Homo sapiens, for its high relevance, and Mus musculus, a historically common and technically advanced model organism. We aim to provide a holistic understanding of these in vitro models by tracing the historical background of the progress made in stem cell biology and discussing the fundamental underlying principles. At each developmental stage, we present corresponding in vitro models that recapitulate the in vivo embryo and further discuss how these models may be used to model diseases. Through a discussion of these models as well as their potential applications and future challenges, we hope to demonstrate how these innovative advances in stem cell research may be further developed to actualize a model to be used in clinical practice.Recent discoveries in stem cell and developmental biology have introduced a new era marked by the generation of in vitro models that recapitulate early mammalian development, providing unprecedented opportunities for extensive research in embryogenesis. Here, we present an overview of current techniques that model early mammalian embryogenesis, specifically noting models created from stem cells derived from two significant species:  Researchers create synthetic embryos, advancing our understanding of early human development and offering new possibilities for disease modeling. In a significant breakthrough, scientists have generated synthetic embryos using stem cells, providing a more accurate model of early human development. These models, including blastoids and gastruloids, mimic the structure and function of early embryos and can be used to study the molecular mechanisms governing embryogenesis. By replicating various stages of development, synthetic embryo models can help researchers investigate congenital diseases and potential clinical applications. As this technology continues to evolve, it may lead to new insights into human biology and the development of novel therapies for a range of conditions. Drosophila embryos, these researchers found key genes associated with regulating several significant early developmental processes, such as segmentation and polarization in Drosophila. Their groundbreaking discoveries in the genetic control of embryonic development laid the foundation for a deeper understanding of the molecular mechanisms governing the formation of multicellular organisms2.The 1995 Nobel Prize in Physiology or Medicine, awarded to Edward B. Lewis, Christiane Nüsslein-Volhard, and Eric F. Wieschaus, marked a turning point in the field of modern developmental biology. By introducing mutations in 3, ethical concerns about using human embryos4, and physical inaccessibility due to the intrauterine development of in vivo human embryos.Over the past three decades, substantial progress has been made in developmental biology ever since the foundation of several techniques, such as genetic engineering techniques, wherein various animal model systems were actively utilized to further enhance the understanding of genetic functions within the processes of embryonic development. However, studying human development has remained a significant challenge due to the inherent differences between conventional model organisms and humans5, which are three-dimensional, self-organizing structures that mimic various human organs. This remarkable progress has expanded the boundaries of human biology research, allowing the investigation of human biological processes in a more physiologically relevant context6. In addition to organoids, recent advances in stem cell research have even facilitated the development of early embryonic models that mimic human embryos. Early embryonic models provide an opportunity to investigate molecular mechanisms that regulate human embryogenesis with better scalability and in a relatively ethical manner, thereby overcoming the major limitations of using natural human embryos10.Recently, the rapid advancement of stem cell research has enabled the generation of stem cell-derived human organoids11, which possess the ability to self-renew and differentiate into a variety of specific cell types of the body. The potency of ESCs varies depending on their epigenetic traits and culture environment12. These shifts in potency allow the induction of various in vitro models, including synthetic whole embryo models that incorporate all significant cell types, such as those derived from both the embryo and extraembryonic tissues8. This review describes the process by which stem cells, including ESCs in relation to their potency, are utilized to create these in vitro models.Early embryonic models were generated by using embryonic stem cells (ESCs)This review aims to explore developments in stem cell research, focusing on stem cell-based in vitro early embryonic developmental models. Overall, this review article aims to provide a comprehensive analysis of the new era of stem cell research, examining the potential uses of developmental-stage-specific embryo models for disease modeling and possible clinical applications. By providing a thorough discussion of these technologies, we hope to inspire further research and advancements in the field.13. Based on their origin, they are divided into two major categories: ESCs and adult stem cells (AdSCs). ESCs are derived from the inner cell mass (ICM) of blastocysts and can differentiate into any type of cell in the body14, whereas AdSCs are found in various tissues in our body and help maintain tissue homeostasis but can differentiate into only a limited number of cell types relative to ESCs15. Early embryonic in vitro models were all derived from ESCs or induced pluripotent stem cells (iPSCs)16 of equivalent potency. This section focuses on ESCs, which serve as the foundation for creating in vitro models, and analyzes their diverse potencies  from the ICM of blastocysts, marking the beginning of modern stem cell research21. Building on the advancements in mESC research, the scientific focus shifted to human ESCs (hESCs), leading to a significant breakthrough in 1998 when Thomson et al. successfully derived and cultured hESCs in vitro by isolating them from the ICM of human blastocysts14. Both mouse and human ESCs possess the capacity for self-renewal and pluripotency, which allows them to maintain an undifferentiated state while proliferating in culture and to differentiate into any cell type of the three germ layers 20.The development of in vitro culture methods also played a crucial role in the establishment of ESCs. Evans pioneered the use of irradiated chick embryo fibroblasts as feeder cells to culture mammalian stem cells in vitro, which enabled the maintenance and expansion of pluripotent stem cells (PSCs)22. mESCs, for instance, have a tendency to form a dome-like structure, whereas hESCs typically exhibit a flattened shape22. Both mESCs and hESCs express the pluripotency-related transcription factor octamer-binding transcription factor 4 (OCT4), albeit with distinct regulatory mechanisms. In mESCs, OCT4 expression is regulated by its distal enhancer, while in hESCs, its expression is mainly regulated by its proximal enhancer24.Although initially established mESCs and hESCs share certain traits, they also display distinct differences in morphological, transcriptional, and epigenetic features26. These cells, derived from the epiblast of post-implantation embryos, were termed mouse epiblast stem cells (mEpiSCs) and shared morphology and a variety of epigenetic properties similar to hESCs26. Continuous investigations have revealed that the differences between mESCs and hESCs originate from the fact that these ESCs represent different stages of development, thereby influencing their slightly different differentiation potentials.As subsequent research progressed, a method was developed to culture mESCs that exhibited characteristics similar to hESCs12. These states have distinct molecular and functional characteristics, and understanding their differences is crucial for optimizing the use of ESCs in modeling early embryos in vitro.Mammalian ESCs are known to exist in a continuum of configurations, which at either end of the spectrum lie two different pluripotent states: naïve and primed27 , which activates the Janus kinase-signal transducer and activator of transcription 3 (JAK-STAT3) signaling pathway31. The development of specific culture conditions to derive and maintain mEpiSCs facilitated further investigation into the signaling pathways that regulate primed pluripotency, including the fibroblast growth factor/extracellular signal-regulated kinase (FGF/ERK) and Activin/Nodal pathways26.The first mESCs established were in the naïve state32 and transforming growth factor-beta (TGF-β)/Activin/Nodal pathways33, which are known for maintaining primed pluripotency. The application of small molecules to target these pathways enabled the conversion of primed hESCs into a state resembling naïve pluripotency34.The establishment of naïve hESCs required the identification of culture conditions that could support the naïve state while suppressing the primed state. This requirement led researchers to focus on modulating signaling pathways, including the inhibition of the FGF/ERK35. They showed that inhibiting the FGF/ERK, TGF-β, and WNT/β-catenin pathways while simultaneously activating the LIF/STAT3 signaling pathway could prompt the conversion of primed hESCs to a naïve state36. These newly established naïve hESCs exhibited key features of naïve pluripotency, including the acquisition of epigenetic states similar to those of naïve mESCs, the expression of naïve-specific markers, and the attainment of an enhanced differentiation potential35.After years of extensive efforts, the landmark achievement of establishing naïve hESCs was made by Gafni et al. in 2013. Gafni and colleagues successfully established naïve hESCs using a combination of small molecules and growth factorsThe early stages of mammalian embryogenesis, leading up to implantation, encompass a series of conserved, systematic steps across various species, including ovulation, fertilization, cleavage, and implantation. Upon ovulation, the oocyte travels through the oviduct, where fertilization takes place in the ampulla. As the embryo travels toward the uterus, it undergoes asynchronous cleavages. At this juncture, the mammalian genome is activated, facilitating embryonic development through the expression of early transcribed proteins.40. Researchers have endeavored to model this process by generating human eight-cell-like cells (8CLCs) to probe the mechanism of totipotency41. In 2022, Mazid et al. developed a method to produce 8CLCs from human PSCs (hPSCs), revealing key roles of developmental pluripotency associated 3 (DPPA3), a master regulator of DNA methylation in oocytes, and tetrapeptide repeat homeobox 1 (TPRX1), a eutherian totipotent cell homeobox (ETCHbox) family transcription factor, in this process. Using this embryo model, the researchers demonstrated that 8CLCs can contribute to embryonic and extraembryonic lineages, providing a valuable resource for the study of the earliest stages of human embryogenesis.In humans, major waves of zygotic gene activation occur at the 8-cell stage, whereas in mice, activation arises at the 2-cell stage 42. This compact embryo matures into a 16-cell morula, where its inner cells later become constituents of the ICM, and its outer cells give rise to the trophectoderm (TE)43. The ICM is situated on one side within the TE, culminating in the formation of the blastocyst. In essence, the blastocyst comprises two primary cell populations: the ICM and the TE.Compaction is one of the most significant events of mammalian cleavage. In mice, after the embryo reaches the 8-cell stage, compaction occurs as blastomeres express cell adhesion proteins and coalesce into a sphere of cellsThe peri-implantation period designates the phase during which the blastocyst is unattached in the uterus, preceding its implantation into the uterine wall, and differentiates into several fundamental structures. The nascent cells of the ICM segregate to form the primitive endoderm (PrE) and the epiblast. These intricate processes, which are fundamental to early embryonic development, have historically been difficult to study in depth. However, recent advances in stem cell research have now made it possible to create in vitro models that closely mimic these early stages of development Fig. .Fig. 2Sc21, stem cell research has significantly advanced. With the subsequent discovery of trophoblast stem cells (TSCs)44 and extraembryonic endoderm stem cells (XENs)45, researchers have been equipped with essential cellular tools for the creation of in vitro mouse embryo-like structures known as mouse blastoids. This marked a pivotal breakthrough in embryonic research, signifying the first successful attempt to replicate an entire embryo in vitro.Since the isolation of mESCs in the 1980s46. Following this breakthrough, numerous research groups have also developed similar in vitro structures that mimic in vivo blastocysts by employing a variety of methods48. Mouse blastoids are generated by aggregating various types of stem cells, including pluripotent and extraembryonic stem cells, in non-adherent hydrogel microwells, utilizing the self-organizing property of the mammalian embryo43. These cells are cultured with a blend of morphogens that influence the specification of the epiblast and TE lineages. Through this self-assembly process, in vitro structures that strikingly resemble in vivo blastocysts are created.Rivron et al. set a precedent by successfully generating the first mouse blastoids49. Moreover, Y-27632, a Rho-associated protein kinase (ROCK) inhibitor, is utilized to prevent cell apoptosis during this complex process.A pivotal event in successful blastoid formation is cavitation, underpinned by TE formation. TE formation is fostered by inhibiting the Hippo and TGF-β pathways. Concurrently, the addition of LIF and inhibitors of mitogen-activated protein kinase kinase (MEK) and glycogen synthase kinase 3 (GSK3) (known as 2i) helps maintain the pluripotency of ESCs by activating the STAT3, ERK, and WNT signaling pathways51. The precise activation of transcription factors within blastoids is essential for directing the self-organization and lineage segregation of ESCs and TSCs into a fully functional blastoid structure. These blastoids share several characteristics with natural blastocysts, such as the capacity to acquire apical‒basal polarity and tight junctions.In just 3 to 4 days, blastoids successfully take shape, exhibiting features such as the blastocoel cavity, ICM, and TE layer, the organization of which closely replicates the spatial organization seen in in vivo blastocysts. As cells start the process of self-assembly into blastoids, additional transcription factors, including GATA binding protein 6 (GATA6) and caudal type homeobox 2 (CDX2), are activated. These transcription factors govern lineage specification and cell differentiation, with GATA6 promoting the formation of the PrE lineage and CDX2 playing a crucial role in inducing the proper differentiation of the TE lineage54. This significant capability allows the formation of human blastoids without necessitating the mixture of ESCs and TSCs57. In a parallel manner, human extended pluripotent stem cells (hEPSCs)59, cultured to embody developmental potency for embryonic and extraembryonic cell lineages, have shown the potential to generate blastoids60.Expanding upon strategies used in the creation of mouse blastoids, researchers have successfully generated human blastoids by harnessing the unique properties of hPSCs. Human naïve ESCs, notable for their increased plasticity and lower lineage barrier compared to naïve mESCs, have the capacity to differentiate into both embryonic (epiblast) and all extraembryonic (TE and hypoblast) lineages55, and similar results can be achieved with standard microwells or pyramid wells60. It should be noted that the use of a single cell type, such as naïve hPSCs or EPSCs, is not the only option to induce blastoid formation in the medium. Indeed, blastoids can also be generated through a combination of hEPSCs and TE-like cells derived from EPSCs61. Similarly, 8CLCs have been used to derive blastoids that exhibit both morphological and transcriptomic similarities to human blastocysts41.Several techniques have been developed to generate human blastoids. For instance, human blastoids can be generated by aggregating naïve hPSCs or hEPSCs in rounded microwells using centrifugation62. These human blastoids bear transcriptomic and morphological resemblances to human blastocysts, and remarkably, some have even shown the potential to initiate events akin to implantation.Another breakthrough was the direct reprogramming of human somatic cells, which was achieved by infecting human dermal fibroblasts with a virus capable of inducing the expression of OCT4, SRY-box 2 (SOX2), Kruppel-like factor 4 (KLF4), and c-MYC, which in turn generated induced blastoids, or iBlastoids55, allowing the observation of post-implantation processes. In compliance with the International Society for Stem Cell Research (ISSCR) guidelines63, which allow human experimental culture up to 14 days, these human blastoids could potentially be implanted into a synthetic endometrial lining for the purpose of investigating post-implantation events. The implantation of blastoids revealed that this process took place in the polar TE region, which likely became polarized due to its nascent relation to epiblast-like cells55. After implantation, sustained proliferation of epiblasts, TEs, and PrE-like cells was observed55.Blastoids have the potential to be implanted into a hormone-stimulated endometrial layer64. Therefore, further analysis is required to determine how accurately these in vitro models represent the in vivo context, and ongoing research should focus on increasing their similarity.Although several blastoids have cellular compositions similar to those of human in vivo blastocysts, it is necessary to investigate them more closely to determine whether they precisely replicate the in vivo state. The similarity of most of the blastoids created thus far to in vivo blastocysts has been validated using single-cell transcriptomics. Occasionally, however, the cells formed in blastoids do not match those of human early blastocysts but rather those of later developmental phases (such as gastrulation after embryonic day (E) 14)Gastrulation is one of the most important processes of the developing mammalian embryo, where it incorporates several combinations of cell movements in newly positioning the embryonic tissue to form the three germ layers—ectoderm, mesoderm, and endoderm—which set a platform that further enables the navigation of cellular lineage specification by placing the tissues accordingly throughout the anterior-posterior (AP), dorsal-ventral, and lateral (left and right) axes. By appropriately positioning the embryonic tissue layers, organizing axes, and facilitating progressive specialization, the embryo is able to undergo processes such as organogenesis and limb morphogenesis that lead to the formation of more complex structures.65. Following their discovery, other studies have likewise continued to generate 3D aggregates that were able to recapitulate early embryonic differentiation and thus were collectively termed “embryoid bodies (EBs)”. When EBs derived from ES cells of mouse blastocyst ICM origin were cultured under in vivo-like conditions with appropriate developmental cues, they acquired the ability to differentiate into all three germ layers67.Going back to mark the initiation of the creation of synthetic embryos, as mentioned earlier, developmental biologists Martin and Evans found that ES-cell-like 2D clonal pluripotent teratocarcinoma cells suspended into aggregates self-organize themselves to acquire a cellular organization pattern similar to that of the differentiation of the early mouse embryoTaking the potency of pluripotent stem cells to advantage, the addition of various signaling factors parallel to those that work at the corresponding developmental stage to EBs coaxed the formation of models that recapitulate the early development of the embryo. Due to the established role of signaling factors around this stage of embryonic development, small molecules added to induce the growth of in vitro models are short-range and similar among different protocols. Agonists of the FGF, BMP, and WNT signaling pathways —key signaling pathways that govern development—as well as inhibitors  are used to induce or inhibit characteristics of the developing embryo  induces the formation of a progressive elongating domain similar to that of the in vivo tail bud of the embryo, as well as the specification of the endoderm, which all lead to simulating aspects of the gastrulation period of the embryo69. Additionally, the precise timing regulated by WNT/β-catenin and Nodal signaling induces symmetry breaking in gastruloids even without the presence of extraembryonic tissue or localized signaling, allowing further polarization to take place, which is evidenced by distinct T/Brachyury expression70. Reducing FGF signaling yielded a diminished tail phenotype in the embryo models, indicating that the inducement of this signaling pathway is important for elongation71. With the knowledge of biological mechanisms underlying embryonic development and the use of key signaling factors, several models that mimic aspects of gastrulation were generated, starting from micropatterned 2D models (“2D gastruloids”), which encompass the endoderm, mesoderm, and ectoderm76, to more advanced free-floating 3D gastrulation models (“3D gastruloids”).As such, incorporating signaling pathways into in vitro cell aggregates yields models that recapitulate important hallmarks of gastrulation. For example, symmetry breaking is achievable in EBs in vitro, where the local activation of the WNT signaling pathway induces AP polarity and the formation of a primitive streak-like region, resulting in differentiation into a mesendodermal compartment 69. Consisting of only 300 mouse ES cells, the EB was cultured in vitro to create a 3D gastruloid, where hallmarks of early mouse embryonic development, such as symmetry breaking and axial organization, were observable under the activation of WNT/β-catenin signaling, which further caused germ layer specification and minor axial elongation upon the addition of specific morphogens such as CHIR99021 and Activin A77. The usual 3D gastruloids are cultured through an initial few-hour-to-day EB suspension, whereafter they are then further matured under shaking culture conditions while adding certain morphogens to induce multi-axial organization and a temporal pattern similar to the gene expression of the embryo. Some gastruloids also acquire in vivo patterning integrity by expressing the collinear Hox gene pattern throughout the AP axis77.Early works to recapitulate aspects of gastrulation were conducted using mouse stem cells. The first absolute 3D gastruloid was created by van den Brink and colleagues in 201478, since elevated expression of genes of the posterior mesoderm was observable with the inducement of WNT signaling78. However, WNT signaling is key in breaking axial symmetry in embryonic development, where its set dose induces primitive streak markers and spontaneously breaks AP symmetry. The aforementioned AP axis establishment enables the organization of embryonic tissues. Despite genetic studies that state that this process relies on the embryo’s exposure to extraembryonic spatiotemporally located signals—WNT/β-catenin and Nodal signaling—studies performed with mouse gastruloid models proved that this might not necessarily be the case. Mouse ESC-based gastruloids induced without surrounding extraembryonic tissues showed localized T/Brachyury expression with polarity and extension to one side, suggesting the notion of AP axis development. Thus, this discovery suggested that extraembryonic tissues are not necessarily required for the embryo to undergo self-patterning69.Through the advancement of the creation of mESC embryonic models, adjustments in the culture conditions of these models enabled more developed structures as well as new discoveries. Although mouse gastruloids effectively mimic embryo-like axial morphogenesis and patterning, they lack regions such as the anterior embryonic region (which includes the brain). To improve this inaccurate depiction of the in vivo embryo, studies have inhibited WNT signaling during the early stages of development to induce gastruloids with anterior neural tissue79.Gastrulation occurs at approximately 16 days post-fertilization (dpf) in human embryos, and in vitro biomimetic models that recapitulate this stage are also achievable by utilizing primed hESCs and hiPSCs, enabling the formation of human gastruloids without the violation of major ethical issues. As in the case of initial murine gastruloid models, human gastruloids were also developed under 2D conditions that portrayed aspects of gastrulation, such as the development of primitive streak-like structures and the three germ layer domains80. For example, the addition of BMP and WNT agonists was found to be crucial in the establishment of AP polarity and the elongation of the tail bud81. Adding extraembryonic compartments resulted in models that more closely resembled the in vivo gastrulating embryo82 by mimicking attachment and AP symmetry breaking and further exhibiting human gastrula-specific cells. 3D aggregates that broke symmetry formed extensions along the AP axis with organized germ layers84. Through the development of the technology of generating human gastruloids, specific gastruloids that addressed parts of the early stages of post-implantation human embryonic development were created, such as the processes of primitive streak formation80, neurulation86, and somitogenesis87.The development of 3D human gastruloids facilitated the study of human development, as they more accurately depict the in vivo gastrulation process than earlier 2D models. Human PSCs embedded in the matrix, such as by utilizing the commercial extracellular matrix (ECM) formula Matrigel, and cultured under conditions that are supplemented with the right morphogens construct aggregates that undergo symmetry breaking and gene expression patterning88. Simpler models that recreate oscillatory movements were also generated89. Inducing AP symmetry breaking in hPSC models allowed scientists to study the mechanisms underlying human somitogenesis.At E7.5 in mice and approximately 20 dpf in humans, the presomitic mesoderm (PSM) condenses and gives rise to somites along the lateral sides of the central neural tube in an anterior-to-posterior fashion. These somites later form structures that act as anchors that hold parts of the embryo, such as the vertebrae, skeletal muscles, cartilage, and dermis. Shortly after somitogenesis has begun, organogenesis is launched, with the heart being the first organ to be generated. Models that mimic somitogenesis, as well as organogenesis, have been created over recent years . Segmentation clock waves are observable in mesoderm-based hPSC models (termed “axioloids” or “somatoids”) that mimic segmentation and somitogenesis in the human embryo90. More specifically, the anterior foregut, midgut, and hindgut were induced in the gastruloid through the creation of a primordium that covers the overall region of the structure91. Furthermore, several gastruloid models, such as trunk-like structure (TLS) gastruloids (using mESCs)92 and elongating multi-lineage organized (EMLO) gastruloids (using hiPSCs)84, portray gut tube-like structures.Gastruloids have also been able to acquire regions that resemble the gut tube. The formation of the AP and dorsal-ventral axes enables the patterning of the primitive gut tube in mouse gastruloids39. Stem cell models that resemble these early “egg-cylinders” were generated by combining mESCs with TSCs and incorporating tissues such as those that resemble the extraembryonic ectoderm without recapitulating the blastocyst stage93. Adding XEN cells that resemble extraembryonic endoderm cells to these models (ETX or iETX embryos) enabled the production of a visceral endoderm-like epithelium that lines the in vitro conceptus95. When synthetic embryos were constituents of extraembryonic compartments, they could initiate implantation95 and further induce gastrulation, neurulation, and organogenesis96.Instead of models that recapitulate only a small aspect of the early developing embryo, attempts were made to model its overall characteristics. In mice, the cylindrical shape of the post-implantation embryo is due to the polar TE invagination and actomyosin contractility that give rise to the extraembryonic ectoderm97 and roller culture systems98. By taking advantage of these systems, the process of generating whole embryos in vitro was conducted. Due to the aforementioned ISSCR regulation restricting the culture of human embryos to a maximum of 14 days in vitro for research purposes (the “14-day rule”), attempts to create synthetic whole embryos in long-term culture conditions were generally proceeded using mESCs.Several culture techniques that assist in establishing proper embryonic development ex vivo were created, such as circulator systems99, mouse naïve ESCs were cultured until E8.5, which yielded a mouse synthetic whole embryo model that was able to undergo gastrulation and to form organ precursors100.To create synthetic whole embryo models, an environment similar to in vivo conditions is needed. With the adaptation of the electronically controlled ex utero roller device that previously cultured a mouse embryo until E11 by the incorporation of the ex utero culture medium (EUCM), which is a mixture of 50% rat serum, 25% Dulbecco’s modified Eagle’s medium (DMEM), and 25% human umbilical cord blood serum62. Recently, there have been strides toward creating a human synthetic whole embryo that can model more advanced developmental processes mimicking post-implantation stages while maintaining the morphological integrity of a natural embryo.Attempts to create human synthetic whole embryos were also made. For human synthetic whole embryo models, blastoids are currently the most advanced tool, mimicking the entire pre-implantation stage of the human embryo. Although researchers have made attempts to push these blastoids toward the post-implantation stages in vitro, they have not been able to match them with the development of in vivo blastocysts102. They drew on strategies previously used to create a mouse synthetic whole embryo, which involved differentiating embryonic and extraembryonic cells separately before aggregating them to produce a post-implantation embryo. Weatherbee et al. generated two types of extraembryonic-like cells from hESCs through the overexpression of transcription factors: GATA6 and SRY-box transcription factor 17 (SOX17) for the hypoblast and GATA3 and transcription factor AP-2 gamma (TFAP2C) for TSCs101. On the other hand, Oldak et al. utilized ectopic expression of lineage-promoting transgenes to induce the formation of the hypoblast and trophoblast102.Two groups of scientists have recently reported their attempts at creating human post-implantation embryo models101. Oldak et al. reported the formation of the bilaminar disc, epiblast lumenogenesis, amniogenesis, primordial germ cell specification, yolk sac formation, and the expansion of extraembryonic mesoderm in their model102. While the creation of human synthetic whole embryo models is expected to continue to evolve, it is essential to closely monitor these developments within the framework of ethical considerations and international agreements.Both groups reported human synthetic whole embryo models that replicated the hallmarks of 13-14 dpf human embryos. Weatherbee et al.’s model consisted of structures such as the amnion, extraembryonic mesenchyme, and primordial germ cell-like cellsOne of the main advantages of synthetic embryos is their feasibility for modeling diseases that arise during the early stages of embryonic development. Thus, congenital diseases that occur due to initial abnormal development are of interest. Furthermore, because synthetic embryo models such as gastruloids can be easily manipulated to imitate specific stages of development, diseases that occur at a particular event could be observed. Although only a few- considering the subject’s relatively recent advancement into the field, attempts have been made to incorporate synthetic embryoids to model human diseases. This section includes an overview of models that recapitulate key processes of embryonic development with respect to disease modeling Fig. .Fig. 4Co103. Several models that mimic the heart have been made to recapitulate aspects of cardiogenesis108; however, to fully understand the dynamics of heart development, observing the organ alone is insufficient. Taking advantage of the gastruloid characteristic of encapsulating multiple cell types of all germ layers, cardiogenesis development is more accurately portrayed by gastruloid models since the formation of the heart necessitates complex interactions such as those among the organ, cardiac progenitor cells, and endothelial cells. Recent studies have mimicked cardiogenesis by manipulating embryo models by incorporating well-known cardiogenic growth factors, such as vascular endothelial growth factor 165 (VEGF-165), FGF2, and ascorbic acid. Rossi et al. captured the process of cardiogenesis via mESC gastruloids, in which the spatiotemporal accuracy was highly conserved compared to its in vivo counterpart109. The model incorporates both cardiac progenitors of the first heart field (FHF) and the second heart field (SHF), which anteriorly organize into a cardiac crescent-like region and form tissues that imitate the beating of the heart.Congenital heart disease (CHD) is one of the leading causes of death in newborns110 to the “elongating multi-lineage organized” (EMLO) gastruloids that they had previously generated111, creating EMLOC gastruloids that enable the remodeling of both cardiogenesis and neurogenesis to explain the complex interconnected lineages between the processes. Various developmental features that resemble those of the in vivo heart were observed, including the formation of the heart tube, putative outflow tract, ventricular wall, and epicardium, as well as the differentiation of cardiomyocytes. This neuro-cardiac model may offer a platform to provide further understanding of cardiac diseases such as neural-based heart arrhythmia diseases and congenital heart diseases.Expanding upon synthetic embryo models that recapitulate only cardiogenesis, new models that include both cardiac muscle and neurons to portray neuro-cardiac lineages have been made. Olmsted and Paluh have added the aspect of cardiogenesisTbx6) gene to generate gastruloids (TLSs)92. The resulting phenotype was the loss of somites and ectopic neural tubes, which correlates with the in vivo phenomenon of PSM transdifferentiation and ectopic neural tube formation at the expense of somites and PSM. Van den Brink et al. performed a screening test that showed that decreased levels of FGF signaling resulted in the generation of fewer somites, which was similar to the defects observed in Fgf mutant mice, and that incorporating 10% Matrigel may induce somitogenesis in gastruloids71.Vertebrates acquire segmented structures through a process called somitogenesis. This process occurs in early development when somites, blocks of paraxial mesoderm, form on either side of the neural tube and the notochord in a bilateral position along the AP axis. Presenting phenotypes similar to those of mouse embryos, mouse gastruloids have been offered as appropriate models to study vertebrate segmentation. For instance, genetic alteration of gastruloids yielded phenotypes that matched those of previous knockout mouse embryo models. Veenvliet et al. used mESCs deleted with the T-box transcription factor 6 (88. They introduced loss-of-function mutations in the segmentation clock gene hes family bHLH transcription factor 7 (HES7) and the marker for the anterior PSM mesoderm posterior bHLH transcription factor 2 (MESP2), which are associated with segmentation defects of the vertebrae (SDV), and generated axioloids with them. Two different HES7 knockout hiPSC lines were used to create axioloids, and their phenotypes were assessed. There were losses of segments and rostro-caudal patterning, as well as the formation of the epithelial somites. Regarding the oscillatory activity of the segmentation clock, Yamanaka and colleagues found a definite loss of HES7 oscillation. In axioloids derived from iPS cell lines introduced with a point mutation in HES7, there were also losses of rostro-caudal patterning, yet these models managed to express normal HES7 oscillation in the tail bud as well as a few somitic mesoderm markers. MESP2 is known to be mutated in patients with SDV. Yamanaka and colleagues also assessed the effects of axioloids with MESP2 knockout. MESP2-knockout axioloids also lacked segments and epithelial somites and exhibited abnormal rostro-caudal patterning, despite their normal elongation. However, MESP2 knockout did not result in the loss of oscillation of HES7. As such, these axioloids serve as models that offer insights into the segmentation development of the human body and a platform to study the pathogenesis of the spine.Aberrations in somitogenesis can lead to several diseases, such as vertebral malformations and spinal defects. Specifically, Yamanaka et al. created axioloids that recapitulated human somitogenesis in vitro and applied them to investigate congenital diseases associated with the human spine81. With exposure to doses of the WNT agonist CHIR99021, the elongation of hPSC aggregates and the formation of NMP cells could be observed, as well as the presence of the mesoderm and neuroectoderm. Neural tubes were also induced in mouse TLSs created by Veenvliet et al. when cultured in a 5% growth factor-reduced Matrigel condition, as well as by the addition of the WNT agonist CHIR99021 and the BMP signaling inhibitor LDN19318992.Neurulation is a highly orchestrated and complex morphogenetic process in which the neural plate fuses to form the neural tube, the precursor of the brain and spinal cord of the central nervous system. When the process of neurulation is disrupted, severe congenital abnormalities such as neural tube defects (NTDs) arise. Several studies have shown that gastruloids are capable of recapitulating the formation of the neural tube. Libby et al. generated a human neural tube gastruloid that demonstrated aspects of early spinal cord development, such as axial elongation, neuromesodermal progenitor (NMP) cell population maintenance, and neuroepithelial cell generation85. In this model, Karzbrun and colleagues incorporated three small-molecule inhibitors—the ROCK inhibitor Y-27632, the HSP-90 inhibitor novobiocin, and the NTD-associated drug valproic acid—to test the suitability of these gastruloids for modeling NTDs. The application of the small molecules resulted in morphological abnormalities, including less curvature of the tube and overall folding defects, which indicated that their model may indeed be utilized to represent the neural tube to study defects to some extent.Furthermore, neurulation was depicted in partially 3D gastruloids induced by 2D micropatterning by Karzbrun et al., where a neural-tube-like structure was detectableHistorically, investigating human development has been an ongoing endeavor with significant limitations, including restricted understanding due to the complexity of the process, the lack of appropriate models (not to mention the inaccessibility of human embryos), and ethical concerns. Herein, we have illustrated how the generation of synthetic models of early mammalian development has assisted in overcoming these challenges.112.Through the discovery of stem cells and research progress made due to their manipulation, the scope of human embryo experimentation, once available only through direct practice on authentic embryos, became more diverse. By inducing human stem cells to become structures characterized by organized systems, the need for human embryos has diminished, thereby compensating for the inaccessibility of human embryos due to the shortage of samples and ethical issues. Ethical concerns are inevitable; accordingly, regulations have been made by officials such as the ISSCR with recurring discussions among scientistsSynthetic models do not act merely as models that recapitulate early development; they also provide a platform for which numerous diseases, and their underlying mechanisms, could be further studied. Since these early embryo models may reliably replicate the embryo’s state at certain developmental stages, specific diseases that arise at specific time points may be investigated by recreating a developmentally similar embryonic structure with temporal accuracy. For example, blastoids allow the further study of implantation mechanisms, which then may suggest possible factors associated with implantation failures, and gastruloids may model congenital diseases, including but not limited to those stated above. Current mammalian embryo models are able to recapitulate in vivo embryos of Carnegie stages 1 to 10, in which diseases that arise may be modeled.114. Furthermore, teratogenicity could be tested through quantifiable gastruloids, which yield statistical robustness, and further applications may be tested, such as species-specific reactions to the molecules115.As a shared advantage of in vitro structures, synthetic embryo models may be produced in bulk, offering compatibility with systems that require large amounts of samples, such as drug screening and toxicological assays. By using stem cell-based models, researchers have studied the effects of small molecules on mammalian developmentNonetheless, a limitation that remains is the total inducement of the synthetic whole embryo in vitro. Several attempts have been made to create synthetic whole embryos, yet results show that there is still a need for further understanding of developmental mechanisms, given the morphological difference of the cultured in vitro embryo with little resemblance to the in vivo embryo and the fact that these synthetic embryos cannot further develop after a set time—not to mention their incompetence to be born.64 has revealed that several in vitro models are composed of cells that differ significantly from the transcriptome of their in vivo counterparts. For modes of fidelity validation, the current tools we have to validate the integrity of cell lineages in in vitro models are largely limited to single-cell analyses. At present, single-cell transcriptomics represents the most advanced tool to assess the fidelity of these models. However, even if parts of the in vitro model display gene expression patterns similar to those of its in vivo counterpart, reliance on single-cell transcriptomics alone is insufficient. The number of genes considered per cell in a single-cell analysis ranges from as few as 200 genes to an average of 2000–6000 genes, suggesting that comparisons of similarities between in vitro models and their in vivo counterparts cannot be completely accurate considering the approximate 30,000 genes in the human genome. Much like how the invention of telescopes unveiled previously unseen galaxies and microscopes provided insights into cellular structures, the advancement of tools that govern degrees of accuracy for analyzing differences between in vitro and in vivo embryos will enable us to see new perspectives that lie within the two objects. Thus, there is a clear need for a more sophisticated tool capable of accurately discerning even the subtlest differences. Such innovation would enable the creation of in vitro models that more closely—if not exactly—mirror the in vivo embryo. Further technological improvements would enhance investigations in modeling diseases and pragmatic applications of these models to clinical areas, such as cell-type-specific transplantation in regenerative medicine.Furthermore, it should be noted that many in vitro early embryogenesis models representing various developmental stages and dimensions often lack fidelity to in vivo conditions in their cellular composition. Recent workGiven that previous works were conducted on less relevant structures via 2D culture and that the field of stem cell and developmental biology has made substantial progress since these advancements in pursuit of generating proper mammalian in vitro embryos that accurately recapitulate developmental stages elicit anticipations of further research improvements and discoveries. The availability of once-seemingly impossible technical applications of lineage-specific stem cell differentiation via these advances will enable further research into discovering new mechanisms that underlie the development of the embryo.


Current guidelines propose therapeutic algorithms based on left ventricular ejection fraction values and clinical presentations; however, these guidelines do not specify which of the four pillar drugs to start first. Some authors suggest starting with SGLT2i and Betablockers, others with ACE-inhibitors or ARNI, while one of the most recent approaches proposes starting with low-dose combination of all four drugs .Harrison’s Principles of Internal Medicine for 12 editions, and founding editor of Heart Disease, is the most frequently cited author in cardiology. He is considered one of the leading scientists in the world in research on cardiovascular diseases. In 2021, he compared sodium glucose cotransporter 2 inhibitors (SGLT2i) to statins [Eugene Braunwald, distinguished Hersey Professor of Medicine at Harvard Medical School (HMS), editor of The nephroprotective effect was demonstrated in subsequent studies showing how the eGFR reduction per year was attenuated by dapagliflozin independently of the presence of HF, suggesting an independent nephroprotective action of SGLT2i .Furthermore, SLT2i has been proposed to improve even erythropoietin secretion, improving anemic status, which is typical of CKD patients .SGLT2i are renoprotective, and the protective effects on the kidney seem to predominate over all the other mechanisms of action of SGLT2i inhibitors.The mechanism of action of SGLT2i in HF is different from that of other HF therapies that focused on reverse left ventricular remodeling, blocking the neurohormonal activation , or enhancing the natriuretic peptide system (such as neprilysin inhibitors) . These oOther reflections beyond the nephroprotective effect lead us to suggest SGLT2i as a first-line drug in the treatment of HF: SGLT-2i does not need up-titration (most HF drugs need titration in order to provide a larger benefit) .SGLT-2i side effects are very minor, making gliflozins extremely well-tolerated drugs .SGLT2i use is not accompanied by hypotension, bradycardia, or hyperkalemia that often accompany the use of currently recommended treatments for heart failure .The cardiologists may consider dose reduction of thiazide or loop diuretics at the time of SGLT2i initiation to avoid excessive diuresis and volume depletion (SGLT2i may induce osmotic diuresis via glucosuria) .SGLT2i treatment has been demonstrated to improve cardiovascular outcomes in patients with HF over a wide range of LVEF, regardless of diabetic status, and has a strong renoprotective effect . In factInhibition of renal glucose reabsorption affects blood pressure and improves the hemodynamic profile and the tubule glomerular function .Natriuresis derived by Empagliflozin is not associated with neurohormonal activation, potassium loss, or renal impaired function  .The benefits of SGLT2i may also be due to favorable hemodynamic and metabolic effects on LV . The dirThe addition of SGLT2i to OMT in patients with HFrEF resulted in a greater improvement in RV systolic function .Finally, recognition by physicians that the benefits of SGLT-2i use on clinical outcomes outweigh the risks will result in the integration of SGLT-2is into clinical practice and lead to improved patient care and outcomes .


How are studies that explicitly aim to emulate a target trial reported?In this systematic review of 200 studies that explicitly aimed to emulate a target trial, reporting was inconsistent, and studies often did not report all necessary information related to the emulation of the target trial.Inconsistent reporting of studies that explicitly aim to emulate a target trial may impair the appraisal, synthesis, and implementation of study findings. This systematic review assesses the reporting of observational studies that explicitly aimed to emulate target trials, hypothetical randomized trials that could answer causal questions of interest. Observational  studies that aim to emulate a randomized trial  are increasingly informing medical and policy decision-making, but it is unclear how these studies are reported in the literature. Consistent reporting is essential for quality appraisal, evidence synthesis, and translation of evidence to policy and practice.To assess the reporting of observational studies that explicitly aimed to emulate a target trial.We searched Medline, Embase, PsycINFO, and Web of Science for observational studies published between March 2012 and October 2022 that explicitly aimed to emulate a target trial of a health or medical intervention. Two reviewers double-screened and -extracted data on study characteristics, key predefined components of the target trial protocol and its emulation , and other items related to the target trial emulation.A total of 200 studies that explicitly aimed to emulate a target trial were included. These studies included 26 subfields of medicine, and 168 (84%) were published from January 2020 to October 2022. The aim to emulate a target trial was explicit in 70 study titles (35%). Forty-three studies (22%) reported use of a published reporting guideline . Eighty-five studies (43%) did not describe all key items of how the target trial was emulated and 113 (57%) did not describe the protocol of the target trial and its emulation.In this systematic review of 200 studies that explicitly aimed to emulate a target trial, reporting of how the target trial was emulated was inconsistent. A reporting guideline for studies explicitly aiming to emulate a target trial may improve the reporting of the target trial protocols and other aspects of these emulation attempts. Moreover, it facilitates the interpretation of effect estimates and promotes meaningful discourse concerning potentially discrepant findings observed across studies.Analyses of observational  data can be used to estimate the causal effect of interventions when randomized clinical trials are unavailable or infeasible. Bias in observational analyses may be limited by conceptualizing them as attempts to emulate target trials, ie, hypothetical randomized trials that would answer causal questions of interest.10 The target trial emulation framework, introduced by Hernán and Robins4 in 2016, provided a template for reporting and conducting studies that aim to emulate target trials. The framework outlines items to be reported in the protocol of a target trial and its emulation, including: eligibility criteria, treatment strategies, assignment procedures, follow-up period, outcome(s), causal contrast(s), and analysis plan. Since the introduction of the framework, several articles have been published to assist researchers in conducting these studies and educating clinicians and other end users to interpret their findings.14 However, there is limited understanding of how researchers have implemented the target trial framework when reporting observational analyses with the explicit aim to emulate a target trial.Since at least the 1950s, the notion of observational analyses as attempts to approximate the goals of randomized clinical trials has underpinned many comparative studies in health, medicine, and related fields.15This review aimed to (1) describe how studies that explicitly aimed to emulate a target trial were reported and (2) examine whether these used published reporting guidelines. The findings of this review will be used to inform the development of a reporting guideline for studies explicitly aiming to emulate a target trial.PRISMA) reporting guideline.16 The protocol was prospectively registered on the Open Science Framework (OSF).18 Protocol deviations are reported in eAppendix 1 in This review is reported according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses  to October 20, 2022. We excluded studies that did not investigate a medical intervention; did not include human participants; were not written in English; only described the protocol of a study emulating a target trial, ie, a protocol of a planned study without results; or for which the full text was unavailable.We included observational studies that explicitly aimed to emulate a target trial of a medical intervention; eAppendix 3 in We de-duplicated all records identified through searches in Endnote version 20 and imported into Excel version 2206 (Microsoft Corp). In duplicate, reviewers  independently performed screening of identified records for eligibility at the level of title and abstract and full text. Disagreements were resolved through discussion.Data were extracted in duplicate  and compiled into a standardized spreadsheet piloted with 3 included studies. Disagreements were resolved by the lead author (H.J.H.) or through discussion. We did not blind reviewers to the journal article or study authors.18We extracted information about the (1) characteristics of the included studies, (2) key protocol components that characterize the target trial approach, and (3) further items that may be important to report in studies emulating a target trial. The complete data extraction spreadsheet and code used are available on OSF.Treatment strategy refers to any health care intervention including treatments, preventative interventions, and no change to current treatments, remaining consistent with the language used by Hernán and Robins.4 If a study investigated prevention of a given outcome in healthy individuals, the subfield of medicine was designated based on the outcome investigated. Each treatment strategy included in an article  was counted separately. We classified treatment strategies defined by the authors as no treatment or usual care as no change to current treatment approach(es).Characteristics of the included studies were year of publication, subfield of medicine defined based upon included population, data source , sample size , primary outcome, and type of treatment strategy being compared. 4 We considered a study to have specified how the target trial was emulated if all the previously listed protocol items were reported; these items are operationalized in eAppendix 4 in the We extracted whether each study reported the eligibility criteria, treatment strategies, assignment procedures, follow-up period, outcome(s), causal contrast(s), and analysis plan of the emulation of the target trial. These items and their definitions were informed by the target trial framework from Hernán and Robins.4 These included:Treatment strategies: type of treatment strategy , aspects of treatment strategies described .Analysis plan: method(s) used to emulate randomization, description and selection of potential confounding variables, statistical and causal assumptions that relate to analyses, sensitivity analyses.Other: study registration, rationale for the target trial emulation, reporting guideline used (referred to as a guideline hereafter). We only included a guideline if it was referenced as guiding the reporting of a study.We deemed a study to report the assumptions underlying their analyses only when the assumption(s) were described in the text or in a cited reference. When authors reported that no residual confounding was assumed, we took this as equivalent to reporting an assumption of conditional exchangeability. We did not regard practices that may assess a causal assumption  as reporting the assumption. We did not assess the appropriateness of authors’ reported assumptions. Items we extracted that were not included in commonly used guidelines are listed in eAppendix 5 in Further details of specific protocol components that may be important to report in studies emulating a target trial were chosen based on expert knowledge and recommendations from methodological papers on the target trial emulation framework.We cleaned and analyzed data in R version 4.2.0  using tableone, openxlsx, tidyverse, and readxl packages for data management and visualization. We summarized categorical variables using counts and percentages. Continuous variables were summarized using mean and SD or median and interquartile range. Post hoc, we assessed the reporting of how the target trial was emulated stratified by whether a guideline was used.220 All reasons for excluding records after full-text review are given in eAppendix 6 in We retrieved 3133 unique records, of which 200 were included in the review .2,22,23,220 168 (84%) were published from January 2020 to October 2022 ,218 cardiology (30 [15%]),214 and oncology (30 [15%]).211 One hundred and thirty-one studies (66%)220 used data from electronic health records, claims databases, or linked data sources. The treatment strategies most frequently investigated were pharmacological (228 of 435 [52%]) and no change to current treatment approach(es) . All extracted characteristics of included studies are displayed in Of the 200 included studies,ber 2022 .22,23,25220 completely reported how the target trial protocol was emulated. Eighty-seven studies (44%)220 provided both the protocol of the target trial and described how it was emulated (One-hundred and fifteen studies (58%)emulated . The folemulated .218 reported in the title that the study aimed to emulate a target trial; 180 (90%)220 did so in the Methods section. Twenty studies (10%) 216 reported the study was prospectively registered, 16 of these 20 (80%)216 also provided information on how to access the registration. One hundred and twenty-six studies (63%)217 specified whether a randomized clinical trial could be feasibly conducted; 61 (31%)216 stated that the randomized clinical trial was possible. Of the studies that stated a randomized clinical trial was possible, uncertainty in the generalizability of available trial findings was the most common reason for the target trial emulation (22 of 61 [36%]).215 Forty-three studies (22%)215 reported using a guideline, most commonly (29 of 43 [67%]) the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline.222 There were no qualitative differences between the reporting of the target trial emulation when studies were stratified by guideline use (eAppendix 7 in Seventy studies (35%)220 reported the set of variables that authors had decided to adjust for  in analyses, and 77 (39%)218 reported how these variables were selected. One hundred and thirty-one studies (66%)220 reported conducting a sensitivity analysis for statistical or causal assumptions; the most frequent (42 of 131 [32%]) was the use of a different approach to confounding adjustment . One hundred and fifty-eight studies (79%)219 reported that causal interpretation rests on the assumption that the comparison groups were comparable  given the variables included in the analysis analysis . Twenty-220 published from 2013 to 2022, the majority of which (168 [84%]) were published between January 2020 and October 2022. The studies spanned 26 fields of medicine and mostly used sources of data that were routinely collected, such as electronic health records, health insurance claims data, or these data linked with other data sources. While the publication of studies explicitly aiming to emulate a target trial is increasing, only 58% of included studies completely reported how the target trial protocol was emulated.This systematic review summarized items reported in observational studies that explicitly aimed to emulate a target trial. We included 200 studies223 Nguyen et al223 systematically reviewed the risk of bias in observational studies investigating the effectiveness of interventions using the ROBINS-I tool,224 a risk of bias tool informed by the target trial framework. The authors found that only 3% of these observational studies (2 of 77) completely specified all items of the protocol of the (implicit or explicit) target trial. A much larger proportion of our sample of studies that explicitly aimed to emulate a target trial reported how the target trial was emulated; however, many were still incompletely reported. It appears the guidance from Hernán and Robins4 and previous work228 has been used inconsistently or perhaps misinterpreted, leaving key elements of the target trial and its emulation unreported.3Our finding that studies aiming to emulate a target trial inconsistently reported the emulation of the target trial is similar with results of previous systematic reviews of observational studies that did not explicitly aim to emulate a target trial.232 For example, in mid-2022, the UK National Institute of Health and Care Excellence released “Real-World Evidence Framework,”229 which emphasizes the importance of using the framework of a target trial when estimating treatment effects for regulatory decision-making using observational data.229 Considering the emerging role of studies explicitly emulating a target trial within the health care decision-making framework, it is critical these studies are consistently and transparently reported. Once a target trial is emulated, unmeasured confounding may be a primary concern with observational analyses informing decision-making.233 We found that only 73 studies (37%) reported potential unmeasured confounders. It is unlikely all confounders would be measured in a given analysis, therefore the robustness of findings from a target trial emulation may be better assessed if potentially important unmeasured confounders are reported.Our review shows there has been an increase in the publication of studies that explicitly aim to emulate a target trial. This trend could be attributed to the growing influence of such studies in shaping policy and regulatory decisions.234 and if actively implemented, can improve reporting consistency and completeness.237 None of the included studies identified specific reporting guidance for studies that aimed to explicitly emulate a target trial, and the authors are not aware of any guidance for studies emulating a target trial published or under development,238 suggesting no formal guidance has been published. Twenty-two percent of studies cited general 222 and potentially inappropriate guidelines .239 The use of guidelines was comparatively lower than seen in similar reviews of other types of observational studies, in which observed rates of guideline use ranged from 46% (67 of 147)240 to 47% (68 of 88).241 The lower use of guidelines observed in our review may reflect authors’ uncertainty on the most appropriate guideline when reporting a study that used the target trial framework.Guidelines have been developed to address inconsistent reporting,224 of the quality and robustness of findings from a study emulating a target trial is impaired when such analyses are poorly reported, leaving readers unable to understand the quality and conduct of the emulation. Similarly, findings of studies emulating a target trial are frequently compared with those of randomized clinical trials.245 Differences in effect estimates between target trial emulations and randomized clinical trials may arise due to various factors.246 Transparent reporting of the target trial protocol and how it was emulated may aid in understanding these differences and optimize the usefulness of these studies for decision-making.Despite the growing number of studies using the target trial framework, reporting was inconsistent. Consistent and transparent reports are important for these studies given their emerging role in decision-making. For example, critical appraisal15 guidelines are available to support authors reporting studies emulating a target trial. Commonly used guidelines 222 do not include items that relate to the protocol of the target trial4 or key items of the target trial emulation . Reporting of these items was not improved when authors followed guidelines such as STROBE (eAppendix 7 in 248No established, consensus-based249 including screening studies and extracting data in duplicate. We prospectively registered this systematic review18 and reported the findings in line with the PRISMA 2020 reporting guideline.16We used a sensitive search strategy to ensure all relevant studies were captured and followed recommended systematic review methods,251 Using the target trial framework is neither necessary nor sufficient for obtaining valid causal effect estimates from observational analyses; however, the framework may guide the implementation of sound principles of causal inference and study design. Second, we prespecified the reporting items to be extracted based on published recommendations for the specification of the target trial protocol and its emulation.4 Therefore, our ratings for these items may be skewed toward a particular way of reporting studies explicitly emulating a target trial. Third, we did not assess the appropriateness of the methods of included studies, only their reporting.This study has several limitations. First, we only included studies that explicitly stated that they aimed to emulate a target trial; therefore, our findings may present a more positive view of reporting practices compared with all observational analyses comparing interventions.In this systematic review, reporting of studies that explicitly emulate a target trial was inconsistent, with several opportunities to improve the reporting of key items. A guideline expanding on the current recommendations may facilitate consistent and transparent reporting, improving the appraisal, synthesis, and implementation of study findings in clinical practice and health policy.


Chronic liver disease or repeated damage to hepatocytes can give rise to hepatic fibrosis. Hepatic fibrosis (HF) is a pathological process of excessive sedimentation of extracellular matrix (ECM) proteins such as collagens, glycoproteins, and proteoglycans (PGs) in the hepatic parenchyma. Changes in the composition of the ECM lead to the stiffness of the matrix that destroys its inherent mechanical homeostasis, and a mechanical homeostasis imbalance activates hepatic stellate cells (HSCs) into myofibroblasts, which can overproliferate and secrete large amounts of ECM proteins. Excessive ECM proteins are gradually deposited in the Disse gap, and matrix regeneration fails, which further leads to changes in ECM components and an increase in stiffness, forming a vicious cycle. These processes promote the occurrence and development of hepatic fibrosis. In this review, the dynamic process of ECM remodeling of HF and the activation of HSCs into mechanotransduction signaling pathways for myofibroblasts to participate in HF are discussed. These mechanotransduction signaling pathways may have potential therapeutic targets for repairing or reversing fibrosis. Mechanical forces can regulate cell function and direct cell behavior, and cells can also maintain homeostasis by responding to mechanical stimuli . Cells sIn most organs of the human body, the ECM is mainly composed of the interstitial matrix and the basement membrane, the latter comprising the epithelial cell basement membrane and the endothelial cell basement membrane . The ECMIn addition, ECM also contains MMPs, TIMPs, lysyl oxidases (LOX), and lysyl oxidase-like enzymes (LOXLs) and a series of growth factors, which play an important role in the formation and regression of fibrosis. MMPs are zinc-dependent endopeptidases, which can degrade all components of ECM and a large number of non-matrix proteins, and participate in regulating the activities of pro-inflammatory factors, growth factors, chemokines, and so on . TIMPs aThe progression of chronic liver disease (CLD), including alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and hepatitis B and C viral (HBV/HCV), is associated with the development of HF. HF formation is a continuous process and can be classified as portal fibrosis (F1), moderate fibrosis (F2), severe fibrosis (F3), and cirrhosis (F4) according to the Metavir score. It is well known that the main feature of HF is that persistent liver injury leads to excessive deposition of ECM proteins, which mainly include fibrous proteins  and GAGs (such as HA and heparin sulfate). In the process of fibrosis formation, the deposition of ECM proteins changes the content and composition of ECM components. For example, compared with a healthy liver, the number of ECM proteins in the fibrotic liver increases by 6 times , and theAs one of the main components of ECM structure, almost all types of collagen are increased during ECM remodeling in HF. For example, the synthesis and deposition of type I collagen in the fibrotic liver can be 8 times higher than in the healthy liver . In an eGAGs and PGs, as another major molecular type of ECM, are also involved in extracellular matrix remodeling in HF. GAGs are linear polyanionic animal heteropolysaccharides, which can be classified into four groups according to the structure of disaccharide units: heparin/heparin sulfate, chondroitin/chondroitin sulfate, keratin sulfate, and HA . PGs arein vivo study in mice found that lysyl oxidase-like 1 (Loxl1) expression was elevated only in the late stages of HF, and inhibition of Loxl1 expression significantly reduced elastin crosslinking, thereby preventing HF progression  and hepatocytes. Under normal physiological conditions, HSCs are resting, storing vitamin A in the form of retinyl esters in their nuclear droplets, also known as hepatic fat-storing cells . When thIn addition to the activation of HSCs by changes in ECM components, transforming growth factor-β1 (TGF-β1) is also involved in the activation process and plays a key role . TGF-β1 Moreover, HSCs can migrate and aggregate to stiff areas through mechanotransduction pathways (described below), further stiffening the extracellular environment . TherefoIn vitro experiments have shown that the effect of mechanical forces on macrophage phenotype is bidirectional, and the application of circulatory pull to macrophages adhering to the scaffold can observe that the M2/MI ratio increases at 7% strain and decreases at 12% strain, suggesting that lower tension may maintain microenvironmental homeostasis, while abnormal stretching may initiate an inflammatory response , natural killer cells (NK), and natural killer T cells (NKT), etc. Studies have pointed out that macrophages may sense extracellular mechanical stimulation by activating and recruiting into the liver, thus secreting specific cytokines and chemokines to activate HSCs and participate in the progression of HF. Macrophages in the liver can be divided into resident macrophages, Kupffer cells, and macrophages derived from bone marrow monocytes. In the event of liver injury, Kupffer cells have the sentinel function of phagocytosing harmful environmental substances and regulating the liver immune response, while monocyte-derived macrophages are cells that produce inflammatory cytokines, which help to timely regulate the liver inflammatory response and wound healing response . In addiresponse . One sturesponse  has showresponse . A recenresponse  showed tresponse . TGF-β iresponse . TGF-β cresponse . The PDGresponse . Tumor nresponse . TherefoCurrently, biomechanics have been extensively studied in the lung, kidneys, and liver. Mechanotransduction refers to the process by which cells activate cellular pathways and affect their functions by converting mechanical stimuli generated by the extracellular environment into biochemical signals . IntraheIn vivo experiments in mice, the experimental activation of YAP/TAZ promoted the regeneration of damaged organs, and overactivation led to liver fibroblasts entering a fibrotic state, which intensified the inflammatory fibrosis of the liver and promoted the occurrence of liver tumors, leading to poor prognosis (Yes-associated protein (YAP) and TAZ  are downstream molecules of the Hippo pathway, which are involved in liver development, regeneration, and injury repair . In vivorognosis . YAP/TAZrognosis .The Hippo-YAP/TAZ signaling pathway consists of a kinase cascade, mammalian sterile line 20-like kinase 1  and mammalian sterile line 20-like kinase 2 , large tumor suppressor protein 1 (LATS1) and large tumor suppressor protein 2 (LATS2), linker proteins SAV1 (SAV1), MOB kinase activators 1A (MOB1A) and MOB kinase activators 1B (MOB1B), and YAP/TAZ. MST1/2 plays a central role in the regulation of the activity of YAP and TAZ proteins. The MST1/2 kinase cascade can phosphorylate YAP and TAZ proteins to inactivate them and cannot interact with TEAD transcription factors to activate their target gene transcription . The celIn addition, YAP/TAZ is also regulated by focal adhesions (FAs), but the signal from it inhibits the Hippo pathway, to activate YAP/TAZ protein and lead YAP/TAZ to enter the nucleus of HSCs to increase the expression of target genes so that cells can proliferate and secrete ECM to adapt to the stiffened external environment .Rho-ROCK is a signaling pathway upstream of Hippo. Rho guanosine triphosphatases (Rho GTPases), belonging to the Ras family, are small signal G proteins that can regulate actin dynamics and actin polymerization by regulating a variety of downstream effector proteins, including ROCK proteins, and they play different functions according to cell types, such as actin-cytoskeleton assembly, cell contraction, and stress fiber formation . In addiThe cytoskeleton is an important means of mechanical force transmission in cells, and Rho directs morphological changes associated with HSCs activation by regulating the actin cytoskeleton . Stress in vitro experiment, it was found that 50 mmHg hydrostatic pressure can significantly induce HSCs to acquire profibrotic properties through the RhoA/ROCK signaling pathway, and ROCK1 mediates the activation of this pathway  and carbon tetrachloride-induced HF in mice (in vitro and HF in vivo (Myocardin-related transcription factors (MRTFs) are mechanosensory that link actin dynamics, including MRTF-A and MRTF-B, the former interacting primarily with serum response factor (SRF) in the nucleus and subsequently activating target gene transcription . MRTF-A  in mice . CCG-142 in mice . Shi et  in vivo . These rIt can be seen from the above description that there are interactions and crosstalk between the above three mechanotransduction signal pathways in the regulation of HF. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase in the cytoplasm that plays an important role in cellular behavior  and is considered an important component involved in early events in cell-ECM interactions . FAK is Extracellular mechanical forces stimulate integrin β1 to activate the FAK signaling pathway. Activated FAK promotes cell migration and invasion and mediates the activation of HSCs, differentiation into myofibroblasts, and inhibition of apoptosis . A studyThe Wnt/β-Catenin signaling pathway is one of the highly conserved signaling pathways activated in the evolution of species and can regulate cell proliferation and cell polarity, control cell fate, maintain homeostasis in embryos, and affect the growth and development of tissues and organs . HoweverThe stiff ECM can enhance the expression of each member of the Wnt/β-Catenin signaling pathway in bone marrow mesenchymal stem cells and primary chondrocytes, but the activation of β-Catenin by mechanical signals does not depend on Wnt signaling, but through the activation of integrins forming IAC or activating the FAK pathway, the β-Catenin accumulated in the cytoplasm then translocates to the nucleus to bind to the Wnt1 promoter region, promote gene transcription, and finally form positive feedback to the Wnt/β-Catenin signaling pathway; that is, the activation of β-Catenin by integrins leads to the increased expression of Wnt1, which in turn activates the Wnt/β-Catenin signaling pathway . MoreovePrevious studies  have shoPiezo1 and Piezo2 are members of a novel class of mechano-activated cation channels (MACs) that mediate cellular biological behaviors, are expressed differently in different cell types, and regulate physiological functions differently . Piezo1 At present, the research on Piezo1 ion channel mechanotransduction mainly focuses on the tumor, skin scars, and atherosclerosis , and relIt has been found that SDC-4 can participate in mechanotransduction, activate mitogen-activated protein kinase (MAPK) and protein kinase C-α (PKC-α) signals in response to directly applied mechanical tension , and canThe transcriptional coactivator YAP/TAZ, also known as a convergent effector of the Hippo pathway, is widely recognized for its novel role as a nuclear mechanosensor in organ homeostasis and cancer processes. Agrin is known for its ability to aggregate acetylcholine receptors (AChR) at the neuromuscular junction, can act as an ECM sensor, and has functions to stabilize FAs and promote HCC , and actin vitro culture system using adjustable matrix stiffness, OPN expression was significantly upregulated in HCC cells with increased matrix stiffness, accompanied by phosphorylation of GSK-3β and expression of nuclear β-catenin, and analysis of integrin β1 knockdown further suggested that high matrix stiffness upregulated OPN expression in HCC cells by activating integrin β1/GSK-3β/β-catenin signaling pathway (Osteopontin (OPN), also known as secreted phosphoprotein 1 (SPP1), is a highly negatively charged cytokine-like protein that regulates ECM turnover and remodeling . After t pathway . In a co pathway . The speFibrosis is a complex process involving a variety of cell types and a wide range of soluble factors , and HF is an inevitable process of liver cirrhosis and the main risk factor for the development of HCC . Matrix Liver ECM stiffness is the main extracellular force that generates mechanical forces, and treatment of ECM stiffness can effectively reduce matrix stiffness and even inhibit or reverse HF. Changes in ECM components often cause an imbalance in matrix generation and degradation. From this perspective, we can search for the influencing factors of the imbalance and intervene with them to achieve the purpose of reducing the stiffness of liver ECM, thereby inhibiting the activation of HSCs and completing the remission or reversal of fibrosis.In the normal liver, MMPs and TIMPs are in dynamic balance, so ECM can be expressed and degraded normally. The biochemical basis of HF is the metabolic imbalance of the ECM. If the production of ECM is greater than degradation, HF will form and develop; otherwise, HF will resolve and reverse. When HF occurs, TIMP-1 inhibits the activity of MMPs, which prevents collagen degradation and promotes HF. Therefore, the degradation of collagen  is the key to the reversal and regression of HF. In a study of the effect of the farnesoid X receptor agonist obeticholic acid (OCA) combined with the dipeptide kinase-4 inhibitor sitagliptin on a rat model of HF, the comLOX has the effect of increasing tissue stiffness and resisting ECM degradation, which is achieved by collagens crosslinking to resist the degradation of MMPs, thereby promoting excessive ECM accumulation in the liver and limiting the reversal of the process. In normal physiological processes, LOX mainly maintains the tensile strength and structural integrity of most tissues, and its protein expression is strictly regulated . In chroIn addition to the fact that cells are stimulated by and respond to their external signaling molecules, biomechanics can also be the reason for cell behavior changes . HSCs anThe Rho-ROCK signaling pathway is one of the mechanical signaling pathways of HSCs perception and effect, which participates in the activation of HSCs, and inhibition of this pathway can inhibit the activation of HSCs and improve HF. P160ROCK (Y27632) is a pyridine derivative that can specifically inhibit the ROCK protein kinase family . In the YAP is a transcriptional coactivator and one of the key downstream proteins in the Hippo signaling pathway, but it is also cross-linked with the Rho signaling pathway and is also a potential therapeutic target. Influenced by the positive feedback of matrix stiffening, YAP is activated and subsequently translocated to the nucleus to direct the expression of genes such as cell proliferation, apoptosis, and motility . StudiesThe TGF-β1/Smads and PI3K/Akt pathways are involved in HSCs activation and autophagy. TGF-β1 is secreted by activated HSCs and is one of the main factors that induce collagen production and lead to matrix deposition and can upregulate TIMPs. QuercetBiological and biomechanical studies have shown that the overexpression of FAK and the increase of FAK activity play an important role in the occurrence and development of HF. FAs are discrete multiprotein complexes formed at internal sites of the cell membrane by cells attached to a stiff matrix, are a major component of the interaction between ECM and cells, and are responsible for sensing and translating mechanical stimuli generated by ECM to the cellular cytoskeleton, whereas FA assembly/disassembly and integrin-mediated mechanotransduction can respond to ECM stiffness . FAK phoin vitro or animal models for matrix stiffness intervention have achieved initial results, a lot of work is needed to further verify their efficacy in the future.Chronic liver injury can induce activation of HSCs and secretion of excessive ECM protein through inflammatory response, destroy ECM homeostasis, lead to abnormal deposition of ECM, increase matrix stiffness, and promote the occurrence and development of HF. The stiff ECM alters the local cell tension and regulates the biological behavior of HSCs through mechanotransduction pathways. At present, mechanotransduction pathways have been found to have potential and application prospects in the diagnosis and treatment of HF, but our understanding of mechanosensing and intercellular mechanotransduction mechanisms is still limited. In addition, in the course of future studies, we also need to take into account that mechanotransduction and biochemical signaling in the liver interact and crosstalk to produce synergistic effects on HF. A better understanding of how hepatocytes  sense matrix stiffness and translate matrix mechanical cues into cellular responses, as well as the interactions and cross-talk between mechanotransduction and biochemical signaling in the liver, could provide us with more opportunities to develop targeted drugs or inhibitors that target altered ECM mechanisms in HF. More importantly, in future studies, we also need to consider combination strategies and drugs targeting biochemical and mechanical signals to improve clinical outcomes in patients with HF. At present, although some


Cannabis is well established to impact affective states, emotion and perceptual processing, primarily through its interactions with the endocannabinoid system. While cannabis use is quite prevalent in many individuals afflicted with psychiatric illnesses, there is considerable controversy as to whether cannabis may worsen these conditions or provide some form of therapeutic benefit. The development of pharmacological agents which interact with components of the endocannabinoid system in more localized and discrete ways then via phytocannabinoids found in cannabis, has allowed the investigation if direct targeting of the endocannabinoid system itself may represent a novel approach to treat psychiatric illness without the potential untoward side effects associated with cannabis. Herein we review the current body of literature regarding the various pharmacological tools that have been developed to target the endocannabinoid system, their impact in preclinical models of psychiatric illness and the recent data emerging of their utilization in clinical trials for psychiatric illnesses, with a specific focus on substance use disorders, trauma-related disorders, and autism. We highlight several candidate drugs which target endocannabinoid function, particularly inhibitors of endocannabinoid metabolism or modulators of cannabinoid receptor signaling, which have emerged as potential candidates for the treatment of psychiatric conditions, particularly substance use disorder, anxiety and trauma-related disorders and autism spectrum disorders. Although there needs to be ongoing clinical work to establish the potential utility of endocannabinoid-based drugs for the treatment of psychiatric illnesses, the current data available is quite promising and shows indications of several potential candidate diseases which may benefit from this approach. Nabiximol (brand name Sativex), a 1:1 combination of Δ9-THC and cannabidiol (CBD) extracted from the cannabis plant administered via oromucosal spray, is approved for use in Europe and Canada.Currently available direct CB1R agonists, all of which are relatively low efficacy agonists, approved by the US FDA for use in humans include the orally administered compounds nabilone (brand name Cesamet) and dronabinol (synthetic ΔOrthosteric antagonists of the CB1R have been identified. Rimonabant (brand name Acomplia), is a high affinity, brain penetrant, inverse agonist that was approved in Europe for a short time to treat symptoms of metabolic disorder and contribute to the cessation of smoking  bind to other regions of the receptor and modulate orthosteric ligand binding and/or signaling. Positive allosteric modulators (PAMs) enhance orthosteric agonist signaling, while negative allosteric modulators (NAMs) inhibit agonist signaling. Thus, PAMs can activate the eCB system, but will do so selectively by potentiating CB1R activity only at receptors with orthosterically bound eCBs, while NAMs will dampen CB1R activity driven by orthosteric CB1R agonists.Several CB1R PAMs have been identified and characterized. 6-Methyl-3-[2-nitro-1-(thiophen-2-yl)ethyl]-2-phenyl-1H-indole (ZCZ011), increases binding of orthosteric agonists and enhances CB1R signaling , responsible for approximately 80% of 2-AG hydrolysis to arachidonic acid in brain 6 and ABHD12   could also function as an intracellular transport protein for the eCBs. SCP-2 binds to both AEA and 2-AG with Early studies of the characteristics of neuronal accumulation of AEA supported the existence of a plasma membrane protein transporter that participates in facilitated diffusion to promote AEA movement across membranes . The remainder of this review will focus on the current state of knowledge for each of these conditions to highlight where the field is at and where it is moving.Preclinical studies demonstrate that modulating the eCB system influences positive reinforcement, anxiety, stress-induced craving, and relapse to substance use , attenuated the subjective effects of smoked cannabis . We recently described evidence supporting this approach for the treatment of CUD: early-phase clinical studies with AEF0117 show that it is well tolerated in both healthy volunteers and people who use cannabis daily  and reduces the positive subjective and reinforcing effects of smoked cannabis , but have not been shown to significantly reduce cannabis use in clinical trials , rimonabant administration  produced significantly greater tobacco abstinence rates and less weight gain relative to placebo. Yet as was seen in the metabolic disorder studies, rimonabant also increased the incidence of adverse events , and the likelihood of suicidality compared to placebo when taken for 1 year  in which a cytosine is replaced by an adenine, resulting in an amino acid substitution in the protein where a proline is replaced by a threonine  . Despite the well-defined behavioral diagnostic features of ASD, the biological markers, etiology, and contributing pathophysiological mechanisms of this brain disorder remain unclear. Importantly, there is a high prevalence of co-occurring conditions such as, intellectual disability, anxiety, seizure susceptibility, developmental delays, sleep disturbances, gastrointestinal disorders, and attention-deficit/hyperactivity disorder with ASD which results in a highly heterogenous clinical population and a complicated landscape for the development of pharmacological interventions  accounts for approximately 1 to 6% of all ASD cases making it the most common, known monogenetic form of ASD . NLGNs are postsynaptic cell adhesion molecules that regulate synaptic transmission and neuronal development  is the most well-studied environmental factor model of ASD and has been extensively used to investigate eCB dysregulation in ASD. Behaviorally, VPA-exposed animals have deficits in social communication, social play, obvious stereotypies, and increased anxiety along with changes in AEA and 2-AG tone. In one VPA model, changes in DAGL and MAGL were found alongside behavioral changes in sociability, anxiety, and nociception , diminished ultrasonic vocalizations, and increased self-grooming (Meyza et al., Collectively, these varying animal models of ASD have some common outcomes. FAAH inhibition with URB597 or AM404 are effective pathways to modulate AEA signaling and producing changes in social domain across etiopathologies of ASD. However, the given the breadth of social functions altered by AEA signaling, more work is needed to understand how to achieve specific and sensitive modulation of social behaviors. Inhibition of 2-AG signaling with JZL184 and CB2R activity with AM630 modulates anxiety and motor ability. Finally, CB1R modulation largely results in amelioration of cognitive deficits although alterations in CB1R activity can also impact aggression and social behaviors.Compared to the range of data available on the pharmacological approaches to target the eCB system in ASD animal models, there are considerably fewer studies that have translated these preclinical findings on eCB biology to the autistic human population. The first reports of eCB dysregulation in autistic humans solely assessed cannabinoid receptor and enzyme expression. Siniscalco and colleagues, showed that in peripheral blood mononuclear cells collected from autistic children mRNA and protein levels of CB2Rs were upregulated compared to neurotypical controls (Siniscalco et al., The studies reviewed indicate major progress of cannabinoid treatment in ASD; however, it is still unclear which cannabinoids are well suited to target core features of ASD. Moreover, since only exogenous cannabinoids have been tested in this clinical population the effectiveness of direct modulation of eCB signaling is unknown. Studies in co-occurring conditions, like social anxiety which directly modulate AEA signaling through FAAH inhibition, may offer greater specificity of target engagement without activating the euphorigenic or off-target effects commiserate with exogenous cannabinoid intervention (Ahmed et al., The eCB system represents a novel target for the development of a new class of drugs for psychiatric illnesses that has not previously been explored in depth. While still in the early days of translation to humans and clinical trials, preclinical data have been very encouraging and the initial studies in humans have largely supported the predictions made from rodent work. As discussed in this paper, there are several key areas where this development has advanced significantly, with some promise. More so, there appear to be several targetable components of the eCB system that have provided some optimism see . First, MAGL inhibitors are still in very early stages of clinical development, so it will be important to see how data from future trials comes out as while 2-AG is a more efficacious agonist at CB1R then AEA is, that may in turn bring issues related to psychoactivity and CB1R desensitization that have not been present with FAAH inhibitors. Currently, most of the focus of MAGL inhibitors has been on neurological and neuroinflammatory conditions, however this therapeutic application could still hold benefit for psychiatric conditions, many of which are associated with neuroinflammation, such as major depression.While CB1R antagonists are too problematic as straight therapeutic approach, as was learned from the rimonabant trials and the subsequent development of adverse psychiatric side effects, more tailored approaches to dampening CB1R signaling could hold some promise. A recently developed analog of pregnenolone has shown some positive outcomes for reducing cannabis intoxication and could be a novel approach to treating CUD as well. A multi-site clinical trial is currently underway to evaluate this specifically. Interestingly, there is also clinical development with these agents for the management of cognitive issues related to neurodevelopmental disorders and so these agents may also represent a novel drug class for disorders including ASD.While there may still be a place for direct CB1R agonists for the treatment of conditions such as chronic neuropathic pain and multiple sclerosis, the potential risk these may carry for exacerbating psychiatric conditions, particularly psychosis and schizophrenia and possibly bipolar disorder, will likely limit their utility for the treatment of psychiatric conditions.Overall, this is an exciting time for eCB-based therapeutics, with several recent positive trials emerging for psychiatric conditions with drugs that amplify eCB activity, there is a renewed interest in the therapeutic potential of this system. While disorders such as major depression, bipolar disorder and schizophrenia may not represent psychiatric illnesses that will benefit from this approach, there is some optimism now that SUD, anxiety and other non-major depression stress-related psychiatric disorders and ASD may represent a cluster of disease states that could be alleviated through eCB based medications.


Cell by the Sinclair lab and collaborators entitled “Loss of Epigenetic Information as a Cause of Mammalian Aging” [1] implicates heritable changes in gene expression as the basis for aging, a postulate consistent with the emerging information theory of aging. Sinclair’s group and colleagues induced epigenetic changes, i.e., DNA and histone modifications, via double-strand breaks (DSBs) catalyzed by the I-Pol endonuclease at specific genomic loci. The genomic DNA breaks, introduced without inducing insertion or deletion mutations (indels) in a mouse model, were targeted to 19 non-coding regions and one region in ribosomal DNA (rDNA), the latter shown to not have a significant effect on the function or transcription of rDNA [1]. With that experimental model in place, the authors present experimental evidence supporting a model that epigenetic changes drive aging via this inducible DNA break mechanism. After demonstrating the phenotypic alterations of this accelerated aging, they attempt to reverse selective phenotypes by resetting the altered epigenetic landscape. Establishing a causal relationship between epigenetic changes and aging, and how this connection might be manipulated to overturn cellular features of aging, is provocative and merits further study.The recently published article in  Earlier observations by in vitro and in vivo aging.To induce site-specific chromosomal DSBs in mammalian cells, vice versa? To address this, they devised a mechanism to induce epigenetic changes, specifically changes to 1) methylation patterns at specific CpG islands, cystine guanine dinucleotides occurring in gene promoters that are commonly methylated, and 2) acetylation and methylation of histones H3 and H4 , previously shown to induce the formation of pluripotent stem cells from somatic cells . Remarkain vitro and in vivo aging are impressive. The results were not exclusively associated with transcriptional or epigenetic changes, but also macroscopic organismal performance in behavioral tests, both cognitive and activity-based. Moreover, both neurological degeneration and muscular atrophy associated with natural and accelerated aging were detected  tissue-specific epigenetic reversal, 2) cognitive and muscular aging reversal via the same tests used in the initial experimental validation, 3) alterations to lifespan, and 4) resistance to disease or other hazards in the mouse model. Collectively, addressing these parameters would further support the idea of extending lifespan by epigenetic manipulations.The study by Yang et al. was performed exclusively with mouse models, and it will be exciting to see if these findings would apply to other model organisms or humans. Importantly, an assessment of age-related clinical features and their reversibility by an epigenetic mechanism would provide new hope for research in the treatment of age-related diseases.