Regen med therapeutic opportunities for fighting COVID‐19

Abstract This perspective from a Regenerative Medicine Manufacturing Society working group highlights regenerative medicine therapeutic opportunities for fighting COVID‐19. This article addresses why SARS‐CoV‐2 is so different from other viruses and how regenerative medicine is poised to deliver new therapeutic opportunities to battle COVID‐19. We describe animal models that depict the mechanism of action for COVID‐19 and that may help identify new treatments. Additionally, organoid platforms that can recapitulate some of the physiological properties of human organ systems, such as the lungs and the heart, are discussed as potential platforms that may prove useful in rapidly screening new drugs and identifying at‐risk patients. This article critically evaluates some of the promising regenerative medicine‐based therapies for treating COVID‐19 and presents some of the collective technologies and resources that the scientific community currently has available to confront this pandemic.

City between 1 March, and 2 April, 2020, hospitalization risks were greatest for those aged ≥75 years, with body mass index >40, and with heart failure. 2 New syndromes are still being described, including skin symptoms and systemic inflammatory syndromes, such as sepsis in adults and a Kawasaki-like syndrome in children. [3][4][5] While the SARS-CoV-2 genome is 96% identical to bat coronavirus and shares 79.6% sequence identity to SARS-CoV, 6 the COVID-19 mutant virus is believed to have a different diagnosis and prognosis from the precedent forms. SARS-CoV-2 has a four-phase infection paradigm. Phase 1 includes cell invasion and viral replication through binding of the viral spike protein to a cell surface receptor called ACE2. 7 Phase 2 includes replication of SARS-CoV-2 in the lung and immune system activation. In more severe cases, phase 3 includes pneumonia, and in the most severe cases, phase 4 includes acute respiratory distress syndrome, a cytokine storm, sepsis, and multiple organ failure. A cytokine storm is characterized by cascades of inflammatory cytokines being released systemiclly, including IL-6, which has been tied to increased damage in the lungs and other organs. 8,9 Current standard of care treatment for COVID-19 patients across these phases can be found on the CDC website, where guidelines are provided for the medical management of COVID-19 and have been published by the National Institutes of Health (NIH). 10 Regenerative medicine is uniquely poised not only to provide solid understanding of the infection mechanism and ways to prevent it, but also to introduce innovative treatments other than drugs.  Intravenous MSC infusion showed a reduction in inflammatory cell types and cytokines, such as tumor necrosis factor, and an increase in anti-inflammatory cytokines, such as interleukin 10 (IL-10). Gene expression profiles showed that MSCs were angiotensin-converting enzyme 2-negative (ACE2-) and transmembrane serine protease 2-negative (TMPRSS2-), which indicated MSCs were resistant to COVID-19 infection. 13 MSCs have also been shown to assist with sepsis 14,15 and reduce inflammation. 16 The goals of regenerative medicine-to repair, regenerate, and restore missing function or tissue-might drive the investigation of several regenerative therapies for COVID-19-recovered patients. In a retrospective, observational study on critically ill patients with SARS-CoV-2 pneumonia, 17 the authors reported that most patients had organ function damage, including 67% with acute respiratory distress syndrome (ARDS), 29% with acute kidney injury, 29% with cardiac injury, and 29% with liver dysfunction. Moreover, type II pneumocytes in the lung are a major target of SARS-CoV, propagating new virus and suffering widespread damage. Type II pneumocytes express ACE2 and associated enzymes transmembrane protease serine 2 (TMPRSS2) and Cathepsin L (CTSL). 18 Lung progenitor cells (CD34 + Oct4+) can be preferentially infected by SARS-CoV (2003 version) compared with more mature pneumocytes. 19 Taken together, these findings mean that as COVID-19-infected lungs try to replace damaged and dead lung cells, the replacement stem and progenitor cells may also be targeted by the virus in a one-two punch. This may be behind the long periods of time it takes for patients to recover, as well as the lung scarring that may reduce lung capacity, perhaps permanently for some patients. After the pandemic is past, there will be a lasting population of patients who need long-term therapy to regain lost lung function.
Any treatment that can boost the regenerative power of lung tissues by replacing these critical stem cells will help a large new patient population.

| SMALL ANIMAL MODELS FOR COVID-19
The use of the standard laboratory mouse has been limited because of the mismatch in the ACE2 receptor sequence between mouse and humans. Mice genetically engineered to carry the human ACE2 gene Compared with mouse models, Syrian hamsters present more human-like disease symptoms and pathogenesis with viruses like Ebola, 20 and their immune responses are more similar to humans. 21 Studies of SARS-CoV in Syrian hamsters noted viral replication in the lungs with clearance in 7 days; however, the hamsters did not progress to ARDS. 22  When infected with SARS-CoV-2, ferrets have elevated temperatures, lethargy, and appetite loss but do not progress to ARDS. There are also few research facilities capable of working with ferrets, which have complicated animal husbandry requirements and a limited commercial supply. 23 Each animal model offers advantages that will be useful, not only for the testing of therapies, but also for understanding related co-morbidities. Interested readers are referred to this excellent reference, which compares 10 different animal models in the search for the best model to study COVID-19. 24

| ADVANCED ORGANOID SYSTEMS FOR MODELING COVID-19
Organoids can model organ function in-vitro, providing a more accessible, faster, and higher throughput screening tool than in vivo models.
There are a few methods to engineer organoids, including manual 3D culture, liquid handling, or 3D bioprinting. All of these methods can use ECM in the process, which results in cell assembly that recapitulates organ function on a small scale and can help model different clinical features of COVID-19. 25 Using organoids to model organ function and infection by the SARS-CoV-2 virus can be a valuable tool to gather more insights on its viral tropism and pathogenesis, as well as serve as a platform to find potential treatment strategies for COVID- 19. While initial studies with SARS-CoV-2 have used Vero E6 cells, using models of the lung, heart, kidney, and intestine can be crucial to new therapies.
Lung organoids have previously been used to model infections by different pathogens. Hui et al. developed human lung organoids that recapitulate the bronchial airway, including cilia, goblet cells, club cells, and basal cells. 26 They then successfully infected these organoids with the influenza virus, analyzing replication competence, tissue tropism, and host responses. Their results were comparable to those observed in human ex vivo bronchus cultures, offering a viable alternative for modeling viral infections in the lung.

Similarly, Monteil et al used SARS-CoV-2-infected human kidney
organoids to investigate the effectiveness of human recombinant soluble ACE2 (hrsACE2) in inhibiting viral growth. 27 Liver organoids were also utilized to investigate how this tissue is infected and damaged by SARS-CoV-2. Zhao et al found evidence to support the hypothesis that COVID-19 patient liver damage might be resultant from cholangiocyte injury and consequent bile acid accumulation caused by the viral infection. 28 These results indicate that organoids are a powerful tool in understanding mechanisms through which SARS-CoV-2 affects liver tissue. Immune organoid culture can also contribute to understanding COVID-19 immune responses. 31 Organoids are a useful tool to investigate and characterize organ function and disease progression, and they should be further explored as a high-throughput, highly specific platform for COVID-19 drug screening.
Human in vitro tissue-on-a-chip platforms enable the direct use of human heart cells to evaluate potential effectiveness of existing or new COVID-19 drugs ( Figure 2). Additionally, using cells from a diverse patient population, including both sexes, may help classify the cardiovascular and pulmonary risk of COVID-19 drugs. Additionally, there is need to screen repurposed drugs for COVID-19 for toxicity, and an excellent review has evaluated current therapeutic drugs for the treatment of COVID-19 patients. 32 Tissue chips using lung airway epithelium that express high levels of ACE2 and TMPRSS2 have been used to screen repurposed drug candidates for inhibition of SARS-CoV-2 viral entry. 27 When used to assess seven clinically approved drugs (chloroquine, arbidol, toremifene, clomiphene, amodiaquine, verapamil, and amiodarone), only toremifene and amodiaquine were found to inhibit viral entry. These results suggested that human tissue chip technology screening assays could be used to study human disease pathogens and expedite drug repurposing. 33 Figure 2 highlights how organ toxicity of COVID-19 drugs could be evaluated using these organoid tissue chips, where advances in connecting these organ chips to achieve multi-tissue interactions will be important to detect unanticipated drug toxicity. 34 Taken together, employing an advanced organoid tissue chip can reduce the cost and time needed to develop new treatments, due to the ability to test so many compounds across these different organ systems, which could be created for thousands and thousands of patients. This idea is in fact being taken to a new level by the NIH, which has deployed funding opportunities for clinical trials on a chip.
The future will harness these tissue chips and use large data repositories to essentially perform clinical trials using patient-derived organoid models.

| REGEN MED THERAPIES FOR COVID-19
Patients suffering from severe COVID-19 have increased proinflammatory cytokine levels, including increased IL-6 levels that can be part of the cytokine storm or cytokine release syndrome (CRS). 35   Rather than quashing immune responses, boosting immune responses using natural killer (NK) cells also has been proposed as a treatment for severe COVID-19. Celularity Inc. recently received FDA approval to initiate a trial in 86 COVID-19 patients. Celularity's placental stem cell-derived NK cells may be able to target and eliminate virally infected cells; however, there are risks that NK cells may add to COVID-related inflammation and that they might not be able to detect which cells are infected. 48 Overall, regenerative medicine offers tremendous hope for treating COVID-19, and initial clinical trial results are promising. However, properly designed and conducted clinical trials, with appropriate enrollment, controls, and evaluation, are still needed.

| TECHNOLOGIES AND RESOURCES TO FIGHT COVID-19
With great challenges come great opportunities for the scientific community. Several resources have been made available to combat COVID-19 over a short period of time since the deceleration of the pandemic. In the future, we envision doing more with less, fully automating standardized workflows, using artificial intelligence (AI) to mine data and develop learning and predictive algorithms and using modular GMP environments to optimize manufacturing processes.
Social distancing requirements will be imposed on laboratories in response to the COVID-19 pandemic, so automation will improve the standard workflow in the laboratory and minimize the use of people and personal protective equipment. Recent publications discuss automation and standardization of processing for a potential miRNA marker candidates. 49,50 For instance, the Revos Tissue Processor (Thermo-Fisher) and high resolution slide scanners, such as those from 3DHISTECH, reduce labor time and maintain efficiency during reduced staff schedules. It will also be advantageous to pair these technologies with archiving solutions like the ARCOS platform from EPREDIA for efficient data storage and recovery and potentially to be combined with machine learning and predictive algorithms. With remote access to images, research can continue even during limited access to a facility. Building upon this, the adoption of cloud-based AI solutions further enables remote access to data. One such ecosystem, a spin-off from Memorial Sloan Kettering Hospital, has assisted in accelerating biomarker discovery by PAIGE.AI. 51 Cell therapies production models are moving toward closed systems to reduce contamination risks. Closed bioreactors are gaining traction for cell expansions steps. Closed, modular cGMP cell production environments that can enclose all the cell production steps from start to finish are available and will be implemented in a post-COVID-  55 The Lancet also has a resource center focused on COVID-19 and provides free access. 56 The journal Nature's newsletter, Nature Briefing, is a weekly gathering of the latest information. 57 COVID-specific literature web search engines, such as COVID Scholar, 58 have also appeared.
Utilizing these free reliable resources will give you the latest, substantiated scientific developments under way to combat COVID-19.
The US FDA has done an outstanding job in response to COVID- 19. The FDA has fast-tracked potential treatments (~60 day review) and has been very responsive in approving Emergency Use Authorizations. They also have a dedicated specific resources: Coronavirus Disease 2019 (COVID-19), 59 Emergency Use Authorizations, 60 and Coronavirus Treatment Acceleration Program (CTAP). 61 Additional resources to get the complete picture of the developments taking place are also available at the following organizations: World Health Organization (WHO), 62 Centers for Disease Control and Prevention (CDC), 63 John Hopkins, 64 Chinese CDC, 65 and Gates Notes. 66 The Atlantic has developed The COVID Tracking Project 67 to focus on the incredibly important topic of testing and compiles the most accurate data available. Eva Garland Consulting LLC has done a great job of compiling and continuously updating the grants opportunities. 68 CellTrials.org also provides a free report on all registered COVID-19 trials. 69 COVID-evidence 70  Page 73 and shared with RMMS working group co-leads to continue meaningful discussions within the working groups and also to plan future webinars and perspective articles to disseminate these important regenerative medicine resources to the entire scientific community.
Those wishing to become involved in RMMS working groups are encouraged to visit our website (http://regenmedmanufacturing.org/ membership/) and sign up to be a member.
The scientific community continues to work diligently and cooperatively to combat COVID-19 and the coronavirus. Through collaboration, reliance on real-world evidence, and a willingness to roll up our sleeves and get to work, we will overcome this pandemic together.

| CONCLUSIONS AND FUTURE DIRECTIONS
Regenerative medicine is poised to make a true difference in the fight against COVID-19. We have reviewed some of the advances in organoid systems to model COVID-19 symptoms that could be used for developing new treatments or screening at-risk patients. We have also covered some of the effects regenerative medicine-based therapies may have on treating COVID-19-related symptoms, such as immunomodulatory effects or even regenerative effects. We have discussed technologies and resources that are currently available to confront COVID-19. It is times like these when we must all come together, share our talents and resources, and develop the best methods, technologies, and capabilities to address global health challenges.

ACKNOWLEDGMENTS
We thank Sarah Alter and Priya Baraniak for their suggestions to the therapeutic section of this perspective article and technical review.
We thank Emily Gregg for her graphic art capabilities in designing