Teratogenic Potential in Cultures Optimized for Oligodendrocyte Development from Mouse Embryonic Stem Cells

Abstract

We describe a rapid and efficient 5-step program of defined factors for the genesis of brain myelin-forming oligodendrocytes (OLs) from embryonic stem cells (ESCs). The OLs emerge on the same time frame in vitro as seen in vivo. Factors promoting neural induction (retinoids, noggin) are required, while exogenous Sonic hedgehog is not. In contrast we were unable to generate OLs by trans-differentiation of ethically neutral mesenchymal stem cells, indicating a requirement for cis-differentiation via neural ectoderm for OL genesis. In the ESC-derived cultures, our optimized protocol generated a mixed population with 49% O4(+), Olig2(+) OL lineage cells. These cultures also retained pluripotential markers including Oct4, and an analysis of embryoid body formation in vitro, and allogeneic grafts in vivo, revealed that the ESC-derived cultures also retained teratogenic cells. The frequency of embryoid body formation from terminal differentiated OL cultures was 0.001%, 100-fold lower than that from ESCs. Our results provide the first quantitative measurement of teratogenicity in ESC-derived, exhaustively differentiated allogeneic grafts, and demonstrate the unequivocal need to purify ESC-derived cells in order to generate a safe population for regenerative therapy.

Transplantation of Neural Stem Cells Clonally Derived from Embryonic Stem Cells Promotes Recovery After Murine Spinal Cord Injury

“…These approaches have shown some functional success, but an EB intermediate in the generation of NSCs carries a serious risk of non-neural lineage ESCs from the EB persisting after transplant [25]. Non-neural lineage cells-particularly those expressing endoderm lineage genes and pluripotency markers-carry a distinct potential for tumorigenesis [23,25,26]. Tumors and teratomas have been observed in in vivo transplants of NSCs derived from ESCs using the EB formation step [26,27], indicating a clear need for an alternative approach to generate safe NSCs from ESCs.…”

Adjunctive MSCs enhance myelin formation by xenogenic oligodendrocyte precursors transplanted in the retina

“…They have also been reported to generate neurons and glia, although reports of trans-differentiation have generated considerable controversy [9]. It remains possible that trans-differentiation requires a longer time frame than the studies here, although to date we have not been successful in deriving OPCs from MSCs in vitro [10]. This is in sharp contrast to the myelin-forming ability of transplanted brain glia, where stem-derived allografts and xenografts promote myelin repair, and as little as 7% cell replacement is sufficient for functional recovery [6].…”

“…Methods: MSCs were isolated from C57BL/6-EGFP mouse bone marrow and expanded as described [2]. OPCs were isolated from newborn Sprague Dawley rat brain [5] and expanded in R1236 media [10]. For detection, OPCs were labeled by transfection of a membraneanchored GFP reporter (construct details are available upon request).…”

“…At present the least problematic resource are OPCs derived in vitro from pluripotential stem cells, and considerable efforts have been made to generate OPCs from both mouse and human ESCs. We have established an efficient method of sequential culture conditions for generating OPCs from murine ESCs [10], and our studies suggest that the timing of OPC differentiation in vitro can reflect the normal developmental program in vivo. However, our analysis also suggests that sequential differentiation does not eliminate all teratogenic cells in these cultures and ESCderived cell types must be purified in order to generate a safe graft population.…”

Teratoma showing the features of retinal structure: A case of sacrococcygeal teratoma

“…This finding may aid the development of efficient methods to solve the problem of condition setting in retinal transplantation (12)(13)(14) using embryonic stem (ES) cell (15) or induced pluripotent stem (iPS) cell (16) technology, which may cause teratogenic transformation.…”