Selective growth and expansion of human corneal epithelial basal stem cells in a three-dimensional-organ culture

Papini, Sandra; Rosellini, Alfredo; Nardi, Marco; Giannarini, Claudio; Revoltella, Roberto P.

doi:10.1111/j.1432-0436.2005.07302006.x

Cited by 22 publications

(24 citation statements)

References 22 publications

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“…Contamination of corneal epithelial elements was excluded by no expression of CK-3 by the conjunctival epithelial cells, in line with previous reports (Joseph et al, 2004, Papini et al, 2005. Probably in response to the culture conditions, all samples from different donors revealed that early in culture the most superficial levels sharply detached, including the positive CK-19 and less intensively positive CK13/CK-10 cells and goblet cells which were all released in the medium, while the inner basal and part of the parabasal cell compartment containing p63 and nestin positive cells remained stably anchored to the submucosa.…”

Section: Isolation and Expansion Of Epithelial Basal Stem/precursor Csupporting

confidence: 90%

“…It was probably generated from the basal and parabasal keratinocyte precursor cells that remained attached to the fragment at T1. These cells were rapidly adapting to the culture conditions entering into an active proliferative phase (judging by the increasing Ki-67 staining and constantly low apoptotic cells), as reported in general in squamous epithelia maintained in similar cultures (Michelini et al, 2004;Papini et al, 2005). The basal precursor cells of the conjunctival epithelium kept for at least 2 weeks in culture within their natural environment, seem to have a less complex structure with simple patterns that generate a single phenotypic lineage and apparently have more autonomy with less dependence in extraepithelial influences for their pattering and survival.…”

Section: Isolation and Expansion Of Epithelial Basal Stem/precursor Cmentioning

confidence: 74%

“…Tissue fragments and dispersed epithelial cells were maintained in EpiLife medium as described in Papini et al, 2005.…”

Section: Culturesmentioning

confidence: 99%

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Human conjunctival epithelial precursor cells and their progeny in 3D organotypic culture

Rosellini¹,

Papini²,

Giannarini³

et al. 2007

Int. J. Dev. Biol.

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KEY WORDS: epithelial basal precursor, stem cell, niche, epithelium regeneration, interlamellar leukocytesThe conjunctival epithelium is a stratified, non-keratinized epithelium containing goblet epithelial cells. It can be distinguished by the corneal epithelium by the expression of different cytokeratins, mucins and by the presence of glycocalix (Meller et al., 2002). In the human conjunctiva, goblet cells secreting gel-forming mucins, can occur singly or in greater numbers lining epithelial infoldings or crypts (Shatos et al., 2003). Decrease or loss of mucin/ glycocalix production (Paladino et al., 2004;Pflugfelder et al., 1997) generate squamous metaplasia, which may lead to dry eye and ocular surface diseases. In the conjunctiva, putative epithelial stem cells are concentrated mainly in the fornix region, but can be found also along the whole inner epithelium layer (Lavker et al., 1998). Conjunctival epithelial precursor cells are bipotent cells giving rise to either goblet and non-goblet keratinocytes and good candidate to be conjunctival stem/precursor cells (Wei et al., 1997;Pellegrini et al., 1999). Conjunctival epithelial stem/precursor cells can be maintained and expanded ex vivo, if cultivated upon appropriate matrix components, on which both goblet and Int. J. Dev. Biol. 51: 739-743 (2007) doi: 10.1387/ijdb.062198ar non-goblet keratinocytes could be expanded (Santos et al., 2005;Connon et al., 2006).Intact small fragments of conjunctiva were cultivated in an appropriate medium and analysed at different time intervals [prior to culture (T0), after 7 days (T1) and 14 days(T2)] for histology and for the expression of different cellular markers. 3D-organotypic cultures on spongesConjunctival fragments on gelatin-supports, revealed good maintenance of 3D-tissue-architecture and cellular heterogeneity throughout the whole period of cultures. At each time in culture, the stromal compartment appeared well maintained, with few capillaries that remained apparently intact and remarkably unAbbreviations used in this paper: 3D, three dimensional; RT-PCR, reverse transcriptase-polymerase chain reaction. 740 A. Rosellini et al.

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Section: Isolation and Expansion Of Epithelial Basal Stem/precursor Csupporting

confidence: 90%

Section: Isolation and Expansion Of Epithelial Basal Stem/precursor Cmentioning

confidence: 74%

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Human conjunctival epithelial precursor cells and their progeny in 3D organotypic culture

Rosellini¹,

Papini²,

Giannarini³

et al. 2007

Int. J. Dev. Biol.

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“…[6][7][8][9][10][11] Circumvention of these limitations is achieved by human keratinocyte immortalization, [12][13][14] and generation of stratified epithelia in interactive 3D cell cocultures as described for skin, 15 oral cavity, 16,17 and cornea. [18][19][20][21][22] Following immortalization, the resulting keratinocyte cell line should express most of the biomarkers, assigning to tissue homeostatic parameters such as differentiation, to reflect an authentic in vitro model system, suitable as an animal experiment substitute. Regarding this issue, we have recently shown that HPV 16 E6/E7-immortalized human corneal keratinocytes (IHCK) exposed to various biomechanical external cues are still capable to express the above-mentioned biomarkers, critical for corneal tissue homeostasis.…”

mentioning

confidence: 99%

“…This is important to obviate variations of cell/tissue-innate biomarker expression, hampering interpretation of results with respect to their transferability to the in vivo situation. In light of these critical issues, the aforementioned cornea models suffer from nonuniformity, since they comprise immortalized/primary cell combinations [19][20][21] or in the case of uniformity, 18 consist of only primary cells, which can hardly be used for reproducible in vitro testing, due to the previously described growth limitations.…”

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confidence: 99%

Natural Corneal Cell-Based Microenvironment as Prerequisite for Balanced 3D Corneal Epithelial Morphogenesis: A Promising Animal Experiment-Abandoning Tool in Ophthalmology

Schulz

Beck

Laird

et al. 2014

Tissue Engineering Part C: Methods

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To achieve durable recognition as a promising animal experiment-abandoning tool in ophthalmology, in vitro engineered tissue equivalents of the human cornea should exhibit proper morphogenesis. Regarding this issue, we were seeking for the natural cell microenvironment fulfilling the minimum requirements to allow human corneal keratinocytes to develop a balanced epithelial morphology with regular spatial appearance of tissue homeostatic biomarkers. Hence, we established cocultures of 3D cell-based collagen scaffolds comprising immortalized corneal keratinocytes combined with a gradual cornea-derived in vivo-like cell microenvironment, together with immortalized stromal fibroblasts alone (nonholistic) or fibroblasts and immortalized endothelial cells (holistic). With matched non-holistic microenvironments revealing mostly flattened cells and putative apical cell ablation foci at day 6, and 9 in HE stains, holistic counterparts yielded proper epithelial stratification with cell flattening restricted to apical layers. Concordantly, RT 2 -PCR showed a tremendous increase in gene expression for progressive and terminal biomarkers of corneal keratinocyte differentiation, cytokeratin (CK) 12, and filaggrin (FIL), in response to nonholistic environments, while involucrin (INV) was moderately but significantly upregulated. Although visible, this increase was moderate in corneal keratinocytes with a holistic environment. On the protein level, indirect immunofluorescence revealed that only epithelia of holistic environments showed diminishment in CK19, counteracted by CK12 rising over time. This time-dependent progression in differentiation coincided with declined proliferation and tissue-regular focus of differentiation biomarkers inv and fil to suprabasal and apical cell layers. Our novel findings suggest the interplay of native tissue forming cell entities, important for balanced corneal epithelial morphogenesis. In addition, they provide evidence for a holistic cell microenvironment as a prerequisite for development of an in vitro engineered corneal epithelial tissue equivalent, exhibiting a regular appearance of tissue homeostatic biomarkers. Such equivalents will be promising tools in ophthalmology, for example, for mechanistic studies in basic research and/or testing of generics or preclinical validation of innovative cornea-tailored biomaterials, desired for regenerative strategies.

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Ex vivo organ culture of adipose tissue for in situ mobilization of adipose‐derived stem cells and defining the stem cell niche

Yang

Kim

Choi

et al. 2010

Journal Cellular Physiology

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In spite of the advances in the knowledge of adipose-derived stem cells (ASCs), in situ location of ASCs and the niche component of adipose tissue (AT) remain controversial due to the lack of an appropriate culture system. Here we describe a fibrin matrix-supported three-dimensional (3D) organ culture system for AT which sustains the ASC niche and allows for in situ mobilization and expansion of ASCs in vitro. AT fragments were completely encapsulated within the fibrin matrix and cultured under dynamic condition. The use of organ culture of AT resulted in a robust outgrowth and proliferation in the fibrin matrix. The outgrown cells were successfully recovered from fibrin by urokinase treatment. These outgrown cells fulfilled the criteria of mesenchymal stem cells, adherence to plastic, multilineage differentiation, and cell surface molecule expression. In vitro label retaining assay revealed that newly divided cells during the culture resided in interstitium between adipocytes and capillary endothelial cells. These interstitial stromal cells proliferated and outgrew into the fibrin matrix. Both in situ mobilized and outgrown cells expressed CD146 and alpha-smooth muscle actin (SMA), but no endothelial cell markers (CD31 and CD34). The structural integrity and spatial approximation of CD31(-)/CD34(-)/CD146(+)/SMA(+) interstitial stromal cells, adipocytes, and capillary endothelial cells were well preserved during in vitro culture. Our results suggest that ASCs are natively associated with the capillary wall and more specifically, belong to a subset of pericytes. Furthermore, organ culture of AT within a fibrin matrix-supported 3D environment can recapitulate the ASC niche in vitro.

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Selective growth and expansion of human corneal epithelial basal stem cells in a three-dimensional-organ culture

Cited by 22 publications

References 22 publications

Human conjunctival epithelial precursor cells and their progeny in 3D organotypic culture

Human conjunctival epithelial precursor cells and their progeny in 3D organotypic culture

Natural Corneal Cell-Based Microenvironment as Prerequisite for Balanced 3D Corneal Epithelial Morphogenesis: A Promising Animal Experiment-Abandoning Tool in Ophthalmology

Ex vivo organ culture of adipose tissue for in situ mobilization of adipose‐derived stem cells and defining the stem cell niche

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