Secretion and Organization of a Cornea-like Tissue In Vitro by Stem Cells from Human Corneal Stroma

Du, Yiqin; SundarRaj, Nirmala; Funderburgh, Martha L.; Harvey, S.; Birk, David E.; Funderburgh, James L.

doi:10.1167/iovs.07-0587

Cited by 104 publications

(123 citation statements)

References 29 publications

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“…Our observations agree with recent literature, 80 where stem cells from human corneal stroma cultured in the serumfree medium secreted fibrillar collagen only if cultured in three-dimensional pellets (i.e., forced stratification). Collagen fibrils produced in the stratified 1% ITS þ 1% FBS culture appear denser and more aligned than those of Du et al's pellet cultures.…”

Section: Ecm Structuressupporting

confidence: 93%

“…Collagen fibrils produced in the stratified 1% ITS þ 1% FBS culture appear denser and more aligned than those of Du et al's pellet cultures. 80 This difference may be due to our culture substratum (glass instead of pellet), medium formulation (we employed more glucose and ascorbic acid than Du et al, plus insulin and serum), or cell type.…”

Section: Ecm Structuresmentioning

confidence: 99%

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Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells

Bueno

Saeidi

Melotti

et al. 2009

Tissue Engineering Part A

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The in vitro production of highly organized collagen fibrils by corneal keratocytes in a three-dimensional scaffold-free culture system presents a unique opportunity for the direct observation of organized matrix formation. The objective of this investigation was to develop such a culture system in a glass substrate (for optical accessibility) and to directly examine the effect of reducing serum and=or increasing insulin on the stratification and secretion of aligned matrix by fourth-to fifth-passage bovine corneal stromal keratocytes. Medium concentrations of 0%, 1%, or 10% fetal bovine serum and 0% or 1% insulin-transferrin-selenium were investigated. Highresolution differential interference contrast microscopy, quick-freeze=deep-etch, and conventional transmission electron microscopy were used to monitor the evolution, morphology, and ultrastructure of the cell-matrix constructs. In a medium containing 1% each of serum and insulin-transferrin-selenium, stromal cells stratified and secreted abundant and locally aligned matrix, generating the thickest cell-matrix constructs (allowing handling with forceps). The results of this study have the potential to significantly advance the field of developmental functional engineering of load-bearing tissues by (i) elucidating cues that modulate in vitro cell secretion of organized matrix and (ii) establishing an optically accessible cell culture system for investigating the mechanism of cell secretion of aligned collagen fibrils.

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Section: Ecm Structuressupporting

confidence: 93%

Section: Ecm Structuresmentioning

confidence: 99%

Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells

Bueno

Saeidi

Melotti

et al. 2009

Tissue Engineering Part A

View full text Add to dashboard Cite

show abstract

“…For instance, the formation of cellcell junctions appears to be critical to the production of organized collagen in corneal stem cell cultures. 45 However, the lack of time-resolved imaging limits the ability to determine when and how cell-cell connections are produced. Such information and the ability to mechanically manipulate the substrate could lead to methods that promote formation of cell-cell junctions and enhanced organization of the resulting matrix.…”

Section: Previous Bioreactor Designsmentioning

confidence: 99%

Design and Performance of an Optically Accessible, Low-Volume, Mechanobioreactor for Long-Term Study of Living Constructs

Paten

Zareian

Saeidi

et al. 2011

Tissue Engineering Part C: Methods

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Currently available bioreactor systems used by tissue engineers permit either direct, high-magnification observation of cell behavior or application of mechanical loads to growing tissue constructs, but not both simultaneously. Further, in most loading bioreactors, the volume of the dead space is not minimized to reduce the cost associated with perfusion media, exogenous stimulatory/inhibitory agents, proteases, and label. We have designed, developed, and tested a bioreactor that simultaneously satisfies the combined requirements of providing (i) controlled tensile mechanical stimulation, (ii) direct high-magnification imaging capability, and (iii) low dead-space volume. This novel mechanostimulatory (uniaxial tensile loading) bioreactor operates on an inverted microscope and permits continuous optical access (up to 600 · ) to a loaded, growing construct for extended periods of time (weeks). The reactor employs an adjustable reaction chamber in which the dead space can be reduced to < 2 mL. The device has been used to cultivate our human primary corneal fibroblastderived, tissue-engineered system for up to 14 days. Using the instrument we have successfully recorded (i) the process of fibroblasts populating, growing to confluence, and stratifying on different substrates; (ii) recorded complex and organized cell sheet motions; and (iii) recorded the behavior of a subpopulation of what appear to be degradative/catabolic cells within our fibroblast culture. The device is capable of providing detailed, long-term, dynamic images of mechanically stimulated cell/matrix interaction that have not been observed previously.

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“…In culture, these side population cells showed clonal growth and could be differentiated to express keratocyte, chondrogenic and neurogenic markers (139). The same group has concomitantly showed that while these undifferentiated corneal stromal stem cells predominantly express generic stem cell related genes (Bmi-1, Kit, Notch-1, Six2, Pax6, ABCG2, Spag10, p62/OSIL) in adherent cultures, when passaged in suspension in serum free medium with FGF2 and insulin, they form spheroid pellets, in which keratocyte-like cells secrete an ordered ECM and express mRNAs of known (keratocan, PTGDS, ALDH3A1) and potential (FLJ30046/SLAIN, CxAdR, PDK4, MTAC2D1, F13A1) keratocyte markers (140).…”

Section: Stromal Stem Cellsmentioning

confidence: 99%

Stem cells of the adult cornea: From cytometric markers to therapeutic applications

et al. 2008

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The cornea is a major protective shield of the interior of the eye and represents two thirds of its refractive power. It is made up of three tissue layers that have different developmental origins: the outer, epithelial layer develops from the ectoderm overlying the lens vesicle, whereas the stroma and the endothelium have mesenchymal origin. In the adult organism, the outermost corneal epithelium is the most exposed to environmental damage, and its constant renewal is assured by the epithelial stem cells that reside in the limbus, the circular border of the cornea. Cell turnover in the stromal layer is very slow and the endothelial cells probably do not reproduce in the adult organism. However, recent experimental evidence indicates that stem cells may be found in these layers. Damage to any of the corneal layers leads to loss of transparency and low vision. Corneal limbal stem cell deficiency results in severe ocular surface disease and its treatment by transplantating ex vivo expanded limbal epithelial cells is becoming widely accepted today. Stromal and endothelial stem cells are potential tools of tissue engineering and regenerative therapies of corneal ulcers and endothelial cell loss. In the past few years, intensive research has focused on corneal stem cells aiming to improve the outcomes of the current corneal stem cell transplantation techniques. This review summarizes the current state of knowledge on corneal epithelial, stromal and endothelial stem cells. Special emphasis is placed on the molecular markers that may help to identify these cells, and the recently revealed mechanisms that could maintain their ''stemness'' or drive their differentiation. The techniques for isolating and culturing/ expanding these cells are also described. '

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Secretion and Organization of a Cornea-like Tissue In Vitro by Stem Cells from Human Corneal Stroma

Abstract: Scaffolding-free pellet culture of hCSSC induces keratocyte gene expression patterns in these cells and secretion of an organized stroma-like ECM. These cells offer a novel potential for corneal bioengineering.

Cited by 104 publications

References 29 publications

Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells

Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells

Design and Performance of an Optically Accessible, Low-Volume, Mechanobioreactor for Long-Term Study of Living Constructs

Stem cells of the adult cornea: From cytometric markers to therapeutic applications

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