Real tears
An organoid of the lacrimal gland grown in vitro successfully took root in a rat
Ismail Yunusov, N+1
Japanese biologists have grown and successfully transplanted a lacrimal gland organoid from multipotent epithelial cells of the eye to a rat. This organoid is similar to lacrimal glands in morphology, immune markers and expression of certain genes.
Article by Hayashi et al. Generation of 3D lacrimal gland organoids from human pluripotent stem cells is published in the journal Nature.
The lacrimal gland is the main exocrine gland of the eye. It secretes lacrimal fluid — the most important component of the tear film and various protective proteins, for example, lactoferrin, lysozyme, which are involved in antimicrobial protection.
The cells of the lacrimal gland of the fetus are capable of regeneration, but in adults this activity is minimal. It is difficult to study this gland because the organ is inaccessible for biopsy and cell isolation. Therefore, the gland is a target for drug development and research in the field of regenerative therapy.
Ryuhei Hayashi and Toru Okubo from the Osaka University Graduate School of Medicine in 2017, together with colleagues, talked about the organoid of the eye they created — SEAM (self-formed ectodermal autonomous multi-zone). SEAM partially repeated the mechanisms of development of the real eye and contained progenitor cells for the cell lines of the eye. Now they have published an article in which they talk about a lacrimal gland organoid grown from SEAM cells.
Development of glands on days 4, 15 and 39. A drawing from an article in Nature.
After that, the researchers decided to find out which cells from the third SEAM zone are the precursors of the lacrimal gland organoid. To do this, they used the method of flow cytometry with the addition of antibodies that interact with the stem epithelial cells of the eye (anti-ITGB4, anti-SSEA-4 and anti-CD200). The analysis showed that 90 percent of the cells are negative for CD200, while 60 percent are positive for ITGB4, and SSEA-4. Collectively, these characteristics relate to the stem epithelial cells of the eye. Therefore, the researchers concluded that the cells that formed the lacrimal gland organoid belong to the line of stem epithelial cells of the eye. After that, the authors analyzed the entire SEAM and obtained the following results: approximately 70 percent were positive for CD200, and four cell populations were isolated from the remaining CD200.
These populations were then transferred to the medium for further cultivation, and among all of them only one formed a multi-process form. Therefore, the researchers decided that this cell line is a precursor for lacrimal gland organoids.
After selecting the optimal environment for budding and branching of glands from this cell line, the researchers analyzed the single-stranded RNA of these cells. He showed that on the 20th day of development, the cells began to synthesize functional proteins of the lacrimal glands: lipocalin-2 and β-defensin. In addition, large clusters of cells, such as myoepithelial, acinar and ductal, have formed.
To test the hypothesis that the precursors of the lacrimal gland developed from the stem epithelial cells of the eye, biologists conducted frame-by-frame microscopy to observe the formation of colonies and identified two cell forms: flat and domed. By studying the genes associated with the development of glands, the researchers realized that they are more expressed in dome-shaped cells. Also, knockout of one of these genes showed a decrease in budding and branching in the glands. The reverse situation was observed in flat cells: markers of corneal progenitor cells were found there to a greater extent. These data indicate that the initial hypothesis about the origin of the precursors of the lacrimal gland was correct.
After the researchers grew the lacrimal gland organoid, they transplanted it into the connective tissue next to the rat's eye (its lacrimal gland was previously removed). Four weeks after the operation, the transplanted organoid was preserved and formed structures similar to ducts. Unlike organoids grown in vitro, transplanted ones showed clear formation of ducts in the second week, and in the fourth they became more pronounced.
Lactoferrin-producing cells that could not be seen in vitro were found in some parts of the transplanted organoid. Analysis of gene expression after transplantation showed that the expression of the LYZ and LTF genes (encode tear film proteins) increased 3000 times in transplanted compared to non-transplanted ones.
Based on the data obtained, biologists came to the conclusion that the expression level of certain genes and markers in the transplanted organoid and the native lacrimal gland is the same.
According to the authors of the article, the obtained data and successful transplantation open up prospects for research on the development of the lacrimal gland and the treatment of certain diseases, for example, Sjogren's syndrome. In addition, such grown organoids can be used in the development of drugs and the study of pathologies of the lacrimal glands.
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