Human Endometrial Stromal Stem Cells Differentiate into Megakaryocytes with the Ability to Produce Functional Platelets

Abstract

Human endometrium is a high dynamic tissue that contains endometrial stromal stem cells (hESSCs). The hESSCs have been differentiated into a number of cell lineages. However, differentiation of hESSCs into megakaryocytes (MKs) has not yet been investigated. The aim of this study was to investigate the feasibility of MK generation from hESSCs and subsequent production of functional platelets (PLTs). In our study, hESSCs were cultured from endometrial stromal cells as confirmed by positive stromal cell specific markers (CD90 and CD29) and negative hematopoietic stem cell markers (CD45 and CD34) expression. Then, hESSCs were differentiated in a medium supplemented with thrombopoietin (TPO) for 18 days. The MK differentiation was analyzed by flow cytometry and confocal microscopy. The differentiation medium was collected for PLT production analysis by flow cytometry, transmission electron microscopy and functional measurements. Our results show: 1) MKs were successfully generated from hESSCs as identified by expression of specific markers (CD41a: 1±0.09% and 39±3.0%; CD42b: 1.2±0.06% and 28±2.0%, control vs. differentiation) accompanied with reduction of pluripotent transcription factors (Oct4 and Sox2) expression; 2) The level of PLTs in the differentiation medium was 16±1 number/µl as determined by size (2–4 µm) and CD41a expression (CD41a: 1±0.4% and 90±2.0%, control vs. differentiation); 3) Generated PLTs were functional as evidenced by the up-regulation of CD62p expression and fibrinogen binding following thrombin stimulation; 4) Released PLTs showed similar ultra-structure characteristics (alpha granules, vacuoles and dense tubular system) as PLTs from peripheral blood determined by electron microscopic analysis. Data demonstrate the feasibility of generating MKs from hESSCs, and that the generated MKs release functional PLTs. Therefore, hESSCs could be a potential new stem cell source for in vitro MK/PLT production.

Unraveling the Molecular Signature of Extracellular Vesicles From Endometrial-Derived Mesenchymal Stem Cells: Potential Modulatory Effects and Therapeutic Applications

“…In bibliography, different populations of endometrial stem cells have been described (Chan et al, 2004) and different names have been used for these cells, such as menstrual bloodderived stromal/mesenchymal cells, or endometrial-derived stromal/mesenchymal stem cells (endMSCs) (Kyurkchiev et al, 2010;Gargett et al, 2016). Additionally, different laboratory procedures have been established for in vitro isolation and expansion (Schüring et al, 2011;Wang et al, 2012;Rossignoli et al, 2013). Nowadays, menstrual blood-derived endMSCs can be easily isolated by a non-invasive method, without any painful procedure and their in vitro expansion can be achieved by simple, and reproducible methods (Sun et al, 2019).…”

Human adipose tissue‐derived stromal cells can differentiate into megakaryocytes and platelets by secreting endogenous thrombopoietin

“…Mesenchymal cells are distinct from hematopoietic cells, but Wang et al . and our group have demonstrated that MKs and platelets can be generated from mesenchymal cells such as endometrial stromal cells, subcutaneous adipose tissue cells, and preadipocyte cell lines . Mesenchymal cells constitute an advantageous cell source for manufacturing platelets, because differentiation of these cells into MKs and platelets does not require gene transfer.…”

The Endometrium as a Source of Mesenchymal Stem Cells for Regenerative Medicine1

“…Moreover, the differentiated megakaryocytes were able to release functional platelets that can bind fibrinogen after thrombin stimulation in vitro. Ultrastructural assessment of these platelets showed the expression of alpha granules, and a dense tubular system similar to their endogenous circulating counterparts [77]. Further in vivo studies will help to delineate the functionality of HEDSC derived platelets and their potential clinical utility.…”

“…The vast differentiation potential of HEDSC was further supported by an in vitro study, in which HEDSC were differentiated into megakaryocyte-like cells that are able to produce functional platelets [77]. When cultured in media containing thrombopoietin, up to 30% of the cells expressed megakaryocyte markers CD41a and CD42b.…”