Modified methods for efficiently differentiating human embryonic stem cells into chondrocyte-like cells.

INTRODUCTION: Human articular cartilage has a poor regenerative capacity. This often results in the serious joint disease- osteoarthritis (OA) that is characterized by cartilage degradation. An inability to self-repair provided extensive studies on AC regeneration. The cell-based cartilage tissue engineering is a promising approach for cartilage regeneration. So far, numerous cell types have been reported to show chondrogenic potential, among others human embryonic stem cells (hESCs). MATERIALS AND METHODS: However, the currently used methods for directed differentiation of human ESCs into chondrocyte-like cells via embryoid body (EB) formation, micromass culture (MC) and pellet culture (PC) are not highly efficient and require further improvement. In the present study, these three methods for hESCs differentiation into chondrocyte-like cells in the presence of chondrogenic medium supplemented with diverse combination of growth factors (GFs) were evaluated and modified. RESULTS: The protocols established here allow highly efficient, simple and inexpensive production of a large number of chondrocyte-like cells suitable for transplantation into the sites of cartilage injury. The most crucial issue is the selection of appropriate GFs in defined concentration. The obtained stem-derived cells reveal the presence of chondrogenic markers such as type II collagen, Sox6 and Sox9 as well as the lack or significantly lower level of pluripotency markers including Nanog and Oct3/4. DISCUSSION: The most efficient method is the differentiation throughout embryoid bodies. In turn, chondrogenic differentiation via pellet culture is the most promising method for implementation on clinical scale. The most useful GFs are TGF-ß1, -3 and BMP-2 that possess the most chondrogenic potential. These methods can also be used to obtain chondrocyte-like cells from differentiating induced pluripotent stem cells (iPSCs).

Comparison of the early response of human embryonic stem cells and human induced pluripotent stem cells to ionizing radiation.

Despite the well-demonstrated efficacy of stem cell (SC) therapy, this approach has a number of key drawbacks. One important concern is the response of pluripotent SCs to treatment with ionizing radiation (IR), given that SCs used in regenerative medicine will eventually be exposed to IR for diagnostic or treatment­associated purposes. Therefore, the aim of the present study was to examine and compare early IR­induced responses of pluripotent SCs to assess their radioresistance and radiosensitivity. In the present study, 3 cell lines; human embryonic SCs (hESCs), human induced pluripotent SCs (hiPSCs) and primary human dermal fibroblasts (PHDFs); were exposed to IR at doses ranging from 0 to 15 gray (Gy). Double strand breaks (DSBs), and the gene expression of the following DNA repair genes were analyzed: P53; RAD51; BRCA2; PRKDC; and XRCC4. hiPSCs demonstrated greater radioresistance, as fewer DSBs were identified, compared with hESCs. Both pluripotent SC lines exhibited distinct gene expression profiles in the most common DNA repair genes that are involved in homologous recombination, non­homologous end­joining and enhanced DNA damage response following IR exposure. Although hESCs and hiPSCs are equivalent in terms of capacity for pluripotency and differentiation into 3 germ layers, the results of the present study indicate that these 2 types of SCs differ in gene expression following exposure to IR. Consequently, further research is required to determine whether hiPSCs and hESCs are equally safe for application in clinical practice. The present study contributes to a greater understanding of DNA damage response (DDR) mechanisms activated in pluripotent SCs and may aid in the future development of safe SC­based clinical protocols.

Genetic stability of pluripotent stem cells during anti-cancer therapies.

Regenerative medicine is a rapidly growing field that holds promise for the treatment of many currently unresponsive diseases. Stem cells (SCs) are undifferentiated cells with long-term self-renewal potential and the capacity to develop into specialized cells. SC-based therapies constitute a novel and promising concept in regenerative medicine. Radiotherapy is the most frequently used method in the adjuvant treatment of tumorous alterations. In the future, the usage of SCs in regenerative medicine will be affected by their regular and inevitable exposure to ionizing radiation (IR). This phenomenon will be observed during treatment as well as diagnosis. The issue of the genetic stability of SCs and cells differentiated from SCs is crucial in the context of the application of these cells in clinical practice. This review examines current knowledge concerning the DNA repair mechanisms (base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end-joining) of SCs in response to the harmful effects of genotoxic agents such as IR and chemotherapeutics.