N-cadherin is essential for retinoic acid-mediated cardiomyogenic differentiation in mouse embryonic stem cells

Contraction forces developed by cardiomyocytes are transmitted across the plasma membrane through end-to-end connections between the myocytes, called intercalated disks, which enable the coordinated contraction of heart muscle. A component of the intercalated disk, the adherens junction, consists of the cell adhesion molecule, N-cadherin. Embryos lacking N-cadherin die at mid-gestation from cardiovascular abnormalities. We have evaluated the role of N-cadherin in cardiomyogenesis using N-cadherin-null mouse embryonic stem (ES) cells grown as embryoid bodies (EBs) in vitro. Myofibrillogenesis, the spatial orientation of myofibers, and intercellular contacts including desmosomes were normal in N-cadherin-null ES cell-derived cardiomyocytes. The effect of retinoic acid (RA), a stage and dose-dependent cardiogenic factor, was assessed in differentiating ES cells. all-trans (at) RA increased the number of ES cell-derived cardiomyocytes by approximately 3-fold (at 3 x 10(-9) M) in wt EBs. However, this effect was lost in N-cadherin-null EBs. In the presence of supplemented at-RA, the emergence of spontaneously beating cardiomyocytes appeared to be delayed and slightly less efficient in N-cadherin-null compared with wt and heterozygous EBs (frequencies of EBs with beating activity at 5 days: 54+/-18% vs. 96+/-0.5%, and 93+/-7%, respectively; peak frequencies of EBs with beating activity: 83+/-8% vs. 96+/-0.5% and 100%, respectively). In conclusion, cardiomyoyctes differentiating from N-cadherin-null ES cells in vitro show normal myofibrillogenesis and intercellular contacts, but impaired responses to early cardiogenic effects mediated by at-RA. These results suggest that N-cadherin may be essential for RA-induced cardiomyogenesis in mouse ES cells in vitro.     

Factors influencing the differentiation of embryonal carcinoma and embryo-derived stem cells

The effects of aggregation, retinoic acid, and medium conditioned by Buffalo rat liver (BRL) cells, alone and in combination, on the differentiation of PSA4TG12 embryonal carcinoma and E14 embryonal stem cells are reported. The observations indicate that BRL-conditioned medium has more than one effect on the differentiation process, that retinoic acid has at least two effects which operate in different concentration ranges, and that both agents influence the choice of differentiation pathway as well as the extent of differentiation.     

Cell-cell interaction can influence drug-induced differentiation of murine embryonal carcinoma cells

When cultured in the presence of either retinoic acid (RA) or dimethyl sulfoxide (DMSO), aggregates of the P19 line of mouse embryonal carcinoma (EC) cells differentiate and the spectrum of cell types formed depends on the drug dose. It is shown here the EC cells rapidly lose their colony-forming ability when cultured as aggregates in the presence of DMSO. This loss of plating efficiency (PE) also occurs rapidly following RA treatment. Loss of PE has been used as a quantitative procedure for assessing the rate of drug-induced differentiation. The relationship between drug dose and loss of PE is much steeper for DMSO than for RA, suggesting that these two drugs affect different stages of the differentiation decision-making apparatus. Mutant EC cell lines (D3 and RAC65) do not differentiate in the presence of drug-inducers (DMSO and RA, respectively). Neither differentiation-deficient mutant has an altered ability to form gap junctions. When D3 and P19 cells were mixed within the same DMSO-treated aggregates, the D3 cells remained undifferentiated and the P19 cells differentiated much less efficiently than if they were cultured in the absence of the D3 cells. When RAC65 and P19 cells were mixed in RA-treated aggregates, each cell responded to the drug as though the other were absent. Thus RA behaves as a cell-autonomous inducer of differentiation, whereas DMSO-induced differentiation seems to be mediated by interactions between neighboring cells.     

Sodium butyrate induces histone hyperacetylation and differentiation of murine embryonal carcinoma cells

Cells from embryonal carcinoma (EC) lines 6050AJ and PCC4.aza 1R differentiate in response to treatment with sodium butyrate as well as retinoic acid (RA) or hexamethylenebisacetamide (HMBA). Murine 6050AJ EC cells exposed to sodium butyrate possess hyperacetylated forms of histones H4 and altered forms of histones H2a and H2b, whereas histones from cells treated with other inducers appear to be unaffected. These results might indicate that the mechanism by which sodium butyrate promotes differentiation of EC cells is different from the ways in which RA and HMBA act. Differentiation-defective PCC4(RA)-1 EC cells fail to respond to RA, presumably because they possess minimal amounts of active binding protein for RA (cRABP). Sodium butyrate treatment of these cells results in only a modest level of differentiation. On the other hand, exposure to sodium butyrate plus RA leads to extensive differentiation. As is the case with 6050AJ cells, PCC4(RA)-1 cells treated with sodium butyrate also contain hyperacetylated histones. Furthermore, these cells now possess high levels of cRABP. The latter observations suggest that sodium butyrate has the ability to reactivate a silent cRABP gene in PCC4(RA)-1 cells and thereby lead to extensive differentiation via the retinoid pathway when RA is added.     

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.     

Phosphatidylinositol transfer protein in murine embryonal carcinoma cells during retinoic acid-induced differentiation

Phosphatidylinositol transfer protein (PI-TP) was studied in P19 embryonal carcinoma (EC) cells at different stages of retinoic acid (RA) induced differentiation. Western blot analysis indicated an increased expression of PI-TP (35 kDa) during differentiation. Western blots of isoelectric focusing gels showed that the 35 kDa band consisted of the PI-carrying form of PI-TP (pl 5.5) and of a novel, more acidic form of PI-TP (pl 5.4), levels of both of which increased during differentiation. These increased levels were not reflected in the in vitro PI-transfer activity of the cytosolic fraction nor in the mRNA levels as analyzed by northern blotting. By using indirect immunofluorescence it was shown that PI-TP is localized in the cytoplasm and associated with perinuclear Golgi structures and that this distribution is slightly affected during RA-induced differentiation. Immunoprecipitation of PI-TP from [³²P]P_i labeled cells demonstrated that the level of phosphorylation of PI-TP is high in undifferentiated P19 EC cells and low after 5 days of RA-induced differentiation. These results strongly suggest that changes in the levels of PI-TP are intimately connected with changes in the growth characteristics of P19 EC cells during RA-induced differentiation. It remains to be established to what extent this connection is governed by the recent finding that PI-TP is an essential cytosolic factor in stimulating phospholipase C activity.     

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.     

Mitochondrial metabolism directs stemness and differentiation in P19 embryonal carcinoma stem cells

The relationship between mitochondrial metabolism and cell viability and differentiation in stem cells (SCs) remains poorly understood. In the present study, we compared mitochondrial physiology and metabolism between P19SCs before/after differentiation and present a unique fingerprint of the association between mitochondrial activity, cell differentiation and stemness. In comparison with their differentiated counterparts, pluripotency of P19SCs was correlated with a strong glycolytic profile and decreased mitochondrial biogenesis and complexity: round, low-polarized and inactive mitochondria with a closed permeability transition pore. This decreased mitochondrial capacity increased their resistance against dichloroacetate. Thus, stimulation of mitochondrial function by growing P19SCs in glutamine/pyruvate-containing medium reduced their glycolytic phenotype, induced loss of pluripotent potential, compromised differentiation and became P19SCs sensitive to dichloroacetate. Because of the central role of this type of SCs in teratocarcinoma development, our findings highlight the importance of mitochondrial metabolism in stemness, proliferation, differentiation and chemoresistance. In addition, the present work suggests the regulation of mitochondrial metabolism as a tool for inducing cell differentiation in stem line therapies.Embryonal carcinoma cells, including the P19 cell line, are pluripotent cancer stem cells (CSCs) derived from pluripotent germ cell tumors called teratocarcinomas. These have been described as the malignant counterparts of embryonic stem cells (ESCs) and are considered a good model to study stem cell (SC) differentiation. The P19 cell line can be maintained as undifferentiated cells (P19SCs) or differentiated (P19dCs) to any cell type of the three germ layers. Similar to ESCs, P19 cells differentiate with retinoic acid (RA) in a dose-dependent manner and depending on growth conditions.¹ Although differentiation generally yields a mixed population of differentiated cells, P19 cells grown in monolayer and treated with 1 μM RA primarily differentiate in endoderm or mesoderm, while retaining their immortality.^{2, 3}Although some therapeutic approaches for regenerative medicine and to targeting CSCs are based on differentiation⁴ and mitochondrial-targeted therapies,^{5, 6} very little is known about the role of mitochondrial metabolism in SC maintenance and differentiation.⁷ Several mitochondrial characteristics that distinguish transformed cells from healthy cells have been described,⁸ including increased mitochondrial transmembrane electric potential (Δψm), which may result from decreased mitochondrial ATP production under normoxia.⁹ Similarly, normal SCs primarily rely on glycolysis for energy supply, although the exact mechanism how this occurs in the presence of oxygen and the relationship between SC metabolism and cell fate control is not yet completely understood.¹⁰Given the mitochondrial involvement in stemness and differentiation,¹¹ one can ask whether manipulation of mitochondrial physiology results in an improvement of therapy efficacy. Therefore, characterizing the metabolic and mitochondrial profiles of both SCs and differentiated cells holds promise in order to explain the resistance of cancer cells expressing an embryonic signature to mitochondrial-targeted therapies. In the present work, we have two tandem hypotheses: (a) metabolic and mitochondrial remodeling accompanies P19SC differentiation and (b) P19SC differentiation results in a higher susceptibility to mitochondrial-directed therapies.     

The in vivo differentiation of murine neuroblastoma

C1300 neuroblastoma was implanted with regenerating skeletal muscle to study the role of tissue interactions during tumor cell differentiation. Combined tumor-muscle implants, placed subcutaneously or within diffusion chambers were compared with control tumors implanted without muscle. Neuroblastoma implanted with injured muscle undergoes a partial neuronal differentiation. The tumor cells lose their normal round cell configuration and develop numerous cytoplasmic processes. Accompanying these outward changes are an increased content of microtubules in the neuritic processes; the appearance of glial-like processes containing abundant microfilaments; and the occurrence of growth vesicles identical to those of the growth cones of normal neurites. Although the implants usually contain large numbers of regenerated myofibers, tumor cell differentiation is not dependent upon the presence of these newly formed fibers. Tumor differentiation occurs equally well on the surfaces of degenerating muscle fragments, fibrin deposits and on the membrane surfaces of the diffusion chambers. These observations suggest that non-specific cell surface phenomena, rather than neuromuscular interactions were primarily responsible for the tumor cell differentiation in vivo.     

Cloning of murine gelsolin and its regulation during differentiation of embryonal carcinoma cells

The regulation of gelsolin levels during differentiation of the murine embryonal carcinoma cell line, PC-13, was investigated using nucleic acid and immunological probes. A cDNA clone, Mu-319, which contained the entire coding sequence for the cytoplasmic form of murine gelsolin was isolated using a polyclonal antibody. Gelsolin was detected in several cell lines but was not detectable in three undifferentiated embryonal carcinoma cell lines. Levels of gelsolin mRNA increased 10-fold during the differentiation of the murine embryonal carcinoma cell line, PC-13. Differentiation of PC-13 was accompanied by changes in cell shape, from small indistinct cells to large flat cells. The accumulation of gelsolin mRNA in PC-13 cells began 12-24 h after addition of the differentiation-inducing agents. In comparison, 2-5A-dependent RNase activity showed a 40-fold increase beginning after 24 to 36 h and c-fos mRNA were shown to increase about 9-fold beginning 36 to 60 h after induction of differentiation. The levels of gelsolin per se, as determined by immunoreactivity were also shown to increase with differentiation of PC-13 cells. These results suggest that gelsolin may play a role in the restructuring of actin filaments which accompanies the dramatic changes in cell shape during differentiation.     

Visceral-endoderm-like cell lines induce differentiation of murine P19 embryonal carcinoma cells

When P19 embryonal carcinoma (EC) cells were cocultured with cells from one of several established visceral-endoderm-like cell lines, the EC cells were rapidly induced to aggregate and differentiate, into cell types including mesoderm-derived cardiac and skeletal muscle. Neither parietal-endoderm- nor mesoderm-like cell lines induced aggregation or differentiation of EC cells in coculture, although a cell line with both parietal and visceral endoderm characteristics induced aggregation but not differentiation. Also, without the feeder cells aggregates of P19 failed to differentiate, provided that serum in the culture medium had been previously passed over dextran-coated charcoal to remove lipophilic substances, which may include endogenous retinoids. All experiments were carried out using serum treated in this way. Taken together, the results demonstrated that aggregation was necessary, but not sufficient, to make P19 EC cells differentiate. Direct contact between the two cell types was not necessary, since even when separated by an agar layer in cocultures, aggregates of P19 still differentiated. Medium conditioned by cells of the END-2 line, a visceral-endoderm-like derivative of P19, was particularly potent in inducing endodermal and mesodermal differentiation of single P19 aggregates, confirming the involvement of a diffusible factor secreted specifically by visceral-endoderm-like cells in this process.     

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.     

Cytoplasmic and nuclear protein synthesis during in vitro differentiation of murine ICM and embryonal carcinoma cells

Protein syntheses during in vitro differentiation of inner cell masses (ICM) isolated from mouse blastocysts and of pluripotent embryonal carcinoma cells (ECC) were compared by two-dimensional electrophoretic analysis of [³⁵S]methionine-labeled cells. While most of the polypeptides found in ICM, ECC, and embryoid bodies (EB) derived from them were common to all four preparations, some distinct differences were noted. More polypeptides changed in intensity during the differentiation of ICM than during the differentiation of ECC. Analysis of ECC prior to differentiation revealed that only some of the polypeptides abundant in ICM were present, while at the same time, some of the polypeptides abundant in ICM-EB were being synthesized. These data indicate that ECC represent cells further advanced in development than the cells of ICM isolated from 4-day-old blastocysts. The EB derived from ECC also differ from those from ICM. Comparison of EB derived from ICM and ECC with cells of the parietal yolk sac line, PYS, indicates that all three synthesize two polypeptides abundant in EB. These two polypeptides can, therefore, be used as biochemical markers of parietal entoderm differentiation. Pluripotent ECC synthesize small amounts of characteristic EB proteins and the 10-nm filament protein (also found in PYS cells but not in EB). This indicates that small numbers of differentiated or differentiating cells are present in pluripotent ECC cultures.     

Adipose-derived stem cells modified genetically in vivo promote reconstruction of bone defects

AimsBone defects induced by different causes are difficult to replace and repair. We sought to repair bone defects by transplantation of genetically modified adipose-derived stem cells (ADSC) and acellular bone matrix (ACBM).MethodsWe constructed the biologic material of ACBM and evaluated its mechanical properties, general biocompatibility and biosafety. ADSC isolated from minipigs were cultured in vitro and then transfected by recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human vascular endothelial growth factor (rhVEGF) plasmids, respectively. Subsequently, the compounds of ACBM/ADSC/rhBMP-2/rhVEGF were used to repair bone defects of the ulna in minipigs. X-ray examination, radionuclide bone imaging and single photon emission computerized tomography (SPECT) were employed to monitor the therapeutic effects 2, 4, 8 and 12 weeks after operation. Histologic experiments were carried out 12 weeks after operation.ResultsACBM had no or weak antigenicity and the natural mechanical properties of ACBM were preserved. In vitro, ADSC transfected by rhBMP-2 and rhVEGF, respectively, could release rhBMP-2 or rhVEGF for at least 4 weeks. The X-ray, radionuclide bone imaging and SPECT examinations indicated that the compound of ACBM/ADSC/rhBMP-2/rhVEGF had better treatment effects on bone defects compared with the controls.ConclusionsScaffolds, seed cells and bioactive factors are key points in tissue engineering. This research indicates that ACBM is a good biologic material for tissue repair, and ACBM/ADSC/rhBMP-2/rhVEGF can accelerate bone formation significantly.