Trophoblast stem cells - methods of isolation, histological and cellular characteristic, and their possible applications in human and animal models

The placenta is a part of the multifunctional feto-maternal unit formed during pregnancy, unique to mammals, but also defined as “the least understood organ” [1, 2]. Due to its anatomical complexity and constant structural changes observed throughout the pregnancy, its abnormalities are tough to study [3]. While the major part of the placenta is removed from the uterus after childbirth, the second and even more interesting part, involved in the regulation of placental invasion, is not available for research because of its localization at the border of the uterine wall [4]. Properties of the placenta include its endocrine activity, its responsibility for gas exchange, transfer of nutrients, and elimination of waste products [1, 5, 6]. Furthermore, it produces several steroid hormones like estrogen or progesterone, simultaneously provides essential communication between mother and fetus, acts as an efficient immune barrier, and plays the main role in the regulation of fetal growth [1,5,7, 8, 9, 10, 11]. Placental defects lead to numerous pregnancy disorders including fetal growth restriction, preeclampsia, and even miscarriages [11, 12].

Whereas the embryo arises from the embryonic tissue or epiblast, the primary part of the placenta derives from trophoblast lineage which has three basic cell subpopulations: the cytotrophoblast (CT), extravillous trophoblast (EVT), and syncytiotrophoblast (ST) [5, 6, 13]. All of these cell types arise from the trophectoderm of the blastocyst [6, 14]. Proliferative cytotrophoblast cells retain their plasticity, so they can develop into either extravillous trophoblast or syncytiotrophoblast cell lineages [6]. Some of the EVT cells invade the endometrium and are actively involved in the remodeling of the decidual spiral arteries that enables contact between the maternal and fetal circulations [6, 11, 15]. ST cells, because of their direct contact with maternal blood, are responsible for nutrient/ gas exchange and production of human chorionic gonadotropin which is a specific marker of pregnancy [6, 11]. ST and CT form specific structures called villi, which emerge from the chorion. Primary villi contain only the trophoblast cells, while the secondary villi contain trophoblast and extraembryonic mesoderm [16].

While embryonic stem cells (ESCs) originate from embryonic lineage and can evolve into cells of all three germ layers: ectoderm, endoderm, and mesoderm, trophoblast stem cells (TSCs) can differentiate into all trophoblast subtypes. [8, 17, 18, 19, 20]. TSCs are self-renewing, multipotent cells that are necessary for the formation of the placenta, specifically, the fetal part of this organ [21, 22, 23]. It has been established that TSCs derive from the trophectoderm structures. In mice, they can be directly isolated from blastocysts or extraembryonic tissues early after the implantation [20, 24, 25, 26, 27, 28, 29, 30]. TSCs, indeed, are becoming popular for studying early placentation in murine models [31]. Unfortunately, there are no readily available sources of human trophoblast stem cells, therefore animal models play a key role in current scientific approaches [32]. Mice are used as the most common source of TSCs in these studies [23, 27, 33]. However, there are reports for other animals too, for example, rabbits [12]. The existence of human trophoblast stem cells had for long remained uncertain, but now there is evidence that human TSCs can be isolated and maintained in vitro under specific conditions. Undoubtedly, this discovery has great scientific value [6]. Nonetheless, due to ethical considerations, the isolation of human TSCs still remains rather complicated [6, 34].

There are several methods of derivation and maintenance of trophoblast stem cells in mice. For instance, TSCs can be obtained from outgrowths of a blastocyst’s polar trophectoderm as well as extraembryonic ectoderm [4]. Usually, special feeder cells and additional substances like fibroblast growth factor 4 protein (FGF4), heparin or fetal bovine serum, and other cell culture media are used in TSC cultures [20, 23]. We would like to summarize one of the widely accepted isolation protocols [35, 36]. Firstly, it is necessary to prepare mouse embryonic fibroblasts (MEFs) as feeder cells. The next step is the collection of murine uteri. Dissected uteri should be placed in a small volume of TS medium in a 60 mm plate which should flush them out. Later, the blastocysts must be collected and transferred into a clean 12-well plate containing TS medium. Blastocysts should be placed into separate wells in a 12-well plate containing the mitomycin C-treated MEFs feeders and incubated at 37°C, 5% CO₂. Small cell growth should be noticed on day 3, at which point the cultures must be fed with fresh TS medium supplemented with FGF4/Heparin. On day 4 or 5, the blastocyst should be ready for disaggregation. The cultures demand washing with phosphate-buffered saline (PBS), then trypsin/EDTA solution should be added. Later, the aforementioned outgrowth should be separated into small clumps, the trypsinization process must be stopped by a mix of TS medium with FGF4/Heparine and MEF-CM (MEF-conditioned medium). Between days 6 to 10, TSC colonies emerge, and between days 15 and 20, reach up to 50% confluency. At this point it undergoes the next washing with PBS and addition of trypsin/EDTA. The next step is once again halting the trypsinization and transferring the material to new 6-well plates. After one or two passages (3-5 days between passages), TSCs are cultured with a few MEF feeders. To purify TSCs population, cultures must be incubated at 37°C, 5% CO₂ for 30 minutes after the trypsinization. Afterward, the supernatant should be collected – only TSCs should remain in the collected cell suspension, MEFs should have been attached to the culture plates. TSCs populations must be once again transferred into new plates and treated with the mix of TS medium with FGF4/Heparin and MEF-CM. TSCs can be identified by immunostaining for the stem cell marker – CDX2 (caudal type homeobox 2) [35]. When feeders and fibroblast growth factor 4 are absent, the expression of CDX2 disappears, signifying that cells have begun their differentiation. It should be highlighted that the described protocol has multiple possible modifications. For instance, Kubaczka et al. modified that method using a different cell medium, called TX medium which consists only of chemically defined ingredients. That enabled them to avoid using fetal bovine serum and embryonic-fibroblast-conditioned medium which contributed to substantial standardization of culture conditions [37].

In their recent study Okae et al. have found that human TSCs require different culture conditions to sufficiently adhere to the culture plates. It was established that the optimal culture medium should include: CHIR99021 (a Wingless/Integrated (WNT) activator), epidermal growth factor (EGF), A83-01 and SB431542 (transforming growth factor-beta inhibitors), valproic acid (a histone deacetylase inhibitor), and Y27632 (a Rho-associated protein kinase inhibitor). All these substances together facilitated long-term cultures of human blastocysts and proliferative CT cells. Both cell lines can differentiate into three major trophoblast lineages. Owing to that fact, scientists suggested that proliferative CT and blastocyst-derived cell lines are human trophoblast stem cells [6]. This ground-breaking discovery can redefine previous, unsuccessful attempts to isolate human TSCs.

The relatively recent experience gained from analyses of animal experimental TSCs models has eventually enabled the first successful isolation of undifferentiated human trophoblast cells. It should be highlighted that the first in vitro cultures of the human trophoblast stem cells open new perspectives for further molecular analyses focused on elucidating the genesis of abnormal placentation which leads to the development of various gestational pathologies such as preeclampsia or fetal growth abnormalities. Furthermore, these observations may facilitate the explanation of issues associated with abnormal implantation and consequent infertility. Such ground-breaking insights could potentially be translated to humans and applied as novel experimental treatment strategies. Anticipated advances in laboratory techniques and optimization of culture conditions may contribute to the invention of better protocols of TSC isolation from term placentas obtained during Caesarean sections, which in turn could eliminate potential ethical controversies connected with manipulation of human blastocysts or use of terminated fetal tissue for research purposes.

The isolation of TSCs is possible in both human and animal models. However, there are some essential differences in both the culture requirements and transcriptomic profiles of human and murine TSCs. The establishment of human TSCs models, though it raises ethical concerns, certainly sheds new light on further investigations in the area of molecular aspects of early embryonic development and reproductive failures, which may potentially lead to great advancements in future treatment strategies in several gestational pathologies.