In Vitro Neural Differentiation of Human Embryonic Stem Cells Using a Low-Density Mouse Embryonic Fibroblast Feeder Protocol

Abstract

Human embryonic stem cells (hESCs) have the capacity to self-renew and to differentiate into all components of the embryonic germ layers (ectoderm, mesoderm, endoderm) and subsequently all cell types that comprise human tissues. HESCs can potentially provide an extraordinary source of cells for tissue engineering and great insight into early embryonic development. Much attention has been given to the possibility that hESCs and their derivatives may someday play major roles in the study of the development, disease therapeutics, and repair of injuries to the central and peripheral nervous systems. This tantalizing promise will be realized only when we understand fundamental biological questions about stem cell growth and development into distinct tissue types. In vitro, differentiation of hESCs into neurons proceeds as a multistep process that in many ways recapitulates development of embryonic neurons. We have found in vitro conditions that promote differentiation of stem cells into neuronal precursor or neuronal progenitor cells. Specifically, we have investigated the ability of two federally approved hESC lines, HSF-6 and H7, to form embryonic and mature neuronal cells in culture. Undifferentiated hESCs stain positively for markers of undifferentiated/pluripotent hESCs including surface glycoproteins, SSEA-3 and 4, and transcription factors Oct-3/4 and Nanog. Using reduced numbers of mouse embryonic fibroblasts as feeder substrates, these markers of pluripotency are lost quickly and replaced by primarily neuroglial phenotypes with only a few cells representing other embryonic germ layer types remaining. Within the first 2 weeks of co-culture with reduced MEFs, the undifferentiated hESCs show progression from neuroectodermal to neural stem cell to maturing and migrating neurons to mature neurons in a stepwise fashion that is dependent on both the type of hESCs and the density of MEFs. In this chapter, we provide the methods for culturing pluripotent hESCs and MEFs, differentiating hESCs using reduced density MEFs, and phenotypic analyses of this culture system.

Paracrine and Epigenetic Control of Trophectoderm Differentiation from Human Embryonic Stem Cells: The Role of Bone Morphogenic Protein 4 and Histone Deacetylases

“…Standard pluripotent hESCs are maintained by growing them on mouse embryonic fibroblasts (MEF) and routinely passaging them to prevent unwanted spontaneous differentiation. A reduction in MEF-secreted pluripotency factors by hESC culture on low-density MEFs encourages spontaneous neurectoderm differentiation [19][20][21][22], whereas bone morphogenic protein 4 (BMP4) supplementation to MEFs at low density [23,24] produces TE from pluripotent hESCs. However, the factors responsible for initiating human TE differentiation from pluripotent hESCs, that is TE induction, are incompletely understood.…”

Cryopreserved mouse fetal liver stromal cells treated with mitomycin C are able to support the growth of human embryonic stem cells

“…Thus, the number of adherent cells was significantly reduced when compared with the other groups when the same number of cells were seeded. Taking into account that the density of the feeder cells may influence the maintenance of hESCs (6,26–28), cell numbers were increased in the −70°C 60 day group in order to provide a number of adherent cells that was consistent with those in the other groups. Even though the cell number was supplemented, it was observed that the proliferation rate of the hESCs was slower in the −70°C 60 day group than that of the other groups from the beginning of the third generation; hESC colonies on the feeder were evidently differentiated (loosely arranged with no clear boundary, thin clumps and morphological heterogeneity).…”

Stem Cells for Temporomandibular Joint Repair and Regeneration

“…To date, several diverse cell types have been derived from hESCs, including cardiomyocytes [66,67], endothelial cells [68][69][70], chondrocytes [71][72][73][74][75], osteoblasts [76][77][78], neurons [79][80][81], keratinocytes [82,83], and hepatocytes [84].…”