Matthew J. Branch

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

Mesenchymal Stem Cells in the Human Corneal Limbal Stroma

PURPOSE Peripheral and limbal corneal stromal cells (PLCSCs), which contain keratocytes, have a complex phenotype. Knowledge of keratocyte cell properties, function, and origin is limited. Evidence available thus far has suggested both mesenchymal stromal and hematopoietic characteristics. Multipotent mesenchymal stromal cells (MSCs) are found in an increasing number of tissues and are the subject of considerable interest and investigation in the disciplines of tissue engineering, immunology, gene therapy, and oncology. METHODS Isolated PLCSCs were characterized by markers aldehyde dehydrogenase and keratocan, cultured, and analyzed against a set of criteria for the identification of MSCs developed by the International Society of Cellular Therapy (ISCT). PLCSCs were directly compared to fetal liver MSCs (flMSCs). Additional cell surface markers were also used to quantify differentiation, which was also performed on both cell types. RESULTS PLCSCs were found to be plastic adherent, displayed the correct profile and proportions of CSMs, and demonstrated trilineage potential in accordance with the ISCT guidelines. Furthermore, PLCSCs displayed a high degree of similarity to flMSCs and this likeness extended into the non-ISCT MSC cell surface markers and trilineage differentiation, which were often but not always comparable. CONCLUSIONS Herein we report a novel observation that PLCSCs conform to all the ISCT criteria and are therefore MSCs. Furthermore, this study has identified the limbal stroma as yet another MSC niche and presents a new perspective on the role of the PLCSC.

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.

The collagen matrix of the human trabecular meshwork is an extension of the novel pre-Descemet's layer (Dua's layer)

Background The trabecular meshwork (TM) located at the angle of the anterior chamber of the eye contributes to aqueous drainage. A novel layer in the posterior part of the human cornea has recently been reported (the pre-Descemets layer (Duas layer (PDL)). We examined the peripheral part of this layer in relation to the origin of the TM. Methods The PDL and TM of 19 human donor eyes and one exenterated sample were studied. Samples were examined by light and electron microscopy (EM) for tissue architecture and by immunohistology for four matricellular proteins, five collagen types and CD34. Results EM revealed that beams of collagen emerged from the periphery of PDL on the anterior surface of the Descemets membrane and divided and subdivided to continue as the beams of the TM. Long-spacing collagen was seen in the PDL and TM. Trabecular cells (CD34-ve) associated with basement membrane were seen in the peripheral part of the PDL and corresponded to the start of the separation of the collagen lamellae of PDL. Collagen VI was present continuously in PDL and extended into the TM. Matricellular proteins were seen predominantly in the TM with only laminin extending into the periphery of PDL. Conclusions This study provides an insight into the origins of the collagen core of the TM as an extension of the PDL of the cornea. This finding adds to the knowledge base of the TM and cornea and has the potential to impact future research into the TM and glaucoma.

Augmented Dried versus Cryopreserved Amniotic Membrane as an Ocular Surface Dressing

Purpose Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material. Methods AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays. Results Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM. Conclusions Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability and is a superior substrate to conventional cryopreserved AM. In addition this product is stable and easily transportable allowing it to be globally wide reaching for use in clinical and military sectors.

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.

The use of innovative scaffolds in the development of corneal stroma-derived stem cell therapies for future corneal regeneration strategies [Abstract]

This is an accompanying abstract of a poster presented at 4th TERMIS World Congress Boston, Massachusetts September 8–11, 2015. Final publication is available from Mary Ann Liebert, Inc., publishers https://www.liebertpub.com/doi/pdf/10.1089/ten.tea.2015.5000.abstracts

Isolation of adult stem cell populations from the human cornea.

Corneal blindness is a leading cause of vision loss globally. From a tissue engineering perspective, the cornea represents specific challenges in respect to isolating, stably expanding, banking, and effectively manipulating the various cell types required for effective corneal regeneration. The current research trend in this area focuses on a combined stem cell component with a biological or synthetic carrier or engineering scaffold. Corneal derived stem cells play an important role in such strategies as they represent an available supply of cells with specific abilities to further generate corneal cells in the long term. This chapter describes the isolation protocols of the epithelial stromal and endothelial stem cell populations.