Limits...
Mesenchymal cells from limbal stroma of human eye.

Polisetty N, Fatima A, Madhira SL, Sangwan VS, Vemuganti GK - Mol. Vis. (2008)

Bottom Line: The profile was further confirmed by RT-PCR.We 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.

View Article: PubMed Central - PubMed

Affiliation: Sudhakar Sreekanth Ravi Stem Cell Biology Laboratory, LV Prasad Eye Institute, Kallam Anji Reddy Campus, Hyderabad, India.

ABSTRACT

Purpose: Mesenchymal 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.

Methods: Fresh 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.

Results: Spindle 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-L(CD105, 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.

Conclusions: We 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.

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Related in: MedlinePlus

Colony Formation Unit (CFU) assay of MC-L and MSC-BM. The figure shows the crystal violet stained colonies of stromal cells – MC-L (A) and MSC-BM (B) in T75 flasks.
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f2: Colony Formation Unit (CFU) assay of MC-L and MSC-BM. The figure shows the crystal violet stained colonies of stromal cells – MC-L (A) and MSC-BM (B) in T75 flasks.

Mentions: When plated at 2 cells/cm2, MC-L in culture showed a colony forming efficiency between 30%–40% at passage 2 (Figure 2A), 10%–15% at passage 3 (P3), and 8% at P4 while MSC-BM showed a colony forming unit (CFU) of 20% at passage 2 (Figure 2B), 8%–12% at passage 3, and 2%–4% at passage 4. At P5, the cells showed no colony forming ability illustrating that the colony forming ability decreases with increasing passages.


Mesenchymal cells from limbal stroma of human eye.

Polisetty N, Fatima A, Madhira SL, Sangwan VS, Vemuganti GK - Mol. Vis. (2008)

Colony Formation Unit (CFU) assay of MC-L and MSC-BM. The figure shows the crystal violet stained colonies of stromal cells – MC-L (A) and MSC-BM (B) in T75 flasks.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268845&req=5

f2: Colony Formation Unit (CFU) assay of MC-L and MSC-BM. The figure shows the crystal violet stained colonies of stromal cells – MC-L (A) and MSC-BM (B) in T75 flasks.
Mentions: When plated at 2 cells/cm2, MC-L in culture showed a colony forming efficiency between 30%–40% at passage 2 (Figure 2A), 10%–15% at passage 3 (P3), and 8% at P4 while MSC-BM showed a colony forming unit (CFU) of 20% at passage 2 (Figure 2B), 8%–12% at passage 3, and 2%–4% at passage 4. At P5, the cells showed no colony forming ability illustrating that the colony forming ability decreases with increasing passages.

Bottom Line: The profile was further confirmed by RT-PCR.We 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.

View Article: PubMed Central - PubMed

Affiliation: Sudhakar Sreekanth Ravi Stem Cell Biology Laboratory, LV Prasad Eye Institute, Kallam Anji Reddy Campus, Hyderabad, India.

ABSTRACT

Purpose: Mesenchymal 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.

Methods: Fresh 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.

Results: Spindle 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-L(CD105, 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.

Conclusions: We 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.

Show MeSH
Related in: MedlinePlus