Inflammation is one of the main features of

Inflammation is one of the main features of neurodegenerative diseases (Campbell, 2004). Having methods available that generate human astrocytes that are responsive to inflammation allows for the modeling of this component of the disease using patients\' cells. Previous studies reported the generation of reactive astrocytes after a 7-day treatment with IL-1β or TNF-α (Holmqvist et al., 2015; Roybon et al., 2013), but, as the authors used the ELISA assay or protein arrays to quantify chemokine and cytokine production, it is difficult to assess what proportion of the cellular population became effectively reactive. In our hands, classical protocols using iPSC-derived NPCs (Chandrasekaran et al., 2016; Tyzack et al., 2016) did not generate astrocytes responsive to pro-inflammatory stimuli using the FACS-based assay described in this study. Here we report a method for differentiating astrocytes using intermediate GPCs that were generated in a serum-containing medium that is reminiscent of the McCarthy-de Vellis astrocyte culture protocol (McCarthy and de Vellis, 1980). Because astrocytes contact blood vessels in vivo, it is possible that the serum contains unknown signals necessary for the differentiation of astrocytes responsive to inflammatory stimuli in culture.
Experimental Procedures
Author Contributions
Acknowledgments For the production of the iPSCs, the authors would like to acknowledge financial support from Janssen Pharmaceuticals. This work was supported by the Paul G. Allen Family Foundation, Bob and Mary Jane Engman, The JPB Foundation, The Leona M. and Harry B. Helmsley Charitable Trust grant #2012-PG-MED002, Annette C. Merle-Smith, R01 MH095741 (F.H.G.), U19MH106434 (F.H.G.), and The G. Harold & Leila Y. Mathers Foundation. This work was supported by the Flow Cytometry Core Facility of the Salk Institute with funding from NIH-NCI CCSG: P30 014195; the Next Generation Sequencing Core Facility of the Salk Institute with funding from NIH-NCI CCSG: P30 014195; the Chapman Foundation and the Helmsley Charitable Trust and by The Razavi Newman Integrative Genomics and Bioinformatics Core Facility of the Salk Institute with funding from NIH-NCI CCSG: P30 014195. This research was also supported by the Swiss-NSF outgoing PD fellowship (K.C.V.), Lynn and Edward Streim fellowship (K.C.V.), EMBO long-term fellowship (B.N.J.), the Bettencourt Schueller Foundation (B.N.J.), and the Philippe Foundation (B.N.J.). The authors would like to thank M.L. Gage for editorial comments.
Introduction Demyelinating diseases, injuries, and conditions, including pediatric leukodystrophies, white matter strokes, radiation-induced damage after cancer therapy, and spinal cord injury (SCI), are characterized by the loss or dysfunction of oligodendrocytes, the metabolic enzymes primarily responsible for myelin production in the CNS (Goldman and Kuypers, 2015). Without myelin insulating sheaths, axons are unable to properly conduct electrical impulses (Griffiths, 1998), resulting in impaired neurological and motor function, among other outcomes (Kassmann et al., 2007; Lappe-Siefke et al., 2003). No cure exists for demyelinating conditions, and current treatment regimens can only manage disease symptoms or slow the rate of demyelination (Helman et al., 2015; Najm et al., 2015). Due to their ability to differentiate into myelinating oligodendrocytes, oligodendrocyte precursor cells (OPCs) have emerged as candidates for cell therapy (Archer et al., 1997; Windrem et al., 2004, 2008). Specifically, unlike mature oligodendrocytes, which are fibrous, fragile, non-proliferative, and non-migratory, OPCs are more resistant to mechanical stress, highly proliferative, and migratory, and can become myelinogenic after dispersal and subsequent maturation (Goldman and Kuypers, 2015). Human pluripotent stem cells (hPSCs) have the capacity to self-renew and differentiate into all neural lineages and thus represent a promising and, in principle, scalable source for OPCs (Kang et al., 2007; Nistor et al., 2005), unlike primary human tissue (Uchida et al., 2000). In fact, OPCs generated from human embryonic stem cells (hESCs) were used in the first hESC-based clinical trial (Alper, 2009; Keirstead et al., 2005) and are undergoing further clinical development for SCI (Priest et al., 2015). Notably, there have been numerous advances that have enabled the production of OPCs from hPSCs, including several impressive efforts that applied key insights from developmental biology studies (Goldman and Kuypers, 2015). However, additional advances in their manufacturing would help OPCs reach their full therapeutic potential. For instance, current methods for differentiating OPCs from hPSCs can take more than 100 days to complete (Wang et al., 2013); entail undefined components, such as serum or Matrigel, which pose inherent reproducibility and safety challenges (van der Valk et al., 2010); and require purification strategies that can affect cell yield and viability (Diogo et al., 2012; Douvaras et al., 2014). Moreover, the large-scale manufacturing of clinical-grade OPCs could face further challenges considering that, to date, two-dimensional (2D) surfaces have been exclusively harnessed for their production (Czepiel et al., 2015; Douvaras and Fossati, 2015; Hu et al., 2009; Keirstead et al., 2005; Wang et al., 2013). For example, there are currently >200,000 patients with SCI, with an annual new incidence of 17,000 in the United States alone (Singh et al., 2014). Given that current clinical trials for SCI are dosing up to 20 million cells, producing sufficient cells for this target using 2D surfaces could pose a challenge, requiring an estimated 16,000 T75 flasks per year for culture densities of 300,000 cells/cm2. Thus, the field would benefit from the development of scalable processes for generating OPCs from hPSCs.