Preeclampsia is a complex hypertensive disorder in pregnancy which can be lethal and is responsible for more than 70,000 maternal deaths worldwide every year. Besides the higher risk of unfavorable obstetric outcomes in women with preeclampsia, another crucial aspect that needs to be considered is the association between preeclampsia and the postpartum cardiovascular health of the mother. Currently, preeclampsia is classified as one of the major risk factors of cardiovascular disease (CVD) in women, which doubles the risk of venous thromboembolic events, stroke, and ischemic heart disease. In order to comprehend the pathophysiology behind the linkage between preeclampsia and the development of postpartum CVD, a thorough understanding of the abnormal uteroplacental vascular remodeling in preeclampsia is essential. Therefore, this review aims to summarize the current knowledge of the defective process of spiral artery remodeling in preeclampsia and how the resulting placental damage leads to excessive angiogenic imbalance and systemic inflammation in long term CVD. Key molecular factors in the pathway—including novel findings of microRNAs—will be discussed with suggestions of future management strategies of preventing CVD in women with a history of preeclampsia. Preeclampsia is a subtype of hypertensive disorders of pregnancy (HDP), defined as hypertension (systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg) newly developed at or after 20 weeks of pregnancy with at least one of following conditions: proteinuria , maternal organ dysfunction , or uteroplacental dysfunction  , and their receptors . Vascular endothelial growth factor (VEGF) is highly expressed in decidual cells and invading cytotrophoblasts in normal pregnancy, which leads to endothelial cell proliferation for newly developing capillaries in uteroplacental circulation . MoreovePlacental growth factor (PlGF), a member of the VEGF family, is another proangiogenic factor that binds to Flt-1 which augments the angiogenic effect of VEGF. PlGF exerts not only direct effects on endothelial cells, but also indirect effects on nonvascular cells with pro-angiogenic activity by altering the functioning of immune cells; it recruits monocytes and activates macrophages which can release angiogenic factors, and encourages proliferation of mesenchymal fibroblasts and attracts myeloid progenitors to develop sprouts and collateral vessels . MoreoveAs a VEGF receptor, Flt-1 is highly expressed in the invading extravillous trophoblasts in the first trimester, which implies that VEGF-Flt-1 interactions lead to early trophoblast invasion . As gestSoluble endoglin (sEng), a coreceptor for transforming growth factor-β1 (TGF-β1), is another antiangiogenic factor released by the placenta that acts in synergy with sFlt-1. Endoglin (Eng) is an angiogenic receptor expressed mainly on the surface of placental syncytiotrophoblast and endothelial cells which serves as a co-receptor of angiogenic TGF-β signaling . TGF-β iActivin A is a dimeric glycoprotein belonging to the TGF-β family produced by the placenta and fetal membranes . In pree+6 weeks of gestation) was related to development of preeclampsia, which requires further confirmation with large-scaled studies [Hypoxia inducible factor (HIF) is a heterodimer consisting of HIF1-α and HIF2-α subunits, which are regulated by oxygen, and a constitutively expressed HIF1-β subunit. In a hypoxic environment, HIF-1 regulates transcription of various genes, including VEGF, TGF-β3, and NOS, by binding at their promotor and enhancer regions . HIF exp studies .MicroRNAs(miRNAs) are small (<25 nucleotides), single-stranded, non-coding RNAs that regulate gene expression by inhibiting translation. These molecules bind to the untranslated lesion of a target gene and silence their expression . During Specifically, miR-210 has been reported to be overexpressed in placentas of preeclampsia . StudiesAdditionally, miR-125b is known to be an anti-angiogenic factor which decreases VEGF expression when it is overexpressed . A recenThe role of miR-21 in preeclampsia has been also newly studied, since it regulates the forkhead box M1 protein (FOXM1), which is expressed in cytotrophoblasts for proliferation and differentiation, responsible for the early placental development . In factAs discussed so far, the key mechanism of preeclampsia is disrupted spiral artery remodeling resulting in insufficient blood flow to the placenta, which in turn leads to RUPP. RUPP leads to a change in the level of several factors released by the placenta—mainly an increase of pro-inflammatory and anti-angiogenic factors and a decrease of pro-angiogenic factors—which eventually spread into the maternal circulation ; this reCardiovascular diseases (CVD) are currently considered as the leading cause of death globally . Women wRecently, a history of HDP has been designated as an independent risk factor for future cardiovascular events and incorporated in guidelines for risk stratification of stroke and CVD ,105. HowThe nationwide register-based cohort study of 1.5 million women who had delivered in Denmark from 1978 to 2012 has reported that women with gestational hypertension had the highest risk of developing chronic hypertension after pregnancy, followed by women with severe preeclampsia and moderate preeclampsia. In fact, of women in their 20 s, 14% of nulliparous women with HDP developed hypertension in the first 10 years after pregnancy compared with 4% of nulliparous women with normotensive pregnancy. The corresponding percentages of developing hypertension after HDP and without HDP in their forties were higher as expected: 32% and 11%, respectively. In fact, in a year after delivery, women with HDP had 12-fold to 25-fold higher rates of hypertension compared to women with normotensive pregnancy—which remained high as 3 to 10-fold in the first 10 years after pregnancy and twice higher when it passes 20 years postpartum. The decreasing trend in the relative risk of developing chronic hypertension as time passes from delivery (compared to controls) seem to be due to the natural behavior of hypertension in which its baseline risk increases with age. Therefore, it can be concluded that the risk of hypertension associated with HDP is highest shortly after an affected pregnancy and persists for more than 20 years . The PROThe American Heart Association has reported the Heart Disease and Stroke Statistics—2021 Update with an enhanced focus on adverse pregnancy outcomes (APO). APOs include HDP, gestational diabetes, preterm delivery, and small for gestational age. These interrelated disorders are associated with long-term risk of cardiometabolic disease in maternal and offspring . While nCarotid intima-media thickness (CIMT) is a well validated, non-invasive marker of subclinical atherosclerotic disease. It involves ultrasound evaluation of the thickness of the intimal and medial carotid arterial wall. Data have shown that an increased CIMT confers an elevated risk of coronary atherosclerotic lesions and future cardiovascular disease in both histological and epidemiological studies . Althougp = 0.0001) [A prospective study of women with late onset preeclampsia reported a significant increase of 108μm in CIMT compared with those in normotensive pregnancy  . While e 0.0001) . On the  0.0001) . DespiteThe coronary artery calcification (CAC) Score assessed by coronary computed tomography (CCT) is another important predictor for CVD events. Calcification is related to arterial stiffness associated with an increased risk of CVD, and the St. Francis Heart study determined that for a CAC score > 100 Agaston units (AU), the relative risk for atherosclerotic CVD was 9.6  . SignifiAtheromatous plaque is formed through a chronic, inflammatory progression of atherosclerotic disease without overt symptoms. It can result in a sudden occurrence of coronary artery disease (CAD), such as myocardial infarction, unstable angina pectoris and cardiac death . The preIn addition, the risk ratio of cerebrovascular disease (CD) in women with previous preeclampsia ranged from 1.53 to 3.13 ,139,140.Although the mechanisms still remain elusive, it is evident that the risk of CVD rises after HDP, which can occur early in the postpartum period. As of today, various guidelines for postpartum follow-up are suggested. The American College of Obstetricians and Gynecologists (ACOG) recommends that all women with HDP have an initial check with their obstetrician within three weeks postpartum, followed by another visit at three months postpartum, and annual cardiovascular checkups consequently . A recenThe key mechanism of preeclampsia is defective spiral artery remodeling resulting in inadequate blood flow to the placenta, which in turn leads to RUPP. RUPP leads to rise in pro-inflammatory and anti-angiogenic factors and decrease in pro-angiogenic factor in maternal circulation which in turn results in endothelial dysfunction leading to disrupted maternal hemodynamics. The association between HDP and postpartum CVD has become a well-accepted fact, especially among women with a history of preeclampsia. Risk factors of CVD mostly develop in the early postpartum period and the most vulnerable timing for CVD events is the decade after delivery, in which the related circulating factors persist in maternal circulation. Therefore, women with a history of HDP should be considered for risk evaluation before the current cardiovascular screening guidelines. Although the exact linking mechanisms between HDP and CVD are still left for further research, primary prevention strategies to reduce cardiovascular disease in women should always include risk stratification based on their obstetrical history.


Successful human reproduction relies on the well-orchestrated development of competent gametes through the process of meiosis. The loading of cohesin, a multi-protein complex, is a key event in the initiation of mammalian meiosis. Establishment of sister chromatid cohesion via cohesin rings is essential for ensuring homologous recombination-mediated DNA repair and future proper chromosome segregation. Cohesin proteins loaded during female fetal life are not replenished over time, and therefore are a potential etiology of age-related aneuploidy in oocytes resulting in decreased fecundity and increased infertility and miscarriage rates with advancing maternal age. Herein, we provide a brief overview of meiotic cohesin and summarize the human genetic studies which have identified genetic variants of cohesin proteins and the associated reproductive phenotypes including primary ovarian insufficiency, trisomy in offspring, and non-obstructive azoospermia. The association of cohesion defects with cancer predisposition and potential impact on aging are also described. Expansion of genetic testing within clinical medicine, with a focus on cohesin protein-related genes, may provide additional insight to previously unknown etiologies of disorders contributing to gamete exhaustion in females, and infertility and reproductive aging in both men and women. Infertility, a disease defined by the failure to achieve a successful pregnancy after 12 or more months of regular, unprotected intercourse or due to an impairment of a person’s capacity to reproduce , is estiIntentional delays in childbearing until later maternal age has been an increasing phenomenon over several decades. In the United States, between 1970 and 2006, the proportion of first births to women age 35 or over increased nearly eight times . This trWith later maternal age, women experience a gradual decrease in fecundity and increased rates of miscarriage . These pThroughout a woman’s reproductive life, successful meiosis requires homologous recombination-mediated DNA repair and proper segregation of chromosomes and sister chromatids to yield haploid oocytes. Sister chromatid cohesion, mediated by cohesin rings which tether the two sister chromatids of replicated chromosomes, is essential to ensure that these processes are carried out correctly. Loss or weakening of chromosome cohesion with advancing maternal age has been proposed as a leading cause of age-related aneuploidy in oocytes . TherefoUnderstanding the etiology of aneuploidy with advancing maternal age requires an understanding of female mammalian meiosis. Mammalian meiosis is a specialized form of cell division characterized by a single round of DNA replication, followed by two rounds of chromosome segregation resulting in haploid gametes. Segregation of homologous chromosomes occurs during meiosis I (MI) and segregation of sister chromatids occurs during meiosis II (MII) . Female Meiotic prophase is the first and longest stage of mammalian meiosis in which the homologous chromosomes must pair, synapse, and undergo meiotic recombination to generate a crossover event. Meiotic prophase I include unique processes which are distinct from mitotic prophase. Following pre-meiotic DNA replication, meiotic prophase I initiates and progresses through various stages including leptonema, zygonema, pachynema, diplonema, and diakinesis . PairingRecombination during prophase I occurs at the DNA level, between two non-sister chromatids . ProgramChromosome cohesion is established during pre-meiotic S phase and early prophase I via loading of cohesin rings/complexes. While mitotic and meiotic cohesin subunits are widely conserved in diverse species , three tThe most well-defined mechanism of cohesin removal during meiosis involves Separase-induced proteolytic cleavage . During Cohesin removal also depends on a non-proteolytic mechanism known as the prophase pathway, which precedes the action of Separase. The prophase pathway is orchestrated by an interaction between PDS5B, Sororin and wings apart-like (WAPL) protein . WAPL faThe proper step-wise removal of the cohesin rings is essential to ensure correct chromosome segregation during MI and MII. In sexually mature females, follicle-stimulating hormone (FSH) promotes growth of a dominant follicle in the ovary. Proliferation of granulosa cells surrounding the follicle leads to estrogen production which ultimately triggers the pre-ovulatory luteinizing hormone (LH) surge . The LH Cohesin established in females during fetal life is not replenished over time during postnatal development. Oocyte cohesin expression is restricted to pre- and early meiotic prophase stages and allows for complete fertility and prevents aneuploidy . AdditioPrimary ovarian insufficiency (POI) is defined by oligomenorrhea or amenorrhea prior to age 40 in conjunction with elevated serum FSH levels in the menopausal range, as defined by the reporting laboratory, drawn on two separate occasions at least 1 month apart . AffectiSTAG3, the index patient case(s) have presented with primary amenorrhea, elevated FSH levels in menopausal range, and streak gonads on ultrasound. These findings are consistent with early gonadal dysgenesis in young girls with otherwise normal 46XX karyotypes.Recent studies of families with POI from various ethnic backgrounds have identified several variants in the gene encoding the meiosis-specific cohesin protein STAG3 as a potential etiology for their diagnosis . InteresSTAG3 resulting in a frameshift mutation and subsequent premature stop codon  has POI on the more severe end of the spectrum with diagnosis at a young age with primary amenorrhea, elevated FSH levels, and streak gonads on ultrasound consistent with a clinical picture of gonadal dysgenesis.The first report of mutant cohesin leading to POI was reported by op codon . Ultimatrm cells . TherefoSTAG3 variant identified in a family with POI was by STAG3 gene. Furthermore, STAG3–both affected sisters were homozygous for this mutation which resulted in a premature stop codon and thus, predicted a truncated protein (p.[Ser227∗]). Interestingly, the 87-year-old paternal grandmother and 48-year-old maternal aunt of the sisters were both carriers of this mutation and did not suffer from amenorrhea or infertility, though they did go through premature menopause at age 40 and 37 years old, respectively.The second report of a STAG3 (c.1573 + 5G > A) which was predicted to result in a frameshift mutation and premature stop codon. The unaffected parents and brother in this family were heterozygous carriers for the mutation (STAG3 variants on families with POI. STAG3 (c.305delC) which yielded a premature stop codon predicting a truncated protein. The second variant was a T to G substitution in exon 7 (c.659T > G) which led to a missense mutation. Interestingly, this variant was also identified in the unaffected mother who proceeded through menopause at age 51. STAG3 (c.291dupC and c1950C > A) leading to POI in a 21-year-old Brazilian woman. Compound heterozygosity for these variants was felt to be the mode of inheritance given the rarity of both variants and their impact on the transcript and protein; however, the affected patient was adopted and therefore parental DNA was not available to confirm this assumption.In addition, two sisters affected with POI from a Han Chinese family were found to have a homozygous donor splice site mutation in mutation . AdditioREC8 variant  from their mother and a variant in GDF9  from their father. Both variants were predicted to be damaging by the Polyphen2 database , a tool which predicts the potential impact of amino acid changes on the structure and function of a protein. The other patient who presented with primary amenorrhea was diagnosed with POI at age 13 and was found to have a variant in SMC1β  as well as BMP15 , both of which were predicted to be damaging by the Polyphen2 database. An additional SMC1β variant  was identified in one patient with secondary amenorrhea and POI diagnosed at age 22, yet she was also found to have two variants in the oocyte-specific gene, NOBOX. The findings of these studies demonstrate that variants in genes encoding cohesin-associated proteins may play a role in POI pathogenesis. Moreover, digenic and perhaps polygenic inheritance likely plays a role in the timing of onset of POI as well. Future studies of patients with POI, and their evaluation for potential etiologies, may need to consider these candidate genes, and specifically STAG3, given multiple affected families across various ethnic backgrounds have been identified to harbor these variants.Additional cohesin-ring components have also been implicated in human POI, specifically, SMC1β and REC8 . BouillyThe vast majority of meiotic errors detected in human pregnancies result from errors in female meiosis and are a leading cause of pregnancy loss . MeiosisRegarding trisomy 21, both increased maternal age as well as altered recombination events, regardless of age, have been associated with meiotic errors . The absn = 1,437 parents). DNA samples were genotyped on the HumanOmniExpressExome-8v1-2 array. These genotypes were then used to determine (1) if the non-disjunction error was maternally derived and (2) if this occurred during MI or MII. Subgroup analyses of mothers vs. fathers, MI mothers vs. fathers, MII mothers vs. fathers, and MI mothers vs. MII mothers were performed. Given that defects in cohesin proteins may play a role in PSSC and aneuploidy, the authors chose to assess candidate genes for meiosis-specific cohesin  within the subgroup analyses. Of the analyzed genes involved in the meiotic cohesin complex in this GWAS, there was a statistically significant association with a missense variant in RAD21L in the all mothers vs. fathers (OR 0.67) and MI mothers vs. fathers (OR 0.67) subgroup analyses, suggesting variants in this gene may be a risk factor for non-disjunction, more commonly in MI. None of the other candidate genes for cohesin-associated proteins demonstrated significance.A recent study by RAD21L in male mice leads to infertility whereas in female mice there is apparent age-related infertility . This occurs in approximately 1% of men of reproductive age . ApproxiSTAG3 variants in a cohort of men with azoospermia. n = 33). This cohort included 31 unrelated men and two brothers from a consanguineous family. After sequencing data was filtered, one patient was found to harbor two variants in STAG3 (compound heterozygote). The STAG3 variants included a frameshift insertion which generated a premature stop gain in the 597 amino acid; this is predicted to result in a protein lacking the entire armadillo-type domain of STAG3 which is involved in DNA and protein interactions. The other STAG3 variant identified was a novel splicing variant located on exon 23, an indispensable site for normal splicing. The patient’s unaffected fertile brother was only a heterozygous carrier for the frameshift mutation. Upon histologic analysis of testis biopsies including immunohistochemistry, the authors concluded that meiotic entry occurred with normal frequency, however, meiosis was noted to arrest as there was evidence of DSB formation, but failure to complete chromosome pairing. Therefore, it appears that STAG3 mutations in this patient resulted in complete bilateral meiotic arrest (MA) of spermatogenesis resulting in NOA.An alternative approach, utilizing information known from mouse models, has recently identified STAG3 in a different cohort of men with idiopathic NOA with MA (n = 28). In this analysis, the full coding region of STAG3 was sequenced directly. These authors identified two compound heterozygous variants in STAG3  in exon 13 leading to complete bilateral MA in an otherwise healthy human male. The c.1262T > G variant was paternally inherited and is a missense variant that changes a highly conserved neutral amino acid (Leucine) to a basic amino acid (Arginine) likely affecting protein folding and post-translational modifications. This variant is predicted to be disease-causing by all utilized prediction programs. The c.1312C > T variant was maternally inherited and is a nonsense substitution which introduces a premature stop codon (p.Arg438Ter). The index patient had bilateral small testis volumes (12–13 mL) and normal testosterone levels. The most developed germ cell type in any seminiferous tubule was the primary spermatocyte and a microdissection testicular sperm extraction (microTESE) procedure did not yield any sperm from this patient. Analysis of meiotic chromosome spreads from the spermatocytes of the affected patient found that no meiotic cells progressed through the zygonema stage of meiotic prophase I, which is consistent with MA. Additionally, compound heterozygosity was confirmed by Sanger sequencing of the patient’s parents. His brother inherited the c.1262T > G variant and spontaneously conceived two children.In similar fashion, STAG3–one splicing variant and one frameshift deletion in exon 16 which led to a premature stop codon at the 558th amino acid affecting the armadillo-type domain.In another recent study by STAG3 variants negatively impact protein function and lead to human male infertility due to MA. Similar to observations in females harboring variants in STAG3 have been shown to be associated with POI as previously described. With further evidence of the important role of cohesin-associated STAG3 in meiotic events in both males and females, consideration can be made for analysis of STAG3 in men and women with otherwise “idiopathic” NOA or POI, respectively.These studies are the first to demonstrate RAD21L is transcribed abundantly in testis and localized to the lateral and axial elements of the synaptonemal complex playing an essential role in homologous chromosome synapsis during meiotic prophase I. Male mice deficient in RAD21L are defective in homologous chromosome synapsis which in turn leads to zygonema arrest and subsequent azoospermia with male sterility in mice , which resulted in the removal of the final 41 amino acids of the protein. His fertile brother was found to be a heterozygous carrier. The affected patient had bilaterally small testis and histology demonstrated complete MA. In a targeted gene approach, RAD21L coding region in a Japanese cohort with 38 men with NOA due to MA and 140 men with NOA due to Sertoli cell-only syndrome (SCOS) and compared these men to 200 fertile controls. The RAD21L coding region was sequenced, and three variants were found . The distribution of two of the variants  was significantly different between the NOA patients with either MA or SCOS compared with the fertile controls. The authors postulated that these amino acid substitutions may play a role in disruption of spermatogenesis in Japanese patients, however, the function of the SNPs in those positions were predicted to be benign via the PolyPhen2 database. Therefore, the relationship between these identified SNPs and a mechanistic cause of NOA has yet to be determined.In the study by In addition to its role in sister chromatid cohesion and chromosome segregation, cohesin has been implicated in genome stability owing to its role in DNA repair and recombination. DNA damage arises continuously by various endogenous and exogenous factors and cells must possess mechanisms to repair these lesions. One mechanism is via homologous recombination (HR). In this process, BRCA2 (BRCA2 DNA repair associated) and PALB2  are key regulators and promote RAD51 activity which is essential for HR. Failure to repair DNA properly may lead to cell apoptosis or cancer . Cohesinin vitro. PDS5B associates with BRCA2 in early S-phase and depletion of PDS5B compromises the localization of both RAD51 and BRCA2 to the nucleus essentially disturbing HR and leading to increased cell sensitivity to DNA damaging agents , and all-cause mortality . RegardiMore recent reports have also implicated male factor infertility in diseases of aging and mortality . EisenbeAn underlying link connecting male and female infertility with overall health and lifespan remains uncertain due to the multifactorial pathogenesis of infertility and aging as well as heterogeneity of the literature. However, the use of infertility, specifically the most extreme versions including POI in females and NOA in males, may provide a biomarker for further counseling on genetic implications for current attempts at conception as well as the future health of patients and their offspring.Expansion of genetic testing within clinical medicine, and specifically reproductive medicine, may offer additional insight into previously unknown etiologies of disease. Specifically focusing on cohesin protein-related genes may inform the diagnosis of disorders contributing to gamete exhaustion in females, as well as infertility and reproductive aging in both men and women. The regulation of cohesin loading and removal requires the coordination of various kinases and phosphatases and further studies are needed to gain a better understanding of these mechanisms. Given that protein kinases are an emerging class of drug targets, identification of genetic defects in cohesin-related genes or regulators may serve as potential targets for future therapeutic studies of reproductive aging.RB, MS, and MB-E collected the references and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Recently, several new drugs have been introduced to treat obstructive lung diseases associated with inflammation , including long-acting bronchodilators, corticosteroids and monoclonal antibodies. Despite these new medicines, there are still patients who might benefit from a more selective approach to treatment given that this may be accompanied by fewer adverse effects, especially when a combination of drugs is required to manage severe disease. The possibility that inhibiting certain phosphodiesterase (PDE) isoenzymes (in particular PDE3 and PDE4) and, thereby, increasing the concentration of cyclic adenosine monophosphate (cAMP) in specific tissues and organs, may have therapeutic benefit has gained general acceptance over the last 30 years. Indeed, PDEs have become attractive drug targets because an increase in cAMP can relax smooth muscle and suppress inflammation.Beute et al., PDE3 plays a critical role in basophil and mast cell degranulation and, therefore, its inhibition by a small molecule inhibitors such as enoximone may be a viable treatment option in allergic airway inflammation. Similarly, another selective PDE3 inhibitor RPL554 (ensifentrine) has been intensively tested during last years and exhibits both bronchodilator and anti-inflammatory activity in vitro and in vivo assays. The benefits of inhaled ensifentrine in clinical studies with asthma and COPD patients is reviewed by Phillips and showed predominantly bronchodilator effects without reliable evidence for anti-inflammatory activity usually associated with PDE4 inhibition (The articles in this series of mini-reviews focus on the involvement of various PDE isoforms in the mechanisms underlying inflammatory and other airway diseases. As described by hibition . This ca2-adrenoceptor agonist (LABA), a long-acting muscarinic receptor antagonist and an inhaled corticosteroid. However, roflumilast has a low therapeutic ratio and ways to improve effectiveness and tolerability are needed. Possibilities include the development of dual- or multi-specificity agents. Another potential approach to improve the tolerability and therapeutic index is to develop PDE4 inhibitors for inhaled administration. Several examples exist and some have advanced to clinical trials for the treatment of asthma and COPD. Phillips, in his brief update, summarizes the pharmacological and clinical details of several inhaled PDE4 inhibitors (e.g. CHF6001) as well as those which failed to demonstrate sufficient efficacy in clinical trials. With this in mind, Stellari et al. have described the anti-inflammatory activity of the novel PDE4 inhibitor, CHF6001, showing specifically that topical administration efficiently disrupts NF-κB activation associated with lipopolysaccharide challenge, an effect that may be relevant to the prevention of exacerbations in patients with COPD.Currently, roflumilast is the only selective PDE4 inhibitor that has been approved for the treatment of a particular subset of patients with COPD. Disease guidelines recommend that roflumilast be used as an add-on therapy in people categorized as high risk, having severe, symptomatic COPD in whom exacerbations occur despite regular treatment with a combination of a long-acting βKawamatawong and Janosova et al., review the potential use of selective PDE4 inhibitors in non-COPD respiratory diseases, providing evidence of their efficacy in controlling symptoms in allergic rhinitis/rhinosinusitis and diseases associated with mucus hypersecretion. However, few PDE4 inhibitors have progressed to phase II and III clinical trials for these indications because of weak clinical efficacy and adverse effects. The treatment of respiratory diseases associated with non-Th2  inflammation such as non-Th2 asthma, asthma-COPD overlap, and non-Th2 allergic rhinitis represent a clear, unmet clinical need for which PDE4 inhibitors could be effective.The articles by Joskova et al.. In their review, they conclude that selective or dual PDE inhibitors may represent important tools in supporting normal cilia function and effective mucokinetic activity. For example, PDE1A inhibitors potentiate the cilia-stimulatory effects of β2-adrenoceptor agonists and this is mediated by cAMP; in contrast, PDE4 inhibitors become effective in the presence of LABAs to stimulate ciliary beating. This makes the mucociliary clearance in pathological airway conditions more effective. Furthermore, dual PDE3/4 inhibitors could provide additional benefits and show a more rapid response when taken with β2-adrenoceptor agonists; however, their cilia-stimulatory effect is probably mediated predominantly by PDE4 inhibition. The role of PDE inhibitors in airway cilia-driven transport may help in prevention of progressive loss of pulmonary function often observed despite current therapy.More light into the mechanisms governing ciliary motility and mucociliary clearance in respiratory disorders and the involvement of PDE is provided by Despite the fact that PDE inhibitors have been studied for decades, their full therapeutic potential in respiratory diseases after systemic and local  administration is still unclear. Therefore, a continued effort to collect sufficient evidence about the efficacy and safety of these molecules in animal models and in the clinical setting is essential.


Purpose of Review: Atherosclerosis is the principal cause of cardiovascular diseases (CVDs) which are the major cause of death worldwide. Mechanical force plays an essential role in cardiovascular health and disease. To bring the awareness of mechanosensitive Piezo1 role in atherosclerosis and its therapeutic potentials we review recent literature to highlight its involvement in various mechanisms of the disease.Recent Findings: Recent studies reported Piezo1 channel as a sensor, and transducer of various mechanical forces into biochemical signals, which affect various cellular activities such as proliferation, migration, apoptosis and vascular remodeling including immune/inflammatory mechanisms fundamental phenomenon in atherogenesis.Summary: Numerous evidences suggest Piezo1 as a player in different mechanisms of cell biology, including immune/inflammatory and other cellular mechanisms correlated with atherosclerosis. This review discusses mechanistic insight about this matter and highlights the drugability and therapeutic potentials consistent with emerging functions Piezo1 in various mechanisms of atherosclerosis. Based on the recent works, we suggest Piezo1 as potential therapeutic target and a valid candidate for future research. Therefore, a deeper exploration of Piezo1 biology and translation towards the clinic will be a novel strategy for treating atherosclerosis and other CVDs. Following their discovery, a number of studies were conducted on Piezo proteins architecture, their, cellular distribution in mammals, physiological functions and associated pathologies, and molecular mechanisms were demonstrated Mechanotransduction is the translation of mechanical forces into biochemical signals, which is necessary for potent cellular physiology. The molecular identity of mammalian mechanosensitive channels remained poorly understood, until the evolutionary discovery of Piezo channel family, which shed light on this mystery , and (3) the TM blade-constituting mechanosensory portion 50, and for broad structural concept in recent paper Piezo is the family of genes, Piezo1 and Piezo2, and are large transmembrane proteins that represent a special class of mechanically activated (MA) cation channels. Human (MA) channel molecular identity was mysterious for long, until the revolutionary discovery of Piezo 1& 2, which may increase our understanding of cell signaling and mechano-transduction, bringing new opportunities for understanding and targeting theses signaling mechanisms involved in health and disease. Piezo1 protein identical to that of humans was initially discovered in a mouse. Piezo1 proteins are collectively arranged to form a three blade-like propeller, it is inserted within the lipid bilayer which makes the central ion pore that senses mechanical forces Piezo1, the similar mutating of R2482H leads to delayed inactivating kinetics and is correlated with dehydrated hereditary stomatocytosis Piezo1 and Piezo2 gain of function mutating could be mapped into the structures, demonstrating that these models cover multiple of the functionally essential subunits and, furthermore, provides for a structural interpreting of the disease causative processes.As widely expressed in various cell types including cardiomyocytes, EC, VSMCs, and immune cells, Piezo1 play a wide range of roles in cellular physiology as well as pathology. Regarding this, researchers were curious in exploring the structures and/or domain of the channel involves in its function in health and disease. Despite revelation of general structure of Piezo1 by cryo-electron microscope (cryo-EM), the exact structural regions/subunits involved in activating as well as consequent inactivating of Piezo1 in correlation with its function are to be clarified. Through electrophysiology various researches study the possible structural domains to explore Piezo1 activation Previous studies demonstrated Piezo1 activation mechanisms as in the concept of “Force-from-lipids” Piezo1 was reported to function in sensing and transducing of various mechanical forces, such as membrane tension, stretching ex vivo experiment was found to be restrained or absent after endothelial Piezo1 knockdown. These findings proved Piezo1 as bona fide shear sensor and it is required for transducing shear forces into biochemical signals. Despite its recognition as shear sensor and backed by bountiful evidences, we still have a fundamental question of what is the precise mechanism of Piezo1 shear sensing and what are the exact structures and domain involved in Piezo1 activation by shear stress, other mechanical and chemical stimulus? This is of course a critical and difficult question to address due to the technical limitations and infancy of Piezo research. Several studies disclosed lately, the mechanism and domains that may be involved in the modulation of channel activation and inactivation of the channel as discussed in Section 5.Shear stress resulting from blood flow is important phenomenon and vital for cardiovascular physiology. Piezo1 is well recognized and specialized shear stress sensor as proved by many evidences. Regardless of Piezo1 research infancy, the physiological and pathological relevance of Piezo1 in various aspects of cardiovascular was earlier reported Atherosclerosis is a multifactorial and complex disease process of the vasculature involving various mechanisms and pathways in its pathogenesis. Accumulating evidences suggested the role of immune/inflammatory and other cellular process like cell proliferation, migration, apoptosis and remodeling in atherosclerotic pathogenesis in vitro, and found increased Piezo1-dependent cyclical hydrostatic pressure evokes proinflammatory gene expression in control, compare to Piezo1 disrupted cells. Furthermore, application of mechanical force to monocytes and macrophage initiates robust and specific pattern of Piezo1-dependent expression of proinflammatory and chemoattractive molecules ex vivo. Interestingly, absence of proinflammatory cytokines and chemokines expression was observed in the lack of Piezo1 ex vivo model. In a study of flow model, endothelial Piezo1 or Gαq/Gα11 specific depletion in mouse, exhibit diminished inflammatory signals and activation of integrin with regression of atherosclerosis in athero-susceptible region 2+ signaling may regulate a number of vital cellular activities 2+ signals control important cellular functions like mitosis, differentiating and migration, expression and functioning of several endocrine secretion as well as transcription molecules,  and ECM rearrangement including its synthetization and degrading In cardiovascular, mechanical forces mediated by Piezo1, regulates various cellular activities like proliferation, migration, apoptosis. Occurrence of cellular proliferation, migration and apoptosis in macrophage, SMCs, and EC is crucial mechanism in the development of atherosclerosis plaque Smooth muscle cells. Cellular proliferation is a fundamental process involved in vascular health and disease -related apoptosis-inducing ligand (TRAIL) in human umbilical vein endothelial cells (HUVECs) and cancer cells. Hope et al. reported the massive apoptosis elevation in number of cell lines  after application of both Piezo1 agonist Yoda1 and Trail, which happen through Piezo1 dependent signaling via Ca2+ influxes which triggers calpain, while application of GsMTx-4 decreased shear induced TRAIL stimulation Smooth muscle cells. Mechanical force affects apoptosis and viability of vascular cells including ECs and VSMCs as they are exposes to different forms of mechanical stress of various intensity and magnitudes. Currently, Piezo1 is recognized as important player in mechanosignaling. Growing number of reports are increasingly proving the relevance of mechanical forces in cardiovascular apoptosis Other cell types. Recent study reported that in alveoli, amid (ARDS) acute respiratory distress syndrome, stimulation of Piezo1 by mechanical stretching result in elevated intracellular Ca2+ evoked type II pneumocytes apoptosis, whereas, reduction in apoptosis was observed in the absence of Piezo1, which are in association with Bcl-2 pathway ex vivo model using shear, revealed Piezo1 mechanical force mediation of Ca2+ evoked Osteosarcoma cell apoptosis and inhibit proliferation. Additionally, another study in vivo reported growth encouragement of these cells after abrogation of Piezo1 gene expression in immunodeficient mouse Apart from mechanical forces, Piezo1 can chemically be gated. Recent studies, through high throughput screening technologies, small chemical compounds such as Yoda1 BolbostemmaAs new cation channel with versatile cardiovascular functions involving complex mechanisms and related pathway its pharmacology is still immature. Till date, GsMTx-4 . Additionally, another therapeutic idea through Piezo1 is also promising approach, in which cellular responses can be mechanically regulated through modulation of the mechanosensory genes-circuit. Recent study disclosed an amazing novel immunotherapeutic innovation called chimeric antigen T- cell receptor (CAR-T cell), with efficacy against multiple soft tumors. For aimed and regulated deliverance, mechanosensitive CAR T cell was manipulated through regulating of mechanosensory capacity of Piezo1, in order that mechanical perturbation by ultrasound wave could trigger the CART T cells within selected area. It is a therapeutic approach with high geotemporal accuracy which could be employed for remote control stimulating of various cell species Aforementioned works including many not cited here, indicated the significance of Piezo1 in various pathophysiological processes, specifically cardiovascular. Due to the infancy of both Piezo1 and its pharmacology research, a lot is not yet known, but its functions are still emerging. The realization of Piezo1 and its pharmacology Table  provides, cell specificity, and structure-function-relationship are required for elucidating how Piezo1 regulates particular disease process specifically atherosclerosis, to develop a high precision therapeutic approach through rational design of specific Piezo1 agonists and antagonists for obtaining effective therapies as well as avoiding or minimizing unwanted effects.Piezo1 discovery, emergence of its roles in various aspects of cardiovascular and rapid development of its research demonstrated a promising potential of the field towards novel therapeutic innovation for clinical application targeting atherosclerosis and other CVDs. Regardless of the infancy of the field, recent advancement in the realization of Piezo1 topographical structure and the outstanding breakthrough of its pharmacological agents indicate Piezo1 as doubtless target for novel therapeutic approaches in cardiovascular. Despite lack of potent agonists and specificity of Piezo1 blockers, both agents are widely accepted and employed as tools in Piezo1 research. Among the challenges of Piezo1 research are; wide range of expression and functions in multiple cell types, limited knowledge of Piezo1 functions in association with other proteins, such as interplay with cytoskeletal structures 2+ dependent signaling. Hence, through calcium, Piezo1 may contribute to atherosclerosis, though further study is required for this concept. Additionally, Piezo1 mechanical triggering leads to intracellular Ca2+ elevation and might consequently trigger the Akt/MTOR pathways through calmodulin (CaM)/CaM dependent protein kinases II (CaMII) which enhances cell proliferation and migration Due to relevance of mechanical forces in cardiovascular disease, mechanotransduction has attracts much attention. The role of Piezo1 mechanotransduction in vascular, immune/inflammatory cells, indicates that Piezo1 is involved in atherosclerosis, therefore Piezo1 is a promising candidate for therapeutic innovation against atherosclerosis and other CVDs. It may be engaged in number of unexplored pathophysiological mechanisms through different signaling. For example, Piezo1 is key transducer of mechanical cues into Ca


Both type 1 and type 2 diabetes are characterized by a progressive loss of beta cell mass that contributes to impaired glucose homeostasis. Although an optimal treatment option would be to simply replace the lost cells, it is now well established that unlike many other organs, the adult pancreas has limited regenerative potential. For this reason, significant research efforts are focusing on methods to induce beta cell proliferation (replication of existing beta cells), promote beta cell formation from alternative endogenous cell sources (neogenesis), and/or generate beta cells from pluripotent stem cells. In this article, we will review (i) endogenous mechanisms of beta cell regeneration during steady state, stress and disease; (ii) efforts to stimulate endogenous regeneration and transdifferentiation; and (iii) exogenous methods of beta cell generation and transplantation. The majority of these cases represent type 2 diabetes (T2D); whereas type 1 diabetes (T1D) constitutes approximately 5–10% of all cases of diabetes. In both T1D and T2D, there is a marked reduction in functional beta cell mass. In individuals with T1D, the primary cause of beta cell loss is through the autoimmune destruction of beta cells. In T2D patients, both a reduction in beta cell mass and impaired beta cell insulin secretory function have been observed [According to the latest figures from the International Diabetes Federation (IDF), approximately 463 million adults are living with diabetes, and this number is estimated to rise to 700 million individuals by 2045  or normalizing to total pancreatic weight (for beta cell mass)—confusingly, beta cell mass and volume tend to be used interchangeably in the literature . In T1D,Although a reduction in beta cell mass has predominantly been attributed to beta cell death, studies in mouse models of both T1D and T2D have suggested that beta cell dedifferentiation and/or transdifferentiation can also contribute to beta cell loss ,11,12. TWith the increasing focus on beta cell dysfunction and loss as a contributor to the progression of both T1D and T2D, extensive efforts have been made to identify mechanisms to replace functional beta cell mass. As discussed below, many studies have explored how beta cell populations are established and expanded during different critical windows of life, including fetal development, adult basal function, as well as injury and stress conditions, with the goal of developing therapeutics that can promote the induction of these mechanisms to restore beta cell mass in afflicted individuals. These studies have met with some success and, although several controversies remain, they have revealed important insights into the biology of rodent and human beta cell development and growth.The establishment of beta cell mass in rodents and humans occurs predominantly during embryonic and early postnatal stages ,17,18. IThere have been many studies, primarily in rodents, to identify endogenous extrinsic regulators of beta cell mass during the highly proliferative neonatal period, with hopes that these factors could be harnessed to promote beta cell proliferation on demand. In particular, factors supplied from the islet extracellular microenvironment have been shown to be necessary for beta cell proliferation. Some of the earliest studies focused on components produced by the extracellular matrix (ECM) and vascular structures surrounding the islets. In vitro studies identified endothelial cells as a source of laminins that promoted proliferation of the immortalized mouse insulinoma 6 (MIN6) cell line and suggested beta1-integrin was required for this response . SubsequNeonatal pancreatic pericytes, which are components of the islet vasculature, have also been shown to be necessary for neonatal beta cell proliferation. Using an in vivo diptheria toxin ablation strategy to delete pericytes in neonatal mice, Ephstein et al. showed the depletion of islet-associated pericytes significantly reduced beta cell proliferation . FurtherIslet associated macrophages have also been implicated in promoting neonatal beta cell proliferation. Mussar et al., used in vivo depletion and reconstitution experiments in mice to demonstrate that a CCR2-positive subpopulation of macrophages were transiently enriched in the neonatal pancreas and produced the growth factor IGF2 to promote proliferation . The depOnce formed, beta cells are extremely long-lived with limited evidence of expansion in basal conditions ,34. HoweThe case for neogenesis, especially in basal conditions, has been much more controversial, with almost an equal number of studies providing evidence to support the neogenesis hypothesis as there are refuting it (reviewed in ). One maAlthough there has been an increase in access to human cadaveric tissue in recent years, tools to investigate beta cell mass in humans remains extremely limited. Live imaging is very challenging due to the position of the pancreas in the abdomen, the small size of islets, and the lack of specific beta cell markers. Significant research efforts have explored the use of non-invasive imaging modalities, including positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI), and although these methods have been moderately successful for monitoring transplanted islet mass, they have been less efficacious for measuring endogenous beta cell mass . PanAlthough regeneration in the adult pancreas in basal conditions is limited, it has been shown that in conditions associated with metabolic stress, such as pregnancy and/or obesity, the endocrine compartment can compensate by expanding beta cell mass, including through the increase in beta cell size (hypertrophy), altering the balance between beta cell proliferation and apoptosis, and/or producing new beta cells through neogenesis or transdifferentiation . These mReversible beta cell mass expansion occurs during pregnancy to compensate for progressive insulin resistance; failure to compensate results in gestational diabetes. Interestingly, 70% of women who develop diabetes while pregnant will go on to become diabetic later in life, emphasizing the importance of adaptive changes in beta cell mass for preventing both gestational and T2D . In rodeMany studies have explored the mechanisms underlying beta cell expansion in the rodent pregnancy model, aiming to identify and harness signaling factors that induce beta cell mass expansion that could represent a powerful mechanism to promote beta cell expansion to treat beta cell loss in diabetes. Some of the earliest studies correlated beta cell proliferation with the onset of placental lactogen and/or prolactin secretion ,50. AddiObesity is associated with peripheral insulin resistance that requires increased insulin production to prevent the development of hyperglycemia. This increase is primarily achieved by a compensatory increase in beta cell mass and insulin output that allows the maintenance of glucose homeostasis; T2D ensues when this compensation fails ,58,59,60One challenge in the field has been to understand the intracellular pathways that facilitate or—more often—impede beta cell proliferation. In one approach, transcriptional analysis demonstrated the differential regulation of cell cycle genes required for cell proliferation between the C57BL/6 mice that can increase their beta cell mass and BTBR mice—a strain that is unable to increase beta cell mass sufficiently to meet the insulin demand and as a result develops diabetes . AlthougIn addition to animal models of long-term stress conditions, such as pregnancy or insulin resistance, artificial models of beta cell mass expansion involving experimentally induced injury have become powerful tools to study tissue remodeling in the pancreas. These studies have been instrumental in characterizing the response to acute stress and have revealed previously unappreciated cellular plasticity in both the rodent and human pancreas. + progenitor, although only a subset of cells re-activated Neurog3 and to a lesser extent than previously described [+ cells were able to contribute to neogenesis [Partial duct ligation (PDL) is a model of severe pancreatic injury that has been used in rodents for many decades to investigate the plasticity of the pancreas. The model is generated by ligating one of the main ducts responsible for draining enzymes secreted by acinar cells to cause the inflammation and atrophy of approximately 50% of the acinar cells proximal to the ligature. Several studies have shown that PDL also results in an expansion of beta cell mass ,74, thouescribed . Howeverogenesis . To dateogenesis ,80,81.Experiments involving partial pancreatectomy (Ppx) in rats showed that in conditions of chronic hyperglycemia, the remaining beta cells downregulate the expression of key beta cell associated genes such as insulin, GLUT2 and glucokinase and upregulate the expression of so-called “disallowed genes”  in response to this acute stress ,83,84. TOne of the more surprising observations in recent years—first in genetic mouse models and now with some validation in human tissues—is the remarkable plasticity of adult endocrine cells. Several mouse genetic models in which pancreatic transcriptional regulators have been deleted have revealed robust transdifferentiation between the alpha and beta cell lineages ,90,91,92Although beta cell replication is a rare event, evidence that it can be stimulated in certain conditions raises the hope that the augmentation of beta cell proliferation could be a potential regenerative therapy. Studies of natural and artificial models of beta cell mass expansion in rodents have begun to shed light on the molecular mechanisms involved, providing essential knowledge required to translate these findings to utilize proliferation as a therapeutic that promotes human beta cell proliferation.One obvious area of interest is cell cycle regulators and how they can promote or suppress beta cell replication. In rodents, Georgia and Bhushan demonstrated that Cyclin D2 was uniquely required for beta cell replication in the early postnatal period. It was expressed at high levels in beta cells up to two weeks postnatally but was downregulated in adult beta cells . FurtherSeveral studies have also explored the differential expression of cell cycle regulators in rodent versus human islets to understand why rodent beta cells have increased replicative potential. A comprehensive evaluation of cell cycle regulators in human islets revealed that although cell cycle regulators required for entrance into cell cycle are expressed in beta cells, they are sequestered in the cytoplasm . AdditioIn efforts to understand and perhaps harness the upstream regulators of the cell cycle machinery, several groups have explored the role of transcription factors. One of the most promising regulators of cell cycle initiation and progression in mouse and human beta cells is the forkhead box transcription factor FOXM1. Several studies have demonstrated the importance of FOXM1 for beta cell proliferation in rodents following injury or in pregnancy ,108. FurNKX6.1, an essential pancreatic homeobox transcription factor, is highly expressed in rodent beta cells during the secondary transition, a period of embryogenesis when beta cell mass expands significantly . StudiesA large number of studies in rodent models and immortalized cell lines have characterized the intracellular signaling pathways within the beta cell that could control proliferation in response to mitogenic signals. These studies have been extensively reviewed in a set of three comprehensive perspective articles ,117,118.In general, the differences in molecular signaling pathways regulating beta cell proliferation in rodents versus humans has made it difficult to translate findings from animal research into clinical therapies. For this reason, efforts are shifting towards high throughput methods of screening small molecules on human islets and or induced pluripotent stem cell (iPSC)-derived islets for potential efficacy. Impressively, three different groups identified several unrelated small molecules—adenosine kinase inhibitor 5-iodotubercidin (5-IT), Harmine and GNF4877—that each promoted beta cell replication through the inhibition of dual-specificity tyrosine-regulated kinase-1a (DYRK1A) ,120,121.Another high throughput screening method used a combination of NKX6.1, VGF and proliferative readouts to ensure that GSIS was not impaired upon the induction of replication to identify GNF-9228, a small molecule that stimulated human beta cell proliferation, GSIS and promoted the survival of rat insulinoma cells . Interes6 islet equivalents (IE) collected from two to three individual donors [Although the many endogenous approaches addressing decreased beta cell mass in diabetic patients are encouraging, and our knowledge of the mechanisms involved in stabilizing beta cell mass continues to increase, there remains no true cure for diabetes. Currently, the closest approximation to a cure is whole pancreas or pancreatic islet transplantation. Whole pancreas transplantation has been accomplished in several thousand diabetic subjects and has achieved long-term insulin independence and good metabolic control in many individuals ,129. Altl donors . They pol donors ,134. AltThe severe shortage of donor islets remains a major hurdle to overcome. The Edmonton protocol requires several donors, and even when donor pancreata are available, the yield of high-quality islets recovered is often very low. The ability to culture ex vivo islets long-term would enable banking and even pooling of low yield islets preps, however, isolated islets tend to rapidly deteriorate under conventional culture conditions. This is likely due to oxygen depletion and consequent necrosis in the core of the islets, and as such, varying the oxygen concentration so that it more closely resembles the in vivo environment could be a key to achieving long-term culture ,136,137.With the challenges associated with procuring sufficient donor islets for transplantation treatments, beta cells derived from human pluripotent stem cells have emerged as a viable and promising alternative. In the past ten years, the field has achieved remarkable advancements in their ability to generate functional beta-like cells for cell replacement therapy, although—as discussed below—several challenges remain.Mesenchymal stem cells (MSCs) are a somatic cell population that is present in many different perinatal and adult tissues  are derived from the inner cell mass (ICM) of the embryo at the blastocyst stage of development . They arTo truly harness the potential of human pluripotent stem cells (hPSCs) (either hESCs or iPSCs) as a therapy for diabetes, there is a need to develop reproducible and efficient protocols that consistently promote their directed differentiation into the different islet cell types, and primarily beta cells. Despite significant progress, even the most advanced differentiation protocols fail to produce a full complement of mature islet cells that are phenotypically and functionally similar to those found in healthy human islets. Additionally, these protocols suffer from inconsistent reproducibility between differentiations and, similar to cadaveric islets, there are also issues with cell survival upon transplantation.The most successful protocols that have been developed for differentiating beta cells from hPSCs faithfully recapitulate the developmental cues that a cell would normally receive in an in vivo context . As our MAFA or UCN3—two genes that are normally associated with adult functionally mature beta cells.More recently, however, additional studies from Velazco-Cruz and Nair developed protocols that enhanced the functionality of sBCs in vitro by altering culture conditions and/or recapitulating endocrine cell clustering. In both studies, these protocol modifications resulted in the production of more phenotypically mature sBCs in vitro with dynamic glucose stimulated insulin secretion (dGSIS) profiles that were comparable to human islets ,154. IntOne ongoing challenge in the generation of mature sBCs is the identification of specific markers that indicate the production of mature human beta cells. As mentioned above, one such marker, UCN3, is postulated to be a key marker of mature of human beta cells. In mice, UCN3 is highly and specifically expressed in adult beta cells where it is required for glucose stimulated insulin secretion ,156. DetThe advent of single cell RNA sequencing (scRNAseq) technology in recent years has also revealed the extensive heterogeneity of sBC cultures and has shed light on the differentiation processes that give rise to them. In 2019, Veres et al. published transcriptional profiling of over 100,000 human cells at various stages of in vitro beta cell differentiation . Their eAlthough, as described above, there has been tremendous progress with the in vitro generation of human sBCs, protecting these cells upon transplantation will be the next hurdle to overcome. Similar to transplanted cadaveric islets, sBCs undergo extensive cell death upon transplantation. A study by Feleo et al. demonstrated that culturing sBCs in physiological oxygen concentrations and supplementing the transplanted cells with amino acids prevented hypoxia-induced death and improved cell survival . Based oIn addition to developing protocols to improve sBC graft survival post-transplantation, it is also necessary to protect transplanted cells from immune rejection. With the advent of using individualized patient-derived iPSCs as the source for beta cells, this has become less of an issue; however, this remains a costly and time-consuming effort. Furthermore, for individuals with T1D, the transplanted sBCs also need to be protected from recurrent autoimmune destruction. Historically, immunosuppression has been the predominant therapy used to protect the graft, although it has its associated risks . More reFinally, an exciting area of emerging research is the use of gene editing technologies to engineer immune privileged beta cells. This approach has been used successfully to overexpress the immune checkpoint protein programmed death ligand 1 (PD-L1) in iPSC-derived human islet-like organoids to protect them from xenograft and allogenic rejection . AdditioIn summary, major inroads have been made towards the development of regenerative therapies for the treatment of diabetes . As with


Mammalian epidermis is a striking example of the role of lipids in tissue biology. In this stratified epithelium, highly specialized structures are formed that leverage the hydrophobic properties of lipids to form an impermeable barrier and protect the humid internal environment of the body from the dry outside. This is achieved through tightly regulated lipid synthesis that generates the molecular species unique to the tissue. Beyond their fundamental structural role, lipids are involved in the active protection of the body from external insults. Lipid species present on the surface of the body possess antimicrobial activity and directly contribute to shaping the commensal microbiota. Lipids belonging to a variety of classes are also involved in the signaling events that modulate the immune responses to environmental stress as well as differentiation of the epidermal keratinocytes themselves. Recently, high-resolution methods are beginning to provide evidence for the involvement of newly identified specific lipid molecules in the regulation of epidermal homeostasis. In this review we give an overview of the wide range of biological functions of mammalian epidermal lipids. The evolution of an impermeable barrier that could preserve the internal aqueous environment of the body away from water in ancestral reptiles was one of the most important events that allowed the colonization of dry land by vertebrates. That barrier, the “cornified” or “horny” epidermis, leveraged the physical properties of hydrophobic lipid and protein molecules to achieve the separation between the “wet” inside and the “dry” outside. Since then, over the course of evolution, the epidermis has acquired numerous additional structures and adaptations. However, the core physical mechanisms through which it exerts its most fundamental functions as well as its basic architecture have remained similar in all terrestrial vertebrates .stratum corneum   .In the basal and lower spinous layers, phospholipids are most common, with high levels of phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines and sphingomyelins . This phAs keratinocytes progress through differentiation and become more specialized, their lipid and protein makeup change. Starting from the upper spinous layer and in the granular layer the keratinocytes begin forming cross-linked bundles of keratin fibers and filaggrin in their cytoplasm. Their organelles begin degenerating and they assemble lamellar bodies (LB), also known as membrane-coating granules, lamellar granules, or Odland bodies . LB haveEarly characterization of the contents of LBs revealed that they are mostly made up of lipids and are specifically rich in glycolipids (mostly glucosylceramides), phospholipids and cholesterol as well as smaller amounts of sterol esters, ceramides, and fatty acids . An espeBesides lipid molecules, LBs contain several hydrolytic enzymes such as carboxypepdidase, cathepsin B, acid hydrolase, as well as acid lipase, β-glucosidases, phospholipase A, sphingomyelinase, ceramidases, and steroid sulfatase that play an important role during the subsequent differentiation steps .stratum corneum: they form an extremely resistant protein shell, the cornified cell envelope, just beneath their plasma membrane, made of crosslinked involucrin, loricrin, small proline-rich proteins and other proteins. The nucleus starts to degenerate and ultimately disappears, as the cytoplasm becomes filled with keratin bundles and the cell becomes flattened. The LBs approach the apical plasma membrane of the cell, their bounding membrane finally fusing with it, releasing the LB contents in the extracellular space , cholesterol (∼25%), and fatty acids (∼10%), the main constituents of the intercellular lipid “mortar” of the cornified layer , which agulation .The chemical changes in lipids are accompanied by morphological ones, as the disk-like bilayers seen in the LBs fuse to become a series of broad lamellar sheets . The corstratum corneum acts as an inhibitor of these proteases. The concentration of cholesterol sulfate is highest in the granular layer, and from there it gradually decreases along the thickness of the cornified layer due to the action of steroid sulfatase. The lower concentration of cholesterol sulfate near the surface thus allows the proteases to become active and enables the eventual desquamation of the corneocytes , hydroxylation profile , and presence of an ω-esterified fatty acid moiety  . Based oThe functional importance of such lipid complexity is underscored by several studies that attempt to replicate the ultrastructure of cornified layer lipid lamellae in chemically defined model membranes . It is fBesides its role as an impermeable physical barrier between the internal environment of the body and the outside world, the epidermis also actively participates in the protection of the organism against potential threats from opportunistic pathogenic micro-organisms . Given iThe surface of the epidermis is a generally inhospitable environment for microbes owing to its mild acidity (pH 4-5.5), scarcity of water and poor availability of essential metabolites like phosphate . Moreovestratum corneum and free fatty acids in the sebum , wax esters (∼25%), squalene (∼12%), and fatty acids (∼10%). In contrast, mouse sebum is mostly made up of wax esters (∼70%), with a much smaller fraction of triglycerides (∼6%) and no fatty acids or squalene . Among hactivity . Among tactivity . Undecylactivity . Oleic  and lack efficacy against Gram-negative bacteria . This has been associated with the fact that the lipopolysaccharide-coated outer membrane of Gram-negative bacteria represents an effective barrier against penetration of hydrophobic compounds  most effective in boosting activity  and biochemical – either protein or lipid – factors of both host and microbial origin shapes the composition of the skin microbiota . All theIn addition to their direct antimicrobial activity, fatty acids contribute to the acidification of the surface of the skin – the so-called “acid mantle”  – which A fascinating example of the crosstalk between the immune system and the epidermal “shield” is the ability of epidermis-resident innate lymphoid cells to limit the growth of sebocytes and influence the presence of antimicrobial fatty acid in the sebum. To be retained in the tissue, innate lymphoid cells require secretion of chemokines and cytokines by the epithelium. If the lymphoid cells are lost, the epidermis reacts by producing more sebum and increasing sebaceous antimicrobial lipid content, in turn regulating the composition of the commensal microbiota . In anotSome antimicrobial lipids have anti-inflammatory activity. Sapienic acid inhibits gene expression resulting from lipopolysaccharide stimulation in murine macrophages . Though Lipids from numerous different classes and cellular origins can work as paracrine or autocrine mediators through cell-surface receptors to help determine the type of adaptive immune response that is triggered after an external stimulus perturbs the physiological equilibrium of the skin or to intervene in the regulation of epidermal cell behavior .Eicosanoids are a heterogeneous group of lipids derived from the metabolism of C20 poly-unsaturated fatty acids (PUFAs) such as arachidonic acid (C20:4ω6), dihomo-γ-linolenic acid (C20:3ω6), and eicosapentaenoic acid (C20:5ω3). These molecules, together with some related PUFAs, can be found in the epidermis and they can become substrates of a set of different enzymes. Cyclooxygenases can generate prostanoids . Lipoxygenases can give rise among others to leukotrienes, a variety of hydroxy-fatty acids , and resolvins. In addition, cytochrome P450 enzymes lead to the production of such molecules as dihydro-eicosatetraenoic acids (DHETs) and epoxyeicosatetraenoic acid (EET). All these lipid species are known autocrine or paracrine biological mediators, with some of them being active in the epidermis .2 is one of the main mediators of inflammation in skin. PGE2 exerts its action by binding to four different G-protein-coupled receptors (GDPR), termed EP1–4 . In a murine model, ablation of EP4 receptor impaired Langerhans cell migration toward the lymph nodes following antigen stimulation and the ability of Langerhans cells to stimulate T lymphocytes, suggesting a positive modulation of these processes by PGE2 , ultimately leading to cellular mobilization (2 is able to induce expression of β-defensin-3 (hBD-3) in keratinocytes by engaging a src/MEK/ERK/c-Fos signaling axis, through DP2, and can thus bolster the antimicrobial defense of the epidermis. Moreover, since hBD-3 can trigger mast cell activation and further release of PGD2, this leads to the establishment of a positive feedback loop of communication between mast cells and keratinocytes  stimulation. In this case, the release of Langerhans cells is impaired by PGD2 binding to prostaglandin receptor DP1 and is dependent on the subsequent adenylate cyclase activation and rise in cAMP levels . Increased Gsα signaling is proposed to lead to cytoskeletal rearrangements and inhibition of cellular movement  – one of the key enzymes for the generation of leukotrienes – the major source of leukotriene production in the epidermis is thought to be the infiltrating immune cells . Leukotrmovement .4) from immune cells . The most prominent members of this class of compounds are arachidonic acid metabolites N-arachidonoyl-ethanolamine (anandamide) and 2-arachdonoylglycerol (2-AG). Anandamide can work as a partial agonist for CB1 and CB2 receptors, while 2-AG functions as a full ligand for both receptors  which can also be activated by endocannabinoids  as well as intracellularly . Due to its relatively low hydrophobicity and presence of a polar headgroup, S1P can exit the membrane space and move into solution but is not able to readily flip-flop across membrane leaflets. S1P is mostly found in serum and interstitial fluid, is strongly released during platelet degranulation, and exerts its actions in a paracrine or autocrine manner through GPCRs , arising from the action of sphingomyelin deacylase on sphingomyelin, has biological activity in epidermal cells. SPC is sufficiently polar to be able to move between different membranes and enter solution; it is thought to exert its effects through low-affinity binding to S1P receptors as well as intracellularly . SPC hasoduction . Consistoduction .For nearly a century of research, lipids have played a center-stage role in studies of the structural integrity and protection of the skin barrier. However, recent evidence is beginning to reveal an even more dynamic role for lipid mediators in signaling events that are key to the maintenance of epidermal homeostasis. Lipid molecules can act in an autocrine, paracrine or intracellular fashion to modulate several aspects of epidermal cell behavior, particularly keratinocyte differentiation, and the extent of their action has likely been underappreciated so far. In this respect, it is important to understand the enormous diversity of lipid species present in the epidermis and how this impacts the biology of the skin.stratum corneum corneocyte is influenced by several cellular signaling pathways that control the balance between proliferation and differentiation, exit from the stem cell compartment and progression through the epidermal layers. Some of the prominent signaling molecules involved in these processes include β-catenin, extracellular-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPK), phosphoinositide-3-kinase (PI3K)/RACα serine/threonine-protein kinase (Akt) and PKC. More specifically, accumulation of β-catenin in the nucleus promotes expansion of the stem cell compartment  transcription factor action  can enhance melanin production by increasing tyrosinase expression following p38/ERK1/ERK2 and CREB activation downstream of CBanocytes .As mentioned above, the PKC pathway can be directly regulated by lipids. Depending on the isoform, PKC activity is modulated by both intracellular calcium levels and presence of diacylglycerol/lipids (-α -β -γ), by diacylglycerol/lipids alone , or by allosteric interaction with other proteins (-ζ -ι/λ) . The twoThe specific acyl residues contained in diacylglycerol, as well as its hydrophilicity/hydrophobicity profile, can influence the degree to which PKC becomes regulated . In suppSphingolipids are one of the most functionally multifaceted lipid classes in the epidermis. They play a fundamental role in the formation and the structure of the epidermal barrier and also participate in the protection of the epidermis. Additionally, several sphingolipid species take part in epidermal signaling . These ide novo by the condensation of palmitate and serine catalyzed by serine palmitoyl transferase and the subsequent activity of ceramide synthase. Alternatively, they can be derived from either sphingomyelin through the action of sphingomyelinases or glucosylceramides through the action of glucocerebrosidase. They can be mainly found in the ER membranes (where their de novo synthesis occurs) or at the plasma membrane. Due to their physico-chemical properties they are unable to transfer freely between different cellular compartments, needing either vesicular transport or protein mediators to move across membranes. They can, however, flip-flop between membrane leaflets, though this may too be subject to regulation  ceramides can inhibit their proliferation and induce differentiation  ceramides and endogenous ceramides can directly regulate the expression of certain differentiation-related genes in keratinocytes. One example is glucosylceramide transporter ABCA12, involved in the translocation of glucosylceramides into lamellar bodies, whose transcription is enhanced by ceramide through increased PPARβ/δ expression . Another PPARβ/δ . C2 ceraoduction . Ceramidechanism .in vivo not only, as predicted, leads to disruption of epidermal homeostasis but has also a surprising impact on keratinocyte differentiation. Eliminating alkaline ceramidase, an enzyme responsible for ceramide degradation, in mouse skin leads to an abnormal accumulation of ceramides, which in turn compromise the barrier function of the epidermis and cause alterations in the final stages of keratinocyte differentiation and an increase in the epidermal stem cell population of the hair follicle , an enzyme responsible for ceramide synthesis, in mice produces an expansion of the hair follicle stem cell compartment . The difIn contrast to ceramides, glucosylceramide has mitogenic effects on epidermal keratinocytes when administered subcutaneously in mice . Convers2, causing the release of arachidonic acid from phospholipids and an increase of 15-deoxy-PGJ2; this leads to activation of PPARα or -β/δ, which promotes expression of Src and stimulates STAT1/3-mediated expression of the β-defensin genes (Ceramides-1-phosphate (C1Ps) are sphingolipid mediators that generally exert opposing effects to those of ceramides , and phosphorylation by ceramide kinase is usually considered a way in which ceramides can be inactivated. C1Ps can be found in the Golgi apparatus and the plasma membrane. They are limited in their mobility between membranes, and owing to their polar head group are unlikely to flip-flop between membrane leaflets . In kerain genes .2 antagonizes the proliferative signals coming from the insulin receptors by activating PKCδ, causing dephosphorylation of Akt and leading to inhibition of cyclin D2 and upregulation of cyclin-dependent kinase inhibitors p21 and p27Kip1  also inhibits proliferation and promotes differentiation of keratinocytes . Binding p27Kip1 . Moreoventiation .In addition to proliferation inhibition and in sharp contrast to its role in Langerhans cells, S1P stimulates keratinocyte migration. This effect is enacted by S1P receptors through transforming growth factor-β (TGF-β)-receptor I-mediated phosphorylation and activation of Smad3 . In factvia assembly of an intracellular signaling complex, causing the stimulation of C/EBPα-dependent transcription, and ultimately leading to increased expression of the cathelicidin LL-37 and enhanced epidermal protection  appears to promote an abnormal route of differentiation, with increased intracellular calcium levels and activation of transglutaminase 1, at the same time as inhibiting the expression of cornified cell envelope proteins such as profilaggrin and loricrin . SPC alsin vivo has been controversial, they have been observed in artificial bilayers mimicking the lipid composition of the plasma membrane (Sphingomyelins (SMs) are synthesized in the Golgi apparatus and found at the plasma membrane. Their double aliphatic chain makes them insoluble in water and their polar phosphocholine group impairs flip-flopping between membrane leaflets . Unlike membrane . Early smembrane ; more remembrane .Lysophosphatidic acids (LPAs) are the simplest phospholipid, formed by a glycerol backbone attached to a phosphate group, a fatty acid, and a hydroxyl moiety. LPA can be synthesized intracellularly by glycerophosphate acyltransferase, acylglycerol kinase or phospholipase A. Alternatively, it can be generated extracellularly by the secreted lysophospholipase D autotaxin. Degradation of LPA is mainly carried out by lipid phosphate phosphatases. The single aliphatic chain of LPA makes it sufficiently soluble to leave membranes .1–6 – through which it exerts its biological activity. Some of these receptors belong to the same family as S1P receptors, and S1P and LPA share some structural similarity   . The SRFor (SRF) . The incor (SRF) .Other lipid-sensitive proteins that have been shown to be involved in the regulation of keratinocyte differentiation are peroxisome proliferator-activated receptors (PPARs) and liver X receptors (LXRs). When activated by a lipid ligand, PPARs or LXR heterodimerize with retinoid X receptor (RXR) and regulate gene expression. Three PPAR isoforms  exist, all of which are expressed in keratinocytes: while PPARβ/δ expression is constitutive, PPARα and PPARγ levels increase with differentiation . Both LXin vitro in human keratinocytes and in vivo in mouse epidermis , which in turn is able to elicit transcription of the profilaggrin gene . This meWhile we have presented evidence that separate lipid classes play a role in signaling to impact keratinocyte proliferation, differentiation and other processes, this is inevitably an oversimplification. The lipid species produced in the cell and the enzymes that modify them form an extremely intricate network and it is difficult to predict the multiple perturbations that ensue from a specific intervention, such as depletion of a single lipid modifying enzyme . Lipid sin vitro keratinocyte differentiation system combined with a lipid-modifying enzyme siRNA screen to evaluate the changes in cellular lipid composition occurring during differentiation and to try to understand which changes might be important for the early phases of this process.Despite these challenges, newly developed techniques or refinement of established methods are beginning to shed more light on the potential contribution of endogenous cellular lipid subspecies to a variety of cellular processes. Prompted by the finding that the alteration of the ceramide profile in mouse epidermis could lead to an effect on the stem cell compartment , our labin vitro , while maintaining its fundamental microstructure and functional characteristics  whose influence on cellular behavior is still poorly understood and has only recently begun to be investigated. A combination of lipid enzyme targeting and lipidomics has identified individual lipid subspecies from multiple classes that can influence keratinocyte differentiation in vitro, underscoring the still little-explored regulatory potential of the cellular lipid repertoire (In the case of the modulation of keratinocyte differentiation, it often remains unclear to what degree the activity of endogenously generated individual lipid mediators is important and at what stage of the pertoire . Such copertoire .in vivo. Technological advances will be key to answering these complex questions. In this respect, implementation of recently developed, sophisticated techniques, like single-cell lipidomics (Challenges still remain to explore some of the more elusive aspects of lipid biology, for example how the cellular distribution and/or traffic of bioactive lipids among the various membranous organelles may play into their functionality, or how much individual lipid species or lipid features matter for epidermal homeostasis pidomics , alreadypidomics . We are MV and FW wrote and edited the text. MV designed the figures. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Over the past years, brain development has been investigated in rodent models, which were particularly relevant to establish the role of specific genes in this process. However, the cytoarchitectonic features, which determine neuronal network formation complexity, are unique to humans. This implies that the developmental program of the human brain and neurological disorders can only partly be reproduced in rodents. Advancement in the study of the human brain surged with cultures of human brain tissue in the lab, generated from induced pluripotent cells reprogrammed from human somatic tissue. These cultures, termed brain organoids, offer an invaluable model for the study of the human brain. Brain organoids reproduce the cytoarchitecture of the cortex and can develop multiple brain regions and cell types. Integration of functional activity of neural cells within brain organoids with genetic, cellular, and morphological data in a comprehensive model for human development and disease is key to advance in the field. Because the functional activity of neural cells within brain organoids relies on cell repertoire and time in culture, here, we review data supporting the gradual formation of complex neural networks in light of cell maturity within brain organoids. In this context, we discuss how the technology behind brain organoids brought advances in understanding neurodevelopmental, pathogen-induced, and neurodegenerative diseases. This is in contrast to the observed in the mouse brain, which presents mixed symmetrical/asymmetrical divisions. This difference may explain the increased size of the human cortex in comparison to the mouse cortex. Also, genetic mutations that cause microcephaly in humans do not produce similar effects in the mouse brain, demonstrating the fine regulation in the growth of the cortex during evolution  to model neurological disorders. The idea is to paint a picture of organoid functionality based on maturity and how that can be applied in the context of disease modeling.Distinct cell types represented in a human brain organoid could be addressed by proteomic and transcriptomic analyses  (2.5 months) when frequent calcium spikes were observed after the addition of exogenous glutamate  were highly enriched in 1-month brain organoids resembling cortical domain . They have a typical morphology of tangential migration neurons that is suggestive of interneurons. Also, GABA positive (vGAT) cells were observed in regions resembling medial ganglionic eminence (MGE)  generate interneurons that migrate tangentially into the dorsal forebrain  at 3.5 months, while mature cells positive for parvalbumin were described only after 6 months of culturing  and GABA-synthesizing enzymes (GAD1 and GAD2) in these organoids at 2.5 months. Immunostaining for calbindin, GAD67, and other markers indicated the presence of medium spiny neurons in the striatal spheroid  has been extensively used as neuron-like cells. Under differentiation, these cells usually do not express the axonal component, but dendritic and either dopaminergic or cholinergic markers are abundantly expressed  and the vesicular acetylcholine transporter VACHT after 1 month in culture , about 28% of oligodendrocytes (MBP+) interacted with NF-H+ processes, and different stages of myelination were observed under electron microscopy  after extracellular electrical stimulation, spontaneous and stimulated spiking, and burst events . While organoids at 2.5–3.5 months showed spontaneous and asynchronous calcium transients, suggestive of an immature network, cultured neurons showed a synchronized pattern for calcium transients together with bursts in response to glutamate, which was blocked by GABA and glutamate antagonists , which are mediated by depolarizing GABAergic responses typically found in immature neurons Ben-Ari, . In rodeEven though gaining access to a more complex functional integration of microcircuits is challenging, advances in the assembloid methodology have provided recordings of functional neural circuits in a more sophisticated way. Owing to the need to assess neural projections extension, interneurons migration and integration, the dorsal-ventral forebrain axis was recreated and tracked with fluorescent reporters. Meanwhile, well-documented cell migration has been observed in assembloids  were detected around 1–2 months of culture, and synchronized calcium activities enhanced after adding GABAIn a recent study, Andersen and collaborators showed cortico-spinal connections when combining cortico-spinal organoids with iPSC-derived skeletal muscle organoids. After optogenetic stimulation, cortical organoids were able to contract skeletal muscle organoids . OPCs in these oligodendrocyte spheroids expressed voltage-dependent sodium and potassium channels from about 3.6–5.8 months in culture that were not observed in multipolar cells, an expected progression as OPCs mature. Potassium currents seemed to be inactivated slowly, with complementary differences in input capacitance and resistance. Cells of the oligodendrocytic lineage were sensitive to glutamate release, an indication that they express ionotropic glutamate receptors that are activated in human oligodendrocyte spheroids ; formation of protein aggregates characteristic of neurodegenerative pathologies, and differential susceptibility to pathogens have hindered to address important aspects of developmental and neurodegenerative disorders; and pathogen invasion into the brain [reviewed in Gerakis and Hetz ; MarshalPrimary microcephaly is characterized by a reduction of brain size caused by insufficient production of neural progenitors and neurons during fetal development + cells in patient organoids  is the most common cause of human primary microcephaly. iPSCs derived from a patient with a mutation in the Aspm gene formed smaller embryonic bodies compared to control. Reduction in neuroepithelial regions was observed in cortical organoids together with reduced staining for Pax6, Sox2, and ZO-1 as well as fewer proliferative Ki67+ and pH3+ cells. On day 65 of development (~2 months), Aspm mutant organoids showed disorganized lamination as seen by Tbr2 immunostaining. Calcium signaling could be observed at day 85 (2.8 months) of development and showed no differences in transient frequencies, but mutant organoids were less synchronized than controls, suggesting that those cells were not able to develop mature circuits  is a rare neurodevelopmental disorder characterized by speech and motor disabilities, along with cognitive impairment, which start to appear after a period of normal development. Mutations in the methyl-CpG-binding protein 2 (MeCP2) gene are the cause of Rett syndrome, and the spectrum of severity can vary depending on the site of mutation. Since the MeCP2 gene is located in the X chromosome, female cells present a mosaic pattern of expression  of development. Increased proliferation in detriment of neuronal generation was suggested as the cause of delayed development observed in 2D neuronal cultures  and 2.2 months (64–67 days). Alterations in neuronal activity were measured using patch-clamp at 1.5 and 2.6 months (81 days), revealing a reduction in the firing frequency and abortive-like action potential in mature MeCP2 mutant neurons. The fusion of region-specific organoids from MGE and cortex revealed defective interneuron migration in MeCP2 mutant assembloids at day 41 (1.3 months) (fusion of assembloids occurred at day 13)  is a frequent neurodevelopmental disorder characterized by impairments in social interaction, communication, and behavioral flexibility  is a chromatin remodeling factor, which was identified as a top candidate gene for ASD from independent exome-sequencing studies and is also involved in schizophrenia and intellectual disabilities , and MPPED1  and in the new coronavirus SARS-CoV-2, and how brain organoids helped to elucidate aspects of viral infection in the brain tissue.www.who.int). ZIKV experimental infection in brain organoids induced cell death and reduced proliferation. Also, a decrease in neuronal cell-layer volume and in organoid size resembling microcephaly phenotypes was observed following infection , and inhibition of TLR3 reduced the phenotypic effects of ZIKV infection , Motor Neuron Disease (MND), and Frontotemporal Dementia  and Alzheimer's Disease (AD) are the most common progressive, age-related neurodegenerative conditions. Distinct brain areas are affected by PD and AD. PD affects mainly the striatum, resulting in motor impairment. The entorhinal cortex is the first to be affected in AD, resulting in cognitive decline. Importantly, protein aggregation is a pathological hallmark of these diseases, which could be replicated in AD is characterized by progressive neurodegeneration and memory loss and is the most prevalent cause of dementia accounting for 60–80% of cases. The presence of senile plaques, constituted by amyloid-β peptides (Aβ) and neurofibrillary tangles (NFTs), formed by phosphorylated tau, are two major neuropathological hallmarks in AD which often precede the onset of symptomatic dementia by decades  accounts for <5% of AD cases and appears at an early onset (~40 years old) when compared to sporadic AD (SAD), which accounts for the majority of cases and develops later in life (>60 years old) , leading to non-convulsive epileptic discharges  in the midbrain. A pathological hallmark of PD is α-synuclein deposition in Lewy bodies. Accumulation of α-synuclein generates synaptic dysfunction which leads to neurodegeneration  the specific organoid protocol to use in order to have a model that is enriched in the cell type(s) that is (are) most relevant to what they wish to study; (2) time in culture—the age of the organoid—to allow for the target cell types to differentiate; (3) the proper time in culture to allow for cell maturity and full functionality. This way, if all 3 items mentioned are thoroughly considered, brain organoids can be successfully applied to brain disease modeling. In the context of neurological disorders, iPSC-derived from healthy subjects, patients carrying mutations, or genomic editing offer a variety of phenotypes to be studied. With advances in obtaining brain organoids, the prototype of a room for growing and maturing brain organoids could be conceived, similarly as cell culture or animal facilities in research institutes. In the near future, we hope that the establishment of robust, reproducible methodological settings as well as functional assessments at accessible costs will propel science in the brain organoid field.https://hca-organoid.eu) aims to compare organoids' cell type composition with that of the developing human brain using single-cell sequencing (Bock et al., The evolving field of brain organoids allowed the investigation of human diseases, leading to the molecular understanding of human brain tissue that was otherwise inaccessible. While reproducibility is a challenge, current initiatives to set standards through the comparison across multiple labs and over many replicates are ongoing. The Organoid Cell Atlas project (The application of novel analytical technologies is key to the improvement of brain organoids as a model. Following single-cell sequencing, single-cell proteomics is an emerging technology that can be applied to disclose biological processes taking place during development, as it can reveal effective protein amounts in cells and post translational modifications which are relevant for protein function (Goto-Silva and Junqueira, Lack of throughput is still a limitation in current organoid protocols. Implementation of high throughput, microfluidic and compartmentalized platforms may allow handling cultures from multiple individuals (Renner et al., in vitro findings and patient phenotypes is among the major challenges in having brain organoids to model disease. The connections among distinct brain regions reproduced in brain assembloids can help us understand alterations in the connectivity of neural networks in different conditions. A robust model to address connectivity and neuron activity can bring in vitro research closer to the neuroimaging field so that we can start translating connectivity measurements from human brains to human brain organoids.Filling the gaps between LOP and PFL drafted the outline. LOP, PFL, and LG-S wrote the manuscript. LOP prepared the figures. The manuscript was then reviewed and edited by SKR. All authors have approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.