Enriched Protein Screening of Human Bone Marrow Mesenchymal Stromal Cell Secretions Reveals MFAP5 and PENK as Novel IL-10 Modulators The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Milwid, Jack M et al. “Enriched Protein Screening of Human Bone Marrow Mesenchymal Stromal Cell Secretions Reveals MFAP5 and PENK as Novel IL-10 Modulators.” Molecular Therapy 22, 5 (May 2014): 999–1007 © The American Society of Gene & Cell Therapy As Published http://dx.doi.org/10.1038/MT.2014.17 Publisher Elsevier BV Version Final published version Citable link http://hdl.handle.net/1721.1/111793 Terms of Use Creative Commons Attribution-NonCommercial-NoDerivs License Detailed Terms http://creativecommons.org/licenses/by-nc-nd/4.0/ Cell therapy © The American Society of Gene & Cell Therapy original article Enriched Protein Screening of Human Bone Marrow Mesenchymal Stromal Cell Secretions Reveals MFAP5 and PENK as Novel IL-10 Modulators Jack M Milwid1,2, Jessica S Elman1, Matthew Li1–3, Keyue Shen1, Arjun Manrai2, Aaron Gabow4, Joshua Yarmush6, Yunxin Jiao1, Anne Fletcher5, Jungwoo Lee1, Michael J Cima3, Martin L Yarmush1,6 and Biju Parekkadan1,7 1Department of Surgery, Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA; 2Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; 3David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; 4Computational Biology Center, Memorial Sloan-Kettering Cancer Center, New York, New York, USA; 5Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA; 6Department of Biomedical Engineering, Rutgers University, Piscataway, New Jersey, USA; 7Harvard Stem Cell Institute, Cambridge, Massachusetts, USA The secreted proteins from a cell constitute a natural bio- Insulin, a secreted pancreatic hormone, has been supplemented logic library that can offer significant insight into human for decades as a first-line therapy for regulating the uptake of glu- health and disease. Discovering new secreted proteins cose by target cells in diabetic patients (2011 US sales: $9.8B).5,6 from cells is bounded by the limitations of traditional sep- Interferon-α and interferon-β are widely used to treat hepatitis C aration and detection tools to physically fractionate and virus infection and multiple sclerosis, respectively (collective 2011 analyze samples. Here, we present a new method to sys- US sales: $2.5B).6,7 Proinflammatory cytokines, such as tumor tematically identify bioactive cell-secreted proteins that necrosis factor (TNF)-α, have become mega-blockbuster phar- circumvent traditional proteomic methods by first enrich- maceutical targets for patients with autoimmune diseases (2011 ing for protein candidates by differential gene expression US sales of TNF-α inhibitors: $6.8B).6,8 Total US sales in 2011 of profiling. The bone marrow stromal cell secretome was protein drugs based on native secreted proteins exceeded $32B.6 analyzed using enriched gene expression datasets in Identification of secreted proteins has primarily relied on combination with potency assay testing. Four proteins hypothesis-driven interrogation and is limited to known biologi- expressed by stromal cells with previously unknown anti- cal pathways.9 Open-ended screening approaches that assume inflammatory properties were identified, two of which less about the underlying biology are impractical because estab- provided a significant survival benefit to mice chal- lishment of libraries of all known secreted proteins is cost- lenged with lethal endotoxic shock. Greater than 85% prohibitive; probing the vast interactome is intractable; and not of secreted factors were recaptured that were otherwise all proteins synthesized in the human body can be made recom- 10 undetected by proteomic methods, and remarkable hit binantly. A bottom-up approach to access and dissect a finite rates of 18% in vitro and 9% in vivo were achieved. library of secreted proteins involves the analysis of conditioned medium (CM) from a cell. Traditional biochemical methods for Received 15 July 2013; accepted 30 January 2014; advance online characterizing CM, such as liquid chromatography (LC) and mass publication 18 March 2014. doi:10.1038/mt.2014.17 spectrometry (MS), have led to the discovery of many important INTRODUCTION proteins including interferon and hepatocyte growth factor.11,12 Cells can communicate by secreting proteins that act on neigh- These methods are, however, labor intensive because of the need boring or distant target cells to regulate function and phenotype. to grow billions of cells in order to generate liters of CM and elimi- Identification of these communication signals has led to a deeper nate serum and albumin contaminants for sensitive analysis.13,14 understanding of physiological processes involved in health and These challenges have restricted discovery efforts in academic and disease, including cell proliferation,1 differentiation,2 motility,3 and industrial laboratories. New methods to elucidate secreted pro- apoptosis.4 Secreted proteins have also become important in the teins and the pathways they influence can accelerate the discovery development of some of the most successful therapies in history. of new signaling molecules for biological research and therapeutic These therapies are generally designed to either increase the natu- testing. ral levels by administration of recombinant proteins or decreas- A new, simple method for discovering potential secreted ing the level by use of neutralizing agents, such as antibodies. protein communication pathways is reported here. The method Correspondence: Biju Parekkadan, Department of Surgery, Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA. E-mail: [email protected] Molecular Therapy vol. 22 no. 5, 999–1007 may 2014 999 © The American Society of Gene & Cell Therapy Profiling of BM-MSC Secretions (referred to as enriched protein screening (EPS)) circumvents the evaluate the bulk anti-inflammatory activity of BM-MSC-CM need for LC/MS by using comparative gene profiling to enrich for (Figure 2a). PBMCs were incubated with BM-MSC-CM and then active secreted proteins that can be recombinantly screened in stimulated with lipopolysaccharide (LPS) to instigate a proinflam- bioassays (Figure 1). A human peripheral blood mononuclear cell matory response. LPS-stimulated production of IL-10 by human (PBMC) inflammation assay was used to screen candidates natu- PBMCs increased significantly when the cells were preincubated rally expressed by human bone marrow mesenchymal stromal cells with BM-MSC-CM (Figure 2b; Supplementary Figure S2a). (BM-MSCs)15 as a proof of concept. BM-MSCs are stem-like cells Upregulation of IL-10 release was dose dependent (Figure 2c), that are being explored clinically as a therapeutic for a variety of suggesting that the receptors responsible for IL-10 release were inflammatory and autoimmune diseases.16 The implementation of not saturated in this range of CM. Digestion of BM-MSC-CM EPS led to the discovery of two novel immunomodulatory proteins, with proteinase K attenuated the IL-10 response, confirming the microfibrillar-associated protein 5 (MFAP5) and unprocessed pro- causative role of proteins in the CM (Supplementary Figure S2b). enkephalin (PENK), that when administered to mice subjected to BM-MSC-CM also suppressed inflammatory interferon (IFN)-γ endotoxemic shock, reversed the cytokine storm and provided a production in this assay (Supplementary Figure S3). Boosting of significant survival benefit comparable to the gold-standard anti- IL-10 was further used as an assay endpoint because it is less prone inflammatory biologic, monoclonal anti-TNF-α antibodies. to nonspecific assay artifacts, such as suppressed IFN-γ expression from cytotoxicity. The sensitivity of the PBMC assay was tested RESULTS using fractions of BM-MSC-CM based on size (Supplementary Development of a human blood potency assay to Figure S4a) and charge (Supplementary Figure S4b), and clear analyze BM-MSC–secreted factors delineation of active regions confirmed sufficient detection sen- BM-MSCs have been extensively reported to have anti-inflam- sitivity of the assay. matory properties when used in a transplantation setting.17 Interleukin (IL)-10 is increased in the serum of inflamed rodents Genomic enrichment of BM-MSC–secreted proteins after treatment with BM-MSCs or a bolus of concentrated based on potency BM-MSC-CM.18 A potency assay was developed to recapitulate A comparative gene expression scheme was developed to rationally this IL-10 response in vitro using primary human PBMCs to enrich candidate proteins that contribute to the anti-inflammatory Cell 1 12 Hit 1 Hit 2 Hit 3 Hit 4 Cell 2 Cell 3 Assay response Assay 3N Cell N Cell 1Cell 2 2 > 1 > ... > N Vehicle Cell N Protein products Potency assay screening Comparative profiling Enriched candidate screen Refined candidate screen Figure 1 Enriched protein screening (EPS). A generalized schematic of EPS methodology. Protein products are derived from various cell types in the form of conditioned media and screened for activity in an in vitro potency assay. Based on the activity of the conditioned media from the cells, differential