RegenLab
Cell Biology

Investigating MSC (mesenchymal stem cell) Potency Metrics by Integrating Imaging Modalities with Lipidomic Analysis

2024-08-17

Title:
Investigation of MSC Potency Metrics via Integration of Imaging Modalities with Lipidomic Characterization
Investigation of MSC potency metrics via integration of methods

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 and revealed that these features can distinguish functional MSC subpopulations induced by immune stimulation. Using DPC microscopy and MALDI-MSI techniques, the authors explored the association between morphological changes and the activation of specific lipid classes, indicating that this could contribute to optimizing the MSC manufacturing process.

Background:
Because MSCs (mesenchymal stem cells) have immunosuppressive effects, they are promising for regenerative medicine; however, their efficacy varies with different donors and cell proliferation stages. This study aimed to understand functional heterogeneity at the single-cell level and to improve clinical application by integratively evaluating the morphology and lipidomic profiles of MSCs.

Methods:
Using label-free differential phase contrast (DPC) microscopy and MALDI-MSI, the authors simultaneously acquired the morphology and lipidomic profiles of MSCs. They compared morphological and lipidomic changes between IFN-γ-stimulated MSCs and untreated controls.

Results:
In IFN-γ-stimulated MSCs, strong correlations were observed between specific morphological features (e.g., compactness, perimeter, major axis length) and lipids (particularly PC, LysoPC, TAG, etc.). These features can serve as metrics for identifying functional MSC subpopulations.

Discussion:
This study showed that MSC morphology and lipidomics can potentially be used to identify functional subpopulations. This is expected to improve the MSC manufacturing process in regenerative medicine.

Novelty Compared to Previous Studies:
This study performed integrated analysis of morphology and lipidomics at the single-cell level and demonstrated that this is a new approach for elucidating the functional heterogeneity of MSCs.

Limitations:
A limitation of this study is that the number of detectable lipids is limited because the signal of single-cell lipidomics is low. In addition, because large datasets are required, further research is needed.

Potential Applications:
This method can potentially be applied to identifying functional MSC subpopulations and optimizing the manufacturing process.

Supplementary Information:
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Q&A:

Q: How were single-cell lipids analyzed?
A: Single-cell lipid analysis was performed using the following methods.

  1. MALDI-MSI (Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging):
    Using MALDI-MSI technology, the lipid profiles of MSCs were obtained at the single-cell level. In this technique, MSCs were seeded onto indium tin oxide (ITO)-coated slides, and an IFN-γ-stimulated group was compared with an untreated control group. In MALDI-MSI, lipid peaks corresponding to specific m/z values were extracted from the spectral data and imaged.
  2. Co-registration:
    The morphological images obtained by DPC microscopy and the lipid images obtained by MALDI-MSI were integrated, and co-registration was performed to associate the morphology and lipid profile of each cell. In this process, morphological features were matched with lipid peaks.
  3. Statistical analysis:
    To identify differentially expressed lipids in the obtained lipid profiles, principal component analysis (PCA) and logistic regression analysis were performed. In addition, high-resolution Fourier-transform ion cyclotron resonance (FTICR) and ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) were also used for accurate mass identification of the spectra.

Through this approach, the changes in the lipid profile of MSCs induced by IFN-γ stimulation at the single-cell level were revealed.

Q: What is MALDI-MSI?
A: MALDI-MSI (Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging) is a technique that uses a mass spectrometer to visualize the spatial distribution within a biological sample. Specifically, it is performed in the following steps.

  1. Sample preparation:
    The sample (usually thinly sectioned tissue or cells) is placed on a special substrate (e.g., an indium tin oxide–coated slide). Next, a compound called a matrix is uniformly coated onto the sample surface. This matrix plays the role of making it easier to absorb the energy of the laser light.
  2. Laser irradiation:
    The matrix on the sample is irradiated with a laser, and that energy causes ions to be released from the sample surface. This process is called “desorption.”
  3. Ionization:
    The released molecules are ionized, becoming detectable by the mass spectrometer. This is called “ionization.”
  4. Mass analysis:
    The ionized molecules are guided into the mass spectrometer, separated based on the mass-to-charge ratio (m/z), and detected. This yields mass spectra of the various compounds in the sample.
  5. Imaging:
    In MALDI-MSI, mass spectra are acquired at each position on the sample, and based on that data the spatial distribution of compounds is imaged. This makes it possible to visually confirm how specific molecular species are distributed within the sample.

MALDI-MSI is widely used to analyze in detail the structure and chemical properties of biological tissues and cells, and is particularly suited to revealing the spatial distribution of molecules such as lipids and proteins.

Q: How many types of lipids were identified?
A: MALDI-MSI analysis identified a total of 54 specific lipid peaks. Among these, between IFN-γ-stimulated MSCs and untreated control cells, 20 lipids were significantly more abundant in the IFN-γ-stimulated cells, and 6 lipids were significantly more abundant in the control cells. The remaining 26 lipids showed no significant change between the two groups.

Q: Is 54 types a small number?
A: Fifty-four types of lipids are generally not few in single-cell-level MALDI-MSI analysis, but depending on technological advances and analysis conditions, it is also possible to identify even more lipids.

In single-cell analysis by MALDI-MSI, because the signal intensity of mass spectrometry is low, the number of detectable lipids can be limited. In single-cell analysis in particular, because the amount of material obtained from the sample is very small, highly sensitive measurement is needed to identify more lipids.

For this reason, the number of 54 types is considered a reasonable number for single-cell MALDI-MSI analysis, but with future technological progress and combination with other complementary analytical methods (e.g., ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS)), it may be possible to identify even more lipids.

Q: Why was IFN-γ used for stimulation?
A: The reason for stimulating with IFN-γ (interferon gamma) is to investigate the immune responsiveness of MSCs (mesenchymal stem/stromal cells). Specifically, there are the following reasons.

  1. Promotion of the immune response:
    IFN-γ is a cytokine that plays an important role in the immune system and has a strong stimulatory effect on many cells, including MSCs. By stimulating MSCs with IFN-γ, MSCs more readily exert immunosuppressive and immunomodulatory functions, and the cellular responses and changes at that time can be examined in detail.
  2. Elucidation of functional heterogeneity:
    MSCs can show different immune responses depending on the donor and culture conditions. By stimulating with IFN-γ, changes in the lipids and morphology related to the immunosuppressive function of MSCs can be observed, and the functional differences between different subpopulations can be revealed.
  3. Improvement of clinical application:
    “Priming” (pretreatment) of MSCs with IFN-γ is used as a strategy to enhance therapeutic efficacy in the clinic. Understanding the cellular response to stimulation can help with the quality control and optimization of the manufacturing process of MSCs.

In this way, stimulation with IFN-γ is an important step in research that evaluates the immune responsiveness of MSCs and enhances the efficacy of MSCs in regenerative medicine and immunotherapy.

Q: Specifically, what kinds of lipids were increased?
A: In IFN-γ-stimulated MSCs, the following lipids were confirmed to be increased.

  1. Ceramide-1-phosphate (C1P):
  1. Phosphatidylcholine (PC):
  1. Lysophosphatidylcholine (LysoPC):
  1. Triglyceride (TAG):

These lipids play roles related to immune function and cell membrane structure in IFN-γ-stimulated MSCs, and they have been confirmed to increase particularly when immune responsiveness is heightened. The increase in these lipids is thought to affect the immunosuppressive function and cell morphology of MSCs.

Q: What is the relationship between these lipids and the immune response?
A: The lipids increased in IFN-γ-stimulated MSCs include those that play important roles in the immune response. Below, the relevance of each lipid to the immune response is explained.

  1. Ceramide-1-phosphate (C1P):
  1. Phosphatidylcholine (PC):
  1. Lysophosphatidylcholine (LysoPC):
  1. Triglyceride (TAG):

Q: How do these lipids affect immune cells? Are the lipids taken up? Do they stimulate receptors?
A: These lipids affect immune cells through various mechanisms. Below, the specific mechanisms by which each lipid affects immune cells are explained.

  1. Lipid release and receptor stimulation:
    The lipids secreted by MSCs are released mainly via small extracellular vesicles (EVs) called exosomes and microvesicles. These vesicles contain lipids, proteins, RNA, and so on, and can interact with other cells.
  1. Direct uptake of lipids:
    Lipids released from MSCs are also sometimes taken up directly by immune cells. This uptake includes processes in which lipids are incorporated as part of the cell membrane or used as an energy source.
  1. Indirect changes in signal transduction:
    The effects of lipids can also be exerted indirectly through changes in signal transduction inside and outside immune cells.

Q: Is the understanding that the lipids are secreted from MSCs?
A: No, the preceding explanation was somewhat misleading. While there are cases in which lipids are secreted from MSCs as exosomes or vesicles, the lipids referred to in this study function mainly as MSCs’ own cell membrane or internal metabolites.

Accurate understanding:
The role of the lipids in this study is mainly related to the function and morphology of the MSCs themselves. Specifically, MSCs are thought to affect the immune response through the following processes.

  1. Changes in the cell membrane composition and function of MSCs:
  1. Adjustment of the substances secreted by MSCs:
  1. Direct interaction between MSCs and immune cells:

Summary:
The role of the lipids in this study is that MSCs use lipids in their own cell membrane and interior to induce changes related to immune function, and that this ultimately affects the immunomodulatory function that MSCs exhibit toward immune cells. Therefore, these lipids mainly affect the function of the MSCs themselves and play an important role when MSCs interact with immune cells.