Perspective: The promise of multi-cellular engineered living systems.

Recent technological breakthroughs in our ability to derive and differentiate induced pluripotent stem cells, organoid biology, organ-on-chip assays, and 3-D bioprinting have all contributed to a heightened interest in the design, assembly, and manufacture of living systems with a broad range of potential uses. This white paper summarizes the state of the emerging field of "multi-cellular engineered living systems," which are composed of interacting cell populations. Recent accomplishments are described, focusing on current and potential applications, as well as barriers to future advances, and the outlook for longer term benefits and potential ethical issues that need to be considered.

miR-214 is Stretch-Sensitive in Aortic Valve and Inhibits Aortic Valve Calcification.

miR-214 has been recently found to be significantly downregulated in calcified human aortic valves (AVs). ER stress, especially the ATF4-mediated pathway, has also been shown to be significantly upregulated in calcific AV disease. Since elevated cyclic stretch is one of the major mechanical stimuli for AV calcification and ATF4 is a validated target of miR-214, we investigated the effect of cyclic stretch on miR-214 expression as well as those of ATF4 and two downstream genes (CHOP and BCL2L1).

Disturbed Flow Increases UBE2C (Ubiquitin E2 Ligase C) via Loss of miR-483-3p, Inducing Aortic Valve Calcification by the pVHL (von Hippel-Lindau Protein) and HIF-1α (Hypoxia-Inducible Factor-1α) Pathway in Endothelial Cells.

Objective- Calcific aortic valve (AV) disease, characterized by AV sclerosis and calcification, is a major cause of death in the aging population; however, there are no effective medical therapies other than valve replacement. AV calcification preferentially occurs on the fibrosa side, exposed to disturbed flow (d-flow), whereas the ventricularis side exposed to predominantly stable flow remains protected by unclear mechanisms. Here, we tested the role of novel flow-sensitive UBE2C (ubiquitin E2 ligase C) and microRNA-483-3p (miR-483) in flow-dependent AV endothelial function and AV calcification.

Implementation of a Biomedical Engineering Research Experience for African-American High School Students at a Tier One Research University.

Enriching science experiences and competencies for underrepresented students during high school years is crucial to increasing their entry into the science pipeline and to improving their preparedness for success in college and STEM careers. The purpose of this paper is to describe the implementation of project ENGAGES, a high school STEM year-long research program for African–American students, mentored by graduate students and postdoctoral researchers at Georgia Tech. It aims to provide an authentic research experience and expose student to the possibility and benefits of attaining an advanced degree and careers in STEM fields.

Biomanufacturing of Therapeutic Cells: State of the Art, Current Challenges, and Future Perspectives.

Stem cells and other functionally defined therapeutic cells (e.g., T cells) are promising to bring hope of a permanent cure for diseases and disorders that currently cannot be cured by conventional drugs or biological molecules.

Identification of side- and shear-dependent microRNAs regulating porcine aortic valve pathogenesis.

Aortic valve (AV) calcification is an inflammation driven process that occurs preferentially in the fibrosa. To explore the underlying mechanisms, we investigated if key microRNAs (miRNA) in the AV are differentially expressed due to disturbed blood flow (oscillatory shear (OS)) experienced by the fibrosa compared to the ventricularis. To identify the miRNAs involved, endothelial-enriched RNA was isolated from either side of healthy porcine AVs for microarray analysis.

Engineering as a new frontier for translational medicine.

The inclusion of engineering ideas and approaches makes medicine a quantitative and systems-based discipline that facilitates precision diagnostics and therapeutics to improve health care delivery for all.

A study of a three-dimensional PLGA sponge containing natural polymers co-cultured with endothelial and mesenchymal stem cells as a tissue engineering scaffold.

The interaction between vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in a complex hemodynamic and mechanical environment plays an important role in the control of blood vessel growth and function. Despite the importance of VSMCs, substitutes are needed for vascular therapies. A potential VSMC substitute is human adult bone marrow derived mesenchymal stem cells (hMSCs).

A global assessment of stem cell engineering.

Over the last 2 years a global assessment of stem cell engineering (SCE) was conducted with the sponsorship of the National Science Foundation, the National Cancer Institute at the National Institutes of Health, and the National Institute of Standards and Technology. The purpose was to gather information on the worldwide status and trends in SCE, that is, the involvement of engineers and engineering approaches in the stem cell field, both in basic research and in the translation of research into clinical applications and commercial products. The study was facilitated and managed by the World Technology Evaluation Center.

Influence of mesenchymal stem cells on the response of endothelial cells to laminar flow and shear stress.

The interactions between endothelial cells (ECs) and smooth muscle cells (SMCs) in a complex hemodynamic environment play an important role in the control of blood vessel function. Since autologous SMCs are not readily available for the tissue engineering of a blood vessel substitute, a substitute for SMCs, such as human adult bone marrow-derived mesenchymal stem cells (MSCs), is needed. The objective of this study was to use a three-dimensional coculture model of the blood vessel wall, comprised of ECs and MSCs, to determine how the presence of MSCs affects EC function.

Fluid shear stress pre-conditioning promotes endothelial morphogenesis of embryonic stem cells within embryoid bodies.

Pluripotent embryonic stem cells (ESCs) are capable of differentiating into all mesoderm-derived cell lineages, including endothelial, hematopoietic, and cardiac cell types. Common strategies to direct mesoderm differentiation of ESCs rely on exposing the cells to a series of biochemical and biophysical cues at different stages of differentiation to promote maturation toward specific cell phenotypes. Shear forces that mimic cardiovascular physiological forces can evoke a myriad of responses in somatic and stem cell populations, and have, thus, been studied as a means to direct stem cell differentiation.

Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro.

Background Aims: Multipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown.

Methods: MSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined.

Strain magnitude-dependent calcific marker expression in valvular and vascular cells.

Aortic valve disease and atherosclerosis tend to coexist in most patients with cardiovascular disease; however, the causes and mechanisms of disease development in heart valves are still not clearly understood. To understand the contributions of the magnitude of cyclic strain (5% hypotension, 10% physiological, and 15% hypertension) in calcification, we used a model system of tissue-engineered collagen gels containing human aortic smooth muscle cells and human aortic valvular interstitial cells, both isolated from noncalcific heart transplant tissue. The compacted collagen gels were cultured in osteogenic media for 3 weeks in a custom-designed bioreactor and all assessments were performed at the end of the culture period.

Differentiation patterns of embryonic stem cells in two- versus three-dimensional culture.

Pluripotent stem cells are attractive candidates as a cell source for regenerative medicine and tissue engineering therapies. Current methods of differentiation result in low yields and impure populations of target phenotypes, with attempts for improved efficiency often comparing protocols that vary multiple parameters. This basic science study focused on a single variable to understand the effects of two-dimensional (2D) versus three-dimensional (3D) culture on directed differentiation.

Dynamic shear stress regulation of inflammatory and thrombotic pathways in baboon endothelial outgrowth cells.

Endothelial outgrowth cells (EOCs) have garnered much attention as a potential autologous endothelial source for vascular implants or in tissue engineering applications due to their ease of isolation and proliferative ability; however, how these cells respond to different hemodynamic cues is ill-defined. This study investigates the inflammatory and thrombotic response of baboon EOCs (BaEOCs) to four hemodynamic conditions using the cone and plate shear apparatus: steady, laminar shear stress (SS); pulsatile, nonreversing laminar shear stress (PS); oscillatory, laminar shear stress (OS); and net positive, pulsatile, reversing laminar shear stress (RS). In summary, endothelial nitric oxide synthase (eNOS) mRNA was significantly upregulated by SS compared to OS.

Bioengineering and the cardiovascular system.

The development of the modern era of bioengineering and the advances in our understanding of the cardiovascular system have been intertwined over the past one-half century. This is true of bioengineering as an area for research in universities. Bioengineering is ultimately the beginning of a new engineering discipline, as well as a new discipline in the medical device industry.

Effects of shear stress on germ lineage specification of embryonic stem cells.

Mechanobiology to date has focused on differentiated cells or progenitors, yet the effects of mechanical forces on early differentiation of pluripotent stem cells are still largely unknown. To study the effects of cellular deformation, we utilize a fluid flow bioreactor to apply steady laminar shear stress to mouse embryonic stem cells (ESCs) cultured on a two dimensional surface. Shear stress was found to affect pluripotency, as well as germ specification to the mesodermal, endodermal, and ectodermal lineages, as indicated by gene expression of OCT4, T-BRACHY, AFP, and NES, respectively.