Cysteine-Rich Intestinal Protein 1 is a Novel Surface Marker for Myometrial Stem/Progenitor Cells

Abstract

Uterine fibroids are benign tumors that develop in the myometria of most women with unknown etiology. Myometrial stem/progenitor cells (MyoSPCs) have been proposed as the cells of origin for fibroids because uterine fibroids are almost always clonal. We previously identified SUSD2 as a possible MyoSPC marker, but the relatively poor enrichment in stem cell characteristics of SUSD2+ over SUSD2- cells compelled us to find better discerning markers for more rigorous analysis. We combined bulk RNA-seq of SUSD2+/- cells with single cell RNA-seq to identify markers capable of further enriching for MyoSPCs. We observed seven distinct cell clusters within the myometrium, with the vascular myocyte cluster most highly enriched for MyoSPC characteristics and markers, including SUSD2. CRIP1 expression was found highly upregulated in both techniques and was used as a marker to sort CRIP1+/PECAM1- cells that were enriched for colony forming units and were able to differentiate into mesenchymal lineages, suggesting that CRIP1+/PECAM1- cells could be used to better understand the etiology of uterine fibroids.

A systems-based approach to uterine fibroids identifies differential splicing associated with abnormal uterine bleeding

“…Similarly, our integrated analysis (Supplementary Fig. 20) of single-cell myometrium datasets 54,55 showed elevated signalling pathways in UF-patient myometrium, including uniquely existing pathways such as TGF-b (right panel in Fig. 6c).…”

Unraveling the Molecular Landscape of Uterine Fibroids, Insights intoHMGA2and Stem Cell Involvement

“…Our initial objective was to examine whether HMGA2 overexpression enhances stem cell/progenitor-like properties in a differentiated myometrium cell line. Primary myometrial cells were immortalized (MyoN-TERT) and subsequently depleted for stem/progenitor cells via fluorescence-activated cell sorting (FACS) (Figure 1a and 1b), utilizing the cell surface marker CRIP1, a putative myometrium stem/progenitor cell marker recently identified in our laboratory (26). Among the MyoN-TERT cells, 73.7% of the MyoN-TERT were CRIP1 - (Figure 1b) and were sorted for downstream experiments.…”

“…Among these, 304 genes were downregulated, while 405, including HMGA2 (Log 2 FC = 2.2, FDR = 3.4 x 10 -55 ), were upregulated (Table S1). Additionally, CSPG4 , a gene associated with mesenchymal stem cells (26), was significantly upregulated (Log 2 FC = 0.4, FDR = 3.1 x 10 -2 ) in HMGA2hi cells (Figure 2c). However, other putative myometrial stem/progenitor-related genes such as PDGFRβ (32), SUSD2 (32), MCAM (32), CD44 (19), and CD34 (33) did not exhibit significantly (FDR > 0.05) higher expression in the HMGA2hi cells compared to control cells, except for ITGA6 (also known as CD49f ) (33), which was downregulated (Figure S1).…”

“…Transcriptomic analysis of HMGA2hi cells further revealed increased stemness, as evidenced by a significant increase in the expression of another mesenchymal marker, CSPG4 (26), along with enrichment for two stem-related pathways. Several studies have demonstrated the capability of myometrial and fibroid stem/progenitor cells to differentiate into various cell types (26,32,36).…”

“…HEK293T cells were transfected with hTERT plasmid, Addgene #85140 (24) and helper plasmids (25) to prepare hTERT lentivirus. Myometrial cells were dissociated and isolated from a fibroid-free myometrium patient (MyoN) as previously described (26) and incubated at 37°C overnight in growth medium (DMEM/F12, 10% FBS) prior to transduction with lentiviral preparations. The subsequent day, transduced MyoN cells were selected with 50 µg/mL of hygromycin for 1 week.…”