JADICELL -Mesenchymal Stem Cells: A New Piece in the Puzzle of COVID-19 Treatment

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JADICELL -The role of mesenchymal stromal cells in immune modulation of COVID-19: focus on cytokine storm

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The role of mesenchymal stromal cells in immune modulation of COVID-19: focus on cytokine storm

https://stemcellres.biomedcentral.com/a ... 20-01849-7

Abstract
The outbreak of coronavirus disease 2019 (COVID-19) pandemic is quickly spreading all over the world. This virus, which is called SARS-CoV-2, has infected tens of thousands of people. Based on symptoms, the pathogenesis of acute respiratory illness is responsible for highly homogenous coronaviruses as well as other pathogens. Evidence suggests that high inflammation rates, oxidation, and overwhelming immune response probably contribute to pathology of COVID-19. COVID-19 causes cytokine storm, which subsequently leads to acute respiratory distress syndrome (ARDS), often ending up in the death of patients. Mesenchymal stem cells (MSCs) are multipotential stem cells that are recognized via self-renewal capacity, generation of clonal populations, and multilineage differentiation. MSCs are present in nearly all tissues of the body, playing an essential role in repair and generation of tissues. Furthermore, MSCs have broad immunoregulatory properties through the interaction of immune cells in both innate and adaptive immune systems, leading to immunosuppression of many effector activities. MSCs can reduce the cytokine storm produced by coronavirus infection. In a number of studies, the administration of these cells has been beneficial for COVID-19 patients. Also, MSCs may be able to improve pulmonary fibrosis and lung function. In this review, we will review the newest research findings regarding MSC-based immunomodulation in patients with COVID-19.

Introduction
The city of Wuhan was the origin of coronavirus disease (COVID-19), a severe acute respiratory syndrome with SARS-CoV-2 as its causative agent. Presently, COVID-19 infection has spread to all continents of the world [1]. Due to unknown reasons, COVID-19 infection has been widely distributed in various geographical regions with high population densities [2]. Moreover, the profile of symptoms and severity of COVID-19 infection show extensive variation in different parts of the world [3]. Worldwide assessments suggest that only 3.4% of those infected with SARS-CoV-2 have perished as a result of COVID-19, which also shows high difference in various parts of the world [4].

Constant fever, non-productive cough, dyspnea, myalgia, fatigue, normal or reduced WBC counts, hyperferritinemia, and radiographic evidence of pneumonia are among the clinical signs of patients with COVID-19, which are similar to the symptoms of infection by other members of this family, namely SARS-CoV and the Middle East respiratory syndrome-related coronavirus (MERS-CoV) [5,6,7]. The mortality rate of the new coronavirus, known as SARS-CoV-2, is high because of insufficient knowledge about the pathogenesis of COVID-19, and no specific treatment has been recognized for it [8]. On the other hand, the response to COVID-19 infection can be overwhelmed in many patients. When SARS-CoV-2 enters into the lungs, it unleashes an immune response, attracting immune cells to the region attacked by the virus and resulting in localized inflammation [9]. In some cases, excessive or unchecked levels of cytokines are released that can be fatal due to an overreaction of the immune system, which is referred to as a cytokine storm [10]. The cytokine storm can trigger organ injury and cause edema, gas exchange dysfunction, acute respiratory distress syndrome (ARDS), acute cardiac injury, and secondary infection, which can be potentially fatal [11].

Consequently, the inhibition of cytokine storm is a main factor in the treatment of patients who are infected with SARS-CoV-2. Currently, available therapies for COVID-19 include non-specific antiviral drugs, antibiotics used for the treatment of secondary bacterial infections, sepsis, and reduction of inflammation [12]. A large number of anti-inflammatory medications have been developed, including NSAIDs, glucocorticoids, chloroquine/hydroxychloroquine, antagonists of inflammatory cytokines (such as IL-6R monoclonal antibodies, TNF inhibitors, IL-1 antagonists), and Janus kinase JAK inhibitors [13, 14]. However, in severe cases of ARDS, it is a difficult task to treat the cytokine storm induced by the virus. The findings suggest that stem cell-based therapy is applicable to treat infected patients.

Mesenchymal stromal cells and their features
Mesenchymal stromal cells (MSCs) are the cells with the unique ability to exert suppressive and regulatory effects on the immune system [15]. MSCs have been the focus of research because evidence has indicated that MSCs are able to migrate to and return from damaged tissues, exercise potent anti-inflammatory and immune regulatory activities, support the regeneration and repair of tissues, resist against apoptosis, inhibit tissue fibrosis, and decrease tissue injury [16]. MScs are able to migrate to site of lesion and differentiate into tissue-specific active cells such as lung, smooth muscle, and nerve cells [17]. Following intravenous or intra-arterial infusion of MSCs, these cells are primarily trapped in capillary beds of the liver and lungs [18]. MSC homing processes are not fully realized but are known to involve a variety of molecules such as chemokine receptors, including CCR2, CCR4, CCR7, CCR10, CXCR5, CXCR6, and CXCR4, adhesion proteins, and matrix metalloproteinase (MMPs), namely molecules also implicated in the well-known process of leukocyte extravasation [19, 20]. Hypoxia and inflammation are frequent indications of an injured tissue capable of affecting paracrine features of MSCs, which are mainly mediated via VEGF, FGF2, IGF-1, and HGF [21].

When MSCs are trapped in the lungs, a wide range of soluble mediators are secreted by them, including antimicrobial peptides, anti-inflammatory cytokines, extracellular vesicles, and angiogenic growth factors [22]. The release pattern of anti-inflammatory mediators is unique to the inflammatory lung environment, which is adjusted by differential damage and pathogen-associated molecular receptors that are expressed on MSCs [23], namely TLRs (toll-like receptors). As for COVID-19, TLRs are stimulated by viral unmethylated CpF-DNA (TLR9) as well as viral RNA (TLR3), leading to sequential cellular signaling pathways and the activation of MSCs [24].

On the other hand, inflammation leads to nuclear factor-kappa B (NF-κB) and c-Jun NH2-terminal kinase (JNK) signaling, which is also controlled through the factors secreted by MSCs. In addition, lung damage improves during the response of MSCs to oxidative stress, cytoprotection, and phosphoinositide 3-kinase/protein kinase B (P13K / Akt) signaling pathway [25]. Administration of BM-MSCs alleviated lung injury in a preclinical study via potentiating the PI3K/Akt signaling pathway [26, 27].

For example, the release of IL-1ra through MSCs inhibits IL-α/β activity via generating TSG-6, which is followed by the downregulation of NF-κB signaling and reduced production of inflammatory cytokines. Secretion of prostaglandin E2 (PGE2) is another efficient way to decrease inflammation by MSCs, which is a function of IL-10 production as a strong anti-inflammatory cytokine. Khakoo et al. showed that MSCs prevent PKB signaling of target cells via a contact-dependent way [28].

MSCs encounter a complex setting specified by various chemical and physical stimuli while moving toward an injured tissue and the microenvironment impacts MSCs’ behavior [29]. MSCs are able to release many types of cytokines through paracrine release or direct interaction with immune cells, which leads to immunomodulation [30]. These cells have the capacity to interact with immune cells in innate and adaptive immune systems [31]. Besides, MSC-mediated immunosuppression depends on the combined reaction of chemokines, inflammatory cytokines, and effector factors, as along with the microenvironment and the rate of inflammatory stimulus [32]. Owing to their powerful immunomodulatory ability, MSCs might have beneficial effects for preventing or attenuating the cytokine storm of SARS-CoV-2 infection [33, 34]. This paper tries to explain the significant role of MSCs in secreting important factors for immune regulation in COVID-19.

The SARS-CoV-2 infection and cytokine storm
ARDS caused by cytokine storm is the main mortality factor in COVID-19 [35]. The lethally uncontrolled systemic inflammatory response is stimulated by the secretion of a large number of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-2, IL-6, IL-7, IL-12, IL-18, IL-33, interferon (IFN)-α, IFN-γ, tumor necrosis factor-α (TNFα), granulocyte colony-stimulating factor (GSCF), interferon-γ inducible protein 10 (IP10), monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein 1-α (MIP1A), and transforming growth factor-beta (TGF-β) such as chemokines by immune effector cells within coronavirus infection (Fig. 1) [36,37,38].



Concussion and future perspective
Given the prevalence of COVID-19 and its complications such as cytokine storm, which is followed by ARDS and death of patients, finding a way to treat and improve the patients is of high importance [116]. As mentioned in this paper, there is no specific therapy for this virus and supportive therapies as well as non-specific antiviral drugs are mainly used for this purpose. Today, cell therapy is a modern method for treating a variety of diseases and several studies have been conducted in recent months to treat the SARS-CoV-2 virus using stem cells, suggesting the application of MSCs or immune cells such as NK cells [33, 117, 118]. According to research on MSC-based therapy, the safety and immunomodulatory role of MSCS in ARDS have been approved [82]. MSCs can secrete factors that improve the lung microenvironment, inhibit immune system overactivation, promote tissue repair, rejuvenate alveolar epithelial cells, inhibit pulmonary counteracting fibrosis, or improve function in damaged lung tissue because of SARS-CoV-2 infection [119, 120].

Many issues related to the application of MSCs, including the ideal dose and optimum timing of MSC delivery, should be further explored. In several animal models of human diseases, the use of secretory exosomes from MSCs has been claimed to mimic the beneficial effects of MSCs in antiviral therapy for influenza virus, reducing virus replication in lungs and virus-induced release of pro-inflammatory cytokines [121, 122]. Experimental studies and ongoing randomized trials will play an essential role in the clarification of the therapeutic potential of MSCs, which further our understanding of how MSCs interact with lung tissue infected by SARS-CoV-2.
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Re: JADICELL -ReeLabs Approved to Conduct First and Only Stem Cell Trial Against COVID-19 in India

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ReeLabs Approved to Conduct First and Only Stem Cell Trial Against COVID-19 in India
September 20, 2020 By Cade Hildreth (CEO) Leave a Comment

ReeLabs received permission from the Central Licensing Authority to conduct the first and only trial in India using MSCs from placenta and umbilical cord tissue against COVID-19.
There are numerous publications that have documented the role of stem cell against COVID-19. Currently there are around 23 stem cell trials in progress in US, UK, Japan, Germany, China, Spain, France and various other developed countries and hitherto published literature is extremely encouraging.

India is now all set to join this elite list.

In a significant, potentially path-breaking development, ReeLabs Private Limited, India’s biggest stem cell banking and research company, have been approved by Central Drugs Standard Control Organisation (CDSCO) and Drugs Controller General of India (DCGI) to conduct a stem cell trial against COVID-19, the first one in India.

ReeLabs to Conduct 1st Stem Cell Trial Against COVID-19
The trial is registered with the Clinical Trial Registry of India (CTRI) and will use intravenous administration of a patented stem cell product (mixture of umbilical cord and placenta derived mesenchymal stem cells) obtained from ReeLabs Private Limited.

In Phase I of the project out of the 20 patients selected in the moderate to severe category, 10 will get 100 million mesenchymal stem cells (MSCS) intravenously and 10 patients will get 200 million stem cells intravenously. The cells will be injected intravenously over forty minutes with a speed of 40 drops per minute.

ReeLabs also recently granted an US patent that describes their technology in detail.

The reason why moderate to severe patients deteriorate clinically to become severely critical is due to the ‘Cytokine storm’. MSCs by the virtue of their paracrine mechanisms reduce the harmful effect of the Cytokine storm by: (i) reducing the level of the harmful pro-inflammatory cytokine TNF-alpha and (ii) increasing the level of the protective anti-inflammatory cytokines IL-10. MSCs also improve the pulmonary microenvironment and lung function by differentiating into different types of alveolar epithelial cells.

Dr. Abhijit Bopardikar MD, Director ReeLabs stated:
“The sheer magnitude of the COVID-19 pandemic has sent shock waves throughout the world due to which ReeLabs has immediately recalibrated its priorities. We are delighted to be involved in two significant clinical trials against COVID-19, using our patented product (mixture of cultured placental and Cord derived mesenchymal cells) and using Convalescent Plasma. The world has started serious activity to overcome the devastation and India cannot be left behind. In fact we would like to lead the way whilst working out tangible solutions to treat multitude of both, actively infected patients of COVID-19 as well as patients suffering from post COVID-19 complications. The result of these trials shall be major milestones for the Asian subcontinent.”

Leveraging Stem Cells Against COVID-19
ReeLabs has offered to supply the patented stem cell product for free to all patients infected by COVID- 19 in the country even after completion of the study. Given that India has a population of 1.4 billion, representing approximately 18% of the global population of 7.8 million, this is a major commitment.

Group ReeLabs, Mumbai, India is the largest, state-of-the-art stem cell enterprise of Asia covering over 5000 square meter of laboratory space. The enterprise has launched two FDA approved clinical trials.
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Which COFEPRIS approval processes were necessary to commercialize your products?

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Q: Which COFEPRIS approval processes were necessary to commercialize your products?

A: The Mexican market is plagued by charlatanism and introducing a level three development requires a long and complex process to ensure that the product is 100 percent safe. COFEPRIS is still pretty young and expands according to the new innovations brought by Mexican developers. However, this does not happen often. There have been significant advances in the regulation of stem cells but we are still missing a general norm on the subject that includes cells, tissue and genetic therapies. These regulations already exist in the US and Europe, which provides us with guidelines as these countries have marketed many therapies in the past 10 years. I would not consider the stem cell market young because its development and regulation have been strengthening for 20 years.

Despite COFEPRIS’ young experience in the field, it has strict requirements. The commission provides the sanitary registration for the use of stem cells in Mexico to those who comply with three licenses: procurement of source tissue, stem or progenitor cell storage bank and regenerative medicine. However, we do expect to see stronger regulations regarding quality control frameworks for the production of stem cell systems because we know how delicate and complex this process is. Mexico already has 20 laboratories working with mesenchymal stem cells and they all need to be working under the same guidelines and quality criteria.

Q: The FDA approved a clinical trial of mesenchymal stem cells to treat COVID-19. How can Exomelab use its expertise to exploit this possible solution?

A: Exomelab has been working on a protocol for the application of mesenchymal stem cells on the treatment of viral sepsis caused by the SARS-CoV-2 virus. We submitted this protocol to CONACYT and the results will determine if they will support the initiative.

The importance of stem cells is in how they can help the lungs in the immunomodulation process. COVID-19’s severe damage to the respiratory system destroys many healthy cells within the body and this worsens in people suffering from any chronic disease, such as diabetes or obesity. One of the molecules that the virus uses to enter the body is the angiotensin receptor 2, which is the antagonist of molecule ASR that is related to the construction of blood vessels. Combined with a molecule that dilates the vein, this results in a massive impact on the human body as it inflates the system and compromises the respiratory system.

Chinese research has shown that the use of three mesenchymal stem cell doses of 50 million each applied on days one, three and nine, allowed patients to overcome the most critical stages of COVID-19 within days.

Q: What are Exomelab’s near-term goals?

A: Exomelab is working on the commercialization of our device through the brand Skin Care Exocell owned by Laboratorios GEM. We are also working on the creation of the International Society of Exosome-Based Medicine, which is developing quickly as it has attracted a great deal of attention and gathered important actors from many parts of the world. The idea of this society is to create a professional scientific forum to organize congresses and courses that encourage the use and development of exosome-based therapies.

Q: How is Exomelab innovating in patient care and treatment in the field of regenerative therapy?

A: Despite being a new company in the market, our expertise as a research team began around 1997 in the field of cells, organs and tissue transplantation. We began generating biotechnological platforms for isolating mesenchymal stem cells and as a result of these efforts, we decided to create this company. Through our research discoveries, we generated information on how stem cells can help the organism to renew cells in organs and tissues afflicted by any chronic degenerative disease.

Mesenchymal stem cells can be isolated from many tissue sources, such as bone marrow, adipose tissue, body fat, endometrial tissue, and from embryonic attachments, such as placenta, the amniotic membrane and the umbilical cord. Endometrial tissue was the most important source for our studies. Women eliminate great quantities of stem cells per month through menstruation. Gemlabs laboratory has patented a device that isolates the menstrual flow from the first two days of the period to recover stem cells. Through an in vitro process, we are able to obtain millions of stem cells and bioactive elements from these cells, including exosomes and microvesicles. After isolating all these molecules, we combine them through tissue engineering into a biological scaffolding of collagen and calcium alginate that results in a patch that can be used on any damaged tissue.

As we grew as a company, I started to specialize my research in Type II diabetes mellitus, specifically on diabetic foot as a result of a vascular tissue failure that causes sores that can even lead to the loss of a foot. We saw a great opportunity for mesenchymal stem cells to regenerate damaged tissue in patients suffering from diabetic foot. The patch stimulates the regeneration of tissue because it allows a process of revascularization, meaning the creation of new blood vessels and arteries. Moreover, this patch allows the wound to regenerate completely, meaning that it can even grow back hair, regenerate sweat and sebaceous glands. It also regenerates the nervous system, which allows a return of sensitivity. This patch prevents the formation of a scar or a fibrotic tissue. We started preclinical trials at PEMEX’s hospital and in 2019 we received the patent for this level three device development and also its sanitary registration with COFEPRIS.

The patch can act on any damaged tissue, independently of the person’s healthcare background. This regenerative process is an unprecedented development because of the quality of the final result. Neither scientists nor doctors are used to seeing a healing process that does not create fibrotic scars but truly regenerates the tissue from within.
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Re: JADICELL -‘Combination of human umbilical cord mesenchymal stem cell transplantation with IFN-γ treatment synergisti

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‘Combination of human umbilical cord mesenchymal stem cell transplantation with IFN-γ treatment synergistically improves the clinical outcomes of patients with rheumatoid arthritis’’

https://ard.bmj.com/content/early/2020/ ... 8762?rss=1

We thank Ma et al1 for their interest in our recent report titled ‘Combination of human umbilical cord mesenchymal stem (stromal) cell transplantation with IFN-γ treatment synergistically improves the clinical outcomes of patients with rheumatoid arthritis’.2 Ma et al1 brought up an important issue regarding the safety profile of intramuscular infusion of interferon (IFN)-γ, which may initiate further immune reactions.3 4 As previously described, recombinant human IFN-γ monotherapy is known to be safe but ineffective in treating rheumatoid arthritis.5 6 Furthermore, the safety of IFN-γ-primed mesenchymal stem (stromal) cells (MSCs) remains unknown, as there has been no such clinical research report addressing this issue. Therefore, for the subject’s maximum safety considerations, the clinical protocol was MSC transplantation (MSCT) plus intramuscular infusion of IFN-γ, instead of IFN-γ-primed MSCs, and as we have anticipated no new or unexpected safety issues were reported for either treatment group for up to 1 year. Indeed, in future studies, interpreting the safety of the IFN-γ-primed MSC protocols would be more appropriate than that of recombinant IFN-γ monotherapy from a translational standpoint.

As to the question of whether MSCT plus intramuscular infusion of IFN-γ would further modulate serum levels of IFN-γ in patients, all patients who received intramuscular infusion of IFN-γ had a transient increase in serum IFN-γ level within 24 hours after infusion, which gradually decreased during the subsequent follow-up. However, as we described in our previous study that there are huge individual variations in the baseline serum IFN-γ levels,7 we did not list the IFN-γ data. In addition, with regard to the serum level of proinflammatory cytokines, consistent with our previous study,7 there was a significant decrease in the serum levels of tumour necrosis factor-α and interleukin (IL)-6 among patients of the MSCT plus IFN-γ group, while no significant changes in IL-1β, IL-2R and IL-8 levels were observed. Unfortunately, we did not find that such a proinflammatory cytokine combination affected the immunosuppressive functions of MSCs, as observed in the in vitro study.

Finally, we agreed that it is possible that the flow cytometry (FC) gauged percentages of CD3+ and IFN-γ+ MSCs could differ using different control techniques for gating. In our study, isotype controls were used for identification of the background binding caused by antibody isotypes. As there are only two kinds of fluorochromes involved in the FC experiment, a single positive control technique was more adequate as a compensation control technique than the fluorescence minus one control technique, which is suitable for multiple fluorochromes (≥3) FC study.8 9
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JADICELL -Scalable manufacturing of allogeneic cell therapy products using Vertical-Wheel bioreactors

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Scalable manufacturing of allogeneic cell therapy products using Vertical-Wheel bioreactors

https://www.regmednet.com/webinars/scal ... oreactors/
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JADICELL -Aegle Therapeutics Announces $4M Financing to Fund Groundbreaking Stem Cell Exosome Clinical Trial

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Aegle Therapeutics Announces $4M Financing to Fund Groundbreaking Stem Cell Exosome Clinical Trial

Posted by: Exosome RNA Administrator in Headlines, Industry News January 9, 2020 0 1,003 Views

Aegle’s capital raise will fund the first clinical trial in the U.S. using exosomes isolated from allogeneic mesenchymal stem cells as therapy.

Aegle Therapeutics Corp., a first in class biotechnology company isolating extracellular vesicles, including exosomes (“EVs”), secreted by mesenchymal stem cells as therapy, today announced the closing of a $4M financing. Aegle’s platform technology is initially being developed to treat dystrophic epidermolysis bullosa (“DEB”), a rare pediatric skin blistering disorder. The investment was led by Boca Raton-based New World Angels, with participation from Tellus BioVentures, DEFTA Healthcare Technologies and DeepWork Capital, as well as exiting investors including OceanAzul Partners, LLC.

Aegle’s technology is based on decades of work conducted by Dr. Evangelos Badiavas, M.D., Ph.D., Aegle’s founder. Aegle’s proprietary isolation process safely and efficiently isolates EVs secreted by allogeneic bone marrow derived mesenchymal stem cells. In preclinical research, Aegle’s EVs demonstrated similar regenerative functionality to their parent cells, opening up the potential for “cell-free” therapy. Additionally, Aegle’s EVs carry specific proteins and mRNA that may prove essential for the treatment of DEB. Aegle’s technology is a platform technology with many potential indications in and beyond dermatology.

“We are very excited to close this first institutional financing round with such knowledgeable, high-caliber biotech investors,” said Aegle CEO Shelley Hartman. “The new funding validates our business plan and allows us to advance AGLE-102 into the clinic as well as expand the capabilities and opportunities of this cutting-edge platform.”

Aegle’s IND for the treatment of DEB patients has been cleared by the FDA. The Company anticipates beginning clinical trials in DEB in the first half of 2020.

“We are very excited to participate in advancing this pioneering research into the clinic. We expect that in addition to DEB, there are many other potential applications for this platform technology,” said Dr. David Schimmel of New World Angels who will join the Board of Directors, along with Lonnie Moulder at Tellus BioVentures and Elona Baum of DEFTA Healthcare Technologies.

Lonnie Moulder, founder of Tellus BioVentures, added ,”We are pleased to support the advancement of this very promising science into clinical trials by Aegle’s highly capable management team.”
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Re: JADICELL Regenerative Orthopedics - understanding how it will help you and your low back pain ?

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Regenerative Orthopedics - understanding how it will help you and your low back pain ?

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JADICELL -Shattering barriers toward clinically meaningful MSC therapies

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Shattering barriers toward clinically meaningful MSC therapies

https://advances.sciencemag.org/content/6/30/eaba6884

Abstract
More than 1050 clinical trials are registered at FDA.gov that explore multipotent mesenchymal stromal cells (MSCs) for nearly every clinical application imaginable, including neurodegenerative and cardiac disorders, perianal fistulas, graft-versus-host disease, COVID-19, and cancer. Several companies have or are in the process of commercializing MSC-based therapies. However, most of the clinical-stage MSC therapies have been unable to meet primary efficacy end points. The innate therapeutic functions of MSCs administered to humans are not as robust as demonstrated in preclinical studies, and in general, the translation of cell-based therapy is impaired by a myriad of steps that introduce heterogeneity. In this review, we discuss the major clinical challenges with MSC therapies, the details of these challenges, and the potential bioengineering approaches that leverage the unique biology of MSCs to overcome the challenges and achieve more potent and versatile therapies.
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Re: JADICELL -Human Mesenchymal Stromal Cell Secretome Promotes the Immunoregulatory Phenotype and Phagocytosis Activity

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JADICELLS -Human Mesenchymal Stromal Cell Secretome Promotes the Immunoregulatory Phenotype and Phagocytosis Activity in Human Macrophages

Cells 2020, 9, 2142; doi:10.3390/cells9092142 www.mdpi.com/journal/cells
Article
Human Mesenchymal Stromal Cell Secretome
Promotes the Immunoregulatory Phenotype and
Phagocytosis Activity in Human Macrophages
Minna Holopainen 1,2,*, Ulla Impola 1
, Petri Lehenkari 3
, Saara Laitinen 1,† and Erja Kerkelä 1,†
1 Finnish Red Cross Blood Service, FI-00310 Helsinki, Finland; ulla.impola@bloodservice.fi (U.I.);
saara.laitinen@bloodservice.fi (S.L.); erja.kerkela@bloodservice.fi (E.K.)
2 Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental
Sciences, University of Helsinki, FI-00014 Helsinki, Finland
3 Department of Anatomy and Surgery, Institute of Translational Medicine, University of Oulu and Clinical
Research Centre, FI-90014 Oulu, Finland; petri.lehenkari@oulu.fi
* Correspondence: minna.holopainen.fin@gmail.com
† These authors contributed equally to this paper.
Received: 31 August 2020; Accepted: 21 September 2020; Published: 22 September 2020
Abstract: Human mesenchymal stromal/stem cells (hMSCs) show great promise in cell therapy due
to their immunomodulatory properties. The overall immunomodulatory response of hMSCs
resembles the resolution of inflammation, in which lipid mediators and regulatory macrophages
(Mregs) play key roles. We investigated the effect of hMSC cell-cell contact and secretome on
macrophages polarized and activated toward Mreg phenotype. Moreover, we studied the effect of
supplemented polyunsaturated fatty acids (PUFAs): docosahexaenoic acid (DHA) and arachidonic
acid, the precursors of lipid mediators, on hMSC immunomodulation. Our results show that unlike
hMSC cell-cell contact, the hMSC secretome markedly increased the CD206 expression in both
Mreg-polarized and Mreg-activated macrophages. Moreover, the secretome enhanced the
expression of programmed death-ligand 1 on Mreg-polarized macrophages and Mer receptor
tyrosine kinase on Mreg-activated macrophages. Remarkably, these changes were translated into
improved Candida albicans phagocytosis activity of macrophages. Taken together, these results
demonstrate that the hMSC secretome promotes the immunoregulatory and proresolving
phenotype of Mregs. Intriguingly, DHA supplementation to hMSCs resulted in a more potentiated
immunomodulation with increased CD163 expression and decreased gene expression of matrix
metalloproteinase 2 in Mreg-polarized macrophages. These findings highlight the potential of
PUFA supplementations as an easy and safe method to improve the hMSC therapeutic potential.
Keywords: cell therapy; immunomodulation; polyunsaturated fatty acid; CD206; phagocytosis
1. Introduction
Mesenchymal stromal/stem cells (MSCs) show great promise in cell therapy, such as in the
treatment of graft-versus-host disease [1,2] and Crohn’s disease [3,4]. MSCs have diverse
immunomodulatory effects, which are mediated via cell-cell contact and secreted paracrine factors,
such as extracellular vesicles (EVs), tryptophan-degrading enzyme indoleamine-2,3-dioxygenase and
lipid mediator prostaglandin E2 (PGE2) [5–7]. MSCs are able to, e.g., inhibit the proliferation of T cells
and promote the generation of regulatory T cells [5,8]. Moreover, MSCs polarize macrophages toward
a more anti-inflammatory phenotype by increasing the expression of multiple cell surface markers,
such as CD206, and by enhancing their phagocytosis activity [9–12].
Cells 2020, 9, 2142 2 of 18
A tight classification of macrophages into different subtypes is redundant due to their plastic
and rapidly changing phenotype giving rise to a heterogeneous population [13]. Yet for simplicity,
macrophages are typically classified into classically activated, proinflammatory M1 phenotype and
to wound healing, anti-inflammatory M2 phenotype. Regulatory macrophages (Mregs) represent an
immunoregulatory phenotype that produce anti-inflammatory cytokines, such as interleukin (IL)-10
and transforming growth factor β1 (TGF-β1), potently suppress T-cell function and promote
regulatory T-cell phenotype [14,15]. Interestingly, Mregs are also investigated as a potential adjunct
therapy in renal transplantations (clinicaltrials.gov: NCT02085629). Although the effects of MSCs on
monocytes and type M1 and M2 macrophages have been intensively studied, less is known about the
effects of MSCs on Mregs. In our previous study, we observed that human bone marrow-derived
MSCs (hBMSCs) and hBMSC-derived EVs (hBMSC-EVs) enhanced the anti-inflammatory phenotype
of Mregs [16]. Both hBMSC cell-cell contact and EVs decreased the production of IL-23 and IL-22,
which are up-regulated in inflammation and promote T helper 17 cell maintenance and proliferation,
respectively. The hBMSCs and EVs also increased the production of PGE2 [16], which is an essential
lipid mediator in MSC function and induces the MSC-mediated skewing of macrophages toward an
anti-inflammatory phenotype [11,17].
EVs are small (majority < 300 nm) lipid-bilayered particles secreted by cells through exocytosis
and membrane budding. EVs carry intracellular messages by transporting lipids, proteins, nucleic
acids, carbohydrates or their metabolites and can mediate immunological effects [18]. Intriguingly,
MSC-EVs are able to mediate the therapeutic response of MSCs and have been investigated in various
in vivo models, such as acute kidney injury [19], stroke [20,21] and sepsis [22]. Thus, MSC-EVs have
emerged as a cell-free therapeutic option for MSCs.
The immunomodulatory response of MSCs resembles the resolution of inflammation, the active
dampening phase of inflammation [23]. Lipid mediators, especially the specialized proresolving
mediators (SPMs) promote the resolution of inflammation [24] by, e.g., reducing neutrophil
trafficking, increasing macrophage polarization toward anti-inflammatory phenotype and
macrophage efferocytosis of apoptotic neutrophils. Polyunsaturated fatty acids (PUFAs), such as n-3
docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and n-6 arachidonic acid (AA) are
precursors to lipid mediators [25]. DHA is a precursor to resolution-phase SPMs such as D-series
resolvins, maresins and protectins and EPA to E-series resolvins. AA is a precursor to proresolving
lipoxins, but also for prostaglandins (PGs), thromboxanes and leukotrienes with mainly
proinflammatory functions.
We have previously demonstrated that the phospholipid, fatty acid and, importantly, lipid
mediator profiles of hBMSCs can be modified with the supplementation of PUFAs [26,27]. hBMSCs
cannot efficiently synthetize these long-chained PUFAs from n-6 and n-3 precursors rendering the
PUFA supplementation into the culture medium essential to ensure a sufficient level of precursors
for lipid mediator biosynthesis [26]. Interestingly, we also observed that PUFA supplementation to
hBMSCs caused the subsequent remodeling of the phospholipid membrane of hBMSC-EVs [27].
Furthermore, EPA and DHA supplementation to murine MSCs increases their immunomodulatory
capacity in allergic asthma [28] and sepsis [29,30] models, highlighting the importance of PUFA
modifications on MSC immunomodulation.
In this study, we investigated the effect of hBMSC cell-cell contact and secretome on polarized
and activated Mregs. Moreover, we investigated the immunomodulatory effect of hBMSCs on Mregs
after DHA or AA supplementations, which alter the downstream lipid mediator profile of hBMSCs.
Our results demonstrate that the hBMSC secretome skewed macrophages toward a more antiinflammatory and proresolving phenotype. This effect was even more pronounced by the secretome
of DHA-modified hBMSCs. Strikingly, we show for the first time that the hBMSC secretome
enhanced the Candida albicans phagocytosis activity of macrophages by increasing the CD206
expression.
Cells 2020, 9, 2142 3 of 18
2. Materials and Methods
2.1. hBMSC Culture and PUFA Supplementation
The patient protocols of the hBMSC isolation were approved by the Ethical Committee of
Northern Ostrobothnia Hospital District (ethical approval number: Oulu University hospital
EETTMK 21/2011). The hBMSCs were collected from upper femur metaphysis of adult patients after
receiving a written informed consent and characterized as described previously [31]. The cells have
been characterized according to the guidelines of the International Society of Cell & Gene Therapy
[32]. The cells expressed typical MSC markers and lacked the expression of hematopoietic stem cell
markers, and the differentiation toward osteoblasts and adipocytes was also tested (data not shown).
hBMSCs derived from three donors were inspected in this study.
The cells were thawed, cultured [27] and PUFAs supplemented [26] as described previously. In
brief, the hBMSCs at confluence of 80–90% were supplemented with ethanol (purity ≥99.5%, Altia
Industrial, Rajamäki, Finland) as a control, DHA or AA (Cayman Chemical, Ann Arbor, MI, USA) as
bovine serum albumin (BSA, Merck, Darmstadt, Germany) conjugates. Prior to the PUFA
supplementation, the medium was changed to medium containing only 5% fetal bovine serum (FBS,
Thermo Fisher Scientific, Waltham, MA, USA) in contrast to 10% FBS in the proliferation medium.
The PUFAs dissolved in ethanol were added into 1.5 mM BSA-Dulbecco's phosphate buffered saline
(DPBS, Thermo Fisher Scientific) solution, vortexed and immediately added to the cell culture
medium. The final PUFA concentration supplemented to the cells was 50 µM. After 24 h, hBMSCs
were detached and 50,000 cells/well were added into Mreg polarization assay.
2.2. Mreg Polarization Assay
The use of anonymized peripheral blood mononuclear cells (PBMCs) from blood donors in
research is in accordance with the rules of the Finnish Supervisory Authority for Welfare and Health
(Valvira, Helsinki, Finland). The layout of the assay is described in Figure 1 and macrophages derived
from six different donors were used in the assay. The Mregs were cultured as described in [16] with
certain changes. In brief, 2× 106–4 × 106 PBMCs were plated on 12-well plates (Corning™ Costar™ flat
bottom, Thermo Fisher Scientific), incubated for 1–2 h and washed with DPBS. The attached
monocytes were incubated in 1.5 mL RPMI-1640 medium (Thermo Fisher Scientific) with 10% FBS
(Merck), GlutaMAX™ supplement (Thermo Fisher Scientific) and 5 ng/mL macrophage colonystimulating factor (M-CSF, PromoCell, Heidelberg, Germany) for 6 days at 37 °C, 5% CO2. This
medium is referred as polarization medium and macrophages obtained in these conditions are
referred as Mreg-polarized macrophages from here onwards. At day 6, the medium was changed
into the polarization or activation medium [polarization medium with 25 ng/mL interferon (IFN)-γ
and 10 ng/mL lipopolysaccharide (LPS, both from Merck)]. Macrophages cultured in the activation
medium are referred as Mreg-activated macrophages. The next day, 50,000 control-hBMSCs, DHAhBMSCs or AA-hBMSCs were added to the bottom of wells (referred as hBMSC cell-cell contact) or
to inserts (Corning™ Transwell™, pore size 0.4 µm, Thermo Fisher Scientific) (referred as the hBMSC
secretome).
Cells 2020, 9, 2142 4 of 18
Figure 1. The layout of macrophage polarization assay. Mreg, regulatory macrophage; hBMSC,
human bone marrow-derived mesenchymal stromal cell; DHA, docosahexaenoic acid; AA,
arachidonic acid; M-CSF, macrophage colony-stimulating factor; IFN, interferon; LPS,
lipopolysaccharide.
At day 10, the medium was centrifuged at 300 g for 15 min and the supernatant was snap frozen
and stored at −80 °C. The cells were washed with DPBS and either detached for flow cytometry with
0.75 µL 4 °C Macrophage Detachment Solution DFX (PromoCell) or scraped into 600 µL RLT lysis
buffer (Qiagen, Hilden, Germany) for quantitative polymerase chain reaction (QPCR). The RLT
samples were snap frozen and stored at −80 °C.
2.3. Determination of Cytokine Production
The medium samples were thawed on ice and analyzed with human tumor necrosis factor
(TNF)-α, IL-10 and IL-23 DuoSet enzyme-linked immunosorbent assays (ELISA) (all from R&D
Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. The absorbance was
measured with CLARIOstar® microplate reader (BMG Labtech, Ortenberg, Germany).
2.4. Macrophage Phenotyping with Real-Time Quantitative PCR
Only the macrophages with hBMSC secretome samples (hBMSCs cultured on an insert) were
analyzed with real-time QPCR, because cell-cell contact samples included both macrophages and
hBMSCs. The RNA was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s
protocol. The concentration and purity of RNA was measured with NanoDrop ND-1000
spectrophotometer (Thermo Fisher Scientific). RNA was converted to complementary DNA with
High Capacity cDNA RT Kit (Thermo Fisher Scientific). The gene expression was analyzed with realtime QPCR (CFX96™ Real-Time Systems and C1000™ Thermal Cycler, Bio-Rad, Hercules, CA, USA)
using TaqMan® Gene Expression assays and TaqMan® Universal Master Mix II (Thermo Fisher
Scientific). The following genes were analyzed: TGFB1 (ID: Hs00998133_m1), MMP2 (ID:
Hs01548727_m1), DHRS9 (ID: Hs00608375_m1), STAT3 (ID: Hs00374280_m1) and STAT1 (ID:
Hs01013996_m1). HPRT1 was used as the reference gene. Samples were analyzed as duplicates and
the results were analyzed with CFX Manager™ 3.0 (Bio-Rad) and with the 2−ΔΔCt method using HPRT1
as the reference gene [33]. The relative gene expression levels are expressed as log2 fold change
relative to the Mreg-polarized or Mreg-activated macrophages cultured without hBMSCs.
2.5. Macrophage Phenotyping with Flow Cytometry
The antibody staining was performed as described in Hyvärinen et al. (2018) using anti-human
antibodies PE-CF594-CD86 (clone 2331 FUN-1), APC-CD206 (clone 19.2), BV421-CD163 (clone
GHI/61), PerCP-Cy™5.5-CD90 (clone 5E10) (from BD Biosciences, San Diego, CA, USA), FITC-HLADR (clone L243), BV 510™-CD274 (PD-L1, B7-H1, clone 29E.2A3), PE/Cy7-CD120b (TNFR2, clone
3G7A02) (from BioLegend, San Diego, CA, USA) and PE-MERTK (clone HMER5DS, Thermo Fisher
Scientific). Cell viability was determined with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit
(Thermo Fisher Scientific). Data was acquired with BD FACSAria IIU (BD Biosciences) flow
cytometer using FACSDiva™ (v8.0.1, BD Biosciences) and analyzed with FlowJo® (v10, BD
Biosciences). Gating strategy and doublet discrimination is depicted in Figure S1. Fluorescence
positive cells were determined by using isotype controls and CD90 positive cells were excluded from
the analysis (Figure S1). The results are presented as median fluorescence intensity (MFI) and
frequency of positive cells and as their log2 fold change values relative to the Mreg-polarized or
Mreg-activated macrophages cultured without hBMSCs.
2.6. Yeast Heat-Inactivation and CFSE-Staining
Lyophilized C. albicans pellets (ATCC® 10231™, Microbiologics, Saint Cloud, MN, USA) were
dissolved in 1 mL NaCl Peptone Broth solution (Merck) for 30 min at 37 °C according to the
Cells 2020, 9, 2142 5 of 18
manufacturer’s protocol. The yeast solution was incubated for 1 h in 80 °C to kill the cells [34]. Yeast
solution (1 × 107 cells/mL) was stained with 2 µM carboxyfluorescein succinimidyl ester (CFSE,
Thermo Fisher Scientific) for 15 min at 37 °C. The stained yeast cells were washed with RPMI 1640
and centrifuged at 1000 g for 5 min. The pellet was suspended with polarization medium at
concentration of 5 × 106 cells/mL and filtered. The CFSE-stained C. albicans were stored o/n at 4 °C.
The staining was verified with imaging flow cytometry (Amnis® ImageStream®X Mark II, Luminex
Corporation, Austin, TX, USA). The viability of heat-killed yeast was tested by the absence of growth
on agar plates after 48 h incubation at 37 °C.
2.7. Phagocytosis Assay with Imaging Flow Cytometry
Polarized macrophages were cultured with control-hBMSCs on inserts as described above.
Macrophages derived from five different donors were employed in the phagocytosis assay. On day
10, the hBMSC inserts were removed and the medium was replaced with 1 mL polarization medium
containing heat-killed CFSE-stained C. albicans at low (5 × 105 cells/well) or high (1.25 × 106 cells/well)
concentration. The plates were centrifuged at 30 g for 1 min to synchronize the phagocytosis in all
wells and incubated for 45 min at 37 °C, 5% CO2. The cells were washed with DPBS, detached, stained
with PE-CF594-CD86 and APC-CD206 and analyzed with imaging flow cytometry.
Amnis® ImageStream®X Mark II 12-channel imaging flow cytometer with the software
INSPIRE® (Luminex Corporation) was used for data collection and analysis. Acquisition settings
were as follows: Excitation lasers 405 (off), 488 (15 mW), 642 (150 mW) and 785 (6.75 mW) were
applied for the excitation of fluorochromes and laser Channels (Ch) 01 and Ch09 (bright field, BF),
Ch06 (scattering channel, SSC), plus fluorescence channels Ch02, Ch04 and Ch11 were activated for
signal detection.
All acquisition settings were the same in all experiments. Single cells were separated from debris
and aggregates in the BF channel using the IDEAS features aspect ratio and area. Samples were
acquired at 60× magnification with low flow rate/high sensitivity. At least 2000 events of gated single
cells for each sample were collected. Single color controls were used to create a compensation matrix.
Unlabeled cells and isotype control samples were used to determine the auto fluorescence and the
non-specific background.
Compensated data files were analyzed using algorithms available in the IDEAS® analysis
software (v6.2.188.0). Positive events were gated based on cell morphology and the intensity values
of each fluorescence channel and cell BF images (Figure S2). Gating and compensation values were
used as analysis template for all experimental files. This batch processing of all files assured the
comparisons of each experiment with the other.
2.8. Statistical Analysis
Non-parametric tests were performed due to the non-normal distribution of parameters. The
results are expressed as medians with interquartile ranges (IQR). Pairwise statistical testing was
conducted with Wilcoxon signed rank test. Groupwise statistical testing was conducted with
Kruskal-Wallis rank sum test and post hoc pairwise comparisons using Dunn's test for multiple
comparisons with Mreg-polarized or Mreg-activated macrophages cultured without hBMSCs. All
statistical tests were conducted with R version 3.5.1 and the PMCMR package [35,36] and p-value <
0.05 was considered significant.
3. Results
3.1. Phenotype of Polarized and Activated Macrophages
The phenotypes of macrophages were determined by flow cytometry using markers for T-cell
activation costimulatory molecule CD86, major histocompatibility complex class II cell surface
receptor human leukocyte antigen (HLA)-DR, mannose receptor CD206, scavenger receptor CD163,
programmed death-ligand 1 (PD-L1, also known as CD274), tumor necrosis factor receptor 2 (TNFR2)
and Mer receptor tyrosine kinase (MerTK) (Tables S1 and S2). hBMSCs were excluded using CD90
Cells 2020, 9, 2142 6 of 18
labeling and were detectable only in the samples with hBMSC cell-cell contact (Figure S1). The
median frequency of dead cells of Mreg-polarized macrophages was 1.6% (IQR 2.5) and 1.9% (IQR
5.4) for Mreg-activated macrophages.
The phenotype of Mreg-polarized macrophages is shown in Tables S1 and S2. We observed
considerable donor-specific variation in response to Mreg polarization conditions and subsequently
in the expression of phenotype markers between different buffy coat donors. Approximately, 78% of
these macrophages were positive for CD86, 99.7% for HLA-DR, 35% for CD206, 6% for CD163, 59%
for PD-L1 and <1% for both TNFR2 and MerTK. The phenotype of Mreg-activated macrophages
(mature Mregs) was similar to that of Mreg-polarized macrophages (Tables S1 and S2).
Approximately 97% of the activated macrophages were positive for CD86, 99.9% for HLA-DR, 23%
for CD206, <1% for CD163, 92% for PD-L1, negative for TNFR2 and <1% for MerTK. In general, the
expression levels of phenotype markers were similar between the two macrophage types, but the
Mreg-activated macrophages had a higher frequency of CD86+ (pairwise Wilcoxon signed rank test,
p = 0.031) and PD-L1+ cells (p = 0.031) than Mreg-polarized macrophages.
Both macrophage types produced TNF-α and IL-10 (Table 1). The production of TNF-α
increased in Mreg-activated macrophages compared with Mreg-polarized macrophages (pairwise
Wilcoxon signed rank test, p = 0.031) while the production of IL-10 remained at similar levels. The
production of IL-23 was negligible. The donor-specific variation was high in the cytokine production.
Both Mreg-polarized and Mreg-activated macrophages expressed TGF-β1, matrix
metalloproteinase (MMP)-2, human Mreg marker dehydrogenase/reductase 9 (DHRS9) [15], signal
transducer and activator of transcription (STAT)3 and STAT1, determined with QPCR. There were
no statistically significant differences in gene expression between the two macrophage types (not
shown). Expression was, again, variable depending on the donor.
3.2. hBMSC Secretome Skews Mreg-Polarized and Mreg-Activated Macrophages toward an AntiInflammatory and Proresolving Phenotype
The phenotype of both Mreg-polarized and Mreg-activated macrophages was modified
especially by the hBMSCs secretome. The effect of hBMSC cell-cell contact and secretome on the cell
surface protein expression of Mreg-polarized macrophages is shown in Figure 2 and Tables S1 and
S2. Strikingly, the hBMSC secretome increased the log2 fold change of both CD206 MFI (KruskalWallis test with all hBMSC conditions, p < 0.001; post hoc test p-values are described in Figure 2) and
frequency of CD206+ cells (p = 0.002) regardless of the PUFA supplementation. The secretome of
control- and DHA-hBMSCs elevated also the log2 fold change of PD-L1 MFI (p < 0.001) and DHAhBMSCs the log2 fold change of CD163 MFI (p = 0.039), but the effect was donor-dependent.
Interestingly, hBMSC cell-cell contact with all PUFA modifications decreased the log2 fold change of
HLA-DR MFI (p = 0.001).
The effect of hBMSC cell-cell contact and secretome on the cell surface protein expression of
Mreg-activated macrophages is shown in Figure 3 and Tables S1 and S2. Similar to Mreg-polarized
macrophages, the log2 fold change of CD206 MFI and the frequency of CD206+ cells increased in the
Mreg-activated macrophages by the secretome of hBMSCs with all PUFA modifications (KruskalWallis test of all hBMSC conditions p < 0.001; post hoc test p-values are described in Figure 3) and
with AA supplementation (p < 0.001), respectively. Moreover, the secretome elevated the log2 fold
change frequency of MerTK+ cells regardless of the PUFA supplementation (p = 0.014). The
expression was still low (<15% positive cells) and the increase was variable between donors. The cellcell contact had an effect on only the log2 fold change frequency of HLA-DR+ cells, which was slightly
decreased regardless of the PUFA modification (p < 0.001). The most drastic change in both Mregpolarized and Mreg-activated macrophages was the elevated CD206 expression, which is depicted in
Figure 4 (presenting the CD206 data in more detail without the log2 fold change values).
Cells 2020, 9, 2142 7 of 18
Figure 2. The effect of hBMSC cell-cell contact and secretome on the cell surface the phenotype of Mreg-polarized macrophages. The median fluorescence intensities (MFI,
on the left in each panel) and frequencies of positive cells (on the right in each panel) were determined with flow cytometry investigating the expression of (A) CD86, (B)
HLA-DR, (C) CD206, (D) CD163, (E) PD-L1 and (F) MerTK. The effect of hBMSCs is visualized with log2 fold changes calculated against macrophages cultured without
hBMSCs (represented by the zero line) for each individual donor. The differences among groups were determined with Kruskal-Wallis rank sum test and post hoc using
Dunn's test (the latter presented in the figures). The results are expressed as log2 fold changes as medians with interquartile ranges; n = 6 biological replicates. Ctrl, control;
DHA, docosahexaenoic acid; AA, arachidonic acid.
Cells 2020, 9, 2142 8 of 18
Figure 3. The effect of hBMSC cell-cell contact and secretome on the cell surface the phenotype of Mreg-activated macrophages. The median fluorescence intensities (MFI,
on the left in each panel) and frequencies of positive cells (on the right in each panel) were determined with flow cytometry investigating the expression of (A) CD86, (B)
HLA-DR, (C) CD206, (D) CD163, (E) PD-L1 and (F) MerTK. The effect of hBMSCs is visualized with log2 fold changes calculated against macrophages cultured without
hBMSCs (represented by the zero line) for each individual donor. The differences among groups were determined with Kruskal-Wallis test and post hoc using Dunn's test.
The results are expressed as log2 fold changes as medians with interquartile ranges; n = 6 biological replicates. Ctrl, control; DHA, docosahexaenoic acid; AA, arachidonic
acid.
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Table 1. Effect of hBMSC cell-cell contact and secretome with Mreg-polarized and Mreg-activated macrophages to cytokine production.
Concentration, pg/mL (IQR)
Cell-Cell
Contact
Cell-Cell
Contact
Cell-Cell
Contact Secretome Secretome Secretome
Cytokine Mreg-Polarized
Mreg-Polarized
+ControlhBMSC
Mreg-Polarized
+DHA-hBMSC
Mreg-Polarized
+AA-hBMSC
Mreg-Polarized
+ControlhBMSC
Mreg-Polarized
+DHA-hBMSC
Mreg-Polarized
+AA-hBMSC p-value a
TNF-α 123.5 (131.5) 247.6 (199.7) 165.4 (278.8) 183.6 (177.2) 181.9 (434.9) 228.0 (273.8) 192.8 (350.3) 0.999
IL-10 39.5 (81.5) 48.0 (31.7) 36.9 (55.4) 36.1 (31.4) 32.5 (40.5) 29.1 (29.6) 53.6 (27.7) 0.975
IL-23 0.0 (0.0) 0.3 (0.9) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.015
Mreg-activated
Mreg-activated
+controlhBMSC
Mreg-activated
+DHA-hBMSC
Mreg-activated
+AA-hBMSC
Mreg-activated
+controlhBMSC
Mreg-activated
+DHA-hBMSC
Mreg-activated
+AA-hBMSC p-value a
TNF-α 1591.6 (1898.5) 2106.7 (1825.5) 1887.1 (2041.1) 1899.1 (1672.3) 2025.5 (1512.2) 1950.5 (1895.9) 1823.6 (1959.4) 0.998
IL-10 31.0 (69.9) 45.5 (70.4) 39.6 (54.4) 33.9 (76.7) 31.5 (88.2) 45.0 (57.1) 33.3 (69.6) 0.871
IL-23 0.0 (0.1) 0.4 (2.7) 0.4 (1.0) 0.0 (0.2) 0.1 (0.2) 0.0 (0.4) 0.1 (0.4) 0.757
DHA, docosahexaenoic acid; AA, arachidonic acid; TNF, tumor necrosis factor; IL, interleukin; IQR, interquartile range. a The statistical significance of variation between
groups was determined using the Kruskal-Wallis rank sum test.
Cells 2020, 9, 2142 10 of 18
Figure 4. The secretome of hBMSCs increases the expression of CD206. (A) The upper panel describes
the results of Mreg-polarized macrophages and (B) the lower panel the results of Mreg-activated
macrophages. The median fluorescence intensities (MFI, left panel) and frequencies of positive cells
(middle panel) were determined with flow cytometry. The representative histograms are presented
on the right. The differences among groups were determined with Kruskal-Wallis rank sum test and
post hoc using Dunn's test. These results are also described as log2 fold changes in Figures 2 and 3.
The results are expressed as medians with interquartile ranges; n = 6 biological replicates. Ctrl, control;
DHA, docosahexaenoic acid; AA, arachidonic acid.
The hBMSCs had no effect on the cytokine production in this experimental setting (Table 1). In
the coculture with Mreg-polarized macrophages, the production of IL-23 increased in by the cell-cell
contact of control-hBMSCs (Kruskal-Wallis test p = 0.015; post hoc Dunn's test, p = 0.002); however,
the production of IL-23 was at the detection limit and this result should be interpreted with caution.
The expression of most genes investigated remained unaltered by the hBMSC secretome (Figure 5).
Nevertheless, DHA-hBMSCs resulted in a decreased MMP-2 gene expression in the Mreg-polarized
macrophages (Kruskal-Wallis test p = 0.031; post hoc Dunn's test, p = 0.007) and AA-hBMSCs caused
a similar trend. There was also a trend of increased gene expression of the Mreg marker DHRS9 in
DHA and AA-hBMSCs coculture.
Cells 2020, 9, x 11 of 18
Figure 5. The effect of hBMSC secretome on the gene expression of Mreg-polarized and Mregactivated macrophages. The gene expression of macrophage phenotype markers was investigated
with QPCR. The effect of hBMSCs on (A) Mreg-polarized and (B) Mreg-activated macrophages is
visualized with log2 fold changes calculated against macrophages cultured without hBMSCs
(represented by the zero line) for each individual donor. The differences among groups were
determined with Kruskal-Wallis rank sum test and post hoc using Dunn's test. The results are
expressed as medians with interquartile ranges; n = 4 biological replicates. Ctrl, control; DHA,
docosahexaenoic acid; AA, arachidonic acid.
3.3. Phagocytosis Assay
The C. albicans phagocytosis activity of Mreg-polarized macrophages was assessed with/without
hBMSCs secretome and the results analyzed with imaging flow cytometry (Figure 6). The
macrophages elicited a donor-dependent CD86 and CD206 expression. In contrast to the previous
Cells 2020, 9, x 12 of 18
results, hBMSC secretome did not increase the CD206 expression in all of the macrophages derived
from different PBMC donors (non-responders n = 2, Figure S3). However, in three cases, the CD206
increased by 1.9 to 4.2 fold when compared with the non-responders, which elicited a 0.7 to 0.8-fold
decrease in the CD206 expression. The results of the CD206 responders are reported in Figure 6. In
the responders, the phagocytosis of low concentration (5 × 105 cells/well) C. albicans increased by 1.6
to 5.8 fold in the hBMSC secretome group when compared with Mreg-polarized macrophages alone.
In general, the levels of ingested C. albicans were higher in the high concentration (1.25 × 106 cells/well)
group and the phagocytosis increased by 1.2 to 3.1-fold in the hBMSC secretome group when
compared with Mreg-polarized macrophages alone.
Figure 6. hBMSC secretome improves the C. albicans phagocytosis activity of Mreg-polarized
macrophages in a CD206-mediated manner. (A) The frequency of CD86 and CD206 positive cells and
the phagocytosis of CFSE-stained C. albicans were determined with imaging flow cytometry, n = 3
biological replicates. (B) A representative imaging flow cytometry image of a macrophage that has
phagocytosed C. albicans. Low C. albicans concentration, 5 × 105 cells/well; high C. albicans
concentration, 1.25 × 106 cells/well; CFSE, CFSE-stained C. albicans.
4. Discussion
We investigated the effect of hBMSC cell-cell contact and secretome to the phenotype of
immunoregulatory macrophages, which were either polarized (Mreg-polarized) or polarized and
activated (Mreg-activated) toward Mreg phenotype. Moreover, we supplemented hBMSCs with
PUFAs DHA or AA prior to the macrophage coculture to elucidate if PUFAs induced changes in the
immunomodulatory capacity of hBMSCs. The hBMSC secretome increased the expression of antiinflammatory and proresolving phenotype markers in both macrophage types with all hBMSC
modifications (control, DHA and AA). In particular, we observed a substantial increase in the CD206
expression. Furthermore, the hBMSC secretome increased donor-dependently the phagocytosis
activity of Mreg-polarized macrophages in C. albicans phagocytosis assay, a result which was
associated with the increased CD206 expression. Intriguingly, the DHA-supplemented hBMSCs
induced the most prominent anti-inflammatory changes in Mreg-polarized macrophages while AA
supplementation had only a slight effect.
Our aim was to study macrophages both in a more naïve stage (Mreg-polarized macrophages)
and as mature Mregs (Mreg-activated macrophages) [37] in order to elucidate the effect of hBMSCs
on macrophages at different polarization stages. The phenotypes of these two macrophage types
were similar, but the Mreg-activated macrophages manifested a more classically activated phenotype
with increased expression of CD86 and PD-L1. Moreover, the Mreg-activated macrophages produced
Cells 2020, 9, x 13 of 18
more TNF-α than Mreg-polarized macrophages as expected due to Toll-like receptor engagement by
LPS.
Previously, we demonstrated that hBMSC cell-cell contact and hBMSC-EVs enhanced the antiinflammatory properties of mature Mregs by decreasing the production of IL-23 and IL-22 and
increasing the PGE2 production [16]. In the current study, we did not detect a decrease in the IL-23
production, which was very low when measured with ELISA (previously analyzed with ProcartaPlex
Immunoassay), but we observed other anti-inflammatory effects of hBMSCs on these
immunoregulatory macrophages. Additionally, the experimental setup and the markers investigated
of these two studies were slightly different.
The hBMSC secretome enhanced the anti-inflammatory properties of both Mreg-polarized and
Mreg-activated macrophages especially by increasing the CD206 expression. CD206, also known as
mannose receptor, is a pattern recognition receptor, which binds to the glycan structures on microbes
[38]. Previous studies have shown that human MSC cell-cell contact or secretome can increase the
expression of CD206 and overall anti-inflammatory properties of monocytes or M1 macrophages
[9,11]. Moreover, human MSC-derived EVs can increase the CD206 and anti-inflammatory properties
of murine macrophages [39]. The secretome consists of secreted cytokines, lipid mediators and other
molecules and also includes EVs. Thus, our results with macrophages polarized toward Mregs are in
agreement with previous studies investigating monocytes or other macrophage subtypes and CD206
expression. In our preceding study with Mregs, we did not observe an increase in the CD206
expression with hBMSC-EV addition [16]; however, the experimental settings of our two studies are
not directly comparable. Previously, the EVs added to Mregs were derived from unstimulated
hBMSCs and given in two doses. In the current study, the hBMSCs produced the EVs and additional
soluble factors constantly in a stimulated environment with macrophage coculture.
Next, we investigated if the increased CD206 expression translated to an increased phagocytosis
activity of yeast C. albicans by Mreg-polarized macrophages in the presence of hBMSC secretome. The
cell surface of C. albicans is covered in terminal mannose residues that are recognized by CD206 of
macrophages [38,40]. Interestingly, when the hBMSC secretome increased the CD206 expression in
the responder macrophages, the phagocytosis of C. albicans was also increased indicating an
association between these two hBMSC secretome-mediated phenomena. The effect was PBMC donordependent, because the donors responded to the polarization and hBMSC secretome differently. Both
human and murine MSCs have been shown to induce the phagocytosis of macrophages in various
assays [10,12,41] but to our knowledge this is the first study showing that MSCs induce the
phagocytosis of C. albicans. One of the key aspects in the resolution of inflammation is the clearance
of pathogens and apoptotic cells via phagocytosis and efferocytosis [24]. Phagocytosing macrophages
become more prevalent during resolution of inflammation when the cell debris and microbes are
cleared away in order to achieve homeostasis [24] emphasizing the ability of hBMSC secretome to
promote the proresolving phenotype of macrophages.
In addition to CD206, the PD-L1 expression increased by the hBMSC secretome in Mregpolarized macrophages. When PD-L1 binds to its receptor, a co-inhibitory receptor programmed
death 1 (PD-1), T-cell activation and proliferation are inhibited and the immune response is
attenuated [42]. PD-L1 is expressed on the surface of proresolving macrophages [43], which indicates
that the hBMSC secretome skews the macrophage phenotype in an anti-inflammatory direction.
Supporting our results, MSCs have been shown to increase the PD-L1 expression in M2-type
macrophages [10].
Strikingly, in Mreg-activated macrophages, the hBMSC secretome increased the expression of
MerTK, which was generally negative in the cells. The different PBMC donors; however, responded
in a different manner. MerTK is a marker of anti-fibrotic M2c macrophages [44], it is important in the
clearance of apoptotic cells [45] and induces SPM production in macrophages [46]. Interestingly, EVs
from cardiosphere-derived cells are able to increase the MerTK expression of macrophages via the
transfer of microRNA-26a [47], which may indicate that the EVs in the hBMSC secretome are
mediating the increased MerTK expression. It has been well established that EVs mediate a large
proportion of the immunomodulatory effects of MSCs [48]. Although we did not examine the effect
Cells 2020, 9, x 14 of 18
of EVs alone, we can hypothesize that observed anti-inflammatory and proresolving effects of
hBMSC secretome were at least in part mediated by the EV fraction.
The secretome of hBMSCs had a larger impact on the phenotype of macrophages than the
hBMSC cell-cell contact. Mainly, the cell-cell contact lowered the HLA-DR expression of Mregpolarized and, to a certain extent, Mreg-activated macrophages. HLA-DR is a proinflammatory
marker and is involved in the T-cell activation via antigen presentation. In agreement with our result,
human MSCs can diminish the HLA-DR expression of macrophages and monocytes [49,50]
indicating that the hBMSC cell-cell contact also rendered the macrophage phenotype more antiinflammatory.
The PUFA supplementation of hBMSCs prior the coculture with macrophages resulted in a more
pronounced anti-inflammatory phenotype of Mregs. In particular, the secretome of DHA-hBMSCs
increased the CD163 expression and decreased the gene expression of gelatinase MMP-2 in Mregpolarized macrophages while control- or AA-hBMSCs had no effect. CD163 is a hemoglobin
scavenger receptor inducing an anti-inflammatory response and its increased expression is one of the
major changes in the macrophage switch to alternatively activated phenotype [51]. On the other hand,
M1 macrophages secrete MMP-2 to induce the degradation of extracellular matrix and recruitment
of inflammatory cells to the site of tissue injury [52]. Previous studies have shown that MSCs are able
to decrease the gene expression of MMP-2 of macrophages in vitro [53] and in vivo [54] corroborating
with our results. The secretome of AA-hBMSCs did not have a significant effect on the PD-L1
expression in Mreg-polarized macrophages while control and DHA-hBMSCs increased this
expression. This result hints that AA supplementation could lower the immunosuppressive
properties of hBMSCs, because PD-L1 is important in suppressing the T-cell mediated immune
response [42]. Contrastingly, the increase in CD206 expression in Mreg-activated macrophages was
the most prominent with AA-hBMSC secretome.
Previous in vivo studies have reported that EPA-supplemented murine MSCs had superior
therapeutic potential when compared with control MSCs [28,30]. Moreover, DHA-supplemented
murine MSCs increased the survival in an in vivo sepsis model when compared with AAsupplemented MSCs [29]. In this study, we observed more prominent anti-inflammatory changes in
Mregs with DHA-hBMSCs than with control or AA-hBMSCs; however, the effect was visible in only
two phenotype markers. The lack of a substantial effect of PUFA supplementation to hBMSC
immunomodulation in this in vitro assay may be due to different reasons. Firstly, the PUFA
remodeling of hBMSC membranes takes place relatively quickly, beginning already after 2 hours
after PUFA supplementation and leading into prominent remodeling after 24 hours [27]. In the
current in vitro setting, the medium in the 3-day macrophage-hBMSC culture contained 10% FBS.
Even though this FBS most likely has modified the cell membranes of hBMSCs and lowered the PUFA
content of the membranes, we hypothesized that the initial modifications in the hBMSC membranes
would suffice to induce profound changes already at the beginning of the coculture. This hypothesis
is supported by the studies, where the EPA- and DHA-supplemented MSCs demonstrated improved
therapeutic potential [28–30], even though the cell membranes of these cells would most likely be
remodeled in vivo.
Additionally, our in vitro setting focused on changes in polarized and activated Mregs and DHA
and AA supplementation. Although limited in this experimental setting, PUFA modifications could
still have an impact in hBMSC immunomodulatory properties in other immunological settings.
Moreover, EPA supplementation has been shown to be beneficial for MSC immunomodulation
[28,30]. EPA is the precursor to PGE3, which is less proinflammatory than PGE2 [55] and could be one
of the underlying reasons for the pronounced effect of EPA supplementation to MSC
immunomodulation. Thus, the effects of PUFA-modified hBMSCs call for further investigations. If
PUFA supplementations of MSCs prove out to be beneficial, these supplementations represent an
easy and safe way to improve the therapeutic response of MSCs.
The macrophage assays were conducted with primary human PBMCs from individual donors.
We acknowledge the challenges of using primary PBMC-derived macrophages as a model due to
their high plasticity and variability in responding to cytokines and activation. However, by
Cells 2020, 9, x 15 of 18
employing primary human cells, we achieve a more realistic setting than by the use of cell lines,
representing a more physiologically accurate situation and acknowledging the varying responses of
the patients in the clinic. Indeed, the magnitude of the response to different assay conditions and
subsequently, the phenotypes of macrophages derived from individual donors were variable. The
hBMSC secretome induced prominent differences in Mreg-polarized and Mreg-activated
macrophages. Some of the changes in phenotype markers, as in CD206, were clear in all individual
donors by the hBMSC secretome highlighting the significance of this finding. It is also noteworthy
that although some of the effects were small, all of the changes skewed macrophages toward an antiinflammatory and proresolving direction. Moreover, we investigated the effects of hBMSCs derived
from three different donors to multiple buffy coat derived PBMCs, which enhances the robustness of
our findings.
To conclude, our results demonstrate that the hBMSC secretome can modify macrophages
toward immunoregulatory and proresolving phenotype, especially by increasing CD206, PD-L1 and
MerTK expression. Moreover, by increasing the CD206 expression, the secretome increased the C.
albicans phagocytosis activity of Mreg-polarized macrophages. According to our hypothesis, hBMSCs
skew macrophages toward a proresolving phenotype that facilitate wound healing and restore
homeostasis. Interestingly, DHA-hBMSCs also increased the expression of CD163 and decreased the
gene expression of MMP-2 in Mreg-polarized macrophages indicating that the DHA modifications
have an impact on the immunomodulatory properties of hBMSCs. These findings highlight the
potential of PUFA supplementations as an easy and safe method to improve the hBMSC therapeutic
potential.
Supplementary Materials: The following are available online at www.mdpi.com/2073-4409/9/9/2142/s1, Figure
S1: Gating strategy and CD90 exclusion, Figure S2: Gating strategy in imaging flow cytometry, Figure S3: The
phagocytose assay results from the CD206 non-responders, Table S1: Effect of hBMSC cell-cell contact and
secretome on the median fluorescence intensity of phenotype markers on Mreg-polarized and Mreg-activated
macrophages, Table S2: Effect of hBMSC cell-cell contact and secretome on the frequency of positive cells of
phenotype markers on Mreg-polarized and Mreg-activated macrophages.
Author Contributions: Conceptualization, M.H., S.L. and E.K.; Data curation, M.H. and U.I.; Formal analysis,
M.H. and U.I.; Funding acquisition, M.H. and S.L.; Investigation, M.H., S.L. and E.L; Methodology, U.I.;
Resources, P.L. and S.L.; Supervision, S.L. and E.K.; Visualization, M.H.; Writing—original draft, M.H.;
Writing—review & editing, M.H., U.I., P.L, S.L. and E.K. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was supported by Finnish Cultural Foundation (M.H.) and Clinical State Research Funding
[EVO/ VTR grant, Finland] (M.H.).
Acknowledgments: We thank Kati Hyvärinen, Lindsay Davies and Kaarina Lähteenmäki for helpful advice in
designing the experiments. We also thank Birgitta Rantala, Lotta Andersson and Lotta Sankkila for excellent
technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.
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Re: JADICELL -Mesenchymal stem cell-derived exosomes: Hope for spinal cord injury repair

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Mesenchymal stem cell-derived exosomes: Hope for spinal cord injury repair

https://www.liebertpub.com/doi/abs/10.1 ... .2020.0133

Abstract
Spinal cord injury (SCI) is a devastating medical condition with profound social and economic impacts. While research is ongoing, current treatment options are limited and do little to restore functionality. However, recent studies suggest that mesenchymal stem cell-derived exosomes (MSC-exosomes) may hold the key to exciting new treatment options for SCI patients. Mesenchymal stem cells (MSCs) are self-renewing multipotent stem cells with multi-directional differentiation and can secrete a large number of exosomes (vesicles secreted into the extracellular environment through endocytosis, called MSC-exosomes). These MSC-exosomes play a critical role in repairing SCI through promoting angiogenesis and axonal growth, regulating inflammation and the immune response, inhibiting apoptosis, and maintaining the integrity of the blood-spinal cord barrier. Further, they can be utilized to transport genetic material or drugs to target cells, and their relatively small size makes them able to permeate the blood brain barrier. In this review, we summarize recent advances in MSC-exosome themed SCI treatments and cell-free therapies to better understand this newly emerging methodology.
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