Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after, but not before, 14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair.     

Novel method of murine embryonic stem cell-derived osteoclast development

Murine embryonic stem (mES) cells are self-renewing pluripotent cells that bear the capacity to differentiate into ectoderm-, endoderm-, and mesoderm-derived tissues. In suspension culture, embryonic stem (ES) cells grow into spherical embryoid bodies (EBs) and are useful for the study of specific gene products in the development and function of various tissue types. Osteoclasts are hematopoietic stem cell-derived cells that participate in bone turnover by secreting resorptive molecules such as hydrochloric acid and acidic proteases, which degrade the bone extracellular matrix. Aberrant osteoclast function leads to dysplastic, erosive, and sclerosing bone diseases. Previous studies have reported the derivation of osteoclasts from mES cells; however, most of these protocols require coculture with stromal cell lines. We describe two simplified, novel methods of stromal cell-independent ES cell-derived osteoclast development.     

The effect of matrix composition of 3D constructs on embryonic stem cell differentiation

The use of embryonic stem (ES) cells as unlimited cell source in tissue engineering has ignited the hope of regenerating any kind of tissue in vitro. However, the role of the material in control and guidance of their development and commitment into complex and viable three-dimensional (3D) tissues is still poorly understood. In this work, we investigate the role of material composition and structure on promoting ES cells growth and differentiation, by culturing mouse ES cell-derived embryoid bodies (EBs) in various semi-interpenetrating polymer networks (SIPNs), made of collagen, fibronectin (FN) and laminin (LM). We show that both composition and strength of the supportive matrix play an important role in EBs development. High collagen concentrations inhibit EBs cavitation and hence the following EBs differentiation, by inhibiting apoptosis. The presence of FN in 3D collagen constructs strongly stimulates endothelial cell differentiation and vascularization. Conversely, LM increases the ability of ES cells to differentiate into beating cardiomyocytes. Our data suggest that matrix composition has an important role in EBs development and that it is possible to influence stem cell differentiation toward preferential pattern, by modulating the physical and biochemical properties of the scaffold.     

Ultrastructural analysis of mouse embryonic stem cell-derived chondrocytes

Pluripotent embryonic stem (ES) cells cultivated as cellular aggregates, so called embryoid bodies (EBs), differentiate spontaneously into different cell types of all three germ layers in vitro resembling processes of cellular differentiation during embryonic development. Regarding chondrogenic differentiation, murine ES cells differentiate into progenitor cells, which form pre-cartilaginous condensations in the EB-outgrowths and express marker molecules characteristic for mesenchymal cell types such as Sox5 and Sox6. Later, mature chondrocytes appear which express collagen type II, and the collagen fibers show a typical morphology as demonstrated by electron-microscopical analysis. These mature chondrogenic cells are organized in cartilage nodules and produce large amounts of extracellular proteoglycans as revealed by staining with cupromeronic blue. Finally, cells organized in nodules express collagen type X, indicating the hypertrophic stage. In conclusion, differentiation of murine ES cells into chondrocytes proceeds from the undifferentiated stem cell via progenitor cells up to mature chondrogenic cells, which then undergo hypertrophy. Furthermore, because the ES-cell-derived chondrocytes did not express elastin, a marker for elastic cartilage tissue, we suggest the cartilage nodules to resemble hyaline cartilage tissue.     

Double labeling of mRNA and protein markers in cultured embryoid bodies

Summary In vitro suspension cultures of embryonal carcinoma or embryonic stem cells (EC/ES) generate cell aggregates termed as embryoid bodies (EBs). EBs have been analyzed to study the mechanisms of cellular differentiation in vitro. The multipotency of EC/ES cells to differentiate into various cell types as well as the expression of many marker genes provides a valuable in vitro model system to study the mechanisms of cellular differentiation. Here we present a procedure for a mRNA detection of a specific gene using double labeling-mRNA probe and an antibody against cellular marker proteins. This double labeling analysis in combination with a culture of EBs provides a useful approach to analyze several mechanisms of cellular differentiation from multipotent EC/ES cells.     

Effects of three-dimensional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells

Embryonic stem (ES) cells have the ability to self-replicate and differentiate into cells from all three germ layers, holding great promise for tissue regeneration applications. However, controlling the differentiation of ES cells and obtaining homogenous cell populations still remains a challenge. We hypothesize that a supportive three-dimensional (3D) environment provides ES cell-derived cells an environment that more closely mimics chondrogenesis in vivo. In the present study, the chondrogenic differentiation capability of ES cell-derived embryoid bodies (EBs) encapsulated in poly(ethylene glycol)-based (PEG) hydrogels was examined and compared with the chondrogenic potential of EBs in conventional monolayer culture. PEG hydrogel-encapsulated EBs and EBs in monolayer were cultured in vitro for up to 17 days in chondrogenic differentiation medium in the presence of transforming growth factor (TGF)-beta1 or bone morphogenic protein-2. Gene expression and protein analyses indicated that EB-PEG hydrogel culture upregulated cartilage-relevant markers compared with a monolayer environment and induction of chondrocytic phenotype was stimulated with TGF-beta1. Histology of EBs in PEG hydrogel culture with TGF-beta1 demonstrated basophilic extracellular matrix deposition characteristic of neocartilage. These findings suggest that EB-PEG hydrogel culture, with an appropriate growth factor, may provide a suitable environment for chondrogenic differentiation of intact ES cell-derived EBs.     

Cultivation of human embryonic stem cells without the embryoid body step enhances osteogenesis in vitro

Osteogenic cultures of embryonic stem cells (ESCs) are predominately derived from three-dimensional cell spheroids called embryoid bodies (EBs). An alternative method that has been attempted and merits further attention avoids EBs through the immediate separation of ESC colonies into single cells. However, this method has not been well characterized and the effect of omitting the EB step is unknown. Herein, we report that culturing human embryonic stem cells (hESCs) without the EB stage leads to a sevenfold greater number of osteogenic cells and to spontaneous bone nodule formation after 10-12 days. In contrast, when hESCs were differentiated as EBs for 5 days followed by plating of single cells, bone nodules formed after 4 weeks only in the presence of dexamethasone. Furthermore, regardless of the inclusion of EBs, bone matrix formed, including cement line matrix and mineralized collagen, which displayed apatitic mineral (PO4) with calcium-to-phosphorous ratios similar to those of hydroxyapatite and human bone. Together these results demonstrate that culturing hESCs without an EB step can be used to derive large quantities of functional osteogenic cells for bone tissue engineering.     

Lentiviral rescue of vascular endothelial growth factor receptor-2 expression in flk1-/- embryonic stem cells shows early priming of endothelial precursors

The vascular endothelial growth factor (VEGF) family and its receptors are important for vascular development and maintenance of blood vessels, as well as for angiogenesis, the formation of new vessels. Loss of VEGF receptor-2 (VEGFR-2; designated Flk-1 in mouse) results in arrest of vascular and hematopoietic development in vivo. We used lentiviral transduction to reconstitute VEGFR-2 expression in flk1-/- embryonic stem (ES) cells. VEGF-induced vasculogenesis and sprouting angiogenesis were rescued in transduced ES cultures differentiating in vitro as EBs. Although the transgene was expressed in the pluripotent stem cells and lacked linage restriction during differentiation, the extent of endothelial recruitment was similar to that in wild-type EBs. Reconstitution of VEGFR-2 in flk1-/- ES cells allowed only precommitted precursors to differentiate into functional endothelial cells able to organize into vascular structures. Chimeric EB cultures composed of wild-type ES cells mixed with flk1-/- ES cells or reconstituted VEGFR-2-expressing ES cells were created. In the chimeric cultures, flk1-/- endothelial precursors were excluded from wild-type vessel structures, whereas reconstituted VEGFR-2-expressing precursors became integrated together with wild-type endothelial cells to form chimeric vessels. We conclude that maturation of endothelial precursors, as well as organization into vascular structures, requires expression of VEGFR-2. Disclosure of potential conflicts of interest is found at the end of this article.     

Enhanced chondrogenic differentiation of murine embryonic stem cells in hydrogels with glucosamine

Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). We have previously reported that hydrogels provide EBs a supportive environment for in vitro chondrogenic differentiation and three dimensional tissue formation [Hwang NS, et al. The Effects of three dimensional culture and growth factors on the chondrogenic differentiation of murine ES cells. Stem Cells 2006;24:284–91]. In this study, we report chondrogenic differentiation of murine ES cells encapsulated in photopolymerizing poly(ethylene-glycol)-based (PEG) hydrogels in the presence of glucosamine (GlcN), an amino monosaccharide found in chitin, glycoproteins and glycosaminoglycans such as hyaluronic acid, chondroitin sulfate and heparin sulfate. We examined the growth and differentiation of encapsulated EBs in standard chondrogenic differentiation medium containing 0-, 2-, and 10-mm GlcN. Morphometric analysis and examination of gene and protein expression indicated that treatment of hydrogel cultures with 2-mm GlcN for 21 days significantly increased EB size, levels of aggrecan mRNA, and tissue-specific extracellular matrix accumulation. GlcN can induce multiple aspects of cell behavior and optimal GlcN concentrations can be beneficial for directing the differentiation and tissue formation of ES cells.     

Biomimetic three-dimensional cultures significantly increase hematopoietic differentiation efficacy of embryonic stem cells

Stem cell-based tissue engineering is a promising technology in the effort to create functional tissues of choice. To establish an efficient approach for generating hematopoietic cell lineages directly from embryonic stem (ES) cells and to study the effects of three-dimensional (3D) biomaterials on ES cell differentiation, we cultured mouse ES cells on 3D, highly porous, biomimetic scaffolds. Cell differentiation was evaluated by microscopy and flow cytometry analysis with a variety of hematopoiesis- specific markers. Our data indicate that ES cells differentiated on porous 3D scaffold structures developed embryoid bodies (EBs) similar to those in traditional two-dimensional (2D) cultures; however, unlike 2D differentiation, these EBs integrated with the scaffold and appeared embedded in a network of extracellular matrix. Most significantly, the efficiency of hematopoietic precursor cell (HPC) generation on 3D, as indicated by the expression of various HPC-specific surface markers (CD34, Sca-1, Flk-1, and c-Kit) and colony-forming cell (CFC) assays, was reproducibly increased (about 2-fold) over their 2D counterparts. Comparison of static and dynamic 3D cultures demonstrated that spinner flask technology also contributed to the higher hematopoietic differentiation efficiency of ES cells seeded on scaffolds. Continued differentiation of 3D-derived HPCs into the myeloid lineage demonstrated increased efficiency (2-fold) of generating myeloid compared with differentiation from 2D-derived HPCs.     

丁酸钠、激活素A和地塞米松诱导小鼠胚胎干细胞分化为胰腺外分泌细胞

目的： 探讨丁酸钠、激活素A （activin A）和地塞米松诱导小鼠胚胎干细胞（ES细胞）分化为胰腺外分泌细胞的可行性,并对诱导作用进行比较。方法： 小鼠 ES细胞悬浮培养为拟胚体后,以不同浓度的丁酸钠（1 mmol/L,2 mmol/L,3 mmol/L）诱导分化,通过RT-PCR检测不同时点胰腺特异性外分泌基因的表达水平,确定丁酸钠诱导ES细胞向胰腺外分泌细胞分化的最佳浓度和作用时间。进一步单独或联合应用丁酸钠、activin A、地塞米松诱导ES细胞分化,并通过细胞形态学变化、RT-PCR和免疫荧光检测观察不同诱导方案对胰腺外分泌基因和蛋白表达的影响,确定最佳诱导方案。结果：1 mmol/L丁酸钠能明显促进胰腺外分泌基因amylase、chymotrypsinogen、elastase1、elastase2和carboxypeptidase的表达,随着丁酸钠浓度的增加,丁酸钠的诱导作用逐渐减弱;1 mmol/L丁酸钠诱导第3 d后可检测到amylase、chymotrypsinogen、elastase1、elastase2和carboxypeptidase的表达,在第5 d外分泌基因mRNA表达水平达到高峰,随后逐渐下降。与自发对照组相比,单独应用丁酸钠、activin A、地塞米松诱导ES细胞分化,均能提高amylase、chymotrypsinogen、elastase1、elastase2和carboxypeptidase的表达水平。但联合应用丁酸钠、activin A、地塞米松诱导后,ES细胞形态更为均一,上述胰腺外分泌基因的表达进一步增强;免疫荧光结果显示amylase表达为阳性。结论： 低浓度的丁酸钠、activin A以及地塞米松均可以诱导小鼠ES细胞胰腺外分泌基因的表达,多种诱导因子的联合作用能明显提高胰腺外分泌细胞的诱导效率。     

The notch response inhibitor DAPT enhances neuronal differentiation in embryonic stem cell-derived embryoid bodies independently of sonic hedgehog signaling.

During development of the neural tube, inhibition of the Notch response as well as the activation of the Sonic Hedgehog (Shh) response results in the formation of neuronal cell types. To determine whether Shh and Notch act independently, we tested the effects of the Notch inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) on neuralized, embryonic stem (ES) cell-derived embryoid bodies (EBs), while varying the levels of Shh pathway activation. Shh-resistant EBs were derived from Smo null ES cells, while EBs with constitutive high level of Shh pathway activation were derived from Ptc1 null ES cells. Intermediate levels of Shh pathway activation was achieved by the addition of ShhN to the EB culture medium. It was found that DAPT-mediated inhibition of the Notch response resulted in enhanced neuronal differentiation. In the absence of Shh, more interneurons were detected, while the main effect of DAPT on EBs with an activated Shh response was the precocious loss of ventral neuronal precursor-specific markers.     

Hematopoietic differentiation of embryoid bodies derived from the human embryonic stem cell line SNUhES3 in co-culture with human bone marrow stromal cells

Human embryonic stem (ES) cells can be induced to differentiate into hematopoietic precursor cells via two methods: the formation of embryoid bodies (EBs) and co-culture with mouse bone marrow (BM) stromal cells. In this study, the above two methods have been combined by co-culture of human ES-cell-derived EBs with human BM stromal cells. The efficacy of this method was compared with that using EB formation alone. The undifferentiated human ES cell line SNUhES3 was allowed to form EBs for two days, then EBs were induced to differentiate in the presence of a different serum concentration (EB and EB/high FBS group), or co- cultured with human BM stromal cells (EB/BM co-culture group). Flow cytometry and hematopoietic colony-forming assays were used to assess hematopoietic differentiation in the three groups. While no significant increase of CD34+/CD45- or CD34+/CD38- cells was noted in the three groups on days 3 and 5, the percentage of CD34+/CD45- cells and CD34+/ CD38- cells was significantly higher in the EB/BM co-culture group than in the EB and EB/high FBS groups on day 10. The number of colony-forming cells (CFCs) was increased in the EB/BM co-culture group on days 7 and 10, implying a possible role for human BM stromal cells in supporting hematopoietic differentiation from human ES cell-derived EBs. These results demonstrate that co-culture of human ES-cell-derived EBs with human BM stromal cells might lead to more efficient hematopoietic differentiation from human ES cells cultured alone. Further study is warranted to evaluate the underlying mechanism.     

Adipogenesis of murine embryonic stem cells in a three-dimensional culture system using electrospun polymer scaffolds

A mechanistic understanding of adipose tissue differentiation is critical for the treatment and prevention of obesity and type 2 diabetes. Conventional in vitro models of adipogenesis are preadipocytes or freshly isolated adipocytes grown in two-dimensional (2D) cultures. Optimal results using in vitro tissue culture models can be expected only when adipocyte models closely resemble adipose tissue in vivo. Thus the design of an in vitro three-dimensional (3D) model which faithfully mimics the in vivo environment is needed to effectively study adipogenesis. Pluripotent embryonic stem (ES) cells are a self-renewing cell type that can readily be differentiated into adipocytes. In this study, a 3D culture system was developed to mimic the geometry of adipose tissue in vivo. Murine ES cells were seeded into electrospun polycaprolactone scaffolds and differentiated into adipocytes in situ by hormone induction as demonstrated using a battery of gene and protein expression markers along with the accumulation of neutral lipid droplets. Insulin-responsive Akt phosphorylation, and beta-adrenergic stimulation of cyclic AMP synthesis were demonstrated in ES cell-derived adipocytes. Morphologically, ES cell-derived adipocytes resembled native fat cells by scanning electron and phase contrast microscopy. This tissue engineered ES cell-matrix model has potential uses in drug screening and other therapeutic developments.     

Scalable production of embryonic stem cell-derived cardiomyocytes

Cardiomyocyte transplantation could offer a new approach to replace scarred, nonfunctional myocardium in a diseased heart. Clinical application of this approach would require the ability to generate large numbers of donor cells. The purpose of this study was to develop a scalable, robust, and reproducible process to derive purified cardiomyocytes from genetically engineered embryonic stem (ES) cells. ES cells transfected with a fusion gene consisting of the alpha-cardiac myosin heavy chain (MHC) promoter driving the aminoglycoside phosphotransferase (neomycin resistance) gene were used for cardiomyocyte enrichment. The transfected cells were aggregated into embyroid bodies (EBs), inoculated into stirred suspension cultures, and differentiated for 9 days before selection of cardiomyocytes by the addition of G418 with or without retinoic acid (RA). Throughout the culture period, EB and viable cell numbers were measured. In addition, flow cytometric analysis was performed to monitor sarcomeric myosin (a marker for cardiomyocytes) and Oct-4 (a marker for undifferentiated ES cells) expression. Enrichment of cardiomyocytes was achieved in cultures treated with either G418 and retinoic acid (RA) or with G418 alone. Eighteen days after differentiation, G418-selected flasks treated with RA contained approximately twice as many cells as the nontreated flasks, as well as undetectable levels of Oct-4 expression, suggesting that RA may promote cardiac differentiation and/or survival. Immunohistological and electron microscopic analysis showed that the harvested cardiomyocytes displayed many features characteristic of native cardiomyocytes. Our results demonstrate the feasibility of large-scale production of viable, ES cell-derived cardiomyocytes for tissue engineering and/or implantation, an approach that should be transferable to other ES cell derived lineages, as well as to adult stem cells with in vitro cardiomyogenic activity.     

小鼠胚胎干细胞的体外培养及诱导分化成酪氨酸羟化酶阳性神经元

探索小鼠胚胎干细胞 (ES)在体外培养及向酪氨酸羟化酶阳性神经元诱导分化的可能性。将小鼠胚胎干细胞在含有白血病抑制因子 (LIF)的ES培养基中扩增 ,并通过以下几个步骤 :胚胎体的形成、巢蛋白 (Nestin)阳性细胞的筛选、Nestin阳性细胞的体外扩增及撤除碱性成纤维细胞生长因子等后观察向酪氨酸羟化酶阳性神经元的分化。结果表明小鼠胚胎干细胞在含有LIF的特定培养基中能够稳定传代并保持不分化状态 ,经过无血清培养基的筛选和培养 ,在SonicHedgehog(SHH)及成纤维细胞生长因子 (fibroblastgrowthfactor 8,FGF8)等细胞因子的作用下能定向分化成酪氨酸羟化酶阳性神经元。这种方法有望为帕金森病等神经变性病的细胞移植治疗提供充足的细胞来源。