Therapeutic potential of amniotic fluid-derived cells for treating the injured nervous system

Therapeutic potential of amniotic fluid-derived cells for treating the injured nervous

system

Kerry Rennie1, Julie Haukenfrers

1, Maria Ribecco-Lutkiewicz

1, Dao Ly

1, Anna Jezierski

1,3

Brandon Smith1, Bogdan Zurakowski

1, Marzia Martina

2,3, Andrée Gruslin

3,4, Mahmud

Bani-Yaghoub1,3

1Neurogenesis and Brain Repair, National Research Council Canada, Bldg. M-54, Ottawa,

ON, K1A 0R6, Canada

2Synaptic Therapies and Devices, National Research Council Canada, Bldg. M-54, Ottawa,

ON, K1A 0R6, Canada

3Department of Cellular and Molecular Medicine, Faculty of Medicine, University of

Ottawa, Ottawa, Canada

4Department of Obstetrics and Gynecology, Faculty of Medicine, University of Ottawa,

Ottawa, Canada

Corresponding Author:

Dr. Mahmud Bani

Department of Translational Bioscience,

National Research Council Canada,

Bldg. M-54, Ottawa, ON, K1A 0R6,

Canada

Email: [email protected])

Phone: (613) 993-5723

Keywords: amniotic fluid, brain injury, cell-based therapy, gap junctions, micro-RNAs,

stem cells

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Abstract

There is a need for improved therapy for acquired brain injury, which has proven resistant

to treatment by numerous drugs in clinical trials, and continues to represent one of the

leading causes of disability worldwide. Research into cell-based therapies for the treatment

of brain injury is growing rapidly, but the ideal cell source has yet to be determined.

Subpopulations of cells found in amniotic fluid, which is readily obtained during routine

amniocentesis, can be easily expanded in culture, have multipotent differentiation capacity,

are non-tumourigenic, and avoid the ethical complications associated with embryonic stem

cells, making them a promising cell source for therapeutic purposes. Beneficial effects of

amniotic fluid cell transplantation have been reported in various models of nervous system

injury. However, evidence that amniotic fluid cells can differentiate into mature, functional

neurons in vivo and incorporate into the existing circuitry to replace lost or damaged

neurons is lacking. The mechanisms by which amniotic fluid cells improve outcomes after

experimental nervous system injury remain unclear. However, studies reporting the

expression and release of neurotrophic, angiogenic and immunomodulatory factors by

amniotic fluid cells suggest they may provide neuroprotection and/or stimulate endogenous

repair and remodelling processes in the injured nervous system. In this paper, we address

recent research related to the neuronal differentiation of amniotic fluid-derived cells, the

therapeutic efficacy of these cells in animal models of nervous system injury, and the

possible mechanisms mediating the positive outcomes achieved by amniotic fluid cell

transplantation.

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1. Introduction

Acquired brain injury, most commonly resulting from traumatic brain injury (TBI)

or stroke, is one of the leading causes of death and disability worldwide. Although the brain

is capable of a limited degree of endogenous repair and functional recovery, acquired brain

injury commonly leads to neurological deficits. Globally, approximately one-quarter to

one-half of brain injury patients exhibit substantial disability six months after moderate to

severe injury, and many are disabled even after mild brain injury (De Silva et al., 2009).

Unfortunately, the deficits are long-lasting for many brain injury patients. For instance, it is

estimated that 4% of Canadians are currently living with the long term cognitive, emotional

and/or sensory/motor sequelae of an acquired brain injury (The Ontario Brain Injury

Association 2011).

Despite decades of research and development leading to the testing of a multitude of

potentially neuroprotective compounds, translation to the clinic has been difficult. Although

neuroprotectants have been approved for clinical use in some countries (e.g. Japan), in

many countries there remains no approved clinically effective treatment for preventing

neuronal loss or repairing the brain after injury, beyond general strategies for promoting the

return of blood supply to the damaged area and reducing brain pressure in the immediate

post-injury phase (Auriel and Bornstein 2010; Gross et al. 2010; Labiche and Grotta 2004).

As a result, there is growing interest in novel therapeutic approaches, such as cell-based

therapy, to repair tissue damage and restore neurological function after acquired brain

injury (Bjorklund and Lindvall 2000; Schouten et al. 2004). In this paper, we will focus on

the growing body of research related to the potential use of human amniotic fluid (AF)-

derived cells as a therapy for nervous system injury.

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2. Mechanisms of acquired brain injury

Two of the most frequent and heavily studied forms of acquired brain injury are TBI

and stroke. Although they have different etiologies, TBI and stroke share many common

mechanisms of injury (Bramlett and Dietrich 2004; Leker and Shohami 2002; Werner and

Engelhard 2007). Both are characterized by a primary phase, in which there is rapid

necrotic death of cells at the core of the injury (in TBI, where the mechanical impact is

centered, and in stroke, where cerebral perfusion falls below a critical threshold). Cells in

the area surrounding the injury core (the penumbra) may be dysfunctional, but remain

viable for a short period. However, the penumbra region is vulnerable to a secondary phase

of injury that evolves in response to the primary injury. Within a few hours, the primary

infarct can expand to encompass nearly the whole penumbra, and damage may continue to

progress for weeks or months after the initial insult (Hossmann 2006; Leker and Shohami

2002; Liu et al. 2010; Park et al. 2008).

Secondary injury occurs via multiple parallel and interacting pathways. The

excessive release and/or inadequate astrocytic uptake of glutamate following the primary

injury results in the overstimulation of glutamate receptors. In particular, activation of the

NMDA receptor allows excessive entry of calcium into the cell, which activates several

calcium-sensitive enzymes, including proteases and phospholipases, which directly damage

cellular proteins and lipids (Kalia et al. 2008; Lo et al. 2003; Waxman and Lynch 2005). In

addition, a prolonged rise in intracellular calcium levels stimulates the formation of reactive

oxygen species, which also damage DNA and impair the integrity of cell and organelle

membranes by initiating lipid peroxidation. Calcium overload, paired with the production

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of free radicals, results in an increase in the permeability of the inner mitochondrial

membrane, impairing the production of energy substrates by oxidative phosphorylation. In

addition, the disruption of ion gradients across the mitochondrial membrane leads to

mitochondrial swelling, damage of the outer mitochondrial membrane, and the release of

cytochrome c into the cytoplasm. Cytochrome c initiates an apoptotic cell death program,

culminating in the activation of the apoptosis factor Caspase 3, which cleaves a number of

key cellular proteins, leading to the death of the cell ( reviewed in Bramlett and Dietrich

2004; Doyle et al. 2008; Hossmann 2006; Leker and Shohami 2002; Mergenthaler et al.

2004). In addition, elevated calcium concentration in the axon results in the destruction of

structural and transport proteins, leading to axonal swelling and synaptic disconnection.

Such white matter injury can be widespread throughout the brain even after a localized

ischemic or traumatic event, which may explain the broad nature of neurological symptoms

observed after focal injury (Bramlett and Dietrich 2004; Leker and Shohami 2002).

TBI and ischemia both stimulate an inflammatory response, with infiltration of

immune cells into the brain. Invading neutrophils release oxygen free radicals and

proteolytic enzymes, which damage resident cells. In addition, there is an upregulation in

the production of pro-inflammatory cytokines by activated microglia, resulting in further

damage (Bramlett and Dietrich 2004; Lakhan et al. 2009; Leker and Shohami 2002; Loane

and Byrnes 2010). Astrocytes proliferate, undergo morphological alterations, and exhibit

changes in the expression of multiple proteins including intermediate filament proteins such

as glial fibrillary acidic protein (GFAP), in a process known as reactive astrogliosis

(Sofroniew 2009). Over time, reactive astrocytes form a glial scar, isolating the injury site

from the surrounding healthy tissue to prevent the spread of damage, but also limiting the

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capacity for axonal regeneration and migration of neuroblasts into the injury site (Fitch and

Silver 2008; Silver and Miller 2004). Figure 1 summarizes the evolution of

neuropathological changes occurring after brain injury.

The neuropathological features observed in TBI and stroke described above also

apply to other brain injury models. We (Bani-Yaghoub et al. 2008; Jezierski et al. 2012;

Tay et al. 2011) and others (Frontczak-Baniewicz et al. 2008; Jadhav et al. 2007) have used

surgically-induced brain injury models to mimic the incidental damage inflicted on the

brain during neurosurgical procedures. Such damage can occur, for instance, during the

excision of brain tumours or the removal of epileptic foci. As in TBI and stroke, the injury

site is characterized by the infiltration of macrophages (Frontczak-Baniewicz et al. 2008;

Frontczak-Baniewicz et al. 2011), invasion by activated microglia, the formation of a glial

scar (Frontczak-Baniewicz et al. 2011), the apoptotic death of neurons (Sulejczak et al.

2008), and an expanding zone of infarction over time (Frontczak-Baniewicz et al. 2011).

Figure 2 illustrates some of these changes after surgical lesion to the mouse motor cortex.

3. The challenge of reconstructing functional brain tissue

Both TBI and stroke can result in pan-necrosis (the loss of all cellular elements)

within the injury core, as well as the loss or damage of selectively vulnerable cell types in

surrounding areas (Katchanov et al. 2003; Kotapka et al. 1991; Lowenstein et al. 1992).

Amongst surviving neurons, many synaptic connections are lost after injury (Brown et al.

2008; Campbell et al. 2012), compromising the integrity of circuits which may support

sensory, motor, or cognitive functions. Repair of this type of damage is not a trivial feat,

owing to the diversity of cell types and the complexity of the circuitry involved (Chiu and

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Rao 2011). The morphological complexity of the tissue lost or damaged after traumatic or

ischemic injury to the cortex can be appreciated by Golgi-Cox staining (see Figure 3) which

emphasizes the enormity of the task of reconstructing functional brain tissue.

Excitatory pyramidal neurons, which extend long axonal projections to connect

with cells in distant brain regions, make up the majority (70-80%) of neocortical neurons

(Markram et al. 2004). While pyramidal neurons (Figure 3; arrows) have common

structural and physiological characteristics, they differ in terms of their location within the

cortical laminae and their patterns of connectivity (Markram et al. 2004; Molyneaux et al.

2007). For instance, pyramidal projection neurons can be classified as associative,

commisural, or corticofugal, based on their predominant output to other neocortical areas,

the contralateral hemisphere, or subcortical targets, respectively (Molyneaux et al. 2007).

Each pyramidal neuron also receives input from distinct cortical and subcortical regions

(Aronoff et al. 2010; Miyashita et al. 1994; Porter and White 1983), and each input arrives

at a layer-specific area of the cell's dendritic arbour (Spruston 2008; Zilles 1990). In

addition to pyramidal neurons, the cortex contains local-circuit interneurons (Figure 3;

arrowheads), which make up 20-30% of the neuron population. This is a diverse group of

cells which differ in terms of morphology, physiology, connectivity, neurochemistry,

molecular characteristics and location within the cortical laminae (Wonders and Anderson

2006).

Pyramidal cells and interneurons form an intricate circuitry within the cortex.

While pyramidal neurons provide the major output from the cortex, interneurons serve to

modulate cortical excitability, provide lateral inhibition, and affect the timing and

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synchronization of neuronal activity across clusters of pyramidal neurons (Markram et al.

2004). It is estimated that each cortical pyramidal neuron makes roughly 1 x 104 synapses

(Huttenlocher 1990), and that there are approximately 1-2 x109

synapses per mm3 of

cortical neuropil (DeFelipe et al. 1999). This sophisticated circuitry arises over a long

period of time during development (months to years in humans) and involves the

elaboration of dendritic trees and axonal arbourizations, and the formation of synapses

(Huttenlocher 1990). Refinement of the circuitry by pruning of overabundant synapses

reportedly continues over decades in some cortical regions (Petanjek et al. 2011).Thus, a

wide variety of cells, which normally exist as part of a complex functional circuit formed

over a prolonged period of development, are lost after injury.

4. Limitations of endogenous brain repair after injury

In the aftermath of traumatic or ischemic brain injury, a number of processes are

initiated that might represent the brain’s attempt to mount an endogenous repair response

(Wieloch and Nikolich 2006). For instance, an increased proliferation of neural stem and

progenitor cells residing in the subventricular zone has been reported in several models of

injury and ischemia (Arvidsson et al. 2002; Chen et al. 2003; Chirumamilla et al. 2002; Jin

et al. 2001; Parent et al. 2002; Ramaswamy et al. 2005; Zhang et al. 2001). Although a

small proportion of these cells may successfully migrate to the damaged cortex or striatum,

the rate of survival of newly born cells is quite low, likely due to the cytotoxic environment

surrounding the injury. Furthermore, the majority of surviving cells differentiate into glia,

with only a small number of cells becoming neurons (Arvidsson et al. 2002; Kernie and

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Parent 2010; Parent et al. 2002). Thus, while the adult brain is capable of supporting the

integration of newly born neurons into pre-existing circuitry, the generation of new neurons

to replace dead or damaged ones is not a widespread phenomenon (Chiu and Rao 2011),

and endogenous neurogenesis does not appear to occur on a scale that is sufficient to

replace a substantial portion of cells lost due to injury (Kernie and Parent 2010).

One reason for the lack of endogenous cell replacement after injury may be that the

specification of neuronal subtypes is a highly complex process that is not normally

recapitulated in the adult brain. During development, different cell types establish the

telencephalon in a spatiotemporal manner, encounter different extracellular signals, and

express different transcriptional programs, all of which influence their eventual neuronal

phenotype (Molyneaux et al. 2007; Sansom and Livesey 2009; Wonders and Anderson

2006). The signals that promote differentiation into particular neuronal subtypes, as well as

axonal extension towards specific targets, may not be present in the adult brain. The lack of

appropriate signals for neuronal specification and axonal targeting in the adult likely

hampers the ability of endogenous cells to replace the full complement of neuronal

subtypes lost after injury and to re-establish functional circuits.

Structural plasticity after brain injury also occurs in the form of axonal sprouting

and synaptogenesis (Carmichael 2003; Fitch and Silver 2008). Both TBI and ischemia

stimulate the expression of growth-promoting genes which mediate signaling within growth

cone membranes, reorganization of cytoskeletal elements, and axonal sprouting

(Carmichael et al. 2005; Stroemer et al. 1995). Subsequently, immunoreactivity for

synaptophysin, a protein found in synaptic vesicles, increases, suggesting that synapse

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formation occurs in the penumbra (Stroemer et al. 1995). However, the inhibitory

environment surrounding an injury may be non-permissive for efficient sprouting and

synaptogenesis. Within the glial scar, growth-inhibitory molecules such as tenascin,

semaphorins, ephrins, and chondroitin sulfate proteoglycans are produced by reactive

astrocytes (Fitch and Silver 2008; Galtrey and Fawcett 2007; Gervasi et al. 2008). As

regenerating axons approach the glial scar, dystrophic endings form on the axon terminal,

and growth is halted (Silver and Miller 2004). Structural plasticity in the post-injury brain

is therefore limited.

5. Amniotic fluid as a source of cells for cell-based therapy

The notion that newly born cells may replace some of those lost or damaged by TBI

or stroke has stimulated an interest in developing cell-based therapies aimed at enhancing

the repair of damaged tissue after nervous system injury. Stem cells from various sources,

including embryonic stem (ES) cells, embryonic and adult neural stem (NS) cells and stem

cells derived from other adult sources such as adipose tissue and bone marrow have been

investigated as a potential therapy for experimental brain injury (reviewed in Boncoraglio

et al. 2010; Burns et al. 2009; Harting et al. 2008; Hess and Borlongan 2008; Honmou et

al. 2012; Jain 2009; Miljan and Sinden 2009; Stabenfeldt et al. 2011; Yu and Morshead

2011). Ultimately, the choice of the most appropriate cell source will be governed by

several factors, including accessibility, ability to generate sufficient quantities of cells,

safety, ethical restrictions and of course, efficacy. Each of the cell types that have

commonly been employed to date in preclinical testing has advantages and drawbacks. ES

cells, although possessing a high capacity for differentiation, have the potential to induce

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tumour formation in the brain (Molcanyi et al. 2009; Riess et al. 2007). In addition, the

ethical debate over the use of cells from discarded embryos has not been resolved and poses

a barrier to the development of clinically applicable therapy. In contrast, while adult stem

cells pose a reduced risk of tumorigenicity and fewer ethical restrictions, difficulties

expanding adult stem cells from some sources in culture have limited their use (Wright et

al. 2006), although advances in the understanding of culture conditions that promote

expansion may overcome this limitation (eg. Ribeiro et al. 2012). Because each of these

cell types is associated with shortcomings that may impede the development of clinically

useful cell-based therapies, there is a demand for further research into alternative sources of

cells for use in regenerative medicine.

Cells derived from amniotic fluid (AF) have a number of characteristics that make

them attractive candidates as a potential source of cells for tissue engineering and cell

replacement strategies (Kaviani et al. 2001; Wilson et al. 2012). As a result, AF cells have

been extensively studied in a variety of experimental models of injury and disease over the

past decade (reviewed elsewhere; Joo et al. 2012; Klemmt et al. 2011; Parolini et al. 2009).

AF, which is routinely obtained by amniocentesis for prenatal screening and diagnosis,

contains a heterogeneous population of cells derived from fetal skin, gastrointestinal,

respiratory and urinary tracts (Fauza 2004). The suggestion that AF might contain

multipotent fetal-derived cells (Prusa and Hengstschlager 2002) led to the identification of

a small subpopulation of actively dividing AF cells which express OCT4, a marker of

pluripotent stem cells (Prusa et al. 2003). Subsequently, AF-derived cells selected based

on the expression of stem cell factor (C-KIT, CD117) were found to express OCT4 along

with a number of markers of somatic stem cells such as CD29, CD44, CD73, CD90 and

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CD105 (De Coppi et al. 2007). The multi-lineage potential of AF-derived cells was

reported by De Coppi et al (2007), who demonstrated that clonal cell lines derived from C-

KIT+ AF cells were capable of differentiating into cell types representing all three germ

layers. A recent genome-wide analysis of AF cells has shown that these cells have a distinct

transcriptional signature, compared with other stem cells (Maguire et al. 2013). Unlike stem

cells derived from adult sources, AF cells are easily cultured and can be used to generate a

large number of cells. For instance, we have shown that a minimum of 2-4x108

cells can be

produced from a single cell clone after only 9 passages (Figure 4A) while maintaining their

karyotypic stability (Figure 4B). An important feature of these cells is that they express

markers of the neural lineage, such as Nestin, Brn-2 (pou3f2) and Neurofilament (see

Figure 5A-D), suggesting that distinct populations of AF cells may have a higher potential

to differentiate along the neural lineage. The use of more specific markers (e.g. for neural

progenitors, neurons and glia) will provide a better understanding of the differentiation

capacity of these cells.

In general, transplanted AF cells may contribute to nervous system repair in two

different ways. Exogenous cells may serve as direct replacements for lost or damaged cells,

which requires that the cells differentiate into the appropriate neuronal subtypes, acquire

functional properties of the desired cell types, and integrate into the existing circuitry.

Another possibility is that transplanted cells may provide support for surviving host cells,

offer protection from the toxic environment surrounding the injury, and/or stimulate

endogenous repair mechanisms (Boucherie and Hermans 2009; De Feo et al. 2012; Einstein

and Ben-Hur 2008). Of course, these possibilities are not mutually exclusive. Here, we

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evaluate the potential for using human AF cells for both cell replacement, and for support

functions in nervous system injury.

6. The application of AF cells as a neuron replacement strategy

Criteria for classifying a cell as a neuron have been previously described (Song et

al. 2002; Yang et al. 2011) and can be used to evaluate the neuronal differentiation capacity

of AF-derived cells. To be regarded as neurons, AF-derived cells should exhibit typical

neuronal morphology, including cellular polarization and the extension of neurites from the

cell body. This should be accompanied by the expression of neuron-specific gene products,

such as neurofilaments, microtubules, microtubule-associated proteins (MAP), and synaptic

proteins. In addition, putative neurons must display electrophysiological and neurochemical

characteristics that would enable intra- and intercellular communication. Thus, they should

be capable of maintaining a stable resting membrane potential and generating action

potentials with characteristic changes in membrane voltage when stimulated. Furthermore,

differentiated neurons should show evidence of synapse formation, by electrophysiological

recording or electron microscopic examination of pre- and post-synaptic specializations

(Song et al. 2002; Yang et al. 2011).

AF-derived cells have repeatedly been reported to acquire neuronal morphology and

express neuronal markers, including beta III tubulin, NeuN, MAP2, neurofilament, and

neuron-specific enolase (NSE) under conditions of neural induction in vitro (Bossolasco et

al. 2006; De Coppi et al. 2007; Jezierski et al. 2010; Kim et al. 2007; Mareschi et al. 2009;

Prusa et al. 2004; Tsai et al. 2006; Tsai et al. 2004). However, in the majority of reports,

neuronal differentiation has been assessed only by changes in morphology and expression

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of neuronal markers, and these changes do not prove the neuronal functionality of the cells.

It has been reported that certain conditions used to induce neuronal differentiation, such as

serum withdrawal and addition of DMSO, can induce morphological changes in cells due to

disruption of the actin cytoskeleton, causing them to resemble neurons and express

neuronal antigens possibly as a stress response (Croft and Przyborski 2006; Lu et al. 2004;

Neuhuber et al. 2004). These changes do not appear to represent true neuronal

differentiation, as the neuronal characteristics may be unstable (Croft and Przyborski 2006).

For instance, when C-KIT+ AF cells were cultured in neural induction media (N2 media

plus 3-isobutyl-1-methylxanthine or 2’-O-dibutyryl-cAMP) a small population of cells

developed neuronal morphology and expression of BIII-tubulin (Prasongchean et al. 2012).

However, these changes were reversible upon removal of the induction media, indicating

that the cells had not undergone true neuronal differentiation (Prasongchean et al. 2012).

Although most studies use growth factors to induce neuronal differentiation (e.g. De Coppi

et al. 2007), rather than serum withdrawal, DMSO treatment or the neural induction media

used by Prasongchean et al (2012), these results highlight the need for functional studies to

validate the neuronal differentiation capacity of AF cells.

A limited number of studies have evaluated the functional properties of AF cells

after culture in neuronal differentiation media. Tsai et al. (2006) reported that after

dopaminergic induction, AF cells produced dopamine in response to depolarization induced

by high K+ stimulation, and work by De Coppi et al. (2007) suggested that neuronally

differentiated AF cells were capable of secreting l-glutamate. However, as pointed out by

Toselli et al (2008), this characteristic is not exclusive to neurons. Electrophysiological

studies suggest that after neuronal induction, AF cells express a barium-sensitive K+

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channel (De Coppi et al. 2007) and a tetrodotoxin-sensitive voltage-gated sodium channel

(Mareschi et al. 2009), both of which are characteristic of neurons. However, as of yet, the

generation of action potentials by neuronally differentiated AF cells has not been

demonstrated, and evidence that AF cells can form functional synapses after neural

induction is completely lacking. Therefore, the ability of AF cells to differentiate into

mature functional neurons requires further investigation.

In a handful of in vivo studies, AF cells have been transplanted into the brain or

peripheral nerves after various types of injury (Bigini et al. 2012; Cipriani et al. 2007; De

Coppi et al. 2007; Donaldson et al. 2009; Pan et al. 2007; Pan et al. 2006; Prasongchean et

al. 2012; Rehni et al. 2007). In some cases, the phenotypes of transplanted cells were not

examined, so it is unclear whether the cells underwent neuronal differentiation. In other

studies, histological examination has generally failed to provide evidence that AF cells

differentiated into mature neurons. For example, the majority of human cells detected at 10,

30 and 90 days after transplantation of human AF cells into the striatum of rats subjected to

focal ischemia expressed the astrocytic marker GFAP (Cipriani et al. 2007). Although a

few human cells expressing doublecortin (a marker of immature neuroblasts) were found 10

days after transplantation, no cells expressing the neuronal marker beta III tubulin were

found at any time point (Cipriani et al. 2007). In another study, AF cells transplanted into

the striatum of rats treated with the dopaminergic toxin 6-OHDA failed to acquire a

dopaminergic phenotype, and survived for only a short period (less than three weeks) in the

brain (Donaldson et al. 2009). The ability of AF cells to integrate into host circuitry, and

express functional characteristics of neurons in vivo, has not been verified. Thus, there is so

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far little evidence that AF-derived cells can be used to directly replace lost or damaged

neurons in the injured nervous system.

7. The application of AF cells for neuroprotection and repair

Even for cell types which have more convincingly been demonstrated to

differentiate into neurons in vitro and in vivo, it is not clear that replacement of lost cells is

the underlying mechanism for the improvements observed after their transplantation into

brain-injured animals (Boucherie and Hermans 2009; van Velthoven et al. 2009). In the

field of regenerative medicine, attention has shifted somewhat to the ability of transplanted

cells to provide neuroprotection and to stimulate repair of damaged nervous tissue

(Boucherie and Hermans 2009; Carletti et al. 2011; Chen et al. 2007; De Feo et al. 2012;

Farin et al. 2009; van Velthoven et al. 2009).

In fact, despite the limited evidence for neuronal differentiation of AF cells in vivo,

a number of studies have demonstrated beneficial effects of AF cell transplantation in

animal models of nervous system injury (summarized in Table 1). For instance, the

administration of AF cells of human or rat origin into a surgically-induced sciatic nerve gap

or nerve crush injury in rats has repeatedly been reported to result in significant functional

improvement, as well as enhanced recovery of compound action potential amplitude and

conduction latency (Cheng et al. 2010; Pan et al. 2009a; Pan et al. 2007; Pan et al. 2009b;

Pan et al. 2006; Pan et al. 2009c). Transplanted AF cells were not observed in the injury

site a month after surgery, and did not penetrate into the regenerating nerve, suggesting that

the observed improvements could not be accounted for by cell replacement (Pan et al.

2007). In a model of embryonic spinal cord thoracic crush injury, Prasongchean et al.

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(2012) demonstrated that transplantation of C-KIT+ AF cells into the spinal cord at the

time of injury improved survival, even though grafted cells were negative for the neural

markers DCX and GFAP. In vitro co-culture of spinal cord slices with AF cells in a

transwell system prevented cell death and axonal degeneration, further supporting the

notion that the survival-promoting effect of AF cells was not due to differentiation and

integration into the tissue, but rather, due to secreted factors released by AF cells

(Prasongchean et al. 2012). AF cells injected into the lateral ventricles of mice three days

after focal ischemia induced by middle cerebral artery occlusion were shown to improve

somatosensory and cognitive function four days later (Rehni et al. 2007). Although the

authors did not examine the phenotype of injected cells, the fact that improvements were

observed after only a few days suggests that the beneficial effects of AF cells were likely

not due to cell replacement, since the differentiation and integration of transplanted cells

would presumably require a longer time.

7.1. Paracrine effects of AF cells

If AF cells do not replace lost or damaged cells in the injured nervous system, what

accounts for the beneficial effects of AF cell transplantation observed in the above

experiments? Studies using other stem cell types have suggested that transplanted cells may

enhance the survival of host neurons through the release of trophic factors, stimulate

endogenous repair through the recruitment of progenitor cells and promotion of neurite

outgrowth, and render the peri-lesion environment less toxic and more favourable to

regeneration by modulating the immune response and scar formation (Chiu and Rao 2011).

To date, the mechanisms by which damaged neural tissue may benefit from AF cells have

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yet to be clearly identified. However, AF cells have been shown to express and secrete a

number of factors that could potentially support neuroprotective and/or reparative

functions. For instance, AF cells secrete vascular endothelial growth factor (VEGF),

stromal cell derived factor- 1 (CXCL12) and IL-8, all of which regulate angiogenesis

(Mirabella et al. 2011; Yoon et al. 2010), and in fact, the pro-angiogenic capacity of AF

cells has been demonstrated in mouse hindlimb ischemia (Mirabella et al. 2011) and

ischemic skin flap (Mirabella et al. 2012) models. Enhanced angiogenesis is also beneficial

in animal models of stroke (Fan and Yang 2007; Gertz et al. 2012; Ma et al. 2012),

traumatic brain injury (Li et al. 2012; Xiong et al. 2010) and nerve injury (Pereira Lopes et

al. 2011). Thus, the secretion of pro-angiogenic factors by AF cells might contribute to

improved function in models of nervous system injury. In addition, RT-PCR analysis has

indicated that some AF cells express a number of neurotrophic factors, such as BDNF,

GDNF, CNTF, NGF and NT-3 (Pan et al. 2007). Since neurotrophic factors have

frequently been shown to be neuroprotective in ischemic stroke models (Beck et al. 1994;

Duarte et al. 2012; Ferrer et al. 1998; Kiprianova et al. 1999; Kitagawa et al. 1998a;

Kitagawa et al. 1998b; Miyazaki et al. 1999; Schabitz et al. 1997; Wang et al. 1997;

Yamashita et al. 1997), traumatic brain injury (Minnich et al. 2010), and peripheral nerve

injury (Fine et al. 2002; Kokai et al. 2011; Wood et al. 2009), it is possible that some of the

beneficial effects of AF cells may be explained by the release of trophic factors. AF cells

have also been shown to secrete a number of immune-modulating cytokines such as Il-6,

and growth related oncogene (GRO) and monocyte chemotactic protein (MCP) family

members (Moorefield et al. 2011) which might serve to limit the damage after nervous

system injury. Whether secretion of angiogenic, trophic, or immune-modulating factors

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actually does account for the positive effects of AF cell transplantation in neural injury

models remains to be determined. This issue warrants further investigation, in order to

capitalize on the beneficial properties of AF cells.

Beyond their natural capacity for providing protection and promoting repair of

neural tissue, AF cells can also be engineered to deliver specific factors to sites of nervous

system damage (see Figure 6). For instance, Cheng et al. (2010) transplanted AF cells

expressing human GDNF into the site of a sciatic nerve crush injury. Transplantation of

GDNF-transduced AF cells resulted in improved electrophysiological and functional

recovery, and prompted an upregulation of GDNF receptor expression at the distal end of

the crushed nerve (Cheng et al. 2010). We are currently evaluating the possibility that

GDNF-expressing AF cells might also be useful for the treatment of surgically-induced

brain injury.

7.2. Transfer of small molecules from AF cells to host tissue

An additional potential mechanism by which AF cells might modulate damage or

mediate repair in the injured nervous system is through the direct transfer of small species

to at-risk cells via gap junction channels. Gap junction channels are permeable to many

small molecules, including metabolites, amino acids, nucleotides and second messengers

(Harris 2007). One type of molecule of particular interest, owing to their capacity to

regulate multiple targets, are microRNAs (miRs). miRs are short (21-23 nucleotide) non-

coding RNA molecules that interact mainly with the 3’UTR of mRNA transcripts, resulting

in translational repression or degradation of the target mRNA (Bartel 2004; Nelson et al.

2003). A number of miRs, including miR-223 (Harraz et al. 2012), miR-181c (Zhang et al.

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2012a), and miR-21 (Buller et al. 2010; Zhang et al. 2012b), have been shown to confer

neuroprotection in in vitro or in vivo models of neural injury, and thus the delivery of

specific miRs to the injured nervous system may represent a valid therapeutic strategy.

Previous work has shown that miRs can pass between pairs of glioma cells (Katakowski et

al. 2010), cardiac myocytes (Kizana et al. 2009) and from bone marrow stromal cells to

breast cancer cells (Lim et al. 2011) likely through gap junctions composed of connexin 43

(Cx43), to affect target genes in the receiving cell. We have hypothesized that AF cells

might also be capable of transferring miRs to host cells in the injured brain via gap

junctions to promote cell survival or repair.

AF cells form gap junction channels made of CX43 (Figure 5D and Jezierski et al.

2012). Dye transfer experiments in our laboratory indicated that AF cells can form

functional gap junctions with other AF cells and with cortical cells in vitro (Jezierski et al.

2012). Interestingly, the expression of Cx43 is upregulated after brain injury (Haupt et al.

2007; Ohsumi et al. 2010; Rouach et al. 2002), possibly increasing the likelihood that

transplanted cells might be able to establish gap junctional communication with vulnerable

host tissue.

To test the capacity of AF cells to transfer miRs through gap junctions, we injected

a fluorescently labelled morpholino mimic of miR-21 into individual AF cells, and

observed the successful transfer of fluorescent signal to neighbouring AF cells (Figure 7A-

D) and cortical astrocytes (Figure 7E-G) in vitro. The transfer of miRs from AF cells to

host cells therefore seems plausible. Whether AF cells can in fact transfer endogenous miRs

to injured neural tissue, which miRs are transferred, and whether this occurs on a scale

sufficient to impact the expression of miR target genes in the receiving cell remains to be

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determined. An alternative approach might be to harness the ability of AF cells to transfer

miRs by engineering HAF cells to produce specific miRs that are expected to have

protective or reparative functions.

8. Future Directions

The reported neural differentiation potential of AF–derived cells has stimulated an

interest in developing AF cell-based therapies for treating damaged or diseased neural

tissue. Research on the ability of AF cells to replace lost or damaged neurons in vivo is still

in its infancy, and major questions remain to be answered. One issue which needs further

exploration is whether certain subpopulations of AF cells might be more suitable for use in

neural applications than others. For instance, Arnhold et al (2011) reported enhanced

neuronal differentiation capacity of C-KIT- AF cells relative to C-KIT

+ cells, although

these differences have not yet been examined in vivo. Furthermore, the question of whether

AF cells should be pre-differentiated towards a neuronal fate or towards specific neuronal

subtypes in vitro prior to transplantation should be addressed. In order to do so, a better

understanding of the culture conditions needed to direct AF cells towards specific neuronal

subtypes will be necessary.

On the other hand, it is possible that AF cells may provide benefit independent of

any neuronal differentiation or integration into the host circuitry, and neuronal

differentiation of AF cells may not be required in order to achieve positive outcomes. In

fact, research thus far supports this idea, as transplantation of AF cells resulted in improved

outcomes after peripheral nerve injury and brain ischemia, without directly replacing lost

cells (Pan et al. 2007; Pan et al. 2006; Rehni et al. 2007). However, the mechanisms by

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which AF cells provide neuroprotection and/or stimulate repair require further research.

Factors secreted by AF cells, and the conditions under which this occurs, should be

explored in greater detail.

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Acknowledgement:

The authors would like to thank Dr. Marianna Sikorska, Dr. Robert Monette and Tanya

Comas for discussions and technical assistance.

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Table 1. Applications of AF cells from different sources in animal models of nervous

system injury or disease. The effects of AF cells administered via various routes on the

behavioural and histological outcomes associated with each model are described. Where

possible, an indication of whether the cells survived in vitro and expressed neuronal

markers is included.

Model Source of AF cells

Route of administration

Outcomes (behavioural and histological) AF cell survival and phenotype Reference

MCAO (rat) Human

Intra-striatal injection one week post MCAO

Not examined

Few GFAP+ AF cells in striatum 30 and 90 days post-injection. Very few DCX+ AF cells 10 days post-injection. No βIII-tubulin+ AF cells at any time point.

Cipriani et al. 2007

MCAO (mouse)

Mouse ICV three days post MCAO

Improved neurological severity score, cognitive score, and somatosensory score 4 days after cell transplantation. No assessment of extent of brain damage.

Not examined Rehni et al. 2007

MCAO (rat) Rat IV, 35 days after MCAO

Reduced infarct volume and improved cognitive and motor functions one month after transplantation.

Not examined Tajiri et al. 2012

Thoracic crush injury (E2.5 chick embryo)

Rat

Implanted in the spinal cord at the time of injury

Reduced hemorrhage and increased survival of chick embryos after injury.

Grafted cells were negative for neural markers DCX, GFAP and Tuj1.

Prasongchean et al. 2012

Sciatic nerve resection (rat)

Human

AF cells in Surgicel and fibrin glue implanted in the surgical site

Improved ankle angle when walking, improved compound muscle action potential and nerve conduction velocities 8 weeks after transplantation.

Not examined Pan et al. 2006

Sciatic nerve crush (rat)

Rat

AF cells in Surgicel and fibrin glue implanted in the surgical site

Improved sciatic function index, ankle angle, compound muscle action potential and nerve conduction latencies 1, 2 and 4 weeks after transplantation

AF cells observed around the crush site, but not penetrating into the nerve. Cells did not survive for more than a month.

Pan et al. 2007

Sciatic nerve crush (rat)

Human

AF cells in Matrigel or in Surgicel/fibrin glue implanted in the surgical site

Effects of AF cells on motor and electrophysiological recovery were augmented using GDNF-transduced AF cells, or co-treatment with hyperbaric oxygen, granulocyte-colony stimulating factor or fermented soybean extract

Cell phenotype not examined

Cheng et al. 2010; Pan et al. 2009a; 2009b; 2009c

6-OHDA Parkinson's disease model (rat)

Human Intrastriatal injection

Not examined

AF cells were only detected in the brain of 1 rat (out of 12). AF cells observed at 3 days, but not at 3 weeks after transplant, and expressed markers of neural progenitors and neurons, but not dopaminergic neurons.

Donaldson et al. 2009

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Abbreviations: AF: amniotic fluid; ALS: amyotrophic lateral sclerosis; DA: dopamine; DCX: doublecortin; E:

embryonic day; GDNF: glial cell-derived neurotrophic factor; GFAP: glial fibrillary acidic protein; ICV:

intracerebroventricular; IV; intravenous; MCAO: middle cerebral artery occlusion; OB: olfactory bulb; 6-OHDA:

6-hydroxydopamine; TH: tyrosine hydroxylase.


Human

Injection into the medial forebrain bundle and ventral tegmental area

CD44+ AF cells reduced apomorphine-induced rotational behaviour

CD44+ AF cells survived for at least 3 weeks, and expressed TH

Liu et al., 2011


Human Instrastriatal injection

Improved bladder function and survival of host DA neurons 14 but not 28 days after AF cell injection.

AF cells survived for less than 28 days. No assessment of cell phenotype.

Soler, 2012

Genetic twitcher model of leukodystrophy (mouse)

Human ICV Not examined

Survival of grafted cells for at least 2 months, found throughout the brain, including hippocampus, OB and periventricular areas in both wt and twitcher mice. Cell phenotypes not examined.

De Coppi et al., 2007

Genetic wobbler model of ALS (mouse)

Human ICV No effect on mouse survival. AF cells remained in the ventricular system and did not migrate into parenchyma.

Bigini et al., 2012

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Figure Legends

Figure 1: Time course of events occurring after brain injury. The approximate temporal

sequence of various neuropathological and reparative processes triggered by brain injury or stroke

is depicted on a timeline spanning from hours to weeks. Shading of the bars indicates the relative

intensity of the process over time, with the darkest colouring indicating the peak intensity of each

process. Necrotic cell death begins within minutes of the insult and is followed by the initiation of

apoptosis (programmed cell death), which can continue for weeks, expanding the zone of infarction.

This is accompanied by morphological and gene expression changes in both microglia (microglial

activation) and astrocytes (astrogliosis). Growth inhibitory genes are expressed in the tissue

surrounding the injury site, preventing axonal extension and neuroblast migration within the

damaged area. In response to the injury, the brain launches endogenous repair processes, including

an increase in the expression of growth-promoting genes, the formation of synapses

(synaptogenesis), and the production of new neurons (neurogenesis).

Figure 2: Mouse model of surgically induced cortical injury. To produce the cortical injury, the

part of the skull overlying the left motor cortex was removed. Injury to the motor cortex was

performed with a sterile needle to remove neural tissue to a depth of 1mm, using the

following coordinates: from AP -0.25mm to -1.0mm, Lat +0.7mm to AP +1.25mm to

+3.0mm, Lat +2.4mm relative to Bregma. Full details of the surgical procedure were

previously described (Jezierski et al., 2012). This method produces a consistent injury with

only slight variability in lesion placement across animals. The changes shown here have

been consistently observed across multiple studies. A: A 3D reconstruction of a lesioned mouse

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brain showing the area of damage in the left primary motor cortex (orange). B: Cresyl violet

staining of a typical coronal section from a lesioned mouse brain showing the extent of damage in

primary motor cortex. C-D: Immunostaining for neurons (MAP2; red) and astrocytes (GFAP;

green) in sham (C) and lesioned (D) mouse motor cortex one week after surgery. Loss of neurons

and neurites as well as an intense astrocytic reaction in the peri-lesion cortex are evident in the

lesioned mouse, compared to sham. E-F: Immunostaining for microglia/macrophages (Iba-1; green)

in sham (E) and lesioned (F) cortex one week after surgery demonstrates the strong inflammatory

response that occurs after surgical lesion. G-H: TUNEL (green) staining at the border of the injury

site indicates that while very low levels of cell death (almost none) are seen in sham brains (G) cell

death continues to occur even 4 weeks after lesion (H). Blue staining in C-H is Hoechst. Scale bar:

2mm (B); 100µm (C-F); 50µm (G-H).

Figure 3: Complexity and diversity of cellular morphology and connectivity in mouse motor

cortex. Golgi-Cox staining of mouse motor cortex reveals the complexity and diversity of cellular

morphology and connectivity within the cortex. Saline-perfused mouse brains (n=4) were fixed in

Golgi-Cox solution (Glaser and van der Loos 1981) for 14 days, rinsed several times in distilled

water, and transferred to 30% sucrose. 200µm sections were cut on a vibratome and mounted on

gelatinized slides. The sections were developed by incubating in ammonium hydroxide followed by

Kodak film fix, and then were dehydrated, cleared and coverslipped. Complete details of the

procedure were previously described (Frechette et al. 2009). The images shown here are

representative images of a non-injured hemisphere taken from a single animal. A. A low

magnification view of motor cortex, showing diverse cell types throughout the cortex. Several

pyramidal neurons with long apical processes and extensively branched basal trees can be seen

(indicated with arrows), as well as structurally varied interneurons (arrowheads) .B. A pyramidal

neuron, demonstrating the highly branched morphology of the basal dendrites and long apical

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process. C. A high magnification image of spines on the basal dendrites of a pyramidal neuron.

Scale bar: 200µm (A); 50µm (B); 3µm (C).

Figure 4. Proliferative potential and karyotypic stability of AF cells

A. Samples of amniotic fluid were obtained from the Ottawa Hospital (Ottawa, ON, Canada)

following routine amniocentesis carried out on pregnant women at 15 to 35 weeks gestation with

written consent and performed under the guidelines established by the Ottawa Hospital and

National Research Council Canada - Research Ethics Boards. In brief, AF was diluted with PBS

and spun at 250 x g for 5 mins; pellets were re-suspended in expansion media composed of DMEM

supplemented with 20% FBS. The cells were subsequently plated in a 10 cm dish, passaged by

trypsinization and serially expanded with a split ratio of 1:3 at 70% confluence (Jezierski et al.

2010). For the generation of single cell clones, a single cell suspension was prepared by gentle

trypsinization and individual AF cells were deposited, by Fluorescence Activated Cell Sorter

(FACS), with a cell density of one cell per well of a 96-well plate in 100ul expansion media.

Resulting clones (e.g. F5) were then sub-cultured first into 24-well plates and thereafter expanded

serially to 10cm plates and subsequently used for a number of cellular and molecular purposes. B.

Single clone (F5) cells were shown to preserve their karyotypic stability after multiple passages in

culture. Single clone F5 cells were grown in DME + 20% FBS until they reached about 70%

confluence. They were then incubated for 4h in medium containing 0.1µg/ml Karyomax Colcemid

solution (Invitrogen), harvested by trypsinization, and spun at 175xg for 5 minutes at room

temperature. Cells were then incubated for 20 minutes at room temperature in 10ml of hypotonic

solution (0.075M KCl) and fixed with fresh methanol/acetic acid fixative (3:1 vol/vol). The fixed

cell suspension was dropped onto a clean glass slide. Slides were incubated with Hoechst (1 µg/ml)

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stain for ten minutes and mounted with Vectashield (Vector). Pictures of the metaphase spreads

were taken using a 40x objective (Axiovert Zeiss).

Figure 5. Expression of neural markers by AF cells

A. RT-PCR analysis of the mRNA harvested from AF single cell clone F5 cells shows the

expression of Nestin (Nes), Brn2 (pou3f2) and Neurofilament Light Chain (NFL). β-ACTIN (ACTB)

and undifferentiated human nTera2 (NT2) cells were used as internal and positive controls,

respectively. NTC, No Template Control. B. Western blotting confirms the expression of NESTIN

protein isoforms in F5 clone and the parent population, AF sample 1 (AF*) compared to another AF

Sample (AF**), suggesting that some AF samples harbour the potential to differentiate along the

neural lineage. β-ACTIN (ACTB) and undifferentiated human NT2 cells were used as internal and

positive controls, respectively, whereas HEK293 cells were used as a negative control. C-D.

Immunocytochemistry for NES expression in F5 cells. Hoechst was used as a counter stain. Scale

bar: 25 µm

Figure 6. GDNF-secreting AF cells

A. AF cells were infected with Tet07-CMV-GDNF-DsRed vector to constitutively secrete GDNF.

These GDNF-secreting cells can be seeded on biodegradable, biocompatible polymer scaffolds

(Bani-Yaghoub et al. 2008) or injected directly into tissue of interest to deliver GDNF. B. AF cells

infected with GDNF-DsRed and DsRed backbone control in vitro. Scale bar: 50µm.

Figure 7. Transfer of morpholino mimic of miR-21 from AF cells to neighbouring AF and

cortical astrocytes in vitro

Cell-to-cell transfer of a fluorescently labeled morpholino mimic of miR 21 (GeneTools) between

adjacent AF cells was assessed using a whole-cell patch-clamp technique. Cells were cultured on

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coverslips and bathed in a solution containing 135mM NaCl, 5mM KCl, 2mM MgCl2, 2mM CaCl2,

10mM HEPES and 10mM glucose (pH 7.4). Whole-cell patch-clamp experiments were performed

at room temperature using borosilicate pipettes (resistance 3-5MΩ) filled with a solution containing

130mM K+-gluconate, 10mM KCl, 2mM MgCl2, 10mM HEPES, 0.5mM EGTA, 4mM ATP,

0.2mM GTP (pH7.4). Morpholino oligos were added to the patch pipette solution to concentrations

of 0.25-0.5mM. Whole-cell patch-clamp recordings were monitored using an Axopatch 1D

amplifier (Axon Instrument Inc) under visual control using differential interference contrast and

infrared video microscopy (IR-DIC; Olympus), Whole-cell access resistance ranged from 5 to 15

MΩ and was monitored throughout the experiments. The cell-to-cell transfer of morpholino oligos

was recorded using a Fluo-View FV1000 Olympus confocal microscope (Olympus). Images were

acquired every minute using a 40X water immersion objective (NA 0.8), laser excitation

wavelength 559nm and BP wavelength 570-640nm. A. The morpholino was injected into a single

AF cell (asterisk; image acquired 1 minute post-injection) and B. The cell-to-cell morpholino

transfer was recorded in real-time (Supplementary Video 1) between adjacent AF cells (arrows;

image acquired 40 minutes post-injection). C. Phase contrast showing patch pipette microinjection

into a single AF cell. D. CX43 expression (arrowhead) at the cell-cell boundary between the patch-

clamped AF cell and adjacent morpholino-receiving cells. CX43 expression was determined by

immunocytochemical staining of the cells, which were fixed after completion of the morpholino

transfer experiment. E-G. Similarly, miR-21 morpholino transfer from patch-clamped AF cell

(asterisk) to mouse cortical astrocytes (arrows) was recorded between minute 1 (E) and 40 (F) post-

injection. As seen in the video (supplementary material), transfer of the morpholino was observed in

the receiving cell as early as 5 minutes post-injection. Scale bar: 25 µm

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Supplementary Figure 1. miR expressing AF cells were engineered by retroviral infection. EGFP

was used as reporter gene in the same vector [miR-132F1R1/pMX clone 22; original vector pMIR-

REPORT Luciferase (Applied Biosystems)]. A. miR-132 EGFP expression, B. phase, C. Merged,

Scale bar: 150 µm

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21 3 4 5 6 7 8 9 10 11 12

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Amniocentesis

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Passage 1

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OutputApproximate CellNumber at 70%

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SampleCollection

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AF Cells

GDNF LentivectorGDNF-secreting AF Cells

Transcription

CMV GDNF DsRed

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Therapeutic potential of amniotic fluid-derived cells for treating the injured nervous system

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Transcript of Therapeutic potential of amniotic fluid-derived cells for treating the injured nervous system