Gain-of-function mutation in ubiquitin ligase KLHL24 causes desmin degradation and dilatation in hiPSC-derived engineered heart tissues

The start codon c.1A>G mutation in KLHL24, encoding ubiquitin ligase KLHL24, results in the loss of 28 N-terminal amino acids (KLHL24-ΔN28) by skipping the initial start codon. In skin, KLHL24-ΔN28 leads to gain of function, excessively targeting intermediate filament keratin-14 for proteasomal degradation and ultimately causing epidermolysis bullosa simplex (EBS). The majority of patients with EBS are also diagnosed with dilated cardiomyopathy (DCM), but the pathological mechanism in the heart is unknown. As desmin is the cardiac homolog of keratin-14, we hypothesized that KLHL24-ΔN28 leads to excessive degradation of desmin, resulting in DCM. Dynamically loaded engineered heart tissues (dyn-EHTs) were generated from human-induced pluripotent stem cell–derived (hiPSC-derived) cardiomyocytes from 2 patients and 3 nonfamilial controls. Ten-fold lower desmin protein levels were observed in patient-derived dyn-EHTs, in line with diminished desmin levels detected in patients’ explanted heart. This was accompanied by tissue dilatation, impaired mitochondrial function, decreased force values, and increased cardiomyocyte stress. HEK293 transfection studies confirmed KLHL24-mediated desmin degradation. KLHL24 RNA interference or direct desmin overexpression recovered desmin protein levels, restoring morphology and function in patient-derived dyn-EHTs. To conclude, presence of KLHL24-ΔN28 in cardiomyocytes leads to excessive degradation of desmin, affecting tissue morphology and function, which can be prevented by restoring desmin protein levels

Dynamic loading of human engineered heart tissue enhances contractile function and drives a desmosome-linked disease phenotype

The role that mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes. However, most EHT systems cannot model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained contractile shortening of >10%. To do this, three-dimensional (3D) EHTs were integrated with an elastic polydimethylsiloxane strip providing mechanical preload and afterload in addition to enabling contractile force measurements based on strip bending. Our results demonstrated that dynamic loading improves the function of wild-type EHTs on the basis of the magnitude of the applied force, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we used hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy due to mutations in the desmoplakin gene. We demonstrated that manifestation of this desmosome-linked disease state required dyn-EHT conditioning and that it could not be induced using 2D or standard 3D EHT approaches. Thus, a dynamic loading strategy is necessary to provoke the disease phenotype of diastolic lengthening, reduction of desmosome counts, and reduced contractility, which are related to primary end points of clinical disease, such as chamber thinning and reduced cardiac output

Characteristics and Immunomodulating Functions of Adipose-Derived and Bone Marrow-Derived Mesenchymal Stem Cells Across Defined Human Leukocyte Antigen Barriers

BackgroundVascularized composite allotransplantation opens new possibilities in reconstructive transplantation such as hand or face transplants. Lifelong immunosuppression and its side-effects are the main drawbacks of this procedure. Mesenchymal stem cells (MSCs) have clinically useful immunomodulatory effects and may be able to reduce the burden of chronic immunosuppression. Herein, we assess and compare characteristics and immunomodulatory capacities of bone marrow- and adipose tissue-derived MSCs isolated from the same human individual across defined human leukocyte antigen (HLA) barriers.Materials and methodsSamples of omental (o.) adipose tissue, subcutaneous (s.c.) adipose tissue, and bone marrow aspirate from 10 human organ donors were retrieved and MSCs isolated. Cells were characterized by flow cytometry and differentiated in three lineages: adipogenic, osteogenic, and chondrogenic. In mixed lymphocyte reactions, the ability of adipose-derived mesenchymal stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (BMSCs) to suppress the immune response was assessed and compared within individual donors. HLA mismatched or mitogen stimulations were analyzed in co-culture with different MSC concentrations. Supernatants were analyzed for cytokine contents.ResultsAll cell types, s.c.ASC, o.ASC, and BMSC demonstrated individual differentiation potential and cell surface markers. Immunomodulating effects were dependent on dose and cell passage. Proliferation of responder cells was most effectively suppressed by s.c.ASCs and combination with BMSC resulted in highly efficient immunomodulation. Immunomodulation was not cell contact-dependent and cells demonstrated a specific cytokine secretion.ConclusionWhen human ASCs and BMSCs are isolated from the same individual, both show effective immunomodulation across defined HLA barriers in vitro. We demonstrate a synergistic effect when cells from the same biologic system were combined. This cell contact-independent function underlines the potential of clinical systemic application of MSCs

Encapsulation of growth factors in acellular nerve allografts for peripheral nerve regeneration

Thesis (M.A.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at [email protected]. Thank you.Background: Nerve injuries can occur due to trauma, tumor removal, and accidental surgical resection. Standard care for a peripheral nerve transection involves excising a portion of a non-essential sensory nerve (i.e., an autograft), such as the sural nerve. This donor nerve is then transplanted into the defect. Although autografts are considered the clinical standard of peripheral nerve repair, they also pose significant disadvantages for patients. Nerve autografts contribute to loss of sensory function and the production of neuromas at the donor site. Surgeons are limited in the number and diameter of nerves that can be harvested. This can complicate trauma situations in which multiple peripheral nerve injuries are sustained. When an autograft is not available, nerve guides are often used to reconstruct defects less than 3 em. To date, there is no commercially available guide that is Food and Drug Administration (FDA)-approved to repair long gap peripheral nerve defects (i.e. gaps > 3 em). Our research has aimed to counteract this deficit in care by investigating nerve conduit therapies that are able to promote repair over long gaps. The combination of synthetic poly(caprolactone) with a native nerve guide structure and a neurotrophic factor delivery system should promote peripheral nerve repair over long gaps. Methods: Poly(caprolactone) nerve guides were fabricated with an internal drug delivery system in the form of double-walled microspheres. Double-walled microspheres were encapsulated with glial cell line-derived neurotrophic factor (GDNF), a prominent growth factor indicated in nerve repair. The poly(caprolactone) was embedded with double-walled microspheres and wrapped around a decellularized nerve allograft to form a novel nerve guide with synthetic and native components. A prototype of this novel guide was developed and scanning electron microscopy was used to further assess nerve guide structure. Results: Scanning electron microscopy revealed smooth and round microspheres with a double-walled morphology. Scanning electron microscopy also revealed the presence of intact endoneurial tubules within the acellular nerve allograft. Conclusion: A novel synthetic-native nerve guide with an internal neurotrophic factordelivery system was created to support repair of large peripheral nerve defects (i.e. defects greater than 3 centimeters)

Delivery of adipose-derived stem cells in poloxamer hydrogel improves peripheral nerve regeneration

INTRODUCTION Peripheral nerve damage is associated with high long-term morbidity. Because of beneficial secretome, immunomodulatory effects, and ease of clinical translation, transplantation with adipose-derived stem cells (ASC) represents a promising therapeutic modality. METHODS Effect of ASC delivery in poloxamer hydrogel was assessed in a rat sciatic nerve model of critical-sized (1.5 cm) peripheral nerve injury. Nerve/muscle unit regeneration was assessed via immunostaining explanted nerve, quantitative polymerase chain reaction (qPCR), and histological analysis of reinnervating gastrocnemius muscle. RESULTS On the basis of viability data, 10% poloxamer hydrogel was selected for in vivo study. Six weeks after transection and repair, the group treated with poloxamer delivered ASCs demonstrated longest axonal regrowth. The qPCR results indicated that the inclusion of ASCs appeared to result in expression of factors that aid in reinnervating muscle tissue. DISCUSSION Delivery of ASCs in poloxamer addresses multiple facets of the complexity of nerve/muscle unit regeneration, representing a promising avenue for further study. Muscle Nerve 58: 251-260, 2018

Long-gap peripheral nerve repair through sustained release of a neurotrophic factor in nonhuman primates.

Severe injuries to peripheral nerves are challenging to repair. Standard-of-care treatment for nerve gaps \u3e2 to 3 centimeters is autografting; however, autografting can result in neuroma formation, loss of sensory function at the donor site, and increased operative time. To address the need for a synthetic nerve conduit to treat large nerve gaps, we investigated a biodegradable poly(caprolactone) (PCL) conduit with embedded double-walled polymeric microspheres encapsulating glial cell line-derived neurotrophic factor (GDNF) capable of providing a sustained release of GDNF for \u3e50 days in a 5-centimeter nerve defect in a rhesus macaque model. The GDNF-eluting conduit (PCL/GDNF) was compared to a median nerve autograft and a PCL conduit containing empty microspheres (PCL/Empty). Functional testing demonstrated similar functional recovery between the PCL/GDNF-treated group (75.64 ± 10.28%) and the autograft-treated group (77.49 ± 19.28%); both groups were statistically improved compared to PCL/Empty-treated group (44.95 ± 26.94%). Nerve conduction velocity 1 year after surgery was increased in the PCL/GDNF-treated macaques (31.41 ± 15.34 meters/second) compared to autograft (25.45 ± 3.96 meters/second) and PCL/Empty (12.60 ± 3.89 meters/second) treatment. Histological analyses included assessment of Schwann cell presence, myelination of axons, nerve fiber density, an

Long-gap peripheral nerve repair through sustained release of a neurotrophic factor in nonhuman primates

Severe injuries to peripheral nerves are challenging to repair. Standard-of-care treatment for nerve gaps >2 to 3 centimeters is autografting; however, autografting can result in neuroma formation, loss of sensory function at the donor site, and increased operative time. To address the need for a synthetic nerve conduit to treat large nerve gaps, we investigated a biodegradable poly(caprolactone) (PCL) conduit with embedded double-walled polymeric microspheres encapsulating glial cell line-derived neurotrophic factor (GDNF) capable of providing a sustained release of GDNF for >50 days in a 5-centimeter nerve defect in a rhesus macaque model. The GDNF-eluting conduit (PCL/GDNF) was compared to a median nerve autograft and a PCL conduit containing empty microspheres (PCL/Empty). Functional testing demonstrated similar functional recovery between the PCL/GDNF-treated group (75.64 ± 10.28%) and the autograft-treated group (77.49 ± 19.28%); both groups were statistically improved compared to PCL/Empty-treated group (44.95 ± 26.94%). Nerve conduction velocity 1 year after surgery was increased in the PCL/GDNF-treated macaques (31.41 ± 15.34 meters/second) compared to autograft (25.45 ± 3.96 meters/second) and PCL/Empty (12.60 ± 3.89 meters/second) treatment. Histological analyses included assessment of Schwann cell presence, myelination of axons, nerve fiber density, and g-ratio. PCL/GDNF group exhibited a statistically greater average area occupied by individual Schwann cells at the distal nerve (11.60 ± 33.01 μm2) compared to autograft (4.62 ± 3.99 μm2) and PCL/Empty (4.52 ± 5.16 μm2) treatment groups. This study demonstrates the efficacious bridging of a long peripheral nerve gap in a nonhuman primate model using an acellular, biodegradable nerve conduit

Evaluation of Senescence and Its Prevention in Doxorubicin-Induced Cardiotoxicity Using Dynamic Engineered Heart Tissues

Background: Doxorubicin is an essential cancer treatment, but its usefulness is hampered by the occurrence of cardiotoxicity. Nevertheless, the pathophysiology underlying doxorubicin-induced cardiotoxicity and the respective molecular mechanisms are poorly understood. Recent studies have suggested involvement of cellular senescence.Objectives: The aims of this study were to establish whether senescence is present in patients with doxorubicin-induced cardiotoxicity and to investigate if this could be used as a potential treatment target.Methods: Biopsies from the left ventricles of patients with severe doxorubicin-induced cardiotoxicity were compared with control samples. Additionally, senescence-associated mechanisms were characterized in 3-dimensional dynamic engineered heart tissues (dyn-EHTs) and human pluripotent stem cell–derived cardiomyocytes. These were exposed to multiple, clinically relevant doses of doxorubicin to recapitulate patient treatment regimens. To prevent senescence, dyn-EHTs were cotreated with the senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol.Results: Senescence-related markers were significantly up-regulated in the left ventricles of patients with doxorubicin-induced cardiotoxicity. Treatment of dyn-EHTs resulted in up-regulation of similar senescence markers as seen in the patients, accompanied by tissue dilatation, decreased force generation, and increased troponin release. Treatment with senomorphic drugs led to decreased expression of senescence-associated markers, but this was not accompanied by improved function.Conclusions: Senescence was observed in the hearts of patients with severe doxorubicin-induced cardiotoxicity, and this phenotype can be modeled in vitro by exposing dyn-EHTs to repeated clinically relevant doses of doxorubicin. The senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol prevent senescence but do not result in functional improvements. These findings suggest that preventing senescence by using a senomorphic during doxorubicin administration might not prevent cardiotoxicity.</p