Mesenchymal Stromal Cells to Treat Brain Injury (Bioengineering in Neurological Disorders) Part 1

Introduction

Brain injury occurs from either a traumatic (mechanical), ischemic (decreased oxygen; accounts for 83% of stroke cases), or hemorrhagic (ruptured blood vessel; accounts for 17% of stroke cases) insult to the brain. Stroke and traumatic brain injury (TBI) are major contributors worldwide to both deaths and persistent disabilities. Stroke is the third leading cause of death (behind heart disease and cancer) in the United States, with 137,000 Americans dying from stroke each year (Heron et al., 2009). Stroke is the leading cause of serious, long-term disability in the United States. Currently, 795,000 people have a stroke each year and 15-30% of survivors have a permanent disability (Roger et al., 2011). Annually, 1.7 million people sustain a TBI in the United States, resulting in 52,000 deaths and over 124,000 permanent disabilities each year (Faul et al., 2010). Annual direct (e.g., medical) and indirect (e.g., loss of productivity) costs to the United States are $41 billion and $60 billion for stroke and TBI, respectively (Finkelstein et al., 2006; Roger et al., 2011).

Though the etiology differs between traumatic and ischemic injury, there are many similarities in their pathology (Bramlett & Dietrich, 2004; Leker & Shohami, 2002). The primary insult initiates a cascade of secondary events such as edema, excitotoxicity, and increases in free radicals, which act to spread the injury to surrounding tissue (for reviews of the pathology, see Greve & Zink, 2009 for TBI and Mitsios et al., 2006 for ischemic stroke). Note that ischemia is part of the secondary injury response for TBI (Coles, 2004; Garnett et al., 2001). The brain attempts to repair and regenerate, but depending on such factors as injury severity, age of onset, and prior injuries, these endogenous attempts are often insufficient to restore normal function. A treatment that limits the spread of secondary damage and/or promotes repair and regeneration is needed. Current clinical treatment practices for TBI primarily aim to reduce intracranial pressure in an effort to minimize brain damage caused by swelling. For ischemic stroke, the only FDA-approved treatment is breaking down blood clots with tissue plasminogen activator. However, patients must meet strict criteria for receiving this therapy, including a 4 hour time window and no evidence of the following: bleeding, a severely elevated blood pressure or blood sugar, recent surgery, low platelet count, or end-stage liver or kidney disorders. Numerous pharmacological treatments that seemed promising in animal models have failed in clinical trials (Maas et al., 2010; O’Collins et al., 2006). Patients with brain injury vary widely with respect to demographics, severity of injury, location of injury, and co-morbidity factors making clinical trials challenging. Most treatments previously tested involved pathways that are both deleterious and beneficial, making the dosage and timing critical to not interfere with normal homeostasis or reparative mechanisms in the brain. Furthermore, these treatments targeted single mechanisms, which may not be enough in light of the multi-faceted pathology. Therapies that currently seem more promising, such as progesterone administration (Wright et al., 2007) and cell transplantation, address multiple pathological events.

Mesenchymal stromal cells to treat brain injury

Mesenchymal stromal cells (MSCs)

Mesenchymal stem cells are multipotent cells that can differentiate into cells of the mesoderm germ layer. These cells can be isolated from adipose tissue, amniotic fluid, placenta and umbilical cord, though are most commonly and efficiently derived from adult bone marrow. Marrow-derived cells that adhere to tissue-culture plastic in vitro are a heterogeneous population of cells that contain mesenchymal stem cells, but the entire population is more correctly defined as mesenchymal stromal cells (Horwitz et al., 2005). As we learn more about these cell populations, the terminology evolves and the acronym MSC is used (and sometimes misused) for mesenchymal stem cell, mesenchymal stromal cell, multipotent stromal cell, and marrow stromal cell. For the purposes of this topic, we will not distinguish amongst these cell populations and use MSC as a general acronym.

Using MSCs to treat brain injury

MSCs are an attractive cell source for transplantation because they are relatively easy to obtain, expand, and manipulate in vitro. In addition, adult human MSCs do not have the tumorigenicity risks that pluripotent cells carry. Ample preclinical data demonstrate that MSC transplantation promotes functional recovery following experimental cerebral ischemic or TBI (for review, see Li & Chopp, 2009 or Parr et al., 2007). Autologous MSC therapy has already shown promise for treating clinical stroke (Battistella et al., 2011; Honmou et al., 2011; Lee et al., 2010; Suarez-Monteagudo et al., 2009) and TBI (Cox et al., 2011; Zhang et al., 2008). Collectively, these trials demonstrate that transplanting MSCs either intra-arterially, intravenously, or intracerebrally is safe and no cell-related adverse events were reported. These groups also indicate that some patients receiving MSCs had improved functional outcome; however, these hints at efficacy must be cautiously interpreted because these were primarily safety trials and were not designed to show robust efficacy.

Important considerations for using MSCs in the clinic include timing (acute versus chronic), delivery route (most commonly intravenous, intra-arterial, or intracerebral), and donor source (autologous versus allogeneic). There are advantages and disadvantages for each of these issues, which are outlined in Table 1. According to www.clinicaltrials.gov (searched in August 2011; summarized in Table 2), there are 11 ongoing clinical trials worldwide using MSCs (either primary or derivatives) to treat stroke. Of these 11 studies, 5 are using autologous MSCs and the other 6 are using allogeneic MSCs from either bone marrow, placenta (1 study) or umbilical cord (1 study). Two of the trials are injecting cells directly into the injured brain (either into the injury cavity or the peri-infarct tissue), 1 trial is injecting cells into the carotid artery, and the other 8 are injecting MSCs intravenously. With regard to timing, 2 of the trials are delivering the cells during the acute phase (within 72 hours post-stroke), 7 trials during the sub-acute phase (between 4 days and 6 weeks post-stroke), and 2 studies are delivering cells during the chronic phase (over 6 months post-stroke). As trials more definitively reveal that MSCs transplantation is both safe and effective for treating brain injury in humans, issues of delivery timing and route and donor source, as well as dosage and the use of immunosuppression will need to be more carefully compared.

Table 1. Clinical considerations for using MSCs to treat brain injury

Sponsor	Country	Start Date	Phase		Donor	Cell Description	Route	Timing (post-stroke)	Follow-up	dinicaltrials.gov ID
National Cardiovascular Center	Japan	2008-May	I/Ha	12	Auto	Bone marrow mononuclear cells	IV	7-10 days	30 days	NCTÛ1Û28794
CellMed AG, a subsidiary of BTG pic.	Germany	2008-October	I/II	20	Alio	GLP-1 CellBeads: alginate microcapsules with mesenchymal cells transfected to secrete glucagon like peptide-1	IC	acute	6 months	NCT01298830
University of Texas Health Science Center	USA	2009-January	I	30	Auto	Bone marrow mononuclear cells	IV	24-72 hours	5 years	NCT00859014
University of California, Irvine	USA	2010-January	I	33	Auto	Bone marrow mononuclear cells OR Cultured marrow mesenchymal stromal cells	IV	Mononuclear: 4 days; Mesenchymal: 23 days	90 days	NCT00908856
University Hospital, Grenoble	France	2010-August	π	30	Auto	Mesenchymal stem cells	IV	Up to 6 weeks	24 months	NCT00875654
Stempeutics Research Pvt. Ltd.	Malaysia	2010-December	I/II	78	Alio	Cultured adult mesenchymal stem cells	IV	Up to 10 days	12 months	NCT01091701
SanBio, Inc.	USA	2011-January	I/Ha	18	Alio	SB623: modified marrow stromal cells	IC	6-24 months	24 months	NCT01287936
Stemedica Cell Technologies, Inc.	USA	2011-February	I/II	35	Alio	Adult mesenchymal bone marrow stem cells	IV	Beyond 6 months	12 months	NCT01297413
Celgene Corporation	USA	2011-March	na	44	Alio	Human placenta-derived cells PDA001-(cenplacel-L)	IV	1 ORI and 8 days	24 months	NCT01310114
Aldagen	USA	2011-March	Π	100	Auto	ALD-401: derived from bone marrow	IA	13-19 days	12 months	NCT01273337
General Hospital of Chinese Armed Police Forces	China	2011-April	Π	120	Alio	Umbilical cord mesenchymal stem cells	IV	1st TP: 10-21 days (hemorrhage) OR 724 days (ischemic); 2nd TP: lumbar puncture 7d after 1st TP	12 months	NCT01389453

# Px= planned number of patients to enroll; Auto=autologous; Allo=aüogeneic; 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

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 topic, 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.

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 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.