Modulation of sarcomere organization during embryonic stem cell-derived cardiomyocyte differentiation

Myofibrillogenesis - sarcomeres - mouse embryonic stem cells - cardiomyocytes - beta1 integrin Mouse embryonic stem (ES) cells, when cultivated as embryoid bodies, differentiate in vitro into cardiomyocytes of ventricle-, atrium- and pacemaker-like cell types characterized by developmentally controlled expression of cardiac-specific genes, structural proteins and ion channels. Using this model system, we show here, (I) that during cardiac myofibrillogenesis sarcomeric proteins are organized in a developmentally regulated manner following the order: titin (Z-disk), alpha-actinin, myomesin, titin (M-band), myosin heavy chain, alpha-actin, cardiac troponin T and M-protein, recapitulating the sarcomeric organization in the chicken embryonal heart in vivo. Our data support the view that the formation of I-Z-I complexes is developmentally delayed with respect to A-band assembly. We show (2) that the process of cardiogenic differentiation in vitro is influenced by medium components: Using a culture medium supplemented with glucose, amino acids, vitamins and selenium ions, we were able to increase the efficiency of cardiac differentiation of wild-type, as well as of beta1 integrin-deficient (beta1-/-) ES cells, and to improve the degree of organization of sarcomeric structures in wild-type and in beta1-/- cardiac cells. The data demonstrate the plasticity of cardiogenesis during the differentiation of wild-type and of genetically modified ES cells.     

Characterization of human embryonic stem cell-derived hematopoietic progenitor phenotype

Differentiation of human embryonic stem cells (hESCs) into hematopoietic lineages using various methods has been reported. However, the phenotype that precisely defines the hematopoietic progenitor compartment with clonogenic activities has yet to be determined. Here, we measured and characterized progenitor function of subfractions of cells prospectively isolated from human embryoid bodies (hEBs) during hematopoietic differentiation basing on surface markers CD45, CD34, CD43, and CD38. We report that hematopoietic progenitors predominantly resided in the CD45⁺ subset. CD43⁺ cells lacking CD45 expression were largely devoid of progenitor activity. However, progenitor activity and multipotentiality was more enriched in CD45⁺ cells co-expressing CD43. CD45⁺ subset co-expressing CD34 but lacking CD38 expression (CD45⁺CD34⁺CD38^-) were further enriched for CFU capacity compared to the CD45⁺CD34⁺CD38⁺ subset. Our study demonstrates a role of CD43 in enriching hematopoietic progenitors derived from hEBs and reveals a hierarchical organization of hESC-derived hematopoietic progenitor compartments defined by phenotypic markers.     

Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro

The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based lentivirus vector pseudotyped with the vesicular stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoietic cells in vitro. An oncoretrovirus vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to efficient transduction of ES cells. Lentivirus vector integration was verified in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoietic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system can be used to analyze lentivirus vector design for optimal expression in hematopoietic cells and for gain-of-function experiments during ES cell development in vitro.     

Forced expression of HoxB4 enhances hematopoietic differentiation by human embryonic stem cells

HoxB4 has been shown to enhance hematopoietic engraftment by hematopoietic stem cells (HSC) from differentiating mouse embryonic stem cell (mESC) cultures. Here we examined the effect of ectopic expression of HoxB4 in differentiated human embryonic stem cells (hESCs). Stable HoxB4-expressing hESCs were established by lentiviral transduction, and the forced expression of HoxB4 did not affect stem cell features. HoxB4-expressing hESC-derived CD34+ cells generated higher numbers of erythroid and blast-like colonies than controls. The number of CD34+ cells increased but CD45+ and KDR+ cell numbers were not significantly affected. When the hESC derived CD34+ cells were transplanted into NOD/SCID beta 2m-/- mice, the ectopic expression of HoxB4 did not alter their repopulating capacity. Our findings show that overexpression of HoxB4 in differentiating hESCs increases hematopoietic colony formation and hematopoietic cell formation in vitro, but does not affect in vivo repopulation in adult mice hosts.     

Shear stress during early embryonic stem cell differentiation promotes hematopoietic and endothelial phenotypes

Pluripotent embryonic stem cells (ESCs) are a potential source for cell‐based tissue engineering and regenerative medicine applications, but their translation into clinical use will require efficient and robust methods for promoting differentiation. Fluid shear stress, which can be readily incorporated into scalable bioreactors, may be one solution for promoting endothelial and hematopoietic phenotypes from ESCs. Here we applied laminar shear stress to differentiating ESCs using a 2D adherent parallel plate configuration to systematically investigate the effects of several mechanical parameters. Treatment similarly promoted endothelial and hematopoietic differentiation for shear stress magnitudes ranging from 1.5 to 15 dyne/cm² and for cells seeded on collagen‐, fibronectin‐ or laminin‐coated surfaces. Extension of the treatment duration consistently induced an endothelial response, but application at later stages of differentiation was less effective at promoting hematopoietic phenotypes. Furthermore, inhibition of the FLK1 protein (a VEGF receptor) neutralized the effects of shear stress, implicating the membrane protein as a critical mediator of both endothelial and hematopoietic differentiation by applied shear. Using a systematic approach, studies such as these help elucidate the mechanisms involved in force‐mediated stem cell differentiation and inform scalable bioprocesses for cellular therapies. Biotechnol. Bioeng. 2013; 110: 1231–1242. © 2012 Wiley Periodicals, Inc.     

Gene expression profiles during early differentiation of mouse embryonic stem cells

Background  

Understanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration. Embryonic stem (ES) cell differentiation can be triggered by embryoid body (EB) formation, which involves ES cell aggregation in suspension. EB growth in the absence of leukaemia inhibitory factor (LIF) leads EBs to mimic early embryonic development, giving rise to markers representative of endoderm, mesoderm and ectoderm. Here, we have used microarrays to investigate differences in gene expression between 3 undifferentiated ES cell lines, and also between undifferentiated ES cells and Day 1–4 EBs     

Differential expression of glucose transporter isoforms during embryonic stem cell differentiation

In mouse blastocysts six facilitative glucose transporter isoforms (GLUT)1-4, 8 and 9 are expressed. We have used the mouse embryonic stem (ES) cell line D3 and spontaneously differentiating embryoid bodies (EB) to investigate GLUT expression and the influence of glucose during differentiation of early embryonic cells. Both ES cells and EBs (2d-20d) expressed GLUT1, 3, and 8, whereas the isoforms 2 and 4 were detectable exclusively in EBs. Differentiation-associated expression of GLUT was analyzed by double staining with stage-specific embryonic antigen (SSEA-1), cytokeratins (CK18, 19), nestin, and desmin. Similar to trophoblast cells in mouse blastocysts the outer cell layer of endoderm-like cells showed a high GLUT3 expression in early EBs. In 20-day-old EBs no GLUT3 protein and only minor GLUT3 mRNA amounts could be detected. A minimal glucose concentration of 5 mM applied during 2 and 8 days of EB culture resulted in up-regulated GLUT4, Oct-4 and SSEA-1 levels and a delay in EB differentiation. We conclude that GLUT expression depends on cellular differentiation and that the expression is modulated by glucose concentration. The developmental and glucose-dependent regulation of GLUT strongly suggests a functional role of glucose and glucose transporters in ES cell differentiation and embryonic development.     

Evaluation of hollow fiber culture for large-scale production of mouse embryonic stem cell-derived hematopoietic stem cells

Hematopoietic stem cells (HSCs) have the ability to differentiate into all types of blood cells and can be transplanted to treat blood disorders. However, it is difficult to obtain HSCs in large quantities because of the shortage of donors. Recent efforts have focused on acquiring HSCs by differentiation of pluripotent stem cells. As a conventional differentiation method of pluripotent stem cells, the formation of embryoid bodies (EBs) is often employed. However, the size of EBs is limited by depletion of oxygen and nutrients, which prevents them from being efficient for the production of HSCs. In this study, we developed a large-scale hematopoietic differentiation approach for mouse embryonic stem (ES) cells by applying a hollow fiber (HF)/organoid culture method. Cylindrical organoids, which had the potential for further spontaneous differentiation, were established inside of hollow fibers. Using this method, we improved the proliferation rate of mouse ES cells to produce an increased HSC population and achieved around a 40-fold higher production volume of HSCs in HF culture than in conventional EB culture. Therefore, the HF/organoid culture method may be a new mass culture method to acquire pluripotent stem cell-derived HSCs.     

胚胎干细胞来源的拟胚体分化早期血管平滑肌标志物的表达时相

目的:研究胚胎血管发育早期SMα-actin、SM22α、myocardin、平滑肌肌球蛋白重链(SMMHC)的表达规律,并初步探讨在此阶段血小板源性生长因子-BB(PDGF-BB)对血管平滑肌细胞(VSMCs)分化的影响。方法:采用转染平滑肌特异性蛋白SM22α启动子控制下表达增强型绿色荧光蛋白(GFP)报告基因载体的胚胎干细胞制备拟胚体(EBs),用免疫荧光染色、RT-PCR、Western blot分析SMα-actin、SM22α、myocardin、SMMHC的表达时相;然后分别用0μmol/L(对照组)、10μmol/L、50μmol/L AG1296(血小板源性生长因子受体抑制剂)处理EBs,观察三组SMα-actin、SM22α、myocardin、SMMHC在基因及蛋白水平上的表达变化。结果:胚胎血管发育早期SMα-actin、myocardin、SM22α、SMMHC分别在EBs第0(胚胎干细胞)、8、11、13d开始有表达。AG1296三种浓度处理后SMα-actin、myocardin、SM22α、SMMHC蛋白表达及myocardin、SM22α和SMMHC mRNA表达均无明显差异。结论:EBs发育过程中存在着自发的VSMCs分化,SMα-actin表达最早,依次为myocardin、SM22α、SMMHC;PDGF-BB对EBs分化早期VSMCs标志物表达的调控可能不是必要的。