Px= planned number of patients to enroll; Auto=autologous; Allo=allogeneic; IV=intravenously;

IC=intracerebral (cavity or peri-infarct tissue); IA=intra-arterial (carotid); TP=transplant

Table 2. Ongoing clinical trials for using MSCs to treat stroke

## **3. Mechanisms of action underlying beneficial effects**

Transplanting stem cells is attractive because they can potentially differentiate into multiple cell types and replace cells lost to injury or disease. MSCs normally give rise to cells along the mesodermal lineage (including bone, cartilage, and adipose tissue); however, there are reports suggesting that they can transdifferentiate into neural cells in certain *in vitro* (Sanchez-Ramos *et al.*, 2000; Woodbury *et al.*, 2000) and *in vivo* (Kopen *et al.*, 1999; Munoz-Elias *et al.*, 2004) environments. Though some studies show a small percentage of donor MSCs express neuronal markers in the injured brain, there is little evidence that these cells functionally incorporate into the endogenous neuronal circuitry. In fact, there is a decidedly lack of evidence that neuronal replacement is the primary mechanism of action for MSC therapy; moreover, there are data demonstrating artifacts associated with MSC to neuron transdifferentiation (Barnabe *et al.*, 2009; Lu *et al.*, 2004; Neuhuber *et al.*, 2004; Phinney & Prockop, 2007; Wells, 2002). There is also the possibility that MSCs replace supporting glial cells (astrocytes, oligodendrocytes, or microglia), which outnumber neurons 10:1 in the brain (reviewed in Boucherie & Hermans, 2009). However, ample evidence shows that benefits and functional recovery occur rapidly and persist long after the donor cells are gone, indicating permanent cell replacement is not required. The most likely governing mechanism is that MSCs provide trophic support to the injured brain, which augments endogenous repair and regeneration pathways. Trophic support, by definition, acts through secreted molecules called trophic factors. MSCs may act as mini-pumps delivering beneficial factors to their microenvironment. Using cells as pumps is preferred to actual engineered pumps because they can deliver a plethora of factors at the site of injury in physiologic concentrations and also respond to the needs of the injured tissue with appropriate feedback. Trophic factors can either directly or indirectly (via a mediator cell) promote neuroprotection (enhance cell survival through repair) or neuroregeneration. MSCs also secrete factors that augment angiogenesis – another important aspect of regeneration after brain injury. An additional likely mechanism of action contributing to the benefit of MSCs is immunosuppression. MSCs can affect immune cells via secreted factors, which would fall under trophic support. For the purposes of this chapter, we will treat it as a separate category since targeting immune functions indirectly promotes recovery compared to acting directly on neural or vascular cells. There is a great deal of overlap between these functions and these categories are fluid. Figure 1 summarizes hypothesized mechanisms of action for MSCs in the injured brain, which are mediated by secreted factors and direct cell-cell contacts.

#### **3.1 Terminology**

Trophic support classically means to provide nutrition, but the definition has been expanded to include promoting cellular growth, survival, differentiation, or migration. Similarly, the terms "trophic factor" and "growth factor" have also become more inclusive. Neurotrophic factors are trophic factors acting specifically on neural cells, i.e., promoting the growth, survival, differentiation, or migration of primarily neurons, but also glial cells (astrocytes, oligodendrocytes, microglia and Schwann cells). The name neurotrophin is sometimes used synonymously with neurotrophic factor; however neurotrophins specify a family of four structurally-related proteins: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). The

(Haynesworth *et al.*,

ELISA of Conditioned Medium

Conditioned Medium

Immunoblotting of Conditioned Medium

Conditioned Medium

Conditioned Medium

Conditioned Medium

(Potian *et al.*, 2003) Cytokine Array of

(Kinnaird *et al.*, 2004) ELISA or

(Arnhold *et al.*, 2006) ELISA of

(Crigler *et al.*, 2006) ELISA of

(Wang *et al.*, 2006) ELISA of

1996)

**Reference Detection Method Trophic Factors Found Abbreviation**

Granulocyte colony stimulating

Granulocyte-macrophage colony

Macrophage colony stimulating

Granulocyte colony stimulating

Granulocyte-macrophage colony

Monocyte chemoattractant

Monocyte chemoattractant

Vascular endothelial growth

Glial cell line-derived

factor G-CSF

stimulating factor GM-CSF Interleukin-11 IL-11 Interleukin-6 IL-6 Leukemia inhibitory factor LIF

factor M-CSF Stem cell factor SCF

factor G-CSF

stimulating factor GM-CSF Growth related oncogene- GRO Interleukin-6 IL-6 Interleukin-8 IL-8

protein-1 MCP-1 Oncostatin M OSM Transforming growth factor- TGF

Angiopoietin-1 ANG-1 Fibroblast growth factor-2 FGF-2 Interleukin-6 IL-6

protein-1 MCP-1 Platelet derived growth factor PDGF Placental growth factor PlGF

factor-A VEGF-A

Brain derived neurotrophic factor BDNF

neurotrophic factor GDNF Nerve growth factor NGF

Brain derived neurotrophic factor BDNF Interleukin-11 IL-11 Nerve growth factor NGF Stromal derived factor-1 SDF-1

Hepatocyte growth factor HGF Insulin-like growth factor-1 IGF-1 Vascular endothelial growth factor VEGF

Angiogenin Angiogenin

term cytokines was initially used to distinguish factors that had specific immunomodulatory properties (produced by and act on immune cells), such as interleukins, lymphokines, and interferons. However, it is now known that many classic cytokines are also produced by and act on non-immune cells. Chemokines are a subclass of cytokines that promote chemotaxis (cell movement in response to a chemical concentration gradient). In general, as more functions are discovered about these proteins, definitions and classifications broaden and the terms are often used interchangeably. While trophic factors commonly refer to soluble proteins, extracellular matrix (ECM) proteins that are immobilized in the intercellular space also fall into this category since they direct cell growth, survival, differentiation, and migration.

Fig. 1. Summary of likely mechanisms of action for MSCs in the injured brain, highlighting the interconnectivity.

#### **3.2 Trophic support**

Transplanted MSCs augment host repair and recovery primarily through direct and indirect trophic support. MSCs secrete a plethora of factors that are known to promote neural cell survival and regeneration through paracrine signaling to neural, vascular, and immune cells. An overview of relevant trophic factors found to be secreted by human bone marrowderived MSCs *in vitro* is provided in Table 3. Which of these factors are secreted in the injured brain is under current investigation. Research is also ongoing to determine the exact or even the most critical mechanism(s) governing the beneficial effects of MSC transplantation. For now, we make a leap of knowledge based on existing evidence. There are numerous studies demonstrating that transplanted MSCs promote certain aspects of

term cytokines was initially used to distinguish factors that had specific immunomodulatory properties (produced by and act on immune cells), such as interleukins, lymphokines, and interferons. However, it is now known that many classic cytokines are also produced by and act on non-immune cells. Chemokines are a subclass of cytokines that promote chemotaxis (cell movement in response to a chemical concentration gradient). In general, as more functions are discovered about these proteins, definitions and classifications broaden and the terms are often used interchangeably. While trophic factors commonly refer to soluble proteins, extracellular matrix (ECM) proteins that are immobilized in the intercellular space also fall into this category since they direct cell growth, survival,

Fig. 1. Summary of likely mechanisms of action for MSCs in the injured brain, highlighting

Transplanted MSCs augment host repair and recovery primarily through direct and indirect trophic support. MSCs secrete a plethora of factors that are known to promote neural cell survival and regeneration through paracrine signaling to neural, vascular, and immune cells. An overview of relevant trophic factors found to be secreted by human bone marrowderived MSCs *in vitro* is provided in Table 3. Which of these factors are secreted in the injured brain is under current investigation. Research is also ongoing to determine the exact or even the most critical mechanism(s) governing the beneficial effects of MSC transplantation. For now, we make a leap of knowledge based on existing evidence. There are numerous studies demonstrating that transplanted MSCs promote certain aspects of

differentiation, and migration.

the interconnectivity.

**3.2 Trophic support**

recovery (e.g., decrease apoptosis, increase neurogenesis, synaptogenesis, and angiogenesis) in the injured brain. Concurrently, there are other studies showing that factors known to be secreted by MSCs are involved in mechanisms that promote these same aspects of recovery. The assumption is that some combination of these pro-recovery mechanisms occurs when MSCs are transplanted into the injured brain and that MSC-secreted factors are essential for these effects. Table 4 reviews potential beneficial mechanisms of action for repair and regeneration of the injured brain provided by MSC-secreted factors. The table provides references that demonstrate that the protein of interest enhances either 1) neuroprotection, 2) neural stem/progenitor cell proliferation or migration, 3) neural stem/progenitor cell differentiation, 4) neuritogenesis or synaptogenesis, 5) angiogenesis, or 6) another mechanism involved in recovery (such as reducing inhibitory components of the glial scar). While these entries are based on a thorough search, it is not intended to be completely exhaustive. Also, only the beneficial aspects of the various growth factors are presented. Some factors that enhance one pathway act as inhibitors in another (e.g., the proinflammatory molecule interleukin-17 potentiates neuronal cell death but supports angiogenesis). Since these studies often examine pathways individually, it is not clear which are the primary mechanisms that occur when (if) the molecule is secreted by MSCs in the injured brain. Further, the exact timing and concentration of the trophic factor are likely

**Promotes Neuroregeneration**

**↑ NSC Differentiation**

*et al.*, 2009)

neuronal (\*Bath & Lee, 2010)

> astrocytic (\*Sabo *et al.*, 2009)

> astrocytic (\*Sabo *et al.*, 2009)

astrocytic (Gajavelli *et al.*, 2004)

**↑ Neurite Outgrowth or Synapse Formation**

(\*Hansen *et al.*, 2008)

(Gascon *et al.*, 2005; \*Lipsky & Marini, 2007; \*Lykissas *et al.*, 2007)

(Gratacos *et al.*, 2001)

**↑Angiogenesis Additional**

Restore BBB (Nag *et al.*, 2011)

(\*Distler *et al.*, 2003)

(\*Distler *et al.*, 2003)

in presence of VEGF (\*Distler *et al.*, 2003)

> (Qin *et al.*, 2011)

critical in determining to which pathways they contribute.

**↑ NSC Proliferation or Migration**

(\*Ohab & Carmichael, 2008)

(\*Bath & Lee, 2010; \*Schabitz *et al.*, 2007)

> (Chou *et al.*, 2006)

ANG-2 (Liu *et al.*, 2009) neuronal (Liu

**Neuroprotection (↓Apoptosis)**

ANG-1 (\*Hansen *et al.*, 2008)

BDNF (\*Lykissas *et*

BMP-2 (Iantosca *et al.*, 1999)

BMP-4 (Iantosca *et al.*, 1999)

BMP-7 (Yabe *et al.*,

*al.*, 2007)

2002)

Angiogenin

**Soluble Growth Factors**

Table 3. Factors secreted *in vitro* by human bone marrow MSCs that may affect neural recovery.

(Potapova *et al.*, 2007) ELISA of

(Schinkothe *et al.*, 2008) Cytokine Array of

(Tate *et al.*, 2010) Cytokine Array of

(Lai *et al.*, 2010) Immunofluorescence

Conditioned Medium

of Extracellular Matrix

Conditioned Medium

Conditioned Medium

**Reference Detection Method Trophic Factors Found Abbreviation**

Monocyte chemoattractant

Vascular endothelial growth

Granulocyte colony stimulating

Monocyte chemoattractant

Tissue inhibitors of

Vascular endothelial

Heparin-binding epidermal

Platelet derived growth factor-

Vascular endothelial growth

Table 3. Factors secreted *in vitro* by human bone marrow MSCs that may affect neural recovery.

Angiogenin Angiogenin Bone morphogenetic protein-2 BMP-2 Interleukin-6 IL-6 Interleukin-8 IL-8 Interleukin-11 IL-11

protein-1 MCP-1

factor VEGF

Angiopoietin-2 ANG-2 Fibroblast growth factor-4 FGF-4 Fibroblast growth factor-9 FGF-9

factor G-CSF Growth related oncogene GRO Hepatocyte growth factor HGF Interleukin-8 IL-8 Interleukin-11 IL-11 Interleukin-17 IL-17

protein-1 MCP-1 Neurotrophin-4/5 NT-4/5 Oncostatin M OSM Placental growth factor PlGF

metalloproteinase-1 TIMP-1

growth factor VEGF Bone morphogenetic protein-4 BMP-4

Bone morphogenetic protein-7 BMP-7 Dickkopf-1 DKK-1 Fibroblast growth factor-7 FGF-7

growth factor-like growth factor HB-EGF Hepatocyte growth factor HGF Interleukin-6 IL-6 Monocyte chemoattractant protein -1 MCP-1

AA PDGF-AA

factor VEGF Collagen I Collagen I

Decorin Decorin Fibronectin Fibronectin Laminin Laminin Perlecan Perlecan

recovery (e.g., decrease apoptosis, increase neurogenesis, synaptogenesis, and angiogenesis) in the injured brain. Concurrently, there are other studies showing that factors known to be secreted by MSCs are involved in mechanisms that promote these same aspects of recovery. The assumption is that some combination of these pro-recovery mechanisms occurs when MSCs are transplanted into the injured brain and that MSC-secreted factors are essential for these effects. Table 4 reviews potential beneficial mechanisms of action for repair and regeneration of the injured brain provided by MSC-secreted factors. The table provides references that demonstrate that the protein of interest enhances either 1) neuroprotection, 2) neural stem/progenitor cell proliferation or migration, 3) neural stem/progenitor cell differentiation, 4) neuritogenesis or synaptogenesis, 5) angiogenesis, or 6) another mechanism involved in recovery (such as reducing inhibitory components of the glial scar). While these entries are based on a thorough search, it is not intended to be completely exhaustive. Also, only the beneficial aspects of the various growth factors are presented. Some factors that enhance one pathway act as inhibitors in another (e.g., the proinflammatory molecule interleukin-17 potentiates neuronal cell death but supports angiogenesis). Since these studies often examine pathways individually, it is not clear which are the primary mechanisms that occur when (if) the molecule is secreted by MSCs in the injured brain. Further, the exact timing and concentration of the trophic factor are likely critical in determining to which pathways they contribute.

 While the initial ischemic or mechanical insult causes immediate necrotic death, secondary cell death primarily occurs through apoptosis. MSCs secrete multiple factors known to promote neural cell survival (see Table 4). Human MSCs have been shown to rescue neural cells following *in vitro* injury (e.g., oxygen glucose deprivation, glutamate toxicity) via secreted soluble factors (Tate *et al.*, 2010; Zhong *et al.*, 2003) and ECM proteins (Aizman *et al.*, 2009). There are several reports of decreased apoptotic markers and enhanced preservation of neural cells in the injury penumbra when transplanting MSCs following experimental ischemic stroke (Li *et al.*, 2010; Li *et al.*, 2002; Xin *et al.*, 2010) or TBI (Kim *et al.*, 2010; Xiong *et al.*, 2009). For example, delivering human MSCs intravenously 1 day following experimental cerebral ischemia in rats led to significant reduction in apoptotic cell death in the injury penumbra as well as functional behavioral recovery (Li *et al.*, 2002). This study also found an increase in BDNF and NGF in the ipsilateral hemisphere of MSC-treated rats at 7 days post-stroke; however, they did not distinguish whether these trophic factors were produced by the donor or host cells. Li *et al.* (2010) show that transplanting human MSCs into the injury penumbra 1 week following experimental cerebral ischemia in monkeys decreased apoptotic cell death and the lesion volume. Human MSCs transplanted into the injury cavity 1 week following experimental TBI in rats lead to enhanced cell survival in the hippocampus and improved functional recovery, and this was further improved when the MSCs were delivered within a collagen I scaffold (Xiong *et al.*, 2009). Kim *et al.* (2010) found that delivering human MSCs intravenously 1 day post-TBI in rats improved functional recovery and enhanced host cell survival by increasing pAkt and decreasing caspace-3 cleavage. Further, this group reports increases in BDNF, NGF, and NT-3 in the MSC-treated brains, though they did not distinguish between donor or host origin. Clearly, exogenous MSCs provide neuroprotection following brain injury and this is one probable mechanism of action for

After brain injury, the brain attempts to regenerate by resorting to a developmental-like state with increased neurogenesis, neurite outgrowth, synaptogenesis, re-myelination, re-formation of the blood brain barrier, and angiogenesis. Once thought to be unable to regenerate, it is now known that neural stem cells persist in the normal adult brain (neurogenic zones include the subventricular zone in the lateral ventricles and the subgranular zone in the dentate gyrus of the hippocampus). After an ischemic or traumatic injury, endogenous neural stem cells proliferate, migrate to the site of injury, and differentiate into neurons and glia (Kernie & Parent, 2010). Neuroplasticity is the reorganization of neuronal circuitry by changing the number and/or strength of neurites and synapses. Such remapping occurs throughout life for learning and memory formation, and compensatory plasticity occurs in the spared tissue following brain injury (Nishibe *et al.*, 2010). Neuroregeneration collectively includes neural stem/progenitor cell proliferation, migration and differentiation, neurite outgrowth, and synapse

**3.2.1 Neuroprotection**

their benefit.

formation.

**3.2.2 Neuroregeneration**

\*Indicates review article; NSC=Neural stem/progenitor cell; Growth factor abbreviations are defined in Table 3

Table 4. Evidence of MSC-secreted factors promoting neuroprotection or regeneration.

#### **3.2.1 Neuroprotection**

56 Advanced Topics in Neurological Disorders

**Promotes Neuroregeneration**

**↑ NSC Differentiation**

**↑ Neurite Outgrowth or Synapse Formation**

(Yi *et al.*, 2010)

(Erskine *et al.*, 2011; Jin *et al.*, 2006)

*al.*, 2004) (\*Sottile, 2004)

2004)

(Einheber *et al.*, 1996; \*Pires Neto *et al.*, 1999)

(\*Colognato and Yurchenco, 2000; \*Pires-Neto 1999)

**↑Angiogenesis Additional**

↑MSC homing (Bantubungi *et al.*, 2008; Erlandsson *et al.*, 2004)

↑ MSC homing (Ponte *et al.*, 2007; Son *et al.*, 2006) and survival (Kortesidis *et al.*, 2005)

↓ glial scar (Davies *et al.*, 2004)

Restore BBB (Hunter *et al.*, 1992);↓glial scar (Hou *et al.*, 2005)

(Sun *et al.*, 2006)

(\*Beck & Plate, 2009; \*Fan & Yang, 2007)

(\*Greenberg & Jin, 2005; \*Shibuya, 2009)

(\*Sottile, 2004)

(\*Sottile, 2004)

(\*Bix & Iozzo, 2008; Lee *et al.*, 2011)

SCF

SDF-1

TGF

**Matrix Proteins**

Fibronectin

Perlecan

Table 3

**Neuroprotection (↓Apoptosis)**

(Dhandapani *et al.*, 2005; Erlandsson *et al.*, 2004; Li *et al.*, 2009)

(\*Buisson *et al.*, 2003; Lu *et al.*, 2005)

2003)

(Sakai *et al.*, 2001; Tate *et al.*, 2007)

2008)

(\*Bix & Iozzo, 2008; Lee *et al.*, 2011)

Laminin (Hall *et al.*,

TIMP-1 (Tan *et al.*,

VEGF (Jin *et al.*, 2000; Sun *et al.*, 2003)

**↑ NSC Proliferation or Migration**

(Bantubungi *et al.*, 2008; Erlandsson *et al.*, 2004; Zhao *et al.*, 2007)

(\*Ohab & Carmichael, 2008; Thored *et al.*, 2006)

(Ma *et al.*, 2008; Mathieu *et al.*, 2010)

(Sun *et al.*, 2003; Wang *et al.*, 2007a; Wang *et al.*, 2007b)

Decorin (Davies *et al.*,

oligodendrocyt ic (Hu *et al.*, 2009)

neuronal (Boote Jones & Mallapragada, 2007; Tate *et al.*, 2004)

\*Indicates review article; NSC=Neural stem/progenitor cell; Growth factor abbreviations are defined in

Table 4. Evidence of MSC-secreted factors promoting neuroprotection or regeneration.

(\*Henderson & Copp, 1997; Tate *et al.*, 2004; Testaz & Duband, 2001)

(Hall *et al.*, 2008; \*Perris & Perissinotto, 2000; Tate *et al.*, 2004)

Collagen I (Ma *et al.*, 2004) neuronal (Ma *et*

Following the initial insult, secondary injury mechanisms persist and cause cell death to surrounding tissue. While the initial ischemic or mechanical insult causes immediate necrotic death, secondary cell death primarily occurs through apoptosis. MSCs secrete multiple factors known to promote neural cell survival (see Table 4). Human MSCs have been shown to rescue neural cells following *in vitro* injury (e.g., oxygen glucose deprivation, glutamate toxicity) via secreted soluble factors (Tate *et al.*, 2010; Zhong *et al.*, 2003) and ECM proteins (Aizman *et al.*, 2009). There are several reports of decreased apoptotic markers and enhanced preservation of neural cells in the injury penumbra when transplanting MSCs following experimental ischemic stroke (Li *et al.*, 2010; Li *et al.*, 2002; Xin *et al.*, 2010) or TBI (Kim *et al.*, 2010; Xiong *et al.*, 2009). For example, delivering human MSCs intravenously 1 day following experimental cerebral ischemia in rats led to significant reduction in apoptotic cell death in the injury penumbra as well as functional behavioral recovery (Li *et al.*, 2002). This study also found an increase in BDNF and NGF in the ipsilateral hemisphere of MSC-treated rats at 7 days post-stroke; however, they did not distinguish whether these trophic factors were produced by the donor or host cells. Li *et al.* (2010) show that transplanting human MSCs into the injury penumbra 1 week following experimental cerebral ischemia in monkeys decreased apoptotic cell death and the lesion volume. Human MSCs transplanted into the injury cavity 1 week following experimental TBI in rats lead to enhanced cell survival in the hippocampus and improved functional recovery, and this was further improved when the MSCs were delivered within a collagen I scaffold (Xiong *et al.*, 2009). Kim *et al.* (2010) found that delivering human MSCs intravenously 1 day post-TBI in rats improved functional recovery and enhanced host cell survival by increasing pAkt and decreasing caspace-3 cleavage. Further, this group reports increases in BDNF, NGF, and NT-3 in the MSC-treated brains, though they did not distinguish between donor or host origin. Clearly, exogenous MSCs provide neuroprotection following brain injury and this is one probable mechanism of action for their benefit.

## **3.2.2 Neuroregeneration**

After brain injury, the brain attempts to regenerate by resorting to a developmental-like state with increased neurogenesis, neurite outgrowth, synaptogenesis, re-myelination, re-formation of the blood brain barrier, and angiogenesis. Once thought to be unable to regenerate, it is now known that neural stem cells persist in the normal adult brain (neurogenic zones include the subventricular zone in the lateral ventricles and the subgranular zone in the dentate gyrus of the hippocampus). After an ischemic or traumatic injury, endogenous neural stem cells proliferate, migrate to the site of injury, and differentiate into neurons and glia (Kernie & Parent, 2010). Neuroplasticity is the reorganization of neuronal circuitry by changing the number and/or strength of neurites and synapses. Such remapping occurs throughout life for learning and memory formation, and compensatory plasticity occurs in the spared tissue following brain injury (Nishibe *et al.*, 2010). Neuroregeneration collectively includes neural stem/progenitor cell proliferation, migration and differentiation, neurite outgrowth, and synapse formation.

decreased lesion volume and functional recovery. Collectively, these data illustrate that exogenous MSCs promote neuroregeneration following brain injury by directly affecting neural stem/progenitor cells and neurons and/or by reducing inhibitory glial scar

Another important aspect of regeneration is angiogenesis, which is the formation of new blood vessels from existing vasculature. In the adult, angiogenesis occurs after injury to help supply the damaged tissue with oxygen and nutrients. The process includes basement membrane disruption, endothelial cell migration and proliferation, three-dimensional tube formation, maturation, and stabilization by vascular smooth muscle cells. Each step is regulated by multiple cytokines and ECM molecules (for review, see Distler *et al*., 2003 or Fan & Yang 2007). Studies show that MSC-conditioned medium enhances endothelial cell proliferation (Kaigler *et al.*, 2003) and promotes angiogenesis *in vitro* and *in vivo* (Kinnaird *et al.*, 2004). Transplanting MSCs increases angiogenesis following experimental ischemic stroke (Omori *et al.*, 2008; Onda *et al.*, 2008; Pavlichenko *et al.*, 2008) and TBI (Xiong *et al.*, 2009). Potential pro-angiogenic factors secreted by MSCs are provided in Table 4. Notably, there is overlap between factors that promote angiogenesis and neurogenesis/neuritogenesis (reviewed in Emanueli *et al.*, 2003 and Lazarovici *et al.*, 2006).  After injury, there is increased permeability of the BBB leading to edema (reviewed in Nag *et al.*, 2011). Part of the repair process includes restoring the BBB, and regeneration includes formation of the BBB for new vasculature. Specific MSC-secreted factors such as ANG-1, FGF-2, and laminin may be involved in reforming the

There is a potent immune response following ischemic and traumatic brain injury. The innate immune response is a part of the normal wound healing process; however, persistent inflammation can become cytotoxic. In addition to interacting with neural and vascular cells, MSCs communicate with immune cells and are now known to be immnosuppressive. Examining the interactions of MSCs with immune cells *in vitro* reveals that MSCs suppress T cell proliferation and activation, inhibit B cell proliferation and IgG production, prevent dendritic cell differentiation and migration, and shift the cytokine secretion profile of dendritic cells, helper T cells, and natural killer cells towards anti-inflammatory (reviewed in Mezey *et al.*, 2010 and Nauta & Fibbe, 2007). Interestingly, studies that separate the MSCs from the immune cells using semi-permeable membranes indicate that soluble factors are critical for these effects. Candidate immunomodulatory factors secreted by MSCs include interleukin-6 (IL-6), transforming growth factor (TGF), prostaglandin E2, hepatocyte growth factor (HGF), indoleamine 2,3-dioxygenase (IDO), and monocyte colony stimulating factor (M-CSF) (reviewed in Mezey *et al.*, 2010 and Nauta & Fibbe, 2007). Moreover, ECM proteins, such as fibronectin, also interact with immune cells (Mosesson, 1984; Nasu-Tada *et al.*, 2005). Since shifting to a less inflammatory environment may facilitate neural repair and

components.

**3.2.3 Angiogenesis**

BBB following injury.

**3.3 Immunomodulation**

There are multiple *in vitro* studies showing that MSCs direct neuroregenerative processes. Bai *et al.* (2007) show that mouse neural stem cells had increased migration and neuronal and oligodendrocytic differentiation when they were cultured with either human MSCs or MSC-conditioned medium, indicating that soluble proteins are responsible for these effects. In related work, co-culture of human MSCs with rat neural stem cells revealed that MSCs promote differentiation into primarily astrocytes and oligodendrocytes (Robinson *et al.*, 2011). However MSC-conditioned media promoted primarily oligodendrocytic differentiation (Robinson *et al.*, 2011), indicating that matrix components or direct cell-cell contact also account for the effects of MSCs on neural stem cell differentiation. Indeed, Aizman *et al.* (2009) demonstrate that human MSC-derived ECM promotes differentiation of cortical cells into neurons, astrocytes and oligodendrocytes and also enhances neuronal neurite networks compared to single ECM proteins. Transplantation of MSCs augments endogenous regeneration following experimental ischemic stroke (Bao *et al.*, 2011; Li *et al.*, 2010; Li *et al.*, 2002; Xin *et al.*, 2010; Yoo *et al.*, 2008) and TBI (Mahmood *et al.*, 2004; Xiong *et al.*, 2009). For example, both Bao *et al.* (2011) and Yoo *et al.* (2008) show that intracerebral transplantation of human MSCs 3 days following experimental cerebral ischemia in rats increases proliferation and migration of host neural stem cells and also decreases their apoptosis, thus enhancing neurogenesis. They also report enhanced behavioral recovery, and Bao *et al.* demonstrate increases in BDNF, NT-3, and VEGF in the brains of MSC-treated rats, though they do not identify the source of these cytokines. Xin *et al.* (2010) found that intravenous delivery of mouse MSCs 1 day following experimental stroke in mice lead to increases in axon fiber density, synaptogenesis and myelination. Following experimental TBI in rats, transplanted rat MSCs promoted increased proliferation and neuronal differentiation in neurogenic zones along with improved motor and sensory recovery (Mahmood *et al.*, 2004). Xiong *et al.* (2009) also report that transplanting human MSCs intracerebrally 1 week post-TBI in rats leads to increased axonal fiber length and that the fiber length was directly proportional to performance on the behavior tasks. Multiple trophic factors secreted by MSCs may contribute to enhancing neuroregeneration (see Table 4).

The glial scar that forms following brain injury acutely acts to sequester the injury. Cellular components of the glial scar include reactive astrocytes, which help buffer excess glutamate and secrete neurotrophic factors, and activated microglia/macrophages which clear out dead tissue and secrete neurotrophic factors. However, extracellular components of the glial scar that persists adjacent to the injury site have been found to inhibit neurite extension (e.g., neurocan, Nogo protein), thus limiting regeneration (for review, see Properzi *et al.*, 2003). Transplantation of MSCs helps overcome this glial scar limitation following experimental stroke (Li *et al.*, 2010; Li *et al.*, 2005; Pavlichenko *et al.*, 2008; Shen *et al.*, 2008) and TBI (Zanier *et al.*, 2011). Following ischemic stroke, rats treated with rat MSCs transplanted intravenously had decreased glial scar thickness at both the acute (3 and 6 days post-stroke; Pavlichenko *et al.,* 2008) and chronic (4 months post-stroke; Li *et al.,* 2005) phases. Along with decreased glial scar thickness, these studies report decreased lesion volume, enhanced regeneration, and functional recovery for animals treated with MSCs. Shen *et al.* (2008) show a decrease in neurocan (an inhibitory chondroitin sulphate proteoglycan) and enhanced axonal outgrowth in the injury penumbra when ischemic rats were treated with rat MSCs. Zanier *et al.* (2011) transplanted human umbilical cord blood-derived MSCs MSCs into the traumatically injured mouse brain and observed a decrease in reactive astrocytes in the glial scar region along with decreased lesion volume and functional recovery. Collectively, these data illustrate that exogenous MSCs promote neuroregeneration following brain injury by directly affecting neural stem/progenitor cells and neurons and/or by reducing inhibitory glial scar components.

#### **3.2.3 Angiogenesis**

58 Advanced Topics in Neurological Disorders

There are multiple *in vitro* studies showing that MSCs direct neuroregenerative processes. Bai *et al.* (2007) show that mouse neural stem cells had increased migration and neuronal and oligodendrocytic differentiation when they were cultured with either human MSCs or MSC-conditioned medium, indicating that soluble proteins are responsible for these effects. In related work, co-culture of human MSCs with rat neural stem cells revealed that MSCs promote differentiation into primarily astrocytes and oligodendrocytes (Robinson *et al.*, 2011). However MSC-conditioned media promoted primarily oligodendrocytic differentiation (Robinson *et al.*, 2011), indicating that matrix components or direct cell-cell contact also account for the effects of MSCs on neural stem cell differentiation. Indeed, Aizman *et al.* (2009) demonstrate that human MSC-derived ECM promotes differentiation of cortical cells into neurons, astrocytes and oligodendrocytes and also enhances neuronal neurite networks compared to single ECM proteins. Transplantation of MSCs augments endogenous regeneration following experimental ischemic stroke (Bao *et al.*, 2011; Li *et al.*, 2010; Li *et al.*, 2002; Xin *et al.*, 2010; Yoo *et al.*, 2008) and TBI (Mahmood *et al.*, 2004; Xiong *et al.*, 2009). For example, both Bao *et al.* (2011) and Yoo *et al.* (2008) show that intracerebral transplantation of human MSCs 3 days following experimental cerebral ischemia in rats increases proliferation and migration of host neural stem cells and also decreases their apoptosis, thus enhancing neurogenesis. They also report enhanced behavioral recovery, and Bao *et al.* demonstrate increases in BDNF, NT-3, and VEGF in the brains of MSC-treated rats, though they do not identify the source of these cytokines. Xin *et al.* (2010) found that intravenous delivery of mouse MSCs 1 day following experimental stroke in mice lead to increases in axon fiber density, synaptogenesis and myelination. Following experimental TBI in rats, transplanted rat MSCs promoted increased proliferation and neuronal differentiation in neurogenic zones along with improved motor and sensory recovery (Mahmood *et al.*, 2004). Xiong *et al.* (2009) also report that transplanting human MSCs intracerebrally 1 week post-TBI in rats leads to increased axonal fiber length and that the fiber length was directly proportional to performance on the behavior tasks. Multiple trophic factors secreted by MSCs may

The glial scar that forms following brain injury acutely acts to sequester the injury. Cellular components of the glial scar include reactive astrocytes, which help buffer excess glutamate and secrete neurotrophic factors, and activated microglia/macrophages which clear out dead tissue and secrete neurotrophic factors. However, extracellular components of the glial scar that persists adjacent to the injury site have been found to inhibit neurite extension (e.g., neurocan, Nogo protein), thus limiting regeneration (for review, see Properzi *et al.*, 2003). Transplantation of MSCs helps overcome this glial scar limitation following experimental stroke (Li *et al.*, 2010; Li *et al.*, 2005; Pavlichenko *et al.*, 2008; Shen *et al.*, 2008) and TBI (Zanier *et al.*, 2011). Following ischemic stroke, rats treated with rat MSCs transplanted intravenously had decreased glial scar thickness at both the acute (3 and 6 days post-stroke; Pavlichenko *et al.,* 2008) and chronic (4 months post-stroke; Li *et al.,* 2005) phases. Along with decreased glial scar thickness, these studies report decreased lesion volume, enhanced regeneration, and functional recovery for animals treated with MSCs. Shen *et al.* (2008) show a decrease in neurocan (an inhibitory chondroitin sulphate proteoglycan) and enhanced axonal outgrowth in the injury penumbra when ischemic rats were treated with rat MSCs. Zanier *et al.* (2011) transplanted human umbilical cord blood-derived MSCs MSCs into the traumatically injured mouse brain and observed a decrease in reactive astrocytes in the glial scar region along with

contribute to enhancing neuroregeneration (see Table 4).

Another important aspect of regeneration is angiogenesis, which is the formation of new blood vessels from existing vasculature. In the adult, angiogenesis occurs after injury to help supply the damaged tissue with oxygen and nutrients. The process includes basement membrane disruption, endothelial cell migration and proliferation, three-dimensional tube formation, maturation, and stabilization by vascular smooth muscle cells. Each step is regulated by multiple cytokines and ECM molecules (for review, see Distler *et al*., 2003 or Fan & Yang 2007). Studies show that MSC-conditioned medium enhances endothelial cell proliferation (Kaigler *et al.*, 2003) and promotes angiogenesis *in vitro* and *in vivo* (Kinnaird *et al.*, 2004). Transplanting MSCs increases angiogenesis following experimental ischemic stroke (Omori *et al.*, 2008; Onda *et al.*, 2008; Pavlichenko *et al.*, 2008) and TBI (Xiong *et al.*, 2009). Potential pro-angiogenic factors secreted by MSCs are provided in Table 4. Notably, there is overlap between factors that promote angiogenesis and neurogenesis/neuritogenesis (reviewed in Emanueli *et al.*, 2003 and Lazarovici *et al.*, 2006).  After injury, there is increased permeability of the BBB leading to edema (reviewed in Nag *et al.*, 2011). Part of the repair process includes restoring the BBB, and regeneration includes formation of the BBB for new vasculature. Specific MSC-secreted factors such as ANG-1, FGF-2, and laminin may be involved in reforming the BBB following injury.

#### **3.3 Immunomodulation**

There is a potent immune response following ischemic and traumatic brain injury. The innate immune response is a part of the normal wound healing process; however, persistent inflammation can become cytotoxic. In addition to interacting with neural and vascular cells, MSCs communicate with immune cells and are now known to be immnosuppressive. Examining the interactions of MSCs with immune cells *in vitro* reveals that MSCs suppress T cell proliferation and activation, inhibit B cell proliferation and IgG production, prevent dendritic cell differentiation and migration, and shift the cytokine secretion profile of dendritic cells, helper T cells, and natural killer cells towards anti-inflammatory (reviewed in Mezey *et al.*, 2010 and Nauta & Fibbe, 2007). Interestingly, studies that separate the MSCs from the immune cells using semi-permeable membranes indicate that soluble factors are critical for these effects. Candidate immunomodulatory factors secreted by MSCs include interleukin-6 (IL-6), transforming growth factor (TGF), prostaglandin E2, hepatocyte growth factor (HGF), indoleamine 2,3-dioxygenase (IDO), and monocyte colony stimulating factor (M-CSF) (reviewed in Mezey *et al.*, 2010 and Nauta & Fibbe, 2007). Moreover, ECM proteins, such as fibronectin, also interact with immune cells (Mosesson, 1984; Nasu-Tada *et al.*, 2005). Since shifting to a less inflammatory environment may facilitate neural repair and

Aizman, I., Tate, C.C., McGrogan, M. & Case, C.C. (2009). Extracellular Matrix Produced by

Alzheimer, C. & Werner, S. (2002). Fibroblast Growth Factors and Neuroprotection.

Araujo, D.M. & Cotman, C.W. (1993). Trophic Effects of Interleukin-4, -7 and -8 on

Arnhold, S., Klein, H., Klinz, F.J., Absenger, Y., Schmidt, A., Schinkothe, T., Brixius, K.,

Bai, L., Caplan, A., Lennon, D. & Miller, R.H. (2007). Human Mesenchymal Stem Cells

Bantubungi, K., Blum, D., Cuvelier, L., Wislet-Gendebien, S., Rogister, B., Brouillet, E. &

Bao, X., Wei, J., Feng, M., Lu, S., Li, G., Dou, W., Ma, W., Ma, S., An, Y., Qin, C., Zhao, R.C. &

Barnabe, G.F., Schwindt, T.T., Calcagnotto, M.E., Motta, F.L., Martinez, G.,Jr, de Oliveira,

Bath, K.G. & Lee, F.S. (2010). Neurotrophic Factor Control of Adult SVZ Neurogenesis. *Developmental Neurobiology,* Vol.70, No.5, pp. 339-349, ISSN 1932-846X; 1932-8451 Battistella, V., de Freitas, G.R., da Fonseca, L.M., Mercante, D., Gutfilen, B., Goldenberg,

*Brain Research,* Vol.600, No.1, pp. 49-55, ISSN 0006-8993

*Journal of Cell Biology,* Vol.85, No.6, pp. 551-565, ISSN 0171-9335

*Neurosciences,* Vol.37, No.3, pp. 454-470, ISSN 1095-9327; 1044-7431

*One,* Vol.4, No.4, pp. e5222, ISSN 1932-6203; 1932-6203

1792-1803, ISSN 1529-2401; 0270-6474

Bone Marrow Stromal Cells and by their Derivative, SB623 Cells, Supports Neural Cell Growth. *Journal of Neuroscience Research,* Vol.87, No.14, pp. 3198-3206, ISSN

*Advances in Experimental Medicine and Biology,* Vol.513, pp. 335-351, ISSN 0065-2598

Hippocampal Neuronal Cultures: Potential Involvement of Glial-Derived Factors.

Kozlowski, J., Desai, B., Bloch, W. & Addicks, K. (2006). Human Bone Marrow Stroma Cells Display Certain Neural Characteristics and Integrate in the Subventricular Compartment After Injection into the Liquor System. *European*

Signals Regulate Neural Stem Cell Fate. *Neurochemical Research,* Vol.32, No.2, pp.

Schiffmann, S.N. (2008). Stem Cell Factor and Mesenchymal and Neural Stem Cell Transplantation in a Rat Model of Huntington's Disease. *Molecular and Cellular*

Wang, R. (2011). Transplantation of Human Bone Marrow-Derived Mesenchymal Stem Cells Promotes Behavioral Recovery and Endogenous Neurogenesis After Cerebral Ischemia in Rats. *Brain Research,* Vol.1367, pp. 103-113, ISSN 1872-6240;

A.C., Keim, L.M., D'Almeida, V., Mendez-Otero, R. & Mello, L.E. (2009). Chemically-Induced RAT Mesenchymal Stem Cells Adopt Molecular Properties of Neuronal-Like Cells but do Not have Basic Neuronal Functional Properties. *PloS*

R.C., Dias, J.V., Kasai-Brunswick, T.H., Wajnberg, E., Rosado-de-Castro, P.H., Alves-Leon, S.V., Mendez-Otero, R. & Andre, C. (2011). Safety of Autologous Bone Marrow Mononuclear Cell Transplantation in Patients with Nonacute Ischemic Stroke. *Regenerative Medicine,* Vol.6, No.1, pp. 45-52, ISSN 1746-076X; 1746-0751 Bauer, S., Rasika, S., Han, J., Mauduit, C., Raccurt, M., Morel, G., Jourdan, F., Benahmed, M.,

Moyse, E. & Patterson, P.H. (2003). Leukemia Inhibitory Factor is a Key Signal for Injury-Induced Neurogenesis in the Adult Mouse Olfactory Epithelium. *The Journal of Neuroscience : The Official Journal of the Society for Neuroscience,* Vol.23, No.5, pp.

**5. References**

1097-4547

353-362, ISSN 0364-3190

0006-8993

regeneration, immunomodulation is another feasible therapeutic mechanism of action for transplanted MSCs. Note that many immunomodulatory factors also have potential roles for directly promoting neural cell survival and regeneration (see Table 4). Likewise, NGF, the prototypic neurotrophic factor, has been shown to be anti-inflammatory (Villoslada & Genain, 2004). The interaction between angiogenesis and inflammation is also welldocumented (for review, see Jackson *et al.*, 1997 or Noonan *et al.*, 2008), which further underscores the complexity and interrelatedness of these recovery mechanisms.

## **3.4 Challenges of identifying critical factors and mechanisms**

Cell transplantation is a dynamic treatment that can target multiple therapeutic mechanisms. Advantages of transplanting cells compared to pharmaceutical treatments include the ability to 1) easily localize the treatment to the affected tissue, 2) supply a variety of trophic factors at physiologic concentrations, 3) persist long enough to alter the microenvironment of the injured brain tissue; and 4) interact with host cells. The beneficial effects of transplanted MSCs have been corraborated *in vitro* and *in vivo* and some potential pathways have been identified as described above. It is probable that a combination of multiple mechanisms of action synergistically contribute to improve functional recovery. While this ability to intervene along multiple pathways is desirable for a robust treatment, it makes identifying key mechanisms and factors challenging. Clarifying critical mechanisms of action would allow for treatments to be optimized to best facilitate these roles. Furthermore, difficulty pinpointing key mechanisms is a hurdle for developing potency assays for the clinical use of MSCs. Potency assays are critical for ranking and qualifying different cell lots on their ability to promote recovery. Another complication for determining potency of cells *ex vivo* is that transplanted cells interact with the host cells via paracrine signaling and possibly direct cell-cell contact. MSCs alter the secretion profile of host neural and immune cells, such as astrocytes and microglia (Gao *et al.*, 2005; Xin *et al.*, 2010), which further acts to promote repair and regeneration. Additionally, the secretion profile of MSCs is a function of the microenvironment and changes in the presence of injured brain tissue (Chen *et al.*, 2002a, 2002b). Thus, there is a complex and dynamic web of players involved in MSC-mediated effects. Ideally, potency assays would be easily reproducible *in vitro* assays, however the interplay between donor cells and the host environment is difficult to model *in vitro*. Elucidating critical aspects of this therapy will be the focus of intense research for years to come.

### **4. Conclusion**

Stroke and TBI are major contributors to death and persistent disability, and treatments that effectively promote repair and regeneration are desired. Cell transplantation is a promising treatment for brain injury, and MSCs are an attractive cell source due to their technical and safety advantages. Pre-clinical i*n vivo* data show that transplanting MSCs enhances neuroprotection, promotes regeneration and/or suppresses inflammation. MSCs secrete numerous soluble and insoluble factors that are known to benefit the injured brain, which are likely crucial to the mechanisms of action governing MSC-mediated recovery. MSCs aid injured brain tissue by targeting multiple, non-mutually exclusive pathways, which is an advantage for a potential treatment, but a challenge for elucidating critical mechanisms and factors.

#### **5. References**

60 Advanced Topics in Neurological Disorders

regeneration, immunomodulation is another feasible therapeutic mechanism of action for transplanted MSCs. Note that many immunomodulatory factors also have potential roles for directly promoting neural cell survival and regeneration (see Table 4). Likewise, NGF, the prototypic neurotrophic factor, has been shown to be anti-inflammatory (Villoslada & Genain, 2004). The interaction between angiogenesis and inflammation is also welldocumented (for review, see Jackson *et al.*, 1997 or Noonan *et al.*, 2008), which further

Cell transplantation is a dynamic treatment that can target multiple therapeutic mechanisms. Advantages of transplanting cells compared to pharmaceutical treatments include the ability to 1) easily localize the treatment to the affected tissue, 2) supply a variety of trophic factors at physiologic concentrations, 3) persist long enough to alter the microenvironment of the injured brain tissue; and 4) interact with host cells. The beneficial effects of transplanted MSCs have been corraborated *in vitro* and *in vivo* and some potential pathways have been identified as described above. It is probable that a combination of multiple mechanisms of action synergistically contribute to improve functional recovery. While this ability to intervene along multiple pathways is desirable for a robust treatment, it makes identifying key mechanisms and factors challenging. Clarifying critical mechanisms of action would allow for treatments to be optimized to best facilitate these roles. Furthermore, difficulty pinpointing key mechanisms is a hurdle for developing potency assays for the clinical use of MSCs. Potency assays are critical for ranking and qualifying different cell lots on their ability to promote recovery. Another complication for determining potency of cells *ex vivo* is that transplanted cells interact with the host cells via paracrine signaling and possibly direct cell-cell contact. MSCs alter the secretion profile of host neural and immune cells, such as astrocytes and microglia (Gao *et al.*, 2005; Xin *et al.*, 2010), which further acts to promote repair and regeneration. Additionally, the secretion profile of MSCs is a function of the microenvironment and changes in the presence of injured brain tissue (Chen *et al.*, 2002a, 2002b). Thus, there is a complex and dynamic web of players involved in MSC-mediated effects. Ideally, potency assays would be easily reproducible *in vitro* assays, however the interplay between donor cells and the host environment is difficult to model *in vitro*. Elucidating critical aspects of this therapy will be

Stroke and TBI are major contributors to death and persistent disability, and treatments that effectively promote repair and regeneration are desired. Cell transplantation is a promising treatment for brain injury, and MSCs are an attractive cell source due to their technical and safety advantages. Pre-clinical i*n vivo* data show that transplanting MSCs enhances neuroprotection, promotes regeneration and/or suppresses inflammation. MSCs secrete numerous soluble and insoluble factors that are known to benefit the injured brain, which are likely crucial to the mechanisms of action governing MSC-mediated recovery. MSCs aid injured brain tissue by targeting multiple, non-mutually exclusive pathways, which is an advantage for a potential treatment, but a challenge for elucidating critical mechanisms and

underscores the complexity and interrelatedness of these recovery mechanisms.

**3.4 Challenges of identifying critical factors and mechanisms**

the focus of intense research for years to come.

**4. Conclusion**

factors.

Chou, J., Harvey, B.K., Chang, C.F., Shen, H., Morales, M. & Wang, Y. (2006).

Coles, J.P. (2004). Regional Ischemia After Head Injury. *Current Opinion in Critical Care,*

Cox, C.S.,Jr, Baumgartner, J.E., Harting, M.T., Worth, L.L., Walker, P.A., Shah, S.K., Ewing-

Crigler, L., Robey, R.C., Asawachaicharn, A., Gaupp, D. & Phinney, D.G. (2006). Human

Davies, J.E., Tang, X., Denning, J.W., Archibald, S.J. & Davies, S.J. (2004). Decorin Suppresses

Dempsey, R.J., Sailor, K.A., Bowen, K.K., Tureyen, K. & Vemuganti, R. (2003). Stroke-

Deng, J., Zou, Z.M., Zhou, T.L., Su, Y.P., Ai, G.P., Wang, J.P., Xu, H. & Dong, S.W. (2011).

*Neurological Sciences,* Vol.32, No.4, pp. 641-651, ISSN 1590-3478; 1590-1874 Dhandapani, K.M., Wade, F.M., Wakade, C., Mahesh, V.B. & Brann, D.W. (2005).

*Sciences,* Vol.240, No.1-2, pp. 21-29, ISSN 0022-510X

*Neurology,* Vol.198, No.1, pp. 54-64, ISSN 0014-4886

No.5, pp. 1226-1242, ISSN 0953-816X

pp. 586-597, ISSN 0022-3042

1-8, ISSN 1872-6240; 0006-8993

Vol.10, No.2, pp. 120-125, ISSN 1070-5295

396X

Neuroregenerative Effects of BMP7 After Stroke in Rats. *Journal of the Neurological*

Cobbs, L., Hasan, K.M., Day, M.C., Lee, D., Jimenez, F. & Gee, A. (2011). Autologous Bone Marrow Mononuclear Cell Therapy for Severe Traumatic Brain Injury in Children. *Neurosurgery,* Vol.68, No.3, pp. 588-600, ISSN 1524-4040; 0148-

Mesenchymal Stem Cell Subpopulations Express a Variety of Neuro-Regulatory Molecules and Promote Neuronal Cell Survival and Neuritogenesis. *Experimental*

Neurocan, Brevican, Phosphacan and NG2 Expression and Promotes Axon Growth Across Adult Rat Spinal Cord Injuries. *The European Journal of Neuroscience,* Vol.19,

Induced Progenitor Cell Proliferation in Adult Spontaneously Hypertensive Rat Brain: Effect of Exogenous IGF-1 and GDNF. *Journal of Neurochemistry,* Vol.87, No.3,

Bone Marrow Mesenchymal Stem Cells can be Mobilized into Peripheral Blood by G-CSF in Vivo and Integrate into Traumatically Injured Cerebral Tissue.

Neuroprotection by Stem Cell Factor in Rat Cortical Neurons Involves AKT and NFkappaB. *Journal of Neurochemistry,* Vol.95, No.1, pp. 9-19, 0022-3042; 0022-3042 Distler, J.H., Hirth, A., Kurowska-Stolarska, M., Gay, R.E., Gay, S. & Distler, O. (2003).

Angiogenic and Angiostatic Factors in the Molecular Control of Angiogenesis. *The Quarterly Journal of Nuclear Medicine,* Vol.47, No.3, pp. 149-161, ISSN 1125-0135 Du, H., Li, P., Pan, Y., Li, W., Hou, J., Chen, H., Wang, J. & Tang, H. (2010). Vascular

Endothelial Growth Factor Signaling Implicated in Neuroprotective Effects of Placental Growth Factor in an in Vitro Ischemic Model. *Brain Research,* Vol.1357, pp.

Ultrastructural Distribution of the Alpha 8 Integrin Subunit in Developing and Adult Rat Brain Suggests a Role in Synaptic Function. *Journal of Comparative*

Vascularization and Neurogenesis by Neurotrophins. *British Journal of*

(2008). Wnt-3a and Dickkopf-1 Stimulate Neurite Outgrowth in Ewing Tumor Cells

Einheber, S., Schnapp, L.M., Salzer, J.L., Cappiello, Z.B. & Milner, T.A. (1996). Regional and

Emanueli, C., Schratzberger, P., Kirchmair, R. & Madeddu, P. (2003). Paracrine Control of

Endo, Y., Beauchamp, E., Woods, D., Taylor, W.G., Toretsky, J.A., Uren, A. & Rubin, J.S.

*Neurology,* Vol.370, No.1, pp. 105-34, ISSN 1096-9861; 0021-9967

*Pharmacology,* Vol.140, No.4, pp. 614-619, ISSN 0007-1188

Bechara, C., Chai, H., Lin, P.H., Yao, Q. & Chen, C. (2007). Growth Related Oncogene-Alpha

Beck, H. & Plate, K.H. (2009). Angiogenesis After Cerebral Ischemia. *Acta Neuropathologica,*

Bendfeldt, K., Radojevic, V., Kapfhammer, J. & Nitsch, C. (2007). Basic Fibroblast Growth

Bix, G. & Iozzo, R.V. (2008). Novel Interactions of Perlecan: Unraveling Perlecan's Role in

Boote Jones, E.N. & Mallapragada, S.K. (2007). Directed Growth and Differentiation of Stem

Bouhy, D., Malgrange, B., Multon, S., Poirrier, A.L., Scholtes, F., Schoenen, J. & Franzen, R.

Bramlett, H.M. & Dietrich, W.D. (2004). Pathophysiology of Cerebral Ischemia and Brain

Buisson, A., Lesne, S., Docagne, F., Ali, C., Nicole, O., MacKenzie, E.T. & Vivien, D. (2003).

Buschmann, I.R., Busch, H.J., Mies, G. & Hossmann, K.A. (2003). Therapeutic Induction of

Chen, X., Katakowski, M., Li, Y., Lu, D., Wang, L., Zhang, L., Chen, J., Xu, Y., Gautam, S.,

Chen, X., Li, Y., Wang, L., Katakowski, M., Zhang, L., Chen, J., Xu, Y., Gautam, S.C. &

*of Neuroscience Research,* Vol.69, No.5, pp. 687-691, ISSN 0360-4012

*Clinical Research,* Vol.13, No.6, pp. RA87-90, ISSN 1234-1010

Vol.117, No.5, pp. 481-496, ISSN 1432-0533; 0001-6322

No.7, pp. 1509-1521, ISSN 1097-4547; 0360-4012

Vol.24, No.2, pp. 133-150, ISSN 0271-678X

*Neurobiology,* Vol.23, No.4-5, pp. 539-550, ISSN 0272-4340

0270-6474

1059-910X

1530-6860; 0892-6638

7322

0919-6544

(GRO-Alpha): Roles in Atherosclerosis, Angiogenesis and Other Inflammatory Conditions. *Medical Science Monitor: International Medical Journal of Experimental and*

Factor Modulates Density of Blood Vessels and Preserves Tight Junctions in Organotypic Cortical Cultures of Mice: A New in Vitro Model of the Blood-Brain Barrier. *The Journal of Neuroscience,* Vol.27, No.12, pp. 3260-3267, ISSN 1529-2401;

Angiogenesis. *Microscopy Research and Technique,* Vol.71, No.5, pp. 339-348, ISSN

Cells Towards Neural Cell Fates using Soluble and Surface-Mediated Cues. *Journal of Biomaterials Science.Polymer Edition,* Vol.18, No.8, pp. 999-1015, ISSN 0920-5063 Boucherie, C. & Hermans, E. (2009). Adult Stem Cell Therapies for Neurological Disorders:

Benefits Beyond Neuronal Replacement? *Journal of Neuroscience Research,* Vol.87,

(2006). Delayed GM-CSF Treatment Stimulates Axonal Regeneration and Functional Recovery in Paraplegic Rats Via an Increased BDNF Expression by Endogenous Macrophages. *The FASEB Journal,* Vol.20, No.8, pp. 1239-1241, ISSN

Trauma: Similarities and Differences. *Journal of Cerebral Blood Flow and Metabolism,*

Transforming Growth Factor-Beta and Ischemic Brain Injury. *Cellular and Molecular*

Arteriogenesis in Hypoperfused Rat Brain Via Granulocyte-Macrophage Colony-Stimulating Factor. *Circulation,* Vol.108, No.5, pp. 610-615, ISSN 1524-4539; 0009-

Mahmood, A. & Chopp, M. (2002a). Human Bone Marrow Stromal Cell Cultures Conditioned by Traumatic Brain Tissue Extracts: Growth Factor Production. *Journal*

Chopp, M. (2002b). Ischemic Rat Brain Extracts Induce Human Marrow Stromal Cell Growth Factor Production. *Neuropathology,* Vol.22, No.4, pp. 275-279, ISSN

Glezer, I. & Rivest, S. (2010). Oncostatin M is a Novel Glucocorticoid-Dependent

Gratacos, E., Checa, N., Perez-Navarro, E. & Alberch, J. (2001). Brain-Derived Neurotrophic

Greenberg, D.A. & Jin, K. (2005). From Angiogenesis to Neuropathology. *Nature,* Vol.438,

Greve, M.W. & Zink, B.J. (2009). Pathophysiology of Traumatic Brain Injury. *The Mount*

Hall, P.E., Lathia, J.D., Caldwell, M.A. & Ffrench-Constant, C. (2008). Laminin Enhances the

Hamanoue, M., Takemoto, N., Matsumoto, K., Nakamura, T., Nakajima, K. & Kohsaka, S.

Hansen, T.M., Moss, A.J. & Brindle, N.P. (2008). Vascular Endothelial Growth Factor and

Haynesworth, S.E., Baber, M.A. & Caplan, A.I. (1996). Cytokine Expression by Human

Henderson, D.J. & Copp, A.J. (1997). Role of the Extracellular Matrix in Neural Crest Cell

Heron, M., Hoyert, D.L., Murphy, S.L., Xu, J., Kochanek, K.D. & Tejada-Vera, B. (2009).

Honda, S., Kagoshima, M., Wanaka, A., Tohyama, M., Matsumoto, K. & Nakamura, T.

Honmou, O., Houkin, K., Matsunaga, T., Niitsu, Y., Ishiai, S., Onodera, R., Waxman, S.G. &

Horwitz, E.M., Le Blanc, K., Dominici, M., Mueller, I., Slaper-Cortenbach, I., Marini, F.C.,

*Statistics System,* Vol.57, No.14, pp. 1-134, ISSN 1551-8922

*Research,* Vol.32, No.2, pp. 197-210, ISSN 0169-328X

1807, ISSN 1460-2156; 0006-8950

No.7070, pp. 954-959, ISSN 1476-4687; 0028-0836

9533

2507

9541

ISSN 1090-2139; 0889-1591

Vol.9, pp. 71, ISSN 1471-2202

ISSN 0022-3042

ISSN 0360-4012

Barrier Function. *Journal of Cell Science,* Vol.112, Pt 12, pp. 2049-2057, ISSN 0021-

Neuroinflammatory Factor that Enhances Oligodendrocyte Precursor Cell Activity in Demyelinated Sites. *Brain, Behavior, and Immunity,* Vol.24, No.5, pp. 695-704,

Factor (BDNF) Mediates Bone Morphogenetic Protein-2 (BMP-2) Effects on Cultured Striatal Neurones. *Journal of Neurochemistry,* Vol.79, No.4, pp. 747-755,

*Sinai Journal of Medicine, New York,* Vol.76, No.2, pp. 97-104, ISSN 1931-7581; 0027-

Growth of Human Neural Stem Cells in Defined Culture Media. *BMC Neuroscience,*

(1996). Neurotrophic Effect of Hepatocyte Growth Factor on Central Nervous System Neurons in Vitro. *Journal of Neuroscience Research,* Vol.43, No.5, pp. 554-564,

Angiopoietins in Neurovascular Regeneration and Protection Following Stroke. *Current Neurovascular Research,* Vol.5, No.4, pp. 236-245, ISSN 1875-5739; 1567-2026

Marrow-Derived Mesenchymal Progenitor Cells in Vitro: Effects of Dexamethasone and IL-1 Alpha. *Journal of Cellular Physiology,* Vol.166, No.3, pp. 585-592, ISSN 0021-

Migration. *Journal of Anatomy,* Vol.191, Pt.4, pp. 507-515, ISSN 1469-7580; 0021-8782

Deaths: Final Data for 2006. *National Vital Statistics Reports : From the Centers for Disease Control and Prevention, National Center for Health Statistics, National Vital*

(1995). Localization and Functional Coupling of HGF and c-Met/HGF Receptor in Rat Brain: Implication as Neurotrophic Factor. *Brain Research.Molecular Brain*

Kocsis, J.D. (2011). Intravenous Administration of Auto Serum-Expanded Autologous Mesenchymal Stem Cells in Stroke. *Brain,* Vol.134, No.Pt 6, pp. 1790-

Deans, R.J., Krause, D.S., Keating, A. & International Society for Cellular Therapy.

Via a Frizzled3- and c-Jun N-Terminal Kinase-Dependent Mechanism. *Molecular and Cellular Biology,* Vol.28, No.7, pp. 2368-2379, ISSN 1098-5549; 0270-7306

*and Cellular Biology,* Vol.28, No.7, pp. 2368-2379, ISSN 1098-5549; 0270-7306 Erlandsson, A., Larsson, J. & Forsberg-Nilsson, K. (2004). Stem Cell Factor is a

Erskine, L., Reijntjes, S., Pratt, T., Denti, L., Schwarz, Q., Vieira, J.M., Alakakone, B., Shewan,

Fan, Y. & Yang, G.Y. (2007). Therapeutic Angiogenesis for Brain Ischemia: A Brief Review.

Faul, M., Xu, L., Wald, M.M. & Coronado, V.G. (2010). *Traumatic brain injury in the United*

Finkelstein EA, Corso PS, Miller TR. 2006. *The Incidence and Economic Burden of Injuries in the*

Forsberg-Nilsson, K., Behar, T.N., Afrakhte, M., Barker, J.L. & McKay, R.D. (1998). Platelet-

Frontini, M.J., Nong, Z., Gros, R., Drangova, M., O'Neil, C., Rahman, M.N., Akawi, O., Yin,

Gajavelli, S., Wood, P.M., Pennica, D., Whittemore, S.R. & Tsoulfas, P. (2004). BMP Signaling

Gao, Q., Li, Y. & Chopp, M. (2005). Bone Marrow Stromal Cells Increase Astrocyte Survival

Anaerobic Insult. *Neuroscience,* Vol.136, No.1, pp. 123-134, ISSN 0306-4522 Garnett, M.R., Blamire, A.M., Corkill, R.G., Rajagopalan, B., Young, J.D., Cadoux-Hudson,

Gascon, E., Vutskits, L., Zhang, H., Barral-Moran, M.J., Kiss, P.J., Mas, C. & Kiss, J.Z. (2005).

Gillis, P., Savla, U., Volpert, O.V., Jimenez, B., Waters, C.M., Panos, R.J. & Bouck, N.P.

*Neuroscience,* Vol.21, No.1, pp. 69-80, ISSN 0953-816X

*Neurosci Res,* Vol.53, No.5, pp. 521-530, ISSN 1097-4547; 0360-4012

*Experimental Neurology,* Vol.188, No.2, pp. 205-223, ISSN 0014-4886

*Research,* Vol.301, No.2, pp. 201-210, ISSN 0014-4827

*United States*. Oxford University Press, New York, NY

965, ISSN 1097-4199; 0896-6273

1557-1890

0156

ISSN 0897-7151

Control, Atlanta, GA

Via a Frizzled3- and c-Jun N-Terminal Kinase-Dependent Mechanism. *Molecular*

Chemoattractant and a Survival Factor for CNS Stem Cells. *Experimental Cell*

D. & Ruhrberg, C. (2011). VEGF Signaling through Neuropilin 1 Guides Commissural Axon Crossing at the Optic Chiasm. *Neuron,* Vol.70, No.5, pp. 951-

*Journal of Neuroimmune Pharmacology,* Vol.2, No.3, pp. 284-289, ISSN 1557-1904;

*States: emergency department visits, hospitalizations, and deaths 2002-2006*. Centers for Disease Control and Prevention, National Center for Injury Prevention and

Derived Growth Factor Induces Chemotaxis of Neuroepithelial Stem Cells. *J*

H., Ellis, C.G. & Pickering, J.G. (2011). Fibroblast growth factor 9 delivery during angiogenesis produces durable, vasoresponsive microvessels wrapped by smooth muscle cells. *Nature Biotechnology*, Vol.29, No.5, pp. 421-427, ISSN 1546-1696; 1087-

Initiates a Neural Crest Differentiation Program in Embryonic Rat CNS Stem Cells.

via Upregulation of Phosphoinositide 3-kinase/threonine Protein Kinase and Mitogen-Activated Protein Kinase kinase/extracellular Signal-Regulated Kinase Pathways and Stimulate Astrocyte Trophic Factor Gene Expression After

T.A. & Styles, P. (2001). Abnormal Cerebral Blood Volume in Regions of Contused and Normal Appearing Brain Following Traumatic Brain Injury using Perfusion Magnetic Resonance Imaging. *Journal of Neurotrauma,* Vol.18, No.6, pp. 585-593,

Sequential Activation of p75 and TrkB is Involved in Dendritic Development of Subventricular Zone-Derived Neuronal Progenitors in Vitro. *The European Journal of*

(1999). Keratinocyte Growth Factor Induces Angiogenesis and Protects Endothelial

Barrier Function. *Journal of Cell Science,* Vol.112, Pt 12, pp. 2049-2057, ISSN 0021- 9533

Johe, K.K., Hazel, T.G., Muller, T., Dugich-Djordjevic, M.M. & McKay, R.D. (1996). Single

Joseph D'Ercole, A. & Ye, P. (2008). Expanding the Mind: Insulin-Like Growth Factor I and Brain Development. *Endocrinology,* Vol.149, No.12, pp. 5958-5962, ISSN 0013-7227 Kaigler, D., Krebsbach, P.H., Polverini, P.J. & Mooney, D.J. (2003). Role of Vascular

Kernie, S.G. & Parent, J.M. (2010). Forebrain Neurogenesis After Focal Ischemic and

Kim, H.J., Lee, J.H. & Kim, S.H. (2010). Therapeutic Effects of Human Mesenchymal Stem

Kinnaird, T., Stabile, E., Burnett, M.S., Lee, C.W., Barr, S., Fuchs, S. & Epstein, S.E. (2004).

Kopen, G.C., Prockop, D.J. & Phinney, D.G. (1999). Marrow Stromal Cells Migrate

Kosaka, N., Kodama, M., Sasaki, H., Yamamoto, Y., Takeshita, F., Takahama, Y., Sakamoto,

Kumar, R., Yoneda, J., Bucana, C.D. & Fidler, I.J. (1998). Regulation of Distinct Steps of

Lai, Y., Sun, Y., Skinner, C.M., Son, E.L., Lu, Z., Tuan, R.S., Jilka, R.L., Ling, J. & Chen, X.D.

Lazarovici, P., Marcinkiewicz, C. & Lelkes, P.I. (2006). Cross Talk between the

Cells. *Tissue Engineering,* Vol.9, No.1, pp. 95-103, ISSN 1076-3279

9369

4571

ISSN 0006-4971

pp. 1484-1485, ISSN 1530-6860; 0892-6638

Vol.12, No.4, pp. 749-757, ISSN 1019-6439

ISSN 1557-8534; 1547-3287

1095-953X; 0969-9961

1557-9042; 0897-7151

Factors Direct the Differentiation of Stem Cells from the Fetal and Adult Central Nervous System. *Genes & Development,* Vol.10, No.24, pp. 3129-3140, ISSN 0890-

Endothelial Growth Factor in Bone Marrow Stromal Cell Modulation of Endothelial

Traumatic Brain Injury. *Neurobiology of Disease,* Vol.37, No.2, pp. 267-274, ISSN

Cells on Traumatic Brain Injury in Rats: Secretion of Neurotrophic Factors and Inhibition of Apoptosis. *Journal of Neurotrauma,* Vol.27, No.1, pp. 131-138, ISSN

Marrow-Derived Stromal Cells Express Genes Encoding a Broad Spectrum of Arteriogenic Cytokines and Promote in Vitro and in Vivo Arteriogenesis through Paracrine Mechanisms. *Circulation Research,* Vol.94, No.5, pp. 678-685, ISSN 1524-

Throughout Forebrain and Cerebellum, and they Differentiate into Astrocytes After Injection into Neonatal Mouse Brains. *Proceedings of the National Academy of Sciences of the United States of America,* Vol.96, No.19, pp. 10711-10716, ISSN 0027-8424 Kortesidis, A., Zannettino, A., Isenmann, S., Shi, S., Lapidot, T. & Gronthos, S. (2005).

Stromal-Derived Factor-1 Promotes the Growth, Survival, and Development of Human Bone Marrow Stromal Stem Cells. *Blood,* Vol.105, No.10, pp. 3793-3801,

H., Kato, T., Terada, M. & Ochiya, T. (2006). FGF-4 Regulates Neural Progenitor Cell Proliferation and Neuronal Differentiation. *The FASEB Journal,* Vol.20, No.9,

Angiogenesis by Different Angiogenic Molecules. *International Journal of Oncology,*

(2010). Reconstitution of Marrow-Derived Extracellular Matrix Ex Vivo: A Robust Culture System for Expanding Large-Scale Highly Functional Human Mesenchymal Stem Cells. *Stem Cells and Development,* Vol.19, No.7, pp. 1095-1107,

Cardiovascular and Nervous Systems: Neurotrophic Effects of Vascular Endothelial Growth Factor (VEGF) and Angiogenic Effects of Nerve Growth Factor

(2005). Clarification of the Nomenclature for MSC: The International Society for Cellular Therapy Position Statement. *Cytotherapy,* Vol.7, No.5, pp. 393-395, ISSN 1465-3249

Hou, S., Xu, Q., Tian, W., Cui, F., Cai, Q., Ma, J. & Lee, I.S. (2005). The Repair of Brain Lesion

Hu, J., Deng, L., Wang, X. & Xu, X.M. (2009). Effects of Extracellular Matrix Molecules on the

Hunter, D.D., Llinas, R., Ard, M., Merlie, J.P. & Sanes, J.R. (1992). Expression of s-Laminin

Iantosca, M.R., McPherson, C.E., Ho, S.Y. & Maxwell, G.D. (1999). Bone Morphogenetic

Iihara, K., Hashimoto, N., Tsukahara, T., Sakata, M., Yanamoto, H. & Taniguchi, T. (1997).

Jackson, J.R., Seed, M.P., Kircher, C.H., Willoughby, D.A. & Winkler, J.D. (1997). The

Jin, K., Mao, X.O. & Greenberg, D.A. (2006). Vascular Endothelial Growth Factor Stimulates

Jin, K., Mao, X.O., Sun, Y., Xie, L., Jin, L., Nishi, E., Klagsbrun, M. & Greenberg, D.A. (2002).

Jin, K., Sun, Y., Xie, L., Childs, J., Mao, X.O. & Greenberg, D.A. (2004). Post-Ischemic

Jin, K.L., Mao, X.O. & Greenberg, D.A. (2000). Vascular Endothelial Growth Factor: Direct

*Journal of Neurobiology,* Vol.66, No.3, pp. 236-242, ISSN 0022-3034

*Comparative Neurology,* Vol.323, No.2, pp. 238-251, ISSN 0021-9967

*Metabolism,* Vol.17, No.10, pp. 1097-1106, ISSN 0271-678X

Vol.11, No.6, pp. 457-465, ISSN 0892-6638

*Neuroscience Methods,* Vol.148, No.1, pp. 60-70, ISSN 0165-0270

1465-3249

50-57, ISSN 0168-0102

0360-4012

0270-6474

8424

399-408, ISSN 0271-678X

(2005). Clarification of the Nomenclature for MSC: The International Society for Cellular Therapy Position Statement. *Cytotherapy,* Vol.7, No.5, pp. 393-395, ISSN

by Implantation of Hyaluronic Acid Hydrogels Modified with Laminin. *Journal of*

Growth Properties of Oligodendrocyte Progenitor Cells in Vitro. *Journal of Neuroscience Research,* Vol.87, No.13, pp. 2854-2862, ISSN 1097-4547; 0360-4012 Huang, X., Choi, J.K., Park, S.R., Ha, Y., Park, H., Yoon, S.H., Park, H.C., Park, J.O. & Choi,

B.H. (2007). GM-CSF Inhibits Apoptosis of Neural Cells Via Regulating the Expression of Apoptosis-Related Proteins. *Neuroscience Research,* Vol.58, No.1, pp.

and Laminin in the Developing Rat Central Nervous System. *The Journal of*

Proteins-2 and -4 Attenuate Apoptosis in a Cerebellar Primitive Neuroectodermal Tumor Cell Line. *Journal of Neuroscience Research,* Vol.56, No.3, pp. 248-258, ISSN

Platelet-Derived Growth Factor-BB, but Not -AA, Prevents Delayed Neuronal Death After Forebrain Ischemia in Rats. *Journal of Cerebral Blood Flow and*

Codependence of Angiogenesis and Chronic Inflammation. *The FASEB Journal,*

Neurite Outgrowth from Cerebral Cortical Neurons Via Rho Kinase Signaling.

Heparin-Binding Epidermal Growth Factor-Like Growth Factor: Hypoxia-Inducible Expression in Vitro and Stimulation of Neurogenesis in Vitro and in Vivo. *The Journal of Neuroscience,* Vol.22, No.13, pp. 5365-5373, ISSN 1529-2401;

Administration of Heparin-Binding Epidermal Growth Factor-Like Growth Factor (HB-EGF) Reduces Infarct Size and Modifies Neurogenesis After Focal Cerebral Ischemia in the Rat. *Journal of Cerebral Blood Flow and Metabolism,* Vol.24, No.4, pp.

Neuroprotective Effect in in Vitro Ischemia. *Proceedings of the National Academy of Sciences of the United States of America,* Vol.97, No.18, pp. 10242-10247, ISSN 0027-

Lu, P., Blesch, A. & Tuszynski, M.H. (2004). Induction of Bone Marrow Stromal Cells to

Lu, Y.Z., Lin, C.H., Cheng, F.C. & Hsueh, C.M. (2005). Molecular Mechanisms Responsible

Ma, M., Ma, Y., Yi, X., Guo, R., Zhu, W., Fan, X., Xu, G., Frey, W.H.,2nd & Liu, X. (2008).

Ma, W., Fitzgerald, W., Liu, Q.Y., O'Shaughnessy, T.J., Maric, D., Lin, H.J., Alkon, D.L. &

Maas, A.I., Roozenbeek, B. & Manley, G.T. (2010). Clinical Trials in Traumatic Brain Injury:

Mahmood, A., Lu, D. & Chopp, M. (2004). Marrow Stromal Cell Transplantation After

Mehler, M.F., Rozental, R., Dougherty, M., Spray, D.C. & Kessler, J.A. (1993). Cytokine

Mezey, E., Mayer, B. & Nemeth, K. (2010). Unexpected Roles for Bone Marrow Stromal Cells

Minamino, K., Adachi, Y., Okigaki, M., Ito, H., Togawa, Y., Fujita, K., Tomita, M., Suzuki, Y.,

Neovascularization. *Stem Cells,* Vol.23, No.3, pp. 347-354, ISSN 1066-5099 Mitsios, N., Gaffney, J., Kumar, P., Krupinski, J., Kumar, S. & Slevin, M. (2006).

*Diseases,* Vol.16, No.2, pp. 129-135, ISSN 1601-0825; 1354-523X

*Neurosurgery,* Vol.55, No.5, pp. 1185-1193, ISSN 1524-4040; 0148-396X Mathieu, P., Piantanida, A.P. & Pitossi, F. (2010). Chronic Expression of Transforming

*Neurovascular Research,* Vol.4, No.2, pp. 143-151, ISSN 1567-2026

*Research,* Vol.77, No.2, pp. 174-191, ISSN 0360-4012

*BMC Neuroscience,* Vol.9, pp. 117, ISSN 1471-2202

Vol.190, No.2, pp. 276-288, ISSN 0014-4886

No.3, pp. 200-201, ISSN 1423-0216; 1021-7401

Vol.362, No.6415, pp. 62-65, ISSN 0028-0836

126, ISSN 1878-7479

1015-2008

Neurons: Differentiation, Transdifferentiation, Or Artifact? *Journal of Neuroscience*

for Microglia-Derived Protection of Sprague-Dawley Rat Brain Cells during in Vitro Ischemia. *Neuroscience Letters,* Vol.373, No.2, pp. 159-164, ISSN 0304-3940 Lum, M., Turbic, A., Mitrovic, B. & Turnley, A.M. (2009). Fibroblast Growth Factor-9 Inhibits

Astrocyte Differentiation of Adult Mouse Neural Progenitor Cells. *Journal of Neuroscience Research,* Vol.87, No.10, pp. 2201-2210, ISSN 1097-4547; 0360-4012 Lykissas, M.G., Batistatou, A.K., Charalabopoulos, K.A. & Beris, A.E. (2007). The Role of

Neurotrophins in Axonal Growth, Guidance, and Regeneration. *Current*

Intranasal Delivery of Transforming Growth Factor-beta1 in Mice After Stroke Reduces Infarct Volume and Increases Neurogenesis in the Subventricular Zone.

Barker, J.L. (2004). CNS Stem and Progenitor Cell Differentiation into Functional Neuronal Circuits in Three-Dimensional Collagen Gels. *Experimental Neurology,*

Past Experience and Current Developments. *Neurotherapeutics,* Vol.7, No.1, pp. 115-

Traumatic Brain Injury Promotes Cellular Proliferation within the Brain.

Growth Factor-Beta Enhances Adult Neurogenesis. *Neuroimmunomodulation,* Vol.17,

Regulation of Neuronal Differentiation of Hippocampal Progenitor Cells. *Nature,*

(Or MSCs): A Real Promise for Cellular, but Not Replacement, Therapy. *Oral*

Zhang, Y., Iwasaki, M., Nakano, K., Koike, Y., Matsubara, H., Iwasaka, T., Matsumura, M. & Ikehara, S. (2005). Macrophage Colony-Stimulating Factor (M-CSF), as Well as Granulocyte Colony-Stimulating Factor (G-CSF), Accelerates

Pathophysiology of Acute Ischaemic Stroke: An Analysis of Common Signalling Mechanisms and Identification of New Molecular Targets. *Pathobiology : Journal of Immunopathology, Molecular and Cellular Biology,* Vol.73, No.4, pp. 159-175, ISSN

(NGF)-Implications in Drug Development. *Current Pharmaceutical Design,* Vol.12, No.21, pp. 2609-2622, ISSN 1381-6128

Lee, B., Clarke, D., Al Ahmad, A., Kahle, M., Parham, C., Auckland, L., Shaw, C.,

Lee, J.S., Hong, J.M., Moon, G.J., Lee, P.H., Ahn, Y.H., Bang, O.Y. & STARTING

*Cells (Dayton, Ohio),* Vol.28, No.6, pp. 1099-1106, ISSN 1549-4918; 1066-5099 Leker, R.R. & Shohami, E. (2002). Cerebral Ischemia and Trauma-Different Etiologies Yet

Li, J., Zhu, H., Liu, Y., Li, Q., Lu, S., Feng, M., Xu, Y., Huang, L., Ma, C., An, Y., Zhao, R.C.,

Li, J.W., Li, L.L., Chang, L.L., Wang, Z.Y. & Xu, Y. (2009). Stem Cell Factor Protects Against

Li, Y., Chen, J., Chen, X.G., Wang, L., Gautam, S.C., Xu, Y.X., Katakowski, M., Zhang, L.J.,

Li, Y., Chen, J., Zhang, C.L., Wang, L., Lu, D., Katakowski, M., Gao, Q., Shen, L.H., Zhang, J.,

Li, Y. & Chopp, M. (2009). Marrow Stromal Cell Transplantation in Stroke and Traumatic

Lipsky, R.H. & Marini, A.M. (2007). Brain-Derived Neurotrophic Factor in Neuronal

Liu, X.S., Chopp, M., Zhang, R.L., Hozeska-Solgot, A., Gregg, S.C., Buller, B., Lu, M. &

No.21, pp. 2609-2622, ISSN 1381-6128

3005-3023, ISSN 1558-8238; 0021-9738

6240; 0006-8993

879X

0894-1491

ISSN 0027-8424

3940

*Reviews,* Vol.39, No.1, pp. 55-73, ISSN 0165-0173

Vol.59, No.4, pp. 514-523, ISSN 0028-3878

Vol.1122, pp. 130-143, ISSN 0077-8923

(NGF)-Implications in Drug Development. *Current Pharmaceutical Design,* Vol.12,

Fidanboylu, M., Orr, A.W., Ogunshola, O., Fertala, A., Thomas, S.A. & Bix, G.J. (2011). Perlecan Domain V is Neuroprotective and Proangiogenic Following Ischemic Stroke in Rodents. *The Journal of Clinical Investigation,* Vol.121, No.8, pp.

collaborators. (2010). A Long-Term Follow-Up Study of Intravenous Autologous Mesenchymal Stem Cell Transplantation in Patients with Ischemic Stroke. *Stem*

Similar Mechanisms: Neuroprotective Opportunities. *Brain Research.Brain Research*

Wang, R. & Qin, C. (2010). Human Mesenchymal Stem Cell Transplantation Protects Against Cerebral Ischemic Injury and Upregulates Interleukin-10 Expression in Macacafascicularis. *Brain Research,* Vol.1334, pp. 65-72, ISSN 1872-

Neuronal Apoptosis by Activating AKT/ERK in Diabetic Mice. *Brazilian Journal of Medical and Biological Research,* Vol.42, No.11, pp. 1044-1049, ISSN 1414-431X; 0100-

Lu, M., Janakiraman, N. & Chopp, M. (2002). Human Marrow Stromal Cell Therapy for Stroke in Rat: Neurotrophins and Functional Recovery. *Neurology,*

Lu, M. & Chopp, M. (2005). Gliosis and Brain Remodeling After Treatment of Stroke in Rats with Marrow Stromal Cells. *Glia,* Vol.49, No.3, pp. 407-417, ISSN

Brain Injury. *Neuroscience Letters,* Vol.456, No.3, pp. 120-123, ISSN 1872-7972; 0304-

Survival and Behavior-Related Plasticity. *Annals of the New York Academy of Sciences,*

Zhang, Z.G. (2009). Angiopoietin 2 Mediates the Differentiation and Migration of Neural Progenitor Cells in the Subventricular Zone After Stroke. *The Journal of Biological Chemistry,* Vol.284, No.34, pp. 22680-22689, ISSN 1083-351X; 0021-9258 Lopez-Lopez, C., LeRoith, D. & Torres-Aleman, I. (2004). Insulin-Like Growth Factor I is

Required for Vessel Remodeling in the Adult Brain. *Proceedings of the National Academy of Sciences of the United States of America,* Vol.101, No.26, pp. 9833-9838,

Ohab, J.J. & Carmichael, S.T. (2008). Poststroke Neurogenesis: Emerging Principles of

Omori, Y., Honmou, O., Harada, K., Suzuki, J., Houkin, K. & Kocsis, J.D. (2008).

Onda, T., Honmou, O., Harada, K., Houkin, K., Hamada, H. & Kocsis, J.D. (2008).

Opanashuk, L.A., Mark, R.J., Porter, J., Damm, D., Mattson, M.P. & Seroogy, K.B. (1999).

Parr, A.M., Tator, C.H. & Keating, A. (2007). Bone Marrow-Derived Mesenchymal Stromal

Pavlichenko, N., Sokolova, I., Vijde, S., Shvedova, E., Alexandrov, G., Krouglyakov, P.,

pp. 369-380, ISSN 1073-8584

Vol.1236, pp. 30-38, ISSN 0006-8993

*Metabolism,* Vol.28, No.2, pp. 329-340, ISSN 0271-678X

*Neuroscience,* Vol.19, No.1, pp. 133-146, ISSN 0270-6474

Vol.40, No.7, pp. 609-619, ISSN 0268-3369

Vol.25, No.7, pp. 1761-1768, ISSN 1066-5099

No.7, pp. 3426-3434, ISSN 0022-1767

0100-879X

Migration and Localization of Immature Neurons. *The Neuroscientist,* Vol.14, No.4,

Optimization of a Therapeutic Protocol for Intravenous Injection of Human Mesenchymal Stem Cells After Cerebral Ischemia in Adult Rats. *Brain Research,*

Therapeutic Benefits by Human Mesenchymal Stem Cells (hMSCs) and Ang-1 Gene-Modified hMSCs After Cerebral Ischemia. *Journal of Cerebral Blood Flow and*

Heparin-Binding Epidermal Growth Factor-Like Growth Factor in Hippocampus: Modulation of Expression by Seizures and Anti-Excitotoxic Action. *The Journal of*

Cells for the Repair of Central Nervous System Injury. *Bone Marrow Transplantation,*

Fedotova, O., Gilerovich, E.G., Polyntsev, D.G. & Otellin, V.A. (2008). Mesenchymal Stem Cells Transplantation could be Beneficial for Treatment of Experimental Ischemic Stroke in Rats. *Brain Research,* Vol.1233, pp. 203-213, ISSN 0006-8993 Perris, R. & Perissinotto, D. (2000). Role of the Extracellular Matrix during Neural Crest Cell Migration. *Mechanisms of Development,* Vol.95, No.1-2, pp. 3-21, ISSN 0925-4773 Phinney, D.G. & Prockop, D.J. (2007). Concise Review: Mesenchymal stem/multipotent

Stromal Cells: The State of Transdifferentiation and Modes of Tissue Repair-- Current Views. *Stem Cells,* Vol.25, No.11, pp. 2896-2902, ISSN 1549-4918; 1066-5099

Diverse Roles in the Growth and Guidance of Central Nervous System Axons. *Brazilian Journal of Medical and Biological Research,* Vol.32, No.5, pp. 633-638, ISSN

Domenech, J. (2007). The in Vitro Migration Capacity of Human Bone Marrow Mesenchymal Stem Cells: Comparison of Chemokine and Growth Factor Chemotactic Activities. *Stem Cells,* Vol.25, No.7, pp. 1737-1745, ISSN 1066-5099 Potapova, I.A., Gaudette, G.R., Brink, P.R., Robinson, R.B., Rosen, M.R., Cohen, I.S. &

Doronin, S.V. (2007). Mesenchymal Stem Cells Support Migration, Extracellular Matrix Invasion, Proliferation, and Survival of Endothelial Cells in Vitro. *Stem Cells,*

Activity of Mesenchymal Stem Cells: Functional Discrimination between Cellular Responses to Alloantigens and Recall Antigens. *Journal of Immunology,* Vol.171,

Pires Neto, M.A., Braga-de-Souza, S. & Lent, R. (1999). Extracellular Matrix Molecules Play

Ponte, A.L., Marais, E., Gallay, N., Langonne, A., Delorme, B., Herault, O., Charbord, P. &

Potian, J.A., Aviv, H., Ponzio, N.M., Harrison, J.S. & Rameshwar, P. (2003). Veto-Like

Mosesson, M.W. (1984). The Role of Fibronectin in monocyte/macrophage Function. *Progress in Clinical and Biological Research,* Vol.154, pp. 155-175, ISSN 0361-7742 Mudo, G., Bonomo, A., Di Liberto, V., Frinchi, M., Fuxe, K. & Belluardo, N. (2009). The FGF-

Nag, S., Kapadia, A. & Stewart, D.J. (2011). Review: Molecular Pathogenesis of Blood-Brain

Nasu-Tada, K., Koizumi, S. & Inoue, K. (2005). Involvement of beta1 Integrin in Microglial

Neuhuber, B., Gallo, G., Howard, L., Kostura, L., Mackay, A. & Fischer, I. (2004).

Neuss, S., Becher, E., Woltje, M., Tietze, L. & Jahnen-Dechent, W. (2004). Functional

Nishibe, M., Barbay, S., Guggenmos, D. & Nudo, R.J. (2010). Reorganization of Motor Cortex

*European Journal of Neuroscience,* Vol.7, No.2, pp. 344-350, ISSN 0953-816X Noonan, D.M., De Lerma Barbaro, A., Vannini, N., Mortara, L. & Albini, A. (2008).

Numasaki, M., Fukushi, J., Ono, M., Narula, S.K., Zavodny, P.J., Kudo, T., Robbins, P.D.,

O'Collins, V.E., Macleod, M.R., Donnan, G.A., Horky, L.L., van der Worp, B.H. & Howells,

Oh, J., McCloskey, M.A., Blong, C.C., Bendickson, L., Nilsen-Hamilton, M. & Sakaguchi, D.S.

*Cancer Metastasis Reviews,* Vol.27, No.1, pp. 31-40, ISSN 0167-7659

Growth. *Blood,* Vol.101, No.7, pp. 2620-2627, 0006-4971; 0006-4971

*Research,* Vol.88, No.13, pp. 2798-2809, ISSN 1097-4547; 0360-4012

Vol.59, No.3, pp. 467-477, ISSN 0364-5134

through PKA. *Glia,* Vol.52, No.2, pp. 98-107, ISSN 1098-1136; 0894-1491 Nauta, A.J. & Fibbe, W.E. (2007). Immunomodulatory Properties of Mesenchymal Stromal

Cells. *Blood,* Vol.110, No.10, pp. 3499-3506, ISSN 0006-4971

*Stem Cells,* Vol.22, No.3, pp. 405-414, ISSN 1066-5099

pp. 4585-4595, 1529-2401

204, ISSN 0360-4012

Vol.37, No.1, pp. 3-23, 1365-2990; 0305-1846

2/FGFRs Neurotrophic System Promotes Neurogenesis in the Adult Brain. *Journal of Neural Transmission,* Vol.116, No.8, pp. 995-1005, ISSN 1435-1463; 0300-9564 Munoz-Elias, G., Marcus, A.J., Coyne, T.M., Woodbury, D. & Black, I.B. (2004). Adult Bone

Marrow Stromal Cells in the Embryonic Brain: Engraftment, Migration, Differentiation, and Long-Term Survival. *The Journal of Neuroscience,* Vol.24, No.19,

Barrier Breakdown in Acute Brain Injury. *Neuropathology and Applied Neurobiology,*

Chemotaxis and Proliferation on Fibronectin: Different Regulations by ADP

Reevaluation of in Vitro Differentiation Protocols for Bone Marrow Stromal Cells: Disruption of Actin Cytoskeleton Induces Rapid Morphological Changes and Mimics Neuronal Phenotype. *Journal of Neuroscience Research,* Vol.77, No.2, pp. 192-

Expression of HGF and HGF receptor/c-Met in Adult Human Mesenchymal Stem Cells Suggests a Role in Cell Mobilization, Tissue Repair, and Wound Healing.

After Controlled Cortical Impact in Rats and Implications for Functional Recovery. *Journal of Neurotrauma,* Vol.27, No.12, pp. 2221-2232, ISSN 1557-9042; 0897-7151 Nobes, C.D. & Tolkovsky, A.M. (1995). Neutralizing Anti-p21ras Fabs Suppress Rat

Sympathetic Neuron Survival Induced by NGF, LIF, CNTF and cAMP. *The*

Inflammation, Inflammatory Cells and Angiogenesis: Decisions and Indecisions.

Tahara, H. & Lotze, M.T. (2003). Interleukin-17 Promotes Angiogenesis and Tumor

D.W. (2006). 1,026 Experimental Treatments in Acute Stroke. *Annals of Neurology,*

(2010). Astrocyte-Derived Interleukin-6 Promotes Specific Neuronal Differentiation of Neural Progenitor Cells from Adult Hippocampus. *Journal of Neuroscience*

Schabitz, W.R., Steigleder, T., Cooper-Kuhn, C.M., Schwab, S., Sommer, C., Schneider, A. &

Schinkothe, T., Bloch, W. & Schmidt, A. (2008). In Vitro Secreting Profile of Human

Schmidt, A., Ladage, D., Schinkothe, T., Klausmann, U., Ulrichs, C., Klinz, F.J., Brixius, K.,

Sehara, Y., Hayashi, T., Deguchi, K., Zhang, H., Tsuchiya, A., Yamashita, T., Lukic, V.,

Shang, J., Deguchi, K., Ohta, Y., Liu, N., Zhang, X., Tian, F., Yamashita, T., Ikeda, Y.,

*Neuroscience Research,* Vol.89, No.1, pp. 86-95, ISSN 1097-4547; 0360-4012 Shen, L.H., Li, Y., Gao, Q., Savant-Bhonsale, S. & Chopp, M. (2008). Down-Regulation of

ISSN 1524-4628; 0039-2499

ISSN 1547-3287

2098, ISSN 0021-9738

Vol.1151, pp. 142-149, ISSN 0006-8993

909-916, ISSN 1573-6830; 0272-4340

No.7, pp. 2165-2172, ISSN 1524-4628; 0039-2499

Stimulating Factor After Focal Cerebral Ischemia. *Stroke,* Vol.34, No.3, pp. 745-751,

Kuhn, H.G. (2007). Intravenous Brain-Derived Neurotrophic Factor Enhances Poststroke Sensorimotor Recovery and Stimulates Neurogenesis. *Stroke,* Vol.38,

Mesenchymal Stem Cells. *Stem Cells and Development,* Vol.17, No.1, pp. 199-206,

Arnhold, S., Desai, B., Mehlhorn, U., Schwinger, R.H., Staib, P., Addicks, K. & Bloch, W. (2006). Basic Fibroblast Growth Factor Controls Migration in Human Mesenchymal Stem Cells. *Stem Cells,* Vol.24, No.7, pp. 1750-1758, ISSN 1066-5099 Schneider, A., Kruger, C., Steigleder, T., Weber, D., Pitzer, C., Laage, R., Aronowski, J.,

Maurer, M.H., Gassler, N., Mier, W., Hasselblatt, M., Kollmar, R., Schwab, S., Sommer, C., Bach, A., Kuhn, H.G. & Schabitz, W.R. (2005). The Hematopoietic Factor G-CSF is a Neuronal Ligand that Counteracts Programmed Cell Death and Drives Neurogenesis. *The Journal of Clinical Investigation,* Vol.115, No.8, pp. 2083-

Nagai, M., Kamiya, T. & Abe, K. (2007). Potentiation of Neurogenesis and Angiogenesis by G-CSF After Focal Cerebral Ischemia in Rats. *Brain Research,*

Matsuura, T., Funakoshi, H., Nakamura, T. & Abe, K. (2011). Strong Neurogenesis, Angiogenesis, Synaptogenesis, and Antifibrosis of Hepatocyte Growth Factor in Rats Brain After Transient Middle Cerebral Artery Occlusion. *Journal of*

Neurocan Expression in Reactive Astrocytes Promotes Axonal Regeneration and Facilitates the Neurorestorative Effects of Bone Marrow Stromal Cells in the Ischemic Rat Brain. *Glia,* Vol.56, No.16, pp. 1747-1754, ISSN 1098-1136; 0894-1491 Shen, Y., Inoue, N. & Heese, K. (2010). Neurotrophin-4 (ntf4) Mediates Neurogenesis in

Mouse Embryonic Neural Stem Cells through the Inhibition of the Signal Transducer and Activator of Transcription-3 (stat3) and the Modulation of the Activity of Protein Kinase B. *Cellular and Molecular Neurobiology,* Vol.30, No.6, pp.

Therapeutic Aspects of Vascular Endothelial Growth Factor. *The FEBS Journal,*

Ogihara, T., Kaneda, Y. & Morishita, R. (2006). Gene Transfer of Hepatocyte Growth Factor Gene Improves Learning and Memory in the Chronic Stage of

Shibuya, M. (2009). Brain Angiogenesis in Developmental and Pathological Processes:

Shimamura, M., Sato, N., Waguri, S., Uchiyama, Y., Hayashi, T., Iida, H., Nakamura, T.,

Cerebral Infarction. *Hypertension,* Vol.47, No.4, pp. 742-751, ISSN 1524-4563

Vol.276, No.17, pp. 4636-4643, ISSN 1742-4658; 1742-464X

Properzi, F., Asher, R.A. & Fawcett, J.W. (2003). Chondroitin Sulphate Proteoglycans in the

Qin, L., Kim, E., Ratan, R., Lee, F.S. & Cho, S. (2011). Genetic Variant of BDNF (Val66Met)

Robinson, A.P., Foraker, J.E., Ylostalo, J. & Prockop, D.J. (2011). Human stem/progenitor

Robinson, S., Tani, M., Strieter, R.M., Ransohoff, R.M. & Miller, R.H. (1998). The Chemokine

Roger, V.L., Go, A.S., Lloyd-Jones, D.M., Adams, R.J., Berry, J.D., Brown, T.M., Carnethon,

Sabo, J.K., Kilpatrick, T.J. & Cate, H.S. (2009). Effects of Bone Morphogenic Proteins on

Sadohara, T., Sugahara, K., Urashima, Y., Terasaki, H. & Lyama, K. (2001). Keratinocyte

Sakai, T., Johnson, K.J., Murozono, M., Sakai, K., Magnuson, M.A., Wieloch, T., Cronberg, T.,

Sanchez-Ramos, J., Song, S., Cardozo-Pelaez, F., Hazzi, C., Stedeford, T., Willing, A.,

Schabitz, W.R., Kollmar, R., Schwaninger, M., Juettler, E., Bardutzky, J., Scholzke, M.N.,

*Neuro-Signals,* Vol.17, No.4, pp. 255-264, ISSN 1424-8638; 1424-862X

*Medicine,* Vol.7, No.3, pp. 324-30., ISSN 1546-170X; 1078-8956

*Experimental Neurology,* Vol.164, No.2, pp. 247-256, ISSN 0014-4886

Neural Precursor Cells and Regulation during Central Nervous System Injury.

Growth Factor Prevents Ischemia-Induced Delayed Neuronal Death in the Hippocampal CA1 Field of the Gerbil Brain. *Neuroreport,* Vol.12, No.1, pp. 71-76,

Isshiki, A., Erickson, H.P. & Fassler, R. (2001). Plasma Fibronectin Supports Neuronal Survival and Reduces Brain Injury Following Transient Focal Cerebral Ischemia but is Not Essential for Skin-Wound Healing and Hemostasis. *Nature*

Freeman, T.B., Saporta, S., Janssen, W., Patel, N., Cooper, D.R. & Sanberg, P.R. (2000). Adult Bone Marrow Stromal Cells Differentiate into Neural Cells in Vitro.

Sommer, C. & Schwab, S. (2003). Neuroprotective Effect of Granulocyte Colony-

*Transactions,* Vol.31, No.2, pp. 335-336, ISSN 1470-8752; 0300-5127

*Development,* Vol.20, No.2, pp. 289-300, ISSN 1557-8534; 1547-3287

No.2, pp. 775-783, ISSN 1529-2401; 0270-6474

ISSN 0270-6474

1524-4539; 0009-7322

ISSN 0959-4965

Central Nervous System: Changes and Synthesis After Injury. *Biochemical Society*

Polymorphism Attenuates Stroke-Induced Angiogenic Responses by Enhancing Anti-Angiogenic Mediator CD36 Expression. *The Journal of Neuroscience,* Vol.31,

Cells from Bone Marrow Enhance Glial Differentiation of Rat Neural Stem Cells: A Role for Transforming Growth Factor Beta and Notch Signaling. *Stem Cells and*

Growth-Regulated Oncogene-Alpha Promotes Spinal Cord Oligodendrocyte Precursor Proliferation. *The Journal of Neuroscience,* Vol.18, No.24, pp. 10457-10463,

M.R., Dai, S., de Simone, G., Ford, E.S., Fox, C.S., Fullerton, H.J., Gillespie, C., Greenlund, K.J., Hailpern, S.M., Heit, J.A., Ho, P.M., Howard, V.J., Kissela, B.M., Kittner, S.J., Lackland, D.T., Lichtman, J.H., Lisabeth, L.D., Makuc, D.M., Marcus, G.M., Marelli, A., Matchar, D.B., McDermott, M.M., Meigs, J.B., Moy, C.S., Mozaffarian, D., Mussolino, M.E., Nichol, G., Paynter, N.P., Rosamond, W.D., Sorlie, P.D., Stafford, R.S., Turan, T.N., Turner, M.B., Wong, N.D., Wylie-Rosett, J. & American Heart Association Statistics Committee and Stroke Statistics Subcommittee. (2011). Heart Disease and Stroke Statistics--2011 Update: A Report from the American Heart Association. *Circulation,* Vol.123, No.4, pp. e18-e209, ISSN Stimulating Factor After Focal Cerebral Ischemia. *Stroke,* Vol.34, No.3, pp. 745-751, ISSN 1524-4628; 0039-2499

Sun, Y., Jin, K., Xie, L., Childs, J., Mao, X.O., Logvinova, A. & Greenberg, D.A. (2003). VEGF-

Swartz, K.R., Liu, F., Sewell, D., Schochet, T., Campbell, I., Sandor, M. & Fabry, Z. (2001).

Taga, T. & Fukuda, S. (2005). Role of IL-6 in the Neural Stem Cell Differentiation. *Clinical Reviews in Allergy & Immunology,* Vol.28, No.3, pp. 249-256, ISSN 1080-0549 Tan, H.K., Heywood, D., Ralph, G.S., Bienemann, A., Baker, A.H. & Uney, J.B. (2003).

Tate, C.C., Fonck, C., McGrogan, M. & Case, C.C. (2010). Human Mesenchymal Stromal

Tate, C.C., Garcia, A.J. & LaPlaca, M.C. (2007). Plasma Fibronectin is Neuroprotective

Tate, M.C., Garcia, A.J., Keselowsky, B.G., Schumm, M.A., Archer, D.R. & LaPlaca, M.C.

Terauchi, A., Johnson-Venkatesh, E.M., Toth, A.B., Javed, D., Sutton, M.A. & Umemori, H.

Synapses. *Nature,* Vol.465, No.7299, pp. 783-787, ISSN 1476-4687; 0028-0836 Testaz, S. & Duband, J.L. (2001). Central Role of the alpha4beta1 Integrin in the

Thored, P., Arvidsson, A., Cacci, E., Ahlenius, H., Kallur, T., Darsalia, V., Ekdahl, C.T.,

Vana, A.C., Flint, N.C., Harwood, N.E., Le, T.Q., Fruttiger, M. & Armstrong, R.C. (2007).

Vasse, M., Pourtau, J., Trochon, V., Muraine, M., Vannier, J.P., Lu, H., Soria, J. & Soria,

*Neuroscience,* Vol.27, No.1, pp. 22-31, ISSN 1095-9327; 1044-7431

*Brain Research,* Vol.896, No.1-2, pp. 86-95, ISSN 0006-8993

0021-9738

1044-7431

1555-3892; 0963-6897

ISSN 0014-4886

8388

No.3, pp. 739-747, ISSN 1066-5099

pp. 975-988, ISSN 0022-3069

ISSN 1079-5642

Induced Neuroprotection, Neurogenesis, and Angiogenesis After Focal Cerebral Ischemia. *The Journal of Clinical Investigation,* Vol.111, No.12, pp. 1843-1851, ISSN

Interleukin-6 Promotes Post-Traumatic Healing in the Central Nervous System.

Tissue Inhibitor of Metalloproteinase 1 Inhibits Excitotoxic Cell Death in Neurons. *Molecular and Cellular Neurosciences,* Vol.22, No.1, pp. 98-106, ISSN

Cells and their Derivative, SB623 Cells, Rescue Neural Cells via Trophic Support Following in Vitro Ischemia. *Cell Transplantation,* Vol.19, No.8, pp. 973-984, ISSN

Following Traumatic Brain Injury. *Experimental Neurology,* Vol.207, No.1, pp. 13-22,

(2004). Specific beta1 Integrins Mediate Adhesion, Migration, and Differentiation of Neural Progenitors Derived from the Embryonic Striatum. *Molecular and Cellular*

(2010). Distinct FGFs Promote Differentiation of Excitatory and Inhibitory

Coordination of Avian Truncal Neural Crest Cell Adhesion, Migration, and Survival. *Developmental dynamics,* Vol.222, No.2, pp. 127-140, ISSN 1097-0177; 1058-

Kokaia, Z. & Lindvall, O. (2006). Persistent Production of Neurons from Adult Brain Stem Cells during Recovery After Stroke. *Stem Cells (Dayton, Ohio),* Vol.24,

Platelet-Derived Growth Factor Promotes Repair of Chronically Demyelinated White Matter. *Journal of Neuropathology and Experimental Neurology,* Vol.66, No.11,

C. (1999). Oncostatin M Induces Angiogenesis in Vitro and in Vivo. *Arteriosclerosis, Thrombosis, and Vascular Biology,* Vol.19, No.8, pp. 1835-1842,

Shimazaki, T., Shingo, T. & Weiss, S. (2001). The Ciliary Neurotrophic factor/leukemia

Shirakura, M., Inoue, M., Fujikawa, S., Washizawa, K., Komaba, S., Maeda, M., Watabe, K.,

Death in Gerbils. *Gene Therapy,* Vol.11, No.9, pp. 784-790, ISSN 0969-7128 Shyu, W.C., Lin, S.Z., Yang, H.I., Tzeng, Y.S., Pang, C.Y., Yen, P.S. & Li, H. (2004). Functional

Smadja, D.M., d'Audigier, C., Weiswald, L.B., Badoual, C., Dangles-Marie, V., Mauge, L.,

*vascular biology*, Vol.30, No.12, pp. 2544-2552, ISSN 1079-5642; 1524-4636 Solaroglu, I., Tsubokawa, T., Cahill, J. & Zhang, J.H. (2006). Anti-Apoptotic Effect of

Son, B.R., Marquez-Curtis, L.A., Kucia, M., Wysoczynski, M., Turner, A.R., Ratajczak, J.,

Metalloproteinases. *Stem Cells,* Vol.24, No.5, pp. 1254-1264, ISSN 1066-5099 Sottile, J. (2004). Regulation of Angiogenesis by Extracellular Matrix. *Biochimica Et Biophysica*

Suarez-Monteagudo, C., Hernandez-Ramirez, P., Alvarez-Gonzalez, L., Garcia-Maeso, I., de

Sugiura, S., Lahav, R., Han, J., Kou, S.Y., Banner, L.R., de Pablo, F. & Patterson, P.H. (2000).

Sun, L., Hui, A.M., Su, Q., Vortmeyer, A., Kotliarov, Y., Pastorino, S., Passaniti, A., Menon,

*Neuroscience,* Vol.143, No.4, pp. 965-974, ISSN 0306-4522

*Acta,* Vol.1654, No.1, pp. 13-22, ISSN 0006-3002

*Neuroscience,* Vol.27, No.3, pp. 151-161, ISSN 0922-6028

Brain. *Cancer Cell,* Vol.9, No.4, pp. 287-300, ISSN 1535-6108

457-466, ISSN 0953-816X

pp. 7642-7653, ISSN 1529-2401; 0270-6474

0009-7322

Inhibitory factor/gp130 Receptor Complex Operates in the Maintenance of Mammalian Forebrain Neural Stem Cells. *The Journal of Neuroscience,* Vol.21, No.19,

Yoshikawa, Y. & Hasegawa, M. (2004). Postischemic Administration of Sendai Virus Vector Carrying Neurotrophic Factor Genes Prevents Delayed Neuronal

Recovery of Stroke Rats Induced by Granulocyte Colony-Stimulating Factor-Stimulated Stem Cells. *Circulation,* Vol.110, No.13, pp. 1847-1854, ISSN 1524-4539;

Evrard, S., Laurendeau, I., Lallemand, F., Germain, S., Grelac, F., Dizier, B., Vidaud, M., Bièche, I. & Gaussem, P. (2010). The Wnt Antagonist Dickkopf-1 Increases Endothelial Progenitor Cell Angiogenic Potential. *Arteriosclerosis, thrombosis, and*

Granulocyte-Colony Stimulating Factor After Focal Cerebral Ischemia in the Rat.

Ratajczak, M.Z. & Janowska-Wieczorek, A. (2006). Migration of Bone Marrow and Cord Blood Mesenchymal Stem Cells in Vitro is Regulated by Stromal-Derived Factor-1-CXCR4 and Hepatocyte Growth Factor-c-Met Axes and Involves Matrix

la Cuetara-Bernal, K., Castillo-Diaz, L., Bringas-Vega, M.L., Martinez-Aching, G., Morales-Chacon, L.M., Baez-Martin, M.M., Sanchez-Catasus, C., Carballo-Barreda, M., Rodriguez-Rojas, R., Gomez-Fernandez, L., Alberti-Amador, E., Macias-Abraham, C., Balea, E.D., Rosales, L.C., Del Valle Perez, L., Ferrer, B.B., Gonzalez, R.M. & Bergado, J.A. (2009). Autologous Bone Marrow Stem Cell Neurotransplantation in Stroke Patients. an Open Study. *Restorative Neurology and*

Leukaemia Inhibitory Factor is Required for Normal Inflammatory Responses to Injury in the Peripheral and Central Nervous Systems in Vivo and is Chemotactic for Macrophages in Vitro. *The European Journal of Neuroscience,* Vol.12, No.2, pp.

J., Walling, J., Bailey, R., Rosenblum, M., Mikkelsen, T. & Fine, H.A. (2006). Neuronal and Glioma-Derived Stem Cell Factor Induces Angiogenesis within the

Xin, H., Li, Y., Shen, L.H., Liu, X., Wang, X., Zhang, J., Pourabdollah-Nejad, D.S., Zhang, C.,

Xiong, Y., Qu, C., Mahmood, A., Liu, Z., Ning, R., Li, Y., Kaplan, D.L., Schallert, T. & Chopp,

Yabe, T., Samuels, I. & Schwartz, J.P. (2002). Bone Morphogenetic Proteins BMP-6 and BMP-

Yamada, M., Tanabe, K., Wada, K., Shimoke, K., Ishikawa, Y., Ikeuchi, T., Koizumi, S. &

Yan, Y.P., Sailor, K.A., Lang, B.T., Park, S.W., Vemuganti, R. & Dempsey, R.J. (2007).

Yi, J.J., Barnes, A.P., Hand, R., Polleux, F. & Ehlers, M.D. (2010). TGF-Beta Signaling

Yoo, S.W., Kim, S.S., Lee, S.Y., Lee, H.S., Kim, H.S., Lee, Y.D. & Suh-Kim, H. (2008).

Yung, H.S., Lai, K.H., Chow, K.B., Ip, N.Y., Tsim, K.W., Wong, Y.H., Wu, Z. & Wise, H.

Zanier, E.R., Montinaro, M., Vigano, M., Villa, P., Fumagalli, S., Pischiutta, F., Longhi, L.,

Zechel, S., Werner, S., Unsicker, K. & von Bohlen und Halbach, O. (2010). Expression and

Zhang, Z.X., Guan, L.X., Zhang, K., Zhang, Q. & Dai, L.J. (2008). A Combined Procedure to

Injury. *Cytotherapy,* Vol.10, No.2, pp. 134-139, ISSN 1477-2566; 1465-3249

*Neuroscientist,* Vol.16, No.4, pp. 357-373, ISSN 1089-4098; 1073-8584

*Journal of Neurochemistry,* Vol.78, No.5, pp. 940-951, ISSN 0022-3042

6203

6240; 0006-8993

ISSN 0360-4012

1097-4172; 0092-8674

1424-8638; 1424-862X

No.6, pp. 1213-1224, ISSN 0271-678X

*Medicine,* Vol.40, No.4, pp. 387-397, ISSN 1226-3613

*Critical Care Medicine,* ISSN 1530-0293; 0090-3493

Zhang, L., Jiang, H., Zhang, Z.G. & Chopp, M. (2010). Increasing tPA Activity in Astrocytes Induced by Multipotent Mesenchymal Stromal Cells Facilitate Neurite Outgrowth After Stroke in the Mouse. *PloS One,* Vol.5, No.2, pp. e9027, ISSN 1932-

M. (2009). Delayed Transplantation of Human Marrow Stromal Cell-Seeded Scaffolds Increases Transcallosal Neural Fiber Length, Angiogenesis, and Hippocampal Neuronal Survival and Improves Functional Outcome After Traumatic Brain Injury in Rats. *Brain Research,* Vol.1263, pp. 183-191, ISSN 1872-

7 have Differential Effects on Survival and Neurite Outgrowth of Cerebellar Granule Cell Neurons. *Journal of Neuroscience Research,* Vol.68, No.2, pp. 161-168,

Hatanaka, H. (2001). Differences in Survival-Promoting Effects and Intracellular Signaling Properties of BDNF and IGF-1 in Cultured Cerebral Cortical Neurons.

Monocyte Chemoattractant Protein-1 Plays a Critical Role in Neuroblast Migration After Focal Cerebral Ischemia. *Journal of Cerebral Blood Flow and Metabolism,* Vol.27,

Specifies Axons during Brain Development. *Cell,* Vol.142, No.1, pp. 144-157, ISSN

Mesenchymal Stem Cells Promote Proliferation of Endogenous Neural Stem Cells and Survival of Newborn Cells in a Rat Stroke Model. *Experimental & Molecular*

(2010). Nerve Growth Factor-Induced Differentiation of PC12 Cells is Accompanied by Elevated Adenylyl Cyclase Activity. *Neuro-Signals,* Vol.18, No.1, pp. 32-42, ISSN

Leoni, M.L., Rebulla, P., Stocchetti, N., Lazzari, L. & Simoni, M.G. (2011). Human Umbilical Cord Blood Mesenchymal Stem Cells Protect Mice Brain After Trauma.

Functions of Fibroblast Growth Factor 2 (FGF-2) in Hippocampal Formation. *The*

Deliver Autologous Mesenchymal Stromal Cells to Patients with Traumatic Brain

Villoslada, P. & Genain, C.P. (2004). Role of Nerve Growth Factor and Other Trophic

Vincent, V.A., Robinson, C.C., Simsek, D. & Murphy, G.M. (2002). Macrophage Colony

Wang, L., Li, Y., Chen, X., Chen, J., Gautam, S.C., Xu, Y. & Chopp, M. (2002). MCP-1, MIP-

Wang, M., Crisostomo, P.R., Herring, C., Meldrum, K.K. & Meldrum, D.R. (2006). Human

Wang, Y., Jin, K., Mao, X.O., Xie, L., Banwait, S., Marti, H.H. & Greenberg, D.A. (2007a).

Wang, Y.Q., Guo, X., Qiu, M.H., Feng, X.Y. & Sun, F.Y. (2007b). VEGF Overexpression

Weiss, T.W., Samson, A.L., Niego, B., Daniel, P.B. & Medcalf, R.L. (2006). Oncostatin M is a

Widera, D., Holtkamp, W., Entschladen, F., Niggemann, B., Zanker, K., Kaltschmidt, B.

Wilkins, A., Chandran, S. & Compston, A. (2001). A Role for Oligodendrocyte-Derived IGF-

Woodbury, D., Schwarz, E.J., Prockop, D.J. & Black, I.B. (2000). Adult Rat and Human Bone

Wright, D.W., Kellermann, A.L., Hertzberg, V.S., Clark, P.L., Frankel, M., Goldstein, F.C.,

*FASEB Journal,* Vol.20, No.13, pp. 2369-2371, ISSN 1530-6860; 0892-6638 Wells, W.A. (2002). Is Transdifferentiation in Trouble? *The Journal of Cell Biology,* Vol.157,

0079-6123

0022-3042

R880-4, ISSN 0363-6119

pp. 740-747, ISSN 0360-4012

No.1, pp. 15-18, ISSN 0021-9525

Vol.61, No.4, pp. 364-370, ISSN 0360-4012

ISSN 0360-4012

0171-9335

1097-6760

1491

5332

Factors in Brain Inflammation. *Progress in Brain Research,* Vol.146, pp. 403-414, ISSN

Stimulating Factor Prevents NMDA-Induced Neuronal Death in Hippocampal Organotypic Cultures. *Journal of Neurochemistry,* Vol.82, No.6, pp. 1388-1397, ISSN

1, IL-8 and Ischemic Cerebral Tissue Enhance Human Bone Marrow Stromal Cell Migration in Interface Culture. *Hematology,* Vol.7, No.2, pp. 113-117, ISSN 1024-

Progenitor Cells from Bone Marrow Or Adipose Tissue Produce VEGF, HGF, and IGF-I in Response to TNF by a p38 MAPK-Dependent Mechanism. *American Journal of Physiology.Regulatory, Integrative and Comparative Physiology,* Vol.291, No.4, pp.

VEGF-Overexpressing Transgenic Mice show Enhanced Post-Ischemic Neurogenesis and Neuromigration. *Journal of Neuroscience Research,* Vol.85, No.4,

Enhances Striatal Neurogenesis in Brain of Adult Rat After a Transient Middle Cerebral Artery Occlusion. *Journal of Neuroscience Research,* Vol.85, No.1, pp. 73-82,

Neuroprotective Cytokine that Inhibits Excitotoxic Injury in Vitro and in Vivo. *The*

& Kaltschmidt, C. (2004). MCP-1 Induces Migration of Adult Neural Stem Cells. *European Journal of Cell Biology,* Vol.83, No.8, pp. 381-387, ISSN

1 in Trophic Support of Cortical Neurons. *Glia,* Vol.36, No.1, pp. 48-57, ISSN 0894-

Marrow Stromal Cells Differentiate into Neurons. *Journal of Neuroscience Research,*

Salomone, J.P., Dent, L.L., Harris, O.A., Ander, D.S., Lowery, D.W., Patel, M.M., Denson, D.D., Gordon, A.B., Wald, M.M., Gupta, S., Hoffman, S.W. & Stein, D.G. (2007). ProTECT: A Randomized Clinical Trial of Progesterone for Acute Traumatic Brain Injury. *Annals of Emergency Medicine,* Vol.49, No.4, pp. 391-402, 402.e1-2, ISSN

**4**

*China*

**Development of Foamy Virus Vectors for**

The complexities of neurological diseases make it difficult to develop effective methods to treat them. In addition, it is difficult for the drugs used in neurological diseases to reach target cells at a sufficient concentration due to the blood-brain barrier, which may lead to side-effects in other organs, even though newly developed drugs are targeted directly to the origin of the diseases. Nevertheless, as the field of gene delivery gradually matures, new ways to deliver therapies for diseases become possible, and there are reasons to believe that

There are two main classes of vectors used for gene delivery: nonviral vectors and viral vectors (Gardlik et al., 2005). As viral vectors display a higher efficiency and specificity than nonviral vectors, they have been developed as therapeutic gene transfer carriers for various organ systems throughout the body in which the nervous system is involved (Glorioso et al., 2003). The four major viral vector systems used for neurological diseases are recombinant lentivirus (LV), adenovirus (Ad), herpes-simplex virus (HSV), and recombinant adenoassociated virus (rAAV) (Manfredsson et al., 2010). However, the utilization of these vectors

Foamy viruses (FVs), widespread, complex retroviruses with a large genome approximately 12-13 kb, are syncytial viruses containing two different promoters termed IP (internal promoter) and P (promoter) (Löchelt et al., 1995). Due to the specific structure of the viruses and their innocuousness in both human and primate infections, FV vectors have potential as a safe and capable vehicle with a larger capacity for transgenes (Williams, 2008). It has been reported that FV are potentially safer than gammaretroviruses and lentiviral vectors (Trobridge et al., 2009). In this review, we discuss the use of FV vectors as a safe and efficient carrier to deliver therapeutic genes for neurological disorders and other diseases.

gene therapy would be an optional treatment for neurological diseases.

is limited by safety concerns because of their pathogenicity (Zhang et al., 2010).

**1. Introduction**

 \*

Corresponding author \*\*Equal contribution to this work **Gene Therapy for Neurological**

**Disorders and Other Diseases**

Yingying Zhang1\*\*, Guoguo Zhu1\*\*, Yu Huang1\*\*,

*1School of Basic Medical Sciences, Wuhan University, Wuhan 2Centre for Medical Research, Wuhan University, Wuhan*

Xiaohua He1,2\* and Wanhong Liu1,2\*

## **Development of Foamy Virus Vectors for Gene Therapy for Neurological Disorders and Other Diseases**

Yingying Zhang1\*\*, Guoguo Zhu1\*\*, Yu Huang1\*\*, Xiaohua He1,2\* and Wanhong Liu1,2\* *1School of Basic Medical Sciences, Wuhan University, Wuhan 2Centre for Medical Research, Wuhan University, Wuhan China*

## **1. Introduction**

78 Advanced Topics in Neurological Disorders

Zhao, L.R., Singhal, S., Duan, W.M., Mehta, J. & Kessler, J.A. (2007). Brain Repair by

Zhong, C., Qin, Z., Zhong, C.J., Wang, Y. & Shen, X.Y. (2003). Neuroprotective Effects of

Zhu, W., Cheng, S., Xu, G., Ma, M., Zhou, Z., Liu, D. & Liu, X. (2011). Intranasal Nerve

2584-2591, 1524-4628; 0039-2499

3940

Hematopoietic Growth Factors in a Rat Model of Stroke. *Stroke,* Vol.38, No.9, pp.

Bone Marrow Stromal Cells on Rat Organotypic Hippocampal Slice Culture Model of Cerebral Ischemia. *Neuroscience Letters,* Vol.342, No.1-2, pp. 93-96, ISSN 0304-

Growth Factor Enhances Striatal Neurogenesis in Adult Rats with Focal Cerebral Ischemia. *Drug Delivery,* Vol.18, No.5, pp. 338-343, ISSN 1521-0464; 1071-7544

> The complexities of neurological diseases make it difficult to develop effective methods to treat them. In addition, it is difficult for the drugs used in neurological diseases to reach target cells at a sufficient concentration due to the blood-brain barrier, which may lead to side-effects in other organs, even though newly developed drugs are targeted directly to the origin of the diseases. Nevertheless, as the field of gene delivery gradually matures, new ways to deliver therapies for diseases become possible, and there are reasons to believe that gene therapy would be an optional treatment for neurological diseases.

> There are two main classes of vectors used for gene delivery: nonviral vectors and viral vectors (Gardlik et al., 2005). As viral vectors display a higher efficiency and specificity than nonviral vectors, they have been developed as therapeutic gene transfer carriers for various organ systems throughout the body in which the nervous system is involved (Glorioso et al., 2003). The four major viral vector systems used for neurological diseases are recombinant lentivirus (LV), adenovirus (Ad), herpes-simplex virus (HSV), and recombinant adenoassociated virus (rAAV) (Manfredsson et al., 2010). However, the utilization of these vectors is limited by safety concerns because of their pathogenicity (Zhang et al., 2010).

> Foamy viruses (FVs), widespread, complex retroviruses with a large genome approximately 12-13 kb, are syncytial viruses containing two different promoters termed IP (internal promoter) and P (promoter) (Löchelt et al., 1995). Due to the specific structure of the viruses and their innocuousness in both human and primate infections, FV vectors have potential as a safe and capable vehicle with a larger capacity for transgenes (Williams, 2008). It has been reported that FV are potentially safer than gammaretroviruses and lentiviral vectors (Trobridge et al., 2009). In this review, we discuss the use of FV vectors as a safe and efficient carrier to deliver therapeutic genes for neurological disorders and other diseases.

<sup>\*</sup> Corresponding author

<sup>\*\*</sup>Equal contribution to this work

Development of Foamy Virus Vectors for

**<IP_ADDRESS> IP**

gene to the late structural gene.

**2.1.2 Structural genes**

uncertain.

is initiated by the 5' LTR promoter start site (Li et al., 1999).

Gene Therapy for Neurological Disorders and Other Diseases 81

subregion) structure forms the LTR. The LTR promoter, containing a component of the U3- R-U5 region, is a traditional retroviral promoter. The prototypical HFV U3 region is approximately 777 nt and includes a TATA box, transcription regulatory signals and several BREs (Bel1 transactivator response elements) that are located at ~89-116 nt, ~284-327 nt, ~348-360 nt, ~418-454 nt and ~506-559 nt, respectively (Lee et al., 1993; Verdin et al., 1995). As there are a number of BRE regions, Choy, Kerr et al. suggested that the Bel1 regions regulate gene expression by integrating with intracellular transcription factors and transcription initiation complexes without binding directly to BREs and act as enhancers that work together to make a change (Lee et al., 1993; Choy et al., 1993; Kerr et al., 1993; Yang et al., 1997; Löchelt et al., 1994)). The negative control region in the RU5 inhibits the gene expression of HFV, and the viral mRNA R-U5 forms a stable secondary structure (Nabel et al., 1987). There is a splice donor site (SD) 51 nt downstream of the transcription site of the R region, where the posttranscriptional splicing of the *gag*, *pol*, *env*, and *bel* genes

All FVs possess a promoter in the U3 region of the LTR. In addition to the LTR promoter P, they also contain another unique internal promoter, termed IP (Löchelt et al., 1993). The internal promoter is located at the end of *env* gene. These two promoters require Tas for high-level expression (Löchelt et al., 1994). Compared with the LTR promoter, Tas has a higher affinity for the IP (Kang et al., 1998). In the early stage of viral infection, the IP initially regulates the expression of Tas transcription through an LTR cis-acting effect and the participation of as yet unknown cytokines. Next, Tas affects the IP through its selfactivating mechanism to elevate its synthesis level; when the Tas protein reaches a certain level, the activated LTR initiates the transcription and expression of the structural gene and performs transformation of gene transcription and expression from the early non-structural

Following the 5' LTR of FVs, there are three structural genes (*gag*, *pol*, *env*). The newly synthesized 74/78 kDa precursor protein encoded by the *gag* gene is located in the nuclei of the infected cells (Schliephake et al., 1994), and it is cleaved into the matrix, capsid, and nucleocapsid structural protein by the viral protease (Saïb et al., 1995). The nucleocapsid (NC) domain of Gag protein lacks Cys-His boxes found in all other retroviral Gag proteins, but its C terminus possesses three glycine-arginine motifs (GR boxes) that are required for RNA packaging (Stenbak et al., 2004; Yu et al., 1996; Maurer et al., 1998). The *pol* gene overlaps the *gag* gene, and its product does not contain any Gag protein determinant; however, unlike other conventional retroviruses, there is no Gag-Pol fusion protein synthesized in the course of viral transcription, but the underlying mechanism remains unknown (Stenbak et al., 2004; Yu et al., 1996). The precursor protein encoded by the *env* gene, which is approximately 130 kDa, is cleaved into the SU and TM subunits during the process of maturation (Saïb et al., 1995; Aguzzi, 1993). Linial et al. found that the extracellular virions of HFV require the Env protein for further production but that the formation of intracellular particles seems to be independent of the Env protein (Baldwin et al., 1993). Unfortunately, the role of Gag, Pol, and Env in the assembly of the FVs remains

## **2. Characteristics of foamy virus (Figure 1)**

## **2.1 Construction**

FVs were first described 50 years ago and are widely distributed in the natural world (Loh et al., 1992). They have been isolated from several mammals, including nonhuman primates, cattle, cats, and horses.

(1) IFP35 can interact with the bovine Tas (BTas) and arrest the replication of BFV; and Bel-1 interacts with -enolase, then represses the activation of LTR and IP;(2) Tas can be acetylated by p300, and activate the virus replication;(3) PML inhibits the binding of Tas to IP and LTR.

Fig. 1. The genomic organization of FVs and Tas of FVs interacted with other proteins in cells

Once infected by FVs, life-long latency of the infected cell is obtained (Maxine et al., 2000). FVs contain the largest genome and possess the most complex gene structure of all the retroviruses. For example, the human foamy virus (HFV, also called primate foamy virus (PFV)) contains LTR (long terminal repeat) sequences at the 5'and 3' ends of its genome, and there are three structural genes between these two LTRs that are found in all viral genomes: *gag*, *pol*, and *env* (Saïb et al., 1995). Several ORFs (open reading frames), often two or three, are located between the *env* and the LTR at the 3'end of all sequenced FVs, which encode autologous transactivators, including Tas, an important transactivator (Linial, 2000). The Tas protein is required by both the promoter (P) in the U3 region of the 5' LTR and another unique internal promoter (IP) at the end of the *env* gene for high-level expression (Linial, 2000).

#### **2.1.1 Promoters**

#### **<IP_ADDRESS> LTR**

Long terminal repeats (LTRs), located at the 5' and 3' end of the viral genome, are noncoding regions that exist in all types of retroviruses. The LTR is required for the integration of the viral genome into the host genome and is related to the replication, kinetics, tissue tropism, and pathogenicity of the virus. A U3-R-U5 (unique 3' subregion-repeat subregion-unique 5' subregion) structure forms the LTR. The LTR promoter, containing a component of the U3- R-U5 region, is a traditional retroviral promoter. The prototypical HFV U3 region is approximately 777 nt and includes a TATA box, transcription regulatory signals and several BREs (Bel1 transactivator response elements) that are located at ~89-116 nt, ~284-327 nt, ~348-360 nt, ~418-454 nt and ~506-559 nt, respectively (Lee et al., 1993; Verdin et al., 1995). As there are a number of BRE regions, Choy, Kerr et al. suggested that the Bel1 regions regulate gene expression by integrating with intracellular transcription factors and transcription initiation complexes without binding directly to BREs and act as enhancers that work together to make a change (Lee et al., 1993; Choy et al., 1993; Kerr et al., 1993; Yang et al., 1997; Löchelt et al., 1994)). The negative control region in the RU5 inhibits the gene expression of HFV, and the viral mRNA R-U5 forms a stable secondary structure (Nabel et al., 1987). There is a splice donor site (SD) 51 nt downstream of the transcription site of the R region, where the posttranscriptional splicing of the *gag*, *pol*, *env*, and *bel* genes is initiated by the 5' LTR promoter start site (Li et al., 1999).

#### **<IP_ADDRESS> IP**

80 Advanced Topics in Neurological Disorders

FVs were first described 50 years ago and are widely distributed in the natural world (Loh et al., 1992). They have been isolated from several mammals, including nonhuman primates,

(1) IFP35 can interact with the bovine Tas (BTas) and arrest the replication of BFV; and Bel-1 interacts with -enolase, then represses the activation of LTR and IP;(2) Tas can be acetylated by p300, and

Fig. 1. The genomic organization of FVs and Tas of FVs interacted with other proteins in

Once infected by FVs, life-long latency of the infected cell is obtained (Maxine et al., 2000). FVs contain the largest genome and possess the most complex gene structure of all the retroviruses. For example, the human foamy virus (HFV, also called primate foamy virus (PFV)) contains LTR (long terminal repeat) sequences at the 5'and 3' ends of its genome, and there are three structural genes between these two LTRs that are found in all viral genomes: *gag*, *pol*, and *env* (Saïb et al., 1995). Several ORFs (open reading frames), often two or three, are located between the *env* and the LTR at the 3'end of all sequenced FVs, which encode autologous transactivators, including Tas, an important transactivator (Linial, 2000). The Tas protein is required by both the promoter (P) in the U3 region of the 5' LTR and another unique internal promoter (IP) at the end of the *env* gene for high-level expression (Linial,

Long terminal repeats (LTRs), located at the 5' and 3' end of the viral genome, are noncoding regions that exist in all types of retroviruses. The LTR is required for the integration of the viral genome into the host genome and is related to the replication, kinetics, tissue tropism, and pathogenicity of the virus. A U3-R-U5 (unique 3' subregion-repeat subregion-unique 5'

activate the virus replication;(3) PML inhibits the binding of Tas to IP and LTR.

**2. Characteristics of foamy virus (Figure 1)**

**2.1 Construction**

cells

2000).

**2.1.1 Promoters**

**<IP_ADDRESS> LTR**

cattle, cats, and horses.

All FVs possess a promoter in the U3 region of the LTR. In addition to the LTR promoter P, they also contain another unique internal promoter, termed IP (Löchelt et al., 1993). The internal promoter is located at the end of *env* gene. These two promoters require Tas for high-level expression (Löchelt et al., 1994). Compared with the LTR promoter, Tas has a higher affinity for the IP (Kang et al., 1998). In the early stage of viral infection, the IP initially regulates the expression of Tas transcription through an LTR cis-acting effect and the participation of as yet unknown cytokines. Next, Tas affects the IP through its selfactivating mechanism to elevate its synthesis level; when the Tas protein reaches a certain level, the activated LTR initiates the transcription and expression of the structural gene and performs transformation of gene transcription and expression from the early non-structural gene to the late structural gene.

#### **2.1.2 Structural genes**

Following the 5' LTR of FVs, there are three structural genes (*gag*, *pol*, *env*). The newly synthesized 74/78 kDa precursor protein encoded by the *gag* gene is located in the nuclei of the infected cells (Schliephake et al., 1994), and it is cleaved into the matrix, capsid, and nucleocapsid structural protein by the viral protease (Saïb et al., 1995). The nucleocapsid (NC) domain of Gag protein lacks Cys-His boxes found in all other retroviral Gag proteins, but its C terminus possesses three glycine-arginine motifs (GR boxes) that are required for RNA packaging (Stenbak et al., 2004; Yu et al., 1996; Maurer et al., 1998). The *pol* gene overlaps the *gag* gene, and its product does not contain any Gag protein determinant; however, unlike other conventional retroviruses, there is no Gag-Pol fusion protein synthesized in the course of viral transcription, but the underlying mechanism remains unknown (Stenbak et al., 2004; Yu et al., 1996). The precursor protein encoded by the *env* gene, which is approximately 130 kDa, is cleaved into the SU and TM subunits during the process of maturation (Saïb et al., 1995; Aguzzi, 1993). Linial et al. found that the extracellular virions of HFV require the Env protein for further production but that the formation of intracellular particles seems to be independent of the Env protein (Baldwin et al., 1993). Unfortunately, the role of Gag, Pol, and Env in the assembly of the FVs remains uncertain.

Development of Foamy Virus Vectors for

express Tas and Bet but virus will not.

**2.2 Replication and transcription**

1996).

(Mergia, 1994).

**3.1 Replication-competent HFV**

(Nestler et al., 1997).

Gene Therapy for Neurological Disorders and Other Diseases 83

clarifies that the LTR does not initiate the synthesis of viral protein, whereas the expression of Bet is induced by IP (Linial, 2000). Transcription may be different in diverse cell types. Additionally, the combination site of Tas for the internal promoter IP is different from that for the LTR promoter, and the required cytokines are different. High expression of the virus would be suppressed under conditions in which the IP promoter was active in various cell types, yet cytokines, which the LTR promoter requires, exist in a select few cells. The transcription of integral provirus is mainly initiated by IP and will

As mentioned previously, FVs are a member of the spumavirus genus of the retroviridae family. It is believed that the replication and transcription mechanisms of various family members are similar (Coffin, 1990). However, Yu et al. found that although HFV possesses a replication pathway with features of both retroviruses and hepadnaviruses, there are also unique features contained in the assembly and reverse transcription pathway (Yu et al.,

At an early stage of viral infection, Tas is expressed due to the high level of transcription of IP, which in turn transactivates the expression of IP and LTR, leading to the expression of accessory and structural genes, respectively, inducing late stage viral infection (Löchelt et al., 1994). The Gag-Pol fusion protein is synthesized during the transcription of other retroviruses. However, as discussed above, the *pol* gene product in foamy viruses does not contain any Gag protein determinant, which means there is no Gag-Pol fusion protein synthesized in the course of viral transcription. Therefore, unknown mechanisms exist in the synthesis of *pol* gene products. As there are two promoters present in the genome of foamy virus, it is presumed that IP functionally replaces the post-transcriptional regulatory protein of other retroviruses and thus provides a temporal pattern with stage-specific promoters

Although FVs infection can result in a unique foam-like cytopathic effect and syncytia in cell cultures, they are nonpathogenic in naturally or experimentally infected animals, and the pathogenicity to their hosts remains unknown. However, more and more research groups are attracted to FVs due to the lack of pathogenicity; thus, increased attention has been

The first described FVs vectors (pFOV-1, pFOV-3 and pFOV-7) were replicationcompetent. Noncoding sequences in *bel-2* and *bel-3* were deleted, and two termination codons and a *bel-1* splice donor site mutation were introduced into these vectors (Schmidt et al., 1995). The vector pFOV-7, containing the herpes virus type 1 (HSV-1) thymidine kinase (tk), the *E.coli* cytosine deaminase (cd) and the polynucleotide phosphorylase (*pnp*) genes, was introduced into tumor cells and promoted their death

given to the development of FVs as vectors of target gene delivery.

**3. The development of human foamy virus (HFV) (Figure 2)**

### **2.1.3 Regulatory genes**

There are several ORFs encoding accessory genes or regulatory genes that are located between the *env* and the LTR and are therefore named *bel* genes. These ORFs encode autologous transactivators, including *bel-1*, *bel-2*, *bel-3* and *bet*.

### **<IP_ADDRESS> Tas**

The regulatory gene *bel-1*, which is approximately 902 bp in length, is located in the nucleus of expressing cells and is essential for HFV replication. FVs transactivators of different genera have been named differently; the simian FVs transactivator, for example, was termed *taf*. However the First International Foamy Virus Meeting determined that these transactivators, including Taf and Bel1, should be designated Tas (Zou et al., 1996). The activity of the functional IP and P of FVs depends on this transactivator. Transcription starts from the integration of Tas and the IP, leading to transactivation of the IP. However, the LTR promoter will not be activated until the level of Tas reaches a certain level (Linial, 2000). In view of the fact that the Tas protein plays an important role in the transcription and replication of FVs, it has received increasing attention and interest among virologists.

The interactions between Tas and other cellular molecules are complex. For example, Tas can be acetylated by p300 and PCAF (Bodem et al., 2007). Recently, an interaction between Tas and transcriptional coactivators, such as HATs, was reported. Studies show that only acetylated Tas enhances viral transcription (Bodem et al., 2007). Interferon-induced proteins (IFPs) complete multiple functions through various interferon signals. IFP35, which is approximately 35 kDa, is a member of the IFP family. It interacts with the bovine foamy virus Tas regulatory protein, and its overexpression disturbs viral gene transcription and replication of the bovine foamy virus Tas protein. Furthermore, IFP35 plays a crucial role in the interferon-induced anti-foamy virus host response (Tan et al., 2008). First discovered by Lohman and Mayerhof in 1934 (Lohman et al., 1934), α-enolase was later shown to be a highly conserved enzyme that catalyses the transformation of 2 phosphoglyceric acid to enolphosphopyruvate in the Embden-Meyerhof-Parnas pathway (Wold, 1971). α-Enolase interrupts the transactivation of Tas with the promoters during viral transcription (Wu et al., 2009). The promyelocytic leukemia protein (PML) is a cell growth and tumor suppressor gene, which is expressed in the nucleus and whose expression is increased significantly in the response to interferon (IFN) (Wang et al., 1998). Regad et al. found that PML inhibits the binding of Tas to the IP and LTR by direct interaction with Tas (Regad et al., 2001).

#### **<IP_ADDRESS> Bet**

Bet is a second IP regulating protein. Generated by the combination of the spliced mRNA of the Tas region and the Bel2 ORF (Linial, 2000), overexpression of Bet prevents the virus from infecting the host and inhibits viral integration at the initial stage of viral infection (Bock et al., 1998). A variant RNA can be generated by the LTR promoter, which has the ability to generate Bet protein fragments that, after reverse transcription, could induce the expression of a △Tas provirus (Saïb et al., 1993). Because of the deficiency of Tas in the provirus, △Tas plays a significant role in the preservation and latency of FVs, which clarifies that the LTR does not initiate the synthesis of viral protein, whereas the expression of Bet is induced by IP (Linial, 2000). Transcription may be different in diverse cell types. Additionally, the combination site of Tas for the internal promoter IP is different from that for the LTR promoter, and the required cytokines are different. High expression of the virus would be suppressed under conditions in which the IP promoter was active in various cell types, yet cytokines, which the LTR promoter requires, exist in a select few cells. The transcription of integral provirus is mainly initiated by IP and will express Tas and Bet but virus will not.

### **2.2 Replication and transcription**

82 Advanced Topics in Neurological Disorders

There are several ORFs encoding accessory genes or regulatory genes that are located between the *env* and the LTR and are therefore named *bel* genes. These ORFs encode