Stem Cell Therapy Lecture 9 PDF

ADVANCED THERAPEUTIC TECHNOLOGIES LECTURE 9 03.12.24 STEM CELL THERAPY Stem Cells (SCs) are a population of undifferentiated cells characterized by the ability of extensive proliferation and a high degree...

ADVANCED THERAPEUTIC TECHNOLOGIES LECTURE 9 03.12.24 STEM CELL THERAPY Stem Cells (SCs) are a population of undifferentiated cells characterized by the ability of extensive proliferation and a high degree of differentiation potential. Differentiation is the process of maturation to its final state as one of the cells of the body. The body is made up of about 200 different kinds of specialized cells (such as muscle cells, nerve cells, fat cells, and skin cells). All cells in the body come from stem cells. Once the differentiation pathway of a stem cell has been decided (the stem cells are said to be committed), it can no longer become another type of cell on its own. However, this concept was challenged by “pushing” the cells to revert the differentiation process with the use of transcription factors or chemical compounds, as in the case of Induced Pluripotent Stem Cells. Stem cells can be classified by their potential into: o Totipotent stem cells derive from the zygote (2-, 4-, or 8-cell stages), and they may give rise to both all three germ layers formed during embryogenesis (ecto-, endo-, and mesoderm) and extraembryonic tissue, such as the placenta. The fertilized egg and the cells that immediately arise in the first few divisions after fertilization are “totipotent.” This means that, under the right conditions, they can generate a viable embryo. However, we are not able to keep them as such because, within a matter of days, totipotent cells change, they become pluripotent. None of the currently studied embryonic stem cell lines are alone capable of generating a viable embryo, they are, in fact, pluripotent, not totipotent. o Pluripotent stem cells are able to differentiate in all of the cells of the body apart from the extraembryonic tissues. They are the main focus of therapies. Within a matter of weeks, pluripotent cells become multipotent. o Multipotent stem cells, also known as adult or tissue stem cells, only give rise to a limited range of cells within a tissue type. They reside in tissues of the body where they are involved in repair and replacement. They are widely used in research and already used to treat patients, but they are, in general, very difficult to isolate. Tissue-specific stem cells have been found in several organs that need to continuously replenish themselves, such as the blood, skin, adipose tissue, and gut (in the crypts), and have even been found in other, less regenerative organs such as the brain, heart, kidneys, and muscles (satellite cells). These types of stem cells represent a very small population and are often buried deep within a given tissue, making them difficult to identify, isolate, and grow in a laboratory setting. In muscles, in addition to satellite cells, several cell types contribute to muscle growth, homeostasis, and regeneration, including pericytes, mesenchymal stromal cells (e.g., Pw1+ Interstitial Cells, Fibro Adipogenic Progenitors, Twist2+ cells), immune cells as well as connective tissue cells. 1 Stem cells are unique cells that are defined by two properties: - Self-renewal – they can divide to make more of themselves - differentiation – they can generate specialized cell types such as skin, muscle, or blood cells Adult stem cells normally remain quiescent (non- dividing) for relatively long periods until they are activated in response to the physiological turnover and tissue damage by signals to maintain tissues. When activated, they divide through symmetric or asymmetric cell division. Through this process, they are able to maintain a pool of stem cells and differentiate into the desired cell types by the formation of a progenitor cell. Progenitor cells, in contrast to stem cells, are already far more specific, they are committed to differentiate into their "target" cell. The ability to self-renew is given by the constitutive expression of telomerase. This enzyme was discovered by two women who got the Nobel Prize for it, and it has the ability to elongate and prolong cell divisions. It binds to the 3’ end of the telomers sequence, along with an RNA template, and catalyzes the addition of bases, restoring the length of the telomere. DNA polymerase extends and seals the DNA strands. If the stem cells do not have the necessary levels of telomerase, self-renewal is disrupted. Stem cells reside in a niche, defined as dynamic and specialized microenvironment that regulates stem cell pool maintenance and tissue repair, by providing support and signals regulating self-renewal and differentiation. As regards the niche signalling, this one consists of 3 main types of molecular signals: integral membrane proteins such as integrins, localized secreted ECM components and soluble proteins surface such as growth factors and cytokines The best characterized niche is the hematopoietic one (the one in the image), located in the bone marrow and characterized by the expression of β-catenin, VCAM/ICAM, stromal cell-derived factor 1 (SDF-1), Angiopoietin- 1 (Ang-1), Adipocyte-derived stem cell factor (SCF), Thrombopoietin (THPO) and Osteopontin (OPN); however several organs are provided with stem cell niches such as gut, testis and skin. Due to their role in the modulation of stemness and in order to limit the changes that stem cells undergo following their isolation from the body, hence allowing their characterization, a lot of research has been performed to reproduce stem cell niches (mimicking the natural microenvironment in which stem cells grow) in vitro. As a result of this necessity, niche bioengineering was born; it consists of a specialized field within bioengineering that focuses on designing and manipulating the microenvironment or "niche" composed of physical, chemical, and biological factors, such as extracellular matrices, mechanical forces, oxygen levels, and signaling 2 molecules) in order to make it the more similar to the natural one; this field is thus fundamental for the development of strategies, devices and novel techniques that are able to mimic local environments for the controlled, ex vivo culture of pluripotent or multipotent cells. PLURIPOTENT STEM CELLS Pluripotent stem cells are stem cells that can give rise to all the embryonic tissue, but not to the placenta indeed none of the currently studied embryonic stem cell lines are alone capable of generating a viable embryo (since they are not totipotent); there are 2 types of pluripotent stem cells: - embryonic stem cells (ESC) → only exist at the earliest stages of embryonic development (early-stage of morula-16 cell stage, or from blastocyst-64 cell stage), indeed they are isolated from the inner cell mass of blastocysts; once isolated, these cells can be grown on a fibroblast feeder layer forming embryonic stem cell cultures. - induced pluripotent stem cells (iPSC) → have been made in the lab by converting tissue-specific cells, such as skin or adipose cells, into cells with the same properties as embryonic stem cells. The first attempt to produce pluripotent stem cells was based on somatic cell nuclear transfer, performed in 1962 by Gurdon that demonstrated that the nucleus from a differentiated tadpole intestinal epithelial cell was capable of generating a fully functional tadpole upon transplantation to an enucleated egg. This represented a paradigm shift in the understanding of cellular differentiation and of the plasticity of the differentiated state since, during the first half of the 20th century, was thought that the mature cells were permanently locked into the differentiated state, and unable to return to a fully immature, pluripotent stem cell state, while Gourdon’s experiments proved that it was actually possible to go back to the stem cell state. Gurdon’s discovery introduced a new research field centered on somatic cell nuclear transfer (SCNT) as a method to understand reprogramming and how cells change as they become specialized. In 1997, the first cloned mammal, the sheep Dolly, was born after SCNT from an adult mammary epithelial cell into an enucleated sheep egg (Wilmut et al., Nature 1997). The experimental strategy by Ian Wilmut and Keith Campbell was based on Gurdon’s work in Xenopus, but with additional technical adaptation. For example, one important modification was that nuclei used for transplantation in mammals came from mammary gland epithelial cells induced to enter quiescence, which makes them better suited to synchronize with the early developing embryo. This strategy has also been applied to humans. From fibroblasts, somatic cell nuclear transfer has been used to generate pancreatic stem cells, hematopoietic cells, cardiomyocytes, hepatocytes and neurons. 3  Pluripotency can be detected by analyzing the expression of specific markers: Oct4, Sox2, Kif4, C-Myc and Nanog, which are upregulated in embryonic stem cells and downregulated in differentiated cells. These markers are also known as Yamanaka factors, because they were used by the scientist to convert differentiated cells in undifferentiated cells, known as iPSCs (induced-PSCs). Yamanaka became interested in the pluripotent state in part by studying pluripotent embryonic stem (ES) cells, first cultured and characterised by Martin Evans,(Nobel Prize in Medicine 2007). All 24 genes encoding these transcription factors were introduced in one step into skin fibroblasts and a few of them actually generated colonies that showed a remarkable resemblance to ES cells. The number of genes were reduced, one-by-one, to identify a combination of only four transcription factors, sufficient to convert mouse/human embryonic fibroblasts to pluripotent stem cells (2006). He demonstrated that it is possible to induce in vitro a reversal of the normal phenomenon that occurs when PSCs become differentiated. Amplify the knowladge about stem cells is importanto to: - Increase our understanding of how our body works and how diseases occur. Studying stem cells in bone marrow, central nervous system, pancreas, heart and other organs allows better understanding of how our body works and diseases develop. - Generate new healthy cells to replace diseased cells (regenerative medicine). Stem cells can be guided to becoming specific cells that can be used to regenerate and repair diseased or damaged tissues. - Test new drugs for safety and effectiveness. Before using investigational drugs in people, researchers can use some types of stem cells to test drugs. APPLICATIONS Hematopoietic stem cells are used to treat patients with disorders of the blood, including some cancers. They have the ability to divide and multiply (self-renew) and to differentiate in different types of mature blood cells. After more than 50 years of research and clinical use, hematopoietic stem cells have become the best studied stem cells and, more importantly, they have seen widespread clinical use. Stem cells are also employed in cell therapy for the treatment of diabetes. This disease is experiencing a dramatic increase in prevalence, with 800 million people worldwide currently dependent on insulin (mostly type II diabetes). A recent publication has presented evidence of favourable outcomes associated with beta-cell transplantation. Beta cell replacement therapies are based on the principles of solid organ pancreas transplantation and cellular transplantation therapy. Pancreas transplantation approach has been demonstrated to achieve long-term insulin independence with excellent metabolic control for individuals with Type one diabetes. However, this approach presents two significant challenges: the invasiveness of the procedure, given its complexity, and the necessity for lifelong immunosuppression following transplantation. Cellular transplantation and islet transplantation offer the potential for a less invasive strategy for beta-cell replacement without the necessity for a major surgical procedure. However, the requirement for lifelong immunosuppression remains a significant challenge. The primary challenges associated with these two procedures pertain to the limited availability of donors. 4 The application of Beta cell replacement to obtain insulin independence in people with type 1 diabetes requires an unlimited source of insulin-producing cells and the ability to block the pathological immune response. Promising strategies to address both goals are encapsulation strategies (macro-, micro- encapsulation or scaffold/hydrogel), and human pluripotent stem cells (hPSCs). The encapsulation of islets has been pursued as a potential strategy to protect them from the immune response, with the goal of eliminating the need for immunosuppressive agents. However, there are some limitations associated with this approach, including compromised access to sufficient oxygen and nutrients for the cells and a compromised exit of insulin/glucagon across the generated barrier. Moreover, the foreign body response to implanted biomaterials consists of inflammatory events and wound healing processes that lead to fibrosis. The cellular and collagenous deposition isolate the device from the host. This can interfere with sensing of the host environment, lead to painful tissue distortion, cut off nourishment (for implants containing living, cellular components) and ultimately lead to device failure. (slide 32, 33 e 35 fino a 47 per vedere articoli riguardo la tecnica di incapsulazione con dati e relativi miglioramenti)). To sum up: - The transplantation of pancreatic islets may restore autonomous insulin function in patients with type 1 diabetes. - Encapsulation technologies based on microcapsules or microencapsulation devices aim to protect transplanted islets from immune responses and to prolong their survival. - The transplantation of encapsulated islets is often limited by insufficient nutrient transfer, lack of revascularization and excessive fibrosis. - Encapsulation technologies may be combined with immunomodulatory and gene-editing strategies to bypass challenges associated with host immune responses. These are two studies that tested the safety of PEC-Direct in immunosuppressed patients with T1D and reported the first clinical evidence for formation of functional Beta cells in the implant. (vedete slides da 35 a 47). As previously mentioned, human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold tremendous promise for both basic biology and cell-based therapies due to their unlimited in vitro proliferation capacity and their potential to generate all tissue types. More specifically, a strategy to provide stem cells for diabetes treatment is to use patients’ adult cells (fibroblasts) to produce iPSCs. There are two ways to do that: - Using Yamanaka factors, therefore the genetic overexpression of transcription factors. - Using chemical stimulation, which consists of the exposure to small-molecule chemicals as reprogramming factors to generate CiPSCs (chemically induced pluripotent stem cells). 5 The generated iPSCs have been used to generate deriving insulin-producing Beta- like cells in vitro with an improved insulin secretion. The CiPSC-islets produced showed transcriptomic identity, composition and insulin secretion comparable to those of human islets. The safety and efficacy of CiPSC-islets were evaluated in two systematic preclinical studies in nonhuman primates, in which amelioration of diabetes and no tumorigenicity during long-term observation were demonstrated, indicating marked therapeutic potential for diabetes treatment. These studies established a foundation for the clinical translation of CiPSC-islets in human patients. Thanks to these strategies, as previously anticipated, recently a 25-year-old woman with type 1 diabetes started producing her own insulin less than three months after receiving a transplant of reprogrammed stem cells. She is the first person with the disease to be treated using cells that were extracted from her own body. I think that this is another article related to this strategy. 6 Later, clinical studies were performed in nonhuman primates, these cells were injected in the patient. In the graph, it is possible to analyze the insulin requirement and the fasting blood glucose (FBG) in the days post transplantation. The insulin dose pre-transplantation was 54 ± 0.9 units/day (mean ± SD). The arrow on day 75 indicates the starting day of complete insulin independence. Instead, the FBG before transplantation was 210 ± 59 mg/dL (mean ± SD). The dashed line at 126 mg/dL indicates the threshold for diabetes diagnosis as specified by ADA recommendations. Moreover, it was considered the proportion of time spent in the five glycemic ranges (very high, high, target, low, and very low, as specified by the ADA professional practice committee) measured by CGM, before transplantation (baseline) and after transplantation. Percentages of time-in- target glycemic range (TIR) are indicated in the green bars: these graph shows that after 3/4 months post transplantation, the percentage of glucose tends to be in target. Another measured value is the HbA1c level pre and post transplantation: the patient’s HbA1c was slightly lower than anticipated for her TIR, a variation that has been reported for patients with chronic liver disease. The transplanted patients were also analyzed with MAGNETIC RESONANCE to highlight the presence of patchy and small nodular-like high-signal areas (indicating the location of transplanted CiPSC-islets). During regular monitoring within 365 days after transplantation, this signal remained stable in terms of location, shape, size, and MR signal intensity. To date, no fatal events have been reported in clinical cell transplantation therapies for type 1 diabetes related to the persistence of undifferentiated cells, tumorigenesis, or unregulated pancreatic hormone secretion due to ABERRANT DIFFERENTIATION when using stem cells. However, as the observation periods have been relatively SHORT, typically spanning only a few years, continuous and careful monitoring will remain essential. Many tumor biomarkers were measured in patients venous blood samples pre and post transplantation follow-ups, without showing alterations. Cell therapy was performed also for TYPE 2 DIABETES. Endodermal progenitor cells (EP), display a proliferative capacity similar to ESCs yet lack teratoma-forming ability. In addition, EP cell lines generate endodermal tissues representing liver, pancreas, and intestine, both in vitro and in vivo. Intrahepatic implantation of islet tissue differentiated in vitro from autologous endoderm stem cells was performed on a 59-year-old man with a 25-year history of T2D. The three major clinical outcomes, such as the glycemic targets, the reduction of exogenous insulin and the levels of fasting and meal-stimulated circulating C-peptide/insulin were monitored throughout the first 116 weeks, by showing a safe and effective treatment for the patient. 7 Another cell therapy was performed to treat AGE RELATED MACULAR DEGENERATION (AMD). AMD is the most common cause of severe loss of eyesight among people 50 and older. AMD affects the central vision, and with it, the ability to see fine details. In AMD, a part of the retina called the macula is damaged. In advanced stages, people lose their ability to drive, to see faces, and to read smaller prints. The study is based on the autologous graft of induced pluripotent stem cell (iPSC)–derived retinal pigment epithelial (RPE) cells to make a transplantation in a woman with age-related macular degeneration. Fibroblasts purified from a skin-biopsy specimen from the patient were transduced with vectors containing the genes of five “reprogramming” transcription factors (OCT3/4, SOX2, KLF4, GLIS1, and L- MYC), thus inducing a state of pluripotency. iPSC-RPE sheet (white arrow) that was transplanted under the fovea was readily visible on the day after surgery. This study reported a successful submacular transplantation of induced pluripotent stem- cell derived retinal pigment epithelial cells. However, some concerns were raised from scientific society regarding the analysis of the fluorescein angiogram in the paper, that highlighted, according to the paper’s author, that there was no neovascularization. Indeed, scientific society proposed that the survival of RPE requires the perfusion of vessels (as also showed in previous data), and, in the same fluorescein angiogram, these scientists were able to notice some small vascular bulbs under the transplanted iPSC-RPE sheet. This structure could indicate the growth of new vessels or the reperfusion of existing vessels, but it is not possible to determine if the vessels are benign or pathogenic. 8 ADVANCED THERAPEUTIC TECHNOLOGIES LECTURE 10: 05/12/2024 Cell therapy side effects The first and most used stem cell therapy is transplantation of bone marrow that can be of two types: autologous and allogenic, based on the source of the stem cells. In an autologous transplant, the term “auto,” meaning self, signifies that the patient serves as their own donor. Stem cells are harvested from the patient, typically before intensive treatments like chemotherapy, and then reintroduced into their body to restore bone marrow function. On the other hand, allogeneic transplants involve stem cells from another person, with “allo” meaning other. This donor could be a related individual, such as a sibling, or an unrelated but suitably matched donor. Matching is critical in allogeneic transplants to reduce the risk of complications, such as graft- versus-host disease, where the donor's cells attack the patient’s body. Allogenic hematopoietic cell transplantation (allo-HCT) is a potentially curative therapy for hematological malignancies, such as leukemias and lymphomas, through the introduction of healthy donor hematopoietic stem cells into a recipient. A critical component of its effectiveness is the graft-versus-leukemia (GVL) effect, which is mediated by alloreactive T cells from the donor. These T cells recognize and target residual malignant cells that might remain after pre-transplant conditioning, significantly reducing the risk of relapse. The GVL effect is primarily localized to lympho-hematopoietic tissues, where blood-forming and lymphoid cancer cells reside, ensuring that the therapeutic immune response is focused on areas with the highest concentration of cancer cells. Alloreactive T cells can also mediate a severe complication known as graft-versus-host disease (GVHD). GVHD is a common immune response in which donor T cells recognize the recipient's healthy tissues as foreign and attack them, leading to tissue damage and inflammation. This condition frequently affects target organs, including the gut, liver, lungs, and skin, where the damage can manifest as specific clinical symptoms. Acute GVHD, which typically develops within the first three months post-transplantation (although delayed cases are possible), is driven by donor immune effector cells attacking the recipient's tissues. The primary symptoms of acute GVHD include skin rashes, often with erythema or peeling; severe diarrhea due to gastrointestinal involvement; and hyperbilirubinemia, which indicates liver dysfunction and may result in jaundice. Acute graft-versus-host disease (GVHD) starts during the early phase of allogeneic stem cell transplantation when the treatment used to prepare the patient, known as the conditioning regimen, damages their tissues. This damage releases inflammatory signals that activate the patient’s immune cells, called antigen- presenting cells (APCs). These cells then alert the donor’s T cells, which recognize differences in tissue markers (HLA) between the donor and recipient. In response, the donor T cells attack the patient’s tissues as if they were invaders. During the later phase of GVHD, donor CD8+ T cells become highly active and directly kill the patient’s cells, causing the damage seen in acute GVHD. GVHD is classified into four grades of severity: I (mild), II (moderate), III (severe), and IV (very severe). The grading depends on the specific symptoms and their intensity in affected organs. For skin GVHD, it’s based on the extent of the rash; for liver GVHD, it’s determined by the level of bilirubin in the blood; for lower gastrointestinal (GI) GVHD, the volume of diarrhea is assessed; and for upper GI GVHD, it’s measured by persistent nausea. Chronic graft-versus-host disease (cGVHD) develops in 30–70% of patients following allogeneic stem cell transplantation, with its likelihood influenced by factors like the type of conditioning regimen, the source of the donor cells, the degree of HLA matching, and the preventive measures used to reduce GVHD. Unlike acute GVHD, which primarily targets the skin, liver, and gastrointestinal tract, chronic GVHD can involve almost any organ in the body, making it a more systemic and unpredictable complication. The first-line treatment for chronic graft-versus-host disease (cGVHD) is corticosteroids, which help control inflammation. For patients whose cGVHD does not respond to steroids (steroid-refractory cGVHD), the FDA has approved several targeted therapies. Ibrutinib, the first agent approved for this condition, is a Bruton's tyrosine kinase (BTK) inhibitor and plays a dual role by suppressing the activation of B-cells and T-cells, which are key drivers of immune responses in GVHD. Ruxolitinib, a JAK 1/2 inhibitor, targets an early intracellular signaling pathway crucial in GVHD development. By inhibiting JAK1/2, it reduces the transcription of pro-inflammatory cytokines, limits the migration of neutrophils that mediate inflammation, and downregulates the expression of MHC class II, curbing the immune system's overactivation. Belumosudil, an inhibitor of Rho-associated coiled-coil kinase 2 (ROCK2), specifically downregulates pro-inflammatory cytokines such as IL-17 and IL-21 in T-cells. These cytokines are central to chronic inflammation and tissue damage in GVHD. Together, these therapies target distinct pathways to control inflammation, reduce immune dysregulation, and mitigate the severity of GVHD. T cells use T cell receptors (TCRs) to identify and bind to antigens on cancer cells. This binding activates a signaling cascade inside the T cell, preparing it to attack. TCRs are specialized proteins designed to recognize antigen fragments presented by major histocompatibility complex (MHC) molecules on cell surfaces. Each TCR has two main extracellular regions: the Variable (V) region, which binds to the antigen/MHC complex, and the Constant (C) region, which is closer to the cell membrane. The Constant region connects to a transmembrane segment and a short cytoplasmic tail that helps transmit activation signals, making the TCR a vital component in targeting cancer cells. Chimeric Antigen Receptor (CAR) technology represents a groundbreaking advancement in immunotherapy, revolutionizing how T cells are engineered to target cancer. In 1992, researchers presented the first genetically modified T cell, which combined the intracellular signaling domain of CD3 zeta (CD3ζ), a key molecule involved in T cell activation, with the antigen-binding region of an antibody. This fusion created a synthetic receptor that equipped T cells with the ability to recognize and attack cells expressing specific target antigens, such as those found on cancer cells. These modified receptors, now known as first-generation CARs, significantly enhanced T cells' ability to "navigate" and bind to tumor cells, improving their targeting precision. However, their initial effectiveness was limited due to insufficient activation and persistence. The subsequent addition of a costimulatory domain, such as CD28 or 4-1BB, to the synthetic receptor marked the evolution to second-generation CARs. This innovation greatly improved T cell activation, survival, and cytotoxicity, making them more effective in eradicating cancer cells. Today, CAR-T cell therapy continues to evolve, offering hope for treating previously intractable cancers. In CAR-T cell therapy, a patient’s T cells are extracted from their blood and genetically engineered in a laboratory to produce CARs on their surface. These CARs enable the T cells to specifically recognize and bind to antigens on cancer cells, targeting them for destruction. Once modified, the CAR-T cells are multiplied in the lab and then reintroduced into the patient through an infusion. Inside the body, these cells continue to grow and actively seek out and destroy cancer cells. The patient’s response to treatment is closely monitored using tests such as CT scans, bone marrow biopsies, and blood tests to evaluate the therapy's effectiveness and ensure the CAR-T cells persist and function as intended. There are currently 6 commercial CAR-T products approved (the first was in 2017), divided in two categories, against B cell lines and CD19. The use of CAR-T cell therapy has shown significant potential in treating various cancers, particularly hematological malignancies. Despite its success, questions remain regarding the long-term potential and clonal stability of the infused CAR-T cells, as their behavior over extended periods is not yet fully understood. Notably, a landmark observation was made in two patients with chronic lymphocytic leukemia (CLL) who received CD19- redirected CAR-T cells. Remarkably, these engineered T cells remained detectable in their systems for over ten years after the initial infusion. During this period, both patients experienced sustained remission, highlighting the durability and effectiveness of CAR-T cell therapy in some cases. CAR-T cell therapy, while highly effective against certain cancers, can cause serious side effects due to on-target off-tumor effects. This occurs when CAR-T cells attack healthy cells that also express the target antigen, not just cancer cells. These adverse effects can vary widely in severity and affect multiple organ systems, depending on the distribution of the targeted antigen in normal tissues. For example, targeting antigens like CD19, which is present on both cancerous and normal B cells, can lead to B cell aplasia or immune deficiencies. The manifestations of these side effects are diverse, ranging from mild symptoms to life-threatening complications, such as cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Even with significant progress in CAR-T cell therapy, many aspects of the underlying causes of its side effects are still not fully understood, making diagnosis and treatment challenging. Managing these toxicities often requires a careful approach, where doctors must control the side effects without reducing the effectiveness of the CAR-T cells. CRS is the most frequently observed complication, also the first, characterized by a systemic inflammatory response due to the massive release of cytokines by activated CAR-T cells and other immune cells. Symptoms can range from mild fever and fatigue to severe hypotension, organ dysfunction, and life-threatening complications. ICANS involves neurological symptoms caused by the immune system's effects on the central nervous system, leading to manifestations such as confusion, aphasia, seizures, or even coma in severe cases. Lastly, HLH/MAS is a rare but severe hyperinflammatory condition driven by overactivation of macrophages and T cells, resulting in widespread immune-mediated tissue damage and high levels of inflammatory cytokines. After CAR-T cell infusion, several cytokines are elevated as part of the immune response. These include interferon-gamma (IFN-γ) and TNF-α, which help activate immune cells to target and kill cancer cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the production of white blood cells, while interleukin 6 plays a role in immune regulation but can also drive inflammation. Additionally, perforin and granzymes are released to directly kill cancer cells. The second most common side effect of CAR-T cell therapy is neurotoxicity, ICANS. This condition occurs when the CAR-T cells cause activation of the endothelial cells and disrupt the blood-brain barrier (BBB), allowing immune cells and proteins to enter the brain. During acute neurotoxicity, the cerebrospinal fluid (CSF) often shows elevated protein levels and an increased number of white blood cells, indicating BBB breakdown. Symptoms of neurotoxicity can range from mild confusion and headaches to severe complications like seizures, requiring careful monitoring and management. Signs of CAR-T cell-related neurotoxicity can range from mild symptoms such as dysgraphia, impaired attention, headache, sleep disorder, anxiety, myoclonus, motor dysfunction, to more severe symptoms, including encephalopathy, aphasia, delirium, tremors, seizures, and cerebral edema. Secondary hemophagocytic lymphohistiocytosis (sHLH), also known as macrophage activation syndrome (MAS), is a serious complication that can occur after CAR-T cell therapy, as well as after allogeneic and autologous hematopoietic stem cell transplantation. This condition is characterized by an overwhelming immune response, leading to hyperinflammation. Diagnosing sHLH/MAS requires clinical suspicion, supported by signs like fever, low blood cell counts (cytopenia), and multi-organ failure. In severe cases, neurological dysfunction, acute kidney injury, and acute respiratory distress can indicate a poor prognosis, making early recognition and management crucial to improving outcomes. Management of sHLH/MAS often involves immunosuppressive treatments, including corticosteroids and other targeted therapies, to reduce the overactive immune response and prevent further organ damage. Early studies on glucocorticoid-resistant graft-versus-host disease (GVHD) highlighted the central role of IL-6 in cytokine release syndrome (CRS). IL-6 is a key pro-inflammatory cytokine that plays a major role in driving the systemic inflammation observed in CRS. In response to these findings, tocilizumab, an IL-6 receptor antagonist, has been developed as a therapeutic intervention to block IL-6 signaling and manage CRS and has been approved by FDA for the treatment. Incidence of second malignancies after CAR-T cell therapy is possible. Long-term adverse effects of CAR-T cell therapy can include the development of certain types of T-cell cancers. These include T-cell lymphoma, T-cell large granular lymphocytosis, peripheral T-cell lymphoma, and cutaneous T-cell lymphoma. Among the 14 reported cases with sufficient data, these cancers have typically appeared within two years of receiving CAR-T therapy, with the onset ranging from 1 to 19 months. While these cases are relatively rare, their occurrence highlights the need for ongoing monitoring and evaluation of patients who have undergone CAR-T therapy, as the long-term impact on immune cell function remains an area of concern. The generations of CAR T cells differ in the signaling domains they incorporate. 1st generation CAR T cells contain only the CD3ζ signaling domain, which activates T cells to kill cancer cells. 2nd generation CAR T cells include an additional costimulatory domain, such as CD28, to enhance T cell activation and persistence. 3rd generation CAR T cells have two costimulatory domains (e.g., CD28 and 4- 1BB) for even stronger T cell activation. 4th generation CAR T cells incorporate a cytokine gene (e.g., IL-12) to further boost the immune response. 5th generation CAR T cells contain a cytokine receptor (e.g., IL-2R) to enhance T cell proliferation and survival. Each generation of CAR T cells has its own advantages and potential side effects. The development of CAR T cell therapy is an ongoing field of research with the aim of improving its efficacy and safety. Emerging engineering strategies are being developed to improve CAR-T cell therapies in terms of safety, specificity, and efficacy. One approach involves modifying CAR-T cells to secrete proinflammatory cytokines, which can help overcome the immunosuppressive environment of solid tumors. Another strategy focuses on enhancing CAR-T cells' ability to differentiate between cancerous and healthy cells, reducing the risk of harming healthy tissues. Additionally, some CAR-T cells can be engineered with a control mechanism that allows activation or deactivation through drugs or small molecules, preventing excessive T cell activation and reducing the risk of toxicity or exhaustion. False advertising and exaggerated claims continue to be a problem, especially with unproven stem cell "treatments." These so-called miracle cures are often promoted without scientific proof of their safety or effectiveness, leading people to spend money on treatments that could be harmful. Some of the most common tactics include: 1. Filing patent applications: This strategy is used to give the impression of innovation and ownership, signaling that a clinic or company has developed something new. However, just filing a patent doesn't prove the technology is effective or successful. 2. Listing clinical trials on government websites: By listing trials on official platforms like clinicaltrials.gov, companies can make their work seem like part of legitimate clinical research. This can give the false impression that their therapies are thoroughly tested, even if the trials are in early stages or have flaws. 3. Publishing papers in predatory journals: Some companies publish research in low-quality journals that charge authors to publish without proper peer review. While these journals may seem credible, they don't meet the standards of respected scientific publications, creating a false sense of scientific approval for their treatments. These tactics, while sometimes providing a veneer of scientific legitimacy, can be misleading and raise ethical concerns. Consumers and investors may be persuaded by these superficial markers, which might not always reflect the true scientific merit or clinical efficacy of the treatments. EXAMPLE 1-A patient reported that she had found the stem-cell clinic through its listing on Clinical-Trials.gov. She was under the impression that she was participating in a clinical trial and that she had met the criteria of the trial. She paid $5,000 for the bilateral procedure. The consent form indicated the risk of blindness. The market for scientific legitimacy in the context of medical treatments, particularly in emerging fields like CAR-T therapy or other novel biotechnologies, is driven by the need for clinical and scientific validation. Clinics and companies offering these treatments often seek to enhance their credibility and appeal to potential patients or investors by using various forms of evidence that suggest scientific legitimacy, even if the actual scientific support may be lacking or weak. These tactics are often referred to as "tokens of scientific legitimacy" and can serve as persuasive marketing tools. 2-A 66-year-old man underwent intrathecal infusions for residual deficits from an ischemic stroke at stem-cell clinics in China, Argentina, and Mexico. The treatments involved infusions of mesenchymal, embryonic, and fetal neural stem cells. These therapies are marketed as potential solutions for stroke recovery, with the idea that stem cells can repair damaged brain tissue. However, the use of these types of stem cells, particularly in unregulated commercial clinics, raises concerns about safety and effectiveness. While some stem cells, like mesenchymal cells, have regenerative properties, the scientific evidence for their ability to treat stroke is limited, and there are risks, such as infections or tumor formation, especially with embryonic or fetal stem cells. Neuropathological analysis revealed a densely cellular, highly proliferative, primitive neoplasm with glial differentiation.