differentiation of mouse embryonic stem cells in vitro: iii. morphological evaluation of tissues...

DEVELOPMENTAL DYNAMICS 197:217-226 (1993)

Differentiation of Mouse Embryonic Stem Cells In Vitro: 111. Morphological Evaluation of Tissues Developed After Implantation of Differentiated Mouse Embryoid Bodies UNA CHEN AND MARIE KOSCO Basel Institute for Immunology, CH-4005-Base1, Switzerland

ABSTRACT Mouse embryonic stem cells (ES) were allowed to differentiate in a liquid culture system. After 23 weeks, complex cystic embryoid bodies developed. These bodies were composed of several structures identified as cardiac muscle and yolk sac blood islands as well as cup- shape compartments containing a mixed population of hematopoietic stem cells. When these cystic embryoid bodies were implanted into adult mice, either subcutaneously or under the kidney capsule, they developed into various tissues. These included bone, blood vessels, cardiac muscle, nerves, and skin with hair follicles. In addition, highly differentiated, complicated tissues resem- bling intestinal epithelium with mucus glands or salivary glandular tissue were derived. The ES tissues from these in uitro developed embryoid bodies developed quickly within 2 to 3 weeks of implantation. This is in contrast to a minimal of 6 weeks for teratocarcinomas derived from embryonic carcinoma cells and/or the direct implantation of undifferentiated embryonic stem cells. Moreover, we found that there are different types of tissue developed upon different sites of implantation. The data suggest a local environment andlor growth factors are influential for ES tissue development. This system provides a possible means to purify and identify stem cells that give rise to specific tissues, and to study the factors regulating the commitment of these stem cells. 0 1993 Wiley-Liss, Inc.

Key words: ES cells, In uitro differentiation, Tis- sue stem cells

INTRODUCTION Several systems are currently used to study the dif-

ferentiation of embryonic tissues and the mechanisms of neoplasia. The most extensive studies have been car- ried out with benign and malignant teratomas (see reviews: Stevens and Roscoe, 1958; Damjanov and Solter, 1974; Damjanov et al., 1987). These teratomas arise spontaneously, or can be chemically induced or derived from embryos transplanted into extra-uterine tissue of adult mice (Damjanov et al., 1979). The teratomas contain tissue from all three primary germ layers (Martin, 1975). However, the particular tissue which develops

0 1993 WILEY-LISS. INC

appears to depend upon the source of the tumor and its history of development. Connective tissue and neurons are found in most mouse teratomas; smooth, striated and cardiac muscle or adipose tissues are found in a lower frequency; and cartilage and bone with bone marrow as well as various types of epithelium are often observed (see review by Damjanov et al., 1987). In ova- rian teratomas, the precursors of teeth have even been encountered. Although this approach is informative, the studies are limited by the fact that the tissue giving rise to the teratoma can not easily be manipulated or altered.

Differentiation of certain teratoma cell lines in vitro can lead to embryoid bodies containing yolk sac-like structures (Martin, 1975; Martin and Evans, 1975). The attachment of these structures to the solid phase provided by a tissue culture plate leads to several dis- tinct cell types (Martin, 1975). However, the potential usage of this system for differentiation is limited by the ability of the culturing conditions to mimic the in vivo environment (Martin, 1975; Robertson, 1987).

An alternative approach is to combine in vitro differentiation of mouse embryonic stem cells (ES) with subsequent implantation and development in vivo. The ES cells are derived from the inner cell mass (ICM) of blastocysts which retain their pluripotency (Gossler et al., 1986; Thompson et al., 1989; Zijlstra et al., 1989; Schwartzberg et al., 1989; Koller et al., 1989; DeChiara et al., 1990). ES cells have been shown to differentiate in vitro to form blood islands equivalent to the in vivo yolk sac stage (Doetschman et al., 1985; Robertson, 1987). Martin (1981) first reported that injection of undifferentiated ES cells subcutaneously gave rise to tumors containing cartilage and epithelial tissues (Mar- tin, 1981; Magnuson et al., 1982). In addition, she was also able to show that in vitro, differentiation of these ES cells produced giant cells, neurons, endothelial cells, cartilage, and tubules with a granular appearance (1981). More recently, we have been able to culture ES cells to the fetal liver equivalent stage using a

Received March 3, 1993; accepted June 22, 1993. Address reprint requestslcorrespondence to Dr. Una Chen, Basel

Institute for Immunology, Grenzacherstrasse 487, Postfach, CH-4005- Basel, Switzerland.

218 CHEN AND KOSCO

liquid organ culture system (Chen, 1992). Cardiac muscle, yolk sac blood islands, and a cup-shaped structure possibly containing hematopoietic cells have been observed (defined as ES fetuses, Chen et al., 1992). In order to further characterize the totipotency of these cells, cystic embryonic bodies were implanted into extra-uterine sites of adult mice. This report describes the development of various tissues in vivo derived from such ES cells differentiated in vitro.

RESULTS ES Tissues Developed From Subcutaneous Implantation

In order to study the development of tissues derived from in vitro cultures, embryoid bodies were subjected to conditions which promote differentiation (see Exper- imental Procedures for definition of these parameters). The resulting embryoid bodies were collected a t weekly intervals and evaluated for SSEA-1 expression. Undif- ferentiated ES cells used as the positive control labeled well while the embryoid bodies were negative by day 14 (data not shown). A set from each time point was individually implanted subcutaneously into one of 40 young adult male Balblc nulnu mice (10 mice per time point, 10 embryoid bodies per mouse). At this point, the embryoid bodies measured 1-2 mm in diameter. The survival rate of the surgical procedure was 90% and 3 weeks later 7 0 4 0 % . Gross ES structures ranging from 3-25 mm in length were demonstrable by 3 weeks. The mice were then sacrificed, the appropriate tissues and organs removed, frozen sections prepared, and the histology characterized by light microscopy. The frequency of gross ES structures which developed per mouse was 1-2 structures if they originated from ES embryoid bodies obtained at day 17-18 or day 25-26 of culture. No gross ES structures developed when the embryoid bodies were cultured first for less than 2% weeks or more than 5 weeks. Interestingly, although subcutaneous structures did not develop in the early points, 20% of the mice presented abnormal enlargement of the testes. This was not seen in the other groups.

Using these strategies, implanted embryoid bodies developed in vivo into two types of teratomas: 1) a simple ES tissue (Figs. 1 and 2) which develops into tissues of the local micro-environment (e.g., dermis and epidermis), and 2) a more complicated structure containing several different tissues haphazardly arranged (Figs. 2,3, and 4). No correlation occurred between the time after differentiation in culture (i.e., day 17-18 or 25-26) and development of either simple vs. complicated structures.

As shown in Figure 1, some of the embryoid bodies (2 out of 15 examined histologically) had developed subcutaneously into the simple form of tissues (3-6 mm in length). They presented structures of the integument: an epidermis of keratinized stratified squamous epithelium with an underlying dermis and hypodermis con-

taining numerous hair follicles (“F” in Fig. lB,C) and mast cells (“Ma” in Fig. 1C; see also Fig. 3F). Numer- ous cells (arrows, Fig. 1D,E) positive for Mac-1 were also present. Using immunocytochemistry, these cells were determined to be of ES cell origin (Fig. lD, single green color Mac-1; Fig. lE , double color; for red [donor MHC class I] plus green [Mac-11 gives orange color).

Upon evaluating the donor vs. host contribution of the tissue which developed within the teratomas, the majority expressed MHC class I molecules of the donor phenotype (Figs. lE , 2A,C). Occasional cells of host origin were observed in these regions (Figs. 2B,D). In the simple ES tissue developed under the skin, an extensive exchange of donor and host cells was evident (Figs. 2A,B). In addition, many of the cells with characteristics of macrophages and mast cells were of donor origin and were scattered in both the host and donor derived dermis (Figs. lD,E).

A more complicated structure ranging in size from 10-25 mm also developed in 13 out of the 15 teratomas examined histologically. These more complicated ES tissues developed muscle, epithelial, and connective tissue (Figs. 3 and 4). Vessels containing cells possibly of hematopoietic origin (HE, Fig. 3B) as well as blood (Fig. 3A; BV, Fig. 3C) were also present. Stratified squamous epithelium giving rise to several keratinocysts (K) formed by the production of keratin (k) were also identified (Figs. 3E, 4A,C) as well as clusters of melanin containing cells (Me, Fig. 3D). Mast cells were present within the peripheral tissue surrounding the tissue (Fig. 3C,F). Two highly differentiated com- binations of tissue were also identified: 1) bone development, and 2) pseudostratified epithelium containing sero-mucous glands. As shown in the left corner of Fig- ure 4A, endochondral ossification appears to have occurred producing a bone consisting of a diaphysis (B) and two ephiseal (E) ends. The cavity within the diaphysis contained cells which morphologically resemble bone marrow (BM, Fig. 4B). Nearby (Fig. 4A,C), a keratinocyst (K) was partially surrounded by pseudostratified and simple columnar epithelium containing many goblet cells (g). These cells appear to be carrying out an exocrine function by secreting substances into the lumen by a holocrine method (i.e., loss of cell mem- brane as well as secretion product; Fig. 4D). In some areas, microvilli could be observed (data not shown). Cartilage was observed in 3 out of 13 ES tissues, whereas bone development was observed in 2 out of 13 ES tissues and goblet cell containing epithelium was identified in 6 out of 13 ES tissues (Table 1). Compar- ison of other tissues are also shown in Table 1. The majority of the cells within these tissues was of donor origin (Fig. 2C), although a few host cells appeared to have migrated into the area (Fig. 2D).

The Development of ES Tissues Under the Kidney Capsule

One to two embryoid bodies were implanted under the left kidney capsule of 20 Balbk nude mice. The

DIFFERENTIATION OF TRANSPLANTED ES CELLS 219

Fig. 1 . Histological section demonstrating the development of a simple ES tissue from ES cells implanted subcutaneously. A illustrates the differentiation of donor cells (arrow) into a cystic structure within the skin of the host. B,C are higher magnifications depicting the components of this tissue which include keratinized stratified squamous epithelium and connective tissue containing hair follicles (F) and mast cells (ma). D, E

are immunofluorescent micrographs localizing cells positively stained with the FITC-labelled monoclonal antibody, anti-MAC-1 (D), together with a Texas-Red labelled anti-H-2Kb (donor haplotype, E) to determine the origin of the cells. Note that in E, the cells are double labelled (yellow) demonstrating their donor origin. A ( x 7.5), B ( x 35), and C ( x 150) are stained with Grunewald-Meyer-Giemsa; D and E ( x 150).

survival rate following surgery was 70%. After 3 weeks of implantation, 50% of the mice survived. From two operations performed, 14 separate ES tissues were obtained, most of which were used for single cell analysis in other experiments (Chen, 1992). Two of these ES

tissues were examined for histology. Disruption of the other 12 ES tissues for single cell suspension was rel- atively easy as compared to dissociating the subcutaneously derived ES tissues. The reason for this was reflected in the histology as shown in Figure 5. The

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Fig. 3. Histological section of a complicated ES tissue which developed after transplantation of ES cells subcutaneously into a Balbic nuhu mouse. A shows the entire ES tissue and the areas enlarged in B-F. Note the many luminal areas which developed within this area. In B, the luminal structures can be seen to be hollow or filled with cells, possibly of hematopoietic origin (HE). Blood vessels (BV) are apparent in C whereas

melanin containingiproducing cells are abundant in D. In E, keratinocysts (k) are observed. Mast cells (ma) can be seen in F deep into the layer of skeletal muscle (see inset for higher magnification showing striations characteristic of the skeletal muscle). Grunewald-Meyer-Giemsa staining. A ( x 3), 6 (x45), C ( x 15), D and € ( x 35), and F ( x loo), insert ( x 400).

kidney-capsule ES tissues had a thin outer covering while the subcutaneous ES tissues were made up of stratified squamous epithelium with underlying fibro- elastic connective tissue (Fig. 1). The tissues shown in Figure 5 were 4-6 mm in length. Similar to the complicated but generally unorganized structures observed

with the subcutaneous implants, several types of epithelial and connective tissues developed. Upon gross evaluation, both types of implants gave rise to ES tissues which contained many luminal structures. Many of the cysts were composed of either moist keratinizing stratified squamous, or non-ciliated cuboidal epithe-

222 CHEN AND KOSCO

Fig. 4. Enlargement of the two central boxes in Figure 3A. In A, the presence of a boney structure (B) with an epiphyseal end (E) and a keratinocyst (K) with simple pearls of keratin (k) are evident. B is a higher magnification showing the cells (BM) contained within the bone, possibly representing bone marrow. In C , an example of the gut-like epithelium

seen in many of the teratomas IS shown Note the pseudostratified and columnar nature of the cells as well as the intermittent goblet cells (9) D provides a higher definition of these structures where secretion of their products appears to be occurring (H, holocrine secretion) A ( x 45), B ( x 200), C ( x go), and D ( x 200).

lium (Fig. 5). Present within several of the lumens were mitotic cells (Mi, Fig. 5C). Cartilage (Ca, Fig. 5D) which appeared to be undergoing endochondral bone development was also observed. Melanin containing cells (Me, Fig. 5E) were scattered between various undifferentiated cell types. In addition, there were several areas within each ES tissue whose cellular organization could not be identified as any particular structure.

Fig. 5. Histological section of a kidney and the ES tissue which developed peripherally following implantation of ES cells beneath the capsule. A shows kidney and ES tissue. Note the numerous luminal areas. B demonstrates the various tissues which had developed and are enlarged in the remaining panels. Stratified squamous epithelium is indicated in B (arrow). Cells with various morphologies including mitotic figures (Mi) are seen within the lumen of a vessel made of non-ciliated cuboidal epithelium in C. Cartilage (Ca) and melanin (Me) producingicontaining cells are apparent in D. A higher magnification of these melanin positive areas are shown in E. Grunewald-Meyer-Giemsa staining. A ( x 9.5), B ( x 45), C (x350), D (x150), and E (x350).


Fig. 5.

224 CHEN AND KOSCO

TABLE 1. Frequency of Various Tissue Types Identified in the ES Tissues Which Developed

Following Subcutaneous Implant of ES Stem Cells

Tissue type Epithelium

Cuboidal Pseudostratified without holocrine secretion Pseudostratified with holocrine secretion Columnar without holocrine secretion Columnar with holocrine secretion Simple squamous Stratified squamous

Without keratinocysts With keratinocysts

Connective tissue Cartilage Bone

Without bone marrow With bone marrow

Connective tissue with glandular-like structures

Cardiac Smooth Skeletal

Nerve tissue Neurons" Astrocytesd

Miscellaneous Melanin containing areas

Muscle

(13)"

7113b 11/13 6113 2113 2113 0113

9113 10113

3113

2113 1113 6113

2113 2113 0113

11/13 12113

3113 - Mast cells 4113

"Number of ES tissues examined for histology; others were used for single cell analysis or other experiments. 'Number of positive ES tissuesinumber of total ES tissues. "Neurons were identified with the staining of mAb anti-neurofilament 68 KD (Debus et al., 1983). dAstrocytes were identified with the immunofluorescent staining of mAb anti-glial fibrillary acidic proteins (GFAP) (Debus et al., 1983).

EXPERIMENTAL PROCEDURES Culture of ES Cells and the Selection of ES Fetuses

ES male cells of strain 129 origin were a gift from Rolf Kemler, Max Planck Institute for Immunobiology, Freiburg, Germany. The liquid culture system for in vitro differentiation has been described elsewhere (Chen, 1992). In brief, ES cells were cultured in suspension without feeder cells (defined as day 0) using ES differentiation medium (i.e., DMEM, supplemented with 15% preselected heat inactivated FCS, 10-4M 2-mercaptoethanol, and antibiotics). About 1-2 x lo5 cells were grown per 60 x 15 mm hydrophobic tissue culture dish (Heraeus, Heusenstamm, Germany). These cellular clusters were cultured at 37°C in a hu- midified incubator with air and 5% CO, for 36 days. The medium was changed daily. Within 4-5 days, the cell clusters expanded three-dimensionally such that simple embryoid bodies could be observed. Four to five days later, complicated structures developed inside some of the bodies consisting of pulsating cardiac muscle and/or yolk sac blood islands. At this stage of development (between day 8-10), the structures are re- ferred to as cystic embryoid bodies (Doetschman et al.,

1985; Robertson, 1987). At the same time or slightly after the appearance of cardiac muscle and blood islands, some cystic embryoid bodies developed a cup- shaped structure containing lymphoid-like cells. These continued to develop until day 36 when no more changes appeared morphologically in the cultures. The structures used for implantation were taken at day 11- 12, day 17-18, day 25-26, and day 35-36 of culture.

Implantation of ES Fetuses Into Adult Mice Four- to six-week-old male nulnu Balbic mice (Roche

Animal Colony, Kaiseraugst, Switzerland) were used for implantation. This was performed with mice under general anaesthesia administered by intraperitoneal injection of Avertin (8 mg per 0.4 ml per 10 g of body weight; Ardreich, Steinheim, Germany). Forty mice received implants subcutaneously in the lumbar region superficial to the spinal column using general surgical procedures. Another group of 10 mice received implants under the capsule of their left kidney using the procedure described in Robertson (1987). Three weeks post surgery, most of the mice were sacrificed. Organs and tissues were removed and prepared for cryostat sectioning. Histological evaluation of the tissues was conducted on Giemsa stained sections using a Zeiss Ax- iophot light microscope.

Characterization of Cell Types by Immunohistochemistry

To evaluate the presence and distribution of host vs. donor macrophages within the subcutaneously implanted ES tissues, sections were incubated with the following antibodies: FITC-labelled M1/70, which recognizes Mac-1 antigen on macrophages (Springer et al., 1979); biotin-labelled B8-24-3, which recognizes H-2Kb class I molecules on all cells of the H-2' haplotype (do- nor phenotype; Koehler et al., 1981); biotin-labelled 15- 5-5, which recognizes H-2Dd class I molecules on cells of the H-2Dd haplotype (host phenotype; Ozato et al., 19801, and Texas Red-labelled streptavidin (Southern Biotechnology, Birmingham, AL). To evaluate the distribution of donor vs. host derived tissues, biotin-labelled B8-24-3 (anti-H-2Kb, donor phenotype, as above) and Texas Red-labelled streptavidin (as above), and biotin-labelled 15-5-5 (anti-H-2Dd, host phenotype, as above), and FITC-labelled streptavidin (Amersham, Arlington Heights, IL) were used. Neurons were identified with the staining of FITC labeled mAb anti-neurofilament 68 KD (Debus et al., 1983). Astrocytes were identified with the immunofluorescent staining of FITC labeled mAb anti-glial fibrillary acidic proteins (GFAP) (Debus et al., 1983). Immunofluorescence was evaluated under UV illumination on a Zeiss Axioscope 20 using the appropriate excitation filters.

DISCUSSION The implantation of cultured embryoid bodies into

nude mice can give rise to simple and complicated ES tissues. Out of the 10 embryoid bodies subcutaneously


implanted per mouse, at least one developed into an ES tissue in each mouse. On occasion, two separate ES tissues were also observed but more often the single structure seen appeared to be a fusion of two or more embryoid bodies. Interestingly, although embryoid bodies of one particular time group were all collected and implanted on the same day, only 1 to 2 ES tissues developed per mouse and varied in size between animals. The difference in size may reflect different envi- ronmental factors in vitro and in vivo, causing variable rates of growth. Also, the possibilities exist that some of the implanted embryoid bodies fused and some of the individual cells may have migrated elsewhere. Alter- natively, some of the embryoid bodies may have been absorbed.

The tissues that these ES structures were composed of varied and no definitive difference could be corre- lated with tissue development derived from embryoid bodies from day 17-18 vs. day 25-26 cultures. Struc- tures derived from ectoderm (epidermis, dermis), me- soderm (muscle, cartilage, bone), and endoderm (glandular epithelium) were obtained. In one example shown in Figure 1, the subcutaneously located ES tissue gave rise to highly organized skin whereas the others were arranged in no particular fashion. ES tissues arising from subcutaneous implants and those under the kidney capsule upon gross examination displayed many luminal structures. Out of the fourteen ES tissues dissected from the kidney capsule implants, none consisted of a dense capsule or gave rise to hair-like structures. The observation that many of the subcutaneous implants gave rise to stratified squamous epithelium hints at the possibility that factors derived from the local environment influenced the array of tissues produced. Interestingly, the ES tissue which gave rise to skin was the most superficially placed subcutaneous implant (see Fig. 1A). The pseudostratified epithelium with goblet cells varied as to whether microvilli could be observed. However, cilia was never associated with this tissue. These morphological characteristics suggest that either gut associated epithelium or salivary glandular tissue was developing. Since the normal an- atomical location of this tissue is absent in these ES tissues, the key to its final differentiated structure is left open. The factors leading to such differentiation are currently under investigation.

The particular stage of development and differentiation reached by the cells a t the time of implant is a question we would like to address. The evidence suggests that the cells have achieved a lineage specific or committed stage since: (1) the tissue which develops is fairly complicated and differentiated; (2) the structures develop within a period of 2-3 weeks; this differs from the minimal 6 week incubation time required for tissue development of teratocarcinoma cell lines following implantation (see review by Damjanov et al., 1979,1987); (3) there is an assortment of tissue types ranging from simple epithelium to bone development; and (4) neither the embryoid bodies after 2 weeks of culture nor the ES

tissues stain with antibodies against SSEA-1. The expression of this antigen is lost when the ES cells differentiated (Solter and Knowles, 1978). Another fact which argues for a lineage pre-determined stage vs. a totipotent set of cells is that we do not observe the induction of all tissue types. Since our liquid culture system requires the cells to differentiate in vitro to a state where hematopoietic cells, cardiac cells, and a yolk-sac are present, we feel that much of our implanted population is probably no longer totipotent.

The development of bone and bone marrow within the more complicated ES tissues was also interesting. Bone and cartilage are known to provide essential el- ements for the development of hematopoietic stem cells (see reviews in Zipori, 1989). Bone and cartilage, on the other hand, already develop from embryoid bodies implanted from the earlier culture time (day 17-18). More work needs to be done in order to identify the nature of the stromal and lymphoid compartment in this area.

Using transplanted immature mouse embryos between the stem cell to egg-cylinder stage or using embryonic teratocarcinoma cell lines, several investiga- tors have shown the development of various tissues in vivo (see reviews by Damjanov et al., 1979, 1987). These include many observed with our systems. The advantage of the system presented here is that the ES cell cultures can be manipulated in vitro and the subsequent effect on differentiation and development of tissues observed. The observation of implanted cystic embryoid bodies which mature into the various tissues suggests that it may be possible to obtain untrans- formed precommitted tissue specific stem cells by com- bining in vitro cultures of ES cells with subsequent implantation into adult mice. This should provide a means to dissect the pattern of development for lineage specific stem cells and committed precursor cells. In addition, these studies may facilitate future applica- tions for somatic gene therapy in skin injury, and hair follicle and bone development.

ACKNOWLEDGMENTS We are grateful for the excellent technical assistance

of Hoyan Mok and Katherine Haft, the gift of antibodies by Uwe Staerz, the graphic work of Hans-Peter Stahlberger, and the preparation of this manuscript by Jenny Zangger and Nicole Schoepflin. We acknowl- edge the generous gifts of embryonic stem cells used in this study from Rolf Kemler, and discussions in estab- lishing ES cell technology by Rolf Kemler, Colin Stew- art, and Elizabeth Robertson. We appreciate the discussion and reading of this manuscript by Shigeo Ekino, Beat Imhof, J im Kaufman, Klaus Karjalainen, and Charley Steinberg. We also would like to thank Andreas Szakal and Caroline Jackson, Medical College of Virginia, and Harald Stein, University of Free Ber- lin, for helpful discussions in evaluating the histological preparations. The Base1 Institute of Immunology was founded and is supported by F. Hoffmann-La Roche, Ltd.

226 CHEN AND KOSCO

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