BACKGROUND AIMS:Over the past 20 years, USA regulation 21 CFR Part 1271 has served as the cornerstone regulation for human cells, tissues, and cellular and tissue-based products (HCT/Ps) in the United States. Designed to ensure public health safety while fostering innovation, the framework has significantly shaped the development and oversight of regenerative medicine. METHODS:This paper examines the evolution of 21 CFR Part 1271 since its inception, highlighting key amendments, milestones, and regulatory challenges. It explores its impact on the industry, focusing on case studies that underscore its influence on clinical translation and commercialization. RESULTS:The analysis shows that 21 CFR Part 1271 addresses ongoing controversies, such as compliance and enforcement challenges, and discusses how emerging technologies like exosomes, gene editing, and bioprinting may reshape its future. CONCLUSION:By reflecting on two decades of regulatory experience, this paper provides insights into the successes and limitations of 21 CFR Part 1271 and proposes pathways for its modernization to address the growing complexity of regenerative medicine.

Peripheral nerve injury (PNI) is characterized by a loss of cellular and axonal integrity that can lead to limited functional recovery. Because many PNIs are not amenable to repair with traditional techniques, cell therapies have emerged as a treatment option. Exosomes, which can be secreted by Schwann cells (SC), carry cellular signaling molecules that facilitate intercellular communication. Our laboratory and others have success using SC-derived exosomes in preclinical PNI models; however, there is no study that directly compares recovery from different PNIs after exosome treatment. Thus, the currently study investigated if SC-derived exosomes can effectively treat different types of PNI, as measured by axonal regeneration and functional recovery. Adult male Fischer rats were divided into several treatment groups, including nerve transection, reversed autograft, conduit + exosomes, nerve crush, and nerve crush + exosomes. Animals underwent functional assessment through the duration of the experiment and at the conclusion (11 weeks), electrophysiological and histological characteristics were assessed. Results indicate an injury-specific effect of SC-derived exosome treatment. Specifically, exosome treatment improved axon regeneration/myelination, muscle recovery, and gait characteristics for severe, large-gap injuries. These SC-exosome based effects were not observed in crush injuries. Taken together, the results of the current study indicate that there may be differences in recovery based on injury type after PNI and treatment with SC-derived exosomes, potentially due to differences in exosome retention/distribution at the injury site. Future studies should explore how exosomes are distributed following administration across various PNI models, as their therapeutic effects may be more pronounced in upstream regions.

Peripheral nerve injury (PNI) is characterized by a loss of cellular and axonal integrity, often leading to limited functional recovery and pain. Many PNIs are not amenable to repair with traditional techniques; however, cell therapies, particularly Schwann cells (SCs), offer the promise of neural tissue replacement and functional improvement. Exosomes, which carry cellular signaling molecules, can be secreted by SCs and have shown promise in PNI. Our laboratory has had success using SCs in preclinical and clinical treatment settings. Transplanted cells have several known limitations, which exosomes mitigate. To that end, the current study investigated if implanted SC-derived exosomes in conduits, conduits with SCs, reverse autograft, or empty conduits comparably improve axonal regeneration and pain outcomes 16-weeks after repair of a long gap PNI in adult rats. Results show that there were no differences between groups in the von Frey filament testing or in the Hargreaves test. Electrophysiological testing showed a significant difference between the injured (ipsilateral) and uninjured (contralateral) limbs while histological assessment showed a significant difference between axonal counts in different areas of the conduit. Based on the results of the current study, more research is needed to understand the therapeutic role of exosomes in PNI.

INTRODUCTION:There is limited long-term clinical outcome data supporting the use of cell-based therapy to treat heart failure. The HYPERION study (NCT03071835) followed long-term outcomes of patients with ischemic cardiomyopathy (ICM) and non-ischemic cardiomyopathy (NIDCM) who received mesenchymal stromal cells (MSC). We hypothesized that improved cardiac parameters predict longer event-free survival. METHODS:We performed a Kaplan-Meier analysis to examine event-free survival as the primary outcome. Time-to-event information was captured from all eligible participants. Endpoint events were defined as death (all-cause), Left Ventricular Assist Device (LVAD) placement, or Heart Transplant. Subjects were categorized based on increase in Left Ventricular Ejection Fraction (LVEF) or decrease in Left Ventricular End Diastolic Volume (LVEDV) for comparisons within disease etiologies. RESULTS:There were 134 men and 21 women, with mean age 60.0 ± 11.0 years. There were 121 (78%) with ICM and 34 (22%) with NIDCM. By the end of long-term follow-up (~13 years), 38 (24.5%) subjects had deceased, 5 (3.2%) received LVAD, and 8 (5.2%) underwent heart transplantation. Post-therapy increase of ≥5% LVEF was associated with longer event-free survival in NIDCM (HR:0.31; 95%CI, 0.11,0.86; P = .025), but not ICM (HR:1.14; 95%CI, 0.47,2.72; P = .776). Conversely, reduction in left ventricular end-diastolic volume (LVEDV) was associated with longer event-free survival in ICM (HR:0.16; 95%CI, 0.05, 0.55; P = .008) but not NIDCM (HR:0.35; 95%CI, 0.1,1.2; P = .098). ICM improvers had LVEDV of 225.7 ± 95.9 mL at baseline and 209.0 ± 100.6 mL by year 5 (P = .046). NIDCM improvers had LVEF of 27.2 ± 8.9% at baseline and 36.1 ± 11.6% by year 5 (P = .018). CONCLUSION:In this long-term observational cohort analysis, improvement of LVEF and/or reduction in LVEDV was associated with survival benefits among subjects with NIDCM and ICM, respectively. In both etiologies the respective improvements are sustained for up to 5 years, providing evidence that cell-based therapy may be a promising and durable treatment option for patients with heart failure.

Background Hypoplastic left heart syndrome (HLHS) requires a series of 3-staged palliative operations, in which the right ventricle (RV) assumes systemic circulation under increased pressure-overload conditions. These patients have a shortened lifespan, often due to RV dysfunction. Neonatal cardiac progenitor cells (nCPCs) improve RV performance in animal models of pressure overload-induced RV dysfunction, as is experienced by HLHS patients. Objectives The authors conducted a phase 1 trial to assess the impact of autologous nCPCs injected into the RV myocardium during stage 2 operation. The primary endpoints were safety and feasibility, with efficacy assessed by evaluating RV size and function over a 1-year period. Methods The study enrolled HLHS patients in 2 groups. Group A consisted of 9 consecutive patients who received nCPC injections during their stage 2 operation. Group B involved a multicenter, randomized, double-blind trial comparing nCPC injections (n = 8) to standard-of-care (SOC) treatment (n = 8). The treatment arm received nCPC injections into the RV myocardium. All caregivers and cores except the surgeon were blinded to treatment. Results The trial met its primary safety and feasibility endpoints. However, the primary efficacy endpoint—changes in RV size or function measured by cardiac magnetic resonance and echocardiography—was not achieved. Despite this, secondary outcomes indicated potential clinical benefits. In group B, the mean major adverse cardiac events rates per 100 person-days were higher in the SOC arm (0.23) compared to the nCPC arm (0.00; P = 0.013) after stage 2 operation. The total 1-year in-hospital length of stay due to cardiac and vascular diseases was 15 days and 2 days per 100 person-days for the SOC and nCPC arms, respectively (P = 0.035). Mechanistic studies revealed that, on average, patients in the nCPC arm exhibited noticeable increases from baseline in plasma levels of VEGFC, VEGFD, TNF-α, and monocyte chemotactic protein-1 compared to the SOC arm by postoperative day 5, though the differences were not statistically significant after false discovery rate adjustment. Whereas subject numbers were insufficient to show significance for the composite endpoint of death or listed for transplantation for group B, when combining both groups A and B, no events were seen in the combined nCPC arms and 3 events were in the SOC arm (log-rank P = 0.005). Conclusions Injecting nCPCs into the RV during the stage 2 operation for HLHS patients appears to be safe but does not improve RV function. These findings warrant the initiation of a phase 2 trial. (The CHILD Trial: Hypoplastic Left Heart Syndrome Study [CHILD]; NCT03406884)

Traumatic brain injury (TBI) triggers a series of pathophysiological events, contributing significantly to secondary injury and long-term functional deficits. While exosome therapy is beginning to emerge as a promising avenue for various injuries, its efficacy in TBI, using preclinical models that mimic the biomechanics of human acceleration/deceleration TBI, remains largely unexplored. This study investigated the capacity of human Schwann cell-derived exosomes (hSC-Exo) to improve outcomes in a model of moderate fluid percussion injury (FPI). We found that jugular infusion of hSC-Exo 30 ​min after trauma attenuated acute proinflammatory responses in the ipsilateral cortex and hippocampus 24 ​h post-TBI, as demonstrated by a reduction in levels of key inflammasome components, and decreased activation of the STAT3/pSTAT3/SOCS3 pathway. Furthermore, exosome treatment mitigated subacute histopathological changes, including a significant decrease in cerebral edema and contusion volumes at 72 ​h post-injury. Immunohistochemical analysis revealed a decrease in microglial activation, characterized by a shift toward a more ramified morphology. Importantly, hSC-exosome therapy led to the preservation of both sensorimotor function subacutely and cognitive performance at chronic time points. Flow cytometry analysis of peripheral blood at 21 days post-TBI demonstrated a reduction in circulating neutrophils, indicating an attenuation of chronic systemic inflammation. These findings highlight the multifaceted therapeutic benefits of hSC-Exo in a clinically-relevant FPI model, targeting both acute and chronic neuroinflammatory processes to promote functional recovery. This study provides new evidence to support hSC-exosomes as a therapeutic strategy for TBI, and emphasizes the translational potential of human exosomes for treating acute and progressive neurological injury.

There is a growing body of evidence that the delivery of cell-derived exosomes normally involved in intracellular communication can reduce secondary injury mechanisms after brain and spinal cord injury and improve outcomes. Exosomes are nanometer-sized vesicles that are released by Schwann cells and may have neuroprotective effects by reducing post-traumatic inflammatory processes as well as promoting tissue healing and functional recovery. The purpose of this study was to evaluate the beneficial effects of human Schwann-cell exosomes (hSC-Exos) in a severe model of penetrating ballistic-like brain injury (PBBI) in rats and investigate effects on multiple outcomes. Human Schwann cell processing protocols followed Current Good Manufacturing Practices (cGMP) with exosome extraction and purification steps approved by the Food and Drug Administration for an expanded access single ALS patient Investigational New Drug. Anesthetized male Sprague-Dawley rats (280-350g) underwent PBBI surgery or Sham procedures and, starting 30 min after injury, received either a dose of hSC-Exos or phosphate-buffered saline through the jugular vein. At 48h after PBBI, flow cytometry analysis of cortical tissue revealed that hSC-Exos administration reduced the number of activated microglia and levels of caspase-1, a marker of inflammasome activation. Neuropathological analysis at 21 days showed that hSC-Exos treatment after PBBI significantly reduced overall contusion volume and decreased the frequency of Iba-1 positive activated and amoeboid microglia by immunocytochemical analysis. This study revealed that the systemic administration of hSC-Exos is neuroprotective in a model of severe TBI and reduces secondary inflammatory injury mechanisms and histopathological damage. The administration of hSC-Exos represents a clinically relevant cell-based therapy to limit the detrimental effects of neurotrauma or other progressive neurological injuries by impacting multiple pathophysiological events and promoting neurological recovery.

Bronchopulmonary dysplasia (BPD) and neurodevelopmental impairment are among the most common morbidities affecting preterm infants. Although BPD is a predictor of poor neurodevelopmental outcomes, it is currently uncertain how BPD contributes to brain injury in preterm infants. Extracellular vesicles (EVs) are involved in interorgan communication in diverse pathological processes. ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) is pivotal in inflammasome assembly and activation of inflammatory response. We assessed expression profiles of the alveolar macrophage (AM) markers CD11b, CD11c, and CD206 as well as ASC in EVs isolated from the plasma of preterm infants at risk for BPD at 1 week of age. We found that infants on higher fraction of inspired oxygen therapy (HO2⩾30%) had increased concentrations of AM-derived EV-ASC compared with infants on lower fraction of inspired oxygen (LO2<30%). To assess the function of these EVs, we performed adoptive transfer experiments by injecting them into the circulation of newborn mice. We discovered that mice that received EVs from infants on HO2 had increased lung inflammation, decreased alveolarization, and disrupted vascular development, the hallmarks of BPD. Importantly, these EVs crossed the blood-brain barrier, and the EVs from infants on HO2 caused inflammation, reduced cell survival, and increased cell death, with features of pyroptosis and necroptosis in the hippocampus. These results highlight a novel role for AM-derived EV-ASC in mediating the lung-to-brain cross-talk that is critical in the pathogenesis of BPD and brain injury and identify potential novel targets for preventing and treating BPD and brain injury in preterm infants.

Schwann cells are essential for the maintenance and function of motor neurons, axonal networks, and the neuromuscular junction. In amyotrophic lateral sclerosis, where motor neuron function is progressively lost, Schwann cell function may also be impaired. Recently, important signaling and potential trophic activities of Schwann cell-derived exosomal vesicles have been reported. This case report describes the treatment of a patient with advanced amyotrophic lateral sclerosis using serial intravenous infusions of allogeneic Schwann cell-derived exosomal vesicles, marking, to our knowledge, the first instance of such treatment. An 81-year-old male patient presented with a 1.5-year history of rapidly progressive amyotrophic lateral sclerosis. After initial diagnosis, the patient underwent a combination of generic riluzole, sodium phenylbutyrate for the treatment of amyotrophic lateral sclerosis, and taurursodiol. The patient volunteered to participate in an FDA-approved single-patient expanded access treatment and received weekly intravenous infusions of allogeneic Schwann cell-derived exosomal vesicles to potentially restore impaired Schwann cell and motor neuron function. We confirmed that cultured Schwann cells obtained from the amyotrophic lateral sclerosis patient via sural nerve biopsy appeared impaired (senescent) and that exposure of the patient’s Schwann cells to allogeneic Schwann cell-derived exosomal vesicles, cultured expanded from a cadaver donor improved their growth capacity in vitro. After a period of observation lasting 10 weeks, during which amyotrophic lateral sclerosis Functional Rating Scale-Revised and pulmonary function were regularly monitored, the patient received weekly consecutive infusions of 1.54 × 10 12 (×2), and then consecutive infusions of 7.5 × 10 12 (×6) allogeneic Schwann cell-derived exosomal vesicles diluted in 40 mL of Dulbecco’s phosphate-buffered saline. None of the infusions were associated with adverse events such as infusion reactions (allergic or otherwise) or changes in vital signs. Clinical lab serum neurofilament and cytokine levels measured prior to each infusion varied somewhat without a clear trend. A more sensitive in-house assay suggested possible inflammasome activation during the disease course. A trend for clinical stabilization was observed during the infusion period. Our study provides a novel approach to address impaired Schwann cells and possibly motor neuron function in patients with amyotrophic lateral sclerosis using allogeneic Schwann cell-derived exosomal vesicles. Initial findings suggest that this approach is safe.

Introduction Peripheral nerve injury (PNI) occurs in approximately 3% of all trauma patients and can be challenging to treat, particularly when injury is severe such as with a long-segmental gap. Although peripheral nerves can regenerate after injury, functional recovery is often insufficient, leading to deficits in the quality of life of patients with PNI. Although nerve autografts are the gold standard of care, there are several disadvantages to their use, namely a lack of autologous nerve material for repair. This has led to the pursuit of alternative treatment methods such as axon guidance channels (AGCs). Second-generation AGCs have been shown to be able to deliver growth-enhancing substrates for nerve repair directly to the injury site. Although our laboratory has had success with second-generation AGCs filled with Schwann cells (SCs), SCs have their own set of issues clinically. Because of this, we have begun to utilize SC-derived exosomes as an alternative, as they have the appropriate protein markers, associate to axons in high concentrations, and are able to improve nerve regeneration. However, it is unknown how SC-derived exosomes may react within second-generation AGCs; thus, the aim of the present study was to assess the ability of SC-derived exosomes to be loaded into a second-generation AGC and how they would distribute within it.Materials and Methods A total of 4 dry second-generation AGCs were loaded with SC-derived exosomes that were derived from green fluorescent protein (GFP)-labeled SCs. They were subsequently frozen and sliced before imaging.Results Here, we present findings that SC-derived exosomes can be loaded into second-generation AGCs through our established loading method utilizing negative pressure and are able to survive and equally distribute along the length of the AGC.Conclusions Although only 4 second-generation AGCs were utilized, these findings indicate a potential use for SC-derived exosomes within second-generation AGCs to treat severe PNI. Future research should focus on exploring this in greater detail and in different contexts to assess the ability of SC-derived exosomes to survive at the site of injury and treat PNI.

BACKGROUND AIMS:In this article we aimed to provide an expert synthesis of the current status of Schwann cell (SC)therapeutics and potential steps to increase their clinical utility. METHODS:We provide an expert synthesis based on preclinical, clinical and manufacturing experience. RESULTS:Schwann cells (SCs) are essential for peripheral nerve regeneration and are of interest in supporting axonal repair after spinal cord injury (SCI). SCs can be isolated and cultivated in tissue culture from adult nerve biopsies or generated from precursors and neural progenitors using specific differentiation protocols leading to expanded quantities. In culture, they undergo dedifferentiation to a state similar to "repair" SCs. The known repertoire of SC functions is increasing beyond axon maintenance, myelination, and axonal regeneration to include immunologic regulation and the release of potentially therapeutic extracellular vesicles. Recently, autologous human SC cultures purified under cGMP conditions have been tested in both nerve repair and subacute and chronic SCI clinical trials. Although the effects of SCs to support nerve regeneration are indisputable, their efficacy for clinical SCI has been limited according to the outcomes examined. CONCLUSIONS:This review discusses the current limitations of transplanted SCs within the damaged spinal cord environment. Limitations include limited post-transplant cell survival, the inability of SCs to migrate within astrocytic parenchyma, and restricted axonal regeneration out of SC-rich graft regions. We describe steps to amplify the survival and integration of transplanted SCs and to expand the repertoire of uses of SCs, including SC-derived extracellular vesicles. The relative merits of transplanting autologous versus allogeneic SCs and the role that endogenous SCs play in spinal cord repair are described. Finally, we briefly describe the issues requiring solutions to scale up SC manufacturing for commercial use.

Background and Aim An essential aspect of ensuring availability and stability of mesenchymal stem/stromal cells (MSCs) products for clinical use is that these cells are cryopreserved before individual infusion into patients. Currently, cryopreservation of MSCs involves use of a cryoprotectant solution containing dimethyl sulfoxide (DMSO). However, it is recognized that DMSO may be toxic for both the patient and the MSC product. In this Production Assistance for Cellular Therapies (PACT) and Biomedical Excellence for Safer Transfusion (BEST) Collaborative study, we compared a novel DMSO-free solution with DMSO containing cryoprotectant solutions for freezing MSCs. Methods A DMSO-free cryoprotectant solution containing sucrose, glycerol, and isoleucine (SGI) in a base of Plasmalyte A was prepared at the University of Minnesota. Cryoprotectant solutions containing 5-10% DMSO (in-house) were prepared at seven participating centers (five from USA, one each from Australia and Germany). The MSCs were isolated from bone marrow or adipose tissue and cultured ex vivo per local protocols at each center. The cells in suspension were frozen by aliquoting into vials/bags. For six out of the seven centers, the vials/bags were placed in a controlled rate freezer (one center placed them at -80°C freezer overnight) before transferring to liquid nitrogen. The cells were kept frozen for at least one week before thawing and testing. Pre- and post-thaw assessment included cell viability and recovery, immunophenotype as well as transcriptional and gene expression profiles. Linear regression, mixed effects models and two-sided t-tests were applied for statistical analysis. Results MSCs had an average viability of 94.3% (95% CI, 87.2-100%) before cryopreservation, decreasing by 4.5% (95% CI: 0.03-9.0%; p: 0.049) and 11.4% (95% CI: 6.9-15.8%; p<0.001), for MSCs cryopreserved in the in-house and SGI solutions, respectively. The average recovery of viable MSCs cryopreserved in the SGI was 92.9% (95% CI: 85.7-100.0%), and it was lower by 5.6% (95% CI: 1.3-9.8%, p<0.013) for the in-house solution. Additionally, MSCs cryopreserved in the two solutions had expected level of expressions for CD45, CD73, CD90 and CD105 with no significant difference in global gene expression profiles. Conclusion MSCs cryopreserved in a DMSO-free solution containing sucrose, glycerol, and isoleucine in a base of Plasmalyte A had slightly lower cell viability, better recovery, and comparable immunophenotype and global gene expression profiles compared to MSCs cryopreserved in DMSO containing solutions. The average viability of MSCs in the novel solution was above 80% and, thus, likely clinically acceptable. Future studies are suggested to test the post-thaw functions of MSCs cryopreserved in the novel DMSO-free solution.

Background: Cell-based therapy has emerged as a promising avenue for post-stroke recovery. A significant challenge lies in tracking the distribution and engraftment of transplanted cells within the target cerebral tissue. To address this, we turn to the potential of Brain MRI detection of mesenchymal stem cells (MSCs), achieved by labeling these cells with superparamagnetic iron oxide (SPIO). This is the first report of a technique to label canine MSCs using a commercially available SPIO, Molday ION Rhodamine B (MIRB), to optimize both viability and labeling efficacy for transplantation purposes." Method: Canine MSCs were incubated with addition of different MIRB concentration from 0, 10, 20, 30 mu g Fe/ml. The cellular uptake of MIRB was confirmed through the analysis of fluorescent images and flow cytometry. The morphological characteristics of MSCs were assessed via microscopic visualization. Cellular viability was evaluated using both a cellometer and flow cytometry. Result: Fluorescent microscopic images of all MIRB incubated MSCs groups show >70% labeled cells with homogenous signal intensity. Notably, the morphology of MSCs remained unaltered in the 10 mu g Fe/ml group compared to the control group. Furthermore, among the labeled groups, the 10 mu g Fe/ml concentration exhibited the highest viability when assessed using two different flow cytometry methods (95.3%, p < 0.05). Conclusion: This study successfully labels canine MSCs with MIRB. The optimal concentration of 10 mu g Fe/ml demonstrates optimal viability, labeling efficacy, and preserved cellular morphology.

Background Hematopoietic acute radiation syndrome (H-ARS) occurring after exposure to ionizing radiation damages bone marrow causing cytopenias, increasing susceptibility to infections and death. We and others have shown that cellular therapies like human mesenchymal stromal cells (MSCs), or monocytes/macrophages educated ex-vivo with extracellular vesicles (EVs) from MSCs were effective in a lethal H-ARS mouse model. However, given the complexity of generating cellular therapies and the potential risks of using allogeneic products, development of an “off-the-shelf” cell-free alternative like EVs may have utility in conditions like H-ARS that require rapid deployment of available therapeutics. The purpose of this study was to determine the feasibility of producing MSC-derived EVs at large scale using a bioreactor and assess critical quality control attributes like identity, sterility, and potency in educating monocytes and promoting survival in a lethal H-ARS mouse model. Methods EVs were isolated by ultracentrifugation from unprimed and lipopolysaccharide (LPS)-primed MSCs grown at large scale using a hollow fiber bioreactor and compared to a small scale system using flasks. The physical identity of EVs included a time course assessment of particle diameter, yield, protein content and surface marker profile by flow-cytometry. Comparison of the RNA cargo in EVs was determined by RNA-seq. Capacity of EVs to generate exosome educated monocytes (EEMos) was determined by qPCR and flow cytometry, and potency was assessed in vivo using a lethal ARS model with NSG mice. Results Physical identity of EVs at both scales were similar but yields by volume were up to 38-fold more using a large-scale bioreactor system. RNA-seq indicated that flask EVs showed upregulated let-7 family and miR-143 micro-RNAs. EEMos educated with LPS-EVs at each scale were similar, showing increased gene expression of IL-6, IDO, FGF-2, IL-7, IL-10, and IL-15 and immunophenotyping consistent with a PD-L1 high , CD16 low , and CD86 low cell surface expression. Treatment with LPS-EVs manufactured at both scales were effective in the ARS model, improving survival and clinical scores through improved hematopoietic recovery. EVs from unprimed MSCs were less effective than LPS-EVs, with flask EVs providing some improved survival while bioreactor EVs provide no survival benefit. Conclusions LPS-EVs as an effective treatment for H-ARS can be produced using a scale-up development manufacturing process, representing an attractive off-the-shelf, cell-free therapy.

Introduction: The long-term clinical outcomes of cell-based therapies to treat ischemic (ICM) and non-ischemic dilated cardiomyopathy (NIDCM) are unknown. We therefore prospectively followed patients (pts) with ICM/NICDM treated by targeted transcatheter endocardial mesenchymal stromal cell (MSC) injections (TESI). Methods: Of the 155 pts enrolled in prior MSC TESI studies, 111 were eligible to be contacted. Of these, 47 (42%) agreed to participate and have periodic follow-up assessments (Cardiac MRI/CT, 6MWT, labs, PE, EKG, and MLHFq) for up to 13 years following their index procedure. Time-to-event information was captured from all 155 former participants. Endpoints included event-free survival, changes in left ventricular ejection fraction (EF), and ventricular remodeling. Clinical endpoint events were defined as the composite of: Death (all-cause mortality), Left Ventricular Assist Device (LVAD) placement, or Heart Transplant. Descriptive statistics were used to classify the sample, and Kaplan-Meier survival analysis was generated to examine time-to-event trends. Results: There were 134 men and 21 women, mean age 60.0±11.0 years, including 121 (78%) with ICM, and 34 (22%) with NIDCM. In pts with ICM, mean EF at baseline was 31 %±10.8% and after 1 year was 32.6%±11.8% (P=0.08). In pts with NIDCM baseline EF was 27%±10% and increased to 34%±13% after 1 year (P=0.002). Overall, 40% of pts had a decrease in LVEF, 26% had <5% increase, 34% had a ≥5% increase, and 17% had a ≥10% increase in LVEF after 1 year. Increases of ≥5% LVEF post-therapy were associated with longer event-free survival in NIDCM (OR: 2.57; 95% CI: 0.6, 11.1) but not ICM (OR: 1.25; 95% CI: 0.4, 3.6). Conversely, reductions in LVEDV were associated with longer event-free survival in ICM (OR: 7.23; 95% CI: 1.9, 27.1) but not NIDCM (OR: 1.88; 95% CI: 0.4, 10.0). Conclusion: In this long-term observational cohort analysis, improvement of EF and/or reduction in LVEDV was associated with survival benefits among pts with NIDCM and ICM respectively. Future studies are needed with placebo controls to determine which pts are most likely to respond to MSC therapy. Clinical Trial Registration: URL: http://www.clinicaltrials.gov.; Unique ID: NCT03071835.

Abstract The safety and benefit of intra-arterial (IA) allogeneic Mesenchymal Stem Cells (MSCs) administered within 1-2 days of stroke in rodent models hold transformative potential for treating acute ischemic stroke (AIS). However, the lack of large animal studies of this approach is a significant gap in predicting success in human clinical trials, given the small size of the rodent brain and arterial vasculature and the lower white-to-gray-matter ratio. Another critical gap is the inability to extrapolate IA MSC dose ranges of MSCs and their therapeutic index from rodent models in a predictable manner. Here we employed an endovascular canine stroke model to evaluate the safety and efficacy of escalating IA doses of allogeneic MSCs at 10, and 40 million (m), and 80m MSCs versus phosphate buffer saline (PBS) controls. The IA MSC group showed safety on the primary endpoints of no worsening of neurological deficit and no new ischemia on imaging up to 40m dose. There was a significantly higher improvement in the treatment group in primary efficacy endpoints of neurological function and reduction in infarct volume on MRI and on the secondary endpoint of recovery of corticospinal tract caliber and fractional anisotropy on DTI compared to controls at 15- and 30-days post-stroke. A significant increase in neuronal survival was also seen in the IA MSC group vs the PBS control. Upon further dose escalation to 80m, there was worsened neurological score and worsened infarct at 4 days post-injection. Thus this study shows the safety and efficacy of IA MSCs over a dose range of 10-40m in a large animal model and its therapeutic index. In conclusion, these findings suggest a high translational success of IA allogeneic MSCs for AIS and provide critical information for the design of future clinical trials.

Background Although cell therapy provides benefits for outcomes of heart failure, the most optimal cell type to be used clinically remains unknown. Most of the cell products used for therapy in humans require in vitro expansion to obtain a suitable number of cells for treatment; however, the clinical background of the donor and limited starting material may result in the impaired proliferative and reparative capacity of the cells expanded in vitro . Wharton’s jelly mesenchymal cells (WJ MSCs) provide a multitude of advantages over adult tissue-derived cell products for therapy. These include large starting tissue material, superior proliferative capacity, and disease-free donors. Thus, WJ MSC if effective would be the most optimal cell source for clinical use. Objectives This study evaluated the therapeutic efficacy of Wharton’s jelly (WJ) and bone marrow (BM) mesenchymal stromal cells (MSCs) in chronic ischemic cardiomyopathy in rats. Methods Human WJ MSCs and BM MSCs were expanded in vitro , characterized, and evaluated for therapeutic efficacy in a immunodeficient rat model of ischemic cardiomyopathy. Cardiac function was evaluated with hemodynamics and echocardiography. The extent of cardiac fibrosis, hypertrophy, and inflammation was assessed with histological analysis. Results In vitro analysis revealed that WJ MSCs and BM MSCs are morphologically and immunophenotypically indistinguishable. Nevertheless, the functional analysis showed that WJ MSCs have a superior proliferative capacity, less senescent phenotype, and distinct transcriptomic profile compared to BM MSC. WJ MSCs and BM MSC injected in rat hearts chronically after MI produced a small, but not significant improvement in heart structure and function. Histological analysis showed no difference in the scar size, collagen content, cardiomyocyte cross-sectional area, and immune cell count. Conclusions Human WJ and BM MSC have a small but not significant effect on cardiac structure and function when injected intramyocardially in immunodeficient rats chronically after MI. Graphical Abstract

People living with HIV (PLHIV) are at a higher risk of having cerebrocardiovascular diseases (CVD) compared to HIV negative (HIVneg) individuals. The mechanisms underlying this elevated risk remains elusive. We hypothesize that HIV infection results in modified microRNA (miR) content in plasma extracellular vesicles (EVs), which modulates the functionality of vascular repairing cells, i.e., endothelial colony-forming cells (ECFCs) in humans or lineage negative bone marrow cells (lin− BMCs) in mice, and vascular wall cells. PLHIV (N = 74) have increased atherosclerosis and fewer ECFCs than HIVneg individuals (N = 23). Plasma from PLHIV was fractionated into EVs (HIVposEVs) and plasma depleted of EVs (HIV PLdepEVs). HIVposEVs, but not HIV PLdepEVs or HIVnegEVs (EVs from HIVneg individuals), increased atherosclerosis in apoE−/− mice, which was accompanied by elevated senescence and impaired functionality of arterial cells and lin− BMCs. Small RNA-seq identified EV-miRs overrepresented in HIVposEVs, including let-7b-5p. MSC (mesenchymal stromal cell)-derived tailored EVs (TEVs) loaded with the antagomir for let-7b-5p (miRZip-let-7b) counteracted, while TEVs loaded with let-7b-5p recapitulated the effects of HIVposEVs in vivo. Lin− BMCs overexpressing Hmga2 (a let-7b-5p target gene) lacking the 3′UTR and as such is resistant to miR-mediated regulation showed protection against HIVposEVs-induced changes in lin− BMCs in vitro. Our data provide a mechanism to explain, at least in part, the increased CVD risk seen in PLHIV.

SummaryAmyotrophic Lateral Sclerosis (ALS) is a terminal condition with accelerated loss of motor neurons (MN), resulting in the progressive paralysis of affected patients. ALS is either sporadic (90%) or genetically transmitted (10%) and affects cortical (pyramidal) and spinal cord (lower) MN, axons, and respective muscle endplates. ALS research has focused on MN survival, and current FDA-approved therapies provide only small patient survival benefits. This study reports the intravenous (IV) delivery of serial infusions of allogenic Schwann cell-derived extracellular vesicles (SCEV). The recipient had transient clinical stabilization during treatment but deteriorated rapidly during a pause in the infusions. There were no SCEV infusion-related adverse events observed. Allogeneic SCEV appeared safe for IV delivery in this case and may have therapeutic potential.

ABSTRACTBackgroundHypoplastic left heart syndrome (HLHS) survival relies on surgical reconstruction for the right ventricle (RV) to provide systemic circulation. This leads to substantially increased loads on the RV, wall stress, maladaptive remodeling and dysfunction, which in turn can increase risk of death or transplantation.ObjectivesWe conducted a phase I multicenter trial to assess safety and feasibility of intra-operative MSC injection in HLHS patients to boost RV performance in the systemic position.MethodsAllogeneic MSCs were directly administered by intramyocardial injections during the second stage palliative operation. The primary endpoint was safety.ResultsTen patients received intramyocardial injections of allogeneic MSCs (Lomecel-B). No patients experienced major adverse cardiac events (MACE). All subjects were alive and transplant-free at 1 year following, and experienced growth comparable to healthy control historical data. Cardiac magnetic resonance imaging (CMR) revealed improving tricuspid regurgitant fraction (Baseline: 0.45±0.19; 6 mo.: 0.32±0.06; 12 mo.: 0.06±0.09), while global longitudinal strain (Baseline: -24.39±6.99; 6 mo.: -20.55±3.05, p > 0.05 vs baseline; 12 mo.: - 23.88±4.6, p>0.05 vs baseline) and RV ejection fraction (EF; baseline: 62.62±5.99; 6 mo.: 53.69±9.56; 12 mo.: 52.31±5.63: p=NS for change over time) were unchanged. Computational modeling identified 167 derived RNAs specific to circulating exosomes originating from transplanted MSCs corresponding to RVEF changes and identifying potential mechanistic underpinnings.ConclusionsIntramyocardial MSCs appear safe in HLHS patients, and may favorably affect RV performance. Circulating exosomes of transplanted MSC-specific provide novel insight into bioactivity. Conduct of a controlled phase trial is warranted and is underway.Condensed AbstractThe ELPIS phase I trial was designed to assess safety and feasibility of intramyocardial injection of allogeneic MSCs into the RV during second stage palliation of HLHS. There were no incidences of major adverse cardiac events (MACE) or other safety concerns, and there was a 100% transplant-free survival at 1-year follow-up, supporting the safety and feasibility of this approach. The ELPIS results are important for advancing MSC therapy for all ages and congenital heart conditions, and warrant further investigation in a controlled Phase II trial powered for efficacy.