Endometrial regenerative cells: a novel stem cell population

Abstract

Angiogenesis is a critical component of the proliferative endometrial phase of the menstrual cycle. Thus, we hypothesized that a stem cell-like population exist and can be isolated from menstrual blood. Mononuclear cells collected from the menstrual blood contained a subpopulation of adherent cells which could be maintained in tissue culture for >68 doublings and retained expression of the markers CD9, CD29, CD41a, CD44, CD59, CD73, CD90 and CD105, without karyotypic abnormalities. Proliferative rate of the cells was significantly higher than control umbilical cord derived mesenchymal stem cells, with doubling occurring every 19.4 hours. These cells, which we termed "Endometrial Regenerative Cells" (ERC) were capable of differentiating into 9 lineages: cardiomyocytic, respiratory epithelial, neurocytic, myocytic, endothelial, pancreatic, hepatic, adipocytic, and osteogenic. Additionally, ERC produced MMP3, MMP10, GM-CSF, angiopoietin-2 and PDGF-BB at 10-100,000 fold higher levels than two control cord blood derived mesenchymal stem cell lines. Given the ease of extraction and pluripotency of this cell population, we propose ERC as a novel alternative to current stem cells sources.

Figure 1

Morphology of Cultured ERC. A) Morphology of freshly isolated menstrual blood mononuclear cells. B) Fibroblast-like morphology of menstrual blood mononuclear cells after 2-week cell culture. C) Clonal population of menstrual cells after plating in 96 well plate 1 week after cloning. D) The same population 2 weeks after cloning.

Figure 2

Pluripotent Differentiation. ERC were cultured under appropriate differentiation media as described in Materials and Methods and assessed for differentiation using the indicated staining methods. A) Adipocytic differentiation, yellow indicates lipid vacuoles stained by AdipoRed. B) Osteocytic differentiation, red indicates calcium stained by Alizarin Red. C) Myocytic differentiation, green indicates alpha actinin stain. D) Skeletal muscle differentiation, green indicates skeletal myosin stain. E) Endothelial differentiation, green indicates CD34 stain. F) Endothelial differentiation, green indicates CD62 stain. G) Hepatocytic differentiation, morphology resembles hepatic body. H) Hepatocytic differentiation, green indicates albumin stain. I) Pancreatic differentiation, green indicates insulin stain. J) Neuronal differentiation, green indicates Nestin stain. K) Neuronal differentiation, green indicates GFAP stain. L) Respiratory epithelial differentiation, green indicates Prosurfactant protein C stain. M) Cardiogenic differentiation, green indicates Troponin I stain.

Figure 3

Normality of Karyotype. ERC were cultured for the indicated cell passage numbers and assessed for lack of karyotypic abnormalities. A. ERC at Passage 10 (38 doublings). B. ERC at Passage 18 (68 doublings). Both detailed analyses shows normal karyotypes of 46, XX.