This document summarizes research on differentiating human pluripotent stem cells into neuronal and glial cells. It discusses protocols for generating several neural cell types, including dopaminergic neurons, motor neurons, GABAergic neurons, cholinergic neurons, retinal cells, and oligodendrocytes. These differentiation techniques aim to provide functional cells for applications in disease modeling, drug discovery, and regenerative medicine for conditions like Parkinson's, ALS, retinal degeneration, and multiple sclerosis. However, improving differentiation efficiency and safety is still needed, especially for induced pluripotent stem cells.

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Neuronal and glial differentiation of human pluripotent stem cells
Diana Catarina Santos*
*Mestrado Integrado em Engenharia Biomédica
Bioengineering Department - Instituto Superior Técnico
Av. Rovisco Pais, 1049-001 Lisboa
e-mail: diana.c.santos@ist.utl.pt / dianassantos@hotmail.com
KEYWORDS: Induced Pluripotent Stem Cells (iPSCs), Embryonic Stem Cells (ESCs), Neuronal differentiation,
Neuronal Progenitors (NPs), glial differentiation.
ABSTRACT
In vitro differentiation from human embryonic stem cells (hESCs) and induced pluripotent stem
cells (iPSCs) is a recent and promising technique for the achievement of mature neuronal and
glial cells (functional neurons, astrocytes and oligodendrocytes) that can be used for drug
discovery, disease modeling and regenerative medicine applications. Moreover, differentiation of
iPSCs results in patient-specific cell, avoiding transplantation rejection and controversial issues
associated with hESCs. However, iPSCs are related to lower differentiation efficiency and
tumorigenesis risk. Better protocols for generation of cells restricted to neural cell lines are
needed in order to get safest and efficient therapies.
INTRODUCTION
Human pluripotent stem cells (hPSCs) are defined as self-renewable cells that have the potential to
differentiate in several types of cells of the three germ layers, giving rise to any of the cell types of
the organism [1]. In order to ensure pluripotency of cells, International Stem Cell Banking Initiative
(ISCBI) proposed an exhaustive set of tests, including nuclear and surface markers expression
analysis, spontaneously differentiation test of EBs in vitro and in vivo in the three germ layers of the
embryo, teratoma formation and karyotype analysis, gene expression profile and microbiological
tests [2,3].
Both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent
populations that have been derived worldwide and are being used in drug discovery, modeling of
diseases and are a promising source for regenerative medicine, for instance to neurological diseases
treatment, such as alzheimer, parkinson, autism and schizophrenia [1,4,5].
Being pluripotent ESCs and iPSCs can generate, under specific culture conditions, neuronal
differentiated cells as functional neurons, glial cells and oligodendrocytes [6]. Recently, iPSC have
been shown to have the potential of differentiation in dopaminergic (DA) neurons and motor
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neurons, even so associated to an oncogenic risk [7]. The process of reprogramming patient’s

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somatic cells in iPSCs in vitro, generating neuronal populations represents an unlimited source of
cells for transplantation, being a personalized therapy (Figure 1) [1,7,8].
Although the optimal source for stem cells with neural potential remains controversial, the use of
human neuronal stem cells (hNSCs), isolated from the neuroectoderm, is also promisor in the
treatment of neurological disorders [6,7].
PSCS: ESCS AND IPSCS
While hESCs are isolated from the inner mass of the blastocyst (with 5-6 days), hiPSCs are somatic
adult cells that suffer a process of “de-differentiation” by genetic reprogramming, becoming
embryonic stem cells-like.
Since ESCs are associated to ethical problems, other sources of PSCs were sought. Until now,
several techniques have been developed in order to restore the ability of differentiation on an already
differentiated nucleus. Firstly, in 1962, reprogramming by nuclear transfer was performed, wherein
John Gurdon replaced the genetic material of unfertilized eggs, collected from frogs, by its somatic
cell chromatin, from frog’s intestinal cells. This approach required oocytes availability and, once
again, it was associated with ethical problems. Years later, the fusion of somatic cells to ESCs was
performed, resulting in tetraploid embryonic stem cells-like. Meanwhile, the discovery of
transcription factors, which guide the cell into a specific lineage, lead to direct reprogramming of
somatic cells in PSCs, with huge advantages related to donor-specificity, availability, simplicity and
reproducibility. Yamanaka showed for the first time, in 2006, that mouse somatic cells can be forced
into a pluripotent state using transcription factors [9-12].
In spite of differences between ESCs and iPSCs lines have been reported in serveral studies, in terms
of gene expression and DNA methylation, studies having in account higher numbers of clones defend
that iPSCs and ESCs are very similar and difficult to distinguishe. In terms of differentiation
potential it seems that iPSCs have lower potentials comparing to ESCs and greater variation on the
differentiation levels. Once more, some studies reported non-generation of teratoma by iPSCs,
contrarly to others. Careful comparison may allow the conclusion that these variations are due to
distinct technical procedures, in particular in the order of reprogramming factors addition. Yamanaka
is convinced that ESCs and iPSCs are very similar, which may be indicative of the non-existance of
ESCs under physiological conditions. From his point of view ESCs are also artificial cells formed by
the culture procedure [10].
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The major challenge in using pluripotent cells for cell-based therapy is to produce a homogeneous,
large and renewable population of lineage-committed cells, free from other lineage cells.
REPROGRAMMING PROCESS OF IPSCS
Transcription factors octamer-binding transcription factor ¾ (Oct¾), sex determining region Y-box 2
(Sox2), krupel-like factor 4 (Klf4) and myeocytomatosis oncogene (c-Myc), known also by the four
Yamanaka factors, are considered to be essential factors for somatic cell reprogramming into a
pluripotent state, while Nanog has been reported to be dispensable. However Klf4 and c-Myc are
related to tumors development, their functions are balanced by each other, since Klf4
antiproliferation characteristic is inhibited by c-Myc and apoptosis induction provoked by c-Myc is
inhibited by Klf4 [12,13].
Even though these four factors are capable of reprogramming somatic cells, the efficiency of the
process remains low, less than 1% of fibroblasts become true iPSCs [10,13].
In order to introduce the reprogramming stem cell factors into adult cells, different approaches has
been used, influencing the quality of iPSCs and the efficiency of the reprogramming process [12].
Viral transfection with retrovirus and lentivirus has been widely used, although there were reported
some cases of cancer development, due to the development of insertional mutagenesis and low
efficiency results [7,12]. Thus integration-free techniques, using plasmid, recombinant proteins,
small molecules, adenovirus and sendai virus strategies are highly required in order to avoid
mutagenesis [8,10,12]. Although, the efficiency of reprogramming using integration-free methods is
lower than that using vector integration into the genome. Chemical compounds that promote
reprogramming are being studied, in order to improve efficiency rates and thereby to substitute the
four factors of Yamanaka. Before human clinical trials, the optimal method for somatic cell
reprogramming should be achieved, guarantying the patient’s safety [12].
NEURONAL DEVELOPMENT
When the embryo has about 3weeks, the neural tube formation occurs from the neural plate, in vivo.
Human ESCs express SOX2 factor, which is essential to maintain its pluripotency. When these cells
are differentiating into neural progenitor (NP) cells, SOX2 expression is maintained, however Nestin,
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Figure 1. Potential applications of iPSCs. In spinal muscular atrophy (SMA) patients, motor neurons die progressively leading to
poor life quality. SMA-specific iPSCs could, by on one hand lead to the identification of novel drugs that prevent the abnormal
death of motor neurons in patients and on the other hand be differentiated into healthy motor neurons, for further transplantation to
the patient. [Adapted –[12]].
SOX1, SOX3, PSA-NCAM and MUSAASHI-1 become expressed, serving as markers of neuronal
commitment, as well as neural rosette formation [6].
hPSCs can be cultured in co-culture with stromal cells, such as PA6, usually resulting in the
achievement of midbrain dopaminergic neurons. Also they can be detached from the feeder layers
and aggregated in suspension culture to form embryoid bodies, resulting in the achievement of
ectodermal germ layer formation. Posteriorly, in order to have more differentiated populations,
aggregates are passed into a culture of neuronal growth factors. Neural rosettes become more
differentiated into neural plate-like rosettes and primitive neuroepithelial cells posteriorly. These
cells can be isolated and grown in neurospheres, in suspension, on an appropriated medium culture to
commit them into neurons, astrocytes and oligodendrocytes [14].
Neurons positive for β3-tubulin are firstly produced, followed by glial fibrillary acidic protein
(GFAP) positive astrocytes and finally oligodendrocytes.
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The timings of these events are similar in vitro and in vivo (Figure 2). There are several studies
focused on the direct differentiation of hPSCs on the mature neural cell.
Human iPSCs and hESCs differentiation efficiency seems to be different, since hiPSCs present lower
levels of PAX6 and teratoma formation in vivo, suggesting a low potential of differentiation of
iPSCs, which can be due to the use of viral vectors for the transfection of growth factors [15].
Figure 2. Temporal events in the differentiation, in vivo and in vitro, of human PSCs (ESCs and iPSCs). Neurons are generated in
the second month, astrocytes in the third month, and oligodendrocytes in the fourth month. [Adapted –[1].
NEURONAL COMMITEMENT FROM PSCS
Neurodegenerative diseases are targeted by many studies concerning the differentiation of PSCs.
The in vitro produced neurons have a huge potential for neuronal replacement applied to
neurodegenerative diseases, such as alzheimer, parkinson, spinal cord injury (SCI) and stroke
[11,16].
1. Dopaminergic (DA) neurons
Parkinson's disease, still untreatable, is characterized essentially by progressive degeneration
of dopaminergic (DA) neurons and leads to movements lost and to cognitive problems in
later stages. Thus, scientific community seeks urgently for an efficient therapy [11].
Functional DA neurons are being derived effectively both from ESCs and iPSCs, in a similar
way [1]. It has been reported that fibroblasts growth factor 8 (FGF8) and sonic hedgehog
(SHH) are crucial factors for the differentiation in midbrain DA neurons (Figure 3) [1]. Also
the co-culture of PSCs with PA6/MS5 stromal cells or midbrain astrocytes can induce the
differentiation in DA neurons [1,17]. The addition of glial cell line-derived neurotrophic
factor (GDNF), which is a neuroprotectant agent, has been shown to enhance the yield of DA
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neurons produced. In vivo experiments have proven the potential of these DA neurons to
medical applications, since in transplanted rats the movement errors were corrected [1].
2. Spinal Cord Motor Neurons
Neurodegenerative diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy
lead to poor life conditions, since patients rapidly lost their strength, the capacity of eat and
speak and their muscle becomes atrophied.
Retinoic acid (RA) and sonic hedgehog (SHH) have been shown to play an important role in
differentiation from PSCs into spinal motor neurons, located at the caudal and ventral part of
the neural tube (Figure 3) [11]. In vitro differentiation of spinal motor neurons is similar in
time to what happens in vivo. Grafted motor neurons derived from PSCs appear to be
functional and when transplanted in vivo (mouse and chick models) and they show high
levels of survival. Nonetheless, human in vivo experiments are needed, in order to confirm
the functionality of neurons, for further use in clinical applications. iPSCs can be a very
clever way to discover the mechanisms behind motor neuron degeneration [1].
3. GABAergic and cholinergic neurons
Previous work showed that the inhibition of WNTs signaling pathway and/or activation of
SHH lead to the conversion of PAX6 positive neural precursors to ventral progenitors,
generating GABAergic neurons, located in the striatum, and cholinergic neurons, located in
the basal forebrain [18]. Depending on the SHH concentration, different mature neural cells
are obtained. GABAergic neurons are the population with highest yield of production, about
87%, when cultured in appropriated concentrations of SHH. For low levels of SHH and
WNTs only GABAergic neurons are produced, but for high levels of SHH also cholinergic
projection neurons are obtained, however still in low percentages (Figure 3). The co-culture
of the progenitors with astrocytes has showed to improve the production of cholinergic
neurons. In vivo studies have reported the improvement of treatment for learning and memory
defects, in animals transplanted with cholinergic neurons. In hESCs BMP9 and NGF seem to
be necessary to induce cholinergic fate. Functional studies in vivo are required [1].
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4. Retinal precursors/neurons
Diseases associated with vision loss, as it is the case of age-related macular degeneration,
also deserve special attention since they affect an elevated percent of the whole population.
As all the other neuron types referred, also retinal cells can be differentiated from ESCs and
iPSCs, since they differentiate from the primitive anterior neuroectoderm, in vivo. Once
more, the events comprising the neuronal in vitro differentiation into retinal cells are similar
to those in vivo [1]. WNTs and Nodal antagonists have been shown to promote the
differentiation of retinal progenitors into retinal cells. For instance, retinal pigment epithelia
derived in vitro seems to be functional and has similar morphologies compared to native cells
[19].
Apart from the difficulty in the process, the use of neural progenitors (NPs), instead of matured cell
in transplantation cases, is a promising alternative to the complete differentiation of PSCs, in the
central nervous system (CNS) diseases treatment. Use of dibutyryl cyclic AMP (dbcAMP) and
interferon-gamma (IFN-γ) combined with NPs seems to enhance neuronal differentiation, since the
resulting cells express higher levels of β-III tubulin and present morphological differentiation [16].
OLIGODENDROCYTES COMMITEMENT FROM PSCS
At the moment, patients with demyelinating diseases, such as multiple sclerosis, characterized by
damage in the myelin sheath of neurons, are taking immunosuppressive drugs that extend their
quality of life and reduce pain. Once again, neuronal differentiation of hESCs or hiPSCs can
revolutionize the treatment of such diseases, performing not only the symptoms attenuation but the
effective damage reparation [20]. ESCs are a source for oligodendrocytes progenitor cells (OPCs)
generation that presents variable efficiency and stability results. Besides, they are not a suitable
source due to its availability and ethical concerns. Recent studies in mouse have shown that iPSCs
are also capable of oligodendrocytes differentiation with stability over 67 passages, when cultured in
an appropriated medium [21]. SHH is also involved in the differentiation of progenitors in
oligodendrocytes, in the ventral brain and spinal cord, since it has been shown that inhibition of SHH
leads to the non-generation of OPCs. FGF2, in mouse models, seems to promote SHH production,
leading to oligodendrocytes formation. In contrast, in human cells FGF2 appears to develop the
inverse function, inhibiting OPCs production. The events comprising the differentiation process also
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in this case are similar to those in vivo. The production of OPCs efficiency is low due to low rates of
progenitor’s proliferation [1].
ASTROCYTES COMMITEMENT FROM PSCS
Astrocytes are very promisor for cell-based gene therapy, as delivery vehicles, since they are the
most abundant cell type in the brain and spinal cord and are very important for central nervous
system function, supporting neurons [22]. Besides, astrocytes are more readily modified for drug
screening compared to other cell lines [1]. When transplanted to brain, these cells have been shown
to migrate along white matter tracts.
Gliomas, characterized by tumor mass development, are still a challenge disease which seeks for
better treatments. PSCs derived astrocytes represent an efficient source for gliomas treatment,
accordingly to previous mouse studies [22].
A recent study found that both hiPSCs and hESCs differentiate firstly in neurons and after in
functional astrocytes, phenotypically indistinguishable. Hedgehog (Hh) inhibitor induces the
differentiation of ESCs in astrocytes, while the same is done by the absence of CNTF, on hiPSCs
[23]. Since neurons are the most produced cell lineages, it is necessary to suppress neurogenesis and
promote gliogenesis, which is done by EGF, diminuishing β-III tubulin expression. Besides, ESCs
provide an abundant differentiation in astrocytes, which is not so clear for iPSCs [1].
Figure 3. Neuronal subtype specification in vivo and vitro. In the presence of a low concentration of SHH, the NE become
GABAergic projection neurons. With higher SHH concentration, the NE are fated to basal forebrain cholinergic neurons (BFCNs) and
GABAergic interneurons. Under RA and SHH presence NE differentiate to motor neurons (MNs). In the presence of FGF8 and SHH,
the primitive NE produce mDA neurons. [Adapted – [1]].
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FINAL REMARKS/FUTURE PRESPECTIVES
Derivation of ESCs and iPSCs, in animal and human studies, appear to be suitable for further clinical
applications, especially in neurological disorders, disease modeling and drug discovery. Molecular
and functional equivalence to ESCs should be further investigated, since it has been a controversial
issue until now, and it could affect the potential therapeutic utility [12]. The major problem
associated to iPSCs seems to be the reprogramming method, reported to be associated with
oncogenic potential [11]. Therefore the potential abnormalities associated to these cells should be
clarified, in order to verify if is it due to the reprogramming factors or to already existent mutations
on the patients somatic cells [10,11]. Small molecules are an alternative to reduce viral factors, only
to OCT4 and KLF4 [11]. Once more, this method should be improved, in order to apply these cells
in the clinical practice for disease treatment, without risks for the patient.
Moreover better protocols for ESCs and iPSCs are needed to achieve higher numbers of clones
formed and better specificity of those clones. Also the functionality of the achieved cells should be
tested in vivo.
REFERENCES
[1] Liu, H. and S. C. Zhang (2011). "Specification of neuronal and glial subtypes from human pluripotent
stem cells." Cellular and molecular life sciences : CMLS 68(24): 3995-4008;
[2] Martí, M., L. Mulero, et al. (2013). "Characterization of pluripotent stem cells." Nat. Protocols 8(2):
223-253;
[3] Patani, R., C. R. Sibley, et al. (2012). "Using human pluripotent stem cells to study post-transcriptional
mechanisms of neurodegenerative diseases." Brain Research 1462(0): 129-138;
[4] Yin, D., T. Tavakoli, et al. (2012). Comparison of Neural Differentiation Potential of Human
Pluripotent Stem Cell Lines Using a Quantitative Neural Differentiation Protocol. Human Embryonic
Stem Cells Handbook. K. Turksen, Humana Press. 873: 247-259;
[5] Shi, Y., P. Kirwan, et al. (2012). "Directed differentiation of human pluripotent stem cells to cerebral
cortex neurons and neural networks." Nat. Protocols 7(10): 1836-1846;
[6] Dhara, S. K. and S. L. Stice (2008). "Neural differentiation of human embryonic stem cells." Journal of
Cellular Biochemistry 105(3): 633-640;
[7] Imamura, K. and H. Inoue (2012). "Research on neurodegenerative diseases using induced pluripotent
stem cells." Psychogeriatrics 12(2): 115-119;
[8] Lu, H. F., S.-X. Lim, et al. (2012). "Efficient neuronal differentiation and maturation of human
Pag. 9

10. Stem Cell Bioengineering Journal STEM
REVIEW ARTICLE CELL
Journal
January 2013
pluripotent stem cells encapsulated in 3D microfibrous scaffolds." Biomaterials 33(36): 9179-9187;
[9] http://www.stembook.org/node/514;
[10] Yamanaka, S. (2012). "Induced Pluripotent Stem Cells: Past, Present, and Future." Cell stem cell 10(6):
678-684;
[11] Chamberlain, S., X.-J. Li, et al. (2008). "Induced pluripotent stem (iPS) cells as in vitro models of
human neurogenetic disorders." Neurogenetics 9(4): 227-235;
[12] Stadtfeld, M. and K. Hochedlinger (2010). "Induced pluripotency: history, mechanisms, and
applications." Genes & Development 24(20): 2239-226;
[13] Takahashi, K. and S. Yamanaka (2006). "Induction of Pluripotent Stem Cells from Mouse Embryonic
and Adult Fibroblast Cultures by Defined Factors." Cell 126(4): 663-676;
[14] Li XJ, Zhang SC (2006). “In vitro differentiation of neural precursors from human embryonic stem
cells”. Methods Mol Biol 331:169–177;
[15] Yu J, et al (2007). “Induced pluripotent stem cell lines derived from human somatic cells”. Science
318:1917–1920;
[16] Zahir T, et al (2009). “Neural stem/progenitor cells differentiate in vitro to neurons by the combined
action of dibutyryl cAMP and interferon-gamma”. Stem Cells Dev 8(10):1423-32;
[17] Park IH, et al (2008) “Disease-specific induced pluripotent stem cells”. Cell 134(5):877-86;
[18] Li XJ, et al (2009). “Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal
telencephalic neuron types from human embryonic stem cells”. Development 136:4055–4063;
[19] Kagiyama Y,et al (2005). “Extraocular dorsal signal affects the developmental fate of the optic vesicle
and patterns the optic neuroepithelium”. Dev Growth Differ 47:523–536;
[20] Czepiel, M., V. Balasubramaniyan, et al. (2011). "Differentiation of induced pluripotent stem cells into
functional oligodendrocytes." Glia 59(6): 882-892;
[21] Onorati, M., S. Camnasio, et al. (2010). "Neuropotent self-renewing neural stem (NS) cells derived
from mouse induced pluripotent stem (iPS) cells." Molecular and Cellular Neuroscience 43(3): 287-
295;
[22] Emdad L., et al (2012). “Efficient differentiation of human embryonic and induced pluripotent stem
cells into functional astrocytes”. Stem cells dev 21(3):404-10;
[23] Yuan SH, et al. (2011). “Cell-surface marker signatures for the isolation of neural stem cells, glia and
neurons derived from human pluripotent stem cells”. PLoS One 6(3).
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