The influence of hypoxia and IFN-γ on the proteome and metabolome of therapeutic mesenchymal stem cells
Introduction
Mesenchymal stem cells (MSCs) are multipotent cells that are found in a wide variety of tissues [1,2]. Indeed, it is reasonable to expect that most, if not all, tissues have resident MSC-like cells, which participate in local regeneration upon injury or disease. Because these endogenous cells exist in limited numbers, many investigators have explored administration of large numbers of exogenous MSCs to enhance their therapeutic effect [3]. These cells are most often given by intravenous injection, with the expectation that they can home to affected sites and have long-ranging impact by secreting paracrine factors [4].
The expression profile and phenotype of MSCs residing in healthy tissues or expanded under basal culture conditions are generally different from those expressed by therapeutic MSCs. Upon administration, MSCs enter the diseased microenvironment of the patient and react to the new cues that are present. Two of these cues, which are common to most ailments, are inflammation and hypoxia [5]. Hypoxia specifically connotes an oxygen tension that is lower than physoxia, which is 3–9% oxygen in most tissues [6]. It affects cell behavior because hypoxia inducible factors (HIF) become hydroxylated at higher oxygen levels and face proteasomal degradation, but at lower oxygen tensions, they are more stable (specifically the alpha subunit), and serve as transcription factors. While the signaling pathways for inflammation and hypoxia start off as being distinct, they have many opportunities for cross-talk, as has been reviewed elsewhere [5,7,8].
In acute injury (such as myocardial infarction or stroke), a blood clot leads to a transient decrease in tissue perfusion, and inflammatory cells rush in to tackle foreign invaders and remove damaged tissue. In this situation, the hypoxia and inflammation may be short lived (days to a week), contingent upon successful resolution of the injury [9]. However, in many chronic disease states these two factors co-exist for prolonged periods of time. For example, in rheumatoid arthritis, the synovial joint space is characterized by regions of hypoxia and inflammation, which are thought to influence local cell survival, angiogenesis, and energy metabolism [9]. Another autoimmune disease – inflammatory bowel disease – also exhibits chronic inflammation, with oxygen gradients at the intestinal mucosa that lead to regions of hypoxia. Curiously, these hypoxic regions are relatively protected compared with the rest of the intestinal tract, as the HIF pathway seems to promote epithelial survival while inducing neutrophil apoptosis [9]. Lastly, in solid tumors, hypoxia and inflammation facilitate tumor vascularization and immune escape [7].
In order to understand how MSCs serve as reparative and regenerative cells, we explored how they would respond to a generic pathological microenvironment in which hypoxic and inflammatory cues were provided in vitro. To simulate hypoxia, we chose an incubator setting of 1% O2, as this is a lower oxygen tension than the physoxic state of most tissues without being anoxic [10], and to simulate inflammation, we used interferon-gamma (IFN-γ). While many pro-inflammatory cytokines and TLR agonists have been shown to affect MSC behavior, the response of MSCs to IFN-γ is considered to be a standard benchmark of cell function by the International Society for Cellular Therapy (ISCT), and IFN-γ is more natural to a generic inflammatory setting than polyI:C, for example [[11], [12], [13], [14], [15]]. We then analyzed the proteome and metabolome of adipose-derived MSCs upon 48hr exposure to control conditions, hypoxia (1% O2), inflammation (IFN-γ), or both cues together (a scenario we refer to as “dual priming”).
Section snippets
Methods
MSC culture and priming The protocol for MSC priming follows our previously published work [16]. Briefly, frozen vials of adipose-derived MSCs from fully de-identified commercially obtained human lipoaspirates (LaCell, New Orleans, LA) were tested for tri-lineage differentiation as well as positive expression of in vitro MSC surface markers, as previously published [14]. All experiments were done using passage 5 MSCs from 3 separate cryovials to generate biological triplicates. This passage
Overview of data
MSCs used for proteomic and metabolomic analyses had a viability of >94%, based on trypan blue exclusion, with no significant differences based on exposure to hypoxia or IFN-γ. While we did not observe a change in morphology indicative of undesired differentiation, exposure to IFN-γ did increase the average cell diameter (Fig. S1), as has been observed by others [19]. Our prior studies have also shown minimal changes to MSC surface marker expression, with the exception that IFN-γ leads to
Discussion
Several meritorious studies have been dedicated to identifying the protein expression profile of MSCs derived from different tissue sources [30,31]. However, if basic culture conditions are used, none of these profiles represented how MSCs would behave in a clinical setting, because MSCs are highly responsive to microenvironmental cues, which change in the settings of injury and disease. Since inflammation and hypoxia are common to many pathological environments, we sought to better understand
Disclosures
HMW and GVN are founders of a start-up company Immplacate Inc that uses the technology described in this paper. RDS is a scientific advisor for Immplacate.
Author contributions
The raw data required to reproduce these findings will be made available to interested investigators.
Data availability
All experimental data necessary to reproduce the findings from this study will be made available to interested investigators.
Acknowledgments
The authors gratefully acknowledge the funding support of NIH (grants EB002520 and EB025765 to GVN), the New York State Stem Cell Science Board (NYSTEM, contract #C029159 to LMB) and the Columbia-Coulter Biomedical Accelerator Program (to HMW and GVN).
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2022, CytotherapyCitation Excerpt :Because immunomodulation is one of the main reasons MSCs are used as a potential therapy for pulmonary disorders, enrichment with proteins related to the immune system after hypoxia pre-conditioning might indicate a greater capacity to contain inflammation and adapt the immune response [35]. Other previous study analyzing the proteome of MSCs under hypoxic conditions (1% oxygen for 48 h) have also reported enrichment of pathways related to cell metabolism, matrix organization, etc. [36]. However, here the authors covered some less described pathways and biological processes related to the immune system and extracellular matrix organization in bone marrow-derived MSCs pre-challenged with hypoxia (1% oxygen for 48 h).