Functional heterogeneity is a significant barrier to the clinical translation of many cellular therapies, including mesenchymal stromal cells (MSCs). Though MSCs have shown promise in treatment of immune diseases, the mechanisms of action and critical quality attributes (CQAs, predictors of function) in different therapeutic settings are largely unknown. The overall goal of the Marklein Lab is to develop innovative approaches incorporating high-throughput, therapeutically relevant single cell profiling to assess cellular heterogeneity and accelerate translation of MSC therapies. To accomplish this goal, the Marklein Lab is focused on the following projects:
- Harness high-throughput morphological screening technologies to optimize MSC manufacturing. Significant heterogeneity exists among research labs in terms of manufacturing processes, which arise from differences in donor, tissue source, culture media, cryopreservation techniques, and culture vessels. As the CQAs for MSCs are largely unknown, it is difficult to assess the effects of these manufacturing changes on MSC function in a rapid, high throughput manner. This project will combine high content imaging with screening libraries of microenvironmental cues to create ‘morphological landscapes’ that reveal manufacturing conditions that enhance MSC function (e.g. differentiation or immunomodulation), limit functional heterogeneity, and ensure safety.
- Engineering multipotent MSC-derived extracellular vesicle therapies. MSC-EVs have been shown to modulate immune cells associated with diseases such as osteoarthritis, diabetes, Parkinson’s Disease, and Alzheimer’s Disease. However, due to MSC heterogeneity and unknown CQAs it is challenging to manufacture MSC-EVs at a large scale and predict their therapeutic potential. Priming (i.e. preconditioning) MSCs with disease-relevant stimuli such as inflammatory cytokines or hypoxia has been shown to enhance MSC function and reduce heterogeneity, but there are no standardized methods for priming MSCs or predicting the effects of different priming methods on MSC-EV function. In this project, we will perform a morphological screen of MSCs to an array of inflammatory signals to identify morphological ‘hits’ that will be subsequently explored based on their effect on MSC-EV composition (protein, lipids, nucleic acids) and modulation of immune cells involved with neuroinflammation: microglia, macrophages, and T cells. Through this work, we will identify priming conditions that result in differential uptake of EVs by cell-types, as well as devise combination MSC-EV therapies to achieve a ‘multipotent’ therapeutic effect. Finally, we will explore differences in manufacturing primed MSC-EVs in 2D versus 3D bioreactor contexts and seek to optimize manufacturing of high quality MSC-EVs using larger scale culture formats.
- Determination of MSC mechanism of action through comprehensive single cell profiling. MSCs can modulate immune cells such as T cells, macrophages, NK cells, B cells, and microglia, but there are no rapid, standardized approaches to quantify their disease-relevant mechanisms of action. Furthermore, MSCs derived from different sources and manufactured using different processes can possess markedly different immunomodulatory function. This project aims to better characterize the immunomodulatory properties of MSCs and MSC-EVs derived from different donors, tissues, and manufacturing conditions using advanced single cell profiling techniques. Using high dimensional flow/mass cytometry, we will explore MSC modulation of immune cell subsets within a PBMC-MSC co-culture to identify specific subpopulations that are modulated by direct contact and indirect contact (secreted factors, EVs). Using a different approach, high content imaging of microglia/macrophages will be used to better understand their heterogeneity (beyond the traditional M1-M2 binary activation model) based on morphological responses to inflammatory cues and treatment with MSC therapies. Together, this project will resolve the functional data with comprehensive secretomic, transcriptomic, and surface marker characterization of MSCs/MSC-EVs to elucidate mechanism of action and help inform manufacturing.