Embryonic stem cell lines derived from blastocysts by a simplified technique

DEVELOPMENTAL

BIOLOGY 101,225-228

(1984)

Embryonic Stem Cell Lines Derived from Blastocysts by a Simplified Technique HELENA

R. AXELROD’

Sloan Kettering Institute fw Cancer Research, Laboratory of Lkvelopmental Received March 2, 1983;accepted in revised

Genetics, New York, New Ywk 19104

form June SO,1982

We have isolated euploid, pluripotent stem cell lines directly from mouse hlastocysts by a simple culture technique. Our method permits cell lines to be derived from individual embryos, without the use of ovariectomy, immunosurgery and conditioned medium. We cultured individual intact blastocysts in MicroTest plates on top of a feeder layer of lethally irradiated ST0 mouse fibroblasts in a lo-p1 volume of Dulbecco’s modified Eagle’s medium (DMEM)* supplemented with 10% fetal calf serum (FCS). The cell lines maintain a stable complement of 40 chromosomes, form embryoid bodies and differentiate both in culture and in solid tumors. INTRODUCTION

Existing mouse embryonal carcinoma (EC) cell lines derived from teratocarcinoma tumors, while exhibiting multipotentiality, fail to participate at appreciable frequency in the normal development of chimeric embryos (Papaioannou, 1979; Stewart and Mintz, 1981; Rossant and McBurney, 1982) and most of them are not euploid (McBurney, 1976; Nicolas et aL, 1976; Cronmiller and Mintz, 1978) with a few exceptions (Mintz and Cronmiller, 1981; Rossant and McBurney, 1983). It is possible that these deviations from normalcy are caused or aggravated by the length of the procedures used to isolate these cell lines from tumors. Since EC cells closely resemble the cells of the embryonic ectoderm of the early postimplantation embryo (Martin et ok, 1977, 1978; Evans et aL, 1979; Howe et al, 1980) it should be possible to isolate these cells directly from the embryo. Recently Evans and Kaufman (1981) and Martin (1981) described the successful isolation of pluripotent embryonic stem cells (ESCs) from inner cell masses (ICMs) of blastocysts. Evans and Kaufman (1981) subjected pregnant 129 strain mice to ovariectomy, combined with hormone treatment, in order to delay blastocyst implantation. They reasoned that the delay would increase the number of ICM cells in each embryo, and thus enhance their chance to grow in vitro. Martin (1981) isolated ESCs from immunosurgically isolated ICMs of late blastocysts of both outbred and inbred mice. She cultured approxi Present address: The Wistar Institute, 36th Street at Spruce, Philadelphia, Pa. 19104. *Abbreviations used: EC, embryonal carcinoma; EB, embryoid bodies; ESC, embryonic stem cell; ICM, inner cell mass; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; EDTA, ethylenediaminetetraacetic acid.

imately 30 ICMs together in the presence of concentrated conditioned medium produced by a PSA-1 embryonal carcinoma cell line. Factors in the conditioned medium were thought to stimulate and promote the division of the stem cells. We describe the establishment of continuous stem cell lines directly from preimplantation mouse embryos without the use of concentrated conditioned medium, immunosurgery, or ovariectomy. MATERIALS

AND METHODS

Embryos and Culture Conditions CF1 outbred females (Charles River, Mass.) were superovulated by injecting 7.5 IU of Gestyl (Organon, N. J.) followed 48 hr later with 5 IU of Pregnyl (Organon, N. J.), and caged with males. Males used in these experiments were either C3HDiSn/C57BLlO - to/ Rb(16.17)7 Bnr, or tw73/C57BL10 Rb(16.17)7 Bnr. The Rb(16.17)7 Bnr stock was generously provided by A. Gropp. Embryos were flushed from uteri approximately 90 hr after the injection of Pregnyl. Whole, unhatched blastocysts were individually placed on top of a feeder layer of lethally irradiated (6000 rad) ST0 fibroblasts (Martin and Evans, 1975) in MicroTest wells of Terasaki plates (Falcon, Cat. No. 3034). They were cultured in 10 gl of Dulbecco’s modified Eagle’s medium (DMEM) containing 1 g/liter glucose and 110 mg/liter pyruvate (Grand Island Biological Co., Cat. No. 430-1600) and supplemented with 10% fetal calf serum (FCS) (Grand Island Biological Co.) in a humid, 6% COz incubator at 37°C. When large healthy growths appeared (4 to 7 days later) (Fig. la), they were partly dissociated with 0.05% trypsin and 0.025% ethylenediaminetetraacetic acid

225 0012-1606/34 $3.00 Copyright Q 1984 hy Academic Press, Inc. All righta of reproduction in any form reserved.

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FIG. 1. (A) Egg-cylinder-like growth of a blastocyst ICM after culture for 5 days. The size of the growth was approximately twice the size of the original blastocyst, and was judged ready for passage. (B) A small colony of CEM-5, cultured for 11 passages on ST0 feeders. CEM5 was derived from a blastocyst cultured in regular medium (DMEM + 10% FCS).

(EDTA) for 5 min at 3’7°C. The colonies were picked off the feeder layer with a mouth-regulated, finely drawn pipet. The partly dissociated clumps were seeded individually onto ST0 in 6-mm wells of 96-well plates (Costar) containing approximately 50 ~1 of DMEM + 10% FCS. They were cultured for ‘7-14 days until growing colonies of EC-type cells were seen. These colonies were partly dissociated and subcultured at approximately ‘I-day intervals into increasingly larger culture dishes. For determining the effects of conditioned medium, embryos were cultured in its presence for the initial culture period and three subsequent passages. Preparation of Conditioned Medium PSA-1 EC cells (Martin and Evans, 1975) were grown to 50-75s confluency on ST0 feeders in DMEM + 10% FCS. They were washed five times with Dulbecco’s phosphate-buffered saline, and incubated for approximately 24 hr in serum-free DMEM supplemented with 10 mg/ ml transferrin and 10 mg/ml insulin. The conditioned medium was collected and centrifuged to remove cell debris. It was concentrated 30- to 40-fold and desalted by ultrafiltration through a UM05 Diaflo Membrane using a stirred cell (Amicon, Danvers, Mass.). The concentrate was sterilized by passage through a 0.22 pm filter, supplemented with 10% FCS and 0.1 mM 2-mercaptoethanol, and stored at -80°C in small aliquots. The concentrate was diluted before use with DMEM containing 10% FCS and 0.1 mM 2-mercaptoethanol. The final concentration of conditioned medium used to culture embryos was fourfold greater than the original. The preparation procedure was based on the protocol

used by Martin (1981), but the method of concentration and desalting was different, RESULTS AND DISCUSSION

Establishment of ESC Lines from Embryos We have established cell lines with typical EC morphology from immunosurgically isolated ICMs (prepared according to the method of Solter and Knowles, 1975), early blastocysts and late blastocysts (Table 1). Blastocysts and ICMs grown in regular medium produced outgrowths 73 to 81% of the time, whereas only 40% of the ICMs grown in conditioned medium continued to proliferate. The EC-like morphology of the growing cells was clearly seen by the second or third passage. Although colonies of differentiated cell types such as endoderm, fibroblasts, and trophoblasts could be seen at the beginning, these cells failed to survive and were lost. However, once the EC-type colonies were consistently seen after two or three subcultures, they usually grew indefinitely and without substantial differentiation. ICMs cultured in regular medium gave rise to ESC lines just as frequently as those grown in conditioned medium (Table 1, and Martin, 1981). Therefore, addition of conditioned medium did not appear to be necessary in our culture system. Furthermore, early blastocysts grown in regular medium also produce ESC lines efficiently. There was a reduction in efficiency when late blastocysts were used, but due to the small sample size it is not clear whether the difference is significant. Our findings do not contradict, or directly test, the assumntions of Martin (1981) . I or Evans and Kaufman

227

BRIEF NOTES TABLE 1 GROWTH AND KARYOTYPE OF CELLS FROM INDIVIDUAL EMBRYOS Percentage

and fraction

of growing

embryos

No. of subcultures Embryo

type

Conditionedb

ICM

Regular”

ICM Blastocyst Blastocyst

Culture medium

(early)d (late)e

Regular Regular

0

1

2

N”

88%

40% (8120)

U/8)

100% um

WV

81% (17/21)

44% (7/17)

86% (617)

73% (22/30)

(2;:)

78% (25/32)

28% (7125)

Efficiency of recovery’

(24

CEM-2 (41)

CEM3 (80940)

W’3

(zf

CEM-1 (40)

CEM-4 (ND)

100%

100%

100%

@/a

e/a

cw

CEM-6 (40)

CEM-7 (40)

14% (l/7)

100% (l/l)

14% (l/7)

CEM-5 (40)

29%

29%

Cell line name (chromosome No.)

33%

Note. ND = not done. ’ N = more than 6 passages and 100 divisions. b DMEM conditioned by PSA-1 cells, concentrated four-fold and supplemented with 10% FCS and 0.1 mM 2-mercaptoethanol. ’ DMEM supplemented with 10% FCS. d Blastocysts flushed from uteri at 3’/4! days (90 hr after Pregnyl), whose cavities were at least half the volume of the embryo. e Blastocysts as in (d), cultured overnight in DMEM + 10% FCS in the absence of feeders, and then seeded in Teraaaki wells with feeders. fPercentage of ESC lines recovered from embryos growing after the first subculture.

(1981) used as a basis for developing their methods of ESC isolation. There are most probably growth factors which are produced and required by ESCs as suggested by Martin (1981). We may have circumvented the need for an external source of these factors by culturing embryos in very small volumes. In such circumstances, blastocysts may be able to produce sufficient concentrations of factors for self-stimulated growth. It is also quite possible that an increase in ICM cell number may increase the possible chances of ESC recovery as reported by Evans and Kaufman (1981).

The ESC lines will be useful for the analysis of the developmental process in vitro. They have already shown their responsiveness to aggregation signals for differentiation into a wide variety of cell types. It is likely that they will also be capable of responding to chemical inducers. The most useful types of cells to use in these studies are those which have genetic alterations affecting development. Mutants of the T/t complex, A7, and the Albino region would be interesting because of their effects on early stages of embryogenesis. One tw5 homo-

Characterization of ESC Lines Two clonal lines of CEM-7, derived from single cells, formed embryoid bodies (EB) when cultured in suspension, and differentiated into a variety of cell types and tissues when grown in vitro and in vivo as tumors (data not shown). These cell lines show the multipotentiality characteristic of embryonic stem cells. Three of the ESC lines contained a stable, normal karyotype of 40 chromosomes (Table 1). A clonal derivative of CEM-7 showed the banding pattern of a euploid male (Fig. 2). However, CEM-1 and CEM-2 contained a Robertsonian translocation, and were thus trisomic or tetrasomic. CEM-3 consisted of a mixture of tetraploid and diploid cells. It is possible that some of these karyotype abnormalities may result from improper segregation of the Rb’7 translocation in some of the embryos.

FIG. 2. Giemsa-banded chromosomes from CEM-7 cl1 cell line derived from a blastocyst. This cell line was cloned from a single cell. The karyotype is 40, XY in over 70% of the cells.

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DEVELOPMENTALBIOLOGY

zygous mutant cell line (Magnuson et aZ., 1982) as well as several homozygous diploid cell lines of parthenogenetic origin (Kaufman et aL, 1983) have already been isolated. It is hoped that the relative simplicity of our methods for generating stem cell lines from blastocysts will further encourage the in vitro study of early mouse embryo development.

VOLUME101, 1984

MARTIN,G. (1981). Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. NatL Acad Sci. USA 73,7634-7638. MARTIN, G., and EVANS, M. J. (1975). Differentiation of clonal lines of teratocarcinoma cells: Formation of embryoid bodies in vitro. Proc. NatL Acad Sci USA 72, 1441-1445. MARTIN, G., WILEY, L. M., and DAMJANOV,I. (1977). The development of cystic embryoid bodies in vitro from clonal teratocarcinoma stem cells. Dev. Bid 61.230-244. MARTIN, G. R., SMITH, S., and EPSTEIN,C. J. (1978). Protein synthetic patterns in teratocarcinoma stem cells and mouse embryos at early We are grateful to Dr. Dorothea Bennett for her intellectual and stages of development. Dar. Biol 66,8-16. financial support of this work. We appreciate the efforts of Eric Lader MCBURNEY,M. (1976). Clonal lines of teratocarcinoma cells in vitro: in karyotyping and Daniella Simon in G-banding the chromosomes Differentiation and cytogenetic characteristics. J. CeU PhysioL 89, of our cell lines. We also thank Dr. Davor Solter and Dr. David E. 441-456. Axelrod for helpful criticism of the manuscript. This work was supMINTZ,B., and CRONMILLER, C. (1981).ME’IT-1: A karyotypically normal ported by Grant HD-10668 from the National Institutes of Health in vitro line of developmentally totipotent mouse teratocarcinoma awarded to Dr. Bennett, Grant CA-18470 from the National Institutes cells. Somat. Cell Genet. 7, 489-505. of Health awarded to Dr. B. B. Knowles, and Grant CA-25875. NICOLAS,J.-F., AVNER, P., GAILLARD,J., GUENET,J. L., JACOB,H., and JACOB,F. (1976). Cell lines derived from teratocarcinomas. Cancer REFERENCES Res. 36,4224-4231. PAPAIOANNOU,V. E. (1979). Interactions between mouse embryos and CRONMILLER, C., and MINTZ,B. (1978). Karyotypic normalcy and quasiteratocarcinomas. In “Cell Lineage, Stem Cells and Cell Determinormalcy of developmentally t&potent mouse teratocarcinoma cells. nation” (N. Le Douarin, ed.), pp. 141-155. Elsevier/North-Holland, Dev. Bid 67,465-477. New York. EVANS, M. J., LOVELL-BADGE,R. H., STERN, P. L., and STINNAKRE, M.-G. (1979). Cell lineages of the mouse embryo and embryonal ROSSANT,J., and MCBURNEY,M. W. (1982).The developmental potential carcinoma cells; Forssman antigen distribution and patterns of proof a euploid male teratocarcinoma cell line after blastocyst injection. tein synthesis. In “Cell Lineage, Stem Cells and Cell Determination” J. Em&go1 Exp. MorphoL 70,99-112. (N. Le Douarin, ed.), pp. 115-129.Elsevier/North-Holland, New York. ROSSANT,J., and MCBURNEY,M. W. (1983). Euploid teratocarcinoma EVANS, M. J., and KAUFMAN, M. H. (1981). Establishment in culture lines differ in their ability to differentiate normally after blastocyst of pluripotent cells from mouse embryos. Nature &or&n) 292,154injection. In “Teratocarcinoma Stem Cells” (L. M. Silver, G. R. 156. Martin, and S. Strickland, eds.), Cold Spring Harbor Conferences HOWE,C. C., GMUR,R., and SOLTER,D. (1980). Cytoplasmic and nuclear on Cell Proliferation, in press. protein synthesis during in vitro differentiation of murine ICM and SILVER,L. M., ARTZ, K., and BENNETT,D. (1979). A major testicular embryonal carcinoma cells. Dev. Bid 74,361-363. cell protein specified by a mouse T/t complex gene. Ce0 17.275-284. KAUFMAN, N. H., ROBERTSON,E. J., HANDYSIDE,A. H., AND EVANS, SOLTER, D., and KNOWLES,B. B. (1975). Immunosurgery of mouse M. J. (1983). Establishment of pluripotent cell lines from haploid blastocyst. Proc Nat1 Acad Sci USA 72, 5099-5102. mouse embryos. J. Eminyol Exp. Morph 73,249-261. MAGNUSON,T., EPSTEIN,C. J., SILVER,L. M., and MARTIN, G. R. (1982). STEWART,T. A., and MINTZ, B. (1981). Successive generations of mice produced from an established culture line of euploid teratocarcinoma Pluripotent embryonic stem cell lines can be derived from tw6/tw5 cells. Proc NatL Acad Sci USA 73.63146318. blastocysts. Nature (London) 298.750-753.