Genetic stability during derivation and culture of human embryonic stem cells to ensure safety for clinical applications

Abstract Human embryonic stem cells (hESCs) could potentially provide a renewable source of different types of cells for cell therapy applications. Recently, mesenchymal stem cells (MSCs) have been derived from hESCs either through co-culturing with murine OP9 bone marrow stromal cell line or directly from hESCs without co-culturing with OP9 cells. Although the latter methodology is clinically advantageous over co-culturing with an animal cell layer those mesenchymal cells were reported to be positive for SSEA4. SSEA4 is a marker of undifferentiated hESCs and thus the presence of this marker on hESC-derived cells could potentially be problematic for clinical applications. We have recently achieved a novel and reproducible methodology for deriving a pure population of SSEA4-/CD73+ MSCs from federally approved hESC lines H1 and H9. To initiate the differentiation of hESCs to MSCs, we cultured undifferentiated hESCs on matrigel plates in murine embryonic fibroblast conditioned media with media changes every 3 days. Under these culture conditions a portion of embryonic stem cells differentiated into fibroblast looking cells. Through a multi-step process which involved the use of a culture methodology similar to what is being used to culture bone marrow (BM)-derived MSCs, and passaging cultured cells at defined time points we were able to derive a pure population of cells that were uniformly positive for MSC marker CD73 in about a 4-weeks period. These cells had fibroblast/mesenchymal looking morphology, and expressed cell surface marker antigens similar to what has been reported for adult human BM-derived MSCs: they are positive for CD29, CD44, CD54, CD71, CD90, glycophorin A, CD105, and were negative for hematopoietic markers such as CD34 and CD45. Similar to adult BM-derived MSCs these cells express HLA class-I antigens but not class-II antigens. Using established differentiation protocols we could differentiate the hESC-derived CD73+ MSCs into adipocytes, osteocytes, and chondrocytes as verified by immunohistochemistry and RT-PCR assays. So far we have grown these CD73+ MSCs up to passages 15–18. These cells retained their differentiation potential, and were karotypically normal when tested at passage 12. Most importantly, we did not observe any MSCs that were double positive for CD73 and SSEA4 antigen at any time point during our experiments. MSCs from a variety of fetal and adult sources are in various stages of clinical trials with some encouraging preliminary results. Our hESC-derived MSCs that are very similar to adult BM-derived MSCs regarding their growth and morphologic properties, immunophenotypic characteristics, differentiation potential, and importantly are devoid of hESC marker SSEA4 could potentially provide a novel source of MSCs for clinical applications.

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The Effect of Genetic Stability Molecules on the Movement of Human Embryonic Stem Cells

Nanoscience and Nanotechnology Letters ◽

10.1166/nnl.2020.3095 ◽

2020 ◽

Vol 12 (2) ◽

pp. 245-255

Author(s):

Xiaoxiang Zhou ◽

Yu Liu

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Genetic Stability ◽

Embryonic Stem ◽

Snp Analysis ◽

Chromosome 5 ◽

Type Iv Collagenase ◽

Type Iv ◽

Before And After

Objective: To give full play to the role of human embryonic stem cells (hESCs) in biomedicine, innovate the treatment model of human diseases, and study the effect of genetic stability molecules of hESCs on movement. Methods: hESCs were cultured by mechanical passage, type IV collagenase passage and 0.25% EDTA trypsin passage respectively to find the best passage way of hESCs culture; hESCs were differentiated into endometrial like cells through embryoid pathway in vitro, and the morphology of endometrial like cells was observed under light microscope; the genomic DNA of human embryonic stem cells was detected by SNP chip, and its components were analyzed. The SNP analysis of the samples before and after the induction of differentiation were used to extract the DNA of the endometrial epithelioid cells of hESCs, and the DNA microarray results before and after the induction of hESCs were detected. Results: type IV collagenase passage method took a long time and was not suitable to control the digestion time and inoculation density; 0.25% EDTA trypsin digested hESCs into a single cell state, which was not conducive to continuous growth after being inoculated in the feeder layer; mechanical passage method could cut into different sizes of clumps according to the needs, and could remove differentiated or deviated cells, which was more suitable for hESCs passage; light microscopically, the cells grow in a single layer and adhere to the wall, and the cells extending to the surrounding irregularity are spindle shaped. With the extension of differentiation time, the central part of the clone is loose, the cells fall off, and the surrounding cells grow intensively to the outer part of the clone. The mutant gene involves 1131 mutations and 24 chromosome mutations, of which the mutation rate of chromosome 19 is the largest, and it involves the length of chromosome 5. It is longer than that of other chromosomes, and there are many genes carried by it, which results in the exon mutations on chromosome 5. Conclusion: mechanical subculture is the best way for hESCs culture, and long-term subculture will affect the genetic stability of human embryonic stem cells.

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