Title:
Investigation of MSC Potency Metrics via Integration of Imaging Modalities with Lipidomic Characterization
Journal Name & Publication Year:
Cell Reports, 2024
First and Last Authors:
Priyanka Priyadarshani, Luke J. Mortensen
First Affiliation:
School of Chemical, Materials, and Biomedical Engineering, University of Georgia, Athens, GA, USA
Abstract:
This study analyzed the morphological and lipidomic profiles of MSCs at the single-cell level, revealing that these features can distinguish functional subpopulations of MSCs in response to immune stimulation. Using DPC microscopy and MALDI-MSI techniques, we explored the correlation between morphological changes and the activation of specific lipid classes, suggesting potential contributions to optimizing the MSC manufacturing process.
Background:
MSCs are promising for regenerative medicine due to their immunosuppressive effects, but their efficacy varies depending on donors and cell expansion stages. This study aimed to understand functional heterogeneity at the single-cell level by integratively evaluating MSC morphology and lipidomic profiles, with the goal of improving clinical applications.
Methods:
We simultaneously acquired MSC morphological and lipidomic profiles using label-free differential phase contrast (DPC) microscopy and MALDI-MSI. We compared morphological and lipidomic changes between MSCs stimulated with IFN-γ and untreated controls.
Results:
In MSCs stimulated with IFN-γ, a strong correlation was observed between specific morphological features (e.g., compactness, perimeter, major axis length) and lipids (particularly PC, LysoPC, TAG). These features could serve as indicators for identifying functional subpopulations of MSCs.
Discussion:
This study demonstrated the potential use of MSC morphology and lipidomics in identifying functional subpopulations, which could improve the MSC manufacturing process for regenerative medicine.
Novelty Compared to Previous Studies:
This study provides a novel approach by integrating morphological and lipidomic analyses at the single-cell level, showing that these combined metrics can elucidate the functional heterogeneity of MSCs.
Limitations:
The primary limitation of this study is the low signal intensity in single-cell lipidomics, which restricts the number of detectable lipids. Additionally, larger datasets are necessary for further research.
Potential Applications:
This approach could be applied to identify functional subpopulations of MSCs and optimize their manufacturing processes.
Supplementary Information:
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Q&A:
Q: How was the single-cell lipid analysis performed?
A: The single-cell lipid analysis was conducted using the following methods:
- MALDI-MSI (Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging):
The lipid profiles of MSCs at the single-cell level were obtained using MALDI-MSI. MSCs were seeded on indium tin oxide (ITO)-coated slides, and lipid profiles were compared between IFN-γ-stimulated and untreated control groups. Specific lipid peaks corresponding to certain m/z values were extracted from the spectral data and visualized. - Co-registration:
The morphological images obtained by DPC microscopy were integrated with the lipid images from MALDI-MSI to correlate cellular morphology with lipid profiles. This process matched morphological features with lipid peaks. - Statistical Analysis:
Principal component analysis (PCA) and logistic regression analysis were performed to identify differentially expressed lipids from the obtained lipid profiles. Additionally, high-resolution Fourier-transform ion cyclotron resonance (FTICR) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were used for precise mass identification of the spectra.
This approach revealed changes in the lipid profiles of MSCs in response to IFN-γ stimulation at the single-cell level.
Q: What is MALDI-MSI?
A: MALDI-MSI (Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging) is a technique that visualizes the spatial distribution of biomolecules within a sample using a mass spectrometer. The procedure involves the following steps:
- Sample Preparation:
The sample (usually thinly sliced tissue or cells) is placed on a special substrate (e.g., indium tin oxide-coated slides). A matrix compound is then uniformly coated on the sample surface, which facilitates the absorption of laser energy. - Laser Irradiation:
The matrix on the sample is irradiated with a laser, causing ions to be released from the sample surface, a process known as “desorption.” - Ionization:
The released molecules are ionized and made detectable by the mass spectrometer, a process referred to as “ionization.” - Mass Spectrometry:
The ionized molecules are directed into the mass spectrometer, where they are separated and detected based on their mass-to-charge ratio (m/z). This yields the mass spectra of various compounds in the sample. - Imaging:
MALDI-MSI captures mass spectra at each position on the sample and creates images based on the spatial distribution of specific molecular species. This enables visual confirmation of how particular molecules are distributed within the sample.
MALDI-MSI is widely used for detailed analysis of the structural and chemical properties of biological tissues and cells, particularly in revealing the spatial distribution of lipids and proteins.
Q: How many types of lipids were identified?
A: A total of 54 specific lipid peaks were identified through MALDI-MSI analysis. Among these, 20 lipids were significantly increased in IFN-γ-stimulated MSCs, and 6 lipids were significantly higher in untreated control cells. The remaining 26 lipids showed no significant changes between the two groups.
Q: Is 54 types of lipids considered a small number?
A: While 54 types of lipids are not generally considered few in single-cell MALDI-MSI analysis, it is possible to identify more lipids depending on technological advances and analysis conditions.
In single-cell analysis, the number of detectable lipids can be limited due to low signal intensity in mass spectrometry. Given the small amount of material available from single cells, higher sensitivity measurements are required to identify a larger number of lipids.
Therefore, 54 lipids is a reasonable number for single-cell MALDI-MSI analysis, though further technological advancements or complementary analytical methods (e.g., ultra-high-performance liquid chromatography-tandem mass spectrometry, UHPLC-MS/MS) could enable the identification of more lipids.
Q: Why was IFN-γ used for stimulation?
A: IFN-γ (Interferon-gamma) was used to stimulate MSCs to investigate their immune responsiveness. Specifically, there are the following reasons:
- Promotion of Immune Response:
IFN-γ is a cytokine that plays a crucial role in the immune system, with a strong stimulatory effect on many cells, including MSCs. By stimulating MSCs with IFN-γ, their immunosuppressive and immunomodulatory functions are more readily exhibited, allowing detailed study of cellular responses and changes. - Understanding Functional Heterogeneity:
MSCs can exhibit different immune responses depending on donors and culture conditions. IFN-γ stimulation allows observation of lipid and morphological changes related to the immunosuppressive function of MSCs, clarifying functional differences between different subpopulations. - Improving Clinical Applications:
Preconditioning MSCs with IFN-γ is a strategy used to enhance therapeutic efficacy in clinical settings. Understanding cellular responses to stimulation can aid in optimizing MSC quality control and manufacturing processes.
Thus, IFN-γ stimulation is an important step in evaluating MSC immune responsiveness and enhancing their effectiveness in regenerative medicine and immunotherapy.
Q: Which specific lipids were increased?
A: The following lipids were confirmed to be increased in MSCs stimulated with IFN-γ:
- Ceramide-1-phosphate (C1P):
- m/z 688.4: CerP 33:2;O6
- Phosphatidylcholine (PC):
- m/z 758.6 [M+H]+/780.5 [M+Na]+: PC (34:2)
- m/z 732.6: PC (32:1)
- m/z 804.6: PC (36:4)
- m/z 504.3: PC (16:0/18:1)
- Lysophosphatidylcholine (LysoPC):
- m/z 546.3: LysoPC(18:0)
- Triglyceride (TAG):
- m/z 789.6: TG 43:1;O2
These lipids play roles related to immune function and cell membrane structure in MSCs stimulated with IFN-γ, and they are particularly confirmed to increase during heightened immune responsiveness. The increase in these lipids is believed to influence the immunosuppressive function and cell morphology of MSCs.
Q: What is the relationship between these lipids and immune responses?
A: The lipids that increased in IFN-γ-stimulated MSCs play important roles in immune responses. The relationship between each lipid and immune response is explained below:
- Ceramide-1-phosphate (C1P):
- Role: C1P is involved in sphingolipid metabolism, has anti-apoptotic effects, and plays a crucial role in cell migration and inflammation regulation. It promotes immune cell activation and movement, and it is known to modulate inflammatory responses.
- Phosphatidylcholine (PC):
- Role: PC is a major component of cell membranes and is important for initiating immune responses via receptor-mediated pathways and lipid raft formation. Metabolites of PC are involved in immune cell signaling, differentiation, and proliferation.
- Lysophosphatidylcholine (LysoPC):
- Role: LysoPC is involved in membrane remodeling and cellular signaling, affecting inflammation and immune cell functions. LysoPC is known to regulate gene expression, promoting cell proliferation and differentiation, and it can induce the production of pro-inflammatory cytokines.
- Triglyceride (TAG):
- Role: TAG serves as an important energy source, and its metabolites, such as fatty acids and glycerol, are necessary for immune cell differentiation and function. TAG metabolites are involved in the synthesis of membrane phospholipids and play a role in immune cell signaling pathways.
Q: How do these lipids affect immune cells? Are they taken up by immune cells or do they stimulate receptors?
A: These lipids influence immune cells through various mechanisms. The specific mechanisms by which each lipid affects immune cells are explained below:
- Lipid Release and Receptor Stimulation:
Lipids secreted by MSCs are primarily released through small extracellular vesicles such as exosomes and microvesicles, which contain lipids, proteins, and RNA, and can interact with other cells.
- Exosome Release: MSCs release exosomes that contain lipids. When exosomes are taken up by immune cells, the lipids and other components within them can directly affect the recipient cells.
- Receptor Stimulation: Specific lipids (e.g., LysoPC and PC) bind to receptors on the surface of immune cells, initiating signaling cascades. For example, LysoPC signals through G-protein coupled receptors (GPCRs)
to transmit signals to immune cells, inducing chemotaxis or cytokine secretion. Additionally, PC promotes the clustering and activation of receptors by facilitating lipid raft formation.
- Direct Uptake of Lipids:
Lipids released from MSCs can also be directly taken up by immune cells, where they are integrated into the cell membrane or utilized as an energy source.
- Endocytosis: Immune cells take up lipids released from MSCs through endocytosis. The internalized lipids are used in membrane reconstruction or energy metabolism, contributing to immune cell activation and functional changes.
- Membrane Integration and Signaling: Once integrated into the immune cell membrane, lipids affect the physical properties and signaling pathways of the membrane, altering the sensitivity and responsiveness of the immune cells and inducing specific immune responses.
- Indirect Changes in Signaling:
The impact of lipids can also be exerted indirectly through changes in intracellular and extracellular signaling in immune cells.
- Generation of Signaling Molecules: Metabolic products of lipids, such as diacylglycerol and prostaglandins, regulate immune cell signaling and alter cell functions.
- Induction of Pro-inflammatory Cytokines: Some lipids activate intracellular signaling cascades within immune cells, promoting the production of pro-inflammatory cytokines, which in turn activate or recruit other immune cells, enhancing the overall immune response.
Q: Are these lipids secreted from MSCs?
A: Not exactly. The previous explanation might have been slightly misleading. While lipids can be secreted from MSCs as exosomes or vesicles, the lipids mentioned in this study primarily function as components of the MSCs’ own cell membranes or internal metabolites.
Correct Understanding:
The role of lipids in this study is mainly related to the functions and morphology of MSCs themselves. Specifically, MSCs are thought to influence immune responses through the following processes:
- Changes in MSC Membrane Composition and Function:
- Lipid Raft Formation: Lipids such as PC and LysoPC present in the MSC membrane form regions known as lipid rafts, which promote the organization and clustering of immune receptors. This clustering facilitates immune cell activation or suppression when MSCs come into contact with immune cells.
- Changes in Membrane Fluidity and Structure: Changes in the lipid composition of the MSC membrane alter the physical properties (e.g., fluidity, flexibility) of the cell membrane, influencing the immunomodulatory functions of MSCs.
- Regulation of MSC Secretions:
- Secretion of Cytokines and Growth Factors: Lipid metabolism within MSCs influences the types and amounts of cytokines and growth factors they secrete. These secretions directly or indirectly affect immune cells, modulating immune responses.
- Direct Interactions Between MSCs and Immune Cells:
- Cell-Contact Mediated Signaling: When MSCs come into direct contact with immune cells, the lipids in the MSC membrane play a role in modulating signaling to immune cells. This interaction can trigger activation, differentiation, or suppression of immune cells.
Summary:
The role of lipids in this study primarily involves MSCs utilizing their own membrane lipids or internal metabolites to induce immune-related changes. This ultimately affects the immunomodulatory functions of MSCs when they interact with immune cells. Therefore, these lipids are mainly involved in the functions of MSCs themselves, playing a critical role in MSC-immune cell interactions.
