title: Establishing human lacrimal gland cultures with secretory function.
abstract: OBJECTIVE
  Dry eye syndrome is a multifactorial chronic disabling disease mainly caused by the functional disruptions in the lacrimal gland. The treatment involves palliation like ocular surface lubrication and rehydration. Cell therapy involving replacement of the gland is a promising alternative for providing long-term relief to patients. This study aimed to establish functionally competent lacrimal gland cultures in-vitro and explore the presence of stem cells in the native gland and the established in-vitro cultures.
  
  
  METHODS
  Fresh human lacrimal gland from patients undergoing exenteration was harvested for cultures after IRB approval. The freshly isolated cells were evaluated by flow cytometry for expression of stem cell markers ABCG2, high ALDH1 levels and c-kit. Cultures were established on Matrigel, collagen and HAM and the cultured cells evaluated for the presence of stem cell markers and differentiating markers of epithelial (E-cadherin, EpCAM), mesenchymal (Vimentin, CD90) and myofibroblastic (α-SMA, S-100) origin by flow cytometry and immunocytochemistry. The conditioned media was tested for secretory proteins (scIgA, lactoferrin, lysozyme) post carbachol (100 µM) stimulation by ELISA.
  
  
  RESULTS
  Native human lacrimal gland expressed ABCG2 (mean±SEM: 3.1±0.61%), high ALDH1 (3.8±1.26%) and c-kit (6.7±2.0%). Lacrimal gland cultures formed a monolayer, in order of preference on Matrigel, collagen and HAM within 15-20 days, containing a heterogeneous population of stem-like and differentiated cells. The epithelial cells formed 'spherules' with duct like connections, suggestive of ductal origin. The levels of scIgA (47.43 to 61.56 ng/ml), lysozyme (24.36 to 144.74 ng/ml) and lactoferrin (32.45 to 40.31 ng/ml) in the conditioned media were significantly higher than the negative controls (p<0.05 for all comparisons).
  
  
  CONCLUSIONS
  The study reports the novel finding of establishing functionally competent human lacrimal gland cultures in-vitro. It also provides preliminary data on the presence of stem cells and duct-like cells in the fresh and in-vitro cultured human lacrimal gland. These significant findings could pave way for cell therapy in future.
authors.affiliation: Sudhakar and Sreekant Ravi Stem Cell Biology Laboratory, Professor Brien Holden Eye Research Centre, L. V. Prasad Eye Institute, Hyderabad, India.
authors.forename: Shubha
authors.initials: S
authors.lastname: Tiwari
authors.forename: Mohammad Javed
authors.initials: MJ
authors.lastname: Ali
authors.forename: Murali M S
authors.initials: MM
authors.lastname: Balla
authors.forename: Milind N
authors.initials: MN
authors.lastname: Naik
authors.forename: Santosh G
authors.initials: SG
authors.lastname: Honavar
authors.forename: Vijay Anand P
authors.initials: VA
authors.lastname: Reddy
authors.forename: Geeta K
authors.initials: GK
authors.lastname: Vemuganti
mesh_terms: D000080924:Aldehyde Dehydrogenase 1 Family; D002217:Carbachol / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007070:Immunoglobulin A / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D016130:Immunophenotyping; D007527:Isoenzymes / Q000378:metabolism; D007765:Lacrimal Apparatus / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D007781:Lactoferrin / Q000235:genetics / Q000378:metabolism; D009113:Muramidase / Q000235:genetics / Q000378:metabolism; D011247:Pregnancy; D050697:Retinal Dehydrogenase / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D007070:Immunoglobulin A; D007527:Isoenzymes; D002217:Carbachol; D000080924:Aldehyde Dehydrogenase 1 Family; C510223:ALDH1A1 protein, human; D050697:Retinal Dehydrogenase; D009113:Muramidase; D007781:Lactoferrin