Raymond Hickey, Ph.D.
Curative ex vivo Hepatocyte-directed Gene Editing in a Mouse Model of Hereditary Tyrosinemia Type 1
CaitlinVanLith; Rebekah Guthman; Clara Nicolas; Kari Allen; DongJin Joo; Scott Nyberg; Joseph Lillegard; Raymond Hickey.
Mayo Clinic, Rochester, Minnesota.
Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). Our work has previously shown that ex vivo hepatocyte-directed gene therapy using an integrating lentiviral vector to replace the defective Fah gene can cure liver disease in small and large animal models of HT1. In this study, we hypothesized that ex vivo hepatocyte-directed gene editing using CRISPR/Cas9 could be used to correct a mouse model of HT1 in which a single point mutation results in loss of FAH function. To achieve high transduction efficiencies of the target hepatocytes, we utilized a lentiviral vector (LV) to deliver both the Streptococcus pyogenes Cas9 nuclease and target guide RNA, and an adeno-associated viral vector to deliver a 1.2kb homology template (AAV-HT). Cells were isolated from Fah-/- mice and cultured in the presence of LV and AAV vectors. Transduction of cells with LV-Cas9 induced significant indels at the target locus, and correction of the point mutation in Fah-/- cells using AAV-HT was completely depended on LV-Cas9. Next, hepatocytes transduced ex vivo by LV-Cas9 and AAV-HR were then transplanted into syngeneic Fah-/- mice that had undergone two-thirds partial hepatectomy or sham hepatectomy. Recipient animals were cycled on/off the protective drug NTBC to provide a selective advantage for corrected FAH+ cells to proliferate. Interestingly, a significant improvement was observed in weight stability off NTBC between animals that received partial hepatectomy or not. After six months, mice were euthanized and thorough biochemical and histological examinations performed. All transplanted mice became weight stable off NTBC. Biochemical markers of liver injury were significantly improved over non-transplanted control animals. Histological examination of mice revealed normal tissue architecture and immunohistochemistry showed robust repopulation of recipient animals with FAH+ cells, with increased corrected cells in mice that had undergone partial hepatectomy. In summary, this is the first report of ex vivo hepatocyte-directed gene repair using CRISPR/Cas9 to demonstrate curative therapy in an animal model of liver disease.
Viola Vogel, Ph.D.
Unraveling the Secrets of How the Mechanobiology of Extracellular Matrix Regulates Cell and Tissue Functions
Department of Health Science and Technology; Institute of Translational Medicine, ETH Zürich, Switzerland
Extracellular matrix (ECM) fibers are far more than passive scaffolds to anchor cells. While cells need to be anchored to ECM to survive, the role of ECM in guiding developmental processes, tissue homeostasis, and aging has long been underestimated. Also how ECM orchestrates wound healing and the deterioration of healthy toward pathological tissues, including fibrosis and cancer, remains poorly understood. Mechanobiology is a rapidly growing field, and many novel mechanisms have recently been deciphered on how mechanical factors can switch protein functions, and thus cell signaling. Inquiring how the biochemical and physical crosstalk between ECM and cells drives tissue morphogenesis is thus timely, as this might inspire new thoughts on how to better exploit the mechanobiology of ECM in tissue engineering, regenerative medicine as well as for diagnostic and therapeutic applications.
Bruno Péault, Ph.D.
Perivascular Presumptive Mesenchymal Stem Cells: Identity, Diversity, Versatility
MRC Centre for Regenerative Medicine, Edinburgh, UK, and Dept of Orthopaedic Surgery, University of California, Los Angeles, USA
Mesenchymal stem cells are favourite regenerative cells in multiple protocols of cell therapy and tissue engineering. MSCs have been tested in about 800 clinical trials; this interest is justified by the diverse and robust potentials deployed by these cells to achieve tissue (re)generation and repair: MSCs can be tissue progenitors but also, via cell contact and secretion of trophic factors, stimulate angiogenesis, regulate immune/inflammatory reactions and support lineage committed stem cells. Moreover, mesenchymal stem cells are easy to derive and expand from virtually any vascularized organ, leaving a choice of convenient, abundant and dispensable sources such as adult abdominal fat and foetal appendages at birth. On the other hand, indirect selection by adherence and proliferation in culture has obscured the biologic characteristics of innate mesenchymal stem cells. MSCs being by definition long-term cultured cells, their native embryonic origin, identity, lineage affiliation, tissue distribution, frequency and – importantly – natural role in tissue homeostasis and repair has remained unknown decades after their initial discovery. In the past ten years, the very identity of native mesenchymal stem cells has been gradually uncovered, revealing a perivascular origin for these elusive regenerative cells. Natural MSCs are pericytes, encircling capillaries and micro-vessels, and adventitial cells surrounding large arteries and veins. Prospective identification of innate MSCs now opens the possibility of using highly purified, uncultured and precisely characterized mesenchymal stem cells for cell therapies. We will discuss the cellular and molecular characterization of human MSCs, the natural role of subsets of these cells in the fibrotic degeneration or repair of skeletal and cardiac muscles, and the use thereof in diverse cell therapy protocols.
Cellular and Molecular Regulation of Mammalian Heart Regeneration
Ahmed I. Mahmoud
Department of Cell and Regenerative Biology, University of Wisconsin-Madison
Heart failure is the leading cause of death in the world due to the inability of the adult mammalian heart to regenerate following injury. Lower vertebrates, such as zebrafish are capable of complete and efficient regeneration of the myocardium following injury. Similarly, we demonstrated that neonatal mice are capable of regenerating their hearts within a short period after birth but lose this potential in the first week of life. Adult mammals lack this cardiac regeneration potential, yet how and why mammals lose this regenerative potential remains unclear. Thus, our overarching goal in the laboratory is to dissect the molecular underpinnings of regeneration in the neonatal heart so that we can explore potential avenues to activate this process in adult humans.
Polycaprolactone-chitosan Nanofibers Electrospun on Decellularized Bovine Pericardium as a Regenerative Biomaterial for Heart Valve Tissue Replacement
Jahnavi Mudigonda; Muralidhar Padala
Division of Cardiothoracic Surgery, Emory University School of Medicine, Atlanta, GA
Objective: Glutaraldehyde (GA) fixation is a crucial step in preparing decellularized bovine pericardium (BP) for use in bioprosthetic heart valves (HVs). GA crosslinks collagen and imparts substantial mechanical strength and durability to the tissue, that is lost during decellularization. However, GA is also cytotoxic, pro-calcific and often leads to accelerated degeneration and failure of bioprosthetic HVs. In this study, we sought to develop a new strategy by electrospinning polycaprolactone-chitosan nanofibers (PCL-CH-NF) onto the surface of decellularized BP to obtain the same mechanical strength as GA treated tissue, but without the use of any cytotoxic agents. We demonstrate that this strategy results in a material that has equivalent mechanical strength to fresh BP, and could potentially enable cellular infiltration and material regeneration.
Methods: Fresh BP was decellularized with 1% sodium deoxycholate, and analyzed for acellularity (DNA estimation and presence of nuclei on H&E stain) and extracellular matrix (ECM) damage (histology and scanning electron microscopy-SEM). PCL-CH-NF were electrospun onto the decellularized BP, along the native collagen fibers in the BP. Nanofiber attachment to the decellularized tissue was analyzed with SEM, and tensile testing was performed to compare the stiffness and yield strength of fresh BP, decellularized BP and PCL-CH-NF electrospun decellularized BP.
Results: The nanofiber layer thickness was 70±10 nm, with good attachment of the nanofibers to the underlying matrix and without delamination. H&E, DAPI and trichrome staining demonstrated acellularity in the decellularized BP and PCL-CH-NF covered decellularized BP, with preserved ECM architecture. The yield stress was 40 MPa for fresh BP, 22 MPa for decellularized BP and 50 MPa for PCL-CH-NF covered decellularized BP, indicating that the electrospun nanofibers restored the mechanical strength after decellularization.
Conclusion: Electrospinning polymeric nanofibers onto decellularized BP can reliably increase the mechanical strength of the tissue, without the cytotoxic effects of GA and thus lower risk of degradation.
Medial Hypoxia and Adventitial Vasa Vasorum Remodeling in Human Ascending Aortic Aneurysm
Marie Billaud (1-4); Jennifer C. Hill (1); Tara D. Richards (1); Jeffrey Nine (5); Thomas G. Gleason (1-4); Julie A. Phillippi (1-4)
Department of Cardiothoracic Surgery, University of Pittsburgh, Pittsburgh, PA (1); McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA (2); Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA (3); Center for Vascular Remodeling and Regeneration, University of Pittsburgh, Pittsburgh, PA (4); Department of Pathology, University of Pittsburgh, Pittsburgh, PA (5)
Human ascending aortic aneurysm pathophysiology involves elastin degeneration, and loss of smooth muscle cells in the media provoked by mechanisms that are mostly unknown. We recently uncovered down-regulation of several key pro-angiogenic factors in the adventitia from aneurysmal aortic specimens. In this study, we investigated the role of the adventitial microvascular network (vasa vasorum) and hypothesized that the vasa vasorum is disrupted in patients with ascending aortic aneurysm and is associated with medial hypoxia. Morphometric analyses of hematoxylin and eosin-stained human aortic cross-sections revealed evidence of vasa vasorum remodeling in aneurysmal specimens, including reduced density of small (<50 µm) vessels, increased lumen area and thickening of smooth muscle actin-positive layers. Gene expression of hypoxia-inducible factor 1α and its downstream targets, vascular endothelial growth factor and metallothionein 1A, were down regulated in the adventitia of aneurysmal specimens when compared with non-aneurysmal specimens. Immunodetection of glucose transporter 1, a marker of chronic tissue hypoxia, revealed minimal expression in non-aneurysmal specimens and high local accumulation within regions of medial elastin degeneration in aneurysmal specimens. This was accompanied with increased protein expression of glucose transporter 1 in the media of aneurysmal specimens when compared to non-aneurysmal specimens. These data reveal vasa vasorum remodeling in the aortic adventitia of patients with thoracic aortic aneurysm, associated down-regulation of angiogenic and hypoxia-related gene targets in the adventitial layer and evidence of chronic hypoxia in the aortic media. The noted vasa vasorum remodeling, coupled with deficiency of pro-angiogenic gene targets in the adventitia could contribute to medial degeneration through malnourishment of the aortic media, and a compromised ability to undergo vasculogenesis in the adventitia.
Characterize Coronary Revascularization During Heart Regeneration in Zebrafish
Jisheng Sun; Jinhu Wang
Division of Cardiology, Emory University School of Medicine
Humans, like all mammals, lack a natural ability to replace lost cardiac contractile tissues after injury. This inability to regenerate can lead to heart failure, the major cause of morbidity and mortality. Zebrafish efficiently regenerate functional myocardium following cardiac injury and represent a useful model for heart repair. We are interested to understand how regenerative responses to injury have been optimized in non-mammalian vertebrates like zebrafish, to discover new targets that underlie regenerative deficiencies in mammals. We have investigated the regenerative biology of two major adult cardiac tissues: the myocardium and the epicardium. We found that adult zebrafish can fully regenerate lost myocardium and reverse signs of heart failure within several weeks, and the epicardium is required for myocardial regeneration. Currently, we are defining roles of non-myocardial cells like perivascular cells and mechanisms of coronary revascularization during heart regeneration, utilizing genetic manipulation and live imaging techniques. In preliminary studies, we employed deep sequencing, in situ hybridization and BAC transgenic technology in search for novel genetic markers specific for perivascular cells and coronary endothelial cells, and candidate genes upregulated after heart injury. We have identified a new transgenic strain that specifically marks perivascular cells of coronary vessels, and also identified a novel transgenic strain that specifically labels coronary vessels. We have developed an ex vivo system to monitor robust coronary vascularization in heart surface. We will address central questions about requirements of perivascular cells for cardiac regeneration and regulation of coronary revascularization with bunch of new tools.
Tissue Specific Muscle Extracellular Matrix Hydrogels Improve Skeletal Muscle Regeneration in vivo over Non-matched Tissue Sources
Jessica L. Ungerleider (1); Dylan Zawila (2); Juhi Madan (1); Samuel R. Ward (1, 3, 4); Karen L. Christman (1, 5)
Bioengineering, University of California, San Diego (1); Biomedical Engineering, University of Minnesota (2); Orthopedic Surgery, University of California, San Diego (3); Radiology, University of California, San Diego (4); Sanford Consortium for Regenerative Medicine (5)
Decellularized extracellular matrix (ECM) hydrogels present a novel, clinical intervention for a myriad of regenerative medicine applications. In this paradigm, the source of ECM is typically the same tissue to which the treatment is applied; however, the need for tissue specific ECM sources has not been rigorously studied. We hypothesized, in a muscle regeneration model, that tissue specific ECM improves regeneration and function through preferentially stimulating physiologically relevant processes (e.g. progenitor cell proliferation and differentiation). One of three decellularized hydrogels: tissue specific skeletal muscle, non-specific cardiac muscle, and non mesoderm-derived lung, or saline were injected intramuscularly two days after notexin injection in male adult C57BL/6 mice (n=7 per time point) and muscle was harvested at days 5 and 14 for histological and gene expression analysis. All injectable hydrogels were decellularized using detergent methods and were controlled for donor characteristics (i.e. species, age). At day 5, the skeletal muscle ECM hydrogel significantly increased the density of Pax7+ and myogenin+ satellite cells in the muscle. Gene expression analysis at day 5 showed that both cardiac and skeletal muscle ECM hydrogels increased expression of genes implicated in muscle hypertrophy/growth. By day 14, both skeletal muscle and cardiac muscle ECM hydrogels improved muscle regeneration over saline and lung ECM hydrogels as shown through a shift in fiber cross sectional area distribution towards larger fibers (average fiber areas – saline: 564 ± 64 µm2; lung ECM: 545 ± 65 µm2; cardiac ECM: 623 ± 80 µm2; skeletal muscle ECM: 695 ± 94 µm2; data are mean ± SEM). This data indicates a potential role for muscle-specific regenerative capacity of decellularized, injectable muscle hydrogels. Further transcriptomic analysis of whole muscle mRNA is ongoing to understand mechanisms of tissue specificity in decellularized ECM hydrogels. This will lead to a greater understanding of the need (or lack thereof) for tissue specificity in regenerative medicine applications and elucidate potential ECM-mediated tissue repair mechanisms in vivo.
Synthetic Bioadhesive Matrix Facilitates Muscle Stem Cell Transplantation and Engraftment in Dystrophic Diaphragm
Woojin M. Han (1,2); Shannon E. Anderson (1,3); Mahir, Mohiuddin (1,3); Young C. Jang (1,3,4); Andrés J. García (1,2)
Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology (1); Woodruff School of Mechanical Engineering, Georgia Institute of Technology (2); Coulter Department of Biomedical Engineering (3); School of Biological Sciences, Georgia Institute of Technology (4)
Duchenne muscular dystrophy (DMD) is a devastating genetic disorder that affects approximately 1 in 3,500 males. A leading cause of death in DMD is diaphragm muscle deterioration, and current respiratory care, such as mechanically assisted ventilation, remains palliative. A therapeutic strategy targeting the diaphragm muscle to restore the respiratory capacity is in a critical need. DMD is caused by the absence of dystrophin, a structural protein that provides support between the muscle fiber cytoskeleton and the extracellular matrix. Restoration of dystrophin through muscle satellite cell transplantation improves the muscle function, but direct cell delivery is limited by sub-optimal survival and engraftment. Furthermore, cell delivery strategies designed to target the anatomically deep-seated and dimensionally thin diaphragm muscle have not been developed. The objective of this work is to engineer a synthetic matrix to facilitate the delivery and engraftment of muscle satellite cells into the dystrophic diaphragm muscles. We engineered a synthetic matrix that supports primary satellite cell survival, proliferation, and differentiation using hydrogels based on PEG-4MAL macromers. Satellite cells cultured in the RGD-presenting hydrogels formed significantly larger MyoD+ 3D myogenic colonies after 6 days of culture compared to colonies formed in RDG, YIGSR, and C16-presenting hydrogels (p<0.0001). While no differences in EdU incorporation were observed, cell survival was significantly higher in the RGD-presenting hydrogels compared to RDG-presenting hydrogels (p<0.05), suggesting that RGD-presenting hydrogels promote cell survival and subsequently stimulate the formation of large myogenic colonies. Myogenic colonies formed in the RGD-presenting hydrogels exhibited significantly lower cell packing density compared to RDG, YIGSR, and C16-presenting hydrogels (p<0.0001), indicating cellular migration. When primed for differentiation, the cells in the RGD-presenting hydrogels formed significantly higher multinucleated myotubes compared to the cells in the RDG-presenting hydrogels (p<0.0001). The optimal material stiffness (G’ ~175 Pa) and mesh size (30 nm) of the hydrogel was also determined by modulating the PEG-4MAL macromer density to further enhance the satellite cell function in 3D. We further developed a delivery strategy to firmly integrate the engineered hydrogel to the inferior surface of the dystrophic diaphragm. Fluorescently-labeled hydrogel delivered to the diaphragm remained localized to the site of delivery, whereas fluorescently-labeled, uncrosslinked hydrogel precursor solution resulted in a non-specific distribution to other internal organs including the large intestine, stomach, and liver. Finally, GFP+ satellite cells delivered to the diaphragm using the engineered hydrogel survive, proliferate, and engraft in vivo.
Biomimetic Sponges Support Myogenic Activity in a Murine Volumetric Muscle Loss Model
Andrew Dunn (1); Gabriel Haas (1); Madison Marcinczyk (1); Muhamed Talovic (1); Robert Scheidt (1); Anjali Patel (1); Mark Schwartz (1); Katherine R Hixon (1); Hady Elmashhady (1); Sarah H McBride-Gagyi (2); Scott A Sell (1); Koyal Garg (1)
Department of Biomedical Engineering, Parks College of Engineering, Aviation, and Technology, Saint Louis University (1); Department of Orthopedic Surgery, Saint Louis University (2)
Musculoskeletal injuries are among the most common and frequently disabling injuries sustained by athletes and soldiers. Most of these injuries involve volumetric muscle loss (VML), defined as the as the surgical or traumatic loss of muscle tissue with resultant functional impairment. While skeletal muscle is remarkably regenerative, VML injuries are irrecoverable in humans and animal models due to the complete loss of indispensable regenerative elements such as basal lamina and resident satellite cells. Currently, there are no approved therapies for the treatment of muscle tissue following trauma, presenting a significant opportunity to develop tissue engineered scaffolds for muscle tissue regeneration.
To improve regeneration of skeletal muscle, we have developed biomimetic sponges composed of collagen, gelatin, and laminin (LM)-111 that were crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Collagen and LM-111 are crucial components of the muscle extracellular matrix and were chosen to impart bioactivity whereas gelatin and EDC were used to provide mechanical strength to the scaffold. Morphological and mechanical evaluation of the sponges showed porous structure, water-retention capacity and a compressive modulus of 590kPa. In vitro testing revealed that compared to pure gelatin sponges, the biomimetic sponges supported greater C2C12 myoblast infiltration, myokine (VEGF, IL-6, and IGF-1) production and myogenic marker (MyoD and myogenin) expression.
The biomimetic sponges were implanted in a mouse model of VML. At 2 weeks post-injury, the biomimetic sponge treated VML injured muscles showed constructive remodeling at the site of injury with the elevated presence of satellite (Pax7+), endothelial (CD31+) and inflammatory (F4/80+) cells compared to untreated VML injured muscles. The sponge treated muscles showed several small diameter myosin+ myofibers in the defect region and a higher quantity of centronucleated myofibers per muscle section. In support, the protein expression of MyoD and myogenin was also higher on the sponge treated injured muscles. However, the expression of heat shock protein (HSP)-70, a marker of cellular stress was lower with sponge treatment. Taken together, these results suggest that implantation of the biomimetic sponges is able to promote myogenic activity in the VML injured muscles. Future studies will evaluate the extent to which biomimetic sponges can improve muscle regeneration and force production at one-month post-VML injury.
Evaluation of the Host Immune Response to Decellularized Lung Scaffolds Derived from Wild Type or -Gal Knockout Pigs in a Non-human Primate Model
Elizabeth C. Stahl (1,2,3); Clint D. Skillen (1,2); Brandon B. Burger (1,2); Bryan N. Brown (*1,2,3)
McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA (1); Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA (2); Cellular and Molecular Pathology Training Program, University of Pittsburgh School of Medicine, Pittsburgh, PA (3)
The development of -Gal negative porcine tissues is an important step to overcome challenges of xenogeneic organ transplantation for human patients, as decellularization strategies alone do not completely remove -Gal epitopes. Humans and Old-World monkeys, such as the rhesus macaque, do not express the -Gal epitope and thus develop antibodies against -Gal, leading to complement activation and hyperacute rejection following exposure to porcine tissues.
In the present study, lungs from wild type or -Gal knockout pigs (GalSafe®) were harvested and decellularized to remove cellular antigens and implanted subcutaneously in a rhesus macaque model (n=8). Native porcine lungs and a sham injury group were included as controls. The scaffolds were explanted at 1, 2, 4, and 8 weeks then fresh scaffolds were re-implanted into sensitized hosts to evaluate adaptive immunity. Histological analyses (H&E, Masson’s Trichrome, and Verhoff-Van Gieson staining) were evaluated qualitatively and quantitatively. Markers of inflammation and wound healing were also examined using immunohistochemistry (CD4, CD8, CD45, CD20, CD68, CD86, CD31, CD1, CD206, and alpha-smooth muscle actin).
The GalSafe® decellularized lung implant performed similarly to the wild type decellularized lung implant in the acute study, and induced more CD3+ T-cells, primarily CD4+ helper subsets, compared to the other implantation groups. However, upon re-implantation into sensitized hosts, the GalSafe® decellularized lung implant had reduced CD3+ T-cell infiltration and no change in CD20+ B-cell levels, likely a result of reduced antibody-mediated immunological memory, while other groups had a more robust adaptive immune response. Overall, the GalSafe® decellularized lung implant performed similarly to the wild type decellularized construct in an acute setting, and shows promise as a potential strategy to overcome chronic immune challenges in xenotransplantation and regenerative medicine applications.
Min Kyoung Sun
Strain-promoted Alkyne–Azide Cycloaddition Derivatized Chondroitin Sulfate Glycosaminoglycan Matrices for Neural Tissue Engineering
Min Kyoung Sun (1); Pradeep Chopra (2); Yang Liu (3); Peter A. Kner (3); Geert-Jan Boons (2); Lohitash Karumbaiah (1)
Regenerative Bioscience Center, University of Georgia (1); Department of Chemistry, University of Georgia (2); Department of Electrical and Computer Engineering, University of Georgia (3)
Chondroitin sulfate glycosaminoglycans (CS-GAGs) are important regulators of neuronal homeostasis in the brain extracellular matrix. However, their use as scaffolds for neural tissue engineering has been underexplored. We have previously demonstrated that photopolymerized CS-GAG matrices consisting predominantly of monosulfated GAGspromoted NSC efficacy and enhanced neuroprotection after a severe traumatic brain injury (TBI). In this study, we use copper-free “click” chemistry to fabricate porous CS-GAG matrices capable of maintaining the undifferentiated state of neural stem cells (NSCs), or selectively facilitating neuronal differentiation as desired.
Polyethylene glycol (PEG) amine functionalized dibenzocyclooctynol (DIBO) was covalently coupled to the carboxyl group on the glucuronic acid on the CS-GAG chain at a molar ratio of 0.6:1. Aliphatic azide functionalized PEG is coupled to the CS-GAG chains similarly to yield azido-CS. 2.5% w/v DIBO-CS and 1% w/v azido-CS in basal media were combined to yield porous hydrogels. For neuronal differentiation studies, NSCs were encapsulated in L1 peptide functionalized hydrogels. Azide tagged L1 peptides (Az-PSITWRGDGRDLQEL, 5mM) are reconstituted with azido-CS to yield L1-azido-CS, and cross-linked with DIBO-CS as described above. Hydrogels are allowed to crosslink for minimum of five minutes at room temperature. Reaction products were validated using 1H NMR, and material properties were characterized using SEM, rheology, and swelling and degradation assays. Human induced pluripotent (HIP) NSCs were suspended in cell culture medium and either seeded on top of pre-fabricated hydrogels, or encapsulated within hydrogels. Layered cell-laden hydrogel complexes to mimic the layered architecture of the cerebral cortex were fabricated by dispensing hydrogel solutions containing NSCs, with and without L1 peptide, sequentially into fluorinated ethylene propylene tubes (0.8mm inner diameter). The interaction between differentiated neurons and undifferentiated NSCs encapsulated in layered hydrogel constructs was evaluated after immunohistochemical staining and validation of tri-lineage markers using light sheet fluorescence microscopy (LSFM).
The study demonstrates the potential utility of differentially functionalized and tunable CS-GAG “click” hydrogel matrix layers of defined structural properties to study NSC maintenance and differentiation, and as scaffolds for functional neural tissue repair after a severe TBI.
Therapeutic Efficacy of Intra–articular Delivered Encapsulated Human Mesenchymal Stem Cells in Osteoarthritis
Jay M. McKinney (1,2); Thanh N. Doan (1,2); Lanfang Wang (3); Juline Deppen (3,4); Ananthu Pucha (2); Rebecca D. Levit (3); Nick J. Willett (1,2,4,5)
Department of Orthopaedics, Emory University (1); Department of Orthopaedics, Atlanta Veteran’s Affairs Medical Center (2); Department of Medicine, Division of Cardiology, Emory University (3); Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University (4); Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology (5)
Osteoarthritis (OA) is a chronic disease of the joints that leads to degeneration of articular cartilage surfaces. Mesenchymal stem cells (MSC) present a promising treatment to target the disease, relying on their regenerative capacity along with their immunomodulatory and anti-inflammatory properties. However, many questions remain as to the mechanism of action of these cells following intra-articular delivery: paracrine action versus cellular engraftment. Cellular encapsulation presents a promising means to study the paracrine factors of MSCs, independent of cellular engraftment, as the alginate microencapsulation provides a mechanical barrier between the encapsulated cells and the native host tissue. The objective of this study was to quantitatively assess the efficacy of encapsulated hMSCs as a disease modifying therapeutic for OA. OA was surgically induced in rats via the medial meniscus transection (MMT) surgery. The efficacy of hMSC intervention was assessed using Lewis Rats with MMT (n=7-8 per group). Intra-articular injections of hMSCs, or controls (saline, empty capsules or non-encapsulated hMSCs), were given 1-day post-op and animals were euthanized at 3 weeks. Micro-structural changes in the articular cartilage were assessed using equilibrium partitioning of an ionic contrast agent based micro-computed tomography (EPIC-ɥCT). We hypothesized that encapsulated hMSCs would have a therapeutic effect, via paracrine mediated action, on OA progression. Quantitative analysis of articular cartilage, via EPIC-ɥCT, showed attenuated increases in cartilage thickness and surface roughness for the encapsulated hMSC condition in comparison to all other groups. Osteophyte volumes, defined as thickening and partial mineralization of cartilaginous tissue at the marginal edge of joints, were augmented in the encapsulated hMSC group in comparison to all other MMT conditions, except non-encapsulated hMSCs. Furthermore, encapsulated hMSCs yielded increased mineralized osteophyte volumes compared to all other groups. This is the first study to demonstrate that the paracrine signaling properties of hMSCs, independent of direct cellular engraftment, can exert a chondroprotective role in OA. However, these protective effects were countered by enhancements of osteophyte volumes. These augmented tissue volumes are especially relevant in clinical application as many clinical trials are currently ongoing and these findings have yet to be reported.
Microfluidic Poly(ethylene glycol)-based Islet Encapsulation Enables Transplantation in Highly Vascularized Site
Jessica D. Weaver; Devon M. Headen; Maria M. Coronel; Michael Hunckler; Andrés J. García
Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology
Encapsulation is a promising biomaterials-based method to reduce immune destruction of transplanted islets in the absence of chronic immunosuppression. A primary limitation of this technology is large capsule diameter, which limits transport of nutrients and insulin through the hydrogel barrier. In addition, large capsule diameter limits graft delivery to the intraperitoneal cavity, resulting in a non-retrievable graft with poor proximity to vascular exchange, resulting in poor graft performance. Herein, we describe a microfluidic, synthetic poly(ethylene glycol) (PEG)-based encapsulation system which allows the reproducible generation of capsules with a 200 µm smaller diameter than traditional electrostatic alginate techniques, and molecular transport properties comparable to gold-standard alginate polymer. This reduced diameter enables encapsulated islet transplantation to isolated and retrievable transplant sites, such as the murine epididymal fat pad (EFP), which enhances islet proximity to vasculature and demonstrates improved syngeneic islet performance in vivo over larger alginate capsules. Moreover, we demonstrate via longitudinal in vivo islet imaging that reduced capsule size confers comparable protection from allo- and autoimmune attack when compared with gold standard alginate encapsulated controls, and in vivo graft monitoring allowed quantification of the degree of graft protection imparted by biomaterial encapsulation alone. In sum, PEG-based microfluidic islet encapsulation protects grafts from direct antigen recognition comparably to gold-standard alginate, while reducing graft size to facilitate safer and more effective encapsulated cell transplantation strategies.
Catalina Pineda Molina
4-Hydroxybutyrate Modulates Activation of Macrophages and Induces Endogenous Expression of Antimicrobial Peptides
Catalina Pineda Molina (1,2); George S. Hussey (1,3); Jonas Eriksson (1,3); Michael A. Shulock (1,2); Laura L. Cardenas Bonilla (1); Ross M. Giglio (1,2); Riddhi M. Gandhi (1,2); Angela Ramirez (1); Brian M. Sicari (1,3); Ricardo Londono (1,4); Stephen F. Badylak, D.V.M., Ph.D., M.D. (1,2,3)
McGowan Institute for Regenerative Medicine (1); Department of Bioengineering, University of Pittsburgh (2); Department of Surgery, University of Pittsburgh (3); School of Medicine, University of Pittsburgh (4)
The molecule 4-hydroxybutyrate (4HB) is a naturally occurring bioactive endogenous short chain fatty acid (SCFA) that has been used for the production of polymeric mesh materials for soft tissue repair applications. The 4HB monomer has been exhaustively studied for its role as metabolite and modulator of the neurotransmitter g-aminobutyric acid (GABA) in the central nervous system (CNS). Other functions of 4HB within non-CNS tissues have been less studied; however, this SCFA is a hydroxylated form of butyrate, a known histone deacetylase (HDAC) inhibitor secreted by commensal bacteria within the gastrointestinal tract. Butyrate reportedly exerts its immunomodulatory functions by suppressing pro-inflammatory macrophages and promoting antimicrobial peptide (AMP) secretion. However, the immunomodulatory effects of 4HB upon cells of the immune system and the ability of 4HB to induce the expression of AMP have not been studied. The present study evaluated the effect of 4HB upon macrophage phenotype and the expression of AMP, and the molecular mechanisms by which such effects are mediated.
The monomer 4HB was used in-vitro to evaluate the activation of markers associated with the pro-inflammatory (iNOS+) and anti-inflammatory (Fizz-1+, Arginase-1+) phenotype of murine bone marrow-derived macrophages. The expression of the AMP cathelicidin LL-37 and β-defensins were also determined. The molecular mechanisms involved in the expression of AMP were evaluated by gene expression and inhibition immunoassays to target intermediate proteins within the pathway. In addition, a surgical mesh material composed of poly (4-hydroxybutyrate) (P4HB) was evaluated in-vivo using a rat bilateral partial thickness abdominal wall defect model. Immunolabeling quantification was used to determine the immunomodulatory effects of P4HB upon macrophage phenotype, using CD-68+/CD206+ (anti-inflammatory), and CD-68+/CD-86+ (pro-inflammatory) markers, and cathelicidin LL-37 expression, at 3, 7, 14, 21, and 35 days post-implantation.
The results show the ability of 4HB to promote an anti-inflammatory, regulatory macrophage phenotype and increased expression of AMP. The associated molecular mechanisms involve transcriptional activation of the genes codifying the AMP through the MAP-kinase pathway. The results expand the understanding of the biologic activity of 4HB in cells of the immune system, and its potential to promote a constructive modulatory effect for regenerative medicine applications.
Stimulation of Exosome Producing Cells Alters Immune Modulation
Seth Andrews (1,2); Timothy Maughon (1,2); Steven Stice (1)
Regenerative Bioscience Center, University of Georgia, Athens, Georgia (1); College of Engineering, University of Georgia, Athens, Georgia (2)
Exosomes, small membrane bound vesicles secreted from nearly all cells, have recently attracted interest as delivery vehicles for therapeutics. Stem cell generated exosomes are of particular interest, as they have been shown to recapitulate some of the effects of the cells themselves. Stem cells are responsive to their environment, with mesenchymal stem cells (MSCs) changing to an “activated” phenotype in the injury microenvironment. This study examined the effects that acidity, hypoxia, and inflammation have on exosome production in neural (NSC) and mesenchymal stem cells, as well as the immune modulatory potency of those exosomes, compared to other cell secreted factors.
Human NSCs and MSCs were cultured in serum free media under inflammatory, acidic, hypoxic, or normal culture conditions. Conditioned media was collected for ultrafiltration to isolate exosomes. Both exosome depleted media and exosomes were frozen at -20C. Vesicle size distribution and concentration were measured via Nanosight. Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors, stained with CFSE, and stimulated with anti CD3/CD28 Dynabeads for three days in the presence or absence of exosomes or conditioned media. The PBMCs were then harvested and stained for CD4, CD8, and CD25, or CD4, CD8, TNF-a, and IFN-y. Flow cytometry was used to obtain the mean percent positive and mean intensity for CFSE, CD25, TNF-a, and IFN-y in CD4+ and CD8+ cells as a measure of the immune modulation of T cells.
Preconditioning with different aspects of the wound microenvironment had dramatically varying effects on exosome production in two stem cell types. MSC exosome production was greatly increased by acidic or inflammatory culture (p<0.05, p<0.01 respectively), while NSCs had little response to the same environment. Hypoxia had little effect on the number of exosomes produced by either cell type. These findings underscore the importance of the cell microenvironment on exosome release, and the immunomodulatory potency of the exosomes will be investigated further by the previously mentioned flow cytometry experiments.
Ipsita Banerjee, Ph.D.
Engineering Micro-vascularized Pancreatic Islet Organoids from Human Pluripotent Stem Cells (hPSCs)
Joseph Candiello (1); Taraka Sai Pavan Grandhi (6); Jacob Dale (8,9); Suzanne bertera (10); Jason Beare (8,9); Kaushal Rege (6,7); Jay Hoying (8,9); Prashant N. Kumta (1,2,3,4,5); Ipsita Banerjee (2,1,5)
Department of BioEngineering, University of Pittsburgh (1); Department of Chemical Engineering, University of Pittsburgh (2); Department of Mechanical Engineering and Material Science, University of Pittsburgh (3); Center for Complex Engineered Multifunctional Materials, University of Pittsburgh (4); McGowan Institute for Regenerative Medicine, University of Pittsburgh (5); Biomedical Engineering, Arizona State University (6); Chemical Engineering, Arizona State University (7); Cardiovascular Innovation Institute, University of Louisville (8); Department of Physiology, University of Louisville (9); Alleghany Health Network (10)
An emerging area in tissue engineering is the development of three dimensional engineered constructs, organoids, which are comprised of multiple organ-specific cell populations capable of recapitulating an in-vivo organ system’s structure and function in an in-vitro setting. Engineering tissue specific organoids from human pluripotent stem cells (hPSCs) has resulted in successful reproduction of similar organ functionality from renewable cell source for a variety of target organ systems. Such systems have included derivation of intestinal, brain, and renal organ models. Development of organoid systems requires organ-specific parenchyma cell source and a platform for self-organization and lineage specific induction of chosen cell types. Additionally, reproducing the organ-specific functional vasculature during reproduction of the complex organ structure is extremely vital for maintaining nutrient supply and appropriate organ function. This is particularly crucial while engineering pancreatic islet organoids, since a dense fenestrated intra islet vasculature is vital for supporting glucose delivery and insulin response.
We have developed a novel hydrogel which promotes spontaneous aggregation of pre- differentiated hPSC derived pancreatic progenitor cells (hESC-PPs) into a 3D organoid which demonstrated functional insulin production in vitro as well as in vivo mouse model. The resulting spheroids are readily recoverable and amenable for size and cellular composition tuning. This hydrogel mediated aggregation allowed direct integration of isolated microvessel fragments within the hESC-PP organoids. Continued culture of the multicellular islet organoids promoted microvascular expansion and formation of vascular networks, especially with inclusion of supporting stromal cell populations. Pancreatic phenotype of the vascularized organoids was strengthened, demonstrated by the gene expression of key pancreatic maturation markers (NKX6.1, PDX1, and INS). Moreover, the intra-organoid vasculature demonstrated an increase in islet endothelial specific API gene expression and PLVAP, an indicator of increased endothelial diaphragm and fenestration, key elements of islet vascular development. Implantation of the vascularized organoids under mouse kidney capsules showed resulted in rapid engraftment of the organoids and inosculation with host vasculature, as confirmed with dextran infusion. Both microfragment derived vessels (GFP positive) and host vessels could be detected in the implanted organoids. In conclusion, we believe the results present a major step in the in-vitro production of microvascularized hPSC islet organoids, likely to enhance function and foster faster in-vivo integration, while also being conducive for organ-on-a-chip applications.
Modulating MSC Secretome to Assess Cell Donor Variability
Gilad Doron (1); Johnna Temenoff (1,2); Robert Guldberg (2,3)
Walter H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology (1); Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology (2); George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology (3)
Because of their ability to secrete cytokines and growth factors that modulate the innate immune response, mesenchymal stem cells (MSCs) have been used in several clinical trials to reduce symptoms and promote tissue healing in multiple disease states (Squillaro et al. Cell Transplant, 2016). Yet despite their clear therapeutic potential, MSC-based therapies have demonstrated a range of efficacy. This inconsistency has largely been attributed to variability of the secretome among MSCs from different donors cultured under varying conditions (Mendicino et al. Cell Stem Cell, 2014). Thus, there is a need to characterize the secretome and predict the potency of MSCs from individual donors before they are used in clinical trials in order to achieve consistent therapeutic outcomes. In this work, we used a multivariate secretome characterization of MSCs cultured under different physical conditions in order to distinguish between MSCs from individual donors.
To characterize the secretomes from individual donors, we analyzed their conditioned culture media for over 40 different immunomodulatory factors. MSCs isolated from bone marrow aspirates from 4 donors were either separately formed into 500-cell aggregates and placed in suspension culture or plated at 13,000/cm2 in static culture. After 4 days in culture, conditioned media was collected and quantified for various immunomodulatory factors using magnetic bead-based cytokine assay kit (HCYTMAG-60K-PX41, Millipore), individual PGE-2 and TSG-6 ELISAs (R&D Systems) and a previously-established IDO enzymatic activity assay (Agaugué et al. J Immunol 2006). Secretome data was normalized to the total number of cells in culture, determined by an automated cell counter (Countess II, Invitrogen).
Of the 43 screened immunomodulatory factors, 18 of the factors were found to be secreted in detectable amounts by MSCs from all donors in both monolayer and aggregate cultures. Formation into 500-cell aggregates increased the secretion of the majority of detectable immunomodulatory factors in 3 of the 4 cell donors. Although no secretory differences were found between cell donors in monolayer culture, formation of MSCs into aggregates revealed differences in the secretion of 10 of the 18 detectable factors (p<0.05). Applying multivariate statistical analysis (partial least squares discriminant analysis) created a model from the secretome data that could significantly discriminate among the secretory profiles of MSC aggregates from all but 2 donors (p<0.05).
This study demonstrates that secretome analysis can be used to characterize MSC donor-to-donor variability. Additionally, this work shows that amplification of secretome through cellular aggregation can increase the sensitivity of this method to assess donor variability. Because MSCs are thought to act through secreted factors, in the future, secretome analysis and modeling may become an important metric in determining differences in cell potency.
Kristi Anseth, Ph.D.
Development and Application of Photoadaptable Hydrogels for Culturing Intestinal Organoids and Directing Crypt Formation
Department of Chemical and Biological Engineering and the BioFrontiers Institute, University of Colorado Boulder
Hydrogels possess a number of material properties that render them useful as synthetic extracellular matrices for 3D cell culture or cell delivery systems for regenerative medicine; these properties include cytocompatibility, ease of functionalization, and physical properties similar to many soft tissues. Many synthetic hydrogel systems are highly tunable, both in terms of their mechanical and biochemical properties, and when combined with advanced processing methods, experimenters can create hydrogels with gradients, patterned ligands or other hierarchical structures found in native tissues. While the ability to control and manipulate hydrogel properties across many size scales has been powerful, the native extracellular matrix (ECM) is a dynamic environment, particularly during development, wound healing, and disease, and biological and mechanical signals can change dramatically with time. The ability to dynamically tune hydrogel properties can be lost in many synthetic ECM mimics, especially if they are crosslinked and functionalized by irreversible covalent bonds. In an effort to capture the variable nature of native cellular microenvironments, we have synthesized hydrogels containing allyl sulfide functionalities that are capable of rapid photoinduced network reorganization. In the presence of a photoinitiator, the allyl sulfide containing crosslinks can undergo a light-mediated addition fragmentation chain-transfer (AFCT) reaction, which allows for in situ manipulation of cell-laden matrices. This talk will highlight results where the AFCT reaction is leveraged to erode, stiffen/soften, or induce transient plasticity in the presence of intestinal crypt stem cells, and subsequently highlight the 2-photon spatial temporal control of the gel properties to direct the growth of intestinal organoids and their crypt structures.
Randolph Ashton, Ph.D.
Engineering Human CNS Morphogenesis in 2- and 3D: Controlled Induction of Neural Rosette/Tube Formation
Gavin T Knight (1,2); Carlos Marti-Figueroa (1,2); Nisha Iyer (2); Brady S Lundin (1,2); Frank Seipel (1,2); Stephanie Cuskey (2); Randolph S Ashton (1,2)
Department of Biomedical Engineering, University of Wisconsin, Madison WI, USA (1); Wisconsin Institute for Discovery, University of Wisconsin, Madison WI, USA (2)
Human pluripotent stem cell (hPSC) derived neural organoids provide unprecedented potential to recapitulate human brain and spinal cord tissues in vitro. However, organoid morphogenesis relies upon spontaneous emergence of biomimetic tissue structures in the absence of normal developmental constraints and morphogen signaling centers. This inherently limits the reproducibility and biomimicry of organoid cytoarchitecture and potentially impedes maturation and interconnectivity of developing tissue structures. As an initial step in overcoming these limitations, we have begun investigating how spatial and temporal control of tissue morphology and morphogen gradients can be used to instruct controlled neural organoid morphogenesis in 2- and 3D. Using micropatterned culture of hPSC-derived neural stem cells (NSCs), we have identified biophysical parameters necessary to induce the emergence of tissues with a singular neural rosette cytoarchitecture (>80% efficiency), which resembles a 2D cross-section of the developing neural tube. Additionally, we are (1) applying microfluidic morphogen gradients to pattern ventral thru dorsal progenitor domains within arrayed neural rosette tissues, and (2) have engineered culture substrates that enable spatiotemporal control of the tissues’ radial expansion while maintaining an apical, polarized neuroepithelial zone. Similar concepts are also being applied in 3D to generate biomimetic human neural tube analogs using a sacrificial lattice biomaterial platform. Ultimately, we aim to meld engineered culture platforms and organoid derivation protocols to enable reproducible derivation of brain and spinal cord tissues with organotypic cell phenotype diversity and biomimetic cytoarchitecture.
Jason Burdick, Ph.D.
Engineered Hydrogels to Enable Tissue Regeneration
Department of Bioengineering, University of Pennsylvania
Hydrogels represent a class of biomaterials that have great promise for the repair of tissues, particularly due to our ability to engineer their biophysical and biochemical properties. Hydrogels can provide instructive signals through material properties alone (e.g., mechanics, degradation, structure) or through the delivery of therapeutics that can influence tissue morphogenesis and repair. Importantly, hydrogel design should reflect both the clinical context and the natural healing cascades of the damaged tissue. Here, I will give examples of the design of hydrogels based on hyaluronic acid (HA) for the repair of musculoskeletal tissues that have limited natural repair processes.
Towards application in cartilage repair, we have developed hydrogels that introduce numerous biochemical signals to mediate stem cell chondrogenesis. These include binding to receptors (e.g., CD44) through the use of HA backbones or the introduction of peptides (e.g., HAVDI) that mimic n-cadherin interactions found during development. We have utilized engineered screening platforms to probe the influence of these various chemical signals on stem cell fate, as well as developed 3D printing technology to translate the signals into scaffold environments. Towards meniscus repair, multi-polymer fibrous hydrogels that permit control over scaffold porosity and therapeutic release via the engineering of specific fiber populations have been developed. For example, multi-polymer fibers were designed with fibers that selectively release collagenase to provide an environment permissive to cell recruitment, chemotactic signals to actually recruit cells, and stable fibers for structural support. When investigated in tissue repair, each fiber population was important to the success of repair tissues.
Diffusion-based Model with Fibrous Scaffolds to Predicting Released Gasotransmitter Concentration Available to Cells
Shawn Rottmann (1); Kenyatta S. Washington (1); Ronald-Dean R. Allado (1); Nawodi Abeyrathna (2); Yi Liao (2); Chris A. Bashur (1)
Department of Biomedical Engineering, Florida Institute of Technology (1); Department of Chemistry, Florida Institute of Technology (2)
Gasotransmitters such as carbon monoxide (CO) are cell-signaling molecules produced naturally in the body that have the potential to improve endothelialization in engineered vascular grafts at appropriate concentrations. Endogenous levels are anti-inflammatory and necessary for vascular function, high levels are pro-inflammatory, and levels in between have been had seemingly conflicting results between studies and different tissues. Thus, CO dose and its impacts need to be better understood. We have previously incorporated CO releasing molecules into electrospun scaffolds to provide controlled, local delivery. The goal of this study is to develop a two-part theoretical model to simulate diffusion of CO and other drugs through fibrous scaffolds commonly used for vascular tissue engineering, and predict the temporal concentration of drug available to cells. The fibrous scaffold was generated and voxelized within the Blender 3D creation suite and the diffusion model was developed in Fortran using the DLOSDE solver. Individual voxels were assigned as a fiber, a cell, or interstitial fluid and the diffusion across the boundary was defined (e.g., diffusivity, permeability) for each voxel transition. Model validation was performed. As drug components are released from fibers, only a fraction of the initial concentration that enters the interstitial fluid in vivo, or culture media in vitro, is available to cells. For simulated CO release from 50:50 PLGA fibers, release occurred quickly (i.e., for 80% release, 17 s from the fibers and 3 min to leave from a boundary vascular sink). Albumin release from the same system was much slower, as expected, and our simulated results showed that the concentration in the interstitial fluid stayed lower than the fast-releasing CO. In preliminary simulations, we have also demonstrated that mesh configuration impacts CO concentrations in the interstitial fluid, and our model suggests that more cell-fiber contact area leads to a higher concentration within the cell. Experimental validation of model release results is ongoing. We are also currently investigating the impact of the intracellular consumption term and a modified boundary for in vitro culture on diffusion. Overall, we hope that the concentration of delivered CO around the cell predicted with this model could provide a value that is difficult to obtain experimentally and will help make delivery results from in vitro culture relevant for the clinical situation.
Emulsion Inks for 3D Printing Tissue Engineering Scaffolds
The University of Texas at Austin
In this study, we describe a new solid freeform fabrication (SFF) technology capable of printing curable emulsion inks to form porous polyHIPE foams with hierarchical porosity. Briefly, HIPE material is deposited layer-by-layer using an open source 3D printer equipped with a syringe and motor-actuated plunger. Emulsions inks are rapidly cured after deposition by constant UV irradiation to form rigid constructs with interconnected porosity in a method we term Cure-on-Dispense (CoD) printing. 3D printed polyHIPE constructs benefit from the tunable pore structure of emulsion templated materials and the fine control over complex geometries of 3D printing that is not possible with traditional manufacturing techniques. Propylene fumarate dimethacrylate (PFDMA) was selected to fabricate bone grafts using this technology due to its established biocompatibility, osteoconductivity, and good compressive properties. Scaffolds fabricated from PFDMA emulsion inks displayed compressive modulus and yield strength of approximately 1 and 15 MPa, respectively. A decrease in infill (from 100% to 70%) resulted in a five-fold increase in permeability; however, there was also a corollary decrease in mechanical properties. In order to generate scaffolds with increased permeability without sacrificing mechanical strength, a biomimetic approach to scaffold design was used to reinforce the highly porous emulsion inks with a dense cortical shell of thermoplastic polyester. Herein, we report an open source method for creating hybrid multi-material scaffolds with emulsion inks and reinforced with a shell of thermoplastic poly(ϵ-caprolactone) (PCL) or poly(lactic acid) (PLA). A multi-modal printing setup was first developed that combined paste extrusion and high temperature thermoplastic extrusion with high positional accuracy in the dual deposition. Scaffolds printed with a PCL shell displayed compressive modulus and yield strength of approximately 30 and 3 MPa, respectively. Scaffolds printed with a PLA shell showed compressive modulus and yield strength of approximately 100 and 10 MPa, respectively. By combining this new paste extrusion of emulsion inks with traditional thermoplastic extrusion printing, we have created scaffolds with superior strength that promote cell viability and proliferation of human mesenchymal stem cells. The development of this technique shows great promise for the fabrication of a myriad of other complex tissue engineered scaffolds.
Krishanu Saha, Ph.D.
Precision Nanomedicines Using Controlled Nonviral Assembly Around CRISPR-Cas9 Ribonucleoproteins
Amr A. Abdeen (1); Guojun Chen (1,2); Jared Carlson-Stevermer (1,3); Yuyuan Wang (1); Pawan Shahi (4); Ruosen Xie (1); Lucille Kohlenberg (1); Madelyn Goedland (1); Kaivalya Molugu (1); Meng Lou (1); Bikash Pattnaik (3,4); Shaoqin Gong (1,2,3,6); Krishanu Saha (1,3)
Wisconsin Institute for Discovery, University of Wisconsin-Madison (1); Materials Science and Engineering, University of Wisconsin–Madison (2); Biomedical Engineering, University of Wisconsin-Madison (3); Pediatrics, University of Wisconsin-Madison (4); Ophthalmology and Visual Sciences, University of Wisconsin-Madison (5); Chemistry, University of Wisconsin–Madison (6)
CRISPR ribonucleoproteins (RNPs) can generate programmable gene edits, however imprecise editing and efficient delivery to human cells are key challenges. Here we describe novel biochemical techniques to assemble various biomolecules and coatings with nanoscale precision around a RNP. First, by modifying the single guide RNA (sgRNA) with a short "S1m" RNA aptamer, we developed a modular strategy, termed a “S1mplex,” to assemble Cas9 RNPs with up to three biotinylated moieties. Using S1mplexes with biotinylated short oligonucleotides greatly improves the ratio of precise to imprecise editing by up to 18-fold over conventional methods, while assembly with fluorescent molecules allows selection and enrichment for cells with multiplexed gene edits. Second, we developed synthetic coatings for nonviral delivery of RNPs to mammalian cells. The “nanocapsule” coating strategy encapsulates a single Cas9 RNP into a novel cell-degradable thin polymeric shell that can be decorated with cell targeting ligands and other biomolecules. Nanocapsules frequently outperformed commercial cationic delivery reagents, while having significantly lower toxicity and higher stability. In human pluripotent stem cells in vitro and via subretinal injection into mice in vivo, robust gene editing (up to 25%) is observed with nanocapsules. The “polyplex” coating strategy is a pre-polymerized cationic polymer that coats and assembles with S1mplexes and nucleic acids. Polyplexes also have improved cytotoxic properties and enable nonviral delivery of CRISPR machinery with high levels of precise gene edits (up to 38%). Combined, these platform technologies - which utilize chemically-defined, off-the-shelf reagents - have significant promise for regenerative medicine applications in vitro (e.g., drug discovery, disease modeling) and in vivo (e.g., somatic gene editing).
Testing Thousands of Nanoparticles in vivo using DNA Barcodes
Kalina Paunovska, Cory D. Sago, Anton Bryksin, James E. Dahlman
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology / Emory
Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology
All DNA and RNA therapies are limited by one problem: drug delivery. DNA or RNA must be avoid clearance by the immune system, kidney, and spleen, access the target cell in a complex microenvironment, and enter the cytoplasm. Engineers and chemists have designed thousands of chemically distinct nanoparticles to target RNA or DNA. After nanoparticles are synthesized, they are sceened in vitro, which may not mimic in vivo delivery. To test thousands of nanoparticles in vivo, we designed 2 high throughput nanoparticle / DNA barcoding platforms, named Fast Identification of Nanoparticle Delivery (FIND), and JOint Rapid Dna Analysis of Nanoparticles (JORDAN).
Using the JORDAN system, we have measured how well over 1,200 nanoparticles deliver genetic drugs in vivo. We focused on 3 fundamental questions. First, does in vitro LNP delivery predict in vivo LNP delivery to the microenvironment? Second, does LNP delivery change within the microenvironment of a target tissue? Finally, how does a given biological perturbation affect delivery to different cell types within a microenvironment? Our data demonstrate that barcoded LNPs can elucidate fundamental questions about in vivo nanoparticle delivery, and identify nanoparticles for in vivo gene therapies.
Addressing Manufacturing Challenges with Platform Technologies
Joshua G. Hunsberger
RegenMed Development Organization
Within the near future, the promise of regenerative medicine will be realized. This promise includes technologies that will enable the repair, replacement, or regeneration of tissue that has been compromised by injury or disease. One of the main barriers that must be overcome before regenerative medicine can become a standard of care is the development of a clinical manufacturing industry. For this industry to operate in a cost effective manner and at the required scale, automation strategies, standards for raw material and clinical products, quality assurance protocols, and many additional processes will need to be developed and validated. RegenMed Development Organization (ReMDO) is a non-profit organization that is leading an advanced biomanufacturing initiative to address all aspects of industry scale clinical manufacturing. Currently, two ReMDO programs have been funded through the Medical Technology Enterprise Consortium . The first program is developing a serum-free, chemically defined cell culture medium for expanding the human cell populations required for regenerative medicine cell based therapies and tissue engineered products. The second program is developing a tunable bioink system that will be compatible with all of the current 3D bioprinting technologies. Herein, current progress made by each of these programs are reviewed. Additionally, the contributions that each of these platform technologies bring to the field of regenerative medicine clinical manufacturing is described.
Sarah Heilshorn, Ph.D.
Adaptable Biomaterials for Expansion and Transplantation of Stem Cells
While neural progenitor cells (NPCs) and their progeny have significant therapeutic promise, the difficulty and cost of expanding and delivering a large number of NPCs remain significant barriers to widespread clinical use. Recently, 3D hydrogels have been proposed as in vitro culture platforms for the expansion of stem cell populations to overcome the space limitations of 2D culture. However, very little is known about which 3D material properties are required to maintain NPCs in an undifferentiated state for expansion. Using a family of protein-engineered biomaterials, we demonstrate that 3D matrix stiffness does not correlate with the maintenance of NPC stemness over a broad range of matrix mechanical properties (E~0.5-50 kPa). In contrast, matrix degradability strongly correlated with the expression of NPC stem markers and NPC proliferation in three different biomaterial systems. Our results have identified matrix remodeling as a previously unknown requirement for maintenance of NPC stemness in 3D hydrogels and suggest that adaptable biomaterials will be useful for expansion and transplantation of therapeutically relevant numbers of NPCs.
Dermal Fibroblasts from Regenerative Mouse Model Defy Common Mechanobiology Dogma
Daniel C. Stewart (1); P. Nicole Serrano (2); Andrés Rubiano (3); Ryosuke Yokosawa (3); Justin Sandler (3); Jason O. Brant (2); Malcolm Maden (2); Chelsey S. Simmons (1,3)
J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida (1); Department of Biology, University of Florida (2); Department of Mechanical and Aerospace Engineering, University of Florida (3)
Researchers at UF recently demonstrated full-thickness skin regeneration in adult Acomys mice, describing a unique regenerative mammal. Fibroblasts play a major role in mammalian wound healing by contracting the wound and replacing damaged tissue, and regenerative fibroblasts from Acomys could inform future regenerative therapies. In response to biochemical and mechanical signals, fibroblasts mature into myofibroblasts that exhibit higher contractile strength and upregulated a-smooth muscle action (aSMA) expression. We hypothesized that dermal fibroblasts from regenerative Acomys skin may not upregulate aSMA nor contractility, in contrast to normal, scarring mammals, and here, we have characterized their surprising, regenerative phenotype. Dermal fibroblasts (DFs) were isolated from Acomys and Mus and cultured on soft silicone substrates to investigate aSMA activation, on polyacrylamide (PA) gels for quantitative assessment of cell contractility, and in 3D collagen gels to assess remodeling of "microtissues".
Classic mechanobiology paradigms suggest stiffer substrates will promote myofibroblast activation, but we do not see this in Acomys DFs (aDFs). Though Mus DFs (mDFs) increase organization of aSMA fibers as substrate stiffness increases, aDFs assemble very few aSMA-positive fibers on substrates ranging from kPa to GPa. mDFs caused silicone wrinkling on soft gels, but aDFs did not, so we quantified contractility with PA traction force microscopy. aDFs (1.6 ± 0.6 pJ) had substantially lower traction forces than mDFs (3.1 ± 0.8 pJ, p = 0.08), reflecting a functional difference in myofibroblast activation.
In addition to increased aSMA expression and contractility, normal mammalian myofibroblasts produce excess collagen that can permanently scar. On 2D glass substrates, both aDFs and mDFs expressed ColI but with a greater intensity in mDFs. Only mDFs expressed ColIII which is surprising as ColIII is thought to be more “regenerative” collagen. In 3D ColI gels, mDFs spread as expected but aDFs remain rounded in appearance. mDFs increase matrix stiffness over 7 days as measured by custom indentation system, while aDFs do not remodel surrounding gel.
The African Spiny Mouse, Acomys, has been shown to have unique tissue regeneration abilities, including regenerating entire layers of skin without any scarring. Compared to their common mouse counterpart, aDFs show profound differences in their in vitro behavior, generating less contractile force, upregulating minimal aSMA in response to cell stiffness, and depositing different levels of ECM proteins. These data suggest that alterations in mechanotransduction pathways in aDFs may be related to their regenerative phenotype.
Mechanical Tension Regulate Fibroblast Functional Diversity That Is Propagated By Exosomes
Natalie Templeman (1); Monica Fahrenholtz (1); Hui Li (1); Xinyi Wang (1); Alexander Blum (1); Emily Steen (1); Yao Ning (1); K. Jane Grande-Allen (2); Paul L. Bollyky (3); Sundeep G. Keswani (1); Swathi Balaji (1)
Department of Surgery, Baylor College Of Medicine and Texas Children’s Hospital (1); Department of Bioengineering, Rice University (2); Department of Medicine-Infectious Disease, Stanford University (3)
The physiologic response to injury involves a dynamic interplay between mechanical forces, inflammatory factors and extracellular matrix (ECM) cues that result in fibrotic scarring. Fibroblasts are central to this process and they exhibit functional diversity which may contribute to the heterogeneity of how humans scar. Biomechanical tension induces a pro-fibrotic phenotype in fibroblasts, characterized by increased inflammatory cytokine and ECM production. The mechanism of pro-fibrotic signaling between fibroblasts that “educate” the scar fibroblasts to perpetually retain a “scarring” phenotype that underlie how scars remain scars is not fully elucidated. We hypothesize that mechanical forces and inflammation regulate fibroblast functional diversity that is propagated by exosomes.
Murine fetal(fetalFb, E14.5) and adult fibroblasts(adultFb, 8 wk) were cultured on silicone membranes +/-10% static strain (1,3,6,12,24 hours) and analyzed for genes that encode exosome synthesis (RAB27ab, SMPD3), and fibrogenic potential (CD26/alpha-SMA/TGF-B). Exosomes were isolated from 6,12h media samples and analyzed for size and count (Zetasizer) before use in a migration assay. An inflammation/fibrosis super-array was performed comparing fetalFB and adultFB, and their respective exosomes. Data is represented as mean+/-SD, (n=5/group/time point); p-value by ANOVA.
Under static conditions, adultFB and fetalFB had significant intrinsic differences in fibrogenic potential, including more alpha-SMA, TGF-B1 and CD26 gene expression in adultFB(p<0.01). Interestingly, fetal fibroblasts had increased expression of RAB27a,b and SMPD3. With tension, alpha-SMA and CD26 expression was unaffected in adultFB, but protein expression of both the markers increased in fetalFB(p<0.05). Tension downregulated RAB27a,b and SMPD3 in both fetalFB(p<0.01) and adultFB (p<0.05), with a more pronounced effect on fetalFB. But more exosomes with increased size distribution were produced by adultFB than fetalFB under static and tension conditions(p<0.05). Tension further resulted in a significant increase in exosome production by adultFB (84.3±8.3vs.74.3±7.4kcps, p<0.05), but not by fetalFB. AdultFB-derived exosomes, as well as fetalFB derived exosome under tension impaired the migration of fetalFB in a scratch wound assay. Super-array data demonstrated significant differences in inflammation-related genes in fetalFB versus adultFB, which will be correlated to the data from their exosomes.
AdultFB display a more fibrogenic phenotype than fetalFB, which is influenced by tension. Exosomes are a likely target for extracellular communication, as their production in adultFBand fetalFB are regulated by tension and can influence fetalFB behavior. These insights into the intrinsic differences between regenerative fetalFB and fibrotic adultFB and their extracellular communication may yield targets to improve post-natal wound repair.
CMaT, MC3M and NCMC – Global Partnerships to Enable Large-scale, Low Cost, and Reproducible Manufacturing of High Quality Therapeutic Cells
Georgia Institute of Technology/Emory University
Cell Manufacturing and Technologies (CMaT) – CMaT’s vision is to transform the manufacture of cell‐based therapeutics into a large‐scale, low‐cost, reproducible, and high quality engineered system for broad industry and clinical use. CMaT will become a visionary and strategic international resource and an exemplar for developing new knowledge, transformative technologies, an inclusive well‐trained workforce and enabling standards for cell production and characterization processes.
Marcus Center for Therapeutic Cell Characterization and Manufacturing (MC3M) - The initial focus of the Marcus Center for Therapeutic Cell Characterization and Manufacturing (MC3M) are in the areas of (a) mesenchymal stromal/stem cells (MSCs) from bone marrow and cord tissue for immune-modulation and tissue-biofabrication, (b) T and B cells for immunotherapies in cancer, infectious and autoimmune diseases; and (c) Cord blood and bone marrow hematopoietic stem cells for regenerative medicine. The Center will implement the National Roadmap for Cell Manufacturing, recently published by the National Cell Manufacturing Consortium and highlighted by the White House.
National Cell Manufacturing Consortium (NCMC) - Over 25 companies and 15 academic institutions collaborated together with government agencies to produce a national roadmap on cell therapy manufacturing. The National Cell Manufacturing Consortium (NCMC) is the first U.S. based national consortium focused on developing, maturing, and implementing technologies that can enable large-scale, cost-effective manufacturing of therapeutic cells. NCMC is established through the Advanced Manufacturing Technologies (AMTech) grant from the National Institute of Standards and Technologies (NIST).
Kia Washington, M.D.
Total Human Eye Allotransplantation: Looking Toward the Future of Vision Restoration Therapy
Kia M. Washington, MD1,2,4, Yang Li,1 Chiaki Komatsu, MD1, Maxine R. Miller, Lin He, MD1, Touka Banaee, MD1 , Yong Wang, MD1, Bing Li, MD1, Edward Davidson, MD1, Wendy Chen, MD, MS1, Joshua Barnett, BS1, Yolandi van der Merwe, B. Eng.1, Mario G. Solari, MD1,2, Andrew W. Eller, MD1, Joel S. Schuman, MD3, and Jose Sahel, MD1
(1)University of Pittsburgh, Pittsburgh, PA, (2)McGowan Institute for Regenerative Medicine, Pittsburgh, PA, (3)New York University, New York, NY, (4)VA Pittsburgh Medical Center, Pittsburgh, PA
Nearly 39 million people suffer from vision loss worldwide. Traumatic, ischemic or degenerative diseases are the main causes of irreversible blindness. The poor prognosis as a result of these conditions results primarily from irreparable damage to the retina and optic nerve. The goal of our research is to reverse blindness through whole eye transplantation. Similar to face and hand transplantation, whole eye transplantation restores form and function with organ donor tissue as it offers the potential to provide viable retinal ganglion cells, which are the cells that carry visual information from the eye through the optic nerve to the brain, to recipients with vision loss. We have created a viable whole eye transplant model in the rat and are using it to explore viability, structural integrity, and functional outcome in the setting of transplantation. In parallel, we have developed a cadaveric human surgical protocol, which serves as a benchmark for optimization of technique, large animal development and ultimately potentiates the possibility of vision restoration transplantation surgery.
YongTae (Tony) Kim
Microengineered Human Blood-brain Barrier for Neurodegenerative Disease Modeling
Song Ih Ahn (1); Jiwon Yom (1); Yoshitaka Sei (1); Candice Hovell (2); YongTae Kim (1,2)
The George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology (1); The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology (2)
Alzheimer’s disease (AD) is the most common neurodegenerative disease that is characterized clinically by progressive cognitive decline associated with neurovascular dysfunction. While several potential pathophysiological processes have been introduced, the exact mechanism remains elusive, and currently there is no cure for AD. One approach to address this challenge is to develop in vitro models of the human blood-brain barrier (BBB) that can mimic the pathophysiological conditions of the brain, particularly for human diseases like AD, which existing animal models cannot accurately recapitulate. While recent progress has provided new approaches to partially reconstitute the microenvironment of the brain, it remains challenging to have physiologically relevant in vitro models of the human BBB due to the physiological complexity. Here we present a new microengineered human BBB model designed to create vascularized 3D glial networks, a crucial structure of the BBB that has not been replicated successfully by existing in vitro models. Our BBB model allowed 3D quad-culture of human brain endothelial cells (HBMECs), human brain vascular pericytes (HBVPs), normal human astrocytes (NHAs), and human microglia (HMs) with the physiological morphology and interaction. Our BBB platform consists of two PDMS microfluidic channels compartmentalized by a porous membrane to mimic the luminal and abluminal regions of a neurovascular unit. HBMECs establish a tightly connected monolayer on a porous membrane in the luminal side, while HBVPs grow on the other side of the membrane in the abluminal region. The abluminal part of the lower center channel has 3D glial cell networks constructed in our custom hydrogel tuned for brain parenchymal tissue, which is enclosed by two side channels designed for media exchange and perfusion. Our customized brain tissue mimicking hydrogel composed of Matrigel and tenascin C allows astrocytes and microglia to exhibit their ramified morphologies and decrease their reactive gliosis markers in astrocytes including nestin, neural cell adhesion molecule (N-CAM), and glial fibrillary acidic protein (GFAP). Moreover, we found that astrocytic end-feets contact the endothelium with aquaporin 4 expression, a representative feature of the human BBB model with 3D glial networks. We also confirmed that our BBB model showed the lowest permeability across the endothelial monolayer with well-established 3D glial networks, as compared to other co-culture systems. We will use this BBB platform to mimic pathophysiological conditions of AD including neuroinflammation and neurotoxic protein (amyloid beta) accumulation, and study the pathological mechanism and explore potential AD therapeutics. Our approach will present a representative example of a cost-effective platform serving as a bridge to animal and clinical studies for identification of new therapeutic targets in neurodegenerative diseases.
Human-on-a-chip Systems for Use in Efficacy and Toxicological Investigations in Pre-clinical Drug Discovery
James J. Hickman
NanoScience Technology, Chemistry, Biomolecular Science and Electrical Engineering, University of Central Florida, Orlando, FL 32826 and Chief Scientist, Hesperos, Inc. Orlando, FL 32826
The utilization of human-on-a-chip or body-on-a-chip systems for toxicology and efficacy that ultimately should lead to personalized medicine has been a topic that has received much attention recently. Currently drug discovery and subsequent regulatory approval for new candidates requires 10-15 years of development time before general availability is granted by either the FDA or EMA. Human-on-a-chip systems could help reduce the cost and time of this process either at the single organ level or by using more advanced systems where multiple organ mimics are integrated to allow organ to organ communication and interaction for mechanistic determinations. Additional characteristics are functional readouts that would enable non-invasive monitoring of organ health and viability for chronic studies that now are only possible in animals or humans. In addition, in order to achieve wide spread adoption of these technologies they should also be low cost, easy to use and reconfigurable to allow flexibility for platforms to be examined with small variation.
Our group, in collaboration with Dr. Michael Shuler from Cornell University, has been constructing these systems with up to 6 organs and have demonstrated long-term (>28 days) evaluation of drugs and compounds, that have shown similar response to results seen from clinical data or reports in the literature. We have accomplished the construction of these systems utilizing mostly 2D systems in serum-free medium with functional readouts that employs a pumpless platform. Microsystems fabrication technology and surface modifications are integrated with protein and cellular components to enable mechanically and electronically interactive functional multi-component systems. A specific embodiment of this technology is the creation of a functional human NMJ system to understand ALS. We have investigated four mutations found in ALS patients; SOD1, FUS, TDP43 and C9ORF72. The models have demonstrated variations of the disease phenotype compared to WT for NMJ stability and functional dynamics. Results of these studies will be presented as well as preliminary results for reversal of the deficits. An integrated cancer, liver, cardiac system, that was developed in collaboration with Roche, will also be presented for efficacy of the anti-cancer drug tamoxifen for a multi-drug resistant cancer line in combination with a PgP inhibitor, as well as off-target toxicity of both parent and metabolites with same recirculating system.
There is currently a focus at the NIH, FDA and EMA to understand how one could validate these systems such that qualification could be granted for their use to augment and possibly replace animal studies. This talk will also give results of six workshops held at NIH as a collaboration between the American Institute for Medical and Biological Engineering (AIMBE) and NIH to explore what is needed for validation and qualification of these new systems.
Mapping 2D Distributions of Extracellular Matrix Proteins from Thin Tissue Sections by MALDI Imaging Mass Spectrometry
Peggi M. Angel(1,2); Susanna Comte-Walters (1,2); Lauren E. Ball (1,2); Kelvin G. M. Brockbank (3,4,5); Richard R. Drake (1,2)
Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, SC (1); Tissue Testing Technologies LLC, North Charleston, SC, USA (2); MUSC Proteomics Center, Department of Pharmacology, Medical University of South Carolina, Charleston, SC (3); Department of Bioengineering, Clemson University, Clemson, SC, USA (4); Department of Regenerative Medicine, Medical University of South Carolina, Charleston, SC USA (5)
Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is an established tool to analyze and map the complex molecular environment of tissues. Here, we describe a new workflow using matrix metalloproteinases (MMPs) with MALDI IMS to access 2D distribution of extracellular matrix protein sequences from formalin or glutaraldehyde fixed histological tissue sections. The technique uses an automated sprayer to apply a thin molecular layer of a selected MMP onto 3-10 µm thick tissue sections mounted on a standard microscope slide. The tissue sections are then incubated in a temperature controlled high humidity chamber, allowing the MMP to digest target proteins to peptides. Peptides are detected from tissue by sampling the tissue in a discrete array pattern using high mass accuracy (to the fourth decimal place) MALDI Fourier Transform Ion Cyclotron Resonance (FT-ICR) IMS. We have additionally characterized MMP digest products by using proteomics using nano liquid chromatography coupled to tandem mass spectrometry to improve identification confidence in MMP peptides derived from fixed tissue sections. The workflow is demonstrated on thin tissue sections from porcine and human heart valve, a commercial human heart tissue microarray, and from human cancer.
We report that spraying bacterial MMP collagenase type III allows detection of multiple types of collagen on a single tissue section, including COL1A1, COL1A2, COL3A1, COL5A1, COL6A2, COL6A3. High mass accuracy proteomics analysis on adjacent tissue sections (typically 5 mm x 5mm x 5 µm) reported 13 collagens when tissue sections are digested by collagenase type III. An additional 37 extracellular matrix proteins were detected e.g., fibronectin, vimentin, EMILIN-1, biglycan with 4 non-ECM proteins PLECTIN, TNS1, PEBP1, PRDX5. Notably, the non-collagen proteins found by proteomics participate in collagen binding, synthesis, and scavenging mainly through direct interactions with collagen, thus we suggest that we are also pulling down the collagen interactome. Additional work done using MMP12 (elastase) showed distinct localization of discrete elastin isoform 2 peptides regionally localized to tissue features. This suggests that the use of MMP12 in a MALDI IMS workflow will allow reporting of unique elastin isoforms relevant to tissue features. Further work is being done to examine other MMPs as ECM targeting enzymes for use in MALDI IMS.
The developed technology provides new access to the study of 2D distributions of extracellular matrix proteins, particularly of collagens and elastin sequences, localized to tissue features.This high throughput and highly multiplexed technological advance should be applicable to any tissue regardless of disease type, tissue origin, or disease status and is thus relevant to all research including basic, translational and clinical applications.
Lori Setton, Ph.D.
Regenerative Medicine for Treating Intervertebral Disc Disorders
Lori A Setton, Ph.D.
Low back pain now ranks as #1 for disease impact in the USA, in part due to intervertebral disc disorders that contribute to pain and disability in affected individuals. Pathological processes for resident cells of the intervertebral disc, the nucleus pulposus cells, contribute to dysfunctional production of inflammatory cytokines and premature cell death that can drive loss of intervertebral disc height, tissue destruction and disc c herniation. Inflammatory cytokines produced by resident cells and recruited monocytes are known to mediate cell death as well as the painful symptoms of intervertebral disc herniation, although systemic treatment with inflammatory antagonists (e.g., tumor necrosis factor “blockers”) has failed to date. In this talk, we will describe our work with engineering substrates and protein-conjugated biomaterials to maintain healthy, biosynthetically active nucleus pulposus cells, factors that can be manipulated to attenuate inflammatory cytokine expression, promote matrix biosynthesis, and control progenitor cell differentiation.
Recapitulating Bone Development for Tissue Regeneration through Engineered Mesenchymal Condensations and Mechanical Cues
Anna M. McDermott (1,2); Samuel Herberg (3); Devon E. Mason (1,2); Hope B. Pearson (2); James H. Dawahare (2); Joseph M. Collins (1,2,4); Mark W. Grinstaff (5); Daniel J. Kelly (6); Eben Alsberg (7); Joel D. Boerckel (1,2,4)
Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA (1); Department of Aerospace and Mechanical Engineering, University of Notre Dame, Notre Dame, IN (2); Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH (3); Department of Bioengineering, University of Pennsylvania, Philadelphia, PA (4); Department of Mechanical Engineering, Trinity Center for Bioengineering, Trinity College Dublin, Dublin, Ireland (5); Department of Biomedical Engineering, Boston University, Boston, MA (6); Department of Orthopaedic Surgery, Case Western Reserve University, Cleveland, OH (7)
Traditional tissue engineering approaches typically employ scaffolds that aim to match the properties of mature tissues, but a more effective paradigm for regenerative medicine may be to reproduce developmental conditions. Natural tissue repair often occurs through this type of developmental mimicry. For example, fracture healing recapitulates bone development through endochondral ossification, resulting in high clinical success rates of 90-95%. In contrast, large bone defects of critical size cannot form a callus, will not heal on their own, and exhibit high complication rates even after intervention.
Therefore, mimicking the endochondral process that occurs in bone development and natural fracture repair may improve regenerative outcome for these challenging defects. Since physical stimuli (i.e., mechanical forces) are critical for proper endochondral ossification during bone morphogenesis and for induction of the endochondral cascade in fracture healing, we hypothesized that mechanical loading would be essential to achieve endochondral defect regeneration. Here, we developed limb bud-mimetic, engineered mesenchymal condensations by high density culture of human bone marrow stromal cells, incorporated with gelatin microspheres for local chondrogenic morphogen presentation (TGF-β1). We found that in vivo mechanical loading, via dynamically tuned fixator compliance, was necessary to restore bone function through endochondral ossification, out-performing the current clinical standard of BMP-2 delivery on collagen sponge. Both early (immediate) and delayed loading (initiation after four weeks of rigid fixation) enhanced bone regeneration compared to continuous stiff fixation, though only delayed loading restored intact bone properties, with an order-of-magnitude greater response to loading than that observed previously for BMP-2-mediated repair. Endochondral regeneration exhibited zonal cartilage and spongiosa mimetic of the native growth plate, with active YAP signaling in human hypertrophic chondrocytes in vivo. Mechanical loading regulated the amount and distribution of cartilage formation and vascular invasion, quantified by dual contrast-enhanced microCT imaging (CA4+ and microfil MV-122, respectively). Both cell devitalization prior to condensation transplantation and morphogen delivery without the cellular anlage failed to induce bone formation, demonstrating transplanted cell function, while cell delivery without morphogen presentation also failed to produce regeneration regardless of loading. Together, this study represents the first demonstration of the effects of mechanical loading on transplanted cell-mediated bone defect regeneration and establishes the importance of in vivo mechanical cues for recapitulation of development for tissue engineering.
Cell Therapy for Cartilage Repair using Human Fetal Cartilage-derived Progenitor Cells (hFCPCs)
Byung Hyune Choi (1); Byoung-Hyun Min (2,3)
Department of Biomedical Sciences, Inha University College of Medicine (1); Department of Molecular Science and Technology, Ajou University (2); Cell Therapy Center, Ajou Medical Center (3)
We have utilized human fetal cartilage-derived progenitor cells (hFCPCs) as a source of cell therapy for cartilage repair. Fetal cartilage tissue was obtained from fetues of GA12-16 with the IRB approval and written consent from donors. hFCPCs were isolated at very high yield and could be culture expanded for more than 20 passages without clear sign of senescence. They also showed evidences of stem cell properties in the colony forming assays, gene expression pattern and multi-potent differentiation ability. In addition, hFCPCs showed an immune- and inflammation-modulating activity in vitro comparable to that of MSCs. We have developed a scaffold-free cartilage fabrication method using hFCPCs, which produced a high quality and injectable artificial cartilage tissue with a pliable mechanical property. We have proven its pre-clinical safety and efficacy in animal models of cartilage defect and are currently setting up a manufacturing process with a quality control scheme. The presentation will deal with these information about hFCPCs and the artificial engineered cartilage.
Acknowledgement: This study was supported by a grant of the Korea Health Technology R&D Project (HI17C2191) funded by the Ministry of Health & Welfare, Republic of Korea.
Biomaterial-based Therapies for Improved Lymphatic Function and the Resolution of Chronic Inflammation in Post-traumatic Osteoarthritis
Thanh Doan (1); Rachit Agarwal (4); Fabrice Bernard (3); Jay McKinney (1); Zhana Nepiyushchikh (4); Allen Liu (3); Robert Guldberg (4); Andrés J. García (4); Brandon Dixon (4); Nick J Willett (1, 2, 3)
Orthopaedics, Emory University (1); Research, Atlanta VA Medical Center (2); Biomedical Engineering, Georgia Institute of Technology (3);
Mechanical Engineering, Georgia Institute of Technology (4)
Osteoarthritis (OA) is a disease associated with chronic unresolved inflammation that is a key driver of disease development and progression. In other peripheral tissues around the body, the lymphatic system serves as the primary mechanism to resolve inflammation. The central dogma in the OA field is that the chronic inflammation is inherently a problem of production, i.e. the tissues of the joint continuously produce inflammatory cytokines, proteases, and other pathologic factors. The clearance of these factors has always been assumed to be a rapid process which is not the rate limiting step in disease progression. The objective of our work was to develop biomaterial-based therapies than can be integrated with imaging modalities to both: 1) elucidate the role of lymphatic function in normal and diseased joints; and 2) promote joint clearance and the resolution of chronic inflammation by targeting lymphatic function and the downstream lymph node. We have utilized novel quantitative near-infrared (NIR) imaging tools to measure: in vivo clearance of intra-articular injected particles within the knee joint, lymphatic pump function from the knee joint, and biodistribution upon clearance from the joint. We have fabricated different sized particles ranging from 2 nm diameter up through ~1 micron diameter and characterized clearance mechanisms of these particles showing size dependent clearance rates and effects on biodistribution (to the draining lymph node or systemic clearance). Utilizing a pre-clinical model of OA in the rat we observed impaired afferent, clearing lymphatic vessel function. Furthermore, we have assessed the effect of a potent vasoconstrictive molecule, endothelin-1 (ET-1), on knee clearance; ET-1 is upregulated in OA though the role in the disease process is unknown. We showed that intra-articular injection of ET-1 can block clearance from the joint and that this effect can be reversed by co-injecting small molecule antagonists of the ET-1 receptors. These data suggest that impaired lymphatic function and clearance may have a role in sustaining chronic inflammation within OA joints and preventing normal clearance and resolution. We have leveraged our understanding of clearance mechanisms and chronic inflammation to design nanoparticles to provide sustained delivery of lipid mediators which resolve inflammation. Loaded particles were injected intra-articularly in the pre-clinical rat OA model and shown to attenuate OA disease development. Together, this work provides evidence that lymphatic function plays a critical role in joint homeostasis and that targeting lymphatics and the resolution of chronic inflammation may be a novel therapeutic approach to treat OA.
Funding Opportunities through the Osteo Science Foundation
Myron R. Tucker
Adjunct Clinical Professor, Oral and Maxillofacial Surgery. Louisiana State University
Osteo Science Foundation, Scientific Liaison
The mission of Osteo Science Foundation is to advance hard and soft tissue regeneration, with a focus on Oral, Cranial and Maxillofacial Surgery in the United States and Canada. The aim is to promote high quality basic and clinical research as well as education that leads to improved outcomes for patients.
The OSF offers three levels of research grants including the Peter Geistlich Award ($100,000 over two years) the Philip Boyne Junior Faculty Award ($50,000 oveer two years) and the Resident Award ($20,000) over two years.
This presentation will focus on the types of research funded by the foundation and the logistics required to apply for and receive grant funding.
Nanostructured Biomaterials Improve Therapeutic Outcomes from Non–viral mRNA Delivery
Andrew S. Khalil (1); Xiaohua Yu (1); Jennifer M. Umhoefer (2); Timothy M. Hacker (3); William L. Murphy (1,4,5,6)
Department of Biomedical Engineering, University of Wisconsin-Madison (1); Department of Biology, University of Wisconsin-Madison (2); Cardiovascular Physiology Core Facility, University of Wisconsin-Madison (3); The Material Science Program, University of Wisconsin-Madison (4); Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison (5); The Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison (6)
Gene delivery is a widely used approach for regulating protein expression in numerous research and regenerative medicine applications. Classic approaches often utilize either non-viral delivery of plasmid DNA (pDNA) or viral vectors for overexpression of therapeutic proteins. However, these methods are typically ill-suited for in vivo applications due to the low non-viral transfer efficiency of pDNA to non-mitotic populations and the risk of insertional mutagenesis and tumorigenicity of viral vectors, respectively. As an alternative, non-viral delivery of messenger RNA (mRNA) is safe and has high gene transfer efficiency in vivo. However, mRNA delivery is limited by short-lived timeframes of expression of therapeutic proteins of interest, often on the order of hours. Here we present a biomaterials-based approach, whereby mineral coatings applied to microparticles (MCMs) provide an efficient and localized non-viral mRNA delivery vehicle for in vivo applications and increase the intended biological response via sequestration and stabilization of the overexpressed protein. Specifically, delivery of mRNA encoding for basic fibroblast growth factor (bFGF) via MCMs increased proliferation in primary human fibroblasts two-fold relative to mRNA delivered without MCMs and increased the duration of this biological response. Additionally, MCM-mediated mRNA delivery required a 44-fold lower production of bFGF to match the extent of cell proliferation elicited by treatment with recombinant bFGF protein. Lastly, MCM-mediated delivery afforded local overexpression of proteins in a murine diabetic dermal wound model and resulted in improved rate of wound closure and healing outcomes relative to recombinant bFGF and delivery of mRNA without MCMs. These findings represent a new biomaterials-based mRNA delivery approach, which leverages the innate advantage of high non-viral mRNA transfer efficiency in non-mitotic cell populations and properties of the nanostructured coating to achieve a prolonged and more robust biological response to an overexpressed therapeutic protein.
Extracellular Matrix Guided Endothelial Differentiation to Support Engineered Thick Tissues
Mikayla L. Hall (1,2); Brenda M. Ogle (1-5)
Department of Biomedical Engineering, University of Minnesota – Twin Cities (1); Stem Cell Institute, University of Minnesota – Twin Cities (2); Lillehei Heart Institute, University of Minnesota – Twin Cities (3); Institute of Engineering in Medicine, University of Minnesota – Twin Cities (4); Masonic Cancer Center, University of Minnesota –Twin Cities (5)
Vascularization is a major challenge in the creation of thick, engineered tissues. This challenge is complicated by the limited efficacy of differentiation of induced pluripotent stem cells (iPSCs) toward endothelial cells, requiring cell sorting to achieve purity. Cell sorting is not ideal for scenarios in which differentiation is desired within a complex 3D construct. For this reason, protocols to induce efficient endothelial differentiation in 3D engineered tissues would be of great benefit and are likely to include the class of proteins with both superior structural capacity and potent biochemical signaling – the extracellular matrix proteins (ECM). Here we use a 3D ECM-based model systems to identify ECM formulations supportive of endothelial differentiation.
Native chemical ligation was used to encapsulate Collagen I, Collagen IV, Laminin 111, Laminin 411, and Laminin 511 with mouse iPSCs in 3D polyethylene glycol (PEG) gels. These proteins were chosen as they represent ECM components of the vascular basement membrane and associated stroma. 3D gels were maintained in minimum essential medium with 10% FBS without additional soluble factors. After 14 days gels were fixed and stained with an antibody to the endothelial marker, CD31. The ratio of CD31 to DAPI staining was measured. All three Laminin subtypes showed higher endothelial differentiation than Collagen I or PEG alone, approaching 70% efficiency. Collagen I inclusion yielded 11±7% CD31+ cells and was not statistically different from PEG gel alone. Laminin 111 inclusion yielded 40±10% CD31+ cells. Laminin 411 showed the highest percentage of endothelial differentiation at 60±10% and Laminin 511 produced 40±20% CD31+ cells. Tube formation was also seen in a portion of Laminin 411 and 511 gels, indicating the formation of more mature endothelial cells with angiogenic potential.
In sum, proteins found in the vascular basement membrane increased endothelial differentiation compared to stromal components. In the best case, with Laminin 411, the efficiency of differentiation exceeded maxima achieved via temporally prescribed soluble factors stimulation (e.g., CHIR99021, vascular endothelial growth factor, etc.). This approach could be quite valuable for creating complex 3D structures since the differentiation stimulus (i.e., insoluble ECM) could be confined to vascular channels without inadvertently influencing the differentiation and maturation of parenchymal cells of the engineered tissue.
Extracellular Matrix Composition Impacts Regenerative Potential and Immune Response in 3D Grafts Designed for Cardiac Tissue Engineering
Whitney L Stoppel (1,2); Breanna M Duffy (1); Gladys A Argueta Xiloj (1); Kelly E Sullivan (1,3); Elizabeth C Porter (4); Jonathan M Grasman (1); David L Kaplan (1); Lauren D Black, III (1,4)
Biomedical Engineering, Tufts University, Medford, MA (1); Chemical Engineering, University of Florida, Gainesville, FL (future affiliation, Fall 2018) (2); Translational Systems Biology within Comparative Biology and Safety Sciences, Amgen Inc, 360 Binney Street Cambridge, MA (current affiliation) (3); Cell, Molecular, and Developmental Biology, Tufts Sackler School of Graduate Biomedical Sciences, Boston, MA (4)
Introduction: Heart failure is a major clinical issue that plagues both young and elderly patients. Many patients undergo reconstructive heart surgery where invasive procedures do not lead to sufficient life-long repair and results in detrimental scar tissue formation and weakening of the surrounding heart muscle, limiting long-term patient outcomes. To ameliorate these issues and improve long-term results, natural, bioactive, degradable, and implantable biomaterial platforms have been extensively evaluated in our group, demonstrating the potential uses for silk-extracellular matrix (ECM) composite sponges in the prevention of negative left ventricular remodeling following myocardial infarction (MI) and in the repair of congenital heart defects in young patients. Our data indicate that the degradation and host immune response following implantation are impacted by the composition of the incorporated ECM. Therefore, we aim to understand the role of the individual proteins present within decellularized ECM and elucidate the importance of the immune response and cell infiltration in the measured remodeling responses to injury in both young and old animals. Previous work has shown that fetal ECM components promote cardiac proliferation1-3 and improve the growth and development of other mature cell types, such as neurons, in 3-dimesions (3D).4 Here, we aim to determine the mechanism by which ECM components lead to positive regenerative capabilities within diseased or damaged cardiac tissue.
Materials and Methods: In vitro analysis of monocyte activation and macrophage polarization in response to ECM composition was evaluated via qPCR and immunohistochemistry. The timeline of fetal development and ECM production in the heart, as well as in other key tissues, were evaluated via ECM enrichment5 and analysis of both cellular and extracellular proteins via western blot and SWATH® analysis using iTRAQ® quantification on an LC-MS/MS. Silk sponges were formed as previously described,6-8 and evaluated in both post-MI remodeling in the rat and right ventricular outflow tract (RVOT) repair in young pigs. In the MI model, heart function was monitored 1, 3, 5, 7, 9, and 11 weeks post-implantation via echocardiography and at 3 and 11 weeks post-implantation via pressure-volume loops analysis. Evaluation of cell infiltration, gene expression, and protein expression were performed via immunohistochemistry, RNA-Seq, and western blot/ LC-MS/MS, respectively. Similar measurements were carried out in RVOT repair model over the course of 1-3 months post-implantation.
Results and Discussion: Our recent investigation of silk-ECM composite patches suggests that ECM composition impacts cell infiltration and remodeling, immune response, and fibroblast activation. In infarct remodeling, LC-MS/MS analysis of tissue from the infarct region suggests that remodeling and ECM deposition in the area varies with injury and treatment method. Immunofluorescence analysis after 3 weeks post-implantation, suggests that ECM composition impacts the timing and rate of remodeling by the host cardiac cells and immune system. In the case of the RVOT repair, we also find that developmental age of the cECM incorporated into the scaffolds altered the remodeling response. The work presented utilizes a variety of methods to evaluate ECM composition within developing fetal porcine tissue paired with in vitro cell response and in vivo implantation data to investigate the regenerative potential of decellularized ECM in a developmental-age related manner.
Conclusion: Understanding what ECM proteins are present during development and evaluating their impact on graft integration into the host is a critical component for the development of materials that can instruct and direct tissue remodeling and regeneration following damage or disease. Our work aims to elucidate the mechanism of ECM-host interactions in cardiac remodeling, enabling improved design of instructive materials for cardiac tissue engineering.
References: 1Williams C, et al., Advanced Healthcare Materials 2015, 4 (10), 1545-1554.; 2Williams C, et al., Acta Biomaterialia 2015, 14 (0), 84-95. 3Williams C, et al., Acta Biomaterialia 2014, 10 (1), 194-204. 4Sood D, et al., ACS Biomater Sci Eng 2016, 2 (1), 131-140. 5Naba A, et al., JoVE 2015, (101), e53057. 6Stoppel WL, et al., Biomedical Materials 2015, 10 (3). 7Rnjak-Kovacina J, et al., Advanced Functional Materials 2014, 24 (15), 2188-2196. 8Rnjak-Kovacina J, et al., ACS Biomater Sci Eng 2015, 1 (4), 260-270.s
Stephen Dalton, Ph.D.
Vascular Engineering using Pluripotent Stem Cells
Center for Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA
Splanchnic mesoderm (SplM) is a transient population of mesoderm progenitor cells that forms during early embryonic development. These progenitor cells give rise to many cell types in the heart and other coelomic organs, including the major vascular lineages. To identify small molecules that promote differentiation of human pluripotent stem cell (hPSC)-derived SplM to vascular cell types we performed a screen and identified compounds that synergize with BMP4 to generate vascular progenitor cells of the mesothelium lineage (MesoTs). hPSC-derived MesoTs are multipotent and generate smooth muscle cells, endothelial cells and pericytes and self-assemble in to vessel-like networks in vitro. MesoT cells transplanted into mechanically damaged neonatal mouse heart migrate into the injured tissue and contribute to nascent coronary vessels in the repair zone. When seeded onto decellularized vascular scaffolds, MesoT cells differentiate into the major vascular lineages and self-assemble into vasculature capable of supporting peripheral blood flow following transplantation. These findings demonstrate the potential utility of MesoT cells in tissue repair and vascular engineering applications.
Footprint-free Reprogramming of Human Adipose-derived Stem Cells
Melany López (1); Binnur Eroglu (2); Roni J. Bollag (3); De-Huang Guo (1); Mahito Nakanishi(4); Carlos M. Isales(1,5); Ali Eroglu(1,6)
Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta University (1); Georgia Cancer Center, Medical College of Georgia, Augusta University (2); Department of Pathology, Medical College of Georgia, Augusta University (3); Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan (4); Orthopaedic Surgery, Medical College of Georgia, Augusta University (5); Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta University, Augusta, GA 30912 (6)
Human adipose tissue can readily be harvested through a clinically safe liposuction procedure, and its stromal compartment harbors multipotent cells known as adipose-derived stem cells (ASCs). ASCs are highly proliferative and expected to be easily amenable to reprogramming into pluripotency. The objective of this study was to investigate the reprogramming efficiency of ASCs under defined (TeSR-E8 medium) and undefined (conventional human embryonic stem cell [hESC] medium consisting of DMEM/F-12 with KnockOut Serum Replacement [KOSR]) conditions and in the presence of four inhibitors (4i: Na butyrate, CHIR99021, PD0325901, and ROCK-1 inhibitor). For reprogramming, ASCs were transfected with a replication-defective Sendai virus vector that remains in the cytoplasm and expresses four transcription factors (i.e., Oct4, Sox2, Klf4, and c-Myc) without integrating into genome. The reprogramming efficiency was assessed based on the ratio of the number of TRA-1-60-positive colonies to the number of plated cells. The pluripotency of the reprogrammed cells was confirmed by verifying demethylation of promoters of Oct4 and Nanog and their endogenous expression, alkaline phosphatase staining, and teratoma assay. Reprogramming was carried out in the following experimental groups: (1) TeSR-E8+4i; (2) TeSR-E8+4i+KOSR; (3) Feeder cell (FC)-conditioned hESC medium+4i; (4) FC-conditioned hESC medium+4i+basic fibroblast growth factor (bFGF); and (5) Non-FC-conditioned hESC medium+4i. The reprogramming efficiency was highest (0.46%) when ASCs were reprogrammed in FC-conditioned hESC medium+4i. In contrast, reprogramming was ineffective (0.0016%) under the defined (TeSR+4i) condition. Adding KOSR to TesR+4i significantly improved the reprogramming efficiency (0.23%) although it still remained significantly lower than that of the FC-conditioned hESC medium+4i group. When bFGF was added to FC-conditioned hESC medium+4i at a concentration present in TeSR-E8 medium, the reprogramming efficiency was decreased (0.32%) and became statistically similar to that of the TeSR+4i+KOSR group. Likewise, when non-FC-conditioned hESC medium+4i was used, the reprogramming efficiency was lower (0.30%) and statistically similar to the TeSR-E8+4i+KOSR group. Taken together, our results indicate that (1) ASCs can be reprogrammed into a pluripotent state with reasonable efficiencies; and (2) FC-released factor(s) and undefined components of KOSR improve the reprogramming efficiency while bFGF negatively affects reprogramming.
This study was supported by a program project grant (P01AG036675).
Modeling Zika Virus Exposure and Screening Therapeutic Compounds with Human iPSC-derived Neural Cells
Zhexing Wen (1,2,3); Christy Hammack (4); Sarah C. Ogden (4); Xuyu Qian (5,6); Miao Xu (7); Emily M. Lee (4); Feiran Zhang (8); Yujing Li (8); Bing Yao (8); Jaehoon Shin (5,9); Wei-Kai Huang (5); Kimberly M. Christian (5,10), Menghang Xia (7); Peng Jin (8), Wei Zheng (7), Hengli Tang (4), Hongjun Song (5,10) and Guo-li Ming (5,10)
Department of Psychiatry and Behavioral Sciences (1); Department of Cell Biology (2); Department of Neurology (3); Department of Human Genetics (8); Emory University School of Medicine, Atlanta, GA 30322, USA; Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA (4); Institute for Cell Engineering (5); Biomedical Engineering Graduate Program (6); The Cellular and Molecular Medicine Graduate Program (9); Johns Hopkins University School of Medicine, Baltimore, MD 21025, USA; National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA (7); Department of Neuroscience, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA (10)
Zika virus (ZIKV), a mosquito-borne flavivirus, is currently reported to be circulating in over 70 countries and territories globally. While ZIKV infection has been linked to microcephaly in newborns and other brain abnormalities such as Guillain-Barré syndrome, how ZIKV impairs brain development and function is unknown. Here we show that three stains of ZIKV, Puerto Rican ZIKV-PR, Asian ZIKV-C and African ZIKV-M, directly infects human induced pluripotent stem cell (hiPSC)-derived cortical neural progenitor cells (hNPCs) with high efficiency. Infected hNPCs further secrete infectious ZIKV particles. Importantly, ZIKV infection increases cell death and dysregulates cell cycle progression, resulting in attenuated hNPC growth. Gene expression analyses of infected hNPCs reveal transcriptional dysregulation, notably of cell cycle-related pathways. In addition, we performed a drug repurposing screen of ∼6,000 compounds and identified leading compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in hiPSC-derived neural cells. Our results thus fill a major gap in our knowledge about ZIKV biology and serve as an entry point to establish a mechanistic link between ZIKV and microcephaly. Our study also provides a tractable experimental model system for investigating the impact and mechanism of ZIKV on human brain development. Of equal importance, our high-throughput screening platform with hiPSC-derived neural cells has led to the identification of therapeutic compounds that either suppress ZIKV infection or ameliorate its pathological effects during neural development, which may have an immediate effect on the development of anti-ZIKV therapeutics.
Spatial Patterning of Liver Progenitor Cell Differentiation Mediated by Cellular Contractility and Notch Signaling
Kerim B. Kaylan; Ian C. Berg; Gregory H. Underhill
Department of Bioengineering; University of Illinois at Urbana-Champaign; Urbana, IL 61801
Liver progenitor cell differentiation and bile duct formation are driven by spatially-dependent and temporally-sequenced cell–cell and cell–factor interactions coordinated by several biochemical signaling pathways, namely Notch and TGF beta. The regionalization of biliary differentiation and morphogenesis near the portal region of the liver has suggested that spatially segregated microenvironmental signals govern this process. Our recent work utilizing biomaterial substrates of defined stiffness suggests that mechanical cues play a previously unrecognized role in liver progenitor differentiation. Here, we used a cell microarray platform that enables the simultaneous analysis of these biochemical and biomechanical microenvironmental cues to define the mechanisms of action and functional overlap of these pathways. We utilized bipotential mouse embryonic liver (BMEL) progenitor cells as a model cell type for examining differentiation mechanisms within the array platform. To present Notch ligand to cells, we printed Fc-chimeric Notch ligands (JAG1, DLL1, DLL4) on a polyacrylamide hydrogel substrate together with collagen I and Protein A/G. We integrated this cell microarray platform with traction force microscopy (TFM) by adding fiducial beads to the polyacrylamide hydrogel and imaging bead displacement before and after cell dissociation. In order to determine the effect of activating Notch signaling on liver progenitor differentiation, we presented Fc-Notch ligands to cells in arrays, inducing biliary differentiation restricted to the edges of patterns as measured by expression of osteopontin. Addition of an inhibitor of Notch signaling prevented peripheral biliary differentiation. Immunofluorescence analysis of expression of the biliary transcription factor SOX9 showed restriction to the island periphery while expression of the hepatocyte transcription factor HFN4A was central. Further, we observed that SOX9 expression increased on stiff substrates while that of HNF4A increased on soft substrates, which implicated biomechanical stimulation as the gradient-forming cue. Finite element modeling (FEM) simulations suggested a radial gradient of mechanical stresses in the circular patterns, which we confirmed experimentally using TFM. Next, we investigated the impact of downstream signaling events in a series of inhibition studies, showing that peripheral biliary differentiation is dependent on Notch, TGF beta, myosin-mediated cell contractility, and ERK signaling. In conclusion, in these studies we have delineated distinct sets of biomechanical cues that coordinate with Notch signaling to guide the fate specification of liver progenitors toward both hepatocyte and biliary epithelial cell fates.
Glycobiology Engineering to Advance Biotherapeutics and Biomaterials
Antonietta Restuccia; Margaret M. Fettis; Shaheen Farhadi; Gregory A. Hudalla
J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida
Carbohydrates are the most abundant organic molecules on Earth, and carbohydrate conjugates (i.e., “glycans”) decorate all human cells and nearly half of all human proteins. Glycobiology is the study of the synthesis, structure, and function of carbohydrates, as well as their biomolecular binding partners. Research in the Hudalla Lab leverages advances in glycobiology to engineer biotherapeutics and biomaterials with new or improved functional properties. For example, we developed an engineered variant of galectin-1, a soluble carbohydrate-binding protein that modulates cell behavior in health and disease, with an effective dose that is 10-fold lower than the wild-type protein. Specifically, we mutated 3 of 4 surface-exposed cysteine residues on galectin-1 to serine residues, and used the remaining cysteine residue to site-specifically crosslink two galectin-1 molecules via a homobifunctional polymer. Together, these modifications yielded a covalent galectin-1 homodimer that is resistant to inactivation in oxidative environments and is more stable under dilute conditions than the native non-covalent homodimer. Additionally, we developed an approach to prolong enzyme biocatalytic activity in proximity to an injection site via conjugation to galectin-3 (Gal3), a protein that recognizes extracellular glycans and glycosaminoglycans. Specifically, we fuse an enzyme onto the N-terminus of Gal3 via DNA recombination, and then engineer these fusion proteins to self-assemble into multivalent “nanoassemblies” via peptide domains that form coiled-coils. Enzyme-Gal3 fusion proteins and nanoassemblies bind carbohydrates with tunable affinity that is dictated by Gal-3 valency. In vivo, Gal3-mediated binding to extracellular carbohydrates slows enzyme diffusion away from an injection site. As a result, enzyme-Gal3 fusion proteins and nanoassemblies provide localized biocatalytic activity that persists over 7-14 days, whereas wild-type enzyme activity is lost within hours. Finally, we developed low-fouling biomaterials based on glycosylated peptides that self-assemble into nanofibrillar hydrogels. High-density carbohydrate display along the nanofiber creates a water shell that inhibits mammalian cell and bacteria adhesion, as well as non-specific protein adsorption. Together, these examples illustrate the enormous potential of carbohydrates and their binding partners to advance biotherapeutics and biomaterials to address a broad assortment of human health challenges.
Allan Dietz, Ph.D.
Therapeutic Potential of Mesenchymal Stromal Cells (MSCs) from the Stromal Vascular Fraction of Adipose Tissue
Allan B. Dietz (1); Andre J. van Wijnen (2); Greg W. Butler (1); Darcie J. Radel (1); Mike Deeds (1); Adam Armstrong (1); Peggy Bulur (1); Sarah Withers (1); Dennis A. Gastineau (1);
Immune, Progenitor, and Cellular Therapeutics, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States (1); Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States (2)
Mesenchymal stromal cells (MSCs) from the stromal vascular fraction of adipose tissue have great therapeutic potential. We developed a manufacturing platform and associated regulatory and pre-clinical support for the rapid implementation of internal investigator-initiated clinical trials for a number of diseases. We have manufactured autologous MSC products from more than 134 patients for 6 clinical trials. The products for all of these trials were autologous MSCs derived from adipose tissue that were manufactured using an identical cell culture platform consisting of Advanced MEM (Gibco Life Technologies, Grand Island, NY) supplemented with human platelet lysate (PLTMax, Mill Creek LifeSciences, Rochester MN). The median age of these patients was 58 years old (range 18 - 80, SD 14) and median adipose biopsy was 0.4 grams (range 0.08 - 5.53, SD 0.89). The median absolute passage 1 cell count was 4.24x106 cells (range 6.5x104 – 4.22x107, SD 8.82x106). We found that the number of cells after passage correlated with adipose biopsy weight (p<0.0001), donor age (p=0.008) and adipose digestion time (p=0.0014), however no correlation was seen with BMI or incubation time before Passage 1. The cells had a median doubling rate of 1.38 per day (range 0.69 - 1.92, SD 0.25). With this protocol, we could routinely generate 500 million cells. Our first time success rate (biopsy to treatment) was 82%, and an overall success rate of 97%. RNA-seq analysis of MSCs from >30 patients revealed remarkable uniformity of the product, compared to the RNA-seq profiles of >200 other human MSCs and stem cell types. Transcriptome analysis reveals that these MSCs have a reproducible cell surface profile of CD markers which have been validated by flow cytometry. In addition to manufacturing data and high-resolution molecular quality control, we will update clinical results on the first four trials to complete enrollment representing more than 80 patients. Overall we have a process where human stromal vascular fraction derived MSCs have been consistently manufactured from adipose tissue independent of age and underlying disease.
Johnny Huard, Ph.D.
The Role of Stem Cell Depletion in Aging and Disease: Implications for Stem Cell Therapy
Johnny Huard, Ph.D.
The University of Texas Health Science Center at Houston (UTHealth)
Aging is characterized by the progressive erosion of tissue homeostasis and functional reserve in all organ systems. Although controversy remains as to the molecular mechanism(s) underlying the process of aging, accumulated cellular damage, including DNA damage, appears to be a major determinant of lifespan as well as age-related pathologies. Moreover, there is evidence that the accumulation of damage in stem cells renders them defective for self-renewing and regenerating damaged tissues. We have demonstrated that a population of muscle progenitor cells(MPCs) isolated from the ERCC1-deficient mouse model of accelerated aging, are defective in their proliferation abilities, differentiation capacity and resistance to oxidative stress. We have observed that intraperitoneal (IP) injections of wild-type (WT)-MPCs into Ercc1 knockout (Ercc1-/-) mice resulted in an improvement in age related pathologies. Although the mechanisms by which the transplantation of WT-MPCs extend the lifespan of these progeria mice is still under investigation, we have obtained evidence that the beneficial effect imparted by the injected cells occur through a paracrine effect that involve angiogenesis. In an attempt to determine whether the defect observed in ERCC deficient MPCs was not exclusive to this progeria model, we have isolated and characterized MPCs from another progeroid mouse models, the zinc metalloproteinase (Zmpste24) knock-out mouse, an animal model of the Hutchinson-Gilford progeria syndrome (HGPS). Similar to ERCC deficient MPCs, we have observed that Zmpste24-/- MPCs have proliferation and differentiation defects, characteristics also observed in MPCs isolated from naturally aged mice. These results suggest that the defect in MPCs is not specific to a particular model of progeria and can also be observed in naturally aged animals. Finally, we have investigated whether a defect in MPCs can also be observed in skeletal muscle disease such as Duchenne muscular dystrophy (DMD), which is a degenerative muscle disorder characterized by the lack of dystrophin expression at the sarcolemma of muscle fibers. Interestingly, DMD patients lack dystrophin from the time of birth; however, the onset of muscle weakness only becomes apparent at 4-7 years of age, which happens to coincide with the exhaustion of the MPC pool. There are several lines of evidence that support this concept including the gradual impairment of the myogenic potential of MPCs isolated from DMD patients during aging, which results in a reduction of muscle regeneration in older DMD patients. In addition to muscle weakness, DMD patients acquire osteopenia, fragility fractures, and scoliosis indicating that DMD may represent a model of premature musculoskeletal aging with a potential dysfunction in MPCs. Here, we report that dystrophin–utrophin double knockout (dko) mice, an animal model of DMD, exhibit a spectrum of degenerative changes in various musculoskeletal tissues including skeletal muscle, bone, articular cartilage, and intervertebral discs. In contrast to that observed with MPCs isolated from the mdx mice (dystrophin deficient and mild phenotype), we have recently shown a defect in the MPCs isolated from dKO mouse. We have observed that the MPC defect from the dKO mouse model appears to be age dependent and not specific to MPC since other stem cell population also appears to be affected. These results taken together support the concept that stem cell exhaustion plays a role in aging and disease.