Laura E. Sidney

Concise review: evidence for CD34 as a common marker for diverse progenitors.

CD34 is a transmembrane phosphoglycoprotein, first identified on hematopoietic stem and progenitor cells. Clinically, it is associated with the selection and enrichment of hematopoietic stem cells for bone marrow transplants. Due to these historical and clinical associations, CD34 expression is almost ubiquitously related to hematopoietic cells, and it is a common misconception that CD34‐positive (CD34+) cells in nonhematopoietic samples represent hematopoietic contamination. The prevailing school of thought states that multipotent mesenchymal stromal cells (MSC) do not express CD34. However, strong evidence demonstrates CD34 is expressed not only by MSC but by a multitude of other nonhematopoietic cell types including muscle satellite cells, corneal keratocytes, interstitial cells, epithelial progenitors, and vascular endothelial progenitors. In many cases, the CD34+ cells represent a small proportion of the total cell population and also indicate a distinct subset of cells with enhanced progenitor activity. Herein, we explore common traits between cells that express CD34, including associated markers, morphology and differentiation potential. We endeavor to highlight key similarities between CD34+ cells, with a focus on progenitor activity. A common function of CD34 has yet to be elucidated, but by analyzing and understanding links between CD34+ cells, we hope to be able to offer an insight into the overlapping properties of cells that express CD34. Stem Cells 2014;32:1380–1389

Hydrogels derived from demineralized and decellularized bone extracellular matrix

Graphical abstract

Characterization of corneal stromal stem cells with the potential for epithelial transdifferentiation

IntroductionThe corneal stroma is being increasingly recognized as a repository for stem cells. Like the limbal and endothelial niches, stromal stem cells often reside in the peripheral cornea and limbus. These peripheral and limbal corneal stromal cells (PLCSCs) are known to produce mesenchymal stem cells in vitro. Recently, a common corneal stromal and epithelial progenitor was hinted at. This study aims to examine the stem cell potential of corneal stromal cells and to investigate their epithelial transdifferentiation ability.MethodsPLCSCs were grown in traditional Dulbecco modified Eagle medium (DMEM)-based keratocyte culture medium and an M199-based medium and analyzed for a profile of cell-surface markers by using flow cytometry and differentiated into mesenchymal phenotypes analyzed with quantitative polymerase chain reaction (qPCR) and histologic staining. PLCSCs in M199 were subsequently divided into subpopulations based on CD34 and CD105 expression by using fluorescence- activated cell sorting (FACS). Subpopulations were characterized by marker profile and mesenchymal differentiation ability. Both whole PLCSCs and subpopulations were also cultured for epithelial transdifferentiation.ResultsCells cultured in M199 demonstrated a more stem-like cell-surface marker profile, and the keratocyte marker CD34 was retained for several passages but absent in cells cultured in DMEM. Cells cultured in M199 also exhibited a greater mesenchymal differentiation potential, compared with DMEM. PLCSCs could be divided into CD34+CD105+, CD34-CD105+, and CD34-CD105- subpopulations, of which CD34+CD105+ cells were the most stemlike with regard to marker expression and mesenchymal differentiation potential. Subpopulations of PLCSCs exhibited differing abilities to transdifferentiate into epithelial phenotypes. Cells that were initially CD34+CD105+ showed the greatest differentiation potential, producing CK3+ and CK19+ cells, and expressed a range of both epithelial progenitor (HES1, FRZB1, DCT, SOD2, ABCG2, CDH1, KRT19) and terminally differentiated (DSG3, KRT3, KRT12, KRT24) genes.ConclusionsCulture medium has a significant effect on the phenotype and differentiation capacity of PLCSCs. The stroma contains a heterogeneous cell population in which we have identified CD34+ cells as a stem cell population with a capacity for mesenchymal and epithelial differentiation.

Keeping an Eye on Decellularized Corneas: A Review of Methods, Characterization and Applications

The worldwide limited availability of suitable corneal donor tissue has led to the development of alternatives, including keratoprostheses (Kpros) and tissue engineered (TE) constructs. Despite advances in bioscaffold design, there is yet to be a corneal equivalent that effectively mimics both the native tissue ultrastructure and biomechanical properties. Human decellularized corneas (DCs) could offer a safe, sustainable source of corneal tissue, increasing the donor pool and potentially reducing the risk of immune rejection after corneal graft surgery. Appropriate, human-specific, decellularization techniques and high-resolution, non-destructive analysis systems are required to ensure reproducible outputs can be achieved. If robust treatment and characterization processes can be developed, DCs could offer a supplement to the donor corneal pool, alongside superior cell culture systems for pharmacology, toxicology and drug discovery studies.

Comparison of Osteogenic Differentiation of Embryonic Stem Cells and Primary Osteoblasts Revealed by Responses to IL-1β, TNF-α, and IFN-γ

There are well-established approaches for osteogenic differentiation of embryonic stem cells (ESCs), but few show direct comparison with primary osteoblasts or demonstrate differences in response to external factors. Here, we show comparative analysis of in vitro osteogenic differentiation of mouse ESC (osteo-mESC) and mouse primary osteoblasts. Both cell types formed mineralized bone nodules and produced osteogenic extracellular matrix, based on immunostaining for osteopontin and osteocalcin. However, there were marked differences in the morphology of osteo-mESCs and levels of mRNA expression for osteogenic genes. In response to the addition of proinflammatory cytokines interleukin-1β, tumor necrosis factor-α, and interferon-γ to the culture medium, primary osteoblasts showed increased production of nitric oxide (NO) and prostaglandin E2 (PGE2) at early time points and decreases in cell viability. In contrast, osteo-mESCs maintained viability and did not produce NO and PGE2 until day 21. The formation of bone nodules by primary osteoblasts was reduced markedly after cytokine stimulation but was unaffected in osteo-mESCs. Cell sorting of osteo-mESCs by cadherin-11 (cad-11) showed clear osteogenesis of cad-11(+) cells compared to unsorted osteo-mESCs and cad-11(-) cells. Moreover, the cad-11(+) cells showed a significant response to cytokines, similar to primary osteoblasts. Overall, these results show that while osteo-mESC cultures, without specific cell sorting, show characteristics of osteoblasts, there are also marked differences, notably in their responses to cytokine stimuli. These findings are relevant to understanding the differentiation of stem cells and especially developing in vitro models of disease, testing new drugs, and developing cell therapies.

Endothelial cell loss following tissue harvesting by pneumodissection for endothelial keratoplasty: an ex vivo study

Aims To study ex vivo the difference in endothelial cell density (ECD) in tissue harvested by pneumodissection for pre-Descemets endothelial keratoplasty (PDEK) and Descemets membrane endothelial keratoplasty (DMEK). Methods Tissue for PDEK and DMEK were obtained from 10 eye bank sclerocorneal discs by trephination after air injection into corneal stroma and big bubble (BB) formation. PDEK tissue was prepared in five corneas after achieving a type 1 BB and DMEK after a type 2 BB in five corneas. Five sclerocorneal discs for each group were used as controls. Endothelial cell counts were obtained from all samples before and after injection using phase-contrast microscopy with an eyepiece reticle. We used paired t test to analyse the results using the GraphPad Prism V.6 software. Results The range of change of ECD before and after injection in the PDEK sample group varied from −9% to +0.2% with an average of −5.36% ±3.8%. The difference was not statistically significant (p=0.0512). On the other hand, the range of change of ECD of the DMEK groups before and after injection varied from −0.4 to −20.6, with an average of −12.44% ±8.11%. This difference was statistically significant (p=0.0456). Also, there was a significant difference between DMEK test samples (postinjection) and their controls (p=0.028). Conclusions Corneal endothelial cell loss in PDEK tissue preparation is no worse, if not slightly better than, in DMEK tissue prepared by pneumodissection. PDEK preparation by pneumodissection represents a viable graft preparation technique.

Expression of Toll-like receptors in human retinal and choroidal vascular endothelial cells

Toll-like receptors (TLRs) are a family of proteins that initiate the innate immune response in reaction to invading microbes. Studies confirm the expression of TLRs in a variety of ocular tissues and cells, and it has also been suggested that selected TLRs may be associated with geographic atrophy and neovascularisation in age-related macular degeneration, diabetic retinopathy and other vascular and inflammatory diseases of the ocular posterior segment. However, TLR expression and localisation in the retinal and choroidal vasculature has not been defined. A better understanding of differential TLR expression in the choroid and retina, particularly in endothelial cells would improve our knowledge of vascular and inflammatory diseases in the posterior segment of the eye. In this study the gene (mRNA) expression of TLRs 1-10 was investigated using RT-PCR and comparative qPCR and the protein expression and localisation of selected TLRs (3, 4, 6 and 9) were examined using western blotting, flow cytometry and immunofluorescent staining. PCR showed gene expression of TLR1-6 and 9 in human choroidal endothelial cells (hCEC) and TLR2-6, 9 and 10 in human retinal endothelial cells (hREC). Western blotting detected TLR3, 4 and 9 proteins in both hCEC and hREC with higher levels in hCEC, whilst TLR6 protein was not detectable in either endothelial cell type. Flow cytometry detected all four TLRs (3, 4, 6 and 9) on the cell surface and intracellularly, TLR6 expression was detectable but low. The expression and localisation of TLR3, 4 and 9 were confirmed by immunofluorescent staining in endothelial cells and whole tissue sections and their functionality tested by expression of IL-6 (ELISA) in response to stimulation with specific TLR ligands. This study has, for the first time, identified the differential expression and localisation of TLRs in intraocular endothelial cells. This profiling will help inform our understanding of different retinal and choroidal vascular diseases, as well as the development of future treatments for intraocular vascular diseases.

Corneal Decellularization: A Method of Recycling Unsuitable Donor Tissue for Clinical Translation?

Abstract 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.

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 reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Expression was evaluated at different stages of CSC culture and compared to another stromal cell type, extracted from Whartons jelly (WJ-MSC). RESULTS In vivo keratocytes did not express cytokeratins. However, cultured CSC expressed epithelial-associated CK3, CK12, and CK19, but not 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 in which 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 recognized.

3D Microfabricated Scaffolds and Microfluidic Devices for Ocular Surface Replacement: a Review

In recent years, there has been increased research interest in generating corneal substitutes, either for use in the clinic or as in vitro corneal models. The advancement of 3D microfabrication technologies has allowed the reconstruction of the native microarchitecture that controls epithelial cell adhesion, migration and differentiation. In addition, such technology has allowed the inclusion of a dynamic fluid flow that better mimics the physiology of the native cornea. We review the latest innovative products in development in this field, from 3D microfabricated hydrogels to microfluidic devices.