Above, X-ray image of severe ARDS in human lungs by J. Heilman

During this SARSv2/COVID-19 pandemic, there are strategies to accelerate advance of our knowledge to defeat this pathogen by way of characterization [1] [2], prevention [3], detection [4] [5] [6] [7], and monitoring [8].  Nevertheless, it is equally important to consider other impacts of the disease, namely the patients who are to endure it for weeks in a hospital setting, those who ultimately do not survive, and those who must care for them [9]. If a therapeutic modality such as extracellular vesicles (EVs) or secretomes from human mesenchymal stem/stromal cells (hMSCs) could reduce the number of hours each patient spent recovering in critical care, it could free up valuable hospital resources, alleviate the exhaustion of the health system, and bypass the ethical challenges of triage conditions.

What do we know so far?

It is now known, as highlighted by Dr. Sanjay Mukhopadhyay, of Cleveland Clinic in this video, that severe COVID-19 cases tend to lead to Acute Respiratory Distress Syndrome (ARDS) and are indeed fatal. Based on data obtained from a study conducted in Wuhan by Zhou and colleagues (Lancet article) [10], of the patients admitted, about 30% develop ARDS and of the non-survivors, 93% had ARDS.

Above, image of Diffuse Alveolar Damage (DAD) presented by Dr. Sanjay Mukhopadhyay, MD, The Cleveland Clinic, COVID-19 can kill: a lung pathologist explains what ARDS means and why it’s important

In an attempt to reduce the fatality rates in severe cases of COVID-19, we took a closer look at the works of two ARDS pioneers; Carolyn Calfee, MD and Michael Matthay, MD both from the University of California, San Francisco, who have been pursuing a breakthrough in critical care treatment and rehabilitation for ARDS.

Peeling back ARDS 

The following section summarizes a two-part lecture provided by iBiology and posted on YouTube.  Dr. Calfee covers Part 1 [11], describing salient features of ARDS pathology and why it remains an unmet medical need today.  Dr. Matthay covers Part 2 [12], providing an overview of the work of his lab and others on mesenchymal stem/stromal cells (MSCs) as a therapeutic modality against ARDS.

Key highlights from Dr. Calfee’s talk:

Clinical indications of ARDS, depicted by a series of X-rays from a clinical case, show that a patient’s lungs presented increasingly visible interstitial opacities due to the filling of fluid in the alveolar space as a result of viral pneumonia [11, 1:03].  By definition, ARDS must meet these three criteria: (1) onset of bilateral lung opacity, (2) hypoxia/low oxygen in the blood, and (3) pulmonary edema that is not due to heart failure [11, 2:42]. ARDS affects 200,000 people in the USA alone and kills 75,000 every year [10, 3:20]. Currently, no therapeutic options are available other than ventilators and fluid management [11, 3:39]. [13] [14] While ventilation with lower tidal volumes improves survival and fluid conversation therapy reduces time on ventilator, these are merely supportive care practices. Unfortunately, despite 25 years of attempts in developing pharmacologic therapies of ARDS, none were successful [11, 8:34]. And while ARDS mortality has improved due to better ventilation mechanics and fluid conservation management, mortality remains high (20-25%) and in cases of unselected patients, 30-40% [11, 9:04]. Enter MSCs. They might be of benefit not via engrafting as originally proposed, but rather exerting multiple effects from their secretomes, anti-inflammatory and antimicrobial properties, and other pathways [11, 9:47].

Key highlights in Dr. Matthay’s talk:

Based on the pathogenesis of lung injury due to ARDS, Dr. Matthay believes that possible mechanisms are due to the fact that MSCs (i) have anti-inflammatory properties, (ii) restore endothelial and epithelial barrier integrity, (iii) enhance the clearance of edema fluid from the lungs, (iv) have antimicrobial properties, (v) decrease the death of some of the endogenous cells in the lungs, and (vi) work by both cell contact dependent and independent mechanisms [12, 1:53]. Initial experiments in mice showed that MSCs increase survival from endotoxin in a severe lung injury model [12, 3:14] [15]. Subsequently, in a clinically more relevant mouse model using live E. coli, mice showed better survival, as well as surprisingly improved clearance of the pathogen [12, 4:17].  It was mediated at least in part via anti-microbial peptides released by MSCs and enhanced monocyte phagocytosis [16] [17].

Human ex vivo organ donor lung perfusion experiments showed that endotoxin-injured lungs were improved in physiologic function by treatment with MSCs, delivered directly to the bronchus [12, 9:10].  Both MSCs and MSC conditioned media returned endothelial permeability in this model system to normal during the four-hour experiment. Pathological alveolar fluid also decreased.  Further, the mechanistic study suggests that these protective effects may be inhibitable by siRNA directed against keratinocyte growth factor (KGF), suggesting KGF as one of the key paracrine factors in MSC’s protective secretome [12, 9:10]  [18] [19]. Further studies in the ex vivo lung model using clinical grade cryo-preserved allogeneic BM-MSC from University of Minnesota, delivered intratracheally (IT) or intravenously (IV), restored alveolar fluid clearance to about two-thirds of normal.  Surprisingly, despite the diminution of neutrophil presence in E. coli-injured lung, there is a dose response of MSCs antibacterial effect by apparent induction of phagocytosis [12, 16:00] [20]

Additional studies done by other groups include:

Work with Dr. Bhattacharya’s group at Columbia University suggest mitochondrial transfer mechanisms from MSCs into endothelial cell barriers.  Here, a cell contact-dependent mechanism via improved bioenergetics may also account for some of the MSC therapeutic mechanism [12, 21:59] [21]. Studies in sheep with Dr. Traber’s group at University of Texas showed potential safety and efficacy characteristics of MSCs introduced into the lung injury model (smoke + P. aeruginosa) of large live animals.  MSCs given at clinically relevant doses for humans reduced pulmonary edema at the high dose over the 24h course of the study, and improved oxygenation with both low and high doses [12, 23:16] [22]. In closing this lecture, Dr. Matthay describes the clinical trial strategy for forthcoming studies in humans through Phases I and II [12, 28:00].

The Phase 2a trial from Drs. Matthay and Calfee’s work with intravenous MSCs in patients with moderate to severe ARDS reported no patient experiencing any of the predefined MSC-related haemodynamic or respiratory adverse events.  Perplexingly, non-statistically significant higher mortality at 28 days was reported in the MSC treatment group. This work is published in Lancet Respiratory Medicine [23].  Media conditioned by cultured MSCs could offer an intriguing alternative vs. direct injection of live MSCs. If the MSC “secretome” could in fact ameliorate the damage of ARDS within mere hours as implied in Matthay’s video [12], a resulting therapy could be beneficial towards both patient and hospital bed shortages.

Role of MSC EVs

As suggested by works of Dr. Calfee, Dr. Matthay and others, what MSCs secrete plays an essential role in mediating lung repair and regeneration in ARDS.  Due to the apparent ability of EVs to maintain many therapeutic properties of their cognate parent cells, the role of MSC exosomes and EVs has been a hot topic amongst researchers in the field [24]. A key advantage of therapeutic EVs lies in the storage and transport logistics, but the challenge in the standardization of methods to evaluate quality and potency remains. Clinical studies of MSC derived EVs and/or exosomes are therefore still in their infancy. Nevertheless, today’s crisis spurs a call to action. In the current 8 trials targeting COVID-19, one trial at Ruijin Hospital is exploring the use of allogeneic adipose MSC derived exosomes via aerosol inhalation.

Maintaining quality of MSC EVs

For product developers and manufacturers, a critical consideration is the quality of EVs produced by the cells during the culture process. Collection of EVs using serum-containing culture media could hamper both the quality and quantity of EVs collected. Commercially available MSC growth media with 10% hPL can contain as high as 18 billion particles/mL, thus it can introduce a high baseline noise level to the final particle count. This drove RoosterBio to develop a solution: low particulate, xeno-free, protein-free and chemically defined media directly suited for EV collection from our cGMP expanded hMSC cellular starting materials. Thus, in contrast to the previous example, the baseline, cell-free measurement of RoosterCollect™-EV contains dramatically reduced particulates.

To accelerate the path of MSC-EVs into the clinic, a clinically relevant, GMP-manufactured RoosterCollect™-EV has thus recently been made available to complement the cGMP hMSC cell banks and xeno free bioprocess expansion media – each with its own type II FDA master file for referencing.

Scale up of MSC and MSC EVs

Looking forward, as translational centers and institutions gear up to treat the devastating effects of COVID-19-induced ARDS with therapies based on MSCs or their secretomes, it will be essential to employ sufficient quantities of high quality, standardized, clinical grade cells and paired media materials that are readily available for rapid GMP production.  Since scalable MSC manufacturing solutions are necessary to meet the rapid response required to COVID-19, we discussed this topic in a previous blog post.

Our other blog posts related to SARS-CoV-2/COVID-19 are linked below:

• Scalable MSC Manufacturing Matters in a Rapid Response to COVID-19 
• Can MSCs Treat Patients with COVID-19?
• “Honey, We Shrunk the Curve!”

References

1. Huang, C., et al., Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet, 2020. 395(10223): p. 497-506. 10.1016/S0140-6736(20)30183-5
2. Walls, A.C., et al., Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell, 2020. 10.1016/j.cell.2020.02.058
3. Chen, W.-H., et al., The SARS-CoV-2 Vaccine Pipeline: an Overview. Current Tropical Medicine Reports, 2020: p. 1-4.
4. Chan, J.F., et al., Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-polymerase chain reaction assay validated in vitro and with clinical specimens. J Clin Microbiol, 2020. 10.1128/JCM.00310-20
5. Zhang, Y., et al., Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP. medRxiv, 2020: p. 2020.02.26.20028373. 10.1101/2020.02.26.20028373
6. Guan, W.J., et al., Clinical Characteristics of Coronavirus Disease 2019 in China. N Engl J Med, 2020. 10.1056/NEJMoa2002032
7. Andersen, K.G., et al., The proximal origin of SARS-CoV-2. Nature Medicine, 2020. 10.1038/s41591-020-0820-9
8. Dong, E., H. Du, and L. Gardner, An interactive web-based dashboard to track COVID-19 in real time. Lancet Infect Dis, 2020. 10.1016/S1473-3099(20)30120-1
9. Gale, J. There Is a ‘Tipping Point’ Before Coronavirus Kills.  . 2020; Available from: https://bloom.bg/2WnQBqS
10. Zhou, F., et al., Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet, 2020. 10.1016/S0140-6736(20)30566-3
11. Calfee, C.S. The Acute Respiratory Distress Syndrome and Treatment with Mesenchymal Stem Cells, Part 1. 2013; Available from: https://youtu.be/SpqFFpmrtmY.
12. Matthay, M.A. The Acute Respiratory Distress Syndrome and Treatment with Mesenchymal Stem Cells, Part 2. 2013; Available from: https://youtu.be/v3tbwhu3Xio.
13. Acute Respiratory Distress Syndrome, N., et al., Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med, 2000. 342(18): p. 1301-8. 10.1056/NEJM200005043421801
14. National Heart, L., et al., Comparison of two fluid-management strategies in acute lung injury. N Engl J Med, 2006. 354(24): p. 2564-75. 10.1056/NEJMoa062200
15. Gupta, N., et al., Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice. J Immunol, 2007. 179(3): p. 1855-63. 10.4049/jimmunol.179.3.1855
16. Gupta, N., et al., Mesenchymal stem cells enhance survival and bacterial clearance in murine Escherichia coli pneumonia. Thorax, 2012. 67(6): p. 533-9. 10.1136/thoraxjnl-2011-201176
17. Krasnodembskaya, A., et al., Human mesenchymal stem cells reduce mortality and bacteremia in gram-negative sepsis in mice in part by enhancing the phagocytic activity of blood monocytes. Am J Physiol Lung Cell Mol Physiol, 2012. 302(10): p. L1003-13. 10.1152/ajplung.00180.2011
18. Frank, J.A., et al., Physiological and biochemical markers of alveolar epithelial barrier dysfunction in perfused human lungs. Am J Physiol Lung Cell Mol Physiol, 2007. 293(1): p. L52-9. 10.1152/ajplung.00256.2006
19. Lee, J.W., et al., Allogeneic human mesenchymal stem cells for treatment of E. coli endotoxin-induced acute lung injury in the ex vivo perfused human lung. Proc Natl Acad Sci U S A, 2009. 106(38): p. 16357-62. 10.1073/pnas.0907996106
20. Lee, J.W., et al., Therapeutic effects of human mesenchymal stem cells in ex vivo human lungs injured with live bacteria. Am J Respir Crit Care Med, 2013. 187(7): p. 751-60. 10.1164/rccm.201206-0990OC
21. Islam, M.N., et al., Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury. Nat Med, 2012. 18(5): p. 759-65. 10.1038/nm.2736
22. Asmussen, S., et al., Human mesenchymal stem cells reduce the severity of acute lung injury in a sheep model of bacterial pneumonia. Thorax, 2014. 69(9): p. 819-25. 10.1136/thoraxjnl-2013-204980
23. Matthay, M.A., et al., Treatment with allogeneic mesenchymal stromal cells for moderate to severe acute respiratory distress syndrome (START study): a randomised phase 2a safety trial. Lancet Respir Med, 2019. 7(2): p. 154-162. 10.1016/S2213-2600(18)30418-1
24. Liu, A., et al., Therapeutic potential of mesenchymal stem/stromal cell-derived secretome and vesicles for lung injury and disease. Expert Opin Biol Ther, 2020. 20(2): p. 125-140. 10.1080/14712598.2020.1689954
25. Heilman, J. Severe ARDS. 2017; Available from: https://commons.wikimedia.org/wiki/File:ARDSSevere.png.
26. Mukhopadhyay, S. COVID-19 can kill: a lung pathologist explains what ARDS means and why it’s important. 2020; Available from: https://youtu.be/vPtH42Lnt_Y.