Stem Cell Growth and Differentiation in Organ Culture: New Insights for Uterine Fibroid Treatment

Round 1

Reviewer 1 Report

In this manuscript, the authors investigated the mechanisms behind normal myometrium homeostasis and the growth of fibroids in tissue slices. More in detail, the authors proposed two different mechanisms. In normal myometrium, a CD49b stem cell asymmetrically divides into two daughter cells. In leiomyomas, MED12  mutated CD49b stem cells symmetrically divide leading to stem cell expansion. Overall, the paper is well written and the data support the authors' conclusions. The figures are clear and self-explanatory. I have no specific comments. For this reason, I suggest accepting the manuscript.

Author Response

We sincerely thank the reviewer for recommending the manuscript to publish in Biomedicines in its present form.

Reviewer 2 Report

In this manuscript, the authors describe new findings obtained from study of tissue culture of uterine leiomyoma. The manuscript has high relevance both for basic and clinical esearchers from the gynecology research area. However, several major concerns can be raised regarding the study presented:

MAJOR

1.       In the tissue culture system, there is not enough cell characterization - what is the proportion of fibroblasts, myofibroblasts, smooth muscle cells (SMC) in the studied slices on different time of incubation?

2.       The description to Fig. S2 contains the following information about MED12 mutations: «Mutant alleles were preferentially expressed at baseline (T0) and after 25 days of culture (T25)». Does it mean that the aberrations remain during all culturing period? Is this observation suitable for all four LM?

3.       How do the findings of MED12 mutations compare with other aberrations including HMGA2 and PLAG1?

4.       How relevant are the data of CD34 marker in the context of the LM development? Figure 2 shows a weak single signal collocolized with CD24 marker which is involved in immune response processes and highly introduced on lymphocytes. Is it possible that a fragment of the endothelium was introduced?

5.       In Fig. 1A demonstrating the expression of ACTA2 there are some weak signals on both sides from arrowhead, what does it mean?

6.       Do all four tumors demonstrate similar pattern of markers? Have you evaluated whether there are the differences in markers (or stages they occur) with driver mutations you have found?

7.       The authors claim that CD73 and CD24 expression can be a new marker for uterine leiomyoma. How can it be compared with previous findings on CD51/CD61 overexpression in uterine leiomyoma?

MINOR

1.       The description to Fig. 1B contains the following: «..cells with strong  (white arrowheads) and weak (arrowheads) signals». I think it should be clarified as both the arrowheads seem to be white.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript by Salas and co-authors explores the mechanisms behind normal myometrium homeostasis and the growth of fibroids.  They suggested two different mechanisms by which stem cells repopulated tissue slices behind normal myometrium homeostasis and the growth of fibroids. 

The study is interesting and well organized. The molecular analysis was well conducted, including those evaluating the stem cell division.

The authors suggest a specific  stem cell division switch, an asymmetric stem cell division process in normal myometrium, ( CD49b stem cells) versus a symmetrical stem cell division in leiomyomas (MED12 mutated CD49b stem cells). However, the description of these results must be improved in order to facilitate their correlation with the model presented in Figure 4. 

Author Response

As suggested by the reviewer, we have now included a more detailed description of the CD49b immunodetection in normal and fibroid slices. A new paragraph is included in pg 6, lines 219-230, and states: “The CD49b⁺ stem cells appeared scattered throughout the myometrium slices at any time point analyzed (T0 and long-term culture), similar to LM at T0 (Figure 2). In these slices, CD49b⁺ cells seemed to be isolated or forming tiny clumps of cells, which were not superior to 3-4 cells, and were always surrounded by unstained cells. On the contrary, in two LM slices at T20, we found clusters formed by hundreds of cells with solid red immunostaining associated with weaker stained cells but still expressing the CD49b stem cell marker (Figure 2B). We did not find CD49b cells in these two tumors at longer incubation times (23, 26, and 29 days of culture). Similarly, we did not find CD49b immunostained cells in the remaining two tumors after long-term culture. This data suggests different mechanisms of stem cell activation in tumor and normal cells. In addition, earlier activation of tumor stem cells may explain their absence in two tumors and longer than 20 days of incubation.”

We also slightly modified a sentence of the discussion section to facilitate correlation with the results (pg.10 lines 343-346):

In LM, MED12 mutant CD49b⁺ stem cells may symmetrically divide, which induces expansion of the stem cell pool resulting in the abundant CD49b⁺ cell clusters observed in tumor slices.

We thank the reviewer for his/her wise comment that helped us better explain the different mechanisms of normal versus tumor cells proliferation and improve the manuscript.

Round 2

Reviewer 2 Report

Thank you for the answers. Now the manuscript is improved.

Reviewer 3 Report

no comments