Procoagulant extracellular vesicles (EV) and platelet activation have been associated with gestational vascular complications. EV-induced platelet-mediated placental inflammasome activation has been shown to cause preeclampsia-like symptoms in mice. However, the effect of EV-mediated placental thrombo-inflammation on trophoblast differentiation remains unknown. Here, we identify that the EV-induced thrombo-inflammatory pathway modulates trophoblast morphology and differentiation. EVs and platelets reduce syncytiotrophoblast differentiation while increasing giant trophoblast and spongiotrophoblast including the glycogen-rich cells. These effects are plateletdependent and mediated by the NLRP3 inflammasome. In humans, inflammasome activation was negatively correlated with trophoblast differentiation marker GCM1 and positively correlated with blood pressure. These data identify a crucial role of EV-induced placental thrombo-inflammation on altering trophoblast differentiation and suggest platelet activation or inflammasome activation as a therapeutic target in order to achieve successful placentation.

Cellular differentiation is a complex but fascinating process in all multicellular organisms. Differentiation can involve changes in numerous aspects of cell physiology; size, shape, polarity, metabolic activity, responsiveness to signals, and changes in gene expression profiles. These changes form the basis for differentiation to occur. The human hemochorial placenta is an intricate apposition of fetal and maternal tissues that is strategically juxtaposed at the interface, with its widespread ‘villous’ or tree-like projections, directly in contact with maternal blood. It is therefore, ideally suited to perform life-sustaining functions such as exchange of nutrients, respiratory gases and metabolic wastes, with the maternal supply. It also plays a central role in the maintenance of the immunologically privileged status of the fetal semi-allograft. Placental development is directed towards the establishment of a continuous nutrient supply to the developing fetus. This requires efficient access of maternal blood to a transporting surface, the multinucleate syncytiotrophoblast layer. This is made possible by the rapid proliferation and ensuing invasion of mononuclear trophoblasts into the maternal uterus and remodeling of the spiral arteries therein. It is interesting to note that in early pregnancy, it is the placenta that first engages its active growth and proliferation and only then, permits the logarithmic growth phase of the embryo. As a developing organ, the placenta undergoes constant tissue remodeling, which is characterized by the functional loss of trophoblast cells by apoptosis. Most of these changes occur at the trophoblast layer of the placental villous that is composed of two cell types: cytotrophoblasts (CT) and syncytiotrophoblasts (ST). The mononuclear cytotrophoblast cells, which are located between the syncytiotrophoblast layer and its basement membrane, proliferate and fuse during trophoblast differentiation to form the overlying multinucleated syncytium. CT are highly proliferating and invasive cells, in contrast to the ST which are non proliferative less invasive and functionally very active. Syncytiotrophoblast cells form the continuous, uninterrupted, multinucleated, epithelium-like surface of the placental villous that separates maternal blood from the villous interior. ST performs a crucial role in feto-maternal exchanges and serves as an endocrine tissue by its ability to synthesize and secrete a variety of hormones such as GnRH, chorionic gonadotrophin (CG), placental lactogen (PL) and steroid hormones involved in the homeostasis during pregnancy. Thus, differentiation of CT into ST serves as an ideal model to study cellular differentiation as morphologically and functionally these cells exhibit highly contrasting features. The molecular basis of cytotrophoblast differentiation has been studied using primary cultures of human trophoblast cells as a model system. Highly purified preparations of mononucleated cytotrophoblast cells can be isolated from preterm and term placental tissue by enzymatic dispersion. The isolated cells from term placental tissue aggregate spontaneously in culture and fuse to form a multinucleated syncytiotrophoblast which synthesizes and secretes placental lactogen (hPL), chorionic gonadotropin (hCG) and other syncytiotrophoblast-specific protein and steroid hormones . These in vitro changes, which recapitulate important activities accomplished by normal cytotrophoblast cells during in vivo maturation, implicate a critical relationship between the differentiation of cytotrophoblast cells into syncytiotrophoblast cells. Though primary cell culture is an ideal model to study these changes, it comes inherently associated with various problems like health risk of handling human tissues, time involved, variability in each placental samples depending on health status of the subject and quite often lack of history of the subject which makes the results from these experiments difficult to reproduce and assess. One way to overcome this is the cell culture model which is a reproducible experimental system and permits the direct observation of time-dependent processes and their experimental manipulation. BeWo cells, the cells which we have used in our study, were derived from human gestational choriocarcinoma. These cells are the highly invasive malignant counterparts of the normal human trophoblast wherein, the limited capacity for cell proliferation is far exceeded. However, they still retain important features of their normal counterpart, like the potential of hormone production and induced differentiation. Differentiation of CT to ST is precisely controlled by different agents such as transcription factors, hormones, growth factors, cytokines and oxygen levels. BeWo cells have been used by other investigators as well as by us and it has been shown that these cells can be induced to differentiate with the agents mentioned above and terminally differentiate into cells which express typical characteristics of the normal differentiating trophoblast; like morphological transition from cytotrophoblast to syncytiotrophoblast-like cells, increased production of protein and steroid hormones (hCG, hPL, estrogens, progesterone); increased activity of cellular alkaline phosphatase and arrested cell proliferation. Since these cells can be triggered by external agents to differentiate, they serve as a useful model for the study of changes that occur during differentiation. Using primary cells and various cell lines including BeWo cells, various groups have attempted to study trophoblast differentiation and the regulators that control this process. The results of such study have only come out with a list of genes or proteins which might be having a role in this process and no functional correlation has been drawn so far from these studies. The members of the syncytin protein family, ADAM (a disintegrin and metalloprotease) proteins may well be some of the main players in the process of trophoblast fusion; some of the requisites of trophoblast fusion being redistribution of phosphatidylserine to the outer leaflet of the plasma membrane and activity of certain intracellular proteases. Clearly, further studies on trophoblast differentiation are needed to answer the question of the precise identity of regulatory proteins and role of these proteins during differentiation. The present study is aimed at gaining insights into the process of trophoblast differentiation and the molecular events which occur during this process. Our aim is also to study the regulated process of differentiation using BeWo cell model and identify the differentially expressed genes and relate the known function of these gene products to changes seen during differentiation process. We have employed the Differential Display Reverse Transcriptase Polymerase Chain Reaction (DD-RTPCR) and Microarray analysis to monitor the changes in gene expression. In CHAPTER 1, a brief account of morphological, biochemical and physiological changes which occur during placentation and trophoblast differentiation is discussed. Various aspects of placental function are discussed in brief, with special reference to the many unique abilities of trophoblast cells that contribute to a successful pregnancy. Detailed accounts of molecular mechanism of cellular differentiation, the models used in these studies and the advantages and drawbacks have been highlighted. The results of the previous studies from our laboratory using different model system and the outcome of the study are also outlined in this chapter. The advantages and disadvantages of the primary cell lines and the ease of handling of continuous cell culture model, BeWo is also presented in this chapter. The aim and objective of our study is to understand the molecular mechanisms underlying the trophoblast differentiation and the literature available is reviewed in the light of the objective and the aims and scope of the present study. The details regarding the materials used and the techniques employed during the entire study are outlined in CHAPTER 2-‘Materials and Methods’. The conditions for culture of BeWo human choriocarcinoma cell line are described and details of procedures employed for the validation of BeWo cells as a model system for monitoring the process of cellular differentiation are mentioned in this chapter. The details of the procedures employed for isolation of RNA, Reverse Transcriptase Polymerase Chain Reaction (RTPCR), Differential Display RT-PCR (DD-RT-PCR), Microarray analysis, Northern Blot analysis and Western Blot analysis are also described. The principle of the MTT assay used for verifying the viability of cells following various treatments is provided along with the working protocol. This chapter also includes protocols of the in vivo studies in rat, the methods employed for rat uterine mince cultures and isolation of rat uterine epithelial cells and dose and duration of the various treatments with steroid hormones and their inhibitors, treatment with protein kinase inhibitors in cell culture system are also described. In addition, this chapter also describes the procedures for transfection of hTERT, silencing of SLPI gene using SiRNA approach, gelatin zymography, MAP Kinase assay, FACS, cloning and expression of SLPI protein and procedure employed for raising antibodies to SLPI in rabbit. Finally, details of statistical tests employed fro anlaysis of data are presented. The results obtained in the present study are presented in 4 chapters(Chapters 3-6), CHAPTER 3 describes the characterization and validation of model system employed- BeWo cells to study human trophoblastic differentiation. BeWo cells under normal culture conditions resemble cytotrophoblasts like cells and when treated with various effectors of differentiation can be induced ot differentiate into syncytiotrophoblasts. We used 10 µM Forskolin to induce differentiation in BeWo cells. Forskollin is known to induce characteristic changes associated with human trophoblast differentiation in these cells. Incubation of BeWo cultures in the presence of 10 µM Forskolin resulted in dramatic morphological biochemical changes intheir cytotrophoblast-like phenotype. Mononuclear cells were seen to fuse to form multinucleate syncytial structures over a period of 72-96 hours in culture. This process was also associated with an increased production of β-hCG, Endoglin and hTERT thereby validating this model system for study of human trophoblastic differentiation. Analysis of cell cycle genes in this system established the arrest of proliferation thus further validating the system. The viability of these cells, during the entire period of culture, was verified using the MTT assay. This chapter discusses the importance of in vitro cell culture systems in the study of human placental development, and also addresses the suitability of these model systems for the study of human trophoblast proliferation and differentiation. One of the important finding of our earlier studies was that arrest of proliferation was a prerequisite for trophoblast differentiation to occur. This conclusion was based on the fact that telomerase expression which is a hallmark of all proliferating cells was down regulated in BeWo cells by 48h as assessed by TRAP (Telomere Repeat Amplification Protocol) assay or RT-PCR analysis for hTERT which is the catalytic subunit of telomerase. Telomerase activity was undetectable by about 96th by which time syncytium formation is normally completed after the addition of differentiation inducing agents like Forskolin, TGF β etc. Although the telomeric holo enzyme consists of many components the subunits which are critical for enzyme action are hTERT and hTR; hTR; hTR which is the RNA component of telomerase is ubiquitously expressed in most cell types including telomerase negative cells such as differentiated somatic cells. Since the BeWo cells can be induced to differentiate into multinucleated ST by addition of Forskolin and periodically the aged ST are eliminated by apoptosis. It is very well documented that the life span of ST is very limited and the ST have to be replaced by the freshly formed ST out of fusion of CT. Considering this, it was of interest to test whether differentiation can be prevented or delayed by extending the expression of telomerase activity. This would further validate our system that one of the requisites for cells to differentiate is down regulation of hTERT in BeWo cells. This was achieved by transfection of BeWo cells with hTERT expression vector. The results of the study clearly established that we were able to over express hTERT in BeWo cells; we also noticed an increase in the proliferation of BeWo cells as assessed by BrdU incorporation. In agreement with this observation is the fact that, in contrast to the empty vector transfected cells, in hTERT transfected group, the cell density appeared to be clearly more at 72 h. That the decrease in the hTERT expression in the control (empty vector transfected) is not due to cell death was established by MTT assay, which indicated that there was no difference in the viability between control and hTERT transfected cells. Further more, results of analysis for a variety of cell proliferation and differentiation markers by RT-PCR and Western blot analysis clearly supports the conclusion that hTERT over expression delays syncytium formation. Although reports are available on the differential expression of genes during differentiation of CT to ST with both primary cell lines as well as BeWo cell line, relatively less is known about the functional importance of differentially expressed genes. In CHAPTER 4, results of our studies to profile the differentially expressed genes during Forskolin induced differentiation in BeWo cells by two approaches DD-RTPCR and microarray analysis and relate the known functions of these genes to changes that occur during the differentiation of CT to ST are presented. We identified several genes that had robust change during differentiation by DD RTPCR and the differential expression of ten transcripts was confirmed by Northern blot analysis. The genes which we identified were SLPI, Elongation factor-1 alpha -1, Prolyl hydroxylase beta, LIMO-4 etc. These genes were either shown to have a role during differentiation of cells or have functional role in the syncytiotrophoblasts. Secretory Leucocyte Protease Inhibitor was one of the differentially expressed transcripts which were significantly up regulated during Forskolin induced differentiation of BeWo cells. SLPI which is a 12 KDa protein reported to exhibit a variety of activities which include inhibition of proteases and elastase, in addition to antibacterial and anti inflammatory activities. It was chosen for our further studies because of its multifunctional role in placenta and also during implantation. Micro array analysis revealed the up-regulation of hCG, hCS, and Endoglin thus validating the experimental system. Several candidate genes that could influence trophoblast differentiation, cell adhesion and cellular proliferation were identified. Genes involved in cellular proliferation include cyclin M3, replication factor 3, signal-induced proliferation-associated gene 1, osteonectin, clusterin, etc clearly indicating a growth-arrested phenotype for the differentiating BeWo cells. Trophoblastic differentiation associated genes included adipose differentiation-related protein, GADD45A, PPAR binding protein, galectin 3, tubulins, collagen, stathmin, etc. The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest or DNA repair or apoptosis. Although we did not observe any change in the p53 mRNA levels, the total protein level as well the phosphorylation status of p53 was up regulated upon differentiation. We confirmed the down regulation of Cyclin D1, D2 and PCNA in differentiated cells and up regulation of CDK inhibitors, P27kip1, P21cip1which are p53 induced genes by RT-PCR and Western blot analysis. Phosphorylation of ser 20 leads to reduced interaction of p53 with its negative regulator MDM2. MDM2 inhibits the accumulation of p53 by targeting it for ubiquitination and proteosomal degradation. Analysis for the phosphorylated status of p53 revealed that specifically the ser 20 phosphorylated p53, was increased upon differentiation. Phosphorylation of ser-392 has been reported to influence the growth repressor function, DNA binding and transcriptional activation of p53 and in agreement with this, western blot analysis revealed an increase in the ser-392 phosphorylated p53. These results suggest that p53, a nuclear protein regulating several genes involved in proliferation and differentiation is playing a pivotal role in growth arrest during trophoblast differentiation. We also noticed that several components of the apoptotic cascade are differentially expressed in cytotrophoblast and the syncytiotrophoblasts layer, and these changes appear to be associated with the stage of apoptosis. Apoptosis is involved in the removal of aging syncytiotrophoblasts and it also promotes cytotrophoblast fusion and formation of the syncytial layer. We found that various apoptosis related genes are up regulated and anti apoptotic genes suppressed following differentiation in our micro array analysis. We identified the involvement of p53 in this process also and chapter 4 deals with this aspect. Genes which regulate the invasive behaviour of trophoblasts which include MMP2, cathepsin K, cystatin N, SLPI and cysterine-rich angiogenic inducer 61, etc. were found to be up regulated following differentiation in our micro array analysis, which establishes these differences in gene expression reflects the physiological changes that occur during placentation. The Co-ordinated regulation of ptoteases and protease inhibitors I (for example SLPI, cystatin B and MMP2) suggests that these genes play an important role in the regulation trophoblast invasion at the uterine-placenta interface in vivo. Our studies revealed that one of the transcripts,namely, SLPI(Secretory leukocyte protease inhibitor) was robustly up regulated as assessed in DDRT-PCR, micro-array, Northern blot and RT-PCR analysis. Considering its importance in implantation, placentation and maintenance of pregnancy several aspects of this multifunctional protein were studied in detail and the results are presented in CHAPTER 5. Studies on the regulation of this transcript in Be-Wo cells revealed that SLPI mRNA is regulated by progesterone in Be-Wo cells. The up regulation of SLPI mRNA by progesterone was specifically inhibited by Progesterone receptor antagonist, RU 486 and estradiol 17β did not have any effect on the expression of SLPI mRNA expression in BeWo cells. The absence of regulation of SLPI by estradiol in BeWo cells was also established by the fact that simultaneous addition of progesterone and aromatase inhibitor, fadrazole did not block the increase in SLPI expression. Interestingly in vivo and in vitro studies using rat uterine minces and rat epithelial cells revealed that SLPI mRNA is regulated by Estradiol 17β and the effect is specifically inhibited by estrogen receptor antagonists such as ICI 182780, Tamoxifen, and Centchorman. Promoter analysis of rat and human SLPI revealed the absence of a consensus progesterone responsive element (PRE) in human and estrogen responsive element (ERE) in rat, suggesting the possibility of a non-genomic action of progesterone or estrogen in the induction of SLPI mRNA. This was confirmed by the observation that induction of SLPI mRNA could be effectively blocked by the addition of Staurosporine, an inhibitor of protein kinase C along with progesterone and estrogen to either BeWo cells or rat uterine epithelial cells. These results suggest that the non-genomic action of steroid hormones may be involved in the induction of SLPI. In the present study, we have also identified the intracellular signaling pathway that regulates SLPI gene expression by using various protein kinase inhibitors. We have also shown that activation of MAP kinase pathway upon progesterone treatment and the involvement of protein kinases in this activation, permitting us to conclude the non genomic action of progesterone in induction of SLPI mRNA in BeWo cells. The results of these studies are presented in detail in Chapter 5. The observation that SLPI expression is markedly increased during differentiation and differentially regulated by progesterone and estradiol, and induction by non genomic pathway prompted us to undertake studies to investigate its role during differentiation. This was accomplished by using SiRNA to silence the expression of SLPI in Forskolin induced differentiating BeWo cells and the results of this study are presented in CHAPTER 6. Different concentrations and combinations of oligos were used to silence the SLPI gene and we found that effective knockdown (>80%) was achieved with SiRNA concentrations ranging from 5-25nM. A combination of oligos also increased the knockdown from 50% to 90% as assessed by RT-PCR and western blot analysis for mRNA and protein levels of SLPI respectively. We found that inhibition of SLPI expression by SiRNA also inhibited the morphological differentiation of BeWo cells. Functionally this was reflected, by increase in the protease activity as assessed by gelatin zymography. It should be noted that SLPI is a protease inhibitor; it inhibits a variety of proteases, including proteases from neutrophils, pancreatic acinar cells and mast cells and SLPI present in the syncytiotrophoblast may have a crucial role in controlling protease activity associated with invasiveness and differentiation. Inhibition of differentiation by silencing the expression of SLPI provides an opportunity to monitor the changes in gene expression where in a single gene has been silenced in contrast to the model employed in chapter 4. We carried out microarray analysis using control (Forskolin treated) and SLPI silenced (Forskolin treated) samples. The results revealed that proliferation and differentiation, apoptosis and inflammatory pathways genes are affected due to SLPI silencing and the results of this study are presented in CHAPTER 7. We confirmed the changes in gene expression by semi quantitative RT-PCR analysis of the some important genes in each pathway. A comparison of the results obtained with that of our earlier microarray analysis which is described in chapter 4 revealed that the changes in levels of expression of the genes involved in cell proliferation, differentiation, apoptosis and inflammation were completely reversed after silencing the expression of SLPI. We have presented in chapter 5 the importance of MAP kinase pathway in Forskolin induced differentiation and the activation of this pathway when SLPI expression is increased following progesterone treatment. Interestingly after silencing the expression of SLPI we found that MAP kinase pathway is affected. It was observed that silencing of SLPI expression resulted in inhibition of activation of MAP kinase as assessed by the phosphorylation status by ELISA and no activation of MAP kinase was observed in SLPI silenced Forskolin treated cells. CHAPTER 8 provides a general discussion of the results obtained in the present study in the light of current understanding the type of genes involved, changes during human trophoblastic proliferation and differentiation and the key players during this process. This chapter also brings out the importance of SLPI during trophoblastic differentiation, placentation, implantation and its regulation by steroid hormones. The highlights and salient features of the present study are summarized in this chapter. In CONCLUSION, the present investigation has led to the identification of specific genes involved in trophoblast differentiation, human placental growth and development. Also evident from this study is the usefulness of the trophoblastic cell culture system for the study of cellular differentiation. We have attempted to relate the gene expression changes to physiological changes that occur during placentation, implantation and pregnancy. Many of the regulatory events that we have described during human trophoblastic differentiation, may not only be restricted to these cells, but may represent common principles/features of cellular differentiation in general. Loss of differentiation is a wide-spread feature of tumor progression, and frequently accompanies aggressive neoplastic behavior. Our studies provide unequivocal evidence to support cellular differentiation as a natural barrier to malignant transformation. Most importantly we have shown that silencing of a single gene can disrupt this differentiation process and the importance of SLPI during differentiation process perse.

Placenta is a transient association of the fetal and maternal tissues, that develops during pregnancy, in most viviparous animals. The evolution of placenta ensured the development of the fetus inside the womb of the mother, providing a protected environment for the development of the fetus, and preventing the loss of progeny due to unfavorable environmental conditions. Because it is strategically poised at the maternal and fetal interface, the placenta is ideally suited to carry out alimentary, respiratory and excretory functions for the developing fetus. In addition, it serves as an immunological barrier preventing the rejection of the fetal semi-allograft, by the maternal immune system. Furthermore, the placenta elaborates a variety of protein, polypeptide and steroid hormones. These include growth factors, growth factor receptors, neuropeptides, opioids, progesterone and estrogen, whose secretion is dependent on the gestational age of the placenta and its differentiation status. The human placenta, adapts itself remarkably to cater to the changing requirements of the developing fetus. For instance, during the first trimester of pregnancy, the placenta is an actively dividing, a highly invasive and a rapidly differentiating organ; while near term, it represents a fully differentiated and a non-invasive unit. Furthermore, the placenta of the first trimester and that at term differ in their hormone profiles, extents of apoptosis, expression of several transcription factors, etc. This dramatic change in the phenotype of the human placenta can be considered to be the outcome of an intrinsically programmed pattern of differentiation, which may be referred to as the functional differentiation of the placenta. It may be hypothesized therefore, that this functional differentiation could be brought about by the differential expression of genes in the first trimester and the term placenta. The objectives of the present study were: 1. To gain an insight into this process of " functional differentiation” by investigating the differential expression of genes in the two developmentally distinct stages during gestation, viz. during the first trimester and at term. 2. To understand the functional relevance of the differentially expressed genes. A general introduction of the human placenta, describing the importance of differential expression in modulating placental function, is discussed in chapter 1. The functions of the human placenta along with a brief description of its development and differentiation are also briefly described. A Differential Display RT-PCR-based (DD RT-PCR) approach, using total RNA from the first-trimester and term placental villi, was employed to display the differentially expressed genes in the first trimester and the term placenta. The display so generated was used to identify a few differentially expressed cDNAs. This study was aimed at understanding the functional significance of the transcripts which were identified from the display, rather than just concentrate on documenting the differences in the gene expression patterns in the first trimester and the term placental tissue. A detailed description of the methodology adopted for performing DD-PCR using placental tissue, discussing the advantages and disadvantages of using differential display PCR, is described in chapter2. The use of DD-PCR for studying differential gene expression in the human placenta was validated by the finding that one of the cDNAs that was differentially expressed in the first trimester placental tissue, is a fragment of β-hCG cDNA. It is well documented that the differential expression of the β-subunit of hCG (human chorionic gondatrophin) during the first eight weeks of gestation is the rate limiting step in the synthesis and secretion of the functional hormone, which comprises the α and the β-subunits. Furthermore, the use of the model system viz., the first trimester and term placental tissue, was also validated for carrying out DD-PCR by ensuring that all placental samples used for DD analysis were free of endometrial contamination. A detailed description of optimization and validation of DD-PCR in human placental tissues is given in chapter 2. Cloning and sequencing of yet another cDNA from the first trimester differential display revealed that it is T-Plastin. T-Plastin is a member of a family of proteins that are involved in actin-bundling. Northern blot analysis and immunohistochemical studies using an antibody generated to a peptide corresponding to human T-Plastin, confirmed its differential expression and localization in the first trimester placenta. Considering the fact that several carcinomas show enhanced expression of T-Plastin, we tested the hypothesis that its differential expression is correlated with the proliferative potential of the first-trimester placenta It was observed that the first-trimester tissue expressed high levels of beta-actin as compared to the term placental tissue. This is in agreement with the up-regulation of beta-actin following mitogenic stimulation/proliferation and during neoplastic transformation or transformation-associated invasive behaviour of cells, two characteristic features shared by the early placenta with cancerous tissues. Based on our studies and available information in the literature, it is proposed that T-Plastin expression in the first trimester placenta is a growth-associated phenomenon which is partially responsible for the tumor-like phenotype of the first trimester tissue. Studies carried out with the partial T-Plastin cDNA clone that was isolated from the first trimester differential display, are presented in chapter 3. Sequencing of yet another cDNA clone identified from the term placental differential display, T-18 revealed that it had no homology to any known sequence in the nucleotide or est databases. The sequence corresponding to this clone was submitted to the GenBank and was assigned an accession number- AF089811. The differential expression of T-18 was confirmed by Northern blot analysis and RT-PCR analysis. Attempts were made to isolate the full-length cDNA corresponding to T-18 from a commercially available library from Clontech. However, repeated trials to identify the clone corresponding to T-18 did not yield any positive results. However, a genome database search revealed that T-18 was a portion of a large contig contained in chromosome 15. Analysis of the annotated gene sequences in and around the region in which T-18 is located in chromosome 15, revealed that there are very few ests reported in this contig and quite a few repeat sequences reported. Interestingly, it was observed that 6 kb downstream of the region in which T-18 is located, there was an est that had homology to a Bcl-2 precursor protein (an evolutionarily conserved, anti-apoptotic protein, capable of conferring protection against death-inducing signals) and the death adaptor protein, CRADD {Caspase and RIP adapter with death domain). Further updating of the ests in the database might probably be of help in the identification of the full-length cDNA corresponding to T-18 and confirm as to whether T-18 is a part of the gene/gene cluster that comprises the afore-mentioned est. An account of the identification and cloning of T-18 from the term placenta and the attempts to isolate the full-length cDNA clone corresponding to T-18 from a term placental cDNA library, is described in chapter 4. In the absence of any information on the identity of T-18, a study to understand the functional significance of T-18 expression was carried out. Since it was not possible to carry out studies pertaining to the temporal expression of T-18 throughout gestation on the human placenta for ethical reasons, alternate animal/organ models were employed to study T-18 expression. Rat placenta and rat Corpus Luteum (CL) were chosen as alternate models for studying T-18 expression as these two organs/tissues underwent dynamic changes in their function throughout pregnancy. For instance, it is well known that CL is the primary source of progesterone for maintaining pregnancy in the rat and that the progesterone secreting capacity of the luteal cells peak on day 16 of gestation and decline thereafter. Interestingly, a common feature among all the tissues that were chosen for investigating the regulation of T-18 expression, is the fact that they underwent apoptosis with increase in gestational age. The expression of T-18, in tissues exhibiting increased incidence of apoptosis suggested that T-18 maybe an apoptosis-associated gene. Using an explant culture model it was demonstrated that placental villi when cultured in vitro underwent spontaneous apoptosis and that the levels of T-18 message increased, under these conditions. Furthermore, this spontaneous induction of apoptosis in explant cultures could be blocked when villi were cultured in the presence of superoxide dismutase, a free radical scavenging enzyme. In addition, the expression of T-18 was shown to be modulated following treatment with SOD, or in response to oxidative stress. These studies clearly indicate a role for T-18 in placental apoptosis and moreover, implicate the usefulness of explant culture to examine the molecular mechanisms involved in placental apoptosis. Furthermore, the expression of the anti- and pro-apoptotic genes, bcl-x and bax respectively, were investigated, in an attempt to elucidate the signalling pathway(s) that led to the activation of an important downstream protease, caspase-3, in placental apoptosis. The present study revealed that induction of apoptosis in the placenta in vitro involved a bcl/bax independent, caspase-3 dependant pathway. The validation of an explant culture model for studying placental apoptosis and data pertaining to the role of T-18, bcl-x, bax and CPP32 in placental apoptosis, in response to oxidative stress, are presented in chapter 5. The last section titled general discussion summarizes the work carried out in this study and proposes a model for the apoptotic mechanism(s) that may be operating in placenta In conclusion, the present study has led to the identification of two developmentally-regulated factors, T-Plastin and T-18 in the first trimester and term placenta, respectively. The differential expression of these genes, in addition to several other molecular players, is proposed to be responsible for the overall functional differentiation of the placenta through the course of gestation.

ROS are frequently associated with deleterious effects caused by oxidative stress. Despite the harmful effects of non-specific oxidation, ROS also function as signal transduction molecules that regulate various biological processes, including stem cell proliferation and differentiation. Here we show that mitochondrial ROS level determines cell fate during differentiation of the pluripotent stem cell line P19. As stem cells in general, P19 cells are characterized by a low respiration activity, accompanied by a low level of ROS formation. Nevertheless, we found that P19 cells contain fully assembled mitochondrial electron transport chain supercomplexes (respirasomes), suggesting that low respiration activity may serve as a protective mechanism against ROS. Upon elevated mitochondrial ROS formation, the proliferative potential of P19 cells is decreased due to longer S phase of the cell cycle. Our data show that besides being harmful, mitochondrial ROS production regulates the differentiation potential of P19 cells: elevated mitochondrial ROS level favours trophoblast differentiation, whereas preventing neuron differentiation. Therefore, our results suggest that mitochondrial ROS level serves as an important factor that directs differentiation towards certain cell types while preventing others.