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. 2015 Apr;100(4):E601-6.
doi: 10.1210/jc.2014-2134. Epub 2015 Feb 6.

Human uterine leiomyoma stem/progenitor cells expressing CD34 and CD49b initiate tumors in vivo

Ping Yin  1 , Masanori Ono, Molly B Moravek, John S Coon 5th, Antonia Navarro, Diana Monsivais, Matthew T Dyson, Stacy A Druschitz, Saurabh S Malpani, Vanida A Serna, Wenan Qiang, Debabrata Chakravarti, J Julie Kim, Serdar E Bulun
Affiliations

Human uterine leiomyoma stem/progenitor cells expressing CD34 and CD49b initiate tumors in vivo

Ping Yin et al. J Clin Endocrinol Metab. 2015 Apr.

Abstract

Context: Uterine leiomyoma is the most common benign tumor in reproductive-age women. Using a dye-exclusion technique, we previously identified a side population of leiomyoma cells exhibiting stem cell characteristics. However, unless mixed with mature myometrial cells, these leiomyoma side population cells did not survive or grow well in vitro or in vivo.

Objective: The objective of this study was to identify cell surface markers to isolate leiomyoma stem/progenitor cells.

Design: Real-time PCR screening was used to identify cell surface markers preferentially expressed in leiomyoma side population cells. In vitro colony-formation assay and in vivo tumor-regeneration assay were used to demonstrate functions of leiomyoma stem/progenitor cells.

Results: We found significantly elevated CD49b and CD34 gene expression in side population cells compared with main population cells. Leiomyoma cells were sorted into three populations based on the expression of CD34 and CD49b: CD34(+)/CD49b(+), CD34(+)/CD49b(-), and CD34(-)/CD49b(-) cells, with the majority of the side population cells residing in the CD34(+)/CD49b(+) fraction. Of these populations, CD34(+)/CD49b(+) cells expressed the lowest levels of estrogen receptor-α, progesterone receptor, and α-smooth muscle actin, but the highest levels of KLF4, NANOG, SOX2, and OCT4, confirming their more undifferentiated status. The stemness of CD34(+)/CD49b(+) cells was also demonstrated by their strongest in vitro colony-formation capacity and in vivo tumor-regeneration ability.

Conclusions: CD34 and CD49b are cell surface markers that can be used to enrich a subpopulation of leiomyoma cells possessing stem/progenitor cell properties; this technique will accelerate efforts to develop new therapies for uterine leiomyoma.

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Figures

Figure 1.
Figure 1.
Identification and characterization of three populations of leiomyoma cells based on the expression of cell surface markers CD34 and CD49b. (A) Differential expression of CD34 and CD49b fractionates the total leiomyoma cells into three subpopulations: CD34−/CD49b−, CD34+/CD49b−, and CD34+/CD49b+ cells. Contour plot is a representative of experiments run with cells from 8 independent patient samples. Values are expressed as mean ± SEM. (B) CD34+/CD49b+ cells were selectively enriched for LMSP cells (left). LMSP cells were rarely detected in the CD34+/CD49b− (middle) or CD34−/CD49b− (right) subpopulations. Dot plot shows one representative experiment repeated with cells from three independent subjects. (C) Percentage of LMSP cells detected by the Hoechst dye exclusion method in total leiomyoma cells and in the CD34+/CD49b+ cell population. Values are expressed as mean ± SEM for three independent subjects. (D) Quantitative real-time RT-PCR was performed to determine the mRNA levels of cell surface markers (CD34 and CD49b), stem cell factors (KLF4, NANOG, SOX2, and OCT4), steroid receptors (ERα and PR), and smooth muscle cell marker α-SMA in the three FACS-sorted subpopulations of leiomyoma cells. Graphs are means ± SEM of representative triplicate experiments, repeated using cells from 3 to 5 independent patient samples. (E) Representative western blots show differential protein expression of CD34 and CD49b between three leiomyoma cell populations. Experiments were repeated with cells from three patient samples.
Figure 2.
Figure 2.
Regeneration of human leiomyoma-like tumors by CD34+/CD49b+ cells. (A) Macroscopic visualization of the regenerated tumor (blue arrows) 8 weeks after engrafting. (B) Quantification of tumor volume. Data shown are mean ± SEM of representative triplicate experiments, repeated using cells from three independent patient samples. (C) Quantification of the percentage of Ki67 positive cells (Ki67 labeling index) in tumors regenerated from CD34+/CD49b+ and CD34−/CD49b− cells. Values are expressed as means ± SEM of representative triplicate experiments, repeated using cells from three patient samples. (D) and (E) Representative images of immunostaining of CD34+/CD49b+ or CD34−/CD49b− cell-reconstituted leiomyoma tissues with antibodies against proliferation marker Ki67 (D) and α-SMA (E). Tissues were counterstained with hematoxylin. Red arrowheads indicate Ki67-positive cells. (F) Immunofluorescent staining of CD34+/CD49b+ or CD34−/CD49b− cell-reconstituted leiomyoma tissues with antibodies against PR (green) and α-SMA (red). Nuclei were stained with Hoechst dye (blue) and white arrowheads indicate PR-positive cells. (G) Trichrome staining to characterize expression of typical extracellular matrix proteins in regenerated tissues.
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