2.3.1.1 Stroke

Stroke is a leading cause of chronic disability and mortality. The only approved ischemic stroke treatment, tissue plasminogen activator, is limited to 15% of patients because of its 4.5-h therapeutic window. Thrombectomy therapy can help in some cases but requires specialized resources, thereby restricting global access for most patients [63]. Several stem cells, including MSCs, NSCs, ESCs, and iPSCs, have been used to treat stroke.

MSCs have been widely studied for treating stroke via intravenous, intra-arterial, or intracerebral injection [64]. MSCs protect the cerebral microvasculature from ischemic–reperfusion injury by reducing inflammation lowering astrocyte and microglial activation, leukocyte infiltration, and the levels of proinflammatory cytokines (interleukin [IL]-1α, IL-1β, IL-6, and tumor necrosis factor [TNF-α]), and increasing the levels of anti-inflammatory cytokines (IL-4, IL-10, and interferon [IFN]-β) [65, 66]. MSCs can home to damaged sites and differentiate into neuron-like cells. Mitochondrial transfer by MSCs aids in stroke recovery by restoring function and enhancing survival, providing neuroprotection, and improving outcomes in stroke models [64]. MSC therapy for stroke is generally safe and shows promise with improvements in motor function, daily activities (higher Barthel Index [BI] scores), disability (lower modified Rankin Scale [mRS] scores), lesion volumes, and brain connectivity. However, these improvements are not consistently observed [5]. A meta-analysis of nine randomized controlled trials (159 MSC-treated patients and 147 controls) by Huang et al. [67] revealed that MSC transplantation improved neurological deficits in ischemic stroke patients but had a limited impact on the BI and mRS. Larger, well-designed phase II trials are needed to confirm the therapeutic benefits of MSC therapy. The therapy shows promise but faces challenges, such as optimal timing for MSC administration. While some studies advocate delivery in the acute phase (within 48 h) for immunomodulation, others highlight the benefits of later administration (up to 1 month) in promoting neurogenesis and neuroplasticity, leaving the ideal timing uncertain.

In addition to MSCs, several NSC lines have been used to treat patients with stroke. The main mechanisms of action of NSCs in stroke include direct replacement of neurons, paracrine effects, angiogenesis, and neurogenesis [68]. Intracerebral injection of human NSC lines, such as NSI-566 and CTX0E03, was well tolerated and improved the mRS, Fugl–Meyer motor score [69], National Institutes of Health Stroke Scale (NIHSS) score, and MRI in patients with ischemic stroke [69, 70]. However, no improvement in the NIHSS score was observed in patients during the subacute-to-chronic recovery phase [71].

ESC treatment improves stroke via angiogenesis and neurogenesis via their secretion [72, 73]. Although preclinical studies have shown benefits in terms of dopaminergic, sensory, and motor functions, clinical trials using ESCs for stroke are rare, likely because of concerns about teratoma formation and malignancy [74]. Only one trial reported improvements in functional recovery and severity of neurological deficits with no complications following ESC therapy [75].

Preclinical studies have demonstrated the role of iPSCs in treating stroke through their ability to replace cells and promote neuroprotection, immunomodulation, angiogenesis, and synapse formation [76]. However, the clinical application of iPSCs for stroke treatment is limited, with one ongoing phase 1 trial (NCT05993884) assessing iPSC-derived endothelial progenitor cells (EPCs) in 27 acute ischemic stroke patients.

2.3.1.2 Traumatic Brain Injury

Traumatic brain injury (TBI) is a severe condition that involves physical damage to brain tissue, particularly in young individuals [66, 77]. The mortality rate for acute severe TBI is as high as 36% [78]. The current guidelines for managing TBI emphasize controlling physiological factors such as blood pressure, intracranial pressure, oxygenation, and nutrition, among others [79].

MSC therapy holds significant promise for treating TBI through various delivery methods, including direct injection, intravenous infusion, lumbar puncture, and stereotactic implantation, by mitigating oxidative stress, reducing neuroinflammation, preventing apoptosis, and alleviating mitochondrial dysfunction. They transfer healthy mitochondria, regulate antioxidants, increase Bcl-2 expression, and reduce microglial activation and proinflammatory cytokines while increasing anti-inflammatory cytokines. MSCs also suppress immune cells, inhibit T-cell proliferation, and polarize microglia to a neuroprotective state, enhancing neuronal survival and tissue repair [77]. The administration of BM-MSCs improved neurological function without toxicity [80], enhanced brain function, consciousness, and motor ability [81], and improved motor function [82]. UC-MSCs delivered via lumbar puncture improved neurological function and self-care [83]. Despite promising evidence, challenges in MSC therapy for TBI include optimizing timing and the lack of standardized protocols. Consistent definitions of the MSC source, TBI severity, and dosage are needed to improve efficacy.

NSCs have also been explored as a treatment for TBI in one clinical trial by Wang et al. [84], who reported improvements in neurological function and increases in the serum levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) without SAEs after the injection of 20–40 × 10⁶ autologous MSC-derived NSCs into 10 TBI patients.

2.3.1.3 Spinal Cord Injury

SCI commonly results in the loss of sensory, motor, and autonomic function below the injury level, with a global incidence of 10.5 per 100,000. Although rapid recovery of neurological function is desired, effective strategies for repairing damaged nerve cells are still lacking [85]. Several cell types, such as MSCs, NSCs, and ESCs, have been used for SCI. Among them, MSCs are the most prevalent.

MSCs show promise for spinal cord repair through anti-inflammatory, neuroregenerative, and vascular-supportive mechanisms. MSCs reduce inflammation by promoting anti-inflammatory M2 macrophages, inhibiting the toll-like receptor 4 (TLR4) and NF-κB pathways, and decreasing inflammasome activity. They also support axonal regeneration via pathways such as the Wnt/β-catenin and PI3K–mTOR pathways while promoting vascular repair by releasing angiogenic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF). These actions aid in restoring nerve function and improving motor recovery in SCI patients [85].

MSCs have been used in multiple clinical trials to treat chronic, acute, and subacute SCI. BM-MSCs are the most popular source of MSCs for SCI [85]. These trials employed various delivery methods, such as intrathecal infusion, intraspinal injection, in situ transplantation, and intravenous injection, with doses ranging from 1 × 10⁶ to 4 × 10⁸ cells. Data suggest that local injections may be more effective than systemic approaches, and thus, intrathecal injection offers a convenient and fast-acting method for multiple MSC doses [86]. Notably, the low concentration of cells used for transplantation (<5 × 10⁷ cells) had outcomes comparable to those of the high concentration of cells (≥5 × 10⁷ cells) [87]. MSC administration has been shown to improve sensation near the injury site, increase American Spinal Injury Association (ASIA) Impairment Scale grades, increase ASIA sensory and motor scores, improve self-care and muscle tone, and significantly improve movement, bowel, and bladder function in patients with chronic complete SCI [85, 87, 88]. MSCs hold promise for SCI treatment, but factors such as dosing, mechanisms, cell survival, and transplantation protocols require clarification. Further research is needed to refine MSC therapy and understand SCI processes.

NSCs have also been used to treat patients with SCI in at least four clinical trials [58]. NSCs have been shown to modulate the inflammatory response by inhibiting reactive macrophages and increasing the expression of growth factors that are beneficial for recovery, such as NGF, BDNF, insulin-like growth factor-1 (IGF-1), and glial cell line-derived neurotrophic factor (GDNF) [89]. The intraspinal injection of NSCs (15–100 × 10⁶ total cells) was proven safe and feasible, with no SAEs. Patients have shown functional recovery and improvements in motor function and spasticity [58].

ESCs can differentiate into neurons and glial cells to replace nonfunctional cells in SCI [89]. Shroff et al. [90, 91] reported that patients with SCI had increased ASIA scores and neurological function, with no SAEs observed. However, the considerable proliferative ability of ESCs poses a risk of tumor formation, which may limit their use in clinical trials.

2.3.1.4 Cerebral Palsy

Cerebral palsy (CP) is a group of permanent movement and posture disorders that are often accompanied by issues such as sensation, cognition, communication, behavior, epilepsy, and musculoskeletal problems. CP is the most common physical disability in children, with an incidence of 1.6 out of 1000 in high-income countries and 3.4 out of 1000 in low- and middle-income countries [92]. Despite over 180 interventions and a 30% reduction in incidence due to prevention efforts, there is still no cure for CP [93]. Several cell types, such as MSCs and ESCs, are currently under investigation for the treatment of CP. MSC therapy for CP has been administered via the intrathecal, stereotactic, and intravenous routes. It has been shown to be safe and to improve motor function, health status, comprehension, quality of life, and overall function in children with CP. However, further randomized controlled trials are needed to determine the optimal dose, frequency, timing, and administration routes [94]. MSC therapy may promote recovery through paracrine effects, involving the secretion of cytokines that reduce inflammation, support neuronal survival, increase angiogenesis, and activate endogenous repair mechanisms. Additionally, MSCs may differentiate into neurons and glial cells to replace damaged cells. However, most data suggest that their main therapeutic effect is establishing a reparative environment conducive to neural recovery rather than through direct cell engraftment [95]. The MSC sources tested to date included UC-MSCs, BM-MSCs, UCB-MSCs, and Wharton's jelly-MSCs, with UC-MSCs being the most commonly used and doses ranging from 2 to 22 × 10⁷ cells in total (1–4 injections) or 1 × 10⁶ cells/kg body weight.

There are few clinical studies on the use of ESC therapy in CP. These findings suggest that ESCs may migrate to hypoperfused areas of the brain, “homing” to affected regions and potentially contributing to neurogenesis in CP. ESC treatment improved motor function and enhanced cognitive function after ESC treatment [96]. An improvement in motor function was also observed in CP patients treated with 1–2 × 10⁷ autologous MSC-derived NSCs [58].

2.3.1.5 Alzheimer's Disease

Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by progressive dementia, memory loss, and cognitive decline. Its brain pathology involves amyloid β (Aβ) plaque accumulation and the intracellular formation of neurofibrillary tangles, leading to cholinergic neuron loss [66, 97, 98].

MSCs are the most commonly used cell type for AD. A recent literature review revealed that MSCs act on AD by reducing Aβ and NFT accumulation and inflammatory cytokines (TNF-a, IL-1b, and ROS) and the polarization of inflammatory M1 microglia into anti-inflammatory M2 microglia, promote neurogenesis by differentiating NSCs into neural progenitor cells, ultimately into neurons, and enhance synapse formation. Additionally, MSCs have paracrine and autocrine effects by releasing cytokines, such as growth/differentiation-15 and galectin-3, and neurotrophic factors, such as VEGF, BDNF, and NGF, to increase neuronal repair [66, 97, 98]. MSCs also transfer functional mitochondria and miRNAs to increase their bioenergetic profile and improve microglial clearance of accumulated protein aggregates [98]. Clinical studies in which MSCs are used to treat AD are limited. To date, MSCs have been administered via stereotactical, intracerebroventricular, or intravenous routes with cell doses ranging from 3.0 × 10⁶ to 9.0 × 10⁷ cells. Recent studies and trials on MSC therapy for AD have reported promising safety profiles with no dose-limiting toxicities or manageable adverse events (AEs) and have shown cognitive stability or improvement in some MRI and biomarker levels in some patients [99-101]. Several ongoing MSC trials for AD are listed on ClinicalTrials.gov, with results pending (NCT03117738, NCT02833792, NCT04684602).

Gene-editing technologies such as clustered, regularly interspaced short palindromic repeats (CRISPR) hold promise for addressing AD. In early-onset AD, CRISPR can correct autosomal-dominant mutations in presenilin 1 and 2 (PSEN1/PSEN2). For late-onset AD, it offers the potential to replace the high-risk APOE4 isoform with the protective APOE2 isoform, potentially reducing the risk of developing AD by up to 40% [102]. Additionally, human cortex-derived NSCs can be engineered to express IGF-1. When these modified NSCs were transplanted into AD model mice, the spatial memory was effectively restored [103]. Clinical trials for gene-edited stem cells are still in the early stages. In one study, autologous fibroblasts genetically modified to express human NGF were implanted into eight AD patients. After 22 months, no long-term AEs were observed, cognitive assessments indicated a slower rate of decline, and PET scans revealed significant increases in cortical glucose metabolism. Autopsy findings also showed robust growth responses to NGF [104]. However, a recent study by Ortega et al. [105] highlighted the challenges of NGF gene therapy, with limited success in clinical trials. These findings underscore the need for further research and development to establish gene therapy as a viable treatment option for AD [105].

2.3.1.6 Parkinson's Disease

PD is a chronic, progressive neurodegenerative disorder characterized by motor impairment, social dysfunction, α-synuclein aggregation, and dopamine deficiency due to neuronal loss in the substantia nigra. Affecting 2–3% of people over 65 years of age, PD led to 5.8 million disability-adjusted life years and 329,000 deaths in 2019, with rates expected to double by 2040. Current treatments focus on symptom management, including medications such as levodopa and dopamine agonists, as well as interventions such as deep brain stimulation and lesion surgery [106]. The main cell types, such as MSCs, NSCs, ESCs, and iPSCs, have been used to treat PD.

MSCs act through multiple mechanisms, including inhibiting α-synuclein transmission, modulating apoptosis (by upregulating Bcl2 and downregulating Bax), and reducing inflammation by decreasing astrogliosis and microgliosis. MSCs also secrete neurotrophic factors such as BDNF, cerebral dopamine neurotrophic factor, and hepatocyte growth factor (HGF), supporting dopaminergic neuron survival and facilitating motor recovery. Additionally, MSCs produce anti-inflammatory cytokines (IL-10 and transforming growth factor-beta [TGF-β]), suppress proinflammatory cytokines (TNF-α, IFN-γ, and IL-1β), and may facilitate mitochondrial transfer to damaged neurons, promoting cellular repair [106-108]. MSC research for PD began in 2010 with a study by Venkataramana et al. [109], where BM-MSCs improved motor function, disease severity, and quality of life (based on the Unified PD Rating Scale (UPDRS), Hoehn and Yahr, and Schwab and England scores), facial expression, gait, and freezing episodes with no SAEs reported. Since then, several studies have been conducted to investigate the potential of MSC therapy in PD. BM-MSCs are feasible for treating mild to moderate PD and are safe and tolerable [110]. UC-MSCs improved UPDRS scores and offered additional cognitive and emotional benefits over BM-MSCs, including reduced anxiety and depression. Both cell types improved motor and daily living functions, highlighting UC-MSCs as promising options [111, 112]. PD patients treated with AT-MSCs also experienced no AEs over 6 months and improved Movement Disorder Society-UPDRS scores, suggesting the potential of AT-MSCs as PD therapy [113]. In PD, advanced glycation end products (AGEs) contribute to dopamine neuron apoptosis, but soluble AGE receptors can counter this effect. Using CRISPR–Cas9, UC-MSCs have been engineered to secrete these receptors, reducing neuronal death and improving motor function in a PD mouse model, demonstrating promising therapeutic potential [114].

NSCs have been tested in two clinical trials for PD. One trial involved transplanting 3 × 10⁷ neural precursor cells into 21 patients, resulting in significant symptom improvement with no major side effects [115]. Another study by Madrazo et al. [116] transplanted 2 × 10⁶ neural progenitor cells into eight patients and reported no AEs or motor function improvements in seven out of eight patients. Other stem cell sources have also been investigated for PD. Two trials in South Korea (NCT06477744, NCT05887466) are testing ESC-derived dopamine progenitor cells, which were completed in 2029 and 2026. A recent case report described a personalized cell therapy approach using autologous iPSC-derived dopaminergic progenitor cells in a PD patient. Clinical and imaging findings have indicated potential benefits, including improvements in motor function and patient-reported symptoms [117]. Another US-based phase 1 trial (NCT06422208) assessing autologous iPSC-derived dopamine neurons is expected to finish in 2026.

2.3.1.7 Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by repetitive behaviors, limited activities, and social communication difficulties. In 2021, the WHO estimated that ASD affects one in 270 people, with a higher prevalence in men, and by 2023, the estimate had increased to one in 100 people globally [118]. Current treatments for ASD, such as psychotropic drugs, therapies, and educational support, can help manage symptoms such as irritability, seizures, and mood disorders but do not modify the underlying condition [119, 120].

The role of MSCs in treating ASD has only recently begun to be explored, and studies suggest that they may support neurogenesis and synaptogenesis, regulate synaptic function and plasticity by secreting growth factors, enhancing synaptic plasticity, restoring neurotransmitter release, and integrating into synaptic networks [121, 122]. In ASD, where there is an imbalance between Th1 and Th2 cells, excessive proinflammatory markers, and low anti-inflammatory responses, MSCs may restore immune balance by inhibiting TNF-α, IL-1β, and IFN-γ and increasing the levels of IL-10 and IL-4 [122]. Additionally, MSCs offer neuroprotection by reducing neural apoptosis, microglial activation, astrocyte proliferation, and oxidative stress [120, 122].

MSC therapy shows promise for ASD treatment, as it is safe and potentially effective. Lee et al. [123] treated an ASD patient with UC-MSCs and reported improved social communication and reduced CARS scores without side effects. Sun et al. [124] administered intravenous MSCs to 12 patients and reported improvements in autism severity and social communication with no AEs. Sharifzadeh et al. [125] used intrathecal BM-MSCs in a trial and reported specific improvements in CGI severity and CARS scores. However, further studies are needed to confirm the benefits of MSC therapy.

CRISPR gene editing offers the potential for addressing the genetic underpinnings of ASD, which is often associated with rare monogenic mutations or complex polygenic influences. The CRISPR strategy creates a versatile experimental platform to systematically explore the role of ASD-associated genes in human cells and represents a potential treatment for ASD [126]. Recently, the US FDA approved a phase I clinical trial for JAG201 (NCT06662188), a gene replacement therapy targeting ASD associated with SHANK3 mutations and Phelan–McDermid syndrome. JAG201 utilizes an adeno-associated virus serotype 9 vector to deliver a functional SHANK3 minigene directly to neurons in the central nervous system. This approach seeks to restore the synaptic function critical for neurodevelopment and maintaining cognitive and motor skills. The trial is expected to conclude in 2031.

2.3.1.8 Amyotrophic Lateral Sclerosis

ALS is a rare, fatal neurological disease affecting upper and lower motor neurons, with an incidence of 0.6–3.8 per 100,000 people [127]. Although ALS is becoming more prevalent, Riluzole remains the only approved treatment, and a cure is still elusive.

MSCs have shown promise in preclinical and clinical studies and are administered intrathecally, intravenously, or via direct spinal cord injection. MSC therapy supports neural health by releasing neurotrophic factors for neuroprotection and neurogenesis and promoting an anti-inflammatory environment in the central neural system (CNS). MSCs can also promote synaptic connection and remyelination of damaged axons and reduce apoptosis. In cerebrospinal fluid, MSCs increase Tregs and Th2 cells and the levels of anti-inflammatory cytokines such as IL-4 and IL-10, decrease activated dendrites, and release TGF-β, promoting CNS homeostasis and transforming microglia from an inflammatory (M1) state to an anti-inflammatory (M2) state. These effects help regulate ALS progression and maintain CNS function [128]. The first clinical trial in which MSCs were used to treat ALS was conducted by Mazzini et al. in 2003 [129]. Since then, multiple studies have explored the therapeutic effects of MSCs on ALS, showing safety profiles without serious side effects. MSC-treated patients experienced slower disease progression, improved forced vital capacity (FVC), increased life expectancy, and improved ALS functional rating scale (ALS-FRC) scores [127, 128].

In addition to MSCs, NSCs are typically given at doses ranging from 5 × 10⁴ to 1 × 10⁵ cells/injection, with 1–5 injections via unilateral or/and bilateral intraspinal injection to treat patients with ALS. NSCs can slow the progression of ALS symptoms and prolong survival time by providing neuroprotection, reducing inflammation and astrocyte activation, and enhancing synaptic plasticity [62]. NSCs have also been demonstrated to migrate and integrate into the spinal cord and differentiate into neural phenotypes to delay the deterioration of motor ability in an ALS rat model. A phase 1 trial revealed that injections of NSCs are safe and tolerable [130], delay disease progression [131], and improve the ALS-FRC or Medical Research Council scores [132]. A phase 1/2 trial using neural progenitor cells modified with a lentiviral vector to express glial-derived neurotrophic factor (CNS10-NPC-GDNF) was conducted in 18 ALS patients. It revealed a trend toward improved motor function in treated limbs, although the efficacy of this approach was limited by incomplete virus penetration and an immune response [133].

2.3.3.1 Type 1 DM

T1DM is an autoimmune disease in which the immune system destroys pancreatic insulin-producing cells, leading to minimal or no insulin production. While exogenous insulin helps control blood sugar, it often fails to prevent complications and may cause poor glycemic control or hypoglycemia [139, 140].

Several clinical trials have evaluated the use of MSCs for treating T1DM, demonstrating that MSC therapy can increase C-peptide levels while reducing insulin requirements and HbA1c levels [141-144]. Despite these findings, a meta-analysis of MSC trials revealed only improved HbA1c, with no significant changes in fasting glucose or C-peptide [145]. Further large-scale studies are needed to confirm these benefits because of the variability in MSC sources, doses, and patient numbers [140].

In vitro, the differentiation of stem cells into insulin-producing cells represents a promising therapeutic strategy for T1DM, with iPSCs and ESCs emerging as ideal candidates for this approach. ViaCyte (ViaCyte Inc. San Diego, CA, USA) developed an immune isolation device to encapsulate pancreatic endodermal cells, which showed promise in controlling diabetes in rodents [146]. However, a clinical trial with 19 T1DM patients revealed high variability in outcomes, likely due to poor vascularization and hypoxia, leading to minimal cell survival after 12 weeks [147]. A modified device (VC-02) with wider pores improved oxygenation but required immunosuppressive therapy due to a lack of immune protection. While some patients showed a C-peptide response, none achieved insulin independence, and the results were limited by insufficient cell engraftment and fibrous tissue formation [148]. Vertex Pharmaceuticals (Boston, MA, USA) conducted a phase I/II trial (NCT04786262) using fully differentiated insulin-producing cells derived from allogeneic PSCs, which also require immunosuppression. One patient achieved insulin independence, with an HbA1c of 5.2%, whereas the second showed only a 30% reduction in insulin needs. Despite promising early data, the need for immunosuppression remains a significant limitation, and further optimization is needed.

Researchers have developed a method to isolate and expand Tregs, which are often dysfunctional in T1DM while preserving their diversity and functionality. In a phase 1 trial with 14 patients, ex vivo-expanded autologous Treg therapy proved safe, with some Tregs persisting for up to 1 year without SAEs. Notably, several patients maintained stable C-peptide levels for more than 2 years, paving the way for a phase 2 trial to assess therapeutic efficacy [149].

2.3.3.2 Type 2 DM

The most common form of diabetes is T2DM, which typically occurs in adults, where the body becomes resistant to insulin or fails to produce enough insulin. MSCs are the most commonly used cell type in clinical trials for treating T2DM. A summary of 18 clinical trials highlights that intrapancreatic and intravenous infusion methods are typically employed, with cell doses ranging from 0.3 to 300 × 10⁶ cells/kg, with 1 × 10⁶ cells/kg being the most frequently used dose. The proposed mechanisms include β-cell regeneration, improved hepatic metabolic homeostasis, reduced insulin resistance, and the regulation of systemic inflammation [150]. MSCs play a role in initiating endogenous insulin production and stimulating the proliferation of β-cells. However, the transdifferentiation of MSCs into β-cells and their transplantation engraftment may not significantly contribute to the restoration of pancreatic function. Instead, MSCs secrete various cytokines and growth factors, including TGF-β and VEGF, which enhance islet function through both paracrine and autocrine mechanisms while facilitating the vascularization process [151]. MSCs also are stimulated by inflammatory cytokines, including TNF-α and IFN-γ, which in turn shift to an immunosuppressive phenotype by inducing the secretion of soluble factors that mediate immunomodulatory activities, such as prostaglandin E2 (PGE2), HGF, indoleamine-pyrrole 2,3-dioxygenase, and IL-10 [152].

MSCs can reduce islet cell apoptosis by decreasing the cleavage of caspase 3 [153]. MSCs can enhance the formation of autophagosomes by clearing impaired mitochondria and increasing the number of insulin granules [154]. MSC-mediated mitochondrial transfer is a mainstay method for rescuing injured cells, restoring mitochondrial functions [155], and repairing renal proximal tubular epithelial cells in diabetic nephropathy in vivo [156]. The mitochondria of MSCs can be transferred to β-cells under hypoxic conditions to increase the insulin secretion rate [157]. MSCs alleviate insulin resistance in T2DM patients by enhancing insulin signaling pathways. These compounds increase GLUT expression and increase the phosphorylation of IRS-1 and AKT in insulin-target tissues. MSCs also inhibit MG53, an E3 ligase that promotes IRS-1 degradation in skeletal muscles, which aids insulin sensitivity. Additionally, MSCs suppress NLRP3 inflammasome formation, reducing inflammation and enhancing IRS-1 and GLUT4 function in hepatic cells, further mitigating insulin resistance [150].

Clinical trials have shown promising potential for MSC therapy in T2DM. As of October 2024, a search on ClinicalTrials.gov using “mesenchymal stem cells” as the treatment and “diabetes type 2” as the condition revealed 25 registered studies, with nine completed [158]. MSC therapy significantly reduces fasting and postprandial blood glucose, HbA1c, and insulin requirements while improving C-peptide levels and insulin resistance with mild and manageable symptoms like fever, nausea, headache, and minor hypoglycemia [159, 160].

Islet transplantation has recently shown promise for treating T2DM. Wu et al. [161] conducted the first-in-human trial of autologous E-islets derived from patient-specific iPSCs to treat T2DM. In a 59-year-old patient with advanced T2DM, islet transplantation improved glycemic control within 2 weeks, resolving severe hyperglycemia and hypoglycemia. By week 32, time in target range reached 99%, HbA1c decreased from 6.6 to 4.6%, and insulin was discontinued by week 11. Fasting C-peptide levels tripled, and no tumors were detected during the 116-week follow-up. This study demonstrated the potential of stem cell-derived islets to restore islet function in late-stage T2DM [161].

Recently, Balboa et al. [162] demonstrated that insulin mutations disrupt β-cell differentiation in a neonatal diabetes model. Using iPSCs derived from affected patients, researchers have applied CRISPR/Cas9 to correct missense mutations in the insulin gene. Single-cell RNA sequencing revealed that, compared with corrected cells, mutant cells presented increased endoplasmic reticulum stress and reduced proliferation, highlighting the potential of gene editing to address β-cell dysfunction [162]. Overall, cell-based therapies show promise for treating diabetes, but further research is needed to confirm their efficacy and clinical applicability.

2.3.4.1 Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a life-threatening lung condition characterized by intense inflammation and fluid accumulation in the lungs’ tiny air sacs (alveoli), preventing adequate oxygen exchange. This inflammatory response, coupled with fluid buildup, impairs the ability of the lungs to fill with air, leading to a significant reduction in blood oxygen levels [167]. ARDS can rapidly progress, leading to organ failure and severe complications, often triggered by infections such as pneumonia and sepsis, trauma, or toxic exposure [168].

Preclinical studies have shown that MSCs effectively reduce inflammation and promote lung tissue regeneration in animal models of ARDS. This anti-inflammatory effect is crucial in ARDS, addressing both lung damage and excessive immune responses. MSC-based therapies have been shown to lower the levels of proinflammatory cytokines such as TNF-α, IL-1α, and IL-6, improve survival rates, and potentially mitigate cytokine storms and inflammation-related lung damage [169]. In response to the limitations of conventional treatments, MSCs also offer promising therapeutic options for managing ARDS because of their tissue repair properties and antimicrobial properties [170]. These attributes are particularly beneficial when infections trigger ARDS, help control inflammation, promote healing, and reduce the risk of further infection. Thus, using MSCs represents a comprehensive therapeutic strategy for improving ARDS outcomes, enabling faster recovery and minimizing lung damage.

Clinical trials on cell therapies for ARDS have evolved, with recent studies emphasizing larger cohorts and standardized outcome measures. Early studies (2013–2015) were relatively rare, often with fewer than 20 participants, and they primarily assessed safety and feasibility. Although some improvements in clinical parameters, such as the lung injury score (LIS) and SOFA score, have been reported, the lack of standardized outcome measures and variability in cell dosing have made comparisons challenging [171, 172]. Since 2017, there has been a shift toward larger patient cohorts and the incorporation of standardized outcome measures, such as the Acute Physiology and Chronic Health Evaluation II score and ventilator-free days [173]. A 2023 study revealed that allogeneic MSCs significantly improved ventilator-free days in patients with moderate to severe ARDS from COVID-19, highlighting the potential of MSC therapy [174]. Despite initial success, further research is crucial to optimize cell types, dosages, and delivery methods to maximize therapeutic benefits for ARDS patients.

2.3.4.2 Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD) is a chronic lung condition that primarily affects premature infants who require prolonged oxygen therapy or mechanical ventilation soon after birth. The pathogenesis of BPD involves oxidative stress and inflammation, compounded by incomplete lung development, leading to damage to fragile developing lungs [175]. BPD results in inflammation, fibrosis, and long-term respiratory problems that may persist into childhood and beyond. Infants with BPD often need ongoing respiratory support and are at increased risk of complications such as recurrent infections and pulmonary hypertension [176].

MSC therapy has emerged as a potential avenue for promoting lung repair. Clinical trials have focused on the potential of MSCs to repair lung tissue and reduce inflammation. MSCs are promising because they address oxidative stress, promote tissue repair, and mitigate inflammation, critical factors in BPD. Before 2018, research on MSC therapy was limited, with studies involving fewer than ten infants and focusing primarily on safety and feasibility. UC-MSCs and UCB-MSCs were selected for early-phase studies because of their accessibility and regenerative potential. These preliminary investigations revealed that MSC therapy was well tolerated and did not cause SAEs (Table S1) [177-179]. However, the lack of standardized outcome measures made cross-study comparisons difficult.

Recent advancements in the field have included trials with larger cohorts of high-risk premature infants, emphasizing both safety and long-term respiratory outcomes. The focus has shifted to quantifiable metrics, such as the duration of mechanical ventilation, oxygen dependency, and the need for respiratory support after discharge (Table S1) [180, 181]. Long-term follow-up remains crucial for evaluating the potential of MSC therapy to mitigate the effects of BPD [181]. A 2020 study administered MSCs to four high-risk infants and reported a lower incidence of severe BPD among treated infants, with improvements such as reduced reliance on respiratory support [178]. These findings provide initial evidence supporting the potential of MSC therapy in high-risk populations (Table S1). Overall, MSC therapy holds promise for severe BPD in premature infants. Future trials should include larger populations, assess long-term safety and efficacy, and determine the optimal dosage, timing, and administration to enhance outcomes and facilitate broader application.

2.3.4.3 COVID-19

COVID-19, caused by the coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and primarily affects the respiratory system, although it can also damage other organs. In severe cases, COVID-19 can trigger a hyperactive immune response known as a “cytokine storm,” leading to acute respiratory distress and organ failure [182]. High-risk groups, including elderly individuals and individuals with preexisting conditions, are more vulnerable to severe complications.

Clinical trials have investigated the use of MSC therapy for critically ill COVID-19 patients, focusing on UC-MSCs owing to their safety profile and ease of accessibility. A systematic review revealed that stem cells are safe and can significantly reduce both mortality and morbidity in COVID-19 patients. Additionally, stem cell infusion has been shown to improve pulmonary function, alleviate symptoms, and reduce inflammation [183].

One phase 2 trial involved 16 severely ill COVID-19 patients who received four doses of UC-MSCs (1 × 10⁸ cells per infusion) to assess the safety and efficacy of MSC therapy. The primary outcomes measured included improvements in oxygenation, reduced progression to critical illness, and decreases in inflammatory markers such as C-reactive protein and IL-6 [184]. The phase 1 trial conducted in 2020 highlighted significant clinical improvements in patients receiving MSC therapy compared with the placebo group. By day 14, MSC-treated patients had improved oxygenation, lower inflammatory marker levels, and no progression to severe respiratory failure, with no significant AEs [185]. A trial involving 12 severe COVID-19 pneumonia patients treated with UC-MSCs at a dose of 2 × 10⁶ cells/kg reported similar results, including reduced mechanical ventilation needs and respiratory stabilization, with no patients requiring escalated care [186].

Several studies have shown the potential of MSC therapy to modulate the hyperinflammatory response, improve oxygenation, reduce lung injury and ventilator dependence, and accelerate recovery in critically ill patients [185, 186]. However, the limited scale and short-term nature of these studies necessitate further investigation. Larger randomized trials with longer follow-up periods are needed to confirm the efficacy and safety of MSC therapy in patients with COVID-19. Research should optimize dosing, timing, and delivery (e.g., intravenous vs. inhaled) to enhance outcomes.

2.3.4.4 Chronic Obstructive Pulmonary Disease

COPD affects approximately 300 million people globally and was responsible for 3.23 million deaths in 2019, making it the third leading cause of death worldwide [186, 187]. It is triggered primarily by long-term exposure to harmful agents such as cigarette smoke and pollutants that cause chronic airway and lung inflammation. This leads to airway narrowing, mucus overproduction, alveolar destruction, impaired gas exchange, airflow obstruction, and trapped air, worsening breathlessness [188]. MSCs may mitigate COPD progression by secreting anti-inflammatory cytokines and MMP inhibitors, which collectively reduce inflammation and protect the lung structure, supporting improved lung function [189].

A systematic review and meta-analysis by Liu et al. [190] explored 20 preclinical studies on MSC treatment for COPD-related lung injuries and showed that MSC administration leads to significant improvements in lung health, as evidenced by metrics such as the mean linear intercept, a measure of lung tissue damage; TUNEL staining, an assessment of cell death in lung tissues; and pulmonary function tests. The meta-analysis revealed that MSCs effectively alleviated airway inflammation and enhanced anti-inflammatory cytokine production, facilitating tissue repair and reducing acute lung injury, underscoring the potential of MSCs to mitigate chronic inflammation, promote lung regeneration, and improve lung function [190].

Clinical trials highlight the significant potential of MSC therapy as a treatment for COPD. MSCs have been demonstrated to be safe and well tolerated, with no serious AEs reported in most cases, reduced inflammation, and increased exercise capacity and quality of life. While some studies have shown only modest improvements in lung function and symptom relief, others have reported substantial benefits, particularly in alleviating dyspnea and increasing 6-min walking test distances (Table S1). Furthermore, combining MSC therapy with advanced techniques such as endobronchial valve placement has synergistic effects, further reinforcing its potential to improve lung function [191].

2.3.7.1 POI/POF

POI describes an early loss of ovarian function in women younger than 40 years. The disease results in decreased estrogen levels, a lack of normal egg development, and infertility in the majority of affected women. The etiology and pathogenesis of POF are complex. Genetic defects, such as Turner syndrome or fragile X syndrome in women with only one X chromosome or those with fragile X chromosomes, are often associated with POF [215, 216]. Other pathogenic genetic variants affecting ovarian development and function from gonadogenesis to folliculogenesis and ovulation have recently been reported [217]. In addition to genetic factors, exposure to toxins and environmental factors, viral and bacterial infection, cancer treatment via radiation, chemotherapy, and pelvic surgery are common causes of POF [218, 219]. Autoimmune disorders, characterized by positivity of autoantibodies against the ovary, abnormal levels of cytokines, and dysfunction of Treg cells and Th17 cells, are also known risk factors for POF [220].

Cell therapy might act to improve POI through several mechanisms. PSCs, including female germline stem cells and iPSCs, can differentiate into several ovarian cell types, such as oocytes, estrogen-sensitive epithelial-like cells, and granulosa-like cells [221-223]. These cells are functionally active, produce estrogen, and promote follicular development. MSCs, while less potent in terms of their differentiation capacity, are able to migrate to inflammatory sites and rescue the compromised cellular environment [224]. MSCs support ovarian function by secreting growth factors, modulating the immune system, reducing oxidative stress, stimulating mitochondrial transfer, promoting angiogenesis, and enhancing ovarian cell survival [225].

Treatment of POI with MSCs has been shown to improve sex hormone levels and ovarian follicle development in female animals [226, 227]. Many studies have investigated the ability of PSCs, including ESCs and iPSCs, and adult stem cells, including MSCs from diverse sources, such as BM, AT, menstrual blood, UC, amniotic fluid, the amniotic membrane, the placenta, and the endometrium, to regenerate damaged ovaries and oocytes [228]. While research on ESCs and iPSCs has been limited to in vitro differentiation and in vivo evaluation in animal models of POI [221, 229], primary clinical data on the safety and efficacy of therapeutic MSCs have been reported (Table S4).

Overall, studies of cell therapy for POI treatment are still in an early phase, with few patients and randomized controlled results. All studies involved intraovarian injection and reported no AEs. Approximately 5–10% of women with POI can naturally conceive [230], whereas cell therapy resulted in different rates of natural conceptions, such as three out of 15 (20%) after BM-MNC treatment [231], two out of 14 (14%) and four out of 61 (7%) in two UC-MSC studies [232, 233], and three out of 15 (20%) after menstrual blood-derived MSC injection (compared with zero out of 16 in the control group) [234].

Cell therapy may enhance other treatments, such as ovarian autotransplantation, to preserve fertility in women with cancer. As cryopreservation and grafting of ovarian tissue often lead to follicle death due to hypoxia and a lack of blood vessels, stimulating revascularization with proangiogenic factors or cell therapy could improve outcomes. MSCs have been shown to support blood vessel formation around grafted ovarian tissue and increase follicle survival [235].

Hence, the efficacy of MSC therapy remains to be further investigated with more advanced control studies. Many issues need to be addressed: the best cell sources, impact of potential genetic/epigenetic modifications, licensing of therapeutic cell products, optimal cell dose and efficacy of repeat dosing, concomitant treatments, and use of biomaterials and scaffolds for cell therapy.

2.3.7.2 Asherman's Syndrome and Endometrial Atrophy

Asherman's syndrome and endometrial atrophy are disorders of the uterus. Asherman's syndrome occurs when scar tissue is formed in the uterine cavity, causing intrauterine adhesions [236], whereas endometrial atrophy is characterized by decreased thickness of the endometrium, the inner epithelial layer of the uterus [237]. Both diseases prevent implantation of fertilized eggs, resulting in infertility. Some studies have explored cell therapy for refractory cases (Table S4). Singh et al. [238] reported in a 5-year follow-up study a short-term increase in endometrial thickness after intrataurine injection of BM-MNCs combined with hormone therapy (n = 25). Six of seven amenorrhea women experienced menses again, and three had successful pregnancies [238]. Similarly, cell therapy using autologous menstrual blood-derived MSCs and the adipose-derived stromal vascular fraction improved endometrial thickness [239, 240]. Ma et al. [240] reported a natural conception and four conceptions via embryo transfer in twelve patients (42% in total) treated with menstrual blood-derived MSCs.

Two studies explored the use of a collagen scaffold combined with UC-MSCs for intrauterine injection in patients with recurrent Asherman's syndrome. In a study of twenty-six patients, therapy increased endometrial thickness and decreased the intrauterine adhesion score, which was correlated with improved endometrial proliferation and neovascularization [241]. Ten patients (38%) became pregnant within a 30-month follow-up, with eight successful pregnancies. These results are supported by a recent study in which endometrial thickness increased 3 months after UC-MSC/collagen treatment [242]. Four of the eighteen women became pregnant, including one natural conception and three successful embryo transfer conceptions that resulted in the birth of three healthy babies.

Furthermore, cell therapies, such as the use of MSCs from AT, have been explored for their ability to manage sexual dysfunction in both females and males because of their ability to enhance the endocrine function of the ovary and Leydig cells [243]. This approach could represent a novel method to complement conventional treatment with sex hormone replacement therapy. However, the efficacy of this method remains to be tested.

2.4.1.1 Heterogeneity of MSCs

The heterogeneity of MSCs arises from factors such as tissue origin, donor variability, and cell isolation and culture methods, leading to phenotypic and functional differences. MSCs from different sources exhibit distinct characteristics, including surface markers, proliferation rates, differentiation potentials, and immunomodulatory properties. This variability complicates standardization, therapeutic prediction, dosing optimization, and quality control (QC) for clinical use [245, 246]. One possible solution is a matching strategy, as hypothesized by Hoang et al. [5]. This approach emphasizes selection of MSCs based on their tissue origin to enhance therapeutic efficacy for specific conditions. For example, BM-MSCs might show promise for neurological injuries because of their neuroregenerative potential, AT-MSCs might be better suited for reproductive and skin conditions, and UC-MSCs might be advantageous for pulmonary diseases because of their strong immunomodulatory and proliferative abilities [5]. Taken together, the complexity of diseases, cell responses, and interactions with the microenvironment underscore the need for ongoing research to identify the most effective MSC types for different clinical applications.

2.4.1.2 Challenges in Clinical Applications

The biodistribution and fate of MSCs in the human body remain unclear because of challenges in tracking them post administration. Understanding how MSCs interact with injured microenvironments further complicates their study. Additionally, species-specific biological differences often hinder the translation of preclinical successes into clinical efficacy, posing a major obstacle to advancing MSC-based therapies [247].

The lack of standardized protocols for timing, dosing, and delivery routes also complicates the comparison of trial outcomes and the optimization of treatment strategies. Efficient stem cell delivery is particularly challenging for neurodegenerative diseases, as the blood–brain barrier (BBB) limits cell homing to the brain, and even when successfully delivered, poor survival and limited migration into host tissues diminish therapeutic efficacy. Additionally, long-term safety concerns, such as delayed AEs or disease recurrence, remain unresolved. Variability in study designs, methodological rigor, and outcome measures further hampers reliable assessment of safety and efficacy, underscoring the need for standardized evaluation methods [248].

Stem cell therapies face significant challenges, including strict regulatory scrutiny as advanced therapy medicine products and the high cost of producing GMP-compliant clinical-grade stem cells. These factors delay clinical translation and limit accessibility, posing barriers to widespread adoption.

2.4.1.3 Potential Complications of MSC Therapy

One significant issue is the occurrence of an instant blood-mediated inflammatory reaction (IBMIR) following the systemic administration of cellular products, which rarely occurs but might result in severe complications. Moll et al. [249] reported that in vitro culture of MSCs significantly increased tissue factor (TF) expression on their surface, causing IBMIR and severe outcomes in vivo. Upon intravascular infusion, TF, which acts as an initiator of the extrinsic coagulation cascade, becomes exposed to plasma coagulant factors, thereby activating them and triggering thrombi formation [250, 251]. Consequently, intravascular infusion of MSCs without appropriate anticoagulant prophylaxis can lead to mild-to-severe thromboembolic events [252]. Other factors, such as the presence of phosphatidylserine on the surface of MSCs, also contribute to thrombotic events [253]. Recently, Moll et al. [250] suggested improving MSC safety criteria by assessing TF levels and hemocompatibility of MSC products. Therefore, it is essential to consider these detrimental effects and develop strategies to mitigate the incidence of IBMIR and thrombosis to ensure safe cell-based therapies.

Furthermore, MSCs have been implicated in promoting tumor growth under certain conditions [254]. Conversely, MSCs are being explored as powerful therapeutic tools for treating cancer [255]. This dual role of MSCs raises concerns when MSC therapy is used for the treatment of diseases, and more studies with long-term follow-up are needed. Finally, the use of allogeneic stem cells introduces immunological risks. Rejection by the host immune system can necessitate immunosuppression, which introduces its own set of complications, including increased susceptibility to infections.

3.2.1.1 Neurological Diseases

Neurological diseases present a significant challenge because of the limited regenerative capacity of the nervous system following injury or illness. This limitation has spurred interest in innovative therapeutic strategies, particularly EV-based therapies, aimed at restoring function in patients with CNS disorders. Although the precise mechanisms remain under investigation, several promising approaches have emerged to enhance neural regeneration by targeting inflammation modulation, angiogenesis promotion, axon regeneration mediation and/or Schwann cell activation [307].

Inflammation plays a significant role in the pathology of various neurological diseases, including TBI, SCI, and neurodegenerative disorders such as AD and PD. Thus, modulating inflammation has emerged as an effective strategy to enhance the microenvironment of damaged nerves. MSC-derived EVs (MSC-EVs) decrease the production of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, which are known to worsen tissue damage and impede repair [308, 309]. Furthermore, MSC-EVs can modulate the immune response by influencing immune cells such as microglia and macrophages. By inducing M2 polarization, these EVs facilitate tissue repair and functional recovery. This mechanism has been observed in TBI models, where BM-MSC-EVs diminished M1 microglial activation and encouraged the polarization of macrophages toward the M2 phenotype by downregulating the expression of inducible nitric oxide synthase (INOS) and upregulating the expression of clusters of CD206 and arginase-1 (Arg1) [310]. This inflammatory modulation supports axon regeneration and functional recovery.

Restricted blood flow and oxygen supply to brain tissue after injury can severely damage both neural and vascular structures. Angioneurogenesis is a process involving the stimultanous regeneration of blood flow and promotion of tissue generation. BM-MSC-EVs have been shown to increase the population of endothelial cells in the ischemic striatum of stroke mice and in both the lesion boundary zone and the dentate gyrus of TBI mice, thereby inducing vascularization and long-term neuroprotection [311, 312]. Similarly, UC-MSC-EVs activate the Akt/mTOR pathway, which enhances neurovascular regeneration and alleviates blood‒spinal cord barrier disruption [313]. These findings underscore the importance of MSC-EVs in facilitating the regeneration of neural and vascular tissue in the injured CNS.

MSC-EVs also contribute to neuroprotection and axonal regeneration by providing neurotrophic factors, protecting against apoptosis and promoting neural plasticity. A variety of neurotrophic factors are released and packaged into MSC-EVs, including BDNF, GDNF, and VEGF, which promote neuronal survival, differentiation, and regeneration [314]. In conditions such as TBI and stroke, MSC-EVs have been shown to prevent neural cell death by promoting the expression of antiapoptotic proteins such as BCL-2 while suppressing the proapoptotic protein Bax [315]. In an SCI model, UC-MSC-EVs effectively relieved nerve tissue damage and promoted regeneration in the central nervous system by activating the Akt/mTOR pathway via the miR-29b-3p/PTEN axis [316]. In addition, MSC-EVs can facilitate axon regeneration following SCI by increasing the proliferation of endogenous stem cells through the ERK pathway [317, 318]. Human placental MSC-derived exosomes markedly increased the proliferation of endogenous neural stem/progenitor cells (NSPCs) with high expression of SOX2+GFAP+, PAX6+Nestin+, and SOX1+KI67+ cells through activation of the MEK/ERK/CREB signaling pathway [318]. However, UC-MSC-EVs not only improved the migration of NSPCs but also induced these cells to proliferate and differentiate via the ERK1/2 signaling pathway [317].

Notably, EVs isolated from BM-MSCs under hypoxic conditions more effectively promoted the proliferation and migration of Schwann cells, supporting their paracrine function through the circRNANkd2/miR-214-3p/mediator complex subunit 19 (MED19) axis. Furthermore, hypoxia-preconditioned BM-MSC-EVs demonstrated enhanced functionality in facial nerve repair and regeneration following facial nerve injury [319]. Additionally, miR-22-3p in AT-MSC-derived exosomes directly inhibited the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), thereby activating the phosphorylation of the AKT/mTOR axis, which promoted Schwann cell proliferation and migration, contributing to the repair of the recurrent laryngeal nerve [320, 321].

The promising use of EV-based therapies for promoting nerve regeneration and functional recovery has led to the initiation of clinical trials. A single-arm phase I clinical trial revealed that the intrathecal administration of allogeneic human UC-MSC-derived exosomes significantly improved various functional scores, including ASIA pinprick and light touch scores, SCIM III total scores, and NBD scores, in patients with subacute SCI compared with baseline levels [296]. In another clinical trial, patients with mild to moderate AD were treated with intranasal AT-MSC-exosomes at three doses of 2, 4, and 8 × 10⁸ particles twice per week (24 treatments in total) [295]. The medium-dose arm showed significant improvements in cognitive function, as evidenced by a decrease in AD Assessment Scale–Cognitive section (ADAS-cog) scores and an increase in Montreal Cognitive Assessment scores at week 12. Additionally, improvements in daily living activities were noted, and hippocampal volume shrinkage was reduced. A dose of 4×10⁸ particles is suggested for further large-scale multicenter clinical trials. Finally, the microRNA (miRNA) and protein expression profiles of AT-MSC-derived exosomes revealed 277 miRNAs and 1443 proteins, identifying potential neurotrophic factors for patients with AD in the current clinical trial [295]. These promising results indicate that EV-based therapies may be potential treatments for neurodegenerative diseases such as SCI and AD.

3.2.1.2 Hepatic Diseases

The liver possesses a unique ability to regenerate and repair itself, making it the only organ in the human body with a remarkable capacity. However, this regenerative ability is limited by the progression of liver disease and liver failure, which also present significant global health challenges. Cell-free treatments based on EVs offer a highly effective alternative to organ transplantation and cellular therapy for treating liver failure. EVs derived from stem cells, such as UC-MSCs, AT-MSCs, and BM-MSCs, have been most frequently reported to possess therapeutic potential for liver failure and liver injury. Additionally, EVs derived from human menstrual blood-derived stem cells, iPSC-derived MSCs, and liver stem cells have also shown protective effects against liver failure [322, 323].

In liver disease, inflammation is a hallmark of various conditions, including acute liver injury, nonalcoholic steatohepatitis, and autoimmune hepatitis. EVs help mitigate liver inflammation through different mechanisms. In acute liver failure, UC-MSC-EVs and AT-MSC-EVs containing miR-17 or lncRNA H19 reduced the expression of NLPR3, caspase-1, IL-1β, and IL-6, which are key factors related to hepatic injury [324, 325]. In addition, UC-MSC-EVs suppressed CCL4-induced liver injury by activating the ERK and IGF-1R/PI3K/AKT signaling pathways [326]. Human UC-MSC-derived exosomes have also been demonstrated to mitigate hepatic ischemia‒reperfusion (I/R) injury through the suppression of oxidative stress and the neutrophil-mediated inflammatory response. This is achieved by inhibiting Beclin1- and FAS-mediated autophagy and apoptosis while also modulating the inflammatory immune response [327]. Furthermore, UCB-MSC-derived exosomes presented anti-apoptotic, prosurvival, and anti-inflammatory effects through the modulation of the GSK3β-mediated Wnt/β-catenin signaling pathway or by influencing the balance between Treg and Th17 cells [328].

Fibrosis, characterized by excessive accumulation of ECM, is a key feature of chronic liver diseases, including cirrhosis. MSC-EVs inhibit hepatic stellate cell activation and proliferation through the delivery of antifibrotic miRNAs such as miR-122 and miR-199 and suppress key fibrogenic signaling pathways such as the TGF-β/Smad and Wnt/β-catenin pathways [329]. These vesicles also enhance liver regeneration by delivering HGFs and miRNAs that promote hepatocyte proliferation, counteracting fibrotic damage. Moreover, MSC-EVs also downregulate the expression of ECM proteins, such as collagen I and fibronectin, while promoting the expression of MMPs [330].

Menstrual blood stem cell-derived exosomes have been shown to inhibit liver cell apoptosis and reduce the number of liver MNCs and the level of the active apoptotic protein caspase-3 in injured livers, resulting in significantly improved liver function [322]. AT-MSC-derived exosomes attenuated liver I/R injury through the PGE2-mediated ERK1/2 and GSK‑3β signaling pathways [331]. In addition, miR-17 or lncRNA H19 in AT-MSC-derived exosomes promoted hepatocyte proliferation through the HGF/c-Met pathway and related downstream channels, thereby increasing the survival rate of rats with ALF [325]. In a rat partial- hepatectomy model, miR-124 in UC-MSC-derived exosomes promoted liver regeneration via the inhibition of Foxg1 [332]. Interestingly, both EVs isolated from normal and damaged tissue significantly attenuated the progression of apoptosis induced by CCL4 and induced the proliferation of hepatocytes by increasing the level of HGF at the site of injury [333].

3.2.1.3 Pulmonary Diseases and Lung Fibrosis

Recent research has elucidated the critical role of EVs in the treatment of various pulmonary diseases, including acute lung injury (ALI), ARDS, BPD, and idiopathic pulmonary fibrosis. EVs act as natural carriers of bioactive molecules, modulating cellular functions and promoting tissue repair. The mechanisms of action of these compounds in pulmonary disease and fibrosis are multifaceted and include inflammation, fibrosis, apoptosis, and tissue regeneration.

MSC-derived EVs are particularly effective in mitigating inflammation by modulating cytokine signaling pathways. These EVs inhibit the activation of key inflammatory pathways, such as the NF-κB and TGF-β pathways, leading to reduced expression of proinflammatory cytokines such as IL-6 and TNF-α, thereby alleviating inflammation in lung tissues [334]. BM-MSC-EVs decrease the infiltration of inflammatory cells, both total white blood cells and neutrophils, and MIP-2 cytokine levels in ALI mice [334]. Similarly, AT-MSC-EVs containing miR-27a-3p are effectively taken up by alveolar macrophages, which facilitates polarization toward the M2 macrophage phenotype, a crucial process in the repair of acute lung injury. Specific miRNAs within EVs, such as miR-21-5p, miR-182-5p, miR-23a-3p, miR-Let-7, and miR-27a-3p, coordinate the response to pulmonary fibrosis by downregulating proinflammatory genes, enhancing phagocytosis and promoting the resolution of inflammation [335]. Additionally, these miRNAs decrease the synthesis of proinflammatory cytokines and inhibit signaling pathways associated with the activation of inflammation, including the NLRP3 inflammasome and NF-κB. Furthermore, AT-MSC-EVs containing miR-127-3p and miR-125b-5p further contribute to M2 macrophage polarization, highlighting their role in mitigating inflammation and supporting tissue repair in pulmonary conditions.

EVs have also proven effective in protecting and regenerating pulmonary epithelial cells, which are crucial for maintaining alveolar integrity and overall lung function. MSC-EVs promote epithelial proliferation, prevent apoptosis, and help restore epithelial integrity. MSC-EVs have been shown to protect pulmonary epithelial cells from the harm caused by ALI/ADRS [336]. BM-MSC-EVs enhance barrier function and inhibit epithelial apoptosis by upregulating GPRC5A, which in turn increases the expression of junction proteins such as E-cadherin, claudin-1, occluding, and ZO-1. Moreover, these EVs deliver miR-425, activating the PI3K/Akt pathway to alleviate hyperoxia-induced ALI. The transfer of lncRNA-p21 via EVs also prevents epithelial apoptosis and ALI by modulating the expression of miR-181 and SIRT1. MSC-EVs that overexpress miR-30b-3p enhance antiapoptotic and proliferative effects, effectively countering LPS-induced alveolar epithelial cell apoptosis. These effects are vital for preventing further lung damage during the inflammatory response.

In the context of pulmonary fibrosis, MSC-EVs play a crucial role by delivering antifibrotic miRNAs, such as miR-29 and miR-21, which target profibrotic genes in myofibroblasts and effectively suppress excessive ECM production [337]. MSC-EVs also inhibit fibrogenic signaling pathways, such as the TGF-β/Smad and Wnt/β-catenin pathways, which are critical for the development of lung fibrosis [334]. Specifically, miR-29b-3p and miR-186 have the potential to inhibit the activation of fibroblasts by regulating FZD6, SOX4, and DKK1 [335]. Moreover, EVs suppress the synthesis of collagen and fibronectin, critical components of the ECM [338]. BM-MSC-EVs reduced the expression of profibrotic factors (TGF-β1, α-SMA, and collagen I/III) through the modulation of miR-23a-3p and miR-182-5p while also inhibiting the NF-κB/Hedgehog pathways. Furthermore, UC-MSC-EVs and EVs derived from bronchial epithelial cells can directly disrupt the TGF-β pathway, thereby inhibiting myofibroblast proliferation and collagen production [338].

The COVID-19 pandemic caused by SARS-CoV-2 highlighted, the significant challenges in managing severe respiratory complications, particularly ARDS. MSC-EVs have emerged as a promising therapeutic approach to mitigate the cytokine storm, restore suppressed antiviral defenses, and repair lung damage associated with mitochondrial dysfunction. To date, clinical trials evaluating MSC-EVs are limited and focus mainly on patients with COVID-19. In a phase I trial involving 24 severe cases of ARDS associated with COVID-19 [297, 300], a single 15 mL intravenous dose of allogenic BM-MSC-EVs (ExoFlo) improved clinical status and oxygenation, as evidenced by an increased PaO₂/FiO₂ ratio, a reduced neutrophil count, and improved lymphopenia [297]. In a subsequent phase II trial, 102 patients with COVID-19-associated moderate to severe ARDS were enrolled at five sites across the United States and received two doses of either 10 or 15 mL of ExoFlo [300]. The results indicated that 60-day mortality was lower in the ExoFlo-15 group than in the placebo group and that the number of ventilation-free days improved [300]. A similar reduction in mortality was observed in patients treated with human placental MSC-derived small EVs, although this treatment did not significantly impact laboratory values [301]. Additionally, nebulized EVs have been shown to be safe and effective for pulmonary diseases [294, 298, 299]. Improved pulmonary lesions were observed via CT imaging in seven patients with severe COVID-19 who received aerosol inhalation of AT-MSC-exosomes at a dose of 2 × 10⁸ particles/day for 5 consecutive days [298]. Nebulized UC-MSC-EVs also promoted the absorption of pulmonary lesions and reduced the duration of hospitalization for mild cases of COVID-19 pneumonia [299]. In summary, MSC-EVs demonstrate a favorable safety profile, the capacity to restore oxygenation, the ability to downregulate the cytokine storm, and the potential to reconstitute immunity.

3.2.1.4 Cardiovascular Diseases

The limited regenerative capacity of cardiomyocytes poses a significant challenge for cardiovascular therapy, particularly in conditions involving extensive damage, such as myocardial infarction (MI) and I/R injury. Emerging EV-based therapies offer promising avenues for cardiac regeneration, leveraging the bioactive cargo of EVs to modulate inflammation, prevent apoptosis, promote cell proliferation, and stimulate tissue repair [339].

Many preclinical studies have shown that MSC-EVs exhibit potent immunomodulatory effects by altering inflammatory pathways and macrophage polarization. BM-MSC-EVs have immunomodulatory effects on macrophages and inhibit TLR4 through the shuttling of miR-182, leading to attenuation of myocardial I/R in a mouse model [340]. Similarly, exosomes released from BM-MSCs containing miR-182-5p not only diminish inflammation or necrosis but also ameliorate cardiac function and reduce MI via Gasdermin D [341]. In another study, BM-MSC-EVs induced the polarization of macrophages to the M2 phenotype via miR-21-5p, which resulted in a decrease in inflammation and promoted heart repair [342]. UC-MSC-EVs enriched with miR-100-5p suppressed FOXO3 to inhibit NLRP3 inflammasome activation and reduce cytokine release, protecting cardiomyocytes from hypoxia/reoxygenation-induced pyrosis and injury [343].

In addition, other studies have indicated that MSC-EVs can mitigate myocardial damage by preventing apoptosis and promoting CM proliferation [344, 345]. BM-MSC-EVs presented with miR-486-5p regulate the PTEN/PI3K/AKT signaling pathway to inhibit the apoptosis of injured cardiomyocytes and repair the myocardial injury caused by I/R [344]. Other studies have shown that the presence of the lncRNA HCP5 in BM-MSC-EVs protects myocardial cells against I/R injury by activating the IGF-1/PI3K/AKT pathway by sponging miR-497 [345]. miR-143-3p in BM-MSC-EVs effectively reduces apoptosis through regulating autophagy via the checkpoint kinase 2 (CHEK2/CHK2)/beclin 2 (BECN2) pathway [346].

AT-MSC-EVs demonstrate robust cardioprotective properties that safeguard the myocardium from I/R injury by activating the Wnt/β-catenin signaling pathway [347], effectively prevent heart damage by targeting Bcl2 binding component 3 (BBC3/PUMA) and the ETS proto-oncogene 1 transcription factor (ETS1) through the miR-221/miR-222 pathway [348], and they also protect cardiomyocytes from oxidative stress-induced apoptosis and offer cardioprotective benefits [349].

PSCs, such as ESCs and iPSCs, possess enormous potential to differentiate into various cardiac cell types, making them promising candidates for cardiac regeneration. ESC-derived EVs promote endogenous natural repair mechanisms and improve cardiac function following MI [350]. iPSC-derived EVs have protective effects on heart cells in vitro, promote angiogenesis, and increase apoptosis in vivo. Injection of iPSC-derived EVs into the mouse ischemic myocardium prevents I/R injury and provides cardioprotective effects [351].

The clinical application of EV-based therapies has shown promising results in CVD treatment. A 59-year-old man with nonischemic cardiomyopathy received three intravenous infusions of the EV-enriched secretome from cardiovascular progenitor cells derived from iPSCs (SECRET-HF) [302]. The patient was classified as New York Heart Association (NYHA) class III and had previously been implanted with a prophylactic internal cardioverter defibrillator. Six months posttreatment with SECRET-HF, his condition improved to NYHA class II. The patient reduced his daily diuretic dosage and had better scores on the Minnesota living with heart failure (MLHF) questionnaire as well as improved echo parameters.

3.2.1.5 Musculoskeletal Disease

Bone and cartilage are composed of connective tissue and are fundamental structures that play a role in controlling body movement, providing mechanical support and mineral storage and protecting internal organs. The repair process following bone damage is highly complex and involves interactions among various cell types and biological signaling pathways. Bone regeneration includes a series of coordinated biological processes referred to as osteoconduction and osteoinduction. Compared with the repair of bone-damaged tissue, the treatment of cartilage damage presents greater challenges and complexities, primarily due to the inherent characteristics of cartilage, which does not regenerate readily. EVs represent a promising new strategy for bone and cartilage reconstruction therapy. They have significant potential in promoting repair and regeneration by regulating the microenvironment at injury sites, stimulating bone and playing proangiogenic roles in various cell types essential for bone formation.

AT-MSC-EVs inhibit the secretion of IL-1β and IL-18 and suppress the NLRP3 inflammasome in osteoclasts, leading to reduced bone resorption and recovery from bone loss in streptozotocin-induced diabetic osteoporosis rats [352]. To protect cartilage and bone from OA, BM-MSC-EVs safeguard chondrocytes from apoptosis and inhibit macrophage activation. Additionally, BM-MSC-EVs restore the expression of chondrocyte markers, including type II collagen and aggrecan, while suppressing the expression of catabolic markers (MMP-13, ADAMTS5) and inflammatory markers (iNOS) [353]. Zheng et al. [354] discovered that exosomes derived from normal primary chondrocytes can restore mitochondrial function and promote the polarization of macrophages to the M2 phenotype. Additionally, intra-articular injection of these exosomes effectively inhibited the onset and progression of OA [354].

In vitro data indicate that BM-MSC-derived exosomes enhance obsteoblast activity, promoting osteogenic differentiation, ultimately facilitating fracture healing via miR-22-5p/Anxa8 axis [355]. In a mouse model of osteoporosis, BM-MSC-EVs that contain MALAT1 enhance osteoblast activity by regulating the miR-34c/SATB2 axis, thereby alleviating osteoporosis [356]. Similarly, exosomes from vascular endothelial cells can reverse glucocorticoid-induced inhibition of osteoblasts by suppressing ferritinophagy-dependent ferroptosis [357]. Hu et al. [358] discovered that BM-MSC-EVs enriched with miR-335 enhance osteoblast differentiation and accelerate fracture healing by targeting VapB and activating the Wnt/β-catenin pathway.

In 2020, a pilot safety study was approved by the USA Institutional Review Board to evaluate the safety of allogenic BM-MSC-EVs (ExoFlo) [303]. The study included a substantial number of Navy SEAL veterans, specifically thirty-three individuals diagnosed with combat-related injuries resulting in moderate to severe OA. These veterans received single 2 mL ExoFlo treatment for various sites of OA: knee (n = 58), shoulder (n = 32), elbow (n = 16), hip (n = 12), ankle (n = 8), and wrist (n = 6). After 6 months of follow-up, no complications were reported, and patients demonstrated improvements in the Brief Pain Inventory, Oswestry Disability Index, Lower Extremity Functional Scale, Upper Extremity Functional Scale, and Quick Dash Scale scores.

3.2.1.6 Skin and Wound Healing

Numerous investigations have indicated that MSC-EVs, which can overcome the inherent limitations of MSCs [359], are advantageous for all phases of the skin repair process, such as hemostasis, inflammation, proliferation, and remodeling [360]. The potential functions of EVs in cosmetic applications and the treatment of various skin conditions, including antiaging, wound healing, and anti-inflammatory effects, have been increasingly revealed. Beneficial EVs can be derived from multiple sources, such as plants, probiotics, and human MSCs [361]. Among them, MSC-EVs have been studied the most for cutaneous applications [361]. Some studies demonstrate a role in antiwrinkle and antiaging [362, 363] of MSC-EVs, which is meaningful for the development of novel cosmetic products. The general mechanism by which MSC-EVs recover the functions of senescent dermal fibroblasts [364] and keratinocytes [365] is mediated by downregulation of oxidative stress and senescence-associated molecules, which leads to increased expression of ECM components, including collagen and elastin [366, 367]. In addition, EVs derived from neonatal human dermal fibroblasts (HDFs) have been utilized to encapsulate and deliver COL1A1 mRNA to the dermis via microneedles to reduce wrinkles in a photodamaged murine skin model [18]. The results demonstrated that COL1A1 mRNA-loaded HDF-EVs could dramatically induce dermal collagen grafts, leading to impressive elimination of wrinkles. Another representative application of MSC-EVs is wound healing [360, 368].

The healing functions of MSC-EVs may be mediated mainly by their miRNAs [369]. For example, miR-233 in BM-MSC-EVs can regulate the M1-like inflammatory phenotype to the M2-like regenerative phenotype of macrophages [370], which is beneficial for the phase transition from inflammation to proliferation. In addition, miR-125a-3p in ADSC-EVs supports angiogenesis [371], whereas miR-126 in BM-MSC-EVs increases new capillary formation [372], miR-192-5p in AT-MSC-EVs can eliminate fibrosis during remodeling [373]. Notably, MSC-EVs have also been shown to exert therapeutic effects on severe cutaneous inflammatory diseases, such as atopic dermatitis and psoriasis [374]. For example, IFN-γ-primed MSC-EVs can reduce both inflammation and the expression of IL receptors for Th2 cytokines while normalizing the skin barrier in an AD murine model [375]. Moreover, IFN-γ-primed UC-MSC-EVs exhaust Th17 cells and decrease the concentration of inflammatory cytokines in psoriasis murine skin [376].

The clinical use of EVs derived from AT-MSCs and platelets has been demonstrated to be safe for skin applications [304-306]. After 12 weeks of treatment with AT-MSC-EVs, patients experienced a reduction in skin wrinkles, along with increased skin elasticity and hydration and a decrease in melanin levels [306]. Additionally, histopathological analysis of facial skin tissues revealed greater deposition of collagen and elastic fibers than on the control side.

3.2.2.1 EVs as Therapeutic Carriers

3.2.2.2 Modified EVs for Targeted Delivery of Therapeutics

While efficient therapeutic loading is a critical determinant of therapeutic efficacy, ensuring the selective delivery of therapeutic-loaded EVs to target tissues is equally important for minimizing off-target effects and toxicity. Strategies for EV engineering or modification have gained attention as promising approaches to enhance tissue targeting. To date, genetic modification, lipid insertion, and chemical ligation are among the most common methods explored in regenerative medicine to optimize EV-targeting capabilities [383].

3.2.2.3 EVs as Gene Therapy Vehicles

EVs could serve as platforms for delivering gene-editing tools, such as CRISPR–Cas9. This technology could be used to correct genetic defects, increase cellular regeneration, and/or facilitate tissue repair in conditions such as inherited diseases or injuries [400]. The CRISPR/Cas9 system consists of a Cas9 nuclease and a single guide RNA (sgRNA), which direct Cas9 to specific DNA sequences for editing. This system can be delivered to target cells in the form of DNA (plasmid), mRNA, or ribonucleoprotein (RNP) complexes [401]. While the use of EVs to deliver CRISPR/Cas9 has been extensively studied for cancer therapy [402], EVs also hold significant potential for applications in regenerative medicine. For example, EVs derived from HEK293T (human embryonic kidney) cells were transfected with Cas9 RNP. These modified EVs were shown to target specific sites on the HGF gene, resulting in the upregulation of endogenous HGF expression in an acute liver injury-induced mouse model [403]. Similarly, Cas9 RNPs were loaded into purified exosomes isolated from hepatic stellate cells (LX-2 cells) through electroporation. These exosomes targeted PUMA (p53 upregulated modulator of apoptosis) and Cyclin E1 (CcnE1), which are important therapeutic targets for acute liver injury and chronic liver fibrosis, respectively [404]. Together, these findings highlight the potential of Cas9 RNP EVs as a promising therapeutic strategy for the treatment of liver diseases.

In another study, Cas9 RNPs were loaded into isolated serum EVs via a transfection kit. These modified EVs caused exon deletion in the dystrophin gene, allowing dystrophin expression in muscle fibers in a DMD mouse model. This approach opens new opportunities for the rapid and safe delivery of CRISPR components to treat DMD [405]. Furthermore, exosomes from human UC-MSCs were modified with a CAQK peptide to target brain injuries. Cas9 DNA was then loaded into these exosomes via electroporation. When administered intravenously to SCI model mice, the exosomes reached the injury site and inhibited the inflammatory response in the spinal cord, promoting recovery after SCI [406]. A recent study successfully produced Cas9 RNP-loaded exosomes, termed NanoMEDIC, from HEK293T cells. These exosomes exhibited exon skipping with an efficiency exceeding 90% in skeletal muscle cells derived from the iPSCs of DMD patients. These results highlight the potential of NanoMEDIC for in vivo genome editing therapy to target DMD [407]. In research conducted by Osteikoetxea et al. [408], Cas9 and sgRNA-containing plasmids were transfected into Expi293F cells to produce Cas9 RNP-loaded EVs. These engineered EVs effectively knocked down the PCSK9 gene in vitro. Low PCSK9 gene expression reduces circulating low-density lipoproteincholesterol, indicating the therapeutic potential of CRISPR/Cas9-loaded EVs for treating atherosclerotic CVD.

3.2.3.1 Inhibiting EV Biogenesis and Release

The list of inhibitors of EV biogenesis and release has been intensively reviewed by Catalano and O'Driscoll [411]. Among the listed inhibitors, GW4869 has been studied relatively extensively for cancer and regenerative therapy. GW4869 is a chemical compound commonly used as an inhibitor of neutral sphingomyelinase (nSMase), an enzyme involved in the formation and release of exosomes [412]. Inhibiting nSMase with GW4869 reduces exosome release from cells. Dinkins et al. [413] reported that GW4869 treatment reduced Aβ plaques in the brains of an AD mouse model, indicating the potential of GW4869 in targeting EV release for the treatment of AD. Similarly, inhibiting exosome release with GW4869 reduces sepsis-induced cardiac inflammation, attenuates myocardial dysfunction, and prolongs survival in an LPS-induced sepsis mouse model [414]. In another study, treatment with GW4869 inhibited exosome release by fibroblasts (CFs), preventing myocardial hypertrophy and cardiac fibrosis in a mouse model of cardiac hypertrophy [415].

3.2.3.2 Blocking EV Uptake

Blocking EV uptake can prevent harmful signals or materials from entering healthy cells. This can be achieved by targeting EV-mediated uptake receptors on the EV surface via specific siRNAs or antibodies. For example, the uptake of EVs from senescent BMSCs by muscle satellite cells contributes to the development of sarcopenia. Therefore, inhibiting EV uptake through blockade of CD81 with CD81-specific siRNA or an anti-CD81 antibody attenuated sarcopenia in aged mice [416].

The major challenges in using EVs as therapeutic targets are related to specificity and safety concerns. As EVs are involved in both physiological and pathological processes, therapy must selectively target disease-related EVs without disrupting the normal function of healthy EVs or causing unintended side effects in healthy tissues. While both in vitro and in vivo studies have shown promise for EV-targeting therapy in disease models, this approach remains far from clinical application. A more comprehensive understanding of EV biogenesis and uptake is needed before this strategy can be translated into an effective treatment.

4.2.1.1 Bioactive Ceramics (Bioceramics)

In recent decades, bioceramics, including hydroxyapatite (HAp), zirconia, alumina, tricalcium phosphates and bioactive glasses, have gained special interest in musculoskeletal regeneration. Owing to their high biocompatibility with cells and good ECM interaction with bone, bioceramics have been frequently used in hard tissue regeneration [455].

4.2.1.2 Polymers

Polymers, both natural and synthetic, are particularly effective for soft tissue regeneration, including cardiovascular and skin tissues, due to the ability to control porosity and degradation rates. Natural polymers such as alginate, gelatin, and collagen are widely used because of their biocompatibility and ECM-mimicking properties. However, alginate often needs to be combined with other materials to support cellular interactions, as it lacks cell-adhesive motifs. Recently, hybrid systems that integrate alginate with cellulose nanofibrils have improved mechanical strength and printability, expanding their use in tissue engineering [456].

Synthetic polymers such as poly(L-lactic acid), poly(L-lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone (PCL), and polyurethane (PU) are also of interest because of the ability to control mechanical properties and degradation rates. Owing to the availability of the click-chemistry technique and versatile poly(ethylene glycol) (PEG), novel copolymer structures with new functions to meet tissue-specific requirements have been created. For example, polymers can be functionalized with peptides to increase cell adhesion. The incorporation of gelatin methacrylate (GelMA) with PEG could further improve cellular interactions, increasing the feasibility of bioprinting applications [457].

4.2.1.3 Decellularized ECM

The decellularized ECM (dECM) has attracted considerable attention in tissue engineering because of its ability to mimic the intricate composition of the native ECM. This intricacy plays a crucial role in providing biochemical cues that direct cell differentiation and tissue-specific functions [458]. Recent advances in the processing of dECM have increased their solubility and printability, enabling fabrication of constructs similar to the native tissue environment. Zhe et al. [459] discussed the utilization of dECM bioinks in 3D bioprinting for tissue engineering by means of creating an enabling environment that permits repair and tissue regeneration.

4.3.3.1 Engineering Total Artificial Heart

Since the first mechanical heart transplant with the Liotta-Cooley mechanical heart, in 1969, which kept the patient alive for 64 h [502], heart engineering has continued to develop. The 1980s saw the development of the total artificial heart (TAH) by the University of Utah. William C. DeVries and Willem J. Kolff developed the Jarvik-7 and transplanted it into a 61-year-old man with chronic congestive heart failure [503]. Despite normal blood pressure and cardiac output, the patient experienced complications and died after 112 days. The TAH has since evolved, with the Jarvik-7 prototype leading to commercial versions like SynCardia, AbioCor TAH, and CARMAT TAH, all US FDA-approved as a bridge to transplant [502, 504]. However, TAH outcomes remain limited, with the longest life extension 1374 days (4 years) [502, 505]. Torregrossa and colleagues [505] reported a 10% device failure rate and 24% of the patients died on device support in addition to other AEs including systemic infections (53%), driveline infections (27%), thromboembolic events (19%), and hemorrhagic events (14%). TAH implants are still limited as temporary transplants due to device size and patient-specific blood circulation needs.

Xenotransplantation of genetically humanized hearts is especially needed for patients with heart failure until suitable cardiac allografts are available. The first xenotransplantation occurred in 1984 with transplantation of a baboon heart into a human infant, who suffered from hypoplastic left heart syndrome [506]. However, the baby survived only 20 days due to graft failure [507]. Subsequently, scientists favored xenotransplanted porcine hearts over nonhuman primates (NHPs) due to the decreased risk of transmitting pathogens like simian immunodeficiency virus, which can transform into HIV [508]. The first cardiac xenotransplantation of wild-type pigs into humans in 1997 resulted in a patient surviving 1 week before hyperacute rejection [509]. Host immune cells recognized xenoantigens on porcine vascular endothelial cells, leading to the complement mediated activation endothelial injury and activation of the coagulation cascade, causing graft failure. Decades later, Dr. Bartley Griffith performed two xenotransplantations of genetically modified pig hearts with patients surviving 2 months and 6 weeks, respectively [508]. While few engineered xeno-TAH strains have been tested due to knowledge limits and ethical concerns, the topic remains promising as a potential solution for millions of patients worldwide.