Lecture 14: Mesenchymal Stem Cells - Clinical Trials and Controversies PDF

🥽 Lecture 14: Mesenchymal stem cells - Clinical trials and controversies List the minimal criteria for defining mesenchymal stromal cells. MSCs form part of the HSC niche in bone marrow The majority of thes...

🥽 Lecture 14: Mesenchymal stem cells - Clinical trials and controversies List the minimal criteria for defining mesenchymal stromal cells. MSCs form part of the HSC niche in bone marrow The majority of these cells are located in a perivascular region in the bone marrow, but they can also be found in smaller numbers in other parts of the bone marrow. Human MSCs: Express markers such as STRO1, CID146, VECAM1, CID271, and CID105. Male MSCs: Express additional markers such as SCAR1 and PDGF receptor. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 1  BM MSCs give rise to cells forming the skeleton MSCs can give rise to: osteoblasts, chondroblasts, adipoblasts, other stromal cells, such as fibroblasts. Bulk stroma vs bona fide (genuine/true) stem cell Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 2  Bona Fide Skeletal Stem Cells: Using the markers previously described, MSCs can be isolated from the bone marrow. They are plated at low density to select for clonogenic cells or single-sorted cells, which are expanded into colonies. These single-cell derived clones can be transplanted into a mouse to form an ossicle (heterotopic bone). The formation of an ossicle demonstrates self-renewal and multipotency, qualifying these cells as skeletal stem cells. Stroma Cells Used in Cell Therapy: MSCs are plated at high density and selected for plastic adherence. These preparations may contain a small number of skeletal stem cells. However, the bulk of the preparation is not stem cells and is more accurately defined as mesenchymal stromal cells. Key Concept: Skeletal stem cells demonstrate self-renewal and multi-potency (e.g., ossicle formation). Mesenchymal stromal cells used in cell therapy lack these defining properties and are not true stem cells. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 3  ISCT definition of MSCs: 1. Plastic Adherence: According to the ISCT definition, MSCs are plastic adherent cells that can proliferate and be passaged in culture up to a certain extent. 2. Phenotype (Markers): These cells express CD73, CD90, and CD105. They lack the expression of hematopoietic markers such as CD11B, CD45, CD19, and HLA-DR. 3. Multipotency: In vitro, when cultured in permissive conditions, these cells can give rise to: Adipocytes, Osteoblasts, Chondrocytes. Because of their supposed multi-lineage differentiation capacity, early studies focused on their potential to differentiate into osteogenic cells that produce bone tissue. This was explored as a treatment for bone fractures or bone diseases. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 4  Understand why this minimal set of criteria is inadequate. Minimal and inadequate criteria The International Society for Cell Therapy (ISCT) has set minimal criteria to define MSCs. 1. Cells must adhere to plastic in standard culture conditions. Limitation: This criterion is inadequate because non-clonal cultures from any connective tissues that adhere to plastic would also meet this definition. 2. MSCs must be positive for CD105, CD73, and CD90. MSCs must be negative for hematopoietic markers. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 5  Limitations: These markers are expressed on many cell types and do not uniquely identify MSCs. 3. MSCs must differentiate into osteoblasts, adipocytes, and chondroblasts in vitro. Limitations: In vitro differentiation is prone to culture artifacts. Chemically directed differentiation does not confirm that the cells are inherently multipotent. MSCs - fact vs fictions: (1) MSCs are not strictly stem cells There is a distinction between bona fide skeletal stem cells and the stromal cells used for cell therapy. Bona fide skeletal stem cells: Can be isolated using MSC markers. Plated at low density or as single cells on plastic dishes to expand clonogenic cells. Demonstrated to form ossicles in animal models, proving their self-renewal and multi-potency. Stromal cells used for cell therapy: Bulk cultured cells, taken from bone marrow and plated at high density on plastic dishes. Expanded adherent cells are infused into patients. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 6  While the preparation might contain small numbers of bona fide stem cells, the majority are not true stem cells. (2) MSCs are not immune privileged Early Understanding: Early studies suggested that MSCs do not express MHC class II or co-stimulatory molecules (e.g., CD80, CD86). This implied that MSCs should not elicit a strong immune response. Recent Findings: MSCs are immunogenic and can elicit both cellular and humoral allogeneic responses. Studies have shown: Generation of antibodies against MSCs. Immune rejection of allogeneic donor MSCs. Implications: MSCs were long thought to be hypoimmunogenic or immune privileged, enabling transplantation across MHC barriers and the creation of off-the-shelf therapies. Recent evidence suggests that MSCs may not be immune privileged. Unresolved Questions: It is unclear whether the rejection of donor MSCs affects the clinical efficacy of allogeneic MSC therapies. The clinical advantage (if any) of using autologous MSCs versus allogeneic MSCs remains unknown. (3) MSCs do not engraft Early Studies: Focused on the potential of MSCs to differentiate into bone or cartilage. Assumed that MSCs would need to engraft into the tissue to form new bone or cartilage. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 7  Findings: Several studies have shown that MSCs encounter a pulmonary impasse, meaning: They get trapped in the lungs. They are short-lived. Autopsy tissues from patients who had undergone MSC treatment in clinical trials indicated: No long-term engraftment. No ectopic tissue formation. Conclusion: MSC function appears to be mediated through a hit-and-run mechanism rather than sustained engraftment in injured tissues. Stemness does not explain how MSCs repair tissues, as there is little evidence that these cells engraft or differentiate at the site of injury. Describe some of the immunomodulatory effects of MSCs. Immunomodulatory effects of MSCs MSCs possess potent immunomodulatory properties, which can be utilized clinically to treat inflammatory conditions. MSCs can modulate the function of multiple immune cell types. Bi-Directional Effects: Example: MSCs inhibit the proliferation and cytokine production of T cells. T cell-derived cytokines, in turn, can enhance the immunosuppressive capacity of MSCs. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 8  Response to Inflammatory Signals: MSCs respond to inflammatory signals, such as: Interferon-gamma (IFN-γ) Tumor Necrosis Factor (TNF) These signals are produced by immune cells, including T cells, during an immune response. MSC Licensing or Activation: This process leads to: Production of inhibitory factors and growth factors. Altered expression of surface molecules. These changes contribute to the modulatory effects of MSCs. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 9  Know why MSCs are being investigated as cell therapy in multiple disease conditions + Describe the major controversies surrounding the use of MSCs. Earlier studies showed that MSCs do not elicit a strong immune response & have anti-inflammatory effect → this has led to a significant increase in clinical trials utilizing MSCs to treat a range of diseases and injuries. Trials included both: Patient-derived (autologous) MSCs & Donor- derived (allogeneic) MSCs. Regulatory Approvals: MSCs are referred to as "living medicine" due to their therapeutic potential. They have received conditional regulatory approval in some countries for the treatment of: Acute graft-versus-host disease. Perianal fistula. The field is awaiting FDA approval in the US for the use of MSCs to treat pediatric steroid-resistant acute graft-versus-host disease. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 10  Paradigm vs Paradox of MSCs Current paradigm: MSCs secrete a multitude of soluble factors that act in a hit-and-run manner. Paradox: 1. Short-lived MSCs: MSCs are cleared from the body relatively quickly after infusion and do not survive long-term. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 11  They also get trapped in the lungs, limiting their ability to reach other tissues directly. 2. Hit-and-Run Mechanism: Despite their short lifespan and limited migration, MSCs appear to have measurable immunosuppressive and anti- inflammatory effects. 3. Challenge: For these effects to occur, their secreted factors would need to: Have a long-lasting presence (long half-life). Work effectively over long distances. Current knowledge does not fully explain how these processes are achieved, hence the paradox. Takeaway: MSCs may not work by directly replacing damaged tissues or maintaining long-term presence but rather by temporarily secreting bioactive molecules that modulate immune and inflammatory responses. The paradox lies in reconciling their short lifespan with the reported therapeutic benefits. Testing MSCs in lung inflammation (allergic asthma) Why Allergic Asthma? When MSCs are injected, most cells go directly to the lungs, where the inflammatory action occurs. Allergic asthma serves as a relevant model for studying MSC effects in the lungs. Mechanism of Allergic Asthma: The allergen is taken up by an antigen-presenting cell (e.g., dendritic cell). The antigen is presented to T cells, which differentiate into TH2 cells. TH2 cells produce TH2 cytokines, including: Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 12  IL-5: Recruits eosinophils to the lungs. IL-4: Essential for B-cell switching and IgE production. IL-13: Causes airway inflammation and goblet cell mucus production. Overall outcome: Symptoms typical of an asthmatic attack. Experiment investigating MSCs immunosuppressive effect SETTING UP: MSC Characterization: MSCs were isolated from bone marrow, umbilical cord, and adipose tissue. Characterized based on ISCT minimal criteria: Adhere to plastic and can be passaged in vitro. Form fibroblastic colonies. Express CD73, CD90, CD105, but lack hematopoietic markers. Capable of in vitro differentiation into adipocytes, osteoblasts, and chondrocytes. Testing MSCs in Asthma Model: Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 13  Used ovalbumin (OVA) as a model allergen. Experimental setup: Mice were sensitized to OVA. Upon intranasal or airway challenge with OVA, mice developed hallmark indicators of asthma. RESULTS: MSC Treatment Reduce Asthma Indicators: MSC treatment in the mouse model of allergic asthma showed a reduction in key indicators and mediators of the disease. IL-5: Critical for eosinophil recruitment. Reduced levels in MSC-treated mice. IL-13: Important for airway hyperreactivity and airway function. Reduced levels in MSC-treated mice. Eosinophils: A critical cellular mediator of asthma. Reduced numbers in MSC-treated mice. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 14  RESULTS: MSC Treatment Improve Lung Inflammation Observations: Normal Lung (Leftmost Panel): No pink goblet cells. Very few purple inflammatory cells. Asthmatic Mouse Lung (Second Panel): Increase in pink goblet cells and purple inflammatory cells. MSC-Treated Asthmatic Mice (Third and Fourth Panels): Reduction in pink goblet cells and purple inflammatory cells. In some regions, pink goblet cells are absent. Conclusion: MSC treatment inhibits lung inflammation in asthmatic mice. RESULTS: MSC Treatment inhibit airway hyperreactivity In asthmatic mice, more mucus leads to increased resistance (Rl) in response to methacholine + reduced elasticity (dynamic compliance - Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 15  Cdyn) MSC-Treated Mice Treated with MSCs derived from bone marrow, umbilical cord, or adipose tissue. All three treatment groups showed a reduction in airway hyperreactivity, with lung resistance and compliance levels improving almost to those of normal mice Tracking the Fate of Injected MSCs: Method: Used a gene from the firefly encoding luciferase, which glows in the presence of its substrate, luciferin. MSCs with the luciferase gene inserted emit a glow in the presence of luciferin. This enabled imaging experiments to: 1. Determine where the cells localize after injection. 2. Track how long the cells persist in the body. Findings: 1. Localization: Injected luciferase-expressing MSCs immediately localized to the lungs. 2. Survival: Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 16  The MSCs remained in the lungs briefly but the signal disappeared completely within 2–3 days, indicating the cells did not survive long in the lungs. Conclusion: Post-MSC injection: MSCs localize to the lungs. MSCs do not survive long in the lungs. Following MSC administration - The ability to induce MSC apoptosis appears to predict a patient's response to MSC treatment. Apoptosis Following Intravenous Administration: Apoptosis: An immunologically silent form of cell death. Involves proteolysis by caspases activated via: Intrinsic pathway. Extrinsic pathway. Evidence from Study (Science Translational Medicine, 2017): MSCs administered into a mouse model of graft-versus-host disease (GVHD) showed increased caspase activity, indicating apoptosis of MSCs post-administration. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 17  Relevance to GVHD Patients: MSC treatment effectiveness in GVHD patients varies: Responders: Patients who responded well to treatment had immune cells that induced apoptosis in MSCs. Non-responders: Patients who did not respond well had immune cells that failed to induce apoptosis in MSCs. Engulfment of Apoptotic MSCs by Phagocytes ⇒ Phagocytes express immunosuppressive molecules Labeling and Tracking Apoptotic MSCs: Apoptotic MSCs were labeled with cell trace violet (a fluorescent dye). After injection into mice, cell trace violet was detected in various populations of lung phagocytes, indicating the engulfment of apoptotic MSCs. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 18  Human Monocyte Co-Culture Experiment: Live Cell Imaging: Co-culture of human monocytes (phagocytic cells) with apoptotic MSCs in a dish. Over time, monocytes engulfed apoptotic MSCs. Immunosuppressive Changes in Phagocytes: Phagocytes that engulfed apoptotic MSCs expressed immunosuppressive molecules: IDO (Indoleamine 2,3-dioxygenase). TGF-beta. PGE2 (Prostaglandin E2). IL-10. They also down-regulated pro-inflammatory molecules: TNF-alpha. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 19  Key Insight: MSCs apoptose and die after infusion. The phagocytes that engulf apoptotic MSCs up-regulate immunosuppressive molecules and down-regulate pro- inflammatory molecules. This switch in the phenotype and function of macrophages and monocytes may drive the immunosuppressive effects of MSC treatment. Depletion of macrophages abrogates the immunosuppressive effects of MSCs. Experimental Setup: Mice in the allergic asthma model were used. Macrophages were depleted prior to MSC treatment using clodronate, which kills macrophages. Macrophage depletion was performed a few days before MSC treatment. Findings: 1. Lung Function Test: MSC treatment (green) improved lung function as expected. Mice depleted of macrophages prior to MSC treatment no longer responded to MSC treatment. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 20  2. Histology of Lung Sections: First Panel: Unsensitized mouse (normal lung). Second Panel: Asthmatic mouse (inflammation present). Third Panel: Asthmatic mouse treated with MSCs (reduced inflammation). Fourth Panel: Asthmatic mouse with macrophage depletion prior to MSC treatment (no reduction in inflammation). Describe some of the proposed mechanisms underlying the immunomodulatory effects of live versus apoptotic MSCs. Understanding the Mechanisms of Live MSCs: 1. Activation and Surface Molecules: MSCs are activated by inflammatory cytokines like interferon- gamma and TNF. MSCs express surface molecules involved in their immunomodulatory effects. 2. Production of Immunomodulatory Molecules: MSCs produce a wide array of anti-inflammatory or immunosuppressive molecules. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 21  3. Extracellular Vesicles: MSCs produce extracellular vesicles, which have been shown to play a role in mediating their immunosuppressive effects. 4. Contributions to Therapeutic Effects: The contribution of these mechanisms to the therapeutic effects of MSCs in vivo is still debated. A large proportion of MSCs undergo apoptosis upon intravenous administration. These apoptotic MSCs are engulfed by phagocytes, which then produce anti-inflammatory molecules. 5. Combined Effects of Live and Apoptotic MSCs: Both live and apoptotic MSCs contribute to the downstream effects of modulating immune cell function. Identify the differences between mesenchymal stem versus stromal cells 1. Differences Between Skeletal Stem Cells and Mesenchymal Stem Cells (MSCs): Skeletal Stem Cells: Progenitors of skeletal tissues. Located in the bone marrow. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 22  Assayed as single cells in vivo. Tested for tissue regeneration through grafted cells. Upon transplantation, they engraft, survive for decades, and are considered true stem cells. Effective in vivo as living cells. Mesenchymal Stem Cells (MSCs): Progenitors of both skeletal and nonskeletal tissues. Isolated from various tissues, not just bone marrow. Assayed as non-clonal cultures in vitro. Defined by chemically induced properties. Used as a cell therapy product for paracrine modulation of host cells. Administered via infusion, and embolized in the lungs, leading to short survival times. Defined as "drug stores" and effective in vivo as dying cells. Unlike skeletal stem cells, MSCs do not function effectively as living cells in tissue regeneration. 2. Minimal Criteria for Defining MSCs: The minimal criteria for MSCs include adherence to plastic, specific surface markers (CD73, CD90, CD105), and the ability to differentiate into certain cell types. The inadequacy of these criteria was discussed in relation to MSCs being defined by their chemically induced properties rather than their true stem cell characteristics. 3. MSCs in Cell Therapy: MSCs are being investigated for cell therapy in various disease conditions. Their immunomodulatory effects are key to their potential therapeutic applications. However, controversies surround their effectiveness and mechanisms of action. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 23  4. Immunomodulatory Effects: MSCs have immunosuppressive or anti-inflammatory effects through interactions with immune cells. Their mechanisms of action involve live MSCs and apoptotic MSCs, which are engulfed by phagocytes that produce anti-inflammatory molecules. 5. Skeletal Stem Cells vs. Mesenchymal Stromal Cells: Skeletal stem cells are effective in vivo as living cells, whereas MSCs are primarily effective as dying cells following infusion. MSCs' embolic fate in the lungs and limited survival highlight differences in their therapeutic effectiveness compared to skeletal stem cells. Takeaways: Understanding the differences between skeletal stem cells and MSCs, the criteria for defining MSCs, their controversial mechanisms, and their immunomodulatory effects is crucial in appreciating their role and limitations in therapy. Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 24 Lecture 14: Mesenchymal stem cells - Clinical trials and controversies 25