88.18 Developing Ex-Vivo Model of Left Ventricular Outflow Tract to Understand Disease Etiology?

Posted on January 31, 2019

K. Brown1, Y. Ning2, M. Kang1, M. Farenholtz2, J. Grande-Allen1, S. Keswani1,2  1Rice University,Bioengineering,Houston, TX, USA 2Texas Children’s Hospital,Department Of Surgery,Houston, TX, USA

Introduction: Turbulent flow in the Left Ventricular Outflow Tract (LVOT) is considered the main cause of Discrete Subaortic Stenosis (DSS). DSS is a heart disease characterized by the formation of a fibrotic membrane encircling the LVOT. Our lab is using DSS patient echocardiographic data to mimic pathologic conditions ex-vivo in a parallel plate flow loop bioreactor. We aim to understand the mechanism of DSS by studying cellular behavior under varying flow conditions. To understand cellular behavior, we must have an appropriate system for cells to adhere to. In this study, we compare the efficiency of cell adherence to different matrices when exposed to flow as modeled on patient echo data.

Methods:  To compare the efficiency of cell adherence with different matrices, we created gelatin, gelatin methacryloyl (gelMA), and fibronectin coatings on functionalized glass slides. Organic residues were removed from glass slides with sulfuric acid. The slides were then treated with 3-(Trimethoxysilyl)propyl methacrylate to functionalize the surface. Endocardial endothelial cells (EECs) were isolated from porcine left ventricle tissue. We then plated 1×10^6 of EECs on each slide and subjected them to flow rates observed in the LVOT.  We analyzed cell adherence using a computational algorithm to assess the efficiency of each matrix with an image analysis pipeline to assess cell morphology and cell counting. CD31 IHC is used to validate endothelial phenotype. 

Results: We created a bioreactor that could mimic flows at high, low, and static conditions. EECs were confirmed to be near 100% endothelial lineage by CD31 and DAPI staining. Cells were seeded onto gelatin, gelMA, and fibronectin matrices. Gelatin was observed to have 100% adherence at static conditions, 80% adherence at low shear, and 75% adherence at high shear. Cells under high shear flow on gelMA matrix showed a dissociation of the CD31 from the cell membrane in response to high shear as compared to low shear.

Conclusion: Cells adhered to gelatin with optimal adherence under high flow. This gel composition in the bioreactor allows us to the investigate flow disturbances in the LVOT. Interestingly, CD31 is suggested to be affected by differential shear forces and may have a role in mechanotransduction in LVOT pathology. We hope this project will open new avenues for studying DSS and other heart diseases influenced by turbulent flow.

88.12 Human Platelet Lysate Improves In vitro Survival of Human Diabetic Mesenchymal Stem Cells

Posted on January 31, 2019

L. P. Brewster1,2, J. Raykin1,2  1Emory University School Of Medicine,Surgery,Atlanta, GA, USA 2Atlanta VA Medical Center,Surgery And Research Services,Decatur, GA, USA

Introduction:  Diabetic persons undergo premature aging of their vasculature leading to an earlier onset and more severe presentations of cardiovascular disease. Thus, they have great need for regenerative therapies. Mesenchymal stem cells (MSC) are one promising regenerative therapy that may help prevent vascular complications of diabetes. The objective of this work is to identify whether MSC survival pathways are improved by platelet lysate (PL), and if so, whether PL gel confers benefit to MSC survival and regenerative function on endothelial cells (EC).

Methods:
MSCs from Diabetic PAD (dMSC) and healthy patients were used. Luminescence studies were performed in MSCs transfected with luciferin lentivirus. Secretome analysis was performed with RayBiotech angiogenesis assays. Akt cell survival pathways were quantified by multiplex analyses.

Results:  dMSCs and healthy MSCs have increased retention and survival (2-3x) in PL gel than that seen in saline injection (most commonly used clinically). EGF expression in dMSCs is significantly increased (>5x; P<.0001) in PL gel over that in control groups. dMSCs had significant down-regulation of pAKT compared to healthy MSCs and MSCs from PAD patients without diabetes. Further, PL drastically changes the secretome profile of dMSCs compared to FBS. (Figure

Conclusion:

Initial concerns with cells from cardiovascular patients have now come into question for PAD patients. Improving dMSC survival could be important to PAD patients. In this work, we show that PL gel improves MSC survival in vivo and dMSC EGF expression, and that dMSCs have Akt signaling defects that may be reversible with PL supplementation.

87.13 Tgf-β Suppression with Wnt Activation Increases Intestinal Stem Cell Proliferation in Vitro

Posted on January 31, 2019

S. M. Zuber1, G. Levin1, C. R. Schlieve1, A. I. Squillaro1, K. L. Fowler1, L. A. Nucho1, T. C. Grikscheit1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA

Introduction:
Intestine organoid units (OU), multicellular epithelial and mesenchymal cell clusters, include stem and progenitor cells and can be cultured long-term in a reduced-factor medium with subsequent generation of tissue-engineered small intestine (TESI) after implantation in vivo. Application of Y-27632 (Rho-Kinase Inhibitor), A-83-01 (Tgf-β Inhibitor), and CHIR99021 (GSK3 Inhibitor) (YAC) small molecules to primary hepatocytes is known to convert terminally differentiated cells to bipotent liver progenitors. Tgf-β suppression and Wnt activation also induce intestinal stem cell proliferation. We therefore hypothesized that addition of YAC inhibitors might increase the proliferation and size of cultured OU to increase TESI yields for future human therapies.

Methods:
Small intestine from 2-week old C57/BL6 mice was harvested to generate OU that were maintained on reduced-factor Matrigel for 5 days (n=6). OU were cultured in DMEM supplemented with 10% fetal bovine serum, non-essential amino acids, and antibiotics in either the presence or absence of YAC small molecules refreshed every two days. Eight random fields in each well were imaged to assess the number and average area of OU (ImageJ software) and evaluated by ANOVA (GraphPad Prism 7). OU were collected on Day 5 either for whole mount or in agarose embedded in paraffin, then sectioned at 8 µm thickness. OU were co-stained for E-cadherin (epithelial marker) and Ki-67 (proliferation marker) for immunofluorescence. Paraffin-embedded sections were imaged with fluorescence microscopy and whole mount fixed OU were imaged with confocal microscopy. For each condition, OU containing a lumen were randomly imaged. The ratio of Ki-67 positive cells and total cells stained with E-Cadherin was measured and evaluated by paired t-test.  

Results:
On day 5, OU treated with YAC were significantly larger than OU cultured in control media (49928 µm2 vs 9529 µm2, p=0.0036) (Figure 1A and 1B). While there was initially a significant increase in number of OU in each well with YAC application after day 1 (170.6 vs 264.5, p<0.0001), there was no significant difference in OU number on Day 5 (p=0.07). There was an increase in intestinal cell proliferation as seen by the significantly higher ratio of Ki-67 positive cells over total cells stained with E-cadherin in our YAC group (0.488 vs 0.124, p=0.0248) (Figure 1C).

Conclusion:

While there is not an increase in the overall number of OU, the addition of YAC small molecules in culture increases both the size and proliferation index of intestine organoid units. Ongoing investigation will determine if this approach increases the yield of resulting tissue-engineered small intestine growth.

87.10 Use of Mesenchymal Stem Cells and Nanosilk to Bioengineer a Patch for Wound Healing and Fetal Interventions

Posted on January 31, 2019

S. A. Hilton1, L. C. Dewberry1, C. Zgheib1, M. M. Hodges1, E. J. Burtch2, J. Jacot2, P. Rozance3, S. Seal4, K. W. Liechty1  1University Of Colorado Denver,Department Of Surgery,Aurora, CO, USA 2University Of Colorado Denver,Department Of Bioengineering,Aurora, CO, USA 3University Of Colorado Denver,Department Of Pediatrics,Aurora, CO, USA 4University of Central Florida,Department of Material Science Engineering, AMPAC And NSTC Center,Orlando, FL, USA

Introduction

Spina bifida encompasses a range of caudal neural tube defects affecting 1 in every 2000 live births in the United States, the most severe being myelomeningocele where the spinal cord herniates out of the back. Amniotic fluid stem cells are an easily obtained source of autologous cells capable of differentiation into all three germ lines. Silk fibroin (SF) is a biocompatible and biodegradable polymer, which can be tailored to different nanostructures for biomedical application. We have tested multiple formulations of nanosilk including a liquid and matte formulation. We have previously shown that the nanosilk liquid improves biomechanical properties of human skin and both liquid and matte formulations and can be used for drug delivery. We hypothesized, we could isolate and differentiate ovine amniotic fluid stem cells and use these cells to populate a nanosilk matte that could be applied to a variety of wound healing applications including fetal repair of myelomeningocele. 

 

Methods

Nanosilk (matte formulation) was created from silk fibroin isolated from the cocoons of Bombyx mori silk worms, cut into small pieces, and dissolved in solution to obtain the viscosity needed for electrospinning nanofibers. Ovine amniotic fluid mesenchymal stem cells (oAF MSCs) were isolated from mid-gestation amniotic fluid collected during fetal intervention. oAF MSCs were cultured with the nanosilk for 7 days.

Nine female 12 week old mice that are bred homozygous diabetic (Db/Db) were used. Db/Db mice were used as this is an established murine model of abnormal diabetic wound healing. A single 8mm wound was made on the dorsal neck skin of each mouse with a punch biopsy. Wounds were treated with one time administration of phosphate buffered saline control or Nanosilk only and Nanosilk + oAF MSCs at the time of wounding.

 

Results

oAF MSC cells were isolated from 7 ewes within 7-21 days after amniotic fluid harvest. oAF MSCs were confirmed with IHC markers (positive for CD29, CD90, CD44, and negative for CD45) and showed the ability to differentiate into multiple cell lineages including adipocytes, osteocytes, and neural cells (Figure 1). Diabetic mice treated with nanofiber had improved wound healing (day 16 compared to day 18). Nanofiber cultured with ovine amniotic stems cells had similar wound healing to the nanofiber. The nanofiber did not incorporate into the wound and acted as a biologic dressing that was shed as the wound healed (Figure 2).

 

Conclusions 

oAF MSCs can be quickly isolated and cultured for incorporation into nanofiber silk patch that can be applied to wounds and possibly for fetal repair of myelomeningocele. The nanofiber silk patch is able to hold the MSCs stable in culture for several weeks and presents a possible delivery method for these cells. No adverse effects on wound healing are seen when the nanosilk or nanosilk + oAF MSCs are used and may have improved wound healing. 

87.09 Varying Degrees of Plication to Optimize Spring-Mediated Intestinal Lengthening

Posted on January 31, 2019

G. Dubrovsky1, A. Thomas2, S. Shekherdimian1, J. Dunn2  1University Of California – Los Angeles,Los Angeles, CA, USA 2Stanford University,Palo Alto, CA, USA

Introduction:
Intestinal lengthening with endoluminal springs has been well studied in animal models, and has been shown to lengthen a segment of intestine by as much as 3-fold. Springs are secured inside the intestine by surgically narrowing the intestine around the spring. This method of intestinal lengthening can be performed within a functional segment of bowel, and does not interfere with normal digestion or peristalsis. However, the amount of intestinal narrowing or plication necessary for lengthening has not been studied. The goal of this study is to determine if lengthening can be optimized by changing the amount of intestinal plication performed to secure an endoluminal spring.

Methods:
Juvenile mini-Yucatan pigs underwent surgical implantation of a nitinol spring within a segment of intestine via enterotomy that was closed. Plication or narrowing of the intestine around the spring with sutures was performed to help secure the spring within the intestine. There were three total groups of pigs; in the first group, the diameter of the intestine was reduced by 50%, in the second group it was reduced by 30%, and in the third group it was reduced by only 10% (FIGURE). Pigs were kept on a liquid diet post-operatively. 3 weeks after the original operation the pigs were euthanized and intestinal segments were examined for longitudinal lengthening and for histological changes.

Results:
All pigs tolerated the surgeries without complications. There were no cases of obstruction or perforation and all springs remained in place at the site of original implantation. While 50% plication resulted in 3-fold lengthening, 30% plication resulted in a 1.8-fold increase in length, and 10% plication resulted in a 1.3-fold increase in length. All lengthened segments showed significant increases in both the thickness of the muscularis propria as well as the depth of crypts as compared to normal segments of intestine.

Conclusion:
Intestinal plication is effective in securing springs within a segment of intestine to achieve intestinal lengthening, and the degree of plication effects the amount of lengthening that can be achieved. 50% reduction in the diameter of the bowel allows for maximal intestinal lengthening, while still avoiding bowel obstruction. These results will be important in guiding potential future therapies in the treatment of short bowel syndrome.
 

87.05 A Bioadhesive Reverse Thermal Gel for Minimally Invasive In Utero Patching of Myelomeningocele

Posted on January 31, 2019

J. R. Bardill1,2, D. Park1, A. I. Marwan1,2  1University Of Colorado Denver,Bioengineering,Aurora, CO, USA 2University of Colorado Denver Anschutz Medical Campus,Pediatric Surgery,Aurora, CO, USA

Introduction:

Myelomeningoceles (MMC) are one of the most common congenital birth defects of the spinal cord. Prenatal repair of these defects became the standard of care, however, open approaches present significant risks to mother and fetus.  Our laboratory has developed a novel Reverse Thermal Gel (RTG) to deliver a minimally invasive patch for MMC coverage. Preliminary studies demonstrated long-term in-vitro gel stability and minimally invasive application in a mouse MMC model with partial defect coverage.

In preparation to transition to the ovine model, an RTG patch with surface adhesion in the amniotic environment and cellular scaffolding properties to integrate with host will be needed.  Here, we demonstrate the chemical synthesis and characterization of a bioadhesive RTG, in-vitro cellular scaffolding properties, and the biological applicability in a mouse MMC defect model.

Methods:

A. Synthesis of Bioadhesive RTG:  Dopamine was conjugated to the RTG polymer backbone and verified using proton nuclear magnetic resonance (H NMR) spectroscopy and infrared (IR) spectroscopy.  Mechanical properties were tested using rheology. B. In-vitro RTG cellular scaffold:  Mouse skin fibroblasts and human epidermal keratinocytes (HEKA) were mixed (separately) with RTG-media solution, followed by gelling at 37°C to encapsulate the cells within RTG.   Fibroblasts were stained for vimentin and alpha smooth muscle actin (α-SMA), and HEKAs were stained with keratin 14 (k14) antibodies.  C. Adhesive RTG injection into mouse MMC defect embryos:  In-vivo applicability was tested in Grhl3 mice to assess MMC defect coverage by the adhesive RTG.  D. Inflammatory response:  Defect tissue was immunostained for CD68 and F4/80 macrophages. 

Results:

H NMR and IR verified aromatic and hydroxyl peaks from dopamine are present in the polymer backbone.  The elastic strength of the adhesive RTG increased 500-1000 Pascal’s compared to the regular RTG.  Dermal fibroblasts expressed vimentin and α-SMA markers.  HEKAs in RTG culture expressed k14 markers. The adhesive RTG was applied to mouse MMC defects by needle injection and improved defect coverage, with an average of 72% defect coverage at harvest.  CD68 and F4/80 macrophage positive cells were not significantly different when comparing adhesive RTG and saline injections in defect embryos.

Conclusion:

We synthesized and characterized a bioadhesive RTG with increased elastic strength.  This adhesive RTG has in-vitro fibroblast and keratinocyte cellular scaffolding properties cultured in an RTG matrix.  Finally, the adhesive RTG can be injected in a minimally invasive manner to improve mouse MMC defect coverage compared to the non-adhesive RTG with minimal macrophage response.  Overall, this work has shown the adhesive RTG is a promising candidate for future application in the ovine model. 

 

 

87.03 Natural History of the Heterogeneous Pulmonary Response Following Tracheal Occlusion

Posted on January 31, 2019

R. Marwan1, S. Al-Juboori1, E. Dobrinskikh2, J. Haines3, R. Marwan1  1University Of Colorado Denver,Pediatric Surgery,Aurora, CO, USA 2University Of Colorado Denver,Medicine,Aurora, CO, USA 3University Of Colorado Denver,Biochemistry And Molecular Genetics,Aurora, CO, USA

Introduction:  Infants with severe congenital diaphragmatic hernia (CDH) offered experimental tracheal occlusion (TO) have inconsistent clinical outcomes. Understanding such clinical variability is crucial to advancing neonatal care but the reasons for such deviations are currently unknown. Our laboratory has proposed a novel concept of heterogeneous topological zones formation in fetal lungs following TO. However, the temporal pattern of airspace morphometry and metabolic landscape changes following TO is not fully elucidated. Our objective is to explore the natural history of heterogeneous changes in fetal lungs following TO.

Methods:  We evaluated fetal lungs after 1 and 4 days following TO; global and local metabolic changes, as well as morphometric indices were examined using mass spectrometry-based metabolomics, Fluorescence Lifetime Imaging Microscopy (FLIM) and Tissue Airspace Ratio (TAR), respectively.

Results: 1 day (D1) after TO, there is an appearance of two distinct morphometric areas varying between small (high TAR values) and large (small TAR values) airspaces, with an overall shift toward smaller airspaces compared to controls. In contrast, by day 4 (D4) TO lungs have a higher frequency of large airspaces (small TAR values) compared to control. D4 TO lungs global metabolomics data demonstrate a significant decrease in glucose and hexose phosphate, and an increase in lactate production, compared to D1 TO lungs. Moreover, there is a suppression of the tricarboxylic acid cycle (TCA) with a significant decrease in citrate and fumarate at D4. These changes are accompanied by significant increase in spermidine and spermine at D4, which are important for cellular growth and proliferation. Locally on FLIM, D1 TO lungs demonstrate two types of heterogenous zones. Namely, zones which have free/bound NADH ratio similar to control lungs, and zones with decreased free/bound NADH ratio- indicating increased OXPHOS in these regions. Similar to the histological findings, FLIM on D4 TO lungs demonstrates appearance of zones with enlarged airspaces, which have metabolic shift towards glycolysis, accompanied by decreased FLIM-surfactant signal only in those areas.

Conclusion: In normal fetal lungs, we report a novel unique natural history of heterogeneous changes; TO leads to heterogeneous pulmonary response. Initially, there is formation of zones with small airspaces, followed by airspace enlargement over time.  While FLIM of D1 TO lungs reveals zones with increased OXPHOS, FLIM in D4 TO lungs demonstrate a shift toward glycolysis in the enlarged airspaces with decreased surfactant production. We speculate that the “best responders” to tracheal occlusion should have bigger lungs with small airspaces and normal surfactant production.
 

67.01 Encapsulation Of Arteriogenic Macrophages Enhances Cell Retention And Ischaemic Limb Perfusion

Posted on January 31, 2019

F. E. Ludwinski1, G. Damodaran1, A. S. Patel1, J. Cho1, S. Jayasinghe2, A. Smith1, B. Modarai1  1King’s College London School Of Medicine,School Of Cardiovascular And Medical Sciences,London, London, United Kingdom 2University College London,Department Of Mechanical Engineering,London, LONDON, United Kingdom

Introduction:
Cell therapy has been proposed as a means of improving perfusion of ischaemic limbs. Direct injection of pro-angiogenic cells has, however, shown only modest outcomes in clinical trials, perhaps because of their loss from the site of injection. Here, we investigate alginate microsphere encapsulation as a means of enhancing retention and improving reperfusion of the ischaemic hindlimb.

Methods:
150μm sodium alginate microspheres, containing 108cells/ml of Tie2-expressing murine bone marrow-derived macrophages (eTie2-iBMMs), were generated using a clinical-grade cell encapsulator. Comparisons were made between eTie2-iBMMs and non-encapsulated cells (nTie2-iBMMs), with respect to: cell viability and phenotype; in vitro pro-angiogenic function after angiopoietin-mediated stimulation of Tie2 (VEGF expression and endothelial tubule formation); cell retention (IVIS biofluorescent imaging); and ability to promote revascularisation in the ischaemic murine hindlimb.

Results:
Encapsulation did not affect Tie2-iBMM viability or phenotype up to 7days in vitro, the expression of VEGF, or formation of endothelial tubules (p=>0.05), although stimulated eTie2-iBMMs secreted significantly greater levels of MCP-1 (p=0.0022). Cell retention was significantly enhanced in ischaemic murine hindlimbs treated with eTie2-iBMMs (p=0.0083) and this was associated with significantly greater limb perfusion over 21 days (p=0.0005).

Conclusion:
Alginate encapsulation of pro-arteriogenic macrophages is not detrimental to their viability or phenotype, and increases their retention and regenerative capacity in the ischaemic hindlimb. Translation of this methodology to a clinical setting may enhance the efficacy of novel cell-based therapies for the treatment of CLI.
 

66.08 Application of Purified Exosome Product Improved Wound Healing In Ischemic Wounds: A Rabbit Model

Posted on January 31, 2019

M. F. Bowers1,2, T. C. Huang1, M. D. Sabbagh1, S. L. Moran1  1Mayo Clinic,Rochester, MN, USA 2Meharry Medical College,Nashville, TN, USA

Introduction: Exsomes are extracellular vesicles that are secreted by cells and execute intracellularcommunication1. Exosomes convey cell communication through the export of their contents. Exosomes are known to contain tissue-specific mRNA, miRNA and proteins. Their ability to transfer these contents has been implicated in physiological processes including stem cell renewal, tumor formation, and inflammation reduction2. One of the standard therapies for advanced and tunneled wounds is application of Integra flowable wound matrix [Integra LifeSciences Corporation, NJ, USA] and a subsequent wound vacuum. Previous literature has shown that PEPs can accelerate cutaneous wound healing by upregulating blood vessel and collagen synthesis. However, the effect of PEPs in ischemic or tunneled cutaneous wounds has yet to be examinated in depth. The aim of this study is determine the effectivenss of purified exosome product (PEP) in healing full-thickness and ischemic wounds. 

Methods: 10 New Zealand White rabbits were used for this study and randomly distributed into thee groups:  Control (Saline + Integra, n=3), 5% PEP +Integra (n=4), and 20% PEP + Integra (n=3). Each underwent a surgical creation of an ischemic wound in the right ear while under general anesthesia. Two of the three ear neurovascular bundles were dissected and ligated within the ear. A circumferential skin tunnel was created and a 2cm full thicknesss wound was created by punch biopsy. Integra with either saline or PEP was applied on the wound for complete coverage and sealed with a Tegaderm film (3M). The incisions were closed with sutures and the ear was wrapped in a light dressing. Wound tissue samples were collected after three weeks and wound healing was analyzed via analyzed histologically and through 3D scanning.  Gene Expression profiles for each wound were quantified via reverse transcriptase real-time PCR. 

Results: Control wounds were indurated while all of treated wounds (n=7) showed less induration and inflammation on observation compared to the control group. Analysis from RT-qPCR data showed that the 5% PEP group had up-regulation of TGF-B, TNF-A, CTGF, and PDGF and down-regulation of COL-1 and FGF-1 compared to the control group. The 20% PEP group had down-regulation of BMP-2, COL-1, TNF-A, CD3, CD31, and TGF-B among others. There was no up-regulation of any genes studied in the 20% compared to control. None of the Control wounds showed evidence of epidermal regeneration of histological analysis while half of the 5% wounds, and all of the 20% wounds showed epidermal regeneration. 

Conclusion: PEP use in conjunction with Integra matrix correlated with morphological improvement in ischemic wound healing vs Integra alone. Along with that, wounds treated with PEP showed gene expression profiles indicative of more mature wound healing. Also, data shows that PEP may enhance epidermal regeneration of ischemic wounds and increase the capillary density of these wounds through an unknown mechanism.    
 

66.05 Human Abdominal Adhesion Fibroblasts Upregulate PDGFRa Surface Expression and EMT Pathway Genes

Posted on January 31, 2019

D. S. Foster1,2, C. D. Marshall1,2, G. Gulati1, R. E. Jones1, A. Titan1,2, A. Nguyen1, A. Salhotra1, R. C. Ransom1, S. Mascharak1, R. E. Brewer1, J. A. Norton1,2, C. J. Kin2, A. A. Shelton2, M. T. Longaker1  1Stanford University,Hagey Laboratory For Pediatric Regenerative Medicine,Stanford, CA, USA 2Stanford University,General Surgery,Palo Alto, CA, USA

Introduction: Intra-abdominal adhesions are a common cause of small bowel obstruction after surgery and cost the U.S. health care system $5 billion annually. Fibroblasts are activated in the context of abdominal surgery and deposit fibrotic scar tissue. Despite the prevalence of adhesions, no effective therapeutics exist for prevention or treatment.

Methods: Human adhesion tissues, along with control peritoneum from patients without surgical history, were harvested from patients undergoing elective operations at Stanford Hospital (Fig. 1A). Adhesion-forming and quiescent fibroblasts were fluorescence-activated cell sorting (FACS)-isolated using an unbiased, lineage-negative approach (Fig. 1B-C). These cells were screened for expression of known fibroblast surface markers. Bulk RNA-seq was conducted; gene expression was compared between adhesion and control cohorts, as well as with bulk RNA-seq data acquired from mouse abdominal adhesions and control specimens. Adhesion specimens were fixed, sectioned and stained. Histological data was compared with control and mouse tissue specimens. All experiments were approved by Stanford University’s IRB and APLAC, as appropriate. 

Results: Human adhesion-forming fibroblasts show significantly increased cell surface expression of PDGFRa compared with non-adhesion fibroblasts (Fig. 1D). PDGFRa is also highly-expressed on the surface of adhesion-forming fibroblasts in mice. Histologically, PDGFRa+ fibroblasts appear to closely associate with collagen fibers (Fig. 1E). Adhesion fibroblasts also express the cell surface marker CD26 in both human (Fig. 1D) and mice. These markers are also associated with scar-forming fibroblasts in skin. Bulk RNA-seq of human adhesion fibroblasts shows significant upregulation of Col24a1 expression compared to control, which is a member of the collagen gene family involved in type 1 collagen regulation and is also highly expressed by mouse adhesion fibroblasts. In addition, multiple genes involved in the epithelial-mesenchymal transition (EMT) pathway are upregulated by both human and mouse adhesion fibroblasts (eg. SPP1, SDC1, Comp, CDH2) (data not shown). These present potential therapeutic targets.

Conclusion: This research identifies PDGFRa-expressing fibroblasts as major contributors in abdominal adhesion formation and provides in depth tissue level and gene expression characterization of adhesion fibroblasts in humans. These data provide insight into key signaling processes responsible for adhesion formation as well as identifying a set of potential therapeutic targets common to human and mice. These targets can be validated preclinically and suggest promise for the development of therapeutics that could change the course of this challenging disease.
 

66.04 DEL-1 Interacts with Integrins of BCSPs to Promote Fracture Healing

Posted on January 31, 2019

D. M. Struck1, T. V. Boyko1, R. K. Mann4, M. T. Longaker4, C. K. Chan4, G. Yang1,2,3  1Stanford University,Department Of Surgery,Palo Alto, CA, USA 2University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA 3Birmingham VA Medical Center,Birmingham, ALABAMA, USA 4Stanford University,Stanford University School Of Medicine,Palo Alto, CA, USA

Introduction:  Of the millions of people who suffer a bone fracture every year, 5-10% will have delayed or impaired healing leading to major economic and quality of life burdens. We have shown the developmentally-regulated endothelial cell locus 1 protein (DEL-1) acts as a growth factor for mouse Bone Cartilage Stroma Progenitors (BCSPs), the skeletal progenitor population most important in fracture healing. DEL-1 is a modular protein with three EGF like repeats, two discoidin domains and an RGD motif. Deletion of Del-1 leads to mice that heal fractures with decreased bone. Based on this phenotype and the presence of an RGD motif, we hypothesized that DEL-1 interacts with integrins at this binding site to promote BCSP proliferation to the fracture callus during bone healing.

Methods:  Polymerase chain reaction mutagenesis was used to create a mutant Del-1 DNA construct where the RGD (Arg-Gly-Asp) sequence was substituted with RAD (Arg-Ala-Asp). Both DEL-1 wildtype (WT) and DEL-1 RAD mutant (Mut) was produced using a baculovirus expression system, and purified with immobilized metal affinity and size-exclusion chromatography. Del-1 knockout mice underwent mid-femoral fractures with internal fixation followed by local application of hydrogels loaded with DEL-1 WT,  DEL-1 Mut or blank controls. Additional controls were wild type mice treated with blank hydrogel. BCSPs were isolated from the fracture callus 7 days after fracture using FACS. Untreated Del-1 knockout BCSPS were also plated on 24 well plates coated with DEL-1 WT, DEL-1 RAD Mutant or BSA control. Proliferation was measured every day for a week.

Results: The addition of DEL-1 WT to Del-1 knockout mice resulted in increased callus formation compared to control (p<0.05). This was correlated with increased BCSPs at the fracture callus (p< 0.05). In vitro, the addition of DEL-1 WT led to increased proliferation of BCSPs (p< 0.05). In contrast, the addition of DEL-1 Mut had no effect on either the size of the fracture callus or the number of BCSPs at the fracture site. In vitro, there was no impact on proliferation of DEL-1 Mut on BCSPs.

Conclusion: DEL-1 WT rescued the phenotype of poor fracture healing in the Del-1 knockout mice leading to increased fracture callus size with increased numbers of BCSPs. This was correlated with stimulation of proliferation in vitro. In contrast, the DEL-1 Mut had no impact on fracture callus size, mobilization of BCSPs or proliferation of BSCPs. Based on the known function of the RGD motif, we conclude that DEL-1 affects BCSPs through binding of integrins using the RGD motif.

 

66.03 Nerve-dependent progenitor expansion is critical in post-injury mandibular repair

Posted on January 31, 2019

R. Jones1, A. Salhotra1, C. Ransom1, D. Foster1, T. Chari1, K. Robertson1, D. Wan1, M. T. Longaker1  1Hagey Laboratory for Pediatric Regenerative Medicine,Division Of Plastic And Reconstructive Surgery, Department Of Surgery,Palo Alto, CA, USA

Introduction:
We have previously established a mouse model of inferior alveolar nerve (IAN) disruption, and demonstrated via fluorescence active cell sorting that IAN denervation impairs skeletal stem cell (SSC) recruitment function after fracture injury.However, the impact of denervation on healing morphology and tissue-specific progenitor activity has yet to be examined.

Methods:
Wild-typemice were subjected to surgical disruption of the right IAN, and two weeks were allowed for complete nerve degeneration. This model of denervation has been validated in our previous studies (data not shown). Using innervated mandibles as a control, mice underwent unicortical osteotomy on the buccal surface of the right mandible (Fig 1a). Hemimandibles were harvested 2 weeks after fracture, then sectioned and stained for analysis. The same surgical protocol was enacted on Actin-CreERT2::Rosa26-VT2/GK3 mice, or actin rainbow mice, which house a multicolor Cre-dependent reporter. Upon Cre recombinase activation by tamoxifen, individual cells randomly and permanently express one of four fluorochromes: eGFP (cytoplasmic green), mCerulean (membrane blue), mCherry (membrane red), and mOrange (membrane yellow). Selection of the ubiquitous actin Cre driver permits visualization of clonal expansion of cells, with clones defined as continuous regions of a single color. Denervated and innervated mandibles from actin rainbow mice were harvested two weeks postoperatively after unicortical fracture and mounted whole. They were examined with stereoscopic microscopy and corrected fluorescence intensity of fracture area was computed in ImageJ.

Results:
Denervated fractures healed with gross morphological abnormalities (Fig 1b). Compared to the compact callus formed in innervated bone healing, denervated tissue exhibits large gaps in bone tissue repair. Fluorochrome intensity was measured and compared between innervated and denervated fracture sites in actin rainbow mandibles. The denervated tissue exhibited significantly less fluorescent signal than the innervated samples in green, red, and merge channels (p= 0.0349*, 0.0079**, and 0.0097**, respectively) (Fig 1c). Images of fluorochrome signals are exhibited (Fig 1d), consistent with a striking fault in clonal expansion of denervated fracture healing tissue.

Conclusion:
With our novel mouse model of mandibular denervation, we show that bony healing of denervated mandibular fracture is morphologically aberrant. This defect is paired with lack of clonal expansion into the zone of healing, indicating that impaired stem/progenitor activity is responsible for deficient injury repair in denervated healing. Our data suggest that mandibular fracture healing is enacted via stem/progenitor cells which exhibit a high degree of nerve dependency.
 

66.02 Sex Differences in Murine Myocutaneous Flap Revascularization

Posted on January 31, 2019

J. S. Brandenburg1, R. M. Clark1, B. B. Coffman4, G. Sharma2, H. J. Hathaway3, E. R. Prossnitz2, T. R. Howdieshell1,3  1University Of New Mexico HSC,Surgery,Albuquerque, NM, USA 2University Of New Mexico HSC,Molecular Medicine,Albuquerque, NM, USA 3University Of New Mexico HSC,Cell Biology,Albuquerque, NM, USA 4University Of New Mexico HSC,Pathology,Albuquerque, NM, USA

 

Introduction:   Sex differences in susceptibility to ischemia/reperfusion injury have been documented in humans.  Premenopausal women have a lower risk of ischemic heart disease than age-matched men, whereas after menopause, the risk is similar or even higher in women.  However, little is known about the effects of sex on cutaneous wound revascularization.

Methods:   A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of 10-12 week old male (n=10) and female (n=10) C57BL6 mice.  Planimetric analysis of digital photographic images was utilized to determine flap viability.  Real-time flap perfusion, ischemia, and functional revascularization was determined by laser speckle contrast imaging (LSCI).  Image analysis of CD31-immunostained sections confirmed flap microvascular anatomy, density, and surface area.  Values are expressed as means ± SEM.  Student’s t test was performed when comparing two groups, and two-way ANOVA used when comparing multiple groups.  Statistical significance was accepted at a p value < 0.05.

Results:  Flaps created on female mice were engrafted to the recipient site resulting in nearly complete viability at post-operative day 10.  In contrast, distal full thickness myocutaneous necrosis was evident at 10 days post-surgery in male mice (percent flap necrosis: female 5.8 ± 0.9% versus male 30.8 ± 4.7%, p < 0.05).  Over the 10 day study interval, LSCI documented functional flap revascularization in all regions of interest (ROI) in female mice, but minimal distal flap reperfusion in male mice (day 10 distal ROI perfusion: female 191.5 ± 9.2 PU versus male 138.9 ± 11.7 PU, p < 0.05).  Day 10 histologic sections immunostained to detect CD31 confirmed significant increases in distal flap vessel count and vascular surface area in female compared to male mice (vessel count: female 127 ± 12 vessels/mm2 versus male 38 ± 6 vessels/mm2, p < 0.05; vascular surface area: female 7,899 ± 85 µ2/mm2 versus male 2,335 ± 40 µ2/mm2, p < 0.05).

Conclusions:  In a graded-ischemia wound healing model, flap revascularization was more effective in female mice.  The recognition and identification of sex-specific wound healing differences may lead to a better understanding of the underlying mechanisms of myocutaneous revascularization and drive novel discovery to improve soft tissue wound healing.

 

64.10 Platelet Rich Plasma Induces M2 Macrophage Polarization and Improves Metrics of Mesh Incorporation

Posted on January 31, 2019

J. Van Eps2,3, X. Wang2, S. Minardi2, E. Tasciotti2, J. Fernandez-Moure1,2  1Hospital Of The University Of Pennsylvania,Traumatology, Surgical Critical Care, And Emergency Surgery,Philadelphia, PA, USA 2Houston Methodist Research Institute,Houston, TX, USA 3Houston Methodist Hospital,Surgery,Houston, TX, USA

Introduction:  Platelet rich plasma (PRP), a growth factor rich form of superconcentrated platelets, has been shown to improve metrics of incorporation (MOI), improve acellular dermal matrix (ADM) strength, and reduce recurrence in ventral hernia repair (VHR) with ADM. Macrophage phenotypic differentiation to the M2 phenotype has been shown to promote ADM incorporation. We hypothesized that PRP would induce a differential pattern of macrophage polarization towards a regenerative phenotype and improve early MOI.

Methods:  Lewis rats underwent chronic hernia creation and subsequent repair with non- crosslinked porcine ADM after 30 days(d). A standardized dose (200μl)  of PRP with a concentration of 1×106 platelets/μl  was applied prior to skin closure. The abdominal wall including mesh was harvested at 3d and 10d. To determine macrophage phenotype, the excised mesh and 2 mm  of surrounding tissue was decellularized and flow cytometry performed at 3d using CD3, CD45, CD206, and antimacrophage marker. Expression of genes associated with an inflammatory (M1) phenotype (IL-1b, iNOS, TNFα) and a regenerative (M2) phenotype (ARG-1, IL-10) were then quantified with RT-PCR. To assess MOI, tissues were fluorescently stained for αSMA and CD31 and the expression of Col1, Col3, VEGF, and vWF at 10d quantified by RTPCR .

Results: PRP treated ADMs had increased percentage of M2 (antimacrophage+CD206+) macrophages at 3d (p<0.05) (Figure 1a). This correlated with a significant increase in expression of M2 associated genes (Figure 1 b-c) and decreased expression of M1 associated genes at 3d (Figure 1d-f). PRP treated ADMs showed increased αSMA and CD31 intensity in histology. This finding correlated with increased Col1, Col3, VEGF, and vWF expression at 10d.

Conclusion: PRP, a growth factor rich blood product, improves metrics of wound healing. We demonstrate that M2 polarization is a responsible mechanism for the clinically relevant findings of improved ADM performance seen in our previous studies. PRP induced a differential pattern of macrophage polarization to the M2 phenotype and was associated with improved MOI. These findings suggest that PRP is a useful adjunct with ADM and may have a putative role in other wound healing scenarios, especially those in which graft incorporation is essential.

 

28.04 The Critical Role of Autophagy and Mitochondrial Remodeling in Vascular Tissue Engineering

Posted on January 31, 2019

K. E. Hekman1, C. He2, J. A. Wertheim1  1Feinberg School Of Medicine – Northwestern University,Department Of Surgery,Chicago, IL, USA 2Feinberg School Of Medicine – Northwestern University,Department Of Cell And Molecular Biology,Chicago, IL, USA

Introduction:
Vascular tissue derived from patient-specific induced pluripotent stem cells (iPSCs) suffers from premature replicative senescence, creating a significant barrier to the advancement of organ and tissue bio-engineering. Viral overexpression of longevity gene sirtuin1 as well as nutrient starvation each attenuate this premature senescence. Both are known mediators of autophagy, which is the process of cellular self-digestion that recycles intracellular components. Selective autophagy of the mitochondria, i.e., mitophagy, regulates accumulation of dysfunctional mitochondria which contribute to endothelial cell (EC) senescence. The mechanisms of premature senescence in reprogrammed iPSC-derived ECs are currently unknown.

Methods:
The iPSC lines ACS1028 and Y6 were subjected to directed differentiation and acquired endothelial lineage markers over 6 days. Cells were then purified by positive selection for VE-cadherin. Autophagy activity was monitored by quantifying the key autophagy protein, microtubule-associated proteins 1A/1B light chain 3B (LC3), through Western blotting. Mitochondrial mass, morphology and membrane potential were evaluated with MitoTracker staining. Endothelial cell function was assessed through light microscopy and quantification of nitric oxide production using the 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate substrate.

Results:
During directed differentiation from iPSCs to ECs, mitochondrial morphology evolved from globular to filamentous, and the membrane potential per mass decreased >50% (p=0.09 ACS1028, p<0.05 Y6). LC3 expression decreased by >40% (p<0.05 Y6), which correlated with increased mitochondrial mass on day 3. Mature iPSC-derived ECs had minimal autophagy activity within 5 days after purification, over which time nitric oxide production also declined by 60% (p<0.05). These iPSC-derived ECs senesced by day 13 after purification.

Conclusion:
The evolution of mitochondrial morphology and membrane potential represent remodeling during EC differentiation from iPSCs. The inverse correlation between mitochondrial mass and autophagy activity suggests this process is mediated by mitophagy. Mature iPSC-derived ECs demonstrate a significant decline in autophagy activity that correlates with the loss of mature EC function, evidenced by decreased nitric oxide synthesis, which precedes the onset of premature replicative senescence. This supports the role of mitophagy in mediating cellular senescence of iPSC-derived ECs, and renders autophagy induction a target for attenuating senescence. Overcoming this premature senescence would enable a more durable supply of patient-specific ECs for diagnostic and therapeutic applications in a wide range of disease states.
 

26.08 Hepatic Progenitor Cells are Generated from a GMP-Compliant Human Induced Pluripotent Stem Cell Line

Posted on January 31, 2019

A. I. Squillaro1,2, A. M. Fode2, L. A. Nucho2, S. M. Zuber1,2, D. F. Chang2, C. R. Schlieve1,2, T. C. Grikscheit1,2,3  1Children’s Hospital Los Angeles,Division Of Pediatric Surgery,Los Angeles, CA, USA 2Children’s Hospital Los Angeles,Developmental Biology And Regenerative Medicine At The Saban Research Institute,Los Angeles, CA, USA 3University Of Southern California,Keck Medical School,Los Angeles, CA, USA

Introduction:  Donor scarcity remains an obstacle for patients requiring orthotopic liver transplantation, although in some inborn errors of metabolism, replacement of an entire organ may not be necessary to prevent the buildup of toxic metabolites. Liver cell transplantation has been proposed for these patients; however, primary liver cells have limited expansion in vitro and still require donor tissue. Human induced pluripotent stem cells (hiPSC) offer a renewable source for cellular therapies that, unlike human embryonic stem cells (hESC), are derived from postnatal tissues. We initially derived hepatic progenitor cells from hESC after 17 days of directed differentiation. However, in order to plan to meet regulatory standards, we next hypothesized that hepatic progenitor cells (HPC) might be be generated from a fully characterized GMP hiPSC line, LiPSC-GR1.1, in a variation of our tested differentiation protocol. 

Methods: LiPSC-GR 1.1 hiPSC were cultured in suspension for 17 days and differentiated into hepatospheres containing HPC. These hepatospheres were then analyzed by H&E and immunofluorescence staining for hepatic nuclear 4α (HNF4α), α- fetoprotein (AFP), and cytokeratin 19 (CK19), a marker for cholangiocytes. Immunofluorescent staining for proliferating cell nuclear antigen (PCNA) evaluated the presence of proliferation. 

Results: H&E revealed formed hepatospheres. Immunofluorescence staining for hepatocyte markers HNF4α, an important regulator in metabolism, and AFP confirmed that hepatic progenitor cells were formed after 17 days of directed differentiation in vitro (Figure 1A). Cholangiocytes were detected in the HPC by the presence of CK19. Active proliferation of cholangiocytes were identified by PCNA co-staining (Figure 1B). 

Conclusion: A fully characterized, GMP-compliant hiPSC line can produce hepatic progenitor cells after 17 days in vitro. The day 17 cells form hepatospheres that contain hepatocytes and cholangiocytes which are actively proliferating. Further investigation of hepatic progenitor cells from this source may provide a functional cellular therapy that salvages patients from congenital metabolic diseases. 

24.03 Orthotopic implantation develop better triple negative breast cancer Patient-Derived Xenograft

Posted on January 31, 2019

M. Okano1, M. Oshi1, K. Takabe1  1Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA

Introduction: Patient-Derived Xenograft (PDX) has come into the limelight of breast cancer research to be used for pre-clinical studies. Some of its weaknesses are its poor engraftment rates and slow growths, which often limits its use as an avatar of the donor patient. Therefore, improvement of the models especially in engraftment and tumor growth are in urgent need. We hypothesized that orthotopically implanted tumors (Ortho) engraft better, grow faster and larger compared from subcutaneously implanted PDX (SQ), which is the standard model. 

Methods: NSG mice were used to generate PDX. 2 tumors were derived from brain metastasis (B-met), and the others were from primary breast cancers. 3 tumors were ER(+)HER2(-) and 7 tumors were triple negative (TN). Both of B-met tumors are ER(+)HER2(-). Tumor “engraftment” was defined as tumorigenesis of palpable tumor after implantation regardless of time it took.

Results:The overall engraftment rate was significantly better in Ortho than SQ (77.8% (n=137/176) vs. 50.7% (n=79/156), p<0.01). Ortho tumors grew remarkably larger than SQ tumors. The mean tumor weight was significantly heavier in Ortho than SQ (0.75g vs 0.14g, p<0.01). Ortho tumors demonstrated more abundant mitotic figures compared with SQ tumors (19.2 vs 7.9, p<0.01). Ortho tumors had more Ki-67 positive cells than SQ tumors (31.5 vs 21.8, p=0.015). The tumor weight was significantly larger when implantation was made to the 2nd or to the 4th mammary fat pad (0.73g vs 0.96g, p=0.02). Tumor engraftment of 1st generation was low (24.8% (n=32/129)), but the rate of 2nd (82.2% (n=46/56)) and 3rd (80.6% (n=58/72)) generation was significantly increased (p<0.001). The time it took for the 1st generation to grow was the longest between 3 generations (1st; 152days, 2nd; 66days, 3rd; 63days, p<0.01). The mean tumor weight was significantly higher in Ortho sites among all generations (1st -3rd) of TN cancer (0.2g, 1.1g vs 0.8g, respectively, p<0.01). ER positive cancer xenograft revealed significantly lower engraftment rate (26.7% (n=12/45) vs 65.1% (n=216/332), p<0.01), slower tumor growth, and lighter tumor weight (0.18g vs 0.47g, p<0.001) than TN xenograft. The xenograft from brain-metastasized breast cancer also showed higher engraftment rate in MP than SQ (94.4% (n=68/72) vs. 69.4% (n=50/72), p<0.01) although was not the organ that brain metastasis originally grew. The brain metastasis tumors also demonstrated higher tumor weight in MP than SQ (0.28g vs 0.54g, p<0.001). The brain metastasis tumors grew faster than primary tumor (52days vs 95days, p<0.01). 

Conclusion:Orthotopical implantation showed better take rate, greater tumor size and weight than heterotopic implantation, regardless of the cancer subtypes and their sources.

 

23.04 Sex Differences in Murine Myocutaneous Flap Revascularization

Posted on January 31, 2019

J. S. Brandenburg1, R. M. Clark1, B. B. Coffman4, G. Sharma2, H. J. Hathaway3, E. R. Prossnitz2, T. R. Howdieshell1,3  1University Of New Mexico HSC,Surgery,Albuquerque, NM, USA 2University Of New Mexico HSC,Molecular Medicine,Albuquerque, NM, USA 3University Of New Mexico HSC,Cell Biology,Albuquerque, NM, USA 4University Of New Mexico HSC,Pathology,Albuquerque, NM, USA

 

Introduction:   Sex differences in susceptibility to ischemia/reperfusion injury have been documented in humans.  Premenopausal women have a lower risk of ischemic heart disease than age-matched men, whereas after menopause, the risk is similar or even higher in women.  However, little is known about the effects of sex on cutaneous wound revascularization.

Methods:   A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of 10-12 week old male (n=10) and female (n=10) C57BL6 mice.  Planimetric analysis of digital photographic images was utilized to determine flap viability.  Real-time flap perfusion, ischemia, and functional revascularization was determined by laser speckle contrast imaging (LSCI).  Image analysis of CD31-immunostained sections confirmed flap microvascular anatomy, density, and surface area.  Values are expressed as means ± SEM.  Student’s t test was performed when comparing two groups, and two-way ANOVA used when comparing multiple groups.  Statistical significance was accepted at a p value < 0.05.

Results:  Flaps created on female mice were engrafted to the recipient site resulting in nearly complete viability at post-operative day 10.  In contrast, distal full thickness myocutaneous necrosis was evident at 10 days post-surgery in male mice (percent flap necrosis: female 5.8 ± 0.9% versus male 30.8 ± 4.7%, p < 0.05).  Over the 10 day study interval, LSCI documented functional flap revascularization in all regions of interest (ROI) in female mice, but minimal distal flap reperfusion in male mice (day 10 distal ROI perfusion: female 191.5 ± 9.2 PU versus male 138.9 ± 11.7 PU, p < 0.05).  Day 10 histologic sections immunostained to detect CD31 confirmed significant increases in distal flap vessel count and vascular surface area in female compared to male mice (vessel count: female 127 ± 12 vessels/mm2 versus male 38 ± 6 vessels/mm2, p < 0.05; vascular surface area: female 7,899 ± 85 µ2/mm2 versus male 2,335 ± 40 µ2/mm2, p < 0.05).

Conclusions:  In a graded-ischemia wound healing model, flap revascularization was more effective in female mice.  The recognition and identification of sex-specific wound healing differences may lead to a better understanding of the underlying mechanisms of myocutaneous revascularization and drive novel discovery to improve soft tissue wound healing.

 

21.05 A Novel Role for the ALX1 Homeobox Transcription Factor in Craniofacial Development

Posted on January 31, 2019

Y. Hu1, J. Pini1, E. Liao1  1Massachusetts General Hospital,Center For Regenerative Medicine,Boston, MA, USA

Introduction:
Frontonasal dysplasia (FND) is a class of congenital disorders caused by incomplete growth and fusion of the central frontonasal protrusion with the paired maxillary facial prominences. Genetic etiologies of FND are unclear, but mutations in ALX1 are associated with FND. ALX1 is expressed in the facial mesenchyme of vertebrate embryos, and in cranial neural crest cells (NCCs) and its cartilage derivatives. However, little is known about this transcription factor’s role during craniofacial development.

Methods:
Using a human pedigree of 4 subjects in a family with heritable FND, we defined an ALX1 gene variant (A, B). Control and patient blood samples were used to generate induced Pluripotent Stem Cells (iPSCs) using Sendai virus transfection of Yamanaka factors. iPSCs were characterized based on their embryonic stem cells (ESCs) properties. Moreover, karyotype, and ALX1 gene sequencing were performed to finalize the iPSCs characterization. Further, iPSCs were differentiated to neural crest cells to study the role of ALX1 in craniofacial formation. To study the requirement of ALX1 in an in-vivo context, CRISPR Cas9 mutagensis and dominant-negative alx1 mRNA overexpression were employed to create alx1 mutant zebrafish. Cell migration, differentiation and gene expression were examined in the mutant alx1 embryos.

Results:

Control and ALX1-/- iPSCs displayed embryonic stem cells properties, such as self-renewal and ESC markers expression. iPSCs from both genotypes displayed normal karyotype. Whole exome sequencing revealed a pathogenic L165F variant. The iPSC were differentiated into neural crest cells (C), and demonstrated phenotypes that reveal their mechanism of developmental anomaly. Notably, we found abnormal expression of neural crest markers and an increase in the sensitivity of these differentiating cells to apoptotic stress (D).

In studying our zebrafish models, we were able to recapitulate our findings in-vitro and observed phenotypes of palate and eye malformation (E). 

Conclusion:
Studies of FND using complementary iPSC and zebrafish models revealed that ALX1 is dispensable for iPSC differentiation into neural crest, but exhibit increased apoptosis during the differentiation process, leading to a fewer number of NCC progenitor cells that contribute to craniofacial morphogenesis. These studies revealed the requirement of ALX1 in craniofacial regulatory networks and provide a framework for how patient derived iPSC can be used to model craniofacial anomalies. Further study of ALX1 function and identification of its target genes will elucidate frontonasal development and innovate future treatments.

02.19 Impact of Hepatic Stellate Cells in Scaffold-Free 3D-Bioprinting of the Liver Model

Posted on January 31, 2019

C. Koehler1, W. Zhang1, J. R. Walsh1, R. A. Naqvi1, A. M. Chen1, E. Gramelspacher1, L. J. Smith2,3, P. Li1, B. Ekser1  1Indiana University School Of Medicine,Transplant Surgery/Department Of Surgery,Indianapolis, IN, USA 2Indiana University School Of Medicine,Department Of Radiology And Imaging Sciences,Indianapolis, IN, USA 3Indiana University School Of Medicine,3D-Bioprinting Core,Indianapolis, IN, USA

Introduction: Hepatic stellate cells (HSC), which compromise ~2-8% of liver cells, are vital to hepatocellular function. Scaffold-free 3D-bioprinting (SF3DBP) offers an avenue for the creation of realistic organ models without the use of biomaterials. Therefore, we hypothesized that co-culturing primary hepatocytes with HSC in SF3DBP liver model would uphold hepatocyte function over time, providing us a better 3D-liver model for research.

Methods: We used freshly thawed primary pig hepatocytes and immortalized pig HSC to generate spheroids with (i) hepatocytes alone, (ii) HSC alone, or (iii) a combination of hepatocytes and HSC. Spheroids were formed using low adhesion plates, then characterized for distance from well center, diameter, roundness, and smoothness. A column of spheroids was printed using a Regenova 3D-bioprinter. Remaining loose spheroids were incubated over two weeks for functionality assays (albumin secretion, mRNA transcription, urea clearance). Optimized spheroids of hepatocytes and HSC were cultured and printed to create a SF3DBP liver model.

Results: Co-cultures of hepatocytes and HSC (2.5:1 ratio) formed spheroids within 48 hours, as did HSC alone spheroids. Spheroids composed of hepatocytes alone failed to form round spheroids (Figure 1). The combination spheroids increased in roundness and decreased in diameter between characterizations over 6 days. Functional assays showed maintenance of albumin secretion and increased urea clearance over 14 days in spheroids formed by combined cells. Real-Time PCR demonstrated increased albumin (hepatocyte marker) and CRBP-1 (HSC marker) mRNA expressions in combination spheroids. Optimized spheroids (hepatocyte/HSC) enabled the creation of a SF3DBP construct on day 4. The 3D construct fused by day 3 and was able to be removed from temporary microneedle support by day 6 (Figure 1).

Conclusion: Spheroids formed by HSC alone proved too large to print at 48 hours. SF3DBP of spheroids (formed by hepatocyte:HSC in 2.5:1 ratio) would be viable by day 6. Optimization in centrifuging and incubation time allowed combination spheroids (2:1 ratio) to print earlier. Being able to print spheroids on day 4 as compared to day 6 increases the utility of future constructs for pharmacological, immunological, and hepatotoxicity testing. Maintenance of functionality of gene expression and albumin secretion emphasizes the utility of the 3D-bioprinted model over a period of 14 days. Further optimization of spheroids using different cell ratios including HSC, hepatocytes, liver sinusoidal endothelial cells, cholangiocytes, and fibroblasts will allow for production and printing of more physiologically accurate liver models.

 

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