Developmental Plasticity of CNS stem cells

Developmental Plasticity of CNS Stem Cells:
Implications for Neurodevelopmental/Psychiatric Diseases

1Developmental Plasticity of CNS stem cells

Abstract

Interest in neurodevelopmental and psychiatric diseases, including autism spectrum
disorders, and metal disorders including schizophrenia, continues to increase in the
medical science as well as with respect to social issues. However, very few effective
biomedical therapies are available for the treatment of such conditions, which may
be attributable to the lack of knowledge of the basic principles governing thedevelopment, shaping and operation of the human brain. These deficits in scientificunderstanding drew my interest to the physiological regulation of the development of
the central nervous system (CNS), leading to the acquisition of higher brain
functions. I hypothesize that impairment of these processes leads to the developmentof various neurodevelopmental and psychiatric diseases. As a first step, I have sought
to experimentally investigate the temporal regulation of developmental plasticity ofstem cells in the CNS, and was able to recapitulate the “first neuron and glia
generation” principle in vitro by inducing the neural differentiation of mouseembryonic stem cells. In introducing this work, I will also suggest how thesefindings might help to deepen our understanding of the human brain and diseases of
the CNS.
2

Developmental Plasticity of CNS stem cells

Introduction:
The Rationale to investigate CNS stem Cells

The human brain, which is composed of a trillion cells including neurons andglia (the name “glia” refer to astrocytes, oligodendrocytes and so on), is one of themost complex biological organizations on this planet. Cellular communities such asneuronal networks and neuron-glia interactions have enabled us to acquire higher
brain functions. I believe that various neurodevelopmental and psychiatric diseases
develop because of impairment of these brain functions, leading to serious social
problems and economic burdens. At present, the media has provided us with a hugeamount of information about neurodevelopmental/psychiatric diseases, including
autism spectrum disorders such as Asperger syndrome, Savant syndrome andattention deficit hyperactivity disorder (ADHD), as well as related mental disorders,such as schizophrenia, from the aspects of both medical science and social issues.
People who suffer from these kinds of neurodevelopmental/psychiatric disorders tend
to have difficulties in their learning abilities or social skills (Osumi, 2006, chap.1).
However, it is also interesting to notice that a certain population of people/children3

Developmental Plasticity of CNS stem cellswith neurodevelopmental disorders are gifted with higher abilities in other fields
such as art, photographic memory, imagination, etc. These people with autism whoare endowed cognitively with “higher functioning” are diagnosed as having
high-functioning autism (HFA) (Rao et al., 2008, p.353). Historically, people whohave had great impacts on human civilization, such as Isaac Newton, Albert Einstein,
Wolfgang Amadeus Mozart, Ludwig van Beethoven, and Lewis Carroll, are said tohave suffered from HFA or Asperger syndrome (Osumi, 2016, chap.1). It is also
unknown why several kinds of neurodevelopmental/psychiatric diseases occur
simultaneously in the same individuals. I suspect that this situation results from theambiguity of diagnosis for these diseases, due to the lack of basic understanding ofthese diseases. To identify concrete mechanisms leading to these diseases and todevelop their innovative therapeutic methods, the normal developmental processes in
the human brain must first be studied and then contrasted with those ofpatient-specific developmental processes. To understand these processes, I came to
be interested in the stem cells present in developing and adult central nervous system(CNS), so-called “CNS stem cells,” which produce a wide variety of cells within the
CNS (Temple, 2001, p.113). Particularly, as a first step to finding a way to4

Developmental Plasticity of CNS stem cells

understand and cure neurodevelopmental/psychiatric diseases, I have tried to confirm
the temporal regulation of the differentiation ability (which I term “developmental
plasticity”) of CNS stem cells using actual data obtained from experiments that I
performed during the summer of 2016.
The precise mechanisms of how a wide variety of neuronal and glial cells areproduced during CNS development largely remain unknown. Previous studies haveshown that both neurons and glia are generated from CNS stem cells in spatially andtemporally regulated fashions. It is also known that a wide variety of neuronalsubtypes are generated according to their spatial and temporal identities. The
developing neural tube (a prototype of the CNS) is known to subdivided into distinct
domains along two perpendicular axes, i.e., anteroposterior (A-P) and dorsoventral(D-V) axes, and each region produces a specific subtype of CNS stem cells(Imaizumi et al., 2015, p.1010). Currently, regional identities of CNS stem cells canbe obtained in vitro during neural differentiation of pluripotent stem cells (PSCs),
including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),by modulating signals involved in development of A-P and D-V axes (Imaizumi et al.,2015, p.1011).
5

Developmental Plasticity of CNS stem cells

Regarding the temporal specification of CNS stem cells, the order of “firstneuron and glia generation” is widely conserved across both CNS regions (Okano
and Temple, 2009, p.112). However, precise mechanisms for how CNS stem cells
acquire their temporal identities remain largely elusive. In the present study, I could
confirm the “first neuron and glia generation” principle in a dish utilizing neuraldifferentiation of mouse ESCs through improvements of the previous protocol(Okada et al., 2008, p. 3087). This method will be applied, in combination withgenome editing and patients-specific iPSCs, to investigate functions of genes that arepotentially involved in the specification of CNS stem cells and to modelneurodevelopmental/psychiatric diseases (Okano and Yamanaka, 2014, p.6) to finally
understand and conquer these diseases.

Experimental Procedures
ESCs Culture and Neural Induction
Mouse ESCs (EB3) were cultured as reported previously (Niwa et al., 2002,
p.1527). Their neural differentiation was performed using a protocol similar to thatof a previously report (Okada, 2008, p.3087). Formation of primary, secondary and6

Developmental Plasticity of CNS stem cells

tertiary neurospheres and subsequent their differentiation was performed according
to the previous method (Okada et al., 2008, p.3087). However, I made the following
modification: undifferentiated mouse ESCs were dissociated and used for embryoid
bodies (EBs) formation in the presence of 150 nM LDN193189 (StemRD), instead of
Noggin protein.
Immunocytochemistry and Cell Counting
Immunocytochemical analysis of cultured cells was performed according to aprevious report (Imaizumi et al., 2015, p.1018) using anti-β-TUBULIN III(Sigma-Aldrich) and anti-GFAP (DAKO Cytomation) as the primary antibodies andobserved using a LSM-710 confocal laser microscope (Carl Zeiss). The number of
-TUBULIN

III-positive

neurons,

GFAP-positive

glia

and

total

cells

(Hochest33342-positive cells) was determined by counting the number of each typeof cell present in 10 visual fields for each slide. For the statistical analysis, theStudent t-test was used (*P < 0.05, ***P< 0.001).

Results and Discussions
Maintenance of mouse ESCs and neural induction
7

Developmental Plasticity of CNS stem cells

To examine the effects of temporal changes in the differentiation potential of
CNS stem cells, I took advantage of a neural induction system consisting of mouse
ESCs (Fig.1), using the EB3 line, which carries a Blasticidin (an antibiotic)-resistantgene under the promoter of Oct4 gene (a pluripotent stem cell marker gene) for
maintenance of undifferentiated and pluripotent state. To induce the differentiation of
EB3 cells, I replaced the medium with Blasticidin-free one and allowed embryoid
bodies (EBs) to form. To obtain efficient neural induction during EBs-formation, I
tried to block the BMP signaling pathway, which inhibits neural development (Smithand Harland, 1992, p.829). Noggin is an endogenous factor involved in neuralinduction by blocking the BMP signal (Zimmerman et al., 1996, p.599); thus, arecombinant Noggin protein was added to cell cultures to induce EBs topreferentially adopt a neural fate in a previous study (Okada et al., 2008, p.3087).
Noggin-treated EBs allow the induction of CNS stem cells, which can then be
expanded using the neurosphere method. However, the recombinant Noggin protein
is an expensive reagent; thus, I added a cheaper inhibitor of the BMP signaling
pathway, LDN193189, which is a small chemical compound that can inhibit BMP
type I receptors ALK2/3 (Yu et al., 2008, p.1363). The replacement of Noggin with8

Developmental Plasticity of CNS stem cells

LDN193189 worked well, and I was able to efficiently induce primary neurosphere
formation from LDN193189-treated EBs.
Changes in Differentiation Potential of CNS stem cells during passage
Next, I examined whether the passage of primary neurospheres into secondary
and tertiary neurospheres could result in a change in the differentiation potential of
CNS stem cells (Figs. 2 and 3). Consequently, I was able to obtain the following data
based on the three independent experiments: i) primary neurospheres almost
exclusively gave rise to neurons, ii) the glial cell differentiation potential of CNS
stem cells increased significantly during passages of neurospheres (Primary <Secondary, Tertiary) (*P < 0.05, ***P< 0.001), iii) the neuronal differentiation
potential of CNS stem cells decreased significantly during cell passages (Primary >Secondary, Tertiary) (*P < 0.05, ***P< 0.001). Interestingly, these results obtained invitro using mouse ESCs-derived CNS stem cells were consistent with the in vivo
situation, i.e., “first neuron and glia generation” (Temple, 2001, p.112).
Evidence showing early CNS stem cells lack a response to gliogenic signals
Since the primary neurospheres were almost exclusively composed ofneurons, but not glia, in the experiments described in Figure 1, it is likely that “early”
9Developmental Plasticity of CNS stem cells

CNS stem cells lack the competency to respond to gliogenic signals (glia-inducing
factors). Alternatively, it is possible that the amount of these factors) was insufficient
to promote glial differentiation. To investigate these possibilities, I examined theeffects of BMP-2, a well-known glia-inducing factor (Nakashima et al., 1999, p.479),
on the glial differentiation of primary neurospheres in the same experiments shownin Figs 2 and 3. Interestingly, primary neurospheres still preferentially gave rise toneurons even in the presence of exogenously added BMP-2. Thus, early CNS stem
cells have little competency to respond to gliogenic signals, consistent with the “firstneuron and glia generation” principle.
Notably, a recent report charactering patients-specific iPSCs derived from Rett
syndrome (a neurodevelopmental disease resulting in mental retardation and autistic
behavior (Hargberg et al., 1983, p.471-479)) indicates that glial differentiation
potential is prematurely induced in patients-derived early CNS stem cells(Andoh-Noda, 2015, p.1). This would provide an evidence showing the investigationof temporal identity of CNS stem cells is essential for understanding
neurodevelopmental/psychiatric diseases. How their impaired temporal identity
could lead to these diseases would be important issues to be clarified in the future.
10

Developmental Plasticity of CNS stem cells

Perspectives: Advanced research I can do by applying this method
In the present study, I treated and characterized wild type ESCs-derived
neurospheres. The method used in the present study is a fundamental strategy forevery kind of experiment using ESC/iPSCs-derived neurospheres, including thosederived from human patient-derived iPSCs. Using this method, I will be able toanalyze the relationship between the differentiation potential and the functions ofspecific genes using transgenic or genetically modified ESCs and iPSCs. Thus, thepresent technology is likely to contribute to the fundamental understanding of
neurodevelopmental/psychiatric diseases, which I hope to continue studying in the
future.

Conclusions
1. The temporal changes in the differentiation potential of CNS stem cells-derived
mouse ESCs were recapitulated in a manner that reflected those occurring in vivo
during CNS development.
2. I could develop an improved method for the neural induction of mouse ESCs
11

Developmental Plasticity of CNS stem cells

using a small chemical BMP-inhibitor.
3. The current method will be useful for the investigation ofneurodevelopmental/psychiatric diseases by analyzing the developmental
plasticity of CNS stem cells using patient-derived iPSCs.

Acknowledgements
I have done almost all the experiments described in the present article by myself at
Department of Physiology, Keio University School of Medicine under the guidanceof the mentor in the summer of 2016, by participating in Global Science Program
Campus program at Keio University which is aimed to host high school students forfacilitating their scientific activities with a support of Japan Science and Technology
Agency. I would like to thank members of Department of Physiology, Keio
University School of Medicine for their kind instructions.

12Developmental Plasticity of CNS stem cells

Figure 1.

Induction of CNS stem cells from mouse ESCs by forming EBs andneurospheres.

Figure 2.

Differentiation of primary (left), secondary (middle), and tertiary (right)

neurospheres into III-TUBULIN-positive neurons (red) and GFAP-positive glia
(green). Quantitative data are shown in Fig.3.

13

Developmental Plasticity of CNS stem cells

Figure 3.

Ratio of neuronal (upper panel) or glial (lower panel) differentiation ofmouse ESCs-derived primary, secondary and tertiary neurospheres based on the
immunocytochemical data.

14

Developmental Plasticity of CNS stem cells

References
Andoh-Noda, T., Akamatsu, W., Miyake, K., Matsumoto, T., Yamaguchi, R.,
Sanosaka, T., Okada, Y., Kobayashi, T., Ohyama, M., Nakashima, K.,
Kurosawa, H., Kubota, T., & Okano, H. (2015). Differentiation of multipotent
neural stem cells derived from Rett syndrome patients is biased toward theastrocytic lineage. Molecular Brain, 8, 31.
Hagberg, B., Aicardi, J., Dias, K., & Ramos, O. (1983). A progressive syndrome ofautism, dementia, ataxia, and loss of purposeful hand use in girls: Rett's
syndrome: report of 35 cases. Annals of Neurology, 14, 471–479.
Imaizumi, K., Sone, T., Ibata, K., Fujimori, K., Yuzaki, M., Akamatsu, W., & Okano,
H. (2015). Controlling the regional identity of hPSC-Derived neurons touncover neuronal subtype specificity of neurological disease phenotypes. Stem
Cell Reports. 5, 1010-1022.
Nakashima, K., Yanagisawa, M., Arakawa, H., Kimura, N., Hisatsune, T., Kawabata,M., Miyazono, K., & Taga, T. (1999). Synergistic signaling in fetal brain by
STAT3-Smad1 complex bridged by p300. Science, 284, 479-482.
Niwa, H., Masui, S., Chambers, I., Smith, A.G., & Miyazaki J. (2002). Phenotypic15

Developmental Plasticity of CNS stem cells

complementation establishes requirements for specific POU domain andgeneric transactivation function of Oct-3/4 in embryonic stem cells. Molecular
and Cellular Biology, 22, 1526–1536.
Okada, Y., Matsumoto, A., Shimazaki, T., Enoki, R., Koizumi, A., Ishii, S., Itoyama,Y., Sobue, G., & Okano, H. (2008). Spatiotemporal recapitulation of central
nervous system development by murine embryonic stem cell-derived neural
stem/progenitor cells. Stem Cells, 26, 3086-3098.
Okano, H., & Temple, S. (2009). Cell types to order: temporal specification of CNS
stem cells. Current Opinion in Neurobiology, 19,112-119.
Okano, H., & Yamanaka, S. (2014). iPS cell technologies: significance andapplications to CNS regeneration and disease. Molecular Brain 7, 22.
Osumi, N. (2016). A neurodevelopmental perspective toward understanding ofautism spectrum. Tokyo: Kodansha. (in Japanese)
Rao, P.A., Beidel, D.C., & Murray, M.J. (2008). Social skills interventions forchildren with Asperger's syndrome or high-functioning autism: a review andrecommendations. Journal of Autism and Developmental Disorders, 38,353-361.
16

Developmental Plasticity of CNS stem cells

Smith, W.C., & Harland, R.M. (1992). Expression cloning of noggin, a newdorsalizing factor localized to the Spemann organizer in Xenopus embryos.
Cell, 70, 829–840.
Temple, S. (2001). The development of neural stem cells. Nature, 414, 112–117.
Yu, P.B., Deng, D.Y., Lai, C.S., Hong, C.C., Cuny, G.D., Bouxsein, M.L., Hong,
D.W., McManus, P.M., Katagiri, T., Sachidanandan, C., Kamiya, N., Fukuda,T., Mishina, Y., Peterson, R.T., & Bloch, K.D. (2008). BMP type I receptor
inhibition reduces heterotopic ossification. Nature Medicine, 14, 1363-1369.
Zimmerman, L.B., De Jesus-Escobar, J.M., & Harland, R.M. (1996). The Spemannorganizer signal noggin binds and inactivates bone morphogenetic protein 4.
Cell, 86, 599–606.

17