NEUROVASCULAR MEDICINE - Pursuing Cellular Longevity for Healthy Aging Part 5 pdf

228 STEM AND PROGENITOR CELLS IN DEGENERATIVE DISORDERS

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231

Chapter 9

MOTONEURONS FROM HUMAN

EMBRYONIC STEM CELLS:

PRESENT STATUS AND FUTURE

STRATEGIES FOR THEIR USE IN

REGENERATIVE MEDICINE

K. S. Sidhu

ABSTRACT

Human embryonic stem (ES) cells are pluripotent and

can produce the entire range of major somatic cell

lineage of the central nervous system (CNS) and thus

form an important source for cell-based therapy of

various neurological diseases. Despite their potential

use in regenerative medicine, the progress is ham￾pered by diffi culty in their use because of safety

issues and lack of proper protocols to obtain puri￾fi ed populations of specifi ed neuronal cells. Most

neurological conditions such as spinal cord injury

and Parkinson’s disease involve damages to projec￾tion neurons. Similarly, certain cell populations may

be depleted after repeated episodes of attacks such

as the myelinating oligodendrocytes in multiple scle￾rosis. Motoneurons are the key effector cell type for

control of motor function, and loss of motoneurons

is associated with a number of debilitating diseases

such as amyotrophic lateral sclerosis (ALS) and spi￾nal muscular atrophy; hence, repair of such neuro￾logical conditions may require transplantation with

exogenous cells. Transplantation of neural progeni￾tor cells in animal models of neurological disorders

and in patients from some clinical trial cases has

shown survival of grafted cells and contribution to

functional recovery. Recently a considerable progress

has been made in understanding the biochemical,

molecular, and developmental biology of stem cells.

But translation of these in vitro studies to the clinic

has been slow. Major hurdles are the lack of effec￾tive donor cells, their in vivo survival, and diffi culty in

remodeling the non-neurogenic adult CNS environ￾ment. Several factors play a role in maintaining their

functions as stem cells. It is becoming increasingly

apparent that the role of developmental signaling

molecules is not over when embryogenesis has been

completed. In the adult, such molecules might func￾tion in the maintenance of stem cell proliferation, the

regeneration of tissues and organs, and even in the

maintenance of their differentiated state. A major

challenge is to teach the naïve ES cells to choose a

neural fate, especially the subclasses of neurons and

232 STEM AND PROGENITOR CELLS IN DEGENERATIVE DISORDER

glial cells that are lost in neurological conditions.

I review the progress that has been achieved with ES

cells to obtain motoneurons and discuss how close

we are to translating this research to the clinics.

Keywords: central nervous system, neuroectoderm,

motoneurons, cell replacement therapy, growth fac￾tors, neural induction.

The development of CNS involves spatial

distribution and networking (circuitry) of

neuronal and glial cells. These anatomical

developments undergo modifi cations dur￾ing functional maturation. Insults, injury, or disease

causes damage or loss of certain elements in the CNS

circuitry that disrupts the neural network. Repair of

these circuits would require sequential reactivation of

the developmental signals in a particular spatial order,

for which the adult mammalian brain and spinal cord

have limited capacity (Steiner, Wolf, Kempermann

2006). Consequently, the adult brain often fails to

repair the neural framework assembled by projection

neurons despite the presence of stem cells or progeni￾tors. These stem/progenitor cells in adult life appear

to be designed for replenishing other parts of the CNS,

because they differentiate primarily into interneurons

and glial cells (Steigner, Wolf, Kempermann 2006).

Most neurological conditions such as spinal cord

injury and Parkinson’s disease involve damages to

projection neurons. In other circumstances, certain

cell populations may be depleted after repeated epi￾sodes of attacks such as the myelinating oligodendro￾cytes in multiple sclerosis. Motoneurons are the key

effector cell type for control of motor function, and

loss of motoneurons is associated with a number of

debilitating diseases such as ALS and spinal muscu￾lar atrophy (Lefebvre, Burglen, Reboullet et al. 1995;

Cleveland, Rothstein 2001). Hence, repair of such neu￾rological conditions may require transplantation with

exogenous cells. Transplantation of neural progenitor

cells in animal models of neurological disorders and

in patients from some clinical trial cases has shown

survival of grafted cells and contribution to functional

recovery. Laboratory investigation into understanding

the biochemical, molecular, and developmental biol￾ogy of stem cells has progressed rapidly in the last few

years. However, until relatively recently, translation

of these in vitro studies to the clinic has been slow.

Neural replacement as a therapy still needs further

laboratory investigations. Major hurdles are the lack

of effective donor cells, their in vivo survival, and dif￾fi culty in remodeling the non-neurogenic adult CNS

environment. Several factors play a role in maintain￾ing their functions as stem cells. It is becoming increas￾ingly apparent that the role of developmental signaling

molecules is not over when embryogenesis has been

completed. In the adult, such molecules might func￾tion in the maintenance of stem cell proliferation,

the regeneration of tissues and organs, and even

in the maintenance of their differentiated state

(Maden 2007).

Derivation of functional neurons from human

embryonic stem cells (hESCs) as surrogate in regen￾erating medicine for treating various neurodegene￾rative diseases is the subject of intensive investigation.

Three basic features of hESCs, that is, self-renewal,

proliferation, and pluripotency, make them immortal,

capable of unlimited expansion and differentiation

into all 230 different type of cells in the body, and

thus hold great potential for regenerative medicine

(Hardikar, Lees, Sidhu et al. 2006; Valenzuela,

Sidhu, Dean et al. 2007). Most published protocols

for guiding the differentiation of these cells result in

heterogeneous cultures that comprise neurons, glia,

and progenitor cells, which makes the assessment of

neuronal function problematic. However, many recent

studies including from our laboratory (Lim, Sidhu,

Tuch 2006) have demonstrated that enough purifi ed

neurons could be generated from hESCs and used

for carrying out gene expression and protein analyses

and for examining whether they can form functional

networks in culture (Benninger, Beck, Wernig et al.

2003; Zhang 2003; Keirstead, Nistor, Bernal et al.

2005; Muotri, Nakashima, Toni et al. 2005; Ben-Hur

2006; Soundararajan, Miles, Rubin et al. 2006; Lee,

Shamy, Elkabetz et al. 2007; Soundararajan, Lindsey,

Leopold et al. 2007; Wu, Xu, Pang et al. 2007; Zeng,

Rao 2007). This review will discuss how recent

advancement in stem cell technology offers hope for

generating potential effective donor cells for replace￾ment therapy with a special emphasis on develop￾mental potentials of ES cells.

POTENTIAL USE OF HUMAN

EMBRYONIC STEM CELLS

Adult stem cells are restricted during development to

a particular fate of the tissue in which they are found.

Brain-derived neural stem cells may generate neurons

and glia. However, the subclasses of neurons and glia

differentiated from neural stem cells depend on the

regions and developmental stages in which the pro￾genitor cells are isolated and expanded. Thus, the

ideal stem cell population would be those that can

generate most or all subtypes of neurons and glial

cells. Presently, the best known cells that possess such

traits are ES cells. ES cells are able to differentiate into

all cell and tissue types of the body. Technology has

been developed to selectively maintain and expand

mouse and human ES cells in a synchronized, undif￾ferentiated state. Compared to adult stem cells, ES

Chapter 9: Motoneurons from Embryonic Stem Cells 233

recently some of the studies have been successful in

purifying enough hESC-derived neurons to carry out

gene expression and protein analyses and examine

whether they can form functional networks in culture

(Lim, Sidhu, Tuch 2006; Lee, Shamy, Elkabetz et al.

2007; Soundararajan, Lindsey, Leopold et al. 2007).

However, different hESC lines behave very differ￾ently in cultures and have variable potential to pro￾duce neurons (Lim, Sidhu, Tuch 2006; Wu, Xu, Pang

et al. 2007).

NEUROECTODERMAL INDUCTION

Neuroectodermal Induction

and Neuronal Specifi cation

The production of neurons involves several sequen￾tial steps precisely orchestrated by signaling events

(Wilson, Edlund 2001). The initial step is the specifi -

cation of neuroepithelia from ectoderm cells, the pro￾cess known as neural induction, which is accomplished

by inductive interaction with nascent mesoderm and

defi nitive endoderm. Despite being a topic of inten￾sive study, there is still no consensus on the mecha￾nisms and signals involved in neural induction. Bone

morphogenetic protein (BMP) antagonism has been

viewed as the central and initiating event in neural

induction. According to this concept, neuroepithelial

specifi cation occurs as a default pathway (Munoz￾Sanjuan, Brivanlou 2002). However, recent fi ndings

challenge this neural default model and indicate some

positive instructive factors, such as fi broblast growth

factors (FGFs) and Wnt. For example, interference

cells can be expanded in vitro with current technol￾ogy for a prolonged period, and yet they retain the

genetic normality. Hence, ES cells can provide a large

number of normal cells for deriving the desired cells

for transplant therapy. A major challenge is to teach

the naïve ES cells to choose a neural fate, especially

the subclasses of neurons and glial cells that are lost

in neurological conditions.

hESCs are pluripotent cells derived from the inner

cell mass of preimplantation embryos (Thomson

1998). Like mouse embryonic stem (ES) cells, theo￾retically they can differentiate into various somatic

cell types (Fig. 9.1) with a stable genetic background

(Thomson 1998; Amit, Carpenter, Inokuma et al.

2000; Reubinoff, Pera, Fong et al. 2000; Thomson,

Odorico 2000; Sidhu, Ryan, Tuch 2008). These

unique features make hESCs a favorable tool for

biomedical research as well as a potential source for

therapeutic application in a wide range of diseases

such as Parkinson’s disease, Alzheimer’s disease, and

spinal cord injuries. Directing ES cells to differentiate

to cells of interest, such as neural lineages, depends

on strategies based on the understanding of mamma￾lian neural development (Lee Lumelsky, Studer et al.

2000; Tropepe, Hitoshi, Sirard et al. 2001; Billon,

Jolicoeur, Ying et al. 2002; Wichterle, Lieberam, Porter

et al. 2002; Ying, Stavridis, Griffi ths et al. 2003).

Mass-scale production of functional neurons from

hESCs for treating neurodegenerative diseases is the

subject of intensive investigation. Most published pro￾tocols for guiding the differentiation of these cells

result in heterogeneous cultures that comprise neu￾rons, glia, and progenitor cells, which makes the assess￾ment of neuronal function problematic. However,

Skin

cells of

epidermis

Neuron

of brain

Gastrula

Pigment

cell Sperm Egg

Skeletal

muscle

cell

Smooth

muscle

(in gut)

Tubule

cell of the

kidney

Skin

Nerves

Eyes Bones Blood

Muscles

Cardiac

muscle

Red blood

cells

Mesoderm

(middle layer) Ectoderm Endoderm

Mesoderm

Endoderm

(internal layer)

Ectoderm

(external layer)

Germ

cells

Lungs

Lining

of gut

Liver Lung cell

(alveolar

cell)

Thyroid

cell

Pancreatic

cell

Zygote

Inner-cell mass

Some Embryonic Cell Types at Gastrulation

Blastocyst

Blastocyst

Rostral

neural

Figure 9.1 Pluripotency in embryonic stem cells and the potential derivation of various lineage-specifi ed cells.