Speaker
Description
Introduction
Mesenchymal stem cells (MSCs) are self-renewing, non-specialized, multipotent cells with great potential to differentiate into various tissue cells. Due to their secretion activity, migration, adhesion and homing abilities, MSCs are currently highly investigated stem cells and the one most commonly used in regenerative medicine. The use of adult MSC is still challenging, since a limited number of these stem cells can be obtained from a single donor and a substantial cell quantity is required for their clinical application. Other challenges include the limited adult MSC proliferation capacity, which may significantly constrain their capabilities of immunomodulation and secretion of bioactive factors. Considering these facts, we differentiated induced pluripotent stem cells (iPS cells) towards mesenchymal stem cells (iMSCs) using the optimized xeno-free culturing protocol. Next, we have compared properties of obtained cells with MSCs isolated from Wharton’s Jelly (WJ-MSCs).
Methodology
We used two protocols based on differentiating medium supplemented with FBS and human platelet lysate respectively. We examined phenotype and properties of acquired iMSCs in terms of their: morphological features (microscopy techniques), expression surface markers (flow cytometry), trilineage differentiation potential, expression of selected factors (RT-PCR) and cell proliferation by use of BrdU assay (flow cytometry). We have also evaluated cellular senescent during long time differentiating process by senescence-associated β-galactosidase (SA-β-gal) activity assay.
Results
IMSCs we obtained show the minimal criteria outlined for MSCs by the International Society of Cellular Therapy (ISCT). The cells adhere to plastic in standard culture conditions and display fibroblast-like morphology and ability to trilineage differentiation towards adipogenic, osteogenic and chondrogenic lineages. Cytometric analysis revealed high expression of specific mesenchymal markers. More than 90% of cells were CD73, CD90, CD105 and CD146 (MSC progenitor marker) positive, whereas they do not express hematopoietic antigens (CD45, CD14, CD19, CD34, CD3). During long term expansion iMSCs show high proliferation potential with insignificant number of SA-β-gal positive cells. The purity of populations has been assessed by reduced expression of OCT-4, SOX-2, Nanog and EpCAM compared to the population of iPS cells. It should be noted that iMSCs injected into mice did not form tumors.
Conclusions:
The relatively easy process of obtaining iMSCs make them an inexhaustible source of MSCs that could be used for clinical applications. Since iMSCs exhibit similar properties as MSCs isolated from natural tissues, they open the wide possibilities of their use as off-the-shelf allogeneic cell-based therapeutics.
This work was supported by research grant 2018/29/B/NZ5/00915 from the National Science Center in Poland.
41883668648
