Reprints Available Directly from the Publisher Photocopying Permitted by License Only Differentiation of Mouse Embryonic Stem Cells to Lympho-hematopoietic Lineages in Vitro

Mouse embryonic stem (ES) cells can differentiate in culture to late stages of many cell lineages. have found culture conditions that are favorable for development in vitro of ES cells into hematopoietic cells at a stage equivalent to day I1-14 of fetal liver development. describe here (1) the growth conditions necessary for maintenance of ES cells in an undifferentiated state, and the conditions that allow differentiation of cystic embryoid bodies that contain precursors of most hematopoietic cell lineages, including lymphoid cells; (2) the development of lymphoid vessels from ES fetuses in vivo; (3) the characterization of lymphoid, erythroid, megakaryoid, and myeloid cells from ES fetuses; and (4) the cloning of cell lines representing lymphoid, myeloid lineage cells from differentiated ES cells.


th ES cells i
vitro (Lindenbaum and Grosveld, 1990;  Wiles and Keller, 1991; Schmitt et al., 1991).

The origin of the pluripotent stem cells and their lineage commitment'to lymphocytes from mouse embryos are important issues in develop- mental biology that have recently received much attention.Stem cells from yolk sacs have been shown to give rise to erythrocytes, megakaryotic cells, and myeloid cells (Labastie et al., 1984).B lymphocytes can also be found after yolk sac stem cells are transferred into animals, but not in in vitro differentiation condition (Melchers, 1977;  Paige et al., 1979; Melchers and Abramczuk,   1980; Chris Paige, personal communication).On the other hand, stem cells obtained from the fetal liver can give rise to erythroid, myeloid, and B-lymphoid cells in vitro (Owen et al., 1975;   Melchers, 1977; Ogawa et al., 1988; Rolink et al.,   1991).Differentiation of T-lymphoid cells requires a thymic microenvironment (see review by von Boehmer, 1990).

Pluripotent stem cells have been difficult to study because they are r re cells found in the het- erogeneous cellular populations of bone marrow and fetal liver.Great progress has been made in obtaining subpopulations of bone marrow from adult and fetal liver that are highly enriched for pluripotent stem cells.Yet, very little is known about how stem cells commit to differentiation and how stem cells self-maintain.This lack of knowledge is at least partially due to the inability to maintain stem cells in vitro.Culture systems that would allow the growth of very primitive stem cells, such as mouse embryonic stem cells and the primitive hematopoietic stem cells, would be invaluable in addressing these issues.

U. CHEN The liquid culture system described in this study allows the development of pluripotent hematopoietic stem cells and.all known hemato- poietic lineages, including lymphoid (T and B)  lineages from ES cells.Because this is an import- ant step for studying lymphohematopoiesis in vitro, and because any deviation from the culture condition described in this study did not give rise to lymphocytes, I therefore describe in detail the differentiation conditions, as well as the growth conditions important for maintaining undifferentiated ES cells.

Differentiation in vitro can be followed under the microscope by observing the growing cells in three dimensions.First, they form simple embryoid bodies within clusters of cells and then cystic embryoid bodies with a complicated inner structure are developed.Finally, the embryoid bodies become "ES fetuses," which contain pulsating cardiac muscle, yolk sac blood islands, and, in addition, novel cup-shaped structures containing lymphoid cells.Further development of these ES fetuses was seen following introduc- tion under the kidney capsule of young adult mice.From selected "ES fetuses," I use a variety of techniques to establish lymphoid and myeloid cell lines.


RESULTS

Differentiation of ES Cells to Simple, Then Cystic Embryoid Bodies, and the Selection of ES Fetuses Among Cystic Embryoid Bodies Mouse ES cells derived from strains 129 (ES-D3) and C57B1/6 (ES-B6) grow two dimensionally on top of nondividing feeder cells, which are pri- mary embryonic fibroblasts.ES cells were removed from feeder cells and allowed to differ- entiate in a liquid suspension culture.Within 24 hr, the cells aggregate and small round struc- tures are formed.After 4-5 days, an outer layer of large endoderm cells is formed; some of them develop cavities.An inner layer of "ectodermal- like" cells is formed.These are simple embryoid bodies.From 8 to 30 days, there is a more compli- cated internal structure, which includes several easily recognizable mesodermal tissues such as blood cell clusters and cardiaclike tissue.These are cystic embryoid bodies (Martin and Evans,   1975; Cudennec and Nicolas, 1977;

etschman
et al., 1985; Gossler et al., 1986; Robertson, 1987).Depending on the origin of the ES cell line and the number of passages in culture, about 0.1-1% of the ES cells develop into cystic embryoid bodies.Under the liquid culture condition described in the Materials and Methods section, which allows a slow and free movement of embryoid bodies, I found that about 30-40% of the cystic embryoid bodies develop into "ES fetuses" (Fig. 1).The ES fetuses differentiated 10x more efficiently in the hydrophobic Heraeus tis- sue culture dish than the bacterial culture dish (see Materials and Methods section for source of dish).Fully formed cystic embryoid bodies, referred to as ES fetuses, are characterized by the presence of pulsating cardiac muscle, yolk sac blood islands, and, most importantly, the cupshaped structures (larg e arrows in Fig. 1) con- taining small round 7-1O-tm diameter lymphoid- like cells (small arrows in Fig. 1).They can be selected at various growth stages of differen- tiation from day 11 to 30.

A) and 2(B), were characterized histologically.As shown in Fig. 2(C), cardiac muscle cells and the cavity formed by the epithelium cells (cup-shape structure under the microscope) are visible.The clusters of cells inside the cavity shown in Fig. 2(D) are identified to be erythroid cells (arrow 5), myeloid cells (arrow 2 and arrow 4), and lymphoid cells (arrow 1).

ES fetuses were subsequently implanted under the kidney capsules of male Balb/c nude mice for 3 weeks, excised, and frozen.As shown in Figs.  2E) to 2(I), immunophenotyping of the cryosections was performed.The sections were double stained for anti-H-2K b (conjugated to Texas Red, to demonstrate the class-I haplotype of ES origin) plus either anti-CD3 (conjugated to FITC) or anti- / (conjugated to FITC).Both T cells and B cells were identified.T cells were identified in Figs. 2(F) and 2(G) by being positively stained with anti-CD3 alone (green color

n Fig. 2(F), solid small arrow) or double stain
d with anti-CD3 plus anti-H-2K b (orange color in Fig. 2(G), solid small arrow).H) and 2(I), by positively stained with anti-/ alone  aStarting 2 weeks after retrovirus infection/lymphokine stimulation, the morphology of cell types was checked periodically for 6 months.The pattern of cell types was stable from about 4 weeks, if they were kept in the culture.Figure 3 shows representative individual hematopoietic cell types established from ES cells.

bLym: 1.ym hocytes; Mo: macrophages; Mono: monocytes; Mega: megakaryocytes; RBC: erythrocytes.

example, the myc and mil oncogenes (Group 4) produced cells mainly of the myeloid lineage.

The cells established with growth factors (Groups 5 and 7) and J2 virus (Group 2) gave rise to both lymphoid and myeloid cells.Abelson (Group 1) and RIM viruses (Group 3) produced the broadest spectrum of cell types.The combi- nation of RIM virus and growth factors (Group 6)  established cell populations that differentiated into plasma cells, and mature T-cells, in adoptive transfer experiments (Chen et al., in preparation).

Characterization of Cell Types by Morphology and In Situ Immunofluorescence Cytocentrifuge preparations of each group were evaluated following staining with Giemsa.Table 1 summarizes the diversity of cell types found in each group after the establishment of stable cell populations, which required a period of 4 weeks to 6 months of culture.I started to examine the morphology of the cell types 2 weeks after "stimulation" (infection and/or growth factors) and found that the phenotypes were stable from 4 weeks onwards.Figure 3 shows the mor- phology of individual cells from one of these cul- tures (Group 6).Cells with morphology charac- teristic of monocytes (Fig 3, arrow 2) and macrophages (Fig. 3, arrow 3) were observed, and many of them were found to cluster around erythrocytes (Fig. 3, arrows 5 and 5').Megakaryocytes (Fig. 3, arrow 4) were also found to be associated with these clusters.Lymphoidlike cells (Fig. 3, arrow 1) with little cytoplasm and dark-blue-staining nuclei were observed, either in clusters or interspersed between irregularly shaped, larger, light-blue-staining myeloid cells.Cells grown in the presence of cytokines and mitogens (Groups 5, 6, and 7) contained a signifi- cant number of mitotic figures, and many of them appeared to be irregular and contained multiple vacuoles, possibly due to spontaneous or virally induced transformation during longterm culture (i.e., up to 6 months).Although the FIGURE 2. Characterization of "ES fetuses."Phase-contrast photomicrographs of one ES fetus. (A) Low magnification (5x)  showing the whole body.C=cup-shape structure.H=heart. (B) Higher magnification (20x) focusing on a cup-shape structure containing a red dot o cells in the middle (arrow). (C) Azura A-Eosin B staining of an ES fetus in plastic embedding from (A) (low magnification, 5x). (D) Hematopoietic cells (magnification 100x) found in the cup-shape structure Shown in (C): arrow 1= lymphocyte; arrow 2=monocyte; arrow 3=mitotic cell; arrow 4=megakaryocyte; and arrow 5=reticulocyte. (E) A Giemsa-stained frozen section of ES fetus under the kidney capsular (arrow), excised from Balb/c nude mice 3 weeks after implantation (magnification 3x).(F to I) Immunophenotyping of lymphocytes (solid small arrow) inside the lymphoid vessels (hollow large arrow) from mature ES fetuses as shown in (E) (magnification 40x).The sections were stained with (F) FITC-labeled anti-CD3 arrow) and (G) anti-H-2K plus rat antimouse IgG-Texas Red (red color, hollow small arrow) antibody (green color, solid small (G) when double stained (CD3/, H-2KD/), the color was orange (solid small arrow).The sections were also stained with (H) anti-/ conjugated with FITC (green color, solid small arrow) and (I) anti-H-2K b/ plus rat antimouse IgG Texas Red (red color, hollow small arrow); and (I) double stained (orange color, solid small arrow); (G and I) Note that some cells expressed only H-2K surface antigen (red color, hollow small arrow). (See colour plate II at the back of this publication).FIGURE 4. Differentiated lymphoid cells derived from ES cells identified by staining of lymphoid-cell-surface markers by indirect immunofluorescence microscopy.Photomicrographs of the cells from Group 6 (see Table 1). (A, C, and E)

Immunofluorescent staining. (B, D, and F) Corresponding phase-contrast photomicrographs. (A) B-lymphoid cells identified by FITC-labeled sheep antimouse t-chain F(ab)'2 fragment. (C) T-lymphoid cells identified by anti-CD3 antibody (500-A2) and second antibody of FITC-labeled rabbi antihamster IgG F(ab)'2 fragment. (E) Second antibody alone (negative control).The staining was performed after six months of culture.


Characterization of Surface Antigen Expression of Cell Populations by Flow Cytometry

As summarized in Table 2 and in one example of a histogram (Fig. 5), cells from each group were first analyzed for expression of B-cell markers such as surface Ig (sIg) and B220 antigens as well as the pre-B-cell marker identified by mAb G-5-2.

A significant amount of sIg cells was present in cells from different groups after retrovirus infec- tion.Interestingly, cells from Group 7 did not express sIg.However, they possessed precursor B-cell markers such as G-5-2.The expression of B220 antigen did not correlate with sIg expression.Also, Thyl antigen was not always present.The reason for this is not clear.It may suggest that it was an unusual phenomenon caused by the viral infection.In most groups, a signifciant number of cells expressed CD3 antigens, although most of them were CD4-, CD8-cells.It is very interesting that Group 7 pos- sessed CD4 / single positive (i.e., CD3-, CD8-) cells.Whether these are the precursor cells resembling stem T cells found in the adult thymus (Wu et al., 1991) remains open.All groups contained cells of the myeloid lineages as defined by the mAb Mac-1.In all groups, most of the cells did not express SSEA-1 antigen, which appeared only at the blastocyst stage (Solter and Knowles,  1978), but retained various portions of embryonic antigen recognizeqt by the mAb $7 (see what fol- lows for a more detailed description of these antibodies).These data suggest that progenitor cells, precursor lymphoid cells, and some T and B cells exist in these mixed populations.

Cloning and Characterization of Lymphoid, Myeloid, and Precursor Stem Cells from ES-Derived Hematopoietic Cells One mixed population of hematopoietic cells established by using RIM virus infection (Group 3) was cloned on irradiated splenic feeder cells.

The cloning frequency was 1 in 10 cells, because they were plated at 0.3 cells per wellm37 clones out of 384 cultures containing growing cells.

According to Poisson's distribution, about 90% of these positive cells could be derived from a single cell.These clones were divided into six subgroups according to three cell-surface-marker expressions (i.e., G-5-2, CD3, and H-2KB).Some clones were characterized in detail by using a panel of mAbs and flow cytometry.As shown in Fig. 6, cells from primitive stem cells to lymphoid and myeloid lineages were established.All clones were grown in an ES differentiation medium (see Materials and Methods section) without cytokines.They have been in continuous culture for 1 year without alteration of growth behavior and phenotype.

All clones have lost the expression of SSEA-1 antigen, which was detected by mAb MC480 (Solter and Knowles, 1978).This antigen expresses on cells of the blastocyst stage of embryogenesis and is mostly lost when embryonic cells differentiate.Some clones have retained embryonic antigens detected by mAb $7.

$7 has been described to detect pro-B cells (Hardy et al., 1989).We found that it detects a wide range of embryonic cells expressed from the blastocyst stage to the midsomite stage on most embryonic tissue (Chen et al., unpublished).Thus, I use this mAb to detect the stage of differ- entiation, the stage where the loss of antigens detected by $7 is roughly equivalent to the stage where increasing expression of H-2 class-I antigen is found.As shown in Fig. 6(A), clone 19.1 is a T-cell clone because it expresses surface antigen of T-cell phenotype, that is, CD3/, obTCR/, ,dTCR-, CD4-, CD8-.Clones 3.8 in Fig. 6(B) is as pre-B-cell clone; it expresses surface markers of precursor B-cell phenotype G52+, B220-, sIg-.Clone 3.2 in Fig. 6(C) is an immature myeloid cell clone; it expresses Mac-1 / and $7 / phenotype.Clone 3.12 in Fig. 6(D) is a possible natural killer (NK) cell or a precursor cell; it expresses NKI.1 surface marker.Other hematopoiesis markers tested are Lyl-, Ack-+, and Pgpl / (histograms not shown).This clone killed sublethally irradiated adult mice (10 out of 10) 10-14 days upon intraperi- toneal injection.Clone 3.25 in Fig. 6(E )   negative control; 2: FITC-labeled goat antimouse /-chain; 3: biotin- labeled G-5-2 and PE-labeled streptavidin; 4: FITC-labeled 14.8 (anti-B220); 5: PE-labelled GK1.5 (anti-CD4); 6: FITC-labeled 500-A2 (anti-CD3); 7: FITC-labeled 53-6-75 (anti-CD8); 8: FITC-labeled T-24 (anti-Thyl); 9: FITC-labeled mouse antirat IgG (second antibody control); 10: FITC-labeled MC480 (anti-SSEA-1); 11: rat IgG antimouse  percentage of positive cells of each individual staining antibody as indicated in each histogram, mAbs used are indicated in each panel an
The Hematopoietic Cells Arose from ES Cells, Not from Feeder Cells Extreme care was taken to ensure that hemato- poietic cells were established from ES fetuses, not from feeder cells.Primary embryonic feeder cells were derived from fibroblasts of nonlymphoid tissues.All the feeder cells were subjected to mitomycin C treatment or irradiation to render them incapable of dividing.The nondividing feeder cells perished after 1-2 passages (4-5 days) in the culture, whereas the differentiation of ES fetuses requires 11-30 days.In control experiments, feeder cells were grown under simi- lar conditions but without ES cells; nothing remotely resembling embryoid bodies .orES fetuses was found.A) and (B).Low levels of H-2 class-I antigens are expressed at the midsomite stage (day 9), and the level of expression remains low until day 13 (Ozato et al., 1985).However, ES-cell-derived hematopoietic cells express H-2Kb, Fig. 7(C), as do cells of strain 129 from which the ES cells were derived; the feeder cells do not express this H-2K b haplotype, Fig. 7(D).In quantitative immunofluoresence measurements by flow cytometry, about half of the cells were H-2K b positive, Fig. 7(E).These cells do not express H-2Dd, which corresponds to the haplotype of the feeder cells, Fig. 7(F).ES cells and feeder cells must be trypsinized to obtain, single cell suspensions, and this procedure destroys the surface antigens; hence, flow cytometric analysis of ES cells and feeder cells -was not feasible.From these results with H-2 typing, I conclude that the hematopoietic cells cannot have arisen from the feeder cells.


DISCUSSION

ES cells are cell lines cloned from the inner cell mass (ICM) of blastocysts.In this paper, I have described an embryonic organ culture system in which these totipotent cells differentiate into ES fetuses that contain various hematopoietic cells includinglymphoid cells.Upon in vivo implan- tation, they develop into more mature lymphoid tissues.I have also decribed the establishment of cell lines by using retroviruses.Mixed popu- lations of myeloid, erythroid, megakaryoid, and lymphoid cells were ide

ral cell types were established.It is intriguing that it is possi
le to infect "ES fetuses" with retroviruses containing oncogenes.Infection seemed to increase the cell mass and life-span of ES fetuses up to 6 months in culture, and the cells obtained from these ES fetuses contained lin- eages of hematopoietic cells.This system has ma.ny advantages for studying developmental immunology.

There are elements described in detail in the Materials and Methods section that are known to be essential for lymphohematopoietic stem cells to differentiate.The most important element is the "healthy" status of "undifferentiated" ES cells.LIF alone does not support any of the ES cell lines used in this study.The STO cell line, either alone or together with LIF, is not as sup- portive as the primary embryonic fibroblasts (data not shown).The periodical examination of the undifferentiated stage of ES cells by the cri- teria described is critical.Differentiation is found if the embryoid bodies are allowed to move freely, detached from a fixed environment (such as hydrophilic plastic surface of a regular tissue culture dish).The use of hydrophobic Heraeus tissue culture petri dishes seems to particularly favor the growth of ES fetuses.The daily feeding with cold medium containing high concen- trations of ood FCS also stimulates the growth.The feeding not only provides the nutrients but also the gentle stirring caused by the change of medium.

The development of ES fetuses from undiffer- entiated ES cells is not synchronous.Embryoid bodies grown in the same petri dish develop at various rates.It is important to select the ES fetuses from the original "master" dish at a criti- cal time when all three of the following struc- tures are present: blo d islands (red in color), pulsating cardiac cells, and cup-shaped struc- ture.It is especially important to look for the cup-shaped feature, which contains lymphoid cells.These selected ES fetuses are then placed in a new petri dish in groups of 5-10 and cultured further for infection with viruses and/or addition of interleukins.Prolonged culture in the master dish does not result in further differen- tiation of the embryoid bodies.Because no exogenous stimulating factors were included in the daily feeding medium, it was likely that the growth and differentiation seen were the result of factors derived from cells inside the embryoid bodies.It appeared that the selected ES fetuses contain "tissue" or its precursors important for lymphoid lineage development.Implantation of ES fetuses into adult animals gives rise to many tissues.Among them, lymphoid vessels contain- ing positive surface markers of T and B cells were identifiable (this study, Fig. 2, and Chen et al., in preparation).
Although Moloney leukemia virus long ter- minal repetitive sequences (LTR) are inactive in the undifferentiated F9 teratocarcinoma (Gorman  et al., 1985) and in ES cells (U.Chen, un- published), the receptors for retrovirus must be generated during differentiation.I have immor- talized cell lines by using retroviruses.Abelson viruses are shown to immortalize precursor B cells and many other tem cells.RIM viruses are known to immortalize plasma cells and were tumorgenic when they were injected intraperi- toneally into pristane primed adult mice (Clynes   et al., 1988), but this is the first indication that RIM may be able to immortalize hematopoietic cells.Preliminary data indicate that the vector was stably integrated into the cell lines (U.Chen et al., unpublished data).The establishment of stable clones of uncommitted stem cells, T cells, pre-B cells, myeloid cells, as well as NK cells,   indicates that lymphoid and myeloid cells exist in the retrovirus-infected hematopoietic cell population derived from ES fetuses.

It is possible to obtain beating cardiac muscle, blood islands, and lymphoid-cell positive clus- ters, the so-called "ES fetuses," from ES lines derived from several different mouse strains.The ES-D3 cell line has been shown to be germ-line transmittable (Gossler et al., 1986;Doetschman et al., 1987).Although t e ES-B6 cell line does not contribute to the germ line in vivo, I have obtained ES fetuses from this line, established hematopoietic cell lines, and found lymphocytes.Thus, the ability of a line to differentiate in vitro is not a predictor of its ability to form chimeras in vivo.

I have demonstrated that a variety of hemato- poietic cells could be developed in this organ cul- ture.It takes only 2 to 3 weeks for undifferen- tiated ES cells to progress to the fetal liver equivalent stage.ES fetuses developed in this culture contain pre-B cells capable of responding to bacterial lipopolysaccharide stimulation (U.Chen and F. Melchers, unpublished).A signif cant amount of these precursor B cells possess G-5-2 surface markers and these mitogenresponding pre-B cells are normally found between day 16-19 of fetal liver in mouse embryos (Melchers, 1977; Rolink et al., 1991;  F. Melchers, personal communication).

The IL-2 and IL-7 responsive lymphocytes appear, at the earliest, at day 12-13 during embryogenesis (B.Imhof, personal communi- cation).Adult thymus provides an environment for precursor cells to mature into intrathymic precursor cells that express mature T-cell marker CD4 (Wu et al., 1991).Fetal thymus appears earl- iest at day 10 of mouse embryogenesis and colon- ization by precursor stem cells is 1 day later, and parallels the development of fetal liver.The find- ings of IL-2-and IL-7-responsive lymphocytes and single CD4 cells from ES fetuse and the establishment of the CD3 obTCR+, CD4-CD8-T-cell clone suggest that either ES fetuses provide thymus-equivalent environment or thymic environment is not neces- FIGURE 7. Identification of H-2 haplotype of ES cells, feeder cells, and hematopoietic cells.Clusters of ES cells were derived from the inner cell mass of blastocysts originated from mouse strain 129 (H-2Kb) (hollow large arrows in A and B).The surrounding feeder cells were fibroblasts derived from the Balb/c origin (H-2Dd) (hollow small arrows in A and solid small arrows in B).Cells in A were treated with (A) fluorescence-labeled anti-H-2K and (B) anti-H-2D antibodies.Hematopoietic cells established from ES fetuses were treated with (C) fluoresence-labeled anti-H-2K (solid small arrows) and (D) anti-H-2D (hollow small arrows) antibodies.These cells were also subjected to quantitative immunofluorescent analysis by flow cytometry. (E) The histogram of the hematopoietic cells stained with anti-H-2K antibody. (F) The histogram of these cells staine

with anti-H-2D antibody.(E and F) The x-axis indicates the log
fluorescence intensity and the y-axis indicates the relative cell number.

sary for the maturation of these cells.The culture conditions described in this paper seem to be fav- orable for the development of lymphocytes equi- valent to these stages.Genetic manipulation of mouse ES cells (Thompson et al., 1989; Mansour et al., 1988;  Koller and Smithies, 1989) and germ-line trans- mission of a specific defect (Schwartzberg et al.,  1989; Thompson et al., 1989; Zijlstra et al., 1989;   DeChiara et al., 1990; Koller et al., 1990) have provided a very powerful tool for studying gene function.However, if the knockout of some genes may affect normal embryonic development and/or the survival of the fetus, germ-line trans- mission cannot be achieved.The in vitro system described here may be able to provide an alter- nate way for studying the function of these genes.ES cells were kept in a humidified incubator (37 , 10% CO2) and fed with DMEM (Gibco, Grand Island, NY, Cat.No. 041-01965M), supplemented with 4.5 g/ml glucose, 1 mM sodium pyruvate, 20 mM L-glutamine, penicillin (100 units/ml) (Gibco), streptomycin (100 units/ml) (Gibco), and 15% heat-inactivated (56 C, 30 min) preselected fetal calf serum (FCS; Seromed, Biochrom KG, Berlin, FRG, Lot No. 2L03, or Boehringer, Mannheim, FRG, Lot No. 60039202).The FCS were selected following two criteria: (a)  they give a high plating efficiency of ES cells at the undifferentiated stage (see Robertson, 1987,   p.74, for detailed procedure).The screening of FCS requires a positive control lot of FCS for comparison, and (b) they are supportive in the splenic lymphoid cell culture system of Mishell  and Dutton (1967) and are classified as "good FCS" b the Mishell's nomenclature (see Shiigi and Mishell, 1975; Mishell et al., 1978).


MATERIALS AND METHODS

ES cells

ere kept undifferentiated (criteria are described in the Results section) i
60x15-mm tis- sue culture dishes (Falcon 3002, Becton Dickin- son, Mountain View, CA) with nondividing feeder cells (see what follows).ES cells were used for less than 20 passages.ES cells, feeder cells, and all reagents are mycoplasma-free.It is neces- sary to maintain the cultures free of mycoplasma; no ES fetuses were formed whenever there was contamination.

Undifferentiated ES cells were identified in this study by (a) morphological appearance, (b) serol- ogy, and (c) ability to differentiate to hemato- poietic lineages in vitro.Morphological charac- terization of ES cells by the criteria of Robertson  (1987) was performed under the phase-contrast microscope.Morphologically, ES cell clusters resemble "pancakes" or "islands," and the edges of the individual cells are not clearly distinguish- able.Once the cells begin to grow three dimen- sionally, and the edges of the clusters begin to be distinct, the culture has already moved toward "chaotic" differentiation.Undifferentiated ES cells express a stage-specific embryonic antigen (SSEA-1), which can be recognized by a mono- clonal antibody, MC-480 Solter and Knowles,   1978).When the cells start to differentiate, the expression of SSEA-1 is lost.Immunotyping provides a very sensitive, reliable, simple, and quick way to identify differentiated ES cells before the apparent morphological disorder.

Feeder Cells for Maintenance of Undifferentiated ES Cell Lines Primary embryonic fibroblasts were used to maintain ES cell lines at the totipotent, undiffer- entiated stage.Embryonic fibroblasts were prepared from day 13-14 mouse embryos as described by Gossler et al. (1986) and Rolf Kemler (Max Planck Institute for Immunobiol- ogy, Freiburg, FRG, personal communication).Head, thymus, fetal liver, gut, and hard tissues were removed.The remaining tissues were minced into small pieces with a pair of scissors, passed through a fine sieve, and digested for 30 min in Trypsin-EDTA (lx) (Gibco).Cells from 10-15 embryos were isolated and seeded in the medium described before and grown in one 150x 15-mm tissue culture dish (Falcon 1058).After overnight culture, the nonadherent cells were removed.Fibroblasts were established after three to four passages.They were either used immedi- ately or frozen in aliquots in liquid nitrogen tanks.All feeder cells were rendered incapable of division, either by treating with mitomycin C (Sigma, St. Louis, MO, 10 flg/ml) for a minimum of 2 hr (Martin and Evans, 1975) or irradiation (3,000

d).LIF alone does not support the ES cells described in this study at the undifferen- tiated stage
The STO cell line, either alone or together with LIF, is not as supportive as the pri- mary embryonic fibroblasts.

In Vitro Differentiation of ES Cells to Simple, then Cystic Embryoid Bodies, and the Selection of ES Fetuses A modification of published protocols (Gossler et   al., 1986; Robertson, 1987) was used.Undifferen- tiated ES cells were removed from feeder cells and cultured in suspension using ES differen- tiation medium (the same medium as specified before with 10-4M 2-mercaptoethanol).Undiffer- entiated ES cells were detached fr.om the plate using trypsin-EDTA solution (Gibco), 1-2x105 cells were transferred to a 60x15-mm hydrophobic tissue culture dish (Heraeus, Heusenstamm, FRG) or if not available, to a 100x15-mm bacterial culture dish (Greine Nuringer, FRG).

The small clusters of cell aggregates were grown in suspension at 37 C in a humidified incubator with air and 5% CO2 (and not 10% CO2) for 30 days with daily change of medium (temperature of medium must be 4 C).The clusters of cells expand three dimensionally to form simple embryonic bodies.Later on, complicated struc- tures develop inside the bodies, which then become cystic embryonic bodies

rom day 11 to 30
cultures were examined, and among cystic embryoid bodies containing complex embryonic structures, ES fetuses were selected by the fol- lowing criteria: the presence of beati