What this paper adds
- More basic research is necessary to optimize the use of stem cells in CP.
- Combinatorial approaches are likely to be the ideal option.
Cerebral palsy (CP) is a chronic childhood disorder described by a group of motor and cognitive impairments and results in a substantial socio-economic burden to the individual, family, and healthcare system. With no effective biological interventions, therapies for CP are currently restricted to supportive and management strategies. Cellular transplantation has been suggested as a putative intervention for neural pathology, as mesenchymal and neural stem cells, as well as olfactory ensheathing glia and Schwann cells, have shown some regenerative and functional efficacy in experimental central nervous system disorders. This review describes the most common cell types investigated and delineates their purported mechanisms in vivo. Furthermore, it provides a cogent summary of both current early-phase clinical trials using neural precursor cells (NPCs) and the state of stem cell therapies for neurodegenerative conditions. Although NPCs are perhaps the most promising candidates for cell replacement therapy in the context of CP, much still remains to be understood regarding safety, efficacy, timing, dose, and route of transplantation, as well as the capacity for combinatorial strategies.
Embryonic stem cell
Induced pluripotent stem cell
Mesenchymal stem cell
Neural precursor cell
Olfactory ensheathing glia
Cerebral palsy (CP) is the most common pediatric developmental disability and is caused by perinatal asphyxia, infection, or preterm birth. In developed countries it occurs in approximately 2.5/1000 live births. The mortality rate among affected children is higher than among their unaffected peers, and comorbid cognitive and sensory deficits, in addition to lifelong motor impairment, are frequent. A late prenatal or perinatal hypoxic–hemodynamic insult is the dominating final common pathway in the pathogenesis of CP. Survivors of preterm birth constitute the largest etiological subgroup of children with CP, and periventricular leukomalacia (PVL) is the most common form of brain injury in this cohort (Fig. 1). Phenotypically, children with CP present with spastic quadriplegia (35%), spastic diplegia (21%), and spastic hemiplegia (31%), with other forms of dyskinetic CP contributing the remainder (13%). Animal models in several species provide evidence for a maturation-dependent vascular predisposition to white matter injury, with a specific sensitivity of the immature oligodendrocyte populations to ischemic and inflammatory insult. Pathologically, PVL preferentially occurs around the lateral ventricles that border the penetrating branches of the major cerebral arteries and spatially overlap descending corticospinal tracts (Fig. 1). Relevant to this, cell replacement therapy aims to replace damaged oligodendrocytes in order to normalize function in affected neuromotor tracts.
Stem cells are multipotential cells that exist in both adult and developing tissue. Their key unifying characteristics are their multipotential capability and their ability to self-renew. Throughout development, pluripotency decreases with increased cellular differentiation (Fig. 2); differentiation is accompanied by changes in both gene expression and the epigenetic profile, leading to a mature cell phenotype. ‘True’ stem cells can form any tissue in the body or amnion and have virtually unlimited proliferative and self-renewal capacity. However, these cells are never used in cell transplantation paradigms, primarily because of their high mobility and propensity for teratoma formation. Instead, more differentiated cells, such as multipotent cells or fate-restricted progenitors, are the types used in preclinical studies. Furthermore, non-stem cells, usually glial cells such as peripheral Schwann cells and olfactory ensheathing glia (OEG), have also been explored for cellular therapy, and will be discussed a posteriori. Here will discuss all types of stem cells along the developmental lineage, with understanding that the cell types currently being explored preclinically primarily consist of late-stage tissue-specific progenitors or more mature cell types.
Totipotent cells are the earliest lineage cells, found within the first few cell divisions of the early blastocyst. They can produce all cell types – including extra-embryonic tissue – but are rarely used for therapeutic purposes owing to the lack of specialization and associated high propensity to the development of teratoma.
After roughly 4 days of cell division, the blastocyst forms an external trophoblast and an inner cell mass, from which develop, respectively, the extra-embryonic tissue and the embryo proper. Pluripotent stem cells are extracted from the inner cell mass of the early segmented blastocyst and can contribute to all germ layers and tissues in the human body, short of placental tissue (which develops from the trophoblast). These are generally referred to as embryonic stem cells (ESCs), and are the subtype most commonly associated with the term ‘stem cells’.
An alternative means of obtaining pluripotent stem cells has recently been elucidated. Induced pluripotent stem cells (iPSCs) can be derived from human somatic (typically skin or blood) tissue, by up-regulating the ‘Yamanaka’ transcription factors OCT4, c-Myc, Sox2, and KLF4 (or substitutes), thereby creating patient-specific cells from adult sources. This potentially reduces the need for tissue donor waiting lists and also circumvents the destruction of an embryo during pluripotent cell production. While iPSCs are not ready for clinical utilization, improvements in production and differentiation methods – such as the use of non-integrating transposon, virus, small molecule, protein, and mRNA systems – as well as non-oncogenic derivation, optimized cell culture methods, and increased understanding of how original somatic cell type affects progeny have led to ‘safer,’ more optimized iPSC lines.
Despite their benefits, pluripotent cells display a high degree of plasticity and are unsuitable for transplantation. For ESCs or iPSCs to be used in successful cell therapy they must be differentiated into multipotent or fate-restricted progenitors. Further to this, direct lineage reprogramming, or ‘trans-differentiation’ of somatic cells to neural subtypes, could potentially extend to cell transplant paradigms in the future.
Later in development, pluripotent stem cells narrow their potential and become multipotent cells. Several types of multipotent stem cells can also be derived from pluripotent sources in vitro via growth factor conditioning (reviewed by Ruff and Fehlings). Multipotent progenitors produce different cell lineages that are restricted to a specific type of tissue. For example, in vivo, mesenchymal stem cells (MSCs) become blood, bone, muscle, and fat, while neural precursor cells (NPCs) differentiate into neurons, astrocytes, and oligodendrocytes (for a review, see Ruff et al.). It is these cells and fate-restricted precursors that hold most clinical promise, as they are associated with a low risk of teratoma formation.
Still further along the developmental pathway exist unipotent progenitor cells; these are usually immature or fate-restricted precursors that differentiate into mature cells.
At this point during development, many ‘stemness’ characteristics have been lost and common nomenclature utilizes the terms progenitor and precursor in lieu of stem cell.
For neural pathology, these unipotent progenitors are typically premyelinating cells such as immature oligodendrocytes or Schwann cells.
The use of stem cells for preventative or (more often) restorative clinical benefit is currently under investigation in human clinical trials. Preclinical animal data suggest three major mechanisms whereby stem cells can mediate neurological repair: (1) they can provide structural support to the damaged and surrounding tissue, (2) they may remyelinate damaged axons, and (3) they can express neurotrophic growth factors. Cell subtype restricts each cell's potential mechanism of repair, and cells of separate lineages exhibit distinct regenerative capabilities (Fig. 3). It is important to note that not all cell subtypes used in preclinical therapy paradigms are stem cells. Indeed, Schwann cells and OEG, two of the most commonly investigated transplant types, are not stem cells, but, in fact, more mature cells. Alternatively, MSCs and NPCs are multipotent progenitors that still possess limited ‘stemness’ characteristics.
Tripotential NPCs exist in the subventricular and hippocampal regions of the adult brain, as well as in the ependymal zone of the adult spinal cord. They can also be created by differentiating pluripotent cells through the default pathway towards neurogenesis. Unipotent glial precursors are also found in the local neural parenchyma. Following neurological damage, intrinsic neural precursor populations are able to expand and home to the site of injury, although endogenous repair is limited. Experimental drug therapy-induced expansion of endogenous neural populations has shown moderate success in vivo. However, it is not yet possible to expand and mobilize large numbers of progenitors pharmacologically that would be comparable to those which can be injected from an expanded exogenous population. Thus, the most common form of NPC introduction following injury involves intraparenchymal injection of adult, pluripotent, or fetal-derived precursors. NPCs are thought to act as myelinating cells, homing to the site of injury and replacing lost or damaged oligodendrocytes; they can promote recovery after transplant into damaged neural tissue, particularly in combination with neurotrophic growth factors, and can also recruit endogenous progenitors to the site of injury. Although NPCs have the potential to differentiate into three different cell types (as previously described) in vitro, when they are transplanted in vivo without preconditioning they almost exclusively differentiate into glial subtype cells, to the exclusion of neurons. In the context of CP, in which demyelination predominates, additional neuronal formation is unwanted because of the potential for aberrant connectivity and resulting neuropathic pain or further motor dysfunction; the observed preferential glial subtype formation is thus ideal for repair and support for other cellular subtypes. Therefore, although their safety in humans has not yet been fully elucidated, NPCs show the most potential for positive functional results when used in neurological injury models and subsequent clinical translation.
Peripheral glial cells such as Schwann cells and OEG are not stem cells but will be discussed here because of their popular utility in cell transplant studies. Schwann cells and OEG have been used for cell replacement and trophic modulation following experimental neural injury. Because they create potent trophic and physical substrates for axonal growth, proximal sensory and propriospinal axons readily enter and rarely leave Schwann cell grafts. Consequently, strategies that employ Schwann cell transplants generally involve co-transplanted growth factors, biomaterials, or cells such as OEG. OEG can be derived from the olfactory bulb in the brain or through the lamina propria of the olfactory mucosa and have shown mixed results in vivo, thought to be partially due to origin or culture conditions (reviewed by Tetzlaff et al.). Schwann cells and OEG are thought to act via remyelination, as well as providing trophic and structural support. However, preclinical and clinical trials with these cells have shown mixed, and sometimes non-replicable, results.[21, 22]
Mesenchymal stem cells exist in the non-hematopoietic CD34- subset of bone marrow and umbilical cord cells. These cells, which constitute a subset of the adherent stromal cell fraction, are most commonly used in transplant studies, particularly in pediatric populations, because of their proven safety through decades of use in the context of leukemia and blood diseases.
Mesenchymal stem cells can be efficiently isolated from a number of sources – including bone marrow and/or aspirates, umbilical cord, placenta, fat, and tooth – and they provide a minimally invasive, autologous source of cells for transplantation. Although the MSCs that have shown safety in the context of leukemia have traditionally been derived from donor bone marrow, this paradigm is shifting toward more convenient cord-derived MSCs. Although MSCs can exhibit neurogenic differentiation in vitro, it is unclear whether this occurs in vivo to any meaningful extent. Transplanted MSCs can create a trophic substrate in vivo, enhancing tissue sparing in penumbral regions and reducing lesion volume, apoptosis, inflammation, and demyelination. MSCs are also reported to mobilize endogenous NPCs, promote angiogenesis, and provide physical scaffolding for elongating axons. However, studies utilizing MSC transplantation report highly variable outcomes. Protocols and cell sources need to be optimized and further study is required in order to isolate and characterize more homogeneous populations and glean reliable, replicable results. Overall, there exists little and conflicting evidence for the efficacy of MSCs in the context of neurological repair.
Because of the increasing popularity of postnatal umbilical cord stem cell harvest and storage, the most easily accessible current source of stem cells in clinical trials is umbilical cord-derived MSCs. Umbilical cord blood and other associated tissue such as placenta and Wharton's jelly are diverse sources of mononuclear progenitor and stem cell types, including MSCs. The ease of obtaining cells from fresh umbilical tissue and their associated autologous nature makes cord- and placental-derived MSCs very appealing. These cells pose little risk to infant or mother and have low immunogenicity; in addition, there use is associated with fewer ethical issues than other cells used in transplant scenarios. Although human umbilical cord blood mononuclear cells have shown promise in animal disease models, there is an overall paucity of data. The few trials that have utilized these cells preclinically show a wide spectrum of results, ranging from normalization of function after several weeks to complete cell disappearance after 24 hours or worsened injury. Furthermore, experimental outcomes are often contradictory, even within the same disease model,[30, 31] and mechanisms of action are not consistent or are not completely elucidated.
The first North American clinical trial in humans using cord blood-derived MSCs is currently under way at Duke University, NC (clinicaltrials.gov identifier NCT01147653) and has shown safety. Similarly, a study from the Sung Kwang Medical Foundation in Korea has combined rehabilitation, erythropoietin, and cord-blood MSC infusion and is the first to report positive results (clinicaltrials.gov identifier NCT01193660).[32, 33] Further study is required to elucidate (1) whether these results are replicable across institutions and (2) what cellular properties can enhance or reduce this regenerative response.
Of the 240 current clinical trials investigating treatments for CP or perinatal stroke, only eight involve stem cells. However, there exists, partly as a result of a lack of transparency from scientists but also because of unwarranted media hype, a supply and demand mismatch that has fostered the development of ‘stem cell tourism’. Clinics abound in Asia, Europe, the Middle East, and Central America that offer ‘stem cell treatments’ that are unregulated, lack legitimate affiliation or accountability, and use technologies without solid scientific foundation. Individuals utilizing such clinics expose themselves to a lack of success, exclusion from legitimate trials, false hope, complications (including mortality), and financial hardship (US$15–30 000 per treatment).
A detailed review of stem cell tourism by Lau et al. reveals that companies (e.g. Beike Biotech, Shenzen, China; ACT, Turks and Caicos Islands; and Emcell, Ukraine) claim to have treated nearly 6000 patients. Lau et al. analyzed the websites of companies offering ‘stem cell therapies’ and reported that 79% of sites described benefits as ‘somewhat or very relevant’ while 74% portrayed risk as ‘very irrelevant’. Furthermore, all of the websites reported benefits from treatment, with only 25% reporting any risk – giving the false impression that stem cell therapy is safe, routine, and effective.
The most common ‘therapies’ consisted of intravenous or intrathecal administration of what companies claim were autologous MSCs (>60%), coupled with several weeks of intensive physiotherapy. There is no way to verify the reliability of these claims, the effect of this intensive physiotherapy alone on outcome, or the purity of the cell suspension.
While there are anecdotal reports of success with stem cell tourism, there are concomitant reports of failures: neuropathic pain, multiple brain tumor formation, neurological injury, and even fatalities.[35-37] Furthermore, the extent of unreported complications in self-regulated stem cell clinics remains unknown.
In animal models, combinatorial approaches have proven to be most effective at establishing enhanced functional connectivity following neural injury and dysmyelination.[38, 39] Strategies employed preclinically in tandem with stem cell transplant or enhancements include bioengineering strategies, rehabilitation, magnetic stimulation, and growth factor/drug administration. While it is agreed in the field of regenerative medicine that combinatorial strategies, as opposed to a single therapeutic approach, provide the most promise for successful functional recovery, there still exists a knowledge gap surrounding the performance and role of stem cells in enhancing neurological recovery. Thus, before they can be studied in concert with other treatment methodologies, their particular role in the regenerative response must be elucidated in clinical trials.
The field of cell transplantation for CP is burgeoning; eight current (one completed with results) phase I clinical trials are using stem cells (all MSCs) for treatment of CP or perinatal stroke (summarized in Table 1). Stem cells are infused either intravenously or intrathecally using single or multiple infusions (www.clinicaltrials.gov). Participants are generally of either sex, aged 1 to 12 years, lack seizures or comorbidities, and have CP of any etiology. The only study which has reported results so far is a double-blind randomized trial of 105 participants of both sexes aged 10 months to 10 years. Participants received (1) active rehabilitation, (2) erythopoietin plus active rehabilitation, or (3) allogeneic umbilical cord blood infusion (intravenous) plus erythropoietin injection plus active rehabilitation. Assessment of neurological and neuromuscular function, brain white matter integrity (using magnetic resonance imaging), and brain glucose metabolism (using positron emission tomography) was undertaken regularly up to 6 months post intervention. Initial data analysis suggests that the primary outcome measures of motor function and standardized gross motor function were improved in group three, the MSC-treated group, compared with other groups. Secondary outcome measures of cognition, neurodevelopment, and brain imaging findings also suggest improvements in the MSC group compared with the other groups (clinical trials identifier NCT01193660). However, further statistical analysis remains to be completed to fully tease out the results and the implications from this promising study.
|Location of trial||Clinicaltrials.gov identifier||Cell source||Criteria||Phase||Delivery|
|1||Sung Kwang Medical Foundation, Korea||NCT01193660||Allogeneic umbilical cord blood, rehabilitation, and erythropoietin||10mo–10y with CP||I (completed with results)||Intravenous|
|2||Royan Institute, Iran||NCT01404663||Bone marrow-derived CD133||4–12y with quadriplegic CP, no seizures||I (completed)||Intrathecal|
|3||Royan Institute, Iran||NCT01763255||Bone marrow derived CD133||4–12y with quadriplegic CP, no seizures||I||Multiple intrathecal|
|4||Hospital Universitario Dr Jose E Gonzalez, Mexico||NCT01019733||Autologous stem cells (CD34+)||1–8y with hypoxia–ischemia||I (completed)||Intrathecal|
|5||Hospital Universitario Dr Jose E Gonzalez, Mexico||NCT01506258||Autologous non-cryopreserved CD34+ cells||48h post birth in encephalopathic neonates, Apgar score <5 at 5min, pH<7||I||Intravenous|
|6||Children's Memorial Hermann Hospital, USA||NCT01700166||Autologous human cord blood derived||6wks to 6y with arterial ischemic stroke||I||Intravenous|
|7||Georgia Health Sciences University, USA||NCT01072370||Autologous umbilical cord blood||1–12y old with CP, no seizures||I/II||Intravenous|
|8||Roberson Foundation/Duke University Medical Center, USA||NCT01147653||Autologous umbilical cord blood||12mo–6y with spastic CP||II||Intravenous|
There are numerous experimental studies illustrating the potential benefits of transplanted NPCs in clinical neurodegeneration.[40, 41] However, there is a relative paucity of data from clinical trials regarding the use of NPCs to treat central nervous system (CNS) disorders, particularly those of childhood. There are currently seven clinical trials in progress involving the use of NPCs (summarized in Table 2) for neurological conditions. Most involve intravenous, intrathecal, or intraparenchymal injection in adults with chronic neurological conditions. However, the first clinical trial in children (StemCells Inc., Newark, CA, USA) with advanced neuronal ceroid lipofuscinosis (Batten disease) has completed early-phase trials. If proven safe in patients with this terminal condition, this trial could provide the basis for further pediatric studies in CP. A second study in children with less advanced Batten disease (using CD133+ cell culture-expanded NPCs) was started, but was discontinued owing to insufficient enrollment of patients meeting study criteria. Further promising clinical studies of NPCs are being carried out in adults with degenerative diseases such as Pelizaeus–Merzbacher disease (a myelination disorder), stroke, thoracic spinal cord injury, and inoperable glioblastoma (for a review, see Trounson et al.). Interestingly, NPCs are being harvested from neurological biopsies of patients with Parkinson disease by the company NeuroGeneration (Los Angeles, CA, USA). It is hoped that the harvested cells can be expanded to produce at least 10% dopaminergic cells which can be used as a source for therapeutic transplantation.
|Company conducting trial||Clinicaltrials.gov identifier||Cell source||Application||Phase||Delivery|
|1||Geron Corp, USA||NCT01217008||ESC-derived oligodendrocyte progenitors||SCI||I||Intraspinal|
|2||StemCells Inc., USA||NCT01321333 (long-term follow-up: NCT01725880)||Fetal derived human NPCs||SCI||II||Intraspinal|
|3||StemCells Inc., USA||NCT00337636, NCT01238315||Neuronal ceroid lipofuscinosis (Batten disease)||I (completed), Ib (ceased due to lack of enrolment)||Intracerebral|
|4||StemCells Inc., USA||NCT01005004 (follow-up: NCT01391637)||Pelizaeus–Merzbacher disease||I/II||Intracerebral|
|5||Reneuron, UK||NCT01151124||Donated fetal brain NPCs||Adult stroke||I||Intracerebral|
|6||Neuralstem Inc., USA||NCT01348451||Fetal human spinal cord NPCs||ALS||I||Intraspinal|
|7||Neuralstem Inc., USA||NCT01772810||SCI||I||Intraspinal|
Neurodegenerative diseases share common pathologies such as neuronal dysfunction, demyelination, and cell death. The potential of NPCs to differentiate into neuronal subtypes and promote neuroprotection and neurogenesis strongly favors their application for neurodegenerative disorders. However, there remain several technical and regulatory hurdles before successful translation can be achieved.
The phenotypically different sources of NPCs used in the literature[46, 47] contribute to variability in efficacy, glial differentiation, and marker expression. Both source and cell culture/sorting parameters can affect behavior in vitro. Therefore, it is currently unknown which NPC sources (fetal derived, adult derived, endogenous, and pluripotent derived) and treatment conditions create the ideal cell type for transplantation. Or, indeed, whether NPCs alone or in concert exert the most beneficial effect.
While there are resident NPCs in the adult and fetal CNS encouraging their application in chronic neurodegeneration paradigms, harvesting human NPCs from fetal or adult CNS is both ethically and technically challenging. NPCs differentiated from pluripotent sources, via variable drug and differentiation protocols, present an alternative source of adult NPCs. Although there are means to derive several cell types from pluripotent progenitors, and indeed some groups are exploring transdifferentiation between two adult cell types via similar mechanisms to iPSCs, much work is still required to optimize conditions and to translate this technology safely to humans.
Another barrier to translation is the limited survival of transplanted exogenous cells. Indeed, there is currently a scarcity of data suggesting that cells survive for longer than a matter of weeks, and several groups have taken measures – including scaffolding, presorting, and modification – to increase cell viability. Although successful experimental transplantation of exogenous NPCs and/or recruitment of endogenous NPCs has been demonstrated in several animal models of CNS disorders, the underlying mechanisms of these processes remain to be elucidated, and it is not known if this phenomenon can be replicated in humans.
The optimal timing, route, and dose of cells are still largely unknown; this is reflected in the variability of these parameters in current clinical trials. Transplanted NPCs are frequently delivered intrathecally or intraparenchymally, with cell dose depending on the site of injury and local microenvironment. Multiple intracerebral injection sites, with slow and prolonged infusion rates, seem to provide maximal incorporation; however, dose–responsiveness has never been studied in humans.
Clinically, current neuroprotective strategies in encephalopathic newborn infants have a narrow optimal therapeutic window of hours after injury, which corresponds with rising immune activation; however, injecting cells at the peak of the inflammatory response to initial injury may inhibit stem cell survival. Furthermore, many children with perinatal complications fail to develop CP later in life. Indeed, most cases of CP are not diagnosed until approximately 2 years of age. Injecting at this chronic time point may be more feasible but potentially requires combinatorial therapies to ensure success.
Although NPCs are generally considered minimally immunogenic and non-tumorigenic in animal models, tumorigenicity and transplant rejection are still major causes for concern when translating therapies to the clinic. In terms of transplantation, the two most promising cell types, MSCs and NPCs, represent two ends of a continuum: MSCs are safe in clinical paradigms, but have not shown consistent preclinical benefit; conversely, NPCs show the most benefit in animal models, but have not yet been demonstrated to be safe in the long term in humans. Since stem cell transplantation is irreversible, all potential side effects must be fully elucidated both experimentally and in tightly controlled early safety trials before moving to humans.
Owing to the multivariate etiology and pathology of CP, and indeed all neurological injury, remyelination alone is not certain to alleviate all disease symptoms. Therefore, combinatorial strategies to combat the disease will be necessary. Some common strategies currently under exploration include bioengineering, rehabilitation strategies (such as constraint-induced movement therapy, treadmill training, virtual reality, gaming, and robot-assisted locomotor training), pharmacological interventions such as baclofen and botulinum toxin as well as transcranial magnetic stimulation.
A solid understanding of stem cell development and biology provides a stable basis for understanding the role of stem cells in regeneration and repair; from this, it is clear that stem cell transplantation is a viable option for neural regeneration. There is a clear clinical demand for stem cell therapy; interested stakeholders can find more information on stem cells for the treatment of neurological conditions at http://www.drfehlings.ca/stem-cell-information/. Further understanding of cells’ developmental lineages and elucidation of the factors that govern their individual and combinatorial functions in cell replacement, scaffolding, and trophic support will form the basis of the future of regenerative medicine. Closely controlled safety studies, using uniform, high-purity, ‘safe’ cell lines and optimizing route, timing, and dose, are crucial to minimizing adverse effects and to understanding these cells’ inherent mechanisms of repair. In chronic injury, it may be that exogenous NPC transplantation is not only required in combination with stimulation of endogenous NPC populations but also carried out in parallel with existing clinical management, as part of a multimodal and multidisciplinary approach. Clinical application of the tenets gleaned from basic research in this field will partly address some of these issues and further guide experimental and clinical studies to optimize application, safety, and the cost-benefit ratio.