Derivation of Mesenchymal Stromal Cells from Pluripotent Stem Cells through a Neural Crest Lineage using Small Molecule Compounds with Defined Media

Abstract

Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton, cornea, peripheral nervous system, and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), further modifications are required to improve the robustness, efficacy, and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions, the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70–80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons, glia, melanocytes, and corneal endothelial cells. In addition, cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine.

Craniofacial Bone Regeneration using iPS Cell-Derived Neural Crest Like Cells

“…iPS-NCLCs cultured in serum-containing medium exhibited MSC phenotype expression of common MSC markers including Gli-1, craniofacial-specific MSC marker 31) , and the multipotency to differentiate into 3 mesenchymal cell lineages. These findings were consistent with previous reports demonstrating that NCs derived from human iPS cells have a capacity to diff erentiate into MSCs 32) . iPS-NCLC-MSCs can also be cryopreserved and retain differentiation and proliferation capacity in long-term culture.…”

“…The safety of iPS-NCLC-MSCs further confi rmed the reduction of some pluripotency genes as shown in our previous study of iPSNCLCs 12) . Consistent with our results, recent studies related to MSCs derived from iPS cells have demonstrated their safety for clinical application 32,[34][35][36] , providing evidence to promote the use of iPS-NCLCMSCs for stem cell therapy. Interestingly, in iPS-NCLC-MSCs in the present study, Sox2 expression level was unchanged compared to that for undiff erentiated iPS cells.…”

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

“…The induction and maintenance of induced neural crest cells (iNCCs) and iMSCs derived from iPSC were previously described (43). FOP-iPSCs used in this study [FOP-iPSCs from patient 1 and 2, previously described as vFOP4-1 and vFOP5-22 (25), respectively] harbor the R206H heterozygous mutation in ACVR1, and gene-corrected resFOP-iPSCs were generated by BAC-based homologous recombination (26).…”

Human Turbinate-derived Mesenchymal Stem Cells Differentiated into Keratocyte Progenitor Cells

“…However, cell-based therapy for keratocyte has severe limitations because keratocytes easily transform to fibroblast that secrete metalloproteinase that induces extracellular remodeling in vivo and in vitro. Recent observations demonstrate that keratocyte progenitor cells derived from neural crest can differentiate into keratocytes without changing to fibroblasts [12,[34][35][36]. Progenitor cells derived from neural crest migrate precisely to the targeted tissue origin pathway.…”