Mycobacterium tuberculosis and HIV co-infections. Applications of current and future co-infection models to address important clinical and research questions are further discussed. Humanized mice have become an important workhorse model for HIV research. Advances that enabled development of a human immune system in immune deficient mouse strains have aided new basic research in HIV pathogenesis and immune dysfunction. The small animal features facilitate development of clinical interventions that are difficult to study in clinical cohorts, and avoid the high cost and regulatory burdens of using non-human primates. The model also overcomes the host restriction of HIV for human immune cells which limits discovery and translational research related to important co-infections of people living with HIV. In this review we emphasize recent advances in modeling bacterial and viral co-infections in the setting of HIV in humanized mice, especially neurological disease, and  Pneumocystis pneumonia, Cytomegalovirus) that are important causes of death in people with low CD4+T cell count due to progression to Acquired Immune Deficiency Syndrome (AIDS) . Theorrhoeae –20. Receorrhoeae , 22. HerMtb infection and reactivation; (2) accelerates development of Mtb drug resistance; (3) compromises TB diagnostics; (4) is associated with drug interactions when combining TB chemotherapy and ART; and (5) causes an aggressive course of TB disease [+ T cell depletion and can even continue following restoration of T cells by ART  reviews . Treatme reviews .Addressing these clinical challenges of the HIV and TB co-syndemic requires a much greater understanding of co-infection pathophysiology. Models that permit evidence-based pathogenesis studies are needed to inform development and implementation of countermeasures to complement ART and antimycobacterial therapies in co-infected people. To address this gap, our research group has developed two novel models of HIV/TB co-infection in HIS mice. As described below, these models of active co-infection and TB relapse offer systems to investigate mechanisms of microbial synergy and test novel interventions to treat co-infection.+T cells in vivo can simulate the effect of severe immune compromise, characteristic of AIDS in murine TB models [+T cell loss-independent mechanisms of dual infection. The development of the HIS mouse as a small animal model of TB [Mtb infection that produced caseous granulomatous inflammation consistent with human disease [Mtb-driven inflammation including organization of human CD3+T cells in the periphery of Mtb granulomas adjacent to human macrophages positive for acid fast bacilli (AFB). A human cellular immune response to Mtb infection was also demonstrated in the lung and periphery of infected HIS mice including activation of human effector cytokines (e.g. TNF-α and IFN-γ) that have defined roles in host defense and maintenance of latency. In two separate reports, HIS BLT mice that were infected with Mycobacterium bovis (M. bovis) BCG (vaccine strain) also were shown to mount an adaptive human immune response and develop lung lesions populated by human T cells and macrophages [Mtb, and not BCG [Disease models that use standard mouse strains are indispensable in the TB research field for basic discovery and development of preventives and therapeutics. Depletion of CD4B models . The resel of TB  was an i disease . Lesionsrophages , 35. Dev not BCG , 34. TheMtb co-infection [Mtb challenge to simulate the progression of TB in the setting of active viral replication in a human immune system. Outcomes in this model reproduced several endpoints observed in co-infected persons including increased mycobacterial proliferation, disruption of organized granulomatous lesions including TB pneumonia, and development of exacerbated pro-inflammatory immune responses. Similar to observations in the rhesus macaque model of SIV/TB co-infection [Mtb lesions in the lungs of HIS mice [Mtb-driven sites of inflammation was shown to precede the increase in lung bacterial load.An acute HIV infection model generated with BLT HIS mice was subsequently used as the platform to develop a model of nfection . HIV infnfection  and lungnfection , HIV + cHIS mice . Interes+ cells at the site of Mtb infection could change the local immune response to the advantage of Mtb pathogenesis. Similarly, the inflammatory host response to Mtb could provide a microenvironment advantageous for replication of HIV. This model sets the stage for carefully controlled experiments to address these and other important questions relevant to understanding co-infection pathophysiology.Although further investigations are needed, these results in an experimentally controlled setting suggest that recruitment or proliferation of HIVMtb infections, PWH are more likely to experience reactivation of latent TB, have greater rates of TB chemotherapy failure, and experience increased rates of recurrence after treatment [A majority of the 10.4 million new TB cases that occur annually are in those who have access to TB drug therapy through public health programs . Clinicareatment , 44, 45.reatment , 47.+T cells is linked to latent TB reactivation, treatment failure, or relapse upon drug therapy completion  and isoniazid (INH) to reproduce a Cornell-like model of latency in HIS BLT mice [Mtb infection with RIF/INH promoted an accelerated rate of regrowth in lung and a site of dissemination (liver). These results are consistent with the increased rates of TB relapse that occur following evidence for clinical cure and are increasingly understood to represent regrowth of the original infectious strain of Mtb in human subjects [BLT mice generated with an NSG background were used to develop a HIS mouse model of TB relapse following development of paucibacillary TB due to anti-mycobacterial drug therapy . In thesBLT mice  similar BLT mice , 55. Infsubjects , 45.Mtb following TB chemotherapy and regrowth driven by HIV.A surprising observation in the HIS mouse model of HIV-mediated TB relapse was that regrowth did not appear to coincide with breakdown of contained granulomas as is generally assumed to occur . InsteadMtb including in the T cell zones of lung granulomas. The increased viral replication correlated with increases in several cytokines that activate the HIV LTR  due to Mtb proliferation. An interesting hypothesis supported by these observations, is that localized inflammation at sites of paucibacillary Mtb containment could promote proliferation of an HIV reservoir. In return, or potentially simultaneously, the compromise of local immunity by HIV may permit growth and dissemination of Mtb away from the localized site. The small animal nature of the HIS mouse is an advantage that could be exploited to investigate these and other mechanisms of microbial synergy. Important caveats of Cornell or Cornell-like models that should be considered are the requirement for drug treatment to generate paucibacillary infection, and reports that suggest rates and kinetics of reactivation can be difficult to reproduce with precision in different groups of animals [HIV replication was also observed to increase in lung and liver of mice with relapsed TB as compared to mice with HIV mono-infection. Interestingly, a marked difference was observed in these tissue compartments despite comparable viral load in the blood . RNA sco animals . The addMtb has long been known to be higher than Mtb-naïve counterparts despite comparable plasma values [N. gonorrhoeae infection [Mtb or N. gonorrhoeae [Mtb. Xu and colleagues similarly demonstrated that vaginal shedding of HIV-1 BaL was increased in mice with N. gonorrhoeae co-infection, an outcome also not reflected in the plasma [Several lines of evidence suggest that plasma viral load, which is used diagnostically to assess viral suppression status, does not always reflect replication in other tissue sites. This question is especially relevant in the setting of co-infections where inflammation and other factors can promote HIV pathogenesis in local tissue sites. Notably, viral shedding in lung lavage of those with concurrent a values . Similarnfection . As prevorrhoeae , 20. We + cells throughout humanized mouse tissues is an important endpoint in the model that enable studies of tissue specific pathogenesis. This facilitates investigation of viral replication and latent reservoirs in difficult to assess compartment such as the CNS, as shown with human brain  [Experimental control of the human immune cell populations represents an important advantage of the HIS mouse. Reconstitution of human immune populations lacking specific compartments is emerging as an important tool for understanding unique roles of cell types in HIV pathogenesis. The development of the myeloid only model (MoM) or T cell only model (ToM) demonstrated importance of the myeloid cells in HIV persistence , 62. Surel (LoM) . Long teMtb isolate. The side effects, complexity, access, and cost of the treatments can lead to compliance issues and treatment failure [Treatment of people with both HIV and other infections can be challenging due to the complexity and sometimes lengthy period of combination therapy. ART requires lifelong treatment of anti-retroviral cocktails that may require modifications when combined with other medications for non-communicable and communicable diseases. TB chemotherapy requires treatment with a multi-drug regimen for 6–18 months depending on the relative drug susceptibility or resistance of the  failure . Pharmac failure , 67.Mtb or mock-infected shortly before initiation of ART. Animals were then provided a daily regimen of combination ART to evaluate any effects of active Mtb infection on viral suppression. A 3 week course of ART led to viral suppression in all Mtb-naïve mice consistent with similar reports [Mtb-infected mice at 3 weeks and in 1/5 mice at 5 weeks post-ART initiation. The effects did not reach significance due to variation and small study size. These preliminary findings could suggest that underlying Mtb infection impacts efficacy of ART in this model. Expanded studies with larger animal numbers and more rigorous assessments through measurements of time to viral suppression kinetics are needed. Long term, HIS models may have application in the study of OI-driven inflammation, changes in tissue pathogen distribution (e.g. reservoirs), or other mechanisms of microbial synergy that may compromise ART efficacy.Initial drug toxicity and drug/drug interaction evaluations can be done in normal rodent models, however, efficacy testing is relegated to expensive non-human primates or HIS animal models. Among these, the HIS mouse has proven to be reliable, cost effective and reproducible. An additionally important advantage of the HIS mouse is the potential to model co-infection disease and treatment outcomes along with drug interactions and efficacy. In preliminary studies Fig. , we have reports . In contReports using HIS mice as models of neurological disease are increasing in the literature. Potential utility of these models is greatest when attempting to emulate the pathophysiology of neurological diseases that require or involve the human immune system. Infection with HIV-1, which leads to HAND, fits that paradigm well , 69. HIVIn patients given ART, HIVE and CNS viral replication virtually disappear while HAND persists. Thus, neuropathological and neurovirologic substrates of HAND remain essentially unknown, which in turn makes it difficult to emulate the clinical picture in animal models. Attempts to reproduce HIVE pathology in HIS mouse models are interesting, and represent an essential step forward . DiseaseSome studies using HIS mice have taken steps to replicate aspects of HIV infection of the CNS. The first and most basic hurdle was to produce CNS infection with HIV-1 in the HIS mouse. This has been demonstrated in several reports that utilized a variety of HIS models –79. ThesThe neuropathological changes observed in many HIS mouse models are usually subdued and do not represent the full neuropathological picture of HIV encephalitis (HIVE). Only moderate inflammatory changes have been produced , 80, 81.Engineering rodent models of CNS impairment that faithfully emulate HIV infected patients with viral suppression appears to be within sight, but many challenges remain. Human astrocytes are infected with HIV-1 and may contribute to the size of the latent HIV DNA pool in the CNS. Current HIS mouse models may be less suitable to study that aspect of HIV latency. As well, the regional distribution of infected human cells within the CNS of HIS mouse models might differ from the CNS of people living with HIV infection. High levels of HIV-1 replication in the human brain tend to occur in deep brain structures such as the basal ganglia and thalamus. The mapping of the latent (not replicating) pool of HIV-1 also needs to be elucidated in these models for correlation with human CNS infection. Comparative neurovirology between human and rodent infections is an important aspect with implications regarding the elucidation of circuit level changes in CNS function in patients with HAND. Perhaps most vexing of all is that populations of HIV infected patients harbor comorbidities with higher prevalence than noninfected populations, particularly including substance abuse and chronic exposure to therapeutic drugs with potentially adverse effects in the CNS. These ongoing complexities suggest that specific HIS mouse models may be required to emulate key aspects of human CNS infection.Further development of HIS mouse models should take the step forward of emulating the CNS inflammatory picture of HIVE, with CNS dysfunction when replication is not suppressed. A key subsequent step involves treating the HIV-infected animals with ART to prevent HIVE and reduce the intensity of CNS dysfunction, while not eliminating it, as observed in ART treated patients. Virally suppressed HIS mice should contain a pool of latent HIV DNA that is comparable to what is observed in human brain specimens including its size, its responsiveness to latency reversing agents, and its neuroanatomical distribution. As well, the HIS mouse response to psychological testing should correspond to systemic anomalies correlated with HAND, such as the association with bacterial gut translocation , systemiPrior to the ART era, CNS forms of several infections such as cryptococcal meningitis, cerebral toxoplasmosis, and tuberculous meningitis were associated with severe clinical outcomes . Neuro-TMtb. Our previously unpublished work shown in Fig. Mtb outcomes. In a limited survey to date, we have occasionally observed acid-fast bacilli (AFB) in the brains of humanized mice after several weeks of Mtb infection  increase the CNS viral load or reservoir size; if systemic or tissue compartment inflammation due to OIs promotes HIV CNS outcomes; whether OIs contribute to HIV Associated Neurological Dysfunctions; and what are the mechanistic bases for increased rates or more severe CNS outcomes of OI in those with HIV? Demonstrations that important endpoints of CNS co-infections could be realized in the HIS mouse models would also support use in translational studies to test clinical interventions.Due to their similar routes of transmission, it is increasingly common for PWH to have a hepatitis virus co-infection. Of the current 39 million PWH , 10% areHIV/HBV co-infection exacerbates morbidity and mortality of both viruses, with death rates of co-infected individuals being 8 times that of HIV infection and nearly 20 times greater than HBV infection . Much ofMuch of the difficulty of studying these co-infections stems from the challenges to establishing animal models of infection. The human-restricted tropism of all three viruses has proven to be nearly insurmountable to gaining insights into single or dual infections. A number of potential models described below have been developed in attempts to overcome this obstacle.−/− IL2Rg−/− mice under the control of the albumin promoter, allowing for the inducible destruction of mouse hepatocytes [+ hematopoietic stem cells (HSC) into the irradiated neonatal mouse liver along with the human hepatocytes [AFC8 mice were originally created to allow for human hepatocyte engraftment into mice to facilitate the study of HCV pathogenesis. A fusion protein consisting of KF506-binding protein (KFBP) and caspase 8 was knocked-in to Balb/c Rag2atocytes . Neonataatocytes . The engrag1−/−/il2rg−/− background with a mutation that disables the fah gene, which encodes fumarylacetoacetate hydrolase [Other liver xenograft models could be similarly adapted to HIV/hepatitis virus co-infection. The FNRG mouse is a NOD/ydrolase . These mydrolase , 96.−/− IL2Rγ−/− NOD.sirpa uPAtg/tg (BRGS-uPA) strain that carries the same hepatocyte-lethal phenotype, but is better able to support establishment of a human immune system due to the expression of human SIRPα [A similar approach was based upon Alb-uPA/SCID (uPA) mice. In the uPA model, the urokinase-type plasminogen activator was placed under the control of the albumin promoter. The accumulation of plasminogen activator results in mouse hepatocyte death, which are subsequently replaced by engrafted human hepatocytes. Strick-Marchand et al. established a Balb/c Rag2an SIRPα . These m. reconstituted TK-NOG mice with human hepatocytes and CD34+ HSCs. The resulting mice showed functional human hepatocytes secreting serum albumin and human CD45+ lymphocyte populations consisting of CD4+ and CD8+ T cells and B cells. These humanized liver and immune system mice were subsequently infected them with HIV. Decreases in CD4:CD8 ratios and serum albumin levels were detected in at 5 weeks post-infection. This successful demonstration of HIV infection in this model makes the TK-NOG-hu-HSC /Hep model a leading candidate for the study of HIV/Hepatitis virus co-infection.The TK-NOG mouse  a NOD/SC+ cord blood HSCs would be particularly useful, given the regional restrictions on use of human fetal tissues. Such HIS-huHEP mouse models would have significant advantages over other potential animal models. SHIV/HCV co-infection of chimpanzees (Pan troglodytes), for example, should be feasible, but would be prohibitively expensive and challenging from a regulatory standpoint in many parts of the world. HIS-huHEP mice are cheaper, more easily housed and enjoy a far greater breadth of available reagents. Other potential animal models lack the ability to study genuine human-infectious pathogens in the context of a human immune response. HIS-huHEP mice afford the opportunity to study native HIV, HBV and HCV, in the appropriate host cells, in the context of a relevant human immune response. Studies of liver fibrosis, immune cell dysfunction, HIV viral reservoirs and a host of other pathologies of co-infection are all uniquely possible in these systems.Hepatic replacement models overcome the challenge of infecting mice with viruses that have a specific tropism for human cells, such as HIV, HBV and HCV. While none of the above models, to our knowledge, has yet been used to examine HIV/HBV or HIV/HCV co-infection, they should all be suitable for that purpose. Models that can establish a human immune system from CD34These models may have some shortcomings, however, particularly with regard to vaccine testing and development, owing to the incomplete reconstitution of the B cell compartment in most HIS mouse models. Also of significant concern is the matter of immune cell compatibility between the tissue donors. Graft vs. host disease is an ongoing issue in HIS mice, with the reconstituted immune system attacking the host animal as non-self. HIS-huHEP mice will likely have similar issues, possibly compounded by graft vs graft disease, with the reconstituted immune system reacting to both the host animal and the engrafted liver if they are not sourced from the same donor.More recently, a simpler model has been shown to be useful in the study of HCV infection. In the HIL model , the injThe HIL model has significant advantages over other HIS-huHEP models. It is much simpler to establish, as it does not require murine hepatocyte ablation. Hepatic progenitor cells have been shown to engraft directly into the mouse liver . It alsoAnother set of models that may be useful in the study of HIV/HBV or HIV/HCV coinfection are those that carry human leukocyte antigen (HLA) genes. HLA transgenic (HLA-tg) mice were developed to study the nature of antigen recognition by T cell receptors in the context of HLA molecules , and lat+ HSCs (HLA-tg/NSG-HIS), hopefully overcoming the limited T cell-dependent B cell activation and cytotoxic T cell killing often observed in reconstituted NSG mice.This model was further adapted to the study of HIV by crossing HLA-tg mice with NSG mice (described above), resulting in HLA-tg/NSG mice . These mThe HLA-tg/NSG-HIS model could be further applied to the study of HIV/HBV or HIV/HCV co-infection. Such models can be chronically infected with HIV, and either pulsed with HLA-specific hepatitis virus antigens or hydrodynamically injected with DNA encoding hepatitis virus proteins. Hydrodynamic injection is a method in which HCV cDNA or an HBV encoding plasmid is injected intravenously in PBS equivalent to ~ 10% of the animals’ weight in less than 10 s . This raHIV-infected, viral protein producing, HLA-tg/NSG-HIS mice could have utility in a number of areas. They would be particularly useful in studying the immune reaction to HBV or HCV proteins in the context of HIV infection, as all the immune dysfunction of HIV infection would be present. Similarly, candidate hepatitis vaccines intended for use in PWH could undergo initial animal testing in HLA-tg/NSG-HIS mice. The effects of HBV or HCV infection on the establishment and maintenance of the latent HIV reservoir could also be examined is this model, as transfected hepatocytes replicate most aspects of chronic viral infection, including long-term inflammation.The HLA-tg/NSG-HIS model would be of more limited utility in studying the mechanisms by which HIV infection exacerbates viral hepatitis, as this model does not replace murine with human hepatocytes. While most viral proteins are expressed and most aspects of infection are reproduced, the lack of viral replication, cell-to-cell spread and dsRNA in infected cells are formidable obstacles to the study of hepatic disease. Such investigations would be best left to the hepatic replacement models described above.Chlamydia trichomonas, N. gonorrhoeae, and Herpes simplex-2 also increase the risk for HIV infection through reductions in barrier effectiveness, activation of HIV co-receptors, and inflammation [Neisseria gonorrhoeae, however, suggest microbial synergy may also occur [N. gonorrhoeae co-infection has been demonstrated, including after viral suppression with ART [A bidirectional relationship between HIV and several sexually transmitted infections (STI) is well established. Beyond the common route of transmission and overlapping epidemiological factors, HIV increases the susceptibility to, and disease outcome of, many STIs . Similarammation . Clinicaso occur , 111. Inwith ART . The mecN. gonorrhoeae co-infection [+ stem cells are well populated with human immune cells. Importantly, human T cells including abundant CD4+CCR5+ T lymphocytes that support productive HIV replication were described. Viral shedding could be easily detected in vaginal washes of mice that had been infected with HIV by an i.p. route. Co-infection with N. gonorrhoeae was observed to increase the time to establishment of HIV proliferation in the reproductive tract, and to markedly increase viral shedding in vaginal washes, compared to HIS mice infected with HIV only. The description of this small animal model of HIV and N. gonorrhoeae supports further investigations of mechanisms driving microbial synergy and provides a translational platform to test microbicides and other clinical interventions. Long term, the demonstration that the HIS mouse repopulates these important human immune components and supports HIV infection in the gut and reproductive tract further supports use of the CD34+ cord blood models for co-infection studies when use of the BLT mouse is limited.An important advance for modeling outcomes of HIV in the reproductive tract has been the demonstration of rectal and vaginal mucosal infections in the HIS mouse , 16, 61.nfection . Xu and Development of HIS models of HIV co-infections with other important pathogens which are transmitted sexually is feasible. Productive infection with herpes simplex virus 2 (HSV-2) and development of human immune responses to the virus was previously demonstrated in an HIS mouse developed using cord blood stem cells . Once deImprovements in engraftment of human immune compartments in xenochimeric mice continue to support expanded applications of these models to HIV research. These advances have now permitted successful exploration of HIV disease in multiple compartments, including co-infections of the respiratory system and reproductive tract, that reproduce important features of the respective human disease. Recent advances in hepatocyte regeneration have further enabled development of hepatitis virus models in HIS mice that could additionally support modeling of HIV co-infections with these important causes of cancer. Many caveats to the use of HIS mice to study HIV as well as HIV associated co-infections remain, most notably the xenochimeric immunity and physiology. Care must be taken to define the research questions as appropriate to the model advantages and limitations. Validation of the outcomes using human specimens and human clinical research is also central to ensuring advances have translational relevance. Long term, HIS mouse models of HIV-associated co-infections are likely to yield key insights into mechanisms of microbial synergies and provide an informative and economical platform for testing clinical interventions.


The COVID-19 pandemic caused disruptions of public life and imposed lockdown measures in 2020 resulted in considerable reductions of anthropogenic aerosol emissions. It still remains unclear how the associated short-term changes in atmospheric chemistry influenced weather and climate on regional scales. To understand the underlying physical mechanisms, we conduct ensemble aerosol perturbation experiments with the Community Earth System Model, version 2. In the simulations reduced anthropogenic aerosol emissions in February generate anomalous surface warming and warm-moist air advection which promotes low-level cloud formation over China. Although the simulated response is weak, it is detectable in some areas, in qualitative agreement with the observations. The negative dynamical cloud feedback offsets the effect from reduced cloud condensation nuclei. Additional perturbation experiments with strongly amplified air pollution over China reveal a nonlinear sensitivity of regional atmospheric conditions to chemical/radiative perturbations. COVID-19-related changes in anthropogenic aerosol emissions provide an excellent testbed to elucidate the interaction between air pollution and climate. As a result, large parts of the manufacturing sector in China halted temporarily, causing a widespread reduction of anthropogenic aerosol emissions after the Chinese New Year holidays . This is illustrated by the rapid drop in atmospheric NO2 concentrations in February 2020 relative to previous years, which attained values of 40–70%  at 550 nm5 , massive public health interventions were implemented in China from January 20209, massivnm5 Fig. . Consequ10. An abrupt reduction or increase of anthropogenic aerosol emissions could therefore affect weather and climate. To quantify potential effects of the COVID-19 lockdown measures on current and future climate, coordinated earth system model experiments have been conducted13. The main conclusion from these perturbation simulations is that the aerosol reduction effects on climate are insignificant or very small on global scale. This might be because the emission reductions are not enough in magnitude and time to have a strong effect on a global climate13. On the other hand, widespread warming over parts of eastern China, South Asia, Europe, and the eastern United States occurs in the emission reduction simulations due to the fast climate response to aerosol reductions14. The ongoing and controversial discussion in recent studies highlights how difficult it is to deduce the magnitude of aerosol effects on climate18 from model simulations as well as from short observational datasets, in particular, in the presence of strong synoptic weather noise19. Whether the overall reduction of certain types of anthropogenic aerosols influenced regional weather and climate conditions still remains unclear, thus analyses on regional scales are required to understand the impact of COVID-19-related emission13.Aerosols are known to alter the energy budget through aerosol-radiation and aerosol-cloud interactions21. The unusual aerosol conditions in early 2020 in China and other parts of the world subsequently, could serve as a testbed to benchmark the performance of state-of-the-art earth system models and their representations of aerosol-radiation and aerosol-cloud interactions. It is therefore paramount to determine whether temporary lockdown measures in China from late winter to spring of 2020 impacted air quality, weather, and climate patterns across East Asia. Hence, we conduct a suite of idealized aerosol perturbation ensemble sensitivity experiments using the Community Earth System Model (CESM) and compare the main results to observations to elucidate whether the COVID-19 lockdown measures impacted East Asian climate.Furthermore, resolving the aerosol effects on weather and climate is necessary for understanding how future implementation of air pollution mitigation strategies nationally and/or internationally will impact regional and large-scale climate conditions22 with ~ 1° × 1° atmospheric and oceanic horizonal resolutions. There are 32 atmospheric vertical levels and 60 oceanic levels. We conduct a control experiment (CTL) and two aerosol perturbation experiments. The aerosol perturbation experiments include a weak anthropogenic aerosol emission experiment (W_AER) and a strong anthropogenic aerosol emission experiment (S_AER).To determine the influence of a short-term reduction in anthropogenic aerosol emissions on regional climate over East Asia we employ the CESM version 2.1.223 and greenhouse gas concentrations24 corresponding to the 2010 CE observational estimates. We then identify the January sea surface temperature (SST) state that shows the closest resemblance (in a root mean squared error sense) between the spin-up simulation and the observed SST anomaly for January 2020 obtained from HadISST25 and ERSST26 datasets. The selected year of the spin-up run is then used as an initial condition for three experiments.To prepare the initial conditions for these sensitivity experiments, we perform a 60-year-long present-day spin-up simulation with annually repeating anthropogenic aerosol emissions23. In the W_AER, we reduce local anthropogenic aerosol emissions over East Asia  in February using the ratio between satellite-derived February NO2 concentrations in 2020 and 2018–2019 ‐based emission values (year 2010) of anthropogenic aerosols originating from energy, industry, residential, transportation, agriculture and waste sectors 019 Fig. a. We chaive Fig. b. This i–14 K) atmospheric temperature initial conditions (micro-perturbations) are applied for the ensemble runs. This approach overcomes potential difficulties in identifying aerosol-cloud interactions statistically from limited observational datasets which exhibit large unforced variability in cloud properties19; but it relies on the specific parameterizations of aerosol-cloud microphysics processes in the CESM.Starting from initial conditions on January 1st we conduct a 20-member ensemble of 1-year-long integrations for each experiment, i.e., CTL, W_AER, and S_AER. Slightly different  with the weekly composite difference between conditions after and before the Chinese New Year holiday. The composite is based on the periods from 2011 to 2020. Although China implemented lockdown measures in Wuhan already on January 23, 202030, these were quite localized initially. Thus, the inclusion of the year 2020 in the composite of Chinese New Year holidays does not affect our conclusions.Regular or occasional reductions of the human and industrial activities can change the concentration of anthropogenic air pollutants. For example, similar to the COVID-19 lockdown, the extra measures taken during the Beijing Olympic Games in summer 2008 decreased PM2.5  by 48–56% over northeastern China2 concentrations) over the eastern China .To better understand the robustness of the observational results Fig.  and idenTo explore the robustness of the cloud response and potential nonlinearity of climate responses to aerosol forcings, we further investigate the AOD and cloud response in the S_AER, which applies unrealistically high anthropogenic aerosol emissions. As expected, AOD increases substantially over China Fig. b. As the31, potentially offsetting the direct CCN effect.To better explain why the drastic changes in CCN in W_AER and S_AER do not translate into larger changes in cloud cover, we hypothesize here that the radiative effect induced by changes in aerosol concentrations leads to anomalous surface warming or cooling, respectively, causing low and high sea level pressure anomalies. This in turn leads to anomalous moisture advection towards China and subsequent changes in clouds32. This process can destabilize the atmosphere and enhance vertical mixing33. Even though we observe the AOD reduction in most parts of China, the increased downwelling shortwave radiation is most pronounced in southeastern China. This mismatch can be explained by increased low-level cloud cover in northeastern China as shown in Fig. To test this hypothesis, we examine the possible contribution of aerosol-induced changes in circulation and moisture transport to cloud formations. In the W_AER, the reduced AOD leads to an increase in downwelling shortwave radiation Fig. a, result13 even on regional scales. Owing to the strongly imposed perturbation, the S_AER simulations show a more robust response: enhanced aerosol loadings reduce downwelling shortwave radiation, leading to surface cooling, anomalous high-pressure and northerly winds , which apply idealized and unrealistically strong air pollution over China, show a climate response that is comparable in magnitude (opposite in sign) to that of the much weaker forcing simulation (W_AER). This result further underscores the complexity and potential nonlinearity of the negative dynamical cloud feedback, which operates in late winter in East Asia. According to the simulations presented here, air pollution in boreal winter has the tendency to strengthen the East Asian winter monsoon, in agreement with previous studies42. How regional-scale weather and climate conditions elsewhere responded to the changes in anthropogenic aerosol concentrations will be left for future work. What is clear is that the observational data collected during the periods of rapid and constrained anthropogenic radiative perturbations in 2020 will become an invaluable future resource to test and calibrate climate and atmospheric chemistry models, which in turn will help in determining the impacts of future long-term air pollution mitigation strategies on our climate system.In 2020 many other countries imposed similar lockdown measures to battle the spread of the COVID-19 disease. Moreover, global transportation decreased, which together led to a temporary improvement of global air quality in 2020Supplementary Information.


Brassica napus genome, providing an opportunity for their utilization in enhancing blackleg resistance.Quantitative resistance (QR) loci discovered through genetic and genomic analyses are abundant in the Brassica napus , even before major resistance genes (R-genes) were extensively explored in breeding programmes. In contrast to R-gene-mediated qualitative resistance, QR reduces blackleg symptoms rather than completely eliminating the disease. As a polygenic trait, QR is controlled by numerous genes with modest effects, which exerts less pressure on the pathogen to evolve; hence, its effectiveness is more durable compared to R-gene-mediated resistance. Furthermore, combining QR with major R-genes has been shown to enhance resistance against diseases in important crops, including oilseed rape. For these reasons, there has been a renewed interest among breeders in utilizing QR in crop improvement. However, the mechanisms governing QR are largely unknown, limiting its deployment. Advances in genomics are facilitating the dissection of the genetic and molecular underpinnings of QR, resulting in the discovery of several loci and genes that can be potentially deployed to enhance blackleg resistance. Here, we summarize the efforts undertaken to identify blackleg QR loci in oilseed rape using linkage and association analysis. We update the knowledge on the possible mechanisms governing QR and the advances in searching for the underlying genes. Lastly, we lay out strategies to accelerate the genetic improvement of blackleg QR in oilseed rape using improved phenotyping approaches and genomic prediction tools.Quantitative resistance (QR) has long been utilized to manage blackleg in  Brassica napus  is a major source of oil for the human diet and industrial applications. After soybean, it is the second most important oilseed crop, with an estimated global production of over 76 million tons in 2017 (FAOSTAT Brassica rapa (AA) and Brassica oleracea (CC), occurring around ~ 7500 years ago . This interaction prompts a cascade of molecular events to effect immunity  have already been functionally tested and cloned  in B. napus genome  is defined as a type of plant immunity conditioned by minor-effect genes, resulting in a distribution of phenotypic values that deviate from the classic Mendelian segregation  and Darmor QR recorded fewer leaf lesions and stem canker compared with lines devoid of QR until the 5th year of the experiment. The study also found that initial avirulence/virulence alleles remained unchanged over the five-year trial in plots planted with lines containing the Darmor background and the inoculum recovered from these lines was significantly fewer compared to non-Darmor lines. This decrease in infection may be attributed to the combined action of R and QR genes against L. maculans: the former restricts pathogen colonization in cotyledons during the early disease development, while the latter prevents further pathogen growth in the petiole and stem. These observations were consistent with the follow-up experiment by Delourme et al. . Polymorphisms in these CRMs were also detected between gene promoters of resistant and susceptible cultivars, indicating that these CRMs have functional roles in disease resistance  to function in plant defence biosynthetic pathways  has been demonstrated by the inconsistent detection of QTL and their variable genetic effects when tested in different environments. For instance, only one out of the 13 QTL for internal infection was consistently detected in a QTL mapping experiment based on a 2-year field evaluation  estimate compared with internal infection rating  , it was only since the availability of DNA markers that thorough genetic investigations have been carried out. These studies have established the complex genetic control of blackleg resistance. Early linkage and QTL mapping using bi-parental crosses for mining blackleg resistance loci were conducted using low-throughput markers including restriction fragment length polymorphism (RLFP) and random amplified polymorphic DNA (RAPD) markers  plants on which most of these mapping studies were conducted. DH populations are genetically uniform, allowing replicated evaluation in multiple environments  and Lm09 (A09) into the susceptible cultivar Yudal resulted in significantly lower stem canker infection in near-isogenic lines (NILs). In contrast, NILs carrying LmC2.1 and LmC2.2 (C02) and LmC4.2 (C04) QTLs exhibited severe stem canker infection comparable to the susceptible checks and hence were considered not effective for reducing blackleg at the mature stage.Several QTL analyses have been conducted to discover and/or confirm the presence of QR alleles in Darmor. Four genomic regions controlling field plant survival (P) and stem canker severity expressed as disease index (I) were located in linkage groups (LGs) 2 (C02), 3 (C04), 5(A09) and 11(A02) of the Darmor/Yudal (DY) genetic map  to create the first three mapping populations, while the fourth population was derived from an AVSapphire/Rainbow cross. Composite interval mapping (CIM) detected one QTL on chromosome C05 of the Caiman/Westar cross, and chromosomes A01/C01 of the Canberra/Westar cross, while multiple QTLs were mapped on chromosomes A01, C01 and LG 1 from the AVSapphire/Westar DH population. In contrast, no significant QTLs were detected in the Rainbow/AVSapphire cross, which might be since they are both resistant cultivars, and there is no segregation of resistance alleles. The A01 QTL was suggested to be the same QTL identified in the Skipton/AgSpectrum DH population, designated as QRlm.wwai-A1a. This region, together with another QTL located on chromosome A10, designated QRlm.wwai-A10, showed consistent effects at both the cotyledon and adult plant stages  to create the DH mapping populations designated as TC (Topas/AG-Castle) and TS (Topas/AG-Sapphire)  has not been identified to date, the identity of this new locus remains to be established. In another study  was initiated by the Multinational Brassica Genome Project . At present, reference genomes for other major species, B. carinata  , enables a genome-wide investigation of genes involved in host defence Metzker . In mostBednarek , b, c. FBednarek  found geBrassica diseases for which resistance is primarily based on QR, including stem rot and black rot, several genes were found to contribute to QR. These include genes functioning in hormone synthesis, production of secondary metabolites, regulation of transcription factors, several metabolic pathways   are categorized as variable genes , as originally proposed by Meuwissen et al. , is a foFusarium head blight of barley  models are originally designed to capture minor-effect loci, more advanced algorithms can incorporate prior biological information such as major effect genes or regulatory regions fitted as fixed effects in several models. These include rrBLUP, and Bayesian models, BayesA, BayesB, BayesCπ, Bayesan R, Bayesan RC and Bayesian LASSO (Heslot et al. The accuracy of genomic prediction is dependent on many factors including trait heritability, linkage LD decay, marker density and size of the training population (Daetwyler et al. As LD decays more rapidly, increased marker density and training population size is required for a more accurate prediction. In this respect, prediction for individuals distantly related to the training population generally results in lower accuracy. In Fikere et al. , predictBrassicas, there are ongoing efforts to increase the quality of genomic resources by capturing as much genetic variation within the species through building pan-genomes/super pan-genomes. The availability of such genomic resources for which thousands of markers can be derived and the development of statistical models that can integrate more biological information and multi-trait data will complement GP's implementation for improving blackleg resistance and other yield-related traits in Brassicas.The rapid development in sequencing is resulting in the discovery of high-quality genome-wide markers. In Brassica germplasm, but also uncovered the complicated genetic and diverse molecular mechanisms controlling QR. This complexity often hinders the full exploitation of blackleg QR in breeding. Recent developments in genomics along with the improvement in phenotyping approaches have helped unravel the genomic organization of blackleg resistance loci in the Brassica genome, resulting to the discovery of genome-wide markers that are facilitating the identification of candidate genes underlying QR through association genetics and comparative genomics. The manipulation of these genes is expected to further elucidate the underlying mechanisms of QR-mediated blackleg resistance.Quantitative resistance was the primary form of blackleg resistance selected in most oilseed rape cultivars before R-genes were explored for breeding. QR has been shown to afford a broad-spectrum control and its modest genetic effects render the blackleg pathogen to less likely evolve; hence, its effectiveness is more durable compared with R-gene-mediated immunity. However, the study of the underlying mechanisms through conventional genetic analysis has been hampered by its sensitivity to GxE interaction. The past decades saw an overwhelming exploration of DNA markers, through QTL mapping and GWAS to study the genetic basis of quantitative resistance against blackleg. These efforts not only resulted in the identification of several loci conferring QR in range of Brassica genome and pan-genome references as more sequence information derived from B. napus lines and morphotypes is generated. These improved genome references will encompass both genic and non-genic regions in Brassica species, which will greatly assist in understanding the overall genomic architecture of important traits including quantitative resistance. In addition, the integration of other omics approaches, such as transcriptomics, phenomics and epigenomics will likely improve the understanding of the genetic and molecular underpinnings of quantitative resistance. When integrated with advanced statistical models, this wealth of biological information will result in a more accurate prediction of the genetic performance of lines in GS, thereby accelerating the improvement of blackleg resistance and other important agronomic traits that directly affect yield in oilseed rape.Advances in genome sequencing are projected to further improve the quality of current L. maculans continues to threaten global oilseed rape production due to its highly dynamic biological nature that adapts to changes in crop production practices brought about by global climate change, the need for more effective and sustainable means of disease control is becoming even more important. This can be achieved through the adoption of improved agronomic practices and systematic deployment of genetic resistance. While the deployment of major R-genes will remain an important component of blackleg management in oilseed rape, their effectiveness could be further enhanced by incorporating QR, resulting in a more durable and sustainable control strategy. Thus, the exploration and utilization of QR in oilseed rape cultivar development will become even more critical and relevant to blackleg disease management.As


Organoids—cellular aggregates derived from stem or progenitor cells that recapitulate organ function in miniature—are of growing interest in developmental biology and medicine. Organoids have been developed for organs and tissues such as the liver, gut, brain, and pancreas; they are used as organ surrogates to study a wide range of questions in basic and developmental biology, genetic disorders, and therapies. However, many organoids reported to date have been cultured in Matrigel, which is prepared from the secretion of Engelbreth-Holm-Swarm mouse sarcoma cells; Matrigel is complex and poorly defined. This complexity makes it difficult to elucidate Matrigel-specific factors governing organoid development. In this review, we discuss promising Matrigel-free methods for the generation and maintenance of organoids that use decellularized extracellular matrix (ECM), synthetic hydrogels, or gel-forming recombinant proteins. Kozlowski, Crook, and Ku review current state-of-the-art techniques in culturing and manipulating organoids without the use of EHS-tumor-derived matrix, the most popular of which is Matrigel by Corning. They cover organoid-based applications of different matrix types, ranging from natural sources , synthetic sources (e.g. PEG-based), and recombinant peptide-based systems. Organoids recapitulate important organ functions in vitro while remaining small in size and often free of interfering cell types such as vascular, nerve, or other undesired epithelial cells. For these reasons, organoids are used to study organ development2 and model various diseases such as cancers3, neural disorders4, and autism5; they are also used as pharmaceutical testing platforms6, model systems for CRISPR-CAS9-mediated treatment of genetic diseases7, and replacement organs for transplantation9. It is possible to culture organoids from induced pluripotent stem cells (iPSCs)10 or adult stem cells from patient tissues11, which may lead to personalized medicine. The wide range of clinical applications of organoids is the subject of a recent review by Drost and Clevers12. Many excellent reviews have been published about different organoid types, such as heart14, brain17, liver19, kidney22, pancreas25, and female reproductive tract26.Organoids are multicellular structures derived from stem and progenitor cells that mimic the function and spatial organization of organs27. In an early report of organoid culture, Sato and colleagues grew murine intestinal Lgr5+ stem cells in high concentrations of Matrigel supplemented with the growth factors WNT, Noggin, R-spondin, and EGF28. This culture system has been widely adapted for other organs such as the colon, stomach, and liver33. Related methods have been used to construct simulated versions of the inner ear34 and pancreas39, and the similarities between pancreatic and liver-organoid-generating cells suggest that methods used for making liver organoids may be applicable to the pancreas40. The many organoid-specific applications of Matrigel-based culture methods have been thoroughly discussed elsewhere43.Many organoids have been cultured in Matrigel, a material derived from the secretion of Engelbreth–Holm–Swarm mouse sarcoma cells and enriched for extracellular matrix (ECM) proteins44. The undefined nature of Matrigel makes it difficult to identify the signals necessary for organoid structure and function; this difficulty is compounded by lot-to-lot variations of Matrigel48. Furthermore, Matrigel may not contain all of the necessary components for proper organoid formation; gut organoids cultured in Matrigel lack the characteristic architecture of mammalian intestines, which could be due to a sub-optimal amount of laminin-511 and the absence of other cell types such as mesenchymal cells50. Finally, it has become increasingly clear that the mechanical properties in three-dimensional (3D) culture systems can have large effects on cell51, organoid52, tissue53, and organ development55. The mechanical properties, such as elastic modulus, pore size, stress relaxation, and creep59, cannot be easily separated from the chemical cues in the Matrigel-based culture systems. Furthermore, the mechanical properties of Matrigel samples are heterogeneous; local regions of such gels have been found to exhibit elastic moduli several times higher than the average modulus of the sample61. Finally, the fact that Matrigel is originated from mouse cells hampers its use in human clinical transplantation due to potential immunogenicity18.Despite its versatility and affordability, Matrigel is extremely complex; proteomic analysis shows that it contains more than 1800 unique proteinsGiven these limitations, there is an emerging need to develop Matrigel-independent organoid culture methods. In this review, we discuss recently-developed Matrigel-free techniques for the culture of organoids. We will review undefined matrices, focusing on ECM derived from decellularized tissues and collagen, and defined matrices, including synthetic polymer hydrogels and engineered ECM proteins Fig. . Table 162. In order to accurately recapitulate the composition, structure, and vascularization of native ECM in organ development, some organoids have been grown in decellularized ECM from human or animal donors. The methods of decellularization used are dependent on the target tissue and not readily generalizable; a number of these methods have been reviewed elsewhere63. While xenogeneic ECM has the potential to cause immune responses, this risk can be greatly reduced by using proper preparation techniques64; similar ECM scaffolds derived from animals are FDA-approved for clinical applications such as heart valve replacement, facial reconstruction, and osteopathic implants65. Decellularized ECM may also provide additional cues that promote regeneration of damaged tissue, ultimately supporting the organoid transplant and promoting its function66. Decellularization approaches have been demonstrated for human kidney67, murine kidney68, murine heart70, human and porcine lung71, and porcine testicular72 tissues, with each type posing unique challenges. To illustrate some of these challenges and methods, we will focus on the decellularization of the liver, gut, and pancreas.In organ development, ECM proteins provide signaling cues, serve as an adhesive substrate, and sequester growth factors Fig. 62. In or73. In contrast, Baptista and colleagues perfused Triton X-100 and ammonium hydroxide through a ferret hepatic vascular network to remove cells. This method preserved the underlying ECM and vasculature while retaining most of the glycosaminoglycans, collagens, and elastins. The decellularized material could be colonized by human fetal liver and endothelial cells to produce a functioning organoid74. An illustration of the method of Baptista and coworkers is shown in Fig. 75. More recently, Saheli and colleagues seeded sheep liver ECM gel with a combination of human hepatocarcinoma cells, mesenchymal stem cells, and umbilical cord stem cells; the resulting tumor organoids had greater hepatocyte function than tumor organoids grown in comparable collagen I-based culture76. Lewis and colleagues observed that growing murine small cholangiocytes (a committed progenitor cell type) in porcine liver ECM gel resulted in the formation of complex, branching structures similar to biliary ducts, and these cells also secreted small amounts of bile. In contrast, cholangiocytes cultured in Matrigel formed cysts while those in collagen I proliferated and spread in all directions without spontaneously forming structures77. Thus, Matrigel alone does not provide all of the needed factors for small cholangiocyte differentiation, whereas a decellularized liver ECM gel appears to be a better alternative.Liver-specific ECM can be obtained from a surgically resected portion of a patient’s damaged liver, or from livers unsuitable for transplantation. Lin and colleagues reported that liver tissue decellularization supported growth and maintenance of rat hepatocytes; however, this method relied on mechanical disruption of resected tissue, which resulted in the loss of organ architecture and vascular networks78. Finkbeiner and colleagues found that undifferentiated human embryonic stem cells (ESCs) could not directly differentiate into HIOs in a decellularized porcine intestinal ECM. However, pre-differentiated HIOs were able to seed onto decellularized porcine intestinal ECM and form correct spatial orientation-mimicking intestine79. Giobbe and colleagues developed a method for decellularizing the porcine small intestine to form an intestinal ECM gel similar to the liver ECM gels discussed in the previous section. The porcine intestinal ECM gel was able to support the formation of enteroids from murine Lgr5+ crypt cells, and from human pediatric stomach and intestinal crypts. The authors were also able to achieve mechanical control of the ECM gel by incorporating poly-acrylamide to achieve different stiffness for two-dimensional (2D) culture of human and mouse enteroids, which may be important for future research. This intestinal ECM gel is also applicable to grow organoids from the liver, stomach, and pancreas80.Decellularized matrices have also been used for the growth of human intestinal organoids (HIOs) derived from pluripotent stem cells (PSCs), as well as enteroids derived from adult crypt stem cells81 and porcine pancreas82. Using mass spectrometry, Bi and colleagues found major differences in protein compositions comparing decellularized rat pancreatic extracellular matrix to Matrigel; Matrigel contains lower levels of collagen V than are normally present in pancreatic ECM. By coating plates with Matrigel plus commercially available collagen V, the authors were able to enhance endocrine differentiation of human iPSCs in 2D culture, compared to Matrigel alone83. One key limitation of this study, however, is that the proteomic profile of the rat pancreas may be very different from that of the human pancreas. The human pancreas presents a challenge in decellularization as it has a higher lipid content than animal models. With specific preparation methods that remove lipids, Sackett et al. found that decellularized human ECM is capable of supporting the survival of undifferentiated hPSCs and their pancreatic lineage derivatives, including insulin-expressing beta-like cells, in vitro81.Decellularized ECM has been prepared from various pancreatic cell sources, such as adult human pancreas84 and as a support for the culture of human and murine intestinal, stomach, and colonic organoids85. Other examples are vitrified collagen I for human intestinal organoids86 and murine renal organoids87, as well as fibrin supplemented with laminin-111 capable of supporting various murine and human epithelial organoids88.A complementary approach to the use of decellularized ECM is naturally-derived proteins, such as collagen I derived from porcine tendon, porcine skin, or bovine lens capsules. Collagen I has been used to form human colorectal carcinoma model organoids in a 3D culture of rabbit colons89. Yui and colleagues showed that embedding intact murine colonic crypts and isolated Lgr5+ progenitor cells in collagen I with hepatocyte growth factor, R-spondin 1, EGF, and Noggin generated organoids that were able to engraft onto damaged mouse intestinal epithelia upon transplantation. In contrast to the TMDU method, the Ootani method uses collagen I gel in which small and large intestinal cells are kept suspended at an air-liquid interface; this method improves oxygenation of the organoid, allows viable murine organoids to be maintained in culture for up to 350 days, and preserves the mesenchymal niche in the organoids90.The Tokyo Medical and Dental University (TMDU) method uses collagen I for intestinal enteroid culture91.Two factors have been shown to affect the differentiation of progenitor cells into organoids in collagen-based matrices: the source of seeded cells, and the spatial arrangement of collagen types around the cells. Isshiki and colleagues reported that the choice between the TMDU and Ootani methods should be governed by the source of cells used to generate the intestinal organoid. More consistent results are obtained for growing intestinal organoids from seeded isolated rat intestinal crypts using the TMDU method, whereas the Ootani method is better for growing rat colon organoid cultures of homogenized tissue92. Collagen I has a high affinity for α2β1 integrin, whereas collagen IV binds more strongly to α1β1 integrin93. Collagen IV tends to occur exclusively in basement membranes93. β4 integrin expressed by intestinal organoids is distributed only at the basal surface94, while β1 integrin is required for proper apical-basal polarization97. A combination of collagen I and fibronectin compared to collagen I alone functionalized within a PEG gel enhanced hepatic differentiation from human mesenchymal stem cells98. Clearly, the culture of organoids must take the 3D spatial positioning of the relevant materials into account.In addition to judicious selection of cell type, cell fate is intimately tied to interactions between cell surface integrins and biochemical cues in the ECM100, human brain101, murine intestinal102, human intestinal103, human pancreatic104, and human and murine vascular105. Capeling and colleagues grew HIOs on an alginate substrate and found that differentiation of human pluripotent cells into HIOs could be supported without the alginate providing chemical cues to the cells. The authors hypothesized that cells create their own niche within the alginate hydrogel by secreting basement membrane proteins and forming mesenchyme, allowing cellular survival and differentiation into HIOs103. Rossen and colleagues demonstrated the development of murine and human vascular organoids in a non-functionalized alginate setting105. Alginate has a number of advantages that make it attractive as a material for further study; it is inexpensive, relatively easy to modify and functionalize, biocompatible, and has been used in a wide range of biological and materials applications108. The mechanical properties of alginate, such as elastic modulus, extensibility, and characteristic relaxation time, can also be easily tuned109. For these reasons, alginate is a promising material for further exploration. However, because alginate is biologically derived, its mechanical properties are still subject to lot-to-lot variability110. Similarly, hyaluronic acid and mixtures of hyaluronic acid and chitosan have been extensively used in the growth and construction of neural organoids116.In addition to organoids grown in naturally-derived proteins, several laboratories have grown a wide range of organoids in polysaccharides such as alginate or alginate-chitosan mixtures. Organoid types grown in alginate include human lung117, which allows for rapid clinical translation.Decellularized ECM-based methods can quickly recapitulate organ function. Many or all of the chemical cues required for the formation of a spatially-defined organ, including difficult-to-introduce glycoproteins, are already present, minimizing the need for additional chemical modification of the ECM. Decellularized ECM retains the compositional differences observed between basal and apical regions. Collagen- and alginate-based materials have been approved by the FDA for a wide variety of applications118 or broad changes in the phenotype of seeded cells that do survive119. Contrarily, myocardial infarct is known to trigger remodeling events that stiffen the ECM and change its chemical composition; yet when mesenchymal stem cells are seeded on infarcted tissue, the cells secrete higher levels of pro-survival and immunomodulatory growth factors120. While myocardial infarct appears to enhance the survival of seeded cells, the negative effects of other diseased tissue on organoid development should not be discounted.Decellularized ECM does have disadvantages. Most importantly, the quantity of ECM that is available for study is limited by the availability of donor animals or humans, and the quality of ECM can be affected by the health of a donor. For example, emphysematous or fibrotic lung tissue has hardened and undergone alterations in its architecture. These alterations can lead to cells failing to survive beyond one week of culture121. A related difficulty is that not all decellularization protocols are equally effective at removing cells or other immunogenic species, which can cause varying host immune responses and failure of implants in clinical trials122. Finally, the occasional need for PSC differentiation into organ-specific progenitor cells that are then introduced into the decellularized matrix requires an additional step.Even with healthy donor tissue, batch-to-batch variability remains. The physical properties of decellularized ECM are difficult to control or modify, which limits the experiments that can be conducted. Decellularized ECM is also chemically undefined; the factors driving differentiation are often unknown. Surface proteoglycans that are necessary for successful organoid formation may be removed by harsh decellularization98, which introduces undefined components into the organoid culture. Furthermore, it is difficult to modify the mechanical properties of these culture systems without altering chemical concentrations. To elucidate the effects of mechanical properties on organoid development, researchers have turned to synthetic hydrogels that have been functionalized with cell-binding cues.Collagen-based culture methods are not limited by donor tissue availability; biomedical-grade collagen can be harvested on an industrial scale from cows and pigs. However, some collagen-based culture methods rely on coculture with supporting cells123. This large number of proteins means that many variables cannot be easily dissected to study the influences of ECM on organoid behavior and development. Synthetic hydrogels are attractive because their mechanical properties, functionality, and erosion rate can be controlled. The matrix metalloprotease (MMP) family of enzymes affects cellular and organoid development by degrading ECM proteins124. By including MMP recognition sites on synthetic hydrogels, it is possible to tune the rate of the hydrogel’s erosion. Manipulating synthetic hydrogels using methods such as electrospinning125, photopatterning127, spraying of microspheres128, inkjet and 3D printing129, or microfluidic channels130 further enables control over the shape and size of the organoids. The ability to exert local control over chemical and mechanical properties allows researchers to duplicate the heterogeneity in stiffness and composition found in organs, generate interfaces between materials similar to those found in the ECM, and duplicate essential elements of material microstructure; each of these controls has implications for organ function and disease132. Synthetic hydrogels can also be made responsive to external stimuli. For example, a thermoreversible hyaluronic acid- poly(N-isopropylacrylamide) (PNIPAAm) based hydrogel that solidifies at 37 oC and re-liquefies upon cooling enabled culture and recovery of human pluripotent stem cells without enzymatically digesting the matrix133. Light-sensitive polyvinyl alcohol matrices have recently been developed for cell culture; these matrices allow for control over the spacing of biochemical cues and the material environment134. Both of these materials may be useful for future organoid studies.Native ECM is complex; it contains over 300 different proteins, each of which has a different biological function and stiffness135. Choi and coworkers reviewed the pore sizes typically used in making tissue-engineering scaffolds, ranging from 5–15 microns for fibroblasts to 200–400 microns for osteoblasts, and developed a method for creating an artificial kidney scaffold using microstereolithography136. However, these studies observed the effects of pore size on mature cells rather than organoid development from multipotent cells. Broguiere and coworkers found that a Matrigel culture system had a pore size smaller than 200 nm, or the resolution limit of their confocal microscope, but that a fibrin-based material had a pore size closer to 4 microns; these materials had comparable elastic moduli and colony-forming efficiency88. To our knowledge, studies showing an explicit connection between pore size and organoid differentiation have not yet been reported.The use of synthetic hydrogels may also open up new avenues by altering the porosity of the scaffold on which the cells are grown. Dye and coworkers found that human lung organoids transplanted in mice could merge together to form airway structures. However, this was only possible if the scaffold was able to degrade, which increased the material’s pore size137 but also having slight differences in viscoelastic properties between white matter and gray matter138. The viscoelastic properties of a material affect matrix remodeling, cell spreading, migration, differentiation, and consequently, organoid fate141. The effects of viscoelastic properties on cell culture and behavior are complex but have been thoroughly reviewed elsewhere142. The effects of materials properties other than stiffness have only recently begun to be explored, and tunable hydrogels will enable more sophisticated experiments to be conducted.Synthetic hydrogels also have readily tunable viscoelastic properties, such as loss modulus and characteristic relaxation time. Many relevant tissues are viscoelastic, with brain tissue in particular not only having a strong dissipative component  in its response to stress145. Synthetic hydrogels can be functionalized with biologically active moieties that permit the growth and spread of cells151; concentration and spacing of these cues can be changed independently155. A striking demonstration of the utility of high-throughput approaches was provided by Ranga and colleagues, who prepared PEG-based gels in 1536-well plates and studied murine ESCs (mESCs) expressing an Oct4-GFP reporter. The authors analyzed 1000 variations of matrix elastic modulus, cell-binding peptides, and matrix susceptibility to MMP degradation and their effects on murine ESC fate156. The optimal conditions (elastic moduli ranged from 2–4 kPa and scaffolds with MMP insensitivity) produce murine neural tube organoids that are more homogenous in colony size and morphology, as well as more polarized, than those grown in Matrigel. The percentage of cells containing an actomyosin contractile ring is used as a metric for the polarity of the cells157.Synthetic polymer-based culture allows organoid formation conditions to be evaluated using high-throughput methods159. Attachment of human iPSCs was successful in assemblies functionalized with collagen I and laminin-521 but not with fibronectin. This result builds on previous work by the authors, in which basic human liver function was recapitulated by a PEG-based scaffold160. To promote iPSC differentiation into human neuronal progenitor cells, Ovadia and colleagues compared photo-crosslinked PEG-based gels that contained chemical cues such as the laminin-derived cell-binding sequences YIGSR and IKVAV, the fibronectin-derived sequences PHSRNG10RGDS and RGDS, and the vitronectin-derived sequence KKQRFRHRNRKG161. The authors found that PEG gels functionalized with YIGSR and PHSRNG10RGDS were permissive for human iPSC survival and differentiation into neural progenitor cells in 3D culture.Cell-binding cues from collagen, fibronectin, or laminin have frequently been added to synthetic hydrogels to allow for organoid growth and differentiation. Ng and colleagues created functional human liver organoids derived from human iPSCs in a colloidal crystal of PEG functionalized with collagen I, fibronectin, or laminin-521+ progenitor cells derived from the intestinal crypt. The material consisted of a PEG gel functionalized with either an RGD fibronectin-derived peptide or a laminin-111-derived peptide162. A stiffer matrix containing an RGD fibronectin-derived peptide promoted survival and proliferation of undifferentiated progenitor cells. In contrast, the softer matrix containing a laminin-111-derived peptide promoted the differentiation of progenitor cells into functional murine and human organoids. The authors found that organoid formation in minimal nutrient conditions was permissible only within a narrow range of matrix stiffness; the optimal elastic modulus was 190 Pa. Making the PEG gels more susceptible to MMP degradation resulted in depolarized organoids with irregular shapes. Major findings of this study are illustrated in Fig. The stiffness of synthetic hydrogels can be controlled and has an effect on organoid formation. Gjorevski and colleagues reported a synthetic matrix for the intestinal organoid culture of murine and human Lgr5164. After successful engraftment, HIOs generated from 4-arm PEG maleimide promoted the healing of mucosal wounds in a mouse colon injury model. Intriguingly, when PEG was crosslinked with dithiothreitol to inhibit matrix degradation, organoid viability at seven days was poor as measured by live-dead staining. This demonstrates a requirement of a degradable matrix for prolonged survival.Cruz-Acuna and colleagues also found that PEG-based materials may need to be soft and degradable in order to support differentiated intestinal organoids. The authors used a 4-arm PEG maleimide to encapsulate and culture Matrigel-derived HIOs. Organoid viability was reduced at high PEG density and matrix stiffness; RGD- or AG73 (CGGRKRLQVQLSIRT)-functionalized PEG matrices promoted greater organoid viability than laminin-derived IKVAV- or type I collagen-derived GFOGER peptides165.Neural organoids present special challenges including a lack of reproducibility, batch-to-batch variation in transcriptional profiles, and susceptibility to small microenvironmental changes that may have a considerable effect on organoid fate168. Therefore, the most useful organoids for toxicity models should include multiple populations. Schwartz and colleagues used a PEG-based gel functionalized with pendant RGD cell-binding domains and crosslinked by MMP-degradable peptides to generate neural organoids169. In this study, cells were introduced in three sequential stages: neural cells were introduced at day 0, vascular and mesenchymal stem cells at day 9, and microglia and macrophage precursors at day 13. The organoids were then exposed to a library of known toxic and nontoxic compounds, and the resulting RNA-seq data of the organoid response were used to build a machine-learning algorithm to assess the neurotoxicity of known and unknown compounds. In a blinded test, nine out of ten tested chemicals were correctly identified as toxic or nontoxic; in contrast, the true positive rate of chemical identification in animal models is between 41 and 71%170.Essential elements of the complexity of the brain must be recapitulated to enable clinical applications of neural organoids. For example, toxicity in brain tissue has many potential causes involving multiple cell types114. The authors found that the differentiation of both normal and Down syndrome iPSCs into neurons was dependent on matrix stiffness; cells could be grown at a softer elastic modulus of ca. 500 Pa but not ca. 1500 Pa, as indicated by higher expression of β-2 tubulin and microtubule-associated protein 2. However, Down syndrome patient-derived iPSCs that were differentiated in the softer gel showed no discernable neurite outgrowth, suggesting a block in the maturation of the differentiated neurons.Brain organoids cultured in hyaluronic acid hydrogels were used to model Down syndrome by Wu and colleagues115. Bejoy and colleagues showed that functionalizing hyaluronic acid with heparin affects neuronal patterning; the addition of heparin favored differentiation of human progenitors into neurons with a hindbrain fate, whereas non-functionalized hyaluronic acid favored a forebrain fate. The authors also established that the stiffness of this hybrid material is relevant to cell fate determination; lower elastic moduli, ca. 300 Pa, led to forebrain development, whereas higher elastic moduli, ca. 1000 Pa, led to hindbrain development116.Hyaluronic acid hydrogels can also be functionalized with various peptides to examine brain organoid differentiation. Lam and colleagues found that the concentration of laminin-derived IKVAV, with 300 μM being the optimal concentration, was critical to neural organoid survival; however, this concentration did not enhance neuronal differentiation173, but the spheroids lack spatial complexity and cell types compared to fully-developed organoids. Without having to functionalize the well substrates, Chen and coworkers used a 3D printed mold to cast polydimethylsiloxane (PDMS) microwells to generate human embryoid bodies that have the potential to differentiate into brain organoids in a suspension culture174. They found that a critical factor affecting differentiation was the ridges of the culture vessel, unlike previous studies using smooth wells. This study represents a new direction towards the generation of organoids, where the shape of the culture vessel might be tuned in order to change cellular phenotype, growth, and differentiation.Neural organoids have also been cultured in Matrigel-free conditions using microfluidic approaches such as microwell printing. A number of groups were able to generate spheroids in chip-based devices175. Amikagels with elastic moduli ranging from 37 to 320 kPa were created, but no chemical signaling peptides were incorporated into the gels. The authors found that stiffer gels drove pancreatic progenitor cells to aggregate, leading to increased differentiation and maturation into beta-like cells; this may have been mediated through paracrine signaling enhanced by cellular proximity. Compared to cells grown in Matrigel, beta-like cells grown in Amikagel produced higher levels of functional beta-cell markers PDX1 and NKX6.1 and were more responsive to a glucose challenge. Their finding challenges the conventional wisdom that cell-binding domains are required for effective organoid formation. The stiffness of the gel is also very high, exceeding the elastic modulus of materials typically used in the culture of bone51. Mechanistically, the authors propose that the culture system is forcing the formation of a compressed organ, rather than serving as a mimetic. A potential disadvantage of this approach is limited control of cellular aggregate sizes and attendant consequences of cell viability of no more than five days. The smallest cell aggregates reported by the authors were ca. 200 microns in diameter; the typical human islet has a diameter of ca. 130 microns176. Larger islets have been known to form necrotic centers because of a lack of oxygen and nutrient diffusion; other studies have found that islets best maintain cellular identity and function when they have a size of ca. 100–150 microns177. Further work should be done in this system to establish long-term viability.Pancreatic organoids have proven difficult to prepare without resorting to Matrigel-based culture. To our knowledge, Candiello and colleagues were the first group to use a synthetic hydrogel, amikacin hydrate crosslinked with poly(ethylene glycol) diglycidyl ether known as Amikagel, to culture hESC-derived islet organoids179. Moreover, many such materials, including PEG and PLGA, have been approved by the FDA for use in human therapeutics. Nguyen and colleagues recently assessed more than 1200 synthetic polymer formulations for toxicity and abilities to promote implant vascularization and endothelial cell network formation180. This work provides a valuable resource for organoid researchers, particularly those concerned with vascularization of organoids postimplantation.A major advantage of using synthetic polymers for organoid culture is that they are amenable to systematic variation in structure and properties and can be used to explore the effects of mechanical and chemical cues on cellular fate181 instead of organoid formation162. Improper spacing of biochemical cues can also lead to cell death182. While the backbone materials of synthetic hydrogels are cheap and can be produced on an industrial scale, functionalization of these materials with precisely-placed, custom-made peptides significantly increases cost and requires expertise in materials science, making these materials less attractive to cell biology labs. There have been a number of studies showing that organoids can be grown on unmodified surfaces such as alginate, but this requires more research183. Further, synthetic hydrogels may degrade into cytotoxic by-products184 or require cytotoxic initiators185, limiting the types of polymers that can be used in cell culture186. Synthetic hydrogels may contain pendant or other unreacted groups, which may be toxic to cells 187. Finally, synthetic hydrogels used as medical implants can trigger foreign body reactions188; similar effects may be seen in immunogen-containing organoids. For these reasons, it may be advantageous to engineer recombinant protein gels.There are several disadvantages of synthetic hydrogels. First, many synthetic hydrogels require the incorporation of biochemical cues such as cell-binding peptides. In the absence of biochemical cues cells may not attach to the hydrogel, leading to anoikis (a type of programmed cell death)Recombinant proteins made by genetically engineered organisms have found wide applications in medicine, food processing, and catalysis. Engineered recombinant protein gels possess major advantages compared to other culture methods: chemical cues can be added with exact definition; chemical and mechanical properties of the gel can be altered independently; polydispersity is low; and degradation rates can be programmed by including appropriate recognition sites for MMP degradative enzymes.189. In a follow-up study, murine cardiomyocyte differentiation  was found to be dependent on the stoichiometric ratio of THPC to protein, which tuned the stiffness of the hydrogel. Among the elastic moduli studied , the softest material favored the proliferation of embryoid bodies that contain mesodermal progenitor cells and promoted rapid cardiomyocyte differentiation phosphonium chloride (THPC) as an amine-reactive crosslinker; this material transiently inhibited contractility of murine ESC-derived cardiomyocytes and enhanced survival of dorsal root ganglia cells from chick embryosion Fig. . Embryoi191.In another follow-up study, a very soft matrix with an elastic modulus of 180 Pa promoted intestinal organoid-forming efficiencies comparable to those observed in collagen I-based matrices. Organoid-forming efficiency was higher when the engineered ECM proteins contained 3.2 mM RGD peptide, compared to no RGD. Interestingly, MMP activity was significantly higher in the stiffer matrices. Inhibition of MMP activity reduced organoid-forming efficiency in the stiffer engineered hydrogel matrices, suggesting that secretion of degradative enzymes in adult intestinal organoids may be a response to overly stiff conditions+ ductal progenitor cells were first proliferated in Matrigel, then transferred to a culture containing aECM-lam but not Matrigel. After 2 weeks, endocrine-acinar organoids were observed, demonstrating that aECM-lam was capable of inducing differentiation of ductal progenitor cells into endocrine and acinar lineages192. A follow-up study established that when Matrigel was added, endocrine and acinar cell development was inhibited while ductal cell formation was promoted193, demonstrating the importance of the ECM microenvironment in pancreatic organoid differentiation. Using aECM-lam, other morphologically-distinct organoids were formed from murine postnatal pancreas194 and sorted adult ductal progenitor cells195. Finally, the exact population of adult progenitor cells capable of giving rise to endocrine/acinar cells in aECM-lam was determined to be ductal cells, which have high levels of CD133 but low levels of CD71 expression195. Collectively, these studies demonstrate the utility of aECM-lam in promoting endocrine and acinar cell differentiation in pancreatic organoid culture and identifying the responsible progenitor population.Recombinant ECM protein has been investigated for pancreatic organoid culture. The Tirrell and Ku groups have jointly developed and studied an artificial elastin-like polypeptide that incorporates an 18-amino acid sequence derived from α1 laminin; this polypeptide has been named artificial (a) ECM-lam Fig. . aECM-la196.Peptide-based hydrogels have recently been employed to model Alzheimer’s disease. Zhang and colleagues used the self-assembling peptide RADA-16 to culture human neuronal cells treated with exogenous amyloid-β oligomers, known contributors to Alzheimer’s disease. A 3D culture in RADA-16 resulted in activation of a p21-activated kinase in response to amyloid-β oligomers. Both the activation and localization patterns of the p21-activated kinase are characteristic of neurons in an Alzheimer’s disease state. In contrast, the corresponding 2D culture did not show this activation and localization, suggesting that the 3D organoid culture of neurons is critical for modeling Alzheimer’s disease197. The authors used HYDROSAP peptide 3D hydrogels with elastic moduli of ~800 Pa (similar to the stiffness of human brain tissue) to culture human fetal neural stem cells198, which were able to differentiate into various lineages including astrocytes, oligodendrocytes, and neurons.The HYDROSAP self-assembling peptide hydrogel is a system recently developed by Pugliese, Marchini, and colleagues. In this system, multi-functionalized and branched self-assembling peptides (SAPs) can generate hydrogels with controllable elastic moduli199. Similar work has shown promise in stem cell differentiation and neural regeneration following spinal injury in vivo202, further demonstrating the utility of peptide-based materials in cell culture.Related work has been performed by Edelbrock and colleagues, with peptide amphiphiles capable of forming long, self-assembled nanostructures within a hydrogel. The peptides contain brain-derived neurotrophic factor (BDNF), which enables the formation of mature neurons via activation of the TrkB pathway. Display of BDNF on the peptide amphiphile is necessary for this effect to be observed205. They can be programmed to degrade and remodel at controlled rates by including protease recognition sites206 or changing crosslinking chemistry207. Protein-based hydrogels can be outfitted with a broad range of chemical functionalities by introducing noncanonical amino acids209; they can also be readily tailored to a wide variety of biomedical contexts211 and made thermally responsive213. The programmability of recombinant proteins has prompted increasing interest in the design of protein-based hydrogels as matrices for organoid culture.Self-assembling peptides and recombinant proteins offer important advantages in organoid culture. Recombinant proteins are molecularly well-defined and can be tuned independently for stiffness, viscoelastic behavior, and chemical functionality218. Ensuring that the recombinant protein is of human origin does not guarantee non-immunogenicity219. Care must be taken to avoid introducing other immunogenic factors, such as bacterial endotoxin. Therefore, proteins for clinical use would preferably be expressed in mammalian expression systems  or in yeasts .Protein-based materials have several disadvantages. First, not all proteins can be recombinantly expressed and ensuring re-folding and functionality of these proteins can be challenging. Certain recombinant proteins and self-assembling peptides are immunogenicAlthough several Matrigel-free techniques have been developed, they have been used in a narrow range of target tissues; expanding the number of tissue types will increase the acceptance of these alternative techniques. The ideal material for organoid culture should allow independent changes in the chemical and mechanical properties so that the effects on organoid growth, development, or morphology can be correlated. It should also functionalize biologically-relevant cell-binding proteins or peptides with ease. Finally, the ideal material should mimic the dynamic nature of the ECM in terms of erosion rate, viscoelasticity, and susceptibility to degradation. Because of these requirements, synthetic materials and programmable recombinant proteins represent fruitful areas of future research.220. A similar process was used by Nair and colleagues using mechanical agitation to keep human cells suspended in culture, resulting in functional beta-like clusters221. Control over the mechanical environment in such a system could be exerted by changing the speed of agitation. This method has the advantage of allowing easy harvesting of cells, which are simply allowed to settle to the bottom of a tube.This review has proceeded with the assumption that a matrix is required to culture organoids, but matrix-free culture systems have also been developed. For instance, Pagliuca and colleagues kept human embryonic stem cells suspended in liquid culture at 70 rpm, added specific growth factors to encourage differentiation, and found that the resulting beta-like cells behaved similarly to mature beta cellsUsing the methods discussed above, we anticipate a gradual shift away from the use of Matrigel in organoid culture and towards methods that enable exact control of the cell’s mechanical and chemical environments with a more precise definition.Further information on research design is available in the Peer Review FileReporting Summary