Fwd: Region-specific generation of cholinergic neurons from fetal human neura...

November 17, 2002

#1

> Region-specific generation of cholinergic neurons from fetal human neural

> stem cells grafted in adult rat

>

> >> Ping Wu1, 2, Yevgeniya I. Tarasenko1, Yanping Gu1, Li-Yen M. Huang2, 3,

>> E. Coggeshall1, 2 & Yongjia Yu4

>>

>> 1. Departments of Anatomy & Neurosciences, University of Texas Medical

>> Branch, Galveston, Texas 77555, USA

>> 2. Marine Biomedical Institute, University of Texas Medical Branch,

>> Galveston, Texas 77555, USA

>> 3. Departments of Physiology & Biophysics, University of Texas Medical

>> Branch, Galveston, Texas 77555, USA

>> 4. Department of Radiation Oncology, University of Texas Medical Branch,

>> Galveston, Texas 77555, USA

>> Correspondence should be addressed to P Wu. e-mail: <A

HREF= " http://www.nature.com/neuro/email_response/email.taf?address=piwu%40utmb.e\

du " >piwu@...</A>

>>

>

> Pluripotent or multipotent stem cells isolated from human embryos or adult

> central nervous system (CNS) may provide new neurons to ameliorate neural

> disorders. A major obstacle, however, is that the majority of such cells do

> not differentiate into neurons when grafted into non-neurogenic areas of

> the adult CNS. Here we report a new in vitro priming procedure that

> generates a nearly pure population of neurons from fetal human neural stem

> cells (hNSCs) transplanted into adult rat CNS. Furthermore, the grafted

> cells differentiated by acquiring a cholinergic phenotype in a

> region-specific manner. This technology may advance stem cellâ€“based therapy

> to replace lost neurons in neural injury or neurodegenerative disorders.

>

> Advances in stem cell research have enabled the isolation and propagation

> of human embryonic stem (ES) cells<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B1 " >1</A> and germ (EG) cells<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B2 " >2</A>. These cells are

> pluripotentâ€”they can become any cell type in the human body, including

> neurons. Multipotent neural stem cellsâ€”another source for neural cellsâ€”

> have also been isolated successfully from either fetal<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B3 " >3-7</A> or adult<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B8 " >8-10</A>

> human central nervous system (CNS). Their properties of self-renewal and

> multipotential differentiation make stem cells an attractive and presumably

> unlimited donor source for cell replacement therapy to treat neurological

> disorders.

> Human and rodent stem cells are able to differentiate into specific

> neuronal types when grafted into either developing CNS<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B11 " >11-14</A> or neurogenic

> areas of the adult CNS<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B15 " >15-17</A>. However, these cells remain undifferentiated

> or become mainly glial cells when transplanted into non-neurogenic regions

> of the adult CNS<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B16 " >16-20</A>, indicating that in vitro priming or some

> differentiation prior to grafting is necessary for these cells to develop

> specific neuronal subtypes. In particular, there have been no reports as

> yet of the generation of a significant number of cholinergic neurons from

> long-term mitogen-expanded human stem cells. As these neurons are centrally

> involved in motor function, learning and memory, they are highly relevant

> to clinical applications. For example, human stem cellâ€“derived cholinergic

> neurons may be used to replace motoneurons lost in amyotrophic lateral

> sclerosis (ALS or Lou Gehrig's disease) or spinal cord injury. Here we

> report a simple yet efficient priming procedure that caused

> mitogen-expanded primary fetal human neural stem cells (hNSCs) to become

> almost pure neurons when grafted into both non-neurogenic and neurogenic

> areas of adult rat CNS. Most importantly, a large number of these

> transplanted cells developed into cholinergic neurons when grafted in

> medium septum and spinal cord.

>

> >> Results

>

> Primed hNSCs become cholinergic neurons in vitro To obtain cholinergic

> neurons, we treated K048 hNSCs with tropic factors or other chemicals that

> are important in the development of cholinergic neurons<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B21 " >21</A>, <A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B22 " >22</A>, including

> recombinant human basic fibroblast growth factor (bFGF), epidermal growth

> factor (EGF), leukemia inhibitory factor (LIF), mouse sonic hedgehog

> amino-terminal peptide (Shh-N), all-trans retinoic acid (RA), nerve growth

> factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3

> (NT-3), neurotrophin-4 (NT-4), natural mouse laminin and heparin. The K048

> cell line was originally derived from the cortex of an 8-week legally

> aborted human fetus<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B5 " >5</A> and mitogen-expanded in vitro without viral or

> chemical treatments for immortalization. In this study, K048 hNSCs have

> been passaged continuously in vitro for over two years (85 passages)

> without changes in their proliferation and differentiation patterns, or in

> their normal diploid karyotype. These long-term epigenetically expanded

> hNSC spheres (19â€“55 passages or 38â€“52 weeks) were plated onto poly-D

> -lysine (PDL) and laminin-coated dishes and treated with the above agents

> in vitro at various concentrations either alone or in combination,

> concurrently or sequentially.

>

> The combination consisting of bFGF, heparin and laminin (abbreviated as

> FHL) had unique effects on fetal hNSCs. Thus, a one-day exposure to FHL,

> with or without Shh-N (combination of Shh-N and FHL abbreviated as SFHL),

> resulted in a rapid spreading of large planar cells in culture (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F1.html " >F\

ig. 1a</A>).

> All other one-day treatments (such as bFGF plus laminin, abbreviated FL),

> in contrast, gave rise to a limited radial spread of spindle-shaped cells,

> with the cells remaining close to the cores of the spheres (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F1.html " >F\

ig. 1b</A>),

> similar to a previous description<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B5 " >5</A>. After 6 days of priming and a 10-day

> further differentiation in medium containing B27, large multipolar

> neuron-like cells were found near the edge of the FHL-primed spheres (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F1.html " >F\

ig.

> 1c</A>), while most of the neuron-like cells were small and bipolar or

unipolar

> in other treatment groups (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F1.html " >F\

ig. 1d</A>).

> Immunocytochemical analyses indicated that the small bipolar or unipolar

> cells (<20 m) were either GABAergic or glutamatergic (data not shown), as

> found previously<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B23 " >23</A>. On the other hand, many of the large multipolar cells

> near the edge of FHL-primed spheres, whose neuronal phenotype was shown by

> immunocytochemical staining using a monoclonal antibody against the

> neuron-specific type III -tubulin (TuJ1; <A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F1.html " >F\

ig. 1e</A>), were cholinergic because

> they showed immunoreactivity to markers specific for cholinergic neurons

> such as Islet-1 (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F1.html " >F\

ig. 1f</A>) and choline acetyltransferase (ChAT; <A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F1.html " >F\

ig. 1g</A>).

> Some of these neurons also expressed synapsin I (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F1.html " >F\

ig. 1h</A>). From five

> independent experiments, we estimated that of the total cell population in

> the monolayer regions, 45.5 4.7% differentiated into TuJ1+ neurons,

> whereas cholinergic (ChAT+) neurons accounted for 27.8 4.2% of the same

> population. However, multiple layers in and around the core of spheres

> prevented absolute quantification of any given phenotype in the total

> population. Furthermore, ChAT+ neurons became undetectable if

> differentiated cells were subjected to further dissociation and re-plating,

> a procedure described previously<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B23 " >23</A>. In addition to the cholinergic neurons,

> significant numbers of small neurons (glutamatergic, 6.3 0.5%; GABAergic,

> 11.3 1.4%), astrocytes (35.2 2.8%) and nestin+ cells (18.9 2.0%) were

> also detected in FHL-primed neurospheres even after the additional 10 days

> of differentiation in vitro. On the other hand, no ChAT or Islet-1 positive

> cells were detected in hNSCs untreated or treated with other reagents, in

> which they either became astrocytes or small glutamatergic or GABAergic

> neurons. Astrocytes and small neurons ranged from 40â€“70% and 10â€“60% of the

> total cells, respectively.

> To determine whether the large cells have the electrical characteristics of

> neurons, resting potentials and action potentials were monitored using the

> whole-cell patch clamp recording technique. Seven days after FHL-priming,

> most of the large multipolar cells (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F1.html " >F\

ig. 1i</A>) had resting potentials (-29.0

> 2.0 mV, n = 6), but no action potentials could be evoked. These resting

> potentials were much more negative (-63.6 3.0 mV, n = 5) 14 days after FHL

> treatment, and action potentials were seen when depolarizing currents were

> injected (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F1.html " >F\

ig. 1j</A>). The action potentials were blocked by 1 M tetrodotoxin

> (TTX, <A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F1.html " >F\

ig. 1k</A>). Thus, our data suggested that the FHL-priming procedure

> directed some of fetal hNSCs in vitro to become functional neurons

>

> To determine whether FHL priming has the same effect on other fetal hNSCs

> apart from K048, we tested the K054 cell line, which is derived from the

> cortex of a 10-week human fetus<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B5 " >5</A>. K054 cells (20 passages for 42 weeks)

> behaved very similarly to K048 cells in terms of their differentiation

> patterns and responses to different priming treatments (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F2.html " >F\

ig. 2aâ€“f</A>). Thus,

> FHL-priming for 6 days followed by a 10-day further differentiation

> resulted in the appearance of cholinergic neurons (30.9 3.1%) in vitro (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F2.html " >

> Fig. 2gâ€“h</A>). Other cell phenotypes included nestin+ cells (17.7 1.8%),

> GFAP+ astrocytes (35.8 4.5%) and TuJ1+ (45.3 4.1%), glutamatergic (5.6

> 0.7%) and GABAergic (8.7 1.1%) neurons. Exclusion of FH produced small

> GABA or glutamate neurons, but not ChAT+ neurons.

>

November 17, 2002