Pluripotent Stem Cells: Cancer Study, Therapy, and Vaccination

Introduction Pluripotent stem cells (PSCs) are promising tools for modern regenerative medicine applications because of their stemness properties, which include unlimited self-renewal and the ability to differentiate into all cell types in the body. Evidence suggests that a rare population of cells within a tumor, termed cancer stem cells (CSCs), exhibit stemness and phenotypic plasticity properties that are primarily responsible for resistance to chemotherapy, radiotherapy, metastasis, cancer development, and tumor relapse. Different therapeutic approaches that target CSCs have been developed for tumor eradication. Results and Discussion In this review, we first provide an overview of different viewpoints about the origin of CSCs. Particular attention has been paid to views believe that CSCs are probably appeared through dysregulation of very small embryonic-like stem cells (VSELs) which reside in various tissues as the main candidate for tissue-specific stem cells. The expression of pluripotency markers in these two types of cells can strengthen the validity of this theory. In this regard, we discuss the common properties of CSCs and PSCs, and highlight the potential of PSCs in cancer studies, therapeutic applications, as well as educating the immune system against CSCs. Conclusion In conclusion, the resemblance of CSCs to PSCs can provide an appropriate source of CSC-specific antigens through cultivation of PSCs which brings to light promising ideas for prophylactic and therapeutic cancer vaccine development. Graphical Abstract


Introduction
Cancer is a major worldwide health issue and leading cause of death. It is estimated that the worldwide cancer rate may surpass 29 million cases annually with approximately 1 out of every 6 deaths in 2040 [1]. Despite outstanding progress in many therapeutic approaches, various factors such as the failure of current treatments, drug resistance [2], delayed diagnosis [3] and absence of a strong immune system in affected patients [4] have led to the lack of a definitive cancer treatment. Most importantly, metastasis post-treatment and recurrence of tumors after resection are the main causes of cancer death [5][6][7]. Therefore, therapeutic strategies, which can treat or prevent tumors from metastasis and recurrence could be an outstanding achievement in cancer therapy.
Currently, the role of cancer stem cells (CSCs) in the propagation of malignant tumors and resistance to therapy has become more accentuated. CSCs comprise a group of cells that can form transplantable tumors with the same Mojgan Barati and Maryam Akhondi authors contributed equally to this work. cellular heterogeneity that exists in their tumor of origin [8]. Beside the ability both to self-renew and to differentiate into several heterogeneous cells with less potency, major disorders related to malignancy such as plasticity, drug resistance, epithelial to mesenchymal transition (EMT) and metastasis, and tumor recurrence are attributed to the existence of CSCs. Many attempts have been made to isolate and characterize CSCs to enable further diagnosis and treatment; however, the lack of specific markers for cellular recognition is a serious challenge in dealing with CSCs [9]. There are several questions that should be answered about CSCs and understanding the origin of these cells may help define a useful model for further studies.
Pluripotent stem cells (PSCs), with an indefinite selfrenewal capacity and the potential to generate all cell types of the body, have many similarities with CSCs. PSCs are usually derived via direct cultivation of early embryos (embryonic stem cells [ESCs]) or from reprogramming of somatic cells (induced PSCs [iPSCs]) [10,11]. These cells have been used as promising sources for cell based therapies in clinical trials of spinal cord injuries, diabetes and agerelated macular degeneration (AMD) treatment (reviewed in [12]). However, PSC-based cancer therapy studies are mainly restricted to derivation of functional cancer fighting cells that include PSC-derived dendritic cells (DCs) [13], lymphocytes [14], and transplantable cells like hematopoietic stem cells (HSCs) [15]. The results of some studies have shown that exposure of different types of cancer cells to the PSC microenvironment could inhibit Nodal expression and suppress the malignant characteristics of these cells [16]. The importance of these PSC-based research priorities signifies a novel vaccination strategy against CSCs that possibly could induce long-term memory against a broad-spectrum of tumors [17].
In this review, we discuss the existing theories that concern the origins of cancer or, more specifically, those of CSCs, their resemblance to PSCs and application of PSCs against tumors as a novel cancer therapy approach.

Cancer Stem Cells (CSCs) as Stem Cell Malignancies: Origin of CSCs
Growing evidence support the idea of representing cancer as a stem cell malignancy and the introduction of CSCs as being responsible for metastasis and tumor recurrence [18,19]. Cell-cell fusion, horizontal gene transfer, genetic instability and the cell microenvironment are proposed mechanisms for these malignancies [20]. Accordingly, there are various assumptions about the origin of CSCs that include disrupted tissue-resident stem cells such as specific adult stem cells and their precursors, dedifferentiated somatic cells, and improperly activated residual embryonic cells in adult tissues.

Residual Embryonic Cells
The presence of cells with a semi-embryonic morphology in cancerous biopsies was initially reported in the nineteenth century. This finding ultimately led to the formation of a hypothesis entitled "embryonic rest of cancer origin" developed by Rudolf Virchow and his student Julius Cohnheim, which suggests that the remnants of embryonic cells persist in adult tissues and remain silent until adulthood [21]. In certain conditions, however, these cells can be activated by the influence of internal and external factors and they play a role in cancer development [22]. The close relation between embryonic and cancer cells was reported in cancers such as nephroblastomas that originate from germ cell tumors [23]. Researchers also identified some cells with embryonic morphology nominated as "very small embryonic-like stem cells" (VSELs). Possibly, during early embryogenesis when primordial germ cells (PGCs) originate from the proximal epiblast, a group of PGCs seats in other tissues where they remain silent. Therefore, it was hypothesized that migrating PGCs from the primary epiblast may be the origin of VSELs [24]. There is a hypothesis that places these cells at the top of the stem cell hierarchy in bone marrow, which results in the production of HSCs, mesenchymal stem cells (MSCs), endothelial progenitor cells, and production of tissue-committed stem cells (TCSCs) in other tissues ( Fig. 1) [25]. Furthermore, it is suggested that VSELs are a type of migratory stem cells that are released from bone marrow into the bloodstream during stressful conditions and participate to regenerate damaged tissues [26]. These cells could be found in bone marrow, brain, pancreas, thymus and intestinal epithelium, and ovarian and testis surface epithelium in human and rodents [25,27]. Improperly, fusion of normal cell with VSEL as a circulating stem cell provides the possibility of formation cancer initiating cell or CSCs in solid tumors [26]. VSELs are also isolated from human ESCs' culture using stem-cell-related markers (CD133, SSEA-4) and germinal lineage markers (DDX4/VASA, PRDM14) [28]. These cells possess many characteristics of pluripotent cells; for example, a prototype of undifferentiated morphology with high chromatin content in the nucleus and high nucleus to cytoplasm ratio; presence of bivalent domains in chromatin; pluripotency marker expressions such as Oct4, NANOG, and SSEA1 (murine) or SSEA4 (humans); and the ability to form spheres and in vitro differentiation into derivatives of the three germ layers [29,30]. Moreover, like ESCs, these cells have elevated telomerase activity and lack MHC class I expression on their surfaces [29,[31][32][33]. These also express germ cell markers such as Stella and Blimp1, as well as epiblast markers Gbx2, Fgf5 and Nodal [24].
To have a historical look at the development of VSELs, it should be referred to the mechanism of PGCs migration in the gastrulating embryo. At the onset of PGC migration, the complete erasure of imprinting marks from DNA of these cells results in downregulation of insulin-like growth factor 2 (IGF2) and upregulation of the tumor suppressor gene H19, which promote and inhibit growth, respectively [34]. Indeed, parental allele-specific expression of Igf2/H19 genes is controlled by differentially methylated regions (DMRs) and imprinting control regions (ICRs) which its regulation relies on DNA methylation. Normally, growth factor gene Igf2 is expressed from the paternal allele and tumor suppressor gene H19 is expressed from the maternal allele. These genes are neighboring genes, and both respond to the same enhancers located at H19's distal end. Briefly, the monoallelic expression of Igf2 is mediated by allele-specific DNA methylation at CTCF-binding sites in ICR, located immediately upstream of the H19 gene. Actually, CTCF (known as CCCTC-binding factor) mediates the insulation at the H19-Igf2 imprinted domain [35]. The methylated paternal ICR inhibits CTCF protein binding, which allows the Igf2 promoter to interact with the enhancers and be transcribed. Unmethylated maternal ICR binds to CTCF and prevents the Igf2 promoter from interacting with the enhancers. As a result, only H19 is transcribed on the maternal chromosome. However, in the late migratory PGCs, and similarly in developing VSELs, the complete erasure of imprinting marks from DNA results in down-regulation of Igf2 and up-regulation of H19 which led to the prevention of uncontrolled proliferation in these cells and explain the quiescent state of VSELs in adult tissues ( Fig. 1) [34]. Improperly, if VSELs in adult testes affected by endocrine disruption and alteration of epigenetic modifications in imprinted genes, they may initiate self-renewal and promote testicular cancer [36]. Furthermore reduced sperm count and infertility can occur as a result of increased VSEL proliferation and impaired differentiation [37]. According to Ratajczak and his colleagues, VSELs can convert to CSCs and promote the formation of teratomas and teratocarcinomas, germinal tumors, or pediatric sarcomas if they achieve relevant mutations [26]. The presence of VSELs in ovarian teratoma and testicular cancer suggests these cells as tumor-initiating stem cells. In these tumors, VSELs express both pluripotency and germinal lineage markers and suggests to play important role in tumor invasion through the activation of EMT [38]. On the other hand, like VSELs, the expression of cancer testis antigens (CTAs; for example: MageB3, Ssbx2, and BORIS) and epiblast/germline-related cells are seen in some tumors [26,38]. It's worth noting that identifying these small putative NANOG-, SOX2-, and SSEA-4-positive cells that are responsible for tumor formation in ovarian cancer is crucial from a clinical point of view as the potential therapeutic targets [39].

Tissue-specific Stem/Progenitor Cells
Tissue-specific stem/progenitor cells, also known as adult stem cells, are a rare cell population that have self-renewal and multi-lineage differentiation abilities. These cells are present in various tissues such as the intestines [40], breasts [41], lungs [42], ovaries [43] and prostate [44] and they function to repair or maintain these tissues. In a "classical model", it has been assumed that the asymmetric mitotic division invariably creates one new stem cell and one daughter cell in each niche space of the adult tissues. However, in a "neutral competition model" it is supposed that each of the daughter cells, and even fully differentiated cells, can re-enter the niche and dedifferentiate to replace lost stem cells. The evidence to support this claim is that, with some rare exceptions such as muscle satellite cells or hair-follicle stem cells, the finding of such tissue-specific stem cells in most tissues of the body is almost impossible [45].
On the other hand, tissue-specific stem/progenitor cells are also considered as a primary suspect of cancer onset. However, definitions of these cells described in some studies, for example the assuming of a quiescent state of these cells during the lifetime of their host [46] and their activation under adverse conditions such as tissue damage that may lead to the conversion of these cells to CSCs is more in line with the definition that applies to the VSELs.
It is generally believed that the proliferation of tissuespecific stem/progenitor cells is precisely under control while they also are prompt enough to take action in confronting any awakening signal [47]. Furthermore, these cells can maintain their homeostasis and survival during differentiation by controlling the metabolic process of autophagy [48]. By receiving different signals from their surrounding cells and the microenvironment, tissue-specific stem/progenitor cells can quit quiescence and regain selfrenewal, proliferation, and the ability to differentiate into different cell types, which enables them to maintain an effective role in regeneration [49]. However, it cannot be ignored the accumulation of genetic and epigenetic mutations that specifically be caused by aging may exert adverse effects on the accuracy of self-renewal and cell survival pathways of these cells [50][51][52]. Moreover, it is also possible that issue-specific stem/progenitor cells may turn into cancer initiating cells by fusion with abnormal cells or by absorbing damaging cellular material from cells trapped by phagocytosis and cause alterations in their normal mechanisms [53]. It is likely that processes such as autophagy can apply changes to the stem cell cellular microenvironment, homeostasis, and maintenance, and affect adult stem cell survival by inducing apoptosis or a cancerous state ( Fig. 2) [54][55][56][57].
On the other hand, CSCs can generate progenitors that have self-renewal ability and less stemness capability, which is similar to multipotent tissue-specific stem/progenitor cells [46,58]. In addition, signaling networks, like the Wnt and Notch pathways, are common between CSCs and multipotent tissue-specific stem/progenitor cells [59,60], and signal transduction occurs through some of the same surface receptors such as CD24 [61], CD44 [62], CD133 [63,64], and CD49f [65,66]. This data suggests a close relationship between these two cell types [42,44,67].

Stem/Progenitor Cells Produced by Reprogramming
In 2006, Takahashi and Yamanaka established a major breakthrough with the generation of iPSCs by reprogramming differentiated somatic cells into the pluripotent state by induction of four transcription factors (TFs)-Oct4 (Pou5f1), Sox2, c-Myc, and KLF4 [11,68]. Since most of these TFs are oncogenes [69], genetic and epigenetic alterations in differentiated somatic cells have been hypothesized to activate dedifferentiation processes in adult cells to convert them to CSCs [70]. In this scenario, the expression of differentiation-specific genes in somatic cells is gradually downregulated and upregulation of stemness-related genes occurs, which leads to the establishment of a pluripotencylike state. This transformation has been observed in basallike human mammary epithelial cells [71] and differentiated airway epithelial cells [72]. Similarly, in intestinal tumors, activation of NF-κB in response to inflammatory pathways leads to activation of Wnt signaling and consequently results in reprogramming of non-stem cell intestinal epithelial cells (IEC) into a pluripotent state [73]. Proto-oncogene activating mutations such as KRas and its downstream target c-Myc are among the other dynamic ways that transform cells into cancer stem-like cells. Interestingly, KRas activating mutations have been detected in over 90% of pancreatic cancers and is one of the key mutations in various cancers [74][75][76].
The tumor microenvironment is another important factor that may contribute to transformation of differentiated cells into stem-like cells. In colorectal tumors, cancer-associated myofibroblasts can induce dedifferentiation pathways by secreting hepatocyte growth factor (HGF) that consequently activates Wnt signaling, which enhances stemness properties in differentiated cells [77]. T-cells and macrophages can direct dedifferentiation of differentiated cells through the secretion of tumor necrosis factor alpha (TNF-α) in the induced inflammatory microenvironment in melanoma. On the other hand, it has been assumed that these microenvironments induce plasticity in tumor cells that lead to changes in certain surface markers and the resistance of the tumor cells to an anti-cancer immune response [78]. In general, the tumor microenvironment increases cellular plasticity, which induces various cell types at various differentiation levels to transform into CSCs (Fig. 3).
Regardless of the cell of origin, the stemness characteristics and the resemblance of CSCs to early developmental cells is significant in the stem cell theory of cancer (Fig. 4). In many ways cancer cells imitate the early developmental pathways at the biological and molecular levels. The activation of initial developmental signaling pathways [79] and the TF expressions  (Table 1). Moreover, the similarities in protein content [79] and metabolism [81] can show the proximity of CSCs and PSCs.

Key Transcription Factors (TFs)
The induction of the main pluripotency-associated TFs Oct4, Sox2, KLF4 and NANOG results in induction of pluripotency in terminally differentiated cells into iPSCs. These TFs are considered to be key controllers of stemness in CSCs [82]. Oct4 plays an important role in germ cell tumor formation and is a diagnostic factor in these tumors. Post-translational modifications of Oct4 increase its stability in breast CSCs [83]. Oct4 is considered to be a renal CSC marker along with high expression of NANOG, and it is responsible for maintenance of CSCs, which negatively affects survival [84]. Oct4 expression increases stemness, drug resistance and migration of pancreatic CSCs (PCSCs) [85]. Similar to Oct4, NANOG plays an important role in maintaining malignant stem cell populations and CSCs of glioblastoma, liver, colon and prostate cancers [86][87][88][89]. Sox2 expression can be seen in squamous cell carcinoma of the esophagus [90] and small cell lung carcinoma [91], and its expression is necessary in maintenance of the CSC microenvironment in glioblastoma and breast cancer [92].
On the other hand, KLF4, is a double-edged sword in cancer development. Although this TF promotes malignancies in bladder urothelial carcinoma and osteosarcoma [93,94], its inhibitory role in colorectal cancer [95], neuroblastoma [96], and nasopharyngeal sarcoma [97] has been reported. KLF4 interaction with c-Myc, a proto-oncogene, and its dissuasive effect on p53, a tumor suppressor, results in an increased proliferation and enhanced reprogramming that can be seen in p53 deficiency. Lack of efficient p53  [98]. KLF4 also triggers induction of telomerization activity in cancer cells, which is similar to ESCs [99]. C-Myc augments cell proliferation in many human malignancies and plays a role in reprogramming of fibroblasts to iPSCs [100]. This factor also affects the enzymes involved in transcription and can increase total transcription levels [101]. Together, these factors form a network of interactions that have two sides-one promotes malignancy and the other is responsible for the pluripotent state of cells. The balance between these two defines the normal cell state.

Signaling Pathways
Self-renewal and proliferation are two characteristics of both CSCs and PSCs; however, unlike PSCs, these mechanisms are not strictly controlled by CSCs. Both cells adjust the self-renewal process through the same signaling pathways (Table 2). However, irregular expressions of key factors in CSCs disrupt their balanced state and make them prone to tumor formation [102].
Notch: During development, the Notch signaling pathway is one of the central pathway that makes contact between adjacent cells; it is necessary for differentiation and determination of the cell fate and homeostasis of adult tissues [59]. The Notch signaling pathway reportedly functions in human ESCs (hESCs); however, it appears to be unnecessary for maintenance of pluripotency in these cells. Conversely, Notch signaling plays an important role in maintenance of the differentiating cell types in hESC cultures [103] such as neural differentiation of hESCs [104], as well as migration and proliferation of retinal pigment epithelial cells [105]. Upon activation by direct cell contact, cleaved Notch intracellular domain (NICD) in conjunction with a complex of proteins affects downstream targets that include AKT, NF-kB, p21, p27 and CyclinD1 [106]. Changes in these targets may result in changes in the cell fate, differentiation and uncontrolled proliferation, and may result in melanomas, medulloblastoma, ovarian and breast cancers [102,[107][108][109][110]. As a cell context dependent pathway, Notch signaling may also act as a tumor suppressor [111]. Several therapeutic approaches target components of this pathway. Delta like (DLL-1, DLL2, DLL4) or Jagged (JAG1, JAG2)  [187][188][189] head and neck squamous cell carcinoma (HNSCC) [190] Small Cell Lung Cancer (SCLC) [191,192] [193,194] Medulloblastoma [123,125,193,195] Colon cancer [196] Breast cancer [197] Chronic Myelogenous Leukemia (CML) [198] T-cell Acute Lymphoblastic Leukemia (T-ALL) [199] pancreatic Ductal Adenocarcinoma (PDA) [200] ligand, Notch receptors (Notch1-4) and ɣ-secretase complex have been studied. However, in CSCs, several pathways are interconnected and suggested therapies should cover all features of signaling [60].
Wnt: Wnt is one of the most conserved signaling pathways that is active in a wide variety of organisms and is essential for maintenance and self-renewal of adult stem cells (i.e., HSCs) and PSCs [112][113][114]. Its first connection to cancer was discovered when Wnt overexpression resulted in tumor formation in mice [115]. Nearly a decade later, mutations in the adenomatous polyposis coli (APC) gene were found to be responsible for hereditary colon cancer syndrome [116]. Normally, in the absence of the Wnt ligand, APC along with Axin and GSK3β form a regulatory complex which degrades β-catenin. Mutated APC activates Wnt signaling by stabilizing β-catenin, which results in T-cell factor (TCF) 4 transcriptional activity [117]. Non-canonical Wnt signaling, which is β-catenin independent, may play a role in cancer malignancy by activating planar cell polarity (PCP) and calcium signaling, and result in cell migration and possible metastasis. However, canonical Wnt signaling has been observed more frequently [116]. Telomerase subunit TERT expression after Wnt activation helps to keep telomerase activity, which also links Wnt to CSCs [99]. Other than colorectal carcinoma (CRC) [118], the role of Wnt signaling in leukemia [114], melanoma [119] liver cancer [120], prostate cancer [121] and triple negative breast cancer [122] has been studied and several drugs against this pathway have entered clinical trials [60].

Hedgehog (Hh):
Hh signaling is responsible for cell fate determination, neuro-ectodermal lineage differentiation, and polarity and pattern formation during organogenesis. Hh ligand binding to the PTCH1/2 receptor and Smoothened activation enables GLI families of TFs to target the expressions of several genes [123,124]. Although this pathway is not a key regulator of hESC self-renewal and maintenance [123], disruptions in the normal activity of downstream targets such as the PTCH1 and SMO genes have been discovered in patients with basal cell carcinoma (BCC), medulloblastoma, colon cancer, breast cancer, chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) [123,125]. Interestingly non-canonical Hh signaling (Shh-Ptch-SMO independent) seems to be important in pancreatic ductal adenocarcinoma (PDAC). Several different therapies that use SMO antagonists and PTCH1 inhibitors have shown that cancer cells are resistant to some of the SMO inhibitors [125]. Li et al. recently reported that some SMO inhibitor substitutes could be used successfully against this resistance and present a novel opportunity for treatment of medulloblastoma [126].

Surface Antigens
Tumor and fetal cells have many surface antigens in common; hence, these markers are called oncofetal antigens and they include carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), cancer-testis antigen (CTA), human placental lactogen (hPL), alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (β-hCG) and placental alkaline phosphatase (PLAP) [127][128][129][130][131]. Exposure of mature animals to embryonic materials has been shown to increase tumor resistance and tumor transplant rejection [127]. The similarity between the serum antibodies of a cancer patient to a pregnant woman can be attributed to the similarities between cancer and embryonic antigens [132,133]. Thus, cancer diagnosis during pregnancy may be difficult because both conditions have elevations in the cancer antigen 15-3 (CA 15-3), squamous cell carcinoma antigen (SCC), CA 125, and AFP markers [134]. Many differentiated tissue antigens lose their expressions after tumor formation, which is called "antigenic simplification" [127]. In return, the tumor cells begin to re-express the embryonic genes/ antigens. More importantly, the immunity against tumors created by embryonic material is both humoral and cellular [17,135]. Based on these findings, one therapeutic approach against cancer could be a vaccine based on embryonic/fetal materials.

Developmental Biology: A New Approach to Cancer Treatment
The aforementioned similarities between fetal and cancer cells, especially malignant ones, show that early developmental programs that are active in ESCs and are responsible for fetal development play a role in cancer formation. As these developmental processes are under strict regulation in an embryo, mimicking those regulations may be effective in finding new therapeutic agents against cancer.

Cancer and Cancer Stem Cell (CSC) Modeling Using Pluripotent Stem Cells (PSCs)
The discovery of the ability to reprogramming of somatic cells into PSCs has developed the view of almost all aspects of biomedical research, including cancer. Recently, similar to iPSC derivation technology, the reprogramming protocols that utilize key pluripotency-associated TFs have been used to induce pluripotency in cancer cell lines. In these studies, melanoma-R545, CML-KBM7, osteosarcoma cell lines- (SAOS2, HOS, MG63), non-small cell lung cancer lines- (H358, H460, IMR90), and gastrointestinal cancer cell lines (GCC) have been placed through dedifferentiation that resulted in the generation of iPSCs. However, some of these cells have karyotype abnormalities and are resistance to anti-cancer drugs [136,137]. On the other hand, in order to observe the cancer formation process, iPSCs were derived from somatic cells that contained germline mutations and subsequently differentiated into lineages that had several early signs of malignancy. This method was used as a model in Li-Fraumeni syndrome [138], myelodysplastic syndrome [139], PDAC [140], and colorectal cancer [141] to determine the role of genetic mutations responsible for these malignancies. In the CSC modeling related studies, it was shown that exposure of normal iPSCs to the mouse Lewis lung carcinoma (LLC) cell line microenvironment and extracellular vesicles could turn them into cells with CSC phenotypes that had high tumorigenic and metastatic potentials once transplanted into nude mice [142,143]. Besides self-renewal, these CSCs could differentiate into endothelial cells that resulted in tumor angiogenesis both in vitro and in vivo [144]. Additionally, drug testing on prostate CSC-like cells derived from iPSCs by the same protocol indicated that these cells are resistant to chemotherapeutic drugs and sensitive to agents that target their stemness [145]. These findings from cancer and CSC modeling with iPSC technology suggest that these cells can help to determine the signaling pathways and mechanisms of tumorigenesis, metastasis, and angiogenesis, and also act as platforms to study and test drugs against stemness and CSC-like characteristics of different types of cancer.
It is important to note that despite the increasing advances in iPSC production and further lineage differentiation with improved protocols, cancer modeling with iPSCs has not yet made much progress. Technically, this limitation can be attributed to two factors. First, some studies have shown that cancer cells are more resistance to reprogramming than normal cells. This resistance may be due to various cancerrelated mutations in cancer cells as the starting material for reprogramming. The second factor is that the process of creating iPSCs from a cancer cell and further lineage differentiation does not work in the cell culture in exactly the same that it does in cancer. As mentioned before, most likely, the cancer starts from VSELs or tissue-specific stem/progenitor cells. Therefore, a proper modeling of cancer with iPSCs that was close to reality must first be able to create progenitor cells in this simulation instead of directly producing the final differentiated cells. Moreover, most differentiated cells from iPSCs are similar to cells in early development rather than phenotypes in adults [146].
Another challenge that limits the modelling of cancer is that iPSCs inherit their starting cells' genetic memory. Therefore, the variable genetic instabilities of the primary cancer cells are captured and amplified in their iPSCs. It may be resolved by isolation of single cells and clonal propagation in order to establishment of a stable original genome.
There are also other challenges related to iPSCs' production per se, such as epigenetic instability, incomplete reprogramming, and line-to line variation, that are true for modeling of cancer with iPSCs, too [146].

Differentiation Therapy by Induction of the Embryonic Microenvironment
There is an overlap between signaling pathways that control developmental processes and tumor progression. Thus, the question arises whether or not cancer cells can respond to embryonic messages. Several studies have been designed to answer this question. By using matrix conditioned with hESCs, Postovit and colleagues showed an increase in melan-A (MLANA) expression in human melanoma cells and a reduction in their invasiveness [147]. The inhibitory nature of the conditioned medium of ESCs and secretory factors released by these cells increased the number of CSCs in the G1 phase and with the cell cycle arrest without inducing apoptosis [148]. Investigation of intercellular interactions between breast cancer cells and ESCs in a 3D-engineered tumor model showed that low molecular weight factors released by pluripotent cells suppressed Stat3 signaling and inhibited cancer cell proliferation and migration [149]. Examination of this microenvironment showed that exposed exosomes were the main factors that affected tumor cells and reduced their invasiveness [150]. Notch signaling is also responsible for the antitumor activity of this specific environment against colorectal cancer [151]. Another important factor in the embryonic microenvironment that regulates cancer cell proliferation and differentiation is Gremlin, a BMP4 antagonist. Inhibition of BMP4 activates senescence in melanoma cells [152]. Various miRNAs have been shown to play roles as tumor inhibitors or as oncogenes in cancer. Melanoma cells exposed to hESCs had increased miR-302 expression. An augmented level of miR302 by Notch4 inhibition causes Nodal reduction. On the other hand, expression of miR-27b, which is responsible for angiogenesis and progression of melanoma in the early stages, is reduced after exposure to the hESCs microenvironment [153]. These studies show the modulation effects of ESCs and their microenvironment on cancer cells, and indicate the potential impact of this approach on cancer treatment. However, the mechanism of these effects is still not clear.
Furthermore, the effects of the embryonic microenvironment on tumor cell progression and metastasis has been investigated in animal models. In a study by Illmensee and Mintz, injection of terato-carcinoma cells into mouse blastocysts not only inhibited the tumor phenotype of the cells, but the terato-carcinoma cells could participate in the formation of normal tissues. In a chick model, the tumor-suppressing activity of the embryonic microenvironment also inhibited sarcoma formation after injection of Rous sarcoma virus [154]. It has been shown that tumorigenicity of the mousemelanoma cells decreased when the cells were exposed to factors secreted by neural crest region and the cells gained specific markers of differentiated cells such as neural marker TUJ1 [155]. Therefore, embryonic microenvironments appear to provide regulatory factors that are lost in cancer cells during transformation and these factors can be used to restructure embryonic signaling in cancer cells and their microenvironment (Fig. 5).

Using Pluripotent Stem Cells (PSCs) as a Cancer Vaccine
One of the new potential application of PSCs in cancer research that has been raised in recent years, is the use of PSCs as a global prophylactic cancer vaccine. This idea may seem strange at first; whereas both PSCs and CSCs have common stemness factors, and just as CSCs, PSCs can pose the same threat for body. But, in fact, the Differentiation therapy. Communication between the cells and their microenvironment is essential for the maintenance of normal tissues or formation of cancers and their metastases. In cancer cells, there is disrupted regulation of cell differentiation, which leads to the exhibition of a tumor promoting phenotype. The embryonic niche or embryonic stem cell (ESC) conditioned-media can promote undifferentiated cancer stem cells (CSCs) to become differentiated, which ultimately inhibits tumor growth. After injection into an early embryo, aggressive CSCs learn to "speak the same language" of the embryonic cells and participate in embryo formation instead of tumor growth. The specific niche of the embryo can change the CSC phenotype toward specific cell lineages related to that niche possibility of large scale cultivation of genetically stable PSCs in vitro, makes available the high amounts of CSCsspecific antigens. In one hand, the conditioned medium of PSCs per se can provide an environment similar to the embryonic environment that promote cancer treatment through differentiation therapy (Fig. 5), and on the other hand, PSCs can be considered as a valuable source to have large amounts of CSCs-specific antigens. Thus far, inactivated PSCs have been used to make prophylactic vaccines against colon [156], lung [17] and ovarian cancer [157] in animal models.
While in the immunosuppressive environment of tumors, cancer cells can suppress the activation of immune cells through various mechanisms, including the recruitment of immunomodulatory MSCs, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs), the attenuation of MHC class I expression, and the use of the PD-1/PD-L1 axis, cancer vaccines based on cancer-specific antigens can educate the immune system against cancer cells, so that it can encounters and eliminates them. The basic idea behind a cancer vaccine is to find the optimal antigen and vaccine delivery system that is capable of stimulates immune cells. As a result of cancer vaccines, immune responses could be guided selectively toward patients' cancerous cells while preventing their normal cells from immune attack, potentially avoiding side effects. For example, DCs as a professional APC can induce tumor-specific cytotoxicity by processing and presenting tumor antigens to T lymphocytes. DCs can load with different type of antigens including peptide, RNA and also with whole tumor antigen via tumor lysate or fusion with tumor cells and present numerous antigens to immune effector CD8 + -and CD4 + -T cells [158,159]. In addition, induction of immunological memory by DCs overcomes cancer cell plasticity and tumor recurrence [160]. However, DC vaccine therapies require an effective and adequate number of DCs to produce the desired results while in most patients, functional DC is difficult to achieve PSCs or their extracellular vesicles can induce antitumor immunity that suppresses cancer cell proliferation and prevents tumor devel-opment. Right: PSC-based vaccines can educate the immune system against the cancer and overcome low or no immunogenicity that exist in the tumor microenvironment due to chemotherapy or the tumor's immunosuppressive state [161].
Based on this idea, it is suggested that the exposure to conditioned medium, lysate, or mitotically inactivated CSCs or PSCs can activate the immune system and makes immunity against both embryonic and cancer cells [162]. This ability of PSCs can be used by introduction of embryonic materials to cancer cells. The studies show significant reductions in tumor growth and activation of both the humoral and cellular immune systems in response to PSCs. These immunities prevent the growth and proliferation of cancer cells, and an increase in APCs and activation of lymphocytes has been also reported [163]. Interestingly, in these studies, hESCs also produced immune responses in rats, and it can be concluded that the immune function of these cells is species-independent. These vaccines did not show any side effects such as autoimmune reactions [164]. A PSC-derived based vaccine for cancer combined with a histone deacetylase inhibitor (HDACi) could modify the immune suppression microenvironment of the tumor by increasing CD4 + or CD8 + tumor infiltrating lymphocytes and reducing T regulatory cells. This combined regimen targets CSCs; hence, it could decrease the incidence of metastasis and improve survival rates [165]. Furthermore, vaccination with ESC exosomes, a derivative of these cells in the presence of GM-CSF, resulted in reduced tumor size and immune response [166]. The enhanced targeting ability of ESC exosome carriers combined with chemotherapeutic agents can act highly reactive against tumors [167]. These studies show how ESCs and their secretome act as tumor suppressors and this can be a potential therapeutic approach. The use of exosomes instead of inactivated cells can be a tremendous step towards a cell-free vaccination against cancers (Fig. 6). Overall, these studies showed that with the antigenic similarity to CSCs, PSCs or PSCs-derived secretome therapeutic cancer vaccines can be highly immunogenic against CSCs. As cancer treatment has always been a challenging issue due to the high rate of tumor recurrence and metastasis, PSC-based therapeutic cancer vaccines can theoretically help to overcome the effects of immunosuppression in the tumor microenvironment.

Conclusion and Future Outlook
Despite multiple therapy attempts, cancer has not been defeated. Tumor cell heterogeneity that causes differences in their biology, metabolism and signaling pathways as well as the effects of the microenvironment on normal cells are challenges that complicate the choice of treatment. Fortunately, recognition of processes such as autophagy has contributed greatly to better identification of cancer cells, and allows for a more effective treatment approach. In order to achieve successful and effective treatment, cancer cells have been targeted based on their recurrence and metastasis. As noted above, there is stemness, pluripotency and the expression of early developmental markers in cancer cells, especially in malignant cancers with poor prognosis. Apart from the assumptions related to the origin of CSCs, the existence of morphological and molecular similarities between cancer cells and early-stage stem cells suggests that early developmental programs are inappropriately activated in cancer; however, the mechanism of this phenomenon is not completely understood. It is assumed that the inappropriate expressions of pluripotency factors and reprogramming of somatic or adult stem cells are the main sources of tumor-like stem cells. There is a defect in developmental biology in these scenarios despite the fact that most developmental biology is observed in embryonic development. Therefore, it seems that a study of embryonic development and its differences and similarities with the developmental processes in cancer can help us to find new therapeutic approaches. Targeting markers and stemness and pluripotency pathways, the use of ESCs as a vaccine, and consideration of embryonic microenvironments in order to regulate developmental phenomena have been investigated to improve cancer treatments.