Regeneration and repair of corneal epithelium rely on a reservoir of unipotent progenitor cells, ... more Regeneration and repair of corneal epithelium rely on a reservoir of unipotent progenitor cells, which is situated within the basal epithelial layer at the corneoscleral limbus. If these cells are lost, corneal surface integrity is disturbed, which may lead to a painful loss of vision. Since the late 1990s cultivated grafts of limbal epithelium are being used therapeutically. Limbal epithelial cells are obtained from the fellow eye or from an allogeneic donor, propagated in culture on different types of carriers, and subsequently transplanted. This process entails removal of progenitor cells from their natural environment. However, surrounding cells and extracellular matrix are widely believed to provide important stimuli for stem cell maintenance and for correct differentiation. Therefore, new approaches aim at providing this so-called stem cell niche ex vivo and following transplantation. Niche factors can also drive transdifferentiation of alternative progenitor cell types towards a corneal phenotype. This permits the use of autologous cells in cases of bilateral limbal stem cell insufficiency. Several biosynthetic substrates have been devised for culture, transdifferentiation and transplantation of donor cells. This work intends to provide an overview of constructs that are currently available and to some extent clinically employed. In addition, a summary is given of novel concepts which aim at integrating putative niche factors into the stem cell carriers to replicate the stem cell niche
A range of alternatives to human donor tissue for corneal transplantation are being developed to ... more A range of alternatives to human donor tissue for corneal transplantation are being developed to address the shortfall of good quality tissues as well as the clinical conditions for which allografting is contraindicated. Classical keratoprostheses, commonly referred to as artificial corneas, are being used clinically to replace minimal corneal function. However, they are used only as last resorts, as they are associated with significant complications, such as extrusion/rejection, glaucoma, and retinal detachment. The past few years have seen significant developments in technologies designed to replace part or the full thickness of damaged or diseased corneas with materials that encourage regeneration to different extents. This review describes selected examples of these corneal substitutes, which range from cell-based regenerative strategies to keratoprostheses with regenerative capabilities via tissue-engineered scaffolds pre-seeded with stem cells. It is unlikely that one corneal substitute will be best for all indications, but taken together, the various approaches may soon be able to supplement the supply of human donor corneas for transplantation or allow restoration of diseased or damaged corneas that cannot be treated by currently available techniques.
PurposeMesenchymal stem cells (MSC) are self-renewing, multipotent cells that are present in many... more PurposeMesenchymal stem cells (MSC) are self-renewing, multipotent cells that are present in many adult tissues, including bone marrow, trabecular bone, adipose, and muscle. The presence of such cells of mesenchymal origin and their role during the wound healing of ocular injuries are currently being explored by many studies worldwide. In this study, we aimed to report the presence of mesenchymal cells resembling bone marrow-derived cells (MSC-BM) in the limbus of the human eye.MethodsFresh limbal tissues obtained from human subjects undergoing limbal biopsy for ocular surface reconstruction were used to establish limbal mesenchymal cell (MC-L) cultures. The spindle cell outgrowths observed in extended limbal epithelial cultures (LECs) and from deepithelialized limbal tissues were serially passaged using a human corneal epithelial (HCE) medium, which contained epidermal growth factor (EGF) and insulin, and supplemented with fetal bovine serum (FBS). MSC cultures were established from human bone marrow samples using Dulbecco’s Modified Eagles Medium (DMEM) supplemented with FBS. The mesenchymal cells from both extended limbal cultures (MC-L) and bone marrow (MSC-BM) were characterized by morphology and immunophenotyping using epithelial, mesenchymal, hematopoietic, and endothelial markers using fluorescent-activated cell sorting (FACS). Selective markers were further confirmed by immunostaining and reverse transcription polymerase chain reaction (RT–PCR). Stromal cells of both origins (limbal and bone marrow-derived) were also evaluated for colony forming ability and population doubling. Attempts were made to differentiate these into adipocytes and osteocytes using conditioned medium.ResultsSpindle cells from extended limbal epithelial cultures as well as de-epithelialized human limbal tissues appeared elongated and fibroblast-like with oval vesicular nuclei. Both MC-L and MSC-BM showed colony forming ability in 14 days of plating. MC-L showed a population doubling of 22.95 while in MSC-BM, it was 30.98. Immunophenotyping of these cells by FACS and immunocytochemistry showed that the MC-L were positive for mesenchymal markers and negative for epithelial and hematopoietic markers similar to MSC-BM. The MC-L phenotype has thus been defined as MC-LCD105, CD106, CD54, CD166, CD90, CD29, CD71, pax −6 +/ p75, SSEA1, Tra-1–61, Tra-1–81, CD31, CD34, CD45, CD11a, CD11c, CD14, CD138, Flk1, Flt1, VE-Cadherin -. The profile was further confirmed by RT–PCR. These cells also showed differentiation into adipocytes and osteocytes.ConclusionsWe demonstrated the presence of mesenchymal cells in the human limbus, similar to the bone marrow-derived MSC-BM. This presence suggests that these cells are unique to the adult stem cell niche.
PurposeLimbal stem cell deficiency is a challenging clinical problem and the current treatment in... more PurposeLimbal stem cell deficiency is a challenging clinical problem and the current treatment involves replenishing the depleted limbal stem cell (LSC) pool by either limbal tissue transplantation or use of cultivated limbal epithelial cells (LEC). Our experience of cultivating the LEC on denuded human amniotic membrane using a feeder cell free method, led to identification of mesenchymal cells of limbus (MC-L), which showed phenotypic resemblance to bone marrow derived mesenchymal stem cells (MSC-BM). To understand the transcriptional profile of these cells, microarray experiments were carried out.MethodsRNA was isolated from cultured LEC, MC-L and MSC-BM and microarray experiments were carried out by using Agilent chip (4×44 k). The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction.ResultsThe microarray analysis revealed specific gene signature of LEC and MC-L, and also their complementary role related to cytokine and growth factor profile, thus supporting the nurturing roles of the MC-L. We have also observed similar and differential gene expression between MC-L and MSC-BM.ConclusionsThis study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells.
Regeneration and repair of corneal epithelium rely on a reservoir of unipotent progenitor cells, ... more Regeneration and repair of corneal epithelium rely on a reservoir of unipotent progenitor cells, which is situated within the basal epithelial layer at the corneoscleral limbus. If these cells are lost, corneal surface integrity is disturbed, which may lead to a painful loss of vision. Since the late 1990s cultivated grafts of limbal epithelium are being used therapeutically. Limbal epithelial cells are obtained from the fellow eye or from an allogeneic donor, propagated in culture on different types of carriers, and subsequently transplanted. This process entails removal of progenitor cells from their natural environment. However, surrounding cells and extracellular matrix are widely believed to provide important stimuli for stem cell maintenance and for correct differentiation. Therefore, new approaches aim at providing this so-called stem cell niche ex vivo and following transplantation. Niche factors can also drive transdifferentiation of alternative progenitor cell types towards a corneal phenotype. This permits the use of autologous cells in cases of bilateral limbal stem cell insufficiency. Several biosynthetic substrates have been devised for culture, transdifferentiation and transplantation of donor cells. This work intends to provide an overview of constructs that are currently available and to some extent clinically employed. In addition, a summary is given of novel concepts which aim at integrating putative niche factors into the stem cell carriers to replicate the stem cell niche
A range of alternatives to human donor tissue for corneal transplantation are being developed to ... more A range of alternatives to human donor tissue for corneal transplantation are being developed to address the shortfall of good quality tissues as well as the clinical conditions for which allografting is contraindicated. Classical keratoprostheses, commonly referred to as artificial corneas, are being used clinically to replace minimal corneal function. However, they are used only as last resorts, as they are associated with significant complications, such as extrusion/rejection, glaucoma, and retinal detachment. The past few years have seen significant developments in technologies designed to replace part or the full thickness of damaged or diseased corneas with materials that encourage regeneration to different extents. This review describes selected examples of these corneal substitutes, which range from cell-based regenerative strategies to keratoprostheses with regenerative capabilities via tissue-engineered scaffolds pre-seeded with stem cells. It is unlikely that one corneal substitute will be best for all indications, but taken together, the various approaches may soon be able to supplement the supply of human donor corneas for transplantation or allow restoration of diseased or damaged corneas that cannot be treated by currently available techniques.
PurposeMesenchymal stem cells (MSC) are self-renewing, multipotent cells that are present in many... more PurposeMesenchymal stem cells (MSC) are self-renewing, multipotent cells that are present in many adult tissues, including bone marrow, trabecular bone, adipose, and muscle. The presence of such cells of mesenchymal origin and their role during the wound healing of ocular injuries are currently being explored by many studies worldwide. In this study, we aimed to report the presence of mesenchymal cells resembling bone marrow-derived cells (MSC-BM) in the limbus of the human eye.MethodsFresh limbal tissues obtained from human subjects undergoing limbal biopsy for ocular surface reconstruction were used to establish limbal mesenchymal cell (MC-L) cultures. The spindle cell outgrowths observed in extended limbal epithelial cultures (LECs) and from deepithelialized limbal tissues were serially passaged using a human corneal epithelial (HCE) medium, which contained epidermal growth factor (EGF) and insulin, and supplemented with fetal bovine serum (FBS). MSC cultures were established from human bone marrow samples using Dulbecco’s Modified Eagles Medium (DMEM) supplemented with FBS. The mesenchymal cells from both extended limbal cultures (MC-L) and bone marrow (MSC-BM) were characterized by morphology and immunophenotyping using epithelial, mesenchymal, hematopoietic, and endothelial markers using fluorescent-activated cell sorting (FACS). Selective markers were further confirmed by immunostaining and reverse transcription polymerase chain reaction (RT–PCR). Stromal cells of both origins (limbal and bone marrow-derived) were also evaluated for colony forming ability and population doubling. Attempts were made to differentiate these into adipocytes and osteocytes using conditioned medium.ResultsSpindle cells from extended limbal epithelial cultures as well as de-epithelialized human limbal tissues appeared elongated and fibroblast-like with oval vesicular nuclei. Both MC-L and MSC-BM showed colony forming ability in 14 days of plating. MC-L showed a population doubling of 22.95 while in MSC-BM, it was 30.98. Immunophenotyping of these cells by FACS and immunocytochemistry showed that the MC-L were positive for mesenchymal markers and negative for epithelial and hematopoietic markers similar to MSC-BM. The MC-L phenotype has thus been defined as MC-LCD105, CD106, CD54, CD166, CD90, CD29, CD71, pax −6 +/ p75, SSEA1, Tra-1–61, Tra-1–81, CD31, CD34, CD45, CD11a, CD11c, CD14, CD138, Flk1, Flt1, VE-Cadherin -. The profile was further confirmed by RT–PCR. These cells also showed differentiation into adipocytes and osteocytes.ConclusionsWe demonstrated the presence of mesenchymal cells in the human limbus, similar to the bone marrow-derived MSC-BM. This presence suggests that these cells are unique to the adult stem cell niche.
PurposeLimbal stem cell deficiency is a challenging clinical problem and the current treatment in... more PurposeLimbal stem cell deficiency is a challenging clinical problem and the current treatment involves replenishing the depleted limbal stem cell (LSC) pool by either limbal tissue transplantation or use of cultivated limbal epithelial cells (LEC). Our experience of cultivating the LEC on denuded human amniotic membrane using a feeder cell free method, led to identification of mesenchymal cells of limbus (MC-L), which showed phenotypic resemblance to bone marrow derived mesenchymal stem cells (MSC-BM). To understand the transcriptional profile of these cells, microarray experiments were carried out.MethodsRNA was isolated from cultured LEC, MC-L and MSC-BM and microarray experiments were carried out by using Agilent chip (4×44 k). The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction.ResultsThe microarray analysis revealed specific gene signature of LEC and MC-L, and also their complementary role related to cytokine and growth factor profile, thus supporting the nurturing roles of the MC-L. We have also observed similar and differential gene expression between MC-L and MSC-BM.ConclusionsThis study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells.
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Papers by Naresh Polisetti