# SAFETY, EFFICACY AND MECHANISMS OF ACTION OF MESENCHYMAL STEM CELL THERAPIES

EDITED BY : Guido Moll, Martin Johannes Hoogduijn and James A. Ankrum PUBLISHED IN : Frontiers in Immunology and Frontiers in Medicine

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# SAFETY, EFFICACY AND MECHANISMS OF ACTION OF MESENCHYMAL STEM CELL THERAPIES

Topic Editors:

Guido Moll, Charité – Medical University of Berlin, Germany Martin Johannes Hoogduijn, Erasmus University Rotterdam, Netherlands James A. Ankrum, The University of Iowa, United States

Citation: Moll, G., Hoogduijn, M. J., Ankrum, J. A., eds. (2020). Safety, Efficacy and Mechanisms of Action of Mesenchymal Stem Cell Therapies. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-600-6

This study has received support under the European Union's Horizon 2020 research and innovation program (Horizon 2020 Framework Program) under the grant agreements no. 733006 (PACE) and no. 779293 (HIPGEN).

# Table of Contents

*06 Editorial: Safety, Efficacy and Mechanisms of Action of Mesenchymal Stem Cell Therapies*

Guido Moll, Martin J. Hoogduijn and James A. Ankrum

*10 Mesenchymal Stromal Cell Therapeutic Delivery: Translational Challenges to Clinical Application*

Henry Caplan, Scott D. Olson, Akshita Kumar, Mitchell George, Karthik S. Prabhakara, Pamela Wenzel, Supinder Bedi, Naama E. Toledano-Furman, Fabio Triolo, Julian Kamhieh-Milz, Guido Moll and Charles S. Cox Jr.

*25 Multiplicity of Mesenchymal Stromal Cells: Finding the Right Route to Therapy*

Alison Wilson, Margeaux Hodgson-Garms, Jessica E. Frith and Paul Genever

Anastazja Andrzejewska, Rusan Catar, Janosch Schoon, Taimoor Hasan Qazi, Frauke Andrea Sass, Dorit Jacobi, Antje Blankenstein, Simon Reinke, David Krüger, Mathias Streitz, Stephan Schlickeiser, Sarina Richter, Naima Souidi, Christien Beez, Julian Kamhieh-Milz, Ulrike Krüger, Tomasz Zemojtel, Karsten Jürchott, Dirk Strunk, Petra Reinke, Georg Duda, Guido Moll and Sven Geissler

*88 Effects of Normothermic Machine Perfusion Conditions on Mesenchymal Stromal Cells*

Jesus M. Sierra Parraga, Kaithlyn Rozenberg, Marco Eijken, Henri G. Leuvenink, James Hunter, Ana Merino, Cyril Moers, Bjarne K. Møller, Rutger J. Ploeg, Carla C. Baan, Bente Jespersen and Martin J. Hoogduijn

*99 Comparison of Mesenchymal Stromal Cells From Different Origins for the Treatment of Graft-vs.-Host-Disease in a Humanized Mouse Model*

Céline Grégoire, Caroline Ritacco, Muriel Hannon, Laurence Seidel, Loïc Delens, Ludovic Belle, Sophie Dubois, Sophie Vériter, Chantal Lechanteur, Alexandra Briquet, Sophie Servais, Gregory Ehx, Yves Beguin and Frédéric Baron

*115 Preclinical Toxicity Evaluation of Clinical Grade Placenta-Derived Decidua Stromal Cells*

Behnam Sadeghi, Gianluca Moretti, Fabian Arnberg, Erik Samén, Bita Kohein, Rusan Catar, Julian Kamhieh-Milz, Sven Geissler, Guido Moll, Staffan Holmin and Olle Ringdén

#### *130 Adipose-Derived Mesenchymal Stem Cells Applied in Fibrin Glue Stimulate Peripheral Nerve Regeneration*

Ruslan Masgutov, Galina Masgutova, Adelya Mullakhmetova, Margarita Zhuravleva, Anna Shulman, Alexander Rogozhin, Valeriya Syromiatnikova, Dina Andreeva, Alina Zeinalova, Kamilla Idrisova, Cinzia Allegrucci, Andrey Kiyasov and Albert Rizvanov

*142 Targeting Mesenchymal Stromal Cells/Pericytes (MSCs) With Pulsed Electromagnetic Field (PEMF) Has the Potential to Treat Rheumatoid Arthritis*

Christina L. Ross, Dennis C. Ang and Graça Almeida-Porada

*154 Cost-Effective, Safe, and Personalized Cell Therapy for Critical Limb Ischemia in Type 2 Diabetes Mellitus*

Bárbara Soria-Juan, Natalia Escacena, Vivian Capilla-González, Yolanda Aguilera, Lucía Llanos, Juan R. Tejedo, Francisco J. Bedoya, Verónica Juan, Antonio De la Cuesta, Rafael Ruiz-Salmerón, Enrique Andreu, Lukas Grochowicz, Felipe Prósper, Fermín Sánchez-Guijo, Francisco S. Lozano, Manuel Miralles, Lourdes Del Río-Solá, Gregorio Castellanos, José M. Moraleda, Robert Sackstein, Mariano García-Arranz, Damián García-Olmo, Franz Martín, Abdelkrim Hmadcha, Bernat Soria and the Collaborative Working Group "Noma Project Team"

#### *173 Dissecting Allo-Sensitization After Local Administration of Human Allogeneic Adipose Mesenchymal Stem Cells in Perianal Fistulas of Crohn's Disease Patients*

Alvaro Avivar-Valderas, Cristina Martín-Martín, Cristina Ramírez, Borja Del Río, Ramón Menta, Pablo Mancheño-Corvo, Maitane Ortiz-Virumbrales, Ángel Herrero-Méndez, Julián Panés, Damián García-Olmo, José Luís Castañer, Itziar Palacios, Eleuterio Lombardo, Wilfried Dalemans and Olga DelaRosa

#### *188 Mesenchymal Stromal Cell Secretome: Influencing Therapeutic Potential by Cellular Pre-conditioning*

Joana R. Ferreira, Graciosa Q. Teixeira, Susana G. Santos, Mário A. Barbosa, Graça Almeida-Porada and Raquel M. Gonçalves

*205 Immunomodulatory Effect of MSC on B Cells is Independent of Secreted Extracellular Vesicles*

Laura Carreras-Planella, Marta Monguió-Tortajada, Francesc Enric Borràs and Marcella Franquesa

Andreas Robert Rudolf Weiss and Marc Hendrik Dahlke

*238 The Necrobiology of Mesenchymal Stromal Cells Affects Therapeutic Efficacy*

Daniel J. Weiss, Karen English, Anna Krasnodembskaya, Johana M. Isaza-Correa, Ian J. Hawthorne and Bernard P. Mahon *250 Enhanced Immunomodulation in Inflammatory Environments Favors Human Cardiac Mesenchymal Stromal-Like Cells for Allogeneic Cell Therapies*

Falk Diedrichs, Meaghan Stolk, Karsten Jürchott, Marion Haag, Michael Sittinger and Martina Seifert

Sofia Oja, Tanja Kaartinen, Marja Ahti, Matti Korhonen, Anita Laitinen and Johanna Nystedt

# Editorial: Safety, Efficacy and Mechanisms of Action of Mesenchymal Stem Cell Therapies

Guido Moll 1 \* † , Martin J. Hoogduijn2 \* † and James A. Ankrum3 \* †

1 BIH Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health (BIH), Berlin, Germany, 2 Nephrology and Transplantation, Department of Internal Medicine, Erasmus MC, University Medical Center, Rotterdam, Netherlands, 3 Roy J. Carver Department of Biomedical Engineering, Fraternal Order of Eagles Diabetes Research Center, Pappajohn Biomedical Institute, University of Iowa, Iowa City, IA, United States

Keywords: mesenchymal stem cells, mesenchymal stromal cells, immunomodulation, regeneration, mechanism of action, safety, efficacy, potency analysis

**Editorial on the Research Topic**

INTRODUCTION

**Safety, Efficacy and Mechanisms of Action of Mesenchymal Stem Cell Therapies**

Edited and reviewed by:

Denise Doolan, James Cook University, Australia

#### \*Correspondence:

Guido Moll<EMAIL_ADDRESS>Martin J. Hoogduijn<EMAIL_ADDRESS>James A. Ankrum<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology

> Received: 11 December 2019 Accepted: 30 January 2020 Published: 18 February 2020

#### Citation:

Moll G, Hoogduijn MJ and Ankrum JA (2020) Editorial: Safety, Efficacy and Mechanisms of Action of Mesenchymal Stem Cell Therapies. Front. Immunol. 11:243. doi: 10.3389/fimmu.2020.00243 Mesenchymal stromal/stem cell (MSC) therapies have been employed in more than 800 registered clinical studies across the globe (1) and there are now >55,000 publications readily available on MSCs (2). Their profound immunomodulatory and regenerative properties have made MSCs one of the most promising and intensely pursued cellular therapies (3). Although meta-analysis of clinical trials with first-generation MSC products has demonstrated safety (4), their clinical efficacy and understanding of the underlying mechanism of action (MoA) still needs to be improved [(1, 5–10); Caplan et al.]. A better understanding of the role of patient parameters and adjunct treatment protocols is key to yield an optimal short- and long-term therapeutic benefit. Indeed, different MSC products, as well as their dosing and delivery, may be tailored for specific clinical indications according to their individual needs (6, 8, 11). To optimize next-generation MSC therapies, efforts are now underway to improve product design and delivery to patients, safety and potency assessment pre- and post-treatment, and the understanding of the exact MoA. These important topics are covered within this article collection and in the following sections we will briefly put into context the 20 articles published within this Frontiers Research Topic: "Safety, Efficacy, and Mechanisms of Action of MSC Therapies".

#### DIVERSIFICATION IN MSC PRODUCTS AND DELIVERY

A great diversification in MSC products, treatment indications, and delivery methods has occurred over the past decade, raising many regulatory questions, and potentially entailing reevaluation of safety and efficacy for new products/applications [(1, 12); Caplan et al.]. Adjustments in manufacturing are manifold, e.g., cell expansion conditions, culture media composition, or cell priming (10). A key issue is the tissue source the MSCs are derived from, with clinical trials in the past 5 years utilizing MSCs from bone marrow (BM), adipose tissue (AT), and perinatal tissue (PT) at almost equal frequency (1).

**6**

Wilson et al. give a great overview on all aspects of MSC heterogeneity, from donor to tissue source, the role of cell isolation and in vitro expansion, and the regulatory considerations related to heterogeneous cell therapy. In line, Ankrum and coworkers, who recently reviewed the MSC manufacturing process for therapy (10), newly define isolation and culture conditions to better prepare MSCs for the challenging in vivo environments they encounter post transplantation in their title "Nature vs. Nurture" (Boland et al.).

In their review, Khan and Newsome provide an exemplary assessment on how the production process can shape the phenotype and functional properties of BM-derived multipotent adult progenitor cells (MAPC R , Athersys Inc, Cleveland Ohio) compared to various conventional BM-MSC products. Andrzejewska et al. employed multi-parameter analysis to decipher the relative impact of in vitro culture aging (early vs. later passage) vs. in vivo donor aging (adult vs. elderly donors and typically associated mild comorbidities) on BM-MSC properties in biobanking approaches.

Caplan et al. summarized how delivery methods shape the outcome of MSC therapy, differentiating between specific types of local and systemic delivery, and they further elaborate on the role of innate and adaptive immune responses, in particular cell product hemocompatibility aspects, on steering the clinical outcome. Along with earlier studies, the authors emphasize the need for prior hemocompatibility testing of cell products, if they are intended to be applied by systemic intravascular delivery [(1); Caplan et al.]. Today it is well-recognized that intravascular delivered MSCs get largely trapped in the microvascular network of the lungs and tissues. Recently developed technology to ex vivo perfuse transplant organs on machine perfusion allows directly delivery of MSC via arterial access. To this end, Sierra Parraga et al. report on the effects of machine perfusion conditions on the survival and functionality of MSCs.

# SAFETY AND EFFICACY OF MSC

Grégoire et al. compared different MSC products derived from the three most commonly employed tissue sources (AT-, BM-, and PT-derived) in a mouse model of acute graft-vs.-host disease (GvHD). Sadeghi et al. present their results on the preclinical toxicity evaluation of clinical grade placenta-derived decidual stromal cells (DSCs) in different preclinical models. Masgutov et al. report their promising preclinical findings on peripheral nerve regeneration upon local delivery of AT-MSCs in fibrin glue. A whole different concept is to target endogenous MSC to induce immunomodulatory and regenerative effects. Ross et al. explored this concept with an anti-inflammatory extremely-low frequency pulsed electromagnetic field (PEMF) to reduce chronic inflammation for treatment of rheumatoid arthritis.

Soria-Juan et al. give a hands-on overview on their many years of experience in treatment of critical limb ischemia and diabetes with cell products, in particular AT-derived MSCs, and their optimal delivery. Avivar-Valderas et al. share their valuable data on allo-sensitization after local administration of allogeneic AT-MSCs (Darvadstrocel formerly Cx601, from Takeda/TiGenix) along with detailed mechanistic side-studies on protection and susceptibility to attack by the complement system.

# MECHANISM OF ACTION (MoA): MULTIFACTORIAL CROSSTALK

MSC's regenerative properties and modulation of the immune system have driven their therapeutic application for a variety of conditions. Importantly, these effects are not mediated by a single MoA; Rather, MSCs modulate different tissue and immune cells through numerous soluble immunomodulatory and trophic factors, different types of subcellular vesicles, and efferocytosis mechanisms (Ferreira et al.; Carreras-Planella et al.; Podestà et al.; Weiss and Dahlke; Weiss et al.). While being mostly studied in isolation, a better understanding on the interaction of these MoA in experimental and in vivo contexts remains lacking. In addition, clarification on the role of host immune cells responding to MSCs is needed, to enable the better identification of patients likely to respond to MSC-based therapies (8).

#### Directionality: Direct Signaling vs. Secondary Crosstalk

A large portion of MSC's therapeutic activity is attributed to direct primary signaling through their secretome, comprising a multitude of cytokines, chemokines, growth factors, and subcellular vesicles. Ferreira et al. give a grand overview on the current knowledge of MSC's secreted mediators and how inflammatory priming influences their release. In line with this, Diedrichs et al. present their results on the clinical development of cardiac-derived MSC products and in particular the impact of interferon-gamma (IFN-g) inflammatory licensing on cell product properties in the context of allogeneic cell therapy. Another elegant study by Carreras-Planella et al. demonstrated in mechanistic fashion that the immunomodulatory effect of MSCs on B-cells is largely independent on extracellular vesicles.

Multiple experts also agree that the MoA of MSCs depends on the secondary crosstalk of therapeutic MSCs with the host tissues and in particular the host recipient immune system [(5, 6, 13, 14); Caplan et al.; Podestà et al.; Weiss and Dahlke; Weiss et al.; Yuan et al.]. Clinical effects may result from a bi-directional crosstalk between MSCs and host cells (as long as MSCs are present), and from the initiation of secondary responses of varying duration, which complicates attempts to model kinetics and dosing in "cell pharmacology" (11). In their review article, Podestà et al. decipher the impact of potential MoAs in their safety and feasibility assessment of MSC therapy for solid organ transplantation, with the aim to promote tolerance to the transplant.

#### Necrobiology: Living, Apoptotic, and Dead Therapeutic Cells

Several contributed reviews elucidate how the metabolism of living cells and the physiology of apoptotic and dead cells, and thus their necrobiology, may contribute to the MoA of MSC therapeutics in vivo (Podestà et al.; Weiss and Dahlke; Weiss et al.; Yuan et al.). Weiss and Dahlke delineate that direct signaling through MSC secreted factors is only part of the equation and elaborate on the role of T cells and monocytes in steering the response to viable and non-viable MSCs. A second review by Weiss et al. further elaborates how the host response to dead or dying cells and subcellular particles, and the concomitant processes of autophagy, apoptosis, mitochondrial transfer, and release of subcellular particles, may affect the therapeutic efficacy and choice of cellular therapeutics. In addition, Yuan et al. give interesting new input on the role of cell metabolism as the missing link between MSC manufacturing and therapy.

#### Cryobiology: Fresh vs. Freeze-Thawed Therapeutic Cells

Regarding cell-host immune interaction, it may also be crucial to differentiate between using fresh from culture-derived metabolically active cells, as compared to freeze-thawed cells readily derived from cryostorage, which may show a transient but reversible impairment of their metabolism and cellular integrity directly after thawing [(15–19); Sierra Parraga et al.; Yuan et al.].

This is exemplified by a contribution from Sierra Parraga et al. who found altered activity of freeze-thawed compared to fresh MSCs in a model of normo-thermic machine perfusion to support transplant kidneys. Oja et al. shared their hands-on experience on how freezing steps in MSC manufacturing impact quality and cell functionality attributes, and how a short-term 24 h culture recovery post thawing can restore the full functionality of the cells. In the past years, comparisons on the effect of fresh vs. freeze-thawed cellular therapeutics have gained greater interest in the cell therapy field as a whole, since this does not only seem to be of interest/relevance for MSC therapeutics, but also for other rapidly expanding fields such as bioengineered chimeric-antigen-receptor (CAR) T-cell therapies (19, 20).

#### Modulating Cell-Host Interaction by Steering Therapeutic Cell Formulation

Not only the general mode of manufacturing, but also the final steps of clinical cell formulation/delivery (e.g., cell harvesting, freezing/recovery post cryobanking, or product formulation and mode of application) could be very decisive for therapeutic safety and efficacy outcome in clinical trials (1, 8, 10, 19).

# REFERENCES

Early preclinical and clinical studies paid rather little attention to these aspects and information from publications is still scarce today. Fortunately, these aspects were discussed in great detail in the studies by Oja et al. and Sierra Parraga et al. Our own experience from early-stage trials indicated that freeze-thawed cells appear to be more prone to activate innate immune cascade systems thus being subject to faster clearance (21), which may influence their in vivo persistence (19). Furthermore, certain cell formulations (e.g., MSCs with low-dose heparin and human albumin instead of human blood type AB plasma) appear to give better clinical responses (22–24).

Thus, the composition of the final cell suspension including prior thawing and washing procedures, may be a key component for positively influencing cellular "pharmacodynamics" in vivo and should be studied with greater attention in order to optimize cellular therapeutics (1, 19, 20).

# AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

# FUNDING

GM's contributions were made possible by the German Research Foundation (DFG) and the German Federal Ministry of Education and Research (BMBF) through the BSRT (GSC203) and BCRT. This study has received support under the European Union's Horizon 2020 research and innovation program (Horizon 2020 Framework Program) under the grant agreements no. 733006 (PACE) and no. 779293 (HIPGEN). JA was supported by the Fraternal Order of Eagles Diabetes Research Center and the Straub Foundation.

# ACKNOWLEDGMENTS

We would like to thank the Editors of Frontiers in Immunology and Medicine for hosting this Research Topic, all authors who contributed manuscripts, and all reviewers who provided insightful feedback.

a systematic review and meta-analysis of clinical trials. PLoS ONE. (2012) 7:e47559. doi: 10.1371/journal.pone.0047559

key to unleash the full clinical potential of MSC therapy? Adv Exp Med Biol. (2016) 951:77–98. doi: 10.1007/978-3-319-45457-3\_7

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Moll, Hoogduijn and Ankrum. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Mesenchymal Stromal Cell Therapeutic Delivery: Translational Challenges to Clinical Application

Henry Caplan1†, Scott D. Olson1 \* † , Akshita Kumar 1 , Mitchell George1 , Karthik S. Prabhakara1 , Pamela Wenzel 1 , Supinder Bedi 1 , Naama E. Toledano-Furman1 , Fabio Triolo1 , Julian Kamhieh-Milz 2 , Guido Moll 3 and Charles S. Cox Jr. 1 \*

1 Department of Pediatric Surgery, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, United States, 2 Department of Transfusion Medicine, Charité Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany, 3 BIH Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany

#### Edited by:

Ulrike Koehl, Hannover Medical School, Germany

#### Reviewed by:

Bjarne Kuno Møller, Aarhus University Hospital, Denmark Reem Al-Daccak, Institut National de la Santé et de la Recherche Médicale (INSERM), France Selim Kuci, University Children's Hospital III, Germany

#### \*Correspondence:

Scott D. Olson<EMAIL_ADDRESS>Charles S. Cox Jr<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology

> Received: 23 May 2019 Accepted: 02 July 2019 Published: 31 July 2019

#### Citation:

Caplan H, Olson SD, Kumar A, George M, Prabhakara KS, Wenzel P, Bedi S, Toledano-Furman NE, Triolo F, Kamhieh-Milz J, Moll G and Cox CS Jr (2019) Mesenchymal Stromal Cell Therapeutic Delivery: Translational Challenges to Clinical Application. Front. Immunol. 10:1645. doi: 10.3389/fimmu.2019.01645 For several decades, multipotent mesenchymal stromal cells (MSCs) have been extensively studied for their therapeutic potential across a wide range of diseases. In the preclinical setting, MSCs demonstrate consistent ability to promote tissue healing, down-regulate excessive inflammation and improve outcomes in animal models. Several proposed mechanisms of action have been posited and demonstrated across an array of in vitro models. However, translation into clinical practice has proven considerably more difficult. A number of prominent well-funded late-phase clinical trials have failed, thus calling out for new efforts to optimize product delivery in the clinical setting. In this review, we discuss novel topics critical to the successful translation of MSCs from pre-clinical to clinical applications. In particular, we focus on the major routes of cell delivery, aspects related to hemocompatibility, and potential safety concerns associated with MSC therapy in the different settings.

Keywords: cellular therapy, mesenchymal stromal cell, clinical translation, safety, cell delivery, hemocompatibility, complement, coagulation

# INTRODUCTION

The study of multipotent mesenchymal stromal cells (MSCs) and their usefulness for treating human injury and disease is almost 40 years old and has evolved through a number of phases roughly defined by the most commonly proposed mechanisms of action (MoA) at work. Interest in MSCs began as a study of bone marrow stromal cells in the 1970s by Friedenstein and contemporaries (1). This grew into an interest in their osteogenic differentiation potential in the late 1980s, and later broadened to trilineage differentiation (bone, fat, cartilage) (2). Cell fusion was briefly considered as a possible mechanism of repair (3–6).  Today, many studies focus on paracrine growth and immunomodulatory factors as key mediators of MSC's therapeutic effect, identified to protect injured tissue and to encourage endogenous repair mechanisms (8). In 2006, the International Society for Cellular Therapy (ISCT) published their first position statement on defining minimal criteria for MSCs (9), followed by several updates

mainly focusing on the refinement of standards for therapeutic efficacy (10–12). Efforts to further refine cell pharmacology and drug delivery are ongoing (13). Thus, it becomes apparent that MSC research has undergone numerous advancements over time, in order to understand and benefit from the interesting properties of these cells. In this review, we will discuss key translational hurdles to clinical applications of MSCs. We will first outline popular cell delivery methods specific to their clinical application and then address newly identified efficacy and safety concerns regarding specific delivery methods (14).

#### ROUTES OF THERAPEUTIC CELL DELIVERY

A number of notable efforts have been made to compare the efficacy of different routes of MSC administration, which has become increasingly difficult with the large number of preclinical and clinical studies that are being published daily (14–18). Despite a number of direct comparisons in animal models and efforts to compare specific routes in a limited number of clinical trials, there is no consensus on the optimal method for MSC delivery (**Figure 1**), with specific limitations or advantages being associated with either method in clinical situations. Numerous methods for delivery of MSCs exist today and it stands to reason that different clinical indications and pathologies will require different delivery routes for optimal therapeutic efficacy (19, 20). Notably, a number of efforts are being made to develop MSCderived exosomes or extracellular vesicles as a new "cell-free" way to recapitulate MSC activity with unique challenges and considerations (21).

Many investigators and industry driven studies rely on practical or logistical considerations (22). Nonetheless, new datadriven approaches for product optimization are currently being tested that integrate desired therapeutic MSC properties with representative simulated microenvironment interactions in vitro in an effort to determine optimal delivery and improve clinical outcomes (23–25). Selecting a suitable delivery route for future studies should also include a consideration of the desired MoA, and whether MSC culture techniques sufficiently highlight that mechanism, and if MSCs can be better primed by an alternative method (26).

Here, we will briefly discuss the rationale behind the most common delivery methods –topical application, intramuscular (IM) or direct injection (DI) into tissues/organs, intravenous (IV) infusion, and intra-arterial (IA) infusion, followed by notable considerations and translational challenges from preclinical to clinical application.

#### Topical Application and Local Injection

Classically, localized topical application or injection of a cell therapy into a specific site or target tissue, e.g., intramuscular or penumbral area of an injury, has been shown to be very useful for precision delivery of MSCs and to increase the engraftment of therapeutic cells at a specific site of interest (14, 27). These strategies are often associated with a tissue replacement strategy or direct paracrine support as a MoA and can be particularly useful when combined with specifically tailored exogenous support systems and biomaterials to guide MSC-host interaction and encourage endogenous therapeutic actions (28, 29).

While the direct differentiation and replacement of host tissue by MSCs has been challenged as a result of a different activity, such as cell fusion or transfer of genetic material (6, 30–34), there are also some notable recent reports of MSCs directly contributing to tissue regeneration, such as in recent work in trachea and esophageal replacement (35, 36).

Topical application of MSCs is the least invasive method of delivery and has demonstrated great potential in the fields of burn medicine and wound care. Topically applied MSCs have improved outcomes, wound healing, and skin graft survival in burn wounds, diabetic-related wounds, and other chronic wounds (37, 38). Using a fibrin polymer spray system, Falanga et al. demonstrated that topically applied MSCs improved wound closure rates in a preclinical model, as well as in patients with chronic non-healing lower extremity wounds (39).

Intra-muscular (IM) delivery of MSCs, like topical application, presents a safe and simple method for cell delivery and, furthermore, leads to improved dwell time compared to other routes such as IV, intra-peritoneal (IP) and subcutaneous cell delivery (40). In the study by Braid et al. IM delivery of MSCs in a mouse model led to survival of human MSCs for up to 5 months after injection. In addition to extended dwell time, IM skeletal muscle fibers provide a highly vascular conduit for local and systemic release of trophic factors and support for MSC paracrine actions (27).

In critical limb ischemia (CLI), for example, MSCs may exert their restorative effects via promotion of angiogenesis and revascularization of ischemic tissue (41). A recent Cochrane analysis of autologous cells treatments, including bone marrow (BM)-MSCs, for CLI found no differences between IA and IM deliveries (42). Furthermore, Soria et al. found that IM delivery may be superior to IA delivery regarding the mitigation of adipose tissue (AT)-derived MSCs prothrombotic properties (43). Interestingly, work by Lataillade et al. has also shown promising effects of local IM-injections of MSCs in dosimetryguided surgery treatment of radiations burns (44), while both, local IM and systemic IV delivery of MSCs and MSC-like cells has led to rescue from lethal radiation in animal models (45, 46).

In addition to topical and IM delivery, early investigative efforts often focused on the potential of MSCs to repair tissues by local engraftment and/or differentiation via direct injection (DI) into the target tissue or organ. Pre-clinical studies in neurological disease, such as stroke, attributed the beneficial effects of MSCs to their ability to engraft and differentiate into neurons and/or glia (47). However, the notion that MSCs can differentiate into functioning neuronal cells was subsequently challenged and appears unlikely (48–50).

Others contended that MSC engraftment facilitated endogenous neurorestorative mechanisms such as promotion of host neural and glial cell remodeling (51, 52). Regardless of the MoA, DI has potential advantage of bypassing the blood-brain barrier to increase delivery of cells into the central nervous system (CNS). For example, a recent Phase 1/2a clinical trial investigated intra-cerebral implantation of the SB623 MSC

cell line in adults with chronic, non-hemorrhagic stroke via magnetic resonance imaging (MRI) stereotactic guidance into the peri-infarct area (53). The authors concluded that MSC implantation was safe, feasible and also improved neurologic outcomes at 12 months. However, important to note, is that the study was limited by patient selection (only 4.7% of screened patients were enrolled) and a lack of a control/placebo group.

Direct injection (DI) of MSCs has also been widely utilized for the treatment of cardiac disease—both acute myocardial infarction (AMI) and ischemic heart failure (IHF)—via open trans-epicardial and catheter-based trans-endocardial injection. There have been numerous preclinical and clinical trials in recent years, but here we will highlight only a few selected studies. Important to note, the notion that MSCs can differentiate into cardiomyocytes or promote cardiac stem cell proliferation and differentiation have largely been abandoned in the past decade. Furthermore, even the existence of resident adult cardiac stem cells has recently been challenged and appears unlikely (54, 55).

The PROMETHEUS trial investigated the use of intramyocardial MSC injections into non-revascularized ischemic myocardium in patients undergoing coronary artery bypass grafting (CABG) for IHF and found improvements in regional myocardial and global left ventricular (LV) function (56). The authors rationalized the use of intra-myocardial injections based on the theory the MSCs exert their effects predominately at the injection site via release of anti-fibrotic matrix metaloproteases and stimulation of neovascularization.

Indeed, the authors found that the effect of MSC injection dropped off as a function of distance from the injection site. Of note, this study involved only 6 patients and no control group. However, a recently published, randomized trial of intramyocardial injection of mesenchymal precursor cells (MPCs) in 159 patients with advanced heart failure undergoing left ventricular assist device (LVAD) placement found that MPC therapy did not demonstrate improvement in the primary outcome, weaning from LVAD support within 6 months (57). The authors also noted that one potential factor for the lack of efficacy may have been the use of trans-epicardial injections, which can lead to significant cell loss.

A systematic review and meta-analysis of MSC delivery methods in preclinical and clinical AMI found that transendocardial injections produced more favorable results in swine models in comparison to direct implantation (intra-myocardial) (58). Furthermore, the trans-endocardial approach allows for a minimally invasive, catheter-based direct implantation of cells into the myocardium and avoids the invasive thoracotomy, and thus additional risks for patient harm, required for transepicardial delivery.

There are several significant risks and considerations unique to DI delivery of MSC. Among them are reports of MSC differentiating into problematic tissue/ectopic tissue formation (59, 60), particularly heterotopic ossification into ectopic bone (61, 62). Additionally, localized DI may prohibit interactions between therapeutic MSCs and potential host secondary signaling systems in the lung, spleen, and peripheral blood, thus limiting their repertoire of therapeutic MoA. Consideration must also be given to the logistics and feasibility of various DI approaches, as delivering sufficiently high cell numbers to the selected target tissue can create significant clinical risk due to required surgeries, such as laminectomies to treat spinal cord injuries (63).

#### Intra-arterial Infusion

There is substantial evidence that MSCs exert their effects largely via direct cell contact and local paracrine effects, as opposed to engraftment and differentiation within the target organ (64, 65). Additionally, upon systemic infusion, the interaction with endogenous inflammatory and tissue repair signals, such as immune cells and the innate immune cascade systems in the bloodstream likely influences MSCs responses, bio-distribution and homing to injured or diseased tissues (14, 66).

Intra-arterial (IA) delivery may prove the most efficacious method in one treatment indication, but may be potentially harmful in another. IA delivery of MSCs allows for infusion of cells within the local vascular system of the target organ without the physical risks of direct implantation and pitfalls of IV administration, especially the trapping of cells within the lung microvasculature, and may thus allow more cells to reach the intended target tissue (17, 65, 67).

Importantly, based on a survey of published results (68), the IA delivery of MSCs for stroke entails the potential risk of cerebral infarcts, caused by emboli of cells in the cerebral microvasculature. Factors such as vascular access, cell size, cell dosage and delivery speed must be considered, especially when delivering cells into coronary or cerebral arteries (69, 70).

In AMI several clinical trials have demonstrated safety and improvements in functional outcomes with the use of intracoronary infusion of MSCs and other BM cell populations (71–73). Here, IA delivery is likely a valid option, as it avoids invasive procedures (which are not part of the usual care in AMI) and ensures delivery of cells to the area of focal tissue injury and hypoxia.

As mentioned above, the SafeCell Heart study demonstrated significant improvements in LVEF with intracoronary MSC delivery (74). IA cell delivery has also been utilized in other pathologies including, but not limited to intra-carotid delivery in stroke, intra-renal delivery for renovascular disease, and intrahepatic delivery for cirrhosis (75–77). However, the efficacy of IA MSC delivery in the above examples is still under investigation.

#### Intra-venous Infusion

The most commonly used method to apply MSCs is via IV infusion, due to the relative ease and limited risk (14). This method results in a large number of MSCs accumulating in the lungs, but also distributing throughout the body and other organs, such as the spleen, throughout 24–48 h (67, 78–80). Similar to intra-arterial delivery of cell therapies, IV administration is most often associated with mechanisms involving secondary signaling effector cell systems and interactions with the host immune system (64, 80, 81).

Most of the attempts at developing a commercial cell therapy have used IV administration in order to facilitate use at multiple centers. Perhaps most widely known, the systemically IV-infused Remestemcel-L (Prochymal) has been developed by Osiris Therapeutics (Now Mesoblast), to primarily treat graftvs.-host disease (GvHD) (82). Other notable efforts also include the use of Multistem, a multipotent adult progenitor cell that is somewhat similar to MSCs, to treat ischemic stroke (83) and our own use of cell therapies to treat traumatic brain injury (84). Common among these approaches is the use of cell therapies to modulate inflammation and activation of the immune system in order to decrease inflammation-related secondary injuries and restore homeostasis.

Among the considerations specific to IV administration are the same concerns about cells generating emboli or thrombi, however, with the advantage of the lung capturing potential vascular obstructions before they can disrupt other organ function. IV infusion certainly may result in limited numbers of cells reaching target tissues, a more transient persistence of cells, and a dilution of paracrine factors reaching target tissues. Finally, while it may be part of MSCs MoA in modulating immune responses (85), IV delivery likely facilitates the rapid removal of clinical MSCs by innate host immune cells (14, 86).

#### Additional Routes of Administration

Several additional routes of MSC administration are notable for consideration for some specific applications. Intra-nasal (IN) administration of MSCs is promising route to treat neurologic pathologies that avoids the risks associated with direct injection of cells into the CNS. Preclinical data has demonstrated efficacy of IN MSCs across a spectrum of CNS disorders, including perinatal ischemic brain injury, subarachnoid hemorrhage, and neurodegenerative disorders such as Parkinson's disease (20, 87–90).

Intrathecal (IT) administration of MSCs, often through lumbar puncture, has shown benefit in preclinical and clinical studies across a wide array of neurologic disorders, including chronic neuropathic pain secondary to spinal cord injury (91–94), amyotrophic lateral sclerosis (ALS) (95–97), and epilepsy (98, 99).

Intravitreal administration of MSCs has demonstrated improvement in outcomes in several animal models of retinal injury and dysfunction (100–104); however, the safety of intravitreal injection of MSCs in humans is still an area of clinical concern (105).

Furthermore, the administration of MSCs during ex vivo perfusion of solid organs, e.g., in kidney transplantation or injury, avoids the trapping of cells in the lung and spleen and permits direct interaction of MSCs with the target tissue (106). Gregorini et al. demonstrated that delivering MSCs during hypothermic machine perfusion improved outcomes in a rat model of ischemic kidney injury (107). Recently, Sierra Parraga et al. demonstrated that machine perfusion of MSCs supports their function and survival, although more work will be required to determine the optimal conditions for perfusion (108). Notably, these strategies may reduce the need for immunosuppression to prevent organ rejection (109).

#### TRANSLATIONAL HURDLES WITH SYSTEMIC AND LOCAL DELIVERY

Any therapeutic modality, whether a new pharmacologic agent or surgical procedure, must not only be efficacious, but also safe for the patient. While the vast majority of preclinical and clinical studies have found MSCs to be safe and well-tolerated (110), the rise of unregulated and unproven stem cell interventions have resulted in several reported clinical adverse events (14, 60). Here we will highlight mechanisms by which MSCs may cause unwanted or adverse reactions, including interactions with the host's innate and adaptive immune system, as well as their tumorigenic potential (**Figure 2**).

### Triggering of Innate Immune Responses by MSCs

Systemically infused MSCs activate the host innate immune cascade systems, such as complement and coagulation, termed the instant blood-mediated inflammatory reaction (IBMIR) (14, 66, 111). IBMIR was first used to describe the procoagulant activity of pancreatic islet cells and hepatocytes in relation to their expression of procoagulant tissue factor (TF/CD142) (112, 113).

MSCs demonstrate similar effects when they contact human blood with variable amounts of procoagulant activity when isolated from different issues (14). They normally reside in the perivascular space around blood vessels, are predominantly excluded from direct contact with blood, and express variable amounts of procoagulant TF, first described on placenta-derived decidual stromal cells (DSCs) (114).

The effects of ex vivo expanded MSCs administered IV into patients include a significant increase in complement C3 activation fragment a (C3a) and the coagulation activation marker thrombin-antithrombin complex (TAT), which was accompanied by a decrease in platelet counts and a significant increase in fibrinolysis marker D-dimer (111, 115). Importantly, MSC's TF expression was shown to increase as cell passage increases, and the procoagulant effect of MSCs increases as their TF expression increases (14).

Furthermore, in a study investigating human BM- and AT-derived MSCs, TF expression and procoagulant activity were measured using flow cytometry and calibrated automated thrombogram and thromboelastography, respectively, demonstrating large donor and tissue variability (116). A causal relationship between MSC-associated TF with clot formation has also been demonstrated in human blood using flow cytometry to measure the density of TF on different types of MSCs (24), finding that MSC induce a dose-dependent change in clotting time and thrombin production based on TF expression.

Several authors demonstrated a reduction in procoagulant activity in MSCs when the cells were diluted or treated with TF pathway blocking reagents (24, 111, 117). Multiple authors also found that heparin can nullify accelerated clotting time due to MSC associated TF in vitro (115, 117–119). These data highlight the importance of monitoring MSC's procoagulant activity and provide a possible clinical solution. Thus, MSCassociated TF must be considered as a safety release criterion prior to administration in patients (14).

While a substantial number of in vitro investigations have demonstrated that MSCs exert a procoagulant effect after blood contact, there are limited reports of MSC-associated thrombotic events in humans in the literature, specifically in established clinical trials (60). One of the first reports involved the use of human placenta-derived MSC-like cells in a Phase 1b/2a study in Crohn's disease. The authors reported that two patients suffered from venous thrombosis after infusion (120), and posit TF expression on clinical-grade therapeutic MSCs as a possible cause (111).

Furthermore, subsequent in vitro studies comparing placentaderived DSCs to BM-MSCs found a 15-fold higher expression of TF in the DSCs (115), which may help to explain the results from the above study. The need for increased caution with perinatal tissue (PT)-derived MSC products is further substantiated by a recent report of thromboembolism in two patients treated with umbilical cord MSC products (121).

Another key report of thrombotic events was in a trial that evaluated the use of autologous AT-derived MSCs to treat patients with critical limb ischemia (CLI) (122). The investigators found that two of the patients, both of whom also had diabetes, developed distal microthrombi after infusion. Interestingly, the investigators found no cases of thrombotic events when using autologous AT-MSCs in non-diabetic patients or autologous BM mononuclear cells (BM-MNCs) from diabetic patients.

It was subsequently demonstrated that AT-MSCs from diabetic patients release higher levels of plasminogen activator inhibitor type 1, reduced levels of tissue plasminogen activator, and lower d-dimer formation in comparison to non-diabetic AT-MSCs, all of which might lead to blunted fibrinolytic activity. Furthermore, these diabetic-derived AT-MSCs upregulated TF expression and displayed altered platelet-derived growth factor (PDGF) signaling, which was abrogated using PDGF-BB treatment (123).

There are other case reports of MSC-associated thrombotic events. A man who presented with chest pain was found to have small bilateral pulmonary emboli 1 month after receiving the last of multiple systemic infusions of AT–derived MSC for herniated cervical discs (124). His parents were reported to also have had MSC infusion for osteoarthritis, and both were found to have small pulmonary emboli, although neither was symptomatic. No evidence of hypercoagulable disease was found in any family member. There was no clear mention of the exact source of the clinical MSCs infused, or if these patients received MSCs in conjunction with a clinical trial. There is an additional media report of a 73 year old man who died from a pulmonary embolism after receiving an infusion of AT-derived stromal cells in Japan (125). It appears that this patient was not enrolled in any published clinical trial, and the company involved has come under intense scrutiny. In both of the latter two cases, there are few details regarding the source, manufacturing process and testing of the cells, as well as details surrounding the treating parties.

While the evidence for MSC-associated thrombotic events is nominal, there is a need to ensure the safety of MSC therapy, including their procoagulant effects (14). Furthermore, many patients who may benefit from MSC therapy—those with inflammatory mediated disorders, diabetes, cancer, cardiac dysfunction, or trauma-related injuries—are likely to have an acquired hypercoagulable state or are at high risk of a thrombotic event secondary to their primary disease process. Therefore, we must continue to monitor the pro-thrombotic effects of MSCs as part of release criteria and in clinical trials.

FIGURE 2 | Translational challenges with systemic and local cell delivery. (A-D) Therapeutic cell production / conditioning (e.g., 2D vs. 3D culture and cytokine priming) and the mode of cell delivery (e.g., systemic intravascular infusion vs. local injection) have a major impact on the cell product's immunogenic properties (shown in A), and consequent rapid triggering of innate and adaptive immune responses (shown in B,C), thus affecting its therapeutic efficacy, engraftment and tumorigenicity (shown in D). The MSC product's immunogenic properties are affected by numerous cell-bound and secreted immunoregulatory mediators (e.g., complement regulators, coagulation regulator TFPI, or regulators of the adaptive immune response, such as co-stimulatory molecule expression, sHLA-G and galectin-1). The cells can also exhibit a number of immunogenic features, such as procoagulant TF-expression, cellular stress signals (e.g., PS), and immunogenic antigens (e.g., allo, xeno, and blood groups). (B) The innate coagulation and complement cascade systems are two of the major effector arms of the instant blood-mediated inflammatory reaction (IBMIR) that can recognize blood-incompatible therapeutic cell features and thus trigger the detrimental thromboinflammation compromising cellular therapeutics. The innate immune cascade systems employ multiple sophisticated molecular sensors (e.g., FVII and FXII, or C1q and MBL, respectively), to recognize aberrant cell surface molecular features on infused therapeutic cells (e.g., TF and PS, respectively), which can trigger innate immune cascade activation and amplification by effector cells (e.g., platelets, PMNs, monocytes/macrophages, and T/B cells), potentially leading to adverse reactions (e.g., cell lysis, inflammation, sequestration and rejection). (C) Innate and adaptive effector cell modulation: triggering of IBMIR and therapeutic cell injury/disintegration promotes the release of various bioactive molecules, in itself and from dying MSCs, upon crosstalk with the responsive host immune system, such as activated clotting factors (e.g., thrombin), anaphylatoxins (C3a and C5a), opsonins (iC3b, and C3d/g), and MSC-derived constituents (e.g., microparticles, cytokines and growth factors) in a highly conditional manner, thus greatly amplifying the initial signal, leading to modulation of multiple effector cell types. This can result in alloimunization and consecutive cellular and humoral responses (e.g., T-cells and B-cells alloantibodies), but also in the induction and release of multiple immunoregulatory and regenerative cell types and mediators (e.g., Tregs, Mregs, TolDCs, MPs, and PMs). (D) A large fraction of the infused therapeutic cells is lost within the first hours to days of infusion due to the triggering of instant innate immune responses, which can be furthermore aggravated by triggering of adaptive immune responses in case of allogeneic cell products. Studies on MSC persistence in vivo have shown prolonged survival, dwell-time, and engraftment by alternative routes of delivery (e.g., local injection in conjunction with biomaterials), although long-term engraftment is very limited and ectopic tissue formation rarely reported. Currently, patient clinical (Continued)

FIGURE 2 | responses are still sub-optimal for many MSC therapeutics leaving room for improvement in long-term survival. AT, antithrombin; FI-FXII / FIa-FXIIa; native and activated coagulation factors I-XII; TF / TFPI, tissue factor and tissue factor pathway inhibitor; C3/5-9, complement component 3 and 5 to 9; C3a/C5a, activation fragment a of complement component 3 and 5; C3b/iC3b/C3d(g), complement component 3 sequential degradation fragments b, inactivated fragment b and d(g); complement regulatory molecules: CD35, complement receptor 1, CD46/MCP, membrane cofactor protein; CD55/DAF, decay accelerating factor; CD59, protectin; FI and FH, complement factor I and H; PS, phosphatidyl-serine; MAC, membrane attack complex; and MHC-II, major histocompatibility complex class-II; sHLA-G, soluble human leukocyte antigen G; MPs and PMs, MSC and immune cell-derived micro-particles and paracrine mediators; Tregs, Mregs and TolDCs, immunogerulatory T-cells, myeloid cells, and tolerogenic dendritic cells.

The complement system, another major part of the innate immune cascade systems, is one of the first lines of defense against foreign pathogens. It is not surprising that systemically infused MSCs activate and interact with the complement system (126). In fact, the complement system appears to play a critical role in initiating the immunomodulatory reaction between infused therapeutic MSCs and host cells (127). What role the complement system plays in MSC's MoA and how it affects their efficacy is currently under investigation.

Early studies by Tu et al. revealed that MSCs constitutively express factor H, a complement inhibitor (128); however, even when augmented by the presence of pro-inflammatory cytokines TNF-α and IFN-γ, the overall production of factor H by MSCs proved relatively insignificant in comparison to overall systemic levels. Subsequently, the same group and others demonstrated that although MSCs express cell-surface complement regulators, MSCs activate the complement system upon contact with human sera in vitro, leading to cytotoxicity in a dose-dependent manner (126, 129, 130).

Blockade of these complement inhibitors led to increased cytotoxicity, while both upregulation of CD55 (a cellsurface complement inhibitor) on MSCs via transfection with recombinant adenovirus, and addition of anti-C5 immunoglobulin significantly reduced MSCs cytotoxicity after contact with serum. Li et al. also determined allogeneic MSCs caused increased complement activation, with associated increased cytotoxicity, in comparison to autologous MSCs (126).

In order to prevent complement-mediated cytotoxicity against therapeutic cell products, investigators have engineered complement-resistant MSCs. Heparin is known to inhibit complement activity, and systemic administration of heparin reduced MSC cell damage after infusion (131). In order to prevent unwanted anti-coagulation effects of systemic heparin administration, the authors then demonstrated that incubation of MSCs with activated heparin led to binding of heparin to the surface of MSCs and furthermore, these "heparin-painted" MSCs showed less surface deposition of complement C3 activation fragment b (C3b/iC3b) and suffered less cell damage after contact with serum.

The proposed mechanism of heparin-mediated protection involves recruitment of factor H binding to the MSC cellsurface. Subsequent "painting" of factor H onto MSCs via preincubation led to decreased complement deposition onto MSCs surface, reduced cell damage, and increased cell survival in vitro and in vivo (132). In addition, MSCs with factor H incubation attenuated C5a release, which significantly reduced complementmediated activation of neutrophils and led to improvement in MSC function and reduced cell damage.

Investigators have also engineered human MSCs via transduction of a retrovirus encoding genes from human cytomegalovirus (HCMV), which downregulated HLA1 expression on MSCs without increased vulnerability to NK killing (133). Furthermore, HCMV is known to incorporate the host-encoded complement inhibitor proteins and upregulate host-encoded CD55 and CD46 in order to evade the innate immune system. Soland et al demonstrated that induction of MSCs with the specific HCMV US2 protein led to upregulated expression of complement inhibitors CD46, CD55, and CD59, and a reduction in complement mediated MSC lysis (129).

While further investigation into engineered MSCs as described above is certainly necessary, the ability to evade or attenuate complement-mediated cell damage and lysis may prove crucial to the efficacy of future MSC therapeutics (86). Further investigation has demonstrated a much more complex relation between MSCs and the complement system, with discrepancies between pre-clinical and clinical results. In a clinical trial of patients receiving MSC infusions for treatment-resistant GvHD, the investigators examined the relationship between complement activation, immunosuppressive potential of the MSCs in vitro and the clinical effectiveness of MSC therapy in vivo (127). MSCs activation of the complement system was found to mediate effector cell activation and modulate their immunomodulatory activity in a multifactorial manner.

This finding was mechanistically substantiated by using in vitro inhibition of complement function, which resulted in decreased CD11b upregulation on effector cells. Furthermore, MSC's ability to activate the complement system was found to correlate with its immunosuppressive potential in vitro: MSCs with increased complement activating properties demonstrated increased suppression of peripheral blood monocyte (PBMC) proliferation in mixed lymphocyte reactions (MLRs) and increased ability to trigger CD11b+ effector cells in whole blood. Surprisingly, the authors found an inverse correlation between the immunosuppressive potential of the MSCs in vitro and their clinical effectiveness in human patients, with average suppressing cells yielding the most beneficial therapeutic effects in vivo.

Importantly, it appears that substantial differences exist with respect to complement activation and potential efficacy of fresh vs. freeze-thawed MSCs upon systemic infusion (86). Freeze thawed MSCs were found to demonstrate increased activation of the IBMIR and susceptibility to complement-mediate cell lysis (130). Similar changes in the immunomodulatory capacity of MSC due to cryopreservation have also been reported elsewhere: such as the alteration of MSC-mediated attenuation of T cell activation, inflammatory cytokine concentrations, and an increased susceptibility of MSC to lysis by mixed immune cells (134, 135).

In the clinical sample evaluated (130), the majority of GvHD patients were treated with freeze-thawed MSCs with a small number of fresh culture-derived MSCs being available for comparison; while the study lacked sufficient power and thus should be considered with caution, as also emphasized in the discussion, the authors noted an interesting trend of improved clinical outcomes with fresh MSCs, especially those delivered at early passage, in comparison to freeze-thawed MSCs (130, 136).

Overall, it is now evident that MSCs activate the complement system upon contact with blood, which appears to be positively correlated with their immunosuppressive ability in vitro. Furthermore, freeze-thawed MSCs, in comparison to fresh MSCs, demonstrated an increased triggering of the IBMIR and associated complement-mediated lysis in vitro, leading to a significant reduction in viable cells (130, 137). What remains unanswered is: (1) how and to what extent complement activation influences the clinical efficacy of systemic MSC therapy, and (2) whether fresh MSCs are subject to decreased complement-mediated lysis and, as a result, are more effective clinically than freeze-thawed MSCs in vivo.

# Triggering of Adaptive Immune Responses: Autologous vs. Allogeneic MSCs

A comprehensive review of the immunobiology of MSCs and the many ways that MSCs interact with the local and systemic immune system in both normal and activated systems is beyond the scope of this review, and has previously been covered in several notable efforts to consolidate the literature (138–141). It would not be an understatement to say that MSCs have extensive possible interactions with every major component of the immune system through a combination of paracrine activity, extracellular matrix remodeling, direct contact-based signaling, and more recently, through the use of extracellular vesicles.

These wide-ranging putative molecular mechanisms have made it incredibly difficult for the field to come to a meaningful consensus regarding the effects of self vs. donor antigens, further complicated by the additional xenogenic antigens introduced during common cell culture techniques (such as expansion in FBS) (137, 142, 143). Until recently, MSCs were widely reported to be immune privileged, enabling their use as an allogeneic therapy without concurrent immunosuppression.

As the field increasingly focused on the immunobiology of MSCs, there was a correlating rise in the number of studies that found that MSCs were not exempt from immune recognition. As summarized in a number of more comprehensive literature reviews (140, 144–146), allogeneic MSCs with poorly matched HLA can and do generate both innate and humoral responses from the immune system, albeit responses that appear to be dependent upon the conditional expression and balance of both immune-activating antigens (such as MHCs) and immune-modifying cytokines, molecules and metabolites, like tumor necrosis factor-inducible gene 6 (TSG-6), galectin-1, prostaglandin E2 (PGE2), and indoleamine 2,3 dioxygenase (IDO).

The eventual immune recognition of allogeneic and HLAmismatched MSCs has become increasingly implicated as a barrier to clinical efficacy (140). On the one hand, the huge number of pre-clinical studies in both xenogeneic and allogeneic systems and clinical studies using allogeneic cells without regard to conventional graft-vs.-host compatibility considerations indicate that MSC efficacy is often independent of the eventual immune "rejection" of donor MSCs, either by virtue of MSC activity occurring prior to immune recognition or perhaps even due to a MoA that includes the host immune system.

In a recent report it was found that MSCs can modulate the immune system by being engulfed by antigen presenting cells, and that the subsequent display of MSC antigens by antigenpresenting cells (APCs) results in a chain of anti-inflammatory activity and downstream therapeutic outcomes (85). On the other hand, the recognition and removal of MSCs by the host immune system may also limit the duration and possible efficacy of a number of MSC MoAs.

Among a number of strategies to evade immune recognition, there is a large amount of interest in the use of biomaterials and engineering techniques to shield MSCs from immune activity to prolong paracrine activity (29, 147, 148), efforts to further decrease the immunogenicity of cells (149–151), sophisticated banking strategies to allow for autologous or HLA-matched cells to treat acute injuries in a timely fashion (152, 153), and conventional pharmaceutical immunosuppression given temporarily, all to provide a larger window for MSC activity in vivo (154, 155).

It is our opinion and that of others (86, 136, 140, 156), that reducing the activity of the host immune system is likely to also reduce the therapeutic efficacy of MSCs, as we feel that many of the pleiotropic effects of MSCs require the participation of the host immune system.

# Cell Engraftment and Tumorigenicity

The potential for malignant transformation of MSCs is of obvious concern. Because MSC therapy involves ex-vivo production and expansion of cell lines, and even allogeneic MSCs have the capacity to escape elements of immune recognition, it is crucial to ensure that transplanted or infused cells do not contain transformed, potentially tumorigenic cells (157).

Concerns for the tumorigenic potential initially came from studies of mouse-derived MSCs transplanted into a mouse model (158). The murine-derived BM-MSCs used in the study were reported to spontaneously transform into malignant fibrosarcomas in multiple organs in vivo after systemic infusion into immunocompromised mice. Of note, this same study also evaluated human MSCs and found no evidence of malignant potential in vitro.

Another study demonstrated that the injection of mouse MSCs with spontaneous p53 mutations led to development of fibrosarcomas at the site of injection in immunocompetent mice (159). Yet, there was no evidence that the transfer of MSCs without such mutations led to tumor formation. Transformed MSCs have also been identified for other non-human species. The authenticity of such findings is difficult to confirm in nonhuman cells, as many of such studies lack true verification by modern stringent methods, such as short tandem repeat (STR) profiling (160).

Additional reports of spontaneous malignant transformation of human MSCs further intensified concerns for the tumorigenic potential of MSCs. Two separate studies identified spontaneous malignant transformations of human MSCs in culture, and injection of these transformed cells led the development of tumors in mice (161, 162). However, the findings from both of these studies have since been retracted, as the MSC cultures in both instances were found to have been contaminated with established malignant human cell lines (163).

While two more recent studies have demonstrated, and confirmed using STR analysis, the development of malignant transformation of MSCs in both cynomolgus macaques and human cell lines (164–166), there are, in contrast, far more studies that have demonstrated that ex vivo expanded human MSCs are rather resistant to malignant transformation, even after long-term culture, development of chromosomal aberrations, and application of physical and chemical stress (167–169), and that they undergo senescence rather than becoming tumorigenic.

Further reports concerning the genetic instability of MSCs appear to be grossly overstated and even misleading (170, 171). In addition, studies of Good Manufacturing Practices (GMP) grade MSCs have also demonstrated a lack of malignant potential in vitro and in vivo (172, 173). While the possibility of rare tumorigenic transformations in MSCs cannot be ignored, careful monitoring of cell cultures, minimization of in vitro expansion length and evaluation for cytogenic aberrations when concerns rise should be considered (174).

Human MSCs have been used clinically for more than two decades, the majority from BM, but also increasingly also from other sources. To date, there are no clinical studies that have attributed MSC therapy to the development of tumors or malignancy. A meta-analysis of 36 studies, including 8 randomized control trials (RCTs), involving 1,012 patients found no evidence of association between MSCs and tumor formation (110). A 2013 report from the ISCT noted that although the risk of tumorigenicity of MSCs had yet to be confirmed or denied, no tumors have been diagnosed in patients that would originate from administered MSCs (175). While it appears that MSC therapy is safe and well-tolerated in human subjects, the risk of tumorigenicity must continue to be studied both in clinical trials with long-term follow up and during the culture/expansion process prior to any therapeutic infusion.

#### COMPARATIVE STUDIES FOR OPTIMAL DELIVERY

Two important considerations exist in determining optimal cell delivery. The first consideration is patient safety. The second consideration is the efficacy of a therapy. If DI, IA, or IV demonstrate similar clinical effectiveness, then the least invasive method of cell delivery is preferred. But do MSCs work differently when delivered DI v. IA v. IV? Here we have selected a few examples of preclinical and clinical studies that have compared cell delivery methods head to head in order to demonstrate potential advantages/disadvantages of one method over another.

In heart disease, DI into myocardium may not provide improved MSC engraftment rates or outcomes over IA infusion. In a model of porcine model of ischemic cardiomyopathy, animals received either surgical implantation, trans-endocardial injection, or intracoronary infusion of autologous MSCs and were euthanized 4 h after infusion (176). DI of MSCs via surgical implantation or trans-endocardial injection led to only 16 and 11% retention of MSCs within the myocardium, respectively. The majority of the cells, around 45%, accumulated in non-target organs for all three delivery methods. Intracoronary infusion led to similar rates of intramyocardial MSC retention as DI (both 11%). IA delivery necessitates the need for patent vasculature and, therefore, may not provide benefit in pathologies such as AMI or ischemic stroke secondary to occluded internal carotid or intra-cerebral arteries.

There have also been investigations of IA vs. IV cell delivery, especially in stroke, as endovascular treatments have become increasingly utilized (70). Byun et al. demonstrated that IA delivery leads to improved cerebral engraftment and outcomes over IV delivery in a rat model of cerebral infarction (177). A meta-analysis of preclinical studies of MSCs in ischemic stroke models found that although DI provided the greatest benefit, all 3 methods of delivery—DI, IA, and IV— consistently demonstrated significant improvement in outcomes (178).

Direct comparison of delivery methods is often lacking in clinical trials due to logistical concerns. Furthermore, pooled meta-analysis and systematic reviews often combine different cell types, related pathologies and delivery methods. Therefore, the following small selection of studies is limited and should not be considered a representative sample.

In stroke, a pooled analysis of clinical trials using multiple cells types, largely BM-MNCs and MSCs, Jeong et al. determined that IA provided increased benefit over DI or IV (179). Furthermore, the SafeCell Heart study, a systematic review and meta-analysis of cell therapy in heart disease, found that IA and intra-myocardial (catheter-directed trans-endocardial) infusions provided significant improvements in LVEF, which were not seen with trans-epicardial or IV cell delivery (74).

In another meta-analysis of preclinical and clinical studies of MSCs in cardiac disease, Kanelidis et al. found that cell delivery method did have an effect on outcomes in AMI, and that trans-endocardial and IV delivery improved outcomes in both swine models and clinical trials, while IA infusion with subsequent intracoronary delivery did not demonstrate significant benefit (58).

Clearly, the currently available data, based on few preclinical studies and limited clinical trials, which are often contradictory, are not sufficient to make any major conclusions as to whether one delivery method is superior to another. However, clinical trials directly comparing cell delivery methods will likely not happen until MSC therapy, via any delivery method, is proven efficacious for a particular pathology.

# CONCLUSION

During the past decades, MSC therapeutics have undergone a continuous transition from proof-of-concept to clinically approved therapies. In order to improve our ability to utilize MSCs therapies, great efforts are ongoing to refine potency assessment, cell pharmacology and drug delivery. Compared to advancements in cell sorting, manufacturing and biobanking, the importance of cell delivery methods and the in vivo effects of MSCs on the human immune and hematologic systems are still largely underappreciated today. Thus, we here discussed key aspects related to the effective and safe delivery of MSCs, in the context of recent clinical studies with focus on different methods of MSC administration. As the growth of MSC-based therapeutics accelerates in private, public, and fringe applications, it is vitally important to remember historical safety concerns, recognize modern clinical risks, and use methodology and delivery consistent with the intended MoA, in order to yield the most effective and safest economically viable therapeutic approaches. We encourage our colleagues to

#### REFERENCES

careful consider their assumptions and commonly used practices to ensure that their long-held views about MSC biology are supported by modern studies.

#### AUTHOR CONTRIBUTIONS

HC, SO, AK, MG, and PW wrote the manuscript. KP, JK-M, and GM worked on illustrations. HC, SO, AK, SB, NT-F, FT, GM, and CC edited the manuscript.

# ACKNOWLEDGMENTS

This study was supported by the NIH 2T32 grant GM0879201- 11A1, the Clare A. Glassell family Pedriatic Surgery Research Fund, the Grace Reynolds Wall Research Fund, and the Bentsen Stroke Center. GM's contributions were made possible by German Research Foundation (DFG) and German Federal Ministry of Education and Research (BMBF) funding through the BSRT (GSC203) and BCRT.

functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials. Cytotherapy. (2016) 18:151–9. doi: 10.1016/j.jcyt.2015.11.008

mesenchymal stromal cell-immune reaction. Sci Rep. (2018) 8:6816. doi: 10.1038/s41598-018-24971-2

contribute to endochondral heterotopic ossification. Bone. (2018) 109:71–9. doi: 10.1016/j.bone.2017.06.014

forced expression of human cytomegalovirus us proteins. PLoS ONE. (2012) 7:e36163. doi: 10.1371/journal.pone.0036163

a study on delivery efficiency. J Cell Mol Med. (2012) 16:2768–76. doi: 10.1111/j.1582-4934.2012.01594.x

**Conflict of Interest Statement:** SO has received research support from Athersys, CBR Systems, Hope Bio and Biostage. CC has received research support from Athersys, Cellvation, CBR Systems, Hope Bio, and Biostage, and is on the Scientific Advisory Board of Cellvation, Biostage, and CBR.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Caplan, Olson, Kumar, George, Prabhakara, Wenzel, Bedi, Toledano-Furman, Triolo, Kamhieh-Milz, Moll and Cox. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Multiplicity of Mesenchymal Stromal Cells: Finding the Right Route to Therapy

Alison Wilson1†, Margeaux Hodgson-Garms 2†, Jessica E. Frith2 \* and Paul Genever 1 \*

1 Department of Biology, University of York, York, United Kingdom, 2 Materials Science and Engineering, Monash University, Clayton, VIC, Australia

Over the last decade, the acceleration in the clinical use of mesenchymal stromal cells (MSCs) has been nothing short of spectacular. Perhaps most surprising is how little we know about the "MSC product." Although MSCs are being delivered to patients at an alarming rate, the regulatory requirements for MSC therapies (for example in terms of quality assurance and quality control) are nowhere near the expectations of traditional pharmaceuticals. That said, the standards that define a chemical compound or purified recombinant protein cannot be applied with the same stringency to a cell-based therapy. Biological processes are dynamic, adaptive and variable. Heterogeneity will always exist or emerge within even the most rigorously sorted clonal cell populations. With MSCs, perhaps more so than any other therapeutic cell, heterogeneity pervades at multiple levels, from the sample source to the single cell. The research and clinical communities collectively need to recognize and take steps to address this troublesome truth, to ensure that the promise of MSC-based therapies is fulfilled.

Keywords: mesenchymal stromal cell, heterogeneity, cell subpopulations, cell-based therapy, single cell technologies

# INTRODUCTION

The term "MSCs" is used to describe a heterogeneous population of stromal cells, the exact nature and composition of which remains the subject of much debate. They are often characterized using criteria proposed by the International Society for Cell Therapy (ISCT) as plastic-adherent cells, expressing a distinct set of surface antigens and with the ability to differentiate in vitro into osteogenic, adipogenic, and chondrogenic lineages (1). This minimal definition, however, is far from definitive. MSCs exhibit unique immunomodulatory properties, support the hematopoietic niche and participate in tissue regeneration through diverse biological activities including engraftment-independent paracrine signaling. Though initially described and sourced from bone marrow we are now able to isolate MSC-like cells from a variety of tissues including adipose tissue, dental pulp, placenta, umbilical cord, and umbilical cord blood.

Although MSCs first appeared in the clinic in 1995 (2) and have since become one of the most clinically studied cell therapy platforms worldwide (3) many fundamental aspects of MSC biology remain undetermined; primarily a direct consequence of the pervasive heterogeneity that manifests itself between MSC donors, tissue sources, culture methods and individual cells within a clonal population. Furthermore, MSCs exhibit a remarkable level of plasticity over time and when presented with different microenvironments (4, 5). MSC multiplicity, and a lack of consensus in the scientific community, complicates MSC characterization and their translation into the clinic.

#### Edited by:

Guido Moll, Charité Medical University of Berlin, Germany

#### Reviewed by:

Evren Alici, Karolinska Institute (KI), Sweden Marcella Franquesa, Germans Trias i Pujol Health Science Research Institute (IGTP), Spain

#### \*Correspondence:

Jessica E. Frith<EMAIL_ADDRESS>Paul Genever<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology

> Received: 26 February 2019 Accepted: 01 May 2019 Published: 16 May 2019

#### Citation:

Wilson A, Hodgson-Garms M, Frith JE and Genever P (2019) Multiplicity of Mesenchymal Stromal Cells: Finding the Right Route to Therapy. Front. Immunol. 10:1112. doi: 10.3389/fimmu.2019.01112

**25**

This review will consider the multilevel origins of heterogeneity in MSCs (see **Figure 1**) and how we should be doing more to identify, track and quantify heterogeneity in MSCs to help determine its biological importance and impact in in vitro and in vivo contexts.

## CHANGE IS THE ONLY CONSTANT (HERACLITUS, 535–475 BC)

MSC heterogeneity has certainly obscured our understanding of MSC biology and, correctly, prompted calls to re-evaluate the use of MSCs in therapy (6–10). However, the origins of heterogeneity are complex, fascinating and a constant theme in biology. It is clear from other work, particularly in microbial systems, that heterogeneity arising in genetically identical populations can have a positive impact on overall population fitness (11– 14). Stochastic fluctuations in gene expression, or "noise," can lead to phenotypic variability in clonal cell populations (11, 15) and "bet hedging" can confer survival advantages on individual cells within mixed communities when faced with environmental change (16, 17). It has been proposed that stochastic nongenetic variations (i.e., those not caused by genetic mutations) contribute to the evolution of tumors using bet hedging-like strategies (18–20) and the dynamic switching between subtly different phenotypes has been shown to influence cell fate in different adult and embryonic stem cell populations (21– 23). Gene expression noise in MSCs is also likely to give rise to individual cells with different characteristics and therefore influence the aggregate function of the population. It is also clear that MSC heterogeneity is due at least in part to the existence of different subpopulations with distinct expression profiles and functional properties (24–26). It has not been determined if discrete stromal subpopulations evolve through stochastic or deterministic means, but many appear to possess properties that support general tissue maintenance [for example, immune control, vascular remodeling, hematopoiesis (25)] that are unrelated to stem cell function. Therefore, the umbrella "MSC" descriptor may actually cover a range of related but distinct cell types that are yet to be fully defined.

#### IMPACT OF DONOR- AND TISSUE-DEPENDENT MSC HETEROGENEITY

Cells that currently meet this broad MSC descriptor have been identified in virtually all post-natal organs and tissues (27) and while bone marrow derived MSCs (BM-MSC) are still considered the gold standard, MSCs are now frequently also isolated from adipose tissue (AT-MSCs) and umbilical cord or cord blood (UC/UCB-MSCs) (28–33). There are welldocumented disparities in proliferation, differentiation potential, surface markers, transcriptional, and proteomic profile of MSCs from different sources (34–36); an overarching consensus is hard to come by. For example, prevailing MSC characteristics such as tri-lineage differentiation potential present contradictory evidence in terms of lineage preference and full tri-lineage capacity (29, 30, 32, 37). Even when derived from the same tissue of origin, MSCs demonstrate prodigious donor-to-donor variation. This may be a factor of donor health influencing MSC availability and function (38, 39). Donor age can also affect self-renewal capacity and differentiation potential, which have been reported to decline in older donors (40–43). However, differences are also apparent in healthy donors of a similar age in proliferation rate, differentiation capacity, and ultimate clinical utility (44) leading to a further addition of complexity when directly comparing samples. It is tempting to speculate that MSC heterogeneity mirrors the diversity of environments from which they may be isolated, the reality is however that our understanding of MSCs in vivo is still in its infancy (8).

The multiplicity of MSCs and the absence of a meaningful consensus on definitions and characterization parameters makes comparing studies within the field difficult and translating them into clinical practice even more so. Because heterogeneity is seldom accounted for, and unique cell populations used in individual research projects are rarely fully defined, many studies are not only difficult to reproduce but difficult to evaluate for comparability and impact within the field. Incomplete knowledge of the characteristics of MSCs in vivo and how these will relate to clinical outcomes further exacerbate the problem when considering quality control requirements for MSCs as therapeutic agents. Changes in the source materials of clinical products, e.g., a different donor, prompt regulatory authorities to require re-characterization and evidence of "comparability." In the event that comparability could not be demonstrated, product from the original and subsequent sources would be considered to be essentially different products. Thus, during clinical development, data on early product iterations could be invalidated, and post-authorization could, in the worst-case scenario, require re-authorization. In conjunction with the need for adequate cell numbers, this represents a major challenge to the acceptance of cell-based therapies as mainstream treatments; the options of extended culture or multiple donors each imply unavoidable heterogeneity. Consequently the manufacture of MSC products using processes that rely on a continuous supply of new tissue donations run the significant risk of supply constraint, interruption, and inconsistencies (10).

#### IN VITRO EXPANSION AND MSC HETEROGENEITY

A typical bone marrow aspirate contains just 0.01–0.001% MSCs (45) and trials for the regeneration of bone and cartilage tissue commonly use in the order of 10 million cells. The need for high levels of culture expansion adds to the challenge of generating an MSC population that retains the ability to differentiate effectively or secrete the appropriate biomolecules to induce a beneficial paracrine response. Banfi et al. investigated the growth kinetics and differentiation potential of MSCs, using fresh isolates from different donors through to passage five, and showed a dramatic decrease in MSC functionality over time (46). MSCs from the same donor and same source (iliac crest marrow aspirate) isolated at different timepoints over a signaling factors.

period of 6 months also show significant variation in growth rates (44). Other studies have confirmed this loss of MSC function, demonstrating reduced proliferation, colony-forming (CFU-f) efficiency, telomere length and differentiation capacity with increasing time in culture (4, 40, 47). With the mounting interest in the use of MSCs for their paracrine effect it is also noteworthy that the secreted output of MSCs has been shown to differ with number of passages (48). This reduction in therapeutic potency at the population level can mask changes within clonal MSCs. Schellenberg et al. assessed MSC clones following expansion and observed a continual decrease in CFUf efficiency and differentiation capacity over time (49). Earlier analyses identified a complex hierarchy of MSC clones at varying stages of potency (50), so it may be that the diminishing clonal potential observed during MSC expansion is driven by subsets of cells reaching their proliferative limit or by entering the hierarchy of different stages through which cells pass during differentiation. Subsequent studies to track individual clones from MSC explant cultures showed that clonal complexity decreased markedly over 12 passages resulting in the clonal selection of a few dominant MSC clones (51).

Given the impact that culture expansion has on MSC fate, the in vitro environment and its influence on MSC properties is worth considering. In the majority of research laboratories, MSCs are expanded as a monolayer using standard tissue culture flasks with a plasma-treated polystyrene surface and medium containing fetal bovine serum. Surprisingly, given the detrimental effects on MSC proliferation, differentiation and paracrine activity of these basic methods, the industrial expansion of MSCs for clinical applications often still retains the same basic features (52). Scale-up can be achieved through the use of multilayered cell culture flasks (cell factories) or culture vessels specifically tailored for use with closed-box and automated systems. More advanced systems use roller bottles, hollow-fiber or stirred tank bioreactors [reviewed by (53)]. A major problem with this approach is that that these in vitro conditions are very different from the in vivo MSC microenvironment, lacking much of the complexity in terms of matrix composition, geometry, mechanical properties and interactions with other cell types. All of these microenvironmental factors are interpreted by the cell and have been shown to impact upon their behavior (54– 59). At its worst, the non-physiological conditions of typical cell cultures can cause mutations or cellular defects (60) but even the best-case scenario results in cells whose behavior is markedly changed. Together, this results in loss of potential from the whole population, but MSC heterogeneity may also be driven by cells responding to local changes in the microenvironment, such as through poorly controlled substrate properties or local changes in oxygen and nutrient concentration driven by the static nature of the setup (61).

It is clear that the requirement for extended in vitro expansion is a major contributor to the heterogeneity of MSC populations. A deeper understanding of the impacts of different environmental cues and the mechanisms by which they drive change, will be integral to the development of technologies for the large-scale production of quality MSC populations for clinical use.

#### CLINICAL EXPERIENCE AND REGULATORY CONSIDERATIONS RELATED TO HETEROGENEOUS CELL THERAPY

MSC heterogeneity is multifactorial and functionally influential. Nonetheless the clinical application of MSCs does not appear to take this into account, with a selection of recent trial publications suggesting a comparatively limited assessment of cellular phenotype (**Table 1**). The criteria established for MSCs by the ISCT (1) are sometimes referenced in these studies but not necessarily met. It is of course possible that additional criteria were specified during manufacture but not published, however publication of more detail would increase our understanding of the MSC phenotypes in clinical use.

Basic requirements for all biological medicines include the necessity to define the identity, the purity and the potency of the product. The developers of cell-based medicinal products must define the "active substance"; the cell type on which the therapeutic action of the product depends. Specification limits must be established for unique identification of the active substance within the product and for quantitation of its purity. Other phenotypes present, for example those arising from a tissue biopsy or culture contaminant, and non-viable cells, are generally regarded as impurities. These impurities should be reduced as far as possible and their content in the finished product limited and defined by specifications. Cellular impurities aside, major regulatory authorities do not always require cell-based medicinal products to consist of a pure population of cells. One of the first authorized cellular therapies was the immunotherapy Provenge (Dendreon Inc), approved by the US Food and Drug Administration (FDA) in 2010 for treatment of certain prostate cancers.  Antigen-presenting cells within the PBMC fraction are activated by the fusion protein, providing a tumor-directed action. The exact composition of the Provenge dose varies depending on the cellular composition of each patient's leukapheresis sample, but may contain, amongst others, T and B lymphocytes and natural killer cells so the therapy is inherently heterogeneous (77, 78). In 2015 the European Union (EU) authorized its first stem cell-based product, Holoclar (Chiesi Farmaceutici SPA, Italy). Holoclar is a population of cultured autologous human corneal epithelial cells containing limbal stem cells (LSCs) intended for treatment of ocular burns. The active substance contains only approximately 3.5% of p63bright LSCs, in a mixed population with transient amplifying meroclones and paraclones and terminally differentiated corneal epithelial cells (79). The extensive heterogeneity of the overall product, which arises from the inherent cellular variation in the patient's biopsy, was justified by evidence of relevant supportive properties provided by the non-stem majority population; these were therefore not considered to be cellular impurities (80).

In 2016, the EU approved Strimvelis [Orchard Therapeutics (Netherlands) BV], a gene therapy for treatment of adenosine deaminase (ADA) severe combined immunodeficiency (ADA-SCID), in which autologous CD34+ hematopoietic stem cells (HSCs) were transduced with ADA cDNA to provide the missing gene sequence. The active substance of Strimvelis includes not only the transduced CD34+ cells, but also the nongenetically modified CD34+ fraction, based on the fact that HSC transplantation is itself a standard treatment for ADA-SCID (81) These examples provide illustrations of the general acceptability, where justified, of heterogeneous cell populations within authorized cellular therapies. In the latter two cases, the heterogeneity specifically contributes to the overall clinical effect of the product and is not merely a consequence of the manufacturing process. The complexity associated with using fundamentally variable starting materials which are then processed, inducing further heterogeneity, implies that the purity of most cell-based products will be challenging to define. The regulators' expectation of quantitation of the population being administered in terms of identity and purity (82, 83) will be difficult to achieve definitively; it is probably more reasonable to demonstrate a degree of reproducibility across product batches and to relate the composition of each batch to those used in clinical trials than to provide exact percentages of each minor cellular component (84). The identification of relevant mechanisms of action will be of crucial importance in determining the acceptability of a degree of heterogeneity, since MSC activity in a specific clinical application should help inform selection of an ideal MSC population, whether this may be a heterogeneous preparation or a specified subset.

The inevitability of MSC heterogeneity and the consequences of culture expansion for the production of cell therapies, discussed earlier, raise key questions for developers of regenerative medicines. Whilst, as illustrated above, there is no obligation to demonstrate that a product contains only the specific cell type of interest, the challenges of definition and identification are accentuated when considering MSCs. The apparent absence of major concerns around cellular heterogeneity in whole organ and HSC transplantation is sometimes highlighted as support for a less rigorous approach to the characterization and control of cell-based therapies. However, acceptance of heterogeneity in these situations may be due in part to the fact that organ and HSC transplants are procedures which are considered to fall within the practice of medicine rather than items externally regulated as medicinal products.

#### FUTURE PERSPECTIVES: EMBRACING CHANGE

In order to advance the clinical utility of MSCs, it is essential that strategies to quantify heterogeneity are agreed. As a starting point, it is important to define the biological properties of the different stromal cell types within a mixed population. It is likely that stem-cell and non-stem-cell fractions are co-extracted using current protocols for MSC isolation. For regenerative therapies, it would seem logical that the stem-cell component

is the essential active ingredient, however non-differentiating stromal cells could play important supporting roles, for example in immune control; precisely why we need a full biological understanding that relates to mechanism of action. This can be achieved by exploiting techniques suitable for phenotyping individual cells, including flow cytometry, electrophysiology, microscopy (in various forms), image/morphometric analysis, lineage tracing, and powerful new single cell-omic technologies. Effective strategies will be required to ensure data are integrated, interpreted correctly and shared. The key to clinical translation will be to develop the most appropriate non-destructive biomarker identification techniques that provide functional discrimination. Reliable subtype-specific biomarkers will also support the development of treatments to target MSCs in situ, potentially negating the need for culture expansion. Alongside these, improved methods for MSC expansion that retain, or even promote selection of the desired MSC properties will be essential for the production of MSC products with a more defined set of characteristics and high therapeutic efficacy. Such technologies will likely incorporate biophysical as well as biochemical cues and provide platforms for scale-up culture in bioreactors. With the role of the paracrine effect of MSCs coming to the fore (85), therapies based on the MSC secretome or MSC-derived extracellular vesicles (EVs) may emerge to complement the MSC therapeutic toolkit. However, different MSC populations (or cells within that population) are still likely to produce different secretomes and so many of the fundamental challenges relating to MSC heterogeneity will remain.

Given the challenges associated with providing consistency in an MSC product from multiple tissue isolates, the generation of MSCs from pluripotent stem cell populations has garnered interest (86–92). The expansion capability of pluripotent cells means that a single clonal population can potentially be manufactured and subsequently differentiated into a virtually limitless supply of MSCs. This type of platform relieves the need for continuous tissue donations, simplifies the subject of donor-donor variation and bypasses many of the sources of MSC heterogeneity that arise when working with ex vivo cells. Induced pluripotent stem cells (iPSC)-derived MSCs offer the potential for large-scale production of more homogenous, off-the-shelf products with limited batch-to-batch variation that could deliver more consistent clinical outcomes. The first phase I clinical trial using iPSC-derived MSCs was completed in 2018 with promising results from Cynata Therapeutics's lead CymerusTMCYP-001 product for the treatment of graft vs. host disease (93), although the full findings have not yet been published. While the clinical use of iPSC-MSCs holds promise, an effective comparison of pluripotent cell-derived MSCs to their adult tissue counterparts is required, with appropriate safety profiling. Clonal immortalized MSC lines (both iPSC-derived and genetically modified adult MSCs) may also be developed for bulk harvesting of secreted products, proteins, and EV cargoes, which could ultimately

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dispense with the need for the transplantation of MSCs as a whole-cell product, however the issue of stochastic heterogeneity arising in clonal cell populations will always persist.

MSCs can offer widespread therapeutic benefits but we must balance enthusiastic demands for clinical progress against the need for better mechanistic understanding. Unraveling MSC multiplicity is the essential first step in that process.

#### AUTHOR CONTRIBUTIONS

AW, MH-G, JF, and PG wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.

#### FUNDING

This work was supported by the Tissue Engineering and Regenerative Therapies Centre Versus Arthritis (21156).

inherited and acquired diseases. Expert Opin Biol Ther. (2005) 5:1571–84. doi: 10.1517/147125<IP_ADDRESS>1

sclerosis: an open label study. Curr Stem Cell Res Ther. (2012) 7:407–14. doi: 10.2174/157488812804484648

for the Human ADA cDNA Sequence. Available online at: https://www. ema.europa.eu/documents/assessment-report/strimvelis-epar-publicassessment-report\_en.pdf (accessed December 14, 2018).

**Conflict of Interest Statement:** JF and MH-G acknowledge funding from Cynata Therapeutics.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Wilson, Hodgson-Garms, Frith and Genever. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Nature vs. Nurture: Defining the Effects of Mesenchymal Stromal Cell Isolation and Culture Conditions on Resiliency to Palmitate Challenge

Lauren K. Boland1,2, Anthony J. Burand1,2, Devlin T. Boyt 1,2, Hannah Dobroski 1,2, Lin Di 1,2 , Jesse N. Liszewski 1,2, Michael V. Schrodt 1,2, Maria K. Frazer 1,2, Donna A. Santillan3 and James A. Ankrum1,2 \*

*1 University of Iowa Fraternal Order of Eagles Diabetes Research Center, University of Iowa, Iowa City, IA, United States, 2 Roy J. Carver Department of Biomedical Engineering, University of Iowa, Iowa City, IA, United States, 3 Department of Obstetrics and Gynecology, Center for Immunology and Immune Based Diseases, Center for Hypertension Research, University of Iowa, Iowa City, IA, United States*

#### Edited by:

*Ulrike Koehl, Hanover Medical School, Germany*

#### Reviewed by:

*Martino Introna, Ospedale Papa Giovanni XXIII, Italy Andrea Hoffmann, Hanover Medical School, Germany*

> \*Correspondence: *James A. Ankrum<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology*

> Received: *26 February 2019* Accepted: *29 April 2019* Published: *10 May 2019*

#### Citation:

*Boland LK, Burand AJ, Boyt DT, Dobroski H, Di L, Liszewski JN, Schrodt MV, Frazer MK, Santillan DA and Ankrum JA (2019) Nature vs. Nurture: Defining the Effects of Mesenchymal Stromal Cell Isolation and Culture Conditions on Resiliency to Palmitate Challenge. Front. Immunol. 10:1080. doi: 10.3389/fimmu.2019.01080* As MSC products move from early development to clinical translation, culture conditions shift from xeno- to xeno-free systems. However, the impact of isolation and culture-expansion methods on the long-term resiliency of MSCs within challenging transplant environments is not fully understood. Recent work in our lab has shown that palmitate, a saturated fatty acid elevated in the serum of patients with obesity, causes MSCs to convert from an immunosuppressive to an immunostimulatory state at moderate to high physiological levels. This demonstrated that metabolically-diseased environments, like obesity, alter the immunomodulatory efficacy of healthy donor MSCs. In addition, it highlighted the need to test MSC efficacy not only in ideal conditions, but within challenging metabolic environments. To determine how the choice of xeno- vs. xeno-free media during isolation and expansion would affect future immunosuppressive function, umbilical cord explants from seven donors were subdivided and cultured within xeno- (fetal bovine serum, FBS) or xeno-free (human platelet lysate, PLT) medias, creating 14 distinct MSC preparations. After isolation and primary expansion, umbilical cord MSCs (ucMSC) were evaluated according to the ISCT minimal criteria for MSCs. Following baseline characterization, ucMSC were exposed to physiological doses of palmitate and analyzed for metabolic health, apoptotic induction, and immunomodulatory potency in co-cultures with stimulated human peripheral blood mononuclear cells. The paired experimental design (each ucMSC donor grown in two distinct culture environments) allowed us to delineate the contribution of inherent (nature) vs. environmentally-driven (nurture) donor characteristics to the phenotypic response of ucMSC during palmitate exposure. Culturing MSCs in PLT-media led to more consistent growth characteristics during the isolation and expansion for all donors, resulting in faster doubling times and higher cell yields compared to FBS. Upon palmitate challenge, PLT-ucMSCs showed a higher susceptibility to palmitate-induced metabolic disturbance, but less susceptibility to palmitate-induced apoptosis. Most striking however, was that the PLT-ucMSCs resisted the conversion to an immunostimulatory phenotype better than their FBS counterparts. Interestingly, examining MSC suppression of PBMC proliferation at physiologic doses of palmitate magnified the differences between donors, highlighting the utility of evaluating MSC products in stress-based assays that reflect the challenges MSCs may encounter post-transplantation.

Keywords: mesenchymal stromal cells, umbilical cord mesenchymal stromal cells, obesity, palmitate, human platelet lysate, fetal bovine serum, culture media, cell therapy

#### INTRODUCTION

Cell bioprocessing, involving the large scale production of high volumes of consistent and efficacious cellular products, is a critical component for the translation of mesenchymal stromal cell (MSC) products from academic laboratories to clinical application (1–4). However, a mismatch in culture conditions between basic academic research and clinical translation often exists, with academic labs often relying on fetal bovine serum (FBS) supplemented media and small 2D culture systems, while clinical-grade products are grown in large batches in xeno-free medias (1, 2, 5, 6). Decisions that may once have seemed trivial or even arbitrary in the academic research environment [e.g., media composition (7–11), cell culture format (2, 12–14), or substrate-stiffness (15, 16)] become critical parameters to control in the large-scale industrial production of clinical-grade cellular therapies (17–19). Increasingly, as cellular therapies have been used to treat patients in clinical trials, it has become clear that adopting good manufacturing practices (GMP) early at the research phase can hasten the transition from the bench to the clinic (2, 4, 19, 20). With the diversification of the MSC field to include MSCs harvested from a number of tissue sources (21–24), it is critical to distinguish variance that is inherent in the cell source vs. variability that is introduced by differences in bioprocessing during initial isolation and in vitro expansion (25).

As findings from the lab transition to the clinic, a number of process-related changes are often needed to produce a clinicalgrade product (2, 3, 18). One of the most glaring differences between MSCs studied in research labs to those produced at an industrial scale for use in patients is a switch from using animal serum as a growth supplement to xeno-free alternatives like human platelet lysate or chemically defined serum (6, 26). There is significant and valid concern surrounding the use of animal derived products in the generation of clinical-grade cell therapies (6, 27, 28). Consequently, transitioning to clinicalgrade production often involves transitioning to a xeno-free culture system (1, 2). Today, a large number of xeno-free alternatives are available, many of which have associated drug master files submitted to the FDA, which can make submission of investigational new drug (IND) applications more streamlined (6, 26, 29). A number of notable advantages have been reported for xeno-free culture systems for MSCs, including enhanced cell yield (9, 10, 30), rapid growth kinetics (5, 10), elimination of xenogeneic pathogens (27), and improved genetic stability over extended culture periods (30, 31). In order to ensure that the transition from pre-clinical to clinical application is successful, it is important to understand how the process differences related to culture environments affect MSC function.

Variability in MSC phenotype due to donor age (32–35), sex (35, 36), tissue of origin (21, 37), and co-morbidities (38–40), as well as time spent in culture (41–45), has been extensively documented. A notable example of MSC donor-specific variance was the observation that certain bioactive secreted factors, namely tumor necrosis factor-inducible gene 6 (TSG6), show a sexual dimorphism with higher quantities secreted from female vs. males bone marrow donors (36). An increasingly important contributor to MSC phenotype is the presence of metabolic disease [obesity, type 2 diabetes, atherosclerosis, and metabolic syndrome (32, 34, 38, 39, 46–51)] within donors. A number of functional defects have been documented in adipose-derived MSCs from donors with metabolic disease, including blunted immunosuppressive potency against activated T cells (32, 38), reduced fibrinolytic activity (39, 46), and a diminished ability to halt the progression of neuroinflammation (40). In addition to the functional defect seen in MSCs isolated from patients with metabolic disease, recent work in our lab has demonstrated that healthy donor MSCs exposed to a "metabolically diseased" environment enriched in the saturated fatty acid, palmitate, are no longer suppressive, but stimulatory when cultured with activated peripheral blood mononuclear cells (52). Interestingly, MSCs from some donors were more sensitive to palmitate exposure than others, despite similar levels of potency in traditional palmitate-free co-culture assays. Collectively, this body of work highlights the need for cell manufacturing processes that consistently generate high quality MSCs and the need for in vitro potency assays capable of predicting MSC performance after transplant into challenging metabolic environments.

Although much of the variance in functional performance of MSCs has been attributed to intrinsic donor characteristics, only more recently has attention been focused on how specific process-related decisions may contribute to variance in MSC performance (1–3, 18, 20, 23, 25). While benefits have been reported for xeno-free systems, they have predominately been focused on improving the cell yield during manufacturing rather than the functional properties of the cells (17, 18). Additionally, the interaction of these systems with inherent donor variability is less well-understood (3, 53). Whether or not xeno-free culture systems produce MSCs that are more or less resilient for use in complex metabolic environments has yet to be determined.

In the present study, we investigated how early and late decisions regarding media supplementation modify the health and function of umbilical cord derived MSCs (ucMSCs). By growing ucMSCs from explant to experiment in one of two media supplements, either standard FBS or human platelet lysate, we were able to compare genetically identical cell preparations that had been acclimated to two different culture environments from isolation to analysis. Notably, this study design allowed us to delineate the contributions of donor intrinsic variability (nature) vs. process-derived variability (nurture). Additionally, we demonstrate for the first time that adapting an in vitro potency assay to mimic a disease environment can reveal the impact of process-related decisions on MSC performance within challenging metabolic environments.

#### MATERIALS AND METHODS

## Umbilical Cord MSC (ucMSC) Isolation

Human umbilical cords were collected through the University of Iowa Women's Health Tissue Repository's Maternal Fetal Tissue Bank with consent from the mothers (IRB#200910784). Samples were provided to the investigators in a coded fashion and this work was deemed to not be human subjects research by the University of Iowa IRB (IRB# 201708749). After delivery, samples were placed at 4◦C in PBS (Biological Industries, Cat #02-023-1A-24) supplemented with 2% Penicillin-Streptomycin solution (P/S, Biological Industries, Cat #03-031-1B) and 2% Amphotericin B (AmpB, Biological Industries, Cat #03-028-1B). Umbilical cord tissue explants were plated within 48 h of delivery. To begin, the external wall of the cord was sterilized with 70% ethanol and pharmacidal spray (Biological Industries, Cat #IC-110100- 05) and placed in sterile PBS. The umbilical cords were then cut into 3 mm cross-sections with a sterile razor and subdivided into 1 mm pieces. Approximately 60 cord pieces were then divided into two dry culture dishes (Corning, Cat #430599), with Wharton's Jelly placed against the plate. Umbilical cord pieces were allowed to adhere for 10–15 min before addition of media. Warm α-minimum essential medium (Biological Industries, Cat #01-042-1A) supplemented with 1% P/S, 2% AmpB, and either 10% FBS (Biological Industries, Cat #04-121-1A-US) or 5% PLTGold Human Platelet Lysate (PLT, Biological Industries, Cat #PLTGOLD100R), was then slowly added before the dishes were placed in a 37◦C incubator. Media was subsequently changed every 2–3 days. After 9 days of outgrowth the cord pieces were removed and if the cells were confluent in their colonies the cells were plated as Passage 1 (P1) in T25s. If not confluent after 9 days, media was changed, and the cells were cultured an additional 3 days before plating into T25 flasks. P1 cells were grown to 80% confluence and grown for one additional passage before cryostorage using serum-free cell freezing medium (Biological Industries, Cat #05-065-1A).

#### ucMSC Expansion and Growth Kinetics

ucMSCs isolated as described above were counted, plated (P1), and grown to 70–90% confluence. Cells were then harvested, counted, plated (P2), and expanded prior to cryopreservation (P3). Population doubling level at each of these splits was calculated using the formula PDL = PDL0+3.322(logP1−logP0), where PDL0 is the population doubling level at seeding, P1 is the total cell yield at harvest, and P0 is the initial cell seeding number. Cells harvested immediately after isolation were declared to have an initial PDL of 0. Time to population doubling was calculated from the initial first three cell harvests. Briefly, total cell yields at each harvest were fit using linear regression in GraphPad Prism 7 software and the linear regression equations were used to calculate the average time to population doubling for each MSC preparation.

Post-cryopreservation, MSCs were thawed at 37◦C until ∼1 mm3 piece of ice remained. The cells were then transferred immediately into the appropriate pre-warmed media condition and allowed to attach overnight. Media was switched to remove cryopreservation media and cells were grown out to 70– 90% confluence. Population doublings after cryostorage were calculated as before from the total cell yields after each cell harvest to confirm consistency of growth kinetics in the medias before and after cryopreservation.

### BSA Preparation and Palmitate-BSA Conjugation

Bovine serum albumin (BSA) and palmitate conjugated to BSA were prepared according to a previously described protocol (52, 54, 55). Briefly, to prepare the BSA solution, 20% (w/v) fatty acid free bovine albumin (MP Biomedicals, Cat #0219477205) was gradually added to Ultra Pure water (Biological Industries, Cat #01-866-1A) at 52◦C with gentle agitation until the BSA was fully dissolved, yielding a transparent yellow solution. To prepare a palmitate-BSA solution, 2.6% (w/v) of palmitic acid (Sigma-Aldrich, Cat #P0500) was added to 0.1 M NaOH (Sigma-Aldrich, Cat #221465) and heated to 70◦C until fully dissolved, yielding a concentration of 100 mM. To conjugate palmitate to BSA, the 100 mM stock of palmitate in 0.1 M NaOH was added to 20% BSA at a ratio of 1:9 and heated at 55◦C for 10 min. After conjugation, both the 20% (w/v) BSA solution and 10 mM Palmitate-BSA solutions were filter sterilized using a 0.45µm PES filter (Celltreat, Cat #229709). Solutions were then aliquoted and stored at −20◦C. Before use, solutions were warmed to 37◦C. The total amount of BSA was held constant in all conditions by mixing ratios of fatty acid free BSA to palmitate-BSA as follows: BSA only (4:0), 0.1 mM palmitate-BSA (3:1), 0.2 mM palmitate-BSA (2:2), and 0.4 mM palmitate-BSA (0:4).

#### ucMSC Validation (Immunophenotyping and Differentiation)

To validate ucMSC identity via immunophenotyping, cells were stained to confirm CD105, CD73, and CD90 expression and the absence of CD45, CD34, CD11b, CD19, and HLA-DR as defined by the MSC minimal criteria (56). Positive markers were confirmed using FITC-CD105 antibody (BD Biosciences, Cat #561443), PE.Cy7-CD73 antibody (BD Biosciences, Cat #561258), and PE-CD90 antibody (BD Biosciences, Cat #A15794) with appropriate isotype controls [FITC Mouse lgG1k (BD Biosciences, Cat #556649), PE.Cy7 Mouse lgG1k (BD Biosciences, Cat #557872), and PE-CD90 Mouse lgG1 (Invitrogen, Cat #GM4993)]. A negative marker cocktail and isotype staining was performed using the PE hMSC Negative Cocktail (BD Biosciences, Cat #562530), as per manufacturer's instructions.

For trilineage differentiation (56), each preparation of ucMSCs (seven donors, maintained in either FBS-media or PLT-media) was differentiated to osteoblasts, chondroblasts, and adipocytes by culturing in osteogenic (Biological Industries, Cat #05-440-1B), chondrogenic (Biological Industries, Cat #05-220- 1B-KT), or adipogenic (Biological Industries, Cat #05-330-1B-KT) differentiation medias, respectively. Briefly, for osteogenic and adipogenic differentiation, ucMSCs were plated at a density of 60,000 cells/well in 24-well plates and grown in either FBS- or PLT-media for 24 h. Cells were then visualized to ensure >80% confluence and media was exchanged to osteogenic or adipogenic differentiation medias. Cultures were maintained for 15–18 days with media exchanges every 3–4 days. Osteogenic lineage was confirmed using both bright-field imaging and Alizarin Red for mineral deposition. For adipogenic lineage confirmation, lipid droplets were identified via AdipoRed staining and fluorescent microscopy. For chondrogenic differentiation, a micromass culture (10 uL of 10 million/mL ucMSCs in chondrogenic differentiation media) was incubated for 2 h to allow for spontaneous spheroid formation in 96-well spheroid plates (Corning, Cat #4515). After 2 h, 200 uL of chondrogenic media was added for subsequent culture. Spheroid cultures were maintained for 18 days with media exchanges occurring every 3–4 days. On Day 18, chondrogenic spheroids were fixed for 5 min with 10% Formalin then mounted in Optimal Cutting Temperature (OCT) Compound. Sectioning of the chondrogenic pellets was performed by the University of Iowa Comparative Pathology Core and tissues were subsequently stained with Safranin O.

#### Metabolic Health and Viability Assays

To assess the metabolic function of ucMSCs exposed to palmitate, we performed an XTT assay (Cell Proliferation Kit-XTT based, Biological Industries, Cat #20-300-1000). For each media condition, 1,000 ucMSCs from every donor were plated into a 96-well plate. All wells had a total media volume of 100 µL. The ucMSCs were treated with 20% (w/v) BSA or 0.1, 0.2, or 0.4-mM palmitate-BSA as described above. After 96 h, Activation Reagent was added to warmed XTT Reagent at a ratio of 1:5,000 and 50 µL of XTT solution was added to the media of each well. The plate was read at 475 and 660 nm absorbance at 1 and 2 h time points by a microplate reader. The background reading was subtracted for all wells, and within each media condition, the background-subtracted reading was normalized to a media-only control.

To assess changes in viability, ucMSCs were incubated at 37◦C with BSA, 0.1, 0.2, or 0.4 mM Palmitate-BSA for 96 h. Both detached cells in the media and adherent cells were collected and pooled for analysis. Cells were pelleted, resuspended in prepared Annexin/PI binding buffer [20 mM HEPES at pH 7.4 (Invitrogen, Cat #A14291DJ), 150 mM sodium chloride (American Bio, Cat #AB01915-10000), and 2.5 mM calcium chloride (Sigma Aldrich, Cat #C8106-500g)], and stained with 90 µg/mL FITC-Annexin V (Biolegend, Cat #640945 and 640906) and 1 mg/mL propidium iodide (Invitrogen, Cat #P3566) to final concentrations of 4.5 and 50µg/mL, respectively, in Annexin/PI binding buffer. Samples were incubated for 30 min in the dark at room temperature. Samples were then diluted with Annexin/PI binding buffer and analyzed via flow cytometry (Accuri C6, BD Biosciences).

# MSC:PBMC Co-culture With Palmitate Challenge

Peripheral blood mononuclear cells (PBMCs) from a deidentified donor were isolated from a leukapheresis reduction cone made available by the DeGowin Blood Center at the University of Iowa Hospitals and Clinics. After isolation, PBMCs were cryopreserved in a solution consisting of 50% RPMI (ThermoFisher, Cat #11875093), 40% FBS (VWR, Cat #97068085), and 10% DMSO (Fisher Scientific, Cat #D128) until use. PBMCs were thawed at 37◦C 1 h prior to staining and plated in RPMI containing 10% FBS, 1% L-glutamine , and 1% P/S. In order to track generational proliferation of the PBMCs, PBMCs were stained with CFSE Cell Division Tracker Kit (Biolegend, Cat #423801) according to the manufacturer's protocol. Briefly, PBMCs resuspended in PBS at 1 million cells per mL were stained with 2 µL of 10 mM CFSE per 10 million cells to yield a final CFSE concentration of 1µM. PBMCs were incubated at 37◦C for 15 min, centrifuged at 500 g for 8 min, resuspended in RPMI to neutralize the dye, and incubated at 37◦C for 30 min. Cells were centrifuged again, resuspended at 1 million per mL, and stored at 37◦C until plating with the MSCs.

All 14 preparations of ucMSCs were harvested after staining the PBMCs, and 50,000 cells were plated into 24-well plates. MSCs were allowed to attach for 1 h. Prior to plating, CFSE stained PBMCs were mixed 1:1 with Human CD3/CD28 Dynabeads (Thermo Fisher, Cat #11132D) to activate T cells within the mixed population. 200,000 stimulated PBMCs were added to each MSC condition to yield an MSC to PBMC ratio of 1:4. Total media volume in each well was set at 750 µL and all co-culture conditions were either treated with BSA alone, or 0.1, 0.2, or 0.4 mM palmitate-BSA to simulate metabolic stress. PBMCs with or without Dynabeads were plated as positive and negative controls for PBMC activation and proliferation. After 5 days, PBMCs were isolated by collecting the non-adherent cell fraction from the plates. These cells were then centrifuged, the supernatants collected, and resuspended in RPMI. Supernatants were stored at −20◦C for future analysis of Granzyme-B levels using a bead-based ELISA per the manufacturer's instructions (BD Biosciences, Cat #560304). PBMC proliferation was analyzed by flow cytometry (Accuri C6, BD Biosciences).

# Media Exchange Experiments

Two vials of P4 ucMSC from donor 4600 isolated with FBS media, were thawed and plated in either FBS (FBS-FBS) or PLT (FBS-PLT) supplemented media. Likewise, two vials of P4 ucMSC from donor 4600 were thawed and plated in either FBS (PLT-FBS) or PLT (PLT-PLT) supplemented media. After culturing for 72 h, the ucMSCs were counted and replated to assess their growth kinetics. The cells were then cultured for another passage Boland et al. MSC Resiliency to Palmitate Challenge

(96 h), lifted, counted, and plated for either co-culture or imaging experiments. For co-culture with PBMCs, each preparation of ucMSCs were plated at a ratio 1 MSC: 4 PBMCs. PBMCs were stained with CFSE and stimulated with Dynabeads, as previously described. The co-culture was incubated for 5 days, followed by flow cytometry analysis. For morphological analysis, ucMSCs were plated at 1,000 ucMSCs/cm2 in 6-well plate and cultured for an additional 48 h. Cells were then fixed for 5 min (10% neutral buffered Formalin, Sigma-Aldrich, Cat# HT501128-4L), followed by permeabilization for 5 min (0.1% v/v Triton R X-100 in PBS, Sigma-Aldrich, Cat#T9284-500 mL), at room temperature. After permeabilization, the cells were stained with ActinRed 555 ReadyProbes Reagent (Cat# R37112, Thermo Fisher Scientific) according to the manufacturer's protocol. Briefly, for every 1 milliliter of PBS, 2 drops of reagent were added. The cells were then incubated with 1 mL of reagent in PBS at 37◦C for 30 min. During the last 10 min of ActinRed staining, 1 ug/mL of Hoechst 33342 (Cat# H3570, Invitrogen) was added. Cells were then imaged using a fluorescent microscope (Leica DMI6000) at 20× magnification.

#### Statistical Analysis

Statistical analysis was pre-planned prior to collection of data and performed within GraphPad Prism 7. For all assays involving multiple donors, each donor was considered an independent "n." For assays involving a single donor, independent replicate experiments were considered as "n." Specific statistical tests used for each data set is listed in the caption of each figure.

# RESULTS

## PLT-Media Enhances ucMSC Growth Kinetics and Overall Cell Yield Compared to FBS-Media