Navigating the Genomic Landscape of Human Adipose Stem Cell-Derived ß-Cells.

Diabetes is a pandemic manifested through glucose dysregulation mediated by inadequate insulin secretion by beta cells. A beta cell replacement strategy would transform the treatment paradigm from pharmacologic glucose modulation to a genuine cure. Stem cells have emerged as a potential source for beta cell (ß-cell) engineering. The detailed generation of functional ß-cells from both embryonic and induced pluripotent stem cells has recently been described. Adult stem cells, including adipose derived, may also offer a therapeutic approach, but remain ill defined. In our study, we performed an in-depth assessment of insulin-producing beta cells generated from human adipose, irrespective of donor patient age, gender, and health status. Cellular transformation was confirmed using flow cytometry and single-cell imaging. Insulin secretion was observed with glucose stimulation and abrogated following palmitate exposure, a common free fatty acid implicated in human beta cell dysfunction. We used next-generation sequencing to explore gene expression changes before and after differentiation of patient-matched samples, which revealed more than 5,000 genes enriched. Adipose-derived beta cells displayed comparable gene expression to native ß-cells. Pathway analysis demonstrated relevance to stem cell differentiation and pancreatic developmental processes, which are vital to cellular function, structural development, and regulation. We conclude that the functions associated with adipose derived beta cells are mediated through relevant changes in the transcriptome, which resemble those seen in native ß-cell morphogenesis and maturation. Therefore, they may represent a viable option for the clinical translation of stem cell-based therapies in diabetes.

Computer-Aided Design and Manufacture of Intraoral Splints: A Potential Role in Cleft Care.

BACKGROUND: Nasoalveolar molding is a nonsurgical modality for the treatment of cleft lip and palate that uses an intraoral splint to align the palatal shelves. Repeated impressions are needed for splint modification, each carrying risk of airway obstruction. Computer-aided design and manufacturing (CAD/CAM) has the ability to simplify the process. As a precursor to CAD/CAM splint fabrication, a proof-of-concept study was conducted to compare three-dimensional splints printed from alginate impressions versus digital scans. We hypothesized that intraoral digital scanning would compare favorably to alginate impressions for palate registration and subsequent splint manufacture, with decreased production times. METHODS: Alginate and digital impressions were taken from 25 healthy teenage volunteers. Digital impressions were performed with a commercially available intraoral scanner. Plaster casts made from alginate impressions were converted to Standard Triangle Language files. Patient-specific matched scans were evaluated for total surface area with the concordance correlation coefficient. Acrylic palatal splints were three-dimensionally printed from inverse digital molds. Subjective appliance fit was assessed using a five-point scale. RESULTS: A total of 23 participants were included. Most subjects preferred digital impression acquisition. Impression methods showed moderate agreement (concordance correlation coefficient 0.93). Subjects rated splints from digital impressions as having a more precise fit (4.4 versus 3.9). The digital approach decreased impression phase time by over 10-fold and overall production time by 28%. CONCLUSIONS: CAD/CAM has evolved extensively over the past two decades and is now commonplace in medicine. However, its utility in cleft patients has not been fully realized. This pilot study demonstrated that CAD/CAM technologies may prove useful in patients requiring intraoral splints.

Differentiation of Adipose Tissue-Derived CD34+/CD31- Cells into Endothelial Cells In Vitro.

In this study, CD34+/CD31- progenitor cells were isolated from the stromal vascular fraction (SVF) of adipose tissue using magnetic activated cell sorting. The endothelial differentiation capability of these cells in vitro was evaluated by culturing them in vascular endothelial growth factor (VEGF) induced medium for 14 days. Viability, proliferation, differentiation and tube formation of these cells were evaluated. Cell viability study revealed that both undifferentiated and endothelial differentiated cells remained healthy for 14 days. However, the proliferation rate was higher in undifferentiated cells compared to endothelial differentiated ones. Upregulation of endothelial characteristic genes (Von Willebrand Factor (vWF) and VE Cadherin) was observed in 2D culture. However, PECAM (CD31) was only found to be upregulated after the cells had formed tube-like structures in 3D Matrigel culture. These results indicate that adipose derived CD34+/CD31- cells when cultured in VEGF induced medium, are capable differentiation into endothelial-like lineages. Tube formation of the cells started 3h after seeding the cells on Matrigel and formed more stable and connected network 24 h post seeding in presence of VEGF.

Cellular Based Strategies for Microvascular Engineering.

Vascularization is a major hurdle in complex tissue and organ engineering. Tissues greater than 200 µm in diameter cannot rely on simple diffusion to obtain nutrients and remove waste. Therefore, an integrated vascular network is required for clinical translation of engineered tissues. Microvessels have been described as <150 µm in diameter, but clinically they are defined as <1 mm. With new advances in super microsurgery, vessels less than 1 mm can be anastomosed to the recipient circulation. However, this technical advancement still relies on the creation of a stable engineered microcirculation that is amenable to surgical manipulation and is readily perfusable. Microvascular engineering lays on the crossroads of microfabrication, microfluidics, and tissue engineering strategies that utilize various cellular constituents. Early research focused on vascularization by co-culture and cellular interactions, with the addition of angiogenic growth factors to promote vascular growth. Since then, multiple strategies have been utilized taking advantage of innovations in additive manufacturing, biomaterials, and cell biology. However, the anatomy and dynamics of native blood vessels has not been consistently replicated. Inconsistent results can be partially attributed to cell sourcing which remains an enigma for microvascular engineering. Variations of endothelial cells, endothelial progenitor cells, and stem cells have all been used for microvascular network fabrication along with various mural cells. As each source offers advantages and disadvantages, there continues to be a lack of consensus. Furthermore, discord may be attributed to incomplete understanding about cell isolation and characterization without considering the microvascular architecture of the desired tissue/organ.

Bioprinting functional tissues.

Despite the numerous lives that have been saved since the first successful procedure in 1954, organ transplant has several shortcomings which prevent it from becoming a more comprehensive solution for medical care than it is today. There is a considerable shortage of organ donors, leading to patient death in many cases. In addition, patients require lifelong immunosuppression to prevent graft rejection postoperatively. With such issues in mind, recent research has focused on possible solutions for the lack of access to donor organs and rejections, with the possibility of using the patient's own cells and tissues for treatment showing enormous potential. Three-dimensional (3D) bioprinting is a rapidly emerging technology, which holds great promise for fabrication of functional tissues and organs. Bioprinting offers the means of utilizing a patient's cells to design and fabricate constructs for replacement of diseased tissues and organs. It enables the precise positioning of cells and biologics in an automated and high throughput manner. Several studies have shown the promise of 3D bioprinting. However, many problems must be overcome before the generation of functional tissues with biologically-relevant scale is possible. Specific focus on the functionality of bioprinted tissues is required prior to clinical translation. In this perspective, this paper discusses the challenges of functionalization of bioprinted tissue under eight dimensions: biomimicry, cell density, vascularization, innervation, heterogeneity, engraftment, mechanics, and tissue-specific function, and strives to inform the reader with directions in bioprinting complex and volumetric tissues. STATEMENT OF SIGNIFICANCE: With thousands of patients dying each year waiting for an organ transplant, bioprinted tissues and organs show the potential to eliminate this ever-increasing organ shortage crisis. However, this potential can only be realized by better understanding the functionality of the organ and developing the ability to translate this to the bioprinting methodologies. Considering the rate at which the field is currently expanding, it is reasonable to expect bioprinting to become an integral component of regenerative medicine. For this purpose, this paper discusses several factors that are critical for printing functional tissues including cell density, vascularization, innervation, heterogeneity, engraftment, mechanics, and tissue-specific function, and inform the reader with future directions in bioprinting complex and volumetric tissues.

Squid Ring Teeth-coated Mesh Improves Abdominal Wall Repair.

BACKGROUND: Hernia repair is a common surgical procedure with polypropylene (PP) mesh being the standard material for correction because of its durability. However, complications such as seroma and pain are common, and repair failures still approach 15% secondary to poor tissue integration. In an effort to enhance mesh integration, we evaluated the applicability of a squid ring teeth (SRT) protein coating for soft-tissue repair in an abdominal wall defect model. SRT is a biologically derived high-strength protein with strong mechanical properties. We assessed tissue integration, strength, and biocompatibility of a SRT-coated PP mesh in a first-time pilot animal study. METHODS: PP mesh was coated with SRT (SRT-PP) and tested for mechanical strength against uncoated PP mesh. Cell proliferation and adhesion studies were performed in vitro using a 3T3 cell line. Rats underwent either PP (n = 3) or SRT-PP (n = 6) bridge mesh implantation in an anterior abdominal wall defect model. Repair was assessed clinically and radiographically, with integration evaluated by histology and mechanical testing at 60 days. RESULTS: Cell proliferation was enhanced on SRT-PP mesh. This was corroborated in vivo by abdominal wall histology, dramatically diminished craniocaudal mesh contraction, improved strength testing, and higher tissue failure strain. There was no increase in seroma or visceral adhesion formation. No foreign body reactions were noted on liver histology. CONCLUSIONS: SRT applied as a coating appears to augment mesh-tissue integration and improve abdominal wall stability following bridged repair. Further studies in larger animals will determine its applicability for hernia repair in patients.

Non-coding RNAs in Various Stages of Liver Disease Leading to Hepatocellular Carcinoma: Differential Expression of miRNAs, piRNAs, lncRNAs, circRNAs, and sno/mt-RNAs.

Hepatocellular carcinoma (HCC) was the fifth leading cause of cancer death in men and eighth leading cause of death in women in the United States in 2017. In our study, we sought to identify sncRNAs in various stages of development of HCC. We obtained publicly available small RNA-seq data derived from patients with cirrhosis (n = 14), low-grade dysplastic nodules (LGDN, n = 9), high grade dysplastic nodules (HGDN, n = 6), early hepatocellular carcinoma (eHCC, n = 6), and advanced hepatocellular carcinoma (HCC, n = 20), along with healthy liver tissue samples (n = 9). All samples were analyzed for various types of non-coding RNAs using PartekFlow software. We remapped small RNA-seq to miRBase to obtain differential expressions of miRNAs and found 87 in cirrhosis, 106 in LGDN, 59 in HGDN, 80 in eHCC, and 133 in HCC. Pathway analysis of miRNAs obtained from diseased samples compared to normal samples showed signaling pathways in the microRNA dependent EMT, CD44, and others. Additionally, we analyzed the data sets for piRNAs, lncRNAs, circRNAs, and sno/mt-RNAs. We validated the in silico data using human HCC samples with NanoString miRNA global expression. Our results suggest that publically available data is a valuable resource for sncRNA identification in HCC progression (FDR set to <0.05 for all samples) and that a data mining approach is useful for biomarker development.

Breast Implant-Associated Anaplastic Large Cell Lymphoma: A Systematic Review.

IMPORTANCE: Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL), a rare peripheral T-cell lymphoma, is increasing in incidence. However, many practitioners who treat patients with breast cancer are not aware of this disease. OBJECTIVES: To assess how BIA-ALCL develops, its risk factors, diagnosis, and subsequent treatment and to disseminate information about this entity to the medical field. EVIDENCE REVIEW: A literature review was performed in an academic medical setting. All review articles, case reports, original research articles, and any other articles relevant to BIA-ALCL were included. Data on BIA-ALCL, such as pathophysiology, patient demographics, presentation, diagnosis, treatment, and outcomes, were extracted. Particular focus was paid to age, time to onset, implant type, initial symptoms, treatment, and survival. The search was conducted in January 2017 for studies published in any year. FINDINGS: After duplicates were excluded, 304 relevant articles were assessed, and 115 were included from the first documented case in August 1997 through January 2017. Thirty review articles, 44 case reports or series, 15 original research articles, and 26 "other" articles (eg, techniques, special topics, letters) were reviewed. A total of 93 cases have been reported in the literature, and with the addition of 2 unreported cases from the Penn State Health Milton S. Hershey Medical Center, 95 patients were included in this systematic review. Almost all documented BIA-ALCL cases have been associated with a textured device. The underlying mechanism is thought to be due to chronic inflammation from indolent infections, leading to malignant transformation of T cells that are anaplastic lymphoma kinase (ALK) negative and CD30 positive. The mean time to presentation is approximately 10 years after implant placement, with 55 of 83 (66%) patients initially seen with an isolated late-onset seroma and 7 of 83 (8%) with an isolated new breast mass. Ultrasonography with fluid aspiration can be used for diagnosis. Treatment must include removal of the implant and surrounding capsule. More advanced disease may require chemotherapy, radiotherapy, and lymph node dissection. CONCLUSIONS AND RELEVANCE: Breast implant-associated anaplastic large cell lymphoma is a rare cancer in patients with breast implants but is increasing in incidence. It is important for all physicians involved in the care of patients with breast implants to be aware of this entity and be able to recognize initial symptoms.

Small Non-coding RNA Abundance in Adrenocortical Carcinoma: A Footprint of a Rare Cancer.

BACKGROUND: Adrenocortical carcinoma (ACC) is a relatively rare, but aggressive type of cancer, which affects both children and adults. OBJECTIVE: Small non-coding RNAs (sncRNAs) play important roles and may serve as biomarkers for disease diagnosis, prognosis and treatment. METHODS: In our study, we sought to identify sncRNAs associated with malignant adrenal tumors. We obtained publicly available, small RNA sequencing data derived from 45 ACC and 30 benign tumors arising from the cortex of the adrenal gland, adrenocortical adenomas (ACA), and compared their sncRNA expression profiles. RESULTS: First, we remapped small RNA-seq to miRBase version 21 to check expression of miRNAs and found 147 miRNAs were aberrantly expressed (p<0.05) in ACC samples compared to ACA samples. Pathway analysis of differentially expressed miRNAs revealed p53 signaling pathways to be profoundly affected in ACC samples. Further examination for other types of small RNAs revealed 16 piRNAs, 48 lncRNAs and 19 sn/snoRNAs identified in ACC samples. Conclusions: Our data analysis suggests that publically available resources can be mined for biomarker development and improvements in-patient care; however, further research must be performed to correlate tumor grade with gene expression.

Concise Review: Bioprinting of Stem Cells for Transplantable Tissue Fabrication.

Bioprinting is a quickly progressing technology, which holds the potential to generate replacement tissues and organs. Stem cells offer several advantages over differentiated cells for use as starting materials, including the potential for autologous tissue and differentiation into multiple cell lines. The three most commonly used stem cells are embryonic, induced pluripotent, and adult stem cells. Cells are combined with various natural and synthetic materials to form bioinks, which are used to fabricate scaffold-based or scaffold-free constructs. Computer aided design technology is combined with various bioprinting modalities including droplet-, extrusion-, or laser-based bioprinting to create tissue constructs. Each bioink and modality has its own advantages and disadvantages. Various materials and techniques are combined to maximize the benefits. Researchers have been successful in bioprinting cartilage, bone, cardiac, nervous, liver, and vascular tissues. However, a major limitation to clinical translation is building large-scale vascularized constructs. Many challenges must be overcome before this technology is used routinely in a clinical setting. Stem Cells Translational Medicine 2017;6:1940-1948.

Bioprinting and Cellular Therapies for Type 1 Diabetes.

Type 1 diabetes mellitus is a chronic autoimmune disease that results from the destruction of beta (ß) cells in the pancreatic islets, leading to loss of insulin production and resultant hyperglycemia. Recent developments in stem cell biology have generated much excitement for ß-cell replacement strategies; ß cells are one of many cell types in the complex islet environment and pancreas. In this Opinion, we discuss recent successful attempts to generate ß cells and how this can be coupled with bioprinting technologies in order to fabricate pancreas tissues, which holds great potential for type 1 diabetes. Possibilities of integrating vascularization and encapsulation in bioprinted tissues are expounded, and future prospects, such as pancreas-on-a-chip, are also presented.

Transplantation of Bioprinted Tissues and Organs: Technical and Clinical Challenges and Future Perspectives.

: Three-dimensional (3D) bioprinting is a revolutionary technology in building living tissues and organs with precise anatomic control and cellular composition. Despite the great progress in bioprinting research, there has yet to be any clinical translation due to current limitations in building human-scale constructs, which are vascularized and readily implantable. In this article, we review the current limitations and challenges in 3D bioprinting, including in situ techniques, which are one of several clinical translational models to facilitate the application of this technology from bench to bedside. A detailed discussion is made on the technical barriers in the fabrication of scalable constructs that are vascularized, autologous, functional, implantable, cost-effective, and ethically feasible. Clinical considerations for implantable bioprinted tissues are further expounded toward the correction of end-stage organ dysfunction and composite tissue deficits.