Page 37 - Annual Report 2020

Cells covering surfaces such as the epidermis or lining spaces like the gastrointestinal and respi- ratory tracts are called epithelial cells, and can be identified due to their genetic expression of certain cytoplasmic filaments called keratins or to a cell surface marker such as epithelial cell adhesion molecule (EpCAM). Additionally, these cells can
be tracked as they move around the body since a green fluorescent protein (GFP) under the control of cytokeratin promoters is available. This enables functional studies because we can use the GFP to visualize, isolate, and culture these cells to investi- gate their stem cell epithelial origin.
Cytokeratin positive cells are frequently found in the blood and bone marrow of patients with epithelial cancers and are attributed to metastasis. To deter- mine the presence of epithelial cells in normal blood and bone marrow, we used immunofluorescence microscopy, genetic labeling, quantitative real time PCR, and fluorescence activated cell sorting (FACS). We have made several interesting findings. First,
we discovered rare but reproducible cytokeratin immunoreactive cells the size of small lymphocytes in blood and bone marrow of untreated adult in vivo. We found that Epithelial Cell Adhesion Molecule (EpCAM) positive cells in whole human blood and
bone marrow constituted 0.083% and 1.94% respec- tively (average of 2 biological replicates) regardless of the number of cells counted. Virtually 100% of the EpCAM positive cells were immunoreactive to pan-cytokeratin as determined by immunofluores- cence microscopy. Second, we found low (8.6 native GFP positive cells per 10^6 cells analyzed), but sig- nificant numbers (p<0.0005) of GFP positive cells
in bone marrow compared with negative controls. Third, quantitative real time PCR demonstrated very low but reproducibly detectable expression of Krt1- 14 mRNA in blood and bone marrow, that when compared with purified epidermal keratinocytes, was 1000x and 100,000x less, respectively. More- over, analysis of fresh bone marrow by flow cyto- metric techniques disclosed multiple subpopula- tions of EpCAM positive cells when compared with hematopoietic and mesenchymal lineage markers. We conclude from these observations that cytoker- atin proteins and mRNA are expressed at low, but reproducibly detectable levels in blood and bone marrow cells thus setting the stage for determining the functions of these novel cells.
This research is significant because its conse- quences for fields of hematology and epithelial biology. We documented and quantified bone
marrow derived epithelial cells in healthy subjects to determine the phenotypes. We will determine whether the bone marrow derived epithelial cells have functional characteristics
of epithelial stem/progenitors in culture and
in vivo models. The experiments outlined here have the potential to open up a new field of research in epithelial biology and hematology and should provide motivation for development of new disease models. The implications of our findings toward understanding the etiology of epithelial cancers in general as well as their diag- nosis and treatment have not escaped our notice.
Recent publications:
Park H., Lad, S., Boland, K, Johnson, K., Readio, N., Jin, Guangchun , Asfaha, S., Patterson, K.S., Singh, A., Yang, X., Londono, D., Singh, A., Trempus, C., Gordon, D., Wang, T.C., and Morris, R.J. Bone marrow-derived epithelial cells and hair follicle stem cells contribute to development of chronic cutaneous neoplasms. Nature Communications, 2018, 10.1038/s41467-018-07688-8.
Morris, R.J., Readio, N., Boland, K., Johnson, K., Lad, S., Singh, A., Singh, A., Holtorf, S., and Skaar, S. Isolation of in vivo epidermal keratinocytes
and their in vitro clonogenic culture. JoVE, 2019, 150, 10.3791/58701.
THE HORMEL INSTITUTE // UNIVERSITY OF MINNESOTA PG 37