Transcriptomic profiling of human cells destined for therapeutic applications
Medrano Trochez, Camila
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Cell therapy is a growing field as many diseases are still untreatable. Potential applications of cell therapies include treating cancers, autoimmune disease, urinary problems, and infectious disease, rebuilding damaged cartilage in joints, repairing spinal cord injuries, improving a weakened immune system, and helping patients with neurological disorders. Numerous clinical trials have recently focused on the use of mesenchymal stromal cells (MSCs) as a cell therapy for various diseases with unmet medical challenges, including graft-vs-host disease, osteoarthritis, autism, and auto-immune diseases. MSCs are multipotent cells that have both regenerative and immunomodulatory capacity, and which are being developed for therapeutic intervention across a variety of inflammatory and immune conditions. MSCs can be isolated from various tissues, such as bone marrow, umbilical cord, and bone marrow aspirate concentrate (BMAC), which introduces tissue dependent variability between MSC-based cell products that also differ according to donor. Furthermore, manufacturing processes vary between sites (both clinical and commercial), leading to process-dependent variability. These sources of variability across the MSC field compound the ability to compare clinical trial results and have contributed to a lack of conclusive historical data to support the potential for clinical efficacy. Overall, there is an obvious need for deep phenotypic characterization of MSCs to compare heterogeneity as a function of tissue-of-origin as well as donor, and to identify potential phenotypic signatures that can be used for biomarkers or quality attributes for MSC-based cell products. My research focused on the study of MSCs as well as BMACs, which contain small amounts of MSC, but are still used in cell therapy owing to their MSC-like properties. This study aims to quantify the impact that diverse cell preparation methods may have on patient outcomes, the transcriptional variability due to the tissue of origin of the cells used, and the mechanism by which these cells modulate the immune system. I also evaluate the gene expression profiles of BMAC cell types from osteoarthritic (OA) patients and non-OA donors. 1. Characterization of the gene expression profiles of bone marrow and umbilical cord tissue-derived MSC. I assess the variability due to tissue of origin, batch effects, and donor effects. Samples derived from the same tissue separate in two major groups corresponding to high quality cells and low-quality cells. The high-quality cells further sub-divide in two subgroups. Cells from each donor belong to one or the other of these subgroups. One of the subgroups contains active cells expressing immunosuppressive genes, while the other subgroup cells overexpress cell cycle genes, suggesting these cells may be more actively undergoing mitosis. The samples from the two different tissues also express qualitatively different gene expression profiles. 2. MSC immunosuppressive capacities. I describe how MSC immunosuppressive capacities are enhanced at the transcriptional level by proinflammatory cytokines. I characterize the gene expression profiles of bone marrow derived MSC exposed to TNF⍺ and IFN𝛾, showing that the MSC activated with IFN𝛾 express higher levels of immunosuppressive genes. Additionally, single cell RNAseq analysis performed on T cells exposed to IFN𝛾-enhanced MSC reveals enrichment in gene activity related to T cell suppression and cell stress related pathways. 3. Characterization of BMAC samples form osteoarthritic patients and non- osteoarthritic donors. Osteoarthritis is a common degenerative disease, with no lasting cure. Even though it is considered a non-inflammatory disease, the immune system plays an important role in OA progression. The constant activation of pro-inflammatory cytokines due to tissue damage provokes deterioration of the cartilage. Cell therapy has been proposed as a treatment for this disease. MSC from different sources are being used to treat OA, such as commercial MSC, bone marrow aspirates concentrate (BMAC) or autologous MSC. This chapter describes my characterization of the transcriptomic profiles of BMAC from OA patients and healthy donors. The results suggest overexpression of immune related pathways in BMAC samples from OA patients compared to samples from non-OA donors. Since pro-inflammatory cytokines play a role in progression of the disease, this study suggests a possible benefit to the use of allogeneic BMAC from non-OA donors over samples from OA patients.