Enteroviruses are single-stranded positive-sense RNA viruses that primarily cause self-limiting gastrointestinal or respiratory illness. In some cases, these viruses can invade the central nervous system, causing life-threatening neurological diseases including encephalitis, meningitis and acute flaccid paralysis (AFP). As we near the global eradication of poliovirus, formerly the major cause of AFP, the number of AFP cases have not diminished implying a non-poliovirus etiology. As the number of enteroviruses linked with neurological disease is expanding, of which many had previously little clinical significance, these viruses are becoming increasingly important to public health. Our current understanding of these non-polio enteroviruses is limited, especially with regards to their neurovirulence. Elucidating the molecular pathogenesis of these viruses is paramount for the development of effective therapeutic strategies. This review summarizes the clinical diseases associated with neurotropic enteroviruses and discusses recent advances in the understanding of viral invasion of the central nervous system, cell tropism and molecular pathogenesis as it correlates with host responses. Picornaviridae  are single-stranded positive-sense RNA viruses of the family aviridae . There aoviruses . Three rningitis  and up tningitis  are specningitis . As we nningitis . NeverthOur current understanding of these non-polio enteroviruses is limited, especially with regards to their neurovirulence. Without an effective treatment strategy to combat or prevent non-polio EV infections of the central nervous system, better understanding of the neuropathogenic process of neurotropic EVs is highly warranted. Elucidating the molecular pathogenesis of these viruses is paramount for the development of effective therapeutic strategies. This review summarizes clinical diseases associated with some of the most common neurotropic enteroviruses and discusses recent understanding of viral invasion into the central nervous system, cell tropism and molecular pathogenesis as it correlates with host responses during neurotropic enterovirus infections.Numerous EVs are linked to debilitating and potentially deadly neurological diseases including aseptic meningitis, encephalitis and AFM. In certain instances, EV infections are associated with the development of neurological sequelae years after the onset of acute disease, as is suspected for post-polio syndrome  and GuilEnterovirus infections are the most common cause of both meningitis and encephalitis in children 17 years of age and younger . MeningiNumerous EVs have been detected in cases of encephalitis, aseptic meningitis or meningoencephalitis – inflammation of both meninges and the brain. The wide diversity of enteroviruses associated with encephalitis or meningitis highlights their collective neuropathogenic potential and speaks to their unique tropism. Specifically, poliovirus, CV , echoviruses  and EV-A71 are commonly implicated in causing both encephalitis and aseptic meningitis . HoweverAcute flaccid paralysis (AFP) is a WHO reportable disease in children under the age of 15. AFP is clinically defined as acute onset of flaccid paralysis of one or more limbs due to an infectious cause . The disease may damage different parts of the body including spinal cord, peripheral nerves, neuromuscular junctions and muscles. Poliovirus was the primary etiology of infectious AFP cases causing poliomyelitis. With current near-universal vaccination strategies designed to eradicate poliovirus, the total number of AFP cases attributed to poliovirus around the world have decreased drastically .A subset of AFP called acute flaccid myelitis (AFM) represents a disease where paralysis and limb weakness typically occurs within a week of respiratory symptoms or fever caused by a non-polio viral infection. In addition to the acute limb weakness, a lesion in the spinal cord gray matter that spans at least one vertebra and often elevated leukocyte counts within the CSF is a classic diagnostic feature . SymptomRecent epidemiological criteria using the Bradford-Hill analyses showed strong support that AFM is primarily caused by EV-D68 . The parSome poliovirus infected patients develop a condition called post-polio syndrome (PPS) that is characterized by muscle weakness and atrophy several decades following acute infection but, in contrast to poliomyelitis, these sequelae tend to be transient and progressive . It is cThe precise mechanism of PPS is unknown and treatment is limited to supportive measures . Muscle Guillain-Barré syndrome (GBS) is an inflammatory immune disorder characterized by rapid-onset muscle weakness due to damage to the peripheral nervous system (PNS). The initial symptoms of GBS are often changes in sensation or pain in the extremities that develop over hours to weeks. In some cases, weakness of breathing muscles during the acute phase of the syndrome requires mechanical ventilation but, if the patient survives this phase, complete recovery is likely . DespiteViral pathogens employ a variety of strategies to gain entry into the CNS  and unco+ peripheral blood mononuclear cells (PBMCs) were found to be highly susceptible to CVB3 infection (in vitro cell lines including Raji (B cell), Jurkat (T cell) and U-937 (monocyte)   implicatonocyte) , EV71 waonocyte) , dendritonocyte)  and PBMConocyte)  while econocyte) . Althougin vitro  functions to restrict entry of large molecules as well as cells and pathogens into the brain from the circulatory system. Similarly, the choroid plexus found in each of the four ventricles of the brain controls the production of CSF and serves as the blood-cerebrospinal fluid barrier (BCSFB), preventing passage of most pathogens into the brain while permitting the delivery of nutrients and removal of metabolic wastes. The BCSFB is a site of CNS immune surveillance, providing a more permissive barrier for immune cells to traverse, while the BBB is more resistant to the movement of both host cells and invasive pathogens . Some pain vitro . Howeverin vitro  indicatidothelia . OverallOne important factor in CNS invasion is the generation of genetic variants, otherwise known as quasispecies, which are produced during viral replication . Enterovin vitro studies showing susceptibility of neural progenitor cells to CVB3  on the cell surface. Ever since CD155 was first identified in 1989 as the poliovirus receptor , the extThe intracellular adhesion molecule 5 (ICAM-5) was identified to facilitate infection by contemporary EV-D68 in otherwise non-permissive Vero cells following a sialic-acid independent mechanism . AlthougAn important factor to consider in neuropathogenicity is that EVs can gain the use of additional receptors through adaptation during replication. Receptor adaptation has been well documented for numerous enteroviruses  that originate at the endoplasmic reticulum and then the E6 cells . HoweverE6 cells  and furtE6 cells . This isE6 cells . In factE6 cells . HoweverE6 cells . AnotherE6 cells . The autE6 cells . Animalsscularly , supportAnother major phase of viral replication is viral protein translation. Several host factors are important in mediating translation of the viral polyprotein by associating with the internal ribosome entry site (IRES) located within the 5′ untranslated region (5′ UTR) of the viral genome. Mutations within the 5′ UTR were shown to modulate neurovirulence of poliovirus , EV-A71 A recent publication identified a neuronal-specific host factor that was implicated in modulating EV-A71 neuropathogenesis in animals. The host factor prohibitin (PHB) was upregulated during EV-A71 infection of the motor neuron cell culture NSC-34 and found to contribute to not only binding of virions for cell entry but mediating viral scaffold formation on mitochondria . InhibitAs Enteroviruses hijack the host cell they are able to manipulate the cell cycle progression to generate a favorable cellular environment for viral replication. EV-A71 and CVA16 infected RD cells stopped cell cycle in the S phase, mediated by the non-structural 3D viral protein . In contin vitro cells and evaluation of these mechanisms within the CNS tissue remains to be thoroughly explored. However, local immune cells of the CNS, such as astrocytes and microglia, were found to be important in mediating protective immune responses as a result of viral infection  which detect conserved parts of pathogens, termed Pathogen-Associated Molecular Patterns (PAMPs), or molecules that are released by damaged cells called Damage-Associated Molecular Patterns (DAMPs)  and TLR7 (detects ssRNA) sensors . A signapro , which ipro . Furtherpro  and subvpro while RIG-I cleavage occurs via 3Cpro (pro of CVB3 cleaved MAVS and TRIF further suppressing type I IFN response (pol) and viral 3Cpro interacts with MDA5 and antagonizes the antiviral immune response  production 3 days post treatment suggesting that IFN-γ does have protective, albeit slight, antiviral effects in neurons  and Type III IFN (IFN lambda) responses are also important to combat EV infections. An initial  neurons . Similar neurons . Similar neurons . To coun neurons . Type II neurons , hepatoc neurons  and gobl neurons . However III IFN . It remaThe adaptive immune response is involved in clearing EVs by generating neutralizing antibodies and cell mediated responses while building a memory to protect the host from secondary exposure. However, little is known about the cellular adaptive immune response to most EVs, particularly regarding infection of the CNS tissue. We will briefly describe the main contributors of immunity which includes neutralizing antibodies, T cells and microglia and how these responses relate to EV infection of the CNS.Neutralizing antibodies produced by B cells during EV infection help control viremia and therefore prevent viral dissemination to other organs and protect the host from subsequent infections. This process was well documented in patients who suffer from agammaglobulinemia and cannot produce antibodies. These individuals were found to have heightened susceptibility to EV infection and CNS invasion that caused chronic neuropathies  proteins to inhibit autophagosome-lysosome fusion, diverting vesicles from degradative autophagy to secretory autophagy, leading to exocytosis and vesicle fusion with the plasma membrane (Attempts to study autophagy flux by monitoring the cargo receptor SQSTM1/p62 have been undermined through the discovery that it is cleaved by viral protease 2A during poliovirus, EV-D68, CVB3 and human rhinovirus 1A . SQSTM1/membrane .Autophagy is a transcriptionally regulated process  and is sA growing number of Enteroviruses are associated with life-threatening neurological diseases including encephalitis, meningitis and AFM. The lack of an effective treatment for non-polio enteroviruses underscores the vital need for better understanding of their neuropathogenesis.Despite extensive studies that have uncovered many important aspects of EV infection of the CNS and resulting pathogenesis, much still remains to be learned with respect to mechanisms of EV neuroinvasion, neurotropism and molecular pathogenicity upon viral entry into the CNS. For example, neurotropic EVs can recognize a diverse range of receptors and attachment factors for cell entry, however, the location of these receptors within CNS tissue are yet to be correlated with observed pathology and clinical symptom manifestation. Although molecular mechanisms of viral-host interaction in terms of innate immunity are well documented, the extent of this interaction in CNS relevant cell-types are mainly understudied. Recent studies have implicated the immune response to be an essential component of EV neuropathogenicity, further emphasizing the need to characterize these immune-specific mechanisms. In terms of viral neurotropism, all tested neurotropic EVs were capable of infecting neurons and/or glia cells but viral propagation was found to be largely dependent on viral strain, cell type and genetics of the host. Further studies are therefore needed to delineate these parameters and identify intrinsic host factors that are key for viral entry and replication within CNS-relevant cell-types. Considering that viral life-cycle is dependent on a multitude of host factors, identifying and characterizing these essential elements will provide inroads into designing novel treatments to abrogate EV infection of the CNS.AnM and AlM conceptualized the review, performed the literature search, and prepared the manuscript. TB provided the critical input and revisions for 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.


Papillary thyroid cancer (PTC) is considered an indolent cancer, but some PTC patients do present with distant metastases and treatment strategies for such patients are not well established. Recently, lenvatinib, an inhibitor of multiple tyrosine kinases, has been introduced to treat patients with advanced PTC but carries a risk of serious adverse events such as hemorrhage. Here, we report a PTC patient with a left adrenal metastasis and lenvatinib-induced hemorrhage who underwent successful surgical resection and was subsequently treated with a lower dose of lenvatinib. The patient has now been in a stable state with no adverse events for nearly two years. This case highlights the importance of surgical resection of metastatic PTC and subsequent lenvatinib therapy, even when the tumor is at an advanced stage. Papillary thyroid cancer (PTC) is the most common type of thyroid cancer. PTC is a differentiated thyroid cancer (DTC) considered to be indolent because distant metastases are seen in only a small minority of patients. Standard therapies for metastatic DTC include treatment with radioactive iodine (RAI) , but patHere, we present a patient with RR-PTC successfully treated by resection of a left adrenal metastasis despite lenvatinib-induced intratumoral hemorrhage. We were able to continue lenvatinib treatment in this patient at a lower dose with no severe adverse effects.A 70-year-old woman was first diagnosed with PTC at the age of 53. She subsequently underwent a total thyroidectomy together with regional lymph node dissection, followed by iodine-131 (I-131) therapy immediately following the initial diagnosis. The TNM classification at the first pathological diagnosis was T4aN1aM0. Recurrence did not occur until 10 years after the patient's initial treatment when multiple lung and supraclavicular lymph node metastases were identified during late follow-up. The patient received stereotactic radiotherapy (58 Gy/29 Fr) for the lymph node metastasis and a second course of I-131 therapy. However, metastases in both lung and lymph node gradually progressed despite such therapy. The patient underwent a third round of I-131 therapy three years after the second. Since then, these tumors have remained unchanged for almost 5 years, indicating stable disease (SD).However, the patient presented with a left adrenal tumor 17 years after her initial treatment for PTC. This grew rapidly over only six months, whereas the lung and supraclavicular lymph node metastases remained stable. We initially considered that the adrenal tumor might not have been a PTC metastasis , but theAccording to a previous report, distant metastases from PTC are rare, with only 71 of 5,969 resected PTC cases presenting with disease spread .Several multitarget kinase inhibitor drugs have been introduced recently for the treatment of RR-DTC. Sorafenib was first approved for this indication in 2013 after a phase 3 trial showed significant improvement in progression-free survival (PFS) compared to placebo . LenvatiIn the PTC case described here, surgical resection of a left adrenal metastasis after lenvatinib-induced hemorrhage was performed. Reports of PTC cases with surgical resection of distant metastases are very limited, but long-term survival after the resection of a solitary pancreatic metastasis of thyroid cancer has been reported . Our patA dose of 8 mg is lower than the recommended initial dose of lenvatinib for RR-DTC. Another successful instance where adverse events of lenvatinib were managed by individualizing the drug schedule in terms of reduced dose and timing, with a significant dose intensity reduction, has also been reported . Our casOur findings suggest that surgical treatment is an option for rapidly growing metastatic tumors of PTC origin, even at an advanced stage with multiple lung metastases. If a hemorrhage induced by lenvatinib can be managed satisfactorily, lenvatinib administration at a lower dose may be continued for the treatment of metastatic RR-PTC.


Transplantation of neural stem cells (NSCs) has been proposed as an alternative novel therapy to replace damaged neural circuitry after ischemic stroke onset. Nonetheless, albeit the potential of these cells for stroke therapy, many critical challenges are yet to be overcome to reach clinical applications. The major limitation of the NSC-based therapy is its inability to retain most of the donor stem cells after grafting into an ischemic brain area which is lacking of essential oxygen and nutrients for the survival of transplanted cells. Low cell survival rate limits the capacity of NSCs to repair the injured area and this poses a much more difficult challenge to the NSC-based therapy for ischemic stroke. In order to enhance the survival of transplanted cells, several stem cell culture preconditioning strategies have been employed. For ischemic diseases, hypoxic preconditioning is the most commonly applied strategy since the last few decades. Now, the preconditioning strategies have been developed and expanded enormously throughout years of efforts. This review systematically presented studies searched from PubMed, ScienceDirect, Web of Science, Scopus and the Google Scholar database up to 31 March 2020 based on search words containing the following terms: “precondition” or “pretreatment” and “neural stem cell” and “ischemic stroke”. The searched data comprehensively reported seven major NSC preconditioning strategies including hypoxic condition, small drug molecules such as minocycline, doxycycline, interleukin-6, adjudin, sodium butyrate and nicorandil, as well as electrical stimulation using conductive polymer for ischemic stroke treatment. We discussed therapeutic benefits gained from these preconditioned NSC for in vitro and in vivo stroke studies and the detailed insights of the mechanisms underlying these preconditioning approaches. Nonetheless, we noticed that there was a scarcity of evidence on the efficacy of these preconditioned NSCs in human clinical studies, therefore, it is still too early to draw a definitive conclusion on the efficacy and safety of this active compound for patient usage. Thus, we suggest for more in-depth clinical investigations of this cell-based therapy to develop into more conscientious and judicious evidence-based therapy for clinical application in the future. Stroke is defined as rapid neurological dysfunction due to the disturbance in cerebral blood supply, with acute onset of clinical signs and symptoms persisting for more than 24 h or until death. It is a leading cause of neuronal impairment and adult disability worldwide in which about 70% of the stroke survivors suffer from reduced work capacity, while the remaining 30% require self-management assistance. About 85% of the stroke cases reported are classified as ischemic stroke, a focal neurological deficit that occurs due to insufficient cerebral blood flow (CBF) triggered by occlusion of blood vessel . CBF lesThe neuronal cell death that occurs following ischemic stroke can be reduced if the blood flow occlusion is removed and tissue reperfusion is restored quickly. Treatment for patients with acute ischemic stroke is guided by the time from the onset of stroke, the severity of neurologic deficit and findings on neuroimaging. It was proved that intravenous thrombolysis with intravenous recombinant tissue plasminogen activator  administration significantly reduced mortality and disability associated with ischemic stroke within 4.5 h after onset . NonetheSurvival, proliferation, migration and differentiation of NSC are the key regenerative processes to promote post ischemic recovery. Reports had shown that patients with ischemic stroke injury are capable of undergoing natural endogenous neurogenesis at the ischemic penumbra region of the brain ,12,13. ETherefore, current NSC-based therapy not only depends on endogenous repair, but also depends on exogenous NSC transplantation from independent sources. The exogenous NSC transplantation approach exhibits therapeutic potential since it is not only capable of directly replacing the damaged brain tissue, but also able to secrete neuroprotective factors to protect brain tissue at risk during the acute phase of the stroke. Reports of exogenous NSC transplantation in ischemic stroke-induced animal models showed promising data, with conclusive efficacy and potential of this therapy. Majority of the studies reported improved prognosis of this disease after exogenous NSC transplantation such as improved neurological functions and strengthened motor skills . BasicalIn this review, a literature search based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline was performed to identify publications studying the potential of preconditioned NSC for ischemic stroke treatment conducted in vitro, in vivo or in both. Four specialized databases: PubMed, ScienceDirect, Web of Science and Scopus were used for the literature search using the keywords “precondition” or “pretreatment” and “neural stem cell” and “ischemic stroke”. The systematic search was conducted up to 31 March 2020. In addition, Google Scholar was searched too to find additional studies of interest. In order to achieve a highly sensitive search, the first 100 hits of Google Scholar sorted by relevance were included in the analysis.Search results were limited to fully documented articles in English following the inclusion and exclusion criteria listed below:i.Full-text articlesii.In vitro studies related to preconditioned NSC for ischemic stroke therapyiii.In vivo studies related to preconditioned NSC for ischemic stroke therapyiv.Intervention clinical trial related to preconditioned NSC for ischemic stroke therapyInclusion criteria:i.Irrelevant titles and abstractsii.Duplicated studiesiii.Review articles/meta-analysesiv.News/editorials/lettersv.Case reportsvi.Non-English languageExclusion criteria:Two independent reviewers screened the articles based on the inclusion and exclusion criteria stated above. For the first screening, the related articles were screened based on their titles and abstracts. Next, the remaining papers were checked for duplications. Finally, the selected full-text articles were checked by another reviewer according to the inclusion criteria for final validation.Study selection: The primary search identified 191 articles, including 25 from PubMed, 138 from ScienceDirect, 24 from Web of Science and 4 from Scopus. The first 100 hits from the ‘Most Relevant’ option in Google Scholar were also added. Among these, 137 documents were published in languages other than English or not a research article that met the inclusion criteria. In addition, 26 manuscripts were indexed in two or more databases and were considered only once, resulting in 128 eligible articles. After an initial screening of titles and abstracts, followed by a full-text analysis and validation, 7 articles that fulfilled all the selection criteria were included in this systematic review. A flowchart illustrating the progressive study selection and numbers at each stage is shown in The 7 articles selected were carefully read and analyzed. Based on the collected literature data, we identified seven major preconditioning strategies applied to enhance NSC-based therapy for ischemic stroke treatment . The the2) is one of the critical factors involved in cell proliferation and morphogenesis, where its level is highly regulated in the human body. It is known that mammalian blastocyst is physiologically exposed to oxygen levels ranging from 1.5–5.3% in the reproductive tract during the early stages of embryonic development [Oxygen  transcriptional complex ,25. HIF Minocycline, a semisynthetic second-generation tetracycline derivative, is an antibiotic used to combat bacterial infection. Besides being used as an antibiotic, minocycline also exhibits anti-oxidant, anti-inflammatory and anti-apoptosis effects in various experimental models of acute neurological injury . Known fPreconditioning of NSCs using minocycline for ischemic stroke therapy has been demonstrated by Sakata and colleagues in 2012 . They isIn addition, the transplantation of minocycline-preconditioned NSCs also was found to facilitate neuroprotection effects in ischemic stroke rats via the upregulation of neurotrophic factors such as (1) glial cell-derived neurotrophic factor (GDNF), which potentially promotes the survival of many types of neurons; (2) brain-derived neurotrophic factor (BDNF), which supports survival of existing neurons and encourages growth and differentiation of new neurons and synapses; (3) nerve growth factor (NGF), which primarily involves in the regulation of growth, maintenance, proliferation and survival of neurons; and (4) VEGF cytokine, which triggers neovascularization. The neuroprotective effects exerted by these neurotrophic factors and cytokines were indicated by the attenuated infarct size and improved functional/behavioral recovery in animals transplanted with the minocycline-preconditioned NSCs, compared to the non-transplanted animal group. Moreover, Sakata and colleagues also proved that these factors were released by minocycline-preconditioned NSCs due to the upregulation of Nrf2. This is because the minocycline-induced neuroprotection was abolished by transfecting the NSCs with Nrf2-small interfering RNA which can block the expression of Nrf2.In brief, this study revealed that preconditioning with minocycline (1) protected NSCs from ischemic reperfusion injury via upregulation of Nrf2 and Nrf2-regulated antioxidant genes, (2) increased the migration, proliferation and differentiation capacity of the NSCs, (3) induced the production of neuroprotective paracrine factors by NSCs and (4) resulted in attenuated infarct size and improved neurological recovery after NSC transplantation into ischemic stroke rat models .Similar to minocycline, doxycycline is a tetracycline-derived antibiotic mainly used against microbial infections. It has prolonged serum half-life between 12 and 25 h that allows fewer daily doses usage, making it safer for patients with renal failure . It also2•−) production in doxycycline-preconditioned NSCs, compared to non-preconditioned NSCs. O2•− is a radical species with exceptionally high reactivity due to its unpaired electron. The presence of O2•− could cause cytotoxicity and damage to DNA [2•− resulted in higher survival of NSC in ischemic stroke models.Since doxycycline is a member of second-generation tetracycline, it shares similar chemical structures and mechanism of actions as minocycline. This antibiotic was tested as a potential NSC preconditioning agent in 2013, where Yousra and colleagues confirmed the effectiveness of this small molecule as a potential strategy to enhance the NSC therapeutic potential for ischemic stroke treatment . This ste to DNA . TherefoIn summary, this study provided in vitro evidence that doxycycline preconditioning reprograms NSCs to tolerate oxidative stress and results in reduced apoptosis and increased cell viability, making the cells a better therapeutic agent for ischemic stroke treatment.Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a crucial role in the pathogenesis of various autoimmune and neurological disorders, including the ischemic stroke. IL-6 is produced by both lymphoid and non-lymphoid cells, causing an antigen-specific immune response and excessive inflammation process that may aggravate ischemic cerebral damage . However2O2) and diethylenetriamine/nitric oxide, the IL-6 preconditioned NSCs survived better (by 28%) compared to the non-preconditioned NSCs, indicating the cytoprotection effects exerted by IL-6 preconditioning. Taken together, these findings confirmed the pivotal role of IL-6 as a potential anti-oxidant agent to protect NSCs against the ischemic reperfusion injury.Based on this knowledge, Sakata and colleagues (2012) performed a series of experiments to elucidate the exact role of IL-6 in NSC preconditioning strategy . To do tFurthermore, IL-6 preconditioning has been reported to significantly increase the tolerance of treated cells against apoptotic processes and hostile environments in vivo. NSCs subjected to IL-6 pre-treatment demonstrated a reduction in grafted-cell death by 62% when transplanted into ischemic stroke mouse models. Moreover, these grafted cells managed to survive in the ischemic brains up to 28 days, with no formation of tumors. Staining using green fluorescent protein (GFP) also revealed extensive grafted cell migration toward the ischemic lesion borders, where the grafted cells were capable of differentiating into neurons, oligodendrocytes and astrocytes. More excitedly, the cortical infarct volume was significantly attenuated in the preconditioned NSC group compared with the non-preconditioned control group. These findings indicated that IL-6 preconditioning successfully increased the survival rate of grafted cells and induced their integration into the impaired neuronal networks after ischemic stroke injury.According to Sakata et al. , the enhAdjudin is an indazole-based compound that has been explored as a non-hormonal male contraceptive since the 1990s. Recently, the studies on adjudin have been expanded to evaluate its biological potentials for other ailments such as anti-cancer, anti-inflammation, anti-neurodegeneration and anti-ototoxicity activities . This smAccording to the study, preconditioning of NSCs with adjudin at 10 and 30 uM for 24 h showed significant reduction in apoptosis rate when exposed to oxidative stress stimuli, compared with non-preconditioned NSCs. The underlying mechanism of adjudin-induced cytoprotection against oxidative damage was supported by the activation of serine/threonine-specific protein kinase (AKT) signalling pathway in adjudin-preconditioned NSCs. AKT signalling is a vital signal transduction pathway that promotes cell survival and growth in response to extracellular signals . When acAdjudin also exhibited potential to enhance the expression of antioxidant genes, which plays important roles in resistance to oxidative stress. The antioxidant genes expressed in adjudin-preconditioned NSCs were SOD2, catalase and glutamate-cysteine ligase catalytic (GCLC). Moreover, adjudin pre-treatment was also able to inhibit the expression of inducible nitric oxide synthase (iNOS), compared to non-preconditioned NSCs. Induction of the high-output iNOS usually occurs in an oxidative stress environment, for example, following an ischemic stroke injury . iNOS alAdjudin’s potential as a neuro-inflammatory modulator also has been demonstrated in this study, where it could block microglial activation, as indicated by less CD11b signal detected by fluorescence microscopy. Besides, the adjudin-preconditoned NSCs also could aid to reduce the inflammatory mediators such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) which were dramatically increased after the ischemic injury in mouse models. It is known that an imbalance or overexpression of pro-inflammatory cytokines can lead to different pathological disorders . TherefoBesides oxidative stress and neuroinflammation, Zhang et al.  also focThe study also showed that adjudin preconditioning induced higher expression of neurotrophic factors such as BDNF, NGF and GDNF, as compared to non-preconditioned NSC. These neurotrophic factors could act to support the survival and growth of neurons. Besides, pre-treated NSCs were also able to promote angiogenesis after transplanted into ischemic stroke mouse model, measured by increased endothelial progenitor cell marker CD31 and increased new blood vessels generation. Angiogenesis is directly linked to neurogenesis because re-establishment of blood circulation in the damaged area is necessary for new neuronal survival and development. This statement is supported by the observation that adjudin-pretreated NSCs greatly reduced infarct volume by as much as 20% compared to untreated NSCs. Meanwhile, animals that received adjudin preconditioned NSCs also showed improved behavioural performance by 25% better than the animals treated with non-preconditioned NSCs.Taken together, this study provided ample preclinical experimental evidences to confirm that adjudin preconditioning is able to enhance the survival rate of NSCs and exhibit better effect on inhibiting oxidative stress and neuroinflammation, maintaining BBB integrity, expressing higher levels of neurotrophic and angiogenesis factors after transplanted into ischemic stroke model. All these effects resulted in stronger therapeutic effects of NSCs for ischemic stroke brain injury treatment, indicated by decreased infarct volume and improved behavioral outcomes in the ischemic stroke animal models.Sodium butyrate (NaB) is a functional molecule produced from microbial fermentation in the mammalian intestine . NaB-bas2), as compared to cells preconditioned with either NaB (7.4 ± 4.9 cells/mm2) or nicorandil (8 ± 3.7 cells/mm2) alone.Preconditioning of NSCs using NaB combined with nicorandil for stroke therapy has been explored by Seyed and colleagues in 2018 . In thisBesides that, they also observed that induction of BDNF in the preconditioned NSCs led to an increase in phosphoinositide 3-kinases (PI3K) activity. PI3K is an enzyme involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. As reported in previous studies, PI3K/Akt signaling stimulates neurite outgrowth . In accoAnti-inflammatory properties of NaB and nicorandil-preconditioned NSCs on ischemic stroke also were demonstrated in this study, where the preconditioning strategy was found to sucessfully inhibit pro-inflammatory mediators  and upregulate anti-inflammatory mediators (IL-10). Besides, this preconditioning strategy is also able to suppress microglial activation. Suppression of microglial activation is critical to inhibit severe neuroinflammatory responses which could contribute to the progression of many chronic neurodegenerative disorders .In summary, this study reported that NSC preconditioning by combined treatment using small molecules NaB and nicorandil enhances the post-engraftment survival rate, increases the expression of neurotrophic factor, increases neurite outgrowth, reduces inflammatory responses, and is thus able to potentiate their reparability in a rodent model of experimental stroke, indicated by the reduced infarct size and improved behavioral performance.Recently, electrical stimulation has garnered interest as an effective physical stimulus to manipulate cell behaviors in vitro and in vivo . There aConductive polymer is a form of biomaterial that has gained more and more attention in cell biology and neural tissue engineering applications due to its electrical excitability. This biomaterial has great biocompatibility and is able to interact continuously with the applied stem cells and their surrounding neural environment, making the NSC a better candidate for ischemic stroke therapy . PreviouIn the study, they reported electrical conductive polymer priming the preconditioned NSCs to promote functional recovery via modulation of paracrine factor vascular endothelial growth factor A (VEGF-A). The activation of the VEGF-A pathway increased blood vessel density via the angiogenesis process, validating its function to restore blood supply to the affected brain. Animal models that received NSCs preconditioned using this strategy also experienced faster and advanced functional recovery as compared to the animal group that received unstimulated NSCs. Interestingly, electrically preconditioned NSCs were not only able to improve the rate of recovery, but also able to enhance long-term function as they found that rats that received electrically preconditioned cells continued to perform better throughout their testing, indicating a long-lasting effect of in vitro electrical preconditioning.Data retrieved from Cochrane library, U.S. Clinical Trial Database  and Chinese Clinical Trial Registry recorded only a few numbers of trials using non-preconditioned NSC as clinical cell lines in ischemic stroke treatment  , NSCs haAnother important aspect that needs to be considered for preconditioned NSCs-based therapy is its long term safety, particularly with regard to the potential of tumorigenicity after stimulation by preconditioning approaches. In order to further enhance the efficacy of stem cell-based transplantation therapy, a combination of NSC engineering strategies with preconditioning strategies is recommended to provide enhanced cell replacement, trophic support, immunomodulatory as well as regenerative potential for better therapeutic effects. In addition, alternative treatments using NSC-derived extracellular vesicles (EV) have also been highlighted recently in neurodegenerative disease. This EV-based therapy has spurred a renewed interest in their utility for a cell-free therapeutics option, where it could avoid problems encountered by NSCs-based therapy such as low cell survival rate and high graft rejection after transplantation.Recent significant progression of NSC-based transplantation therapy has brought it to the forefront of regenerative treatment for ischemic stroke. This cell-based therapy has garnered significant interest as a potential therapeutic agent to compensate the damaged neuronal cells after ischemic injury. Some clinical trials are already underway to study the effects of NSCs transplantation for ischemic stroke, however, there are still many challenges to be optimized before NSCs therapy can be applied clinically. One of the biggest limitations of NSC-based transplantation therapy is the limited survival rate of transplanted cells. The extensive cell apoptosis post-transplantation is mainly due to the deficient oxygen and nutrient availability, mechanical stress and the ongoing acute inflammatory response at the site of the injured tissue. Among the strategies proposed to enhance the NSC therapeutic potential for ischemic stroke therapy is via the preconditioning approaches. This review outlines the preconditioning strategies that had been taken to make post-engraftment stem cell survival and regenerative therapy a closer-to-clinical reality. The preconditioning strategies discussed here were the hypoxic condition, small molecules such as minocycline, doxycycline, IL-6, adjudin, NaB and nicorandil and electrical stimulation using conductive polymer. We compiled concrete in vitro and in vivo evidence endorses that these preconditioning strategies reprograms the NSCs to tolerate oxidative stress, secrete neurotrophic factor, enhance neuroprotection, induce angiogenesis, reduce neuroinflammatory responses, reduce infarct volume and increase neurological function after engraftment. We also identified several key pathways involved in mediating the preconditioned NSCs to exhibit enhanced therapeutic potential for stroke treatment. Among the important pathways discussed here were HIF transcriptional complex, STAT3, AKT and PI3K pathways. All these pathways play significant role in regulating critical cellular biology response and are worthwhile to be further explored in the future. In conclusion, all the preconditioning strategies discussed in this review article exhibited promising potential in the enhancement of NSCs-based transplantation therapy for ischemic stroke. However, there is still a scarcity of evidence on the efficacy of these preconditioned NSCs in human clinical studies, therefore, it is still too early to draw a definitive conclusion on the efficacy and safety of this active compound for patient usage. Thus, future studies on preconditioned NSC-based transplantation therapy are imperative in the ongoing quest for an effective, feasible and safe clinical approach.


POAG is the leading cause of irreversible blindness in African Americans. In this study, we quantitatively assess the association of autosomal ancestry with POAG risk in a large cohort of self-identified African Americans.n = 3830; replication cohort, n = 2135). Population structure was interrogated using principal component analysis in the context of the 1000 Genomes Project superpopulations.Subjects recruited to the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study were classified as glaucoma cases or controls by fellowship-trained glaucoma specialists. POAAGG subjects were genotyped using the MEGA Ex array , consistent with higher degree of African ancestry. Among POAG cases, higher African ancestry was also associated with thinner central corneal thickness (P = 2–4). Admixture mapping showed that local genetic ancestry was not a significant risk factor for POAG. A polygenic risk score, comprised of 23 glaucoma-associated single nucleotide polymorphisms from the NHGRI-EBI genome-wide association study catalog, was significant in both cohorts (P < 0.001), suggesting that both known POAG single nucleotide polymorphisms and an omnigenic ancestry effect influence POAG risk.The majority of POAAGG samples lie on an axis between African and European superpopulations, with great variation in admixture. Cases had a significantly lower mean value of the ancestral component q0 than controls for both cohorts (In sum, the POAAGG study population is very admixed, with a higher degree of African ancestry associated with an increased POAG risk. Further analyses should consider social and environmental factors as possible confounding factors for disease predisposition. On average, African Americans have roughly 20% European ancestry, although ancestry proportions can vary significantly among individuals.,,Although it is known that African Americans are disproportionately affected by POAG, the role genetics and particularly ancestry play in the disease's etiology and progression remains poorly understood after taking into account known risk alleles. Studies on POAG have primarily used self-reporting to define racial categorization, which is often an imprecise measure of genetic ancestry.The goal of this study was to investigate the association between genetic ancestry and POAG in a large, primary cohort of self-identified African Americans. These patients were recruited as part of the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study, a 5-year project investigating the genetic architecture of POAG in self-identified African Americans. Using array-based genotyping data, clinically validated POAG diagnoses, and quantitative endophenotypic data, we conclude that average autosomal genetic ancestry may be a significant risk factor for POAG in African Americans,The POAAGG study population consists of self-identified blacks , aged 35 years or older. Subjects were identified from comprehensive and subspecialty ophthalmology clinics at the University of Pennsylvania and satellites, as well as several neighboring ophthalmology clinics in Philadelphia, Pennsylvania . Following examination by a fellowship-trained glaucoma specialist, each subject was classified as a POAG case, POAG suspect, or control based on previously reported criteria.Genomic DNA for all enrolled subjects was extracted from peripheral blood or saliva, which both demonstrated excellent performance in array-based genotyping in a previous study.DNA was isolated from thawed blood samples using Gentra PureGene kits  and included the optional RNase treatment step. DNA from saliva samples was extracted using the prepIT.L2P reagent  and precipitated with ethanol according to manufacturer's instructions. The saliva DNA samples were RNAse treated by double digestion with RNase A and RNase T and reprecipitated using ethanol according to the manufacturer's instructions. The concentrations of DNA from blood and saliva samples were determined using the fluorescence-based Quant iT dsDNA Board-Range assay kit . Fluorescence was measured with a Tecan Infinite M 200 Pro multimode microplate reader .A 25-µL aliquot of all samples with high-quality DNAs and case/control status were plated for array-based high throughput genotyping. Genotyping was conducted on of 3830 DNA samples  and a replication cohort of 2135 samples , using the Multi-Ethnic Genotyping Array (MEGA)V2 (EX) consortium chip on the Infinium iSelect platform by Illumina FastTrack Services . The replication cohort consists of subjects who were enrolled after genotyping of the discovery samples was completed. The selection criteria for the samples, genotyping array, and QC were the same as for the discovery cohort.MEGA array content was supplemented with 5000 single nucleotide polymorphisms (SNPs) from prior genome-wide association study (GWAS) on POAG, common polymorphisms from POAG-associated genes, and variants detected from whole-genome sequencing of POAAGG subjects. At least one sample per 96-well microtiter plate was genotyped in duplicate for purposes of QC. Genotype calls were generated using the Genome Studio genotyping module (GT). Cluster optimization and reproducibility analysis for paired samples were performed as per standard practices at Illumina FastTrack services.P value of less than 0.00001, and a minor allele frequency of less than 0.01 (rare variants) (Directly genotyped variants and samples were subjected to rigorous QC.ariants) .After individual-level and variant-level QC, 1,108,459 SNPs were analyzed for continental ancestry using the 1000 Genomes Project version 5a dataset. This dataset contains 2506 human samples from 26 global populations grouped into five labeled superpopulations . We removed African American samples from the African superpopulation to achieve a higher resolution of African–European admixture. After removing A->T/T->A/C->G/G->C genetic variants for strand alignment, autosomal genotypes were merged between the POAAGG cohort and the 1000 Genomes Project dataset before LD pruning at an R2 ceiling of 0.2 before principal components from a genetic relationship matrix were extracted using Plink version 1.90.We next investigated two-way admixture among cases and controls using fastSTRUCTURE, which is a software designed to infer population structure from genotyped data.To quantify the relationship between African ancestry and case/control status, we divided the q0 variable into quartiles. The lowest quartile of q0 was associated with the highest degree of African ancestry and the lowest degree of European ancestry under the two-way admixture model from fastSTRUCTURE.We used logistic regression models that adjusted for age and gender to calculate the odds ratio and its 95% confidence interval for the association between the estimated African ancestry from fastSTRUCTURE (q0) and POAG. Among POAG cases, we used linear regression models that adjusted for age and gender to determine the associations between African ancestry and four quantitative phenotypes: IOP, central corneal thickness (CCT), cup-to-disc ratio, and retinal nerve fiber layer thickness.P value passed a Bonferroni-corrected threshold of 1.96–5.To perform admixture mapping in the POAAGG samples, we used ANCESTRYMAP, which estimates the ancestral origin of a locus and then tests whether there is a skew in ancestry associated with case status at each marker.Prior POAG-associated variants were extracted from the NHGRI-EBI GWAS catalog,P values.To develop a multivariable risk model, a logistic regression model was fit on the whole cohort using age, gender, polygenic gene risk score, and ancestry as predictors. The association of each predictor with POAG risk was assessed by odds ratios  and The majority of POAAGG samples lie on an axis between the African and European 1000 Genomes reference samples, with a high degree of variation in ancestry proportions among individuals A. The fin = 3806), glaucoma cases had significantly lower mean values of PC1 compared with controls, consistent with a higher degree of African ancestry .For the subsequent ancestry analysis, a small subset of samples were excluded after QC owing to missing data, resulting in a discovery cohort of 3806 patients and replication cohort of 2011 patients. For the discovery cohort  and replication cohort (P = 3–6) suggesting a higher degree of African ancestry in cases , we found that q0 and PC1 were highly correlated  and replication cohort (P = 0.002), as shown in P = 0.02). The other quantitative traits  were not significantly associated with African ancestry.We observed a significant relationship between African ancestry and thinner CCT in both discovery cohort  and replication cohort (P < 0.001).We constructed a polygenic risk score from 23 glaucoma-associated SNPs from the NHGRI-EBI GWAS catalog . This riP < 0.001 for both).However, when adjusted for this polygenic effect, African ancestry remained as a significant risk factor . This fiWe assessed the association of autosomal genetic ancestry with POAG risk in a large self-identified African American cohort, performing global and local ancestry analyses estimated from array-based genotyping data. We found that a higher proportion of African ancestry in the nuclear genome increased disease risk for subjects, whereas local genetic ancestry was not a significant risk factor, when only considering genetic risk factors for POAG.,,–,In this study, the proportion of African ancestry varied greatly in our cohort, consistent with prior studies.We found that, without considering nongenetic risk factors, genome-wide African ancestry is a risk factor for POAG in our African American cohort. Although many previous studies have shown that individuals of African descent are disproportionately affected by POAG, this study used a quantitative assessment of ancestry rather than self-reporting as an independent variable. An empirical assessment of genetic ancestry is a more precise measure of biologic diversity, allowing analyses of continuous rather than a categorical variables.,,–Consistent with prior studies, greater African ancestry was associated with thinner CCT. It is well-established that healthy African Americans have a thinner CCT than healthy European Americans.APBB2 as significantly associated with POAG in individuals of African ancestry.APBB2 in our polygenic risk score, we attempted to replicate the APBB2 (rs59892895) variant in our case-control POAAGG study and found that the association was not significant (P = 0.335) . These results suggest that inclusion of the novel APBB2 variant would not affect the polygenic risk score for this highly admixed African American population. Further studies with the Genetics of Glaucoma in People of African Descent (GGLAD) Consortium are currently underway to better define genetic similarities and differences between these populations. When examining the multivariable risk model, African ancestry remains as a significant risk factor when adjusted for this polygenic effect, a finding that was validated in our replication cohort. POAG risk in African Americans is likely influenced by the additive effects of polygenic risk at known disease-associated loci and an omnigenic ancestry effect.We also tested the significance of a polygenic gene risk score in our cohort, with results showing a significant polygenic effect with regard to disease risk, independent of ancestry. This finding validates the additive effect of known POAG-associated loci in our African American population. Most recently, a GWAS identified a novel locus in ,The strengths of this study include the use of a genotyping array specifically designed to maximize coverage of genetic variants relevant to African Americans. This custom design may help to avoid the limitations of previous studies that used arrays designed for European populations on African populations, leaving up to 40% of variants not assayed in African populations.,,This study does not address how genetic risk factors interact with social and demographic variables to affect POAG risk in African Americans. Prior studies have shown that social and demographic factors interact with biological factors to affect health outcomes.In summary, we found that higher levels of autosomal-wide African ancestry serve as a risk factor for POAG in self-identified African Americans. Although global ancestry was highly predictive of POAG risk, no specific African ancestry informative markers rose to genome-wide significance. This study illustrates the great genetic diversity among African Americans and the importance of using quantitative assessments of ancestry when the aim is to identify genetic associations. In future studies, it will be important to capture and explore how social and environmental variables interact with genetic risk factors to affect POAG risk. We hope that studying this admixed population will be helpful in mapping POAG genes with prevalence differences between ethnic groups. Furthermore, the finding that the highest degree of African ancestry conferred the greatest POAG risk suggests ancestry analysis could contribute to precision screening of this population.


This study demonstrates that monodisperse entangled polymer melts crystallize via the formation of nanoscale nascent polymer crystals  that exhibit substantial variability in terms of their constituent crystalline polymer chain segments (stems). More specifically, large-scale coarse-grain molecular simulations are used to quantify the evolution of stem length distributions and their properties during the formation of polymer nuclei in supercooled prototypical polyethylene melts. Stems can adopt a range of lengths within an individual nucleus  while two nuclei of comparable size can have markedly different stem distributions. As such, the attainment of chemically monodisperse polymer specimens is not sufficient to achieve physical uniformity and consistency. Furthermore, stem length distributions and their evolution indicate that polymer crystal nucleation  is phenomenologically distinct from crystal growth. These results highlight that the tailoring of polymeric materials requires strategies for controlling polymer crystal nucleation and growth at the nanoscale. The properties of polymeric materials—both macroscopic specimens and nanomaterials—are intimately connected to their crystallinity . In parWhile there have been many experimental studies on polymer crystallization and nucleation as highlighted by recent reviews ,3,4, it ous work . Sadler ous work ,13 conneWith molecular simulations, one can directly study the molecular-level nanoscopic details of polymer crystal nuclei and stems during polymer crystallization. Previous in silico studies have probed stem-related properties and phenomena during crystallization, including the lamellar thickness of nuclei ,15, intrn-C720H1442 chains. The methodological details of the simulations are the same as our previous work [n-C720H1442 chains were represented using the CM and CT coarse-grain beads from the coarse-grain Shinoda-DeVane-Klein (SDK) molecular model [2)3– and –CH2CH2CH3 units along the polyethylene backbones. By comparing SDK-based results for molten polyethylene and polyethylene crystallization to results from experiments and atomistic simulations, we have previously demonstrated that the SDK model is an appropriate model for studying polyethylene systems [In order to quantitatively probe the characteristics of stem length distributions during polymer crystal nucleation, we performed microsecond molecular dynamics simulations of crystal nucleation in monodisperse polyethylene melts composed of ous work , so onlyous work  for moreous work . The n-Car model . For ref systems . Due to  systems ,42. As sn-C720H1442 melt consisting of 400 chains under periodic boundary conditions was prepared and equilibrated at 500 K. Ten independent configurations were selected from the high-temperature simulation and used to generate ten independent metastable melts at 300 K. These melts were in turn quenched to 285 K, and each melt was simulated at 285 K for ∼4 A S or Consistent with previous work ,49,50,51ailed in ,42. In bIt is reasonable to include such single-bead stems based on theoretical considerations and in silico results. First, theoretical treatments ,11,12,13We also performed a substantial detailed analysis of our crystallization simulations to validate the inclusion of the single-bead stems in the current study. For reference, the simulation-based literature on polymer crystallization  lengths, which were estimated using the bond length (CM–CM) and bead width (CM) for the SDK model. Throughout this study, various properties are presented as functions of cluster size . Each of these profiles was constructed by first extracting the specified property for each nucleus, and then performing conditional averaging over the extracted values according to cluster size.The crystal cluster shown in Consistent with previous work ,20, averIn addition to intra-nucleus stem length variations as evidenced by Given that average stem lengths increase with crystal size , relativions see A. In turWe now introduce the concept of the structural dispersity as a physical analog of dispersity (a metric commonly used in synthetic polymer chemistry) in order to place structural heterogeneity in nascent polymer crystals on an equal conceptual footing with chemical heterogeneity arising during polymer synthesis. More specifically, the relative distribution of chain lengths in a polymer sample is commonly captured using dispersity (Đ) as calculated according to:structural) of a nucleus can be calculated based on Equations ,,3), Mi iof 1 see B. ConverAt this point, it is worth noting that it can be challenging to compare molecular weight distributions using Đ values ,53,54 asstructural values. Moreover, in analogy to Equation  ,57. Somestructural and Both Đquations , respectx is the length of a particular constituent stem, s is the standard deviation of the stem length distribution for the cluster. Note that the factor of 3 in Equation ,,7), the Equation A,B. On a1 values A, indicabehavior B. For recrystals .Previous work  has demoThe chemistry community has focused extensively on controlling chain length distributions  during polymer synthesis. However, the continued pursuit of lower chemical dispersities will likely not yield the desired control of polymeric materials unless the chemistry community also develops strategies to guide polymer crystallization down to its earliest stages. Our results demonstrate that controlling crystallization cannot simply take place at macroscopic or mesoscopic scales; crystallization must be controlled down to the nanoscale processes through which it is initiated. Even in monodisperse polymer melts, individual nuclei exhibit substantial nanoscale stem length variations, while nuclei of a given size can have markedly different stem length distributions. Structural heterogeneity and structural dispersity are thus important characteristics of polymer crystal nucleation. Consequently, fully tailoring polymeric materials requires strategies for minimizing intra-nucleus and inter-nucleus structural variations on sub-nanometer and nanometer levels.This study has also revealed that various quantitative properties of stem length distributions  exhibit distinct pre-critical and post-critical behaviors with cross-overs in the vicinity of the critical nucleus size. As such, our quantitative characterizations of stem length distributions and their average evolution reinforce that nucleation is phenomenologically distinct from polymer crystal growth, suggesting that insights from crystal growth studies are not necessarily relevant to attempts to control polymer crystal nucleation. By introducing structural dispersity and numerically analyzing stem length distributions more generally, this study provides a fresh quantitative lens for comparing and understanding both polymer crystal nucleation and growth moving forward. Interestingly, the structural dispersity values obtained in this study for clusters in the vicinity of the critical nucleus size are comparable to dispersity values arising from polymerization reactions. As illustrated by a recent perspective article , it rema


Methods used to categorize functional status to predict health outcomes across post-acute care settings vary significantly.We compared three methods that categorize functional status to predict 30-day and 90-day hospital readmission across inpatient rehabilitation facilities (IRF), skilled nursing facilities (SNF) and home health agencies (HHA).Retrospective analysis of 2013–2014 Medicare claims data . Data were randomly split into two subsets using a 1:1 ratio. We used half of the cohort (development subset) to develop functional status categories for three methods, and then used the rest (testing subset) to compare outcome prediction. Three methods to generate functional categories were labeled as: Method I, percentile based on proportional distribution; Method II, percentile based on change score distribution; and Method III, functional staging categories based on Rasch person strata. We used six differentiation and classification statistics to determine the optimal method of generating functional categories.IRF, SNF and HHA.We included 130,670 (17.7%) Medicare beneficiaries with stroke, 498,576 (67.3%) with lower extremity joint replacement and 111,284 (15.0%) with hip and femur fracture.Unplanned 30-day and 90-day hospital readmission.For all impairment conditions, Method III best predicted 30-day and 90-day hospital readmission. However, we observed overlapping confidence intervals among some comparisons of three methods. The bootstrapping of 30-day and 90-day hospital readmission predictive models showed the area under curve for Method III was statistically significantly higher than both Method I and Method II , using the testing sample.Overall, functional staging was the optimal method to generate functional status categories to predict 30-day and 90-day hospital readmission. To facilitate clinical and scientific use, we suggest the most appropriate method to categorize functional status should be based on the strengths and weaknesses of each method. In many disciplines of medicine, clinical staging refers to hierarchical categories along the continuum of the measured construct. –3 The coWhile continuous scores may provide detailed clinically information for clinicians ,12, catePractitioners and researchers have used functional status categories to present hierarchical levels of patients’ function for decades –10. HoweThe study included 100% Medicare claims data from 2013–2014. We used the following data files: Inpatient Rehabilitation Facility (IRF) and Inpatient Rehab Facility- Patient Assessment Instrument (IRF-PAI) ; SkilledThis study was approved by the University Institutional Review Board (IRB # 16–0014). Additionally, a Data Use Agreement was established with the Centers for Medicare and Medicaid Services prior to all data analyses.We identified 2,953,006 eligible cases using a combination of Medical Severity Diagnosis Related Group codes and ICD-9 procedure codes for three impairment conditions: stroke (061–066), lower extremity joint replacement  and hip/femur fractures (480–482). Using a combination of claims and assessment data, we included only those beneficiaries discharged from a hospital to one of the three post-acute care (PAC) settings: IRF, SNF and HHA. After applying exclusion criteria , the finTo develop and validate the three proposed methods, we used 1:1 ratio to randomly split the study cohort into a development subset  and a testing subset . The development subset was used to develop functional status categories from three methods. The testing subset was used to compare outcome prediction for three methods.We also conducted sensitivity analysis to examine difference of demographics and person-level characteristics before and after excluding 23% of potential patients  after index hospital discharge ,18. We cThe primary variable was functional status categories for two domains (Self-Care and Mobility) generated from three methods (details below). Self-Care and Mobility domains were chosen as these two domains being consistently measured across the PAC settings. Additionally, these two domains are potentially modifiable factors relevant to hospital readmission.Comparable items of the Self-Care and Mobility domains from each assessment were selected based on their conceptual meanings . The number of selected items by assessment was 11 in IRF-PAI (6 Self-Care and 5 Mobility items), 11 in MDS (5 Self-Care and 6 Mobility) and 8 in OASIS (5 Self-Care and 3 Mobility) . We usedFor each impairment condition, we created tertile, quartile and quintile categories based on the co-calibrated summed score distribution for each assessment. Self-Care and Mobility had the same numbers of categories. We generated percentiles first for each assessment, following c-statistics to determine whether to choose tertile, quartile or quintile for each impairment condition at each setting. Based on the c-statistics, quartile was chosen for stroke and lower extremity joint replacement, and quintile was chosen for hip and femur fracture. We first calculated the change score between admission and discharge for each assessment . Secondly, we calculated percentile  based on the change score distribution. Lastly, to increase clinical meaningfulness when interpreting negative, zero and positive change scores, we combined the percentile change score distribution with the following operational definitions: tertiles , quartiles  and quintiles .Same as Method I, Self-Care and Mobility of each assessment had the same number of categories due to the nature of percentile method. Using c-statistics, quartile was selected for stroke and lower extremity joint replacement; quintile was selected for hip and femur fracture. The quintile proportion was found inapplicable for stroke and lower extremity joint replacement as the same functional score was used in more than one category. Using the development subset, for stroke, we generated four categories for Self-Care and three categories for Mobility for all three instruments. For lower extremity joint replacement, we generated three Self-Care and two Mobility categories for IRF-PAI and OASIS; and three Self-Care and three Mobility categories for MDS. For hip and femur fracture, we generated three Self-Care and two Mobility categories for IRF-PAI; two Self-Care and three Mobility categories for MDS; and four Self-Care and three Mobility categories for OASIS .Six indices were used to compare the outcome prediction of the three methods:The c-statistics measure the discrimination ability of the model. We compared the logistic model discrimination using c-statistics with asymptotic 95% confidence intervals. The c-statistic is also known as the AUC, the area under the receiver operating characteristic curves. The AUC is the most commonly used method to evaluate probability of model performance in the context of binary outcomes with higher values indicates better model fit –42.Somer’s D is a nonparametric test to assess the strength and direction of the association between an ordinal dependent variable and an ordinal independent variable. Somer’s D is based on the assumption of a monotonic relationship between the independent and the outcome variables. Higher Somer’s D indicates better model fit .Both AIC and BIC  evaluateThe IDI indicates the difference in discrimination slopes between two models. The IDI measures whether the new model improves the average sensitivity without sacrificing its average specificity . Higher The NRI is a reclassification measure using reclassification tables constructed separately for respondents with and without events  between two models . Higher We stratified all analyses by impairment conditions for both development and testing subsets. First, we constructed a baseline logistic regression model which included sociodemographic variables , health status  and post-acute length of stay. Then, we added three types of functional status to the baseline logistic regression model. We used baseline model to (a) ensure fair comparison conveyed by different functional status categories from three methods, and to (b) examine the magnitude change of outcome prediction by adding functional status variables. The predictive models with three methods of generating functional status categories were examined by AUC, Somer’s D, AIC, BIC, IDI and NRI using the testing sample. To validate the stability of the estimates, a bootstrap procedure with 1000 re-samples was used to statistically compare c-statistics of the three methods using the testing sample. The c-statistics with bootstrapping is a standardized way for model comparison. Each of the three methodologies were later compared using paired t-tests if significant difference existed among methods. We used SAS version 9.4  to perform all analyses.The majority were discharged to SNF , followed by HHA  and IRF  . The meaIn both 30-day and 90-day hospital readmission models, the results of the bootstrapping using testing sample showed that the AUC for Method III was the highest compared with both Method I and Method II for the three impairment conditions .We provided functional status categories generated from Methods I-III  Tables. Generating meaningful categories allow for functional status comparisons and optimal outcome prediction across post-acute settings. This study compared three functional category methods and found the functional staging approach (Method III) generated the relatively optimal prediction for 30-day and 90-day hospital readmission. While the study findings imply that using functional staging approach can be relatively optimal for outcome prediction, it is unclear whether this improvement can also produce superior clinically meaningful levels. To facilitate clinical and scientific use, we suggest the most appropriate method to categorize functional status should be based on the strengths and weaknesses of each approach. For example, Method I may have the advantage of convenience , Method II may have the advantage when reporting functional change and Method III may have the advantage in outcome prediction . The choice of the method requires a delicate judgement and balance between available resource, time demand and study purpose. This study provides preliminary data to guide future healthcare policy reforms  when classifying patients’ self-care and mobility function. We also generated tables of functional categories based on the three methods and plots of function-based readmission risks using functional staging for clinicians and researchers to use.Policymakers are beginning to explore the impact of functional status on classification systems in post-acute risk-adjusted capitation payments ,49,50. RWe acknowledge that patients with varying clinical characteristics and disease severity may benefit differently from various levels of care provided at different types of post-acute settings. However, recent healthcare reform proposals emphasize the need for a unified prospective payment system for post-acute settings . Thus, cThe study findings also indicate that generating more categories is not associated with better outcome prediction. Our results support the notion that the number of functional status categories varies by impairment condition, and using distinct functional levels may be more appropriate than the arbitrary percentile cutoff criteria, where a predefined fixed number of distribution-based categories dictates the categorization. Functional staging consider hierarchical functional levels, thus this empirical approach can classify patients into distinct functional levels.Current evidence regarding the advantages and limitations of different functional category methods remains unclear and largely unexplored. In the emerging environment of value-based care and precision medicine, it is reasonable to ask: are percentile proportional distribution and change score too insensitive to provide accurate functional categories necessary to assess and predict quality outcomes? If the answer is yes, then what are the appropriate approaches? Our study and findings address this question and provide a potential solution for improving rigor in comparative effectiveness studies across post-acute settings.Ongoing demonstration projects of uniform functional assessment, episode-based payment models, and unified payment system across post-acute settings signify the growing need to conduct rigorous post-acute health services and health policy research. This study is the first we are aware of to examine the impact of quality measures based on different categorization methods of functional status. Future study should examine whether different categorization methods of functional status are associated with different provision of care services. It is also important to explore other variables in addition to functional status to optimize outcome prediction accuracy for individual patients. In addition, future study should validate whether our finding can be applied to other quality outcomes, such as successful community discharge for Medicare beneficiaries.This study has limitations related to using Medicare files . For exaCurrent measures and methods examining functional status across post-acute settings vary significantly. To compare effectiveness and quality of care across post-acute settings, identifying an optimal functional category method is imperative. While our study found functional staging approach generated functional categories that explained the largest variances in both 30-day and 90-day hospital readmission prediction, we are uncertain whether functional staging approach can provide clinically meaningful improvement compared to alternative methods. We suggest clinicians, researchers and policy makers execute their best judgments to balance the strengths and weaknesses of each method when categorizing functional status. Additional research is needed to better understand the advantages and the limitations of using functional staging categories to assess and predict other important national quality measures across post-acute settings.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.


The cancer stem cell hypothesis postulates that tumors arise from a few cells with self-renewal capabilities. The identification of stem cell markers, the development of mouse and human tumor organoids and their application in mouse models, allowing lineage tracing, helped to better understand the cancer stem cell model as well as the role of differentiation. This review aims at providing insights on the interplay between cancer stem cells and differentiated cells, as well as the importance of plasticity between the two states.+ cells for cancer progression. On the contrary, the role of Lgr5− cells during this process remains largely unknown. In this review, we explore available evidence pointing towards possible selective advantages of cancer cells organized hierarchically and its resulting cell heterogeneity. Clear evidence of plasticity between cell states, in which loss of Lgr5+ cells can be replenished by dedifferentiation of Lgr5− cells, shows that cell hierarchies could grant adaptive traits to tumors upon changing selective pressures, including those derived from anticancer therapy, as well as during tumor progression to metastasis.The cancer stem cell hypothesis poses that the bulk of differentiated cells are non-tumorigenic and only a subset of cells with self-renewal capabilities drive tumor initiation and progression. This means that differentiation could have a tumor-suppressive effect. Accumulating evidence shows, however, that in some solid tumors, like colorectal cancer, such a hierarchical organization is necessary. The identification of Lgr5 as a reliable marker of normal intestinal epithelial stem cells, together with strategies to trace cell lineages within tumors and the possibility to selectively ablate these cells, have proven the relevance of Lgr5 This l models ,14,15,16+ cells can be rapidly replenished by dedifferentiation of Lgr5− cells after Lgr5+ cell damage or ablation [It is interesting to note, however, that from an evolutionary perspective, the hierarchical organization of cells in rapidly renewing tissues, like the intestinal epithelium during homeostasis, can act as a tumor suppressor mechanism, by minimizing cell divisions of stem cells, in charge of self-renewal, while depositing a higher proliferative burden on transit-amplifying cells destined to terminally differentiate into postmitotic cells that are shed out from the tissue. The fact that in colorectal tumors, cells expressing stem cell markers self-renew and give rise to cells expressing different markers of differentiated lineages ,15,16,17Cells in the human intestinal epithelium, which are exposed to the potentially harmful environment of the microbiome and digestive processes, are continually shed and replaced. This rapid cell renewal safeguards the physiological and structural function of this tissue: actively and passively absorbing nutrients while isolating other tissues from the processes of food degradation. However, high rates of cell division increase the likelihood of DNA-replication errors . Many poHowever, if cells engaged in rapid and repeated cell division are “dismissed from the task” of self-renewal, any mutation in these cells is lost when the mutated cell is lost, preventing its fixation in the tissue . TerminaHowever, although the SC compartment has a lower proliferative rate, most SCs divide daily . If everSince differentiation can act as a tumor suppression mechanism by impeding self-renewal when a cell divides but does not further differentiate, it constitutes a self-renewing clone ,26. IndeAlthough transcriptomic analysis of in vivo colorectal cancer models shows that distinguishable cell compartments in CRCs are less defined—probably because of the loss of niche factor gradients that maintain them in non-neoplastic tissues—cells expressing stem cell markers are still distinguishable from populations expressing differentiation markers of diverse lineages reminiscent of normal tissues ,15,16,17+/CD38−) as normal hematopoietic cells that are able to repopulate NOD/SCID mice upon bone marrow transplantation [CSCs have customarily been defined operationally: the ability to recapitulate tumors upon serial transplantation of cells from human cancers into immunocompromised mice. Recapitulation of the organization implies self-renewal capacities as well as potency of differentiation, reflecting the function of normal stem cells of adult tissues. The proportion of injected mice that developed disease was used to quantify the efficiency of tumor formation. Serial dilution of cancer cells decreased this efficiency, leading to the hypothesis that only a small subset of cells, from a heterogeneous bulk, possess these tumor-initiating capabilities. These findings set the conditions to search for cell surface markers to identify these tumorigenic cells. Fluorescent-activated cell sorting (FACS) enabled the enrichment of cell populations with cells possessing these markers, increasing tumorigenic efficiency with reduced numbers of injected cells. In the field-foundational study, cells from acute myeloid leukemia (AML) patients were injected into non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID). The leukemia-initiating cells could be selected with the same markers CD34+/CD38− aantation , brain tantation , colorecantation ,6,7, panantation , and ovaantation . CSCs of solid malignancies, as defined above, have been controversial ,9,31. Re+ cells from patient samples to show that these cells could give rise to multilineage differentiated tumors upon implantation [In spite of the limitations of serial transplantation, this method yielded several markers with the potential to identify cells with self-renewal and differentiation capabilities in vivo. The first marker identified for colorectal CSCs was CD133 ,7. In paantation ,35.Refinement of the specificity of putative CSC markers can potentially be achieved by considering alternative splicing . This caAPC gene [+ cells in vivo [Subsequent development of surface markers focused on those that reflect the function of signaling pathways known to be essential for stemness. In terms of activated signaling pathways, strong parallels were shown to exist between physiological and pathological conditions in the intestine ,41. Wnt APC gene ,46,47. TAPC gene . HoweverAPC gene . ColorecAPC gene . These cAPC gene . The advAPC gene  enabled  in vivo , initiatAlthough Lgr5 has been proven to be a bona fide marker of CRC-CSC and can be reliably used in available CRC in in vivo and ex vivo models, there is evidence of CRC not expressing Lgr5 ,17. ThisCre-loxP genetic recombination systems to perform lineage tracing. In the T2Lgr5-EGFP-IRES-CreER mouse model, the Cre recombinase is expressed under the control of the cell-specific Lgr5 promoter. This mouse line was crossed with R26R–lacZ reporter mice containing a loxP-STOP-loxP sequence in front of the reporter gene lacZ. The activated recombinase specifically activates the reporter gene expression in cells expressing Lgr5 by excising the STOP sequence. After the STOP sequence is removed, future descendent cells of the LacZ+ stem cells continue to express the reporter LacZ [+ CBC cells: the generation of multiple lineages and long-term self-renewal. Although this tracing cannot be performed in humans, stem cell dynamics have been successfully studied in the human colon when observing the spread of somatic mutations [Groundbreaking experiments with genetic lineage tracing were key to show that the crypt base columnar (CBC) cells at the bottom of the intestinal crypts function as bona fide stem cells . This meter LacZ . Both stutations ,54,55.+ adult stem cells, or crypts containing the stem cells isolated from the intestine, into three-dimensional multicellular tissue models. These organoids are maintained by the self-renewal of the intestinal stem cells, thus mimicking the in vivo properties of the intestine and remaining genetically and physically stable. The essential role of Lgr5+ stem cells in forming organoids was demonstrated using the Lgr5DTR-eGFP mouse model. In this model, Lgr5+ stem cells express eGFP and the diphtheria toxin receptor (DTR), which enabled only the stem cells to be marked and ablated. Lgr5DTR-eGFP tumor organoids treated with the toxin collapsed after losing their stem cells and were unable to regrow upon toxin treatment maintenance, thus demonstrating the necessity of Lgr5+ stem cells [The ex vivo organoids culture was an important development that enabled the further investigation of stem cell functionality . Upon inem cells ,50.+ stem cells were shown to be required for the formation of normal tissue organoids as well as tumor organoids [Methods developed to culture organoids originating from human adult colonic tissue  or humanrganoids . The tumrganoids .+ cells [+ cells or other cell types of interest during tumor progression and metastasis.Cultivation over long periods of time led to the establishment of biobanks containing a large number of differently characterized tumor organoids ,60,61. F+ cells ,15. Subs+ cells and resulted in restriction of tumor growth, although not in tumor regression. After the withdrawal of DT, the tumor was repopulated by Lgr5+ cells, reinitiating tumor growth. These experiments resulted in two important findings: (1) tumor development in this model of colorectal cancer, which is genetically relevant to the most common human colon cancer subtype, was driven by Lgr5+ cancer stem cells, and (2) tumors could be maintained by proliferative Lgr5− cells that were able to replenish the eliminated Lgr5+ cells. In a spontaneous metastasis model derived from orthotopic tumors, the ablation of Lgr5+ cells did not provoke regression of the primary tumor but abrogated the formation of metastases in the liver. In contrast, metastasis developed rapidly in control animals. Ablation of Lgr5+ cells in experimental liver metastasis after injection of tumor organoids into the portal vein also ablated the metastatic tumor burden to barely detectable levels [The influence of stem cells and the result of their elimination in tumor tissue was also demonstrated with the Lgr5-eGFP-DTR transgenic mouse model . In hetee levels . + cells in human samples [+ cells, Shimokawa and colleagues used CRISPR/Cas9-mediated homologous recombination to establish Lgr5-GFP clones or KRT20-GFP clones—marking differentiated cells—of colorectal cancer organoids derived from human colorectal tumors. Transcriptional analysis showed that Lgr5+ cells were enriched for intestinal stem cell markers, while Lgr5− cells had upregulation of differentiation markers. Xenotransplantation of these organoids on the sub-renal capsule of NOG mice showed that the histological structure recapitulated the original tumor tissue structures. They found Lgr5-GFP+ cells in the outer area of the tumors. Complementarily, the KRT20-GFP+ differentiated cells were located in the inner area [CreERT2 recombinase inserted into the Lgr5 allele. Tamoxifen treatment at 1 month after xenotransplantation into the sub-renal capsule and strict temporal follow-up for 31 more days enabled them to reconstruct the clonal trajectories. Shortly after tamoxifen injection, single Lgr5+ clones appeared colored in the outer regions, followed by structure formation demarcated by the color of the initial clone. Within these structures, Lgr5+ cells gave rise to Lgr5− cells. This clearly indicated self-renewal and differentiation capacities of Lgr5+ CSCs. Re-xenotransplantation showed long-term CSC function [+ CSCs in these processes, they inserted a transgene expressing iCaspase9 into the Lgr5 allele, and upon injection of a Caspase 9 dimerizer compound, triggered apoptosis exclusively in Lgr5+ cells. This led to a reduction in tumor size. However, a few days after treatment, Lgr5+ cells reappeared and tumor growth resumed, indicating a plastic process [The lack of reliable antibodies against Lgr5 had previously hampered the study of Lgr5 samples . To enginer area . They esfunction . To test process .+ cells from xenografts exhibited a significantly higher clonogenic potential than Lgr5-eGFP− in vitro, and were more tumorigenic upon transplantation in vivo, mimicking the conventional approach to cancer stemness. An elegant color switch strategy allowed them to trace the fate of Lgr5+ cells in xenotransplants. They observed heterogeneous growth dynamics: some clones expanded constantly, while others divided slowly or even remained as individual cells over prolonged periods of time [+/Ki67-TagRFP− (L+K−) ranged from 20% to 50% of the clones. FACS analysis after xenograft dissociation showed that L-/K-cells displayed downregulation of proliferation genes, upregulation of the cell cycle inhibitor CDKN1A, and expression of markers of terminal differentiation, suggesting that they were terminally differentiated postmitotic cells. L−/K+ cells displayed low levels of stem cell markers and upregulated genes characteristic of early absorptive differentiation, suggesting that they could be in a state analogous to transit-amplifying cells. L+/K− cells showed downregulation of proliferative genes and retained elevated levels of intestinal stem cell markers [A similar approach using organoids established from a panel of human colorectal tumors was undertaken in parallel by Cortina and colleagues . Lgr5-eG of time . Cortina markers . Interes markers  . They found that tumor growth could be driven by a proportionally minor subpopulation, which secreted factors that enhanced the proliferation of other subclones. Breakage of this cooperative equilibrium due to a predominant, “excessively fit” clone that outcompeted the cooperative minority provoked tumor collapse and regression. Using a mathematical model applied to non-cell autonomous-driven tumors, they reported that clonal interference, which restrains the excessive predominance of a highly proliferative clone, can stabilize sub-clonal heterogeneity, thus maintaining the sub-clonal interactions necessary for tumor development and evolution of new traits [In an approach specifically designed to assess intra-tumoral clonal cooperation, under microenvironmental constraints, Marusyk and collaborators analyzed a panel of 18 subclones of the human breast cancer cell line MDA-MB-468 implanted into the mammary fat pad of immunodeficient w traits . These experimental models show that non-cell autonomous relationships—through direct paracrine action or through microenvironmental regulation—between cancer cell subclones may be essential for tumor development. Although they do not show that the involved cells are derived from differentiating hierarchies, they suggest that tumorigenicity is not exclusively driven by a certain cell type. Whether the phenotypic heterogeneity derived from cell hierarchies in colorectal cancers can entail selective advantages of subclones or cooperative relationships is still to be explored.+ cancer stem cells not only showed the typical stem cell signature, but also a reduction in cell-cycle-related signatures, supporting their potential function in chemotherapy resistance [Recapitulation of tumors’ structures upon recurrence after chemotherapeutic treatment have placed CSCs at the core of drug resistance. Even when chemotherapy has reached a high level of efficiency, a small population of cancer cells may still be resistant, which is often referred to as minimal residual disease . This issistance . Putativsistance ,103.Soon after the extension of the hypothesis of CSCs into carcinomas, and the development of panels of markers to identify this cell subpopulation in various cancers, an empirical link was established between this phenotype and the induction of epithelial-to-mesenchymal transitions (EMT) in breast cancer cells . EMT, a The notion that CSCs confer resistance to anticancer therapies is supported by the observation that tumors that have been treated with chemotherapy can develop an increased population of CSCs. This could be due to the enhanced survival of the compartment of CSCs. However, the idea that EMTs induce stem-cell-like phenotypes from epithelial cells in a differentiated state also implies a mechanism of cancer cell plasticity. Since cytotoxic chemotherapeutic agents can induce EMTs in cancer cells, the accumulation of CSCs after therapy could be due to differentiated cells constantly feeding this compartment through EMTs .−/keratin-19+ (Krt19+) progenitor cells mark radio-resistant cells above the crypt. These cells were able to dedifferentiate into Lgr5+ intestinal stem cells, replenishing the pool of intestinal stem cells damaged by radiation. When they specifically targeted Lgr5−/Krt19+ cells with an inducible APC homozygous mutation, these cells initiated autochthonous malignant tumors containing mutated Lgr5+ cells—therefore generated through cell dedifferentiation—that drove tumor development. Irradiation of these mice 24 h after induction of this mutation did not prevent tumor development and rapid mortality. In contrast, tumors initiated by specifically inducing the same APC mutation in Lgr5+/Krt19− intestinal stem cells did not provoke mortality in mice irradiated 24 h after the mutation induction [+) were radiosensitive, while non-stem cell tumor-initiating clones were radio-resistant. Part of the progeny of the latter subsequently dedifferentiated into Lgr5+ cells and reestablished a hierarchical organization to drive tumor development.Nevertheless, therapeutic resistance is not necessarily restricted to CSCs. Asfaha and collaborators showed that in the intestinal crypt, Lgr5nduction . This meIn tumors such as colorectal cancers with clear cellular hierarchies, independently of which cellular state is more prone to resist therapy, the possibility of phenotypic switches from differentiated cells to stem cells appears fundamental to tumor relapse. This plasticity seems therefore an essential consideration for therapeutic development.From a Darwinian-selection perspective of cancer progression, cell hierarchies add a third source of cancer cell fitness to the one derived from genetically fixed and epigenetically stable acquired traits. Plasticity between cell differentiation states ensures adaptability to selective pressures. In this scenario, selective advantages of cancer cells derive not only from genetic or epigenetic makeups that deploy specific hallmarks of cancer, but also from the ability to dynamically deploy different hallmarks that ensure better fitness in changing environments. Cancer cells can self-renew in microenvironments that are permissive enough to drive tumor progression and give rise to differentiated cells endorsed with specific properties. In turn, differentiated cells can also dedifferentiate, giving rise to CSC when required. This enables cancer cells to either contribute to shaping these microenvironments—in accordance with tumor sub-clonal cooperation—or to overcome a plethora of diverse and dynamic selective pressures. In conclusion, the notion of plasticity between cellular states not only supports the CSC hypothesis in colorectal cancers, but also underscores a role for the differentiated states of tumor cells during cancer progression. This expands the scope of therapeutic development beyond stemness to include other cellular functions, amongst which cell plasticity itself is beginning to emerge as the next target.