Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal Limbus
The fate decision of limbal epithelial progenitor cells (LEPC) at the human corneal limbus is determined by the surrounding microenvironment with limbal niche cells (LNC) as one of its essential components. Research on freshly isolated LNC which mainly include limbal mesenchymal stromal cells (LMSC)...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-03-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/23/5/2750 |
| _version_ | 1797474872370135040 |
|---|---|
| author | Naresh Polisetti Lyne Sharaf Ursula Schlötzer-Schrehardt Günther Schlunck Thomas Reinhard |
| author_facet | Naresh Polisetti Lyne Sharaf Ursula Schlötzer-Schrehardt Günther Schlunck Thomas Reinhard |
| author_sort | Naresh Polisetti |
| collection | DOAJ |
| description | The fate decision of limbal epithelial progenitor cells (LEPC) at the human corneal limbus is determined by the surrounding microenvironment with limbal niche cells (LNC) as one of its essential components. Research on freshly isolated LNC which mainly include limbal mesenchymal stromal cells (LMSC) and limbal melanocytes (LM) has been hampered by a lack of efficient protocols to isolate and purify these cells. We devised a protocol for rapid retrieval of pure LMSC, LM and LEPC populations by collagenase digestion of limbal tissue and subsequent fluorescence-activated cell sorting (FACS) using antibodies against CD90 and CD117. The sorted cells were characterized by immunophenotyping and functional assays. The effects of LMSC and LM on LEPC were studied in 3D co-cultures and LEPC differentiation status was assessed by immunohistochemistry. Enzymatic digestion and flow sorting yielded pure populations of LMSC (CD117<sup>−</sup>CD90<sup>+</sup>), LM (CD117<sup>+</sup>CD90<sup>−</sup>), and LEPC (CD117<sup>−</sup>CD90<sup>−</sup>). The LMSC exhibited self-renewal capacity (55.0 ± 4.6 population doublings), expressed mesenchymal stem cell markers (CD73, CD90, CD105, and CD44), and transdifferentiated to adipocytes, osteocytes, or chondrocytes. The LM exhibited self-renewal capacity and sustained melanin production. The sorted LEPC expressed epithelial progenitor markers (CK14, CK19, and CK15) and showed a colony-forming ability. Co-cultivation of LMSC and LM with LEPC resulted in a 4–5-layered stratified epithelium and supported the preservation of a LEPC phenotype, as reflected by increased p63<sup>+</sup> and Ki67<sup>+</sup> cells and decreased CK12<sup>+</sup> cells compared with LEPC monocultures. A highly efficient isolation of pure LM, LMSC, and LEPC populations from a single preparation may allow for direct transcriptomic and proteomic profiling as well as functional studies on native unpassaged LNC, which can be considered as proper equivalents of LNC in vivo. The developed biomimetic 3D co-culture method could provide an experimental model for investigating the functional role of LNC in the limbal stem cell niche. |
| first_indexed | 2024-03-09T20:37:13Z |
| format | Article |
| id | doaj.art-ae2865009d0e46a9a6c3d712be664bb8 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T20:37:13Z |
| publishDate | 2022-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-ae2865009d0e46a9a6c3d712be664bb82023-11-23T23:09:04ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-03-01235275010.3390/ijms23052750Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal LimbusNaresh Polisetti0Lyne Sharaf1Ursula Schlötzer-Schrehardt2Günther Schlunck3Thomas Reinhard4Eye Center, Medical Center-Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, GermanyEye Center, Medical Center-Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, GermanyDepartment of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, GermanyEye Center, Medical Center-Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, GermanyEye Center, Medical Center-Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, GermanyThe fate decision of limbal epithelial progenitor cells (LEPC) at the human corneal limbus is determined by the surrounding microenvironment with limbal niche cells (LNC) as one of its essential components. Research on freshly isolated LNC which mainly include limbal mesenchymal stromal cells (LMSC) and limbal melanocytes (LM) has been hampered by a lack of efficient protocols to isolate and purify these cells. We devised a protocol for rapid retrieval of pure LMSC, LM and LEPC populations by collagenase digestion of limbal tissue and subsequent fluorescence-activated cell sorting (FACS) using antibodies against CD90 and CD117. The sorted cells were characterized by immunophenotyping and functional assays. The effects of LMSC and LM on LEPC were studied in 3D co-cultures and LEPC differentiation status was assessed by immunohistochemistry. Enzymatic digestion and flow sorting yielded pure populations of LMSC (CD117<sup>−</sup>CD90<sup>+</sup>), LM (CD117<sup>+</sup>CD90<sup>−</sup>), and LEPC (CD117<sup>−</sup>CD90<sup>−</sup>). The LMSC exhibited self-renewal capacity (55.0 ± 4.6 population doublings), expressed mesenchymal stem cell markers (CD73, CD90, CD105, and CD44), and transdifferentiated to adipocytes, osteocytes, or chondrocytes. The LM exhibited self-renewal capacity and sustained melanin production. The sorted LEPC expressed epithelial progenitor markers (CK14, CK19, and CK15) and showed a colony-forming ability. Co-cultivation of LMSC and LM with LEPC resulted in a 4–5-layered stratified epithelium and supported the preservation of a LEPC phenotype, as reflected by increased p63<sup>+</sup> and Ki67<sup>+</sup> cells and decreased CK12<sup>+</sup> cells compared with LEPC monocultures. A highly efficient isolation of pure LM, LMSC, and LEPC populations from a single preparation may allow for direct transcriptomic and proteomic profiling as well as functional studies on native unpassaged LNC, which can be considered as proper equivalents of LNC in vivo. The developed biomimetic 3D co-culture method could provide an experimental model for investigating the functional role of LNC in the limbal stem cell niche.https://www.mdpi.com/1422-0067/23/5/2750limbal stem cellslimbal niche cellsmesenchymal stem cellsmelanocyteslimbal epithelial progenitor cellscorneal tissue engineering |
| spellingShingle | Naresh Polisetti Lyne Sharaf Ursula Schlötzer-Schrehardt Günther Schlunck Thomas Reinhard Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal Limbus International Journal of Molecular Sciences limbal stem cells limbal niche cells mesenchymal stem cells melanocytes limbal epithelial progenitor cells corneal tissue engineering |
| title | Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal Limbus |
| title_full | Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal Limbus |
| title_fullStr | Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal Limbus |
| title_full_unstemmed | Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal Limbus |
| title_short | Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal Limbus |
| title_sort | efficient isolation and functional characterization of niche cells from human corneal limbus |
| topic | limbal stem cells limbal niche cells mesenchymal stem cells melanocytes limbal epithelial progenitor cells corneal tissue engineering |
| url | https://www.mdpi.com/1422-0067/23/5/2750 |
| work_keys_str_mv | AT nareshpolisetti efficientisolationandfunctionalcharacterizationofnichecellsfromhumancorneallimbus AT lynesharaf efficientisolationandfunctionalcharacterizationofnichecellsfromhumancorneallimbus AT ursulaschlotzerschrehardt efficientisolationandfunctionalcharacterizationofnichecellsfromhumancorneallimbus AT guntherschlunck efficientisolationandfunctionalcharacterizationofnichecellsfromhumancorneallimbus AT thomasreinhard efficientisolationandfunctionalcharacterizationofnichecellsfromhumancorneallimbus |