Mass transfer limitations in embryoid bodies during human embryonic stem cell differentiation

Due to their ability to differentiate into cell types from all the three germ layers and their potential unlimited capacity for expansion, embryonic stem cells have tremendous potential to treat diseases and injuries. Spontaneous differentiation of human embryonic stem cells (hESCs) is influenced by the size of the differentiating embryoid bodies (EBs). To further understand the dynamics between nutrient mass transfer, EB size, and stem cell differentiation, a transient mass diffusion model of a single hESC EB was constructed. The results revealed that the oxygen concentration at the centers of large EBs (400-μm radius) was 50% lower when compared to that in smaller EBs (200-μm radius). In addition, the concentration profile of cytokines within an EB depended strongly on their depletion rate, with higher depletion rates resulting in cytokine concentrations that varied significantly throughout the EB. A comparison of the results of our model with published experimental data reveals a close correlation between the fraction of cells that differentiate to a given lineage and the fraction of cells exposed to different oxygen or cytokine concentrations. This, along with other data from the literature, suggests that diffusive mass transfer influences the differentiation of hESCs within EBs by controlling the spatial distribution of soluble factors. This has important implications for research involving the differentiation of embryonic stem cells in EBs, as well as for bioprocess design and the development of robust differentiation protocols where mass transfer could be altered to control the cell differentiation trajectory.     

拟胚体贴壁时间对小鼠胚胎干细胞心肌分化的影响

目的观察拟胚体(EB)贴壁时间对小鼠胚胎干细胞(ESC)心肌分化的影响并研究其机制。方法用悬滴培养法促进ESCs形成EBs。EBs在不同的分化天数贴壁,观察搏动EBs百分比,RT-PCR检测Nkx2.5,GATA4和β-MHC mRNA表达,Western blot检测Src家族酪氨酸激酶磷酸化水平。结果分化第3,4天贴壁组搏动EB百分比及Nkx2.5,GATA4,β-MHC表达水平显著低于分化第5,6和7天贴壁组(P<0.05);EB贴壁能够使Src激酶磷酸化水平升高,Src激酶阻断剂PP2能够抑制贴壁诱发的Src激酶磷酸化水平升高(P<0.05);对于分化第4天贴壁组,在分化第4～6天使用PP2能够增加搏动EBs百分比及β-MHC表达水平(P<0.05)。结论 EB贴壁时间是影响ESC心肌分化的一个重要因素。在分化第4天或者之前贴壁可以显著抑制心肌分化,其机制可能是EB贴壁激活了Src激酶。     

In vitro differentiation of mouse embryonic stem cells: enrichment of endodermal cells in the embryoid body

Embryonic stem (ES) cells have the potential to differentiate into all three germ layers, providing new perspectives not only for embryonic development but also for the application in cell replacement therapies. Even though the formation of an embryoid body (EB) in a suspension culture has been the most popular method to differentiate ES cells into a wide range of cells, not much is known about the characteristics of EB cells. To this end, we investigated the process of EB formation in the suspension culture of ES cells at weekly intervals for up to 6 weeks. We observed that the central apoptotic area is most active in the first week of EB formation and that the cell adhesion molecules, except beta-catenin, are highly expressed throughout the examination period. The sequential expression of endodermal genes in EBs during the 6-week culture correlated closely with that of normal embryo development. The outer surface of EBs stained positive for alpha-fetoprotein and GATA-4. When isolated from the 2-week-old EB by trypsin treatment, these endodermal lineage cells matured in vitro into hepatocytes upon stimulation with various hepatotrophic factors. In conclusion, our results demonstrate that endodermal cells can be retrieved from EBs and matured into specific cell types, opening new therapeutic usage of these in vitro differentiated cells in the cell replacement therapy of various diseases.     

Rapid single-step separation of pluripotent mouse embryonic stem cells from mouse feeder fibroblasts

Highly enriched, pure populations of pluripotent mouse embryonic stem (mES) cells are a prerequisite to downstream experimental manipulations. However, the existing preplating method does not allow complete removal of co-cultured mouse embryonic fibroblast (MEF) feeder cells. The primary objective of the current investigation was to develop and validate a rapid, single-step separation technique for the complete removal of MEF feeder cells from mES cells. A discontinuous density gradient was prepared using Histopaque 1119 at incremental percentages from the top to bottom of a test tube (20, 40, 60, and 100% in culture medium). A suspension of mES cells and MEF feeder cells was layered on top of the gradient. After centrifugation at 400 x g, ES cells and MEF feeder cells were segregated discretely in separate layers at the 40/20% and 100/60% density interfaces, respectively. The mES cells were enriched to a purity of greater than 99% with a recovery rate of greater than 90%. The separation did not alter the viability or the differentiation potential of mES cells. This study validates a simple technique that enables the preparation of highly enriched mES cells that are essentially free of contaminating MEF feeder cells. The discontinuous density gradient separation method is inexpensive, efficient, rapid, and reproducible. The method can be readily scaled-up to accommodate large batch preparations, enabling a broad range of processing needs. Overall, this simple technique significantly expedites the recovery and enrichment of mES cells from MEFs.     

Tamoxifen-inducible Cre-mediated calreticulin excision to study mouse embryonic stem cell differentiation

Embryonic stem cells are useful to study the functional aspects of lineage commitment. In this study, we report that using the Cre/loxP system provides a useful tool for studying multifunctional proteins that are involved in stem cell differentiation, such as calreticulin. Calreticulin is a chaperone and a major calcium buffer of the endoplasmic reticulum and it functions during both adipogenesis and cardiomyogenesis. We used both a tamoxifen-inducible and cardiomyocyte-specific alpha-myosin heavy chain promoter-driven Cre/loxP system to study cardiomyogenesis, and a tamoxifen-inducible ubiquitously expressed cytomegalovirus promoter-driven Cre/loxP system to study adipogenesis. Both Cre/loxP systems mimicked the results previously observed using the calreticulin-null stem cell systems. Our results indicate that the tamoxifen-inducible Cre/loxP system is an effective and reliable tool to use for gene ablation in studies on functional aspects of stem cell biology.     

二甲基亚砜诱导胚胎干细胞分化伴随凋亡发生

目的：标准化二甲基亚砜（DMSO）诱导胚胎干细胞（ESC）向心肌细胞分化的方法,及DMSO是否同时诱导细胞凋亡。方法：MTT法确定DMSO的应用剂量,不同条件培养基对ESC进行诱导分化,并在形态学、蛋白质及基因水平鉴定ESC源心肌细胞。利用形态学、流式细胞仪等对细胞凋亡进行观测。结果;DMSO的最佳应用浓度为1％,拟胚体经诱导后跳动率为96．7％。该心肌细胞表达多种心肌蛋白,且肌小节结构发育成熟。DMSO的促分化效应可能与心肌转录因子GATA-4的表达有关,1％DMSO可诱导ESC部分产生凋亡,且具有时间和剂量依赖性。结论：1％DMSO不但能够高效诱导ES-D3分化为心肌细胞,且以时间依赖的方式诱导ES-D3细胞部分凋亡。     

小鼠胚胎干细胞来源的心肌细胞系的建立

目的 建立模拟细胞微环境、促进细胞分化的三维培养体系,以体外诱导和培养小鼠胚胎干细胞 (mES-D3)来源的心肌细胞。方法 将mES细胞通过悬浮培养获得拟胚体（EBs）后转入Matrigel? Matrix 3D培养体系中培养。用形态学观察计数收缩集落的数量和频率;用免疫组织化学和RT-PCR检测心肌细胞分化相关基因的表达。结果 在Matrigel?中培养5d左右,可观察到自发节律收缩的EBs,频率约为40～50次/min;继续培养3d左右,频率约为80～90次/min;到45d左右大部分集落最终收缩。分化为心肌细胞所表达的特异性蛋白cTnT, Desmin, Actin表达呈阳性,并表达心肌特异性基因cTnT, NKX2E, GATA-4和MLC-2v。结论 mES细胞来源的EBs在Matrigel?培养体系中能够分化为心肌细胞,籍此我们建立了一株细胞系。     

Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies

Human embryonic stem cells (hESCs) are generally induced to differentiate by forming spherical structures termed embryoid bodies (EBs) in the presence of soluble growth factors. hEBs are generated by suspending small clumps of hESC colonies; however, the resulting hEBs are heterogeneous because this method lacks the ability to control the number of cells in individual EBs. This heterogeneity affects factors that influence differentiation such as cell–cell contact and the diffusion of soluble factors, and consequently, the differentiation capacity of each EB varies. Here, we fabricated size-tunable concave microwells to control the physical environment, thereby regulating the size of EBs formed from single hESCs. Defined numbers of single hESCs were forced to aggregate and generate uniformly sized EBs with high fidelity, and the size of the EBs was controlled using concave microwells of different diameters. Differentiation patterns in H9- and CHA15-hESCs were affected by EB size in both the absence and presence of growth factors. By screening EB size in the presence of various BMP4 concentrations, a two-fold increase in endothelial cell differentiation was achieved. Because each hESC line has unique characteristics, the findings of this study demonstrate that concave microwells could be used to screen different EB sizes and growth factor concentrations to optimize differentiation for each hESC line.     

胚胎源性干细胞分化研究的现状

目前,对于胚胎源性干细胞的分化研究已成为当今医学界研究的热点,但其分化的分子机制和定向分化的方法一直是人们需要解决的难点问题,近年来无论在分子机制研究还是在定向诱导分化研究方面都有了长足进展。将这三种胚胎源性干细胞从其分化机制及定向分化潜能等方面结合国内外研究进展进行综述。     

The antimalaria agent artemisinin exerts antiangiogenic effects in mouse embryonic stem cell-derived embryoid bodies

Artemisinin is widely used as an agent to treat malaria; the possible antiangiogenic effects of this compound are unknown. In the present study, the antiangiogenic effects of artemisinin were investigated in mouse embryonic stem cell-derived embryoid bodies, which are a model system for early postimplantation embryos and which efficiently differentiate capillaries. Artemisinin dose dependently inhibited angiogenesis in embryoid bodies and raised the level of intracellular reactive oxygen species. Furthermore impaired organization of the extracellular matrix component laminin and altered expression patterns of matrix metalloproteinases 1, 2, and 9 were observed during the time course of embryoid body differentiation. Consequently accelerated penetration kinetics of the fluorescent anthracycline doxorubicin occurred within the tissue, indicating increased tissue permeability. Artemisinin down-regulated hypoxia-inducible factor-1alpha and vascular endothelial growth factor (VEGF) expression, which control endothelial cell growth. The antiangiogenic effects and the inhibition of hypoxia-inducible factor-1alpha and VEGF were reversed upon cotreatment with the free radical scavengers mannitol and vitamin E, indicating that artemisinin may act via reactive oxygen species generation. Furthermore, capillary formation was restored upon coadministration of exogenous VEGF. The data of the present study suggest that the antiangiogenic activity of artemisinin and the increase in tissue permeability for cytostatics may be exploited for anticancer treatment.