Abstract
Cardiac two-dimensional tissues were engineered using biomimetic micropatterns based on the fibronectin-rich extracellular matrix (ECM) of the embryonic heart. The goal of this ...developmentally-inspired, in vitro approach was to identify cell–cell and cell-ECM interactions in the microenvironment of the early 4-chambered vertebrate heart that drive cardiomyocyte organization and alignment. To test this, biomimetic micropatterns based on confocal imaging of fibronectin in embryonic chick myocardium were created and compared to control micropatterns designed with 2 or 20 µm wide fibronectin lines. Results show that embryonic chick cardiomyocytes have a unique density-dependent alignment on the biomimetic micropattern that is mediated in part by N-cadherin, suggesting that both cell–cell and cell-ECM interactions play an important role in the formation of aligned myocardium. Human induced pluripotent stem cell-derived cardiomyocytes also showed density-dependent alignment on the biomimetic micropattern but were overall less well organized. Interestingly, the addition of human adult cardiac fibroblasts and conditioning with T3 hormone were both shown to increase human cardiomyocyte alignment. In total, these results show that cardiomyocyte maturation state, cardiomyocyte-cardiomyocyte and cardiomyocyte-fibroblast interactions, and cardiomyocyte-ECM interactions can all play a role when engineering anisotropic cardiac tissues in vitro and provides insight as to how these factors may influence cardiogenesis in vivo.
Three-dimensional (3D) bioprinting is a transformative technology for engineering tissues for disease modeling and drug screening and building tissues and organs for repair, regeneration, and ...replacement. In this Viewpoint, we discuss technological advances in 3D bioprinting, key remaining challenges, and essential milestones toward clinical translation.
ABSTRACT
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
Vascularized 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.
Samples 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.
All 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.
When human ASCs and BMSCs are isolated from the same individual, both show effective immunomodulation across defined HLA barriers
. 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.
The ultimate goal of cardiac tissue engineering is to generate new muscle to repair or replace the damaged heart. This requires advances in stem cell technologies to differentiate billions of ...cardiomyocytes, together with advanced biofabrication approaches such as 3D bioprinting to achieve the requisite structure and contractile function. In this concise review, we cover recent progress in 3D bioprinting of cardiac tissue using pluripotent stem cell-derived cardiomyocytes, key design criteria for engineering aligned cardiac tissues, and ongoing challenges in the field that must be addressed to realize this goal.
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
-ratio. PCL/GDNF group exhibited a statistically greater average area occupied by individual Schwann cells at the distal nerve (11.60 ± 33.01 μm
) compared to autograft (4.62 ± 3.99 μm
) and PCL/Empty (4.52 ± 5.16 μm
) 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.
The extensive crosstalk between the developing heart and lung is critical to their proper morphogenesis and maturation. However, there remains a lack of models that investigate the critical ...cardio-pulmonary mutual interaction during human embryogenesis. Here, we reported a novel stepwise strategy for directing the simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages within a single differentiation of human-induced pluripotent stem cells (hiPSCs) via temporal specific tuning of WNT and nodal signaling in the absence of exogenous growth factors. Using 3D suspension culture, we established concentric cardio-pulmonary micro-Tissues (μTs), and expedited alveolar maturation in the presence of cardiac accompaniment. Upon withdrawal of WNT agonist, the cardiac and pulmonary components within each dual-lineage μT effectively segregated from each other with concurrent initiation of cardiac contraction. We expect that our multilineage differentiation model will offer an experimentally tractable system for investigating human cardio-pulmonary interaction and tissue boundary formation during embryogenesis.
Here we report the 3D bioprinting of a simplified model of the heart, similar to that observed in embryonic development, where the heart is a linear tube that pumps blood and nutrients to the growing ...embryo. To this end, we engineered a bioinspired model of the human heart tube using freeform reversible of embedding of suspended hydrogels 3D bioprinting. The 3D bioprinted heart tubes were cellularized using human stem cell-derived cardiomyocytes and cardiac fibroblasts and formed patent, perfusable constructs. Synchronous contractions were achieved ∼3-4 days after fabrication and were maintained for up to a month. Immunofluorescent staining confirmed large, interconnected networks of sarcomeric alpha actinin-positive cardiomyocytes. Electrophysiology was assessed using calcium imaging and demonstrated anisotropic calcium wave propagation along the heart tube with a conduction velocity of ∼5 cm s
. Contractility and function was demonstrated by tracking the movement of fluorescent beads within the lumen to estimate fluid displacement and bead velocity. These results establish the feasibility of creating a 3D bioprinted human heart tube and serve as an initial step towards engineering more complex heart muscle structures.
Microelectrode arrays (MEAs) have enabled investigation of cellular networks at sub-millisecond temporal resolution. However, current MEAs are limited by the large electrode footprint since reducing ...the electrode's geometric area to sub-cellular dimensions leads to a significant increase in impedance thus affecting its recording capabilities. We report a breakthrough ultra-microelectrodes platform by leveraging the outstanding surface-to-volume ratio of nanowire-templated out-of-plane synthesized three-dimensional fuzzy graphene (NT-3DFG). The enormous surface area of NT-3DFG leads to 140-fold reduction in electrode impedance compared to bare Au microelectrodes, thus enabling scaling down the geometric size by 625-fold to ca. 2 μm × 2 μm. The out-of-plane morphology of NT-3DFG leads to a tight seal with the cell membrane thus enabling recording of electrical signals with high signal-to-noise ratio (SNR) of > 6. This work highlights the possibility to push the limits of the conventional MEA technology to enable electrophysiological investigation at sub-cellular level without the need of any surface coatings. This presented approach would greatly impact our basic understanding of signal transduction within a single cell as well as complex cellular assemblies.
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