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Post-implantation Differentiation and Proliferation of Cytotrophoblast Cells: In Vitro Models—A Review
Placenta (2000), 21, Supplement A, Trophoblast Research, 14, S45–S49 doi:10.1053/plac.1999.0523, available online at http://www.idealibrary.com on
CELL BIOLOGY Post-implantation Differentiation and Proliferation of Cytotrophoblast Cells: In Vitro Models—A Review O. Genbaceva and R. K. Millerb a
University of California at San Francisco, CA and Paper accepted 16 December 1999
b
University of Rochester Medical Center, NY, USA
Cytotrophoblast cells, specialized placental cells, proliferate early in pregnancy and then differentiate into tumour-like cells that invade the uterus and its vasculature. We have established in vitro models of three-dimensional cultures for anchoring villi and cell islands on extracellular matrix in order to study regulation of cytotrophoblast cell differentiation and proliferation. It has been demonstrated that cytotrophoblast cells from cell islands and cell columns share the same characteristics and that their differentiation is triggered by interaction with the extracellular matrix. The fact that during much of the first trimester maternal blood flow to the placenta is at a minimum, suggests that oxygen tension might regulate cytotrophoblast proliferation and differentiation. Hypoxia, comparable to that encountered by early gestation cytotrophoblast cells in the intervillous space, stimulated the cells to enter the cell cycle and inhibited their differentiation along the invasive pathway. Thus, oxygen gradient and cell–matrix interactions at the maternal–fetal interface play an important role in the regulation of cytotrophoblast proliferation and differentiation. 2000 IFPA and Harcourt Publishers Ltd Placenta (2000), 21, Supplement A, Trophoblast Research, 14, S45–S49
MORPHOLOGICAL ASPECTS OF NORMAL HUMAN PLACENTATION Differentiation of trophoblast cells is initiated during the implantation process. The unique anatomy of the human placenta is due in large part to differentiation of its epithelial stem cells, termed cytotrophoblast cells (CTBs). How these cells differentiate determines whether chorionic villi, the functional units of the placenta, float in intervillous space or anchor the conceptus to the uterine wall. In floating villi, CTBs differentiate by fusing to form multinucleate syncytiotrophoblast whose primary functions—secretion and transport—are ideally suited to their location at the villous surface. In anchoring villi, CTBs also fuse, but many remain as single cells that detach from their basement membrane and aggregate to form cell columns (Enders, 1968). Cytotrophoblast cells at the distal ends of these columns attach to, then deeply invade, the uterus (interstitial invasion) and its arterioles (endovascular To whom correspondence should be addressed: O. Genbacev, Department of Stomatology, University of California at San Francisco, 513 Parnassus, HSW 604, San Francisco, CA 94143-0512, USA. Fax: +1 415 502 7338; E-mail: [email protected], or R. K. Miller, Department of Obstetrics and Gynecology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, N.Y. 14642-8668, USA. 0143–4004/00/0A00S45+05 $35.00/0
invasion) (Pijnenborg et al., 1980). As a result of endovascular invasion, CTBs replace the endothelial and muscular linings of uterine arterioles (reviewed in Damsky and Fisher, 1998). This process enlarges vessel diameter and initiates, by the end of the first trimester of pregnancy, maternal blood flow to the intervillous space and placenta. Cytotrophoblast cells from the cell column of anchoring villi possess some unique features such as: (1) they aggressively invade the uterine wall, but their invasion is limited to decidualized endometrium and the inner part of myometrium; (2) proliferation of these cells is restricted and tightly regulated during differentiation along the invasive pathway; (3) subpopulations of these cells replace maternal endothelium and a portion of the smooth muscle wall, creating a hybrid vasculature. Little is known about the mechanisms that normally regulate human placental development in general, and cytotrophoblast proliferation and differentiation/invasion specifically. This information is vital for understanding how these cells fulfill their many unique functions. In addition, these findings could help to explain the aetiology of pregnancy disorders, infections and mechanisms of adverse effects of different toxic substances. Our goal was to establish in vitro models that mimic the in vivo pattern of cytotrophoblast differentiation and proliferation and to design experiments that would help to understand how these processes are regulated. 2000 IFPA and Harcourt Publishers Ltd
Placenta (2000), Vol 21, Supplement A, Trophoblast Research, Vol. 14
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IN VITRO MODELS
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At 6–8 weeks of gestation the fetal portion of the placenta consists of floating and anchoring villi and cell islands. Anchoring villi detached from the uterine wall retain on their tips remnants of cell columns that are, in vivo, the major sites of differentiation of a population of invasive cytotrophoblast cells (Fisher and Damsky, 1993). We have assumed that by culturing the anchoring villi under appropriate conditions it would be possible to replicate: (1) the in vivo pattern of cytotrophoblast cell differentiation along the invasive pathway and (2) to study factors that are involved in the regulation of proliferation and differentiation of these cells. Intact first trimester villi maintained on a matrix similar to either decidual ECM or basal lamina (Matrigel) gave rise in vitro to an extravillous population of invasive cytotrophoblast cells (Genbacev et al., 1992, 1993a,b,c) and replicated many characteristics observed in vivo (Aplin, 1993; Vicovac et al., 1995; Aplin et al., 1999). The fact that trophoblast differentiation along the invasive pathway in vitro does not occur in the absence of matrix proteins and is triggered by CTB-ECM interactions, suggests that similar mechanisms are operating in vivo, both at intrauterine and at ectopic sites of implantation.
Regulation of trophoblast differentiation and proliferation by oxygen tension During much of the first trimester, there is little endovascular invasion, and the growing embryo is separated from the maternal circulation by the trophoblastic shell (Hustin and Schaaps, 1987; Jaffe and Woods, 1993). The oxygen pressure of the intervillus space was estimated to be 17.96.9 mmHg (Rodesch et al., 1992). As cytotrophoblast cells invade the uterus during first trimester they encounter a steep, positive oxygen gradient. These observations, together with the results of initial experiments on isolated cytotrophoblast cells (Genbacev et al., 1996), suggested that during first trimester of pregnancy oxygen tension might regulate cytotrophoblast proliferation and differentiation along the invasive pathway (Genbacev et al., 1997). Anchoring villi explanted from early gestation (6–8 weeks) placentae onto ECM substrate were maintained for 72 h in a hypoxic atmosphere (2 per cent oxygen or 14 mmHg) or were cultured in a standard tissue culture incubator (20 per cent oxygen or 98 mmHg). Effect of hypoxia on cytotrophoblast cell proliferation was assessed by measuring their ability to incorporate BrdU (thymidine analog) into DNA (to mark S phase of cell cycle) and by counting the total number of cells per outgrowth (Genbacev et al., 1997). It was shown by staining serial sections with cytokeratin antibody (that mark cytotrophoblast cells), that cell columns associated with anchoring villi cultured in 2–4 per cent oxygen were larger than cell columns of control villi cultured under standard conditions. By making serial sections of columns attached to two villi explants
Percentage of control
Explant culture of anchoring villi 200
150
100
50
0
0.5–1
1–2 2–4 8 Percentage oxygen
18
Figure 1. Effect of P2 on proliferation of cytotrophoblast cells from cell columns of anchoring villi. Villous explants maintained under various concentrations of oxygen for 3 days, were fixed, embedded and sectioned. Serial sections from two explants per group were used to count the total number of cytotrophoblast cells per cell column. Variations in cell number per column between two explants within each group were between 10 and 15 per cent. Results are expressed as a percentage of control values (explants cultured in 20 per cent oxygen).
maintained for 72 h in 20 per cent oxygen and two that were maintained in 2 per cent oxygen, it was possible to count the number of cells in each column. Under standard tissue culture conditions, cell columns contained a mean of 51572 cells. In hypoxic conditions, they contained a mean of 1476156 cells. Experiments, in which explants were exposed to different oxygen concentrations, provided evidence that cytotrophoblast proliferation was tightly regulated by oxygen tension (Figure 1). At the concentration of oxygen lower than 2 per cent, cytotrophoblast proliferation was decreased. At the oxygen concentration equal to that encountered in vivo (2–4 per cent), the stimulation of cytotrophoblast proliferation was at a maximum. These results indicated that cytotrophoblast cells ‘sensed’ an oxygen gradient and that they responded to changes in oxygen concentration by a finely tuned mechanism. As changes in proliferative capacity are often accompanied by concomitant changes in differentiation, the effects of hypoxia on the ability of cytotrophoblast cells to differentiate along the invasive pathway were assessed as well. Under standard tissue culture conditions, cytotrophoblast cells migrated from cell columns and modulated their expression of stage specific antigens, as they do during uterine invasion in vivo (Damsky et al., 1992). They began to express integrin 1, a laminin-collagen receptor that is required for invasiveness in vitro. When cultured under hypoxic conditions, cytotrophoblast cells failed to stain for integrin 1 (Genbacev et al., 1996, 1997). Hypoxia also reduced staining for human placental lactogen (HPL), another antigen that is expressed once the cells differentiate along the invasive pathway (Genbacev et al., 1992, 1997). To explore further the effects of low oxygen tension (physiological hypoxia) on cytotrophoblast cell differentiation, explant cultures were also immunostained for E-cadherin (unpublished results). It has been shown that E-cadherin staining was reduced in areas in which invasion is active during the first
Genbacev and Miller: In Vitro Models of Post-implantation
S47
Figure 2. Cell island–floating cell column (CC) from 6–7 week of gestation. The tip of anchoring villi were immunostained with antibodies to cytokeratin (CK; cytokeratin antibody 7D3, Damsky et al., 1992) to mark cytotrophoblast cells (CTB) (A), integrin 5/1 to detect cells from cell columns (B) and Ki67 to mark cells in cell cycle (C). Cell islands (CC) were dissected under the microscope (D) and cultured on Matrigel in the presence of BrdU to detect cytotrophoblast cells (CTBs) in the S phase of the cell cycle (E). Staining with BrdU antibody demonstrated that most of the CTBs were proliferating during the first 4 days in culture. Cells that started to differentiate along the invasive pathway (arrow-right) and to migrate on Matrigel did not stain with BrdU antibodies. ST: syncytiotrophoblast.
half of gestation (Damsky et al., 1992). As expected, in explants cultured in hypoxia, E-cadherin expression in cytotrophoblast cells from the cell column was upregulated compared to control cultures. Taken together, these results suggest that the effects of oxygen tension on cytotrophoblast differentiation could have important implications. Relatively high oxygen tension promotes cytotrophoblast differentiation and could help explain why these cells extensively invade the arterial rather than the venous side of the uterine circulation. Conversely, if cytotrophoblast cells do not gain access to an adequate supply of maternal blood, their ability to differentiate into fully invasive cells may be impaired.
Cell islands—floating cytotrophoblastic columns During the first trimester of pregnancy masses of proliferative cytotrophoblast cells are present not only on the tips of
anchoring villi but also as collections (‘islands’) of mononuclear cells that protrude locally into intervillous space from different areas of both anchoring and floating villi (Boe, 1967; Moe, 1969). In spite of the fact that cell islands were described more than 20 years ago, their role remained obscure. Castellucci et al. (1991) considered cell islands as the villous zone of growth since they are formed from proliferative cytotrophoblast cells that express growth factors and growth factor receptors. As cell islands remain attached to placental villi, we propose to name them ‘floating cytotrophoblastic columns’ (CC). Cytotrophoblast cells from cell islands expressed in vivo most of the markers of cytotrophoblast cells from cell columns: they were cytokeratin (CK) positive [Figure 2(A)], expressed integrin 5 [Figure 2(B)] and Ki67, a marker of proliferative cells [Figure 2(C)]. They did not, however, upregulate integrin 1 and HPL—markers expressed by cytotrophoblast cells from the distal portion of cytotrophoblastic column.
Placenta (2000), Vol 21, Supplement A, Trophoblast Research, Vol. 14
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180
Steroid production per 48 h
160 140 120 100 80 60 40 20 0
2
4 Days in culture
6
Figure 3. Hormone production by cell islands–floating cell columns in vitro. Cell islands (n=135) from 12 placentae (6–8 week) were pooled and cultured on Matrigel coated 24-well plates for up to 6 days. Three wells (15 islands/well) were used for each time point. Culture media was collected after 2, 4 and 6 days, centrifuged, frozen and used to measure progesterone and estradiol production in vitro. Statistically significant differences in progesterone and estradiol production/release were obtained between day 2 and 6 of culture (P<0.05). : Oestradiol (pg/ml/5 mgwwt; ; progesterone (ng/ml/ 5 mgwwt).
We have isolated cell islands [Figure 2(D)] and cultured them on Matrigel [Figure 2(E)]. The yield of cell islands per placenta ranged from two to five up to 60. Great variability in size of cell islands was also observed. The described experiments were performed with ‘small’ cell islands (average size less than 1 mm), dissected from 5–15 placentae, and cultured on Matrigel coated inserts, as described for anchoring villi (Genbacev et al., 1993b). All of the experiments were conducted under standard tissue culture conditions in 20 per cent oxygen. Cell islands attached readily to the matrix, produced an outgrowth by day 2–4 and gave rise to invasive CTBs, as well as anchoring villi. Cytotrophoblast cells from cell islands divided in vitro more rapidly than CTBs from the cell column. They incorporated BrdU (bromodeoxyuridine) during 4 days in culture [Figure 2(E)], as compared to CTBs from anchoring villi that exited cell cycle after 24 h in vitro. As they migrated into Matrigel, CTBs upregulated integrin 1/1 expression, following the same pattern as cells from the distal part of cell columns (Damsky et al., 1992). Some cells started to express placental lactogen (HPL) immunoreactivity. By the sixth day of culture, few cytotrophoblast cells fused and formed giant cells. Cells from cell islands produced and released human chorionic gonadotropin (hCG), progesterone (P) and oestradiol (E). Great variations in hCG production were due to the differences in the proportion of syncytial elements per island. In contrast to hCG, production of progesterone and oestradiol increased from day 2–6 of culture and was not correlated with the presence of syncytium (Figure 3). From these experiments, it has been concluded that cell islands at 6–8 weeks of gestation
are a readily available additional source of extravillous cytotrophoblast cells that can be used to study mechanisms of ECM triggered cytotrophoblast differentiation along the invasive pathway. The role of cell islands in vivo is unknown. By the end of the first trimester of pregnancy, cytotrophoblast cells from cell islands are undergoing a transformation that is characterized by: deposition of fibrin, exit of CTBs from cell cycle, presence of apoptotic cells and reduction of number of CTBs per island. These changes are correlated with the establishment of intervillous blood flow. The physiological role of cell islands, if any, should be, therefore, restricted to early gestation. We hypothesized that cell islands during the first trimester of pregnancy might serve, if they get attached to the uterus, as an additional source of invasive, extravillous CTBs. If this is true, the name ‘floating villi’ might be justified. In vitro data suggest also a possibility that cell islands could also contribute (before the establishment of the blood flow) to the intervillous space pool of hormones, growth factors and/or cytokines. In summary, two in vitro models are proposed to study the regulation of proliferation of cytotrophoblast cells and the differentiation along the invasive pathway. These systems can serve to model pregnancy complications such as ectopic pregnancy, pre-eclampsia and growth retardation and can be used to study underlying mechanisms and possible treatments. They may be also suitable to assess effects of different drugs and xenobiotics and to predict benefits and/or adverse effects in early pregnancy. In combination with molecular biological and genetic analysis, application of these in vitro models may help to study gene expression and differentiation under normal and pathological conditions, such as pre-eclampsia, infectious disease and oxidant stress.
REFERENCES Aplin JD (1993) Expression of integrin 6/4 in human trophoblast and its loss from extravillus cells. Placenta, 14, 203–215. Aplin JD, Haigh T, Jones CJP, Church HJ & Vicovac Lj (1999) Development of cytotrophoblast column from explanted first-trimester human placental villi: Role of fibronectin and integrin 5/1. Biol Reprod, 60, 828–838. Boe F (1967) Studies on the human placenta. I. The cell islands in the young placenta. Acta Obstet Gynecol Scand, 46, 591–603. Castellucci M, Classen-Linke I, Muhlhauser J, Kaufmann P, Zardi L & Chiquet-Ehrismann R (1991) The human placenta: A model for tenascin expression. Histochemistry, 95, 449–458. Damsky CH, Fitzgerald ML & Fisher SJ (1992) Distribution patterns of extracellular matrix components and adhesion receptors are intricately modulated during first trimester cytotrophoblast differentiation along the invasive pathway in vivo. J Clin Invest, 89, 210–220. Damsky CH & Fisher SJ (1998) Trophoblast pseudo-vasculogenesis: faking it with endothelial adhesion receptors. Curr Opin Cell Biol, 10, 660–666. Enders AC (1968) Fine structure of anchoring villi of the human placenta. Am J Anat, 122, 419–452. Fisher SJ & Damsky CH (1993) Human cytotrophoblast invasion. Semin Cell Biol, 4, 183–188. Genbacev O, Schubach SA & Miller RK (1992) Villus culture of first trimester human placenta-model to study extravillus trophoblast (EVT) differentiation. Placenta, 13, 439–461. Genbacev O, DeMesy Jensen K, Schubach-Powlin SA & Miller RK (1993a) In vitro differentiation and ultrastructure of human extravillus trophoblast (EVT) cells. Placenta, 14, 463–475.
Genbacev and Miller: In Vitro Models of Post-implantation Genbacev O & Miller RK (1993b) Three-dimensional culture of placental villus—an in vitro model for peri-implantation. Methods in Toxicology, 3, 205–244. Genbacev O, White T, Gavin C & Miller RK (1993c) Human trophoblast in cultures: models for implantation and peri-implantation toxicology. Reprod Toxicol, 7, 75–94. Genbacev O, Joslin RJ, Damsky CH, Polliotti BM & Fisher SJ (1996) Hypoxia alters early gestation human cytotrophoblast differentiation invasion in vitro and models the placental defect that occur in preeclampsia. J Clin Invest, 97, 540–550. Genbacev O, Zhou Y, Ludlow JW & Fisher SJ. (1997) Regulation of human placental development by oxygen tension. Science, 277, 1669–1672. Hustin J & Schaaps JP (1987) Echographic and anatomic studies of the materno-trophoblastic border during the first trimester of pregnancy. Am J Obstet Gynecol, 157, 162–168.
S49 Jaffe R & Woods JR (1993) Color Doppler imaging and in vivo assessment of the anatomy and physiology of the early uteroplacental circulation. Fertil Steril, 60, 293–297. Moe N (1969) Deposits of fibrin and plasma proteins in the normal human placenta. An immunofluorescence study. Acta Pathol Microbiol Scand, 76, 74–88. Pijnenborg R, Dixon G, Robertson WB & Brosens I. (1980) Trophoblastic invasion of human decidua from 8 to 18 weeks of pregnancy. Placenta, 1, 3–19. Rodesch F, Simon P, Donner C & Jauniaux E (1992) Oxygen measurements in endometrial and trophoblastic tissue during early pregnancy. Obstet Gynecol, 80, 283–285. Vicovac Lj, Jones CJP & Aplin JD (1995) Trophoblast differentiation during formation of anchoring villi in a model of the early human placenta in vitro. Placenta, 16, 41–56.
CELL BIOLOGY Post-implantation Differentiation and Proliferation of Cytotrophoblast Cells: In Vitro Models—A Review O. Genbaceva and R. K. Millerb a
University of California at San Francisco, CA and Paper accepted 16 December 1999
b
University of Rochester Medical Center, NY, USA
Cytotrophoblast cells, specialized placental cells, proliferate early in pregnancy and then differentiate into tumour-like cells that invade the uterus and its vasculature. We have established in vitro models of three-dimensional cultures for anchoring villi and cell islands on extracellular matrix in order to study regulation of cytotrophoblast cell differentiation and proliferation. It has been demonstrated that cytotrophoblast cells from cell islands and cell columns share the same characteristics and that their differentiation is triggered by interaction with the extracellular matrix. The fact that during much of the first trimester maternal blood flow to the placenta is at a minimum, suggests that oxygen tension might regulate cytotrophoblast proliferation and differentiation. Hypoxia, comparable to that encountered by early gestation cytotrophoblast cells in the intervillous space, stimulated the cells to enter the cell cycle and inhibited their differentiation along the invasive pathway. Thus, oxygen gradient and cell–matrix interactions at the maternal–fetal interface play an important role in the regulation of cytotrophoblast proliferation and differentiation. 2000 IFPA and Harcourt Publishers Ltd Placenta (2000), 21, Supplement A, Trophoblast Research, 14, S45–S49
MORPHOLOGICAL ASPECTS OF NORMAL HUMAN PLACENTATION Differentiation of trophoblast cells is initiated during the implantation process. The unique anatomy of the human placenta is due in large part to differentiation of its epithelial stem cells, termed cytotrophoblast cells (CTBs). How these cells differentiate determines whether chorionic villi, the functional units of the placenta, float in intervillous space or anchor the conceptus to the uterine wall. In floating villi, CTBs differentiate by fusing to form multinucleate syncytiotrophoblast whose primary functions—secretion and transport—are ideally suited to their location at the villous surface. In anchoring villi, CTBs also fuse, but many remain as single cells that detach from their basement membrane and aggregate to form cell columns (Enders, 1968). Cytotrophoblast cells at the distal ends of these columns attach to, then deeply invade, the uterus (interstitial invasion) and its arterioles (endovascular To whom correspondence should be addressed: O. Genbacev, Department of Stomatology, University of California at San Francisco, 513 Parnassus, HSW 604, San Francisco, CA 94143-0512, USA. Fax: +1 415 502 7338; E-mail: [email protected], or R. K. Miller, Department of Obstetrics and Gynecology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, N.Y. 14642-8668, USA. 0143–4004/00/0A00S45+05 $35.00/0
invasion) (Pijnenborg et al., 1980). As a result of endovascular invasion, CTBs replace the endothelial and muscular linings of uterine arterioles (reviewed in Damsky and Fisher, 1998). This process enlarges vessel diameter and initiates, by the end of the first trimester of pregnancy, maternal blood flow to the intervillous space and placenta. Cytotrophoblast cells from the cell column of anchoring villi possess some unique features such as: (1) they aggressively invade the uterine wall, but their invasion is limited to decidualized endometrium and the inner part of myometrium; (2) proliferation of these cells is restricted and tightly regulated during differentiation along the invasive pathway; (3) subpopulations of these cells replace maternal endothelium and a portion of the smooth muscle wall, creating a hybrid vasculature. Little is known about the mechanisms that normally regulate human placental development in general, and cytotrophoblast proliferation and differentiation/invasion specifically. This information is vital for understanding how these cells fulfill their many unique functions. In addition, these findings could help to explain the aetiology of pregnancy disorders, infections and mechanisms of adverse effects of different toxic substances. Our goal was to establish in vitro models that mimic the in vivo pattern of cytotrophoblast differentiation and proliferation and to design experiments that would help to understand how these processes are regulated. 2000 IFPA and Harcourt Publishers Ltd
Placenta (2000), Vol 21, Supplement A, Trophoblast Research, Vol. 14
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IN VITRO MODELS
250
At 6–8 weeks of gestation the fetal portion of the placenta consists of floating and anchoring villi and cell islands. Anchoring villi detached from the uterine wall retain on their tips remnants of cell columns that are, in vivo, the major sites of differentiation of a population of invasive cytotrophoblast cells (Fisher and Damsky, 1993). We have assumed that by culturing the anchoring villi under appropriate conditions it would be possible to replicate: (1) the in vivo pattern of cytotrophoblast cell differentiation along the invasive pathway and (2) to study factors that are involved in the regulation of proliferation and differentiation of these cells. Intact first trimester villi maintained on a matrix similar to either decidual ECM or basal lamina (Matrigel) gave rise in vitro to an extravillous population of invasive cytotrophoblast cells (Genbacev et al., 1992, 1993a,b,c) and replicated many characteristics observed in vivo (Aplin, 1993; Vicovac et al., 1995; Aplin et al., 1999). The fact that trophoblast differentiation along the invasive pathway in vitro does not occur in the absence of matrix proteins and is triggered by CTB-ECM interactions, suggests that similar mechanisms are operating in vivo, both at intrauterine and at ectopic sites of implantation.
Regulation of trophoblast differentiation and proliferation by oxygen tension During much of the first trimester, there is little endovascular invasion, and the growing embryo is separated from the maternal circulation by the trophoblastic shell (Hustin and Schaaps, 1987; Jaffe and Woods, 1993). The oxygen pressure of the intervillus space was estimated to be 17.96.9 mmHg (Rodesch et al., 1992). As cytotrophoblast cells invade the uterus during first trimester they encounter a steep, positive oxygen gradient. These observations, together with the results of initial experiments on isolated cytotrophoblast cells (Genbacev et al., 1996), suggested that during first trimester of pregnancy oxygen tension might regulate cytotrophoblast proliferation and differentiation along the invasive pathway (Genbacev et al., 1997). Anchoring villi explanted from early gestation (6–8 weeks) placentae onto ECM substrate were maintained for 72 h in a hypoxic atmosphere (2 per cent oxygen or 14 mmHg) or were cultured in a standard tissue culture incubator (20 per cent oxygen or 98 mmHg). Effect of hypoxia on cytotrophoblast cell proliferation was assessed by measuring their ability to incorporate BrdU (thymidine analog) into DNA (to mark S phase of cell cycle) and by counting the total number of cells per outgrowth (Genbacev et al., 1997). It was shown by staining serial sections with cytokeratin antibody (that mark cytotrophoblast cells), that cell columns associated with anchoring villi cultured in 2–4 per cent oxygen were larger than cell columns of control villi cultured under standard conditions. By making serial sections of columns attached to two villi explants
Percentage of control
Explant culture of anchoring villi 200
150
100
50
0
0.5–1
1–2 2–4 8 Percentage oxygen
18
Figure 1. Effect of P2 on proliferation of cytotrophoblast cells from cell columns of anchoring villi. Villous explants maintained under various concentrations of oxygen for 3 days, were fixed, embedded and sectioned. Serial sections from two explants per group were used to count the total number of cytotrophoblast cells per cell column. Variations in cell number per column between two explants within each group were between 10 and 15 per cent. Results are expressed as a percentage of control values (explants cultured in 20 per cent oxygen).
maintained for 72 h in 20 per cent oxygen and two that were maintained in 2 per cent oxygen, it was possible to count the number of cells in each column. Under standard tissue culture conditions, cell columns contained a mean of 51572 cells. In hypoxic conditions, they contained a mean of 1476156 cells. Experiments, in which explants were exposed to different oxygen concentrations, provided evidence that cytotrophoblast proliferation was tightly regulated by oxygen tension (Figure 1). At the concentration of oxygen lower than 2 per cent, cytotrophoblast proliferation was decreased. At the oxygen concentration equal to that encountered in vivo (2–4 per cent), the stimulation of cytotrophoblast proliferation was at a maximum. These results indicated that cytotrophoblast cells ‘sensed’ an oxygen gradient and that they responded to changes in oxygen concentration by a finely tuned mechanism. As changes in proliferative capacity are often accompanied by concomitant changes in differentiation, the effects of hypoxia on the ability of cytotrophoblast cells to differentiate along the invasive pathway were assessed as well. Under standard tissue culture conditions, cytotrophoblast cells migrated from cell columns and modulated their expression of stage specific antigens, as they do during uterine invasion in vivo (Damsky et al., 1992). They began to express integrin 1, a laminin-collagen receptor that is required for invasiveness in vitro. When cultured under hypoxic conditions, cytotrophoblast cells failed to stain for integrin 1 (Genbacev et al., 1996, 1997). Hypoxia also reduced staining for human placental lactogen (HPL), another antigen that is expressed once the cells differentiate along the invasive pathway (Genbacev et al., 1992, 1997). To explore further the effects of low oxygen tension (physiological hypoxia) on cytotrophoblast cell differentiation, explant cultures were also immunostained for E-cadherin (unpublished results). It has been shown that E-cadherin staining was reduced in areas in which invasion is active during the first
Genbacev and Miller: In Vitro Models of Post-implantation
S47
Figure 2. Cell island–floating cell column (CC) from 6–7 week of gestation. The tip of anchoring villi were immunostained with antibodies to cytokeratin (CK; cytokeratin antibody 7D3, Damsky et al., 1992) to mark cytotrophoblast cells (CTB) (A), integrin 5/1 to detect cells from cell columns (B) and Ki67 to mark cells in cell cycle (C). Cell islands (CC) were dissected under the microscope (D) and cultured on Matrigel in the presence of BrdU to detect cytotrophoblast cells (CTBs) in the S phase of the cell cycle (E). Staining with BrdU antibody demonstrated that most of the CTBs were proliferating during the first 4 days in culture. Cells that started to differentiate along the invasive pathway (arrow-right) and to migrate on Matrigel did not stain with BrdU antibodies. ST: syncytiotrophoblast.
half of gestation (Damsky et al., 1992). As expected, in explants cultured in hypoxia, E-cadherin expression in cytotrophoblast cells from the cell column was upregulated compared to control cultures. Taken together, these results suggest that the effects of oxygen tension on cytotrophoblast differentiation could have important implications. Relatively high oxygen tension promotes cytotrophoblast differentiation and could help explain why these cells extensively invade the arterial rather than the venous side of the uterine circulation. Conversely, if cytotrophoblast cells do not gain access to an adequate supply of maternal blood, their ability to differentiate into fully invasive cells may be impaired.
Cell islands—floating cytotrophoblastic columns During the first trimester of pregnancy masses of proliferative cytotrophoblast cells are present not only on the tips of
anchoring villi but also as collections (‘islands’) of mononuclear cells that protrude locally into intervillous space from different areas of both anchoring and floating villi (Boe, 1967; Moe, 1969). In spite of the fact that cell islands were described more than 20 years ago, their role remained obscure. Castellucci et al. (1991) considered cell islands as the villous zone of growth since they are formed from proliferative cytotrophoblast cells that express growth factors and growth factor receptors. As cell islands remain attached to placental villi, we propose to name them ‘floating cytotrophoblastic columns’ (CC). Cytotrophoblast cells from cell islands expressed in vivo most of the markers of cytotrophoblast cells from cell columns: they were cytokeratin (CK) positive [Figure 2(A)], expressed integrin 5 [Figure 2(B)] and Ki67, a marker of proliferative cells [Figure 2(C)]. They did not, however, upregulate integrin 1 and HPL—markers expressed by cytotrophoblast cells from the distal portion of cytotrophoblastic column.
Placenta (2000), Vol 21, Supplement A, Trophoblast Research, Vol. 14
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180
Steroid production per 48 h
160 140 120 100 80 60 40 20 0
2
4 Days in culture
6
Figure 3. Hormone production by cell islands–floating cell columns in vitro. Cell islands (n=135) from 12 placentae (6–8 week) were pooled and cultured on Matrigel coated 24-well plates for up to 6 days. Three wells (15 islands/well) were used for each time point. Culture media was collected after 2, 4 and 6 days, centrifuged, frozen and used to measure progesterone and estradiol production in vitro. Statistically significant differences in progesterone and estradiol production/release were obtained between day 2 and 6 of culture (P<0.05). : Oestradiol (pg/ml/5 mgwwt; ; progesterone (ng/ml/ 5 mgwwt).
We have isolated cell islands [Figure 2(D)] and cultured them on Matrigel [Figure 2(E)]. The yield of cell islands per placenta ranged from two to five up to 60. Great variability in size of cell islands was also observed. The described experiments were performed with ‘small’ cell islands (average size less than 1 mm), dissected from 5–15 placentae, and cultured on Matrigel coated inserts, as described for anchoring villi (Genbacev et al., 1993b). All of the experiments were conducted under standard tissue culture conditions in 20 per cent oxygen. Cell islands attached readily to the matrix, produced an outgrowth by day 2–4 and gave rise to invasive CTBs, as well as anchoring villi. Cytotrophoblast cells from cell islands divided in vitro more rapidly than CTBs from the cell column. They incorporated BrdU (bromodeoxyuridine) during 4 days in culture [Figure 2(E)], as compared to CTBs from anchoring villi that exited cell cycle after 24 h in vitro. As they migrated into Matrigel, CTBs upregulated integrin 1/1 expression, following the same pattern as cells from the distal part of cell columns (Damsky et al., 1992). Some cells started to express placental lactogen (HPL) immunoreactivity. By the sixth day of culture, few cytotrophoblast cells fused and formed giant cells. Cells from cell islands produced and released human chorionic gonadotropin (hCG), progesterone (P) and oestradiol (E). Great variations in hCG production were due to the differences in the proportion of syncytial elements per island. In contrast to hCG, production of progesterone and oestradiol increased from day 2–6 of culture and was not correlated with the presence of syncytium (Figure 3). From these experiments, it has been concluded that cell islands at 6–8 weeks of gestation
are a readily available additional source of extravillous cytotrophoblast cells that can be used to study mechanisms of ECM triggered cytotrophoblast differentiation along the invasive pathway. The role of cell islands in vivo is unknown. By the end of the first trimester of pregnancy, cytotrophoblast cells from cell islands are undergoing a transformation that is characterized by: deposition of fibrin, exit of CTBs from cell cycle, presence of apoptotic cells and reduction of number of CTBs per island. These changes are correlated with the establishment of intervillous blood flow. The physiological role of cell islands, if any, should be, therefore, restricted to early gestation. We hypothesized that cell islands during the first trimester of pregnancy might serve, if they get attached to the uterus, as an additional source of invasive, extravillous CTBs. If this is true, the name ‘floating villi’ might be justified. In vitro data suggest also a possibility that cell islands could also contribute (before the establishment of the blood flow) to the intervillous space pool of hormones, growth factors and/or cytokines. In summary, two in vitro models are proposed to study the regulation of proliferation of cytotrophoblast cells and the differentiation along the invasive pathway. These systems can serve to model pregnancy complications such as ectopic pregnancy, pre-eclampsia and growth retardation and can be used to study underlying mechanisms and possible treatments. They may be also suitable to assess effects of different drugs and xenobiotics and to predict benefits and/or adverse effects in early pregnancy. In combination with molecular biological and genetic analysis, application of these in vitro models may help to study gene expression and differentiation under normal and pathological conditions, such as pre-eclampsia, infectious disease and oxidant stress.
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