| Abstract |
Normal salivary processes are vital to the health of a patient. The
salivary fluid has important functions such as digestion, lubrification,
antibacterial protection and tooth remineralization. Salivary glands
dysfunction such as hyposalivation can be observed in patients undergoing
radiotherapy for head and neck cancer, and in patients suffering from
Sjogren's syndrome, an autoimmune disease. Symptoms of hyposalivation
include xerostomia (dry mouth), oral infections, tooth decay, pain,
dysgeusia (distortion of taste) and disrupted speech. Numerous drugs and
cell therapies have been proposed to reverse the impaired glandular
secretion. Among the tools used for such research is the HSG cell line,
commonly used in salivary gland research and mistakenly labeled as a
submandibular ductal cell cell line. ATCC has recently released a list of
cell lines cross-contaminated by HeLa, that included HSG. Despite the
notice, some salivary gland research labs still use HSG. In this work, we
describe the methodology for authenticating cell lines, assess the
validity of previous HSG findings and develop a new salivary gland cell
line. Objectives-We examine the authenticity and purity of two samples of
HSG obtained from two independent labs using STR analysis. A literature
review on HeLa cells is conducted to determine the validity of using HeLa-
derived cells, namely HSG, as a model for salivary gland cells to support
past salivary gland research findings that resulted from HSG. Lastly, a
new human submandibular salivary gland cell line will be established and
characterized. Results: STR analysis: HSG-Tran and HSG-Delporte have > 0.80
match with HeLa in DSMZ database. This confirms that both cell lines share
a common ancestry (host) with HeLa. Literature review-based on reports of
the karyotyping, cellular markers and membrane barrier function, HSG
(HeLa) is not an appropriate model of normal human salivary gland
acinar/ducal cells. Due to advanced mutations, it is even fair to say that
HeLa is no longer a valid representation of human biology. Cell line
establishment-The morphology of the SV40LT transfected salivary gland
cells (SMG-hu-1) is highly similar to the non-transfected cells, both with
an epithelial-like cobblestone morphology. SMG-hu-1 cells proliferate
steadily (passage 10+) whereas non-transfected cells enter senescence
after passage 1. Under immunofluorescence, SMG-hu-1 cells are positive for
epithelial markers E-cadherin and cytokeratin. In turn, they are negative
for vimentin (mesenchymal), CD31 (endothelial), alpha-smooth muscle actin
(myoepithelial). AQP5 (serous acinar) staining is inconclusive. RNA
expression reveals CK5, AQP5 and SV40. CFTR and CK19 expression are not
found.
Conclusions: STR analysis reveals that HSG is a HeLa derived cell line.
Furthermore, as genotyping of the original cell line was not performed
during its establishment, it will be impossible to authenticate a cell
line that is supposedly an uncontaminated sample of HSG. Similarly, based
on its characteristics and properties, HeLa derived cells, such as HSG,
are not an accurate model of salivary gland cells. The use of HSG in
salivary gland research should be discontinued and past findings should be
reassessed with an accurate model, such as our newly established human
submandibular salivary gland cell line (SMG-hu-1). SMG-hu-1 cells share
the epithelial morphology of its non-transfected counterpart, but with
significantly higher replicative capabilities. Immunofluorescence reveals
presence of E-cadherin and cytokeratin. PCR and RT-PCR reveals expression
of CK5, AQP5 and SV40. By utilizing this cell line, we look to further
investigate salivary processes in hope to develop a cure to hyposalivation.
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