Mesenchymal cells from limbal stroma of human eye

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.

Figure 1

Morphology of cultured mesenchymal cells from limbus (MC-L) and mesenchymal stem or stromal cells from bone marrow (MSC-BM). The phase contrast microscopic picture of MC-L shows the spindle morphology (magnification: 200X) (A); Cell sphere formation in the MC-L cultures gives the impression of embryoid body formation (magnification: 200X) (B); spindle shaped morphology of MC-L as confirmed by Giemsa stain (Light microscope, magnification: 200X) (C); Culture of MSC-BM (magnification: 200X) (D) showing spindle cell morphology similar to that of MC-L.

Figure 2

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.

Figure 3

Surface marker profile of MC-L and MSC-BM by FACS analysis. FACS analysis of MC-L cells and MSC-BM for a battery of markers shows a close resemblance of MC-L toward mesenchymal phenotype as that of MSC-BM (A) (The purple line in the histograms represents the isotype control). FACS analysis of MC-L for expression of both embryonic stem cell markers (B) and endothelial markers (C) is also shown to be negative.

Figure 4

Mesenchymal phenotype of MC-L by Laser Scanning Confocal Microscopy. LSCM pictures of MC-L show positivity (green fluorescence) for vimentin (20X), CD90 (20X), and CD29 (20X) and negativity for CD11c (40X), CD11a (40X), CD45 (40X), CD34 (20X), and CD25 (20X). The nuclei are counterstained with propidium iodide (red).

Figure 5

Mesenchymal phenotype of MSC-BM by Laser Scanning Confocal Microscopy. LSCM pictures of MSC-BM show positivity for vimentin (20X), CD90 (20X), CD29 (20X) and negativity for CD11c (20X), CD11a (20X), CD45 (20X), CD34 (20X), and CD25 (20X). The nuclei are counterstained with propidium iodide (red).

Figure 6

Epithelial phenotype of LEC and MC-L by Laser Scanning Confocal Microscopy and FACS analysis. LSCM pictures of limbal epithelial cells (LECs) show positivity for K3 (20X) and K14 (20X; A,B), and LSCM pictures of MC-L show negativity for K3 (40X) and K14 (20X; C,D). The nuclei are counterstained with propidium iodide (blue, red). FACS histograms of MC-L confirm the absence of K3 (E) and K14 (F) expression. The blue line represents the test sample and the purple line represents the isotype control.

Figure 7

Reverse transcription polymerase chain reaction analysis of limbal mesenchymal cells and MSC-BM. The figure shows the expression profiles of selected markers in MSC-BM (M), MC-L (L), and LECs (E) as well as the negative control (-ve). MSC-BM and MC-L show negative expression profiles of p63 (1387 bp), corneal cytokeratins, K3 (150 bp) and K12 (193 bp), integrin α9 (123 bp), EGFR (484 bp), and PAX−6 (208 bp) in comparison to LECs. The figure also shows a positive expression of connexin 43 (150 bp) and α-enolase (619 bp) by stromal cells of both origin similar to LECs and an expression of p75 by MSC-BM and LECs (230 bp). The above expression studies have been normalized using GAPDH (498 bp) as the internal control.

Figure 8

Differentiation potential of MC-L and MSC-BM into adipocytes and osteocytes. Adipocyte differentiation of stromal cells shows the presence of oil-red positive lipid laden cells in MSC-BM (A) and MC-L (B). Osteogenic differentiation of these cells shows the presence of alizarin stained calcium deposits in MSC-BM (C) and MC-L (D) indicating calcification of cells. All these images are at a magnification of 20X.