorescence protein (GFP)-expressing ECs, we used a transgenic ESC line in which a GFP reporter was driven by the endothelial-specific promoter fetal liver kinase 1. ESC-ECs were isolated from 11-day embryoid bodies by fluorescence-activated cell sorting. Embedding the aggregated ESC-ECs in a 3-dimensional collagen gel matrix resulted in ESC-EC migration out of the aggregates and coalescence into a capillary network. Time-lapse microscopy revealed EC migration, proliferation, lumen formation, and anastomosis to other capillary vessels during this process, which were reminiscent of angiogenic processes. Vascular endothelial growth factor plays major roles in the induction of ESC-EC angiogenesis in vitro. Blockage of the β1 integrin subunit severely impaired ESC-EC survival and migration. We demonstrate that our in vitro ESC-EC angiogenesis model represents a high-resolution dynamic video-image system for observing the cellular events underlying angiogenic cascades. We also consider this model as an image screening tool for the identification of pro-angiogenic and anti-angiogenic molecules.     

Ultrastructural characterization of mouse embryonic stem cell-derived oocytes and granulosa cells

Germ cells are a unique population of cells responsible for transmitting genetic information from one generation to the next. Our understanding of the key mechanisms underlying germ cell development in vivo remains scarce because of insufficient amounts of cell materials available for conducting biological studies. The establishment of in vitro differentiation models that support the generation of germ cells from mouse pluripotent stem cells provides an alternative means for studying reproductive development. The detection and analysis of stem cell-derived germ cells, however, present technical challenges. Methods for determining the developmental stage of germ cells ex vivo, such as gene expression and/or immunochemical analyses are inadequate, frequently necessitating the use of alternative, elaborate methods to prove germ cell identity. We have generated putative oocytes and granulosa cells in vitro from mouse embryonic stem cells and utilized electron microscopy to characterize these cells. Here, we report on the striking ultrastructural similarity of in vitro-generated oocytes and granulosa cells to in vivo oocytes developing within follicles.     

Hypoxia enhances the generation of retinal progenitor cells from human induced pluripotent and embryonic stem cells

The efficient differentiation of retinal cells from human pluripotent stem cells remains a major challenge for the development of successful and cost-effective cellular therapies for various forms of blindness. Current differentiation strategies rely on exposing pluripotent stem cells to soluble growth factors that play key roles during early development (such as DKK-1, Noggin, and IGF-1) at 20% oxygen (O(2)). This O(2) tension is, however, considerably higher than O(2) levels during organogenesis and may impair the differentiation process. In this study, we examined the effect of mimicking the physiological O(2) tension (2%) on the generation of retinal progenitor cells (RPCs) from human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs). Both cell types were induced to differentiate into RPCs at 20% and 2% O(2). After 3 days in suspension culture as embryoid bodies (EBs), 2% O(2) caused the activation of hypoxia inducible factor responsive genes VEGF and LDHA and was accompanied by elevated expression levels of the early eye field genes Six3 and Lhx2. Twenty-one days after plating EBs in an adherent culture, we observed more RPCs co-expressing Pax6 and Chx10 at 2% O(2). Quantitative polymerase chain reaction analysis confirmed that lowering O(2) tension had caused a rise in the expression of both genes compared with 20% O(2). Our results indicate that mimicking physiological O(2) is a favorable condition for the efficient generation of RPCs from both hiPSCs and hESCs.     

Functional characterization of embryonic stem cell-derived cardiomyocytes using scanning ion conductance microscopy

We report here the novel use of scanning ion conductance microscopy (SICM) to produce surface images of embryonic stem cell-derived cardiomyocytes (ESCM) to identify individual contracting cardiomyocytes among different cell types. By measuring amplitude and rhythm we can quantitate contraction of ESCM. This method gives, within the same experiment, an assessment of the number and position of ESCM within the layer of mixed cell types, as well as an accurate measure of the response of individual ESCM. Using different modulators of contraction as examples we showed how SICM could be used for recording their responses. We subsequently demonstrated that this model can be used to investigate the protective effect of antiarrhythmogenic drugs.     

Immunotherapy with pluripotent stem cell-derived dendritic cells

In vivo transfer of dendritic cells (DC) has proven efficient in the priming of T cells and is regarded as a powerful means of providing anti-cancer immunotherapy. Clinical trials of anti-cancer therapy with DC pulsed with peptide antigens have been carried out in many institutions, although dramatic therapeutic effect has not been observed in most of the trials. Negative regulation of the immune response by DC might be applicable to treatment of autoimmune diseases and transplantation medicine. Currently, the DC used for anti-cancer vaccine therapy are generated from the peripheral blood monocytes of the patients. However, there is a limitation in the number of available monocytes and the potential of monocytes to differentiate into DC varies depending on the individual blood donors. To resolve the issue of the cell source for DC therapy, several groups have developed methods to generate DC from pluripotent stem cells. This review introduces methods to generate functional DC from pluripotent stem cells of mouse and human.     

阿霉素致人诱导性多潜能干细胞来源的心肌细胞损伤模型的建立

目的 利用人诱导性多潜能干细胞来源的心肌细胞（hiPSC-CMs）技术,建立人源的阿霉素心肌细胞损伤模型。 方法 从人诱导性多潜能干细胞分化hiPSC-CMs,再用不同浓度阿霉素对hiPSC-CMs作用24 h后检测其细胞活性、钙瞬变、氧化应激和DNA损伤等表型。 结果 阿霉素诱导hiPSC-CMs细胞活力下降,破坏其钙瞬变,引起氧化应激水平上升,导致线粒体膜电位下降和造成DNA损伤,同时右丙亚胺对阿霉素心肌细胞损伤有保护作用。 结论 利用hiPSC-CMs成功建立了人源阿霉素心肌细胞损伤模型,克服了人心肌细胞难以获得及对药物反应存在种属差异的局限性,更好地用于阿霉素心脏毒性的机制研究及药物筛选。     

人诱导性多能干细胞向神经干细胞分化的方法探讨

目的:探讨人诱导性多能干细胞(iPS细胞)体外定向分化为神经干细胞(NSCs)的方法。方法:人iPS细胞悬浮培养4 d形成的拟胚体(EB)经维甲酸(RA)诱导4 d,诱导后的EB在无血清培养基中筛选并扩增培养。倒置显微镜下观察诱导后细胞的形态变化,RT-PCR检测iPS细胞多能性基因Nanog、Oct4和Sox2的表达,免疫荧光法检测NSCs特异性标志物Nestin、神经元标志物β-tubulinⅢ和神经胶质细胞标志物GFAP的表达。结果:(1)人iPS细胞在饲养层细胞上稳定传代,表达多能性基因Nanog、Oct4和Sox2,去除饲养层后悬浮培养能形成球形EB;(2)经RA诱导后的EB在无血清培养基中筛选培养第1 d贴壁生长,周围爬出细胞;第3 d,在高倍镜下可见中央聚集的细胞呈环形的rosette结构,rosette结构增多到第7 d达高峰。对照组未观察到rosette结构。随后大部分rosette细胞脱离瓶壁悬浮生长,形成神经球样克隆。免疫荧光显示神经球呈nestin阳性表达。贴壁培养的神经球能分化成β-tubulinШ和GFAP阳性细胞。结论:RA诱导结合无血清培养基筛选法能诱导iPS细胞高效分化为NSCs。     

Tenogenic differentiation of human induced pluripotent stem cell-derived mesenchymal stem cells dictated by properties of braided submicron fibrous scaffolds

Tendon and ligament (T/L) engineering is a growing area of research with potential to address the inadequacies of current T/L defect treatments. Our group previously developed braided submicron fibrous scaffolds (BSMFSs) and demonstrated the viability of BSMFSs for T/L tissue engineering. The objective of this study was to investigate the effect of fiber chemistry and braiding angle on BSMFS mechanical properties and in turn, tenogenic differentiation of human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) seeded on BSMFSs subjected to cyclic tensile stimulation in the absence of tenogenic medium. By varying fiber chemistry and/or braiding angle, BSMFSs with a range of mechanical properties were produced. We found that fiber chemistry dictated cell adhesion while braiding angle dictated the tissue-specific lineage commitment of hiPSC-MSCs. Scaffolds braided with large angles better supported hiPSC-MSC tenogenic differentiation as evidenced by the production of T/L-associated markers, downregulation of osteogenic markers, and expression of fibroblast-like, spindle cell morphology compared to scaffolds braided with small angles. Our results demonstrate the importance of substrate properties and mechanical stimulation on tenogenic differentiation. These results also demonstrate the versatility of BSMFSs and the potential of hiPSC-MSCs for T/L tissue engineering.