Serum-free differentiation of murine embryonic stem cells into alveolar type II epithelial cells

Alveolar type II (AT2) epithelial cells have important functions including the production of surfactant and regeneration of lost alveolar type I epithelial cells. The ability of in vitro production of AT2 cells would offer new therapeutic options in treating pulmonary injuries and disorders including genetically based surfactant deficiencies. Aiming at the generation of AT2-like cells, the differentiation of murine embryonic stem cells (mESCs) toward mesendodermal progenitors (MEPs) was optimized using a "Brachyury-eGFP-knock in" mESC line. eGFP expression demonstrated generation of up to 65% MEPs at day 4 after formation of embryoid bodies (EBs) under serum-free conditions. Plated EBs were further differentiated into AT2-like cells for a total of 25 days in serum-free media resulting in the expression of endodermal marker genes (FoxA2, Sox17, TTR, TTF-1) and of markers for distal lung epithelium (surfactant proteins (SP-) A, B, C, and D, CCSP, aquaporin 5). Notably, expression of SP-C as the only known AT2 cell specific marker could be detected after serum-induction as well as under serum-free conditions. Cytoplasmic localization of SP-C was demonstrated by confocal microscopy. The presence of AT2-like cells was confirmed by electron microscopy providing evidence for polarized cells with apical microvilli and lamellar body-like structures. Our results demonstrate the differentiation of AT2-like cells from mESCs after serum-induction and under serum-free conditions. The established serum-free differentiation protocol will facilitate the identification of key differentiation factors leading to a more specific and effective generation of AT2-like cells from ESCs.     

Neural differentiation of murine embryonic stem cells ES

Pluripotent murine embryonic stem (ES) cells can differentiate into all cell types both in vivo and in vitro. Based on their capability to proliferate and differentiate, these ES cells appear as a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the neural differentiation of the ES cells is a pre-requisite for selecting adequately the cells and conditions which will be able to correctly repair damaged brain and restore altered cognitive functions. Different methods allow obtaining neural cells from ES cells. Most of the techniques differentiate ES cells by treating embryoid bodies in order to keep an embryonic organization. More recent techniques, based on conditioned media, induce a direct differentiation of ES cells into neural cells, without going through the step of embryonic bodies. Beyond the fact that these techniques allow obtaining large numbers of neural precursors and more differentiated neural cells, these approaches also provide valuable information on the process of differentiation of ES cells into neural cells. Indeed, sequential studies of this process of differentiation have revealed that globally ES cells differentiating into neural cells in vitro recapitulate the molecular events governing the in vivo differentiation of neural cells. Altogether these data suggest that murine ES cells remain a highly valuable tool to obtain large amounts of precursor and differentiated neural cells as well as to get a better understanding of the mechanisms of neural differentiation, prior to a potential move towards the use of human ES cells in therapy.     

Hepatic differentiation of murine embryonic stem cells.

Murine embryonic stem (ES) cells can replicate indefinitely in culture and can give rise to all tissues, including the germline, when reimplanted into a murine blastocyst. ES cells can also be differentiated in vitro into a wide range of cell types. We have utilized a liver-specific marker to demonstrate that murine ES cells can differentiate into hepatocytes in vitro. We have used ES cells carrying a gene trap vector insertion (I.114) into an ankyrin repeat-containing gene (Gtar) that we have previously shown provides an exclusive beta-galactosidase marker for the early differentiation of hepatocytes in vivo. beta-Galactosidase-positive cells were differentiated from I.114 ES cells in vitro. The identity of these cells was confirmed by the expression of the proteins alpha-fetoprotein, albumin, and transferrin and by the fact that they have an ultrastructural appearance consistent with that of embryonic hepatocytes. We propose that this model system of hepatic differentiation in vitro could be used to define factors that are involved in specification of the hepatocyte lineage. In addition, human ES cells have recently been derived and it has been proposed that they may provide a source of differentiated cell types for cell replacement therapies in the treatment of a variety of diseases.     

Basal medium composition and serum or serum replacement concentration influences on the maintenance of murine embryonic stem cells

The expansion of stem cell numbers while retaining their developmental properties is a bioprocess challenge. We compared the growth rates and embryoid body (EB) formation yields of R1 and EFC murine embryonic stem cells (mESC) cultured in two basal media (DMEM or DMEM:F12) with additions of 1.7–15% fetal bovine serum (FBS) or serum replacer (KOSR). Whereas the basal medium or KOSR dose did not have a significant effect on growth rate for either cell line, increasing doses of KOSR had a significant negative effect on the EB yield of EFC cells. Use of DMEM:F12 and increasing doses of FBS independently and significantly increased the growth rate for both cell lines. DMEM:F12 also significantly increased EB yields for both cell lines. The results show that use of DMEM:F12 and several-fold lower than conventional concentrations of KOSR can efficiently support maintenance of mESC and that KOSR should be dose as well as lot optimized.     

In vitro differentiation of murine embryonic stem cells into keratinocyte-like cells

Embryonic stem (ES) cells are omnipotent; they can differentiate into every cell type of the body. The development of culture conditions that allow their differentiation has made it conceivable to produce large numbers of cells with lineage-specific characteristics in vitro. Here, we describe a method by which murine ES cells can be differentiated into cells with characteristics of epidermal keratinocytes. Keratinocyte-like cells were isolated from embryoid bodies and grown in culture. Potential applications of this method are the in vitro differentiation of cells of interest from ES cells of mice with lethal phenotypes during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments.     

The effect of anti-oxidants and medium composition on isolation and culture of alveolar type II pneumocytes

Alveolar type II pneumocytes were isolated from adult male rabbits and were placed in primary culture. The presence of anti-oxidants throughout the isolation procedure, particularly ascorbic acid and glutathione, was found to enhance in vitro attachment efficiency of cells. The use of a culture medium substituting D-valine for L-valine also significantly enhanced attachment efficiency. Although these cells do not ordinarily proliferate in culture, a low-serum medium containing insulin, transferrin, selenium and hydrocortisone allowed limited proliferation in addition to promoting dome formation in culture.     

Pluripotent differentiation in vitro of murine Es-D3 embryonic stem cells

Although the ES-D3 murine embryonic stem cell line was one of the first derived, little information exists on the in vitro differentiation potential of these cells. We have used immunocytochemical and flow cytometric methods to monitor ES-D3 embryoid body differentiation in vitro during a 21-d period. Spontaneous differentiation of embryoid body cells was induced by leukemia inhibitory factor withdrawal in the absence of feeder cells. The pluripotent stem cell markers Oct-3/4, SSEA-1, and EMA-1 were found to persist for at least 7 d, whereas the primitive endoderm marker cytokeratin endo-A was expressed at increasing levels from day 6. The localization of these antigens within the embryoid bodies suggested that embryonic ectoderm- and primitive endoderm-derived tissues were segregated. Localized expression of class III beta-tubulin and sarcomeric myosin also was detected, indicating that representatives of all three embryonic germ layers were present after induction of differentiation in vitro.     

BMP-4 inhibits neural differentiation of murine embryonic stem cells.

Members of the transforming growth factor-beta superfamily, including bone morphogenetic protein 4 (BMP-4), have been implicated as regulators of neuronal and glial differentiation. To test for a possible role of BMP-4 in early mammalian neural specification, we examined its effect on neurogenesis in aggregate cultures of mouse embryonic stem (ES) cells. Compared to control aggregates, in which up to 20% of the cells acquired immunoreactivity for the neuron-specific antibody TuJ1, aggregates maintained for 8 days in serum-free medium containing BMP-4 generated 5- to 10-fold fewer neurons. The action of BMP-4 was dose dependent and restricted to the fifth through eighth day in suspension. In addition to the reduction in neurons, we observed that ES cell cultures exposed to BMP-4 contained fewer cells that were immunoreactive for glial fibrillary acidic protein or the HNK-1 neural antigen. Furthermore, under phase contrast, cultures prepared from BMP-4-treated aggregates contained a significant proportion of nonneuronal cells with a characteristic flat, elongated morphology. These cells were immunoreactive for antibodies to the intermediate filament protein vimentin; they were rare or absent in control cultures. Treatment with BMP-4 enhanced the expression of the early mesodermal genes brachyury and tbx6 but had relatively little effect on total cell number or cell death. Coapplication of the BMP-4 antagonist noggin counteracted the effect of exogenous BMP-4, but noggin alone had no effect on neuralization in either the absence or presence of retinoids. Collectively, our results suggest that BMP-4 can overcome the neuralizing action of retinoic acid to enhance mesodermal differentiation of murine ES cells.     

Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells

Many applications of human embryonic stem cells (hESCs) will require fully defined growth and differentiation conditions including media devoid of fetal calf serum. To identify factors that control lineage differentiation we have analyzed a serum-free (SF) medium conditioned by the cell line END2, which efficiently induces hESCs to form cardiomyocytes. Firstly, we noted that insulin, a commonly used medium supplement, acted as a potent inhibitor of cardiomyogenesis in multiple hESC lines and was rapidly cleared by medium conditioning. In the presence of insulin or IGF-1, which also suppressed cardiomyocyte differentiation, the PI3/Akt pathway was activated in undifferentiated hESC, suggesting that insulin/IGF-1 effects were mediated by this signaling cascade. Time course analysis and quantitative RT-PCR revealed impaired expression of endoderm and mesoderm markers in the presence of insulin, particularly if added during early stages of hESC differentiation. Relatively high levels of the neural ectoderm marker Sox1 were expressed under these conditions. Secondly, comparative gene expression showed that two key enzymes in the prostaglandin I2 (PGI2) synthesis pathway were highly up-regulated in END2 cells compared with a related, but non-cardiogenic, cell line. Biochemical analysis confirmed 6-10-fold higher PGI2 levels in END2 cell-conditioned medium (END2-CM) vs. controls. Optimized concentrations of PGI2 in a fully synthetic, insulin-free medium resulted in a cardiogenic activity equivalent to END2-CM. Addition of the p38 mitogen-activated protein kinase-inhibitor SB203580, which we have shown previously to enhance hESC cardiomyogenesis, to these insulin-free and serum-free conditions resulted in a cardiomyocyte content of >10% in differentiated cultures without any preselection. This study represents a significant step toward developing scalable production for cardiomyocytes from hESC using clinically compliant reagents compatible with Good Manufacturing Practice.     

Efficient suspension bioreactor expansion of murine embryonic stem cells on microcarriers in serum-free medium

Large numbers of cells will be required for successful embryonic stem cell (ESC)-based cellular therapies or drug discovery, thus raising the need to develop scaled-up bioprocesses for production of ESCs and their derived progeny. Traditionally, ESCs have been propagated in adherent cultures in static flasks on fibroblasts layers in serum-containing medium. Direct translation of two-dimensional flatbed cultures to large-scale production of the quantities of cells required for therapy simply by increasing the number of dishes or flasks is not practical or economical. Here, we describe successful scaled-up production of ESCs on microcarriers in a stirred culture system in a serum-free medium. Cells expanded on CultiSpher S, Cytodex 3, and Collagen microcarriers showed superior cell-fold expansions of 439, 193, and 68, respectively, without excessive agglomeration, compared with 27 in static culture. In addition, the ESCs maintained their pluripotency after long-term culture (28 days) in serum-free medium. This is the first time mESCs have been cultured on microcarriers without prior exposure to serum and/or fibroblasts, while also eliminating the excessive agglomeration plaguing earlier studies. These protocols provide an economical, practical, serum-free means for expanding ESCs in a stirred suspension bioprocess.     

BMP‐4 inhibits neural differentiation of murine embryonic stem cells

Members of the transforming growth factor‐β superfamily, including bone morphogenetic protein 4 (BMP‐4), have been implicated as regulators of neuronal and glial differentiation. To test for a possible role of BMP‐4 in early mammalian neural specification, we examined its effect on neurogenesis in aggregate cultures of mouse embryonic stem (ES) cells. Compared to control aggregates, in which up to 20% of the cells acquired immunoreactivity for the neuron‐specific antibody TuJ1, aggregates maintained for 8 days in serum‐free medium containing BMP‐4 generated 5‐ to 10‐fold fewer neurons. The action of BMP‐4 was dose dependent and restricted to the fifth through eighth day in suspension. In addition to the reduction in neurons, we observed that ES cell cultures exposed to BMP‐4 contained fewer cells that were immunoreactive for glial fibrillary acidic protein or the HNK‐1 neural antigen. Furthermore, under phase contrast, cultures prepared from BMP‐4–treated aggregates contained a significant proportion of nonneuronal cells with a characteristic flat, elongated morphology. These cells were immunoreactive for antibodies to the intermediate filament protein vimentin; they were rare or absent in control cultures. Treatment with BMP‐4 enhanced the expression of the early mesodermal genes brachyury and tbx6 but had relatively little effect on total cell number or cell death. Coapplication of the BMP‐4 antagonist noggin counteracted the effect of exogenous BMP‐4, but noggin alone had no effect on neuralization in either the absence or presence of retinoids. Collectively, our results suggest that BMP‐4 can overcome the neuralizing action of retinoic acid to enhance mesodermal differentiation of murine ES cells. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 271–287, 1999     

Influence of regulatory genes of type 1 human immunodeficiency virus on proliferation and differentiation of murine embryonic stem cells

Spontaneous formation of embryoid bodies and subsequent differentiation of some cells into cardiomyocytes were demonstrated on murine embryonic stem cells of R1 line. The lines of embryonic stem cells were obtained that had been transfected with genetic constructs carrying expressing regulatory genes of the human immunodeficiency virus tat and nef and "green protein" gene (GFP). The transfection of embryonic stem cells with the gene tat stimulated their proliferative activity, while this activity decreased in the cells transfected with the gene nef. The time necessary for the formation of embryoid bodies by all lines of transfected cells was similar to that in the control cells. In the cultures of cells transfected with nef and tat, the number of embryoid bodies and the percentage of embryoid bodies with contracting cardiomyocytes were higher and lower than in the control, respectively. Thus, an inverse correlation was observed between the effects of regulatory genes of the human immunodeficiency virus on proliferation and differentiation embryonic stem cells.     

The effect of extracellular matrix on the differentiation of mouse embryonic stem cells

Embryonic stem cells (ESCs) are promising research materials to investigate cell fate determination since they have the capability to differentiate. Stem cell differentiation has been extensively studied with various microenvironment mimicking structures to modify cellular dynamics associated with the cell-extracellular matrix (ECM) interactions and cell-cell communications. In the current study, our aim was to determine the effect of microenvironmental proteins with different concentrations on the capacity and differentiation capability of mouse ESCs (mESCs), combining the biochemical assays, imaging techniques, Fourier transform infrared (FTIR) spectroscopy, and unsupervised multivariate analysis. Based on our data, coating the surface of mESCs with Matrigel, used as an acellular matrix substrate, resulted in morphological and biochemical changes. mESCs exhibited alterations in their phenotype after growing on the Matrigel-coated surfaces, including their differentiation capacity, cell cycle phase pattern, membrane fluidity, and metabolic activities. In conclusion, mESCs can be stimulated physiologically, chemically, or mechanically to convert them a new phenotype. Thus, identification of ESCs’ behavior in the acellular microenvironment could be vital to elucidate the mechanism of diseases. It might also be promising to control the cell fate in the field of tissue engineering.     

Culture of fetal alveolar epithelial type II cells in serum-free medium

Summary A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle’s minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistance of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment.     

Alginate-PLL microencapsulation: effect on the differentiation of embryonic stem cells into hepatocytes

The emergence of hepatocyte based clinical and pharmaceutical technologies, has been limited by the absence of a stable hepatocyte cell source. Embryonic stem cells may represent a potential solution to this cell source limitation problem since they are highly proliferative, renewable, and pluripotent. Although many investigators have described techniques to effectively differentiate stem cells into a variety of mature cell lineages, their practicality is limited by: (1) low yields of fully differentiated cells, (2) absence of large scale processing considerations, and (3) ineffective downstream enrichment protocols. Thus, a differentiation platform that may be modified to induce and sustain differentiated cell function and scaled to increase differentiated cell yield would improve current stem cell differentiation strategies. Microencapsulation provides a vehicle for the discrete control of key cell culture parameters such as the diffusion of growth factors, metabolites, and wastes. In addition, both cell seeding density and bead composition may be manipulated. In order to assess the feasibility of directing stem cell differentiation via microenvironment regulation, we have developed a murine embryonic stem cell (ES) alginate poly-l-lysine microencapsulation hepatocyte differentiation system. Our results indicate that the alginate microenvironment maintains cell viability, is conducive to ES cell differentiation, and maintains differentiated cellular function. This system may ultimately assist in developing scalable stem cell differentiation strategies.     

胚胎干细胞向造血系统的分化

胚胎干细胞是指从囊胚期的内细胞团中分离出来的尚未分化的胚胎细胞,可分化形成各种组织类型。在合适的条件下,胚胎干细胞可发育成造血干细胞及各类成熟血细胞,为造血干细胞移植及血细胞输注开辟了新的来源,同时也为造血发生及造血调控研究提供了有效可靠的模型。本文将综述ES细胞向造血系统分化的诱导条件、调控机制及应用前景。