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immortalized neuronal cell lines has become feasible. condition indefinitely in vitro, and thus generate
Since then, several other such cell lines have become immortal models that are clonable, easy to access and
available, including an example derived from human manipulate, and that provide unlimited amounts of
tissue, such as the NT2 cell line (Andrews et al., 1984; cells. In this regard, the first attempts to immortalize
Borlongan et al., 1998; Flax et al., 1998). This cell line trisomy 16 mouse tissue using oncogene transfection
is derived from a lung metastasis of a 22-year-old methods were unsuccessful (Frederiksen et al., 1996;
Caucasian male with a testicular germ cell tumor. Kim & Hammond, 1995). These were due fundamen-
This line, when cultured in retinoic acid, and then tally to viability problems or poor stability of neuronal
exposed to mitotic inhibitors for 2 weeks, yields a traits after transformation. As previously described,
typical culture that contains approximately 1020% our laboratory has used an original procedure
neurons, commonly referred to as hNT neurons (Caviedes et al., 1993), which has resulted in a
(Andrews, 1984). successfully established neuronal cell line in perma-
Nevertheless, the problem of stability, variability nent culture from diverse origins of the CNS of
and quantity of cells, in vitro senescence along with normal and trisomic mice. These cultured lines have
de-differentiation, and subsequent loss of function are been demonstrated to retain neuronal traits and be
still a major issue. This is particularly pertinent to stable after eight years (Caviedes et al., 1993; Liberona
conditions of a clear genetic origin. One clear example et al., 1997). These efforts were the first to show
is the trisomy 16 mouse, an animal model of human establishment of continuously growing cell lines
trisomy 21 (Downs syndrome) (Cox & Epstein, 1985; expressing stable neuronal phenotypes and lacking
Cox et al., 1980, 1981). Because this condition glial features. Our experience with these and other cell
presents as non-viable in the mouse, and considering lines comparably demonstrates that, with this proce-
the inherent limitations of primary culture techniques, dure, the lines generated retain differentiated traits,
the establishment of immortalized cell lines appears as some for decades (Allen et al., 2000; Cardenas et al.,
an excellent alternative to preserve the trisomic 1999; Caviedes et al., 1993; Liberona et al., 1997). In
TABLE 1 Brief Description of Some Currently Available Cell Lines from Myocardial Skeletal Muscle and Neuronal Origin
H9c2 Subclone of BDIX Spontaneous Skeletal muscle properties, Hescheler et al., 1991;
embryonic rat fusion, response to Kimes and Brandt, 1976;
heart cell line acetylcholine stimulation. Sipido and Marban, 1991
MHEC5 Mouse heart Spontaneous, Endothelial (hemangioendothelioma), Plendl et al., 1995
malignant tumorigenic. Expresses vascular cell
adhesion molecule-1
CMG Mouse bone Spontaneous Forms myotube-like structures, Makino et al., 1999
marrow stroma sarcomeres, beating after two weeks.
Expression of atrial natriuretic peptide,
brain natriuretic peptide, myosin,
desmin and actinin.
RCVC Adult rat UCHT1 protocol Muscle markers (myoglobin, Caviedes et al., 1993
heart muscle a-sarcomeric actin, a-actinin and desmin),
mature Ca2 + channels.
C2C12 Mouse, skeletal Spontaneous, Fusion into terminal mature, Yaffe, 1968;
muscle after serial passaging contractile myotubes. Expression of Yaffe and Saxel, 1977a, 1977b
ryanodine receptors.
RCMH Adult human, UCHT1 protocol Expression of muscle specific markers, Caviedes et al., 1992;
skeletal muscle functional muscle-specific ionic channels, Liberona et al., 1997, 1998
and IP3 receptors. Fusion, forming
multinucleated structures.
Neuro Mouse brain Serial passage Morphology of mature neurons, Augusti-Tocco and Sato, 1969
blastoma neuroblastoma expression of tyrosine hydroxylase.
(several) C1300
NT2 (Ntera2) Human, testicular Serial passage When cultured in retinoic acid, Andrews, 1984; Andrews et al., 1984;
teratocarcinoma they form hNT postmitotic neurons. Borlongan et al., 1998; Flax et al., 1998
Human cell transplant studies have
been conducted.
CNh, CTb Normal (CNh) and UCHT1 protocol Neuronal markers, Allen et al., 2000, 2002;
trisomy 16 (CTb) cholinergic phenotype. Arriagada et al., 2000, 2003;
Functional neurotransmitter receptors Cardenas et al., 1999, 2002a, 2002b;
(glutamatergic, Cox et al., 1981;
cholinergic). Down syndrome related Frederiksen et al., 1996;
anomalies in CTb cells Kim and Hammond, 1995;
Paula Lima et al., 2002
RCSN Adult rat UCHT1 protocol Neuronal, dopaminergic phenotype. Arriagada et al., 1998, 2002;
substantia nigra Functional glutamatergic receptors. Caviedes et al., 2003
Reduces rotational behaviour in 6 OH
dopamine rats, after striatal implant.
this fashion, mouse trisomy 16 cell lines were of the initial rotation values after 16 weeks post
established from the cerebral cortex (named CTb), implant (Arriagada et al., 2002; Caviedes et al., 2003).
hippocampus (HTk), spinal cord (MTh), and dorsal The effect of the implanted RCSN cell line in the
root ganglion (GTl). Simultaneously, cell lines from improvement of the rotational behavior in 6 OH
these same regions were established from normal, age- dopamine lesioned rats is encouraging in the quest to
matched littermates, which serve as controls. These establish a human substantia nigra cell line, and thus
cell lines retain neuronal markers (Neural specific generate a model that could be applied towards cell
enolase -NSE, Choline acetyl transferase -ChAT, transplant therapy in humans. Taken in summary,
synaptophysin, microtubule associated protein 2 - each of the above cell lines offers significant potential
MAP-2) and lack glial traits (GFAP, galactocerebroside, for evaluation of therapeutic candidates.
S-100). Our prior studies have also shown that these
cells express functional neurotransmitter receptors, as
DRUG DEVELOPMENT AND
shown by intracellular Ca2 + responses to neurotrans-
mitter agonists (Cardenas et al., 1999), and also
CULTURED CELLS
cholinergic function (Allen et al., 2000, 2002; Figure 1 presents a flowchart of the development
Cardenas et al., 2002a, 2002b), and present alterations of cell lines from tissues described herein, and
of the aforementioned functions in a manner similar indicates that manipulation targeted to preserve or
to that observed in similar tissues kept in primary recover stable, differentiated phenotypes is essential to
culture conditions. Interestingly, CTb and HTk cells present a given cell line as a model. Further, Table 1
overexpress the amyloid precursor protein (Arriagada outlines characteristics of the cell lines described
et al., 2000, 2003; Paula Lima et al., 2002), whose gene above. In evaluating these cell types, novel therapeutic
is present in both murine chromosome 16 and human agents can be tested quickly and rigorously such that
autosome 21 conditions. This is considered a major the effects of potential drugs can be evaluated and
link between Downs syndrome and the characteristics their mechanism identified. Many different aspects of
of early onset of Alzheimers-like pathology in these a potential new compound can be evaluated via these
patients (Epstein, 1986). As such, these cell lines are a cultures. Particularly relevant are cultured cells which
potential drug screening tool for anti-amyloidogen- represent a disease state model and those which
ic compounds. demonstrate pertinent genetic and biochemical ab-
Another cell line with potential application to normalities, as well as alterations in receptors, ion
the study and treatment of a neuropathological channels, and receptor signaling pathways. It is these
condition is the RCSN neuronal cell line, derived aspects of the culture cells which enable significant
from the substantia nigra of an adult rat (Arriagada information about a putative therapeutic agents
et al., 1998). By immunohistochemistry, the cell line usefulness in such pathologies. Hallmarks of disease
shows positive neuronal markers (NSE, synaptophy- states which are exhibited in vitro allow for evaluation
sin, MAP-2, neurofilament) and while lacking glial of abnormality mitigation when compounds of
traits (GFAP, S100). RCSN cells possess catecholamin- interest are tested in these cultured systems.
ergic traits: the presence of TH, melanin, and DAT. In myocardial cells such as those discussed
This cell line also exhibits intracellular Ca2 + incre- above, it is possible to evaluate the effects of novel
ments in response to excitatory neurotransmitter agents on parameters yielding beneficial information
agonists (Arriagada et al., 2002; Caviedes et al., as to their potential usefulness in cardiac related
2003). These results suggest the RCSN cell line is a disease states. Similarly, as with the skeletal cell
model for the study of cell mechanisms related to cultures heretofore described, we can ascertain how
Parkinsons like neurodegeneration. Further, using new agents will affect aspects of muscle pathophysi-
stereotaxic surgery, intra-striatal implanted suspen- ologies providing insights as to utility for evaluation of
sions of this cell line into rats previously lesioned in drugs for these anomalies. Lastly, assessment of
the nigrostriatal pathway with 6 OH dopamine, putative new drugs on effects in neuronal cell cultures
revealed a steady reduction in the typical apomor- such as the aforementioned will clearly enable
phine-induced rotational behavior, leveling off at 75% screening and development of therapeutic modalities
that can mitigate the effects of central nervous system pathway with the observation that myocardium,
disorders. Clearly, these are only a snapshot of cultured in the palm of his hand with saliva retained
potential disorders which can be evaluated in cell contractile, could be evaluated in vitro, cell culture
culture. The overall potential of agent evaluation in offers an abundance of promise in drug screening
such cultures is significant. and development.
Of equal, if not greater importance, cell cultures
afford the opportunity to evaluate not only the
positive mitigating effects of novel agents, but they ACKNOWLEDGMENTS
also enable rapid assessment of toxicity profiles in Part of the work reviewed herein was supported
these tissues. Alterations in cell cultures which, when by Fondecyt grants 1980906, 7980058, 1040862,
identified, obviously would result in negative effects in 1020672 (Chile), DID TNAC 10-02/01 and ENL-02/
the whole animal, provide information that will 11 (Univ. of Chile), Fondation J. Lejeune (Paris,
preclude moving a potential drug into animal testing France), and CNRS/Conicyt Exchange Program.
or, even more important, evaluation in humans can be
halted when clear toxicity parameters are identified.
There may be no better example of this type of REFERENCES
evaluation being critical than evaluating compounds Airey, J. A., Baring, M. D., & Sutko, J. L. (1991). Ryanodine receptor
in neuronal tissues/cell culture. For new agents which protein is expressed during differentiation in the muscle cell lines
are being utilized for central nervous system disorders BC3H1 and C2C12. Developmental Biology, 148(1), 365 374.
Allen, D. D., Martn, J., Arriagada, C., Cardenas, A. M., Rapoport, S. I.,
and their effects on neurons, it is paramount that the Caviedes, R., & Caviedes, P. (2000). Impaired cholinergic func-
potential effects of these drugs on the brain be known. tion in cell lines derived from the cerebral cortex of normal and
trisomy 16 mice. European Journal of Neuroscience, 12(9), 3259
Related, agents which have demonstrated utility in 3264.
other disease states, such as anticancer drugs, but do Allen, D. D., Cardenas, A. M., Arriagada, C., Bennett, L. B., Garca, C. J.,
not normally gain access to brain that have newfound Rapoport, S. I., Caviedes, R., & Caviedes, P. (2002). A dorsal root
ganglia cell line derived from trisomy 16 fetal mice, a model for
ability to be delivered to the brain must be evaluated Down syndrome. NeuroReport, 13(4), 491 496.
for potential neurotoxicity. One very key way to Andrews, P. W. (1984). Retinoic acid induces neuronal differentiation of
a cloned human embryonal carcinoma cell line in vitro.
evaluate such is to ascertain the effects of these es- Developmental Biology, 103(2), 285 293.
tablished therapeutic agents in neuronal cell cultures. Andrews, P. W., Damjanov, I., Simon, D., Banting, G. S., Carlin, C.,
Because it is possible to study tissues from a vast Dracopoli, N. C., & Fogh, J. (1984). Pluripotent embryonal
carcinoma clones derived from the human teratocarcinoma cell
array of animals, including humans, the flexibility to line Tera-2. Differentiation in vivo and in vitro. Laboratory
assess how drugs will affect target systems in vitro offers Investigation, 50, 147 162.
Arriagada, C., Caviedes, R., & Caviedes, P. (1998). Establishment and
a great advantage early in screening new compounds. characterization of a neuronal cell line derived from the substantia
Obvious ethical issues related to testing in humans can nigra of the adult rat. Soc. for Neurosci. Abstr., 24(2), 647.1.
be circumvented with early evaluation of new agents. Arriagada, C., Cardenas, A. M., Caviedes, R., Rapoport, S. I., & Caviedes,
P. (2000). Cells of the neuronal cell line CTb, derived from
Further, if adverse effects are determined in vitro, cerebral cortex of trisomy 16 mouse, accumulate amyloid in
reduction in the numbers of animals being unneces- intracytoplasmic vacuole-like compartments. Society for Neuro-
science Abstracts, 180.14.
sarily screened can occur as well. Overall, utility of cell Arriagada, C., Salazar, J., Shimahara, T., Caviedes, R., & Caviedes, P.
cultures overcomes many obstacles in the drug (2002). An immortalized neuronal cell line derived from the
development and screening processes. substantia nigra of an adult rat: application to cell transplant
therapy. In: Ronken, E., G., van Scharrenburg, G. (Eds.).
Clearly, many aspects of this review, though Parkinsons Disease. Amsterdam, Netherlands: IOS Press, 120
focusing on myocardial and skeletal tissues and 132. ISBN 1 58603 207 0.
Arriagada, C., Cardenas, A. M., Segura-Aguilar, J., Caviedes, R., &
neuronal cells, pertain to potential utility of drug Caviedes, P. (2003). Increased expression and altered intracellular
screening and evaluation for other tissue types, and are processing of App in a cell line derived from the cerebral cortex of
a trisomy 16 mouse, a model for Downs syndrome. Society for
particularly relevant to assessing potentially novel
Neuroscience Abstracts, 523.6.
therapeutic agents in cancer cells and tumors. The cell Augusti-Tocco, G., & Sato, G. (1969). Establishment of functional clonal
culture systems described herein offer significant lines of neurons from mouse neuroblastoma. Proceedings of the
National Academy of Sciences of the United States of America,
potential for drug development and screening. Con- 64(1), 311 315.
sidering Sir William Harvey steered us down a Behringer, R. R., Peschon, J. J., Messing, A., Gartside, C. L., Hauschka, S.
D., Palmiter, R. D., & Brinster, R. L. (1988). Heart and bone tumors Delcarpio, J. B., Jr., Lanson, N. A., Field, L. J., & Claycomb, W. C. (1991).
in transgenic mice. Proceedings of the National Academy of Morphological characterization of cardiomyocytes isolated
Sciences of the United States of America, 85(8), 2648 2652. from a transplantable cardiac tumor derived from transgenic
Boheler, K. R., Czyz, J., Tweedie, D., Yang, H.-T., Anisimov, S. V., & Wobus, mouse atria (AT-1 cells). Circulation Research, 69(6), 1591
A. M. (2002). Differentiation of pluripotent embryonic stem cells 1600.
into cardiomyocytes. Circulation Research, 91, 189 201. Earle, W. R. (1943). Production of malignancy in vitro. IV. The mouse
Borlongan, C. V., Tajima, Y., Trojanowski, J. Q., Lee, V. M.-Y., & Sanberg, fibroblast cultures and changes seen in the living cells. Journal of
P. R. (1998). Transplantation of cryopreserved human embryonal the National Cancer Institute, 4, 165 212.
carcinoma-derived neurons (NT2N cells) promotes functional re- Epstein, C. J. (1986). The Consequences of Chromosomal Imbalance,
covery in ischemic rats. Experimental Neurology, 149, 310 321. Principles, Mechanisms, Models. New York: Cambridge University
Cardenas, A. M., Rodrguez, M. P., Cortes, M. P., Alvarez, R. M., Wei, Press.
W., Rapoport, S. I., Shimahara, T., Caviedes, R., & Caviedes, P. Evans, M. J., & Kaufman, M. H. (1981). Establishment in culture of
(1999). Intracellular calcium signals in immortal cell lines derived pluripotentialcells from mouse embryos. Nature, 292, 154 156.
from the cerebral cortex of normal and trisomy 16 fetal mice, an Flax, J. D., Aurora, S., Yang, C., Simonin, C., Wills, A. M., Billinghurst, L.
animal model of human trisomy 21 (Down syndrome). Neuro- L., Jendoubi, M., Sidman, R. L., Wolfe, J. H., Kim, S. U., & Snyder,
Report, 10(2), 363 369. E. Y. (1998). Engraftable human neural stem cells respond to
Cardenas, A. M., Arriagada, C., Allen, D. D., Caviedes, R., Cortes, J. F., developmental cues, replace neurons, and express foreign genes.
Martn, J., Couve, E., Rapoport, S. I., Shimahara, T., & Caviedes, P. Nature Biotechnology, 16, 1033 1039.
(2002a). Neuronal cell lines from the hippocampus of the normal Frederiksen, K., Thorpe, A., Richards, S. J., Waters, J., Dunnett, S. B., &
and trisomy 16 mouse fetus (a model for Down syndrome) exhibit Sandberg, B. E. (1996). Immortalized neural cells from trisomy 16
neuronal markers, cholinergic function and functional neuro- mice as models for Alzheimers disease. Annals of the New York
transmitter receptors. Experimental Neurology, 177(1), 159 170. Academy of Sciences, 777, 415 420.
Cardenas, A. M., Allen, D. D., Arriagada, C., Olivares, A., Bennett, L. B., Frelin, C., Vigne, P., & Lazdunski, M. (1983). The amiloride-sensitive
Caviedes, R., Dagnino-Subiabre, A., Mendoza, I. E., Segura- Na + /H + antiport in 3T3 fibroblasts. Journal of Biological
Aguilar, J., Rapoport, S. I., & Caviedes, P. (2002b). Establishment Chemistry, 258(10), 6272 6276.
and characterization of immortalized neuronal cell lines derived Frelin, C., Vijverberg, H. P., Romey, G., Vigne, P., & Lazdunski, M. (1984).
from the spinal cord of normal and trisomy 16 fetal mice, an Different functional states of tetrodotoxin sensitive and tetrodo-
animal model of Down syndrome. Journal of Neuroscience toxin resistant Na + channels occur during the in vitro develop-
Research, 68(2), 46 58. ment of rat skeletal muscle. Pflugers Archiv, 402(2), 121 128.
Caviedes, R., Liberona, J. L., Hidalgo, J., Tascon, S., Salas, K., & Gonoi, T., & Hasegawa, S. (1988). Post-natal disappearance of transient
Jaimovich, E. (1992). A human skeletal muscle cell line obtained calcium channels in mouse skeletal muscle: effects of denervation
from an adult donor. Biochimica et Biophysica Acta, 1134(3), and culture. Journal of Physiology, 401, 617 637.
247 255. Gonoi, T., Sherman, S. J., & Catterall, W. A. (1985). Voltage clamp
Caviedes, P., Olivares, E., Salas, K., Caviedes, R., & Jaimovich, E. (1993). analysis of tetrodotoxin-sensitive and -insensitive sodium channels
Calcium fluxes, ion currents and dihydropyridine receptors in a in rat muscle cells developing in vitro. Journal of Neuroscience,
newly established cell line from rat heart muscle. Journal of 5(9), 2559 2564.
Molecular and Cellular Cardiology, 25, 829 845. Ham, R. G. (1965). Clonal growth of mammalian cells in a chemically
Caviedes, R., Caviedes, P., Liberona, J. L., & Jaimovich, E. (1994). Ion defined, synthetic medium. Proceedings of the National Academy
channels in a skeletal muscle cell line from a Duchenne muscular of Sciences of the United States of America, 53, 288 293.
dystrophy patient. Muscle Nerve, 17(9), 1021 1028. Harrison, R. G. (1910). The outgrowth of the nerve fiber as a mode of
Caviedes, P., Caviedes, R., Freeman, T. B., Sanberg, P. R., & Cameron, D. protoplasmic movement. Journal of Experimental Zoology, 9,
F. Proliferated Cell Lines and Uses Thereof. International 787 848.
Publication Number WO 03/065999 A2. Filed Feb. 8th, 2002, Harvey, W. (2001). On the Motion of the Heart and Blood in Animals.
publication date: August 14th, 2003. World Intellectual Property Vol. XXXVIII, Part 3. The Harvard Classics, New York: P.F. Collier &
Organization, International Bureau. Son, 1909 1914.
Clark, J. L., Jones, K. I., Gospodarowicz, D., & Sato, G. H. (1972). Growth Hescheler, J., Meyer, R., Plant, S., Krautwurst, D., Rosenthal, W., &
response to hormones by a new rat ovary cell line. Nature. New Schultz, G. (1991). Morphological, biochemical, and electro-
Biology, 236(67), 180 181. physiological characterization of a clonal cell (H9c2) line from
Cognard, C., Constantin, B., Rivet-Bastide, Imbert, N., Besse, C., & rat heart. Circulation Research, 69(6), 1476 1486.
Raymond, G. (1993). Appearance and evolution of calcium Huh, N. E., Pasumarthi, K. B., Soonpaa, M. H., Jing, S., Patton, B., & Field,
currents and contraction during the early post-fusional stages of L. J. (2001). Functional abrogation of p53 is required for T-Ag
rat skeletal muscle cells developing in primary culture. Develop- induced proliferation in cardiomyocytes. Journal of Molecular and
ment, 117, 1153 1161. Cellular Cardiology, 33, 1405 1419.
Cohen, S., & Levi-Montalcini, R. (1957). Purification and properties of a Jacobson, S. L., & Piper, H. M. (1986). Cell cultures of adult
nerve growth-promoting factor isolated from mouse sarcoma cardiomyocytes as models of the myocardium. Journal of
180. Cancer Research, 17(1), 15 20. Molecular and Cellular Cardiology, 18(7), 661 678.
Cox, D. R., & Epstein, C. J. (1985). Comparative gene mapping of human Jaffredo, T., Chestier, A., Bachnou, N., & Dieterlen-Lievre, F. (1991).
chromosome 21 and mouse chromosome 16. Annals of the New MC29-immortalized clonal avian heart cell lines can partially dif-
York Academy of Sciences, 450, 169 177. ferentiate in vitro. Experimental Cell Research, 192(2), 481 491.
Cox, D. R., Epstein, L. B., & Epstein, C. J. (1980). Genes encoding for Jones, H. W., Jr., McKusick, V. A., Harper, P. S., & Wuu, K.-D. (1971).
sensitivity to interferon (IfRec) and soluble superoxide dismutase George Otto Gey (1899 1970): the HeLa cell and a reappraisal of
(SOD-l) are linked in the mouse and man and map to mouse its origin. Obstetrics and Gynecology, 38, 945 949.
chromosome 16. Proceedings of the National Academy of Kidokoro, Y. (1975). Developmental changes of membrane electrical
Sciences of the United States of America, 77, 2168 2172. properties in a rat skeletal muscle cell line. Journal of Physiology,
Cox, D. R., Goldblatt, D., & Epstein, C. J. (1981). Chromosomal 244, 145 159.
assignment of mouse PRGS: further evidence for homology Kim, J. H., & Hammond, D. N. (1995). Septal cell lines derived from the
between mouse chromosome 16 and human chromosome 21. trisomy 16 mouse: generation, characterization, and response to
American Journal of Human Genetics, 33, 145A. NGF. Brain Research, 671(2), 299 304.
Kimes, B. W., & Brandt, B. L. (1976). Properties of a clonal muscle cell line expression during cardiac myocyte differentiation. Physiological
from rat heart. Experimental Cell Research, 98(2), 367 381. Genomics, 9, 145 155.
Kyselovic, J., Leddy, J. J., Ray, A., Wigle, J., & Tuana, B. S. (1994). Plendl, J., Sinowatz, F., & Auerbach, R. (1995). A transformed murine
Temporal differences in the induction of dihydropyridine receptor myocardial vascular endothelial cell clone: characterization of cells
subunits and ryanodine receptors during skeletal muscle devel- in vitro and of tumors derived from clone in situ. Virchows Archiv,
opment. Journal of Biological Chemistry, 269, 21770 21777. 426(6), 619 628.
Lesage, F., Attali, B., Lazdunski, M., & Barhanin, J. (1992). ISK, a slowly Posner, M., Gartland, W. J., Clark, J. L., Sato, G., & Hirsch, C. A. (1970).
activating voltage-sensitive K + channel. Characterization of Development of hormone-responsive cell strains in vitro. Sympo-
multiple cDNAs and gene organization in the mouse. FEBS sium of the Society for Developmental Biology, 29, 114 124.
Letters, 301(2), 168 172. Posner, M., Nove, J., & Sato, G. (1971). Selection procedures for
Levi-Montalcini, R., & Cohen, S. (1960). Effects of the extract of the mammalian cells in monolayer culture. In Vitro, 6(4), 253 256.
mouse submaxillary salivary glands on the sympathetic system of Rensberger, B. (1998). Chapter 1: a particle of life. In: Life Itself,
mammals. Annals of the New York Academy of Sciences, 85, Exploring the Realm of the Living Cell. Oxford Univ Press.
324 341. Rohwedel, J., Maltsev, V., Bober, E., Arnold, H. H., Hescheler, J., &
Levintow, L., & Eagle, H. (1961). Biochemistry of cultured mammalian Wobus, A. M. (1994). Related muscle cell differentiation of
cells. Annual Review of Biochemistry, 30, 605 640. embryonic stem cells reflects myogenesis in vivo: developmentally
Liberona, J. L., Caviedes, P., Tascon, S., Giglio, J. R., Sampaio, S. V., regulated expression of myogenic determination genes and
Hidalgo, J., Caviedes, R., & Jaimovich, E. (1997). Expression of ion functional expression of ionic currents. Developmental Biology,
channels during differentiation of a human skeletal muscle cell 164(1), 87 101.
line. Journal of Muscle Research and Cell Motility, 18(5), 586 Romey, G., Garcia, L., Dimitriadou, V., Pincon-Raymond, M., Rieger, F., &
598. Lazdunski, M. (1989). Ontogenesis and localization of Ca2+
Liberona, J. L., Powell, J. A., Shenoi, S., Petherbridge, L., Caviedes, R., & channels in mammalian skeletal muscle in culture and role in
Jaimovich, E. (1998). Differences in both inositol 1,4,5-trisphos- excitation-contraction coupling. Proceedings of the National
phate mass and inositol 1,4,5-trisphosphate receptors between Academy of Sciences of the United States of America, 86,
normal and dystrophic skeletal muscle cell lines. Muscle Nerve, 2933 2937.
21(7), 902 909. Rous, P. A. (1910). A transmissible avian neoplasm (sarcoma of the
Littlefield, J. W. (1964). Selection of hybrids from matings of fibroblasts common fowl). J. Exp. Med. 12(5), 696 705.
in vitro and their presumed recombinants. Science, 145, 709 Sato, G., Augusti-Tocco, G., & Posner, M. (1970). Hormone-secreting
710. and hormone-responsive cell cultures. Recent Progress in Hor-
Makino, S., Fukuda, K., Miyoshi, S., Konishi, F., Kodama, H., Pan, J., mone Research, 26, 539 546.
Sano, M., Takahashi, T., Hori, S., Abe, H., Hata, J.-I., Umezawa, Sato, G., Clark, J., Posner, M., Leffert, H., Paul, D., Morgan, M., & Colby,
A., & Ogawa, S. (1999). Cardiomyocytes can be generated from C. (1971). Enrichment culture techniques for selection of cell
marrow stromal cells in vitro. Journal of Clinical Investigation, 103, culture strains with desirable physiological and genetic properties.
697 705. Acta Endocrinologica. Supplement (Copenhagen), 153, 126 136.
Marks, A. R., Tempst, P., Hwang, K. S., Taubman, M. B., Inui, M., Sen, A., Dunnmon, P., Henderson, S. A., Gerard, R. D., & Chien, K.
Chadwick, C., Fleischer, S., & Nadal-Ginard, B. (1989). Molecular R. (1988). Terminally differentiated neonatal rat myocardial
cloning and characterization of the ryanodine receptor/junctional cells proliferate and maintain specific differentiated functions
channel complex cDNA from skeletal muscle sarcoplasmic following expression of SV40 large T antigen. Journal of Biological
reticulum. Proceedings of the National Academy of Sciences of Chemistry, 263(35), 19132 19136.
the United States of America, 86(22), 8683 8687. Shamblott, M. J., Axelman, J., Wang, S., Bugg, E. M., Littlefield, J. W.,
Martin, G. (1981). Isolation of a pluripotent cell line from early mouse Donovan, P. J., Blumenthal, P. D., Huggins, G. R., & Gearhart, J.
embryos cultured in medium conditioned by teratocarcinoma D. (1998). Derivation of pluripotent stem cells from cultured
stem cells. Proceedings of the National Academy of Sciences of human primordial germ cells. Proceedings of the National
the United States of America, 78, 7634 7638. Academy of Sciences of the United States of America, 95(23),
Mishina, M., Takai, T., Imoto, K., Noda, M., Takahashi, T., Numa, S., 13726 13731. Erratum in: Proceedings of the National Acad-
Methfessel, C., & Sakmann, B. (1986). Molecular distinction emy of Sciences of the United States of America, 1999, 96(3),
between fetal and adult forms of muscle acetylcholine receptor. 1162.
Nature, 321(6068), 406 411. Sherman, S. J., Lawrence, J. C., Messner, D. J., Jacoby, K., & Catterall, W.
Muller, M. M., Rumpold, H., Schopf, G., & Zilla, P. (1986). Changes of A. (1983). Tetrodotoxin-sensitive sodium channels in rat muscle
purine metabolism during differentiation of rat heart myoblasts. cells developing in vitro. Journal of Biological Chemistry, 258(4),
Advances in Experimental Medicine and Biology, 195(Pt B), 475 2488 2495.
484. Shimahara, T., & Bournaud, R. (1991). Barium currents in developing
New, H. V., & Mudge, A. W. (1986). Calcitonin gene-related peptide skeletal muscle cells of normal and mutant mice foetuses with
regulates muscle acetylcholine receptor synthesis. Nature, muscular dysgenesis. Cell Calcium, 12(10), 727 733.
323(6091), 809 811. Sipido, K. R., & Marban, E. (1991). L-type calcium channels, potassium
Pasumarthi, B. S., & Field, L. J. (2002). Cardiomyocyte cell cycle channels, and novel nonspecific cation channels in a clonal muscle
regulation. Circulation Research, 90, 1044 1054. cell line derived from embryonic rat ventricle. Circulation
Pasumarthi, K. B., Tsai, S. C., & Field, L. J. (2001). Coexpression of Research, 69(6), 1487 1499.
mutant p53 and p193 renders embryonic stem cell-derived Steinhelper, M. E., Jr., Lanson, N. A., Dresdner, K. P., Delcarpio, J. B., Wit,
cardiomyocytes responsive to the growth-promoting activities of A. L., Claycomb, W. C., & Field, L. J. (1990). Proliferation in vivo
adenoviral E1A. Circulation Research, 88, 1004 1011. and in culture of differentiated adult atrial cardiomyocytes from
Paula Lima, AC., Arriagada, C., Toro, R., Cardenas, A. M., Caviedes, R., transgenic mice. American Journal of Physiology, 259(6 Pt 2),
Ferreira, S. T., & Caviedes, P. (2002). Nitrophenols reduce H1826 H1834.
expression of intracellular b amyloid in the CTb neuronal cell Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A.,
line, derived from the trisomy 16 mouse. Society for Neuroscience Swiergiel, J. J., Marshall, V. S., & Jones, J. M. (1998). Embryonic
Abstracts, 483.2. stem cell lines derived from human blastocysts. Science,
Peng, C.-F., Wei, Y., Levsky, J. M., McDonald, T. V., Childs, G., & Kitsis, 282(5391), 1145 1147. Erratum in: Science, 1998, 282(5395),
R. N. (2002). Microarray analysis of global changes in gene 1827.
van der Putten, H. H. A. G. M., Joosten, B. J. L. J., Klaren, P. H. M., & prolonged cultivation of myogenic cells. Proceedings of the
Everts, M. E. (2002). Uptake of tri-iodothyronine and thyroxine in National Academy of Sciences of the United States of America,
myoblasts and myotubes of the embryonic heart cell line H9c2 (2- 61(2), 477 483.
1). Journal of Endocrinology, 175, 587 596. Yaffe, D., & Saxel, O. (1977a). Serial passaging and differentiation of
Wang, Y. C., Neckelmann, N., Mayne, A., Herskowitz, A., Srinivasan, A., myogenic cells isolated from dystrophic mouse muscle. Nature,
Sell, K. W., & Ahmed-Ansari, A. (1991). Establishment of a human 270(5639), 725 727.
fetal cardiac myocyte cell line. In Vitro Cellular and Developmental Yaffe, D., & Saxel, O. (1977b). A myogenic cell line with altered
Biology, 27(1), 63 74. serum requirements for differentiation. Differentiation, 7(3),
Weiss, R. E., & Horn, R. (1986). Functional differences between two 159 166.
classes of sodium channels in developing rat skeletal muscle. Young, D. A., Gavrilov, S., Pennington, C. J., Nuttall, R. K., Edwards, D.
Science, 233(4761), 361 364. R., Kitsis, R. N., & Clark, I. M. (2004). Expression of metal-
Wollenberger, A. (1985). Isolated heart cells as a model of the loproteinases and inhibitors in the differentiation of P19CL6 cells
myocardium. Basic Research in Cardiology, 80(Suppl 2), 9 13. into cardiac myocytes. Biochemical and Biophysical Research
Yaffe, D. (1968). Retention of differentiation potentialities during Communications, 322(3), 759 765.