Efficient Generation of Viral and Integration‐Free Human Induced Pluripotent Stem Cell‐Derived Oligodendrocytes

Abstract

Here we document three highly reproducible protocols: 1) A culture system for the derivation of human oigodendrocites (OLs) from human induced pluripotent stem cells (hiPS) and their further maturation; Key features are the concomitant fate restriction, and lineage specification of hiPS towards the neutral and OL phenotypes. The use of very precise amounts of fresh additives and factors also contributes to the shorter periods of time (56days vs. /200days/75days) necessary to obtain hiPS-derived OLs. Our protocol generates viral and integration free OLs that efficiently commit and move forward in the OL lineage recapitulating all the steps known to occur during in vivo development. 2) Protocols for the isolation, propagation and maintenance of neural stem cells (NSCs); and 3) Protocols for the production, isolation, and maintenance of OLs from perinatal rodent and human brain-derived NSCs. Our unique culture systems rely on a series of chemically defined media, specifically designed and carefully characterized for each developmental stage of OL as they advance from OL progenitors to mature, myelinating cells. We are confident that these protocols bring our field a step closer to efficient autologous cell replacement therapies and disease modeling.

Delayed Maturation of Oligodendrocyte Progenitors by Microgravity: Implications for Multiple Sclerosis and Space Flight

“…Each one considers the specific nutrients, additives, and time windows when the additives should be provided to either make the cells remain at a specific developmental stage, i.e., progenitors (Espinosa et al, 1988), or move forward in the lineage. The most recent culture system aims at instructing human induced pluripotent stem cells (hiPS) to become mature, functional OLs [ 9 ]. We started with embryoid bodies (EBs) and thereafter directed and instructed their commitment to the neural and subsequent OLs phenotype.…”

“…We started with embryoid bodies (EBs) and thereafter directed and instructed their commitment to the neural and subsequent OLs phenotype. Once commitment of hiPS to the OLs stage was reached, the steps of lineage progression were bolstered by the culture systems we have already described [ 9 ]. Besides inducing and maintaining a specific phenotype, the importance of this culture system resides in the fact that we achieved phenotype commitment of hiPS to OLs without using gene transfer [ 10 , 11 , 12 ] or undefined substrates present in other methods, such as a different cell used as a substrate or a different cell-line-derived conditioned medium.…”

Human Neural Stem Cells Flown into Space Proliferate and Generate Young Neurons

“…After splashdown, transport to Long Beach airport, and delivery to UCLA, the NSCs were retrieved from the hardware, plated onto poly-d-lysine-coated flasks in our proprietary stem cell chemically defined medium (STM) [6,7], and allowed to recover from space flight. Subsequently, some of these NSCs were plated in neuronal specification medium (NSM).…”

Application of CRISPR/Cas9 to the study of brain development and neuropsychiatric disease

“…Because many subtypes of neurons and glia have been implicated in neuropsychiatric disease (Gotter et al, 2001), it is crucial to study defined cell types, both alone and in combination, in order to understand how they function in health and disease. While discussed in great detail by others (reviewed in Ho et al, 2015), a variety of protocols have reported differentiation or induction of glutamatergic (Zhang et al, 2013, Ho et al, 2016), GABAergic (Sun et al, 2016), dopaminergic (Theka et al, 2013), and serotonergic (Lu et al, 2016) neurons, as well as astrocytes (Emdad et al, 2012), oligodendrocytes (Espinosa-Jeffrey et al, 2016; Thiruvalluvan et al, 2016), and microglia-like cells (Muffat et al, 2016). …”