Showing papers on "Cellular differentiation published in 1994"

Acquisition of Lymphokine-Producing Phenotype by CD4+ T Cells

Abstract: Naive CD4+ T cells when stimulated produce IL-2 as their major lymphokine. Upon priming, these cells develop into cells that produce either IFN gamma, TNF beta, and IL-2 or IL-4 and its congeners. The former cells are designated TH1-like, and the latter TH2-like. Here we review the regulation of the differentiation of naive CD4 cells into IFN gamma- or IL-4-producers. The dominant factors that determine such differentiation are lymphokines and other cytokines. IL-2 itself appears to be required for naive cells to develop into TH1- or TH2-like cells but is not deterministic of their differentiation fate. If IL-4 is also present during the priming period, the resultant CD4+ T cells produce IL-4 upon restimulation; the development of IFN gamma-producing cells is strikingly inhibited by IL-4. In the absence of IL-4, priming for IFN gamma-production occurs, but this is markedly enhanced by IL-12. The role of IFN gamma in enhancing priming for IFN gamma-production is not fully resolved. In some in vitro systems, it appears to act together with IL-12 to enhance such production. Anti-IFN gamma diminishes priming for IFN gamma production in vivo. Lymphokines also exert a "cross-regulatory" or inhibitory effect. As noted above, IL-4 strikingly diminishes priming for IFN gamma production, although this inhibitory effect is blunted in the presence of IL-12. IFN gamma similarly diminishes priming for IL-4 production; this effect is principally observed when low concentrations of IL-4 are used in the priming culture. Although other factors may play a role in the determination of lymphokine-producing phenotype, such as antigen dose, type of antigen-presenting cell, and expression of accessory molecules and hormones, these effects appear to be secondary to the dominant role of the lymphokines and cytokines.

Spermatogenesis following male germ-cell transplantation

Abstract: In the adult male, a population of diploid stem-cell spermatogonia continuously undergoes self-renewal and produces progeny cells, which initiate the complex process of cellular differentiation that results in mature spermatozoa. We report here that stem cells isolated from testes of donor male mice will repopulate sterile testes when injected into seminiferous tubules. Donor cell spermatogenesis in recipient testes showed normal morpholigical characteristics and produced mature spermatozoa. This methodology, besides opening new avenues of basic research into spermatogenesis and stem-cell self-renewal, may prove useful as a tool for biomedical science and biotechnology.

Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.

Abstract: The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity--suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.

TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors.

Abstract: The secreted polypeptide transforming growth factor-beta (TGF-beta) exerts its multiple activities through type I and II cell surface receptors. In epithelial cells, activation of the TGF-beta signal transduction pathways leads to inhibition of cell proliferation and an increase in extracellular matrix production. TGF-beta is widely expressed during development and its biological activity has been implicated in epithelial-mesenchymal interactions, e.g., in branching morphogenesis of the lung, kidney, and mammary gland, and in inductive events between mammary epithelium and stroma. In the present study, we investigated the effects of TGF-beta on mouse mammary epithelial cells in vitro. TGF-beta reversibly induced an alteration in the differentiation of normal mammary epithelial NMuMG cells from epithelial to fibroblastic phenotype. The change in cell morphology correlated with (a) decreased expression of the epithelial markers E-cadherin, ZO-1, and desmoplakin I and II; (b) increased expression of mesenchymal markers, such as fibronectin; and (c) a fibroblast-like reorganization of actin fibers. This phenotypic differentiation displays the hallmarks of an epithelial to mesenchymal transdifferentiation event. Since NMuMG cells make high levels of the type I TGF-beta receptor Tsk7L, yet lack expression of the ALK-5/R4 type I receptor which has been reported to mediate TGF-beta responsiveness, we evaluated the role of the Tsk7L receptor in TGF-beta-mediated transdifferentiation. We generated NMuMG cells that stably overexpress a truncated Tsk7L type I receptor that lacks most of the cytoplasmic kinase domain, thus function as a dominant negative mutant. These transfected cells no longer underwent epithelial to mesenchymal morphological change upon exposure to TGF-beta, yet still displayed some TGF-beta-mediated responses. We conclude that TGF-beta has the ability to modulate E-cadherin expression and induce a reversible epithelial to mesenchymal transdifferentiation in epithelial cells. Unlike other transdifferentiating growth factors, such as bFGF and HGF, these changes are accompanied by growth inhibition. Our results also implicate the Tsk7L type I receptor as mediating the TGF-beta-induced epithelial to mesenchymal transition.

Generation of lymphohematopoietic cells from embryonic stem cells in culture

Abstract: An efficient system was developed that induced the differentiation of embryonic stem (ES) cells into blood cells of erythroid, myeloid, and B cell lineages by coculture with the stromal cell line OP9. This cell line does not express functional macrophage colony-stimulating factor (M-CSF). The presence of M-CSF had inhibitory effects on the differentiation of ES cells to blood cells other than macrophages. Embryoid body formation or addition of exogenous growth factors was not required, and differentiation was highly reproducible even after the selection of ES cells with the antibiotic G418. Combined with the ability to genetically manipulate ES cells, this system will facilitate the study of molecular mechanisms involved in development and differentiation of hematopoietic cells.

Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy.

Abstract: Bone formation in the embryo, and during adult fracture repair and remodeling, involves the progeny of a small number of cells called mesenchymal stem cells (MSCs). These cells continuously replicate themselves, while a portion become committed to mesenchymal cell lineages such as bone, cartilage, tendon, ligament, and muscle. The differentiation of these cells, within each lineage, is a complex multistep pathway involving discrete cellular transitions much like that which occurs during hematopoiesis. Progression from one stage to the next depends on the presence of specific bioactive factors, nutrients, and other environmental cues whose exquisitely controlled contributions orchestrate the entire differentiation phenomenon. An understanding of the cellular and molecular events of osteogenic differentiation of MSCs provides the foundation for the emergence of a new therapeutic technology for cell therapy. The isolation and in vitro mitotic expansion of autologous human MSCs will support the development of novel protocols for the treatment of many clinically challenging conditions. For example, local bone defects can be repaired through site-directed delivery of MSCs in an appropriate carrier vehicle. Generalized conditions, such as osteoporosis, may be treatable by systemic administration of culture-expanded autologous MSCs or through biopharmaceutical regimens based on the discovery of critical regulatory molecules in the differentiation process. With this in mind, we can begin to explore therapeutic options that have never before been available.

Cloning and characterization of the cDNA encoding a novel human pre-B- cell colony-enhancing factor

Abstract: A novel gene coding for the pre-B-cell colony-enhancing factor (PBEF) has been isolated from a human peripheral blood lymphocyte cDNA library. The expression of this gene is induced by pokeweed mitogen and superinduced by cycloheximide. It is also induced in the T-lymphoblastoid cell line HUT 78 after phorbol ester (phorbol myristate acetate) treatment. The predominant mRNA for PBEF is approximately 2.4 kb long and codes for a 52-kDa secreted protein. The 3' untranslated region of the mRNA has multiple TATT motifs, usually found in cytokine and oncogene messages. The PBEF gene is mainly transcribed in human bone marrow, liver tissue, and muscle. We have expressed PBEF in COS 7 and PA317 cells and have tested the biological activities of the conditioned medium as well as the antibody-purified protein in different in vitro assays. PBEF itself had no activity but synergized the pre-B-cell colony formation activity of stem cell factor and interleukin 7. In the presence of PBEF, the number of pre-B-cell colonies was increased by at least 70% above the amount stimulated by stem cell factor plus interleukin 7. No effect of PBEF was found with cells of myeloid or erythroid lineages. These data define PBEF as a novel cytokine which acts on early B-lineage precursor cells.

Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone

Abstract: Human bone marrow stromal cells were examined for their osteogenic potential in an in vitro cell culture system Dexamethasone (Dex) treatment induced morphological transformation of these cells from an elongated to a more cuboidal shape, increased their alkaline phosphatase activity and cAMP responses to PTH and prostaglandin E2, and was essential for mineralization of the extracellular matrix Dex-induced differentiation of human bone marrow stromal cells was apparent after 2-3 days of treatment and reached a maximum at 7-14 days, as judged by alkaline phosphatase activity, although induction of osteocalcin by 1,25-dihydroxyvitamin D3 was attenuated by Dex Withdrawal of Dex resulted in an enhancement of the 1,25-dihydroxyvitamin D3-induced secretion of osteocalcin, whereas alkaline phosphatase activity and the cAMP response to PTH remained at prewithdrawal levels The steady state mRNA level of osteonectin was not affected by Dex Our results, which demonstrate that Dex conditions the differentiation of human bone marrow osteogenic stromal cells into osteoblast-like cells, support the hypothesis of a permissive effect of glucocorticoids in ensuring an adequate supply of mature osteoblast populations Furthermore, the established human bone marrow stromal cell culture provides a good model of an in vitro system to study the regulation of differentiation of human bone osteoprogenitor cells

Toxicity of 5-aza-2'-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation

Abstract: The deoxycytidine analog 5-aza-2'-deoxycytidine (5-azadCyd) has been widely used as a DNA methylation inhibitor to experimentally induce gene expression and cellular differentiation. Prior to the availability of mutant mice with altered DNA methyltransferase levels, treatment of cells with drugs has been the only means to experimentally manipulate the level of genomic DNA methylation in mammalian cells. Substitution of DNA with 5-azadCyd leads to covalent trapping of the enzyme, thereby depleting the cells of enzyme activity and resulting in DNA demethylation. 5-AzadCyd or 5-azacytidine treatment causes multiple changes in treated cells, including activation of silent genes, decondensation of chromatin, and induction of cellular differentiation, all of which are believed to be consequences of drug-induced demethylation. 5-AzadCyd is highly toxic in cultured cells and animals and is utilized as a potent antitumor agent for treatment of certain human cancers. It has been postulated that the toxicity of the drug in mammalian cells is also due to its inhibition of DNA methylation. The chemistry of the methylation reaction is consistent, however, with an alternative mechanism: the cytotoxic effect of 5-azadCyd may be directly mediated through the covalent binding of DNA methyltransferase to 5-azadCyd-substituted DNA. We have tested this possibility by using embryonic stem cells and mice with reduced levels of DNA methyltransferase due to a targeted mutation of the gene. When exposed to 5-azadCyd mutant embryonic stem cells or embryos were significantly more resistant to the toxic effects of the drug than wild-type cells and embryos, respectively. These results strongly suggest that the cellular DNA methyltransferase itself, rather than the secondary demethylation of genomic DNA, is the primary mediator of 5-azadCyd cytotoxicity. In light of our results, some conclusions from previous studies using 5-azadCyd in order to experimentally manipulate cellular methylation levels may have to be reassessed. Also, our data make clear predictions for cancer treatment: tumor cells with elevated DNA methyltransferase levels would be expected to be susceptible to treatment with 5-azadCyd, whereas tumors with reduced levels of the enzyme would be resistant.

bHLH factors in muscle development: dead lines and commitments, what to leave in and what to leave out.

Abstract: In recent years, skeletal muscle has become an important model for understanding the mechanisms that regulate tissue-specific gene expression. The formation of skeletal muscle during embryogenesis involves commitment of mesodermal progenitors to the myogenic lineage and subsequent differentiation of skeletal myoblasts into terminally differentiated myotubes. Like many cell types, skeletal myoblasts do not express markers of terminal differentiation until they are forced to exit the cell cycle in response to environmental cues. Growth factor signals play a central role in regulating the program for muscle-specific transcription by maintaining myoblasts in a proliferative state that is nonpermissive for the expression of muscle-specific genes. Analysis of the mechanisms that regulate muscle differentiation in tissue culture led to the discovery of the four skeletal muscle-specific regulatory factors, MyoD (Davis et al. 1987), myogenin (Edmondson and Olson 1989; Wright et al. 1989), Myf5 (Braun et al. 1989a), and MRF4 (Rhodes and Konieczny 1989; Miner and Wold 1990; Braun et al. 1990), each of which can activate skeletal muscle gene transcription when expressed ectopically in a variety of nonmuscle cell types. Although many mammalian cell type-specific transcription factors have been identified, the myogenic factors are unique in their abilities to orchestrate an entire program of tissuespecific transcription when introduced into diverse cell types. This activity led to the notion that these factors function as master regulators of muscle cell fate during development. However, recent studies in which these genes have been inactivated through homologous recombination in transgenic mice have resulted in surprising phenotypes (or lack thereof) and have necessitated a reevaluation of the potential roles of these factors in the control of determination and differentiation in the myogenic lineage. Here, we review the models that emerged from studies of the myogenic factors in tissue culture and reconsider the potential functions of these factors in the embryo in light of recent gene-targeting experiments. For more comprehensive reviews on the control of muscle gene expression, the reader is referred to several recent reviews (Olson 1990, 1993; Weintraub et al. 1991; Buckingham 1992; Sassoon 1992; Emerson 1993; Wright 1992).

p53 -dependent apoptosis produced by Rb -deficiency in the developing mouse lens

Abstract: The retinoblastoma tumour-suppressor gene (RB) has been implicated in negative growth regulation, induction of differentiation, and inhibition of cellular transformation. Homozygous inactivation of the Rb gene in the mouse leads to mid-gestational lethality with defects in erythropoiesis and neurogenesis. Here we describe the effects of the Rb-deficient state on the development of the ocular lens. The regional compartmentalization of growth, differentiation and apoptosis in the developing lens provides an ideal system to examine more closely the relationships of these processes in vivo. We demonstrate that loss of Rb function is associated with unchecked proliferation, impaired expression of differentiation markers, and inappropriate apoptosis in lens fibre cells. In addition, we show that ectopic apoptosis in Rb-deficient lenses is dependent on p53, because embryos doubly null for Rb and p53 show a nearly complete suppression of this effect. This developmental system provides a framework for understanding the consequences of the frequent mutation of both RB and p53 in human cancer.

A self-renewing multipotential stem cell in embryonic rat cerebral cortex

Abstract: Neuroectoderm cells in the cortical ventricular zone generate many diverse cell types, maintain the ventricular zone during embryonic life and create another germinal layer, the subventricular zone, which persists into adulthood. In other vertebrate tissues, including skin, intestine, blood and neural crest, stem cells are important in maintaining a germinal population and generating differentiated progeny. By following the fates of single ventricular zone cells in culture, we show here that self-renewing, multipotential stem cells are present in the embryonic rat cerebral cortex. Forty per cent of these stem cells produced all three principal cell types of the central nervous system: neurons, astrocytes and oligodendrocytes. Stem cells constituted about 7% of cortical clones; in contrast, over 80% consisted of small numbers of neurons or glia. We suggest that multipotential stem cells may be the ancestors of other cortical progenitor cells that exhibit more limited proliferation and more restricted repertoires of progeny fates.

Localization of proliferating cell nuclear antigen, vimentin, c-Fos, and clusterin in the postischemic kidney. Evidence for a heterogenous genetic response among nephron segments, and a large pool of mitotically active and dedifferentiated cells.

Abstract: The mechanisms leading to the recovery of the kidney after ischemic acute renal failure are poorly understood. To explore the role played by mitogenesis and dedifferentiation in this repair process and to identify whether the genetic response of the nephron segments reflects the level of susceptibility to injury, the temporal and nephron segment expressions of various proteins implicated in mitogenesis, differentiation, and injury were determined. Proliferating cell nuclear antigen (PCNA), a marker for the G1-S transition in the cell cycle and hence mitogenesis, was detected primarily in the S3 segment of the proximal tubule, with maximal expression at 2 d postischemia. Vimentin, normally present in mesenchymal cells but not epithelial cells, and hence a marker for the state of differentiation, was prominently expressed in the S3 segment 2-5 d postischemia. In the S3 segments in the outer stripe of the medulla cells that stained positively for PCNA also stained positively for vimentin. Clusterin, a marker for cell injury, was expressed primarily in the S3 segment and in the distal tubule with distinct staining patterns in each segment. None of the cells that stained with clusterin antibodies were positively stained with PCNA or vimentin antibodies. Likewise, none of the PCNA or vimentin-positive cells expressed clusterin at detectable levels. Thus, in the S3 segment, where there is significant ischemic injury, surviving cells express markers indicating that they undergo mitogenesis and dedifferentiate in the postischemic period. While there is some expression of c-Fos in the S3 segment, c-Fos was expressed predominantly, at 1 and 3 h postischemia, in the nuclei of the distal nephron, particularly in the thick ascending limb. The data support the view that the mature renal S3 segment epithelial cell can be a progenitor cell.

Skeletal muscle satellite cells

Abstract: Evidence now suggests that satellite cells constitute a class of myogenic cells that differ distinctly from other embryonic myoblasts. Satellite cells arise from somites and first appear as a distinct myoblast type well before birth. Satellite cells from different muscles cannot be functionally distinguished from one another and are able to provide nuclei to all fibers without regard to phenotype. Thus, it is difficult to ascribe any significant function to establishing or stabilizing fiber type, even during regeneration. Within a muscle, satellite cells exhibit marked heterogeneity with respect to their proliferative behavior. The satellite cell population on a fiber can be partitioned into those that function as stem cells and those which are readily available for fusion. Recent studies have shown that the cells are not simply spindle shaped, but are very diverse in their morphology and have multiple branches emanating from the poles of the cells. This finding is consistent with other studies indicating that the cells have the capacity for extensive migration within, and perhaps between, muscles. Complexity of cell shape usually reflects increased cytoplasmic volume and organelles including a well developed Golgi, and is usually associated with growing postnatal muscle or muscles undergoing some form of induced adaptive change or repair. The appearance of activated satellite cells suggests some function of the cells in the adaptive process through elaboration and secretion of a product. Significant advances have been made in determining the potential secretion products that satellite cells make. The manner in which satellite cell proliferative and fusion behavior is controlled has also been studied. There seems to be little doubt that cellcell coupling is not how satellite cells and myofibers communicate. Rather satellite cell regulation is through a number of potential growth factors that arise from a number of sources. Critical to the understanding of this form of control is to determine which of the many growth factors that can alter satellite cell behavior in vitro are at work in vivo. Little work has been done to determine what controls are at work after a regeneration response has been initiated. It seems likely that, after injury, growth factors are liberated through proteolytic activity and initiate an activation process whereby cells enter into a proliferative phase. After myofibers are formed, it also seems likely that satellite cell behavior is regulated through diffusible factors arising from the fibers rather than continuous control by circulating factors.(ABSTRACT TRUNCATED AT 400 WORDS).

Novel insights into erythroid development revealed through in vitro differentiation of GATA-1 embryonic stem cells.

Abstract: Mouse embryonic stem (ES) cells lacking the transcription factor GATA-1 do not produce mature red blood cells either in vivo or in vitro. To define the consequences of GATA-1 loss more precisely, we used an in vitro ES cell differentiation assay that permits enumeration of primitive (EryP) and definitive (EryD) erythroid precursors and recovery of pure erythroid colonies. In contrast to normal ES cells, GATA-1- ES cells fail to generate EryP precursors. EryD precursors, however, are normal in number but undergo developmental arrest and death at the proerythroblast stage. Contrary to initial expectations, arrested GATA-1(-)-definitive proerythroblasts express GATA target genes at normal levels. Transcripts of the related factor GATA-2 are remarkably elevated in GATA-1- proerythroblasts. These findings imply substantial interchangeability of GATA factors in vivo and suggest that GATA-1 normally serves to repress GATA-2 during erythropoiesis. The approach used here is a paradigm for the phenotypic analysis of targeted mutations affecting hematopoietic development.

Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21, WAF1/CIP1, expression in the absence of p53.

Abstract: The melanoma differentiation associated gene, mda-6, which is identical to the P53-inducible gene WAF1/CIP1, encodes an M(r) 21,000 protein (p21) that can directly inhibit cell growth by repressing cyclin dependent kinases. mda-6 was identified using subtraction hybridization by virtue of its enhanced expression in human melanoma cells induced to terminally differentiate by treatment with human fibroblast interferon and the anti-leukemic compound mezerein (Jiang and Fisher, 1993). In the present study, we demonstrate that mda-6 (WAF1/CIP1) is an immediate early response gene induced during differentiation of the promyelocytic HL-60 leukemia cell line along the granulocytic or macrophage/monocyte pathway. mda-6 gene expression in HL-60 cells is induced within 1 to 3 h during differentiation along the macrophage/monocyte pathway evoked by 12-0-tetradecanoyl phorbol-13-acetate (TPA) or 1,25-dihydroxyvitamin D3 (Vit D3) or the granulocytic pathway produced by retinoic acid (RA) or dimethylsulfoxide (DMSO). Immunoprecipitation analyses using an anti-p21 antibody indicate a temporal induction of p21 protein following treatment with TPA, DMSO or RA. A relationship between rapid induction of mda-6 gene expression and differentiation is indicated by a delay in this expression in an HL-60 cell variant resistant to TPA-induced growth arrest and differentiation. A similar delay in mda-6 gene expression is not observed in Vit D3 treated TPA-resistant variant cells that are also sensitive to induction of monocytic differentiation. Since HL-60 cells have a null-p53 phenotype, these results demonstrate that p21 induction occurs during initiation of terminal differentiation in a p53-independent manner. In this context, p21 may play a more global role in growth control and differentiation than originally envisioned.

Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents.

Abstract: Cardiomyocytes differentiated in vitro from pluripotent embryonic stem (ES) cells of line D3 via embryo-like aggregates (embryoid bodies) were characterized by the whole-cell patch-clamp technique during the entire differentiation period. Spontaneously contracting cardiomyocytes were enzymatically isolated by collagenase from embryoid body outgrowths of early, intermediate, and terminal differentiation stages. The early differentiated cardiomyocytes exhibited an outwardly rectifying, transient K+ current sensitive to 4-aminopyridine and an inward Ca2+ current but no Na+ current. The Ca2+ current showed all features of L-type Ca2+ current, being highly sensitive to 1,4-dihydropyridines but not to omega-conotoxin. Cardiomyocytes of intermediate stage were characterized by the additional expression of cardiac-specific Na+ current, the delayed K+ current, and If current. Terminally differentiated cardiomyocytes expressed a Ca2+ channel density about three times higher than that of early stage. In addition, two types of inwardly rectifying K+ currents (IK1 and IK,Ach) and the ATP-modulated K+ current were found. During cardiomyocyte differentiation, several distinct cell populations could be distinguished by their sets of ionic channels and typical action potentials presumably representing cardiac tissues with properties of sinus node, atrium, and ventricle. Reverse transcription polymerase chain reaction revealed the transcription of alpha- and beta-cardiac myosin heavy chain (MHC) genes synchronously with the first spontaneous contractions. Transcription of embryonic skeletal MHC gene at intermediate and terminal differentiation stages correlated with the expression of Na+ channels. The selective expression of alpha-cardiac MHC gene in ES cell-derived cardiomyocytes was demonstrated after ES cell transfection of the LacZ construct driven by the alpha-cardiac MHC promoter region followed by ES cell differentiation and beta-galactosidase staining. In conclusion, our data demonstrate that ES cell-derived cardiomyocytes represent a unique model to investigate the early cardiac development and permit pharmacological/toxicological studies in vitro.

Induction of p21 (WAF-1/CIP1) during differentiation.

Abstract: The recently cloned protein, p21 (WAF1/CIP1) is a downstream effector of p53, and mediates growth arrest by inhibiting the action of G1 cyclin-dependent kinases. Since cellular differentiation is frequently characterized by G1 arrest, we examined whether p21 upregulation occurs in differentiation. We show that p21 expression is triggered by multiple differentiation-inducing agents in hematopoietic and hepatoma cells through a p53-independent pathway. The dramatic rise in p21 levels occurs as an immediate early response to differentiation inducers. The induction of p21 is coupled to the expression of early differentiation markers, and is uncoupled from apoptosis. Finally, evidence is presented that p21 expression is uncoupled from G1 arrest in the presence of deregulated c-myc.

The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth.

Abstract: A remarkable overlap was observed between the gadd genes, a group of often coordinately expressed genes that are induced by genotoxic stress and certain other growth arrest signals, and the MyD genes, a set of myeloid differentiation primary response genes. The MyD116 gene was found to be the murine homolog of the hamster gadd34 gene, whereas MyD118 and gadd45 were found to represent two separate but closely related genes. Furthermore, gadd34/MyD116, gadd45, MyD118, and gadd153 encode acidic proteins with very similar and unusual charge characteristics; both this property and a similar pattern of induction are shared with mdm2, whic, like gadd45, has been shown previously to be regulated by the tumor suppressor p53. Expression analysis revealed that they are distinguished from other growth arrest genes in that they are DNA damage inducible and suggest a role for these genes in growth arrest and apoptosis either coupled with or uncoupled from terminal differentiation. Evidence is also presented for coordinate induction in vivo by stress. The use of a short-term transfection assay, in which expression vectors for one or a combination of these gadd/MyD genes were transfected with a selectable marker into several different human tumor cell lines, provided direct evidence for the growth-inhibitory functions of the products of these genes and their ability to synergistically suppress growth. Taken together, these observations indicate that these genes define a novel class of mammalian genes encoding acidic proteins involved in the control of cellular growth.

Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction.

Abstract: Extracellular matrix (ECM) profoundly influences the growth and differentiation of the mammary gland epithelium, both in culture and in vivo. Utilizing a clonal population of mouse mammary epithelial cells that absolutely requires an exogenous ECM for function, we developed a rapid assay to study signal transduction by ECM. Two components of the cellular response to a basement membrane overlay that result in the expression of the milk protein beta-casein were defined. The first component of this response involves a rounding and clustering of the cells that can be physically mimicked by plating the cells on a nonadhesive substratum. The second component is biochemical in nature, and it is associated with beta 1 integrin clustering and increased tyrosine phosphorylation. The second component is initiated in a morphology-independent manner, but the proper translation of this biochemical signal into a functional response requires cell rounding and cell clustering. Thus, physical and biochemical signal transduction events contribute to the ECM-dependent regulation of tissue-specific gene expression in mouse mammary epithelial cells.