Phenotypic change and induction of cytokeratin expression during in vitro culture of corneal stromal cells

Purpose: Cells of the corneal epithelium and stroma can be distinguished in vivo by different intermediate filaments, cytokeratins for corneal epithelial cells (CEC) and vimentin for keratocytes. Isolated and cultured keratocytes change phenotype, losing expression of keratocyte markers and gaining markers associated with mesenchymal stromal cells (MSC). This study investigates this change in phenotype in relation to intermediate filament expression in cultured corneal stromal cells (CSC) compared to CEC. Methods: Expression of epithelial markers (CK3, CK12, CK19, pan cytokeratin, E-cadherin), keratocyte markers (CD34, vimentin) and MSC markers (CD73, CD90 and CD105) were compared in CEC and CSC by immunocytochemistry and RT-qPCR. Expression was evaluated at different stages of CSC culture and compared to another stromal cell type, extracted from Wharton’s jelly (WJ-MSC). Results: In vivo keratocytes did not express cytokeratins. However, cultured CSC expressed epithelial-associated CK3, CK12 and CK19 but other cytokeratins. Expression of cytokeratins increased as CSC were passaged and decreased as CSC were induced to become quiescent. Comparatively, WJ-MSC, expressed lower levels of CK3, CK12 and CK19, but also stained for pan cytokeratin and expressed KRT5. Conclusions: Cultured CSC undergo phenotypic change during culture, expressing specific cytokeratin filaments normally associated with CEC. Cytokeratin expression begins as cells are cultured on plastic and increases with passage. This discovery may influence the way that differences are discerned between cultured CEC and CSC. Investigators need to be aware that the expression of cytokeratins does not necessarily represent epithelial contamination, and that CEC and CSC may be more related than previously recognised

Relationship Stress: Social Media Edition

Couples involved in a romantic relationships (dating, engaged, married) face a variety of stressors that can determine the quality and sustainability of the relationship. These stressors can include money, children, work schedules, and opinions of family and friends about the relationship or one’s significant other. With arrival of the internet, the use of social media has become a new source of stress among relationships. Altshule (2015) found social media use negatively impacts a relationship when one’s significant other is always on social media, engages in appropriate activity online, or is overly private when online (i.e. hiding online activity from their partner). Bea (2012) found that having a significant other who shares too much information about the relationship online, maintains contact with an ex (e.g. tagging, messaging, accepting a friend request), or who suspiciously monitors the online activity of his/her significant other can negatively affect a romantic relationship. Fritz (2015) found sending tweets to friends or followers of the opposite sex can create a source of stress within a romantic relationship. A four part survey was presented to participants, aimed at examining the relatively new stressor (social media) compared to more traditional stressors in relationships. The survey was provided to college students that were members of the Marshall University Psychology Department Human Subjects Pool (SONA)

Tissue engineering in hostile environments: the effects and control of inflammation in bone tissue engineering

The potential effects of introducing bone regeneration strategies into environments of disease and damage are often overlooked, despite the fact that many of the signalling pathways in inflammation have effects on bone development and healing. Embryonic stem cells (ESCs) are increasingly being used to develop models of disease and have potential in osteogenic-cell based therapies. Osteogenic differentiation strategies for ESCs are well established, but the response of these cells to tissue damage and inflammation has not yet been investigated, particularly in comparison to primary osteoblasts. Here, proinflammatory cytokines were used as part of an in vitro model to mimic elements of skeletal disease, such as rheumatoid arthritis and non-union fractures. The response of osteogenically differentiated mouse embryonic stem cells (osteo-mESCs) to the proinflammatory cytokines interleukin 1-β (IL-1β), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), was compared to that of primary mouse calvarial osteoblasts, already well-described in literature and used as a “benchmark” in this study. Although histology, immunocytochemistry and PCR showed similarities in osteogenic differentiation of the osteo-mESCs and the primary calvarial cells, over 21 days in culture, there were marked differences in the response to the proinflammatory cytokines. Viability of the osteo-mESCs was maintained in response to cytokines, whereas viability of primary cells was significantly reduced. There were marked increases in nitric oxide (NO) and prostaglandin E2 (PGE2) production in primary calvarial cells over the entire 21-day culture period, but this was not seen with osteo-mESCs until day 21. The study then went on to look at the effects of proinflammatory signalling on the in vitro bone formation of the two cell types. Significant differences in the effects of proinflammatory cytokines on bone nodule formation and matrix production were seen when comparing the osteo-mESCs and the calvarial cells. This study demonstrates that while osteo-mESCs share phenotypic characteristics with primary osteoblasts, there are some distinct differences in their biochemistry and response to cytokines. This is relevant to understanding differentiation of stem cells, developing in vitro models of disease, testing new drugs and developing cell therapies. An additional objective in this investigation was to look at tissue engineering strategies as a means of controlling inflammation in bone disease. The primary calvarial osteoblasts were utilised as an in vitro inflammation model, and used to study the effects of anti-inflammatory mediators. Anti-inflammatory-releasing porous scaffolds were manufactured from poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG). The calvarial osteoblast inflammation model was used successfully to show successful release of diclofenac sodium from the PLGA/PEG scaffolds. This study demonstrates that there is much to consider in the development of regenerative strategies for bone disease, particularly the role that the effect and control of inflammation will play in bone healing

Potential of mesenchymal stem cells as topical immunomodulatory cell therapies for ocular surface inflammatory disorders

Ocular surface inflammatory disorders (OSIDs) are a group of highly prevalent, heterogeneous diseases which display a variety of aetiologies and symptoms and are risk factors for serious complications including ocular and cornea impairment. Corneal inflammation is a common factor of all OSIDs, regardless of their cause or symptoms. Current medications include over‐the‐counter lubricating eye drops, corticosteroids, and ciclosporin, which either do not treat the corneal inflammation or have been associated with multiple side effects leading to alternative treatments being sought. Regenerative medicine cell therapies, particularly mesenchymal stem cells (MSCs), have shown great promise for immunosuppression and disease amelioration across multiple tissues, including the cornea. However, for successful development and clinical translation of MSC therapy for OSIDs, significant problems must be addressed. This review aims to highlight considerations, including whether the source of MSC isolation impacts the efficacy and safety of the therapy, in addition to assessing the feasibility of MSC topical application to the cornea and ocular surface through analysis of potential scaffolds and cell carriers for application to the eye. The literature contains limited data assessing MSCs incorporated into scaffolds for corneal administration, thus here we highlight the necessity of further investigations to truly exploit the potential of an MSC‐based cell therapy for the treatment of OSIDs

Tissue engineering in hostile environments: the effects and control of inflammation in bone tissue engineering

The potential effects of introducing bone regeneration strategies into environments of disease and damage are often overlooked, despite the fact that many of the signalling pathways in inflammation have effects on bone development and healing. Embryonic stem cells (ESCs) are increasingly being used to develop models of disease and have potential in osteogenic-cell based therapies. Osteogenic differentiation strategies for ESCs are well established, but the response of these cells to tissue damage and inflammation has not yet been investigated, particularly in comparison to primary osteoblasts. Here, proinflammatory cytokines were used as part of an in vitro model to mimic elements of skeletal disease, such as rheumatoid arthritis and non-union fractures. The response of osteogenically differentiated mouse embryonic stem cells (osteo-mESCs) to the proinflammatory cytokines interleukin 1-β (IL-1β), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), was compared to that of primary mouse calvarial osteoblasts, already well-described in literature and used as a “benchmark” in this study. Although histology, immunocytochemistry and PCR showed similarities in osteogenic differentiation of the osteo-mESCs and the primary calvarial cells, over 21 days in culture, there were marked differences in the response to the proinflammatory cytokines. Viability of the osteo-mESCs was maintained in response to cytokines, whereas viability of primary cells was significantly reduced. There were marked increases in nitric oxide (NO) and prostaglandin E2 (PGE2) production in primary calvarial cells over the entire 21-day culture period, but this was not seen with osteo-mESCs until day 21. The study then went on to look at the effects of proinflammatory signalling on the in vitro bone formation of the two cell types. Significant differences in the effects of proinflammatory cytokines on bone nodule formation and matrix production were seen when comparing the osteo-mESCs and the calvarial cells. This study demonstrates that while osteo-mESCs share phenotypic characteristics with primary osteoblasts, there are some distinct differences in their biochemistry and response to cytokines. This is relevant to understanding differentiation of stem cells, developing in vitro models of disease, testing new drugs and developing cell therapies. An additional objective in this investigation was to look at tissue engineering strategies as a means of controlling inflammation in bone disease. The primary calvarial osteoblasts were utilised as an in vitro inflammation model, and used to study the effects of anti-inflammatory mediators. Anti-inflammatory-releasing porous scaffolds were manufactured from poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG). The calvarial osteoblast inflammation model was used successfully to show successful release of diclofenac sodium from the PLGA/PEG scaffolds. This study demonstrates that there is much to consider in the development of regenerative strategies for bone disease, particularly the role that the effect and control of inflammation will play in bone healing

The Visibility of Galactic Bars and Spiral Structure At High Redshifts

We investigate the visibility of galactic bars and spiral structure in the distant Universe by artificially redshifting 101 B-band CCD images of local spiral galaxies from the Ohio State University Bright Spiral Galaxy Survey. Our artificially redshifted images correspond to Hubble Space Telescope I-band observations of the local galaxy sample seen at z=0.7, with integration times matching those of both the very deep Northern Hubble Deep Field data, and the much shallower Flanking Field observations. The expected visibility of galactic bars is probed in two ways: (1) using traditional visual classification, and (2) by charting the changing shape of the galaxy distribution in "Hubble space", a quantitative two-parameter description of galactic structure that maps closely on to Hubble's original tuning fork. Both analyses suggest that over 2/3 of strongly barred luminous local spirals i.e. objects classified as SB in the Third Reference Catalog) would still be classified as strongly barred at z=0.7 in the Hubble Deep Field data. Under the same conditions, most weakly barred spirals (classified SAB in the Third Reference Catalog) would be classified as regular spirals. The corresponding visibility of spiral structure is assessed visually, by comparing luminosity classifications for the artificially redshifted sample with the corresponding luminosity classifications from the Revised Shapley Ames Catalog. We find that for exposures times similar to that of the Hubble Deep Field spiral structure should be detectable in most luminous low-inclination spiral galaxies at z=0.7 in which it is present. [ABRIDGED]Comment: Accepted for publication in The Astronomical Journa

Brief of Amici Curiae Health Law & Policy Scholars and Prescription Policy Choices in Support of Respondents on the Constitutional Validity of the Medicaid Expansion

The Medicaid expansion in Section 2001(a)(1)(C) of the Patient Protection and Affordable Care Act is one part of Congress’s comprehensive effort to expand access to health care coverage. This expansion is not revolutionary, but builds on many prior statutory amendments to Medicaid. Nor does it alter the voluntary nature of the Medicaid program – as before, States remain free to decline federal funding. The Petitioners and their amici have mischaracterized the expansion to obscure these facts, hoping this Court will unravel this hard-fought legislative enactment. The question presented is whether Congress may offer States generous additional funding for Medicaid, with spending conditions that entirely satisfy the four-part test in South Dakota v. Dole, 483 U.S. 203 (1987)

A Qualitative Investigation of Individual, Interpersonal, and Institutional Contributions to Postpartum Work-Family Balance

Managing personal and professional responsibilities may be challenging during the postpartum period, as employees navigate new roles, responsibilities, and family dynamics. The purpose of this paper was to understand the work/life balance experiences of diverse stakeholders and identify opportunities to improve the work environment. We conducted a series of in-depth focus groups with faculty, staff, and graduate students (n = 22), and in-depth interviews with administrators (n = 10) at a research-intensive university in the United States. A six-phase thematic analysis approach was used to examine the experiences and perspectives of individuals with different roles. Three themes with subsequent subthemes emerged: 1) employee role shapes perspectives on campus policies and practices; 2) confusion about policies exacerbates return-to-work issues; and 3) coworkers and supervisors are the primary sources of postpartum support in this workplace. Employees in our study expressed strong desires for clear, consistent institutional policies to ensure employees’ postpartum return-to-work experiences were not dependent on informal arrangements with coworkers and supervisors. Faculty, staff, and graduate student postpartum needs are shaped by individual, interpersonal, and community factors. Clear communication between individuals who make policy decisions and those who are subject to the policies may reduce employee issues during the postpartum period

Corneal decellularization: a method of recycling unsuitable donor tissue for clinical translation?

Background: There is a clinical need for biomimetic corneas that are as effective, preferably superior, to cadaveric donor tissue. Decellularized tissues are advantageous compared to synthetic or semi-synthetic engineered tissues in that the native matrix ultrastructure and intrinsic biological cues including growth factors, cytokines and glycosaminoglycans may be retained. However, there is currently no reliable, standardized human corneal decellularization protocol. Methods: Corneal eye-bank tissue unsuitable for transplantation was utilized to systematically compare commonly used decellularization protocols. Hypertonic sodium chloride; an ionic reagent, sodium dodecyl sulphate; a non-ionic detergent, tert-octylphenol polyoxyethylene (Triton-X); enzymatic disaggregation using Dispase; mechanical agitation; and the use of nucleases were investigated. Decellularization efficacy, specifically for human corneal tissue, was extensively evaluated. Removal of detectable cellular material was evidenced by histological, immunofluorescence and biochemical assays. Preservation of macroscopic tissue transparency and light transmittance was evaluated. Retention of corneal architecture, collagen and glycosaminoglycans was assessed via histological, immunofluorescence and quantitative analysis. Biocompatibility of the resulting scaffolds was assessed using cell proliferation assays. Results: None of the decellularization protocols investigated successfully removed 100% of cellular components. The techniques with the least residual cellular material were most structurally compromised. Biochemical analysis of glycosaminoglycans demonstrated the stripping effects of the decellularization procedures. Conclusion: The ability to utilize, reprocess and regenerate tissues deemed “unsuitable” for transplantation allows us to salvage valuable tissue. Reprocessing the tissue has the potential to have a considerable impact on addressing the problems associated with cadaveric donor shortage. Patients would directly benefit by accessing greater numbers of corneal grafts and health authorities would fulfill their responsibility for the delivery of effective corneal reconstruction to alleviate corneal blindness. However, in order to progress, we may need to take a step back to establish a “decellularization” criterion; which should balance effective removal of immune reactive material with maintenance of tissue functionality

A thermoresponsive three-dimensional fibrous cell culture platform for enzyme-free expansion of mammalian cells

A three-dimensional thermoresponsive fibrous scaffold system for the subsequent extended culture and enzyme-free passaging of a range of mammalian cell types is presented. Poly(PEGMA188) was incorporated with poly(ethylene terephthalate) (PET) via blend-electrospinning to render the fibre thermoresponsive. Using primary human corneal stromal stem cells as an therapeutically relevant exemplar, cell adhesion, viability, proliferation and phenotype on this fibrous culture system over numerous thermal enzyme-free passages is described. We also illustrate the versatility of this system with respect to fabricating thermoresponsive fibres from biodegradable polymers and for the culture of diverse mammalian cell types including mesenchymal stem cells, colon adenocarcinoma cells and NIH-3T3 fibroblasts. This thermoresponsive scaffold system combines the advantages of providing a physiologically relevant environment to maintain a desirable cell phenotype, allowing routine enzyme-free passaging and expansion of cultured cells, whilst offering mechanical support for cell growth. The system described in this study presents a versatile platform for biomedical applications and more specifically for the expansion of mammalian cells destined for the clinic