Widespread Inhibition of Neuroblastoma Cells in the 13- to 17-Day-Old Mouse Embryo1

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[CANCER RESEARCH 46, 1659-1662, April 1986] Widespread Inhibition of Neuroblastoma Cells in the 13- to 17-Day-Old Mouse Embryo1

Robert S. Wells2 and Karen A. Miotto

University of Colorado Health Sciences Center, Department of Pathology, Denver, Colorado 80262

ABSTRACT mation leads to a sensitive assay which, with appropriate con trols, yields quantitative data (18). The disadvantage is that in Previous experiments have demonstrated specific inhibition of tumor formation after neuroblastoma cells were injected into fragments of 8.5- isolated experiments it is not known whether cells which fail to to 9.5-day embryonic tissue (A. H. PodestÃ et ai. Proc. Nati. Acati. Sci. form tumors do so because they have died due to technical USA, 81: 7608-7611, 1984). The effect was localized to the somite and reasons, because they have been killed specifically, or because appeared specific for neuroblastoma as opposed to a variety of other they have been induced to terminally differentiate. Aside from tumor types. This regulation of neuroblastoma cells was believed to routine controls for cell viability, we have relied on the dem reflect an underlying event in the development of migrating embryonic onstration of a pattern of specific regulation of tumor formation neuroblasts. The current experiments were done to determine the effect as an indicator that there may be an underlying developmental on regulation with further embryonic development. The results indicated mechanism. Such a pattern might include specificity for time that later embryos (13 to 17 days of gestation) have a widespread in development, for location in the embryo, and for the type of inhibitory effect in all tissues tested, including the adrenal gland, testis, cancer cell used as a target. kidney, liver, limb bud, and heart. In contrast a leukemia cell line was Using this tumorigenicity assay with fragments of 8.5- to 9.5- not affected by any of these tissues. In organ culture demonstrable colony formation by neuroblastoma was likewise inhibited, and conditioned day embryo we have previously reported that Cl 300 neuroblas media from one of these embryonic sources (limb bud) slowed but did not toma responds to the region of the somite but not to the forming abrogate growth of neuroblastoma cells. neural crest, the interior of the neural tube, the heart, or the brain (1). Likewise, exposure to fragments of adult liver does not affect tumorigenicity by C1300 neuroblastoma (1). INTRODUCTION The current series of experiments were designed to test the Cells derived from neural crest migrate widely in the mouse effect of further somite development during organogÃ©nesison embryo from the time of crest formation in the dorsal midline the regulation of neuroblastoma. at 8.5- to 9.5 days gestation (1). Ultimately these migratory To this end a variety of tissues from 13- to 17-day embryos cells contribute to a variety of tissues. In particular, some of were isolated and tested for their effect on tumor formation by the cells migrate ventral!y between the paired somites flanking neuroblastoma. Recognizing that most neuroblastomas arise in the neural tube and ultimately contribute to the autonomie the adrenal medullae, a prime focus was the adrenal anlÃ¤geat nervous system, including the adrenal medullae (2-5). Deter the time when it could be first detected and isolated at 13-14 mination of neural crest cells has been studied by a variety of days. At this time the normal migrating neuroblasts have been methods, still it is not clear what mediates the migration path, in the gland for approximately 24 h (23). progressive restriction of developmental capability, or site of colonization in end organs. Some experiments point to an active role for the end points of migration (e.g., Ref. 6) whereas others MATERIALS AND METHODS point to early generation of subpopulations that might occur before migration (e.g., Ref. 7). Methods. The strategy of these experiments was an extension of those previously reported in which the effect of 8.5- to 9.5-day embry Our approach has been to use mouse neuroblastoma cells, derived from a widely studied iransplan table tumor, C1300, as onic tissue or tumorigenicity by C1300 neuroblastoma cells was tested (1). In a typical experiment, 5 cancer cells were injected into fragments a probe of neural crest maturation. This approach was fostered of embryonic tissue, and the fragments containing the neoplastic cells by previous studies in which differentiation of tumor cells, were transferred into adult mice in an appropriate site for subsequent either spontaneous or induced, has been closely accompanied tumor formation. The incidence of tumor formation was compared to by loss of tumor formation (8-12). Also, other tumor types, that for 5 cancer cells transplanted into the same site in the absence of including teratocarcinoma and leukemia, have been shown to embryonic tissue. The adult hosts were syngeneic with the tumor cells, react with their corresponding developmental fields (13-20). whereas the hosts were allogeneic with the embryonic tissues. As The C1300 cell line of neuroblastoma used in our experiments previously shown, the embryonic tissue undergoes homograft rejection can be induced to differentiate (21), and this is accompanied by without interfering with tumor formation by the cancer cells (17). The loss of malignant potential (22). Thus, it was reasoned that if LI 210 leukemia cell line was used as a control in these studies; its lack C1300 neuroblastoma were to respond to a developmental of regulation by a variety of embryonic tissues (1, 18) ensured that loss or damage to the small numbers of cells used in these experiments was mechanism aimed at normal migrating neuroblasts, then there not responsible for any loss of tumorigenicity. would be a concomitant loss of tumor forming ability. Cells and Tissue Culture Conditions. The subline of C1300 used in The use of tumorigenicity as an assay has one clear advantage these experiments was obtained from Prasad (21), maintained by serial over other methods. The built-in amplification of tumor for- culture in Ham's 112 plus 10% fetal calf serum (non-heat denatured fetal calf serum) at 37*C in a 5% CO2 atmosphere, and tested for Received 10/28/85; revised 12/23/85; accepted 1/6/86. The costs of publication of this article were defrayed in part by the payment tumorigenicity in A/J mice (The Jackson Laboratory) by transplanta of page charges. This article must therefore be hereby marked advertisement in tion into the testis. Neuroblastoma cells for experiments were prepared accordance with 18 U.S.C. Section 1734 solely to indicate this fact. by treatment with 2.5% trypsin for approximately 30 s; the trypsin was 1This work was supported in part by a gift from R. J. Reynolds Industries, removed and the cells were resuspended in cloning medium (Ham's Inc., and N1H Grants CA-15823, CA-35367, and CA-36069I. 2To whom requests for reprints should be addressed, at Department of MCDB) supplemented with 5% fetal calf serum (23). LI210 leukemia Pathology, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, cells were serially transplanted as an ascites tumor in DBA/2 mice (The Denver, CO 80262. Jackson Laboratory), washed 3 times from the ascites fluid by slow 1659 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1986 American Association for Cancer Research. EMBRYONIC INHIBITION OF NEUROBLASTOMA speed centrifugaciÃ³n, and resuspended in RI'M I + 5% fetal calf serum Table 1 Results of tumor assay with LI210 leukemia cells exposed to 14-day as reported previously (18). embryo fragments Embryonic tissue fragments, each treated by injection of a single leukemia cell, Embryonic Tissues. Mouse embryos at 13 to 17 days gestation (day were grafted into adult mice. In each set of experiments, single leukemia cells 1 was the day when a vaginal plus was observed) were removed asepti- were grafted into adult mice as a matched set of controls. The adult mice were cally from the uterus and the organs were dissected under a Nikon observed for formation of leukemia. dissecting scope. Organs were identified by their anatomic relationships result cell alone controls (24) and gross appearance. Adrenal glands could first be seen with the Embryonic (no. of tumors/no, of (no. of tumors/ aid of a dissecting microscope, isolated by dissection, and confirmed organAdrenal embryonictransplants)27/35 no. oftransplants)50/65 histologically at 13 days of gestation. Other retroperitoneal organs, (77)" (77) which condensed at the same time and were roughly the same size, Limb bud 16/27(59)13/20(65)5 17/27(63)40/55 NSNS included kidney and testis. The identity of several samples of isolated digits LiverExperimental (73)aNSÂ» organs was confirmed histologically after fixation and paraffin embed " Numbers in parentheses, percentage. ding. Whole adrenals, testis, and kidneys were used. Limb buds or * NS, means a > 0.05. digits thereof were cleanly amputated for use. Comparably sized cubes of liver, lung, and heart muscle (roughly 1 mm in dimension) were obtained from isolated tissues by dissociation in 2.5% trypsin for 20 gland, but other retroperitoneal organs (kidney and testis), liver, min. Cells from 5-10 tissue fragment wells were aliquoted into individ lung, heart muscle, and digits of the limb buds. ual wells of Linbro plates (Falcon) with 0.2 ml of medium. Conditioned Colony Formation by Neuroblastoma Cells Exposed to Em medium was collected at 24-48 h from confluent monolayers. bryonic Tissues in Organ Culture. In attempts to reproduce the Injection of Neuroblastoma Cells into Embryonic Fragments for Tumor effect on growth in vitro, embryonic fragments each treated by or Colony Assay. This was done as described previously (1). Isolated injection of 5 neuroblastoma cells were placed in individual embryonic fragments were pooled in an operating drop of cloning tissue culture wells. After 1-2 weeks in culture, in some cases medium under paraffin oil. Cancer cells were placed in a separate donor the neuroblastoma cells had grown to become clearly identifia drop. By means of a Leitz micromanipulator, 5 cells were aspirated ble colonies of tumor cells against the background of prolifer into an injecting pipet, the tip of which was slightly larger than 1 cell diameter. The cancer cells were then moved to the operating drop where ating embryonic cells; these wells were scored as positive for they were injected into the embryonic tissue (held by a second larger colony formation. In other cases neuroblastoma cells failed to pipet) under direct visual control along with a few cell volumes of proliferate even during an additional 2-week period with refeed- medium. Successfully injected fragments were transplanted into anes ing; these were scored as negative for colony formation. Within thetized adult hosts (experimental group); as controls, unused cancer the framework of these operational definitions it was clear that cells from the donor drop were transplanted in an identical manner. colony formation in the presence of embryonic tissues (adrenal, Transplantation was done with finely drawn, orally controlled pipet testis, and kidney) was reduced as compared to that for cells under direct visual control and with microliter amounts of fluid. Ani cultured in the absence of embryonic tissues (Table 3). Similar mals were observed for at least 2 months and tumors were examined results were obtained for coculture of neuroblastoma cells on histologically. To assay growth in vitro, similar techniques were used, monolayers of cells from dissociated embryonic organs (data except that specimens were transferred to individual wells of Linbro plates containing 0.2 ml of medium and observed for subsequent growth not shown). microscopically (20). The results of tumor or colony assays were ex Effect of Media Conditioned by Embryonic Tissues on the pressed on the basis of whether an animal or a tissue culture well Growth of Neuroblastoma Cells. Medium was obtained from contained an identifiable focus of cancer cells. The ratio of positive confluent primary cultures of embryonic organs diluted to a specimens to total tested was compared between experimental and final concentration of 20% in fresh growth medium and tested control groups using the Mantel and Haenszel modification of the x2 for its effect on colony formation by neuroblastoma cells. The test (25). For comparison of colony areas, colony diameters were same lot of medium, left unexposed to tissue, was used as the measured with an ocular micrometer, areas were calculated as a circle, control. As shown in Table 4, there was no significant effect on and the data were analyzed with the Student t test. the rate at which 5 neuroblastoma cells in a tissue culture well would form a colony. This was true for conditioned media from RESULTS adrenals, testis, kidney, and limb bud digits. However, the colonies appeared to contain fewer cells after 1 week of growth. Technical Validity of the Tumor Assay. To ensure cell ac This was confirmed using limb bud conditioned media. The countability (;.<â€¢..thatthe small numbers of cells were not lost diameter of individual neuroblastoma colonies growing either or killed by the manipulations), 5 leukemia cells were injected in 20% conditioned media or growth media was measured with into tissue fragments and the incidence of tumors was compared an ocular micrometer, and colony area was calculated. As shown to that obtained with 5 cells in the absence of embryonic tissue. in Table 5, each of 3 independent lots of conditioned media Leukemia cells were chosen because they produce tumors rap reduced growth rate of neuroblastoma cells. In these experi idly and have never shown regulation in previous experiments. ments individual cells in limb bud conditioned media again As shown in Table 1 there was no significant difference between formed colonies [45/105 (43%)] at a rate similar to that for experimental and control groups. This was in accord with controls [62/120 (52%)]; the difference is not statistically sig previous experience with similarly sized tissue fragments and a nificant. The underlying assumption was that colony area would variety of tumors, including neuroblastoma (1). be proportional to cell numbers. This seemed reasonable since Tumorigenicity of Neuroblastoma Cells Exposed to Various differences in cellular and colony morphology were slight be Embryonic Tissues. As was done with the leukema cells, 5 tween treated and untreated groups. Under this assumption, C1300 neuroblastoma cells were also injected into each embry the data in Table 5 show a 3-7-fold reduction in cell number onic fragment, and the incidence of tumors was compared to after 1 week of growth in 20% limb bud conditioned media. that obtained with 5 control cells in the absence of embryonic tissue. As shown in Table 2, all tissues tested from 13-14-day embryos as well as from 16-17-day embryos markedly sup DISCUSSION pressed tumor formation by neuroblastoma cells. This included In previous studies with tissues from somite-stage embryos not only the obvious target of neuroblast migration, the adrenal (days 8 to 9 of gestation) isolated somites had a specific inhib- 1660

Table 2 Results of tumor assay with NBP2 neuroblastoma cells exposed to embryo fragments Similar to Table 1, embryonic fragments, each bearing neuroblastoma cells, were tested for tumor formation in adult hosts as compared to 5 neuroblastoma cells in the absence of embryonic tissue. Experimental result (no. of S cell alone controls (no. of Embryonic organ tumors/no, of transplants) tumors/no, of transplants 13- 14-day adrenal (85) 16- 17-dayadrenal13-14-day 8/37(22)6/36(17) 31/41(76)33/36 <0.01<0.01

testis (85) 16-17-daytestis13- 9/38(24)0/26 36/39(92)23/29 <0.01<0.01<0.01<0.01<0.01<0.05

14-daykidney13- (0)2/27 (79)25/45

14-dayliver13- (5)2/12 (56)12/15(80)28/32

14-dayheart1 (17)9/36(25)7/27

digits13-14-day3- 14-day limb bud (88)17/29(59)<0.01

lung6/46(13)" (26)44/52 " Numbers in parentheses, percentage

Table 3 Results of colony assay with NBP2 neuroblastoma cells exposed to 13- 14-day embryo fragments in organ culture 9 and 14 days of gestation, somites will contribute to the Each experimental well contained an embryonic fragment treated by injection mesenchyme of a variety of structures during organogÃ©nesis, of 5 neuroblastoma cells and ultimately was scored for the presence of a growing and organ specific mesenchymal-epithelial interactions have colony of tumor cells. Control wells contained 5 neuroblastoma cells in the profound effects on morphogenesis (6, 27, 28). It seemed pos absence of embryonic tissue. sible that what we originally measured was not the effect of Embryonic Experimental result (no. of Control results (no. of organ positive wells/total) positive wells/total a newly formed somites, but rather the effect of more mature, organ specific mesenchyme derived from them. Adrenal (43)" (98) Testis 2/23 (9) 40/40(100) <0.01 To investigate this possibility, the anlÃ¤geof various organs Kidney25/58 1/21 (5)78/80 39/40 (98)<0.01 <0.01 were tested for their effect on neuroblastoma. As reported here, " Numbers in parentheses, percentage. a wide variety of tissues (isolated between 13 and 17 days of gestation) had dramatic inhibitory effects on tumor formation Table 4 Effect of conditioned media from Â¡4-dayembryonic organs on colony by neuroblastoma cells. This included two tissues, heart and forming efficiency by 5 neuroblastoma cells liver, which although already formed had not been inhibitory medium control at the somite stage of development. In particular, no organ Source of Experimental (no. of (no. of positive wells/ conditionedmediumAdrenal positivea18/21 wells/total total) specific pattern of inhibition was discerned; many tissues inhib glands (86)' ited neuroblastoma as effectively as the adrenal primordium. Testis 15/18(83) 22/30 NS If the mechanism of inhibition by later embryonic tissues (14- Kidney 13/23(57) 22/30 NS to 17-day) is the same for each tissue and the same as for 9-day Limb bud digitsGrowth 22/23 (96)22/30 22/30NS* NS " Numbers in parentheses, percentage. somites, then one might postulate a widespread mechanism * NS, a > 0.05. related to developing somatic mesenchyme that is switched on between 10 and 13 days of gestation. In later embryos, this Table 5 Effect of limb bud (LB) conditioned media on the mean area of might have the effect of globally suppressing growth of cells neuroblastoma colonies which have not reached a proper end organ. It might also mask Experimental wells contained 20% limb bud conditioned media and 5 neuro- organ specific effects on cancer cells. blastoma cells. Control wells contained 20% growth media not exposed to Support for these ideas comes from a variety of observations. embryonic tissue plus 5 neuroblastoma cells. After a period of growth the resulting colonies were scored and the mean area was calculated (see "Materials and For instance, it would seem that not all migratory cells reach Methods"). The results from 3 independent batches of conditioned medium are the appropriate organ, and these misplaced cells then usually shown. fail to grow to a significant extent. For example, primitive germ Medium with 20% LB with 20% cells migrate from the region of the yolk sac to colonize the batch12 conditionedmedia70 growthmedia233 gonad, but some germ cells may be left in the dorsal midline. 157 1072 0.01 Transformation of such ectopie cells could be an explanation 3Experimental 189Control 501a0.025 0.01 for extragonadal germ cell tumors. In addition, late stage em bryos have been said to be a difficult site for tumors to grow in (29). Finally, soluble substances with potent activities on cell ilory effect on tumor formation by neuroblastoma (1). Other growth may be acting during organogÃ©nesis. Later embryos tissues from the same age embryo (including heart, brain, and have been a rich source of ^-transforming growth factor, a neural crest) had no effect, and the somites did not inhibit other substance that inhibits growth of many tumors including mel types of tumor, including lines of melanoma embryonal carci anoma (30). Epidermal growth factor levels are markedly re noma, yolk sac carcinoma, or leukemia. However, there was a duced after 14 days of gestation (31). Pertinent to this sort of puzzling loss in the magnitude of the activity with dissociated argument is the observation that normal embryonic sympathetic somite tissue and activity by the somites was not demonstrated neuroblasts are dependent for growth in culture on a partially in vitro} Why did isolated somites appear to lose activity? purified factor (CMF) and rapidly become dependent on NGF As reported by Norr (26), chick somites did not support (32). Trophic substances such as these may play a role in the sympathoblast differentiation unless the chick somites were programmed cell death that occurs during nervous system de conditioned by factors from ventral neural tube. Also, between velopment (33) and selective cell death may be as important to 3 Unpublished data. understand as differentiation. 1661 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1986 American Association for Cancer Research. EMBRYONIC INHIBITION OF NEUROBLASTOMA

Growth inhibitory activity for neuroblastoma is present in 13. Brinster, R. L. The effect of cells transferred into the mouse Mastocyst on subsequent development. J. Exp. Med., 140:1049,1974. media conditioned by the limb bud. In tissue culture the effect 14. Mintz, B.. and Illmensee, K Normal genetically mosaic mice produced from is primarily seen as slowed growth rate rather than total cessa malignant teratocarrinoma cells. Proc. Nati. Acad. Sci. USA, 72:3585-3589, 1975. tion of growth, and it is not yet clear whether depletion of 15. Papioannou, V. E., McBumey, M W., Gardner, R. L., and Evans, M. J. growth factors or addition of inhibitory factors is responsible. Fate of teratocarcinoma cells injected into early embryos. Nature (Lend.), This same limb bud conditioned medium has a similar effect 258:70-73, 1975. 16. Pierce, B. B., Aguilar, D., Hood, G., and Wells, R. S. Trophectoderm in on another neural crest derived tumor (melanoma).3 In the case control of murine embryonal carcinoma. Cancer Res., 44:3987-39%, 1984. of melanoma the ability of embryonic skin tissue to provide 17. Pierce, G. B., Lewis, S. H., Miller, G. J., Moritz, E., and Miller, P. Tumor- inhibitory conditined media is correlated with the time of arrival Â¡genicityofembryonal carcinoma as an assay to study control of malignancy of normal melanoblasts into area of skin.3 It is possible that by the murine blastocyst. Proc. Nati. Acad. Sci. USA, 76:6649-6651,1979. 18. Pierce, G. B., Pantazis, C. G., Caldwell, J. E., and Wells, R. S. Specificity of tissue specific effects will be masked by global inhibitory effects the control of tumor formation by the blastocyst. Cancer Res., 42: 1082- 1087, 1982. at some stages of development. 19. Webb, C. W., Gootwine, E., and Sachs, L. Developmental potential of myeloid leukemia cells injected into midgestation embryos. Dev. Biol., /"/, 221-224,1984. 20. Wells, R. S. An in vitro assay for regulation of embryonal carcinoma by the REFERENCES blastocyst. Cancer Res., 42:2736-2741,1982. 1. PodestÃ ,A. 11. Mullins, .1..Pierce, G. M. and Wells, R. S. The neurula-stage 21. Prasad, K., Sahnu, S., and Sinha, P. Cyclic nucleotides in the regulation of expression of differentiated functions in neuroblastoma cells. J. Nati. Cancer mouse embryo in control of neuroblastoma. Proc. Nati. Acad. Sci. USA, xi: Inst., 57:619-631,1976. 7608-7611,1984. 22. Prasad, K. N. cAMP-induced differentiated mouse neuroblastoma cells lose 2. Le Douarin, N. Migration and differentiation of neural crest cells. Top. Dev. tumorigenic characteristics. Cytobios, 6:163-166,1972. Biol.,/6:31-85,1980. 23. Agy, P. G., Shipley, G. D., and Ham, R. G. Protein-free medium for C1300 3. Le Douarin, N., Smith, J., and LeLieve, C. S. From the neural crest to the mouse neuroblastoma cells. In Vitro (Rockville), 17:671-680, 1981. ganglia of the peripheral nervous system. Annu. Rev. Physio)., 43:653-671, 24. Rugh, R. The mouse, its reproduction and development. Minneapolis, MN: 1981. Burgess Press, 1968. 4. l Imr>.J. P., Duband, J. L., and Delouvee, A. Pathways and mechanisms of 25. Mantel, H., and Haenszel, W. Statistical aspects of the analysis of data from avian trunk neural crest cell migration and localization. Dev. Biol., 93:324- retrospective studies of disease. J. Nati. Cancer Inst., 22:719-748,1959. 343,1982. 26. Ncirr.S. C. In riini analysis of sympathetic neuron differentiation from chick 5. Weston, J. A. The regulation of normal and abnormal neural crest develop neural crest cells. Dev. Biol., 33:16-38,1973. ment. In. V. M. Ricardi and J. J. Mulverhill (eds.), Advances in Neurology, 27. Cunha, G. R. Epithelial-stromal interactions in development of the urogenital pp. 77-94. New York: Raven Press, 1981. tract. Int. Rev. Cytol., 47:137-194,1976. 6. Bee, J., and Thorogood, P. The role of tissue interactions in the skeletogenic 28. Hall, A. K. Stem cell is a stem cell is a stem cell. Cell, 33:11-12,1983. differentiation of avian neural crest. Dev. Biol., 78:47-62,1980. 29. Whisson, M. E. The interaction of tumor and embryonic tissues in vivo. In: 7. Girdlestone, J., and Weston, J. A. Identification of early neuronal subpopu A. V. S. De Reuck and J. Knight (eds.). Cell Differentiation, CIBA Foun lations in avian neural crest cell cultures. Devel. Biol., 709:274-287,1985. dation Symposium, pp. 219-230. London: J & A Churchill, 1967. 8. Metcalf, D. Regulation of self-replication in normal and leukemic stem cells. 30. Proper, J. A., Bjornson, C. L., and Moses, H. L. Mouse embryos contain UCLA Symp. Mol. Cell. Biol. New Ser., 9:141-156, 1983. polypeptide growth factors) capable of inducing a reversible neoplastic 9. Pierce, G. H . Dixon, F. J., and Verney, E. L. Teratocarcinogenic and tissue phenotype in nontransformed cells in culture. J. Cell. Physiol., 110: 169- forming potentials of the cell types comprising neoplastic embryoid bodies. 174, 1982. Lab. Invest., 9:583-602,1960. 31. Mesiano, S., Browne, C. A., and Thorburn, G. D. Detection of endogenous 10. Pierce, G. IV.and Wallace, C. Differentiation of malignant to benign cells. epidermal growth factor-like activity in the developing chick embryo. Dev. Cancer Res., 31:127-134, 1971. Biol., Â¡10:23-28,1985. 11. Speers, W. C. Conversion of malignant embryonal carcinomas to benign 32. (nughlm. M. D., and Collins, M. B. NGF-independent development of teratomas by chemical induction of differentiation in vivo. Cancer Res., 42: embryonic mouse sympathetic neurons in dissociated cell culture. Dev. Biol., 1843-1849, 1982. 110:392-401, 1985. 12. Strickland, S., and Mahdari, V. The induction of differentiation in teratocar- 33. Easter, S. S., Purves, D., Rakic, P., and Spitzer, N. C. The changing view of cinoma stem cells by retinoic acid. Cell, IS: 393-403, 1978. neural specificity. Science (Wash. DC), 230: 507-511,1985.

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Robert S. Wells and Karen A. Miotto

Cancer Res 1986;46:1659-1662.

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