Shifting patterns of multiple sclerosis treatment in a highly prevalent United States population.

OBJECTIVE: Our objectives were to (1) obtain the prevalence and demography of people with multiple sclerosis (MS) in a representative Colorado population, and (2) to assess the utilization of disease-modifying therapy within this prevalent cohort. METHODS: This is a retrospective, observational study of patients that had contact with the University of Colorado Health System from 2012 to 2020. We queried Health Data Compass, a data warehouse, for patient data and applied the MS Prevalence Workgroup Algorithm to generate a prevalent cohort. We calculated prevalence as of 31 December 2020, and stratified by age, sex, race, and ethnicity. Payer information and treatment exposure were obtained from linked claims from the Colorado All Payers Claim Database. Disease-modifying therapies were classified as highly effective and moderately effective based on the clinical trial, TREAT-MS (NCT03500328). RESULTS: From a population of 1,382,821 individuals, 8557 people with MS were captured. Age-adjusted prevalence of MS as of 31 December 2020 was 572.3 per 100,000 with a mean age of 47.36. Prevalence varied between demographic subgroups, with the lowest prevalence in Hispanic men (215.6) and highest in White (824.1) and Black women (820.1). Overall disease-modifying therapy exposure was 62.4%, with increased highly effective therapy use and a corresponding decrease in moderately effective therapy use on a yearly basis. INTERPRETATION: MS is highly prevalent in a representative Colorado cohort. Overall treatment and proportion of highly effective therapy exposure increased significantly during a critical period of MS therapeutic advances, indicating a shift in disease management driven sharply by the availability of on-label anti-CD20 therapy.

Anti-NMDA Receptor Encephalitis Related to Renal Cell Carcinoma: A Case Report.

Anti-NMDA receptor (NMDAR) encephalitis is characterized by a well-defined neuropsychiatric syndrome and CSF antibodies against the GluN1 subunit of the NMDAR. 40% of cases are related to underlying tumors, the vast majority ovarian teratomas (94%). We report a case of anti-NMDAR encephalitis associated with renal cell carcinoma (RCC). A 20-year-old female presented to the ED with behavioral changes, involuntary movements, tachycardia, and alternating obtundation with agitation which progressed over 3 weeks. Involuntary movements were severe, requiring intubation and sedation for control, and were accompanied by rhabdomyolysis. Brain MRI showed bilateral mesiotemporal T2/FLAIR hyperintensities. Anti-NMDAR antibodies were present in the serum (1:640) and CSF (1:320). Malignancy screening revealed a renal mass concerning for RCC, which was confirmed upon resection. She was started on high dose IV methylprednisolone and plasmapheresis, followed by rituximab. Lack of response led to escalating immunotherapy with cyclophosphamide. Clinical course was complicated by prolonged ICU admission, prolonged sedation, severe dysautonomia and bacteremia. Improvement began 2 months after immunotherapy, and she was discharged to rehabilitation 100 days after admission with mild neuropsychiatric symptoms. Repeat malignancy screenings, including whole-body imaging and transvaginal ultrasound were consistently negative. Herein, we describe a case of definite anti-NMDAR encephalitis in the setting of newly diagnosed RCC. This case illustrates how tumors other than ovarian teratomas may act as immunological triggers, as well as the complex and prolonged symptomatic and immunosuppressive therapies required in severe presentations of anti-NMDAR encephalitis.

Diversity, equity, and inclusion (DEI) in medical education: DEI at the bedside.

BACKGROUND: The ability to recognize and address bias is an important communication skill not typically addressed during training. We describe the design of an educational curriculum that aims to identify and change behavior related to diversity, equity, and inclusion (DEI). "DEI at the Bedside" uses the existing infrastructure of bedside teaching and provides a tool to normalize DEI discussions and develop skills to address bias during a neurology inpatient rotation. METHODS: As part of traditional clinical rounds, team members on an inpatient service shared experiences with DEI topics, including bias. The team developed potential responses should they encounter a similar situation in the future. We report the results of our needs assessment and curriculum development to evaluate the feasibility of incorporating a DEI educational curriculum in the neurology inpatient setting. RESULTS: Forty-two DEI experiences were recorded. Medical students were the most frequent discussants (44%). Direction of bias occurred between healthcare team members (33%), against patients (31%), and patients against healthcare team members (28%). Experiences ranged from microaggressions to explicit comments of racism, sexism, and homophobia. CONCLUSIONS: Based on needs assessment data, we developed a DEI educational curriculum for the inpatient neurology setting aimed to improve knowledge and skills related to DEI topics as well as to normalize conversation of DEI in the clinical setting. Additional study will demonstrate whether this initiative translates into measurable and sustained improvement in knowledge of how bias and disparity show up in the clinical setting and behavioral intent to discuss and address them.

Microfibrous substrate geometry as a critical trigger for organization, self-renewal, and differentiation of human embryonic stem cells within synthetic 3-dimensional microenvironments.

Substrates used to culture human embryonic stem cells (hESCs) are typically 2-dimensional (2-D) in nature, with limited ability to recapitulate in vivo-like 3-dimensional (3-D) microenvironments. We examined critical determinants of hESC self-renewal in poly-d-lysine-pretreated synthetic polymer-based substrates with variable microgeometries, including planar 2-D films, macroporous 3-D sponges, and microfibrous 3-D fiber mats. Completely synthetic 2-D substrates and 3-D macroporous scaffolds failed to retain hESCs or support self-renewal or differentiation. However, synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion, viability, proliferation, self-renewal, and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell-cell contacts and altered colony organization. Moreover, the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment, where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus, synthetic substrates with specific 3-D microgeometries can support hESC colony formation, self-renewal, and directed differentiation to multiple lineages while obviating the stringent needs for complex, exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3-D and for stem cell differentiation in situ and transplantation using defined humanized conditions.

Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase.

Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR-DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescue GRN haploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.

Chronic tibial nonunion in a Rothmund-Thomson syndrome patient.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder caused by biallelic mutations in RECQL4, a helicase involved with chromosomal instability and DNA repair. Patients typically present with a poikilodermatous facial rash, photosensitivity, congenital bony abnormalities, short stature, and have a predilection for osteosarcoma and cutaneous malignancies. We present a 34-year-old male RTS patient, previously diagnosed with osteosarcoma of the right forearm which was successfully treated with resection and chemotherapy, who has had multiple tibial fractures and has suffered from chronic nonunion of the proximal tibias bilaterally for greater than 9 years. The patient subsequently developed generalized lower extremity osteopenia with normal calcium homeostasis and calcitriol levels. As the RTS population continues to reach greater ages we must be mindful of other health concerns that may develop. Bone health is one considerable concern with a large portion of patients having congenital bony abnormalities and many receiving chemotherapy for osteosarcoma. We conclude that screening for bone health and supplementation with calcium and vitamin D may be warranted in RTS patients with a history of fractures and osteosarcoma treatment.

Prevalence of multiple sclerosis and treatment utilization in a large, highly diverse population.

BACKGROUND: Despite advances in algorithms for identifying people with MS (PwMS) in large data sets, limited data exists on regional prevalence, or prevalence and care in minority populations. OBJECTIVES: To report the 7-year (01/01/2012-12/31/2018) prevalence and demographics of MS and disease-modifying therapy (DMT) utilization in a large, diverse population. METHODS: This retrospective analysis used the OneFlorida Data Trust, which captures health data from >15 million Floridians across 10 constituent organizations. A validated algorithm identified subjects with MS. DMTs were identified using RxNorm concept unique identifiers and National Drug Codes. Results were stratified across age, sex, race-ethnicity, and location. RESULTS: Of 6,638,649 adults in the database, the algorithm identified 9681 PwMS. Overall prevalence per 100,000 was 145.83. MS prevalence was considerable in women of all races and ethnicities ranging from 138.86 to 253.76 per 100,000. 52.6% of PwMS had one or more DMT prescription. DMT prescription was more likely in Hispanic PwMS. CONCLUSION: Prevalence analysis of the OneFlorida Data Trust revealed a substantial burden of disease in women of all races and ethnicities. Variation in treatment utilization among demographic subgroups underscores the need for additional studies to assess health care disparities in MS at the population level.

Therapy-related myelodysplasia in a patient with Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder of which approximately 300 cases have been reported in the literature. Patients with RTS often present early in life with skeletal and dental abnormalities, short stature, juvenile cataracts, and a characteristic poikilodermal rash. They are at increased risk for the development of osteosarcoma that usually presents by the second decade of life. The genetic defects underlying RTS are truncating mutations in RECQL4, a gene involved with chromosomal stability. Several cases of primary hematological malignancies have been reported in RTS, but it is unclear whether patients with RTS are at higher risk to develop either primary or secondary hematological malignancies. We report a patient with RTS who presented to our clinic at the age of 7, subsequently developed multifocal and recurrent osteosarcoma that was followed by the development of a myelodysplastic syndrome with subsequent progression to acute myeloid leukemia.

Anti-MOG associated disorder-Clinical and radiological characteristics compared to AQP4-IgG+ NMOSD-A single-center experience.

BACKGROUND: The discovery of two immunoglobulin G (IgG) antibodies against aquaporin 4 (anti-AQP4) and myelin oligodendrocyte glycoprotein (anti-MOG) has led to the distinction of the disorders anti-AQP4 immunoglobulin G positive neuromyelitis spectrum disorder (AQP4-IgG+ NMOSD) and anti-MOG associated disorder (MOGAD). Different clinical and radiological features have been proposed to distinguish these two demyelinating CNS diseases. METHODS: This is a single-center retrospective review at the University of Florida (UF) including all patients with the diagnostic code ICD G36 ("other acute disseminated demyelination") from October 2015 to January 2020 (n=141) and all charts included in the clinical NMOSD database of the UF Division of Neuroimmunology (n=36). A total of 151 cases were reviewed for presence of anti-MOG and anti-AQP4 antibodies and NMOSD diagnostic criteria. Differences in MOGAD and AQP4-IgG+ NMOSD were compared. RESULTS: Of the 151 reviewed patient charts, 11 were consistent with MOGAD and 43 with AQP4-IgG+ NMOSD. Patients with MOGAD were significantly younger at symptom onset compared to patients with AQP4-IgG+ NMOSD (14 [1-33] years vs. 37 [6-82] years; p=0.005). In comparison with AQP4-IgG+ NMOSD, optic neuritis in MOGAD was more frequently associated with bilateral optic nerve involvement: (6/11 [54.5%] vs. 6/43 [13.9%]; p=0.009) and fundoscopic presence of optic disc edema (5/11 [45.5%] vs. 3/43 [7%]; p=0.006). Perineuritis was a common radiological feature in MOGAD (present in 4 cases). In case of myelitis, there was more frequent involvement of the conus medullaris in MOGAD (4/11 [36.4%] vs. 2/43 [4.7%]; p=0.012). Symptomatic cerebral syndrome with supratentorial white matter lesions was seen in MOGAD patients with pediatric onset (pediatric onset: 4/6 [66.7%] vs. adult onset: 0/5 [0%]. In MOGAD, evidence for combined central and peripheral demyelination and increased intracranial pressure was present in one patient each. A preceding inciting event (illness/postpartum) was more frequently identifiable in MOGAD (4/11 [36.4%] vs. 4/43 [7%]; p=0.045). Disability as calculated on the Expanded Disability Status Scale was less severe in MOGAD compared to AQP-IgG+ NMOSD (most severe presentation: 5 [2-7] vs. 7 [1-10]; p=0.015; most recent assessment: 2 [0-5] vs. 5 [0-10]; p=0.045) and patients were more likely to respond to treatment of acute attacks with corticosteroids and/or plasmapheresis (Clinical Global Impression-Global Change scale: 1 [1-4] vs. 3 [1-6]; p=0.001). INTERPRETATION: The study confirms that simultaneous bilateral optic neuritis, presence of optic disc edema, transverse myelitis with conus involvement and a less severe disease course are distinctive features of MOGAD.

Five men with arresting and relapsing cerebral adrenoleukodystrophy.

BACKGROUND: X-linked adrenoleukodystrophy (ALD) is the most common genetic peroxisomal disorder with an estimated prevalence of 1:15,000. Approximately two-thirds of males with ALD manifest the inflammatory demyelinating cerebral phenotype (cALD) at some disease stage, in which focal, inflammatory lesions progress over months to years. Hematopoietic stem-cell transplantation can permanently halt cALD progression, but it is only effective if initiated early. Although most cALD lesions progress relentlessly, a subset may spontaneously arrest; subsequent reactivation of these arrested lesions has not been previously detailed. OBJECTIVE: We describe a novel arresting-relapsing variant of cALD. METHODS: Salient clinical and radiographic studies were reviewed and summarized for cALD patients with episodic deteriorations. RESULTS: We report a series of five unrelated men with spontaneously arrested cALD lesions that subsequently manifested signs of clinical and radiologic lesion progression during longitudinal follow-up. In three of five patients, functional status was too poor to attempt transplant by the time the recurrence was identified. One patient experienced reactivation followed by another period of spontaneous arrest. CONCLUSIONS: These cases emphasize the need for continued clinical and radiologic vigilance for adult men with ALD to screen for evidence of new or reactivated cALD lesions to facilitate prompt treatment evaluation.

Three-dimensional tissue-engineered human skeletal muscle model of Pompe disease.

In Pompe disease, the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) causes skeletal and cardiac muscle weakness, respiratory failure, and premature death. While enzyme replacement therapy using recombinant human GAA (rhGAA) can significantly improve patient outcomes, detailed disease mechanisms and incomplete therapeutic effects require further studies. Here we report a three-dimensional primary human skeletal muscle ("myobundle") model of infantile-onset Pompe disease (IOPD) that recapitulates hallmark pathological features including reduced GAA enzyme activity, elevated glycogen content and lysosome abundance, and increased sensitivity of muscle contractile function to metabolic stress. In vitro treatment of IOPD myobundles with rhGAA or adeno-associated virus (AAV)-mediated hGAA expression yields increased GAA activity and robust glycogen clearance, but no improvements in stress-induced functional deficits. We also apply RNA sequencing analysis to the quadriceps of untreated and AAV-treated GAA-/- mice and wild-type controls to establish a Pompe disease-specific transcriptional signature and reveal novel disease pathways. The mouse-derived signature is enriched in the transcriptomic profile of IOPD vs. healthy myobundles and partially reversed by in vitro rhGAA treatment, further confirming the utility of the human myobundle model for studies of Pompe disease and therapy.

Anti-JCV antibody index does not change during ocrelizumab-treatment.

OBJECTIVE: To prospectively assess anti-JCV antibody index (AI) and its relationship to immunoglobulin levels in ocrelizumab-treated MS patients. METHODS: Monocentric prospective observational study over 24 months assessing anti-JCV AI and immunoglobulin levels in MS patients before and after initiation of ocrelizumab. RESULTS: No significant change in anti-JCV AI titers was observed 458 ± 300 days after initiation of ocrelizumab (n = 45, 0.7 ± 2.21 vs. 0.6 ± 2.06, p = 0.8). Seroconversion occurred in 1/20 initially anti-JCV seronegative patients. There was no correlation between changes in anti-JCV AI and immunoglobulins. CONCLUSION: Treatment with ocrelizumab is not associated with an increase in anti-JCV AI titers.

A case of GFAP-astroglial autoimmunity presenting with reversible parkinsonism.

Autoimmune glial fibrillary acidic protein (GFAP) astrocytopathy is a newly recognized autoimmune central nervous system (CNS) inflammatory disorder, presenting with an array of neurological symptoms in association with autoantibodies against GFAP, a hallmark protein expressed on astrocytes. Limited knowledge is available on the disease pathogenesis and clinical outcome. Here, we report a case of autoimmune GFAP astrocytopathy presenting with encephalomyelitis and parkinsonism. Our patient was a 66-year old male who experienced progressive somnolence, apathy, anxiety, right arm tremor, urinary retention, progressive weakness, and falls over the course of three months, followed by acute delusional psychosis. His neurologic exam on hospital admission was notable for cognitive impairment, myoclonus, rigidity, right hand action tremor, bradykinesia, shuffling gait, and dysmetria. Cerebrospinal fluid examination showed elevated protein, lymphocytic pleocytosis, and one unique oligoclonal band. Magnetic resonance imaging (MRI) revealed non-specific T2/FLAIR hyperintensities in the brain and longitudinally extensive transverse myelitis in the cervical spine. FDG-PET showed a pattern of brain uptake suspicious for limbic encephalitis. Serum and CSF paraneoplastic panel showed presence of GFAP immunoglobulin G (IgG). Treatment with corticosteroids resulted in clinical and radiographic improvement. However, the patient was treated with anti-CD20 immunotherapy due to steroid-dependence. This case exemplifies the recently described neurologic syndrome of autoimmune GFAP astrocytopathy presenting with encephalomyelitis and parkinsonism, reversed by B lymphocyte depletion.

Corticosteroids lead to short-term improvement in cerebral amyloid angiopathy-related inflammation.

BACKGROUND: Cerebral amyloid angiopathy - related inflammation (CAA-ri) is an uncommon manifestation of CAA. METHODS: Single-center, retrospective review of all charts with ICD-code I68.0 (CAA) from 2/2/2016-1/1/2020. RESULTS: Of 152 CAA cases, 13 (8.6%) were consistent with CAA-ri. Corticosteroid-treatment led to short-term reduction in modified Rankin Scale scores (2.6 ± 1.4 vs. 1.6 ± 1.5; p = 0.01) and T2/FLAIR lesion volume (78.1 ± 52.2 cm3 vs. 30 ± 30.9 cm3, p < 0.01) as well as short-term improvement in post-treatment Clinical Global Impression - Global Change scores compared to pre-treatment scores (clinical: 6 ± 1 vs. 2.6 ± 1.3, p = 0.03; radiological: 4.6 ± 1.9 vs. 1.2 ± 0.4, p = 0.03). INTERPRETATION: Corticosteroid-treatment leads to clinical and radiological short-term improvement (class IV evidence).

Proteolytic processing of TAR DNA binding protein-43 by caspases produces C-terminal fragments with disease defining properties independent of progranulin.

Neuronal and glial deposition of misfolded, proteolytically processed, polyubiquitinated and abnormally phosphorylated C-terminal fragments (CTFs) of the TAR DNA binding protein-43 (TDP-43) is a pathological hallmark of frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) and certain cases of amyotrophic lateral sclerosis. We demonstrate that TDP-43 can be proteolytically processed by caspases upon induction of apoptosis to a major 35 kDa and a minor 25 kDa CTF. These fragments are initially soluble, but over time they accumulate as insoluble and pathologically phosphorylated derivatives. However, proteolytic processing appears not to be absolutely required for the deposition of insoluble TDP-43 species, since a caspase resistant mutant of TDP-43 is also converted into insoluble species. Phosphorylation at S409/410 apparently occurs late during the conversion of soluble to insoluble TDP-43, suggesting that phosphorylation is not a prerequisite for aggregation. Loss of function of the progranulin (PGRN) gene causes FTLD-U with TDP-43 positive inclusions and has been suggested to lead to caspase activation and subsequent TDP-43 processing. However, siRNA-mediated knockdown of PGRN in cell culture as well as a PGRN gene knockout in mice failed to cause the formation of the disease characterizing CTFs of TDP-43. Our findings therefore suggest that caspase-mediated processing generates CTFs of similar biochemical properties as those occurring in nuclear and cytoplasmic deposits of FTLD-U patients independent of PGRN levels.

Presence, Diversity, and Enrichment of Respiratory Reductive Dehalogenase and Non-respiratory Hydrolytic and Oxidative Dehalogenase Genes in Terrestrial Environments.

Organohalide-respiring bacteria have been linked to the cycling and possible respiration of chlorinated natural organic matter (Cl-NOM) in uncontaminated soils and sediments. The importance of non-respiratory hydrolytic/oxidative dechlorination processes in the cycling of Cl-NOM in terrestrial soil and sediment, however, is still not understood. This research analyzes the dechlorination potential of terrestrial systems through analysis of the metagenomes of urban lake sediments and cultures enriched with Cl-NOM. Even with the variability in sample type and enrichment conditions, the potential to dechlorinate was universal, with reductive dehalogenase genes and hydrolytic or oxidative dehalogenase genes found in all samples analyzed. The reductive dehalogenase genes detected grouped taxonomically with those from organohalide-respiring bacteria with broad metabolic capabilities, as opposed to those that obligately respire organohalides. Furthermore, reductive dehalogenase genes and two haloacid dehalogenase genes increased in abundance when sediment was enriched with high concentrations of Cl-NOM. Our data suggests that both respiratory and non-respiratory dechlorination processes are important for Cl-NOM cycling, and that non-obligate organohalide-respiring bacteria are most likely involved in these processes.

Accumulation of pathological tau species and memory loss in a conditional model of tauopathy.

Neurofibrillary tangles (NFTs) are a pathological hallmark of Alzheimer's disease and other tauopathies, but recent studies in a conditional mouse model of tauopathy (rTg4510) have suggested that NFT formation can be dissociated from memory loss and neurodegeneration. This suggests that NFTs are not the major neurotoxic tau species, at least during the early stages of pathogenesis. To identify other neurotoxic tau protein species, we performed biochemical analyses on brain tissues from the rTg4510 mouse model and then correlated the levels of these tau proteins with memory loss. We describe the identification and characterization of two forms of tau multimers (140 and 170 kDa), whose molecular weight suggests an oligomeric aggregate, that accumulate early in the pathogenic cascade in this mouse model. Similar tau multimers were detected in a second mouse model of tauopathy (JNPL3) and in tissue from patients with Alzheimer's disease and FTDP-17 (frontotemporal dementia and parkinsonism linked to chromosome 17). Moreover, levels of the tau multimers correlated consistently with memory loss at various ages in the rTg4510 mouse model. Our findings suggest that accumulation of early-stage aggregated tau species, before the formation of NFT, is associated with the development of functional deficits during the pathogenic progression of tauopathy.

Cardiopatch platform enables maturation and scale-up of human pluripotent stem cell-derived engineered heart tissues.

Despite increased use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for drug development and disease modeling studies, methods to generate large, functional heart tissues for human therapy are lacking. Here we present a "Cardiopatch" platform for 3D culture and maturation of hiPSC-CMs that after 5 weeks of differentiation show robust electromechanical coupling, consistent H-zones, I-bands, and evidence for T-tubules and M-bands. Cardiopatch maturation markers and functional output increase during culture, approaching values of adult myocardium. Cardiopatches can be scaled up to clinically relevant dimensions, while preserving spatially uniform properties with high conduction velocities and contractile stresses. Within window chambers in nude mice, cardiopatches undergo vascularization by host vessels and continue to fire Ca2+ transients. When implanted onto rat hearts, cardiopatches robustly engraft, maintain pre-implantation electrical function, and do not increase the incidence of arrhythmias. These studies provide enabling technology for future use of hiPSC-CM tissues in human heart repair.

Dynamic culture yields engineered myocardium with near-adult functional output.

Engineered cardiac tissues hold promise for cell therapy and drug development, but exhibit inadequate function and maturity. In this study, we sought to significantly improve the function and maturation of rat and human engineered cardiac tissues. We developed dynamic, free-floating culture conditions for engineering "cardiobundles", 3-dimensional cylindrical tissues made from neonatal rat cardiomyocytes or human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) embedded in fibrin-based hydrogel. Compared to static culture, 2-week dynamic culture of neonatal rat cardiobundles significantly increased expression of sarcomeric proteins, cardiomyocyte size (∼2.1-fold), contractile force (∼3.5-fold), and conduction velocity of action potentials (∼1.4-fold). The average contractile force per cross-sectional area (59.7 mN/mm2) and conduction velocity (52.5 cm/s) matched or approached those of adult rat myocardium, respectively. The inferior function of statically cultured cardiobundles was rescued by transfer to dynamic conditions, which was accompanied by an increase in mTORC1 activity and decline in AMPK phosphorylation and was blocked by rapamycin. Furthermore, dynamic culture effects did not stimulate ERK1/2 pathway and were insensitive to blockers of mechanosensitive channels, suggesting increased nutrient availability rather than mechanical stimulation as the upstream activator of mTORC1. Direct comparison with phenylephrine treatment confirmed that dynamic culture promoted physiological cardiomyocyte growth rather than pathological hypertrophy. Optimized dynamic culture conditions also augmented function of human cardiobundles made reproducibly from cardiomyocytes derived from multiple hPSC lines, resulting in significantly increased contraction force (∼2.5-fold) and conduction velocity (∼1.4-fold). The average specific force of 23.2 mN/mm2 and conduction velocity of 25.8 cm/s approached the functional metrics of adult human myocardium. In conclusion, we have developed a versatile methodology for engineering cardiac tissues with a near-adult functional output without the need for exogenous electrical or mechanical stimulation, and have identified mTOR signaling as an important mechanism for advancing tissue maturation and function in vitro.