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In vitro identification of the trophoblastic stem cell of the human villous placenta
CURRENT INVESTIGATION
In vitro identification of the trophoblastic stem cell of the human villous placenta ROLAND A. PATTILLO, M.D.* GEORGE O. GEY, M.D. ELEANOR DELFS, M.D . RICHARD F. MATTINGLY, M . D . Baltimore, Maryland, and Milwaukee, Wisconsin An in vitro search for the epithelial stem cell of the placenta has been made. The cytotrophoblast has been selected for tissue cuiture from germinal beds of cell column cytotrophoblast of the first and early second trimester placenta. These beds have been shown by Wislocki and Padykula 9 to represent the generative source of new and expanding placental villi in vivo. The morphologic characteristics of these cell columns confirm their cytotrophoblast composition in hematoxylin and eosin sections. Under the dissecting microscope comparable areas were selected and explanted in tissue cultures. Primary growth of cytotrophoblast with characteristic microvilli migrated from these cell columns and have been followed by daily microscopic observation. HeG was detected in the medium in which these cells WHe grown lor a limited period of time, roughly comparable to the period during which mitosis occurred in cytotrophoblast. It is concluded that these cells represent the functioning cytotrophoblast of the normal placenta.
MAN Y PRE V IOU S reports of growth of the human placenta in tissue culture have recorded a variety of cell types and made some attempts to identify the cell responsible for HeG production. t , 2, 8 The critical report of Gey, Jones, and Hellman 4 identified gonadotropin hormone in the media in which placental tissue was grown, thus establishing the placenta rather than the maternal pituitary as the source of pregnancy gonadotropin. In any organ system the variability and complexity of cell types migrating in vitro from a primary explant often present a diffi-
From the Finney-Howell Cancer Research Laboratory, Department of Surgery, The Johns Hopkins Medical Institutions, Baltimore, and the Department of Gynecology and Obstetrics, Marquettte University, Milwaukee. This research was supported in part by Public Health Contract No. 436452, Damon Runyon Fund, American Cancer Society (E-273-I) and Milwaukee Division and Council fOT Tobacco Research. *Postdoctoral Research Fellow, Department of Surgery, The Johns Hopkins University and Hospital. Present address: Department 0/ Gynecology and Obstetrics, Marquette University, Milwaukee, Wisconsin 53226.
582
Volume 100 Number 4
Trophoblastic stem cell
cult task of identification of the specific cells of interest. Sometimes, very few means are available to make such an identification, especially when functional markers are rapidly deleted. The presence of a marker, such as melanin or collagen, or the elaboration of a functional product, such as hormone or immunoglobulin, represents the few cases in which identification is possible. Some cells may take up the specific products of other celIs and present a false image. The initial question to be answered was which ceIJs of the normal early placenta have proliferative capacity in vivo and in vitro. The work of Tao and Hertig,5 Richart,6 Midgley and Pierce/ and Brewers demonstrating the maturation of Langhans' cytotrophoblast to syncytiotrophoblast established the immature nature of the cytotrophoblast and the end stage character of the syncytiotrophoblast. The potential of the mature
villus in vivo and in vitro for trophoblastic proliferation is small in contrast to the large potential for fibroblastic proliferation from components of the villus stroma. The possible nonepithelial contributions of the villus in vitro include stromal connective tissue, endothelium, Hofbauer cells, and reticuloendothelial cells. The identification of those cells of the placenta which give rise to new villi has been designated by Wislocki as cell column cytotrophoblastO (Fig. I). These primary villi reach maturity (tertiary villi) when connective tissue and vascular endothelium migrate into their cores. P.A.S. stains done on the conceptus of less than one month show an extremely high glycogen deposit in cell column cytotrophoblast which is present to only a limited degree in Langhans' cytotrophoblast. This feature is associated with the more undifferentiated nature of the former cell. Mitotic activity in these columns is abundant. Prior to differentiation, these germinal centers of cell columns have the greatest potential for giving rise to replicating cytotrophoblast.
Fig. 1. Section of normal first trimester placental villi from ectopic pregnancy specimen M 1328. Homogeneous sheets of cell column cytotrophoblast at the blind end comprise the primary villus, the tertiary portion below having a differentiated outer layer with connective tissue and blood velsels having migrated in and formed a core. (Hematoxylin and eosin. x300.)
In
villous placenta
583
Materials and methods Sheets of cell column cytotrophoblast could be identified in first and early second trimester placentas obtained from ectopic pregnancies and therapeutic abortions. These represent the germinal bed of the advancing villus (Fig. I). More or less pure sheets of undifferentiated cytotrophoblast, morphologically similar to the histologic pattern seen in invading choriocarcinoma, could be found in these sheets. The large vesicular nucleus with relatively pale cytoplasm reflected this image. Abundant glycogen was found in these cytotrophoblastic cells in contrast to the Langhans' cytotrophoblast of the tertiary villus. Under the dissecting microscope in the living specimen, the columns could be identified as the broad, blunt, frayed tips of individual villi. Using crossed scalpels with No. 11 blades in a sterile Petri dish, these villi were transected at their base and transferred to reconstituted collagen matrix in
584
Pattillo et 01.
previously prepared collagen-coated roller tubes. The collagen tubes were prepared by a modification of the method of Ehrmann and GeylO and were equilibrated with growth medium. The medium initially consisted of 70 per cent Waymouth's 752/ 1 supplemented with
F.bruary 15, 1968
Am.
J. Obst. & Gynec.
pooled 30 per cent human placental cord serum. It was later detected that the cells showed intense granularity and impaired growth with this medium. At this time the composition was changed to 50 per cent Waymouth's 752/ 1, 40 per cent Gey's balanced salt solution (BSS-G), and 10 per
Fig. 2. Living tissue culture outgrowth from the tip of a column of cytotrophoblast which had been selected under the dissecting microscope from an area comparable to Fig. I. Elongated microvilli extend from the cytotrophoblast of this column. Near the base, two young tertiary villus buds with no growth occurring from their differentiated syncytial coverings. ( XI50.)
Fig. 3. Disrupted villus (below) with outgrowth of granular, triangular, and multiangular cell type from the villus stroma where elongated spindle cells also appeared. (x50. )
Volume 100 Number ..
Trophoblastic stem cell in villous placenta
cent human placental cord serum. Trials using maternal sera and fetal calf sera were not encouraging. Supplements of placental extracts, decidual extracts, and umbilical cord extract with its high arid mucopolysaccharide content were each tried without success. Tissue source. Twenty-one fresh placental specimens were explanted in culture. Spe('i_ mens were received from the Department of Gynecology and Obstetrirs, Marquette School of Medicine at Milwaukee County Hospital, transported in nutrient media at 4° C ., and from the Obstetrical Service, The Johns Hopkins Hospital. The specimens, consisting of 1 to 2 film. sections of placental villi, were placed in sterile Petri dishes containing small pools of growth medium and examined under the dissecting microscope. Individual villi with broad, blunt ends containing clusters of advancing cell column cytotrophoblast were identified. These were transected with crossed scalpels at the base and explanted with sterile curved-tip pipettes onto collagen
slants prepared in roller tubes. The roller tubes were prepared by pi petting 0.75 ml. of dialyzed tropocollagen, previously extracted from the tail tendons of isologous PAG albino rats,* along the lower third of 150 mm. x 16 mm. roller tubes. The collagen was gelated by the vapors of NH 4 0H moistened cotton stoppers exposed for 15 minutes. The reconstituted collagen was washed with four changes of distilled water and equilibrated with balanced salt solution in the roller drum at 37° C. All cultures were explanted on both collagen matrix and directly on to the glass surface of the roller tube. The roller tube glass surface proved unfavorable to the growth of trophoblastic epithelium.
Fig. of. Colony form~d from cytotrophoblast aitt'r migration from a cell column cytotrophoblast tip. (xljO.)
585
Relultl
The transected end of the villus with its advancing cell column cytotrophoblast could easily be identified under the microscope in roller tubes of the living cultures (Fig. 2) . Comparison of the living culture and original tissue from which it was derived (Fig. 1) showed maintenance of the microscopic architecture. The cell column cytotrophoblast migrated directly from the advancing tip and displayed multiple microvilli of the asteroid or burr cell described by Gey in 1938. It was possible to identify this cell type consistently migrating from relatively pure sheets of cytotrophoblast. Growth occurred only at the tip of the advancing villus, not from the side wall of the villus where differentiation to syncytial layer had occurred (Fig. 2). No migration occurred from mature tertiary villi which have been shown by Tao and Hertig and others to undergo maturation from Langhans' cytotrophoblast to syncytiotrophoblast within the villus. Replication in the cell column cytotrophoblast, however, was limited to 2 to 3 cell divisions with a two- to threefold increase in the cytotrophoblast columns. Although these continued to survive for periods up to 3 months, no further replication occurred, and, therefore, we have not succeeded in establishing a strain from the normal trophoblast. When the villus was found disrupted, mi·Philad~lphia
albino Gey Ilrain.
586
Pattillo et 01.
Am.
gration of the second cell type occurred. This was initially a granular, broad spindle, or multiangular cell which multiplied rapidly and could be kept up to 7 months in culture with biweekly subcultures (Fig. 3). Multinucleated giant cells and macrophages were also apparent. Lysis of col-
February 15, 1968 & Gynec.
J. Obst.
lagen was consistently seen with growth of trophoblastic epithelium, thus closely paralleling the pattern of the implanting blastocyst. Honnone assays. Assays were performed using the Delfs' immature rat uterine weight method." HCG was extracted from the tis-
.
,
Fig. 5. Blebbing and ballooning of cell membranes from small buds of early but mature tertiary villus outpouchings. No new cell proliferation occurred from this syncytial surface. (x200.)
Table I. Outcome of placental specimen in tissue culture CultuTe identification
Ve St Ad Hu M132B Ma Po Jo Ac Ma Ha Be Qu Ba Tu Shi Ri Shi Smi Ba Ha Jun Man Ca Hal Ma Se Li Gri Ga Mi Ju St M213A Co Th De Ca Ma Tu
Ori~in
of tissue
Ectopic pregnancy Ectopic pregnancy Ectopic pregnancy Therapeutic abortion Ectopic pregnancy Therapeutic abortion Ectopic pregnancy Ectopic pregnancy Ectopic pregnancy Ectopic pregnancy Ectopic pregnancy Therapeutic abortion Ectopic pregnancy Ectopic pregnancy Ectopic pregnancy Therapeutic abortion Ectopic pregnancy Ectopic pregnancy Therapeutic abortion Ectopic pregnancy Therapeutic abortion
Trimester
First First First Second First First First First First First First First First First First First First First First First First
Growth in culture + + +
+
+ + + + + + + +
I
Type 0/ cellular outgrowth Cytotrophoblast Mesenchymal
+ +
+
+ + +
+ +
+
+
+ + + + +
+ + + + + +
+ +
+
+
+ + + + + + + + + + + + + + + + + + + + +
Volume 100 Number 4
Fill. 6. a, After a prolonged period in culture the
microvilli of the cytotrophobl;ut expanded ,o'nger
~d broader procellel. (X150.) b, Higher power
view of a. (x300.)
Trophoblastic stem cell
In
villous placenta
587
sue culture medium by precipitation with four volumes of 95 per cent ethyl alcohol after five washes with equal volumes of ether to remove steroids. Assays were carried out on fluid which had been in contact with celIs for 3 days. The precipitate was dried and stored at 5° C. until assayed when it was redissolved in normal saline and administered by subcutaneous injection over a 48 hour period. The animals were kilIed at 72 hours and the uteri and ovaries examined for increase in weight above the standard normal for the assay. All assays were positive for gonadotropin on cultures showing cytotrophoblast growth; however, these became negative after 2 to 3 weeks on single un pooled tissue fluid assays. Morphologic characteristics of the cytotrophoblast. A total of 21 specimens from ectopic placentas and therapeutic abortions were explanted in primary culture (Table I). Fifteen of the 21 exhibited characteristic features of the cytotrophoblast. The remaining 6 specimens showed no growth or only fibroblasts with negative hormone assays. The features of the cytotrophoblast are best seen in the early period of migration from transec ted germinal beds of cell column cytotrophoblast at 2 to 3 days. Markedly elongated asteroid processes of the cytotrophoblast extend from the cell column masses as these cells migrated from the central mass. Colonies were formed only from the blind end of the viIIus where the germinal bed of cytotrophoblast had been localized (Figs. 1 and 4). Mature tertiary villi with a differentiated syncytial covering showed no growth or colony formation, although ballooning or blebbing of the cell membrane was readily seen (Fig. 5). After approximately 2 weeks in culture, no additional expansion of the cell column takes place, and the cytotrophoblast begins to extend processes broadly in all directions, giving rise to more asteroid or burrlike processes (Figs. 5 and 6, 12 and b). These are readily identifiable; however, they persist in culture but do not enter mitosis except in the immediate period following initial ex-
588 Pattillo et 01.
plantation. Because of lack of mitosis and replication, subcultures have not been possible, and maintenance of these cells in the same culture vessel up to 3 months has not resulted in adaptation followed by continued replication as has been shown to occur in the malignant cytotrophoblast. 12 In one placental specimen, it was possible to maintain cultures for 7 months with a total of 14 subcultures (Fig. 3). However, these were not the asteroid cytotrophoblast but rather a population of broad polygonal and multiangular type cells, probably derived from the connective tissue core of the villus. Comment
The many attempts to establish stable strains of functioning normal diploid cells in culture have met with no success. The first step in such an endeavor is the accurate identification of the cell in question. This must be followed by devising means to isolate and propagate that particular cell type. The presence of a functional marker or product provides an ideal means of identify-
F,·bruary 15, 1968
Am.
J.
Obst. & Gynec.
ing and following trophoblastic cells. However, some differentiation may prevent the possibility of developing a continuously replicating normal cell strain. On the contrary, the malignant trophoblast because of the already existing neoplastic transformation has the acquired characteristic of continuous replication. It may be postulated that the normal gonadotropin hormone of the normal trophoblast induces its own differentiation. Using this reasoning the normal product of the trophoblast would prevent continuous replication of these cells by inducing their differentiation. This might account for the temporary detection of HCG in the medium followed shortly by cessation of mitosis and decline of hormone production. Because of the limited cell populations which were derived from selection of cell column cytotrophoblast for culture, inadequate numbers were available for a thorough study of their state of differentiation, and this explanation must therefore remain speculative.
REFERENCES
1. Stewart, H. L., et al.: J. Clin. Endocrino!. 8: 175,1948. 2. Thiede, H. A.: AM. J. OBST. & GYNEC. 79: 636, 1960. 3. Soma, H., and Hertig, A.: AM. J. OBST. & GYNEC. 18: 704, 1961. 4. Gey, G. 0., Seegar Jones. G., and Hellman, L. M.: Science 88: 306, 1938. 5. Tao, Tien-Wen, and Hertig, A.: Am. J. Anat. 116: 315, 1964. 6. Richart, R.: Proe. Soc. Exper. BioI. & Med. 106: 829, 1961.
7. Midgley, A. R., and Pierce, G. B.: Science 141: 349, 1963. 8. Brewer, J. I.: Am. J. Anat. 61: 429, 1937. 9. Wislocki. G., and Padykula, H.: In Young, W., editor: Sex and Internal Secretions, Baltimore, 1961, The Williams & Wilkins Company, p. 883. 10. Ehrmann, R. L., and Gey, G. 0.: ]. Nat. Cancer Inst. 16: 1375, 1956. 11. Delfs, E.: Endocrinology 28: 196, 1941. 12. Pattillo, R., and Gey, G. 0.: Science. In press.
In vitro identification of the trophoblastic stem cell of the human villous placenta ROLAND A. PATTILLO, M.D.* GEORGE O. GEY, M.D. ELEANOR DELFS, M.D . RICHARD F. MATTINGLY, M . D . Baltimore, Maryland, and Milwaukee, Wisconsin An in vitro search for the epithelial stem cell of the placenta has been made. The cytotrophoblast has been selected for tissue cuiture from germinal beds of cell column cytotrophoblast of the first and early second trimester placenta. These beds have been shown by Wislocki and Padykula 9 to represent the generative source of new and expanding placental villi in vivo. The morphologic characteristics of these cell columns confirm their cytotrophoblast composition in hematoxylin and eosin sections. Under the dissecting microscope comparable areas were selected and explanted in tissue cultures. Primary growth of cytotrophoblast with characteristic microvilli migrated from these cell columns and have been followed by daily microscopic observation. HeG was detected in the medium in which these cells WHe grown lor a limited period of time, roughly comparable to the period during which mitosis occurred in cytotrophoblast. It is concluded that these cells represent the functioning cytotrophoblast of the normal placenta.
MAN Y PRE V IOU S reports of growth of the human placenta in tissue culture have recorded a variety of cell types and made some attempts to identify the cell responsible for HeG production. t , 2, 8 The critical report of Gey, Jones, and Hellman 4 identified gonadotropin hormone in the media in which placental tissue was grown, thus establishing the placenta rather than the maternal pituitary as the source of pregnancy gonadotropin. In any organ system the variability and complexity of cell types migrating in vitro from a primary explant often present a diffi-
From the Finney-Howell Cancer Research Laboratory, Department of Surgery, The Johns Hopkins Medical Institutions, Baltimore, and the Department of Gynecology and Obstetrics, Marquettte University, Milwaukee. This research was supported in part by Public Health Contract No. 436452, Damon Runyon Fund, American Cancer Society (E-273-I) and Milwaukee Division and Council fOT Tobacco Research. *Postdoctoral Research Fellow, Department of Surgery, The Johns Hopkins University and Hospital. Present address: Department 0/ Gynecology and Obstetrics, Marquette University, Milwaukee, Wisconsin 53226.
582
Volume 100 Number 4
Trophoblastic stem cell
cult task of identification of the specific cells of interest. Sometimes, very few means are available to make such an identification, especially when functional markers are rapidly deleted. The presence of a marker, such as melanin or collagen, or the elaboration of a functional product, such as hormone or immunoglobulin, represents the few cases in which identification is possible. Some cells may take up the specific products of other celIs and present a false image. The initial question to be answered was which ceIJs of the normal early placenta have proliferative capacity in vivo and in vitro. The work of Tao and Hertig,5 Richart,6 Midgley and Pierce/ and Brewers demonstrating the maturation of Langhans' cytotrophoblast to syncytiotrophoblast established the immature nature of the cytotrophoblast and the end stage character of the syncytiotrophoblast. The potential of the mature
villus in vivo and in vitro for trophoblastic proliferation is small in contrast to the large potential for fibroblastic proliferation from components of the villus stroma. The possible nonepithelial contributions of the villus in vitro include stromal connective tissue, endothelium, Hofbauer cells, and reticuloendothelial cells. The identification of those cells of the placenta which give rise to new villi has been designated by Wislocki as cell column cytotrophoblastO (Fig. I). These primary villi reach maturity (tertiary villi) when connective tissue and vascular endothelium migrate into their cores. P.A.S. stains done on the conceptus of less than one month show an extremely high glycogen deposit in cell column cytotrophoblast which is present to only a limited degree in Langhans' cytotrophoblast. This feature is associated with the more undifferentiated nature of the former cell. Mitotic activity in these columns is abundant. Prior to differentiation, these germinal centers of cell columns have the greatest potential for giving rise to replicating cytotrophoblast.
Fig. 1. Section of normal first trimester placental villi from ectopic pregnancy specimen M 1328. Homogeneous sheets of cell column cytotrophoblast at the blind end comprise the primary villus, the tertiary portion below having a differentiated outer layer with connective tissue and blood velsels having migrated in and formed a core. (Hematoxylin and eosin. x300.)
In
villous placenta
583
Materials and methods Sheets of cell column cytotrophoblast could be identified in first and early second trimester placentas obtained from ectopic pregnancies and therapeutic abortions. These represent the germinal bed of the advancing villus (Fig. I). More or less pure sheets of undifferentiated cytotrophoblast, morphologically similar to the histologic pattern seen in invading choriocarcinoma, could be found in these sheets. The large vesicular nucleus with relatively pale cytoplasm reflected this image. Abundant glycogen was found in these cytotrophoblastic cells in contrast to the Langhans' cytotrophoblast of the tertiary villus. Under the dissecting microscope in the living specimen, the columns could be identified as the broad, blunt, frayed tips of individual villi. Using crossed scalpels with No. 11 blades in a sterile Petri dish, these villi were transected at their base and transferred to reconstituted collagen matrix in
584
Pattillo et 01.
previously prepared collagen-coated roller tubes. The collagen tubes were prepared by a modification of the method of Ehrmann and GeylO and were equilibrated with growth medium. The medium initially consisted of 70 per cent Waymouth's 752/ 1 supplemented with
F.bruary 15, 1968
Am.
J. Obst. & Gynec.
pooled 30 per cent human placental cord serum. It was later detected that the cells showed intense granularity and impaired growth with this medium. At this time the composition was changed to 50 per cent Waymouth's 752/ 1, 40 per cent Gey's balanced salt solution (BSS-G), and 10 per
Fig. 2. Living tissue culture outgrowth from the tip of a column of cytotrophoblast which had been selected under the dissecting microscope from an area comparable to Fig. I. Elongated microvilli extend from the cytotrophoblast of this column. Near the base, two young tertiary villus buds with no growth occurring from their differentiated syncytial coverings. ( XI50.)
Fig. 3. Disrupted villus (below) with outgrowth of granular, triangular, and multiangular cell type from the villus stroma where elongated spindle cells also appeared. (x50. )
Volume 100 Number ..
Trophoblastic stem cell in villous placenta
cent human placental cord serum. Trials using maternal sera and fetal calf sera were not encouraging. Supplements of placental extracts, decidual extracts, and umbilical cord extract with its high arid mucopolysaccharide content were each tried without success. Tissue source. Twenty-one fresh placental specimens were explanted in culture. Spe('i_ mens were received from the Department of Gynecology and Obstetrirs, Marquette School of Medicine at Milwaukee County Hospital, transported in nutrient media at 4° C ., and from the Obstetrical Service, The Johns Hopkins Hospital. The specimens, consisting of 1 to 2 film. sections of placental villi, were placed in sterile Petri dishes containing small pools of growth medium and examined under the dissecting microscope. Individual villi with broad, blunt ends containing clusters of advancing cell column cytotrophoblast were identified. These were transected with crossed scalpels at the base and explanted with sterile curved-tip pipettes onto collagen
slants prepared in roller tubes. The roller tubes were prepared by pi petting 0.75 ml. of dialyzed tropocollagen, previously extracted from the tail tendons of isologous PAG albino rats,* along the lower third of 150 mm. x 16 mm. roller tubes. The collagen was gelated by the vapors of NH 4 0H moistened cotton stoppers exposed for 15 minutes. The reconstituted collagen was washed with four changes of distilled water and equilibrated with balanced salt solution in the roller drum at 37° C. All cultures were explanted on both collagen matrix and directly on to the glass surface of the roller tube. The roller tube glass surface proved unfavorable to the growth of trophoblastic epithelium.
Fig. of. Colony form~d from cytotrophoblast aitt'r migration from a cell column cytotrophoblast tip. (xljO.)
585
Relultl
The transected end of the villus with its advancing cell column cytotrophoblast could easily be identified under the microscope in roller tubes of the living cultures (Fig. 2) . Comparison of the living culture and original tissue from which it was derived (Fig. 1) showed maintenance of the microscopic architecture. The cell column cytotrophoblast migrated directly from the advancing tip and displayed multiple microvilli of the asteroid or burr cell described by Gey in 1938. It was possible to identify this cell type consistently migrating from relatively pure sheets of cytotrophoblast. Growth occurred only at the tip of the advancing villus, not from the side wall of the villus where differentiation to syncytial layer had occurred (Fig. 2). No migration occurred from mature tertiary villi which have been shown by Tao and Hertig and others to undergo maturation from Langhans' cytotrophoblast to syncytiotrophoblast within the villus. Replication in the cell column cytotrophoblast, however, was limited to 2 to 3 cell divisions with a two- to threefold increase in the cytotrophoblast columns. Although these continued to survive for periods up to 3 months, no further replication occurred, and, therefore, we have not succeeded in establishing a strain from the normal trophoblast. When the villus was found disrupted, mi·Philad~lphia
albino Gey Ilrain.
586
Pattillo et 01.
Am.
gration of the second cell type occurred. This was initially a granular, broad spindle, or multiangular cell which multiplied rapidly and could be kept up to 7 months in culture with biweekly subcultures (Fig. 3). Multinucleated giant cells and macrophages were also apparent. Lysis of col-
February 15, 1968 & Gynec.
J. Obst.
lagen was consistently seen with growth of trophoblastic epithelium, thus closely paralleling the pattern of the implanting blastocyst. Honnone assays. Assays were performed using the Delfs' immature rat uterine weight method." HCG was extracted from the tis-
.
,
Fig. 5. Blebbing and ballooning of cell membranes from small buds of early but mature tertiary villus outpouchings. No new cell proliferation occurred from this syncytial surface. (x200.)
Table I. Outcome of placental specimen in tissue culture CultuTe identification
Ve St Ad Hu M132B Ma Po Jo Ac Ma Ha Be Qu Ba Tu Shi Ri Shi Smi Ba Ha Jun Man Ca Hal Ma Se Li Gri Ga Mi Ju St M213A Co Th De Ca Ma Tu
Ori~in
of tissue
Ectopic pregnancy Ectopic pregnancy Ectopic pregnancy Therapeutic abortion Ectopic pregnancy Therapeutic abortion Ectopic pregnancy Ectopic pregnancy Ectopic pregnancy Ectopic pregnancy Ectopic pregnancy Therapeutic abortion Ectopic pregnancy Ectopic pregnancy Ectopic pregnancy Therapeutic abortion Ectopic pregnancy Ectopic pregnancy Therapeutic abortion Ectopic pregnancy Therapeutic abortion
Trimester
First First First Second First First First First First First First First First First First First First First First First First
Growth in culture + + +
+
+ + + + + + + +
I
Type 0/ cellular outgrowth Cytotrophoblast Mesenchymal
+ +
+
+ + +
+ +
+
+
+ + + + +
+ + + + + +
+ +
+
+
+ + + + + + + + + + + + + + + + + + + + +
Volume 100 Number 4
Fill. 6. a, After a prolonged period in culture the
microvilli of the cytotrophobl;ut expanded ,o'nger
~d broader procellel. (X150.) b, Higher power
view of a. (x300.)
Trophoblastic stem cell
In
villous placenta
587
sue culture medium by precipitation with four volumes of 95 per cent ethyl alcohol after five washes with equal volumes of ether to remove steroids. Assays were carried out on fluid which had been in contact with celIs for 3 days. The precipitate was dried and stored at 5° C. until assayed when it was redissolved in normal saline and administered by subcutaneous injection over a 48 hour period. The animals were kilIed at 72 hours and the uteri and ovaries examined for increase in weight above the standard normal for the assay. All assays were positive for gonadotropin on cultures showing cytotrophoblast growth; however, these became negative after 2 to 3 weeks on single un pooled tissue fluid assays. Morphologic characteristics of the cytotrophoblast. A total of 21 specimens from ectopic placentas and therapeutic abortions were explanted in primary culture (Table I). Fifteen of the 21 exhibited characteristic features of the cytotrophoblast. The remaining 6 specimens showed no growth or only fibroblasts with negative hormone assays. The features of the cytotrophoblast are best seen in the early period of migration from transec ted germinal beds of cell column cytotrophoblast at 2 to 3 days. Markedly elongated asteroid processes of the cytotrophoblast extend from the cell column masses as these cells migrated from the central mass. Colonies were formed only from the blind end of the viIIus where the germinal bed of cytotrophoblast had been localized (Figs. 1 and 4). Mature tertiary villi with a differentiated syncytial covering showed no growth or colony formation, although ballooning or blebbing of the cell membrane was readily seen (Fig. 5). After approximately 2 weeks in culture, no additional expansion of the cell column takes place, and the cytotrophoblast begins to extend processes broadly in all directions, giving rise to more asteroid or burrlike processes (Figs. 5 and 6, 12 and b). These are readily identifiable; however, they persist in culture but do not enter mitosis except in the immediate period following initial ex-
588 Pattillo et 01.
plantation. Because of lack of mitosis and replication, subcultures have not been possible, and maintenance of these cells in the same culture vessel up to 3 months has not resulted in adaptation followed by continued replication as has been shown to occur in the malignant cytotrophoblast. 12 In one placental specimen, it was possible to maintain cultures for 7 months with a total of 14 subcultures (Fig. 3). However, these were not the asteroid cytotrophoblast but rather a population of broad polygonal and multiangular type cells, probably derived from the connective tissue core of the villus. Comment
The many attempts to establish stable strains of functioning normal diploid cells in culture have met with no success. The first step in such an endeavor is the accurate identification of the cell in question. This must be followed by devising means to isolate and propagate that particular cell type. The presence of a functional marker or product provides an ideal means of identify-
F,·bruary 15, 1968
Am.
J.
Obst. & Gynec.
ing and following trophoblastic cells. However, some differentiation may prevent the possibility of developing a continuously replicating normal cell strain. On the contrary, the malignant trophoblast because of the already existing neoplastic transformation has the acquired characteristic of continuous replication. It may be postulated that the normal gonadotropin hormone of the normal trophoblast induces its own differentiation. Using this reasoning the normal product of the trophoblast would prevent continuous replication of these cells by inducing their differentiation. This might account for the temporary detection of HCG in the medium followed shortly by cessation of mitosis and decline of hormone production. Because of the limited cell populations which were derived from selection of cell column cytotrophoblast for culture, inadequate numbers were available for a thorough study of their state of differentiation, and this explanation must therefore remain speculative.
REFERENCES
1. Stewart, H. L., et al.: J. Clin. Endocrino!. 8: 175,1948. 2. Thiede, H. A.: AM. J. OBST. & GYNEC. 79: 636, 1960. 3. Soma, H., and Hertig, A.: AM. J. OBST. & GYNEC. 18: 704, 1961. 4. Gey, G. 0., Seegar Jones. G., and Hellman, L. M.: Science 88: 306, 1938. 5. Tao, Tien-Wen, and Hertig, A.: Am. J. Anat. 116: 315, 1964. 6. Richart, R.: Proe. Soc. Exper. BioI. & Med. 106: 829, 1961.
7. Midgley, A. R., and Pierce, G. B.: Science 141: 349, 1963. 8. Brewer, J. I.: Am. J. Anat. 61: 429, 1937. 9. Wislocki. G., and Padykula, H.: In Young, W., editor: Sex and Internal Secretions, Baltimore, 1961, The Williams & Wilkins Company, p. 883. 10. Ehrmann, R. L., and Gey, G. 0.: ]. Nat. Cancer Inst. 16: 1375, 1956. 11. Delfs, E.: Endocrinology 28: 196, 1941. 12. Pattillo, R., and Gey, G. 0.: Science. In press.