Astrocytes derived from embryonic or induced pluripotent stem cells

Embryonic stem cells (ESC) are self-renewing, pluripotent cells, which can give rise, by definition, to all cell types of an adult organism. They are obtained from the inner cell mass (ICM) of the blastocyst. Murine ESC were the first time isolated in 1981 by Martin Evans, Matthew Kaufman, and Gail R. Martin (Evans and Kaufman 1981). In 1995, Mar-tin Evans and AusMar-tin Smith described the culture of murine ESC with leukemia-inhibitory factor (LIF), which keeps the cells in their pluripotent, self-renewing state. Human ESC have been isolated later in 1998 by James Thomson from the ICM of residual blastocysts

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of in vitro fertilizations (Thomson et al. 1998). They can be kept in a proliferative, non-differentiating state by the addition of FGF2. ESC are pluripotent, in contrast to the mul-tipotent NS cells or adult stem cells, meaning that they can differentiate into cells of all three germ layers: ectoderm, endoderm, and mesoderm (Martello and Smith 2014). Cells of the nervous system in vivo arise from the ectodermal lineage, which forms the epider-mis and the neural plate. ESC can be differentiated into neural stem cells (NSC) in vitro by the withdrawal of growth factors. This withdrawal of factors, which keep the cells in a pluripotent state, leads to a spontaneous differentiation of the cells. This implies that they might not only form NSC, but also cells of other germ layers. However, NSC can be enriched by a switch to neural proliferation medium containing EGF and FGF2 to expand the desired progenitor cells. Neural stem cells have been generated from murine ESC (mESC) since 2001 (Tropepe et al. 2001). In 2003, a protocol for neural induction of mESC in adherent culture has been described, facilitating the generation of pure popula-tions of NSC (Ying et al. 2003). Luciano Conti and colleagues showed in 2005 that these pure NSC can differentiate both into neurons and astrocytes, also after prolonged expan-sion in EGF and FGF2 (Conti et al. 2005). In 2007, the tripotential differentiation capacity of these cells could be shown by immunocytochemistry detecting oligodendrocytes (Glaser et al. 2007). Human embryonic stem cells (hESC) have been differentiated to neural precursors in 2001 by Su Chun Zhang and colleagues (Zhang et al. 2001). These neural precursors, obtained from neurosphere cultures, differentiated into neurons, astro-cytes, and oligodendroastro-cytes, although glia cells represented only a minor fraction. Their differentiation potential to astrocytes has been reported in 2011: Robert Krencik and col-leagues generated NSC within 21 days from hESC and differentiated them into astrocytes within 160 days. The cells showed an astrocytic phenotype, expressed markers of imma-ture and maimma-ture astrocytes, reacted to AMPA and glutamate, and supported neuronal sur-vival (Krencik et al. 2011). Gupta and colleagues also generated cells from hESC with astrocyte characteristics, and demonstrated neuroprotective effects of their cells (Gupta et al. 2012). The differentiation of human embryonic stem cells to astrocytes is much more complicated compared with mouse systems, since human development proceeds in a different time scale. Although human systems would be desirable, current protocols for the generation of astrocytes require long-term cultures and the differentiated astrocytes are in most cases heterogeneous and not fully mature (Roybon et al. 2013). Thus, for studies, which require pure populations of mature astrocytes, such as metabolic studies

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or microarray profiling, astrocytes generated from murine stem cells are still the best ap-proach.

Induced pluripotent stem cells (iPS) are reprogrammed somatic cells, which regain stem cell characteristics by introducing pluripotency factors. This has been done first in 2006 with mouse fibroblasts by Shinya Yamanaka (Takahashi and Yamanaka 2006), who re-ceived, together with John Gurdon, the noble price for his study in 2012. Several tran-scription factors were introduced by retroviral transduction, and cells were cultured equivalent to embryonic stem cells on feeder layers in ESC medium. Oct3/4 (octamer-binding transcription factor 4), Sox2 (sex determining region Y box 2), and nanog have been known factors for maintaining pluripotency (Sun et al. 2006). However, only Oct3/4 and Sox2 to be essential for the generation of iPS. By introducing them together with c-myc and Klf4 (Kruppel-like factor 4), they got a self-renewing cell population expressing ESC markers, which could be differentiated into neural, muscle, and endodermal tissue cells. This finding has been revolutionary for stem cell research, and other iPS cell lines followed. In 2007, two human iPS lines have been generated. Takahashi and colleagues introduced the same factors in human dermal fibroblasts (Takahashi et al. 2007). At the same time, Yu and colleagues generated human iPS through four factors including Oct3/4, Sox2, and Nanog (Yu et al. 2007). Both cell lines regained embryonic stem cell characteristics, and were capable to differentiate into cells of all three germ layers. Now-adays, the classical reprogramming factors are Oct4, Sox2, Klf4, and c-Myc (Kim et al.

2011).

The generation of human iPS opened up new perspectives in biomedical research: iPS might be used for patient-specific cell-based therapy, reducing immunorejection after transplantation. Nevertheless, there are some limitations of the system, which need to be resolved before using iPS-systems in cell therapy. Some of the factors introduced into somatic cells are very potent oncogenes (e.g., c-myc), holding a risk of tumor formation in patients (Gutierrez-Aranda et al. 2010). Moreover, the integration of factors into the genome might engender genomic mutations (Lin and Wu 2015). And the reprogramming itself is debatable: In many cases, it is incomplete, giving rise to cells that are not equiv-alent to ESC (Jalving and Schepers 2009). Zhao and colleagues, however, showed in 2009 that murine iPS injected in blastocysts gave rise to viable mice (Zhao et al. 2009). There-fore, iPS actually might be suitable substitutes for ESC, although their safe application in cell therapy has still to be proven. In neuroscience, iPS and iPS-derived neurons or glia

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cells could be used for transplantation to substitute for injured cells in several CNS dis-eases such as Multiple Sclerosis, Alzheimer’s, or Parkinson’s disease. Therefore, iPS have been differentiated to a neural lineage since 2008. Wernig and colleagues generated tripotent NSC from murine iPS similar to ESC differentiation (Wernig et al. 2008). They could show that these precursor cells give rise to neurons and glia cells, when transplanted into fetal mouse brain. They also demonstrated therapeutic potential of dopaminergic neuron progenies, but were faced with problems concerning teratoma formation. Cai and colleagues used human iPS-derived dopaminergic neurons for transplantation assays in rats, but also observed tumor formation (Cai et al. 2010). To overcome the problem of teratoma formation, possibilities to enrich and increase purity of the desired cell type need to be developed (Lin and Wu 2015). Yao and colleagues used GFP constructs under the control of a neuronal promoter in mouse iPS to select for neurons prior to transplantation, without observing tumor formation (Yao et al. 2011).

Compared to the differentiation of iPS-derived NSC to functional neurons, the differen-tiation to glia cells lags far behind. A differendifferen-tiation of iPS to functional oligodendrocytes has been reported in 2011 (Czepiel et al. 2011). The generation of mature astrocytes from iPS, at least as pure populations, has not been reported yet and tripotential differentiation capacity of iPS-derived NSC has mainly been demonstrated by the expression of GFAP (Conti et al. 2005). However, the differentiation of iPS, derived from Huntington’s dis-ease patients, towards the astrocyte lineage revealed a vacuolation phenotype, indicating a cell-autonomous mechanism for astrocytes in this neurodegenerative disease (Juopperi et al. 2012). Moreover, the transplantation of iPS-derived astrocyte populations has been shown very recently to have beneficial effects in spinal cord injury (Li et al. 2015).

The generation of astrocytes presented here is mainly based on the protocol described in 2005 (Conti et al. 2005). This seminal work mainly focused on neurogenesis, while the generated astrocytes were hardly characterized. A reason may be that the original protocol requires adaptations and further specifications before high quality, pure astrocytes can be reproducibly obtained. As there is such a great, yet largely unrecognized, potential in the Conti et al. astrocyte generation procedure as basis for future astrocyte/NSC research, we set out in part 1 of this study: First, to define the critical steps of the procedure; Second, to characterize the resulting cells; And third, to demonstrate the usefulness of the new protocol for generating cell populations for inflammation and metabolism studies based on multiple stem cell lineages.

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